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Hanna K

okkonen

OULU 2009

D 1035

Hanna Kokkonen

ASPECTS OF BONESUGAR BIOLOGYPECTIN NANOCOATINGS OF HARDTISSUE IMPLANTS

FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF ANATOMY AND CELL BIOLOGY,UNIVERSITY OF OULU

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 0 3 5

HANNA KOKKONEN

ASPECTS OF BONE SUGAR BIOLOGYPectin nanocoatings of hard tissue implants

Academic dissertation to be presented with the assent ofthe Faculty of Medicine of the University of Oulu forpublic defence in Auditorium A101 of the Department ofAnatomy and Cell Biology (Aapistie 7 A), on 4 December2009, at 11 a.m.

OULUN YLIOPISTO, OULU 2009

Copyright © 2009Acta Univ. Oul. D 1035, 2009

Supervised byProfessor Juha Tuukkanen

Reviewed byDocent Tiina Laitala-LeinonenProfessor Risto Renkonen

ISBN 978-951-42-9319-1 (Paperback)ISBN 978-951-42-9320-7 (PDF)http://herkules.oulu.fi/isbn9789514293207/ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)http://herkules.oulu.fi/issn03553221/

Cover designRaimo Ahonen

OULU UNIVERSITY PRESSOULU 2009

Kokkonen, Hanna, Aspects of bone sugar biology. Pectin nanocoatings of hardtissue implantsFaculty of Medicine, Institute of Biomedicine, Department of Anatomy and Cell Biology,University of Oulu, P.O.Box 5000, FI-90014 University of Oulu, Finland Acta Univ. Oul. D 1035, 2009Oulu, Finland

AbstractThe improvement of implant biocompatibility is constantly under investigation. Titanium is astandard biomaterial that performs well in dental and orthopedic implantations. However,detrimental adverse effects resulting from e.g. biomaterial properties, inflammatory responses andsurgical procedures occasionally occur. Coating the biomaterials aims at increasing the proportionof successful operations.

Pectins, large plant cell wall polysaccharides, are innovative, modifiable, and potentially anti-inflammatory candidates for biomaterial nanocoatings. In this thesis, covalently-grafted pectinfragments (modified hairy regions, MHRs) modified either in vitro (from apple) or in vivo (frompotato) were tested.

Cell culture vessels and titanium substratum coated with the apple-MHRs, MHR-A and afurther-tailored fragment type, MHR-B, were compared with controls for their ability to supportproliferation and differentiation of osteoclasts and osteoblasts. Cells grew and differentiated onMHR-B and on the control surfaces; MHR-A did not perform well in these assays. Genetically-engineered potato MHRs did not support bone cell growth to the same extent as apple MHR-B,but nonetheless the possibility to manipulate cellular proliferation with specific in vivo –modifications of pectins was introduced.

When implanted into rat soft tissues, neither of the apple MHRs provoked severe acuteinflammatory reactions, which indicates good in vivo - tolerance of these botanicalmacromolecules. These studies illustrate the biocompatibility of MHRs, and the directionstowards which they could be further tailored. In terms of clinical use, their tolerability in vivo isespecially significant.

Keywords: biocompatible materials, glycomics, osteoblasts, osteoclasts, pectins,prostheses and implants, titanium

To co-authors James J, the Kid B, Kenguru Köö, Nurmio T, Miljoona P, and Hasslehoff D

6

7

Acknowledgements

This work was carried out at the Department of Anatomy and Cell Biology,

Institute of Biomedicine, University of Oulu, during the years 2004–2009. I

express my sincere gratitude to my mentor, Professor Juha Tuukkanen, DDS,

PhD, for his enthusiastic guidance through this project. I also thank Professors

Hannu Rajaniemi, MD, PhD and Petri Lehenkari, MD, Ph.D. – the heads of the

Department of Anatomy and Cell Biology, as well as its whole personnel,

especially the cluster bomb of our bone group.

All my co-authors as well as the other PectiCoaters are thanked for sharing

their knowledge. Docent Tiina Laitala-Leinonen and Professor Risto Renkonen

are acknowledged for reviewing this thesis, and Dr. Deborah Kaska for its

language revision. Eero Oja, Marja Paloniemi, Anna-Maija Ruonala and Paula

Salmela are acknowledged for their valuable technical assistance. This work was

financially supported by the EC-project PectiCoat (NMP4-CT-2005-517036), The

National Graduate School of Musculoskeletal Disorders and Biomaterials

(TBGS), The Academy of Finland (130795), and The Finnish medical Foundation

Duodecim, and The Foundation of Orthopedics and Traumatology.

My officemates, Mari and Virpi, are acknowledged for the collectively pedant

and seriously academic attitude towards work. I am also grateful to the pope M.

Murtomaa for the spiritual atmosphere in our room. Without you I would never

have accomplished my thesis – this slowly. Annina Sipola is acknowledged for

sharing the practical consequences of it. My co-pöntöt Minna Karjalainen and

Johanna Korvala are thanked for the former, radiant study years at the Department

of Biology. Also all my other friends, especially the “old-timers” Anne, Erika,

Jukke, Markus, Mäkiset, Paakkolanvaarat, Pete, Tuomas and WSQ, deserve my

warmest compliments for providing me the feeling of never being alone. Aside,

hedonistic bubbling deserves to be mentioned, as does all the to-be-collegially -

provided professional relief.

Lopuksi osoitan rakkaille vanhemmilleni Orvokille ja Kaukolle suurimmat

kiitokseni kaikesta. Ilman aina saatavilla olevaa apuanne niin tämä kuin kovin

moni muukin asia olisi jäänyt tekemättä. Myös todellista elämänviisautta edustava

isoäitini, Kotimummo Annikki, ansaitsee lämpimät kiitokset. Ihana tyttäreni Hilla

puolestaan on suurin ja tärkein muistutus siitä, millä on merkitystä.

Oulu, November 2009 Hanna Kokkonen

8

Mennään pois täältä

ennen kuin muuttuu ääni kelloissa;

tihulaismyrskyt, ukkosenjohdattimet ihokarvoissa,

nopeus, tehokkuus, kahviautomaatit, oikeustalojen täydet salit,

connecting people

vaikka kätelläkään ei enää saa

Postilaatikot täysinä Niin laajat kaistat,

ettei sinuun kohta yletä

Kaikki muu sähkö aina jotenkin

lähempänä kuin lämmin EKG:si,

padottujen ionikanavien liioitellut maadoitukset

Annetaan hetken olla

elinten laatikoissa, kirurginterästen

oudoissa kummuissa, jänteiden ääriviivat

anatomian kuvastoissa

(opetellaan ne muuten)

Mennään paikkaan, jossa ei ole kenttää – vain vesi virtaa,

hirvet hengittävät

9

Abbreviations

α-MEM Dulbecco`s modified Eagle`s medium, alpha modification

AGE Advanced glycation end product

ALP Alkaline phosphatase

AMI Aminated surface

ANOVA Analysis of variance

Ara Arabinose

AsAP L-ascorbic acid 2-phosphate

β-GP Phosphate disodium salt pentahydrate

BMP Bone morphogenic protein

bp Base pair

BRU Bone remodeling unit

BSA Bovine serum albumin

BSP Bone sialoprotein

Cbfa1 Core-binding factor alpha 1 (a.k.a. Runx2)

cDNA Complementary deoxyribonucleic acid

CT Calcitonin

D-MEM Dulbecco`s modified Eagle`s medium

ECM Extracellular matrix

EtOH Ethanol

FA Focal adhesion

FBGC Foreign body giant cell

FBS Fetal bovine serum

FCS Fetal calf serum

FESEM Field emission scanning electron microscopy

FGF Fibroblast growth factor

FITC Fluorescein isothiocyanate

FN Fibronectin

GAG Glycosaminoglycan

Gal Galactose

GalA Galacturonic acid

Glc Glucose

GlcA Glucuronic acid

HA Hydroxyapatite

HE Hematoxylin-eosin

HM High-methyl pectin

10

hMSC Human mesencymal stem cells

IL Interleukin

LM Low-methyl pectin

MHR Modified hairy region

MMP Matrix metalloproteinase

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NCP Non-collagenous protein

OCIL Osteoclast inhibitory lectin

OCN Osteocalcin

OPG Osteoprotegerin

OPN Osteopontin

PBS Phosphate-buffered saline

PFA Paraformaldehyde

PTH Parathyroid hormone

PTR Transgenic potato -derived

RANK Receptor activator for NF-κB

RANKL Receptor activator for NF-κB ligand

RG Rhamnogalacturonan

RGD Arginine-glycine-aspartic acid

Rha Rhamnose

RNA Ribonucleic acid

RT Room temperature

RT-PCR Reverse transcriptase polymerase chain reaction

sLRP Small leucine-rich proteoglycan

TCPS Tissue culture polystyrene

TGF Transforming growth factor

Ti Titanium (uncoated)

TNF Tumor necrosis factor

TRACP Tartrate-resistant acid phosphatase

TRITC Tetramethylrhodamine isothiocyanate

WGA Wheat germ agglutinin

WT Wild type

11

List of original publications

This thesis is based on the following articles referred to in the text by their Roman

numerals:

I Kokkonen HE, Ilvesaro J, Morra M, Schols HA & Tuukkanen J (2007) Effect

of Modified Pectin Molecules on the Growth of Bone Cells.

Biomacromolecules 8(2): 509–15.

II Kokkonen H, Cassinelli C, Morra M, Schols HA & Tuukkanen J (2008)

Differentiation of Osteoblasts on Pectin-Coated Titanium.

Biomacromolecules 9(9): 2369–76.

III Kokkonen H, Niiranen H, Schols HA, Morra M, Stenbäck F & Tuukkanen J

(2009) Pectin-Coated Titanium Implants are Well-Tolerated in vivo. Journal

of Biomedical Materials Research: Part A. In press.

IV Kokkonen H, Verhoef R, Kauppinen K, Muhonen V, Jørgensen B, Damager I,

Schols H, Morra M, Ulvskov P & Tuukkanen J (2009) Proliferation of

Osteoblastic Cells on In vivo Engineered Potato Pectin Fragments.

Manuscript.

12

13

Contents

Abstract Acknowledgements 7 Abbreviations 9 List of original publications 11 Contents 13 1 Introduction 15 2 Review of the literature 17

2.1 Bone ........................................................................................................ 17 2.1.1 Bone as an organ .......................................................................... 17 2.1.2 Bone as a tissue ............................................................................ 18 2.1.3 Osteoclasts .................................................................................... 19 2.1.4 Osteoblasts.................................................................................... 20 2.1.5 Intercellular and cell-ECM interactions ....................................... 22

2.2 Carbohydrates in bone biology ............................................................... 24 2.2.1 Sugar molecules in bone and joints .............................................. 24 2.2.2 Sugar molecules and osteoclasts................................................... 25 2.2.3 Sugar molecules and osteoblasts .................................................. 27

2.3 Bone implants ......................................................................................... 29 2.3.1 Biocompatibility: Biological factors ............................................ 29 2.3.2 Biocompatibility: Physicochemical factors .................................. 30 2.3.3 Host responses to implantation ..................................................... 31 2.3.4 Coating of bone implants .............................................................. 32

2.4 Pectins ..................................................................................................... 34 2.4.1 Pectins as candidates for biomaterial applications ....................... 35 2.4.2 Modification of pectins ................................................................. 36 2.4.3 Immunological aspects of pectins ................................................. 38

3 Aims of the present study 39 4 Materials and methods 41

4.1 Cell cultures ............................................................................................ 41 4.1.1 Cell lines ....................................................................................... 41 4.1.2 Primary cultures ........................................................................... 41

4.2 Test materials .......................................................................................... 42 4.2.1 MHR preparations ........................................................................ 42 4.2.2 MHR-coated samples ................................................................... 43 4.2.3 Bone slices .................................................................................... 44

14

4.3 Stainings .................................................................................................. 44 4.3.1 Fluorescent labels ......................................................................... 44 4.3.2 Non-fluorescent stains .................................................................. 45

4.4 Microscopic methods .............................................................................. 45 4.4.1 Light and fluorescence microscopy .............................................. 45 4.4.2 Laser scanning confocal microscopy ............................................ 45 4.4.3 Field emission scanning electron microscopy .............................. 46 4.4.4 Image analyses .............................................................................. 46

4.5 Molecular methods .................................................................................. 46 4.5.1 RT-PCR ......................................................................................... 46 4.5.2 Enzyme activity measurement ...................................................... 47

4.6 In vivo procedures ................................................................................... 47 4.6.1 Animals and implantations ........................................................... 48 4.6.2 Histological sections ..................................................................... 48

4.7 Statistical analyses .................................................................................. 48 5 Results 49

5.1 Interactions between MHRs and bone cells ............................................ 49 5.1.1 Proliferation and differentiation of osteoclasts (I) ........................ 49 5.1.2 Proliferation and differentiation of osteoblasts (I, II, IV) ............. 49

5.2 In vivo effects of MHR-coated implants (III) .......................................... 52 6 Discussion 55

6.1 Effects of pectins on bone cells ................................................................. 55 6.1.1 Osteoclastic responses .................................................................. 55 6.1.2 Osteoblastic proliferation ............................................................. 56 6.1.3 Osteoblastic differentiation ........................................................... 58

6.2 In vivo – testing of inflammatory responses ............................................ 61 6.2.1 Fibrous capsule formation ............................................................ 62 6.2.2 Consistence of the capsules .......................................................... 62

6.3 General discussion and future prospects ................................................. 64 6.3.1 Physicochemical features of MHR-coatings ................................ 64 6.3.2 Sugar aspects ................................................................................ 67 6.3.3 Immunological aspects ................................................................. 69

7 Conclusions 71 References 73 Original publications 93

15

1 Introduction

Bone and dental implants are widely used as substitutive items in various clinical

complications resulting from e.g. erosive diseases and trauma. Materials

contacting living tissues are called biomaterials, whose interactions with the

surrounding tissue should not excessively disturb the homeostatic functions of the

cells. This inertness of biomaterials is defined as biocompatibility. However,

problems in biocompatibility often occur and may lead to e.g. inflammation,

infections or even implant loosening. One approach to improving

biocompatibility is to coat biomaterials with appropriate molecules in order to

diminish any negative effects of implants and/or to enhance desired biological

processes after implantation.

Bone tissue undergoes constant remodeling, which is conducted by bone-

resorbing osteoclasts and bone-forming osteoblasts. It is important that the

biomaterial, which in bone and dental implantations usually is titanium, does not

interfere with the cooperation of these cell types.

The purpose of this thesis was to investigate the feasibility of using the novel

molecular candidates, pectins, as bone and dental implant nanocoatings. Pectins

are large and structurally variable polysaccharides of botanical origin. The

interactions between cells and pectin fragments were studied both in vitro and in vivo, focusing especially on the proliferation and differentiation of bone cells on

pectin coatings as well as on the tendency of pectin fragments to provoke

inflammatory responses. These studies may both augment our basic knowledge of

sugar biology in bone cells and in particular assess the applicability of pectin-

coated implants for clinical purposes.

16

17

2 Review of the literature

2.1 Bone

2.1.1 Bone as an organ

The bony skeleton of higher vertebrates provides mechanical support for the

body, protects the vital organs, and enables movement together with muscles. In

addition to these main anatomical functions, bone takes part in several

physiological processes, e.g. regulation of calcium and phosphate ion balances in

the serum, and production of blood cells (hematopoiesis) in the bone marrow of

long bones.

At the macroscopic level, two structurally different textures can be

distinguished in bones. Compact bone forms a dense cortex that surrounds other

sections of bone that form the bone marrow cavity in the diaphysis area of long

bones. In the metaphysis and epiphysis areas of a long bone the cavity is largely

occupied by a network of metabolically active trabecular (or cancellous) bone

surrounding the bone marrow. Cartilaginous growth plates are located at the

border between the metaphysis and epiphysis. The outmost layer of bone is called

the periosteum, and the inner surface between the cortex and the bone marrow is

the endosteum.

