THE CATHOLIC UNIVERSITY OF AMERICA

Ectopic Bone Matrix Mineralization: Unveiling the

Osteoinductive Nature of Crab Cuticle

A DISSERTATION

Submitted to the Faculty of the

Department of Biomedical Engineering

School of Engineering

Of The Catholic University of America

In Partial Fulfillment of the Requirements

For the Degree

Doctor of Philosophy

By

Tiffany Suella Omokanwaye

Washington, D.C.

2014

Ectopic Bone Matrix Mineralization: Unveiling the

Osteoinductive Nature of Crab Cuticle

Tiffany Suella Omokanwaye, Ph.D.

Director: Otto C. Wilson, Jr., Ph.D.

Large bone defects do not heal spontaneously and often require substitute materials. Ideally, a

bone replacement material should mimic bone tissue from a mechanical, chemical, biological and

functional point of view, and facilitate new bone formation. No single existing synthetic material

possesses all the necessary properties required in an ideal bone implant. Using biomimetic

principles and the kinship among biologically derived hard tissues, crustacean exoskeleton

emerged as a natural material for bone implant because of its similarities to bone in composition,

structure, and function. The purpose of this work is to serve as a preliminary investigation of the

role in which crab shell, from Callinectes sapidus or Chesapeake blue claw crab, can play in

bone healing. Soft tissue implantation studies, in rats, were used to investigate the osteoinductive

potential of the crab cuticle.

Crushed crab cuticle was subcutaneously implanted in the abdominal region of 28-day-old

Sprague-Dawley rats and aged for time periods ranging from 1-30 days by our collaborators at

Howard University. Tissue samples which grew in in the region of the crushed crab shell implant

were harvested and processed for microscopy and transmission electron microscopy (TEM)

analysis. This work focuses on characterizing the crystalline nature and physical characteristics

of the mineral phase which formed in the implant samples. Fascinating structures and

architectures were observed in TEM mode --- collagen fibers with the characteristic 67 nm

banding pattern, collagen bundles, fibroblasts, dark regions of crystal-like particles, and 20 x 40

nm nanocrystals. X-ray microanalysis of 20 x 40 nm nanocrystals showed an average

calcium:phosphorus ratio of 1.81 ± 0.37. Selected area diffraction (SAD) was initially used to

determine the degree of crystallinity of mineral phases. Dark electron-dense regions found

around collagen produced diffraction patterns indicative of amorphous solids. Upon further

inspection using high resolution transmission electron microscopy (HRTEM), approximately 2-4

nm crystalline-nano-building-blocks with lattice spacings of 0.95 nm were revealed.

Nanodiffraction was employed to investigate these 2-4 nm nano-structures with lattice spacings

of 0.95 nm in more detail. Nanodiffraction clearly indicated the particle was a single crystal.

Both the END pattern of the crystalline-nano-building-blocks and the SAD pattern of the 20 x 40

nm nanocrystal were both indexed and found to be of the apatite family. Compellingly, the SAD

pattern of the 20 x 40 nm nanocrystals displayed speckled rings made up of discrete spots. This

suggest that there are many oriented single crystals and that the larger crystals are made up of an

assembly of smaller single crystals. This gives evidence for the mesocrystal model of

crystallization for biologically derived hydroxyapatite (HAP). Arguably, our study is the first of

its kind to find biologically produced HAP crystals approximately 2-4 nm in size with evidence

they assemble to make larger HAP crystals based on the mesocrystal model.

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This dissertation by Tiffany Suella Omokanwaye fulfills the dissertation requirement for the

doctoral degree in Biomedical Engineering approved by Otto C. Wilson, Jr., Ph.D. as Director,

and by Patrick Mehl, Ph.D., Isabel k. Lloyd, Ph.D., Pamela L. Tuma, Ph.D., and Victor Frenkel,

Ph.D. as Readers.

–––––––––––––––––––––––––––––––––

Otto C. Wilson, Jr., Ph.D., Director

–––––––––––––––––––––––––––––––––

Patrick Mehl, Ph.D., Reader

–––––––––––––––––––––––––––––––––

Isabel K. Lloyd, Ph.D., Reader

–––––––––––––––––––––––––––––––––

Pamela L. Tuma, Ph.D., Reader

–––––––––––––––––––––––––––––––––

Victor Frenkel, Ph.D., Reader

iii

Dedication

This dissertation is dedicated to the following people:

the loving memory of my mother and best friend, Sandra Gertrude Hamilton, who

provided me unmeasurable love and support and who emphasized the importance of

education;

my patient husband and other best friend, Olushina Omokanwaye, who was extremely

supportive of my work and who endured many challenges and sacrifices while

completing this dissertation;

my awesome children, Mayowa, Lolade, Ayomide, Ekundayo, and Oladipo, for their

wholehearted attempt to be understanding while their mother spent so much time working

on this dissertation.

iv

Table of Contents Preface.......................................................................................................................................... xiii

Acknowledgements ..................................................................................................................... xvii

Chapter 1 Research Paradigm for Crab Cuticle Osteoinductive Potential ..................................... 1

Hypothesis................................................................................................................................... 1

Methodology ............................................................................................................................... 1

Rationale for using crab cuticle for bone implant study ......................................................... 1

Hypothesis Testing.................................................................................................................. 5

My role .................................................................................................................................... 6

Limitations ............................................................................................................................ 10

Hypothesis Implications........................................................................................................ 13

Chapter 2 Literature Review: Bone and Crab Cuticle and TRIZ .................................................. 15

Introduction ............................................................................................................................... 15

Ideal Bone Implant .................................................................................................................... 18

Biomimetics .............................................................................................................................. 18

Bone and Crab Cuticle Functional Similarities using TRIZ ..................................................... 24

Transferring Biology to Engineering ........................................................................................ 29

General Comparison of Bone and Crab Cuticle ....................................................................... 34

Concluding Remarks ................................................................................................................. 35

Chapter 3 Ties that Bind: Evaluation of Collagen I and α-chitin ................................................ 37

Introduction ............................................................................................................................... 37

Framework of chitin and collagen ............................................................................................ 39

Chitin Forms ............................................................................................................................. 40

Collagen Forms ......................................................................................................................... 42

Chemistry .................................................................................................................................. 43

Polysaccharides and Proteins .................................................................................................... 43

Protein and Polysaccharide Interactions ................................................................................... 48

Proteoglycans ............................................................................................................................ 51

Hierarchy................................................................................................................................... 57

Liquid Crystal Characteristics................................................................................................... 59

Synthesis ................................................................................................................................... 74

v

Properties .................................................................................................................................. 80

Applications .............................................................................................................................. 85

Characterization of Collagen and Chitin................................................................................... 88

Materials and Methods .......................................................................................................... 88

Elemental Analysis ............................................................................................................... 88

Zeta Potential Analysis ......................................................................................................... 89

Thermal Analysis (TGA) ...................................................................................................... 89

Surface Morphology (SEM) ................................................................................................. 89

Results ....................................................................................................................................... 90

Chemical analysis ................................................................................................................. 90

Thermal Analysis (TGA) ...................................................................................................... 93

Zeta potential ........................................................................................................................ 97

Scanning Electron Microscopy (SEM) ................................................................................. 99

Conclusion .............................................................................................................................. 101

Chapter 4 Tools of Biomineralization: Calcium Carbonate and Calcium Phosphate ................. 103

Introduction ............................................................................................................................. 103

Definition ................................................................................................................................ 105

Mineral Function/Strength/ Properties .................................................................................... 107

Biomimetics and biomineralization ........................................................................................ 111

Biomineralization Models ....................................................................................................... 113

Biologically Induced ........................................................................................................... 114

Mesocrystals model ............................................................................................................ 119

Brick and mortar model ...................................................................................................... 122

Cellular Role ....................................................................................................................... 122

Matrix Vesicles (MV) ......................................................................................................... 124

Amorphous Mineral Component ........................................................................................ 128

Mg stabilized amorphous precursors .................................................................................. 130

Effect of lipids/proteoglygans/alkaline phosphates ............................................................ 131

Biopolymers ............................................................................................................................ 131

Stages of Crystallization ......................................................................................................... 133

Crystallization Control ............................................................................................................ 137

Concluding Remarks ............................................................................................................... 139

vi

Chapter 5 Osteoinductive Nature of Blue Claw Crab ................................................................. 141

Introduction ............................................................................................................................. 141

Bone Grafts and Bone ............................................................................................................. 144

Osteoinduction, Osteoconduction, and Osteogenic ................................................................ 149

Ossification ............................................................................................................................. 152

Calcification/Mineralization ................................................................................................... 153

Inflammation ........................................................................................................................... 155

Experimental Section .............................................................................................................. 158

Materials ............................................................................................................................. 158

Crab integument preparation............................................................................................... 158

Characterization .................................................................................................................. 159

In vivo crab shell implantation ........................................................................................... 161

Some Preliminary Results ................................................................................................... 164

Analysis............................................................................................................................... 168

Particle Size ........................................................................................................................ 182

Nanodiffraction ................................................................................................................... 183

Indexing the nanodiffraction pattern ................................................................................... 189

Mathematical Method for Hexagonal ................................................................................. 190

Possible mechanism ................................................................................................................ 212

Concluding Remarks ............................................................................................................... 216

Chapter 6 Final Remarks ............................................................................................................ 219

References ................................................................................................................................... 224

vii

List of Figures

Figure 1-1 Composition percentages of bone (Chai, et al., 2012). ................................................ 4

Figure 2-1 Making an Ideal Bone Graft (Stocker & Wolinetz, 2009). ........................................ 17

Figure 2-2 Expanded description of the field of biomimetics (Sarikaya, 1994). ......................... 20

Figure 2-3 Biological (right column) and Engineering (left column) materials are very different

in the way they are developed (Allen, 2010), (Fratzl, 2007). ....................................................... 22

Figure 2-4 This table depicts bone and crab cuticle system, contribution, level or organization,

medium, parameters & interactions, and resource. (Bottom) This diagram ewxhibits a closer

inspection of the functions of bone and crab cuticle, conflicts to accomplishing those functions,

and the tricks or circumventions around those conflicts. .............................................................. 26

Figure 2-5 A contradiction describes the predicament caused by an attempt to improve one

property of a system that induces the degradation of another property (Stanley, Zlotin,

Bolckmans, & Zusman, 2005). ..................................................................................................... 28

Figure 2-6 How TRIZ works (Stanley, Zlotin, Bolckmans, & Zusman, 2005). .......................... 29

Figure 2-7 Schematic of steps to identify a function/concept/idea and to transfer the function

into engineering solution (Vincent, 2008), (Shimomura, 2010). .................................................. 31

Figure 2-8 Bone and Crab Shell Similarities. .............................................................................. 33

Figure 3-1 Diagram showing the direction of the polymer chains in the three crystallographic

forms of chitin. (a) α-chitin. (b) β-chitin. (c) γ-chitin (Wainwright, 1982). ................................. 41

Figure 3-2 Chemical Structure of Collagen showing the glycine, proline, and hydroxyproline

residues (Brodsky, Werkmeister, & Ramshaw, 2005). ................................................................. 44

Figure 3-3 Fragment of chitin chain (Ehrlich, 2010). .................................................................. 45

Figure 3-4 Arrangement of the protein subunits around the chitin core in the microfibril

perpendicular to the fiber axis and along the fiber axis. The 61 helix of protein subunits repeats in

3.06nm (Blackwell & Weih, 1980). .............................................................................................. 50

Figure 3-5 Proteoglycan aggregate (Kierszenbaum, 2007). ........................................................ 52

Figure 3-6 Hierarchical levels of collagen I in bone and α-chitin-protein matrix in crab

exoskeleton. Depicting points of differences in the types of bonds and triple helix structure of

collagen and linear structure of chitin. .......................................................................................... 58

viii

Figure 3-7 Representation of a typical liquid crystal model. ....................................................... 62

Figure 3-8 Depiction of rod-like liquid crystal phases: smectic, nematic, blue phase, and

cholesteric or chiral nematic. ........................................................................................................ 64

Figure 3-9 Illustration of aspects of lyotropic liquid crsytals. ..................................................... 65

Figure 3-10 Liquid crystalline organization of collagen at different concentrations. .................. 70

Figure 3-11 Collagen and Chitin Metabolism (Cohen, 1993). .................................................... 76

Figure 3-12 Chitin and Collagen Synthesis (Laurent, 1987). ...................................................... 77

Figure 3-13 Collagen and chitin unique features and ones they share. ....................................... 84

Figure 3-14 Collagen and Chitin/Chitosan Tissue Engineering Applications (Monzack,

Rodriguez, McCoy, Gu, & Masters, 2011). .................................................................................. 86

Figure 3-15 Other Biomedical Applications. In the diagram some of the collagen applications

are highlighted in yellow (Prashanth & Tharanathan, 2007). ....................................................... 87

Figure 3-16 Thermogram of Collagen and Chitin. ...................................................................... 95

Figure 3-17 Zeta potential (ξ) of collagen. Collagen trend line has been taken from (Andrade,

Ferreira, & Domingues, 2004). ..................................................................................................... 98

Figure 3-18 Chitin from Crab Exoskeleton SEM Micrographs. ............................................. 100

Figure 3-19 Collagen from Bovine Achilles Tendon SEM Micrographs ............................... 101

Figure 4-1 The materials property chart (Dunlop & Fratzl, 2010). ........................................... 108

Figure 4-2 Biomineral crystallographic properties are highly regulated during biomineralization.

These properties provide some indication of potential processing strategies (Gower, 2008). ... 110

Figure 4-3 There exist three primary divisions of biomimetic materials chemistry inspired by

biomineralization studies (Mann, 1995). .................................................................................... 113

Figure 4-4 Schematic of the mineralized collagen fibrils, the basic constituents of bone.

Nanocrystals of HAP are incorporated between collagen molecules (Dorozhkin & Epple, 2002).

..................................................................................................................................................... 118

Figure 4-5 Illustrative representation of classical and non-classical crystallization (Xu, Ma, &

Colfen, 2007). ............................................................................................................................. 121

ix

Figure 4-6 An illustration of the role of inorganic and organic constituents in controlling

biomineral formation (Gower, 2008). ......................................................................................... 123

Figure 4-7 Diagram outlining a proposed mechanism for bone mineral formation

(Boonrungsiman, et al., 2012)..................................................................................................... 125

Figure 4-8 Hierarchical structural organization of bone: (a) cortical and cancellous bone; (b)

osteons with Haversian systems; (c) lamellae; (d) collagen fiber assemblies of collagen fibrils;

(e) bone mineral crystals, collagen molecules, and non-collagenous proteins (Rho, Kuhn-

Spearing, & Zioupos, 1998). ....................................................................................................... 136

Figure 5-1 Schematic of the design strategy of tissue-engineered biomimetic nanocomposite

bone graft (Murugan & Ramakrishna, 2007). ............................................................................. 146

Figure 5-2 Depiction of the past, present and future of tissue repair (Hench, 1998). ................ 147

Figure 5-3 Diagram illustrating hypothesized mechanisms behind osteoinduction by

biomaterials. Physico-chemical and/or structural properties of osteoinductive biomaterials may

prompt the mechanism responsible for heterotopic bone formation (Barradas, Yuan, van

Blitterswijk, & Habibovic, 2011)................................................................................................ 154

Figure 5-4 Schematic depicting the material, interface, and biological enviroment components.

It emphasizes the importance of the interface. ............................................................................ 156

Figure 5-5 The implantation study has an aim to assess the histological response to the crab

cuticle and determine its potential as as a bone biomaterial. ...................................................... 162

Figure 5-6 Transmission electron microscopy image of in vivo study of demineralized crushed

Calinectes Sapides (Blue Claw) crab shell implanted subcutaneously in abdominal region of 28

day old Sprague–Dawley rats. Aging time was one day and image displays a crab shell particle

in the bottom of the image with a macrophage in close proximity (Wilson, Gugssa, Mehl, &

Anderson, 2012). ......................................................................................................................... 164

Figure 5-7 TEM pictures depicts macrophages surrounding the Bouligand structure of

demineralized crushed Calinectes Sapides (Blue Claw) crab shell (Wilson, Gugssa, Mehl, &

Anderson, 2012). ......................................................................................................................... 165

Figure 5-8 Transmission electron microscopy image of collagen bundles that formed after

subcutaneous implantation of demineralized crab shell for 6 days. The image shows 67 nm

banding pattern and regions of electron dense mineralized particles on the collagen fiber surface

and interspersed in between collagen fibers (Wilson, Gugssa, Mehl, & Anderson, 2012). ....... 166

Figure 5-9 (a) SEM micrograph and a schematic drawing of a cross-sectional surface showing

three different layers in the crab exoskeleton: epicuticle, exocuticle, and endocuticle (Chen, Lin,

McKittrick, & Meyers, 2008); (b) TEM micrograph of demineralized crushed Calinectes Sapides

x

(Blue Claw) crab shell displaying prominent Bouligand nested arc patterns and a schematic

drawing of the Bouligand structure showing the arc pattern on oblique surface. ....................... 169

Figure 5-10 Transmission electron microscopy image of collagen bundles that formed after

subcutaneous implantation of mineralized crab shell for 28 days. Similar to the image of

(Wilson, Gugssa, Mehl, & Anderson, 2012), it depicts collagen fibers with the characteristic 67

nm banding pattern. .................................................................................................................... 170

Figure 5-11 Transmission electron microscopy image Collagenous fibers, collagenous bundles,

and fibroblasts. Fibers oriented in longitudinal directions exhibit the characteristic 67 nm

banding pattern of collagen. Lying between regions of many collagen bundles and fibers,

fibroblasts often present an elongated form. Fibrocytes are essentially mature fibroblasts.

Fibrocytes can easily be recognized as such by their dense nucleus, long and thin cytoplasmic

processes and scarcity of cell organelles (Ebe & Kobayashi, 1972). ......................................... 171

Figure 5-12 Transmission electron microscopy image depicting collagenous fibers, collagenous

bundles, and fibroblasts. Fibers oriented in longitudinal directions exhibit the characteristic 67

nm banding pattern of collagen. Lying between regions of many collagen bundles and fibers,

fibroblasts often present an elongated form. Fibrocytes are essentially mature fibroblasts.

Fibrocytes can easily be recognized as such by their dense nucleus, long and thin cytoplasmic

processes and scarcity of cell organelles (Ebe & Kobayashi, 1972). ......................................... 172

Figure 5-13 Micrograph depicting regions of selected area diffraction. This image contains

collagenous fibers, fibroblasts. ................................................................................................... 176

Figure 5-14 (a) Selected area diffraction (SAD) coincides with 0021 SAD region from figure

13. (b) ) Selected area diffraction (SAD) coincides with 0023 SAD region from figure 13. (c)

Selected area diffraction (SAD) coincides with 0025 SAD region from figure 13. (d) Selected

area diffraction (SAD) coincides with 0028 SAD region from figure 13. All of these SAD images

display broad or diffuse rings which indicate amorphous-like phase. ........................................ 177

Figure 5-15 (a) Transmission electron microscopy image of dark region adjacent to collagenase

matrix and (b) its SAD pattern which shows broad diffuse rings that are indicative of a

amorphous solid. ......................................................................................................................... 179

Figure 5-16 SAD pattern of dark region adjacent to collagenase matrix depicted in figure 5-15

which shows broad diffuse rings that are indicative of a amorphous solid. ............................... 180

Figure 5-17 HRTEM image of potential Carbonated HAP depicting the short range or local

order. ........................................................................................................................................... 184

Figure 5-18 HRTEM image of small-size particle. Determined that nanosized particles of

different size preserve stoichiometric HA-like crystal structure (Biggemann, Prado da Silva,

Rossi, & Ramirez, 2008). ............................................................................................................ 185

xi

Figure 5-19 HRTEM nanodiffraction image of potential Carbonated HAP depicting a single

crystal. ......................................................................................................................................... 188

Figure 5-20 TEM image of possible polycrystalline HAP with interplanar or lattice spacings 197

Figure 5-21 SAD pattern of a polycrystalline HAP ................................................................ 198

Figure 5-22 Elemental Analysis with Weight and Atomic Percentages of oxygen, phosphorus

and calcium. ................................................................................................................................ 203

Figure 5-23 Elemental Analysis with Weight and Atomic Percentages of oxygen, phosphorus

and calcium. ................................................................................................................................ 204

Figure 5-24 Schematic diagrams representing the HAP unit cell (Menéndez-Proupin, et al.,

2011) ........................................................................................................................................... 206

Figure 5-25 Schematic depiction of one hexagonal building unit of HAP (Li, et al., 2007). ..... 210

Figure 5-26 Black lines connect Ca(I) columns in hexagonal networks. Cyan and magenta

triangles connect staggered Ca(II) atoms (Xia, Lindahl, Lausmaa, & Engqvist, 2011), (Boanini,

Gazzano, & A, 2010). ................................................................................................................. 211

Figure 5-27 The HAP unit cell has symmetry and order. Table of average distances of atom shell

from calcium atom via x-ray and EXAFS (Harries, Huskins, & Hasnain, 1986). ...................... 211

Figure 5-28 Diagram for wound healing response to crushed crab carapace (Falanga, 2005),

(Salgado, et al., 2011). ................................................................................................................ 213

xii

List of Tables

Table 1-1 Some osteoinduction papers. Only first author listed. ............................................... 3

Table 1-2 Thickness of skin strata in rat, mice and humans (Godin & Touitou, 2007) .......... 12

Table 3-1 Principal Components of Crustaceans (Crab) Exoskeleton and Bones ................... 38

Table 3-2 Key Articles that Highlight Collagen and Chitin .................................................... 40

Table 3-3 Some Macromolecules (polymers) and their primary biological functions (Smith &

Wood, 1991). ................................................................................................................................ 45

Table 3-4 Molecular Weight of Chitin and Collagen .............................................................. 47

Table 3-5 Description of Cholesteric Geometries ................................................................... 71

Table 3-6 Collagen and chitin empirical formula, tissue distribution, microscopic appearance,

ultrastructure, synthesis site, interaction, and function. ................................................................ 82

Table 3-7 Elemental Composition of Collagen. ...................................................................... 93

Table 4-1 Examples of the Diversity of Biominerals (Gower, 2008). ................................... 106

Table 4-2 Characterization of Biologically Controlled Extra-, Inter-, and Intra-cellular

Mineralization ............................................................................................................................. 116

Table 4-3 Designation of layers in the crab cuticle .............................................................. 137

Table 5-1 The composition of the inorganic and organic phases of bone ............................ 145

Table 5-2 Term [A] calculation for various values of hk ..................................................... 193

Table 5-3 Calculation of l2 and l2/(c/a)2 ................................................................................ 194

Table 5-4 Evaluation of Peaks. ............................................................................................. 194

Table 5-5 Allowed hkl and calculated d spacings ................................................................ 195

Table 5-6 d spacing values calculated for crystalline harvested sample. a (Fleet, Liu, & King,

2004). b (Wilson, Elliot, & Dowker, 1999) ................................................................................. 201

xiii

Preface

Bone tissue engineering seeks to regenerate the lost or damaged tissue by making use of the

interactions between cells and biomaterials. Bone formation is a complex process on which

inorganic biomineral (calcium phosphate) precipitation seems to be associated, initially, with

matrix vesicles and subsequently with organic collagen I fibers (Anderson, 1989). For large bone

defect repair, bone formation far from the host bone bed should occur by osteoinduction, a kind

of bone formation that does not start directly from osteogenic cells. Osteoinductive biomaterials

provide a good environment for cells to form bone (Reis & Weiner, 2004). Therefore, one of the

main goals for bone tissue constructs, when implanted in vivo, is to promote osteoinduction.

Bone formation, in soft tissues where no osteogenic cells exists, gives the true indication of

osteoinduction. This type of bone formation is called ectopic bone formation, heterotopic bone

formation, or heterotopic ossification (Reis & Weiner, 2004).

Skeletal conditions are becoming an increasing health concern in the aging population

(Giordano, Sanginario, Ambrosio, Silvio, & Santin, 2006), and reconstruction of bone defects is

one of the major therapeutic goals in various clinical fields. Clinicians and physical, biological,

and material scientists and engineers strive to develop bone biomaterials with novel properties

that perform like natural bone. The composition and nanostructure are believed to influence

biomaterial and biological environment interactions. Some novel materials are first designed

through exploratory research; next their properties are determined; and then potential uses are

identified. This is typically known as discovery based product development. However, the

scientific world is progressing toward a more application based model where the existing needs

are assessed and a design is established to address specific needs. Despite the method,

xiv

biomaterial design is based on an understanding of the biological material being replaced, the

material used as the replacement, and the material/ biological interface. Biological systems such

as bone and crustacean integument have many length scales of fundamental and structural

significance. Bone and crustacean integument are both naturally derived nanocomposites that

share unique attributes which can be used as sources for many lessons. As lessons are revealed,

crab cuticle emerges as an inspiration for developing functionally advanced biomaterial implants

(FAB). In Chapter 1, biomimetics and design concepts of TRIZ (Theory of Inventive Problem

Solving) are discussed highlighting the functional and general similarities of bone and crab

integument.

Bone and crustacean cuticle are biological materials. The term biological material is very

common, because it includes organic phases, inorganic phases, and composites of both organic

(e.g., protein–protein, protein–polysaccharide) and inorganic (e.g., mineral–protein, mineral–

polysaccharide) phases with amazing diversity of forms, shapes, and dimensions. Bone is a

composite that consist of organic and inorganic phases, collagen and hydroxyapatite,

respectively. Crustacean cuticle is also a composite that consist of organic and inorganic phases,

chitin and calcium carbonate, respectively. Bone and arthropod cuticle possess astonishingly

similar principles in organization: the ability to self-assemble; production of fibrillar and fiber-

like structures with hierarchical organization from nano- up to macro- levels; the capability to

perform the role of scaffolds; and the capacity to serve as templates for biomineralization and

formation of the rigid skeletal structures (Ehrlich H. , 2010). Chapter 2 compares the chitin and

collagen, the organic phases of crab cuticle and bone tissue. Chapter 3 surveys calcium

carbonates and calcium phosphate, the inorganic phases of crab cuticle and bone tissue, as tools

xv

of biomineralization. Thus, chapters 1-3 provides an understanding of biomimetics, TRIZ, the

inorganic-organic phases of bone tissue and how this awareness can be utilized to develop FAB.

Chapter 4 focuses on a general biocompatibility study to determine crab cuticle’s suitability

as a bone substitute material. Natural bone uses osteogenic (progenitor osteoblasts or osteocytes)

cells; osteoconductivity (structural support system); and osteoinductivity (such as growth factors)

to produce a mineralized collagenase matrix that hardens (Stocker & Wolinetz, 2009). Ideally, a

bone replacement material should mimic bone tissue from a mechanical, chemical, biological

and functional point of view, and facilitate new bone formation. No single existing synthetic

material possesses all the necessary properties required in an ideal bone implant. Emerging as

valid therapy approach, bone tissue engineering is based on understanding hard tissue formation

and targets induction of new functional tissues. Using inspiration from Nature and exploiting the

kinship that exists among biologically derived hard tissues, crustacean exoskeleton emerged as a

natural material, similar to bone in composition, structure, and function. The crab carapace is a

candidate material for enhancing the healing, remodeling, and engineering of bone.

The basic premise is that the properties and components of crab cuticle, specifically, the blue

claw crustacean, Callinectes sapidus, found in the Chesapeake Bay, satisfy a number of criteria

to make it a successful bone substitute material. Chapter 4 describes the result of an implantation

study in which crab cuticle has been harvested from Sprague-Dawley rats and analyzed to

provide evidence of mineralized collagen matrix formation in an attempt to assess crab shell’s

osteoinductive nature, bioactivity and viability as a bone material substitute. Special emphasis is

given to the characterization techniques transmission electron microscopy (TEM), high-

xvi

resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), electron

nanodiffraction (END), and electron microanalysis - energy-dispersive spectrometry (EDS).

The final chapter, the epilogue, offers concluding remarks and discusses future directions.

Drawing inspiration from natural materials and examining the process involved in bone

morphogenesis, this research examines the possibility that crab exoskeleton is an osteo-friendly

material with promising bone bioactivity. This dissertation aims to illuminate the osteoinductive

attributes of blue claw crab, Callinectes sapidus, cuticle and to provide the first general

biological performance profile about the suitability of crab cuticle as a bone biomaterial. This is

a novel contribution to the fields of biomimetics, biological material science, orthopedic

medicine, and bone tissue engineering.

xvii

Acknowledgements

Foremost, I would like to thank my God and Savior Jesus Christ for giving me the strength,

wisdom, and perseverance to complete this dissertation.

The completion of this dissertation would not have been possible without the love and support

of my entire family --- parents, aunties, uncles, cousins and friends. Thanks for all the prayers,

for all the words of encouragement, and for all the hours of babysitting. I am especially grateful

for my dad, Sam Hamilton, Jr., for assisting me in his own unique ways. I am particularly

thankful for my Aunties Sinobia Gilliard and Wilhelmena Middleton and Uncle Sam Middleton

for constantly checking on me, listening to me, and helping me in whatever ways I asked.

I thank my dissertation committee led by Dr. Otto C. Wilson, Jr. No words can express how

much I truly appreciate your guidance and direction throughout this process. I am very thankful

for your patience and for your belief in me. My readers, Dr. Mehl, Dr. Lloyd, Dr. Tuma and Dr.

Frenkel, I thank you for your invaluable feedback. I am also very appreciative for Dr. Kilic and

Dr. Namazi serving as secretary and chair.

A special thanks to Dr. Anderson and Dr. Gugssa of Howard University for their invaluable

collaboration. Thank you for the use of your laboratory, animals, supplies, and instrumentation. I

am truly indebted for all of your input, feedback, direction and most importantly time.

The authors wished to thank the Nanoscale Imaging, Spectroscopy, and Properties (NISP)

Laboratory at the Kim Engineering Building at the University of Maryland at College Park, Dr.

Lai and Dr. Shang with my data analysis. We wish to thank Dr. Lloyd for the use of Schimadzu

TGA-50The financial support was granted by NSF DMR Biomaterials Grant # DMR-0645675.

1

Chapter 1 Research Paradigm for Crab Cuticle Osteoinductive

Potential

Hypothesis

My initial research question focused on the issue of whether crab shell possess the ability to

induce bone formation in an osteoinductive manner. As my investigation progressed, my

hypothesis developed along the lines of identifying one of the key features in hard tissue

formation: Mineralization. My hypothesis can be stated as follows. Can the electron dense

regions containing nanophase material which form around the collagen-like fibers produced

after implantation of crushed crab cuticle be identified as a biomineral phase related to bone

formation? The rest of my work focused on this aspect.

Methodology

Rationale for using crab cuticle for bone implant study

Large bone defects do not heal spontaneously and often require substitute materials. Bone is

osteoconductive and osteoinductive with osteogenic cells and this combination produces a

collagen matrix that mineralizes and hardens. Ideally, a bone replacement material should mimic

bone tissue from a mechanical, chemical, biological and functional point of view, and facilitate new

bone formation. No single existing synthetic material possesses all the necessary properties required

in an ideal bone implant.

Using biomimetic concepts, TRIZ and insight from previous studies, crab cuticle surfaced as

contender for another xenograft solution for the healing of large bone defects. Biomimetic and TRIZ

2

help to illuminate crab cuticle similarities to bone in regard to composition, structure, and

function. A literature search unearthed a wealth of information that provided even more

inspiration and credence to the possibility of crab cuticle prospects in bone healing.

The ancient Mayan practice of using nacre as bone implant material (Westbroek & Marin,

1998) serve as one source of inspiration. The literature revealed the use of other marine

organisms for dental and bone implants (Demers, et al., 2002). Additionally, derivatives of crab

shell such as chitin/chitosan have been used in bone tissue engineering investigations. Urist’s

(1965) discovery that demineralized bone matrix induced new bone formation when implanted

intramuscularly certainly peaked our interest. Urist and Strates (1971) presented ‘osteoinduction’

to the scientific and medical communities. Reddi and Anderson (1976) further expounded on

Urist's work and provided a compelling explanation of the functional role of purified organic

bone matrices (Gruskin, Doll, Futrell, Schmitz, & Hollinger Jeffrey, 2012).

Natural bone uses osteogenic (progenitor osteoblasts or osteocytes) cells; osteoconductivity

(structural support system); and osteoinductivity (such as growth factors) to produce a mineralized

collagen matrix that hardens (Stocker & Wolinetz, 2009). Crab exoskeleton has many similarities

to bone, but it does not have osteogenic cells. Osteoconduction relates to the ability to support

bone growth on a structure and our crushed crab shell in our experiments was not going to serve

a scaffold. Osteoinduction relates to the ability to create an in vivo miceroenvironment that

promotes new bone formation. Therefore, osteoinduction is essential to new bone formation and

made it the logical test choice.

Bone formation, through osteoinduction, does not start directly from osteogenic cells and

bone formation at ectopic sites has been found after implantation (Huggins, 1931), (Bertelsen,

3

1944), (Urist & McLean, 1952). Urist continued his groundbreaking research on osteoinduction

and later with the assistance of Strates came to the conclusion that protein, in particular bone

morphogenetic protein (BMP), was involved in the cascade of chemotaxis, mitosis,

differentiation, and finally bone formation (Urist & Strates, 1971).

There have been various publication that have illustrated osteoinduction in many animals by

diverse calcium phosphate biomaterials. Some of these papers are listed below in Table 1-1:

Table 1-1 Some osteoinduction papers. Only first author listed.

Author Title Year

Yamasaki

Ripamonnti

Yang

Ripamonnti

Yuan

Habibovic

Ripamonnti

Heterotopic bone formation around porous

hydroxyapatite ceramics in the subcutis of dogs

The Induction of Bone in Osteogenic Composites of

Bone Matrix and Porous Hydroxyapatite Replicas:

An Experimental Study on the Baboon

Osteogenesis in extraskeletally implanted porous

calcium phosphate ceramics: variability among

different kinds of animals

Osteoinduction in Dorous hydroxyapatite implanted

in heterotopic sites of different animal models

Bone formation induced by calcium phosphate

ceramics in soft tissue of dogs: a comparative

study between porous a-TCP and b-TCP

Osteoinductive biomaterials – properties and

relevance in bone repair

The induction of bone formation by coral-derived

calcium carbonate/hydroxyapatite constructs

1990

1991

1996

1996

2001

2007

2009

4

It could be concluded that osteoinduction is a general phenomenon (Yuan & Groot, 2004).

In 1969, Winter and Simpson (1969) observed bone induction by a sponge consisting of

polyhydroxyethyl methacrylate (poly-HEMA) in the soft tissue of pigs. These researchers noted

that the implanted sponge became calcified prior to bone formation in both pigs and rats. Bone

induction by the polymeric sponge cannot be explained by Urist’s BMPs theory. Crab shell like

this synthetic sponge do not contain nor can they produce BMPs. Thus, this suggest calcification

might play a role in the process of osteoinduction (Habibovic & de Groot, 2007).

The mineral phase makes up approximately 60% bone, figure 1-1. Bone matrix calcification

or mineralization is a critical stage of bone formation. Coupled with probable role of

calcification in osteoinduction and mineral percentage of bone, the presence of mineral became

an integral part of my hypothesis.

Figure 1-1 Composition percentages of bone (Chai, et al., 2012).

5

The biomimetic principles, TRIZ analysis, and literature review, help to solidify the notion

that crab shell might have osteoinductive potentials worth exploring.

Hypothesis Testing

There was a need for a relatively simple model in which osteoinductive potential could be

tested in animals. Across species, numerous general cellular properties are common in all

organisms. With each animal model having unique advantages and disadvantages, no single

animal model would be appropriate for all purposes, nor can a model be dismissed as

inappropriate for all purposes (Pearce, Richards, Milz, Schneider, & Pearce, 2007). The rat

model was attractive and selected based on factors such as the size of the material to be

implanted; cost to acquire and care for animals; tolerance to captivity and surgery; ease of

handling and housing; low maintenance care; resistance to infection and disease; adequate

facilities and support staff; and an existing database of biological information for the species.

The rat model would serve as an approximation of the physiological human clinical condition.

The rat subcutaneous tissue implant site has proven to be a high-through-put, relatively low-cost

screening technique for testing the initial tissue response to new materials.

Bone formation by osteoinduction is initiated by soluble and insoluble signals that trigger a

complex cascade of molecular and cellular morphogenetic processes that ultimately leads to the

sculpture of precisely organized mineralized structures. Bone formation, in soft tissues where no

osteogenic cells exists, gives the true indication of osteoinduction (Yuan & Groot, 2004).

Therefore, a pilot study, using the rat subcutaneous tissue model, offered a rapid, cost-effective

6

means of indicating how crab shell interacts with a living system and how this interaction

produced the desired expectation of bone formation.

The implantation studies were conducted at Howard University with the assistance of Dr.

