Bone Tissue Engineering: A Review in Bone Biomimetics and

Drug Delivery Strategies

Joshua R. Porter, Timothy T. Ruckh, and Ketul C. Popat
Dept. of Mechanical Engineering, School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523
DOI 10.1002/btpr.246
Published online October 12, 2009 in Wiley InterScience (www.interscience.wiley.com).

Critical-sized defects in bone, whether induced by primary tumor resection, trauma, or

selective surgery have in many cases presented insurmountable challenges to the current
gold standard treatment for bone repair. The primary purpose of a tissue-engineered scaffold is to use engineering principles to incite and promote the natural healing process of
bone which does not occur in critical-sized defects. A synthetic bone scaffold must be biocompatible, biodegradable to allow native tissue integration, and mimic the multidimensional hierarchical structure of native bone. In addition to being physically and chemically
biomimetic, an ideal scaffold is capable of eluting bioactive molecules (e.g., BMPs, TGF-bs,
etc., to accelerate extracellular matrix production and tissue integration) or drugs (e.g.,
antibiotics, cisplatin, etc., to prevent undesired biological response such as sepsis or cancer
recurrence) in a temporally and spatially controlled manner. Various biomaterials including
ceramics, metals, polymers, and composites have been investigated for their potential as
bone scaffold materials. However, due to their tunable physiochemical properties, biocompatibility, and controllable biodegradability, polymers have emerged as the principal material in bone tissue engineering. This article briey reviews the physiological and anatomical
characteristics of native bone, describes key technologies in mimicking the physical and
chemical environment of bone using synthetic materials, and provides an overview of local
C 2009 American
drug delivery as it pertains to bone tissue engineering is included. V
Institute of Chemical Engineers Biotechnol. Prog., 25: 15391560, 2009
Keywords: bone tissue engineering, orthopedics, drug delivery, biomimetics

Motivation Behind Bone Tissue Engineering

Bone is a remarkable organ playing key roles in critical
functions in human physiology including protection, movement and support of other critical organs, blood production,
mineral storage and homeostasis, blood pH regulation, multiple progenitor cell (mesenchymal, hemopoietic) housing, and
others. The importance of bone becomes clear in the case of
diseases such as osteogenesis imperfecta, osteoarthritis, osteomyelitis, and osteoporosis in which bone does not function
properly. These diseases along with traumatic injury, orthopedic surgeries (i.e., total joint arthroplasty, spine arthrodesis, implant xation, etc.) and primary tumor resection lead
to or induce bone defects or voids. The clinical and economic impact of treatments of bone defects is staggering.
For example, the number of total joint arthroplasties
(TJAs) and revision surgeries in the US has increased from
700,000 in 1998 to over 1.1 million in 2005.1 Medical
expenses relating to fracture, reattachment, and replacement
of hip and knee joint was estimated to be over $20 billion
(USD) in 2003, and predicted to increase to over $74 (USD)
billion by the year 2015.1,2 Spinal arthrodesis is another
example of a surgery typically requiring substantial bone
Correspondence concerning this article should be addressed to K. C.
Popat at Ketul.Popat@colostate.edu.
C 2009 American Institute of Chemical Engineers
V

repair/replacement. Since 1997, spinal arthrodesis has seen

the highest percentage increase of all muscoskeletal surgeries
requiring hospitalization.3 In 2005 alone, over 300,000 spinal
fusions were conducted costing over $20 billion (USD).
Traumatic bone fractures accounted for 8.5 million physicians visits, almost 1 million of which required hospitalization in 2005.1 Also, in 2005, there were over 3000 pediatric
hospitalizations for bone cancer costing over $70 million
(USD).3
All treatments described above involve bone repair or
replacement. For a variety of reasons (such as bone defect
size, infection, and many others), injured or diseased bone
may not be capable of repairing itself by means of mechanical xation alone which results in a non-union. Each condition or treatment has a different rate of incidence of nonunion. For example, it has been reported that as many as 5
10% of bone fractures relating to football injuries result in a
non-union, thus requiring additional treatment.46 Non-unions
are also problematic in joint arthroplasties.7,8 In cases of primary tumor resection or massive traumatic bone loss, bone
defects are critical-sized and do not heal with mechanical
xation.
For non-union or critical-sized defect scenarios, a substitutionary material must be used to ll the bone defect. The
current gold standard treatment of critical-sized bone defects
is autogenous bone grafting. In this treatment, host bone is
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removed from another site (typically from the pelvis or iliac

crest) and used to ll the defect. However, the complication
rate in autogenous bone grafting is as high as 30% and may
include donor site morbidity, pain, paresthesia, prolonged
hospitalization and rehabilitation, increased risk of deep infection, hematoma, inammation, and restricted availability.915
Another reasonable option for patients and surgeons is the use
of bone tissue from other humans (typically cadavers) called allograft. Allografts may derive from viable (alive) or sterilized
non-viable sources. Many orthopedic allograft procedures have
been FDA-approved and utilized for years. Success in both
autograft and allograft procedures is attributed to the physical
and biological similarity in donor (site or patient) and host
tissue. However, orthopedic allografts carry risks of donor to
recipient infection (rate of incidence as high as 13%)16 and disease transmission, and host immune responses.17,18 As a last
resort, patients requiring bone repair or replacement may also
consider a xenograft, or tissue grafting from a non-human.
Early success in xenotransplantation of a variety of cells, tissues, and organs created optimism and public acceptance of
this emerging technology.19,20 After more than 2 decades of
investigation and global clinical trials, however, bone xenografts are now widely considered to be unsuitable for transplantation due to real and perceived risk of disease or virus
transmission, infection, toxicity associated with sterilization,
immunogenicity, and nally host rejection.2123