Bones ossify either via an endochondral or an intramembraneous

developmental route. In endochondral ossification, a cartilaginous prototype of a

bone is subsequently replaced by mineralized matrix. This endochondral route is

typical for long bones. In contrast, flat bones are developed via intramembraneous

ossification, in which bones are formed and fused together without an

intermediate cartilage model. A special anomaly of bone formation is the so-

called ectopic ossification, which signifies the development of bone in soft tissues

outside the skeleton – a phenomenon manifesting in some relatively rare

syndromes, e.g. osteoma cutis- and fibrodysplasia ossificans progressiva

(Ruggieri et al. 1995, Cortes & Gosain 2006, Lounev et al. 2009, Suda et al. 2009).

Biological processes in bones are regulated by various hormones and local

factors, e.g. parathyroid hormone (PTH), calcitonin (CT), estrogen, 1,25-

dihydroxyvitamin D3 (1,25(OH2)D3), tumor necrosis factor (TNF), transforming

growth factors (TGF-α /-β), interferon gamma (IFN-γ), interleukin-1 (IL-1), and

18

bone morphogenic proteins (BMPs). Besides these molecular regulators,

environmental elements including nutrition and physical exercise, as well as

genetic heritage affect the condition of the skeleton. Age is also an important

factor; the peak bone mass is achieved in late adolescence (Matkovic et al. 1994).

2.1.2 Bone as a tissue

Bone tissue can be classified as a specialized dense connective tissue. The

structural units of cortical bone are called osteons, which represent the regions of

bone remodeling. The center of an osteon is occupied by a Haversian system

consisting of a vascular route in a central canal, which is surrounded by lamellar

layers of osteocytes located in solitary niches, i.e. lacunae. The borders of osteons

are seen as cement lines, visual remnants of previous activities of bone

remodeling.

The extracellular matrix (ECM) of bone is composed of both organic and

inorganic compounds. About 22% of the bone matrix consists of proteins, the

majority being thus composed of inorganic components; 70% mineral salts and

8% water (Lane 1979). The most abundant protein, 95% of the total protein

fraction, in bone ECM is type I collagen, in addition to which bone also contains

minor amounts of collagen types III, V, and XI. Collagen fibers are decorated

with hydroxyapatite [3Ca3(PO4)2(OH)2] crystals making the bone matrix rigid and

durable. As indicated by the chemical formula, the main components of

hydroxyapatite are calcium and phosphate (DeJong 1926), which reflects the

aforementioned physiological importance of bone tissue in the regulation of ion

homeostasis. The remaining non-collagenous proteins (NCP) produced by

osteoblasts, such as fibronectin (FN), osteocalcin (OCN), osteopontin (OPN), and

bone sialoprotein (BSP) play important roles in the interactions between bone

cells and the ECM. Additionally, OPN has been shown to be located abundantly

in the cement lines separating old and new bone matrices (Mulari et al. 2004).

Bone tissue is constantly remodeled in a finely-tuned series of complex

events taking place in so-called bone remodeling units (BRU), a process that

occurs also in vitro (Marcus 1987, Väänänen 1993, Mulari et al. 2004, Andersen et al. 2009). The alternating phases of bone resorption and formation are mediated

by two bone cell types, osteoclasts and osteoblasts, respectively. Imbalance

between the activities of these cell types may result in various diseases, such as

the well-known osteoporosis, which is linked to decreased estrogen levels and

subsequent decreased bone mass predisposing to fractures (Takano-Yamamoto &

19

Rodan 1990). Another condition related to bone cell imbalance is osteopetrosis

(Walker 1973), in which the activity of osteoclasts is hindered resulting in

excessive thickness and also to an elevated fracture susceptibility of cortical bone

(Tuukkanen et al. 2000). Thus a well-balanced and coordinated communication

between bone cell types is crucial to maintain the appropriate functionality of

bone. In addition to osteoclasts and osteoblasts, bone tissue contains osteocytes

and bone lining cells.

2.1.3 Osteoclasts

In the remodeling cycle of bone, the mineralized matrix must be dissolved prior to

the phase of new bone formation. The resorption of bone is mediated by large and

multinuclear cells, osteoclasts, first described by Albert Kölliker (Kölliker 1873).

Osteoclasts differentiate from hematopoietic stem cells and thus share the same

developmental origin with e.g. monocytes / macrophages. Osteoclasts gain their

multinuclear state via fusion of mononuclear precursor cells (Suda et al. 1992,

rana-Chavez & Bradaschia-Correa 2009). The differentiation and activity of

osteoclasts is dependent on certain, either stimulating or inhibiting systemic

factors (e.g. PTH, CT, IL-1, TGF-α and -β, and 1,25(OH)2D3) as well as on the

proximity of osteoblasts. For example, the effects of PTH and 1,25(OH)2D3 on

osteoclasts are affected by osteoblasts (Silve et al. 1982, Narbaitz et al. 1983).

Additionally, osteoblasts produce e.g. IL-1 and -6 and prostaglandin-E2 required

for controlling osteoclast activation, and receptor activator for NF-κB (nuclear

factor kappa-light-chain-enhancer of activated B cells) i.e. RANKL (also denoted

as OPGL, TRANCE or ODF). RANKL can stimulate the differentiation and

resorptive activation of osteoclasts by binding to a RANK receptor of osteoclasts

(Suda et al. 1992, Lacey et al. 1998, Matsuzaki et al. 1998, Yasuda et al. 1999).

Another osteoblast-derived component affecting osteoclasts is osteoprotegerin

(OPG), a protein that also binds to RANK thus hindering the differentiation of

osteoclasts. Additionally, bone marrow stromal cells including osteoblasts secrete

local factors, such as macrophage colony-stimulating factor (M-CSF), required

for osteoclastic maturation (Wiktor-Jedrzejczak et al. 1990).

Osteoclasts are capable of excavating both the organic and inorganic

components of bone in an acidified resorption cavitity surrounded by an

osteoclastic membrane organization called the sealing zone (Lakkakorpi et al. 1991, Lakkakorpi & Väänänen 1991). The sealing zone is an actin-rich ring-like

structure tightly attached to the bone matrix surface, onto which it confines

20

another plasma membrane domain, the endosomally and lysosomally active

ruffled border (Baron et al. 1988, Palokangas et al. 1997). Through this

membrane area the cellular vesicles containing acidifying H+ ions generated by

e.g. carbonic anhydrase II enzyme are released into the resorption lacuna (i.e.

Howship`s lacuna). Vacuolar H+-ATPases (V-ATPases) are pivotal in the activity

of osteoclasts, and the localization of the V-ATPase subunit mRNAs change

according to the phase of the resorption cycle and corresponding polarity of an

osteoclast (Laitala-Leinonen et al. 1996). Interestingly, inhibition of V-ATPases

has been shown to alleviate the processes of rheumatoid arthritis (Niikura et al. 2007). The resulting pH gradient is responsible for dissolving hydroxyapatite,

after which the organic composite – mainly composed of collagen-I fibers –

become accessible to different proteinases, such as collagenases, lysosomal

enzymes, and cathepsin-K. Chondrocyte-produced cathepsin-K has also been

shown to play a role in the pathogenesis of transgenic osteoarthritis mouse models

due to its ability to degrade cartilage as well (Morko et al. 2004). Some matrix

metalloproteinases (MMPs) and other proteinases produced by osteoblasts are

also involved in the initiation of the resorption phase thus predisposing bone

surface to resorption (Chambers & Fuller 1985, Kusano et al. 1998, Roman-

Roman et al. 2003).

The remnants of the resorption are endocytosed into the osteoclast, processed

by intracellular vesicular tartrate-resistant acid phosphatase (TRACP),

transcytosed towards a basolateral functional secretory domain and excreted from

the cell through this membrane region (Salo et al. 1997, Vääräniemi et al. 2004).

Phagocytes are also thought to participate in tidying up the resorbed bone site,

albeit some evidence emphasizes the role of osteoblasts in cleaning the organic

components from the resorption lacunae preceding the deposition of new bone

matrix.

2.1.4 Osteoblasts

After osteoclastic activity, bone-forming osteoblasts migrate to the resorption site

to refill the dissolved lacuna with new bone tissue by first producing an organic

matrix called osteoid, and by subsequently (about 20 days after osteoid

deposition) mineralizing it. Osteoblasts can form mineral by means of two,

probably parallel, mechanisms: nucleation (mineral crystals occupy the hole

regions of collagen-I fibrils) and matrix vesicle production (osteoblasts secrete

21

small organelles in the vicinity of calcium phosphate grains) (Anderson 1984,

Schwartz et al. 1987, Williams & Frolik 1991).

Osteoblasts originate from mesenchymal stem cells, as do cells of connective,

cartilaginous, muscle, and adipose tissue. The maturation route of osteoblasts

proceeds from a preosteoblastic cell to an osteoblast to an osteocyte. Additionally

the bone-lining cells can be regarded as a specific phenotype of the osteoblast

lineage.

Preosteoblasts are the precursors of the differentiated osteoblasts, the

veritable bone-forming cells. Several markers of mature osteoblasts can be

distinguished. For example, the production of alkaline phosphatase (ALP), a

plasma membrane -anchored enzyme indicative of mineralization, is a strong

indication of the transition towards a differentiated osteoblastic phenotype (Stein et al. 1989, Owen et al. 1990, Aubin et al. 1995). Several isoforms of soluble

bone ALP (BALP) can be purified and identified (Magnusson & Farley 2002,

Sharp et al. 2007). The expression of osteocalcin (OCN), i.e. bone gla-protein,

serves as the clearest mineralization marker, and its serum or urine concentration

may be measured in metabolic disease as an indicator of bone turnover rate (Price et al. 1980, Ivaska et al. 2005).

Osteoblast-specific genes require two main transcription factors, core-binding

factor alpha 1, i.e. Cbfa-1 (also known as Runx2 or Osf2) and Osterix, in order to

be transcribed (Ducy et al. 1997, Beck et al. 2001). Osteoblasts are also under the

control of various regulators, e.g. insulin-like growth factors (IGFs) and BMPs

(Ripamonti & Reddi 1992), and they also are capable of auto-regulating their

activity via TGF-β production (Robey et al. 1987).

After the formation of fresh bone tissue, some of the osteoblasts (~ 10–20%)

involved in the synthesis remain enclosed in the matrix. These cells become

osteocytes, the most abundant (90–95%) bone cell type (Frost 1960). Osteocytes

are located in separate lacunae and form an intercellular network via small

cytoplasmic projections extending through tunnel-like structures, the canaliculi.

Through these channels osteocytes embedded in mineralized – and thus

impermeable for diffusion – bone tissue is able to of receive oxygen, nutrients,

and other important molecules from the blood supply of the Haversian system.

Additionally, the tips of the processes contain connexin-based gap junctions,

through which osteocytes communicate electrically and metabolically with each

other and probably with other bone cell types as well (Doty 1981, Donahue et al. 1995).

22

The role of osteocytes in bone biology still remains relatively obscure, but

they seem to be involved in sensing and reacting to mechanical loading of the

bones (Aarden et al. 1994, Reijnders et al. 2007, You et al. 2008, Turner et al. 2009). Osteocytes respond to bone micro-damage and affect subsequent

remodeling (Kurata et al. 2006, Heino et al. 2009). Recently it has been shown

that bone also adapts to mechanical loading via modulating the RANKL

expression of osteoblasts at least immediately after stretching stimuli (Kreja et al. 2008).

Some post-proliferative osteoblastic cells have an inactive appearance at the

bone surfaces. These so-called bone-lining cells are not capable of producing

bone matrix, and they mark bone areas not participating in the remodeling

processes. Once the resorption phase starts, bone-lining cells disengage from the

bone surface. The role of bone-lining cells seems to relate to the production of

osteoclast-activating factors, e.g. cytokines. Additionally, they have been shown

to participate in linking the resorptive and formative remodeling phases by

removing collagen rudiments from resorption lacunae together with MMPs

(Everts et al. 2002). These cells may also revert to active osteoblasts (Chow et al. 1998).

2.1.5 Intercellular and cell-ECM interactions

To sustain the physiological and anatomical balance of bone tissue, the

communication and interactions of different bone cell types must be coordinated

properly. The main steps in the bone remodeling cycle are the activation,

resorption, reversal, and formation phases (Frost 1973), which are illustrated in

Figure 1.

Activation is the starting point for the remodeling of a BRU, during which the

bone surface transforms active and osteoclasts are attracted to, fuse, and

differentiate at the site to be remodeled. As mentioned earlier, both osteocytes as

well as bone-lining cells are thought to participate in the activation phase

(Chambers 1985, Martin 2000). Osteoblasts also play an important role since it

has been shown that osteoclasts cannot initiate resorption at the sites of non-

mineralized osteoid; thus it must become either mineralized or degraded

(proteinases) by osteoblasts before resorption can commence (Jones et al. 1986,

Chambers & Hall 1991). The resorption phase is followed by a reversal phase

characterized by the appearance of macrophages at the resorbed bone site (Raisz

1988). The reversal phase is the link between bone resorption and formation,

23

which implies that it is during this stage when the still largely unknown signals to

osteoblasts become manifested. The macrophages (Raisz 1988) as well as bone-

lining cells and osteoclasts may play a role in signaling the osteoblasts by

secreting certain paracrine factors. Additionally, some factors or components of

the bone matrix revealed during resorption may act as signaling molecules for

osteoblasts so that resorption is always succeeded by the deposition of new bone

tissue, i.e. the formation phase (Mundy & Roodman 1987).

Fig. 1. A schematic illustration of the bone remodeling phases. Modified from

(U.S.Department of Health and Human Services 2004).

Osteoblasts are anchorage-dependent cells. Their attachment and spreading

require so-called focal adhesions (FAs), complex protein aggregates including e.g.

paxillin and vinculin, which form a link between the actomyosin cytoskeleton of a

cell and the NCP proteins of the ECM via integrin receptors (Abercrombie et al. 1971, Ruoslahti & Pierschbacher 1987, Sastry & Burridge 2000). Herewith the

bone ECM affects various cellular functions, such as survival, motility, gene

expression, and proliferation (Damsky & Werb 1992, Huhtala et al. 1995, Moursi

et al. 1997).

Integrins are heterodimeric transmembrane receptors composed of α- and β-

subunits, which bind to a specific tripeptide sequence, arginine – glycine –

aspartic acid (RGD) of NCP protein ligands (Ruoslahti & Pierschbacher 1986,

Hynes 1987). Bone cell types differ in terms of their integrin composition. For

instance, osteoblasts typically express e.g. fibronectin-specific α5β1 integrin as

mesenchymal stem cellhematopoietic stem cell

osteoclastosteoblasts

preosteoblast

osteocytes

osteoblastic stromal cell

lining cells

activationresorptionreversalformation

24

well as α2β1 integrin, which binds to fibronectin and collagen-I. Also variable ανβ-

integrins are involved in osteoblastic development (Lai & Cheng 2005). Integrin

binding launches cellular signaling cascades involving e.g. protein kinases

(Boudreau & Jones 1999, Miranti et al. 1999). Integrin-mediated cell adhesion is

fundamental for osteoblastic functions including proliferation, differentiation, and

mineralization, and thus plays an important role also in the biomaterial field

(Lynch et al. 1995, Moursi et al. 1997, Gronthos et al. 1997, Siebers et al. 2005).

2.2 Carbohydrates in bone biology

Carbohydrate molecules play important roles in several biological processes; they

act as recognition molecules in cell-cell- and cell-ECM communication. For

instance, cell adhesion to ECM molecules involves not only integrins but also

some sugar-containing non-integrin receptors, such as the laminin-binding

dystroglycan (Colognato et al. 2007) and various cell surface proteoglycans

(Adams & Watt 1993). Glycans also act as antigenic and differentiation

determinants. The function of glycans is to fine-tune biological processes; these

complex molecules often located at cell surfaces are very information-dense. The

interactions between glycomolecules and proteins capable of binding them, i.e.

lectins, have been under intense investigation because they may pave the way for

the development of e.g. cancer therapeutics. (Nangia-Makker et al. 2002, Flitsch

& Ulijn 2003, Shriver et al. 2004)

2.2.1 Sugar molecules in bone and joints

In bone tissue, various macromolecules containing sugar components are located

both at bone cell surfaces as well as in the ECM. For example,

glycosaminoglycans (GAGs), such as chondroitin and heparan sulfates, are

common constituents of bone ECM. GAGs are acidic polysaccharide side chains

formed of repeated uronic acid and hexosamine sugars forming larger

proteoglycan aggregates together with a core protein (e.g. perlecan or syndecan),

which thus are strongly anionic. The resulting negative charge is considered to be

crucial for the ionic calcification of the bone matrix since the linking of

hydroxyapatite crystals with collagen fibers is affected by and partly dependent

on the bone ECM ground substance rich in proteoglycans. Proteoglycans also

form major components of articular cartilages and intervertebral discs.