Gugssa under the guidance of Dr. Anderson. Approximately 48 rats were used to address the

question of crab shell influencing bone formation using an osteoinductive model involving

subcutaneous implantation of crushed crab shell. The focus of my study involved analyzing

tissue samples from the crushed crab cuticle implant region that had been aged for 28 days, n =

2. A detailed account of implantation study is provided in the Experimental Section of Chapter 4.

My role

To understand the ramifications of this harvested tissue, the knowledge was acquired about

the similarities between crab cuticle and bone and their constituent parts. My literature search

uncovered many shared characteristics of bone and crab cuticle. Biomimetics and TRIZ helped

illuminated and communicate these similarities.

To get a better understanding of bone and crab shell, their compositions were explored and

some properties were characterized. I performed scanning electron microscopy (SEM), zeta

potential, and thermogravimetric analysis of the organic constituents of bone and crab cuticle.

Carbon, hydrogen, and nitrogen (CHN) analysis were performed by an outside lab, Prevalere

Life Sciences. SEM furnished information about the sample’s surface topography and

composition. Zeta potential analysis was used to ascertain the stability of colloidal dispersions of

7

chitin. TGA were done to determine the thermal decomposition and degradation behavior of our

samples. The CHN analysis provided information such as the purity of our sample.

A technician, Dr. Lai, assisted me with the preparation of samples and operated the SEM

during my sessions at the NISP Laboratory at the Kim Engineering Building at the University of

Maryland at College Park analysis. I communicated the magnification and the areas on the

sample that I wanted to observe. Dr Lai took and saved micrographs I wanted to analyze later.

Some of these images and analysis are reported in Chapter 2.

The zeta potential measurements were performed at Catholic University in Dr. Wilson’s

B.O.N.E. C.R.A.B lab. pH was varied systematically from about 2 to 12 in order to determine

how it influences the ζ-potential of chitin. The data was plotted and the analysis is reported in

Chapter 2.

After training, I performed TGA analysis at Dr. Lloyd’s lab at University of Maryland at

College Park. The data was plotted and the analysis is reported in Chapter 2.

Transmission electron microscopy (TEM) offered broad range of characterization techniques

with high spatial and analytical resolution that could be done on the same small sample. I was

charged/commissioned to create and analysis TEM grids samples from the harvested rat tissue in

the region of crushed crab shell implantation.

I participated in cutting some of the 90 nm samples at Howard University after training was

provided by Dr. Gugssa. The main task of my work was to evaluate the samples for

mineralization. Using a Leica Microsystems Instrument outfitted with a diamond blade, at a

speed of 3-4 mm/s and an approach of 0.5 µm, 490 nm samples were cut. 6-8 490 nm samples

were placed on a slide that contained a drop of water and placed on a hot plate at about 30°C to

8

let dry. The dried samples were stained with 25 % Basic Fuchsin Toludine Blue and allowed to

dry on the hot. The samples were rinsed and allowed to dry again on the hot plate. The stained

dried samples were checked under an optical microscope to verify that a viable sample was

obtained. After a section of viable samples was reached, 90 nm slices were generated using the

same above procedure. 90 nm samples were placed on TEM grids.

Technicians, Dr. Lai and later Dr. Shang, assisted me with the preparation of samples and

operated the TEM during my sessions at the NISP Laboratory at the Kim Engineering Building

at the University of Maryland at College Park analysis. I communicated the technique I wanted

to employ, the magnification and the areas on the sample that I wanted to observe. Dr Lai and

later Dr. Shang took and saved micrographs I wanted to analyze later. Some of these images and

analysis are reported in Chapter 4.

I examined many of the TEM grids produced from our pilot study for many hours. I began to

focus on the TEM grids retrieved from the subcutaneous abdominal region of the rat implanted

with the mineralized crushed crab shell because of the interesting structures and particles

observed. Specifically after observing regions of collagen-like structures with the characteristic

67 nm banding pattern, the main question was are these structures mineralized?

I utilized high resolution transmission electron microscopy (HRTEM), selected area

diffraction (SAD), electron nanodiffraction (END), electron microanalysis - energy-dispersive

spectrometry (EDS) at the NISP characterization facility at the University of Maryland. These

techniques assisted me in answering the mineral presence question.

SAD was used in many regions. SAD can be useful for the determination of lattice parameter

information and unit cell details. Lattice structure gives information about the long range

9

composition of a material. Restrictive factors of SAD are that a good crystal must be found for

high accuracy of crystallographic structures and that only limited information about the

structure’s dynamic behavior can be obtained from one single diffraction experiment. Resolution

is an important limitation of SAD. Small particle size can lead to peak broadening and erroneous

interpretation of a SAD pattern (Ferrell Jr. & Paulson, 1977).

In an effort to locate regions of potential mineral formation, dark sections adjacent to

observed collagen bundles were evaluated. The images depicted a region that appears to harbor

material with crystalline characteristics. To assess its crystalline nature, SAD was performed.

The SAD pattern, displayed diffuse rings. This is typically indicative of amorphous solids.

However, using HRTEM clearly showed lattice spacing with long range order despite the fact

the reflections observed in selected area electron diffraction pattern exhibited broad peaks or

diffuse rings. Once I became aware of the nanodiffraction, Dr. Shang was asked to perform this

technique on the samples that had visible long-range order in HRTEM images even though their

SAD patterns suggested they were amorphous. Nanodiffraction offered an avenue to characterize

the ultrastructure of these nanocrystals. The nanodiffraction image depicted a single crystal. This

established that the nanosized matetial had crystalline characteristics. The nanodiffraction pattern

was indexed with the help of computer software and it was determined that the regular pattern of

widely spaced strong spots came from the apatite family.

I also communicated other areas to perform x-ray microanalysis and SAD. Plate shaped nano

-sized particles with lengths and widths of approximately 40 x 20 nm were observed with lattice

spacing measuring 0.95 nm. X-ray microanalysis was performed on the 40 x 20 nm nano

particles. This revealed an average calcium:phosphorus ratio of 1.81 ± 0.37. SAD was taken of

10

the observed nanocrystals, indexed, and determined to be of the apatite family. Moreover, this is

a very interesting diffraction pattern because it displays speckled rings made up of discrete spots.

This suggest that there are many oriented single crystals in this SAD pattern. One can surmise

that this diffraction pattern displaying many oriented single crystals is evidence for the

mesocrystal model of crystallization. Highly oriented subunits distinguish a mesocrystal from a

randomly oriented polycrystal, and the identifiable nano-sized building units distinguish it from a

single crystal. Often, it is difficult to distinguish a mesocrystal from a single crystal because the

mesocrystal shows an identical scattering pattern and behavior to a single crystal. Mesocrytsals

can be indexed like a single crystal. Therefore, it is difficult to define the border between a single

and mesocrystalline substance. However, mesocrystals were shown to be intermediates between

a classical single crystal and a polycrystal.

Limitations

Obvious strengths of a small pilot study are that the research question can be addressed in a

relatively short space of time, obtaining ethical and institutional approval is easier in small

studies, and it avoids spending too many resources. The main problem with small studies is

interpretation of results, in particular confidence intervals and p-values. Ideally all intervals

should be as narrow as 95%, but usually only large studies can produce such precise results.

Therefore, the data must be interpreted and reported carefully. The lack of statistical significance

does not mean there is no effect. There needs to be a prudent balance between not dismissing

outright what could be a real effect and also not making undue claims about the effect. While

11

small studies can provide results quickly, they do not normally yield reliable or precise estimates.

It is important not to make strong conclusions. Data from small studies should be used to design

larger confirmatory studies (Hackshaw, 2008) making methodical, logistical and financial

estimates. Pilots are rapidly becoming an essential pre-cursor. While there are weaknesses, this

pilot will be used to streamline our study to reduce the waste of resources and time.

No one animal model fully authenticates the processes occurring in human tissue repair. Only

humans can be used to reflect accurately the events of human healing, but even human studies

are subject to the pitfalls of laboratory methods and genetic or environmental variables that may

affect the results (Cohen & Mast, 1990). There is a great deal of concern regarding the ability to

transfer animal research data to the human clinical situation. Rats and humans share the

following skin characteristics: the presence of an epidermis, basement membrane, hair follicles,

and dermis. Among rodents, rat skin has more structural similarities to human tissue. Regarding

the rat skin, permeation kinetic parameters are frequently comparable with human skin, table 1-2

(Godin & Touitou, 2007). Obviously, there are numerous anatomical and physiological

differences between human and rat. Among the differences is the fact that rats do not form

keloids or hypertrophic scars but people of certain ethnic backgrounds, such as African-

Americans and Asians, are predisposed to excessive scarring (Dorsett-Martin & Wysocki, 2008).

Although most mammalian species simulate human healing with collagen deposition as a

predominant feature, the healing process in these animals is certainly not identical to humans.

Another difference in healing between animals and humans is their nutritional requirements.

Unlike humans, who require a dietary supply of ascorbic acid, rats are able to synthesize this

12

Table 1-2 Thickness of skin strata in rat, mice and humans (Godin & Touitou, 2007)

important cofactor needed for the production of collagen. (Cohen & Mast, 1990). It must be

recognized that humans to whom the results are being extrapolated are genetically highly

variable due to cultural, dietary, and environmental differences (Rand, 2008).

Characterizing the structure that were assessed to be collagen based on their characteristic

banding pattern was beyond the scope of my work and would not have provided insight on

mineral presence. The presence of mineral would distinguish the collagen production in this

study from typical wound healing collagen.

The amount of tissue available from harvested tissue is often a limiting factor. The limited

amount of harvested tissue available makes performing various test on each sample an issue. It

would have difficult to perform biological assays such as staining for calcium using alizarin red s

and the von Kossa technique and create TEM grids with such a small amount of harvested tissue

available. Additionally, it is nearly impossible to excise these wounds for any analysis without

including some surrounding normal skin. Therefore, contamination of harvested tissue by

uninjured skin can add a degree of inaccuracy in the evaluation.

Standardization in reporting, could facilitate comparisons and may initiate additional research

that favors the inevitable comparisons between studies. This increased knowledge base would be

13

vital in transferring animal-derived data to human clinical situations (Dorsett-Martin & Wysocki,

2008).

Hypothesis Implications

This was part of a preliminary study. Despite its exploratory nature, this study offers some

insight into osteoinductive potential of the crab cuticle. Conclusive evidence for osteoinduction

is characterized by heterotopic bone formation, that is, bone formation in tissues or organs where

bone does not naturally grow. Conclusive evidence for osteoinduction was not provided in this

study. No firm conclusions were made about crab shell’s osteoinductive potential. Although the

current study is based on a small sample of participants, the findings suggest the following:

The degree of crystallinity changes as bone matrix matures, can be masked by the size of

the crystal, and varies with location;

The results from TEM, HRTEM, XRD, SAD, END, and EDS, in this study indicate that

crushed shell from C. sapidus exhibits some unique interactions in vivo using Sprague–

Dawley rats;

The study indicates that crab cuticle in vivo interactions can result in the formation a

matrix containing collagen and mineral components;

These results further suggest that crab shell may be suitable for use in developing

functionally advanced bone implants;

Since bone formation can take months, this material warrants further study to ascertain its

potential osteoinductive properties fully.

14

Therefore, through the presented hypothesis, we suggest that that crab cuticle may be an

effective material for stimulating bone growth. Further extensive studies are warranted on large

randomized controlled trials to further assess the possible application and provide more

definitive evidence.

15

Chapter 2 Literature Review: Bone and Crab Cuticle and TRIZ

Introduction

Bone and crab cuticle are two dynamic and vital biomineralized composite structures. At first

glance, there appears to be more dissimilarities among these two tissues but when one takes a

closer look, there is an extensive array of similarities and compelling relationships. An

understanding of these similarities and relationships can point to a rich and abundant source of

inspiration for development of functionally advanced biomaterial (FAB) implants.

Large bone defects resulting from a multitude of causes, including aging, trauma, deformity,

and disease, are commonplace. These defects usually do not heal spontaneously and

interventions with bone grafts are often required. Bone grafts are substitute material that is

transplanted to the defective bone area to aid in healing, strengthening or improving function.

The clinical gold standard and preferred method for bone repair is grafts from the individuals

themselves, an autologous graft. Although effective and safe due to the low risk of disease

transmission or immunological rejection, autograft bone is limited by the availability of

sufficient donor tissue and problems with donor site morbidity (Braddock, Houston, Campbell,

& Ashcroft, 2001). There are several categories of bone grafts and substitutes that encompass a

variety materials, material sources and origins (Nandi, et al., 2010). All available grafts have

advantages and disadvantages and no single existing material possesses all the necessary

properties required in an ideal bone implant. Biocompatible implanted materials have provided

options in many cases, but the reaction of the body to these devices is far from perfect.

16

Complications, such as thrombosis, infection inflammatory reaction, impaired function,

loosening, and pain, are unfortunate for the patient and costly to the health care system. But, for

biomaterials researchers, these shortcomings with existing materials also represent opportunities

and challenges to engineer improved therapies. (Ratner, 2001). A suitable bone graft material of

proper quality, that is readily available in unlimited quantities, is still needed (Wise, Trantolo,

Lewandrowski, Gresser, & Cattaneo, 2002). Bone defects can be addressed by tissue engineering

techniques (Braddock, Houston, Campbell, & Ashcroft, 2001) that draw from the best material

scientist known --- Nature. This review will cover biomimetics and the uncanny similarities

between bone and crab cuticle.

The overall goal of using bone as a biomimetic subject is to learn from its unique structure

and function and use this insight to develop bone inspired implants that enhance healing and

remodeling of bone (Wilson Jr., 2008). Adapted as a bone implant material and early

biomimetics, nacre’s ability to integrate with bone was noted as early as 600 A.D. in the ancient

Mayan civilization (Westbroek & Marin, 1998). Drawing from this inspiration and accentuating

the interesting similarities to bone tissue, the crab shell cuticle itself has been underestimated as a

candidate material for engineering of bone. Biomimetics and design concepts of TRIZ (Theory

of Inventive Problem Solving) reveals crab cuticle as candidate material for development of FAB

bone implant. In this paper, bone and crab shell integument functional and general similarities

will be highlighted.

17

Figure 2-1 Making an Ideal Bone Graft (Stocker & Wolinetz, 2009).

18

Ideal Bone Implant

Materials designed by humans pale in comparison to those created by nature (Smith & Wood,

1991). Bone, one of nature’s masterpieces, is a remarkable, living, mineralized, connective

tissue, which is characterized by its strength, its resilience, and its ability to remodel and repair

itself (Hing, 2004). Bone is composed of an organic matrix of fibrous protein and collagen (30–

35% of the weight); inorganic calcium phosphate (65–70% of the weight); and water (Kalfas,

2001). Ideally, a bone replacement material should mimic bone tissue from a mechanical,

chemical, biological and functional point of view, be disease free, and facilitate new bone

formation.

There are three characteristics inherent to natural bone: osteoconductivity; osteogenicity; and

osteoinductivity as depicted in figure 2-1 (Stocker & Wolinetz, 2009). Osteogenesis is the ability

of the graft to produce new bone and is dependent on the presence of live bone cells.

Osteoconduction is the physical property of the graft to serve as a scaffold for viable bone

healing and allows for vasculature and the infiltration of osteogenic precursor cells.

Osteoinduction is the ability of graft material to induce stem cells to differentiate into mature

bone cells (Kalfas, 2001). Thus, to facilitate new bone formation an ideal bone substitute

material should have the characteristics inherent to natural bone.

Biomimetics

Material design and development for any replacement or regeneration application should be

19

based on the thorough understanding of the structure to be substituted. Material researchers can

gain valuable knowledge from mineralized tissues. Typically, the main characteristics of the

route by which the mineralized hard tissues are formed are that the organic matrix is laid down

first and the inorganic reinforcing phase grows within this organic matrix/template. Among the

wide variety of biomineralized materials engineered by living organisms, bone and crab wide

variety of biomineralized materials engineered by living organisms, bone and crab exoskeleton

are organic/inorganic composite structures with analogous mechanical properties. Non-biological

methods have not been able to duplicate the elegance of the biomineral assembly mechanisms or

the rather complex composite microarchitectures of hard mineralized tissues like bone and crab

(Reis & Weiner, 2004).

Uniformity of defining and expressing terms or concepts varies depending on discipline. For

example, cuticle is also known as exoskeleton, integument, shell, or carapace, depending on

discipline. Additionally, crab is also referred to as crustacean or arthropod. Many times these

terms are expressed interchangeably. Biomimetics is no stranger to this phenomenon of non-

uniformity. One definition of biomimetics describes it as the study of how Nature designs,

processes and assembles/disassembles molecular building blocks to fabricate high performance

materials such as mineral-polymer composites (e.g., mollusk shells, bone, etc.) and then applies

these designs and processes to engineer new molecules and materials with unique properties

(Reis & Weiner, 2004). Some relate two ways in which biological and engineering systems can

be related --- bioinspiration and biomimetics: (1) bioinspiration is describes as how inspiration is

gained from nature as a basis for developing engineering solutions to problems which requires an

20

Figure 2-2 Expanded description of the field of biomimetics (Sarikaya, 1994).

Biomimetics

(Nature) Understanding, Transfer, Implementation (Engineering)

Repurposing

existing

biological

materials

Using engineered

materials to mimic

(Structure, Energy, Etc.)

Design

Biorecycling Biomimicking

Fields of Operations

(Structure, Energy, Etc.) Bioduplication

Bioinspiration

Biologistics

Novel Synthesis

& Processing

Mastering

molecular

synthesis and

processing

mechanism

New Materials with Tailored Structures

Existing Materials with

Tailored Purpose

21

understanding of the natural system and the operational envelope within which it operates; and

(2) biomimetics is defined as mimicking nature in some way. Inspiration is considered the

dominant of the two (Allen, 2010). Others divide biomimetics into two categories based on

design approaches --- biomimicking and bioduplication: (1) biomimicking deals with the

investigation of the structure of a biomaterial at all possible length scales of spatial resolution

and then deducing and ultimately mimicking the fundamentals of the biomaterial’s unique design

structure; and (2) bioduplication deals with the mastery of the molecular synthesis and

processing mechanisms of biomaterials and applying these newfound methodologies to produce

new technological materials (Sarikaya, Liu, & Aksay, 1995).

However, the argument can be made that fabrication via biomimetics must first start with

inspiration. Secondly, the biomimetic design process is followed by studying or deducing how

Nature designs, processes, and assembles/disassembles molecular building blocks to

manufacture materials. Then the process involves applying the fundamental knowledge gained

through planning and management. Finally, the process creates a novel purpose, strategy, or

material by techniques such as biorecycling, biomimicking (structure, energy, etc.), or

bioduplication. An expanded definition of biomimetics is provided in figure 2-2.

By studying biological principles of nature, new ways of dealing with engineering problems

can be unlocked. Nature or biology finds the most economical and efficient ways to achieve its

objective in terms of energy, cost, and materials. Biology frequently uses a few simple materials

22

Figure 2-3 Biological (right column) and Engineering (left column) materials are very different

in the way they are developed (Allen, 2010), (Fratzl, 2007).

Biological Material

Light common Elements

H, C, N, O, Si

Ca, Na, P, S Cl, K, ...

Growth

by Biologically Controlled Self-Assembly

(Approximate/Variable Design)

Environmentally Influenced Self-Assembly

Hierachical Structuring at all size levels

Interfaces allow separate control of stiffness & fracture

Adaption of form & structure to the function

Responsive Design

Environmentally Responsive; adaptive in function & morphology; Self-repair

Engineering Material

Many Heavy Elements, Some rare

H, C, N, O, Si

Fe, Ni, Al, Zn, Cr, ...

Fabrication

from Melts, Powders, Solutions, Etc.

(Exact Design)

Externally Imposed Form

Mostly Monolithic; Little or No Hierarchy; Forming Parts & Microstructuring

Materials

Few Interfaces; therefore poor fracture control

Selection of material according to function

Secure Design

Considering possible max load; Very little environmental response; Self-repair (little

to nonexistent)

23

arranged in a particular manner. On the other hand, engineering uses much more energy and

often achieve less impressive results (Allen, 2010). Biological and engineering materials are very

different in how they are developed, as shown figure 2-3.

Natural materials use free energy while constructing and operating under conditions of low

temperature (0-40°C), atmospheric pressure, and neutral pH (Smith & Wood, 1991). Biological

materials use few polymers (protein, polysaccharide, etc.), with ceramics (calcium salts, silica,

etc.), and a few iron-containing ceramics (FeS, Fe3O4, etc.), whereas human-made materials

require high temperatures and hundreds of polymers (Allen, 2010). Consequently, the design

strategies of biological materials are not immediately applicable to the design of novel

engineering materials. One major difference is in the range of choice of elements. Biological

materials consist of relatively few constituent elements, which are used to synthesize a variety of

polymers and minerals. Although the choices are far greater for the engineer, man-made

materials are characterized by the use of many more elements and usually cannot match natural

performance of nature’s materials. Another major difference is the way in which materials are

made. While the engineer selects a material to fabricate a component according to an exact

design, nature grows both the material and the whole organism using principles of biologically

controlled self-assembly. This is certainly a key to the successful use of polymers and

composites as structural materials and provides control over the structure of the material at all

levels of hierarchy (Fratzl, 2007). Biology, like engineering, is reliant on materials for the

structures it makes. These structures have to be cheap and reliable (Allen, 2010).

Fundamental science, such as biology, has the goal to observe, describe, classify and analyze.

24

On the contrary, engineering has the intention to act, prescribe, utilize and manage (Vincent J. F.,

Bogatyreva, Pahl, Bogatyrev, & Bowyer, 2005). Thus, the transfer of a concept, process, or

mechanism from living to nonliving systems is not a straightforward matter. The advantage of a

biomimetic approach is that biology has already done a lot of the design prototyping; the

disadvantage is that every case has to be approached newly (Allen, 2010).

Bone and Crab Cuticle Functional Similarities using TRIZ

Different approaches have been developed to solve the same problems. There is no clear

roadmap or single path to the correct way to solve a given problem. Traditional problem solving

techniques focus on what is needed to solve the problem. Thus, the classical problem solving

loop involves: (1) identifying the problem; (2) exploring information generated from a specific

area of expertise; (3) creating ideas based on a specific background; selecting the best idea(s); (4)

building and testing idea(s); and (5) analysis and evaluation of results.

An innovative approach to creative problem solving involves carefully identifying the ideal

result in terms of a delivered function then working out what is needed to get to that ideal.

Altshuller developed a rigorous set of methods that concentrate on defining the function to be

delivered, the working environment or context in which it is to be delivered, and the resources

available within that environment (Allen, 2010). After studying thousands of patents, he

developed a set of methods based on the idea that the solution is best delivered by altering the

environment, rather than by altering the immediate mechanism of delivery and called this set of

25

methods “The Theory of Inventive Problem Solving” (in Russian, Theoriya Resheniya

Izobreatatelskikh Zadatch, or TRIZ). His approach showed that any branch of technology could

provide a solution to a specific problem, even if that branch does not appear to be related to the

problem or its apparent solution. Defining the innovation in terms of the function it is desired to

deliver and expressing it in simple relatively abstract terms, destroys the barriers between various

areas of knowledge (Allen, 2010).

TRIZ is an available design tool that establishes a system into which all known solutions can

be classified in terms of function. A database or information fund exists along with software

packages that facilitate the use of this tool. This database reflects a concentrated collection of

experience and knowledge that can be used to leverage natural and/or technical insight. To date,

the database contains limited biological information. As biological knowledge increase, the area

where nature/biology meets engineering, shrinks (Vincent & Mann, 2002), improving the

creation and application of biomimetic designs, concepts, or solutions.

The typical TRIZ iterative problem solving process involves: (1) identify or define

problem/function; (2) problem/function analysis and definition, using the database and language

of TRIZ, that provides insightful information; (3) contradiction/conflict analysis using

standardized factors (weight, speed, ease of use, etc.) — TRIZ theory contains 39 design

parameters and 40 generic principles for solving the contradictions derived by Altshuller; (4)

analysis of how contradictions/conflicts were circumvented or eliminated; (5) feasibility analysis

of problem resolutions of conflicts; and (6) iteration, if valid solution not found after conflict

26

Bone Entity (System) Cuticle

Mammal Description (Super-System Crab

Structural Support,

Protection of Organs,

Metabolism of Minerals,

Synthesis of Blood Cells

Contribution to or Action in Protection of Organs,

Structural Support

Organelle Cell Hard

Tissue

Level of Organization Organelle Cell Hard

Tissue

Water (Body Fluid) Medium Water

Responsive to

environmental factors

Parameters and Interactions Responsive to

environmental factors

From Medium Resource From Medium

Function(s)

Figure 2-4 This table depicts bone and crab cuticle system, contribution, level or organization,

medium, parameters & interactions, and resource. (Bottom) This diagram ewxhibits a closer

inspection of the functions of bone and crab cuticle, conflicts to accomplishing those functions,

and the tricks or circumventions around those conflicts.

27

elimination (Yeh-Liang, Hung, & Yin, 2006). These factors, and the resolutions of the conflicts

which was defined, represent a collection of engineering best practice (Vincent, 2005). In short,

TRIZ is a systematic and repeatable method for broadening the field of thinking across

disciplines and for enhancing creative options.

Using aspect of a biological case study conducted by Vincent based on TRIZ analysis of a

relatively simple natural composite material—the outer covering of arthropods or cuticle,

similarities between the functions of bone and crab cuticle can be highlighted. Produced by a

single layer of epithelial cells, this layer of material is charged to provide a large number of

functions such as protection, shape, structure, hinges, barrier, filter, etc. (Vincent, 2005). Bone is

charged with many of the same functions. In his study, a list, not necessarily exhaustive or

unbiased, of functions and associated characteristics of cuticle was generated partly by reference

to literature and partly from experience with insects and insect cuticle over the years. In this case,

a short list of functions, conflicts, and circumventions shared by both bone and crab are

illustrated in figure 2-4. It can be easily demonstrated that bone and crab cuticle use very similar

approaches to the solution of design and integration, functionally speaking. Figure 2-5 illustrates

the idea of a contradiction or mixed messaging. A contradiction characterizes the conflicting

goals materials strive to satisfy.

From the point of view of biomimetics, if, by defining functions and comparing the ways in

which biology and engineering deliver a range of functions, an independent assessment can be

made whether engineering stands any chance of being environmentally benign by comparison to

Nature (Allen, 2010).

28

Figure 2-5 A contradiction describes the predicament caused by an attempt to improve one

property of a system that induces the degradation of another property (Stanley, Zlotin,

Bolckmans, & Zusman, 2005).

TRIZ is versatile and can vary drastically in its level of complexity. TRIZ analysis can not

only illuminate that bone and crab exoskeleton performs many of the same functions but

circumvents many the same contradictions in a similar manner. For example, both bone and crab

cuticle have the property of being stiff. However, bone and crab cuticle have to allow movement

and pass sensory information. These contradictions are circumvented with hinge areas and a

system of pores and vascularity. As one delves deeper into the functions, contradictions, and

circumventions, the level of complexity can vary from a general surface analysis to a more

thorough approach. As an illustration, if the discussion were to continue regarding stiffness,

movement, sensory information flow, pores, etc., the discussion could include the entire sensory

network system --- cells, channels, piezoelectric quality, homeostasis, etc. At the same token, the

list of functions can expand. In essence, TRIZ analysis can start and stop with small building

blocks or it can burgeon into a complex framework, figure 2-6.

29

Figure 2-6 How TRIZ works (Stanley, Zlotin, Bolckmans, & Zusman, 2005).

Transferring Biology to Engineering

Properties of materials allow communication of requirements when selecting materials for

implants. An organized system of property information then becomes a valuable commodity. The

functional knowledge coupled with other property information can form a powerful database that

30

(1) equips with new wisdoms and connections to ask and to possibly answer novel questions; and

(2) has the potential of advancing science, pioneering new methods, and ensuring quality of

biomaterials and other products.

TRIZ has been traditionally used to show how technical solutions compare to each other.

Combining biomimetic concepts with TRIZ allows inspection of the way nature solved problems

and see how that compares with technical solutions to the same or similar problems. TRIZ

establishes a systematic scheme in which problems and their solutions can be defined and

classified. Transferring a concept or mechanism from living to nonliving systems can be

daunting because nature exists within a different putative context than nonliving systems. Bone

represents an example of a biological putative prototype that is continually recycled or repaired

for the duration of its life inside the body and profoundly difficult to replicate. Therefore, some

approach or form of interpretation or translation is needed to make the leap from biology to

technology (Vincent, 2008).

Biomimetics is not a brand-new was of adapting ideas from biology, but its approach is

currently guided by experience. TRIZ introduces a set of common principles that builds on

current successes and has the ability to establish a transparent method for technologists to access

biology. TRIZ is a systems approach for engineering and biology is regarded as a system. TRIZ

involves the transfer of various inventions and solutions from one field to another, while

biomimetics involves the transfer of functions, mechanisms and principles from one field to

another. Therefore, TRIZ is a superior candidate to be an ideal bridge to assist in identifying

biomimetic principles, concepts, and functions and transferring them from nature to

31

Figure 2-7 Schematic of steps to identify a function/concept/idea and to transfer the function

into engineering solution (Vincent, 2008), (Shimomura, 2010).

ENGINEERING *BIOLOGY

Total Mimicry

Partial Mimicry

Abstraction

Mechanics

Physics

Inspiration

_______optimizations________

Identify the challenge/function

Interpret challenge/function in terms of biology

Discover biological model that performs same function or deals with same challenge

Analyze the patterns and processes of the biological model

Transfer biological ideas, functions, processes to engineering *

To

Develop ideas and solutions based on biological model

Biological Model: Function/Property Mimicked Product/Solution Lotus Effect: Superhydrophobicity Paint, Self-Cleaning

Sharkskin: Fluid Resistance Swim Suit

Termite Den: Temperature Maintenance Building Architecture

Plant Burr: Cling Velcro (two-sided fastener)

Whale Fin: Reduce Drag Turbine blades

Kingfishers Beak: Travels Quickly & Smoothly Between Contrary Mediums Bullet Train

32

technology/engineering, as shown in figure 2-7 (Vincent J. F., Bogatyreva, Bogatyrev, Bowyer,

& Pahl, 2006).

Although TRIZ is a promising system for use in biomimetics, there are considerable issues.

Biology and technology solve problems in rather different ways. TRIZ is derived from the

environment of things artificial, non-living, technical and engineering while biology is in the

realm of the living while biomimetics operates across the border between living and non-living

systems. Living organism are highly complex. They have great adaptability and versatility, but at

the expense of the predictability of the system’s behavior. Generally, unpredictability in

technical systems are typically unacceptable and avoided.

Two of the more pronounced challenges between biology and technology deals with

hierarchy and energy. Typically living systems are based on a hierarchical structure. Complexity

and added functionality are achieved by adding levels of hierarchy in Nature, due to processing

limitations current technologies are either not significantly hierarchical, or ignore any

hierarchical structure. The hierarchical approach is also difficult to model in engineering and is

not as well developed as it is in biology. Energy is also a testament to the challenges of

transferring biology to technology. Biological functions and processes are less reliant on energy

(Vincent J. F., Bogatyreva, Bogatyrev, Bowyer, & Pahl, 2006), than their technological

counterparts. As biomimetics and TRIZ tools are developed and used more frequently, functions,

ideas, or concepts can hopefully be readily translated from biology to engineering.

33

Figure 2-8 Bone and Crab Shell Similarities.

Hierarchical structuring at all size levels

Biomineralized composites

Ca2+ based inorganic phase

Each inorganic pahse (hydroxyapatite, calcium carbonate contains 40% Ca2+ by mass

Organic ploymer matrix pahse (collagen, chitin)

Highly loaded inorganic phase (50-70 wt%)

Textured, crystallographic orientation of inorganic phase

Protein and muccopolsaccharide constituents

Relatively hard and damage tolerant

liquid crystalline behavior of orgainc matrix

Structural support role

Cellular control

Self-healing behavior

Piezoelectric propertiies (ability to convert stress to electrical signals)

Similarities between the biomineralized hard tissues bone and crab shell

34

General Comparison of Bone and Crab Cuticle

Crab shell integument shares chemical and structural features that are closely related to bone.

The similarities of bone and crab cuticle are outlined in figure 2-8. Both hard tissues are formed

via biomineralization processes and contain calcium as a major component, with calcium

phosphate present in bone and calcium carbonate in crab exoskeleton. The mineral phase of bone

is often referred to as carbonated apatite because carbonate substitutes readily for both phosphate

and hydroxyl anions in biological hydroxyapatite. On a macroscopic level, bone and crab cuticle

provide structural support and protect internal organs. Bone tissue customarily resides inside the

body while crab integument functions as an external protective armor. These hard tissues both

serve as storage sites for calcium, phosphate, carbonate, and other biologically relevant ionic

species. The nanoscale level of bone and crab shell have features that are truly remarkable from a

materials design viewpoint. Bone and crab cuticle are composed of an organic matrix embedded

with mineral network where proteins contribute to the controlled nucleation and growth of

inorganic phase. In exoskeleton of crustaceans, the polysaccharide chitin associates with calcite

while in bone, the protein collagen associates with calcium phosphate (Giraud-Guille, Belamie,

& Mosser, 2004). Both of these associations produce biomineral crystals that appear as thin

platelets (Weiner & Wagner, 1998). Both bone and crab cuticle serve as a framework for the

activity of specialized cells. Bone cells --- osteoblasts, osteoclasts, and osteocytes --- activities

include orchestrating signalling and producing extracellular matrix proteins that coordinate

cellular and tissue interactions while crab cuticle epidermal cells produce and secrete organic

matrix for maintenance of integument integrity and function through various molt cycles and

35

survival events. Bone has a role in providing a reservoir for the production and storage of

hematopoietic and mesenchymal stem cells. This reservoir of cells in the body suggest that bone

is capable of osteogenesis throughout life (Aubin, 1998).

Hierarchical structure and biomineralization phenomena play an integral role in crab shell and

bone tissue physiology and function. In both hard tissues, the manner in which organic

framework molecules, collagen and chitin, influence biomineral formation (nucleation, growth,

morphology, etc.) is a truly intriguing study. This will be discussed in the next chapter.

A fundamental understanding of bone and crab shell structure and biology can be gained by

studying the chemical, physical, and mechanical structure of biomineralized tissues bone and

crab integument with a focus on the organic phase, the inorganic phase, and the inter-relations

between and among these phases. This expanded understanding is a necessary course to develop

successful bioimplants.

Concluding Remarks

Biology and technology/engineering use very different approaches to the solution of similar

problems or functions of design and integration. However, many of nature’s systems use the

same exquisite and superior design principles. As hard tissue systems are studied, the similarities

between systems such as bone and crab cuticle become obviously clear. Because technological or

engineering designs cannot, to date, match the performance of nature’s materials, biomimetic

design strategies, such as biorecycling allows exploitation of natural environmentally friendly

materials, such as marine wastes (Hirano, Nakahira, Nakagawa, & Kim, 1999). This exploitation

36

takes advantage of the economical and efficient ways nature achieves its objective in terms of

energy, cost, and materials. In addition, most of the design prototyping and fabrication has been

done.

If the desired functionally advanced biomaterials based on crab cuticle are to be produced

through biomimetics, further knowledge is needed about: (1) the micro- and nano- structure; (2)

variations of composition and structural organization of the matrix; (3) responses of phases

(organic matrix, and interfaces) under various mechanical stresses; (4) toughening,

strengthening, and hardening mechanisms; (5) biomineralization; and (6) growth mechanism

based on geometry, crystallography, hierarchy, and size. (Sarikaya, Liu, & Aksay, 1995).

Consequently, as the understanding of how nature designs many of its complex systems is

illuminated, it becomes obviously more transparent that systems and their functions in biology

can be adapted to technological ends. Therefore, biomimetics can offer novel and valid

alternatives to technological problems (Vincent, 2005). As biomimetics, TRIZ and other

techniques evolve, they can further illuminate that the solution to many of our technological

problems has been hiding in plain sight and that nature has a multitude of recommendations.

37

Chapter 3 Ties that Bind: Evaluation of Collagen I and α-chitin

Introduction

Bone and crab cuticle are two dynamic and vital biomineralized composite structures.

Studying the similarities and differences between bone and crab shell at various structural levels

allows a greater understanding of the inter-relationships between biomineralized tissues and how

they function to repair, restore, and regenerate. Bone and crab shell integument is comprised of

an organic component and an inorganic component. Calcium phosphate and calcium carbonate

are the inorganic or mineral constituents of bone and crab cuticle respectively. Likewise,

collagen and chitin are the organic phases of bones and crab cuticle, respectively. Table 3-1

exhibits the principle components of two similar biological systems: crab exoskeleton and bone.

Focusing on the organic constituents, some interesting similarities and some unique qualities can

be elucidated. Thus, chitin and collagen are two fascinating matrix framework molecules that

play a key role in the hierarchical development of hard tissue. For a thorough and systematic

design of a bone inspired bio-implant, it is fundamental to study the influence of individual

components (Daamen, et al., 2003) of biological systems.