General Principles in Bone Tissue Engineering

Concerns including the aforementioned and others surrounding the utilization of autogenous cancellous bone grafts
as the gold standard treatment for critical-sized defects in
bone have motivated the development of a wide variety of
sophisticated synthetic (tissue-engineered) bone scaffolds in
recent years. Advantages to utilizing synthetic bone scaffolds
include: the elimination of disease transmission risk, fewer
surgical procedures, a reduced risk of infection or immunogenicity, and the abundant availability of synthetic scaffold
materials. This section reviews basic principles in bone tissue engineering and the design challenges surrounding the
development of a synthetic scaffold which mimics the complicated physiochemical attributes of bone.
The fundamental concept behind tissue engineering is to
utilize the bodys natural biological response to tissue damage in conjunction with engineering principles. As the role
of cell signaling and subsequent functionality in tissue engineering emerges with greater clarity, tissue engineers are
developing multifunctional bioactive scaffolds. Ideal synthetic scaffolds must be capable of presenting a physiochemical biomimetic environment while biodegrading as native
tissue integrates and actively promote or prevent desirable
and undesirable physiological responses, respectively.11,2426
To address these biomimetic requirements specically, a
synthetic bone scaffold must:
1). provide temporary mechanical support to the affected
area,
2). act as a substrate for osteoid deposition,
3). contain a porous architecture to allow for vascularization and bone in-growth,
4). encourage bone cell migration into the scaffold,
5). support and promote osteogenic differentiation in the
non-osseous, synthetic scaffold (osteoinduction),
6). enhance cellular activity towards scaffold-host tissue
integration (osseointegration),

7). degrade in a controlled manner to facilitate load transfer to developing bone,

8). produce non-toxic degradation products,
9). not incite an active chronic inammatory response,
10). be capable of sterilization without loss of bioactivity,
and
11). deliver bioactive molecules or drugs in a controlled
manner to accelerate healing and prevent pathology.
Multiple bone tissue engineering strategies such as cell
transplantation, acellular scaffolds, gene therapy, stem cell
therapy, and growth factor delivery have been applied to
address the challenging requirements listed above.2731 In
practice, most bone tissue engineering approaches implement
a combination of these strategies. However, two primary tissue engineering strategies have emerged as the most promising approaches.24
1). Before implantation, mesenchymal stems cells (MSCs)
are isolated (typically from the patient), expanded ex vivo,
seeded onto a synthetic scaffold, allowed to produce extracellular matrix (ECM) on the scaffold in controlled culture
conditions, and nally implanted into the osseous defect or
void in the patient (Figure 1).26
2). Implantation of an acellular scaffold immediately after
injury/bone removal.
MSCs are pluripotent cells capable of differentiation into
a number of cell types. Under the inuence of chemicals
such as dexamethasone, ascorbic acid, and b-glycerol phosphate, MSC differentiation can be driven towards bone forming cells, or osteoblasts, which then produce bone ECM
within the scaffold ex vivo. Transplanted scaffolds seeded
with MSCs have been shown to enhance osteogenic capacity
and integrate with native tissue faster than acellular scaffolds
in many preclinical trials.3235
A primary obstacle in translating this technology from the
bench to the bedside is that this technique involves an additional surgery and the patient must wait for the bone graft to
develop in vitro. Therefore, to promote the rapid development of a transplant-ready cellular scaffold, a variety of
novel ex vivo culture techniques have been investigated to
accelerate the cellular production of ECM. Three predominant ex vivo culture techniques utilized in bone tissue engineering are: growth factor delivery, bioreactor systems, and
gene therapy.
The most common ex vivo culture technique involves supplementing the MSCs/osteoblasts with growth factors. A
review of bone-relevant growth factors and their inuence
on MSC and osteoblast phenotypic behavior is provided in a
later section. Briey, growth factors such as platelet-derived
growth factors (PDGFs), bone morphogenetic proteins
(BMPs), insulin-like growth factors (IGFs), and transforming
growth factor-bs (TGF-bs) have an indisputable role in
osteoinduction and osteoconduction. In ex vivo conditions,
growth factors can be delivered by simply adding them to
the culture media or encapsulated in a biodegradable scaffold. However, the short half life and subsequently high dose
required in growth factor delivery has motivated the development of alternative technologies for enhancing MSC performance in bone scaffolds ex vivo.
The concept of gene therapy is similar to growth factor
delivery in that the goal is to increase the local concentration
of osteoinductive and osteoconductive cues for surrounding
cells in vivo, and thereby encourage native MSCs to migrate
into the scaffold, proliferate, differentiate, and begin ECM