25

GAGs have various functions in bone and cartilage tissues. As indicated with

murine calvarial cell and organ cultures, GAGs may modulate the effect of

growth factors (e.g. FGF-2 and TGF-β1) on bone development (Manton et al. 2007a). Heparan- and chondroitin sulfates have also been identified as regulators

of BMP-mediated differentiation of osteoblasts from hMSCs (Manton et al. 2007b). Interestingly, GAGs have recently been shown to be overexpressed in

osteosarcoma, and they may thus act as potent regulators of cancer progression by

controlling the binding of OPG to RANKL (Lamoureux et al. 2009).

In cartilage, GAGs and proteoglycans in general are crucially involved in e.g.

the responses of articular cartilage after joint immobilization – induced atrophies

(Kiviranta et al. 1994, Jortikka et al. 1997, Haapala et al. 1999). Degradation and

repair of intervertebral discs as well as knee articular cartilage also involve

changes in the levels of proteoglycans, including biglycan, decorin and

syndecan-1 (Inkinen et al. 1998, Salminen-Mankonen et al. 2005). Additionally,

the proteoglycan / collagen ratio of the intervertebral disc annulus fibrosus has

been reported to decrease due to mechanical stress in dogs, which enhances the

aggregation between the remaining proteoglycans and hyaluronan (Säämänen et al. 1993). Hyaluronan represents a free, unsulfated form of GAG. In cartilage,

hyaluronan contributes to ECM lubrication and assembly, but its functions in

bone have remained unclear. Recently, however, hyaluronan has been asserted to

be involved in reducing inflammation-related bone resorption by inhibiting the

expression of e.g. prostaglandin-E2 and MMPs in osteoblasts (Hirata et al. 2009).

MMP-1, -8- and -13 are the traditionally-recognized collagenases that function in

rheumatoid arthritis (RA), yet human trypsin-2 also appears to be involved in

collagen-II degradation in RA (Stenman et al. 2005).

2.2.2 Sugar molecules and osteoclasts

Glycobinding proteins are generally known as lectins. In order to enter into bone

tissue from the vasculature, osteoclasts have to recognize and bind to certain

glycoproteins, such as laminin, in the vessel basement membranes. The

subsequent interactions with bone ECM glycoproteins are also crucial for

osteoclastic activity. These glycomolecule-involving interactions are mediated by

e.g. galectin-3, a β-galactoside-specific lectin first identified in the plasma

membrane of chicken osteoclasts (Gorski et al. 2002). Galectin-3 is also been

reported to participate in endochondral bone formation since it functions as a

26

substrate for MMP-9, which controls chondrocyte apoptosis and osteoclast

recruitment in this process (Ortega et al. 2005).

Interestingly, some lectins of botanical origin have specific targets in bone.

For instance, Arachis hypogaea peanut agglutinin (PNA), which recognizes β-D-

galactose(1,3)N-acetyl-D-galactosamine (Gal-β-1,3-GalNac) disaccharide, and

wheat germ agglutinin (WGA), which recognizes N-acetyl(1,4)D-glucosamine

(GlcNAc) as well as terminal neuraminic acid, bind to osteoclastic cells (Illes &

Fischer 1989, Illes et al. 1992). PNA has also been reported to bind to some bone

marrow cells, endosteal mononuclear cells as well as peripheral rat monocytes,

and can be used for osteoclast identification (Väänänen et al. 1986). WGA is

commonly exploited in bone tissue analytics: The resorption pits can be stained

with WGA, which indicates the existence of the sugar epitopes recognized by this

lectin in the resorbed bone matrix (Selander et al. 1994). Furthermore, WGA has

been reported to attenuate ALP activity, whereas neuraminidase enzyme tends to

elevate it (Sharp et al. 2007). Additionally, various BALP isoforms can be

distinguished by their different sensitivity to WGA precipitation (Magnusson &

Farley 2002).

An important sugar moiety found in several cell types is a 9-carbon,

carboxylate group -containing sialic acid, a.k.a. neuraminic acid, which usually is

the most distal sugar molecule in the cell surface glycans. Sialic acids are found

in a wide variety of organisms – also in bacteria, fungi, and plants. Two types of

neuraminic acids – N-acetyl neuraminic acid (Neu5Ac) and N-glycolylneuraminic

acid (Neu5Gc) – are expressed in mammalian tissues with the exception that

humans express only the former due to a deletion mutation in the CMP-Neu5Ac

hydroxylase (CMAH) gene. Basically, in vertebrates, three types of glycan classes

can carry sialic acid moieties: glycosphingolipids, N-glycans and O-glycans.

Among other roles, sialic acids participate in cell-virus and cell-cell fusions;

for example alpha (2,3)-linked- and alpha (2,6)-linked sialic acids take part in

osteoclastogenesis (Takahata et al. 2007). Sialic acids may also influence stem

cell therapies as contaminating xenoantigens. For example, murine Neu5Gc

probably affects the efficacy and survival of transplanted human embryonic as

well as mesenchymal stem cells (Heiskanen et al. 2007). In fact, human serum

naturally contains IgG antibodies against Neu5Gc (Nguyen et al. 2005). Some

inherited diseases, such as Salla disease, also involve sialic acids; the transport of

the cleaved sialic acid residues to lysosomal degradation by a sialin transporter is

usually defective in these syndromes (Morin et al. 2004).

27

The functionality of a specific sialic acid is correlated with its terminal

position and specific structure in cell surface glycochains, and as a consequence,

the lectin recognition and subsequent functions of the “host cell” can be modified

by sialic acid modification or removal (Varki 1997). For example, trafficking of

mesenchymal stem cells to bone can be affected by modifying the sialyl-

containing glycans on the membrane of MSCs (Sackstein et al. 2008). Sialic acids

are recognized by e.g. C-type lectins, selectins and by so-called Siglecs, which are

immunoglobulin-related proteins and form the largest category of sialic acid –

binding molecules (Varki 1997). Selectins usually require so-called a sialyl-

LewisX -motif (SLeX) at the terminus of the glycan to be recognized (McEver

2002, Rosen 2004). This motif is made up of sialic acid residues located in the

vicinity of a α-1-3(4)-linked fucose residue. Sialyl Lewis X -epitopes have also

recently been shown to participate in glycoprofiling the differentiation stage of

bone marrow -derived mesenchymal stem cells (Heiskanen et al. 2009). In

addition, sialyl Lewis X -based glycostructures apparently play a role in

inflammatory conditions by acting as organ-specific epitopes for the L-selectins

of infiltrating leukocytes (Renkonen et al. 2002). In relation to tissue analytics, it

is noteworthy that PNA lectin recognizes the galactose moiety uncovered by

removing the terminal sialic acid residue of glycans.

2.2.3 Sugar molecules and osteoblasts

Variable effects of GAGs on bone cells have been discovered: The heparin

concentration affects the growth and mineralization of osteoblasts (Hausser &

Brenner 2004). Recently it has also been shown that heparan sulfate containing

proteoglycans can act as co-receptors for fibroblast growth factors (FGFs), a

phenomenon underlying e.g. osteoblastic syndecan expression (Song et al. 2007).

In turn, a sulfated hyaluronan has been shown to scale up the expression of ALP

and the cell adhesion molecules N-cadherin and connexin-43 in rat osteoblasts

(Nagahata et al. 2004). Biglycan – a bone ECM small leucine-rich proteoglycan

(sLRP) – is needed for the differentiation of osteoblasts (Chen et al. 2004, Chen et al. 2005, Parisuthiman et al. 2005). Another sLRP, decorin, is also crucial for

collagen fibrillogenesis and bone-implant contact (Klinger et al. 1998, Matsuura et al. 2005). Decorin is also probably responsible for the proper timing of

osteoblast differentiation by delaying mineralization of incompletely fibrillated

collagen matrix (Mochida et al. 2003). Heparan sulfate proteoglycans are also

capable of inhibiting osteoblastic functionality, and this restraint is overcome by

28

the heparanase production of osteoblasts (Kram et al. 2006). Interestingly,

heparan-like polymers synthesized from dextran have also been shown to enhance

tissue repairing of bone defects by probably modulating the effects of heparan-

binding growth factos (e.g. FGF) to osteoblastic cells (Blanquaert et al. 1999). In addition to proteoglycans, osteoblasts produce several glycoproteins in

bone matrix including the aforementioned FN. Sialic acids are often linked to

glycoproteins; for example, proteins characteristic of bone and dentin tissues –

OPN, BSP (which is strictly specific for mineralized tissues), bone acidic

glycoprotein-75 (BAG-75) as well as dentin-specific dentin matrix protein -1

(DMP1) and dentine sialoprotein (DSP) – are rich in sialic acids (Oldberg et al. 1986, Somerman et al. 1988, Butler 1989, Ohnishi et al. 1991, Butler et al. 1997,

Qin et al. 2001). Along with these, osteoblasts express calcium-, collagen-, and

hydroxyapatite-binding osteonectin, which is a phosphorylated, sialic acid –

containing glycoprotein accounting for the greatest proportion of osteoblast-

derived NCPs in bone (Termine et al. 1981). Osteonectin has also been shown to

play a role in breast cancer metastasis to bone (Campo McKnight et al. 2006).

Additionally, the presence of the terminal sialic acid and other glycosylations on

the surface of osteoblasts is crucial for the tethering and proliferation of

hematopoietic progenitor cells (Crean et al. 2004).

An additional perspective on the links between glycomolecules and bone

tissue is represented by sugar-binding sites of osteoblasts. For example, galectin-3

is expressed also in osteoblasts, in which the galectin-3 production is altered by

so-called advanced glycation end-products (AGE) thus probably affecting

osteoblast and bone tissue metabolism (Stock et al. 2003, Mercer et al. 2004).

AGEs are produced in diabetes mellitus and probably involved in the appearance

of osteopenia since accumulated AGEs in bone ECM seem to play a role in the

regulation of osteoblast functions that are dependent on the osteoblast

differentiation phase (McCarthy et al. 1999, McCarthy et al. 2001a, McCarthy et al. 2001b). AGEs also seem to affect osteoblastic attachment via integrins by

modifying collagen-I of the bone ECM (McCarthy et al. 2004).

Osteoblasts also express a membrane-bound osteoclast inhibitory lectin

(OCIL) capable of inhibiting osteoclast formation (Zhou et al. 2001, Hu et al. 2004). OCIL-expression of osteoblasts is regulated by various factors including

e.g. parathyroid hormone -related protein (Zheng et al. 2009). Soluble

recombinant mouse and human OCILs bind several important GAGs (Gange et al. 2004).

29

2.3 Bone implants

Implanting substitutive devices and prostheses into pathologically destroyed or

injured tissues (as a consequence of e.g. osteoarthritis, osteoporosis, or trauma) is

a relatively common and clinically important approach to compensate for the

anatomical loss of the tissue and to restore its biological function. A standard

material in bone and dental implants is titanium (Ti), a metallic element proven to

be sufficiently physiologically inert to be used in clinical implantations. Cobalt-

based alloys have also performed well, whereas the formerly used stainless steel

has proven to be too stiff for orthopedic purposes. A shape-memory alloy, nickel-

titanium (NiTi), has also been widely studied as a hard tissue implant material

(Kapanen et al. 2001, Muhonen et al. 2009). Occasionally, however, implanting

procedures may lead to severe adverse effects. Inefficient implant insertion,

inflammation, and / or infection may prevent healing and result even in implant

loosening. The possible complications usually are consequences of insufficient

biocompatibility between the implant and the cells producing the surrounding

tissue.

2.3.1 Biocompatibility: Biological factors

In the case of hard tissue implants, biocompatibility refers to the interactions

between a biomaterial and bone cells. Biocompatibility is characterized by the

ability of the cells contacting the biomaterial to remain alive and functional; these

functions should not be disturbed by contact with the implant. In a broader

context, biocompatibility signifies that unacceptable harm to the recipient of a

biomaterial is avoided. Biocompatibility is a transient concept since its definition

varies with the progress in biomaterial science (Williams 2008).

In orthopedic and dental implants, osseointegration is a critical factor, which

is dependent on various interactive processes between the contacting cells –

especially osteoblasts – and biomaterials. First, cells have to attach to the

biomaterial surface. Osteoblasts adhere via integrins, the plasma membrane

receptors described above. A commonly used method of detecting integrin-based

cellular attachment in vitro is the fluorescent labeling of the protein components,

e.g. paxillin and vinculin, of FAs. Another, osteoblast-specific indicator of cell

attachment onto biomaterials involves ECM protein heparin-binding sites and

heparan sulfate proteoglycans of osteoblastic plasma membrane. This

proteoglycan-based adhesion is especially mediated by the so-called KRSR

30

(lysine-arginine-serine-arginine) peptide found in the ECM protein vitronectin,

and several studies discuss the use of these components in the improvement of

orthopedic biomaterials (Dalton et al. 1995, Dee et al. 1998, Dettin et al. 2002,

Bagno et al. 2007).

Secondly, after attachment, cells should begin to proliferate. Proliferation is

the phase of vigorous division, growth, and spreading, during which the cells

usually do not express specific differentiation markers. The tendency of a

biomaterial to support proliferation can be detected by e.g. staining the

cytoskeletal actin to visualize the cellular morphology and spreading.

Thirdly, cells have to differentiate into mature osteoblasts capable of

producing the mineral required for the effective osseointegration of a bone

implant. The differentiation competency of osteoblasts can be traced by

investigating certain differentiation markers, such as the expression of

osteoblastic genes (e.g. ALP) and mineral deposition (e.g. quantifying the calcium

produced).

The biocompatibility of bone implants proven by osseointegration is mainly

dependent on the functionality of osteoblasts. In fact, it has recently been

suggested that bone formation on artificial materials does not deviate from the

natural process of bone formation. Bone-implant bonding is created by the

interdigitation of the osteoblast-produced, non-collagenous bone cement with the

sub-micron scale topography of the biomaterial rather than a phenomenon defined

solely by the bioactive chemical features of the material surface (Davies 2007).

The activation of resorbing osteoclasts as such may be undesirable in

implantations. However, in prescreening the biocompatibility of the bone-

contacting biomaterials, osteoclasts serve as a feasible cell model due to their

extremely fine-tuned and sensitive growth requirements and differentiation

processes. Additionally, balanced interactions and communication between

osteoclasts and osteoblasts are crucial around the implants as well; the signals

subsequent to resorption that attract osteoblasts to the site could be beneficial also

in the osseointegration of hard tissue implants.

2.3.2 Biocompatibility: Physicochemical factors

Metal surface microtopography has been reported to strongly affect the cells so

that increased roughness of e.g. the titanium surface seems to promote osteoblast

differentiation in parallel with decreased osteoclast formation (Boyan et al. 2002,

Schneider et al. 2003, Lossdorfer et al. 2004). The porosity of a biomaterial also

31

enhances bone formation around certain biomaterials, such as a recently reported

glass fiber – reinforced composite (Mattila et al. 2009). However, the fixation

between bone and total joint arthroplasties, their subsequent permanency and

possible need for revision variably depend on e.g. the overall implant design,

texture, porosity, polishing, wear debris production, fatigue failure, micromotion

and probably elevated hydrodynamic pressure (Bauer & Schils 1999a, Bauer &

Schils 1999b).

The attachment of the cells onto a biomaterial depends largely on e.g. the

adsorption of proteins onto the implant surface, which is the first important phase

in determining the biocompatibility between tissue and foreign materials. Protein

adsorption from surrounding fluids specifically affects the behavior of the cells

that contact the proteins (Horbett & Weathersby 1981, Horbett 1981, Steinberg et al. 1989). Especially proteins containing the RGD sequence play a definitive role

in determining the biocompatibility of the implants. Protein molecules in the

surrounding fluids as well as those secreted by cells are adsorbed onto the implant

material surface depending, for example, on the wettability – i.e. hydrophobicity /

hydrophilicity – of the surface. The pursuant effects are conditional on the

material surface properties and on the types of proteins adsorbed (Steinberg et al. 1989, Lee & Voros 2005). Many physical properties including the wettability and

electrical charge of biomaterials are linked to the type and the size of the

molecules on a surface, which should be carefully considered when coating

biomaterials. The wettability of a biomaterial may affect integrin receptor

expression of the cells: For instance, human fetal osteoblasts prefer a hydrophilic

to a hydrophobic surface in terms of αvβ3-integrin expression and cell spreading

(Lim et al. 2005). Hydrophobicity is also generally considered as a disadvantage

for biomaterial biocompatibility (Chang et al. 2005).