The key chemical and structural features of chitin and collagen were studied in order to gain

insight into the functional role of important organic matrix material of hard tissue. Both collagen

and chitin are constructed with polymeric fibers. Chitin is the second most abundant natural

38

Table 3-1 Principal Components of Crustaceans (Crab) Exoskeleton and Bones Mineral Organic

Biological Composite Hydroxyapatite

(calcium phosphate)

Calcium Carbonate Collagen Chitin

Bones

X X

Crustacean (Crab) Exoskeleton

X

X

fibrous polymer while collagen is among the first. One of the more conspicuous differences is

the biochemistry of collagen I, a protein, and α-chitin, a polysaccharide. There is a considerable

amount of information about the structure of chitin and collagen. However, all the mechanism

involved in the self-assembly of chitin and collagen molecules into solid fibrous structures stills

lacks complete clarity. Both collagen and chitin fall under the class of molecules described by

Bouligand as ‘biological analogues of liquid crystals’ (Bouligand Y. , 1972). Experiments have

accentuated the importance of liquid crystal characteristics with respect to self-assembly. Thus,

the more intriguing similarities involve hierarchical structuring, liquid crystal characteristics and

self-assembly. Advances made in regard to the identification of molecules involved in cellular

and morphogenetic regulation have provided a window into how collagen, chitin, and similar

biological components interact to form fibrous composites. The chemistry, hierarchy, synthesis,

liquid crystal characteristics, biomedical applications and other properties were evaluated to aid

in a deeper understanding and development of concepts for biological, hierarchical, composite

design. In this work, our focus is on inter-relationships between the biomolecular building blocks

chitin and collagen and their respective roles in the architectural development of molecular

organic matrix (MOM).

39

A growing body of work highlights bone and crab exoskeleton including aspects of collagen

and chitin, see Table 3-2 (Bouligand Y. , 1972), (Giraud-Guille M.-M. , 1994), (Giraud-Guille,

Belamie, & Mosser, 2004), (Belamie, Mosser, Gobeaux, & Giraud-Guille, 2006), (Meyers, Lin,

Seki, Chen, & Bodde, 2006), (Meyers, Chen, Lin, & Seki, 2008). One group highlights the

liquid crystal properties of α-chitin and collagen. However, studies that feature a more

comprehensive view of solely collagen I and α-chitin is limited. This article will examine the

organic constituent of bone and crab exoskeleton, collagen I and α-chitin, respectively. Collagen

I and α-chitin have many similarities as well as differences, and. hence forth these fibrous

composites will be referred to primarily as collagen and chitin. Collagen and chitin work

together on various levels sometimes leading, other times playing a more supportive role, but

always integrally linked in establishing form and functionality in biological structures and

systems. Specifically, collagen interacts with polysaccharides via extracellular matrix (ECM)

(collagen and ECM in bone structure (various roles in cell signalling, biomineralization, etc.);

Chitin interactions with proteins (chitin/protein complex (beta pleated sheets in nacre, etc. This

paper is organized to address the similarities and differences at various level of organization of

collagen and chitin.

Framework of chitin and collagen

Chitin was first isolated from mushrooms in 1811 by the French scientist Henri Braconnot.

In 1823, Odier found the same compound in the cuticles of insects and named it chitin

(Stephen, 1995). At least 10 gigatons (1 × 1013 kg) of chitin are synthesized each year making

40

Table 3-2 Key Articles that Highlight Collagen and Chitin

Author Year Title

Bouligand 1972 Twisted Fibrous Arrangements in Biological Materials and

Cholesteric Mesophases

Giraud-Guille 1994 Liquid Crystalline Order of Biopolymers in Cuticles and Bones

Giraud-Guille 2004 Organic and mineral networks and biomimetic materials

Belamie 2006 Possible transient liquid crystal phase during the laying out of

connective tissues: α-chitin and collagen as models

Meyers 2006 Structural Biological Composites: An Overview

Meyers 2008 Biological materials: Structure and mechanical properties

chitin the second most abundant natural polymer on earth. It is found in the exoskeleton of

crustaceans, cuticle of insects, and cell wall of fungi (Jolles & Muzzarelli, 1999). On the other

hand, collagen appears to have been first reported by Zachariades in 1900 (Slack, 1955).

Collagens are found in all multicellular organisms (Gobeaux, et al., 2008). Chitin and collagen

play an important role in the hierarchical control of the biomineralization processes and in the

structural component that provides support and protection.

Chitin Forms

Three polymorphic forms of chitin (α-, β-, and γ-chitins) have been distinguished due to their

crystal structure that differ in packing and polarities of adjacent chains in successive sheets,

figure 3-1. α-Chitin, the most abundant form found in nature, is arranged in an anti-parallel

41

Figure 3-1 Diagram showing the direction of the polymer chains in the three crystallographic

forms of chitin. (a) α-chitin. (b) β-chitin. (c) γ-chitin (Wainwright, 1982).

configuration; β-chitin is organized in a parallel configuration; and γ-Chitin is a mixture of α-

and β-chitin. The anti-parallel configuration gives a highly ordered crystalline structure with

strong hydrogen bonding between chitin chains, leading to the rigid and insoluble properties of

α-chitin (Meyers, Chen, Lin, & Seki, 2008).

α-Chitin types vary as to the length of their N-acetylglucosamine chains, as to the degree of

crystallinity, and as to the dependence on the presence of accessory inorganic compounds or

proteins. As a consequence, the consistency of the chitinous layers in organisms like arthropods

differs considerably (Jolles & Muzzarelli, 1999). A chitinous exoskeleton is diagnostic of the

crustaceans. It is secreted by a single layer of cells (the epidermis) and gains its stiffness and

structural complexity mainly by being folded and curved in many complex ways (Vincent J. F.,

Arthropod cuticle: a natural composite shell system, 2002).

α-chitin β-chitin γ-chitin

(a) (b) (c)

42

Collagen Forms

More than 28 different types of collagen have been identified (Gobeaux, et al., 2008) and

classified primarily according to their physiological structure. Additionally, considering the

relationship between the inner structure of collagen fibrils, their diameter, their spatial layout and

the functional requirements they have to withstand, Ottani et al. proposed that collagen fibrils of

fibrous connective tissue may also belong to two different, mutually exclusive forms indicated as

“T-type” and “C-type”. The first class, consisting of large, heterogeneous fibrils, parallel tightly

packed, subjected to tensile stress along their axis is found in highly tensile structures such as

tendons, ligaments and bone. The other class, consisting of small, homogeneous fibrils, helically

arranged, resisting multidirectional stresses, is mostly present within highly compliant tissues

such as blood vessel walls, skin and nerve sheaths (Ottani, Raspantib, & Ruggeri, 2001).

Vendors, such as Sigma Aldrich, continue to refer to collagens both by the widely used

‘‘modern’’ type classification system defined by the chain types and by types as defined in the

early 1970s by the researchers who first separated collagen chains (Miller type II cartilage). The

earlier collagen classifications tended to be defined more by the tissue type from which the

collagen had been extracted than the collagen chain content (Abraham, Zuena, Perez-Ramirez, &

Kaplan, 2008). Collagen type I usually consists of three coiled subunits: two α1(I) and one α2(I)

chains forming fibrils of 50 nm in diameter (Kolacna, Bakesova, Varga, Kostakova, & Planka,

2007). The family of fibrillar collagens is of the biochemical types I, II, III, V, and XI (Ottani,

Matin, Franchi, Ruggeri, & Raspanti, 2002). All the approximately 44 genes for 28 collagen

types were described up to now; they are divided according to their supramolecular structure.

43

Alpha-chains descend from a common ancestor and are characteristic for each collagen type: the

chains are numbered according to the respective collagen type. The structure of the heterotrimer

in collagen I is [α1(I)2 α2(I)], and the structure of the homotrimer in collagen I is [α1(I)]3]

(Abraham, Zuena, Perez-Ramirez, & Kaplan, 2008). Collagen type I, the main constituent of

bone, tendon, skin, and vessel walls, is synthesized by fibroblast, smooth-muscle cells,

chondroblasts, odontoblasts, and osteoblasts. Osteoblast synthesize only type I collagen (Kuhn

& Glanville, 1980).

Both α-chitin and collagen I are crystalline. While there are three polymorphic forms of

chitin, there are 28 identified types of collagen defined by chain types. Additionally collagen

can be further classified according to its physiological structure.

Chemistry

Collagen I and α-chtin both consist of relatively few constituent elements: H, C, O, and N

(Meyers, Chen, Lin, & Seki, 2008). The chitin monomer general empirical formula is

[C8H13O5N]n. The empirical chemical formula for collagen is [C12H24O4N3].

Polysaccharides and Proteins

The basic chemistry of collagen and chitin has its origins in the basic building blocks of cells.

Major cellular compounds are lipids, carbohydrates and amino acids; these compounds mostly

44

combine to form nucleotides, polysaccharides, and proteins, Table 3-3. Chitin is a polysaccharide

while collagen is a polypeptide or protein.

Polypeptides or proteins are organic compounds made of amino acids arranged in a linear

chain polymer and joined together by peptide bonds between the carboxyl and amino groups of

adjacent amino acid residues. A particular protein always has a specific number and sequence of

amino acid residues. The basic unit of collagens is a polypeptide consisting of the repeating

sequence (G- X - Y)n glycine (G) is small, and is the only amino acid that fits in the crowded

interior of the triple helix. X is usually proline and Y is usually hydroxyproline. See figure 3-2.

Figure 3-2 Chemical Structure of Collagen showing the glycine, proline, and hydroxyproline

residues (Brodsky, Werkmeister, & Ramshaw, 2005).

45

Figure 3-3 Fragment of chitin chain (Ehrlich, 2010).

Table 3-3 Some Macromolecules (polymers) and their primary biological functions (Smith &

Wood, 1991).

Monomer Polymer Examples Functions

Monosaccharide Polysaccharide Chitin Structural

Amino Acids Proteins Collagen

Enzymes

Hormones

Structural

Catalysis

Messengers

Nucleotides Nucleic acids DNA

RNA

Information

Storage

Information

Transfer,

Structural

46

The basic unit of chitin is long linear polysaccharide chain of N-acetylglucosamine joined

together by glycosidic bonds, figure 3-3. The glycosidic link joins the number 1 carbon of the

N-acetylglucosamine to the number 4 carbon of the second N-acetylglucosamine. Joining the N-

acetylglucosamine monomers in a different way would result in a different polysaccharide. β1-4

linked residues tend to form an extended ribbon. Individual polysaccharides rarely have a

specific Mi but rather show a range or distribution of M.

One of the structural features distinguishing polysaccharides from proteins is the great

variation possible in the configuration of the glycosidic bond between adjacent sugar residues.

The many possibilities for linking allow a variety of shapes and surfaces to be formed from just a

few sugar residues. Using a limited range of N-acetylglucosamine, a range of unique molecular

shapes may be generated. With some exceptions, the function of polysaccharide structure lie in

the uniform and repeating shapes and surfaces rather than size itself. In contrast, the result of

protein folding is to produce a unique but irregular surface, a feature that requires a precise

sequence and fixed number of amino acids. Proteins can produce quaternary structures. Both

proteins like collagen and polysaccharides like chitin can produce a range of unique shapes and

surfaces.

Fibrous protein such as collagen have distinctive amino acid compositions, in which a very

few amino acid residues predominate, and usually there is a short sequence of residues that is

repeated many times. They often contain unusual, modified amino acid residues and tend to be

highly cross-linked, giving them strength. Compared with other fibrous proteins, the

47

composition of collagen is unusual. There is a very high content of proline. Additionally, and

uniquely, a large proportion of the proline in collagen is hydroxylated (Smith & Wood, 1991).

Another difference between proteins and polysaccharides is in the variety of components.

Each polysaccharide typically comprises only a few different types of sugars. Often, there is but

one. Sequences of repeating blocks of mono-, di-, tri-saccharides, etc. produces polysaccharides

of simple structures such as ribbons. There is a limited array of groups on the polysaccharide

surface capable of interaction with other molecules. This can be contrasted with much greater

range of chemical or physical interactions displayed by amino acid side chains on the surface of

a protein. Their very simplicity allows them to interact other polymers to form large molecular

networks with important and desirable properties, especially in the extended linear

conformations.

Although there is a limited range of polysaccharide types, the glycosidic bond between

adjacent residues is not confined to just one group in the sugar ring. In contrast to proteins, a

number of glycosidic bond arrangements are found between sugar residues where identical

Table 3-4 Molecular Weight of Chitin and Collagen

Molecule Molecular Weight (Daltons) Reference

Chitin 1.0 x 106

2.0 x 106

Muzzarelli 1976

Morganti 2008

Collagen 1.4 x 105

3.0 x 105

Ying 2001

Worthington Biochemicals 2009

48

peptide bonds join amino acid residues. The availability of different linkages means that the

preferred conformation or shape of the polysaccharide is dependent on the glycosidic bond, in

addition to the nature of the residues themselves. For example, glucose residues, linked α1-4,

produce a polymer with a shape and solubility quite distinctive from one with identical residues,

linked 1-4, but with a β-configuration (Smith & Wood, 1991). Table 3-4 lists molecular weights

of chitin and collagen.

Protein and Polysaccharide Interactions

Chitin and collagen perform similar structural roles in animals. Collagen often exists in

vertebrates as a complex of ordered collagen (protein) molecules in a matrix of water and

polysaccharide. On the other hand, chitin consists of poly-N-acetyl-glucosamine

(polysaccharide) chains which aggregate into crystallites, these crystallites being embedded in a

matrix of water and protein (Wainwright, 1982).

Chitin always occurs associated with proteins (Trim, 1941)and no sample of purified chitin

has been found to be free of amino acids (Wright & Arthur, 1987). The precise nature of the

interaction of cuticular proteins with chitin fibers and a detailed structure has not yet been

resolved. Certain sequence motifs occur in cuticular proteins from even distantly related species

and such conserved motifs have common and important roles for the proper function of cuticle.

Further work is needed to learn if the assignment of protein class to cuticle type is universal

(Trim, 1941). The relative proportions of protein and chitin in cuticles change during processes

such as cuticulogenesis (Wright & Arthur, 1987).

49

A few models attempt to describe the chitin-protein complex. While studying a rubber like

cuticle (locust prealar arm), Neville compared resilin and chitin content. From the measurements

of the unit cell and the diameter of the crystallite, calculation the percentage of chitin present in

the sample were based on 3 assumptions: (1) the shape of the crystallite in cross-section - round,

square or rectangular; (2) the degree of shrinkage of the specimen during preparation; and (3) the

amount of chitin which is 'disorganized' and outside the periphery of the observed crystallite. He

hypothesized that little chitin is found outside the crystallite, though the possibility that some

may be present cannot be completely eliminated. For cuticle to behave as a composite material it

would be expected that the chitin crystallites are strongly bonded to the protein matrix, and there

is some chemical evidence for this (Neville A. C., 1976). Removal of protein from the cuticle

reduced the heavily stained matrix. Neville, when examining the staining properties of structures

composed of chitin and protein resilin, concluded that cuticle is composed of microfibrils of

chitin in a matrix of proteins. High resolution micrographs of cuticular microfibrils show them

to be poorly stained, not unstained as is usually stated, which indicates that either chitin takes up

some stain or the presence of other material, e.g. protein. Because of the strong bonding between

chitin chains it is assumed that the microfibrillar core consists of crystalline chitin and the only

the peripheral chains are associated with protein. Proteins may also be associated with chitin

chains within the core. Although the microfibrils in many cuticle contain chitin, there is no

convincing evidence that any microfibrilar core is pure chitin (Wright & Arthur, 1987). The

presence of proteoglycan coats around fibrils is very likely in most arthropod carapaces, insects

and crustaceans, and this could be the origin of the examples of epidermal control on fibril

50

Figure 3-4 Arrangement of the protein subunits around the chitin core in the microfibril

perpendicular to the fiber axis and along the fiber axis. The 61 helix of protein subunits repeats

in 3.06nm (Blackwell & Weih, 1980).

direction. The complex transformations of filaments after their secretion by the epidermis in crab

cuticle suggest the presence of associated macromolecules which could be involved in the

control of fibril diameters and of their diverse associations (Bouligand Y. , 2008).

Blackwell and Weih have developed a model for the chitin-protein complex as it occurs in

ovipositors of the ichneumon flies Megarhyssa lunator and M. atrata. The model shows a sheath

of proteins subunits arranged in a helix (6 subunits per turn) around a chitin core, figure 3-4. Not

all cuticular proteins are bound to chitin, some form the protein matrix in which is embedded the

chitin-protein complex. The chitin-protein complexes are ordered structures (Blackwell & Weih,

1980), (Wright & Arthur, 1987). More elaborate modelling and experimental future work is

needed both to correlate the models of chitin–protein interaction proposed with the model

proposed by Blackwell and Weih (Blackwell & Weih, 1980). It is also evident from the data

51

overall that each species has its own characteristic protein binding matrix. Apparently there is no

simple relation between chitin content and the amount of covalently bound protein in the chitin-

protein complex (Austin, Brine, Castle, & Zikakis, 1981).

Proteoglycans

Collagen fibrils of extracellular matrices are known to be surrounded by a continuous coat

made of proteins and polysaccharides, called proteoglycans. This coat is quasi-fractal

construction, associating long chains of hyaluronic acid, with attached globular and linear

proteins, and also second-order lateral chains of chondroitin and keratan sulfates. These branched

structures can be arranged to form a cylindrical protection, a sort of sheath coat around each

collagen fibril. Bouligand theorized that: (1) this was possibly selected as being well devised to

stop the assembly of collagen molecules at a given diameter of fibrils; (2) this might be also a

way to have equal interdistances between fibrils and therefore an even distribution of minerals

between fibrils, as required in fiber-reinforced composites; and (3) these proteoglycans could be

involved in the cell control of fibril direction. This direction is initiated by the liquid crystalline

self-assembly, but might be modulated by links associating the cytoskeleton with integrins,

fibronectins and finally this proteoglycan coat and the collagen itself (Bouligand Y. , 2008). The

specific relationship of proteoglycans with the collagen fibrils in sclera closely resembled that

previously described in tendon and in articular cartilage, lending support to the view that the

association of proteoglycans with collagen may be consistent in a majority of connective tissues,

irrespective of their diverse functional specializations (Young, 1985).

52

Figure 3-5 Proteoglycan aggregate (Kierszenbaum, 2007).

Figure 3-5 shows that the extracellular matrix is comprised of proteoglycans attached to a

backbone of hyaluronic acid that is intertwined among collagen fibrils. Proteoglycans have both

chondroitin-sulfate- and keratin-sulfate-rich regions and link proteins facilitate binding of

hyaluronic acid.

Divergent views also exist as to how the carbohydrate portion is linked to collagen.

Different opinions have been expressed as to the functional role of carbohydrates in the collagen

structure. According to some researchers, the bulky carbohydrate group might direct the regular

stagger of tropocollagen molecules by requiring a particular fit. The polysaccharides of

connective tissue constitute the aqueous phase or ground substance of the extracellular space in

which collagen fibrils are embedded. The ground substance is believed to perform many

functions in the tissue. It provides the actual homeostatic environment of the cells and takes up

their metabolites and acts as a barrier against bacterial invasion. The proportion of the various

polysaccharides in ground substance varies in different tissues. The polysaccharides complexes

53

of connective tissue can be divided into two groups: (1) the glycoproteins consisting of protein

molecules to which monosaccharides or oligosaccharides are covalently bound; and (2),

proteoglycans consisting of polysaccharide-protein complexes, in which the polysaccharide

makes up a major part of the whole molecule (Chichester, 1982). List of polysaccharides or

glycosaminoglycan include hyaluronic acid, chondroitin, chondroitin 4-sulfate, chondroitin 6-

sulfate, dermatan 4-sulfate, keratin sulfate, heparin, and heparin sulfate. These glycosamino-

glycan imparts some degree of flexibility to calcified tissue like bone. The functioning of the

various tissues depends not only on the amount and types of collagen but also on the relative

proportion of other associated constituents. The mucopolysaccharides, associated with collagen,

are largely responsible for the water sorptive and retentive capacity of the tissues and for the

creation of osmotic pressures, at physiological concentration of a magnitude that is important to

the living organism. Polysaccharides also have the ability to affect the diffusion of ions through

the connective tissues (Chichester, 1982).

In the animal kingdom the fibers are usually proteins --- collagen. The fibrous soluble

polymer appears always to be predominantly carbohydrate, although of very varying

composition, especially in the invertebrates. This pattern, of a fiber-reinforced composite

material, has great versatility. By changing the ratio of fiber to soluble polymer, or by altering

the spatial relations between the two, quite different situations can be coped with, without

altering the chemical composition of the participants. Of the two elements, the fibers have

tensile strength, i.e. resistance to forces which would extend them. The interfibrillar polymer

expands the meshwork, and thus provides resistance to compressive forces. Movement of small

54

molecules can take place more or less freely in the polymer compartment, to sustain life in the

cells of the matrix. Apart from their physical roles proteoglycans and glycosamino-glycans are

able to interact physicochemically with, for example, collagen in ways which show considerable

specificity, and which presumably are important in the laying down of the fibrous network as

well as in maintaining its mechanical integrity. Systems of insoluble fibrils and soluble polymers

take stresses of movement and the maintenance of shape (Scott J. E., 1975). Chondroitin

sulphates are produced by sponges, crabs and mammals. The extent to which the sizes and

shapes of the polymer molecules correspond to those of the holes in the fiber meshwork is

clearly important in determining the properties of the tissue. Small-scale or very flexible

polymers would be able to move bodily through the meshwork, resulting in an easily deformable,

inelastic matrix.

Sulphated polysaccharides/glycosaminoglycans can change their structure much more

flexibly than a protein β-sheet. Determining the crystal structure in vitro is still quite problematic

because the crystal structure is influenced by temperature, calcium ion concentration, the

concentration of negative charged acidic macromolecules, carbonate ions, water, and other

cations. Sulphate groups for calcification mostly involve sulphated GAG (and some sulphated

glycoproteins), and these are certainly water soluble (Furuhashi, Schwarzinger, Miksik, Smrx, &

Beran, 2009).

Among the sulfated glycosaminoglycans, heparan sulfates, dermatan sulfates, and chondroitin

sulfates seem to be ubiquitous components of mammalian and other vertebrate tissues as well as

of a variety of cells in culture (Nader, et al., 1984). It has been suggested (Scott & Tigwell, 1978)

55

that hyaluronate and chondroitin sulphate differ from dermatan sulphate in being able to form

interresidue hydrogen-bonded sequences along the carbohydrate chain, which stiffen them.

Chondroitin sulphate and hyaluronate are thus particularly suited to occupying and maintaining

large domains in solution against deforming pressures. The creation of a 'buffer volume' between

the thin fibres that serves to keep them well separated and relatively free of anastomosis may be

a manifestation of this property. As a corollary, dermatan sulphate has more capacity to form

intermolecular liaisons, since it is not involved in intramolecular bonding to the same extent. Its

role in binding specifically to collagen may be aspect of this capacity (Scott, Orford, & W,

1981).

Chondroitin sulphate proteoglycans (CSPGs) in mineralized bone tissue of human, bovine

and other species have been isolated and characterized. It has also been suggested by an electron

microscopy that core proteins of proteoglycans interact with collagen fibrils. Since the occupied

region of collagen fibrils is suggested to be an initiation site of mineralization, proteoglycans

possibly inhibit the mineralization process (Takeuchi, Matsumoto, Ogata, & Shishiba, 1990).

Processed extracellularly by proteolytic cleavages at the N- and C-terminals, collagens are

biosynthesized as procollagens before aggregating efficiently to fibrils. There exist a number of

stages at which collagens might interact with proteoglycans, from procollagen, through various

aggregates, to the complete fibril. The first stages are in solution while the later interactions are

surface phenomena (Scott J. E., 1988).

The proteoglycans are soluble polymers in the simple scheme of connective tissues

consisting of a polypeptide 'core' attached to one or more glycan chains (Poole, 1986).

56

Consisting of repeating disaccharide units , in which one residue is always hexosamine, the

glycan chains are of three types, (1) heparan, (2) keratan, and (3) chondroitin dermatan.

Chondroitin-dermatan glycans contain N-acetylgalactosamine, with D-glucuronic and/or L-

iduronic acid, but contains no L-iduronic acid. Found in all connective tissues, a fourth glycan,

hyaluronan, similar in structure to the chondroitins. Hyaluronan is not attached to a peptide

chain, nor is it sulphated; but it contains glucuronic acid and N-acetylglucosamine and is usually

of molecular mass 105-l07 Da (Scott J. E., 1988), (Stern, Asari, & Sugahara, 2006).

Scott reviewed several model experiments dealing with glycosaminoglycans and summarized

a consensus of the findings: (1) Glycosaminoglycans interact electrostatically with type I

collagen under physiological conditions. Additional short range bonding cannot be excluded.

Linear charge density is important, as is glycosaminoglycan shape (determined by iduronate:

glucuronate ratios). (2) Strongly interacting glycosaminoglycans accelerate fibrillogenesis. (3)

Glycosaminoglycans are not incorporated into types I and II collagen fibrils. (4) Chondroitin

sulphate and dermatan sulphate proteoglycans interact strongly with type I collagen,

electrostatically via glycosaminoglycan chains, and by protein-protein interactions. (5)

Chondroitin sulphate and dermatan sulphate proteoglycans are incorporated into type I collagen

precipitates, if present during fibril growth. (6) Chondroitin sulphate and dermatan sulphate

proteoglycans inhibit fibrillogenesis (Scott J. E., 1988).

Tissues composed of collagen type I possess small amounts of proteoglycans which contain

almost exclusively dermatan sulfate; while tissues containing only collagen type II have high

amounts of chondroitin sulfates. The interaction among macromolecules is also important in the

57

extracellular matrix, and point at collagen-proteoglycan interaction as being essential to tissue

resiliency (Junqueira & Montes, 1983).

Hierarchy

Structural order in collagen occurs at several discrete levels. The primary structure denotes

the complete sequence of amino acids along each of three polypeptide chains. The secondary

structure is the local configuration of a polypeptide chain (Ratner, Hoffman, Schoen, & Lemons,

1996). An essential feature for the formation of the triple helical structure is that in the central

part every third position along the chains is occupied by glycine, which leads to the tripeptide

unit Gly-X-Y (Kuhn & Glanville, 1980). Tertiary structure refers to the global configuration of

the polypeptide chain into the triple helical collagen molecule. The fourth-order or quaternary

structure denotes the repeating supermolecular unit structure, comprising several molecules

packed in a specific lattice, which constitutes the basic element of the five molecule microfibril.

Several microfibrils aggregate end to end and also laterally to form a collagen fiber. Collagen

fibers exhibit a characteristic banding pattern with a period of about 65 nm (Ratner, Hoffman,

Schoen, & Lemons, 1996).

The exoskeleton materials of arthropods are complex composites that are hierarchically

structured and multifunctional. The linear chitin chains align anti-parallel and form α-chitin

crystals at the molecular level. Several of α-chitin crystals which are wrapped by proteins form

nanofibrils of about 2–5 nm in diameter and 300 nm in length. A bundle of chitin–protein

58

Figure 3-6 Hierarchical levels of collagen I in bone and α-chitin-protein matrix in crab

exoskeleton. Depicting points of differences in the types of bonds and triple helix structure of

collagen and linear structure of chitin.

Molecular

unit

Peptide

bonds

Nanofibrils

3-chain

coiled

helix

Aggregation of

fibers

Fibrous Bundles

Structure

Linear

Crystals

Glycosidic

bonds

Collagen Type I α-Chitin

59

nanofibrils then form chitin–protein fibers of about 50–100 nm in diameter. These chitin–protein

fibers align together forming planar layers which stack up helicoidally (Meyers, Chen, Lin, &

Seki, 2008). This structure is called a twisted plywood or Bouligand structure (Bouligand Y. ,

1972).

The basic hierarchical sequence for both collagen and chitin is Molecule: Chain of molecules:

Microfibril: Fibril Aggregation: Fibrous Bundle: ‘Twisted Plywood’. The major difference in

the hierarchical scheme lies within the chain of molecules. Collagen has peptide bond that create

a triple helix while chitin has glycosidic bonds that creates a linear crystal structure. See figure 3-

6.

Liquid Crystal Characteristics

All biological structures form in an environment rich in water. How rigid crystalline

structures are formed in biologically aqueous environments is not fully understood. Chemical

reactions in water can produce a precipitate that is insoluble in water. Crystalline structures can

form in water if the concentration of the substance exceeds its saturation point. The crystalline

precipitate can form a rigid structure under certain conditions. In fact, such processes are

biologically important, especially in the formation of crystalline structures, such as bone, cuticle,

and their building blocks.

Biological solid structures do not allow molecules to diffuse around so that all the chemical

and physical processes necessary for life can take place. Fluids allow molecules to move around

60

in a way that allows interactions with other molecules. Succinctly, biological structures must be

rigid enough to add stability and function properly as well as fluid enough to allow the

movement of molecules and all the necessary processes to take place quickly. Many biological

structures achieve a delicate balance between rigidity, order, and fluidity by liquid crystalline

(LC) structures. Membranes surrounding the outer cell, the nucleus, mitochondria, and the

endoplasmic reticulum of animal cells demonstrate this balance and serve many functions: (1)

confines the contents (rigidity); (2) discriminate between various substances to be transported

(order); and (3) control the quick movements of molecules and rapid reactions (fluidity).

Therefore, it is a safe assertion that liquid crystalline structures are major building blocks within

animal cells.

In the liquid crystal phase, molecules move freely about in much the same manner as in a

liquid, but tend to remain oriented in a certain direction. This partial alignment represents a

degree of order not present in liquids. States of matter are different from each other because the

molecules in each state possess different amounts of positional order and orientational order. In a

solid, the molecules are constrained to occupy only certain positions, creating a phase possessing

positional order. Additionally, the specific positioned molecules in a solid are constrained in

how they orient themselves with respect to other molecules, creating a phase possessing

orientational order. Both positional and orientational orders are lost completely, when a solid

melts to a liquid and the molecules chaotically move and tumble. However, when a solid melts

to a liquid crystal, the positional order may be lost but some of the orientational order remains.

61

Molecules likely to be liquid crystalline at a certain temperature: (1) typically are elongated in

shape (significantly longer than it is wide); (2) have some rigidity in its central region; (3) tend to

have somewhat flexible ends. A representative model of a typical liquid crystal molecule is

depicted in figure 3-7. Elongated molecules customarily have stronger attractive forces when

aligned parallel to one another and they bump into each other less when they all tend to point in

the same direction. The flexibility appears to allow one molecule to position itself more easily

between other molecules as they all randomly move about (Collings, 2002).

Liquid crystals can be classified by their dependence on temperature and/or concentration.

Thermotropic LCs present phase transitions, as temperature is changed. When the liquid crystal

phase is dependent on the concentration and temperature of the LC molecules in a solvent, it is

called a lyotropic LC. A classic example of a lyotropic liquid crystal mixture is one with a

molecule that has end groups with different properties; one end of the molecule shows an affinity

for water, while the other end tends to exclude water. Both thermotropic and lyotropic LCs

consist of organic molecules. Although many compounds exhibit only one LC phase, it is not

unusual for a single substance to possess more than one (Collings, 2002).

Membranes surrounding the outer cell, the nucleus, mitochondria, and the endoplasmic

reticulum of animal cells owe its structure to the LC nature of the bilayer phospholipid/water

mixture. Amphiphilic molecules are composed of hydrophobic (water-fearing) and hydrophilic

(water-loving) parts; and are also known as surfactants. As the amount of amphiphilic (loving

62

Figure 3-7 Representation of a typical liquid crystal model.

both kinds) material is mixed with water, it is possible for: (1) a small amount to go into

solution; and (2) an increasing amount to form micelles or vesicles. The fluid mosaic model is

the standard model for the cell membrane. The concentration of one constituent with respect to

the other is far more important in these lyotropic LCs.

Three liquid crystals classifications are relevant to biological composites: nematic,

cholesteric, and blue-phase, figure 3-8. Long rod-shaped molecules line up in parallel in nematic

liquid crystals. In cholesteric liquid crystals (twisted or chiral nematic), long rod-shaped

molecules have helicoidal distribution that is only apparently layered; in a minute volume the

molecules run almost in parallel. Nonetheless, there is a slight angular change between

neighboring molecules, always in one sense of direction for a specified set of conditions. Blue

phase liquid crystals are similar to cholesteric; however the twist is cylindrical, whereas the twist

is planar in cholesteric liquid crystals. Changes in temperature or in concentration may bring

about a change in plane adopted (Neville A. , 1993). Although many compounds exhibit only

one liquid crystal phase, it is not unusual for a single substance to possess more than one. These

liquid crystal phases are formed by rodlike molecules, the most common and well known LC

phases (Collings, 2002). In 1977, researchers in Indian discovered disk-like molecules also form

63

liquid crystal phases and are called them discotic LCs (Chandrasekhar, Sadashiva, & Suresh,

1977).

There are indications that the crystalline ordering of polymer material and proteins is the first

step in the creation of very hard biological structures such as cuticle and bone. The organic

portions of cuticle and bone are chitin and collagen, respectively. These organic polymers form

fibrous material that share many characteristics of LCs. For example, liquid crystals and fibrous

composites are both mobile, have similar architectures or geometries, can self-assemble, and

have similar optical properties. Fibrous materials can be composed of a single polymer or a

mixture of polymers (Neville A. , 1993).

A polymer is any large molecule composed of a long sequence of repeating units. The

monomer is the basic building block and is the unit repeated in the molecule. Proceeding in a

variety of ways, the polymerization process is the chemical reaction in which these bonds

between monomers form. The polymerization process stops when the number of reactive end

groups decreases to the point where there is little chance for further reactions between them.

Polymer LCs can achieve extremely high orientational order; respond to electric and magnetic

fields; are relatively inexpensive and stable; and can be easily fabricated into thin films.

Chitin and collagen are polymer LCs. Chitin is a polymer consisting of a linear chain of

repeating units. The unit is derived from N-acetyl-glucosamine monomer. Notice that there are

chemical entities (-H, -OH, and -CH2OH) attached to the central part of the monomer. As

reactions occur with chitin’s chemical entities, the linear chain adopts a helical structure, gaining

rigidity and the capacity to produce LC phases. Amino acids form the basic building block for

64

the collagen polymer LC. A typical amino acid possesses two quite different reactive groups.

The carboxyl group (-COOH) on the end of one amino acid can react with the amino group (-

NH2) on the end of another amino acid to form a chain of amino acids. The resulting

macromolecules are called polypeptides. The collagen structure is basically a triple helical

Figure 3-8 Depiction of rod-like liquid crystal phases: smectic, nematic, blue phase, and

cholesteric or chiral nematic.

65

Figure 3-9 Illustration of aspects of lyotropic liquid crsytals.

3.

All Biological

Liquid Crystal

Molecules

66

chain, which causes the macromolecule to be fairly rigid and capable of forming LCs. Because

these macromolecules have helical structures, they interact in a fashion which prefers that the

molecules not be exactly parallel to one another. Consequently, some molecules form chiral

nematic phases. The forces between these large molecules are due to the helical molecules

themselves and to the interactions between these large molecules and the solvent molecules

(Collings, 2002). Chitin and collagen macromolecules must dissolve in the solvent. LC phases

only form when the concentration is high enough so that these macromolecules constantly

interact with one another. This cannot be achieved if the macromolecules do not dissolve in the

solvent to high enough concentrations (Collings, 2002). Chitin and collagen fulfill

therequirements of rigidity and of being solvent-dissolved; thus, they, like all biological

structures, are lyotropics LCs. Characteristics of lyotropics LCs is depicted in figure 3-9.

Works published by Bouligand, Giraud-Guille, and Neville reviewed liquid crystalline order

in biological materials such as chitin, collagen, and other fibrous composites (Bouligand Y. ,

1972), (Giraud-Guille M.-M. , 1994), (Giraud-Guille M.-M. , 1992), (Giraud-Guille M. M.,

1996), (Giraud Guille, Mosser, Helary, & Eglin, 2005), (Neville & Luke, 1971). After studying

fibrous structures such as the cuticles of crabs, Bouligand (1972) found that several fibrous

biological materials are: (1) made of fibrils arranged in highly organized geometric patterns ---

‘twisted plywood model’; (2) solids with elongated molecules that show a strong anisotropy; (3)

still more or less fluid phases between two well defined temperatures and may show

birefringence; and (4) capable of forming LCs in solutions. These vary among the factors that led

Bouligand to postulate that the secretions of fibrous structures pass through a brief liquid phase

67

providing an intriguing mechanism in morphogenesis for certain fibrous materials. Neville also

suggested biological fibrous composites might have developed by self-assembly via a liquid

crystalline phase (Neville A. , 1993). This idea was further strengthen by the work of Giruad-

Guille (1989, 1992). Despite their similarities, notable differences exist between biological

fibrous composites and liquid crystals. The fibrous component consists of small parallel bundles

of molecules (crystallites), while liquid crystals consist of individual molecular chains. Another

difference is that biological composites that have reached fruition exist in a solid state, while

liquid crystals exist in a state with properties intermediate between those of a solid and liquid

(Wright & Arthur, 1987).

