a review of materials, fabrication methods, and strategies used to enhance bone regeneration in...

A Review of Materials, Fabrication Methods, and Strategies Usedto Enhance Bone Regeneration in Engineered Bone Tissues

Brian Stevens,1 Yanzhe Yang,2 Arunesh Mohandas,3,4 Brent Stucker,2 Kytai Truong Nguyen1,3,4

1 Department of Biological and Irrigation Engineering, Utah State University, Logan, Utah

2 Department of Mechanical Engineering, Utah State University, Logan, Utah

3 Department of Bioengineering, University of Texas at Arlington (UTA), Arlington, Texas

4 Joint Biomedical Engineering Program of UTA and University of Texas Southwestern Medical Center at Dallas,Dallas, Texas

Received 17 May 2007; revised 18 June 2007; accepted 26 July 2007Published online 15 October 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30962

Abstract: Over the last decade, bone engineered tissues have been developed as alternatives

to autografts and allografts to repair and reconstruct bone defects. This article provides a

review of the current technologies in bone tissue engineering. Factors used for fabrication of

three-dimensional bone scaffolds such as materials, cells, and biomolecular signals, as well as

required properties for ideal bone scaffolds, are reviewed. In addition, current fabrication

techniques including rapid prototyping are elaborated upon. Finally, this review article

further discusses some effective strategies to enhance cell ingrowth in bone engineered tissues;

for example, nanotopography, biomimetic materials, embedded growth factors, mineraliza-

tion, and bioreactors. In doing so, it suggests that there is a possibility to develop bone

substitutes that can repair bone defects and promote new bone formation for orthopedic

applications. ' 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 85B: 573–582, 2008

Keywords: bone; bone ingrowth; scaffolds; tissue engineering; bone graft

INTRODUCTION

According to the AAOS (American Academy of Orthope-

dic Surgeons), there are �6.3 million fractures each year in

the United States. In addition, the costs associated with

these fractures are extremely expensive. In fact, in 2005

there were more than 500,000 bone graft procedures per-

formed in the U.S. costing �$2.5 billion.1 About 90% of

these procedures were involved with the use of either auto-

graft or allograft bone tissues. The current standard tissue

used is autograft tissue, which is usually harvested from

the iliac crest of the patient. Although autografting has

been a major treatment, it has several limitations including

patient pain, cost, and limited supply. As an alternative,

allografting has been studied due to its abundant source.

However, its drawbacks, including the uncertainty of com-

patibility and disease transmission, have limited its use. As

society finds many more baby boomers on the list of frac-

ture patients, we need to search for better replacements for

use in bone grafting procedures.

To overcome limitations of autografting and allografting,

bone scaffolds or bone engineered tissues made from bio-

compatible materials and bone cells have been developed.

Several bone graft substitutes have been designed over the

last decade, and these bone grafts can be clarified to differ-

ent groups by a base material as suggested by Laurencin

et al.1 These include factor-, cell-, ceramic-, and polymer-

based bone graft substitutes. For example, factor-based

bone grafts consist of natural and/or recombinant growth

factors used alone or combined with other materials,

whereas polymer-based bone grafts are involved with

degradable and nondegradable polymers used alone or

combined with other materials. Many advances, new mate-

rials, and novel approaches to produce bone graft substi-

tutes continue to be discovered and investigated. The

reader is referred to several recent review articles on allo-

graft bone graft substitutes,1,2 allowing this review article

to focus primarily on a promising bioengineering approach,

bone tissue engineering (BTE), to develop bone graft sub-

stitutes for the treatment of bone diseases. In particular,

factors used in bone engineering tissues, techniques

employed to form bone scaffolds, and strategies to enhance

Correspondence to: K. T. Nguyen (e-mail: [email protected])Contract grant sponsors: URCO (Undergraduate Research and Creative Opportu-

nities), Engineering Initiatives at Utah State University, CURI grant at Utah StateUniversity

� 2007 Wiley Periodicals, Inc.

573

bone growth in these scaffolds will be discussed in this

review article.