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Figure 1. Schematic of bone tissue engineering via cell-seeded scaffolds which a cultured ex vivo prior to transplantation.26 Regenerative Medicine II Clinical and Preclinical Applications with contributions by numerous experts. Series: Advances in Biochemical Engineering/Biotechnology, Vol. 94 Volume package, Regenerative Medicine Yannas, Ioannis V. (Ed.) 2005, XII, 232 p.
49 illus., Hardcover ISBN: 978-3-540-22868-4. Figure 1 on page 9. Reproduced with kind permission from Springer Science
1 Business Media.

production. There are two basic gene therapy strategies in

bone regeneration: (1) deliver a gene coding for the production of growth factors or other biological cues directly into
the area of interest in vivo, and (2) seeding genetically modied cells into the scaffold ex vivo.3639 Both strategies have
been extensively tested and proven in animal models and
hold great potential for the future of bone tissue engineering.
A systematic review of gene therapy in bone regeneration
can be found in the literature and is not provided here.40
Bioreactor systems of a variety of designs have also been
utilized to enhance the in vitro performance of osteogenic
cells before implantation. Bioreactors simulate the 3D
dynamic and mechanical in vivo environment and are
designed to provide cells seeded deep within a scaffold with
all necessary nutrients and biological cues to survive, proliferate, differentiate, and produce ECM.41,42 Sikavitsas et al.,
recently demonstrated proof of this concept by showing that
after 16 days of culture, MSC-produced ECM was uniformly
distributed in 3D scaffolds cultured in a ow profusion bioreactor, whereas the ECM was limited to the periphery in
the case of standard static culture condition.43
Despite the enormous potential of this approach for bone
tissue engineering, there are still a number of barriers to
address. The rst and most signicant barrier is that a number
of studies have shown that MSCs which have been extensively cultured ex vivo lose their phenotypic behavior (such
as osteodifferentiation and bone forming capacity) once
implanted in vivo.33,44,45 Secondary challenges confronted in
this approach are related to the relative low concentration of
MSCs in bone marrow and their characteristic low proliferative capacity making it difcult to obtain sufcient cell density in a large scaffold.46,47 In addition to the increased risk
due to a second surgery, there is a need to establish rigorous
sterilization techniques for the cell-seeded scaffold which has
been in culture ex vivo for up to several weeks. Figure 2
below displays a schematic of this approach and critical considerations to make at each stage of the process.
The second main tissue engineering strategy involves implantation of an acellular scaffold immediately after injury/

bone removal (Figure 3). The governing principles of this

approach are the same as the rst approach, however, to
ensure rapid healing, it is even more critical to design a scaffold that mimics native bone tissue by driving local MSC
migration into the scaffold, supporting and promoting osteodifferentiation (osteoinduction), and providing a bioerodable
matrix that enhances MSC production of ECM that eventually integrates with the native tissue and lls the void or
defect (osseointegration).25,28,48,49 Some clear advantages of
this approach are that acellular scaffolds are much easier to
sterilize, they have a shelf-life, and they have the lowest
potential for infection or immunogenicity of all the bone
repair strategies discussed earlier.
Similar to the rst approach, the performance of an acellular scaffold may be substantially enhanced through the incorporation of bioactive molecules which are released in a
controlled manner as the scaffold degrades and native tissue
replaces it. The focus of this review is on this second
approach and the development of an acellular scaffold which
meets the requirements listed earlier. Challenges accompanying this strategy will be discussed in detail throughout this
chapter. In brief, the most important challenges reviewed
and addressed in this work are: (1) the design of a micro
and nanoscale dimensional hierarchy representative of bone,
(2) the incorporation and controlled viable release of bioactive molecules and drugs, and (3) control of bioerosion to
match native tissue synthesis rate.
In the design of an acellular scaffold that mimics the
physiochemical attributes of bone, a clear concept of the
bone biology, physiology, and anatomy of bone is essential.
The following section reviews the molecular and cellular
anatomy and physiology of bone, followed by a brief review
in bone remodeling and natural healing cascades.

Boning up on Biology
Bone anatomy and physiology
Bone is a complex organ which plays a wide variety of
critical roles in human physiology as described earlier.

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Figure 2. First approach in bone tissue engineering: considerations and challenges for each stage.31

Figure 3. Schematic describing the second bone tissue engineering strategy wherein biological molecules and
pharmaceutical agents are encapsulated in an acellular scaffold for release after implantation.