The structure of many other biological materials is undeniably related to that of liquid

crystals, despite they are not at all fluid. In most skeletal tissues, the extracellular matrices are

made up of fibrils which also are stabilized analogues of either nematic or cholesteric liquid

crystals (Bouligand Y. , 2008). Cuticles also fall into this class, non-lamellae cuticle (preferred

orientation of microfibril) corresponds to the nematic phase and helicoidal model to the

cholesteric phase (Wright & Arthur, 1987). They are considered as stabilized analogues of liquid

crystals.

Liquid crystalline self-assembly was observed in concentrated solutions of the main polymers

extracted and purified from these materials: chitin and collagen. Liquid crystals appear to be

involved in most physical aspects of biological morphogenesis. Patterns draw their origin from

properties of liquid crystals, mainly geometrical. Certain patterns are very common in thin

sections of biological tissues. The ‘‘arced fibrils’’ were first described in carapaces of

68

arthropods, mainly insects and crustaceans. The crab carapace also called cuticle is strongly

mineralized, and samples must be decalcified in general before dehydration, embedding and

microtomy. The thin section crustaceans showed a fibrous material made of superposed lamellae,

each one being made of a long series of nested arcs, with concavities oriented along a common

direction. The fibrils are made of chitin, a nitrogenous polysaccharide, associated with diverse

proteins. This was rather surprising to observe a similar organization in so distant biological

materials, differing by their chemical composition (chitin, collagen, DNA), their function and

their phylogenetic position (Bouligand Y. , 2008).

In arthropod cuticle, the helicoidal building block is the chitin polymer. Within each

horizontal layer these rod shaped polymers are packed parallel to each other. These layers of

parallel rods are stacked on top of one another, each layer rotated slightly to adjacent layers. The

angle of rotation between the layers is more or less constant, giving a regular helicoidal structure.

The 3D structure is reminiscent of plywood. This multiply construction gives the helicoid the

ability to resist strain in most directions (Murray & Neville, 1997). Similar patterns were

observed in the sections of compact bone in TEM. The collagen network appears very dense,

with fibrils either arranged in two alternating directions forming an orthogonal plywood, or

regularly changing orientation within the section plane, forming arced patterns. Thus, two

osteon models coexist in compact bone; the first obeys the classical description of osteons

forming orthogonal plywood, the second follows the twisted plywood model, where collagen

fibrils show small and regular changes in their orientation. Arced figures are a direct

consequence of what is described as the twisted plywood model. Parallel and equidistant straight

69

lines represent the molecular directions and from one level to the next, the lines turn by a small

and constant angle; series of nested arcs appear on oblique sides of the model (Giraud Guille,

Mosser, Helary, & Eglin, 2005).

In compact bone osteons, the collagen fibrils form arced patterns as a consequence of their

three-dimensional organization; this structure is analogous to a geometry found in certain liquid

crystals (Giraud-Guille M.-M. , 1992). These self-assemblies were analyzed between crossed

polars in optical microscopy since they form birefringent fluids over long distances. Self-

assembled liquid crystalline phases of collagen generated crimp morphologies (Giraud-Guille &

Besseau, 1998). This characteristic and striking architecture must have a common physical origin

in these phyletically and functionally very different biological materials (Bouligand Y. , 1972).

Bouligand discussed a possible mechanism for a liquid crystalline secretion in

crustaceans and in other arthropod cuticles. Bouligand postulated that the fibrous matrix is

produced by a secretion which is a highly concentrated solution of chitin and proteins. Thin

filaments which associate these two components differentiate at the epidermal cells’ membrane

surface, and align in this plane. This corresponds to an ordered secretion which is liquid

crystalline, since alignment and fluidity are realized at the close contact of epidermis. The

parallelism of filaments with the epidermis involves also that of cholesteric layers themselves

with this epidermis. Bouligand believed that cell membranes create strong boundary conditions,

with a considerable influence on textures. The cuticle layers lie parallel or nearly parallel to the

epidermis and rapidly the shape of the epidermis is maintained by the cuticle itself. Cells

maintain a control at the time of the liquid crystalline secretion, mainly by the faculty of

70

changing molecular orientations, through the interactions between the cytoskeleton and the

extracellular matrices, across some specialized areas of cell membranes. Questions still remain

concerning the differentiation of biological analogues of cholesteric liquid crystals, what comes

from liquid crystallinity of the initial secretion, and what is controlled by cells. As research

continues on liquid crystalline phases of fibrous materials, progression to the answers is expected

(Bouligand Y. , 2008).

Neville (1993) reported that ultrastructural observations of arthropod cuticles above the

epidermal cells reveal uniform series of arced patterns, attesting to fluidity at the time of

Figure 3-10 Liquid crystalline organization of collagen at different concentrations.

Isotropic

20-50 mg/ml

Loose

Cholesteric

50-400

mg/ml

Dense

Cholesteric

400-800

mg/ml

Banded

Structures

800-1000

mg/ml

71

secretion. Indeed, products of neighboring cells join up neatly if the system is liquid; otherwise,

many irregularities and faults would appear along the cell boundaries. Directly visible on the

oblique sides of the pyramid are what appear to be superposed series of parallel nested arcs. The

concavities of the arcs are reversed on opposite sides of the model. In biological systems this

particular geometry has often been described as a twisted plywood, but depending on its

appearance in optical or electron microscopy, or on the research field of the authors, a variety of

Table 3-5 Description of Cholesteric Geometries

Descriptions of Cholesteric Geometries

Terms used by physicists or chemists (liquid crystal and polymer field)

Cholesteric mesophases

Cholesteric texture

Chiral nematic structure

Terms used by microscopists (aspect in optical or electron microscopy)

Fingerprint-like patterns

Arced patterns

Terminology introduced by Bouligund and colleagues in France

Twisted plywood

Multidirectional plywood

Twisted fibrillar phase

Terminology introduced by Neville and colleagues in the United Kingdom

Helicoidal

Helicoidal architecture

Helicoidal arrays

(Giraud-Guille M. M., 1996)

72

descriptions of the cholesteric structure are found in the literature, table 3-5. The possibility of

slowly increasing the collagen concentration of a solution has allowed the following of the

consequent changes in the organization of collagen, figure 3-10. This work shows that a

relationship exists between collagen concentration and the diameter of fibrils and the discrete

rotational angle of subsequent layers in collagen rich matrices (Mosser, Anglo, Helary,

Bouligand, & Giraud-Guille, 2006).

Collagen and chitin can readily form analogues of amphiphilic molecules due to the presence

of reactive groups on its backbone. A variety of amphiphilic copolymers containing hydrophobic

and hydrophilic segments, are very active due to their spontaneous self-assembly behavior in

aqueous media. This is explained by the presence of a great number of different hydrophilic and

hydrophobic reactive groups in these structures. Hydrophilic groups include hydroxyl, carboxy

groups and hydrophobic groups include hydrocarbons chains. The items created by amphiphiles

can be rod-like. These anisotropic self-assembled structures can order themselves in a similar

fashion that liquid crystals do, forming large-scale versions of LC phases.

Amphiphilic properties of polysaccharides such as chitin can be varied by manipulating their

hydrophilic and hydrophobic groups. Since most polysaccharides are polyelectrolytes. Their

hydrophilicity can be controlled by varying charge density and their hydrophobicity can be

controlled by the introduction of branches (alkylation, acylation, grafting, crosslinking)

(Desbrieres & Babak, 2008). Amphiphilic chitin derivatives are soluble in both organic and

aqueous solvents and under appropriate conditions they are able to self-assemble (Aranaz,

Harris, & Heras, 2010).

73

The term ‘‘peptide-amphiphile’’ can be used to describe amphiphilic peptides consisting only

of amino acids such as collagen. It has been shown that linking peptides to synthetic hydrophobic

tails enables them to self-assemble into biomimetic films with highly organized interfaces, and

enhances their ability to attain well-defined and stabilized three-dimensional structures that

effectively promote cell adhesion, spreading, migration, growth and differentiation. By changing

any of the structural segments of an amphiphilic molecule, one can control the morphology,

characteristics, surface chemistry, and function of the molecule. Amphiphiles can self-assemble

into a variety of different structures such as micelles, vesicles, bilayers, ribbons, twisted ribbons,

and patterned membranes to minimize unfavorable interactions with their surroundings. Triple-

helical peptide-amphiphiles are very stable and such model collagen peptide-amphiphiles follow

some of the trends observed for other more common surfactants. The long alkyl chains

characteristic of peptide-amphiphiles provide a convenient mechanism for self-assembly and

association with other hydrophobic substrates. Enabling optimal presentation of active binding

sites, the composition and orientation of molecules on the surface is well controlled. The self-

assembly of the hydrophobic tails of the peptide-amphiphiles is a driving force (hydrophobic

effect) that stabilizes the three-dimensional structure of the peptide headgroup into triple helices,

and a-helices, giving rise to protein-like molecular architectures (Kokkoli, et al., 2006).

Bouligand, Neville and Giraud-Guille outlined a progression of phases that collagen and

chitin undergo to create ordered, fibrous structures. The ordering seems to be in layers, with the

preferred direction in each layer rotating slightly in going from layer to layer. At first the order is

liquid crystalline, but as the concentration of the polymer and protein increases, the material

74

becomes more of a gel. The more rigid a molecule is, the less soluble it is. Crystalline bundles of

parallel polymers can also form, causing the material to harden. Such a substrate seems to be

necessary for minerals to begin to accumulate, thus allowing shells, bone, etc., to form (Collings,

2002).

Synthesis

The biosynthesis and degradation of both collagen and chitin are stepwise procedures

governed by enzymatic activity; these pathways consist of catalytic units, precursors,

biotransformations, polymerization, hydrolysis, and fibrillogenesis. Both collagen and chitin

share many common properties at the molecular level of organization. For example, they both

have components formed in the intracellular and in the extracellular space during the synthesis

process. Both synthesis pathways involve changes in the cytoplasmic matrix, the rough

endoplasmic reticulum, processing in the Golgi apparatus and transport through vesicles

culminating in exocytosis into an assembly zone. Both use enzymes to transform. In the

assembly zone, both collagen and chitin molecules form fibrous, hierarchical structures. Both of

these extracellular fibrous molecules are synthesized in a precursor form and their active

structure is polymeric. Furthermore, the resulting tissue formation, i.e. bone for collagen and

cuticle for chitin, follow a common developmental course, namely that the major organic

constituent collagen and chitin are synthesized, extruded from the cell, and then self-assembled

in the extracellular space before mineralization begins. For this reason, bone and cuticles are

good examples of an ‘‘organic matrix-mediated’’ mineralization process (Cui, Li, & Ge, 2007).

75

Collagen is synthesized by osteoblasts and fibroblast as well as smooth-muscle cells,

chondroblasts and odontoblasts. Osteoblasts synthesize only type I collagen (Kuhn & Glanville,

1980). The biological synthesis, secretion, and assembly into definitive extracellular structures of

collagen can be described briefly as follows. Pre-procollagen chain is the starting point for a

chain of bioconversions which include major steps such as non-lysosomal degradation,

hydroxylation, glycosylation, and disulphide bond formation. This metabolic pathway culminates

in the formation of procollagen. Collagen polypeptides are synthesized on the rough endoplasmic

reticulum as propolypeptides, over 100 amino acid long. The individual polypeptide chains,

called pre-procollagen chains, are synthesized on membrane-bound ribosomes in the cisternae of

the rough endoplasmic reticulum. These chains have three major domains: the α-chain, the

amino-terminal peptide, and the carboxy-terminal peptide. These pro-α-chains are modified after

synthesis by the removal of prepeptide sequences, by hydroxylation of proline and lysine, by

glycosylation of propeptides and by disulfide bond formation. Each α-chain has an amino

terminal and a carboxy terminal that direct the spontaneous assembly of the α-chains into the

triple helix, procollagen. After all these post-translational modifications take place, individual

procollagen molecules are transported to the Golgi apparatus, packaged into granules, and

secreted into the extracellular space by exocytosis. The collagen producing cells like fibroblasts,

for example, secrete enzymes which catalyze the hydrolysis of the extension peptides from

procollagen to produce the triple helix called tropocollagen – a rigid rod 300nm long and 1.5nm

in diameter. The extension peptides at both ends of the molecule must be cleaved before

polymerization can occur and the fibers can be assembled. Tropocollagen molecules

76

Figure 3-11 Collagen and Chitin Metabolism (Cohen, 1993).

ENZYMES

turnover

POLYMERIZATION

PRECURSORS Biotransformation:

Collagen

Hydroxylation

Glycosylation

Disulphide bond formation

Collagen

prolyl-3-hydroxylase

prolyl-4-hydroxylase

lysl hydroxylase

Chitin

chitin synthase

Synthesis Translocation Insertion Activation

Chitin

Phosphorylation

Amination

acetylation

Procollagen formation

UDP-GlcNAc formation

Collagen

Limited Proteolysis

(crystallization?, fibrillogenesis)

ORIENTATION OF MICROFIBRILS

Chitin Hydrogen Bond Formation (crystallization, fibrillogenesis)

SUPRAMOLECULAR ORGANIZATION

(covalent and noncovalent interactions)

(with proteins, with polysaccharides)

DEGRADATION

(Collagen: collagenases)

(Chitin: chitinase, chitobiase, chitodextrinase)

RECYCLING

MINERALIZATION

77

Figure 3-12 Chitin and Collagen Synthesis (Laurent, 1987).

78

spontaneously associate in a quarter-staggered manner, all aligning in the same direction, to form

microfibrils. Microfibrils pack closely together to form mature collagen fibers 50 nm thick with

the characteristic banding pattern (Smith & Wood, 1991).

The biosynthesis of the crustacean cuticle or exoskeleton involves the synthesis and assembly

of component lipids, carbohydrates, and proteins by the epithelial cell layer which lies just

underneath the cuticle. Chitin synthesis is directly governed by an enzymatic activity called

chitin synthase. To provide a flow of substrate molecules for the functional catalytic units, a

cascade of cytoplasmic biotransformations of sugar precursors takes place. Chitin synthesis in

the crustacean epithelial cell begins with the glycosylation of a protein acceptor to form a primer

molecule for chitin synthetase. Catalytic units synthesized at the endoplasmic reticulum

compartment are packaged in clusters which involve the Golgi apparatus. The assembled

multiunits are translocated and integrated into the plasma membrane at the apical compartments

of epidermal cells. Trehalose, glucose, or the polysaccharide glycogen, circulating in the

hemolymph, could be the starting points for a chain of bioconversions which include major steps

such as phosphorylation, amination, and acetylation. This metabolic pathway culminates in the

formation of UDP-GlcNAc, the ultimate substrate of chitin synthase. Transformation of fructose-

6-phosphate to glucosamine-6-phosphate by glutamine-fructose-6-phosphate aminotransferase

can be considered as the first turning point on the pathway to chitin synthesis. Next, there is

molecular organization of the chitin synthase units in the cell membrane, and an intricate process

which involve the translocation of chitin polymers across this barrier into the extracellular space

where chemical modification of the noncrystallized chitin occurs (Cohen, 1993). Synthesis of

79

chitin occurs either intracellularly or at the interphase with the extracellular medium. The

modified chitin molecule associates with other molecules like protein. It has become clear that

protein is covalently attached to the structural polysaccharide, chitin, in the crustacean. The last

step in chitin biotransformation involves the formation of microfibrils at the cell surface,

following the coagulation of adjacent developing chitin chains via extensive hydrogen bonding.

Chitin polymerization and crystallization are coupled; they are consecutive processes. The

resulting chitin-protein structure matures through formation of secondary covalent bonds among

its components, creating chitin fibers (Jolles & Muzzarelli, 1999). Studies done by Horst

supports the conclusion that crustacean chitin is secreted as a glycoprotein complex and that

concurrent protein synthesis is required for chitin deposition to continue (Horst, 1989). Liquid

crystal mobility may account for changes in orientation of the chitin microfibrils and, thus, for

the helicoidal appearance. The cyclic phenomenon of molting in crustaceans involves a

concerted and coordinated flow of hormonally-controlled biochemical events governed by

enzymatic axtivity. One such major event is the degradation of the endocuticular chitin, followed

by the subsequent recycling of the aminosugar monomers into newly-formed polymer units

(Cohen, 1993).

There are differences in the biotransformation processes involved in the synthesis of the

molecules secreted into the extracellular matrix of the cells that make collagen and chitin. The

biotransformation of collagen involves non-lysosomal degradation, hydroxylation, glycosylation,

and disulphide bond formation while chitin’s biotransformation involves phosphorylation

80

amination acetylation. While collagen undergoes limited proteolysis after polymerization, chitin

undergoes hydrogen bonding, figure 3-11.

Figure 3-12 is a schematic representation of cells showing the generalized pathway of

collagen and chitin biosynthesis. The sequential changes in the cytoplasmic matrix, RER, and

processing in the Golgi apparatus and transport through vesicles culminating in exocytosis into

the assembly zone are depicted (Laurent, 1987), (Cohen, 1993), (Jolles & Muzzarelli, 1999).

Properties

Collagen fibers transmit forces, dissipate energy, prevent premature mechanical failure and

provide biological signals to adjacent cells that regulate function. Moreover, collagen is

resorbable, it has high water affinity, low antigenicity, very good cell compatibility and ability to

promote tissue regeneration (Kolacna, Bakesova, Varga, Kostakova, & Planka, 2007). Isolated

collagen fibers have high tensile strength that is comparable to nylon (50-100MPa) and an elastic

modulus of approximately 1 GPa (Enderle, Blanchard, & Bronzino, 2005). Cross-linking

increases with age, and collagen becomes less flexible. Collagen often exists in combination with

other proteins.

The mechanical properties of the cuticle are conferred by the proportion of chitin, by the degree

of sclerotization and by the sequences of its proteins (Iconomidou, Willis, & Hamodrakas, 2005).

Chitin microfibrils, being composed of long molecular chains bound firmly to another,

contribute significantly to the mechanical strength of cuticles and their final orientation appears

to be related to the stresses which will be placed on the cuticle. The helicoidal model gives a

81

plywood structure to the cuticle, each ply (or sheet) being made up of high modulus chitinous

fibrils in a low modulus protein matrix. A plywood is a mechanically resistant material, which

associates several wood sheets, stuck together, with the grain of adjacent layers lying at right

angle to each other, so that the fibers follow two alternating directions (Bouligand Y. , 2008).

This would give a very strong structure, one which would resist bending forces in all

directions. On the other hand microfibrils with preferred orientation provide a structure strong in

tension (when the strain is on chitin) but weak in the direction normal to the chitin chains (when

the strain is on the protein) (Wright & Arthur, 1987). Examples of these arced patterns were

found in diverse tissues of animal and plant groups, mainly extracellular matrices, connective

and skeletal tissues, but also in several intracellular differentiations. Variations are considerable

within these materials, simply in the same crustacean carapace for instance, according to the

position in the body and also according to the distance from the outer surface. This is an aspect

of biodiversity, with considerable changes also between species. Research will be necessary in

this field, and the list of these twisted fibrous materials needs to be completed: the presence of

fibrils is often masked but some appropriate methods, as chemical treatments, staining and use of

contrast agents can make the arcs visible. Polarizing microscopy also is essential to detect these

structures (Bouligand Y. , 2008). Table 3-6 provides microscopic information regarding collagen

and chitin.

Collagen and chitin provide the backbone for mineralized tissue such as bone and crab

exoskeleton (Chen, et al., 2008). Similarites between liquid crystal and collagen and chitin were

82

Table 3-6 Collagen and chitin empirical formula, tissue distribution, microscopic appearance,

ultrastructure, synthesis site, interaction, and function.

(Junqueira & Montes, 1983)

realized by Bouligand, Giraud-Guille, and Neville (Bouligand Y. , 1972), (Giraud-Guille M.-M. ,

1992). Collagen and chitin readily form spherulites in vitro (Krebs, et al., 2004).Other common

features of collagen and chitin are that they are both biodegradable, biocompatible,

multifunctional, non-toxic, play a role in mechanical support, have limited reactivity; highly

insoluble and birefringement (Jolles & Muzzarelli, 1999), (Köster, Evans, Wong, & Pfohl,

2008).

83

Features unique to chitin are that it is a polysaccharide polymer found in crab exoskeleton,

has glycosidic bonds, has a linear chain, and has three forms exist (Jolles & Muzzarelli, 1999).

Another feature unique to chitin is that it is antibacterial. One study proclaimed that chitin

sutures resisted attack in bile, urine, and pancreatic juice (Dutta, Ravikumar, & Joydeep, 2002).

The fungicidal and bactericidal action of chitin appears to be mediated by the electrostatic forces

between the protonated NH2 group and the negative residues at cell surfaces. The number of

protonated NH2 groups present increases with increased degree of deacetylation (DD).

Therefore, the DD influences antimicrobial activity (Tsai, Su, Chen, & Pan, 2002). Features

unique to collagen are protein molecule, peptide bond, triple helix coiled chain with cross-

striation pattern, over 27 other types, found in bone (Köster, Evans, Wong, & Pfohl, 2008). It is

interesting that collagen is not antimicrobial like chitin. The simplest explanation can be

attributed to the structure of collagen; in that it does not afford enough electrostatic forces.

There also exist differences in the swelling behavior of chitin and collagen. Chitin does not

swell in water. Collagen may or may not swell in water depending on which tissue is used to

extract the collagen. For example, bone collagen (which normally calcifies) does not swell in

water whereas tendon collagen (which normally does not calcify) does (Bonucci, 1992). Several

crystalline forms of chitin have been ascribed and they differ in packing and polarity of the

adjacent chains. The failure of chitin to swell in water is explained by the very extensive inter-

and intramolecular bonding. This strong bonding leads to the formation of long microfibrils with

high tensile strengths and accounts for chitin’s fibrous character. The extents of the molecular

84

Figure 3-13 Collagen and chitin unique features and ones they share.

bonding differ in different cuticles which can lead to some variation in the observed chemical

and physical properties of the chitins present in different species (Wright & Arthur, 1987).

Figure 3-13 highlights common and unique features of collagen and chitin.

85

Applications

Collagen and chitin have numerous tissue engineering applications, ranging from structural to

metabolic organs (Monzack, Rodriguez, McCoy, Gu, & Masters, 2011), see figure 3-14. In

addition, a wide variety of other applications, figure 3-15, for chitin and collagen have been

reported over the last four decades (Enderle, Blanchard, & Bronzino, 2005), (Singh & Ray,

2000), (Ma & Elisseeff, 2006), (van Blitterswijk, 2007), (Hin, 2004).

The poor solubility of chitin is the major limiting factor in its utilization, and the investigation

of its properties and structure. Despite these limitations, various applications of chitin have been

reported in the literature. For example, fibers made of chitin have been useful as absorbable

sutures and wound-dressing materials. These chitin sutures resist attack in bile, urine, and

pancreatic juice, which are difficult with other absorbable sutures. It has also been claimed that

wound dressings made of chitin fibers accelerate the healing of wounds by up to 75% (Dutta,

Ravikumar, & Joydeep, Chitin and Chitosan for versatile Applications, 2002). Collagen has been

3 N HCl at the boil for 1.5 h. A typical ratio of 3 N HCl to chitin was 10 ml /g. After acid

viewed as an ideal biomaterial, as it is the major component of the extracellular matrix, and

because it can be processed into a wide variety of structures and shapes (sponges, fibers, films,

3D gels). A vast number of publications can be found in the literature, covering a diversity of

clinical applications, such as general surgery, orthopedics, cardiovascular, dermatology, urology,

dentistry, ophthalmology, plastic and reconstructive surgery. Although these examples

encourage applications of collagen in the biomedical arena, the risk of infection, its low

antigenicity and fast degradation when implanted in the human body are, to some extent, limiting

86

Figure 3-14 Collagen and Chitin/Chitosan Tissue Engineering Applications (Monzack,

Rodriguez, McCoy, Gu, & Masters, 2011).

Eye

Collagen

Chitin (Chitosan)

Muscle

Collagen

Chitin (Chitosan)

Tendon

Collagen

Chitin (Chitosan)

Bone and Cartilage

Collagen

Chitin (Chitosan)

Skin

Collagen

Chitin (Chitosan)

Vessels

Collagen

Chitin (Chitosan)

Heart

Collagen

Chitin (Chitosan)

Adipose

Collagen

Chitin (Chitosan)

Nerve

Collagen

Chitin (Chitosan)

Metabolic Organs

Collagen

Chitin (Chitosan)

87

Figure 3-15 Other Biomedical Applications. In the diagram some of the collagen applications

are highlighted in yellow (Prashanth & Tharanathan, 2007).

Chitin/Chitosan

Collagen

Biorenewable

Biocompatible

Biodegradable

Biofunctional

88

the clinical applications of this natural biomaterial. (van Blitterswijk, 2007), (Ma & Elisseeff,

2006), (Atala, Lanza, Thomson, & Nerem, 2008).

Characterization of Collagen and Chitin

Materials and Methods

Collagen samples were obtained from Worthington and Sigma. Chitin, from crab

exoskeleton, was obtained from Sigma. Chitin samples were purified into chitin crystallite

suspensions or chitin whiskers. The samples were boiled in 5% KOH solution for 2 hours to

remove residual protein. The samples were washed in deionized water until the pH of the

sample was 7. Chitin suspensions were prepared by hydrolyzing the purified chitin sample with

hydrolysis, the suspensions were washed with deionized water by centrifugation and decanting

of the supernatant. This process was repeated several times until the suspension spontaneously

transformed into a colloidal state (Li, Revol, & Marchessault, 1996) and the pH of the

suspension was approximately 6.

Elemental Analysis

Elemental analysis to determine the Carbon, Hydrogen, and Nitrogen composition was

performed by Prevalere Life Sciences, LLC, Whitesboro, NY.

89

Zeta Potential Analysis

A Malvern Zetasizer 3000 was used to measure the zeta potential and conductivity of the

collagen and chitin samples. Samples were created varying the pH. Samples tested consisted

98ml of 0.005 M solution of KNO3; 1 ml of either [0.1, 0.25, 0.5, 0.75, 1] M of HNO3 or [0.1,

0.25, 0.5, 0.75, 1 M] of KOH; and 1 ml of 0.001 wt% of chitin whiskers suspension. One sample

consisted of 99 ml of0.005 M solution of KNO3 and 1 ml of 0.001 wt% of chitin whiskers

suspension. Data points were plotted.

Thermal Analysis (TGA)

Thermogravimetric analysis was carried out on dried samples using a Shimadzu TGA-50H

Thermogravimetric Analyzer (Kyoto, Japan). Heating was performed in an alumina pan in an air

flow (20 ml /min) at a rate of 10 °C/min up to 800°C. Percentage of weight loss was calculated

using the formula 𝑊𝑡

𝑊𝑜× 100. The percentage weight loss was plotted as a function of temperature

for both collagen and chitin in Figure 2.

Surface Morphology (SEM)

Collagen and Chitin samples were sputtered with an ultrathin layer of gold and studied with a

Hitachi SU-70 Schottky field emission gun scanning electron microscope (The Nanoscale

Imaging, Spectroscopy, and Properties (NISP) Laboratory the Kim Engineering Building at the

University of Maryland at College Park).

90

Results

Chemical analysis

Collagen I and α-chtin both consist of relatively few constituent elements: H, C, O, and N

(Meyers, Chen, Lin, & Seki, 2008) . Results of the elemental composition measurements are

shown in Table 3-7. The elemental analysis revealed the nitrogen content of collagen derived

from bovine achilles tendon was 7.03% while the %N of chitin derived from a crab carapace was

14.61. The theoretical values for % N for collagen and chitin were calculated to be 15.33 % and

6.90 %, respectively. According to Muzzarelli, elemental analysis of chitin isolates typically

have nitrogen content close to 7% and N/C ratio 0.146 for fully acetylated chitin (Muzzarelli,

1999). Degree of acetylation (DA) corresponds to the mole fraction of acetyl units within the

polymer chains. The carbon/nitrogen mole ratio varies from 5.145 in completely N-deacetylated

chitosan to 6.861 in the fully N-acetylated chitin. The degree of deacetylation, DDA, was

therefore calculated according to %𝐷𝐷𝐴 =6.861−𝐶/𝑁

6.861−5.145 (Wanchoo, Thakur, & Sweta, 2008)

(Abdou, Nagy, & Ellsabee, 2008)

However, an explanation for increased nitrogen content lies in the source of the chitin [crab].

Studies indicate that nitrogen content can vary based on the diet, season, or moulting stage; in

females, the production of egg cells also creates a nitrogen difference. Nitrogen content appears

to be the highest for intermoult stages and healthy diets (Linton & Greenaway, 2000), (Linton &

Greenaway, 1997). The experimental values of N% are near the theoretical (6.89) calculated for

91

a chitin that is completely deacetylated. The nitrogen content is also a measure of the protein

amount still present in the chitin. A low experimental value of N is indicative of the minimum

amount of protein left. The sample composition and purity of polysaccharides are in the range of

commercial chitins (Cardenas, Cabrera, Taboada, & Miranda, 2004). The quantity and

characteristics of these constituents such as nitrogen can vary between species and between

individuals of the same species as functions of growth stage, gender, feeding, and other

environmental conditions (Diaz-Rojas, et al., 2006). After purification, the nitrogen, carbon and

hydrogen contents as well as N/C ratio in purified chitin significantly increased. After conversion

of the nitrogen content to the chitin content by a theoretical factor of 203/14, which is the

molecular weight ratio of a monomer (C8H13O5N = 203) to nitrogen (N = 14) in the chitin

(general formula [C8H13O5N]n), the chitin contents in crude and purified crab chitin were 77.1

and 90.0%, respectively. It seems that acid and alkaline treatments followed by decolorization

with potassium permanganate were an effective method in improving the purity of purified

chitin. However, purified chitin contained some components other than chitin (Yen, Yang, &

Mau, 2009).

Collagen degrades. One common archeological indictor for extent of collagen degradation is

the atomic carbon-to-nitrogen ratio, C/N. Modern vertebrate collagen has C/N of 2.8. When

C/N of archeological collagen is greater than 3.1, the isotopic values generally fall outside the

observed ranges for modern animals, meaning that the collagen has degraded sufficiently, that it

is no longer an accurate reflection of diet. These changes in C/N must be associated with

changes in molecular structure. The nature of collagen degradation products and pathways is

92

still largely unknown. Infrared spectroscopy and amino acid analysis have shown loss of amide

bonds and of net amino acid content in degraded collagen, but they do not reveal new organic

constituents generated by the degradation process. Collagen undergoes denaturation,

deamination of R group nitrogen, peptide bond hydrolysis, and condensation reactions of the

hydrolyzed amino acid. The hydrolysis, deamination, and condensation reactions appear to be

components of a concerted process driven by bacteria which leave biomarkers in degraded

collagen. Samples with identical C/N may not have undergone exactly the same degradation

process (Hinman, Markarewicz, asara, Cody, & Tuross, 2008).

Theoretically, nitrogen content of collagen with glycine, proline, and hydroxyproline can be

derived from the empirical chemical formula (C12H24N3O4=274). Therefore, the molecular

weight ratio of the collagen molecule, (C12H24N3O4=274), to nitrogen (N=42) is 274/42 or 6.5.

Nitrogen values were reported on a water- and ash-free basis for Achilles Tendon-Bovine

origin; trimmed from extraneous fat and muscle tissue, hashed, and dried in vacuum; total

nitrogen, 16.9 percent (Graham, Waitkoff, & Hier, 1948).

For Collagen, theoretically, Carbon (144/274), Hydrogen (24/274) and Nitrogen (42/274)

should represent approximately 85% of the sample. However the collagen sample is total

54.27% CHN. Degradation, Contamination, Other than oxygen what other elements are present.

For Chitin, theoretically, Carbon (96/203), Hydrogen (13/203) and Nitrogen (14/203) should

represent approximately 60.59% of the sample. However the collagen sample is total 62.35%

CHN. Degradation, Contamination, Other than oxygen what other elements are present.

93

Table 3-7 Elemental Composition of Collagen. Sample

Collagena

% C

52.55

%H

8.76

%N

15.33

%CHN

76.64

C/N

3.43

Collagenb 41.48 5.76 7.03 54.27 5.90

Chitinc 47.29 6.40 6.90 60.59 6.86

Chitind 41.22 6.13 14.61 61.96 2.82

aCollagen, Theoretical bCollagen derived from Bovine Achilles Tendon, Worthington c Chtin, Theoretical dChitin derived from Crab Carapace, Sigma

Thermal Analysis (TGA)

In the thermogram of chitin (figure 3-16), two thermal events could be observed. The first

occurs in the range of 50–100 °C, and is attributed to water loss. The second occurs in the range

of 300–400 °C and could be attributed to the degradation of the polysaccharide structure of the

molecule, including the dehydration of polysaccharide rings and the polymerization and

decomposition of the acetylated and deacetylated units of chitin. Based on the work of

Jayakumar and Tamura, this is the thermal behavior expected of chitin (Jayakumar & Tamura,

Synthesis, characterization and thermal properties of chitin-g-poly(e-caprolactone) copolymers

by using chitin gel, 2008) (Tanodekaewa, et al., 2004).

In the thermogram of collagen (figure 3-16), three thermal events could be observed. The

first occurs in the range 50-100 °C, and is attributed to water loss. The second event occurs in

the range of 250-350 °C and could be attributed to the breakdown of the triple helix molecule in

chitin. The third event occurs in the range of 450- 600 °C and could be attributed to the further

94

denaturation of collagen to gelatin. Similar behavior was observed in frog skin, amniotic

membrane, and calfskin collagen (Shanmugasundaram, Ravikumar, & Babu, 2004).

Both the collagen and chitin molecules are crystalline which is indicated by thermal shrinkage

behavior due to reorganization, chain folding, recrystallization and other structural changes.

However, chitin has two thermal transitions while collagen has three. Both collagen and chitin

have a thermal event associated with the evolution of freely bound water. Chitin is linear

molecule; hence it does not require the extra thermal step that collagen molecules require to

break down its triple helix. In collagen, the transition from an extended, triple-helical structure to

a random coil is thermodynamically first-order transition, characteristic of melting in crystalline

materials. The temperature at which this transition takes place varies in collagens from different

animals. The denaturation temperature has been correlated with the amount of proline and

hydroxyproline and found to higher in collagens with a higher proportion of these amino acids

(Wainwright, 1982).

Collagen denaturation may also be extremely slow because complete unfolding of the helix

may occur in many steps and via different pathways. Type I collagen is thermodynamically

rather than kinetically stable. Apparently, helices confined in fibers cannot melt completely

because their confinement would not allow chains to gain as much entropy as in solution

(Leikina, Mertts, Kuznetsova, & Leikin, 2002).

Products from the proteins consisted of aromatic hydrocarbons, their nitrogen-containing

analogs, nitriles, aniline, and phenols. Like most organic materials subjected to high

temperatures, the two proteins examined produced aromatic hydrocarbons. The study described

95

Figure 3-16 Thermogram of Collagen and Chitin.

confirms the expectation that most, if not all, organic material will produce polynuclear aromatic

hydrocarbons on exposure to high temperatures. The formation of simple pyridine bases, nitriles,

aniline, and aromatic hydrocarbons as a result of protein pyrolysis is again difficult to rationalize

mechanistically, and studies of pyrolysis of individual amino acids may be helpful. Many of the

products in the protein pyrolyzates bear structural relationships to amino acid units comprising

the original proteins (Higman, Schmeltz, & Schlotzhauer, 1970).

In previous studies, difference in collagen denaturation temperatures have been correlated

with proline (Pro) and hydroxyproline (Hyp) content which are believed to play a substantial role

in the stabilization of the triple helix due to the non-covalent bonding of their pyrrolidine ring.

Therefore, the higher value of Pro + Hyp contributed to a higher thermal stability. Purified

0

10

20

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800

Weig

ht

Lo

ss (

%)

Temperature (°C)

Collagen

Chitin

96

collagen usually contains traces of non-collagenous proteins and glycosaminoglycan.

Glycosaminoglycans are negatively charged heteropolysaccharides that serve a function in the

formation of the matrix to hold together the collagen fiber of connective tissue. Therefore, the PS

collagen is not only associated with a high degree of glycosaminoglycan but this also affects

other physico-chemical properties. The amino acid composition directly influenced the thermal

stability of different collagen species (Lin & Liu, 2006).

There were over 4500 data points created for each TG curve. Errors can be introduced in

TGA measurements if there are factors such as external vibrations, excessive external heat, slow

recorder/readout equipment, or the sample is packed tightly.

Results from the programmed operation of a thermo balance are represented by a plot of

weight changes versus temperature of time. The plot is referred to as a TG curve. The horizontal

portion indicates the regions where there is no weight change. From this, thermal stability can be

gauged. Instrumental factors that affect a TG curve include furnace heating rate, recording or

chart speed, furnace atmosphere, geometry of sample holder/location of sensors, sensitivity of

recording mechanism, and composition of sample container. Sample factors that affect a TG

curve include amount of sample, solubility of evolved gases in sample, particle size, heat of

reaction, sample packing, thermal conductivity, and sample properties. The strengths of the TGA

include the fact that any type of solid can be analyzed with minimal sample preparation, a

minimum amount of sample is required, and analysis can be qualitative or quantitative. Some

limitations include that the sample must be initially be a solid and data interpretation may not be

straightforward requiring information from other techniques.