MATERIALS, CELLS, AND SIGNALS USED INENGINEERED BONE TISSUES

Several criteria have been followed to develop an ideal

engineered bone tissue that could attain success in bone

graft procedures. A scaffold needs to be integrated with the

surrounding bone tissue and provide the initial three-dimen-

sional (3D) framework upon which cells may adhere, pro-

liferate, and eventually produce extracellular matrix (ECM)

proteins. For the scaffold to integrate with surrounding tis-

sue, it should mimic the structure and morphology of the

natural bone tissue. The bone structure is composed of

inorganic hydroxyapatite (HA) and the organic matrix con-

taining mostly (�95%) collagen type I. The morphology of

the bone has also been described as a porous (50–90%

porosity) tissue depending on the bone type.3 In addition,

in order to provide a proper 3D structure, several important

requirements for scaffolds are as follows: (i) biocompatibil-

ity, (ii) a design that closely resembles the natural extracel-

lular structure, (iii) a highly porous microstructure with

interconnected pore networks to allow cell in-growth and

reorganization, (iv) appropriate surface chemistry to pro-

mote cellular attachment, differentiation, and proliferation,

(v) sufficient mechanical strength to withstand in vivostresses and physiological loading, and (vi) controlled deg-

radation consistent with sufficient structural integrity until

the newly grown tissue has replaced the scaffold’s support-

ing function. The pore size, distribution, and structure

should also be tailored to produce specific application

requirements as they strongly influence cellular adhesion,

proliferation, and matrix deposition as well as the forma-

tion of blood vessels within the scaffolds to help tissue

ingrowth.4,5 To produce ideal bone scaffolds according to

these requirements, three major factors: materials, cells,

and signals, used in BTE will be discussed in detail in the

following section.

Materials

Several biomaterials, including bioceramics, biopolymers,

metals, and composites, have been used in BTE to form

bone scaffolds. For each material used, there are properties

of resorption, surface reactivity, and biocompatibility that

play into their effectiveness to house cells and enhance

cellular adhesion and proliferation. Of those materials, bio-

ceramics have been well investigated due to their

effectiveness. Bioceramics including hydroxyapatite (HA),

Bioglass, A-W glass ceramic, and b-tricalcium phosphate

closely mimic bone tissues. These materials also provide an

environment where bone ECM proteins are absorbed,

resulting in osteoblasts adhering and proliferating more rap-

idly than other materials including titanium.6–8 An in-depth

review of various mechanisms that are responsible for

increased cell attachment, growth, and bone/implant inte-

gration on bioceramics has been discussed by Ducheyne

and Qiu.7

In addition to bioceramics, several biopolymers have

also been utilized. One of the most widely used materials

from the past decade is a group of synthetic polymers and

their copolymers known as polyesters. These polymers

include polylactic acid (PLA), polyglycolic acid (PGA),

and the copolymer poly (lactic-co-glycolic) acid (PLGA).

These materials have played a large part in BTE due to

their biocompatibility and ease of manufacture into differ-

ing shapes.9 The remarkable property of these polymers is

their ability to support mechanical needs for a wide variety

of applications such as screws and fixation devices in

orthopedics. Degradation is another important property of

these materials. In an ideal case (i.e. an ideal biodegrad-

able material property), as the growth of bone into the

scaffold promulgates and the bone cells naturally build an

infrastructure, the initial supporting scaffold is degraded

and a handoff of mechanical stress and strain is passed

onto the neo-tissue structure.10 In addition to polyesters,

other synthetic polymers also have significance in BTE,

including poly(methyl methacrylate), poly(e-caprolactone),polyhydroxybutyrate, polyethylene, polypropylene, polyur-

ethane, poly(-ethylene terephthalate), polyetherketone, and

polysulfone.

Besides bioceramics and polymers, metallic alloys have

also been used in orthopedic implants for their strength

[100 GPa for an elastic modulus), especially in load-bearing

areas.11 The metal alloys used in BTE include cobalt-

chromium, stainless steel, aluminum, and titanium alloys.