Tissues comprising bone include: mineralized osseous tissue,

marrow, endosteum and periosteum, nerves, blood vessels,
and cartilage. Bones are generally classied by their shape;
long, short, at, or irregular. This discussion focuses on long
bones in view of the fact that they are the primary type
affected by the types of surgical procedures or injuries
described above. Bone consists of inorganic components

[primarily a specic type of calciumphosphate mineral

called hydroxyapatite (HAp), Ca10(PO4)6(OH)2] and a wide
variety of organic components (mostly Type I collagen, but
also an abundance of other proteins described later). The
complex hierarchical physical structure, material properties
of the constituents, the cellular organization, and molecular
cues work in concert to perform the function of bone. In this
section, a brief review on the cellular and molecular organization of bone, bone biomechanics, bone remodeling, and
bone biomimetics are provided for background material requisite for an intelligent design of a synthetic acellular bone
scaffold.
Long bones have three primary regions: the diaphysis,
epiphysis, and epiphyseal plates. The diaphysis is the long
portion of the bone which consists primary of cortical or
compact bone. Cortical bone surrounds the perimeter of
bone, accounts for
80% of the total mass of bone.
Compact (cortical) bone consists of closely packed osteons
or haversian systems. The osteon consists of a central canal,
called the osteonic (haversian) canal, that is surrounded by
concentric rings (lamellae) of matrix. Between the rings of
matrix, the bone cells (osteocytes) are located in spaces
called lacunae. Small channels (canaliculi) radiate from the
lacunae to the osteonic (haversian) canal to provide passageways through the hard matrix. In compact bone, the haversian systems are packed tightly together to form what
appears to be a solid mass. The osteonic canals contain
blood vessels that are parallel to the long axis of the bone.
These blood vessels interconnect, by way of perforating
canals, with vessels on the surface of the bone (Figure 4).50

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Figure 4. General schematic of long bone illustrating the basic anatomy, differences in cortical and cancellous bone and micro-structural features in bone.51

The epiphysis contains mostly cancellous, or spongy, bone

and is covered in articular cartilage which facilitates lowfriction contact with other bones. Cancellous bone is less
dense than compact bone, and consists of plates (trabeculae)
and struts of bone adjacent to small, irregular cavities that
contain red bone marrow. Canaliculi connect to the adjacent
cavities, instead of a central haversian canal, to receive their
blood supply. It may appear that the trabeculae are arranged
in a haphazard manner, but they are organized to provide
maximum strength similar to braces that are used to support
a building. The trabeculae of spongy bone follow the lines
of stress and can realign if the direction of stress changes.
The epiphyseal plate, or growth plate, is only active for
growing bones and is responsible for bone lengthening
through endochondral ossication. The epiphyseal plate ossies when the bone has reached a mature size (typically
between 20 and 30 years).51
Cellular organization and bone remodeling
There are three types of bone cells: osteoblasts, osteocytes, and osteoclasts. Osteoblasts are cells which have differentiated from MSCs whose primary function is to produce
and secrete organic and inorganic bone ECM, known as the
ostoid.52 Osteoblasts are also not terminally differentiated
cells. Osteoblasts have two fates: they become embedded in
their own bone matrix and become osteocytes, or undergo
apoptosis (programmed cell death).53 Osteoblasts are responsible for skeletal architecture in two ways: (1) deposition of
bone matrix and (2) regulation of osteoclast activity.54
Osteocytes remain trapped with the matrix and eventually
stop generating ostoid, but play key role in mechanotransduction. The third cell type in bone is the osteoclast, which
is a multinucleated cell derived from fusion of mononuclear
hemopoietic precursors. The primary function of osteoclasts
is to secrete acids and proteolytic enzymes, which erode
bone ECM under the inuence of chemical cues.52,55 This
process of production and resorption of bone ECM by osteoblasts and osteoclasts plays several important roles in bone
physiology such as bone remodeling.
Bone remodeling is a critical process for maintaining skeletal integrity, healing, blood calcium regulation, and accom-

modation of changes in bone stress proles.55 Bone

remodeling is a complex process by which old bone is continuously replaced by new tissue and occurs in three distinct
phases: resorption (osteoclasts are activated through paracrine pathways to digest bone), reversal (mononuclear cells
appear on the surface), and formation (osteoblasts produce
and secrete ECM).55,56 Bone modeling and remodeling is responsible for adjusting the architecture and hence mechanical
properties of bone as a function of mechanical and chemical
signaling. Hormones (i.e., parathyroid hormone, calcitrol,
glucocorticoids, sex hormones, etc.) and growth factors (i.e.,
IGF, prostaglandins, TGF-bs, BMPs, etc.) drive and regulate
of bone remodeling in complicated biochemical cascades. The
process of bone remodeling is extremely complicated and still
not completely understood.57 However, multiple descriptions
and insights can be found in the literature.55,56,58 One key
mechanism by which osteoblasts work in concert with the
osteoclasts to remodel bone is described here. After osteoblast
differentiation, the cell presents a transmembrane protein,
receptor activator of NF-jB ligand (RANKL), which binds
to its receptor (RANK) on osteoclasts. Osteoblasts can downregulate osteoclast activity through the presentation of a transmembrane decoy receptor for RANKL which inhibits osteoclasts binding. However, hormonal control can promote
osteoclast activity through elevating RANKL expression on
osteoblasts. In this way, osteoblasts play a critical role in both
bone formation and the regulation of plasma calcium levels.52
Bone mechanics and mechanobiology
One very important and commonly overlooked regulator
in bone physiology (especially bone modeling and remodeling) is mechanical stimulation and cellular transduction.
Bone cells are profoundly inuenced by the loads they experience in vivo. The exact mechanisms by which bone cells
respond to mechanical stimulation has become more clear in
recent years, but is still not completely understood. There
are two suggested models: (1) each individual bone cell
experiences direct mechanical forces and the sensation/
response is at the cellular level or (2) certain sensory bone
cells (immobilized osteocytes) sense mechanical forces and
subsequently generate biochemical signals for neighboring