97

Zeta potential

Particles interact according to the magnitude of the zeta potential not their surface charge.

Zeta potential values can be used to predict dispersion stability; gives insight into the range of

pH that the substance can operate over without worrying about issues of aggregation and

fluctuations; and gives insight in long term stability. The higher the value of zeta potential, the

more stable the particle dispersion is likely to be. Zeta potential values of 30mV should

remain stable (Kaszuba, 2008).

Figure 3-17 provides the zeta potential of chitin whiskers as a function of pH. The chitin

whiskers are likely to be stable below ~pH 5. From the study of Li, Revol and Marchessault , the

zeta potential of chitin crystallite suspensions at pH 4 in 10-3 M NaCl is 47 mV with a specific

conductance of 0.180. This is of the same order as at pH 2.8 with a zeta potential value of 44

with a specific conductance of 0.699 (Li, Revol, & Marchessault, 1996). These values show that

chitin whiskers are stable below ~pH 5. The isoelectric point of chitin is ~pH 8. According to

the work of Huang and Chen, the isoelectric point of chitosan is pH 8.7. Chitosan and chitin

whiskers are similar derivatives of chitin.

Figure 3-17 also provides the zeta potential of collagen as a function of pH. The isoelectric

point of collagen is ~pH 9-10 (Gobeaux, et al., 2008), (Andrade, Ferreira, & Domingues, 2004),

which is very similar to the isoelectric point of chitin whiskers.

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Figure 3-17 Zeta potential (ξ) of collagen. Collagen trend line has been taken from (Andrade,

Ferreira, & Domingues, 2004).

As more alkali is added to the suspension the particles tend to become more negatively

charged. As acid is added to the suspension, a point is reached where the charge will be

neutralized. As more acid is added, a buildup of positive charge occurs. There is a point where

the curve passes through zero. The point where the curve passes through zero is called the

isoelectric point and it is where the system is least stable. It can be seen in the chitin curve (blue)

when the dispersion pH below 4 or above 9, there is sufficient charge to confer stability. If the

system is between 4 and 9, the dispersion may be unstable. This instability is most likely around

pHIEP 8 (the isoelectric point). If the system is between 7 and 10, the dispersion may be unstable.

-20

-10

0

10

20

30

40

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Zeta

Po

ten

tial

(m

V)

pH

Chitin

Collagen

99

The trendline for both collagen and chitin indicate that basicity does not improve stability much.

It should be noted that collagen has a greater affinity to be less stable in the basic pH region. This

is probable due to the fact the collagen is more basic molecule with hydroproline, more hydrogen

and amine groups.

The error bar indicates the standard deviation for 3 runs of Zeta Potential measurements

It does not change my evaluation of the information the standard deviation adds to the

information about how the degree in which zeta values within the sample differ from the sample

mean. The collagen trendline was taken from Andrade et al. no standard deviation or error bars

were reported. Many in the literature give a couple values of zeta potentials in regard to pH

instead of a complete trend line. One of those points is typically the isoelectric point. Common

limitations to generating high quality zeta potential measurements are very small particles, very

dilute samples, concentrations that are too high, dissolved solids, or residual reactants.

Scanning Electron Microscopy (SEM)

Chitin SEM micrograph shows chitin fibers [see figure 3-18]. Chitin and collagen [figure 3-

19] SEM micrographs both display lattice-like structures with nucleation sites. The fibers in both

collagen and chitin micrographs were oriented parallel, perpendicular, or a variation in between,

to the surface, on successive planes. Multidirectional or ‘twisting plywood’ system (Neville A.

C., 1993) was observed. Bouligand and Neville observed similar helicoid systems while

examining crustaceans (Bouligand Y. , 1972). One major difference, between the chitin and

100

collagen fibers, is that the collagen fibers of SEM micrographs demonstrate the characteristic 67

nm banding pattern of a collagen fiber (Franchi, et al., 2008).

Advantages of the SEM include its wide-array of applications, detailed topographical

imaging, and the fast nature of the instrument. One disadvantages is the fact the preparation of

samples can result in artifacts. SEMs are limited to solid samples small enough to fit inside the

vacuum chamber and able to handle vacuum pressure. Special training is required to operate an

SEM as well as prepare samples.

Figure 3-18 Chitin from Crab Exoskeleton SEM Micrographs.

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Figure 3-19 Collagen from Bovine Achilles Tendon SEM Micrographs

Conclusion

Collagen and chitin follow the same extraordinary storyline. They do not act alone; collagen

is a triple helix embedded in an inorganic matrix and chitin is embedded in a protein helix.

Compared to typical structures of their kind, both collagen and chitin are unusual. Some other

significant points about collagen and chitin include:

Chitin is a polysaccharide; Collagen is a protein;

Collagen and Chitin associate with proteoglycans;

Collagen and chitin complex composites are hierarchically structured and multifunctional

Microfibril: Fribil Aggregation (Fiber): Fibrous Bundle: ‘Twisted Plywood”;

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Chitin and collagen are lyotropic liquid crystals that form LC phases in solution. Liquid

crystals and fibrous composites such as collagen and chitin are both mobile, have similar

ordered geometries, can self-assemble, and have similar optical properties;

The biosynthesis and degradation of both collagen and chitin are stepwise procedures

governed by enzymatic activity; these pathways consist of catalytic units, precursors,

biotransformations, polymerization, hydrolysis, and fibrillogenesis;

Both collagen and chitin are synthesized by sequential changes in the cytoplasmic matrix,

RER, processing in the Golgi apparatus and transport through vesicles culminating in

exocytosis across the membrane where chemical reactions occur and association into

fibril, fibers, and fibrous bundle;

Collagen and chitin share properties; they are both biocompatible, biodegradable, non-

toxic, highly insoluble, and birefringement;

Chitin and collagen have a wide variety of medical applications.

An argument can be made that collagen is what holds bone together. The same argument can

be made for chitin and crab exoskeleton. Since collagen and chitin share so many similarities

could they be used interchangeably as implants? Previous reports indicate that chitin induced

fine collagen fibers histologically. Chitin was found to enhance synthesis of types I, III, and IV

collagens (Kojima, et al., 2004), (Minami, et al., 1996). If chitin can, does this ability extend to

its source which in this case includes crab shell integument?

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Chapter 4 Tools of Biomineralization: Calcium Carbonate and Calcium

Phosphate

Introduction

A vast array of organisms are known to precipitate minerals (Lowenstam H. A., 1981). The

formation of inorganic minerals by biological systems, e.g. in bone, teeth, and mollusk shells, is

called biomineralization (Neues, Ziegler, & Epple, 2007). Until the early 1980s the field was

known as “calcification,” reflecting the predominance of biologically formed calcium-containing

minerals. As more and more biogenic minerals were discovered that contained other cations, the

field became known as “biomineralization” (Weiner & Dove, 2003), and is characterized by a

remarkable level of molecular control of the particle size, structure, morphology, aggregation,

and crystallographic orientation of the mineral phases. Biomineralization results in complex

materials (Mann, et al., 1993). One of the major challenges in the field of biomineralization is to

understand the mechanism(s) by which biological systems determine which polymorph will

precipitate (Weiner & Dove, 2003). When investigating any mineralized tissue, one must be

cognizant of the complex nature of the material: 1) the chemical nature and crystalline form of

the mineral; 2) the nature and form of the organic matrix; 3) the relationship between the organic

and inorganic components of the tissue and the influence of the matrix on crystal morphology; 4)

the sources of mineral for deposition; 5) the pathways for mineral movement into or out from the

mineralized structures; 6) rates of mineral deposition; and 7) the nature and location of

104

nucleation sites within the matrix and mechanisms for control or cessation of crystal growth

(Roer & Dillaman, 1984).

Biomineralization provides an intelligent strategy to generate functional materials such as

mineral skeletons and cuticles (Tao, Zhou, Zhang, Xu, & Tang, 2009) that exhibit tight structural

control at the nanoscale (Barthelat, 2007). Skeletons and cuticles are formed in a wide variety of

shapes, sizes, and compositions of organic and mineral components (Omelon, et al., 2009).

Mineralization takes time depending on the animal species (Skinner & Jahren, 2003). In nature,

crustacean animals periodically undergo cyclic molt of the exoskeleton, which is essential for

their growth, metamorphosis, and reproduction. The layers in cuticle are deposited and

mineralized at distinctly different stages: premolt, intra-molt, postmolt, and inter-molt (Tao,

Zhou, Zhang, Xu, & Tang, 2009). Calcium carbonate is used by many invertebrate organisms to

mineralize a large variety of structures. The delicately structured exoskeleton or cuticle protects

arthropods from environmental hazards like desiccation, osmotic and mechanical stress, and

predation. In addition, it serves as support for organs, provides sites for muscle attachment and

takes up mechanical tension during muscle contraction. These functions require an adequate

mechanical strength of the cuticle (Neues, Ziegler, & Epple, 2007).

Vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite.

The vertebrate skeleton must satisfy a wide range of demands, including structural integrity,

metabolic activity, growth, and continual repair of wear and damage caused by locomotion

and/or trauma. These demands require that vertebrates continually resorb and rebuild their

mineralized skeleton. The high functionality and metabolic activity of the vertebrate skeleton

105

suggest that apatite offers an advantage to the vertebrate organism that other minerals do not

(Omelon, et al., 2009).

To date, approximately 60 biogenic minerals (i.e. generated through biological processes)

have been identified (Dunlop & Fratzl, 2010), see table 4-1 for examples of biomineral diversity.

The calcium carbonate minerals are the most abundant biogenic minerals, both in terms of the

quantities produced and their widespread distribution among many different taxa. Phosphates

comprise about 25% of the biogenic mineral types. The most abundantly produced phosphate

mineral is carbonated hydroxyapatite (Weiner & Dove, 2003).

The vertebrate skeleton or bone is extremely enterprising and continually being rebuilt,

repaired, and resorbed during growth and normal remodeling (Omelon, et al., 2009). In

Crustacea, molting includes a cycle of demineralization of the old and mineralization of the new

cuticle. Crustaceans and bones are excellent models for the study of dynamic biomineralization

processes (Neues, Ziegler, & Epple, 2007). The detailed processes of biomineralization are as

varied as the organisms themselves (Weiner & Dove, 2003).

Definition

Landis and Glimcher defined biomineralization as the process by which living forms

influence the precipitation of mineral materials. The process creates heterogeneous

accumulations, composites composed of biologic (or organic) and inorganic compounds, with

106

Table 4-1 Examples of the Diversity of Biominerals (Gower, 2008).

Biogenic

minerals

Formula Organism Biological

Location

Biological

Function

Calcium

carbonates

(calcite, vaterite,

aragonite,

amorphous)

CaCO3,

(Mg,Ca)CO3,

CaCOnH2O

Many marine

organisms,

aves, plants,

mammals

Shell, test,

eye lens,

crab cuticle,

eggshells,

leaves, inner

ear

Exoskeleton,

optical,

mechanical

strength,

protection,

gravity receptor,

buoyancy

device, Ca store

Calcium

phosphates

(hydroxylapatite,

dahllite)

Ca10(PO4)6(OH)2,

Ca5(PO4CO3)3(OH),

Ca8H2(PO4)6

Vertebrates,

mammals,

fish, bivalves

Bone, teeth Endoskeleton,

ion store,

cutting/grinding,

protection,

precursor

inhomogeneous distributions that reflect the environment in which they form (Landis &

Glimcher, 1978).

According to a modern definition, minerals are elements or compounds, amorphous or

crystalline, formed through biogeochemical processes. This definition acknowledges that some

biominerals are barely crystalline but avoids a discussion of amorphous. Biominerals have

distinct morphologies and make unique contributions to well-known life forms. Many

biomineralized tissues are composite materials, containing a biologically produced organic

matrix and nano- or microscale amorphous or crystalline minerals. During the processes of

biomineralization the organic material acts in various roles as nucleator, cooperative modifier,

107

and matrix or mold for the mineral ions. The resulting tissue has properties very different from

those of the pure minerals themselves. The stiff mineral prevents the organic matrix (proteins,

peptides, polysaccharides, lipids) from yielding, while the organic matrix hinders crack

propagation (Ehrlich, Koutsoukos, Demadis, & Pokrovsky, 2008).

Mineral Function/Strength/ Properties

With unique combinations of stiffness and strength, hard tissues can serve a variety of

functions: mechanical support (bones) or armored protection (cuticles). In nature, the most

common route taken to make stiff materials out of soft protein networks and tissues is to

incorporate minerals, which are much stiffer (Barthelat, 2007). Therefore, most natural materials

are composites based on biopolymers and some minerals (Dunlop & Fratzl, 2010). The effect of

combining soft proteins with stiff minerals can be seen in figure 4-1, which is a material

properties map for a selection of natural ceramics, biopolymers and their composites. Their

mineral content makes them 100–5000 times stiffer than soft proteins (Barthelat, 2007). Despite

the relative scarcity of these constituents, their combination yields materials with a great

variation in functionality. A particular characteristic of biological composites is their

multifunctionality (Dunlop & Fratzl, 2010).

Many mineralized tissues fulfill structural functions. The vast majority of these, such as

bones, teeth, and shells, utilize crystalline minerals to stiffen and strengthen the tissue (Addadi,

Sefi, & Weiner, 2003). In one example, organic fiber networks are reinforced by mineral, as in

the case of the crab cuticle, which consists of a plywood-like arrangement of chitin fibers and

108

Figure 4-1 The materials property chart (Dunlop & Fratzl, 2010).

109

inclusions of calcite and amorphous calcium carbonate. Another example is bone, in which

collagen fibrils are reinforced by hydroxyapatite platelets of only a few nanometer thickness

(Bouligand, 1972), (Dunlop & Fratzl, 2010). The calcium phosphate mineral phase of bone has

two functions: (1) providing structural stability to the skeleton by infusing the organic matrix of

bone to form a rigid structural material; and (2) operating as a storage site for calcium, inorganic

orthophosphate, sodium, magnesium, carbonate, and other ions. Being involved in both the

biomechanical and metabolic functions of osseous tissue, bone mineral acts as a reservoir

providing the body fluids with these ions to maintain the biologically required levels and as a

detoxifying depository to store ions unwanted in the body fluids (Posner, 1985). The relationship

between toughness and modulus for natural materials is shown in figure 4-1. Materials vary

widely for both properties. The materials property chart illustrates many mechanically important

materials where both stiffness and toughness are large (Dunlop & Fratzl, 2010).

The process of mineral phase formation is regulated by solubility, surface chemistry, and

bioavailability of elemental and molecular species such as calcium, carbonate, and phosphate

(Wilson Jr., 2008). The mineral phase is thought to dominate the stiffness of bone, which

increases more than linearly with mineral content. Mineral platelets deform elastically to almost

twice the bulk failure strain. Load-bearing organs or protective elements require a high modulus

to transmit loads (bone) or to minimize deformation (cuticle). High stiffness is readily achieved

by the incorporation of mineral phases (e.g., calcite and hydroxyapatite) and of stiff, fibrous

elements (collagen or chitin) and through extensive cross-linking. (Dunlop & Fratzl, 2010). The

110

main mechanical feature of hard biological tissues is their ‘hardness’, although ‘stiffness’

(resistance to deformation) may generally be a more accurate term (Barthelat, 2007).

Having formed under controlled conditions, biomineral phases often have properties such as

shape, size, crystallinity, isotopic and trace element compositions quite unlike its inorganically

formed counterpart. The term “biomineral” reflects all this complexity (Weiner & Dove, 2003).

Biology is limited to using a small number of biopolymers, some minerals and ions, and various

cross-linking agents to make its materials. Despite these limitations, natural materials present an

incredible variability in their properties. Mechanical properties of biological composites based

on polymers and mineral, are usually very superior to those of the components (Dunlop & Fratzl,

2010). Bone mineral is an integral part of bone tissue having physical and chemical properties

Figure 4-2 Biomineral crystallographic properties are highly regulated during

biomineralization. These properties provide some indication of potential processing strategies

(Gower, 2008).

111

that directly affect those of the tissue as a whole (Termine J. D., 1972). Figure 4-2 depicts some

properties that are regulated during biomineralization.

Biomimetics and biomineralization

The variation in biological mineralization is enormous. These variations represent alternative

solutions to similar problems. The adaptation of biomineralization processes in the laboratory

highlights the potential of a biomimetic approach to crystal science. In particular, the concept of

molecular recognition at inorganic-organic interfaces involved in nucleation and crystal growth

is providing a perspective in which the classical approaches to inorganic crystallization can be

developed within a structural and sterochemical context. Moreover, the integration of organic

supramolecular chemistry, self-assembly, and inorganic materials chemistry provides the

opportunity to develop rational synthetic routes to products with uniform particle sizes,

nanoscale dimensions, tailored morphologies, crystallographic orientation, and organized

microarchitectures (Mann, et al., 1993).

Mann coined the term “molecular tectonics” and defined it as a process that strives towards

the chemical construction of higher-order architectures, albeit on different length scales.

Furthermore, he proposed that the study of the molecular processes that result in the construction

of higher-order architectures in biomineralization is relevant to the development of new

strategies for the controlled synthesis of organized inorganic and composite materials. He

presented a commentary, using representative examples, on the essential aspects of molecular

112

tectonics that connect materials chemistry and biomineralization, and ultimately their

assimilation within a biomimetic approach (Mann, 1993).

The three constructional processes of biomineralization- supramolecular pre-organization,

interfacial molecular recognition (templating) and cellular processing – represent a sequence of

increasing tectonic complexity. This leads to three main divisions of biomimetic materials

chemistry inspired by studies of biomineralization, which are highlighted in figure 4-3. First, the

specific chemical and structural properties of biomolecules involved in biomineralization

processes can be utilized in synthetic reactions. A second area of biomimetic research is in the

use of living organisms, as intact biosystems, for the fabrication of highly ordered composites. A

third biomimetic approach involves the use of biological concepts as inspiration for new ideas in

the synthesis of inorganic materials with controlled properties, such as uniform particle size,

polymorph selectivity, tailored morphology, oriented nucleation and organized assembly (Mann,

1995).

These materials processing rates are achieved through contrasting assembly strategies that are

instructive to the materials scientist. Understanding the inherent complexity of the molecular

systems controlling the biological synthesis is a major challenge to materials scientists, who want

to copy the structure, property, and performance (function) relations of these elegant structures

(Heuer, et al., 1992).

113

Figure 4-3 There exist three primary divisions of biomimetic materials chemistry inspired by

biomineralization studies (Mann, 1995).

Biomineralization Models

Several theories have been proposed for the mechanism of mineralization of hard tissue such

as bone and cuticle (Lowenstam H. A., 1981), (Mann, 1983), (Anderson, 1984), (Nilsen, 1980).

BIOMIMETIC

MATERIALS CHEMISTRY

Biomolecules

Bioconcepts

Biosystems

proteins

polysaccharides

bone

cuticle

molecular recognition

vectorial regulation

cellular processing

preorganization

114

The classical model of biomineralization considers mineral formation as an amplification process

in which individual atoms or molecules add to existing nuclei or templates, while living

organisms may make use of proteins and peptides to deterministically modify nucleation, growth

and facet stability. This conventional concept for crystal growth has been challenged (Cai &

Tang, 2008). Thus, there is at present no unified theory of the mechanism of mineralization,

especially for bone (Posner, 1985). A discussion of various models is presented below.

Biologically Induced

The basic process of biologically induced mineral formation is characterized by bulk

extracellular and/or intercellular mineral formation, without the elaboration of organic matrices.

"Biologically induced" mineralization results in the minerals having crystal habits similar to

those produced by precipitation from inorganic solutions (Lowenstam H. A., 1981). In this

situation, cell surfaces often act as causative agents for nucleation and subsequent mineral

growth. The biological system has little control over the type and habit of minerals deposited,

although the metabolic processes employed by the organism within its particular redox

environment mediate pH, pCO2 and the compositions of secretion products. These chemical

conditions favor particular mineral types in an indirect way. In some cases, biological surfaces

are important in the induction stage because nucleation often occurs directly on the cell wall, and

the resulting biominerals can remain firmly attached. Heterogeneity is the hallmark of

biologically induced minerals. This heterogeneity includes variable external morphology

115

(typically poorly defined), water content, trace/minor element compositions, structure and

particle size (Lowenstam & Weiner, 1989), (Weiner & Dove, 2003).

Organic matrix-mediated

In “organic matrix-mediated” process, the organism constructs an organic framework or mold

into which the appropriate ions are actively introduced and then induced to crystallize and grow.

The mineral type, orientation of crystallographic axes, and microarchitectures are under genetic

control (Lowenstam H. A., 1981).

“Organic matrix-mediated” was generalized by Mann (Mann, 1983) to “biologically

controlled” mineralization. The organism uses cellular activities to direct the nucleation, growth,

morphology and final location of the mineral that is deposited in “biologically controlled”

mineralization. The degree of control varies across species, and almost all controlled

mineralization processes occur in an isolated environment. The results can be exceptionally

sophisticated, species-specific products that give the organism specialized biological functions.

Biologically controlled mineralization processes can be characterized as occurring extra-, inter-

or intracellularly (Table 4-2). In some cases, mineral formation begins within the cell and then

proceeds outside the cell (Weiner & Dove, 2003).

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Table 4-2 Characterization of Biologically Controlled Extra-, Inter-, and Intra-cellular

Mineralization

Biologically controlled

extracellular mineralization

Biologically controlled

intercellular mineralization

Biologically controlled

intracellular mineralization The cell produces a

macromolecular matrix

outside the cell in an

area that will become

the site of

mineralization. (Matrix

refers to a group of

macromolecules

comprised of proteins,

polysaccharides or

glycoproteins that

assemble to form a

three-dimensional

framework).

The structures and

compositions of these

organic frameworks are

programmed to perform

essential regulating

and/or organizing

functions that will result

in the formation of

composite biominerals.

Almost all structures

that form by

extracellular processes

develop upon a pre-

formed matrix derived

from secretory products

of multicellular

epithelial tissues.

Examples of organisms

believed to mineralize

primarily by

extracellular

biologically controlled

processes include shells

of mollusks and bones

and teeth.

It typically occurs in

single-celled organisms

that exist as a

community.

The epidermis of the

individual organisms

serves as the primary

means of isolating the

site of mineralization.

The epithelial substrate

reproducibly directs the

nucleation and growth

of specific biomineral

phases over large areas

of cell surfaces.

Mineralization between

cells can become so

extensive as to

completely fill the

intercellular spaces, thus

forming a type of

exoskeleton.

The epidermis of

individual organisms

directs the polymorph

and shape of the

biomineral that forms.

An example is found in

calcareous algae that

nucleate and grow

calcite with a c-axis

orientation that is

perpendicular to the cell

surface.

Controlled

mineralization can also

occur also within

specialized vesicles or

vacuoles that direct the

nucleation of

biominerals within the

cell.

These

compartmentalized

crystallization

environments govern the

resulting biomineral

composition and

morphology.

The cell has a high

degree of control upon

the concentrations of

cation and anion

biomineral constituents

in an environment where

an organic matrix may

also be active as a

nucleating template.

The compartment

membrane also regulates

the pH, pCO2 and minor

element compositions.

Indicative of the general

scenario whereby

biominerals form

intracellularly before

eventually becoming

extracellular.

(Watabe, 1974), (Veis, 2003), (Weiner & Dove, 2003).

117

Biogenic minerals found in teeth and bones are synthesized by precise cell-mediated

mechanisms. These hard tissues have superior mechanical properties due to their complex

architecture. It is believed that the initial mineralization phase is amorphous and during the

nucleation and growth process, the initially formed metastable amorphous calcium phosphate

phase transforms into thermodynamically stable crystalline hydroxyapatite in a precisely

controlled manner. The organic matrix-mediated controlled transformation of amorphous

calcium phosphate into crystalline HAP has been confirmed by x-ray diffraction, selected area

electron diffraction (SAED or SAD), Raman spectroscopy, and elemental analysis (Gajjeraman,

Narayanan, Hao, Qin, & George, 2007).

Biomineralization of collagen involves functional motifs incorporated in extracellular matrix

protein\molecules. These molecules have the objective to stabilize the amorphous calcium

phosphate into nanoprecursors and to direct the nucleation and growth of apatite within collagen

fibrils, figure 4-4. For organic-inorganic nanocomposites such as bone, this self-assembly

process determines the framework and spatial constraints for nucleation and propagation of the

reinforcing mineral phase. By itself, type I collagen is insufficient to induce nucleation of

carbonated apatite from transient amorphous calcium phosphate (ACP) phases (Falini &

Fermani, 2004). The organic matrix exerts a great degree of crystallographic control over the

nucleation and growth of mineral particles. Type I collagen, the predominant matrix protein in

bone, possesses self-assembling properties and forms enclosed spaces within which the inorganic

reinforcing phase grows. When the degree of supersaturation of calcium and phosphate ions is

high, thermodynamically stable HAP is formed via intermediate precursor polymorphs such as

118

amorphous calcium phosphate (ACP) or octacalcium phosphate. It is well established that type I

collagen matrix does not have the capacity to induce matrix-specific mineral formation from

metastable calcium phosphate solutions that do not spontaneously precipitate but merely provide

the organizational framework and spatial constraint for crystal deposition. Noncollagenous

matrix macromolecules might be involved in the control of nucleation and growth of the mineral

phase. Type I collagen designates the space in which the crystal grows, and this constrained

space might be necessary for defining the crystal size and morphology (Gajjeraman, Narayanan,

Hao, Qin, & George, 2007).

Figure 4-4 Schematic of the mineralized collagen fibrils, the basic constituents of bone.

Nanocrystals of HAP are incorporated between collagen molecules (Dorozhkin & Epple, 2002).

Crystal of HA

( 50 x 25 x 4 nm3)

Mineralized collagen fibril:

few µm long and

100 nm in diameter

Collagen molecules (polypeptide chains)

300 nm long and 1.5 nm in diameter

119

In mollusks, more than 40 types of ultrastructural arrangement are known where many

crystals are embedded in an organic matrix and mechanical requirement adaptations are

reflected. Polymorphs of calcium carbonate (aragonite, calcite, and vaterite) have been observed

(Falini & Fermani, 2004). The location and polymorphism of the crystals are governed by the

super-saturation inside the compartmentalized space in the chitin. As the organic component, a

role of chitin is to reinforce the mineral phase. This is analogous to collagen, which is the main

organic component of bone. This property, in addition to the inherent biodegradability,

biocompatibility, and good mechanical properties of chitin, has paved the way for the use of

chitin–HA composites as a hard tissue substitute. These composites are useful as a bone material

substitute because they allow the introduction of osteoconductive HA into regions of bone loss,

with the chitin matrix acting as a binder to prevent postoperative migration of the HA particles

(Falini & Fermani, 2004). Other composites consisting of collagen, chitin, calcium phosphates,

and their derivatives have been used as hard tissue substitutes (Ge, Baguenard, Lim, Wee, &

Khor, 2004), (Wahl & Czernuszka, 2006), (Jayakumar, Prabaharan, Nair, & Tamura, 2010).

Because of their properties, these materials have the potential for use for a variety of medical

applications (Singh & Ray, 2000), (Dutta, Ravikumar, & Dutta, 2002).

Mesocrystals model

The concept of mesocrystals, which consist of crystallographic oriented crystallites connected

by polymers or surfactants, has been suggested in the studies of biomineralization. Under the

120

regulation of various organic molecules, the formation of mesocrystals has been previously

observed in the growth processes of CaCO3. In these mesocrystals, the building blocks are the

fully crystallized inorganic phase. The organic molecules connect the adjacent crystallites,

resulting in the superstructures. A crucial condition in the architecture of mesocrystals is the

interaction between crystallites and organic molecules (Cai & Tang, 2008).

Mesocrystals are colloidal crystals composed of crystalline nanoparticles in perfect three-

dimensional crystallographic register so that they scatter like single crystals but are clearly a

nanoparticle superstructure. Consistent with the model of a mesocrystal, nanoparticles were

suggested as building units of biominerals in combination with a brick-by brick formation

mechanism. Studies point to the fact that nano-clustered crystal growth, induced by organic

matrices, is a basic characteristic of biomineralization that enables the production of composite

materials with elaborate morphologies. These examples indicate that mesocrystal formation

plays a role in biomineralization, at least as an intermediate in the production of single crystals

with complex morphologies. This nanoparticle mediated crystallization pathway involving

mesoscopic transformation is called ‘‘non-classical crystallization’’. Contrasting a single

nucleation event to form a single crystal, this pathway involves multiple nucleation events of

nanoparticles which form a nanoparticle superstructure. Non-classical crystallization engages

self-organization of pre-formed nanoparticles to an ordered superstructure, that can later fuse to a

single crystal (Xu, Ma, & Colfen, 2007).

121

Figure 4-5 Illustrative representation of classical and non-classical crystallization (Xu, Ma, &

Colfen, 2007).

In figure 4-5, Pathway a) represents the classical crystallization pathway in which nucleation

clusters form and grow until they reach the size of the critical crystal nucleus growing to a

primary nanoparticle, which is amplified to a single crystal. Pathway b). The primary

nanoparticles can get covered by a polymer or other additive before they undergo a mesoscale

assembly, so they can form a mesocrystal. Pathway c). Mesocrystals can form from pure

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nanoparticles. Pathway d). There also exist the possibility that amorphous particles are formed,

capable of transforming before or after their assembly to complicated morphologies (symbolized

by the question mark in path d) (Colfen & Mann, 2003), (Xu, Ma, & Colfen, 2007).

Brick and mortar model

A new model of ‘‘bricks and mortar’’ concerns the biological aggregation of apatite

nanoparticles. An inorganic phase, amorphous calcium phosphate (ACP), acts as ‘‘mortar’’ to

cement the crystallized ‘‘bricks’’ of nano-HAP. Meanwhile, biological molecules control the

nano-construction. By using HAP nanospheres as the building blocks, highly ordered enamellike

and bone-like apatite are hierarchically constructed in the presence of glycine (Gly) and

glutamate (Glu), respectively. During the progression of biological apatite, the amorphous

‘‘mortar’’ can be eventually turned into the ‘‘brick’’ phase by phase transformation to ensure the

integrity of biominerals (Cai & Tang, 2008)

Cellular Role

It is recognized that bone-related cells (especially osteoblasts and osteoclasts) play key roles

in the physiological formation of bone. Bone-related cells not only are theorized to take part in

the formation of biomineral and construction of bone, they also continuously regulate the

density, regeneration and degradation of bone. So understanding the relationship between bone-

related cells and calcium phosphates is a necessary step in order to elucidate the formation

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Figure 4-6 An illustration of the role of inorganic and organic constituents in controlling

biomineral formation (Gower, 2008).

mechanism of bone (Cai & Tang, 2008). Cells are amply sensitive to their surroundings such that

signaling molecules or macro-, micro-, or nano-scale alterations in topography may elicit diverse

cell behaviors (Stevens & George, 2005).

Cellular processing administers spatial and temporal control over the delivery of the species

and their subsequent organic-inorganic interactions. In the physiological environment, there is a

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requirement for mineral reactants and inorganic impurities can have a pronounced impact on the

process, especially in combination with the soluble organic additives, where promotion of the

amorphous precursor pathway can occur. The soluble additives are considered as having either

“process-directing” or “structure-based” interactions. Structure-based interactions may influence

the crystallographic texture, phase, and orientation (Gower, 2008). Thus, the role of inorganic

and organic constituents in controlling biomineral formation is illustrated in figure 4-6.

Matrix Vesicles (MV)

Bone apatite formation seemingly proceeds via a number of cooperative mechanisms. Current

hypotheses encompasses the utilization of: (i) Matrix vesicles which bud from the plasma

membrane and accumulate calcium (Ca2+) and phosphate (PO43−) ions extracellularly before

associating with the collagenous ECM (2); (ii) Noncollagenous proteins, associated with the gap

zones in collagen, mediate and foster mineral nucleation and its propagation within and along

collagen fibrils; and (iii) Amorphous calcium phosphate and ionic calcium stored in

mitochondria is transported via vesicles to the ECM before converting to more crystalline

apatite. In the depiction, “matrix vesicles” are purple, and collagen-mediated mineralization is

portrayed in the bottom left corner with calcium and phosphate ions highlighted in yellow and

red. Mitochondria are shown in green, and vesicles are orange and blue, with and without

mineral/ions, respectively. “N” identifies the cell nucleus (Boonrungsiman, et al., 2012). Figure

4-7 outlines this proposed mechanism for bone mineral formation.

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Figure 4-7 Diagram outlining a proposed mechanism for bone mineral formation

(Boonrungsiman, et al., 2012).

Anderson (1967) and Bunucci (1967) demonstrated membrane-bounded vesicles in the matrix

of hard tissues having a diameter of about 100 nm. They proposed that the initial crystals

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appeared inside these matrix vesicles (MV). Later two subtypes of these matrix vesicles have

been described, type I having a dark, lysosome-like content and type II having a content

resembling intracellular matrix. Several investigators have proposed that matrix vesicles

constitute the beginning stages of mineralization in bone (Nilsen, 1980).

Matrix vesicles are ubiquitous in the cytoplasm of eukaryotic cells and are diverse in the

human body. These vesicles are involved in a wide range of transport, storage and secretory

functions and are assembled with well-defined compositions. They may undergo a variety of

specific transactions involving fusion events, notably during protein trafficking between cisterna

and the Golgi stack. Matrix vesicles generally adopt a spherical morphology in the absence of

external scaffolds. They are extracellular, membrane-invested, and 100 to 200 nm in diameter.

Matrix vesicles are enriched in Ca and lipids and possess a unique assortment of

phospholipids characterized by an acidic phospholipid with a strong affinity for Ca2+. These

particles which have been described as the initial locus of calcium phosphate mineral deposition

in cartilage, growing bone and predentin of teeth. In addition, matrix vesicles are the initial locus

of calcification in all growing skeletal tissues in which studies of anhydrously prepared tissues

support this assertion.

Matrix vesicles have membranes that degrade during calcification by both enzymatic and

mechanical forces. Degradation explains the low matrix vesicle numbers occurring in calcified

zone of growth plate. These membranes that are replete with phosphatase activities including

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alkaline phosphatase, ATPase, inorganic pyrophosphatase, and 5’-nucleotidase. The outer

membranes provides one of the few protected and controlled internal microenvironments that

exist outside cellular environments in which complex interactions between molecules can occur.

Matrix vesicles are programmed based on increasing evidence, whereby metabolic objectives

of the host cell may be pursued vigorously at a distance from the host cell and can be transferred

into other cells. Vesicle formation remains an area of some disagreement and varied opinion.

Their pattern of phospholipid incorporation strongly suggests that they take their origin from

adjacent cells. Thus, major theories of biogenesis include:

a. budding from plasma membrane ( much supported )

b. cell degeneration

c. extrusion of intracytoplasmic vesicles

d. extracellular subunit self- assembly

(Anderson, 1984), (Mann, 1993), (Anderson, Mulhall, & Garimella, 2010).

Calcium phosphate mineral formation in vertebrate bone formation is a multistep process.

Much evidence indicates that extracellular MV play a key role in the induction of the first solid

phase mineral. Previous biochemical and ultrastructural studies have shown that MV contain

large amounts of calcium and phosphate. However, the initial mineral phase has not yet been

characterized (Wu, et al., 1997). With regard to biomineralization, the enclosed intravesicular

space provides a means of controlling the size and location of discrete intracellular mineral

products, whereas ion pumps in the vesicle membrane are responsible for controlling the

physicochemical conditions (supersaturation, ionic strength, complexation, pH) pertaining to

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crystal nucleation and growth (Mann, Molecular tectonics in biomineralization and biomimetic

materials chemistry, 1993).

Amorphous Mineral Component

Hydroxyapatite formation in aqueous environments has been intensively investigated for

studies both of biomineralization and synthesis of biomedical materials. A multistep strategy of

phase transformation has been suggested and highlights the enlistment of transient amorphous

precursors in biomineralization. Evidence of this, for example, are the first-formed amorphous

calcium carbonate (ACC) in sea urchin spicules, corals, and crustacean cuticles, etc. The

participation of amorphous calcium phosphate (ACP) in the early stages during bone generation

and the subsequent transformation into carbonated hydroxyapatite (HAP) have been clarified in

vivo. The precursor role of ACC and ACP in calcium mineralization has also been confirmed in

vitro by a number of laboratory studies. It is believed that these amorphous phases are the

reservoirs for crystallization and the mechanical strengtheners during biomineral formation (Xu,

Ma, & Colfen, 2007), (Tao, Zhou, Zhang, Xu, & Tang, 2009).