The advantages of metallic alloys include a light-weight na-

ture and strength and biocompatibility. Although metallic

alloys have been used to produce bone prostheses, these

materials have several limitations. These drawbacks are per-

manence, cracking, and the potential of releasing metallic

ions and introducing corrosion products into the body from

these materials, raising concerns for their use.12 Unlike bio-

degradable materials, metals cannot be used to produce a

complete tissue replacement for bone defects because of their

permanent property. In addition, the released (wear) metal

particles have been found to affect the release of inflamma-

tory factors, inhibit bone formation markers, and stimulate

bone loss or resorption. For example, studies have shown

that titanium and its alloy particles inhibit bone-cell prolifer-

ation and reduce bone formation markers.13 An increase

in the release of inflammatory factors such as interleukin-6

and transforming growth factor (TGF-b1) has also been

noted.14,15 Moreover, metal implants have less integration

with the tissues surrounding the implant site because there is

a significant difference in stiffness between metal implants

and bone tissue. These metal implants are often stiffer than

the natural bone, leading to a stress shielding effect that

causes poor osseointegration.

Bioactive composite materials have recently been devel-

oped to incorporate the strengths of two or more distinct

574 STEVENS ET AL.

Journal of Biomedical Materials Research Part B: Applied Biomaterials

materials (metallic, ceramic, and polymeric materi-

als).11,16,17 Most composite materials are used to produce

specific strength, stiffness, or toughness properties that

match natural bone. The cortical bone has a wide range of

mechanical properties: 7–30 GPa for Young’s modulus,

50–150 MPa for tensile strength, and 1–3% for elongation

at fracture.11 Biocompatibility is also provided by at least

one of the constituents making the composite materials bio-

active. For instance, hydroxyapatite reinforced high density

polyethylene (HA/HDPE) composite and PLGA/HA com-

posite materials have been developed to mimic the struc-

ture and match the properties of the bone, suggesting these

materials as good matrices for bone cell differentiation and

mineralization.11,17–19 In vitro and in vivo studies of these

composites found that osteoblast cells adhered to HA pref-

erentially, leading to subsequent cell proliferation, and the

composite implants displayed good bone integration and

osteoconductivity in rabbit models.11 The rationale for

designing composite materials is to provide many attractive

properties that each individual material cannot have. For

example, in the case of metal-matrix composites, the metal

will provide the strength to bear large loads, whereas poly-

meric materials produce the environment for the incorpora-

tion of bioactive molecules and integration of surrounding

bone cells at the implant sites.11,20 In general, several mate-

rials and bioactive composites have been investigated for

bone graft substitutes over the last decade. Each of these

materials has its distinctive characteristics and advantages,

yet it remains a great challenge to produce a material that

is similar to nature and that bone cells like to grown on.

Cells

In addition to materials, cells have been used in BTE for

seeding in bone scaffolds before implantation. Numerous

cell sources, such as embryonic stem cells, bone marrow

stromal cells, and muscle-derived stem cells, have been

investigated in BTE.21–23 One of the most used cell sources

is bone marrow stromal cells.24–26 Adult stem cell sources

such as human bone marrow are used in BTE to produce

autologous bone tissues and to avoid ethical issues and the

immune responses generated by the human body against

cells isolated from different sources.27,28 Bone marrow con-

sists of hematopoietic stem cells and mesenchymal stem

cells, which can differentiate into various cell types includ-

ing osteoblasts. Bone marrow has also become a large

source of osteoblasts because of the availability and ease of

harvesting bone marrow from animals for research purposes

in BTE.

To detect the success of bone cell proliferation and dif-

ferentiation within scaffolds, several assays and biomarkers

have been studied. To assess cell proliferation, DNA assays

are done fluorimetrically using various dyes (e.g. Hoescht,

PicoGreen) bound to the cell DNA, which is proportional

to the cell density.29 3H-thymidine is also used to label

cells and can be detected radioactively when cells prolifer-

ate. To study bone cell differentiation, several biomarkers

exhibiting the osteoblastic phenotype have been used.29 For

example, assays for osteocalcin, an ECM as a bone forma-

tion marker, are performed using Iodine-labeled osteocal-

cin, which can eventually be detected in a gamma counter.