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cells to respond to, in which case mechanostimulation is at

the tissue level.59 This section provides a brief review of the
macroscopic mechanical properties of bone followed by
known and speculated mechanisms by which bone cells and
tissue respond to mechanical stimulation.
Cortical bone (long bone) has been experimentally determined to have a yield strength of 78151 MPa in tension
and 131224 MPa in compression when tested along its longitudinal axis. When tested in its transverse axis, bone has a
yield strength of 5166 MPa in tension and 106131 MPa in
compression. The Youngs Modulus (E) of cortical bone is

1720 GPa along the longitudinal axis and 613 GPa along
the transverse axis. Cancellous bone (also called trabecular
or spongy bone) is more isotropic and soft. The mechanical properties of cancellous bone vary widely and are
reported to be a function of apparent density/porosity of the
trabeculae. The strength and modulus of cancellous bone
vary roughly as the square of the apparent density. Cancellous bone is highly viscoelastic, meaning its mechanical
properties depend on the loading rate. Midrange values for
cancellous bone are: strength of 510 MPa and modulus of
50100 MPa.6063
During skeletal movement, forces are transmitted to cells
via direct strain, hydrostatic pressure, ow-induced shear, and
electric elds.59,64 In vitro studies have shown that osteocytes,
osteoblasts, osteoclasts, and osteoprogenitor cells all respond
to mechanical stimulation. In the case of osteocytes, the 3D
network of proteins and cell processes has been said to present
an ideal architecture for mechanical sensation and stimulation.59 In vitro analysis has revealed that osteocytes increase
their metabolic activity under mechanical stimulation similar
to physiological loading conditions.65 Many researcher have
suggested that uid ow induced shear stress is the primary
mechanism of informing bone cells of loading conditions
(osteocytes and osteoclasts more than osteoblasts).66,67 Fluid
ow also induces an electric eld from movement of charged
particles. Osteoclasts have been shown to migrate and act
based on electrical charges caused by these uid ows as
well.64 This research supports the second model described
earlier suggesting that mechanostimulation is at the tissue
level, and provides the fundamental motivation behind bioreactors which attempt to mimic the dynamic environment in
native tissue. Research has shown that osteocytes which have
been over stimulated release osteoblastic factors that results in
accelerated bone formation by osteoblasts, while understimulation resulted in osteocyte apoptosis and osteoclast recruitment and subsequent bone resorption.64 Although osteocytes
are generally considered to be the primary sensing cell,
studies have also shown that osteoblasts are stimulated to
proliferate and produce prostaglandins, alkaline phosphatase,
collagen, and mineralize a matrix in response to mechanical
loading in vivo.68,69 In vitro studies have shown that osteoblasts reorganize their cytoskeleton, upregulate transmembrane focal adhesion proteins, and express osteoblast-specic
proteins involved in ECM adhesion such as osteopontin under
uid ow stimulation.7072
These known mechanisms of mechanical stimulate in bone
cells and tissue have signicant implications in synthetic
bone scaffold design. Most importantly, to simulate the
dynamic mechanical physiological environment, a synthetic
scaffold must have similar mechanical properties (specically elastic modulus) compared to native bone. Second,
research on mechanostimulation of bone cells can be applied
to the design of bioreactors such as ow perfusion systems

Biotechnol. Prog., 2009, Vol. 25, No. 6

for 3D ex vivo culture of bone cells in synthetic scaffolds as

described earlier.