The macromolecules introduced inside the amorphous phase from the start, or at an

intermediate stage, may fulfill two distinct functions. Some macromolecules may contribute to

the transient stabilization of the amorphous phase that must be broken down or otherwise

neutralized in an accurately orchestrated series of events, to allow for crystallization. Other

macromolecules have the ability to modulate crystal growth and are presumably adsorbed at the

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boundary planes as the crystal grows, influencing crystal growth in certain directions rather than

others. It is easy to overlook the presence of ACC or ACP especially when it co-exists with the

crystalline forms of calcium carbonate (Addadi, Sefi, & Weiner, 2003) or calcium phosphate.

Two modes have been postulated for the transition from an amorphous to a crystalline structure:

direct transformation and heterogeneous growth-transformation. Direct transformation proposes

the reorganization of the internal structure of amorphous particles into ordered crystalline states,

whereas heterogeneous growth transformation predicts that crystals grow incrementally on an

amorphous groundwork, eventually overgrowing the preexisting amorphous particles (Tsuji,

Onuma, Yamamoto, Iijima, & Shiba, 2008).

Control over biomineral properties can be accomplished by regulation of particle size, shape,

crystal orientation, and polymorphic structure. Biogenic minerals are assembled using a transient

amorphous mineral phase, and the stabilization of amorphous precursors during the growth phase

is important in biomineralization because transient amorphous minerals play an important role in

many organisms. Amorphous materials can be molded into various shapes. Moreover, a growing

body of evidence has shown that the shape of the biominerals is often controlled through

molding of solid or gelated amorphous precursors. Transition of amorphous phase to crystalline

phase is carried out in a controlled manner by the organism (Gajjeraman, Narayanan, Hao, Qin,

& George, 2007).

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Mg stabilized amorphous precursors

Although the concentration varies considerably among species, magnesium and phosphate are

found in virtually all crustacean cuticles. Still unknown, the function of calcium phosphate

within the crustacean cuticle might enhance the ACC formation because in vitro experiments

showed that under physiological conditions, low concentrations of phosphate prevent calcite

crystal nucleation and formation. However, it is also possible that ACP is merely formed to alter

the mechanical properties of the cuticle. Differences within the composition of the cuticle

between species convey a chemical adaptation to their different biological requirements. Within

the cuticle, the mineral composition relies on the function and the habitat of the animal. In

combination with a high concentration of organic material and rather low amounts of calcite,

ACC occurs in species that require flexible cuticles (Neues, Ziegler, & Epple, 2007). The

formation of ACC occurs from a highly supersaturated solution when additives such as Mg, are

present that prevent deposition of the crystalline phases. By following the cuticle molt of

crustacean, it was established that magnesium switches off the phase transformation by

stabilizing amorphous precursors and the organic matrices. Both ACC and ACP can be well

stabilized by magnesium. It was found that the subsequent introduction of aspartate (Asp) can

effectively switch on the crystallization of these Mg-stabilized amorphous precursors in the

laboratory. The proposed magnesium-Asp based crystallization switch is another lesson inspired

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from nature and this discovery provides an understanding of intelligent controls in

biomineralization (Tao, Zhou, Zhang, Xu, & Tang, 2009).

Effect of lipids/proteoglygans/alkaline phosphates

There is growing evidence supporting the hypothesis that phospholipids, are associated with

the initial mineralization of bone (Boskey & Posner, 1976). Other investigators have postulated

that the initiation of mineralization also involves the effect of proteoglycans, lipids, and alkaline

phosphatases (Nilsen, 1980).

Biopolymers

An underlying concept in biomineralization concerns the use of organized biopolymers to

exert detailed control over the nucleation and growth, the shape and size, and over the assembly

of crystals and other nanoscale building blocks into complex structures. Specific biopolymers

can dictate a strong influence on both crystal nucleation and growth rates as well as temperature,

pH, ionic strength and composition. One example of the influence of soluble biopolymers on a

crystallization event is the control of the CaCO3 crystal modifications using soluble

macromolecules extracted from the respective layers of a mollusk shell. Proteins can promote the

crystallization of a preferred mineral (Weiner & Dove, 2003). Another example is the

relationship between collagen and crystals which was first described by Robinson and Watson.

Crystal-like particles have been shown to be sequentially orientated in collagen fibrils (Nilsen,

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1980). Glimcher and Krane suggested also that the collagen fibrils were the substrate for

nucleation and that the initial stages showed crystals in "holes" in the collagen fibrils (Nilsen,

1980).

The potential mechanisms for formation of organized biomineralized structures include

oriented crystal growth on templates, the aggregation of nanocrystals by oriented attachment, and

the assembly of inorganic nanoparticles mediated by organic molecules into aggregated

structures (Tao, Pan, Zeng, Xu, & Tang, 2007). The classical model of biomineralization

considers mineral formation as an amplification process in which individual atoms or molecules

are added to existing nuclei or templates, but a model involving aggregation-based growth has

gain traction recently and challenges the conventional concept of crystal growth. The brick and

mortar model attempts to explain the biological aggregation of nano-sized apatite by suggesting

that amorphous calcium phosphate acts as “mortar” to cement the crystallized “brick” of

nanosized HAP while biological molecules control the construction process. However, a new

concept of “mesocrystals” highlights the roles of nano-sized particles in biological crystal

engineering and is emerging as the current state of biomineralization theory (Dorozhkin S. ,

2012).

Nacre is one of the biological composites that contain both ceramic and biomacromolecular

phases. This unique structure is composed of alternating nanometer-scale laminated layers of

thin biomacromolecular matrix and CaCO3 (aragonite) platelets, all highly organized to produce

an excellent multifunctional material for the organism (Sarikaya, Liu, & Aksay, 1995). The

unique crystal structure of aragonite and the arrangement of the aragonite nanoparticles in

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nacre’s platelets make the diffraction patterns of individual platelets show single-crystal

characteristics (Li & Huang, 2009). The tablet/platelet of nacre does diffract as a single crystal

but is made up of a continuous organic matrix which breaks the mineral up into coherent

nanograin which share the same crystallographic orientation (Rousseau, 2011).

Stages of Crystallization

Biogenic minerals may be amorphous, paracrystalline, or crystalline and may occur as a

single unit (such as a single crystal), numerous individual units, or aggregates. Mineralized

skeletal hard tissues are examples of such aggregates. The aggregated units are usually arranged

in an orderly fashion, and when crystalline, the crystallographic axes are partially or fully

aligned. These ordered structures are generally differentiated into a number of microarchitectural

units, each of which is enveloped by an organic matrix. Minerals formed at a particular site may

be (i) retained in place, (ii) transferred intact to other sites, (iii) excreted, (iv) dissolved and

replaced continuously, periodically, or only occasionally, or (v) continuously reconstituted.

Mineralized hard tissue can have a number of functions other than structural and mechanical

ones. It is well known that the endoskeletons of mammals act as a reservoir of calcium and

phosphorus and are intimately linked to the organism's metabolism (Lowenstam H. A., 1981).

The hierarchical structure of bone is illustrated in figure 4-8.

The formation of a mature skeleton composed of various shapes ultimately depends on

cellular control and the deposition of mineral within an organic matrix, and the creation of

specialized tissues. The mineral, a calcium phosphate, closely approximates hydroxylapatite,

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Ca5(PO4)3(OH). The mineral in skeletal tissues is found as part of several different textures, e.g.,

woven, lamellar, haversian, trabecular and cortical bone that can be discriminated optically, or

histologically, and at higher resolutions using TEM or SEM. Bones and bone tissues fulfill their

structural function while protecting the marrow where essential cellular material is generated.

Sufficient bone stiffness is achieved by increasing the amount of calcium phosphate mineral

relative to the organic components in the tissue (Skinner & Jahren, 2003). The state in which

Ca2+ ions and PO43- ions are combined with the carboxylate groups on the organic matrices is the

earliest stage in crystal nucleation (Sato, 2007).

Crustaceans demonstrate drastic temporal differences within the same tissue with regard to

the extent of and capacity for mineralization. Such temporal differences raises questions about

the control of nucleation and mineralization. The cuticle is not homogeneous, but contains four

discrete layers. This observation was made more than a century ago (Williamson, 1860).

However, the terminology of Travis (1963) has come to be widely accepted. These layers from

the most external to the most internal are: the epicuticle, the exocuticle, the endocuticle and the

membranous layer (Roer & Dillaman, 1984).

Calcification of the crustacean cuticle is an extracellular phenomenon. Preparation for the

molt in the calcified crustaceans involves the resorption of the mineral and organic components

of the old cuticle and the simultaneous deposition of elements of the new cuticle. During

premolt, the entire organic matrix of the two outermost layers of the new cuticle (the epi- and

exocuticle) are elaborated beneath the old cuticle. The pre-exuvial layers do not calcify until

after the molt. The bidirectional nature of mineral transport and the sharp temporal transitions in

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nucleating ability of the cuticular matrix provide ideal systems in which to study these aspects of

calcification (Roer & Dillaman, 1984).

During ecdysis, crabs periodically shed their rigid calcified exoskeleton. Immediately after

ecdysis, the cuticle is soft; thereafter the calcite mineral bestows progressive hardness to the

cuticle (Giraud-Guille, 1984). Calcification of the epi- and exo-cuticles begins in the most

external regions and proceeds proximally. Mineral apparently reaches the outer portions of the

cuticle via the pore canals. Calcium is concentrated in the distal portions of the epithelial cells

and appears to be extruded in vertical rows corresponding in position to the pore canals of the

new cuticle. Calcification is concurrent with matrix formation, each organic lamella being

mineralized as it is laid down. Mineral deposition continues through postmolt. Calcification

spreads throughout the exocuticle and eventually is found to pervade the walls of the pore canals.

The end of postmolt is marked by the deposition of the membranous layer and the cessation of

net calcium deposition (Roer & Dillaman, 1984).

The epicuticle is the outermost and thinnest layer of the cuticle. It consists of tanned

lipoprotein impregnated with calcium salts. The exocuticle immediately underlies the epicuticle.

The exocuticle is composed of chitin-protein fibers with biphase of calcite and amorphous

calcium carbonate, stacked in layers of continuously changing orientation. The exocuticle is

hardened by quinone tanning and calcification, with the mineral crystals situated between the

fibers (Roer & Dillaman, 1984). The crystallization in exocuticle is completed in postmolt stage

(Tao, Zhou, Zhang, Xu, & Tang, 2009). The endocuticle is the thickest and the most heavily

calcified layer of the cuticle. The endocuticle is composed of horizontal lamellae of chitin-

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Figure 4-8 Hierarchical structural organization of bone: (a) cortical and cancellous bone; (b)

osteons with Haversian systems; (c) lamellae; (d) collagen fiber assemblies of collagen fibrils;

(e) bone mineral crystals, collagen molecules, and non-collagenous proteins (Rho, Kuhn-

Spearing, & Zioupos, 1998).

protein fibers with continuously changing orientation and is apparently not tanned, but hardened

solely by Ca salts (Roer & Dillaman, 1984). The endocuticle is calcified in late postmolt several

days after ecdysis. A membranous layer is also synthesized during postmolt and its completion

signals the onset of inter-molt. The membranous layer contains chitin and protein but is not

calcified. During the inter-molt stage, the isopod cuticle can be divided into epicuticle,

exocuticle, endocuticle, and membranous layer from outside to inside (Tao, Zhou, Zhang, Xu, &

Tang, 2009), Table 4-3.

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Table 4-3 Designation of layers in the crab cuticle

Sequence of formation Chemical Composition

Preecdysial

layers

1 Epicuticle

2 Pigmented layer

Lipoprotein

Chitin-protein,

CaCO3

Ecdysis

Postecdysial

layers

3 Principle layer

4 Membranous layer

Chitin-protein,

CaCO3

Chitin-protein

(Giraud-Guille, 1984)

Crystallization Control

Kinetic control of crystallization can be achieved by modifying the interactions of nuclei and

developing crystals with solid surfaces and soluble molecules. Such processes influence the

structure and composition of the nuclei, particle size, texture, habit and aggregation, and stability

of intermediate phases. In biomineralization, for example, structured organic surfaces are

considered to play a key role in organic matrix-mediated deposition by lowering the activation

energy of nucleation of specific crystal faces and polymorphs through interfacial recognition.

Soluble macromolecules and organic anions, as well as inorganic ions can also have an important

kinetic effect on crystallization, particularly with regard to polymorph selectivity and habit

modification.

A central tenet of biomineralization is that the nucleation, growth, morphology, and

aggregation (assembly) of the inorganic crystals are regulated by organized assemblies of

organic macromolecules (the "organic matrix"). Control over the crystallochemical properties of

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the bio-mineral is achieved by specific processes involving molecular recognition at inorganic-

organic interfaces.

Mann illuminated the role of molecular recognition in inorganic crystallization systems;

showed how organized organic surfaces can control the nucleation of inorganic materials by

geometric, electrostatic, and stereochemical complementarity between incipient nuclei and

functionalized substrates; and described how analogous interactions are responsible for the

morphological modification of inorganic crystals grown in the presence of anionic organic

additives. The surface lattice geometries, spatial charge distributions, polarity of hydration

layers, defect sites, and stereochemistries of the inorganic phase are all possible features that can

be recognized by the organic matrix (Mann, et al., 1993).

Biomineralization processes exhibit a high level of spatial control as the mineralization

usually takes place in a confined reaction environment. This confined reaction environment is

constructed by biomolecules—mainly polymers. They can build a scaffold for the mineralization

site as collagen in the example of bone. The mineralization event itself is also highly controlled

by biopolymers, which are usually soluble. The task of the soluble or functional matrix is the

control of nucleation, growth, polymorph and orientation of the inorganic compounds. It is

difficult to explore the function of the soluble matrix in biomineralization as it usually represents

a mixture of many biopolymers in tiny quantities so that a model system which mimics soluble

biomineralization polymers is an efficient way to understand biomineralization processes. The

functioning of the insoluble matrix and of the various components of the soluble matrix is a

highly synergetic process and only in some cases, the functions of the individual components of

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the soluble matrix are already revealed. This makes the mimicking of biomineralization

processes difficult as often, only little is known about the formation of a biomineral archetype.

Furthermore, it is difficult to achieve the assembly of the structural matrix over several levels of

hierarchy because the synthetic chemist does not have cells at hand, which carry out this job in

nature. However, biomimetic mineralization can be successful if the formation of the natural

archetype is not yet completely understood. Frequently, a model system, which mimics soluble

and/or insoluble matrix is an efficient way to understand biomineralization processes and to

mimic them. Despite the complexity of the topic, characterization of biomineralization processes

and mechanisms has made significant progress in recent years often catalyzed by new findings or

concepts in this field (Xu, Ma, & Colfen, 2007).

Concluding Remarks

Biomineralization/Calcification/Mineralization/Crystallization is an important process in a

plethora of scientific disciplines. There is not a unified molecular and mechanistic descriptions of

crystal nucleation, growth, morphology. Biomineralization results in complex materials and

composites, such as bones and shells that are characterized by an extraordinary level of

molecular control of the particle size, structure, morphology, aggregation, and crystallographic

orientation of the mineral phases. Attempts to create synthetic materials that would imitate

mineral nucleation and growth have embraced a variety of approaches including biomimetic.

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Understanding biomineralization of biological systems offers a genuine possibility for future

strategies that create higher-order architectures.

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Chapter 5 Osteoinductive Nature of Blue Claw Crab

Introduction

There are many different concepts of engineering tissue. The desired properties of the

construct are defined by the intended clinical use (Kneser, et al., 2002). In 1993, Langer and

Vacanti summarized the early developments in this field and defined tissue engineering as “an

interdisciplinary field that applies the principles of engineering and life sciences toward the

development of biological substitutes that restore, maintain or improve tissue or organ function”

(Langer & Vacanti, 1993). Tissue engineering (TE) uses a set of tools at the interface of the

biomedical and engineering sciences to support the growth of living cells or attract endogenous

cells to aid in tissue formation or regeneration to produce therapeutic or diagnostic benefit. TE

also seeks to understand structure-function relationship in normal and pathological tissues; and to

control cell and tissue responses to injury, physical stimuli, and biomaterials surfaces through

chemical, pharmacological, mechanical, immunological, and genetic manipulation. The key

processes occurring during the in vitro and in vivo phases of tissue formation and maturation are:

(1) cell proliferation, sorting and differentiation; (2) extracellular matrix production and

organization; (3) degradation of the scaffold; and (4) remodeling and potentially growth of the

tissue (Schoen, 2013).

In bone tissue engineering, the emerging trend is to employ materials that play an active role

in regeneration, rather than passive or inert roles. Marine invertebrates offer inexhaustible

inspiration (Ehrlich H. , 2010). Marine sources such as coral, algae, and nacre have been

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evaluated as potential orthopedic or dental implants. Evaluations of corals as potential bone graft

substitutes began in the early 1970s in animals and in 1979 in humans (Demers, et al., 2002).

Nacre investigations dates back as early as 600 A.D. with the ancient Mayan civilization

adapting nacre as a bone implant material (Westbroek & Marin, 1998).

Bone tissue engineering is based on the understanding of tissue formation and regeneration

and integrates knowledge from physics, chemistry, engineering, materials science, biology and

clinical medicine into a comprehensive interdisciplinary approach. The goal of bone tissue

engineering is to regenerate a functional structure containing osteoblast, osteocyte, and osteoclast

cells capable of building and of continuously remodeling the mineralized collagenous

extracellular matrix that functions structurally and biomechanically as bone tissue (Kneser, et al.,

2002).

Bone tissue engineering requires the interaction of cells, growth factors, and extracellular

matrix. Several studies both past and present demonstrate the effectiveness of these elements

(Hing, 2004). In 1889, Senn noticed that decalcified bone can induce healing of osseous defects

(Senn, 1889). Urist (1965) discovered that demineralized bone when implanted intramuscularly

induced new bone formation. In 1971, Urist and Strates presented ‘bone morphogenetic protein’

(BMP) and ‘osteoinduction’ to the scientific and medical communities (Urist & Strates, 1971)

and later Urist coined osteoconduction. By 1976, Reddi and Anderson further expounded on

Urist's work dealing with ‘induced osteogenesis’ (Reddi & Anderson, 1976) and provided a

compelling explanation of the functional role of purified organic bone matrices (Gruskin, Doll,

Futrell, Schmitz, & Hollinger Jeffrey, 2012).

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Large bone defects, resulting from a multitude of causes, including aging, trauma, and

disease, are commonplace. These defects usually do not heal spontaneously and interventions

with bone substitutes are often required. Natural bone uses osteogenic (progenitor osteoblasts)

cells; osteoconduction (structural support system); and osteoinduction (growth factors) to

produce a mineralized collagen matrix that hardens (Stocker & Wolinetz, 2009). Ideally, a bone

replacement material should mimic bone tissue from a mechanical, chemical, biological and

functional point of view, and facilitate new bone formation. None of the existing implant

biomaterials are optimized in terms of performance in bone replacement (Hertz & Bruce, 2007).

Emerging as a valid therapeutic approach, bone tissue engineering is based on understanding

hard tissue formation and targets induction of new functional tissues. Among hard tissue

engineering strategies, the selection of a suitable material is paramount. Surveying the

characteristics of bone, crab carapace materialized as a material similar to bone based on factors

such as high strength, stiffness, and hierarchical structuring. Thus, crab cuticle is another

candidate material for enhancing the healing, remodeling, and engineering of bone. To date, our

lab conducted the first preliminary biocompatibility study to determine crab cuticle’s suitability

as a bone substitute material (Wilson, Gugssa, Mehl, & Anderson, 2012). The paper addresses

the osteoinductive nature of crab cuticle, specifically its ability to facilitate the formation of a

mineralized collagen matrix.

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Bone Grafts and Bone

Bone grafts, synthetic, and natural materials are used to assist and to build bones after

damage from disease, trauma, or aging (Yuan & Groot, 2004). Bone is noted for its strength and

hardness and is characterized by its composite structure consisting of organic matrix (35%) and

inorganic elements (65%). The inorganic constituent of bone consists mainly of calcium

phosphate, considerable amounts of citrate and carbonate ions and traces of magnesium fluoride,

and sodium. The organic matter in bone is largely collagen. The calcium phosphate in bone

resembles mineral hydroxyapatite (HAP) (Yuan & Groot, 2004), and contains carbonates,

sodium, magnesium and fluoride salts (Hing, 2004). See Table 5-1.

A dynamic living tissue, bone consists of osteocytes and osteoblasts, living cells that build

bone. During bone remodeling, bone has osteoclasts, living cells that resorb bone (Yuan &

Groot, 2004). Bone is a remarkable composite, and its unique functional characteristics include a

high level of vascularity, the ability to remodel and repair itself, maintaining mineral

homeostasis and providing a source of stem cells, acting as a mineral and blood cell reservoir

(Hing, 2004).

Even when fully mature, bone is not static. There is a constant repairing, modeling, and

remodeling that continues throughout life (Summerlee, 2002). Bone can suffer from disease and

is subject to deterioration as a result of age or trauma (Hing, 2004). When complicated or large

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Table 5-1 The composition of the inorganic and organic phases of bone

(Murugan & Ramakrishna, 2007)

fractures and defects occur in bone, the healing and repair often fails and requires surgical

intervention. Tissue engineering strategies are explored to repair or restore damaged bone tissue

with the assistance of biomimetics, biomaterials, cells, and/ or signal molecules (Janicki &

Schmidmaier, 2011), figure 5-1. There are three characteristics inherent to natural bone:

osteoconductivity; osteogenicity; and osteoinductivity (Stocker & Wolinetz, 2009).

Autologous bone grafts are the gold standard for current therapeutic strategies. Autologous

bone combines the three characteristics inherent to natural bone to promote growth and

regeneration: osteogenic cells, osteoinductive and osteoconductive properties. Autografts have

limitations such as donor site morbidity caused by surgical procedures from harvesting, risk of

infections, and chronic pain (Janicki & Schmidmaier, 2011). Allografts avoid some of the

disadvantages of autografts; however allografts have other limitations like transmission of

diseases from donor to recipient or immunogenic reactions (Janicki & Schmidmaier, 2011).

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Xengografts are tissues harvested from one species and implanted into a different species (Bauer

& Muschler, 2000). Alloplasts include any synthetically derived graft material not derived from

animal or human origin (Suneelkumar, Datta, Srinivasan, & Kumar, 2008). Allografts,

xenografts, and alloplasts all have disadvantages and limitations. An alternative to auto-, allo-

and xenografts are tissue engineered (Janicki & Schmidmaier, 2011) or biomimetic bone

substitutes. Figure 5-2 depicts the past, present and future of tissue repair. Synthetic materials

have widely broadened the available tools and strategies for bone grafting and vary greatly in

osteoconductivity and osteoinductivity, and in mechanical strength, handling properties, and cost

(Bauer & Muschler, 2000).

Figure 5-1 Schematic of the design strategy of tissue-engineered biomimetic nanocomposite

bone graft (Murugan & Ramakrishna, 2007).

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Bone substitutes can be generally divided into two main groups: biological and synthetic

materials. Biological bone substitutes consist of materials like natural polymers (like collagen

type I or demineralized bone matrix (DBM). Materials like porous metals or bioactive glasses,

synthetic polymers and calcium phosphates rank high amongst synthetic materials (Janicki &

Schmidmaier, 2011) used as bone biomaterials. Bone grafts types have limitations and

disadvantages that motivate the development of alternatives (Stocker & Wolinetz, 2009).

Figure 5-2 Depiction of the past, present and future of tissue repair (Hench, 1998).

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The constraints of common bone defect treatment strategies have triggered exploration of new

biological (Holzapfel, et al., 2013) and biomimetic approaches. The three key ingredients for

both morphogenesis and tissue engineering are inductive signals, responding stem cells, and the

extracellular matrix (Reddi, 2000). The classic paradigm of tissue engineering comprises a cell

seeded scaffold, an in vitro stage of tissue formation, and an in vivo stage of tissue growth and

remodeling or an unseeded scaffold, with biological information such as growth factors, to

attract precursor cells and cause differentiation in vivo (Mendelson & Schoen, 2006). In a

biomimetic approach to tissue engineering, natural materials or lessons from nature are exploited

to solve tissue engineering problems. Natural materials can be used with the aspiration to control

the behavior cells and promote the attachment, migration, proliferation, and differentiation of

cell types of interest.

Materials harvested from natural sources are used extensively in bone tissue engineering.

These materials may be derived from a wide range of sources and possess an equally wide range

of physical and biological characteristics. For example, coral was used as a bone graft material in

the mid 1970’s. Coral’s porous CaCO3 exoskeleton displays a unique ability to bond with bone

(White, et al., 1975). Another natural material chitin is derived from the exoskeletons of

crustaceans such as crab and shrimp and is the second most abundant natural polymer after

cellulose. Chitosan, a derivative of chitin, elicits a minimal foreign body reaction, with little or

no fibrous encapsulation (Monzack, Rodriguez, McCoy, Gu, & Masters, 2011). Chitosan,

chitosan derivatives, and blends of chitosan have been used in bone tissue engineering

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applications. Costa-Pinta et. al. provides an extensive review of chitosan’s role as a scaffold in

bone tissue engineering (Costa-Pinto, Reis, & Neves, 2011).

Osteoinduction, Osteoconduction, and Osteogenic

Giving the limitations associated with current bone graft technologies, implementation of

bone tissue repair strategies aims to incorporate the advantages of allografts and autografts

(Nicoll, 2011)as well as the characteristics inherent to bone. Osteoconduction highlights bone

formation towards the implant from the host bone bed; guided bone formation on material

surfaces resulting in bone bonding (sometimes called “bioactivity”, “osteocoalesce”, and

“osteoingreation”); and the absence of other tissues between the newly formed bone and

biomaterial surface (Yuan & Groot, 2004). A distinction can be made between, osteoconduction

and osteointegration. Both depend on biological factors, but osteoconduction may be rather short

lived and successful osteointegration maintains its bone anchorage over an extended period

(Albrektsson & Johansson, 2001). Ensuring the continuity of the repaired bone both structurally

and mechanically, osteoconductive biomaterials form a biological apatite layer in vivo on its

surface where osteogenic cells can easily attach and form bone on it (Yuan & Groot, 2004).

Osteoconductivity respresents structure and the ability of a biomaterial to serve as scaffolding

to support new bone formation and growth (Stocker & Wolinetz, 2009). Osteoconductive

biomaterials influences bone regeneration. On osteoconductive biomaterial surface, guided bone

formation is limited in its distance spotlighting that osteoconductive biomaterials alone may not

repair large bone defects (Yuan & Groot, 2004). Osteogenicity represents the presence of

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osteoblasts or stem cells that can transform into cells responsible for bone formation (Stocker &

Wolinetz, 2009). Osteoinduction entails the recruitment and stimulation of primitive,

undifferentiated and pluripotent cells to develop into bone-forming cell lineage that ultimately

produce bone (Albrektsson & Johansson, 2001). Conclusive evidence for osteoinduction is

characterized by heterotopic bone formation, that is, bone formation in tissues or organs where

bone does not naturally grow (Barradas, Yuan, van Blitterswijk, & Habibovic, 2011) . Although

the exact processes involved in the mechanism of osteoinduction by biomaterials are still largely

unknown, work by many groups has shown that biomaterials need to meet very specific

requirements in terms of (a) macrostructure, (b) microstructure and (c) chemical composition in

order to be osteoinductive (Habibovic & de Groot, 2007).

This initial part of bone fracture healing response includes osteoinduction, a process that

starts immediately after the injury. Osteoinduction is responsible for the majority of newly

formed bone. Osteoconduction depends to a fairly large extent on previous osteoinduction

(Albrektsson & Johansson, 2001).

The phenomenon of bone formation upon implantation of various tissues heterotopically has

been documented: (Huggins, 1931), (Levander, 1934), (Bertelsen, 1944), (Urist & McLean,

1952), (Moss, 1958). Urist’s discovery that decalcified bone matrix induced bone formation in

muscles of mouse, rat, guinea pig and rabbit, subsequent identification of Bone Morphogenetic

Proteins (BMPs) as inducers of heterotopic bone formation, and definition of autoinduction, or

osteoinduction as “the mechanism of cellular differentiation towards bone of one tissue due to

the physicochemical effect or contact with another tissue” set a benchmark in his field of

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research (Barradas, Yuan, van Blitterswijk, & Habibovic, 2011). Other osteoinduction

definitions have been proposed. Wilson-Hench described osteoinduction as the process by which

osteogenesis is induced (Barradas, Yuan, van Blitterswijk, & Habibovic, 2011).

Biomaterials with intrinsic osteoinductivity possess a great potential as alternatives to

biological approaches to bone regeneration. de novo bone origin is attributed to its osteoinductive

properties rather than to the osteoconductive. The capacity to repair bone defects can differ

greatly among materials from the same family. Apart from the chemical composition of the

material, the geometry and macrostructural properties have been shown to play an important

role. In the case of macrostructure, the most striking example is the importance of porosity.

(Barradas, Yuan, van Blitterswijk, & Habibovic, 2011)

Bone formation by osteoinduction is initiated by soluble and insoluble signals that trigger a

complex cascade of molecular and cellular morphogenetic processes that ultimately leads to the

sculpture of precisely organized multicellular mineralized structures (Ripamonti & Roden,

2010). Turing was the first to describe a system of chemical substances, called morphogens,

account for the main phenomena of morphogenesis (Turing, 1952). Secreted from a group of

cells or organizing center, morphogen molecules can provide spatial information and then moves

away. Cells can detect where they are in respect to the organizing center as the activity of the

morphogen decreases gradually as a function of its distance from the source. The cells are

induced by the morphogens to take on different fates according to their position (Vincent &

Perrimon, 2001). Moreover, it is believed that several mineralized and non-mineralized

extracellular matrices of mammalian tissues contain morphogens that direct specific cell

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differentiation in responding stem cells generating tissue form and function (Ripamonti &

Roden, 2010).

Ossification

Bone forms in a process termed ossification. There are some distinctions that can be made

about bone development in two critical phases: either embryonically or postembryonically.

During embryonic or in utero development, bone tissue starts to form mesenchyme cells. During

the second phase, bone elongate and change shape. There are two types of embryonic

ossification: intramembranous ossification and endochondral ossification. Intramembranous

ossification involves mesenchymal cells migrating, becoming osteoblasts, forming aggregations

in organizing or ossification centers, producing bone matrix that undergoes calcification,

encasing osteoblasts to emerge as osteocytes, growing linear extensions called spicules from the

ossification centers, growing of blood vessels branching around the spicules, fusing spicules in a

network to eventually create flat bones. Endochondral ossification includes long, short, and

irregular bones and involves bone development from a pre-existing cartilage model where a rod

of cartilage develops in the expected final position and mimics the general shape of the adult

bone (Summerlee, 2002).

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Calcification/Mineralization

Usually limited to bones and teeth in vertebrates, mineralization and calcification are terms

used interchangeably to describe the process in which tissues are converted to hard tissues.

Matrix mineralization and intact bone cells characterize physiological mineralization (Robey,

2011). Bone formation or osteogenesis can also occur outside the skeleton. Ectopic ossification

happens when precursor cells inappropriately receive signals to develop into bone cells, to

synthesize extracellular matrix and to create an environment facilitating mineralization. Matrix

mineralization is the final step of osteogenesis (Jahnen-Dechent, 2004). Figure 5-3 hypothesizes

a mechanism behind osteoinduction of biomaterials.

Pathologic calcification is a complex regulated process of mineralization that is similar to

bone formation and remodeling. Pathologic calcification refers to the deposition of calcium

phosphates or other calcific salts at sites, which would not normally have become mineralized.

The sizes of the mineral crystals in pathologic calcification are similar to those in bone; however,

there is much more mineral in the deposits than there is in bone (Ciftcioglu & McKay, 2010).

Pathologic calcification can thus occur before and after osteogenesis (Jahnen-Dechent, 2004).

There are two forms of pathologic calcification: dystrophic calcification and metastatic

calcification. Dystrophic calcification occurs in dying or dead tissue that is able to maintain

normal calcium homeostasis. Dystrophic calcification is distinguished by the lack of identifiable

osteoblasts and osteocytes, and lack of marrow (Robey, 2011). Metastatic calcification is the

deposition of calcium salts in otherwise normal tissues due to abnormal biochemistry with

disruptions in the calcium or phosphorus metabolism (Tam, Wang, & Fan, 2002).

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Figure 5-3 Diagram illustrating hypothesized mechanisms behind osteoinduction by

biomaterials. Physico-chemical and/or structural properties of osteoinductive biomaterials may

prompt the mechanism responsible for heterotopic bone formation (Barradas, Yuan, van

Blitterswijk, & Habibovic, 2011).

Ectopic calcification is defined as biomineralization occurring in tissues where it is not

normally found. Ectopic calcification or ossification can be mistaken for pathological

calcification and can develop at the same site. There are two types of ectopic bone: heterotopic

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and osseous metaplasia. Heterotopic bone is essentially normal osseous tissue found in an

abnormal location that is believed to originate from embryonic cells that persist in tissues during

development. Osseous metaplasia involves the transformation of a cell or tissue from one type to

another and can develop from connective tissues at sites of chronic inflammation (Shehab,

Elgazzar, & Collier, 2002).

Inflammation

All space within the body of an organism is filled; therefore the penetration of a biomedical

device is almost inevitably going to result in injury (Ramsden, 2008).

The interface between the implant and the biological environment is the location of proteins

and other biomolecules that interact with implant as well as the location of the body’s wound

healing process (Dee, Puleo, & Bizios, 2002). Materials implanted into tissues by surgical

techniques involve the creation of a wound. The inflammatory response is an early step in the

wound healing process. Just as the initiation and the magnitude of inflammation are regulated by

the characteristics of the biomaterial, the resolution of the inflammatory process is also

dependent on the physical and chemical properties of the biomaterial. These attributes will

determine the overall resolution of the wound healing process (Greco, 1994) and hence the

bioactivity of the implant.

Inflammation is qualitatively separated into acute and chronic inflammation. Acute

inflammation is a stereotypic response, generally of short duration and lasting less than a week.

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Figure 5-4 Schematic depicting the material, interface, and biological enviroment components.

It emphasizes the importance of the interface.

It is characterized by an increased capillary permeability which results in the exudation of

plasma proteins and emigration of polymorphonuclear neutrophilic leukocytes (PMN) to the site

of inflammation. Chronic inflammation is of longer duration and is histologically associated

with macrophages, lymphocytes, proliferation of connective tissues, and deposition of

matrixproteins (Greco, 1994). The inflammatory response to biomaterials is determined by their

composition, purity, shape of the implant and its surface properties, implantation site, positional

stability at the implantation site, porosity, chemical stability, etc. (Mainil-Varlet, Gogolewski, &

Nieuwenhuis, 1996). The material-interface is an important factor in biological response, figure

ions

proteins

blood

enzymes

coagulation

inflammation

proliferation

remodeling

resolution

Biological Environment Interface Material

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5-4, as it is the place of the first interactions with the biological environment. The time course

varies with severity and nature of the injury (Black, 1999).

Biologically, macrophage are perhaps the most important cell type in chronic inflammation.

Macrophages appear at the site of biomaterial implantation due to their role in wound healing in

response to surgery, as well as in response to the biomaterial itself. As a rule, macrophages can

be seen adhering to the biomaterial within 24 hours after implantation. The number of

macrophages attracted to the implant is in part dependent on the texture of its surface.

Macrophages are capable of producing a variety of growth factors, cytokines, chemotactic

agents, proteases, metabolites, oxygen free radicals, complement components, and coagulation

factors. These macrophage products may have a wide spectrum of effects, from cell death to cell

proliferation, and thus may impact on the course and outcome of the chronic inflammatory

process (Greco, 1994). Macrophages react to and generate, signals that influence growth,

differentiation, and death of other cells (Paul, 2008). The activated macrophage can undergo a

number of transformations. In addition to releasing lysozymes, both internally and externally,

related to attempts to digest foreign materials, activated macrophages synthesize and release a

wide range of biochemical factors that can mediate the activity of many other cells, including

lymphocytes, fibroblasts, osteoblasts, and osteoclasts (Black, 1999).

Osteoinductive materials provide a biological stimulus for recruitment, stimulation and

differentiation of primitive, undifferentiated and pluripotent cells into osteoblast or

preosteoblasts, which are the initial cellular phase of a bone-forming lineage. Bone formation is

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essentially achieved by the interaction of cells, growth factors, and an implant material and the

facilitation of an appropriate cellular response (Di Silvio, 2009).

Experimental Section

Materials

Perfusion solutions for animal experiments were a normal saline solution and a fixative

solution (1:4 ratio). The saline solution consisted in a 0.9% NaCl solution with a checked pH of

7.4. The fixative solution is a 1%w/v Paraformaldehyde , 2%w/v Gluteraldehyde in a 0.1M

cacodylate buffer with pH adjusted to 7.4. The preparation of 200 ml/animal was sufficient for

perfusion. Various sets of C. sapidus (Blue Claw Crab) was purchased from commercial sources.

The Sprague-Dawley female rats were purchased from Harlan (Frederick, MD). The Sprague-

Dawley female rat experimental protocols are following the Animal Welfare Care and Use

Committee (IACUC) directives from Howard University. All animal experiments were

performed at Howard University in the Biology Department under the direction of Dr. Anderson.