Alkaline phosphatase (ALP), an enzyme found in bone tis-

sue, is also studied as a bone formation marker using

ELISA.29 Furthermore, stainings for phosphate (Von Kossa

method), calcium (Alizarin Red S), and ALP (immuno-

staining) have been used to reveal these biomarkers in cell

cultures and sectioned tissues.30

Signals

Besides materials and cells, several signals such as media

additives and chemical cues have been investigated to

guide bone cell differentiation and proliferation in BTE. To

differentiate bone marrow stem cells into osteoblasts, the

medium used for cell cultures and bone scaffolds often

includes chemical differentiation additives. The most prom-

inently used agent is dexamethasone (Dex). Dex has been

shown to promote marrow cell differentiation and express

bone marking proteins such as ALP, osteopontin, and

osteocalcin.31,32 Other additions to bone specific media

include b-glycerolphosphate and L-ascorbic acid.

Chemical cues such as growth factors have also been

used to enhance bone growth within the scaffold. The com-

monly used growth factors include TGFs, bone morpho-

genic proteins (BMPs), pleiotrophin, platelet-derived

growth factors, insulin-like growth factors, and fibroblast

growth factors (FGFs). TGFs with their most common iso-

form, the b-isoform, have been useful in augmenting the

growth of bone tissue.33,34 For example, TGFbs (types 1, 2,3, and 5) stimulate osteoblast proliferation, mineralization,

and matrix structures.35 They also play a key role in bone

resorption and bone formation. In addition to TGFs, BMPs

including BMPs 2–7 also induce the formation of bone,

cartilage, and connective tissues.36,37 For critically sized

bone defects, BMP 2 shows the most promise, while both

BMP 7 and 2 are successful in large defects.36,37 Further-

more, pleiotropin, a cysteine-rich peptide known as a sig-

naling molecule for bone formation, and hyaluron

demonstrate the ability to promote adhesion, migration, and

differentiation of human osteoprogenitor cells.35,38

FABRICATION METHODS

Several fabrication processes have been utilized to manu-

facture bone scaffolds that meet the earlier requirements.

Although various traditional fabrication techniques such as

solvent casting are available; the production of scaffolds

using these methods lacks consistency and reproducibil-

ity.39,40 Recently, with the use of modern techniques like

rapid prototyping (RP), it is possible to mimic naturally-

occurring scaffolds, even with their complex structures.

The purpose of this section is to provide a quick overview

575BONE REGENERATION IN ENGINEERED BONE TISSUES


of some traditional procedures, like material injections and

solvent casting, and to expand upon new fabrication techni-

ques, such as RP, to produce bone scaffolds for BTE.

Material Injections

Injectable pastes have been developed for use as a fixation

material or to fabricate bone constructs for cell ingrowth or

preseeding. Injectables have an advantage over preshaped

scaffolds in that they are minimally invasive for surgical

procedures and can conform to any shape that they are

pressed in to.41 In this method, polymerization can be done

in situ. For example, a poly (propylene fumarate) solution

can be injected and polymerized at the site of a bone defect

to fill in any defect shapes.41–43 Studies have also shown

that the chitosan-calcium phosphate composite is a good

material for an injectable, resorbable scaffold. The chitosan

tends be in a paste form at a pH value of 6.5, and when

injected into the body it undergoes a phase transition and

becomes a solid ceramic at the defect site.44 Furthermore,

an injection of hyaluronan mixed with FGF at the site of a

fracture in a rabbit fibula showed increased bone formation

and good mechanical strength in a short period of time.45

Generally, properties of the injectable materials, such as

viscosity, setting time, and initial mechanical strength, play

a large part in their success. The major advantages of this

method are as follows: (a) the material can fill the gaps in

the defect irrespective of the shape of the defect, (b) it is

possible to incorporate bioactive and therapeutic agents by

mixing them together with the injectable materials, and (c)

there is no need for a surgical procedure to replace the

material.44

Solvent Casting

Solvent casting (i.e. particulate leaching) has shown prom-

ise for its ability to produce scaffolds at room temperature.