2.3.3 Host responses to implantation

Implantation of a biomaterial into tissue always induces a so-called foreign body

reaction at the implantation site. This reaction can be regarded as a phase in a

wound healing reaction series, which usually involves acute/chronic inflammation

and thrombosis to a varying degree. A foreign body reaction is preceded by the

formation of fibrin-replacing fibrous connective tissue (i.e. granulation tissue),

and followed by the formation of a fibrous tissue capsule (i.e. fibrosis) around

injured tissue. The response to implantations is also characterized by the arrival of

monocytes, which subsequently mature into macrophages and fuse to form so-

32

called foreign body giant cells (FBGC). For example OPN is reported to inhibit

the formation of FBGCs to some extent (Tsai et al. 2005). From the carbohydrate

point of view, it is worth mentioning that macrophage fusion is partly mediated by

mannose receptors located on their plasma membranes (McNally et al. 1996,

DeFife et al. 1997).

In contrast to natural (without implants) wound healing, host responses to an

implant include protein adsorption onto the implant surface. This adsorption

might promote chronic inflammation at the implantation site. Additionally,

infections caused by bacteria, often Staphylococcus aureus and skin-associated

Staphylococcus epidermidis that attach to implant surfaces are classified as

adverse outcomes of implantations leading to a risk of implant loosening. In fact,

infection frequencies in total hip and knee joint prosthetic implantations average

between 1.5–2.5% in primary and between 3.2–5.6% in revision operations, of

which S. epidermidis accounts for 19–37.5% and S. aureus for 22–23.6% of the

cases (Lentino 2003, Rohde et al. 2007). (Gristina & Costerton 1985, Anderson

1988)

Chronic inflammation around an implant may also increase the release of

wear particles from the biomaterial surface under mechanical strain over time.

This phenomenon is likely to lead in unwanted osteoclastic activity and bone

resorption around orthopedic implants. Osteoclasts are induced by the

inflammatory cytokines and other factors, e.g. interleukins (IL-1, 6, 11), TNFs,

OPG, and RANK/+, produced by the wear particle -stimulated cells (Konttinen et al. 2005). The resulting peri-implant osteolysis is the most common reason for

aseptic implant failure, which also may be partly mediated by lymphocyte

responses to the metal ions released (Haynes et al. 2004, Hallab et al. 2005).

Additionally, wear debris from a biomaterial may carry adherent, bacteria-specific

endotoxins, e.g. lipopolysaccharide (LPS), which also have been shown in some

cases to stimulate osteoclasts and cytokine secretion (Bi et al. 2001, Gorbet &

Sefton 2005).

In order to minimize undesirable host responses and the subsequent risk of

implant loosening, the modification of biomaterials with various molecular

coatings aims to enhance the biocompatibility of implants.

2.3.4 Coating of bone implants

The clinical improvement of bone implants by various coatings has recently been

under intense investigation. ECM components including various proteins and

33

polysaccharides that contribute significantly to the regulation of tissue

homeostasis, have been investigated as potential biomaterial coatings (Morra

2006, Furth et al. 2007). Several examples of interactions between bone cells and

biomaterial coatings have been reported: For example, collagen – a naturally

occuring protein component of bone tissue – has been tested as a coating

molecule (Morra et al. 2003a). Bioactive coatings of hydroxyapatite (HA) and

other ceramics have been found to contribute positively to joint arthroplasties and

dental implants (Dumbleton & Manley 2004, Alzubaydi et al. 2008).

Pharmacologically-tuned implants have also been prepared; for example, HA-

coated titanium implants have been treated with zoledronate (a bisphosphonate) to

reduce peri-implant osteolysis (Peter et al. 2005). For bone tissue engineering, a

resorbable calcium phosphate (CaP) scaffold has produced promising results

(Lickorish et al. 2007). Also nanosized HA-based composites and bone mineral-

organic mimics are being developed (Huang et al. 2008, Best et al. 2008). The

effect of polysaccharides, e.g. foreign (non-human) chitosan on osteoblasts has

been studied (Bumgardner et al. 2003, Wang et al. 2008). Other examples of

glycomolecules considered for coating applications include hyaluronan (Morra

2005), heparin (Keuren et al. 2003), alginic acid (Morra & Cassinelli 2000), and

dextran hydrogels (Ferreira et al. 2004). Plant-derived starch polysaccharides

have also been tested in the context of bone scaffolds (Salgado et al. 2007).

The substratum to be coated in the case of bone implants is usually titanium,

even though other biomaterials, such as algae-derived hydroxyapatite ceramic

bone substitute, have also provided promising in vitro results (Turhani et al. 2005). Despite the general physiological inertness of Ti, several clinical cases

indicate problems relating to titanium implantations and thus the need to seek

appropriate molecular improvements of this implant material. Furthermore,

coating may augment the beneficial features of Ti, i.e. ability to support bone cell

proliferation and differentiation, as reported with e.g. collagen (Morra et al. 2003a), HA (Goto et al. 2004), FN (Schneider & Burridge 1994), and different

adhesive peptides (Dettin et al. 2009). However, the possibility to precisely

modify and tailor the bioactive coating molecules to improve their

biocompatibility would be a significant advance. Novel candidates for this

purpose are pectins.

34

2.4 Pectins

Pectins were first identified in 1825 (Braconnot 1825a, Braconnot 1825b). These

large and complex polysaccharides form a versatile molecular group of structural

components of taxonomically higher, land-habiting plant cell walls

(dicotyledons), as well as of some algae. Pectins are most abundantly found in the

primary plant cell wall and also in the middle lamella between contiguous plant

cells. Together with hemicellulose molecules, pectins comprise the major (on

average 40% of the dry weight in fruits and vegetables) elements of the plant cell

wall matrix, within which cellulose microfibrils construct a rigid lamellar network

capable of bearing osmotic pressure as well as mechanical stress. The schematic

topography of pectin molecules in a plant cell wall is outlined in Figure 2.

Pectins play several important roles in plants; they participate in e.g.

mechanical support, morphological development, fruit ripening, and

emulsification of plant tissue (Redondo-Nevado et al. 2001, Vincken et al. 2003).

Especially the emulsifying role is noteworthy since the hydrocolloidal gel-

forming property of pectins is widely exploited in e.g. the food industry. This

ability of pectins to gel is due to many negatively-charged sugar units (containing

-COO- groups) in the homogalacturonan region that are prone to bind Ca2+ cations

and form so-called “eggbox” structures. This calcium cross-linking of pectin

subsequently leads to profuse hydration.

Fig. 2. Plant cell wall architecture showing the topography of pectins.

35

The structural details of pectins vary both species- and tissue-specifically but

some elements are common to all pectins. Two major structural components can

be separated: so-called smooth and hairy regions. The smooth region consists of

α-1,4-linked D-galacturonic acid (Fig. 3) residues (α-D-GalA), some of which are

esterified on the carboxyl group with methanol yielding either a high-methyl

(HM) or low-methyl (LM) homogalacturonan chain. In contrast, the hairy region

(also known as the ramified region) contains two alternating sugar residues, α-

1,4-linked D-galacturonosyl and α-1,2-linked L-rhamnosyl moieties, forming a

rhamnogalalacturonan-I (RG-I) backbone. The RG-I backbone might carry

various kinds of chemical groups (e.g. acetyl groups linked to hydroxyl groups of

galacturonic acid residues) and side chains (e.g. rhamnose-linked arabinan and

arabinogalactan as well as partly methanol-esterified xylogalacturonan polymers).

Pectins may also contain a conserved and complex RG-II component attached to

the homogalacturonan region (Ishii & Matsunaga 2001). The complexity and

variability of different pectins follow from the diverse combinations of the many

structural blocks, chemical groups, and side chains, which provide pectins with

varying physico-chemical as well as biological properties. (Doco et al. 1997,

Willats et al. 2001, Vincken et al. 2003, Hinz et al. 2005)

2.4.1 Pectins as candidates for biomaterial applications

Pectins are novel and interesting candidate macromolecules for implant coating

applications. In general, plant-derived polysaccharides hold a great potential in

cell behavior modulation, as already indicated with various soluble botanical

sugars and mammalian cell types (Ding et al. 2003, Brunold et al. 2004, Deters et al. 2005). Besides these, the effects of artificial sugar-grafted polystyrene

substrata have also been assessed (Kim et al. 2003).

Due to their botanical origin, the acquisition and use of pectins would not

raise ethical questions. They are readily available and inexpensive since they are

remnant by-products of e.g. juice manufacture. Additionally, it is noteworthy that

the human body cannot degrade these plant-derived molecules, which apparently

is an intriguing feature in biomaterial implantations. Finally, the prime benefit of

screening pectins as coating molecules is their vast natural diversity combined

with the possibility to specifically tailor them in vitro and in vivo.

36

2.4.2 Modification of pectins

Pectin composition depends on the plant species and tissue types: For example,

four different pectin fractions have been isolated from apple (Schols et al. 1995).

In addition to this natural variation on pectin structures, they can be further

tailored in vitro with pectinolytic enzymes – usually as commercial, micro-

organism -derived enzyme mixtures designed for degrading disruptive pectin gels

and to promote the release of beneficial flavonoids in juice production. After

pectin isolation, it is possible to separate smooth and the RG-I -containing hairy

regions with endopolygalacturonase and rhamnogalacturonan hydrolase.

Polygalacturonases (acting either exo- or endo enzymes) degrade

homogalacturonan without methyl esters, and rhamnogalacturonases cut the

glycosidic bond between rhamnose and galacturonic acid residues, respectively

(Mutter et al. 1996). The charge, wettability, and polarity of these molecules can

easily be controlled by modulating the content of methyl and acetyl groups as

well as the chain length of these molecules with e.g. pectin methyl and acetyl

esterases and specific hydrolases and lyases (pectin lyases can also attack methyl-

substituted homogalacturonan) (Schols et al. 1994, Schols et al. 1995, Mutter et al. 1998a, Mutter et al. 1998b, Daas et al. 2000, van Alebeek et al. 2002, van

Alebeek et al. 2003). These enzymes are strictly specific to certain substructures

of pectin meaning that their actions are not dependent on the composition of the

whole molecule (Schols et al. 1994). Methyl esterases of fungal origin tend to act

randomly, whereas the corresponding plant-derived esterases function merely in a

blockwise manner, although many factors seem to influence this mode of action

(Micheli 2001). Acetyl esters from the homogalacturonan region are removed by

pectin acetyl esterases and from the RG-I region by rhamnogalacturonan acetyl

esterases (Searle-van Leuween et al. 1992). The attack sites of pectin-specific

enzymes are shown in Figure 3.

37

Fig. 3. General structural organization of a pectin molecule and the attack sites of

pectin-specific enzymes. Modified from (Voragen et al. 1995, Morra et al. 2004).

Pectin fragments formed by enzymatic treatments are designated as modified

hairy regions (MHRs). It may be possible to exploit the differences in the

chemical, physical, and biological properties of these molecules in the coating of

medical devices. MHR fragments can indeed be considered as nanocoatings since

they have been deposited onto surfaces to form a 6–10 nm thick coating layer.

MHRs can be covalently linked onto a surface via carbodiimide condensation.

The surface to be coated is first functionalized with allylamine plasma deposition

(aminated surface, AMI), after which a covalent bond is formed between the

deposited amino groups and the carboxyl groups of the MHRs. X-ray

photoelectron spectroscopy (XPS) has been used to verify that the immobilization

of MHRs is successful and uniform. Atomic force microscopy (AFM) has in turn

revealed that the repulsive reaction of MHRs occurs over a shorter range than e.g.

carbodiimide-condensed hyaluronan (Morra et al. 2003b). This probably reflects

the fact that, unlike e.g. the merely hydrogen-bonding hyaluronan, the ionic

repulsion of MHRs could be more significant since here the neutral side chains as

well as acetyl and methyl esterifications are emphasized. (Morra et al. 2004)

Another means of tailoring pectin fragments is their in vivo modification in planta (McCann et al. 2001). For example, the pectin composition of potato

tubers has been engineered by expressing a fungal endo-galactanase cDNA in

transgenic potato plants (Oxenboll et al. 2000).

38

Interestingly, LM pectin hydrogels cross-linked either ionically (with Ca2+

ions) or covalently (with divinylsulfone) have also been recently evaluated for

their ability to promote apatite nucleation in the presence of simulated body fluid

(SBF) – a crucial phase in bone-bonding of biomaterials (Ichibouji et al. 2008,

Ichibouji et al. 2009). In turn, pectin nanogel cross-linked with glutaraldehyde has

shown potential as drug delivery system (Chang et al. 2007).

2.4.3 Immunological aspects of pectins

Implant failures often appear as a consequence of excessive host reactions at the

implanted tissue area. Inflammation represents the innate immune system, in

which monocytes/macrophages as well as other leukocytes play important roles.

In contrast, specific immunological responses always involve the action of

lymphocytes, i.e. antibody-producing B-cells and helper- or cytotoxic T-cells,

which possess immunological “memory” and a coordinated, very exact system of

attacking antigens.

Variable and partially opposing immunological effects of pectins have been

described. For example, citrus pectins are reported to have some anti-

inflammatory effects on LPS-stimulated macrophages, a promising discovery

from the implantation point of view (Chen et al. 2006). The degree of

esterification of pectins also plays a role: According to Salman and colleagues

(2008), citrus pectins with a higher degree of esterification are more prone to

inhibit the production of some pro-inflammatory cytokines and to enhance

production of the anti-inflammatory cytokines of human peripheral blood

mononuclear cells (Salman et al. 2008). On the other hand, some pectins promote

certain immunological responses, such as the stimulation of lymphocytes (Sakurai et al. 1999, Dourado et al. 2004a) as well as complement activation (Wang et al. 1994, Michaelsen et al. 2000a). Interestingly, it has also been shown that, in some

cases, a whole pectin molecule is immunologically inert, whereas its degradation

fragments are not (Wang et al. 2003).

The use of MHRs as a bone implant nanocoating is to a great extent

dependent on how it will be tolerated by human tissues. Neutrality in this respect

is the minimum requirement for clinical applications, but naturally it would be

beneficial if MHRs exhibited active anti-inflammatory features of the MHRs. The

immunological potential of MHRs may partly be determined by the acetyl and

carboxyl content indicating that it may be possibile to adapt the immunological

reactivity of the tailored MHRs.

39

3 Aims of the present study

Modified hairy regions (MHRs) of pectins are new, innovative candidate

macromolecules for the improvement of hard tissue implant biocompatibility.

These covalently attached plant-derived polysaccharides could work as a

functional coating on bone implants since the mammalian tissues are unable to

resorb them. MHRs can also be modified to assume various important surface

structures and properties. To examine the potential of pectins, their ability to

support bone cell growth as well as their probable immunological effects were

investigated.

The aims of the present research are summed up as follows:

1. To assess the interactions between both osteoclastic and osteoblastic cells and

the MHRs of apple pectin. By testing different MHRs (MHR-A and MHR-B)

we aim to reveal those properties of pectins that stimulate the attachment and

growth of bone cells.

2. To test the differentiation capacity of osteoblasts on MHRs grafted onto

titanium in order to gain information about bone formation and

mineralization processes on a pectin-coated bone implant material.

3. To investigate the possible inflammatory reactions induced by the MHR-

coated titanium implants in soft tissue in vivo.

4. To assess the interactions between osteoblastic cells and the MHRs of wild-

type and transgenic potato pectins.

40

41

4 Materials and methods

4.1 Cell cultures

The use of both primary and cell line -derived bone cell cultures (studies I, II, and

IV) are briefly explained in the following chapters.