Chemical cues in bone

An exhaustive review of autocrine and paracrine cell signaling pathways in bone is out of the scoop of this review.
However, an understanding of chemical cues which drive basic cell functionality (such as migration, adhesion, proliferation, differentiation, and ECM production) is required to
design a biospecic scaffold able to direct phenotypic behavior in vivo. In all tissues, the extracellular environment contains a wide variety of molecular signal which can be found
in immobile, insoluble hydrated macromolecules (collagen,
bronectin, etc.), soluble mobilized macromolecules (e.g.,
growth factors, transcription factors, cytokines), and also
from the transmembrane proteins of neighboring cells.73 To
be brief, this section will review only the important roles of
immobilized macromolecules and mobile macromolecules in
directing bone cell behavior.
Bone Matrix Proteins and Bioactivity. In addition to
mineralization, osteoblasts produce a matrix of proteins
which not only serve to structurally support cells but also to
provide a variety of chemical cues which regulate functionality. This section provides a brief review on the organic constituents of bone matrix and the role they play in directing
cellular activity. Bone is
30% organic, 90% of which is
Type I collagen.74 The abundance of collagen in bone is
likely why it has been the most widely utilized natural polymer in bone scaffold development.75 Collagen not only plays
the primary role in the tensile properties of bone, but also
contains peptides which cue bone cells. Collagen in a triple
helix molecule which contains a peptide motif (DGEA)
which is a putative a2b1 integrin binding site for osteoblasts.76 HAp crystals are typically found between individual
collagen bers suggesting the calcium binding capacity of
collagen as well.
The most abundant non-collagenous bone ECM protein is
osteonectin. Osteonectin has multiple Ca2 and collagen binding sites and has been shown to be a potential nucleator of
HAp.77,78 The second most abundant non-collagenous protein
in bone matrix is osteocalcin (OC, a.k.a. Bone Gla Protein).
OC is a vitamin-K dependent protein, which has a Ca2/HAp
afnity, and has also been suggested to play a role in osteoclast migration.79 Osteopontin (OPN, aka BSP-1) is a multifunctional extracellular glycoprotein involved primarily in
cell migration, and regulation of mineral deposition.80 Among
all the non-collagenous matrix proteins found in mineralized
tissue, OPN is unique as it preferentially accumulates at mineralized tissue interfaces (i.e., cement lines and laminae limitantes) and at mineralized tissue/implant interfaces suggesting
a role as an interfacial adhesion molecule, thereby maintaining overall structural integrity of bone and bone/implant
systems.8184 OC and OPN are typical biomarkers used for
mature osteoblast phenotypic behavior.
Bone matrix also contains proteoglycans (i.e., decorin and
biglycan) whose exact role in bone physiology is still being
investigated. Research indicates that these proteoglycans do
inuence cell behavior by blocking adhesion motifs from
RGD-containing molecules and also binding TGF-b.85 The
last class of ECM proteins are glycoproteins. Glycoproteins
are carbohydrates with distinct active domains and play key
roles in cell signaling. Examples of cell-binding

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glycoproteins are: bronectin and vitronectin,86 which both

contain the integrin-binding peptide motif RGD.87,88
Bone cells are known to have numerous adhesion receptors (50 now known) belonging to four basic families: the
integrins, cadherins, selectins, and members of the immunoglobin (Ig) superfamily.89 Of these, integrins have recently
been identied as the principal regulators of cell-matrix
interactions.90 Integrin receptor heterodimers comprise transmembrane a and b subunits that assemble non-covalently by
lateral diffusion to form a metal ion-dependent functional
unit specic to matrix protein ligand motifs89 (e.g., the arginine-glycine-aspartic acid tripeptide RGD) present in
many ECM proteins (e.g., bronectin, vitronectin, bone sialoprotein, osteopontin).11,91 These interactions are highly
regulated and vital to establishing focal adhesion sites key to
establishing cellular adhesion within a scaffold. Upon integrin receptor mediated binding, cytoskeletal components
group to form focal adhesions that provide a cell-matrix
structural link and subsequently activates signaling pathways
directing osteoblast survival, cell-cycle progression, gene
expression, and matrix mineralization.90
Mobile Biochemical Cues in Bone. Mobile cues directing
cell behavior can be produced by local osteoblasts or delivery via the blood stream. Transcriptional and growth factors
such as PDGFs, BMPs, IGFs, and TGF-b have indisputable
roles in the facilitation of osteoblast proliferation, differentiation, and subsequent bone formation and regulation.52,92 In
addition to growth and transcriptional factors, systemic and
local complimentary hormones such as parathyroid hormone
inuence osteoblast proliferation and differentiation.93 Osteoblasts also produce phospholipid membrane-bound matrix
vessels, which aid in nucleation of HAp and mineralization.9497 Matrix vesicles contain calcium, phosphorus, and
alkaline phosphatase (ALP). ALP is ubiquitous in bone,
liver, kidney, placental, and intestinal tissues. Research
has shown that ALP does play a direct role in the induction
of hydroxyapatite deposition on extracellular matrix
proteins.98100 ALP aids in mineralization by hydrolyzing
organic phosphate esters, thus producing an excess of free
inorganic phosphate which initiates the biomineralization
process.99,101 Although ALP activity is an excellent indicator
of osteodifferentiation and mineralization, the exact mechanism of action is still not completely understood.
The local concentration of chemical cues inuencing bone
cells increases substantially in the event of an injury. Bone
tissue injury initiates a cascade of events leading to the
migration of neutrophils, macrophages, and broblasts,
which subsequently express and secrete a variety of cytokines and transcriptional factors which direct MSC migration, protein adsorption, cell adhesion, neovascularization,
brosis, and remodeling/healing.102106
Bone dimensional hierarchy
The remarkable properties of bone depend intimately on
its sophisticated macro, micro, and nanoscale hierarchical
structure (Figure 5).26,107109 For example, the macro-scale
organization of osteons, osteoids, and haversian canals provide long bones with their characteristic mechanical anisotropy. The microscale porosity in bone is ideal for cell
migration and vascularization, whereas the nanoscale features act as a cell and mineral binding architecture. Type I
collagen, for instance, has an approximate diameter of 310
nm and length of 300 lm and binds tiny hydroxyapatite

1545

Figure 5. Schematic of the sophisticated hierarchical structure

of bone.106 From: Figure 1 from Molly M. Stevens
and Julian H. George, Science. 310:11351138 (18
Nov. 2005). Reproduced with permission from American Academy for the Advancement of Science.