In this technique, a scaffold is produced by adding a poly-

mer solution (e.g. PLA or PLGA dissolved in an organic

solvent such as chloroform and methylene chloride) into a

mold containing a solid porogen (e.g. salt crystals).46 These

salt crystals are generally insoluble in the organic solvent.

During subsequent processing the porogen is leached out

by immersing the scaffold in water to form a pore structure

within the scaffold. In this method, the pore size and poros-

ity of scaffolds can be varied by changing the size and

morphology of the salt crystals.46 Scaffold properties, such

as polymer degradation time and mechanical strength, can

also be altered by changing polymer concentration and the

amount of salt crystals. For instance, increasing polymer

concentration has been found to result in induced scaffold

compression modulus,47 whereas scaffolds fabricated with

higher amount of salt crystals with smaller sizes (90%

NaCl of 0–53 lm size) were more brittle than others.48,49

Although solvent casting has been effective to produce

scaffolds sufficiently strong for bone implantation, it lacks

reproducibility and the ability to provide designed pore

geometries and morphologies.50,51 In addition, the thickness

of scaffolds made by this technique should be less than 4

mm to have a uniform pore structure, making it more diffi-

cult to fabricate a large 3D scaffold.52 To overcome this,

lamination of several highly porous membranes has been

used. A successful 3D scaffold made of PLLA and PLGA

was prepared using this laminating technique, and the pores

were interconnected forming a continuous pore structure.53

Rapid Prototyping

To overcome the limitations of conventional fabrication

methods, rapid prototyping (RP) technologies (a.k.a solid

freeform fabrication or additive manufacturing technolo-

gies) have been developed and increasingly applied in

BTE. The process of producing an RP part begins when an

object is scanned using a computed tomography (CT) scan-

ner or modeled in a Computer-Aided Design (CAD) soft-

ware package.54 The CAD file is typically converted to an

STL file, the standard RP file format, which represents the

model as a collection of surface triangular patches.55 The

STL file can be scaled to compensate for shrinkage and/or

rotated and translated along any geometric axis to orient

the part for optimum manufacturing. This file is then numeri-

cally sliced into two-dimensional (2D) cross-sections,

which are then fabricated and stacked in such a manner

that the 3D object is formed and the product is finished.

The accuracy of the 3D object relative to the digital data is

a function of the accuracy of the process which forms and

stacks the 2D cross-sections as well as thickness of the

cross-sections.

RP technologies offer several advantages over conven-

tional scaffold fabrication techniques for producing scaf-

folds, due to the fact that both the geometry and material

composition can be controlled.56 Using the RP manufactur-

ing approach, it is possible to create scaffold geometries,

which are reproducible, reliable, geometrically complex,

and highly-customizable. Because of the additive nature of

RP, tissue engineers are also able to custom design a

desired level of complexity into the scaffold, which is a

capability not available using traditional manufacturing

methods. In addition to the geometric flexibility, some RP

machines have material flexibility such that multiple mate-

rials, including living cells in some cases, can be deposited

to form a tissue engineered structure. These types of tai-

lored multi-material structures are not possible using tradi-

tional methods, which produce only single material

structures.

RP technologies have been used in various applications

including the manufacturing of medical devices, controlled

drug delivery systems, and engineered tissues.56–60 Of

these, engineered tissues is a growing application area for

RP technologies.50,61,62 Various processes have been suc-

cessfully employed to fabricate 3D scaffold structures using

biodegradable materials.4,63,64 In addition, a number of

investigators have successfully produced various artificial

576 STEVENS ET AL.


joints and load-bearing implants from biocompatible mate-

rials such as HA, titanium, and CoCrMo.50,65 Customized

prostheses such as customized tracheobronchial stents and

intervertebral spacers, based on anatomical data from imag-

ing systems including CT, have also been investigated.66

Customized products manufactured in this way are touted

as a significant benefit compared with standard product

sizes, which are not always an ideal fit for the patient.

Various methods for RP have been developed over the

past 20 years starting with the advent of stereolithography

in 1986. Several common RP processes, which have been

used for tissue engineering, are overviewed below to illus-

trate the variety of processes and techniques in RP. The

advantages and disadvantages of common RP techniques

are shown in Table I.