4.1.1 Cell lines

Murine calvarial preosteoblastic MC3T3-E1 (subclone IV) cells (ATCC®, LCG

Promochem) were maintained in (I, IV) or induced to form osteoblasts (II). The

cells were cultured in α-MEM (Dulbecco`s modified Eagle`s medium, α-

modification) supplemented with 10% fetal bovine serum (FBS), 1% L-

glutamine, and penicillin (100 U/ml) – streptomycin (100 µg/ml) antibiotics. In

the mineralization medium, 50 µg/ml L-ascorbic acid 2-phosphate (AsAP) and

10-2 M β-glycerol phosphate disodium salt pentahydrate (β-GP) were added. Cells

were cultured in an incubator (+ 37 ºC, 5% CO2, 95% air) in T25 cell culture

bottles, released with trypsinization, and seeded at a density of 10 000 cells/cm2

on test materials (for 2 weeks in mineralization cultures).

Human mesenchymal cells (hMC) from bone marrow (Cambrex Inc.

Walkersville, Md) were cultured (by Dr. Marco Morra and colleagues, Nobil Bio

Ricerche, Portacomaro, Italy) in D-MEM containing 10% FBS, 1% L-glutamine,

and 100 U/ml penicillin + 100 µg/ml streptomycin antibiotics. Cells were

differentiated to mineralizing osteoblasts with 0.05 mM AsAP, 10 mM βGP, 0.1

µM dexamethasone, and mesenchymal cell growth supplement (MCGS)

additives. Cells were released with trypsinization and cultivated (105

cells/titanium disc) on test materials (II).

4.1.2 Primary cultures

Osteoclasts were differentiated from a bone marrow cell pool from long bones of

1–2 day old Sprague-Dawley rat pups (pit assay; I). Endosteal cells were scraped

into α-MEM (Sigma) containing 10% FBS, 1% L-glutamine, 100 U/ml penicillin

+ 100 µg / ml streptomycin, and 20 mM HEPES buffer. The cells were collected

by centrifugation, resuspended in the medium, and cultured (50 µl cell

suspension/cm2) on test materials for 2–4 days.

42

Primary osteoblasts (II) were differentiated from the bone marrow cell pool

of 12-week-old mice (male C57/BL6). Cells were cultured in α-MEM

supplemented with 10% FBS, 1% L-glutamine, 100 U/ml penicillin + 100 µg/ml

streptomycin, 50 µg/ml AsAP, 10-2 M βGP, and 10-8 M dexamethasone. After

centrifugation, the cells were resuspended in the same medium and cultured on

test materials at a density of 106 cells/cm2 for 2 weeks.

4.2 Test materials

The preparation and covalent grafting of the apple- and potato-derived pectin

fragments (MHRs) used in the studies (I-IV) are briefly explained in the

following chapters.

4.2.1 MHR preparations

Two different MHRs isolated from apple tissue (MHR-A and MHR-B) and

genetically engineered or wild type potato MHRs (MHRPTR_ARA,

MHRP_TRGAL, and MHRP_WT) were tested in this thesis. MHR-A and MHR-B

were obtained by in vitro modification with commercial enzyme preparations

[Rapidase 600 for MHR-A and Rapidase Rliq+ for MHR-B (DSM Food

Specialties)].

As reported in Table 1, MHR-A and MHR-B differ particularly in their acetyl

group content, arabinose amount, and side chain length. MHR-A contains linear,

mainly arabinan-rich side chains composed of on average > 10 arabinan units.

These chains have been digested with arabinanases from MHR-B (which contains

only 1–2 arabinoses in a side chain) resulting thus also in a lower molecular

weight (MW). However, the total sugar molar percentage is greater in MHR-B;

especially xylogalacturonan predominates in this fragment. MHR-B possesses a

lower degree of acetylation, whereas MHR-A has abundant acetyl substitutions.

The level of methylation does not differ significantly. (Morra et al. 2004)

Potato MHRs were produced by in vivo modification in transgenic / wild type

potato (Solanum tuberosum L. cv. Posmo) tubers (cultivated by Dr. Peter Ulvskov

and colleagues, Department of Genetics and Biotechnology, University of Aarhus,

Denmark). Transgenic MHRPTR_ARA reduced in arabinan and MHRPTR_GAL

reduced in galactan were obtained by expressing specific arabinase and

galactanase enzymes in the potato plants, respectively. Wild type potato

43

(MHRP_WT) was also tested. The sugar compositions of the MHRPs are

represented in more details in the study IV.

The MHR preparations were treated and analyzed by Dr. Henk Schols and

colleagues (Laboratory of Food Chemistry, Wageningen University, Wageningen,

The Netherlands).

4.2.2 MHR-coated samples

MHRs were covalently linked (Dr. Marco Morra, Nobil Bio Ricerche,

Portacomaro, Italy) to different test materials: tissue culture polystyrene (TCPS)

dishes (I, IV), objective glasses (I), titanium discs (II) or cylindrical titanium

items (III). Briefly, the test material surfaces were functionalized with amino

groups by allylamine plasma deposition (aminated surface, AMI), which is a

commonly used plasma modification method (Puleo et al. 2002). MHRs were

subsequently linked to the surfaces via covalent bonding between the amino

groups and the carboxyl groups of the MHRs (carbodiimide condensation). The

coating protocol is described in more detail by Morra and colleagues (Morra et al. 2004). Uncoated surfaces were used as controls. Test materials were sterilized

with 10 x solution (Sigma) of penicillin-streptomycin (I, II, IV) or with ethanol

(III).

Table 1. Molar percentages (mol-%) of sugar moieties of the apple-MHRs. Partly

modified from (Morra et al. 2004).

Sugar MHR-A (mol-%) MHR-B (mol-%)

Arabinose 50 11

Galactose 10 20

Galacturonic acid 26 37

Methyl / 100 mol of GalA 40 34

Acetyl / 100 mol of GalA 55 11

Glucose 1 3

Mannose 0 0

Rhamnose 5 11

Rhamnose:GalA 0.19 0.3

Xylose 8 18

% (w/w) sugar 66 78

44

4.2.3 Bone slices

Slices (~1.0 cm2, thickness 0.5–1.0 mm) of cortical bone of bovine femurs were

used as a cell culture control substratum. Briefly, slices were cut, sonicated (3 x

30 s), treated with 70% EtOH (prepared by Mikko Finnilä), and used as a culture

surface for bone resorption assays (I).

4.3 Stainings

For visualizing cells (studies I, II, and IV) and histological sections (study III),

various fluorescent and non-fluorescent staining methods were used.

4.3.1 Fluorescent labels

Before staining, the cells were fixed with 3% paraformaldehyde (PFA) in PBS

(supplemented with 2–4% sucrose) for 10 min at RT and rinsed with 1x PBS.

Cell nuclei were stained with a 1.25 µg/ml solution of DNA-binding Hoechst

33258 fluorochrome (Sigma Chemical Co.) for 10 min at RT (I). F-actin was

labeled with 500 ng/ml phalloidin (Sigma Chemical Co.) conjugated with FITC

(green fluorescence) or TRITC (red fluorescence) for 20 min at 37 ºC (I, II, IV).

Paxillin (I, II, IV) and vinculin (I) dots of FAs were stained with indirect

immunofluorescence labeling. The cells were permeabilized with 0.1% (v/v)

Triton-X-100 in PBS (10 min on ice) and blocked with 0.2% BSA (30 min at RT).

The permeabilized cells were then incubated with a 1:100 dilution of a

monoclonal mouse paxillin (ZYMED Laboratories) – or vinculin – (Sigma

Chemical Co.) specific primary antibody for 45 min and with a secondary

antibody (ALEXA Fluor 568 or ALEXA Fluor 488 goat anti-mouse IgG,

Molecular Probes) for 30 min on ice.

The calcium of osteoblast cultures (II) was labeled with

tetracyclinehydrochloride antibiotic (Sigma-Aldrich), which acts as a calcium

chelator able to bind to nascent, globular calcium deposits. Tetracycline (50

ng/µl) was added into the medium of cell cultures and incubated for 5 days before

fixation.

45

4.3.2 Non-fluorescent stains

Osteoclasts (I) and macrophages (III) were stained for tartrate-resistant acid

phosphatase (TRACP) using a Leukocyte Acid Phosphatase Kit (Sigma)

according to the manufacturer`s instructions. The TRACP5b isoform is expressed

by osteoclasts and TRACP5a by activated macrophages and FBGCs (Efstratiadis

& Moss 1985a, Efstratiadis & Moss 1985b, Kadoya et al. 1994).

Histological sections (III) were stained with hematoxylin-eosin (HE). Briefly,

samples were incubated in aluminum hematoxylin for 13 min, rinsed under

running water for 5 min, differentiated in 70% ethanol containing 0.5% HCl for

15–30 s, rinsed with distilled H2O and subsequently in ammonia water (3 ml NH3

in 1000 ml H2O) for 10 s. After rinsing with distilled H2O for 1–10 min, the

samples were incubated in an eosin solution (0.5% eosin in 25–50% ethanol) for

1.5 min, differentiated by quickly dipping the samples in distilled H2O 3–6 times

and by washing them in 95% ethanol that contained 2 drops of 0.1% ammonium

hydroxide.

4.4 Microscopic methods

For microscopic visualizations, the samples were mounted in glycerol-PBS,

Pertex or Shandon Immu-MountTM (Thermo) and covered with cover slips.

4.4.1 Light and fluorescence microscopy

Light microscopy was used for the investigation of TRACP-stained osteoclasts (I)

and histological sections (III). Hoechst- and phalloidin -stained cells (I) were

visualized with a conventional epifluorescence microscope (Nikon Optiphot II)

using a suitable filter for each fluorochrome. Microscopic images were recorded

with a Sony DXC 930P video camera.

4.4.2 Laser scanning confocal microscopy

Samples stained with phalloidin (I, II, IV), paxillin (I, II, IV) and vinculin (I)

antibodies or tetracycline (II) were visualized with a confocal laser scanning

microscope (LSM 510, Zeiss) using 20×, 40×, 63× and/or 100× objectives.

46

4.4.3 Field emission scanning electron microscopy

For field emission scanning electron microscopy (FESEM) visualization of cells

and sample surfaces (IV), cut circular pieces (Ø 8.0 mm) of sample surfaces were

dehydrated in ascending ethanol series (30% 50% 70% 80% 90%, 10

min in each) and finally twice in 100% (5 min). Samples were subsequently

critical point dried (BAL-TEC CPD 030) and sputtered (Agar High Resolution

Sputter Coater) with platinum (~15 nm Pt coat). Imaging was performed with

Zeiss ULTRA Plus -machinery using 3.0 kV voltage and 4000- , 8000-, or 20000-

fold magnifications.

4.4.4 Image analyses

Microscopic fields of paxillin and/or vinculin (I, IV) or tetracycline (II) data

obtained with the laser scanning confocal microscope were analyzed with MCID

M4 3.0 Rev. 1.1 or with MCID-M5+ (Imaging Research, Inc., Canada) digital

image analyzing system.

4.5 Molecular methods

Molecular methods were used for assessing the ALP production and activity of

osteoblastic cells (study II).

4.5.1 RT-PCR

Total RNA was isolated from osteoblasts (II) with a GenEluteTM Mammalian

Total RNA Kit (Sigma-Aldrich) according to the manufacturer`s instructions.

For RT-PCR, RNA was reverse-transcribed with SuperScriptTM III Reverse

Transcriptase (InVitrogen) to cDNA, which was subsequently PCR-amplified

using gene-specific primers (Sigma Proligo) and DyNAzymeTM EXT DNA

Polymerase (Finnzymes) according to the manufacturer`s instructions. The primer

sequences for ALP were 5`-GCCCTCTCCAAGACATATA-3` (forward) and 5`-

CCATGATCACGTCGATATCC-3` (reverse) demarcating a product size of 372

bp (Qu et al. 1998, Zhao et al. 2005). For β-actin, the forward primer sequence

5`-TGGACTTCGAGCAAGAGATGG-3` and the reverse primer sequence 5`-

ATCTCCTTCTGCATCCTGTCG-3` demarcating a 289 bp product were used

(Wu et al. 2004, Zhao et al. 2005).

47

PCR reactions for both genes started with 30 min at 48° followed by 10 min

at 95º (ALP) or at 94º (β-actin). ALP was produced in 30 cycles of 1 min at 95º, 2

min at 55°, and 1 min at 72º. β-actin synthesis consisted of 23 cycles of 1 min at

94º, 45 s at 62º, and 45 s at 72º. Both reactions were completed with 7 min at 72º.

DNA products were subjected to 1% agarose gel electrophoresis, visualized

with SYBR® Green Nucleic Acid Stain (Fluka), and compared to GeneRulerTM

100 bp DNA ladder (Fermentas). Mouse genomic DNA and pure water were used

as negative controls for the PCR reactions.

4.5.2 Enzyme activity measurement

The specific ALP activity (i.e. the ratio of the absorbance to the cell number) of

hMSCs cultured on coated titanium samples was spectrophotometrically

measured (II) 3, 7 and 10 days after cell seeding (by Dr. Marco Morra, Nobil Bio

Ricerche, Portacomaro, Italy). For cell counting, a MTT (dimethyl thiazolyl

diphenyl tetrazolium salt) assay was used. In this assay, the yellow MTT solution

is reduced by the cells` mitochondrial dehydrogenase enzyme to insoluble blue

formazan thus indicating cell viability (Alley et al. 1988). Briefly, the cells were

incubated in MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium

bromide)-sodium succinate solution for 3 h at 37 °C, after which 6.25% v/v

0.1mol/l NaOH in dimethylsulfoxide was added in order to dissolve the formazan.

The wells were agitated for 5 min until the purple color was evenly distributed,

and the absorbance was evaluated at a wavelength of 560 nm.

For ALP activity measurement, the cells were lysed with lysing solution

(0.2% Triton X-100, Sigma). The lysates were then incubated with the enzyme

substrate mixture solution containing 1 mM MgCl2 x 6H2O, 1.5 mM 2-amino-2-

methyl-1-propanol and 20 mM p-nitrophenylphosphate for 2 h at 37 °C. The

reaction was stopped with 1 M NaOH. The absorbance of the samples was read at

a wavelength of 405 nm with a Tecan Genios microplate reader. Absorbances

were finally compared with standard curve values obtained by the measurements

of p-nitrophenol standard solution. The reaction steps of the specific ALP activity

are described in more details in the study II.

4.6 In vivo procedures

The in vivo tolerability of MHR-coated titanium implants were assessed in the

study III.

48

4.6.1 Animals and implantations

Animals (12-week-old Sprague-Dawley rats) used in the in vivo study (III) were

obtained and handled according to the procedure approved by the Ethical

Committee of the University of Oulu and the County Administrative Board of

Southern Finland.

Coated (MHR-A, MHR-B, AMI) and uncoated (Ti) titanium cylinders (length

6 mm, diameter 1.8 mm) were implanted under the fascia of the latissimus dorsi

muscle, during which the animals were under isoflurane (4.5% 2.5% for

maintaining) gas anesthesia. Buprenorphine (Temgesic; 0,05 mg / kg s.c.) was

used for analgesia before (30 min) and after (for 3 d) the operation. After 1 or 3

weeks, the rats were euthanized with CO2 suffocation and the implants together

with the surrounding tissues were removed for histological section preparation.

4.6.2 Histological sections

Soft tissues surrounding the implants were fixed in neutral formaldehyde solution

(100 ml 36–40% formalin, 900 ml H2O, 4 g NaH2PO4, 6.5 g Na2HPO4) and

dehydrated with an ascending alcohol series: 70% (16 h) 96% (16 h) twice

in 100% (1 h) xylene (twice for 30 min). The samples were embedded in 55–

57 ºC paraffin, and sectioned with a microtome (Microm HM 355 S). For

hematoxylin-eosin (HE) staining (described above), the sections were

deparaffinized and rehydrated in a descending alcohol series (95% absolute

ethanol twice; 3–5 min in each) leading to double xylene incubation (3–5 min).

After another dehydration phase following the HE staining, the sections were

mounted onto glass slides for microscopy. Sections were prepared and stained by

Mrs. Anna-Maija Ruonala and Mrs. Paula Salmela.

4.7 Statistical analyses

Statistical analyses were run with SPSS14.0 software. The normality of the

datasets was determined with a Kolmogorov-Smirnov test. For normal

distributions, a parametric analysis of variance (ANOVA) and a t-test were used.

Kruskal-Wallis and Mann-Whitney tests were used for nonparametric analyses.