Crab integument preparation

Carapace integument from fresh and fresh frozen C. sapidus (Blue Claw Crab) specimens

were removed and thoroughly cleaned with deionized water and allowed to dry. Carapace was

initially crushed using a commercial coffee grinder using a pulse cycle. Subsequent size

reduction was accomplished using a mortar and pestle. Crushed crab shell samples were washed

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and aged in absolute ethanol or 70 vol.% ethanol as an initial step in sterilization. A portion of

the crushed crab shell was demineralized by aging 2 g of dry crab shell in 20 ml of 0.5 M HCl.

The crab shell granules were allowed to react in the HCl for 1–3 h. The demineralized crab shell

was washed with deionized water and finally rinsed with ethanol and allowed to dry in air

(Wilson, Gugssa, Mehl, & Anderson, 2012).

Characterization

Transmission electron microscopy (TEM), High resolution transmission electron microscopy

(HRTEM), selected area diffraction (SAD), electron nanodiffraction (END), electron

microanalysis - energy-dispersive spectrometry (EDS) was performed at the NISP

characterization facility at the University of Maryland; in vitro and in vivo studies were used to

investigate the osteoinductive nature of Blue claw crab.

TEM enabled observation of ultrastructure of our sample and allowed for histological

assessment. HRTEM allowed even finer detailed observation in the nanoscale. SAD was used to

identify crystal characteristics. END offered atomic resolution for nanosized crystal. X-ray

microanalysis was used to determine the elemental composition of the specimen.

TEM has different forms and offers a broad range of characterization techniques with high

spatial and analytical resolution. TEM is the central tool for characterization of nanoscale

materials. The advantages of using TEM is that a small amount of materials is needed, strong

diffraction with matter at an atomic resolution, can handle nano-size crystals, and precise method

of structure determination. Despite all the advantages, TEM bring accompanying drawbacks.

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One drawback to TEM techniques is that many materials require extensive sample preparation to

produce a sample thin enough to be electron transparent, which makes TEM analysis a relatively

time consuming process with a low throughput of samples. Another disadvantage is that the

structure and chemistry of the sample may also be changed during the preparation process.

Additionally, the field of view is relatively small, raising the possibility that the region analyzed

may not be characteristic of the whole sample. The sampling abilities of the instrument is greatly

comprised with the higher resolution. Another problem is TEM displays 2D images of 3D

specimens. A single TEM image has no depth sensitivity. This problem can be circumvented

with a series of images taken a t different tilts to create 3D image. Other disadvantages include

image artifacts and defects. Electron beam damage, particularly in the case with biological

materials, is also a concern (Williams & Carter, 2009).

HRTEM is a form of TEM and has many of the same advantages and disadvantages. HRTEM

maximizes the useful detail in the image but at the cost of sampling abilities. HRTEM, SAD and

END were used in a complementary fashion. SAD and END were techniques employed inside

the TEM. SAD can be useful for the determination of lattice parameter information and unit cell

details. Lattice structure gives information about the long range composition of a material.

Atomic structure is a huge amount of data compared to what any other biochemical technique

could provide. Restrictive factors of SAD are that a good crystal must be found for high accuracy

of crystallographic structures and that only limited information about the structure’s dynamic

behavior can be obtained from one single diffraction experiment. Resolution is an important

limitation of SAD. Small particle size can lead to peak broadening and erroneous interpretation

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of a SAD pattern. If a SAD pattern of a polycrystalline aggregate has many rings, the number of

rings may be too great and closely spaced for effective measurement. Thickness variation and

crystal defects can make complete and specific identification of each small crystal difficult even

impossible (Ferrell Jr. & Paulson, 1977). For nanodiffraction, radiation damage effects can be

extreme and biological samples can be especially vulnerable. However, radiation damage effects

are not of great significance for nanodiffraction provided that care is taken to prevent the beam

from staying for an excessive amount of time in any one position. In nanodiffraction, structurally

ordered substances can become amorphous and loss some of its weight limiting the attainable

resolution (Cowley & Spence, 2000).

EDS or x-ray microanalysis was performed inside the TEM as well. Accurate quantitative

analysis requires calibration of the x-ray analysis system using standards of known composition

and thin specimens. Large differences in the self-absorption and fluorescence of emitted x-rays

will limit the precision of quantitative analysis for some combinations of elements.

In vivo crab shell implantation

Female Sprague-Dawley rats ranging in weight from 400 to 450 g were used in this study.

The animals were housed in plastic cages in a room with the temperature in the range of 70-74ºC

and humidity in the range of 35-45%. All animals were fed a rodent chow (Harlan, Teklad

Laboratory) diet and water.

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Figure 5-5 The implantation study has an aim to assess the histological response to the crab

cuticle and determine its potential as as a bone biomaterial.

In our collaboration with Howard, approximately 48 rats were used to address the question of

crab shell influencing bone formation using an osteoinductive model involving subcutaneous

implantation of crushed crab shell. The focus of my study involved analyzing tissue samples that

were obtained after 28 days of aging. The following protocols were used in implanting,

removing, and preparing tissue samples for analysis.

Each rat was slightly anesthetized with Halothane (Sigma) just for the duration of the crab

shell implantation. The abdominal skin was initially cleaned with ethanol. A subcutaneous

incision was made in the abdominal region of anesthetized twenty eight day old Sprague–

Dawley rats and approximately 10–20 mg of demineralized or untreated crab shell was

Incision Site

Objective

The goal of this work was to assess the histological response

or changes to a crab shell stimuli in a Wistar rat animal model;

Assessment

Consideration of the cellular changes, cell migration, proliferation,

and differentiation;

Determination

The potential of crab shell use as a bone implant.

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implanted. The implant site was closed with stainless steel staples and rinsed with ethanol. The

samples were retrieved at 1, 3, 6, and 28 days, respectively, after implantation.

The protocol for retrieval of the tissue close to the implant was as follows: the rats were

anesthetized slightly with halothane and maintained with temporarily halothane exposure during

the whole protocol. The rat abdominal cavity was opened and then the diaphragm was also

opened to expose the beating heart. The blood was then flushed out from the whole animal by

perfusing the saline solution through the left ventricle until all organs became pale. The saline

solution was replaced by the fixative solution to fix the whole animal and in particularly the

tissue close to the crab shell implant. This tissue from the subcutaneous area of the implantation

was cut-off from the skin side as presenting implants being encapsulated. Tissue for TEM

analysis were post fixed with 1% Osmium Tetroxide (OsO4) then stained with lead acetate

before being dehydrated using increasing ethanol concentration aqueous solutions until a final

dehydration in 100% Ethanol. The samples were then embedded in epoxy in preparation for

ultramicrotoming. Thin sections about 90 nm thick were cut using a diamond knife and mounted

on C coated Cu transmission electron microscopy (TEM) sample grids (Wilson, Gugssa, Mehl,

& Anderson, 2012)

A rat subcutaneous implantation model was used to evaluate the in vivo tissue response,

figure 5-5. The healing response triggered by placement of bone implant in subcutaneous tissue

include the host inflammatory reaction to trauma associated with surgical procedure and

implantation of the whole crab material, the inflammatory and immune reaction to the material

itself, and the cellular processes such as migration, proliferation, and differentiation. The speed

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and degree of this response and incorporation of the implant into host tissue is dependent on

local and systemic factors, namely type of implant material and surrounding tissue integrity, and

the systemic physiological state of the host (Di Silvio, 2009)

Some Preliminary Results

A short term assessment of subcutaneous implantation of demineralized crab shell in the

abdominal region of 28 day old Sprague–Dawley rats results are shown in Figures 5-6 and 5-7.

The cellular events occurring at the material/tissue interface and material itself depicts

macarophages or monocyte type cells in close proximity to a demineralized crushed Calinectes

Sapides (Blue Claw) crab shell particle which displays Bouligand structures, figure 5-7.

Figure 5-6 Transmission electron microscopy image of in vivo study of demineralized crushed

Calinectes Sapides (Blue Claw) crab shell implanted subcutaneously in abdominal region of 28

day old Sprague–Dawley rats. Aging time was one day and image displays a crab shell particle

in the bottom of the image with a macrophage in close proximity (Wilson, Gugssa, Mehl, &

Anderson, 2012).

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Figure 5-7 TEM pictures depicts macrophages surrounding the Bouligand structure of

demineralized crushed Calinectes Sapides (Blue Claw) crab shell (Wilson, Gugssa, Mehl, &

Anderson, 2012).

Figure 5-7 also depicts macrophages surrounding the Bouligand structure. The implantation

time was 1 to 6 days. The nucleus displays marginal and central clumping of chromatin material

indicative of fully differentiated cell in which little active synthesis or energy expenditure is

occurring (Porter & Bonneville, 1973). The macrophage or histiocyte is irregularly shaped, has

considerable cytoplasm, a rounded nucleus and a nucleolus. Characteristic of the macrophage is

the presence of numerous lysosomes, which are related to its phagocytic function. Lysosomes

have a heterogeneous electron-dense content and an abundance of proteolytic enzymes, and are

enclosed by membranes which isolate them from the remainder of the cytoplasm. The lysosomes

participate in the intracellular digestion of phagocytosed material (Laguens & Gomez Dumm,

1969). Macrophages are monocytes that have differentiated after entering tissues at sites of

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infection or inflammation (Janeway, Travers, Walport, & Shomchik, 2005). In responding to

these important mediators, macrophages infiltrate the wounded area and assist in cleaning and

removing damaged tissue debris and foreign particles. Once in the wound site, activated

macrophages release several important growth factors and cytokines. Considered to be the most

important regulatory cell in the inflammatory reaction, macrophages become the predominant

cell type during the latter part of the inflammatory phase. Macrophages phagocytize, digest, and

kill pathogenic organisms; scavenge tissue debris; and destroy any remaining neutrophils.

Macrophages release chemotactic factors that attract fibroblasts to the wound area (Li, Chen, &

Kirsner, 2007).

Figure 5-8 Transmission electron microscopy image of collagen bundles that formed after

subcutaneous implantation of demineralized crab shell for 6 days. The image shows 67 nm

banding pattern and regions of electron dense mineralized particles on the collagen fiber surface

and interspersed in between collagen fibers (Wilson, Gugssa, Mehl, & Anderson, 2012).

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The initial results of the potential of implanted crab shell to induce ectopic bone formation

were reported (Wilson, Gugssa, Mehl, & Anderson, 2012) in figure 5-8. Our idea is based on the

work of Urist (1965) in which he demonstrated that demineralized bone matrix (DBM) possessed

the ability to induce new bone formation in non-osseous sites such as subcutaneous zones or

intramuscular regions. Studies on the osteoinductive properties of DBM and calcium phosphate

have been reported: (1) Reddi and Huggins (1974) (1972) and Reddi and Anderson (1976)

showed that the bone induction process proceeded via an endochondral mechanism in which the

implanted demineralized bone matrix was transformed to a cartilage type plaque by the action of

macrophages and fibroblasts which transformed to chondroblasts to facilitate cartilage tissue

formation. The chondroblasts were in turn transformed to osteoblasts and the cartilage

mineralized to complete the endochondral bone formation process. Studies (Reddi, 1981),

(Sampath & Reddi, 1981), (Reddi, 2000) later showed that DBM contained a family of

osteogenic extracellular matrix (ECM) proteins which came to be known as bone morphogenetic

proteins (BMP) which promoted various activities that are necessary for bone formation. Bone

formation induced by BMP or BMP-containing matrix is the most typical example of

osteoinduction (Reis & Weiner, 2004) (2) Ectopic bone formation was exhibited by inorganic

ceramics such as calcium phosphate and calcium carbonate: Yamasaki (Yamasaki & Sakai,

1992) reported bone formation when hydroxyapatite was implanted subcutaneously in dogs; and

Ripamonti (1991) induced bone formation in Baboons with porous hydroxyapatite obtained from

the conversion of calcium carbonate exoskeletons of coral. Crab shell exoskeleton’s main

constituents are chitin and calcium carbonate. The osteoinductive potential of various materials

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differed based on starting time, amount of bone induced, type of material, geometrical

parameters such as microstructure, the chemistry, and type of host or animal (Reis & Weiner,

2004). As indicated previous, crab cuticle is strikingly similar to bone. Coupled with our initial

assessment of collagen fibers produced with the implantation of demineralized crab shell and

previous studies with calcium phosphate, coral, nacre, and similar materials, the idea for using

crab shell as an osteoinductive bone biomaterial showed tremendous promise. The surface

topography of biomaterials is especially important to guide cellular behavior like cell movement

and adhesion that is regarded as being very important for promoting wound healing and tissue

regeneration (Lamers, et al., 2010).

Analysis

Crab shell or cuticle, replaced at each molt, is secreted by epithelial cells. It varies in

thickness and hardness according to its location. The fully formed cuticle of a crab is a

proteinaceous structure that consist s of an outer later, the epicuticle, and underlying exocuticle

(pigmented layer), endocuticle (calcified layer), and membranous layer (uncalcified layer).

Chitin occurs in all but the epicuticle, and calcium, mainly as CaCO3, but also as Ca3(PO4)2 and

CaSO4, is found to varying degree and, depending on location, in all but the membranous layer.

In the blue crab, calcium occurs in parts of the epicuticle; its presence is species dependent. The

epicuticle is the first line of defense between the animal and its environment; contains lipids,

proteins, and usually calcium; and is relatively impermeable to water and salts (Johnson, 1980).

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Figure 5-9 (a) SEM micrograph and a schematic drawing of a cross-sectional surface showing

three different layers in the crab exoskeleton: epicuticle, exocuticle, and endocuticle (Chen, Lin,

McKittrick, & Meyers, 2008); (b) TEM micrograph of demineralized crushed Calinectes Sapides

(Blue Claw) crab shell displaying prominent Bouligand nested arc patterns and a schematic

drawing of the Bouligand structure showing the arc pattern on oblique surface.

Bouligand Structure

Epicuticle

Exocuticle

Endocuticle

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A prominent feature of crustacean exoskeletons is their well-defined hierarchical

organization, which reveals different structural levels, figure 5-9. A twisted plywood structure,

called Bouligand structure, is created by planes that are stacked in a helicoid. This nesteted arc

pattern is illustrated in the TEM and schematic image shown in figure 5-9. These structures

repeat to form the exocuticle and endocuticle. The same Bouligand structure is also characteristic

of collagen networks in compact bone and other fibrous materials (Chen, Lin, McKittrick, &

Meyers, 2008).

Figure 5-10 Transmission electron microscopy image of collagen bundles that formed after

subcutaneous implantation of mineralized crab shell for 28 days. Similar to the image of

(Wilson, Gugssa, Mehl, & Anderson, 2012), it depicts collagen fibers with the characteristic 67

nm banding pattern.

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Figure 5-11 Transmission electron microscopy image Collagenous fibers, collagenous bundles,

and fibroblasts. Fibers oriented in longitudinal directions exhibit the characteristic 67 nm

banding pattern of collagen. Lying between regions of many collagen bundles and fibers,

fibroblasts often present an elongated form. Fibrocytes are essentially mature fibroblasts.

Fibrocytes can easily be recognized as such by their dense nucleus, long and thin cytoplasmic

processes and scarcity of cell organelles (Ebe & Kobayashi, 1972).

The response produced by subcutaneous implantation of demineralized crab shell in the

abdominal region of 28 day old Sprague–Dawley rats was assessed. The implantation time

spanned 28 days. Transmission electron microscopy image, from the harvested sample, figure 5-

10, displays the characteristic 67 nm banding pattern and collagen bundle. Collagenous fibers,

collagenous bundles, and fibroblasts are depicted in figures 5-10, 5-11, and 5-12. The fibers are

orientated in either cross-section or different degrees of tangential or longitudinal sections. In the

case of the longitudinal sections, the fibers exhibit their characteristic banding pattern.

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Figure 5-12 Transmission electron microscopy image depicting collagenous fibers, collagenous

bundles, and fibroblasts. Fibers oriented in longitudinal directions exhibit the characteristic 67

nm banding pattern of collagen. Lying between regions of many collagen bundles and fibers,

fibroblasts often present an elongated form. Fibrocytes are essentially mature fibroblasts.

Fibrocytes can easily be recognized as such by their dense nucleus, long and thin cytoplasmic

processes and scarcity of cell organelles (Ebe & Kobayashi, 1972).

Fibroblast are the most common cells in all forms of fibrous connective tissue and they

become active following tissue injuries. Fibrocytes are essentially mature fibroblast that have

ceased their active protein synthesis (Ebe & Kobayashi, 1972). Fibroblasts are typically irregular

in form and present in wide individual variations. Fibroblasts are the elongated cytoplasmic

173

profiles containing a nucleus and lying between the tissue fibers (Laguens & Dumm, 1969),

(Hammersen, 1976). Fibroblasts are attracted by macrophages.

Macrophage presence represent the hallmark of the inflammation process. A number of

chemical substances are involved in the initiation and control of inflammation. These chemicals

work in concert. Remodeling consists of the deposition of the matrix and its subsequent changes

over time. It occurs throughout the entire wound repair process. One of the characteristics of

wound remodeling is the change of extracellular matrix composition. The regulation of collagen

synthesis is controlled by several growth factors, including TGF-β and fibroblast growth factor

(Li, Chen, & Kirsner, 2007). In cell culture, osteoblasts are nearly indistinguishable from

fibroblasts. All the genes expressed in fibroblasts are also expressed in osteoblasts, and,

conversely, only two osteoblast- specific transcripts have been identified: one encoding Cbfa1, a

transcription factor, and the other encoding Osteocalcin, a secreted molecule that inhibits

osteoblast function. Thus, genetically, the osteoblast can be viewed as a sophisticated fibroblast

(Ducy, Schinke, & Karsenty, 2000).

Fibroblasts, which are nonimmunogenic, easily expandable, and readily available through a

minimally invasive harvesting procedure, have been under investigation as a potential source of

cells for bone tissue engineering applications because native osteoblasts are typically difficult to

isolate and expand in vitro (Hee & Nicoll, 2006). Rutherford et al demonstrated that cultured

strains of gingival or dermal fibroblasts secrete biologically active BMP-7 in vitro, and when

absorbed to porous, preformed sponges and implanted in vivo, induce ectopic and orthotopic

bone formation. Both the implanted donor and host cells participate in bone formation suggesting

174

that the BMP-transduced fibroblasts in contact with host tissue converted to osteoblasts in vivo

(Rutherford, et al., 2002).

A multipotent progenitor cell is postulated to give rise to fibroblasts and osteoblasts.

Hormonal, cytokine, and metabolic cues influence the stromal cell progenitor’s final

differentiation pathway (Wu, et al., 2000). Osteoprogenitor cells can develop into osteoblasts or

fibroblasts depending on various factors. Several members of the fibroblast growth factor (FGF)

family have an important role in the development of skeletal tissues. FGF signaling pathways

also have important roles in bone and contribute to increased osteoblast differentiation (Valta, et

al., 2006).

In this investigation, selected area electron diffraction and high spatial resolution electron

probe X-ray microanalysis have been used to characterize chemical and structural features of

possible calcium phosphate mineral phases. Selected area electron diffraction is among the most

powerful techniques for studying the nano-, atomic- and molecular level structure of pure

organic and inorganic substances, minerals, polymers, mixtures and materials. It can be used

effectively to investigate poorly ordered or disordered materials, such as most biological tissues

and composite materials that resist detailed analysis by conventional chemical and biochemical

techniques (Neary, et al., 2010). Figure 5-13 depicts regions in which selected area diffraction

(SAD) were performed. TEM can provide information to help identify material characteristics in

terms of chemistry and crystallography as well as morphology and size distributions (Bang,

Trillo, & Murr, 2003) of minerals present. Biominerals and biomineralization confer beneficial

attributes on biological forms by: (i) physical, or macrocontributions, the production of skeletal

175

structures that provide integrity and specificity on the organism, and (ii) chemical, or micro- or

sub-microcontributions that provide a personal storage system from which ions are mobilized,

used, or sequestered to assist with the distribution of nutrients that regulate growth. Availability

of stored nutrient ions is crucial to skeletal and tissue repair activities (Skinner & Jahren,

Biomineralization, 2003).

Levy showed in 1984 that bone mineral was a calcium phosphate carbonate, and DeJong,

demonstrated in 1926 that bone mineral gave an Xray diffraction (XRD) pattern similar to that of

poorly crystalline apatite (Glimcher, Bonar, Grynpas, Landis, & Roufosse, 1981). Most

investigators now agree that the principal mineral constituent of mature bone has a structure

closely resembling that of poorly crystalline hydroxyapatite (PCHA). There is also general

agreement that the crystallinity of bone mineral increases with the age of the bone mineral and is

subject to variability and heterogeneity (Glimcher, Bonar, Grynpas, Landis, & Roufosse, 1981).

The apatite family of minerals crystallizes into hexagonal rhombic prisms and has unit cell

dimensions a = 0.9432 nm and c = 0.6881 nm (Park & Lakes, 2007).

In vertebrate hard tissue, poorly crystalline hydroxyapatite-like calcium phosphate mineral

predominates (Neary, et al., 2010). Bone mineral contains approximately 5 percent of carbon

dioxide by weight. Carbon dioxide is therefore a major constituent, and its state in the solid has

an important bearing on the foregoing discussions of the nature of the crystalline lattice. No

study has presented proof that the carbon dioxide in bone occurs as carbonate, although this has

been almost universally assumed (Neuman & Neuman, 1953).

176

Figure 5-13 Micrograph depicting regions of selected area diffraction. This image contains

collagenous fibers, fibroblasts.

TEM using a field emission gun (FEG) and HRTEM was used to study the crystal

morphology and the structure on the atomic level and data was compared to published x-ray

diffraction data and patterns obtained from the American Mineralogist Crystal Structure

Database. Figure 5-13 displays the regions in which SAD was performed. Indexing diffraction

pattern was accomplished by labeling diffraction rings and spots with appropriate (hkl)

usinginterplanar spacing (d-spacing). The diffraction patterns of figure 5-14 display diffuse

177

Figure 5-14 (a) Selected area diffraction (SAD) coincides with 0021 SAD region from figure

13. (b) ) Selected area diffraction (SAD) coincides with 0023 SAD region from figure 13. (c)

Selected area diffraction (SAD) coincides with 0025 SAD region from figure 13. (d) Selected

area diffraction (SAD) coincides with 0028 SAD region from figure 13. All of these SAD images

display broad or diffuse rings which indicate amorphous-like phase.

rings. This is indicative of amorphous solids. Amorphous solids have a random orientation of

atoms (Neary, et al., 2010).

Amorphous calcium phosphate (ACP) is believed to be the initial solid phase that precipitates

from a highly supersaturated calcium phosphate solution, and can convert readily to stable

178

crystalline phases such as octacalcium phosphate or apatitic products. Its morphological form,

structural model and X-ray diffraction patterns are typical for noncrystalline substances with

short-range periodic regularity. In X-ray diffraction, it was shown to have broad and diffuse

peaks. This pattern is typical for substances that lack long range periodic regularity. Amorphous

phases have short-range order that varies between organisms and is presumably under molecular

control (Zhao, Liu, Sun, & Zhang, 2011).

For 0021 SAD, two d spacing values (d121= 0.2933 nm, d222=0.1956 nm) were calculated

from measured diameter. The diffraction pattern of figure 5-14 displays diffuse rings. This is

indicative of amorphous solids. For 0023 SAD, two d spacing values (d121= 0.2933 nm,

d222=0.1956 nm) were calculated from measured diameter. For 0025 SAD, two d spacing values

(d121= 0.2933 nm, d222=0.1956 nm) were calculated from measured diameter. For 0028 SAD,

two d spacing values (d121= 0.2933 nm, d310=0.2200 nm) were calculated from measured

diameter. The values were consistent with those of Carbonate-hydroxylapatite obtained from the

American Mineralogist Crystal Structure Database and found to be consistent (Fleet, Liu, &

King, 2004). The diffraction patterns of figure 5-14 all display broad, diffuse rings. This is

indicative of amorphous solids that have random orientation of particles (Neary, et al., 2010) and

short range order (SRO).

In an effort to locate regions of potential mineral formation, dark sections adjacent to the

collagen bundles were evaluated. Figure 5-15 depicts a region that appears to harbor material

with crystalline characteristics. To assess its crystalline nature, SAD was performed. The SAD

pattern, figure 5-16, displayed diffuse rings. This is typically indicative of amorphous solids.

179

Figure 5-15 (a) Transmission electron microscopy image of dark region adjacent to

collagenase matrix and (b) its SAD pattern which shows broad diffuse rings that are indicative

of a amorphous solid.

180

Figure 5-16 SAD pattern of dark region adjacent to collagen like matrix depicted in figure 5-15

which shows broad diffuse rings that are indicative of a amorphous solid.

However, nanosize particles can contribute to misleading SAD results. The particles are

definitely in the nanosized range. Is it amorphous, too?

181

Size of the crystalline domains can lead to peak broadening (Weidenthaler, 2011). At first

glance, the sample appears to be amorphous. High resolution-transmission electron microscopy

(HRTEM) was used to provide a complementary characterization technique and a more accurate

picture. The HRTEM image, figure 5-17, disclosed lattice spacing with long range order despite

the fact the reflections observed in selected area electron diffraction pattern exhibited broad

peaks or diffuse rings (figure 5-16). The broadening of reflections and weakening of the

scattered intensity was probably due to the size effect of the nanocrystals. The HRTEM studies

confirm the crystalline character of the nanoparticles depicting long range order. Amorphous or

non-crystalline solids possess short range or local order (Termine & Posner, 1967). Suvorova

and Buffat demonstrated that the XRD method has limitations in the phase analysis of small

particles. Even selected area electron diffraction patterns taken from relatively large samples can

be interpreted erroneously due to size effects (Suvorova & Buffat, 2001).

When diffraction patterns give broad and diffuse rings, it is assessed as amorphous. However,

it remains to be seen whether the material is amorphous or nanocrystalline. The use of electron

probes of nanometer-size scale unleashes the possibility to determine the extent of crystallinity

of nanoparticles when the incident beam focuses and illuminates only a small and thin area of the

specimen (Suvorova & Buffat, 1999). When only this area contributes to the diffraction pattern,

electron nanodiffraction (END) is possible (Cowley, 2004). Figure 5-17 depicts HRTEM of

potential carbonated HAP exhibiting long range order.

Diffraction studies on the detailed structure of biological apatite have been limited by its very

small crystal size, and most of our present understanding is inferred from studies on synthetic

182

material (Fleet & Liu, 2007). Carbonate substitution can cause crystal disorder, resulting in

decrease of apatite crystallinity that is one of the reasons why biological apatites are poorly

crystallized (Wang, Zuo, Huang, Hou, & Li, 2010). Combining the information from the SAD

and HRTEM methods, it was determined that the sample was crystalline with long-range order.

Digitally processing the HRTEM micrographs would facilitate better interpretation of image

contrast.

Particle Size

Bone consists of homogeneous plate-like crystals of biological apatite of 15 – 30 nm wide and

30 – 50 nm long (Dorozhkin S. , 2012). Researchers have investigated calcium phosphate, from

precipitation experiments, with HRTEM (Rangavittal, Landa-Canovas, Gonzalez-Calbet, &

Vallet-Regı, 2000), (Suvorova & Buffat, 2001), (Li, et al., 2007), (Neira, et al., 2009),

(Nudelman, et al., 2010), (Ospina, et al., 2012). However, Biggemann et al determined that

nanosized particles of different size preserve stoichiometric HA-like crystal. In their study, they

synthesized HAP by precipitation and then observed with HRTEM and classified particles based

on size: large (between approximately 25 and 150 nm), medium (between approximately 5 and

20 nm), and small (less than 2.5 nm). The small crystals had a rectangular domain which were

approximately two times the HA unit cell parameters. The particles were observed within

amorphous regions of their sample (Biggemann, Prado da Silva, Rossi, & Ramirez, 2008). The

particles of Biggemann, figure 5-18, represent the smallest synthetic nanostructured HAP

crystals, between 2.1 and 2.3 nm, observed with HRTEM coupled with focal-series restoration. It

183

appears that our study is the first of its kind to find biologically produced HAP crystals ~2 nm in

size while investigating a harvested sample for osteogenesis.

Nanodiffraction

X-ray crystallography is a technique for determining the molecular and atomic structure crystals.

Crystalline materials cause X-ray beams to diffract in many directions (Smyth & Martin, 2000).

X-rays are diffracted in crystals when the Bragg’s law (nλ = 2d sin θ ) conditions

are satisfied (Kasai & Kakudo, 2005). Electron diffraction has become a vital part of

transmission electron microscopy. Diffraction patterns obtained in the TEM can provides

information about crystallinity, lattice parameters, symmetry, grain morphology, orientation, and

amount of phases present. Crystal structure is a prominent characteristic in determining and

controlling properties of a material (Williams & Carter, 2009).

The physical structure of solid materials can be classified as amorphous or crystalline and

depends mainly on the arrangements of the atoms, ions, or molecules. A crystal structure has

atoms or ions arranged in a pattern that repeats itself in three dimensions and it is then referred as

a crystalline solid or crystalline material. Atomic arrangements in crystalline solids can be

described by a three dimensional lattice (the unit cell) with three vectors (a, b, c) originating

from one corner of the cell. The axial lengths a, b, and c and the inter-axial angles α, β, and γ are

the lattice constants of the unit cell. Unit cells of different types can be constructed by assigning

184

Figure 5-17 HRTEM image of potential Carbonated HAP depicting the short range or local

order.

185

Figure 5-18 HRTEM image of small-size particle. Determined that nanosized particles of

different size preserve stoichiometric HA-like crystal structure (Biggemann, Prado da Silva,

Rossi, & Ramirez, 2008).

specific values for axial lengths and inter-axial angles. The Bravais lattices consist of seven

crystal systems: cubic, tetragonal, orthorhombic, rhombohedral, hexagonal, monoclinic, and

triclinic. These seven crystal systems have a total of 14 space lattices (unit cells) based on the

internal arrangements of atomic sites within the unit cells. These arrangements can be describes

as simple, body-centered, or face-centered (Kasai & Kakudo, 2005).

A family of crystallographic planes is: (1) are parallel to each other and equally spaced; (2) is

defined as a set of planes that intersect all lattice points; and (3) fully described using three

integer indices h, k, and l, which are called crystallographic or Miller indices. Miller indices

indicate that the planes belonging to the family (hkl) divide lattice vectors (unit cell edges) a, b,

and c into h, k, and l equal parts, respectively. The distance between neighboring crystallographic

186

planes is called the interplanar distance or d-spacing which is dependent on the Miller indices of

a family and the lengths of the three-unit cell edges (Pecharsky & Zavalij, 2009).

There are several different electron diffraction techniques. Techniques associated with the

TEM, such as selected area diffraction (SAD or SEAD) and convergent beam electron diffraction

(CBED), are the focus of this discussion. CBED developed by Kossel and .Möllenstedt, is the

oldest technique used in TEM. Although SAD is the classic way of relating diffraction contrast

information and provides useful information about crystal, ~0.5 µm is the area of the smallest

selectable diameter. CBED overcomes this obstacle and easily penetrates the nanotechnology

domain (< 100 nm). Over the past 40 years, several micro and naodiffraction have been

developed to overcome the spatial resolution limitations of SAD in a TEM (Williams & Carter,

2009). Electron nanodiffraction (END) (Cowley, 2004), a special form of convergent beam

electron diffraction (CBED), involves obtaining diffraction patterns with a nanometer-scale

beam. Nanometer beams are nosiy due to the picoamp current and require a field-emission gun

(FEG) or a cold FEG source to provide a coherent beam. END affords the opportunity to analyze

individual nanoparticles (Williams & Carter, 2009).

Nanodiffraction or END was pioneered by Cowley (1981), (1984), (1996). The technique of

electron micro- and nanodiffraction was extended to the study of near-amorphous materials for

the study of particles having diameters in the 0.10-0.20 nm range. In a preliminary survey, it was

assumed that atom positions were representative of the state of order of the whole sample

(Monosmith & Cowley, 1983). It was demonstrated that as an electron beam with a diameter of

0.4-0.5 nm is moved within a projected unit cell of a crystal changes can be observed in the

187

nanodiffraction pattern (Cowley, 1981). END has been applied most extensively in the

investigation of metal nanoparticles (Cowley, Janney, Gerkin, & Buseck, 2000), (Quintana,

Cowley, & Marhic, 2004) and carbon nanotubes (Qin, et al., 1997), (Cowley & Sundell, 1997).

Nanodiffraction was employed in the same region where the nanocrystals occurred that

produce the diffuse ring pattern and the result is shown in figure 5-19. This establishes that there

are small particles single crystals with no obvious defects in this sample. Most nanodiffraction

work has been concerned with finding the crystal structure, crystal defects, and sometimes

crystal shapes for nanoparticles in the size range of 1–2 nm. In each case, the regular pattern of

widely spaced strong spots comes from a HAP crystallite. The more closely spaced patterns of

weaker spots come from the HAP. The results from many nanodiffraction patterns from

randomly selected areas may be suggestive, and a great amount of statistical information would

be required for significant results. However, in this case the presence of a crystal lattice can be

used as a reference point for orientations (Cowley, 1999).

The (000), (002), (112), and (222) diffraction spots are indexed and displayed in this

nanodiffraction pattern in figure 5-19. The model proposed here of the hexagonal lattice, as an

explanation of the experimentally obtained diffraction pattern dataset.

The chronicle of ACPs began in 1955 with Robinson and Watson (Robinson & Watson,

1955) suggesting that a considerable portion of newly formed mineral in young bone was not

crystalline but instead similar to an amorphous-like precipitate. By 1960s, Posner and

collaborators (Eanes, Gillessen, & Posner, 1965), (Termine & Posner, 1967) used the data

obtained from x-ray diffraction and electron microscopy to point to the fact that bone mineral is a

188

mixture of amorphous calcium phosphate and crystalline apatite. Futhermore, Poser and Betts

proposed a possible structure for ACP, a Ca9(PO4)6 cluster known as Posner’s cluster, that is

present in crystalline HAP (Betts & Posner, 1974), (Posner & Betts, 1975).

Presently, biomineralization processes considers that every biogenic mineral has a precursor

phase that can be amorphous and/or composed of a different mineral phase. In the case bone

Figure 5-19 HRTEM nanodiffraction image of potential Carbonated HAP depicting a single

crystal.

189

apatite, it has been found that minute round particles of ∼1–2 nm in diameter are present in

conjugation with type I collagen molecule and aggregated small round particles are present in

direct association with early nanocrystal formation. A significant feature observed was that

particle crystallinity was extremely variable. Some particles were crystalline over their entire

extent, while others presented crystalline and amorphous zones of different sizes (Ospina, et al.,

2012). Combining the information from the SAD, HRTEM, and nanodiffraction methods and

supplying a more comprehensive assessment, it was determined that the sample was crystalline

with long-range order.

While hydroxyapatite forms needle-like crystals, the amorphous calcium phosphate phase is

in the form of spherical grains of diameter ~ 300 – 1000 Å. Posner’s clusters are roughly

spherical with a diameter of 9.5 Å and is speculated to be the transient precursor to ACP

formation. This Ca9(PO4)6 cluster allows the ACP to dissociate into these clusters instead of

undergoing complete ionic solvation in ACP to HAP transformation (Yin & Stott, 2003).

Indexing the nanodiffraction pattern

There are conventions for labeling, or indexing, diffraction patterns using the numbers (hkl).

One approach is termed “fingerprinting.” The idea is to match d spacings in the observed

diffraction pattern to known patterns from a standard sample, from calculation, or from a

database. The International Center for Diffraction Data, ICDD, (Fultz, 2013); the Joint

Committee for Powder Diffraction Standards, JCPD (Kasai & Kakudo, 2005); and the American

Mineralogist Crystal Structure Database are example of databases that maintain diffraction

190

patterns from hundreds of thousands of inorganic and organic materials. For each material the

data fields include the observed interplanar spacings for all observed diffraction peaks, their

relative intensities, and their hkl indexing. Software packages are also available to identify peaks

or d spacings in the experimental diffraction pattern, and then search databases to find candidate

materials. The task of indexing a diffraction pattern can be helped with information about

chemical compositions and candidate crystal structures (Fultz, 2013). Thus, diffraction patterns

can be indexed using graphical, mathematical, and analytical techniques as well as databases and

computer software or a combination of these techniques. Sometimes diffraction patterns do not

have all the peaks that they should because the sample specimen could be textured or particle

size could be too small. It should be noted that electron patterns can easily be obtained from very

minute volumes of calcium phosphate solid phases containing only a few solid phase particles

(Landis & Glimcher, 1978).

Mathematical Method for Hexagonal

Mathematical methods require some knowledge of the crystal structure and resulting lattice

parameter ratios such as c/a. This information can be determined graphically using a method

developed by Hull and Davey (Hull & Davey, 1921), (Schneer, 1957). This method allows the

determination of structure even if lattice parameters are unknown.