Stereolithography. Stereolithography (SLA) was the

original RP process introduced in the late 1980’s by 3D

Systems Incorporated. SLA utilizes the ability of photopo-

lymerizable liquid polymers to solidify when ultraviolet

light is focused on the surface of the liquid. A platform

supporting the developing model is brought near the sur-

face of a liquid vat, and a UV laser is used to solidify the

first cross-sectional layer of the model to the platform. By

lowering the platform one cross-sectional layer thickness,

recoating the liquid on top of the first layer, and then pass-

ing the UV light over the new liquid layer, a second layer

is deposited. The process is repeated layer by layer until

completion, after which the prototype is typically cleaned

and completely cured by placing it in an ultra-violet

oven.50,55,68 Several biopolymers have been utilized in

SLA. For example, an aqueous poly(ethylene glycol) (PEG)

hydrogel solution has been processed and research results

have been shown that the hydrogel is capable of preventing

human dermal fibroblast cells encapsulated in the solution

from damage during the SLA process.69 Thus, by combin-

ing the SLA process and biopolymers such as PEG, it is

possible to fabricate complex 3D structures with bioactive

agents embedded within the structure. Although biocompat-

ible materials used in SLA are typically biopolymers, con-

siderable research has been conducted on the use of

bioceramics. A bioceramic suspension is first prepared by

adding bioceramic powder into a photopolymer before SLA

processing. The photopolymer acts as a binder to maintain

the geometry of part during fabrication. The suspension is

cured during the SLA process, and a ‘‘green part’’ is pro-

duced. The ‘‘green part’’ is postprocessed by heating in a

furnace to remove the photopolymer binder and sinter the

bioceramic into a porous network.70

Selective Laser Sintering. In selective laser sintering

(SLS), parts are made by passing a laser over a thin layer

of polymer powder. The laser will raise the temperature of

the powders, which will cause neighboring particles to fuse

together both laterally and to the preceding layer below.

Once a layer of polymer is fused, an elevator platform sup-

porting the part is lowered by the height of the next layer

and a roller applies new powder over the previously proc-

essed layer. Post processing of the final part can include

dissolving out unsintered particles (for instance, using a

calcium phosphate solution) to leave microporosity within

the structure. HA and calcium phosphate particulates with a

polymer binder have been used in SLS as possible implant

materials.51,71 SLS has also been used to manufacture bone

scaffolds from a HA/PLLA composite with PLLA selected

as a binder due to its higher degradation time and lower

melting temperature. Results showed that the elastic modu-

lus of HA/PLLA parts range between 140.47 and 257.27

MPa, whereas the bending strength ranges from 1.57 to

4.05 MPa, which is comparable to the modulus and bend-

ing strength of cancellous bone.72 Other SLS research has

employed HA/HDPE composites.73 The disadvantages of

SLS compared to SLA are a rough surface finish, lower

dimensional accuracy, and porosity.74 However, the ability

to directly sinter biocompatible materials without a high-

temperature post-processing step gives SLS inherent mate-

rial advantages over SLA.

3D Printing. 3D Printing uses ink-jet printing technology

to precisely place a ‘‘binder’’ solution on a bed of powder,

thus gluing the powder together in a cross-sectional layer,

much as the lasers in SLS use heat to bind powder layers.

Conceptually, any powdered material, including polymers,

metals, or ceramics can be fused using an ink-jet head

TABLE I. Advantages and Disadvantages of Common Rapid Prototyping Techniques.39,50,54,55,67