[Ca10(PO4)6(OH)2] crystals (
1050 nm long) and other

nanoscale proteins in the ECM. Both the size and orientation
of the HAp crystals are directed by the collagen template,
and the precise structural relationship between the collagen
and HAp is critical to the resilience and strength of bone.
Research has shown that the utilization of signaling sequences alone is not enough to promote differentiation and bone
formation in vitro, and that the nanoscale organization of
these signals is critical.110

Bone Biomimetics
Bulk material selection criteria
A primary objective in bone tissue engineering is to
mimic native bone tissue, and the rst challenge lies in the
selection of a bulk biomaterial. The bulk material composition plays a critical role in the overall success of the
scaffold. The bulk material must be biocompatible, biodegradable, and have appropriate mechanical properties for
load bearing applications. A variety of materials have been
investigated for synthetic bone scaffolds including metals,
ceramics, polymers, and composites of these. Metals such as
titanium, stainless steel, and cobalt-chromium are biocompatible, strong, processable, and relatively inexpensive. However, metals generally have a modulus higher than bone
which may induce stress shielding and do not biodegrade,
which requires additional surgery and may impede native tissue ingrowth.111114 Stress shielding occurs when a xation
device has an elastic modulus substantially higher than the
native tissue. This shielding prevents the native tissue and
the cells within from mechanical stimulation, a primary
affecter in osteoblast phenotypic behavior.115,116

1546

Research has shown that a synthetic bone scaffold should

maintain its mechanical properties for at least 13 months
after implantation (without shielding cells from mechanostimulation) and then should be totally resorbed through metabolic pathways after
1218 months so that it does not
impeded tissue ingrowth and regeneration.59,117 Because of
the difculties associated with measuring the mechanical
properties of bone and the synthetic scaffold during tissue
regeneration and scaffold degradation in vivo, researchers
have developed 3D computational modeling techniques with
the goal of determining the optimal degradation rate of a
synthetic scaffold. For example, assuming the scaffold degradation rate is proportional to the water penetration rate and
ester bond hydrolysis (polymer degradation), and bone
regeneration rate is governed by the rate equation for trabecular surface remodeling.118120
Considering the limited utility of non-degrading synthetic
bone scaffolds or xation devices, the only realistic options
for bulk biomaterial selection are ceramics and polymers.
Calciumphosphate ceramics are an obvious choice and have
consistently demonstrated excellent cellular and tissue
responses in vitro and in vivo. Calciumphosphate ceramics
were introduced more than 40 years ago as bone substitutes.
These materials are considered bioactive as they bond to
bone and enhance bone tissue formation. The bioactivity of
these ceramics has been attributed to the similarity of their
composition and structure compared with the mineral phase
of bone.121 A detailed explanation of the bioactivity of calciumphosphate materials in terms of chemical and physical
interactions with bone cells and tissue is provided below.
The most common types of calciumphosphate materials
investigated for synthetic bone scaffold development are: hydroxyapatite (HAp), Ca10(PO4)6(OH)2, tricalcium phosphate
(TCP), Ca3(PO4)2, biphasic calcium phosphates (BCP), and
multiphasic bio-glasses.122 On the basis of the composition
and stoichiometry of a calciumphosphate ceramic, important physical properties such as degradation rate, modulus,
and processability can be changed.123126 Research on TCP
materials has revealed that the degradation rate is too rapid
in vivo, while synthetic versions of HAp degrade too slowly
to allow native tissue integration. This phenomenon motivated the development of BCP and bio-glasses which have
tunable (to some degree) degradation rates based on the relative magnitude of TCP (more commonly b-TCP) and HAp
in a composite ceramic.127131
To mimic the physical attributes of bone, synthetic scaffolds must have a high degree of porosity. When ceramics
such as TCP, HAp, and BCP are formed into porous scaffolds, the macroscopic mechanical properties are inadequate
for load bearing surfaces due to the inherent brittleness of
the ceramics. This seriously limits their clinical relevance as
synthetic bone scaffolds.127,132,133
Because of their biocompatibility, tunable degradability,
processability, and general versatility, polymers and polymer-ceramic composites are the principle materials investigated for the development of synthetic bone scaffolds.
Polymers may degrade due to hydrolysis from caused by exposure to physiological aqueous environments or by cellular
and enzymatic pathways. The rate of polymer degradation
can be tuned through copolymerization, and changes made
in the hydrophobicity and/or crystallinity of the polymer.134
Common polymers which have been investigated for bone
repair applications include polyesters, polydioxanone, poly
(propylene fumarate)(PPF), poly(ethylene glycol) (PEG),