The statistical significance was set at a p-value ≤ 0.05. Origin 6.0 or Origin 7.5

software was used for constructing the histograms. Statistical significance is

denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

49

5 Results

5.1 Interactions between MHRs and bone cells

The growth of both bone-resorbing osteoclasts and bone-forming osteoblasts on

different, covalently-linked MHR-types were studied in this thesis.

5.1.1 Proliferation and differentiation of osteoclasts (I)

The in vitro biocompatibility between MHRs and bone cells was preliminarily

screened with osteoclasts. Primary osteoclasts differentiated from the bone

marrow cell pool were cultured on MHR-grafted and on uncoated TCPS as well

as on bone slices as control surfaces. Of the pectin fragments, MHR-B was

preferred by the cells, as indicated by cellular density, morphology, and a greater

number of TRACP-positive (TRACP+) osteoclasts. The total number of TRACP+

cells on MHR-B also exceeded that on uncoated TCPS (p < 0.01) yielding values

statistically similar to those on the bone slice surface. In addition to the total cell

number, osteoclasts containing 1–2 nuclei and 3 or more nuclei (indicated as

multinuclear cells) were calculated separately. Osteoclasts with 1–2 nuclei tended

to reject MHR-A (p < 0.001), whereas they preferred MHR-B even over uncoated

TCPS (p < 0.01) or bone slices (p < 0.05). The number of multinuclear (≤ 3

nuclei) cells did not differ between MHR-A and MHR-B. On bone, the greatest

number of multinuclear TRACP+ cells as well as the proportion of multinuclear

cells/total TRACP+ cells was observed compared to all other sample types. The

multinuclear cells/total TRACP+ cells ratio did not differ between MHRs,

whereas the ratio of TRACP+ cells/the total bone marrow pool did in favor of

MHR-B. Actin ring staining supported the data. On MHR-A, actin rings were not

detected, on MHR-B approximately 1/3 of the cells contained an actin ring, and

on bone all the osteoclasts had formed an actin ring.

5.1.2 Proliferation and differentiation of osteoblasts (I, II, IV)

MC3T3-E1 cells (I, II, IV), primary osteoblasts from mice (II), and human

mesenchymal (hMC) cells from bone marrow (II) were cultured on either coated

TCPS dishes (I, IV) or titanium discs (II) to investigate the ability of apple

(MHR-A and MHR-B) and potato (MHRPTRGAL, MHRPTRARA, MHRPTRWT)

50

modified hairy region fragments to support the attachment, spreading, and/or

differentiation of the bone-forming cells.

On the apple MHR -coated TCPS samples (I), MC3T3-E1 cells attached well

on MHR-B and uncoated TCPS but weakly on MHR-A and aminated surface

AMI. The percentages of the cells containing focal adhesions indicated by

paxillin and vinculin stainings were MHR-A: 0.0% < AMI: 4.1% < TCPS: 72.2%

< MHR-B: 81.5% (the difference between TCPS and MHR-B was not

significant). The FA spot size differences were better-defined. Both the average

area (µm2) and length (µm) of FAs on TCPS (1.45 µm2; 2.11 µm) significantly

exceeded those of MHR-B (0.57 µm2; 1.28 µm).

On titanium (II), MC3T3-E1 cells formed paxillin dots on the MHR-A

coating (as well as on all the other surface types). Primary osteoblasts, however,

formed FAs on every titanium surface type except on MHR-A. In general, MHR-

B and pure titanium supported FA formation best.

Osteoblastic cells showed more proliferation on MHR-B than on MHR-A, as

indicated by the cellular density and morphology. On MHR-B, the confluency and

spreading of the cells strongly resembled that on uncoated TCPS or Ti, whereas

on MHR-A mainly rounded and weakly attached cells were observed. MC3T3-E1

cells on MHR-A -coated titanium were exceptionally spread, however. On

aminated TCPS, the proliferating MC3T3-E1 cells did not form cultures that were

as dense or as well-spread as the cells on MHR-B and TCPS. This was not as

clearly manifested on titanium samples.

In addition to the apple MHRs, adhesion and proliferation of MC3T3-E1 cells

on TCPS grafted with transgenic MHRPTR_ARA (ARA), MHRPTR_GAL (GAL),

and wild-type MHRP_WT (WT) potato pectins were studied in order to

preliminarily test the optional, in vivo -based modification method of pectin

fragments (IV). Three focal adhesion parameters – length (µm), proportional area

(%), and number – of MC3T3-E1 cells were quantified. Cells grew best on TCPS

and AMI controls, while only minor differences were observed between cells

cultured on the wild type (WT) or mutant (ARA, GAL) potato samples. Even

though MC3T3-E1 cells generally seemed to prefer the wild-type potato pectin

surface (compared to either of the mutant samples), the only statistically

significant deviation between the potato samples was the greatest FA dot length

on the GAL-surface (4.37 µm) compared to ARA (4.07 µm; p < 0.01) or WT

(4.17 µm; p < 0.05). On TCPS, the average FA dot length was 4.74 µm, which

represent a significance of p < 0.001 compared to all the potato samples and p <

0.05 compared to AMI. Neither the proportional area nor the number of FA dots

51

differed significantly between potato samples. Instead, the area-% and the FA

number on TCPS exceeded significantly those on all the potato sample types (p <

0.001 compared to ARA and GAL and p < 0.01 compared to WT). These results

are reported in Table 3.

Confocal and FESEM imaging of the samples revealed that osteoblasts

spread on all the surface types to some extent, but spreading was clearly better on

TCPS and AMI controls. Between the potato samples, WT generally performed

the best, and the largest cell-free areas were detected on ARA surfaces.

Visualizing the cells from a tilted angle (50º) verified flattened cell morphology

on all the sample types. Screening the sample surfaces without cells at maximum

magnification did not reveal any micro scale deformations of the sample surfaces.

Table 3. Focal adhesion parameters [mean (± SD)] of MC3T3-E1 cells grown on

different potato MHRs and control samples (IV).

MHRPTR_ARA MHRPTR_GAL MHRP_WT AMI TCPS

FA length (µm) 3.40 (0.35) 4.37 (0.12) 4.17 (0.20) 4.51 (0.24) 4.74 (0.14)

FA area (%) 1.37 (0.01) 1.21 (0.00) 1.51 0.00) 1.94 (0.01) 2.37 (0.01)

FA number 141 (40.17) 120 (39.63) 154 (46.61) 201 (44.99) 229 (50.52)

The differentiation of osteoblasts (MC3T3-E1 cells and primary osteoblasts) on

apple MHR -coated titanium samples (II) was evaluated by quantifying the

proportional areas (%) of tetracycline-stained calcium deposits. On MHR-A, no

calcium could be detected (and was thus excluded from the statistical analyses).

On all the other sample surfaces (MHR-B, AMI, Ti), statistically similar amounts

of calcium deposits were observed, the respective values being 14.0%, 12.7%,

and 14.2% for MC3T3-E1 cells, and 26.6%, 24.4%, and 27.5% for primary

osteoblasts.

The ALP expression of osteoblasts (MC3T3-E1 cells and primary osteoblasts)

grown on titanium discs (II) was screened at different time points with RT-PCR.

With MC3T3-E1 cells, ALP mRNA was not produced by day 3 (except very

dimly on Ti), whereas at day 7 and day 15 ALP is clearly expressed in all sample

types. With primary osteoblasts (whose ALP expression was generally stronger

than that of MC3T3-E1 cells), ALP was also expressed at 10 d and 15 d quite

uniformly with the exception of a fainter product band in the 15 d MHR-A

sample. The specific ALP activity of hMCs was determined on days 3, 7, and 10 by

measuring the enzyme activity per cell number. By day 3, the proliferating cells

52

showed only very low ALP activity. At days 7 and 10, cells grown on MHR-A

exhibited significantly lower (p < 0.01) ALP activity than cells grown on other

sample types (where no statistical differences were detected). By the day 10, the

most supportive sample type for ALP activity was MHR-B, which significantly

(p < 0.01) exceeded all the other sample types.

5.2 In vivo effects of MHR-coated implants (III)

Apple MHR (MHR-A and MHR-B) -coated and aminated (AMI) / uncoated (Ti)

control titanium cylinders were implanted under the latissimus dorsi -muscle

fascia of adult rats. After 1 or 3 weeks, the average fibrous capsule thicknesses

formed around the implants (foreign body reaction) were measured from HE-

stained histological sections (Fig. 4 and Fig. 5) to acertain the in vivo

responsiveness of the MHR-grafted titanium.

After 1 week, the only statistically significant difference in capsule thickness

was the more extensive fibrosis around the MHR-B implant (average capsule

thickness 42.9 µm) compared to other sample types (MHR-A: 33.2 µm, AMI:

32.5 µm, Ti: 32.3 µm). However, this difference was no longer apparent by the 3-

week time point, as indicated by the statistically similar capsule thickness values

(MHR-A: 22.5 µm, MHR-B: 24.2 µm, AMI: 26.4 µm, Ti: 23.7 µm). These results

are reported in figure 4. The decrease in capsule thickness between the 1 week

time point and the end of the 3 week follow up period was statistically significant

only on the MHR-B and Ti implants.

53

Fig. 4. HE stained tissue capsules 1 week (upper panel) and 3 weeks (lower panel)

after implantation. (50 µm scale bar) (III).

54

Fig. 5. Statistical analyses of tissue capsule thicknesses (µm) formed around different

titanium sample types A) 1 week, B) 3 weeks after implantation. (III).

Pathological interpretation of the histological sections suggested that the capsule

formation around the implants was a stromal rather than an inflammatory reaction

since the cell profile of the capsule mainly consisted of

fibroblasts/myofibroblasts. This was further verified with TRACP-staining of the

samples. Only activated macrophages and/or FBGCs show positive TRACP

staining (TRACP+). Foreign body giant cells that would represent a strong

inflammatory reaction were not detected in capsules formed around any of the

sample types. A few activated TRACP-positive mononuclear macrophages were

visualized in all sample types at both time points, but interestingly none of these

were in the actual fibrotic capsule area. Instead, they had cumulated beneath the

peri-implant area or even farther away in loose connective and adipose tissues

perivascularly.

55

6 Discussion

6.1 Effects of pectins on bone cells

Appropriate molecular coatings of hard tissue biomaterials are sought to diminish

adverse effects of the implants and to enhance osteoblast-mediated bone

formation at the implantation site. Our results indicate the short-haired apple

pectin RG-I fragment MHR-B is more biocompatible than the longer-chained

MHR-A. This general difference is the result of variable molecular weights and

compositions of the fragments, which probably generate divergent protein

adsorption profiles on the MHR-coated surfaces. The subsequent discrepancies in

cellular behavior on these surfaces were manifested by both osteoclasts and

osteoblasts.

6.1.1 Osteoclastic responses

The preliminary testing of MHRs included in vitro experiments, i.e. cell cultures

on MHR coatings. Among the bone cell types, osteoclasts are appropriate cells for

screening of the interactions between cells and MHRs. The differentiation of

osteoclasts requires specific growth conditions. The sensitivity of these delicat

cells makes them suitable for testing the biocompatibility and the degree of

tolerance of novel implant coatings.

It is generally thought that cell spreading can be considered as a characteristic

of proliferating cells, whereas rounded cells indicate either differentiation or

apoptosis (Boudreau & Jones 1999, Ma et al. 2005). When culturing a bone

marrow -derived mixed cell pool (pit assay, I) on the MHRs, the cells generally

showed a rounded morphology on MHR-A, and a spread and confluent

appearance on MHR-B. The amount of TRACP+ osteoclasts was significantly

diminished on MHR-A compared with MHR-B, TCPS or bone. MHR-A may

promote differentiation, but the cells on MHR-A might be rounded and

aggregated for other reasons, such as improper attachment and a subsequent

decrease in cell growth. However, Hoechst staining did not reveal massive

apoptosis in cells. This effect of MHR-A on cell morphology could be further

studied by observing the cultures at additional time points.

TRACP+ osteoclasts containing 1–2 nuclei and those containing 3 or more

nuclei (multinuclear) were counted separately. Interestingly, on MHR-B, a greater

56

number of 1–2 nuclear TRACP+ cells were observed in comparison to TCPS and

even bone, and the total number of osteoclasts on MHR-B slightly exceeded that

on TCPS. However, the number of multinuclear osteoclasts was higher on bone

than on any other sample type. Based on these results, MHR-B seems to be

tolerated by osteoclasts and supports their proliferation and initial differentiation,

but terminal differentiation processes are somehow hindered. Indeed, the number

of multinuclear TRACP+ cells did not differ between the two MHRs, or between

MHR-B and TCPS (a generally cell-compatible surface) comparison. Thus it

seems likely that the natural bone substrate offers some factors that allow

osteoclastic differentiation to occur properly. Defining these signals and

components could offer interesting cues for further MHR-tailoring as well.

Actin ring formation affirmed these observations. On MHR-A, actin rings

were not detected, whereas on bone, practically all the osteoclasts showed an

actin ring. On MHR-B a few (~1/3) of osteoclasts had organized a ring. Again,

MHR-B tends to allow the formation of activated – but probably not resorbing –

osteoclasts since actin ring organization can be considered as a marker of the

substratum tolerance. Additionally, it has been shown that active osteoclasts can

flourish and migrate on surfaces even without resorptive phases (Saltel et al. 2004).

The effect of MHRs on the maturation, activation, and resorption capacity of

osteoclasts could be further studied using osteoclastogenesis cultures, in which

osteoclasts progress from a stem cell level to a fully-differentiated state with

appropriate growth factors in vitro. With osteoclastogenesis assay it would also be

possible to study how, if at all, osteoclasts resorb sugar coatings. The ability of

osteoclasts to resorb non-mineralized substrates has been reported with somewhat

contradictory results. Although it has been claimed that osteoclasts resorb only

mineralized material (Chambers et al. 1984), they have also been shown to

dissolve other, even non-biological, substrata in vitro (Jones et al. 1984).

6.1.2 Osteoblastic proliferation

The compatibility between MHRs and bone-forming osteoblasts is of interest

especially with regard to the osseointegrative potential of the coating candidates.

The proliferation and differentiation of osteoblasts were examined on both apple

MHRs (MHR-A and MHR-B) as well as on transgenic (MHRPTR_ARA,

MHRPTR_GAL) and wild type (MHRP_WT) potato MHRs). Osteoblastic

proliferation was examined by screening the cells` ability to adhere and spread on

57

the surfaces, whereas differentiation was evaluated by their competence to excrete

osteoid matrix and to convert to a mineralizing phenotype.

As a proliferative indicator, the abundance and certain parameters of focal

adhesions (FAs) were determined for MC3T3-E1 osteoblastic cells (I, IV)

cultured on MHR-grafted TCPS samples. MC3T3-E1 cells are classified as

immortal (but non-cancerous) cells, which implies that they grow rather

efficiently and hence are appropriate for introductory biocompatibility screening

for anchorage-dependent cells. MC3T3-E1 cells spread and attach well onto both

MHR-B and uncoated TCPS, whereas proliferation is clearly hindered on both

MHR-A and AMI surfaces, as indicated by a diminished density and a rounded

morphology of the cells. The proportion of FA-containing cells did not differ

between MHR-B and TCPS, but interestingly the size of the FAs did. On TCPS,

FAs were larger (calculated both as FA lengths and areas) and the difference was

statistically significant. This may indicate that MHR-B allows the cells to adhere

and spread, but the FAs formed on that surface may not be as fully developed as

on the TCPS control.

Differences in MC3T3-E1 cellular behavior were also observed on the

potato-MHR samples (Table 3.). The TCPS control surface performed the best, as

expected, in terms of all the parameters tested (i.e. FA length, proportional area,

and the total number). Surprisingly, in these samples, the aminated surface

performed nearly as well as TCPS, differing significantly only with regard to the

length of the FAs. The cells also spread well on the AMI surface, unlike with

previous apple MHR cultures. In contrast to the apple MHR, none of the potato

MHRs supported cellular growth to the extent of the controls. The wild type (WT)

MHR appeared to be the best substrate, since it allowed the growth of a more

confluent and spread cell population compared to either of the transgenic samples

(ARA or GAL). However, despite the more detached appearance of the cells on

GAL, the longest FA dots were evident on this surface type. Nonetheless, neither

the proportional area nor the number of FAs on GAL exceeded the corresponding

values on ARA or WT (on which the absolute values were actually higher than on

GAL, but the differences were not statistically significant). Based on these facts it

seems that the galactose-deficient MHR type does not properly support cellular

growth, even though the few formed FAs managed to constitute lengthy dots.