A diffraction pattern from a material typically contains many distinct peaks, each

corresponding to a different interplanar spacing, d. For hexagonal crystals with lattice parameter

a, the interplanar spacing, d, is denoted by:

191

1

𝑑2=

4

3(

ℎ2 + ℎ𝑘 + 𝑘2

𝑎2) +

𝑙2

𝑐2

Equation 1

Combine with Bragg’s law ( = 2dsin)

1

𝑑2=

4

3(

ℎ2 + ℎ𝑘 + 𝑘2

𝑎2) +

𝑙2

𝑐2=

4 𝑠𝑖𝑛2 𝜃

2

Equation 2

Rewrite as

𝑠𝑖𝑛2𝜃 =2

4[4

3(

ℎ2 + ℎ𝑘 + 𝑘2

𝑎2) +

𝑙2

𝑐2]

Equation 3

Or

𝑠𝑖𝑛2𝜃 =2

4𝑎2[4

3(ℎ2 + ℎ𝑘 + 𝑘2) +

𝑙2

(𝑐𝑎⁄ )2

]

Equation 4

192

The lattice parameter a and the ratio of lattice parameters c/a are constant for a given diffraction

pattern. These parameters are highlighted in red.

𝑠𝑖𝑛2𝜃 =2

4𝑎2[4

3(ℎ2 + ℎ𝑘 + 𝑘2) +

𝑙2

(𝑐𝑎⁄ )2

]

Equation 5

=2

4𝑎2[𝐴] +

2

4𝑎2[𝐵]

The pattern can now be indexed by considering the parameters [A] and [B] individually.

4

3(ℎ2 + ℎ𝑘 + 𝑘2) ……………………………………………………[A]

and

𝑙2

(𝑐𝑎⁄ )2…………………………………………………………………[B]

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Table 5-2 Term [A] calculation for various values of hk

k

0 1 2 3

h

0 0.000 1.333 5.333 12.000

1 1.333 4.000 9.333 17.333

2 5.333 9.333 16.000 25.333

3 12.000 17.333 253.333 36.000

Starting with [A], this term only depends on the indices h and k. This value can be calculated by

modulating h and k.

Term [A] is calculated for various values of hk, Table 5-2.

Term [B] can be determined by substituting the known value of c/a ratio. This ratio can be

determined graphically by Hull-Davey method discussed earlier; mathematically; or can be an

estimate based on other experimental data. Based on other experimental data such as TEM

micrographs, Ca/P ratios, it is believed that our diffraction pattern depicts a member of the

hydroxyapatite family. For illustrative purposes, we will use c/a value of 0.728 for carbonate-

hydroxyapatite in the table below:

Term [B] calculated for carbonate-hydroxyapatite (c/a = 0.728).

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Table 5-3 Calculation of l2 and l2/(c/a)2

l l2 l2/(c/a)2

0 0 0.000

1 1 1.885

2 4 7.540

3 9 16.965

4 16 30.160

5 25 47.126

6 36 67.861

Next, the two terms [A] and [B] are permitted by the allowed hkl values are added and ranked

din increasing order. Due to symmetry relationships, h and k are interchangeable. For a

hexagonal system, the structure factor calculation follows the rule: If h + 2k = 3n and l is odd,

then there is no XRD peak. Therefore, remove forbidden peaks and eliminate equivalent ones.

Table 5-4 Evaluation of Peaks.

Indices (hkl) l h + 2k Peak

111 ODD 3 NO

105 ODD 1 3n YES

Etc….

195

The calculated values in the table represent a partial list of the allowed (hkl) reflections and the

interplanar spacing.

Table 5-5 Allowed hkl and calculated d spacings

Allowed hkl Calculated d spacing (nm)

100 0.81811

101 0.52658

110 0.47234

200 0.40905

111 0.38941

201 0.35161

002 0.34403

102 0.31713

120 0.30921

211 0.28205

121 0.28205

112 0.27809

196

The parameters a and c can be calculated from the interplanar spacing equation. The

parameter a can be calculated where l = 0 (i.e., hk0) and similarly the parameter c can be

determined where h=k=0 (i.e., 00l) (Suryanarayana & Norton, 1998). These steps can be used to

generate calculated d spacings for both powder diffraction and nanodiffraction patterns.

Assignments can be made for observed d spacing values for powder diffraction patterns. For

nanodiffraction patterns, indexing begins with the identifying the brightest spot in the center of

the diffraction pattern. This represents the transmitted beam, or the (000) diffraction. Next two

independent diffraction spots nearest to the (000) spot require indexing. Once these

two vectors are determined, linear combinations of them can be used to obtain the positions and

indices of all the other diffraction spots. Much of the work in indexing nanodiffraction patterns

include measuring angles and distances between diffraction spots and then comparing these

measurements to geometrical calculations of angles and distances. The structure rule applies and

certain diffraction spot must be eliminated (Fultz, 2013). Computer programs are useful to help

elucidate the combinations of interplanary spacings.

In nanodiffraction, there are different approaches to indexing patterns. All carry frustrations.

One approach is referred to as “by hand.” This involves guessing the zone axis and its diffraction

pattern. This method is usually perform on diffraction patterns with obvious symmetry, such as a

square or hexagonal array of spots for a cubic crystal. The camera equation, r d = λ L, can be

used to obtain the interplanar distance, d, if the camera constant is known. Once the diffraction

pattern has been identified, all linear combinations of the reciprocal lattice vectors must give

197

Figure 5-20 TEM image of possible polycrystalline HAP with interplanar or lattice spacings

indices of all other spots in the diffraction pattern and the zone axis should be consistent with a

right-handed coordinate system (Fultz, 2013).

Another approach involves indexing the spots first and the zone axis can be obtained at the

end of the procedure. Ratios from the allowed hkl are sought and listed. These ratios are equal to

the ratios of the spot separations. Search for diffractions whose spot spacings are in the ratio of

the measured distances. Choose specific vector families that provide the correct angles in the

Interplanar spacing = 0.95 nm

198

Figure 5-21 SAD pattern of a polycrystalline HAP

diffraction pattern. Computer programs can provide ratio data and simplify the task of indexing.

Fultz presents a more detailed explanation (Fultz, 2013).

To index our nanofiffraction pattern, yet another approach was used. In this case, the scale bar

was used to measure and calculate interplanar spacings. This approach involved measuring

angles and distances between diffraction spots, followed by, matching the observed interspacing

with published data. The d spacings observed were: d002 = 0.3451 nm; d112 = 0.2711 nm; and d222

199

= 0.1921 nm. The camera equation, r d = λ L could be used to determine d when the camera

length and λ are known. Another method that could have been utilized involve using ratios of r’s.

The selection of a basic parallelogram can be made and distance between the diffraction spots, r,

measured. A list of all the allowed reflections along their relative spacing can be generated. The

ratios can be compared to find the appropriate hkl. After indexing a couple diffraction spots, the

diffraction pattern can be completely labeled by vector addition. Computer program can help in

various ways to ease the tasks.

SAD was taken in another region in close proximity to the nanocrystals. Characteristic

interplanar or lattice spacing were displayed, figure 5-20. The crystallites are plate shaped nano -

sized particles with lengths and widths of approximately 40 x 20 nm. The measures lattice

spacing was 0.95 nm. This is representative of resolution of the 100 planes of the hydroxyapatite

crystal lattice. This is in line with previous studies (Selvig, 1970), (Suvorova & Buffat, 2001).

The selected area diffraction is more distinct, indicating either a higher degree of crystallinity,

an increase in particle size, or both. Crystalline solids are arranged in fixed geometric patterns or

lattices. They have an ordered arrangement of units maximizing the space they occupy, and are

practically incompressible. (Neary, et al., 2010). Bone mineral has been shown to contain both

an amorphous and a crystalline solid (Termine & Posner, 1967), (Eanes, Termine, & Posner,

1967).

The diffraction pattern of figure 5-21 displays speckled rings made up of discrete spots. This

is indicative of crystalline solids with a continuum of states from nanocrystalline to poly-

crystalline. This suggest that there are nanosized crystals of different sizes represented in this

200

SAD pattern. In essence the smaller HAP nanocrytsals aggregated to form larger HAP crystals.

This implies that the process of HAP formation in this case is based on the mesocrystal model.

This nanocrystal mediated crystallization involves nucleation events of nanocrystals which

engage self-organization to form an ordered HAP agglomerated crystal superstructure. In

classical models, crystalline materials were classified into single crystal and polycrystal, but

mesocrystals introduce a concept of intermediate states --- between single crystal and polycrystal

(Oaki & Imai, 2013). Nanocrystals 20-50 nm in size were observed. The nanocrystal specimen

can be classified in accordance with crystal sizes. The crystals of ~ 2nm gave two diffuse rings in

SAD patterns which look like ‘amorphous’ patterns but produced a nanodiffraction pattern that

indicated a single crystal. The larger nanocrystals with width ~ 20 nm gave spotted SAD

patterns. Nonetheless, all the nanocrystals were determined to be crystalline whatever the particle

size (Suvorova & Buffat, 1999). The nanodiffraction technique indicate that the nanocrystals can

be recognized as a single crystal and are oriented in the same crystallographic directions. The

nanocrystals and biological macromolecules can be regarded as the nanoscale bricks and mortar,

respectively (Oaki & Imai, 2013). This brick and mortar building describes the behavior of the

inorganic nano-sized crystals in the mesocrystal model. The nanocrystals aggregate into ordered

solid phases via oriented attachment to control the reactivity of nanophase materials in nature

(Dorozhkin S. , 2012). These series of SAD, HRTEM, and nanodiffraction results provide

evidence of growth of our HAP crystal via the mesocrystal model.

For SAD, numerous d spacings were calculated from measured diameter shown in Table 5-6.

The values were more consistent with those of Carbonate-hydroxylapatite obtained from the

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Table 5-6 d spacing values calculated for crystalline harvested sample. a (Fleet, Liu, & King,

2004). b (Wilson, Elliot, & Dowker, 1999)

Carbonate

Hydroxyapatitea

Hydroxyapatiteb Harvested Specimen

hkl dnm dnm dnm

200 0.40906 0.40738 0.40714

111 0.38941 0.38847 0.38000

002 0.34403 0.34444 0.33529

102 0.31713 0.31725 0.31667

211 0.28205 0.28114 0.28500

300 0.27271 0.27159 0.27143

202 0.26329 0.26302 0.26430

301 0.25352 0.25266 0.25333

130 0.22690 0.22598 0.22800

131 0.21549 0.21472 0.21923

222 0.19471 0.19424 0.19000

213 0.18421 0.18408 0.18387

004 0.17201 0.17222 0.17273

151 0.14370 0.14200

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American Mineralogist Crystal Structure Database and found to be consistent (Fleet, Liu, &

King, 2004).

A simple two-phase system in which one phase gradually changes to, or is replaced by,

another can explain the diffuse and poorly defined selected area diffraction pattern that is

obtained from bone mineral (Glimcher, Bonar, Grynpas, Landis, & Roufosse, 1981).

The change from a poorly crystalline hydroxyapatite diffraction pattern to a more highly

crystalline one does not occur abruptly and varies only slightly at different locations within a

single section (Landis & Glimcher, Electron Diffraction and Electron Probe Microanalysis of the

Mineral Phase of Bone Tissue Prepared by Anhydrous Techniques, 1978).

A semiquantitative nondispersive electron probe x-ray microanalysis was used to examine the

region of the sample that displayed lattice spacing of 0.95 nm. This revealed an average

calcium:phosphorus (Ca:P) ratio of 1.81 ± 0.37, figures 5-22 and 5-23. Bone-apatite is

characterized by calcium, phosphate and hydroxyl deficiency and Ca:P ratios reported between

1.37–1.87 (Hing, 2004). While many investigators of calcification agree that mineralization

originates within matrix vesicles (Anderson, 1984), there is some disagreement about the exact

mechanisms of the process. It seems likely that both cellular and physiochemical factors are

involved. Generally amorphous calcium phosphates with a Ca:P ratio varying between 1.44 and

1.55 are believed to be deposited under the control of osteoblasts (Hing, 2004).

In a study conducted on human rib bones, the median reference value for the Ca/P was 2.19.

The individual variation for the Ca/P ratio in rib bone from healthy humans was lower than for

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Element Weight% Atomic%

O K 47.43 66.88

P K 21.31 15.52

Ca K 31.26 17.60

Totals 100.00

Figure 5-22 Elemental Analysis with Weight and Atomic Percentages of oxygen, phosphorus

and calcium.

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Element Weight% Atomic%

O K 37.88 58.28

P K 19.75 15.70

Ca K 42.37 26.02

Totals 100.00

Figure 5-23 Elemental Analysis with Weight and Atomic Percentages of oxygen, phosphorus and

calcium.

Ca and P values taken separately; therefore, Ca/P ratio provides greater reliability (Tzaphlidou &

Zaichick, 2003).

This preliminary investigation has demonstrated the potential of characterizing crystals in

the nanometer scale, and identifying the components via combined analysis of TEM, HRTEM,

SAD, and EDS. With diffraction pattern of broad and diffuse rings and speckled rings made up

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of discrete spots, and d spacing values, and EDS findings, this sample taken from the

implantation of blue claw crab in rats is surmised to contain mineralized collagen matrix. This

sample has regions with a variety of crystalline states or variations in crystallinity. The crystal

structure of apatite allows the incorporation of a wide variety of cations and ions beyond the

essential calcium and phosphate needed for mineral and apatitic bone formation (Skinner &

Jahren, 2003).

Amorphous calcium phosphate was first described by Aaron S. Posner (Posner, Perloff, &

Diorio, 1958). Leng obtained a similar pattern from a HA single crystal in transmission electron

microscopy (TEM) when examining a sample for heterotopic osteogenesis of calcium phosphate

(Leng & Qu, 2002). Posner’s structural studies led to the highly publicized debate over the

nature of the carbonate found in bone and ignited the story of amorphous calcium phosphate.

Termine and Eanes also identified amorphous calcium phosphate (Eanes, Gillessen, & Posner,

1965). Posner and Termine quantified how much ACP was in bone and also determined how

ACP could be stabilized (Termine & S, 1967). Blumenthal, Betts and Lehninger identified ACP

as the mineral in the hepatopancreas of the blue crab (Betts, Blumenthal, Posner, Becker, &

Lehninger, 1975). Boskey studied the mechanism of hard tissue and became convinced that ACP

was not a mandatory precursor to apatite (Boskey A. L., 1997). Lowenstam asserted that in many

non-vertebrates, the initial mineral (whether phosphate or carbonate) is amorphous (Lowenstam

H. A., 1981), and that these amorphous minerals transform to crystalline phases as the animals

mature (Lowenstam & Weiner, 1989).

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Figure 5-24 Schematic diagrams representing the HAP unit cell (Menéndez-Proupin, et al.,

2011)

Arzate et. al. studied the cementoblastoma cell line by transmission electron, high resolution,

scanning, and atomic force microscopy. Cemtoblasts are thought to give rise to progenitors of

each cell type that comprise the periodontium, such as osteoblasts and periodontal ligament

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fibroblasts. This study revealed that the diffraction patterns obtained from the intracellular

material and human cellular cementum were similar, with D-spacings of 3.36 and 2.8, consistent

with those of hydroxyapatite (3.440 and 2.814). To reveal the formed mineral phase, selected

area diffraction and nanodiffraction techniques were used and because of the nanosize of the

formed crystals, patterns of concentric double rings emerged (Arzate, et al., 2000). Another study

by Leng and Qu examined a single crystal of hydroxyapatite from an implanted porous

hydroxyapatite/tricalcium phosphate from dorsal pouches of dogs after 2-months. The diffraction

pattern lead them to identify the particle as an HA single crystal after comparing the diffraction

pattern with the HA lattice structure (Leng & Qu, 2002). To date, this research is the first to

describe the use of nanodiffraction to confirm mineral hydroxyapatite formation from an implant

of crab shell in a Wistar rat. The goal of this paper is not to give a detailed review of the growth

of HAP mineral crystal or the biomineralization process. This is a report of our investigation

with HRTEM and electron nanodiffraction of hydroxyapatite formation from an implant of crab

shell in a Wistar rat.

Mineralization of a tissue refers to the deposition of calcium phosphate mineral phase. This

mineral, often called ‘apatite-like, closely resembles bone mineral in composition and structure

(Ito & Onuma, 2003). The chemical equation for the formation of hydroxyapatite is 10 Ca2+ + 6

PO43- + 2 OH Ca10(PO4)6(OH)2. Stoichiometrically this reaction could be simplified by

dividing by two, it is written to emphasize that Ca10(PO4)6(OH)2 is the formula for a single unit

cell of HAP (Simmer & Fincham, 1995). Figure 5-24 depicts different ways of representing the

HAP unit cell. The primary difference between bone mineral and hydroxyapatite manifest in

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crystallinity and impurity content (Ito & Onuma, 2003). Perfect and pure Hap crystals are

extremely difficult to form in both synthetic processes as well as in vivo in biological systems.

Biological HAP contains a variety of vacancies, interstitials, and trace elements (Matsunaga,

2010) and is subjected to common ion substitutions, PO43- by CO3

2- and OH- by CO32-. These

two are the most common ion substitutions and are called B-type and A-type, respectively. B-

type substitution, the preferential carbonate substitution found in the bone of a variety of species,

is believed to improve bioactivity and greater strength (Chappell & Bristowe, 2007), (Ibrahim,

Mostafa, & Korowash, 2011). B-type carbonate substitution, where carbonate substitutes for the

phosphate ion, is of particular importance because biological apatites contain 4–6% carbonate by

weight and the connection between carbonate concentration and physical properties (Leventouri,

2006). Thus, biological apatites are often described as a calcium deficient HAP modified with

different ion substitutions (Mostafa & Brown, 2007). Hydroxyapatite is among the most stable

calcium phosphate solid phase and can precipitate from a solution having a relatively low

concentration of calcium and phosphate ions (Simmer & Fincham, 1995).

Bone apatite crystals are plate-like shaped with average length, width and thickness of

50×25×4 nm, although a great range of values can be found in the literature (Rossi, et al., 2012).

The Hap crystal structure is comprised of two inequivalent Ca2+ sites: (1) Ca cations at site I

forms triangles coordinated by nine oxygens belonging to six PO43-

that displays a columnar

arrangement; (2) Ca cations at site II are coordinated by six oxygen atoms belonging to five

PO43- anions and one OH- anion (Li, et al., 2007), figures 5-24, 5-25, and 5-26. Substitutions can

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result in changes in the crystal lattice dimension, with a decrease in the a-axis and an increase in

the c-axis (Hench & Best, 2013).

Having similar general atomic arrangements, HAp has two kinds of polymorphism, a more

complex monoclinic (P21/b), and hexagonal (P63/m). The hexagonal form is encountered most

frequently and is involved in bone formation because it allows for much easier exchange of OH-

groups with other anions. However, the monoclinic form is more stable, from the thermodynamic

prospective (Corno, Busco, Civalleri, & Ugliengo, 2006). The hexagonal primitive unit cell of

HAP has a total 44 atoms and the lattice parameters of a=b=0.94081 nm, c=0.68887 nm, with a =

β = 90 and γ = 120 (de Leeuw, 2010). To form the OH group along the c axis, there are two

inequivalent Ca sites (Ca-1 and Ca-2), one P site, four different oxygen sites (O-1 to O-4), and

one H site bonded to O-4. This structure has four possible sites for the OH groups along the c

axis and may undergo disorder in the column (Matsunaga, 2010). The monoclinic unit cell is

obtained from the hexagonal one by doubling the b lattice parameter with an alternating

arrangement of the anion chain (Calderin & Stott, 2003). Singh and Jonnalagadda studied metal

substituted hydoxy apatites’ ability to maintain their structure and temperature at temperatures

reaching 900°C. Using Brunauer–Emmett–Teller (BET), they determined that HAP had a surface

area of 72.5 m2/g and a pore volume of 0.2385 m2/g. Additionally, the degree of crystallinity, Xc,

was calculated by Scherer’s equation and was determined to be 0.795 (Singh & Jonnalagadda,

2013).

Harries, Hukins and Hasnain (1986) analyzed a spectrum recorded above the calcium edge of

hydroxyapatite using Extended X-ray Absorption Fine Structure (EXAFS) to gain structural

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information on the local atomic environment surrounding the calcium ion out to a distance of 0.6

nm. EXAFS spectra have been used to provide quantitative information on relatively simple

crystal structures, distances between atoms, and the near neighbors of absorbing atoms in more

complex systems. Crystallographic data was essential to interpret the EXAFS of hydroxyapatite

providing the initial EXAFS parameters used in simulating the spectrum. In addition, phase

shifts calculated using the ab initio method and local atomic potentials were constructed using

the muffin-tin approximation, choosing a local atomic arrangement consistent with the known

Figure 5-25 Schematic depiction of one hexagonal building unit of HAP (Li, et al., 2007).

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Figure 5-26 Black lines connect Ca(I) columns in hexagonal networks. Cyan and magenta

triangles connect staggered Ca(II) atoms (Xia, Lindahl, Lausmaa, & Engqvist, 2011), (Boanini,

Gazzano, & A, 2010).

Figure 5-27 The HAP unit cell has symmetry and order. Table of average distances of atom shell

from calcium atom via x-ray and EXAFS (Harries, Huskins, & Hasnain, 1986).

Hydroxyapatite

(HAP)

Chemical Formula Ca10(PO4)6(OH)2

Ideal Ca/P ratio 10/6

Density 3.2 g/cm3

Atomic Mass 1004.6 g/mol

Mass of unit cell ~1.67 x 10-21 g

Volume of unit cell ~5.21 x 10-22 cm3

Atom Type Averaged

Coordination

Number

Average

distance of atom

shell from

calcium atom

(nm) (x-ray)

Average

distance of atom

shell from

calcium atom

(nm) (EXAFS)

Oxygen 5.4 0.239 0.237

Oxygen 1.0 0.251 0.256

Oxygen 2.0 0.279 ---

Phosphorus 2.4 0.320 0.311

Calcium 0.8 0.344 0.346

Phosphorus 3.0 0.360 ---

Oxygen 6.6 0.399 ---

Calcium 8.4 0.405 0.395

Oxygen 4.8 0.445 0.445

Oxygen 6.5 0.481 0.469

Calcium 1.5 0.545 ---

Calcium 2.0 0.584 0.574

Calcium 6.0 0.618 0.622

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crystal structure of hydroxyapatite. The inter-atomic distance and the Debye-Waller terms were

refined to obtain the best agreement with experimental data. Their analysis of the average

calcium environment for hydroxyapatite as determined by their x-ray crystallography and

EXAFS is provided in figure 5-27. With image processing to reveal a clearer picture, the

distances between the atoms in figure 5-17 can be compared with the data from figure 5-27.

Possible mechanism

Bone formation induced by osteoinductive biomaterials occur in several steps: (1) attachment

of mesenchymal cells on material surface; (2) proliferation and differentiation of mesenchymal

cells; (3) bone matrix formation by induced osteogenic cells; (4) mineralization of bone matrix;

an (5) bone remodeling to form mature bone. The time for different steps to happen varies with

the osteoinductive potentials of the biomaterial. Within two weeks of implantation, cells

typically attach to biomaterial; within three weeks, cells proliferate and differentiate; within 30

days, bone matrix form; within 45 days, mineralized bone forms; and within 60 days,

remodeling. The processes of osteoinduction will never happen if a non-osteoinductive

biomaterial is implanted in tissue (Reis & Weiner, 2004).

Similar steps are always followed for bone formation induced by different biomaterials

despite the varying starting times. In skeletal development, bone naturally occurs either by direct

replacement of pre-existing connective tissues (intramembranous ossification) or by replacement

of pre-existing cartilage (endochondral ossification). Cartilage formation has not been found in

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Figure 5-28 Diagram for wound healing response to crushed crab carapace (Falanga, 2005),

(Salgado, et al., 2011).

Differentiation

Chemotaxis,

Fibroblastic to

Osteoblast phenotype

Resolution Osteogenic cells,

Collagen matrix release,

Mineralization

Monocytes, Neutrophils

migrate to implant site

after injury

Monocytes become

macrophages and attach to

crab carapace

Fibroblastic cells of the

osteoprogenitor cell line

migrate to site

Signal for phenotype

switch: fibroblast to

osteoblast phenotype

Cells differentiate to

Osteogenic-like cells

Cytokine and other

metabolic cues influence

cells pathways

Osteogenic cells release

collagen matrix and begin

to mineralize.

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bone formation induced by osteoinductive biomaterials; therefore it is supposed that bone

formation induced by osteoinductive materials resembles the intramembranous ossification

development. Starting directly as bone, secondary bone formation occurs when bone formation

takes place at the resorbed bone bed during remodeling. In secondary bone formation, osteogenic

precursor cells attach on the resorbed bone bed, proliferate, differentiate, and form new bone.

Bone formation induced by osteoinductive biomaterials has sequences similar to secondary

bone formation. When bone formation induced by biomaterials begins, osteogenic precursor

cells (mesenchymal cells) attach on osteoinductive biomaterials surface, proliferate and

differentiate to osteogenic cells which then form bone. Therefore, it is more likely that bone

formation induced by osteoinductive biomaterials is a secondary bone formation. Bone repair

happens faster with implants with a high osteoinductive potential. Osteoinductive biomaterials

provide a good environment for osteogenic cells to form bone and induce non-osteogenic cells to

differentiate into osteogenic cells that then form bone (Reis & Weiner, 2004).

In the case of Calinectes Sapides (Blue Claw) crab shell, we postulate that monocytes, closely

following the neutrophils, migrate to the implant. Once in the tissue, the monocytes become

macrophages and attach to the crab shell implant. These attached macrophages release a wide

range of biochemical factors that can mediate the activity of many other cells, including

fibroblasts, osteoblast, and osteoclasts (Ziats, Miller, & Anderson, 1988). It has been shown that

macrophages express osteopontin, a protein needed in bone formation (Freeman & Otto, 2005).

Macrophages influence the migration of multipotent progenitor fibroblastic cells that are of

osteoprogenitor precursor cell line or osteoblast phenotype. These multipotent progenitor

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fibroblastic cells give rise to osteoblasts (Wu, et al., 2000). Several members of the fibroblast

growth factor (FGF) family have an important role in the development of skeletal tissues and

increased osteoblast differentiation (Yun, et al., 2010) (Valta, et al., 2006). These fibroblastic

cells in contact with the crushed crab cuticle proliferate and differentiate to osteogenic cells.

Hormonal, cytokine, and metabolic cues influence the multipotent progenitor cell’s final

differentiation pathway (Wu, et al., 2000). The osteogenic cells release collagen matrix and

begin to mineralize. The macrophage, fibroblastic cells, and the crab cuticle signal each other

releasing biochemical factors that create a microenvironment conducive to bone formation.

Termine and Posner suggested that amorphous calcium phosphate is the first mineral deposited

during the calcification process and that the amorphous bone mineral fraction can act as a

metabolically active, metastable precursor of crystalline bone apatite (Termine & Posner,

Amorphous / Crystallinne Interrelationship in Bone Mineral, 1967). The beginning stages of

matrix mineralization were evidenced in this study.

Other factors may be involved in bone formation induced by biomaterials, including growth

factors other than BMP’s, the surface energy (zeta-potentials), low oxygen, asymmetric cell

division which may occur on a rough surface and cause differentiate into osteogenic cells (Reis

& Weiner, 2004).

Additionally, the importance of the inflammation phase is noteworthy. Inflammation is a

crucial phase in both normal wound healing and bone repair and remodeling. A growing body of

evidence has emerged that suggest that a complex balance exists between regenerative and

damaging inflammatory processes and tissue healing involves overlapping phases of

216

inflammation, renewal, and remodeling (Mountziaris, Spicer, Kasper, & Mikos, 2011).

Inflammation is necessary early in the regenerative process to initiate the repair cascade. If

healing occurs normally, then the inflammatory phase is resolved promptly and tissue

regeneration can occur (Thomas & Puleo, 2011). In reference to bone, the study of the

relationship between inflammatory mediators and bone has been termed “osteoimmunology”

(Graves, 2008).

There are several pathways through which tissue can heal, yet the unique attribute of bone

repair is that it occurs without the development of a fibrous scar. Inflammation, repair, and

regeneration are well- orchestrated, carefully choreographed processes with many interrelated

anatomical, biomechanical and biochemical processes (Marsell & Einhorn, 2011).

We believe that the phases of healing regarding the implanted crushed crab shell resembles a

cross between normal wound healing and bone tissue repair. This process, with overlapping

phases, begins with the inflammation phase after injury, followed by proliferation phase, then a

differentiation phase based on the modification of fibroblast behavior in the presence of the crab

carapace, and finally resolution phase involving the deposition of collagen matrix followed by

mineralization of newly formed fibers. The cascading events are depicted in figure 5-28.

Concluding Remarks

Biology and technology/engineering use very different approaches to the solution of similar

problems or functions of design and integration. However, many of nature’s systems use the

same exquisite and superior design principles. As hard tissue systems are studied, the similarities

217

between systems such as bone and crab cuticle become obviously clear. Exploitation of these

similarities, biorecyclying of crab cuticle takes advantage of the economical and efficient ways

nature achieves its objective in terms of energy, cost, and materials. In addition, most of the

design prototyping and fabrication has been done.

The hypothesis that crab shell may possess the inherent ability to induce bone formation in is

based on unique similarities between the hard tissue bone and crab shell integument. The results

from TEM, HRTEM, XRD, SAD, END, and EDS, in this study indicate that crushed shell from

C. sapidus exhibits some unique interactions in vivo using Sprague–Dawley rats. These results

further suggest that crab shell may be suitable for use in developing functionally advanced bone

implants and this material warrants further study to ascertain its potential osteoinductive

properties.

Thus a few points are highlighted:

1. Degree of crystallinity changes as bone matrix matures

2. Degree of crystallinity can be masked by the size of the crystal

3. Degree of crystallinity varies with location

4. Our study suggests that crab shell cuticle is osteoinductive.

5. Our study indicates that crab cuticle may be an effective material for stimulating bone

growth.

6. Our findings lays the groundwork for further studies.

Assessing crab shell osteoinductive attributes and providing the first general biological

performance profile about the suitability of crab cuticle as a bone biomaterial is a novel

218

contribution to the fields of biomimetics, biological material science, orthopedic medicine,

microbiology, and bone tissue engineering. Furthermore, this process will enhance treatments

options for repair, remodeling, and reestablishing health of bone.

219

Chapter 6 Final Remarks

Biomimetics involves examining the manner in which biological organisms solve problems to

observe how that compares with solutions of the same or similar problems within technical

environment. Using biology as a living database of the solution of technical problems by

organism, TRIZ is a method that allows interpretation or translation from the biological to

technical world by providing focused ideas for the solutions of recognized problems. Biology

can be analyzed and described by TRIZ to complete the concept of biomimetics assisting in the

transfer of nature’s technology and its implementation in the technical world. By comparing the

functionality of biological a man-made polymers, proteins, and polysaccharides, man-made

polymers are not as versatile or responsive as the two biological polymers, protein and

polysaccharide. When considering biological hard tissue, calcium derivatives, carbonates and

phosphates are predominant. A limited range of chemicals suffices for nearly all biological hard

materials (Vincent J. F., Biomimetic materials, 2008). Although biological/engineering TRIZ in

its infancy, it can be used to feature that crab cuticle and bone tissue are the solution to similar

problems. Biomimetics and TRIZ can illuminate and build on the similarities between bone and

crab cuticle sanctioning biology’s ability to transform engineering.

Bone tissue and crab cuticle are nanocomposite that exhibit hierarchical structuring and

contain two distinct constituent phases. Bone tissue has an organic and inorganic phase, collagen

I and calcium phosphate, respectively. Crab cuticle also has an organic and inorganic phase, α-

chitin and calcium carbonate, respectively. Bone and crab cuticle are the sum of their parts.

220

When inspecting the organic constituents (collagen and chitin) of these hard tissues, many

uncanny observations emerge. Collagen and chitin have a similar and engaging life story. They

do not act alone; collagen is a triple helix embedded in an inorganic matrix and chitin is

embedded in a protein helix. Compared to typical structures of their kind, both collagen and

chitin are unusual. Although chitin is a polysaccharide and collagen is a protein, collagen and

chitin associate with proteoglycans. Collagen and chitin complex composites are hierarchically

structured and multifunctional Microfibril: Fribil Aggregation (Fiber): Fibrous Bundle:

‘Twisted Plywood”. Chitin and collagen are lyotropic liquid crystals that are both mobile, have

similar ordered geometries, can self-assemble, and have similar optical properties. The

biosynthesis and degradation of both collagen and chitin are stepwise procedures governed by

enzymatic activity. Both collagen and chitin are synthesized by sequential changes in the

cytoplasmic matrix, RER, processing in the Golgi apparatus and transport through vesicles

culminating in exocytosis across the membrane where chemical reactions occur and association

into fibril, fibers, and fibrous bundle. Collagen and chitin share properties are both

biocompatible, biodegradable, non-toxic, highly insoluble, and birefringement. Both chitin and

collagen have a wide variety of medical applications.

Bone and crab cuticle are naturally fabricated bioceramic composites that are assembled from

readily available materials, typically in aqueous media, at ambient conditions, and to net ordered

shape. The production of hard tissues involves an exquisite level of control of the nucleation and

growth of mineral. The most common mineral being phosphate and carbonate salts of calcium

221

that are used in conjunction with organic polymers such as collagen and chitin to give structural

support to bones and shells. The minerals are formed by normal cellular processes and replicate

to produce precisely organized structures in cells ranging from bacteria to osteoblasts of bone

(Mann, Molecular recognition in biomineralization, 1988).

Biomineralization is not a single process. Every organism has adapted certain strategic

principles to optimize the specific function of its hard tissue to the specific environment in which

it lives (Heuer, et al., 1992). The tools for biomineralization of crab cuticle and bone are calcium

carbonate and calcium phosphate, respectively. Biological systems are teeming with examples of

organic supramolecular assemblies (double and triple helices, multisubunit proteins, membrane

bound reaction centers, vesicles, tubules, etc.), some of which (collagen, cellulose, and chitin)

extend to microscopic dimensions in the form of hierarchical structures. This hierarchical

processing is important in the application of molecular tectonics in biomineralization: organic

architectures produced by molecular processes are used in turn as the building framework for the

synthesis of organized materials, which in turn provide the prefabricated units of complex

microstructures (Mann, Molecular tectonics in biomineralization and biomimetic materials

chmeistry, 1993).

Several theories have been proposed for the mechanism of mineralization of hard tissue. There is

at present no unified theory of the mechanism of mineralization. Theories of mineralization

include, but not limited to, biologically induced, organic matrix-mediated, mesocrystal model,

cell-mediated, matrix vesicles initiation, and a multistep phase transformation involving an

amorphous transient phase.

222

Biomineralization deals with ratios of Ca/P that change as mineralization matures.

Crystallinity varies with bone maturity (Landis & Glimcher, Electron Diffraction and Electron

Probe Microanalysis of the Mineral Phase of Bone Tissue Prepared by Anhydrous Techniques,

1978). Degree of crystallinity can be masked by the size of the crystal and varies with location.

Studies have indicated that the earliest deposits of mineral generate an amorphous solid phase

pattern while more mature deposits generate patterns of very poorly crystalline hydroxyapatite

which gradually approach those of crystalline hydroxyapatite (Landis & Glimcher, Electron

Diffraction and Electron Probe Microanalysis of the Mineral Phase of Bone Tissue Prepared by

Anhydrous Techniques, 1978).

Our study suggests that crab shell cuticle is osteoinductive and may be an effective material

for stimulating bone growth similar to demineralize bone matrix. Clinical outcome assessment

and comparative effectiveness to determine the efficacy of the tissue-engineered bone are

limited. Many of the preclinical models to test efficacy use methods that are invasive or not

readily translated to the assessment of human engineered tissues. Outcomes such as histology

and biomechanical evaluation require sacrifice of the animal and tissue harvest. Improved

noninvasive assessment tools are needed to monitor bone healing to allow effective comparisons

of the relative efficacy of different materials and composites.

The old adage that life is merely advanced aqueous organic chemistry has been superseded

for the most part by a view that embraces the interconnectivity of metal and non-metals in the

biochemical processes (Mann, Molecular tectonics in biomineralization and biomimetic

materials chmeistry, 1993). Embracing the interconnectivity of biological systems can be

223

achieved using biomimetics, TRIZ, and robust information technology tools involving

monitoring, tracking, and reporting for all aspects and levels of the biological system.

Designing biomimetics bone implants using involves transforming needs and requirements

into an integrated system design solution through concurrent consideration of all life-cycle needs

from development, testing, evaluation, tracking, reporting, and disposal. While bone tissue

engineering has capitalized on a number of breakthrough technologies, none have been able to

produce an ideal bone biomaterial. As our ability improves to collect, store, and analyze

information about all the elements of a biological system, more cues will emerge about the true

mechanisms of biological processes such as bone formation. This will undoubtedly lead to

advances in understanding and replicating the best material scientist, nature.

224

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