Method Advantages Disadvantages

Stereolithography Hydrogel materials, high-resolution and accuracy,

liquid build material can easily be removed from

within complex scaffolding

Limited choice of materials, may require furnace

postprocessing (e.g. bioceramics), high material

cost, complex and expensive equipment

Laser sintering Wide range of material choices, good mechanical

properties, lower material cost, good accuracy

Materials may thermally degrade during the process,

undesired porosity, hard to remove trapped powder,

complex and expensive equipment

3D printing Wide range of material choices, low cost, quick

process, multimaterial capabilities through multi

print-heads

Hard to remove trapped materials, low to Medium

resolution, powder particles may not bind well,

binders are always necessary to bind powders

Fusion deposition

modeling

No trapped materials, minimal material waste, low cost Materials may thermally degrade during the process,

lower range of material choices, medium resolution



which passes over and applies droplets of a binder solution

to the powder. A platform is lowered and more powder is

deposited and processed in a layer by layer fashion. The

unbound powder in the void spaces of the prototype can

then be removed by compressed air or by manually brush-

ing it away. A number of studies have investigated the

application of 3D printing technologies for direct and indi-

rect manufacturing of scaffolds from various materi-

als.65,75–77 By varying the composition of the ‘‘glue’’ which

is printed (much like colors can be varied in a traditional

color ink-jet printer), the composition of the resulting scaf-

fold can be varied as well in three dimensions.

Fused Deposition Modeling. Fused deposition model-

ing, another RP technology used for tissue engineering,

uses a small temperature-controlled orifice to extrude fila-

ment material and deposit semimolten polymer onto a plat-

form, which immediately solidifies. At the end of the

deposition of each layer, the platform is lowered so that the

next layer can be deposited. By changing the direction of

each layer and the spacing between materials, scaffolds

with highly uniform internal structures are obtained.64,78

Using multiple heads or other deposition processes simulta-

neously, a multi-material scaffold can be created with com-

plex internal geometry.

Several other variations of RP processes have been

developed and are being used for scaffold fabrication.

Most of these processes are based either on material print-

ing, extrusion, or spraying techniques, which involve mul-

tiple deposition heads or material feeders so that multi-

material scaffolds can be formed, which are capable of

optimizing both geometric and material composition simul-

taneously.67,79–86

STRATEGIES TO ENHANCE BONE GROWTH

To enhance bone growth in scaffolds, several strategies

have been developed. These include generation of nano-

topography on the scaffold surface, development of biomi-

metic materials, formation of mineralized layers on bone

scaffolds, and utilization of bioreactors for cell seeding and

feeding. The principle of these approaches is to mimic the

natural environment in which bone cells grow. Studies on

nanotopography, biomimetic materials, embedded growth

factors, mineralization, and growth environment of bone

scaffolds are discussed in the next section.

Nanotopography

Several studies have shown that bone grafts with nanotopo-

graphic surfaces (or nanotopography) similar to native tis-

sues have better implant-tissue integration.87 In native bone

tissues, bone cells are exposed to substrates and structures

with nanoscale features, such as ECM proteins, minerals,

and pores in membranes and tissues.87 By mimicking this

nanotopography, researchers hope to enhance bone cell

growth and tissue integration. In fact, nanotopographic tita-

nium surfaces obtained by chemical treatment with H2SO4/

H2O2 increased both bone tissue growth and ALP activ-

ity.88 Similarly, TiO2-coatings that contained substantial

‘‘surface pores’’ at dimensions of 15–50 nm promoted the

formation of bone mineral-like calcium phosphate as well

as induced tissue attachment.89,90 Furthermore, calcium and

phosphorus mineral contents are induced more in nano-

phase Ti6Al4V compared to microphase Ti6Al4V.91 One

interesting study investigated the osteoblastic cell response

to nanostructured islands (11, 38, and 85 nm produced by a

polymer-demixing (polystyrene/polybromostyrene) tech-

nique) and found that the smaller nanoisland produced an

increase in cell adhesion and proliferation as well as in

ALP activity.92 Nanotopography was also a significant fac-

tor for differentiation of mesenchymal stem cells (or osteo-

progenitor cells) to osteoblastic phenotype.93 Results from

these studies suggest that nanotopography of bone scaffolds

will stimulate bone formation and enhance bone-implant

integration, leading to better tissue repair and regeneration

at the bone implant-biomaterial interface.