ARA may thus be considered as intermediate between the GAL and WT types

with regard to osteoblastic proliferation. However, none of the potato MHRs

appears to achieve the level of the apple MHR-B, but on the other hand they do

not seem to perform as faintly as the MHR-A fragment.

58

Even though these potato MHRs did not perform appreciably well (but were

not clearly incompatible with cells either), it is important to note that also the in

vivo -produced MHRs affect bone cell behavior. Enzymes expressed in transgenic

plants for RG-I modification offer an alternative method besides the in vitro

enzyme treatments. The results also provide some preliminary information about

the significance of certain monosaccharide units of the MHR molecules. ARA

represents an arabinan-poor (~ 70% decrease) MHR-type, which has been

produced by expressing endo-α-L-1,5-arabinanase in the Golgi membranes of the

potato tuber cells (Skjøt et al. 2002). Correspondingly, lowering the degree of

galactosylation in the GAL sample was obtained by expressing endo-β-1,4-

galactanase resulting in ~ 30% reduction of galactose moieties (Oxenboll et al. 2000). Thus osteoblast attachment seems to react to the depletion of galactose

since on the GAL surface, the FA dots were the longest although paradoxically

they showed the lowest area and number. Thus the galactose deficiency appears to

be relative unsupportive for FA formation, but the few FAs formed managed to

form a long dot structure. On the other hand, the reduction of the arabinose

content did not cause obvious differences. Since the wild type MHR that

contained the default amounts of both arabinose and galactose performed better

than either of the transformants, it may be deduced that both monosaccharides

play some role in osteoblast adhesion. However, molecular analyses (by Dr. Peter

Ulvskov and colleagues, data not shown) have shown that unexpectedly, the

transgenic expressions of arabinanase and galactanase do not affect only the

arabinan and galactan side chains, respectively, but also other structures of the

RG-I molecule. The ARA resembles more the wild type, which might explain the

lack of any statistically significant FA deviations between them. However, in the

GAL sample, the backbone of the RG-I as well as the galactan chains are altered.

As a result, the MHR type deviates more from the WT. The WT and ARA

backbones consist of approximately the same number of Rha residues, whereas

the GAL backbone carries a Rha unit chain that is twice that long. Moreover, the

length of the GalA homogalacturonan chain is nearly double that in WT or ARA.

Thus in vivo modification of MHRs may alter not only the specific side chains,

but also the overall RG-I structure.

6.1.3 Osteoblastic differentiation

MC3T3-E1 cells, primary murine bone marrow cells, and human mesenchymal

stem cells (hMSCs) were differentiated using osteogenic media into mineralizing

59

osteoblasts on apple MHR (MHR-A and MHR-B) -coated titanium (II). Uncoated

(Ti) and aminated (AMI) titanium served as control surfaces. The ability of the

samples to support osteoblastic differentiation was screened by defining the

extent of calcium deposition as well as the ALP gene expression levels of the

cells. As expected, on pure Ti, the cells were both abundant and well-attached. As

in the proliferation cultures on TCPS, MHR-B again manifested better cell

compatibility than MHR-A. Cell confluency on MHR-A still remained the lowest.

However, MC3T3-E1 cells formed FAs on MHR-A (and AMI) surfaces probably

due to differences in the sample surface topography, i.e. higher roughness (Ra ~

0.300 nm) of titanium in comparison to TCPS (Park et al. 2001). The microrough

topography together with a relatively high wettability can produce capillary

effects, which are not characteristics of TCPS surfaces. In contrast, primary

murine osteoblasts did not form detectable FAs on MHR-A. Instead, the primary

cells did not appear to be viable and failed to organize an actin cytoskeleton on

MHR-A.

Quantitative evaluation of osteoblastic mineralization in vitro may often be

difficult, especially when using small culture areas, from which the isolation of

calcium for further analyses may be inadequate. In this thesis, the mineralization

capacity of osteoblasts growing on titanium discs was determined by quantifying

the calcium deposits that were stained in situ with tetracycline, a fluorescent

antibiotic used for labeling calcium in cell cultures among other applications

(Chentoufi et al. 1993, Wan et al. 2002, Mulari et al. 2004). Nevertheless,

tetracycline can diffuse into the cells to some extent and subsequently bind to free

intracellular calcium. This was considered in the microscopic analyses and only

the brightest, clearly extracellular calcium-containing mineral depositions were

measured.

The deposition of calcium by both MC3T3-E1 cells and primary murine

osteoblasts clearly differed on the two MHR samples. On the MHR-A surface,

practically no calcium could be detected – thus reflecting the inability of

osteoblasts to differentiate on this coating. However, on all the other sample

types, i.e. MHR-B, AMI and Ti, equivalent quantities of deposited calcium were

evident. Primary osteoblasts produced calcium more abundantly than MC3T3-E1

cells grown on the corresponding surfaces – an expected feature in primary vs.

cell line cultures. However, when testing metallic biomaterials, it should be kept

in mind that the differentiation progression may also be influenced by properties

other than the nanocoatings. For example, MC3T3-E1 cells have been shown to

commence mineralization later on titanium than on plastic (Matsuura et al. 2005).

60

The quantification of mineral depositions was further evaluated by analyzing

osteoblastic differentiation at the molecular level. The expression of ALP by

osteoblasts was studied either with enzyme activity measurements (hMSCs) or

RT-PCR (MC3T3-E1 cells and primary murine osteoblasts). As demonstrated by

DNA gel electrophoresis with MC3T3-E1 cells, ALP gene expression could not

be detected 3 days after cell seeding on any coating type (except very faintly on

Ti). This is the default situation during the proliferation phase. After a week in

culture, osteoblast differentiation should begin, which indeed can be seen as the

presence of ALP bands on the gel at the 7 day time point. Interestingly, the

intensities of the ALP bands from extracts of MC3T3-E1 cells grown on all the

sample types were approximately equal. At culture day 10, primary osteoblasts

should clearly express the putative differentiation markers. At this time point,

very bright ALP bands were visualized, as expected, and again circa similarly in

all the sample types. Interestingly, on day 15 with both MC3T3-E1 cells (which

also should achieve a mineralizing phenotype by then) and primary cells, ALP

bands could still be detected in all the sample types. With primary cells, the bands

are clearly more intense, which is in agreement with the tetracycline data.

Interestingly, despite the total lack of mineralization on MHR-A, ALP is also

expressed at least on the mRNA level in cells cultured on this sample surface, as

verified by both cell types (though the band was slightly paler with primary cells

compared to the other samples). This might signify that the mineralization process

may be inhibited downstream of ALP gene expression, possibly due to some

physicochemical feature of the MHR-A surface. Still, the ALP expression level

does not always prefigure the amount of mineralization, and often ALP

expression is considered merely as a marker of a bony phenotype (Walboomers et al. 2004). Besides the grafted coating, i.e. MHRs in this case, the properties, such

as surface microtopography, of the bulky material itself may play a role. As an

example, fetal rat calvarial osteoblasts produce more active ALP and mineralized

nodules on rougher titanium surfaces accompanied by decreased proliferation

(Boyan et al. 2002). It should also be noted that, for example, different MC3T3-

E1 subclones may fluctuate in e.g. ALP expression (Wang et al. 1999).

ALP enzyme activity – representing an intermediate protein-level status

between mRNA transcription and final mineralization – was investigated with

hMSCs. Only low levels of specific ALP activity were detected on culture day 3

with all the sample types, which further emphasizes the proliferative, rather than

differentiated, phase of the cells at this early time point. This also matches the

corresponding 3 d MC3T3-E1 RT-PCR results. However, at later time points, ALP

61

activity data revealed differences not apparent with the RT-PCR. At day 7, cells

grown on MHR-A did not perform as well as the other sample types; the specific

activity of ALP was lower in cells on MHR-A than in cells cultured on the other

sample types. This was also true at day 10 further indicating the inability of

MHR-A to support osteoblastic differentiation. Interestingly, the highest ALP

level was seen in cells cultured for 10 days on MHR-B, higher even than that of

cells on uncoated Ti. This difference again was not observed with the RT-PCR.

These deviations between spectrophotometric and RT-PCR outcomes may be

explained by either methodological or biological discrepancies. From a

methodological point of view, quantitative RT-PCR could possibly detect subtle

differences in the expression levels between sample types. However, this would

not change the fact that ALP mRNA is produced at least to some extent also by

cells on the MHR-A sample, which at both the enzyme activity as well as the

mineralization levels manifests the weakest biocompatibility. The biological

explanation suggests that the distinctions in differentiation capacity as well as its

timing on these sample types may become more evident at the level of protein

activity than at the level of gene expression. Furthermore, extracellular

influences, such as the presence of MHR molecules might affect the actual

mineral organization downstream of the protein level. Also the timing of the

differentiation may vary during culture. The ALP activity seems to peak at

different times on different samples, even though mineralization is equivalent by

the end of the 2-week culture time (as indicated by the tetracycline data of MHR-

B, AMI and Ti).

6.2 In vivo – testing of inflammatory responses

In addition to in vitro studies, further investigation of MHRs included in vivo

implantation in order to obtain preliminary indications of how these molecules are

tolerated in a living organism. Preclinical testing is always needed when a new

biomaterial is proposed to assess the possibility of actual clinical use. Also

biomaterials intended for use in hard tissue implants, are usually tested in soft

tissues for inflammatory screening (Jansen et al. 2004, Marques et al. 2005). This

is due to a greater sensitivity, freedom of implant mobility, and the ease of

measuring tissue responses in comparison to e.g. bone. Movement of the implant

may indeed play a significant role; for example, poly-L-lactic acid -coated

implants have been shown to cause different degrees of tissue response in

subcutaneous and periosteal implants (Parker et al. 2002).

62

Titanium samples were implanted into rats under the latissimus dorsi -muscle

fascia. After a 1- or 3-week follow-up period, the thicknesses of the soft tissue

capsules surrounding the implants were measured as indicative of possible

fibrosis reactions. The emergence of activated macrophages and/or FBGCs was

determined with TRACP staining.

6.2.1 Fibrous capsule formation

The amplitude of peri-implant fibrous capsule formation indicates (inflammatory)

reactivity toward an implant. A week after implantation, only the capsule

surrounding the MHR-B -coated titanium sample was statistically significantly

thicker than the capsule around uncoated Ti, as shown in figures 4 and 5.

However, after 3 weeks, the capsule thicknesses did not differ significantly

between any sample types. In general, capsules at 3 weeks were thinner than after

one week, as expected due to a decrease in the initial foreign body reactivity.

Between the two time points of parallel samples, this attenuation was

statistically significant only with MHR-B and Ti. This may indicate a weaker

initial reactivity towards MHR-A and AMI samples and/or more efficient healing

around MHR-B and Ti. Of course, in these interpretations, it should be notified

that the tissue reactivity might also vary between different implantation sites even

in the same animal.

The thicker capsule formation at 1 week with the MHR-B sample probably

resulted from the general tendency of this coating type to support cell growth;

abundant adhesion of fibroblasts would explain the observation. It has been

shown that murine fibroblasts grow well on this MHR-coating (Nagel et al. 2008). On the other hand, the in vitro -proven proclivity of MHR-A to reject

cellular adhesion could account for the temperate capsule formation around this

sample type. In fact, according to the histological sections (Fig. 4), the capsule

texture seemed to be more diffuse and disorganized around MHR-A (and AMI),

whereas it appeared as a more compact and uniform structure around MHR-B and

Ti. More detailed information on the capsule composition was obtained by

pathological interpretations and phagocyte-specific staining.

6.2.2 Consistence of the capsules

No strong or acute inflammations were detected in any of the samples at either

time point. The weak mononuclear reaction perceived 1 week after implantation

63

generally seemed to subside by the 3-week time point. Indeed, according to the

pathological interpretation, the cell layer surrounding the implant cavity appeared

to consist mainly of fibroblasts / myofibroblasts instead of inflammatory cells or

lymphocytes. To verify this, sections were stained for TRACP, a member of the

purple acid phosphatase family that is typically expressed by osteoclasts

(proteolytically processed disulfide-linked heteromeric 5b), activated

macrophages and FBGS (monomeric 5a isoform involved in the generation of

reactive oxygen species).

None of the fibrotic capsules contained any TRACP+ phagocytes. A few

TRACP+, mononuclear macrophages (but not FBGCs) were found farther away

from the peri-implant area, i.e. in connective and adipose tissue areas (mainly

perivascularly). This indicates that the fibrous capsules were formed mainly by

fibroblasts and the collagen produced by them. This was indicated by the absence

of activated inflammatory cells, which should be observed in large numbers in the

vicinity of the implant in strong inflammatory responses, especially if a chronic

inflammation was provoked. In an acute inflammation response, the levels of the

monocytes / macrophages as well as neutrophils are characteristically elevated

(Marchant et al. 1983). In these results, however, the samples at the early 1-week

time point maintained a hale appearance in this respect.

Considering the botanical origin, this surprising inertness of the MHR-

coating in terms of inflammatory reactivity is a promising outcome when

evaluating their clinical potential. Especially auspicious is the attenuation of and

the absence of a foreign body reaction around MHR-B, which showed good

biocompatibility with bone cells in the in vitro experiments. It is noteworthy that

the performance of this MHR after implantation did not significantly deviate from

that of pure titanium, the gold standard of hard tissue biomaterials. However, the

MHR-B coating has been shown to promote the secretion of more pro-

inflammatory cytokines IL-6 and TNF-α from J774.2 macrophages in vitro than

MHR-α, a fragment resembling the MHR-A while resulting in the lowest

proliferation index (Bussy et al. 2008). This offers interesting parallels with the

observations made with osteoclasts, which share the same hematopoietic origin

with macrophages. As mentioned earlier, the most abundant appearance of

osteoclast precursors was detected on MHR-B, whereas the number of terminally

differentiated cells did not differ between the MHRs.

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6.3 General discussion and future prospects

MHR-B coatings performed nearly or even equally as well as pure titanium in

terms of compatibility with bone cells in vitro and inflammatory tolerance in vivo.

Although it is not unusual to obtain a biomaterial that is similar in performance to

titanium, the advantage of pectins is their molecular flexibility. MHRs have the

potential to be modified with multiple enzymes to produce variable types of

fragments. The results of this thesis indicate that by tailoring pectin fragments to a

more MHR-B -like structure, more osseocompatible nanocoatings could be

achieved. Screening the naturally existing sugar molecules of bone tissue could

help direct the modification of pectin fragments towards increasing

biocompatibility.

In addition to bone cells, other cell types, such as human umbilical artery

smooth muscle cells as well as murine fibroblasts, have manifested a similar

preference for MHR-B while repelling MHR-A (Morra et al. 2004, Nagel et al. 2008). Additionally, other MHRs from different plant species, such as carrot and

potato, have been tested. An apple-derived MHR-α, which has been modified

even further to the “MHR-A -like end point”, did not perform well with

fibroblasts, whereas carrot and potato MHRs were intermediate in structure and

performance between MHR-A and -α and MHR-B (Nagel et al. 2008). Thus the

cell compatibility of MHR-B seems to be a universal tendency. Moreover, pectin

fragments of the MHR-A type could have potential in non-adhesive soft tissue

applications (e.g. stents and catheters). Furthermore, the examples of the anti-

inflammatory effects of pectins together with our in vivo tolerance results are

intruiging and require further investigation.

6.3.1 Physicochemical features of MHR-coatings

Observations of this thesis illustrate that tailoring pectin side chains (i.e. so-called

hairs) as well as additional modifications, such as altering the methyl and acetyl

contents of MHRs, may contribute to the regulation of cellular adhesion and

behavior on device surfaces coated with these materials. It is interesting that

plant-derived macromolecules can have such a noticiable effect on the growth,

attachment, and differentiation of mammalian bone cells. This interactive

functional relationship presents an interesting possibility to utilize and connect

biological processes of very distant taxonomical kingdoms.

65

In addition to the bone cells themselves, the growth factors and cytokines

involved in bone biology should immobilize properly on implants. Schierano and

colleagues (2005) have shown that as a property of the bulk material itself, porous

titanium promotes this adsorption and subsequent osseointegration of minipig

tibial implants earlier than does a machined Ti surface (Schierano et al. 2005).

Thus the protein adsorption profiles of the MHRs are another aspect of these

materials that should be studied.

When comparing the compositions (Table I) of the two apple MHRs tested,

some deductions regarding their physicochemical effects can be made. Cell

adhesion and proliferation on a biomaterial depend greatly on the adsorption of

ECM proteins (from the surrounding fluid, i.e. interstitial fluid in vivo or the

serum of the culturing medium in vitro) onto the surface, which controls

subsequent cellular responses e.g. differentiation and even apoptosis. The length

of the RG-I molecule probably plays a role. The shorter-haired structure of MHR-

B may allow cellular attachment due to a lower physical hindrance of protein

adsorption. However, the long-chained MHR-A probably provides a stronger

steric repulsion thus diminishing the accumulation of the proteins (Szleifer 1997).

The most striking difference between the MHR fragments is the degree of

acetylation: the MHR-B fragment is depleted in esterified acetyl groups in

comparison to the MHR-A. Thus the acetyl group is likely to play a pivotal role in

the regulation of cell behavior. Indeed, the general importance of O-acetylation

has been reported in other cases concerning e.g. apoptotic processes (Malisan et al. 2002, Kniep et al. 2006).

Physicochemical features affecting protein adsorption, cell adhesion and

blood coagulation, relate to the wettability of the surface: The availability of

hydrogen bonding of the solvent molecules is widely determined by attractive or

repulsive forces depending on the surface energy of a water-contacting

substratum (Vogler 1999). A hydrophilic surface usually allows for more protein

adsorption and cellular growth. For example, standard TCPS is hydrophobic, but

is usually converted to a hydrophilic surface for cell culturing. Micropatterned

surfaces further illustrate this phenomenon: Human gingival fibroblasts have

shown a distinctively patched growth on air plasma – treated areas of an alginate-

coated substratum (Cassinelli et al. 2002).

In terms of wettability, MHRs offer a tempting candidate for the control of

hydration since this represents one of the main functions of pectins in plant cell

walls (Vincken et al. 2003). Naturally, pectins carry negative charges, but their

polarity and total charge can be altered by side chain modification. For example,

66

methyl groups neutralize negative charges, and they can be removed with enzyme

or alkali treatment. In turn, the presence of acetyl groups renders pectins more

hydrophobic. Thus the acetyl (and methyl) -poorer MHR-B chemically represents

a more hydrophilic fragment type. Interestingly, however, the obvious cellular

preference for hydrophilicity can not be directly deduced with these MHRs since

MHR-B actually produces a more hydrophobic surface than MHR-A when

grafted onto a substratum. This has been verified with water contact angle

measurements of MHR-grafted TCPS samples. This is probably due to masking

of some of the ionic, and thus water-interacting, carboxyl groups that link to the

amino groups deposited in the carbodiimide condensation process. On the other

hand, the longer-chained MHR-A may produce a more wettable hydrogel surface

in aqueous surroundings. (Morra et al. 2004)

Concerning wettability, it should also be considered that the degree of

hydrophilicity / hydrophobicity -preference may vary depending on the

conditions, proteins adsorbed, and cell types. In general, the effect of surface

modifications on the integrin receptor functionality is of primary importance

when anchorage-dependent cells, such as osteoblasts, are concerned (Stephansson et al. 2002, Garcia & Keselowsky 2002, Keselowsky et al. 2005). Fibroblasts and

liver cells seem to prefer a highly wettable surface (Groth & Altankov 1996, De et al. 2002), whereas in other cases, fibroblasts as well as endothelial cells favor a

moderately hydrophilic surface (Niu et al. 1990, Faucheux et al. 2004). The types

of adsorbing proteins may play an important role in determining the biological

responses. FN is usually considered as an adhesion-promoting protein, whereas

albumin has shown contrasting features (Groth & Altankov 1996, Chen et al. 1998). Indeed, the adsorption of FN onto e.g. a titanium surface promotes

osteoblastic growth (Yang et al. 2003). The wettability preference of albumin

adsorption is not clear. According to Roach and colleagues (2005), albumin

adsorbs readily onto a hydrophobic surface (Roach et al. 2005). However, some

reports show a greater affinity of albumin towards a hydrophilic substratum, such

as a wettable vitamin E -coated surface that was subsequently repelled by

MC3T3-E1 cells (Reno et al. 2005). It has also been reported that hydrophobic

chitosan-coated titanium promotes the adhesion of preosteoblasts by favoring the

adsorption of FN and albumin onto the surface (Bumgardner et al. 2003).

Wettability may also influence the conformation of the adsorbed proteins; e.g.

adsorbed albumin has displayed a less organized secondary structure on a

hydrophobic surface (Roach et al. 2005, Ganazzoli & Raffaini 2005). It has also

been demonstrated that on MHR-B, the integrin-binding RGD-sequence of the

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adsorbed FN displays a more available conformation than on MHR-α (Nagel et al. 2008). Interestingly, solubilized MHRs show an effect on fibroblast adhesion

opposite to that of the PS-grafted MHRs probably further reflecting the

importance of the ECM-protein binding accessibility (Nagel et al. 2008).

Different MHRs may thus cause variability in protein folding, and exploring the

tendency of the MHRs to promote or hinder the adsorption of proteins, especially

FN, would clarify this aspect.

A probable explanation for the general cellular preference for hydrophilic

surfaces involves the expression of the genes encoding integrin polypeptides. As

an example, human fetal osteoblasts have grown better on a hydrophilic surface

due to active integrin expression and subsequent FA formation (Lim et al. 2005).

According to Redey and coworkers (2000), coating biomaterials with polar

components could improve the attachment of osteoblasts and thus the

osteoconduction of the implant (Redey et al. 2000). Therefore the polarity of the

tested MHRs should also be taken into account in designing subsequent

modifications. In addition, carbohydrate moieties of the ECM proteins may

contribute to the adsorption profile (Andrade & Hlady 1987). Thus carbohydrate

interactions between the MHRs and ECM proteins, including lectins may be

significant.

6.3.2 Sugar aspects

Despite the widely recognized significance of carbohydrates in biology, the sugar

biochemistry of bone tissue and the use of sugars in the design of biomaterials

still remain relatively unfamiliar areas of glycoscience. Understanding the

interactions between plant polysaccharides and mammalian bone cells is an area

of basic research that could yield information important for hard tissue implant

applications.

Compared to the conventional polysaccharide biomaterial coatings, such as

hyaluronan, modified pectins that focus on the side chain and acetyl and methyl

groups affect the ionic properties of the molecule instead of weak hydrogen bond

-based interactions (Morra et al. 2004). For example, the degree of acetylation of

polysaccharide coating molecules, e.g. chitosan, affects the osteogenic capacity of

bone marrow stromal cells (Amaral et al. 2005). The interactions between MHRs

and osteocytes, the cells representing the final stage of osteoblastic cell

development that are trapped in mineralized bone matrix, would also be

fascinating to assess. Endochondral ossification studies could reveal whether

68

these sugar components act as stimulators, vehicles or inhibitors of bone

formation in the authentic surroundings of animal tissue.

In the bone ECM, sLRPs play a significant role in the natural differentiation

of osteoblasts as well as in the fixation of an implant into bone tissue. Thus

investigating the possible effects of MHRs with the bone ECM glycomolecules

and the “sugar language” of bone cells could offer new insights into the

compatibility of bone implants. Examples of exploitable interactions between the

glycocomponents of biomaterials and bone cells already exist: a

chitooligosaccharide molecule in a soluble form promotes the growth and

differentiation of human osteosarcoma-derived osteoblasts (Ohara et al. 2004).

Added to this, chondroitin sulfate -coated fabrics induced enhanced ALP

production of hMSCs during their differentiation into mature osteoblasts

(Wollenweber et al. 2006). Studies like these are in agreement with the premise

that the gene expression profile and maturation of osteoblasts could be affected by

polysaccharides, such as pectins.

The “sugar language” of bone cells offers another interesting carbohydrate

aspect. Understanding the mechanisms by which osteoclasts and/or the exposed

bone matrix components of a newly formed resorption pit attract osteoblasts to

the BRU site could serve as a valuable tool in implant-designing; the ability to

enhance the arrival of osteoblasts around and onto the bone implant would greatly

assist the healing process by improving contacting bone formation The

aforementioned fact that resorption pits can be specifically stained with WGA

lectin implies that osteoclastic activity probably leaves some carbohydrate signals

at the BRU site. Identification of these sugar molecules could guide the further

tailoring of MHRs to a more osteoblast-inviting format.

Lectins recognize sugar epitopes. Osteoclast inhibitory lectin (OCIL)

belongs to the C-type lectin family and is expressed on the plasma membrane of

e.g. osteoblasts in bone tissue. OCIL binds to sulfated GAGs and a natural killer

cell -associated receptor, and it inhibits the differentiation of osteoclasts that is

important for normal bone remodeling (Kartsogiannis et al. 2008). Thus in further

studies concerning pectins it would be relevant to investigate the possible effects

of different MHRs on lectin expression of bone cells. The possible interactions

between MHRs and OCILs should also be studied to obtain information about the

differentiation of osteoclasts coexisting with osteoblasts. It is noteworthy that

certain dietary pectins are capable of inhibiting galectin-3 -mediated cell-cell

interactions. For example, hemagglutination can be reduced by arabinogalactan-

rich pectin polysaccharides (Sathisha et al. 2007).

69

6.3.3 Immunological aspects

Pectins have been shown to be involved in various – and partially conflicting –

immunological processes. The effects of pectins on the immune system may

result from various mechanisms including the complement system, cytokine

secretion and leukocyte recruitment. For example, a soluble form pectic

arabinogalactan from Vernonia modulates B-cell proliferation and complement

fixation in vitro (Nergard et al. 2006). Dietary pectins have been reported to play

a protective role in e.g. diabetes, heart diseases, and even cancer; it has been

speculated that cancer metastasis could be inhibited with appropriate pectin

fragments blocking galectin-mediated recognition between cancer cells and their

galactose epitopes (Inohara & Raz 1994, Nangia-Makker et al. 2002, Sathisha et al. 2007). Black currant (Ribes nigrum) seed galactans with high MWs tend to

inhibit the adhesion of Helicobacter pylori to human gastric mucosa probably by

blocking specific binding receptors on the bacterial surface (Lengsfeld et al. 2004). In turn, soluble LM apple pectin is capable of activating the apoptosis of

human colon adenocarcinoma cells (Olano-Martin et al. 2003). Pectins also

possess e.g. hypocholesterolemic and heavy metal detoxifying properties (Potter

& Steinmetz 1996, Freedman et al. 2007, Eliaz et al. 2007, Salman et al. 2008).

In some cases, pectins might excessively stimulate the immune system, which

should be taken into careful consideration in implant design. Some pectins from

the leaves of different plants, such as ginsengs (Panax ginseng C.A. Meyer) and

greater plantain (Plantago major) can activate the complement system (Kiyohara et al. 1994, Michaelsen et al. 2000b). These results probably reflect the wound-

healing effect of the leaves of some plant species. Generally plants hold a great

potential in medicine. For example, recently certain plant-derived glucocorticoid

receptor modulators have shown promising results as anti-inflammatory

components (Gossye et al. 2009).

Murine lymphocytes have been shown to be stimulated by e.g. arabinan-rich

almond (Prunus dulcis) pectin (Dourado et al. 2004b) and by root pectin

fragments of a medicinal herb, the so-called Chinese thoroughwax i.e. Bupleurum falcatum (Sakurai et al. 1999). Interestingly, the active structure of the Bupleurum dulcis -fragment (bupleuran 2IIc) is exactly the ramified region of the

polysaccharide containing an RG-core carrying abundant neutral sugar side

chains, which may indicate the structural immunological reactivity of pectins in

general (Sakurai et al. 1999). The immunologically relevant monosaccharide

composition of pectin fragments would also be important to resolve. For instance,

70

alginate rich in mannuronic acid residues induces stronger antibody production

than glucuronic acid -rich alginate when used as a biomaterial component in

microencapsulation of islets of Langerhans (Kulseng et al. 1999). Neither MHR-

A nor MHR-B contains mannose or its derivative mannuronic acid (Table I) – a

noteworthy aspect when considering the fact that macrophages carry mannose

receptors involved in the FBGC fusion process (te Velde et al. 1988, Dadsetan et al. 2004). However, the activity of other structural details of MHRs could be

further evaluated since the immunogenicity of pectins probably is at least partly

defined by their acetyl and carboxyl group content (Wang et al. 2005). The

possibility of controlling the amounts of these chemical groups in MHR-

fragments with enzymes may thus provide a way to modulate their

immunological effects.

71

7 Conclusions

Study I: The cytocompatibility of apple MHRs (MHR-A and MHR-B) was tested

with both osteoclastic and osteoblastic bone cells. Due to their sensitivity,

osteoclasts are especially suitable for the preparatory screening of a biomaterial,

and their ability to differentiate on MHR-B to some extent was a promising result.

Both primary and cell line -derived bone cells proliferated well on MHR-B and

on control surfaces (TCPS and bone). However, the MHR-A coating was clearly

rejected by both cell types. The main outcome of the study was that altering the

structure of the MHRs covalently grafted onto substrate surfaces can influence

bone cell growth.

Study II: After obtaining preliminary data from TCPS-grafted MHRs,

osteblast differentiation on similarly coated titanium samples was tested. This

process is of prime significance in osseointegration. In concordance with previous

observations of osteoblastic proliferation, MHR-B again outperformed MHR-A

by providing a substratum suitable for differentiation of both the osteoblastic cell

line and primary osteoblasts that was comparable with pure Ti, the gold standard

material in hard tissue implantations. Thus this study together with the previous

one illustrate the potential of the MHR-B fragment to support bone cell

proliferation and differentiation, and suggest that future modifications of MHRs

should be modeled after MHR-B.

Study III: Investigating the in vivo responses to MHR-coated and control

titanium samples indicated that these pectin fragments are immunologically

relatively well-tolerated since neither of them provoked significantly stronger

inflammatory reactions in soft tissue surroundings than the pure, widely-used Ti.

The slightly thicker fibrous capsule formed around the MHR-B implant after 1

week subsided after 3 weeks. Thus none of the sample types caused a severe,

acute inflammatory reaction, as verified by the absence of activated macrophages

and / or FBGS in the tissue capsules. This surprisingly good tolerance of

mammalian tissue towards these plant-derived polysaccharides indicates MHRs

hold promise for implant coatings.

Study IV: The effects of in vivo -modified MHRs isolated from transgenic

potato tubers were ascertained by screening the ability of preosteoblasts to

proliferate on them. It was found that reducing the galactose moieties on MHRs

affected the FA formation of the cells. On the other hand, the performance of

MHRs with an arabinan deficiency was similar to the wild type MHR, which was

the most biocompatible potato pectin fragment tested. However, in contrast to the

72

apple MHRs, none of the potato samples exceeded the performance of the control

TCPS surface. Both the transgenic and wild type potato MHRs were less suitable

for the growth of osteoblasts than the apple MHR-B, but were still better than the

apple MHR-A. The results of this study indicate that osteoblastic growth can be

affected by genetically engineered potato pectin fragments.

73

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Original publications

I Kokkonen HE, Ilvesaro J, Morra M, Schols HA & Tuukkanen J (2007) Effect

of Modified Pectin Molecules on the Growth of Bone Cells.

Biomacromolecules 8(2): 509–15.

II Kokkonen H, Cassinelli C, Morra M, Schols HA & Tuukkanen J (2008)

Differentiation of Osteoblasts on Pectin-Coated Titanium.

Biomacromolecules 9(9): 2369–76.

III Kokkonen H, Niiranen H, Schols HA, Morra M, Stenbäck F & Tuukkanen J

(2009) Pectin-Coated Titanium Implants are Well-Tolerated in vivo. Journal

of Biomedical Materials Research: Part A. In press.

IV Kokkonen H, Verhoef R, Kauppinen K, Muhonen V, Jørgensen B, Damager I,

Schols H, Morra M, Ulvskov P & Tuukkanen J (2009) Proliferation of

Osteoblastic Cells on In vivo Engineered Potato Pectin Fragments.

Manuscript.

Reprinted with permissions from American Chemical Society (I, II) and John

Wiley & Sons Inc. (III).

Original publications are not included in the electronic version of the dissertation.

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