Biomimetic Materials

Besides nanotopography, biomimetic materials have been

investigated to increase cell adhesion and proliferation into

a scaffold.94 In nature, osteoblasts often attach or adhere

(spread) onto a surface based on their cell membrane pro-

teins. The main family of these membrane proteins is integ-

rins, which are composed of two chains, an a-chain and b-chain. Osteoblasts have been shown to express a1, a2, a3,a4, a5, a6, av, b1, b3, and b5 integrin subunits. These

integrins mediate cell attachment and regulate cell migra-

tion, growth, differentiation, and apoptosis. Several

attempts have been made to develop materials that mimic

integrin-binding in various biological systems. For exam-

ple, a three peptide sequence of arginine-glycine-aspartic

acid (RGD) bound to several of these cell membrane integ-

rins has been incorporated into a scaffold to enhance bone

cell adhesion and growth.95 In addition, ECM proteins such

as collagen I, laminin, fibronectin, vitronectin, and fibrino-

gen as well as peptides designed from these proteins have

been applied onto scaffolds to induce cell adhesion and

proliferation.94,95 Of these, fibronectin and vitronectin have

been viewed as the optimum adhesion proteins for osteo-

blastic cells.94 Advances in biomimetic materials have been

discussed in detail by Shin et al.96

Embedded Growth Factors

In addition to nanotopography and biomimetic materials,

growth factors may also be used in conjunction with scaf-

fold materials to enhance bone ingrowth in scaffolds. In the

case of nonunion fractures, growth factors, most notably

BMPs, are needed for enhanced healing.97,98 In the past,

delivery of these growth factors using carrier matrices to

the site of concern was needed for successful bone recon-

578 STEVENS ET AL.


struction.33,35,99 Currently, growth factors such as BMPs

have been used for embedding in the scaffold materials.

Embedding growth factors in scaffold materials have dis-

tinct functions: to feed seeded cells and provide locally the

release of growth factors for subsequent bone mass

growth.99,100 These embedded growth factors (BMPs) have

been shown to differentiate the mesenchymal cells into

osteogenic cells and to enhance bone growth.100

Mineralization of Bone Scaffolds

Another strategy to enhance bone ingrowth is generating

mineralized layers on bone scaffolds. Since inorganic min-

erals have been found in biological bone structures,

researchers have manipulated implant surfaces to induce a

bone-like structure (mineralization) in which new bone

may form after implantation or reconstruction proce-

dures.101,102 For instance, implant surfaces modified with

an apatite layer are favorable to induce osteogenesis.102–105

Materials containing the functional groups Si��OH,

Ti��OH, Zr��OH, Nb��OH, and Ta��OH have been

shown to form apatite layers in the presence of an acellular

protein-free simulated body fluid.102,104 As apatite particu-

lates are generated on synthetic materials, new bone may

form on this mineralized layer, and implants may be stabi-

lized by increasing material-bone interface strength.

Growth Environments (Bioreactors)

A novel addition to the field of BTE is the augmentation of

cell proliferation through the use of bioreactors.106–109 Cul-

turing of bone cells has been done traditionally in polysty-

rene flasks with added bioactive reagents such as

dexamethasone and ascorbic acid for bone specific lineages.

Spinner flasks and perfused cartridges have also been used

to enhance cell proliferation in bone scaffolds in various

studies.110 Recently, researchers have used bioreactors for

cell seeding111 and circulation of media through 3D scaf-

folds for waste removal and nutrient diffusion.112–115 Oscil-

lating perfusion of cell suspensions produced higher

seeding efficiency, better cell distribution, and stronger

cell-matrix interactions.111 Bioreactors have also been

shown to increase the proliferation of bone cells.112 The

same group elucidated that increasing shear forces leads to

the enhancement of osteoblastic phenotype expression,

which increases mineralized matrix production.112–114

CONCLUSION

Engineered bone scaffolds have been developed to over-

come the limitations of autografting and allografting. The

development of successful scaffolds requires appropriate

materials, cell sources, bioactive molecules, and fabrication

techniques. Challenges in BTE are being overcome by

applying novel strategies to enhance bone formation, such

as mimicking natural nanostructures. As interdisciplinary

fields donate their expertise, new methods for BTE will

lead to further successes. This can be seen with the appli-

cation of RP technologies to BTE.

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