Biotechnol. Prog., 2009, Vol. 25, No. 6

poly(orthoesters), polyanhydrides, and polyurethanes. An indepth discussion on polymers including degradation

mechanisms and bioactive molecule or drug encapsulation
and release for bone tissue engineering applications is provided below. Polyesters are the most commonly researched
polymers for bone regeneration applications. This is likely
because there are several FDA-approved polyesters with
extensive clinical history. Another favorable characteristic of
polyesters is how easily they can be copolymerized with
variable constituent percentages to ne tune the degradation
rate. Biodegradable polymers, like ceramics generally lack
the mechanical properties required for temporary bone substitutes. Ceramics fail mechanically due to brittleness (hard
material with small elongation to failure), whereas in polymers there is a deciency in the compressive modulus compared with native bone tissue (polymers typically too
soft). This discrepancy may be reduced or eliminated
through incorporation of high modulus micro and/or nanoscale constituents within the polymer matrix. A wide variety
of polymer-ceramic composites have been developed to this
end. The most commonly researched constituent in polymer
composites for bone scaffolds is micro or nanoscale HAp
particles due to its biomimetic and osteogenic properties.
Several inorganic constituents have been utilized to improve
the physical and biological performance of synthetic bone
scaffolds including carbon nanotubes and ceramic or metal
micro and nanobers/spheres.91,135 High modulus dispersed
micro or nanoscale constituents have been shown to improve
tensile strength, modulus, and crack resistance of polymer
scaffolds.136139 Natural polymers such as collagen, glycosaminoglycans, brin, silk, and many others have also been
investigated as a bulk biomaterial for bone scaffold fabrication. Drawbacks for utilizing natural polymers include a
higher risk of infection, xed degradation rates, and immunogenicity. Some researchers, however, have recently
reported successful synthesis of natural polymers such as
collagen in the lab.61
Once bulk biomaterial selection criteria have been considered, a scaffold must be designed and manipulated to mimic the
specic chemical and physical attributes of bone. Both chemical
and physical attributes of the native environment profoundly
inuence osteoprogenitor and osteoblast cell functionality
including bone forming capacity. In the following paragraphs, a
review of basic biomimetic strategies is provided.
Chemical effectors in synthetic bone scaffolds
CalciumPhosphate Materials. The primary constituent
(60%) of bone is calciumphosphate minerals, specically
HAp. Synthetic HAp has been shown to be biocompatible
and promotes osteoblast adhesion and migration/inltration
in vitro.129,130,140143 Dozens of calciumphosphate formulations have been developed and investigated for their
bioactivity [i.e., tricalcium-phosphate (TCP), biphasic calcium phosphate (BCP), hydroxyapatite (HAp), and bioglass
ceramics (BGC)]. Additionally, there have been several successful fabrication techniques (i.e., soaking in simulated
body uid, combustion synthesis, compression/sintering) all
inducing variable biological responses.144146 For simplicity,
all calciumphosphate formulations will be generalized
into one category called calciumphosphate (CaP) materials.
Some CaP formulations (i.e., TCP) have a tunable bioresorption rate (critical attribute of tissue engineering
scaffolds) in addition to be osteoconductive and
biocompatible.123,129,130,140,147

Biotechnol. Prog., 2009, Vol. 25, No. 6

Gui et al., has recently summarized a model for the biointeraction between CaP materials and bone cells. Studies have
demonstrated that CaP materials (specically biphasic CaP)
have superior stability and in vivo osteogenic properties
compared with autologous bone grafts in critical-sized bone
defects.128,148,149 Short- and long-term in vitro and in vivo
studies have conrmed that CaP materials induce osteogenic
differentiation (osteoinduction), promote MSC migration
(osteoconduction), and allow for bony tissue ingrowth and
integration (osseointegration).132,149 However, CaP materials
are most successful as implant coatings considering their mechanical properties exclude them from being viable options
in 3D scaffold applications.150,151
Protein and Peptide Recognition. Immobilized bone
ECM macromolecules act as primary chemical effectors in
cell signaling and functionality. Many bone ECM proteins
contain progenitor and osteoblast integrin binding sites and
growth factor binding sites and present and obvious selection
for developing bone scaffolds.73 Because of the ubiquity and
biocompatibility of Type I collagen and glycosaminoglycans
(GAGs) in bone, they have been extensively investigated for
use in natural and natural/synthetic composite materials bone
scaffolds.75,152156 ECM proteins (specically collagen) provide tensile and bending strength to bone, but no compressive strength. Therefore, bone scaffolds containing natural
proteins must be combined with other constituents to
approach the elastic modulus of bone. In a recent highly biomimetic approach, Ramakrishna et al. fabricated a nanoscale
collagen/HAp composite scaffold. In vitro studies indicated
that osteoblasts seeded on the collagen/HAp scaffold preferentially adhered and mineralized the surface compared with
controls.155 Proteins delivered to the site of an injury via
blood, such as brin and brinogen, have also been investigated as scaffold materials for their hemostatic and cell-binding capacity.157 Several other naturally derived polymers
such as silkworm silk bers exhibit comparable biocompatibility in vitro and in vivo as compared with other commonly
used biomaterials such as collagen and polylactic acid
(PLA).158
Despite the optimism surrounding this approach, several
important barriers remain in the pursuit towards utilizing
full-length natural proteins for the construction of in 3D
scaffolds. First, there is a lack of control over the mechanical
and chemical properties of components. Second, there are
concerns of immunogenicity, and complications associated
with efcient isolation and purication of natural proteins.73
Therefore, many researchers are working towards the incorporation of the biologically signicant regions (peptides) of
natural proteins into synthetic materials whose degradation
and mechanical qualities can be tuned for a specic application. The most common peptide-based strategy involves the
inclusion or deposition of the peptide arginine-glycine-aspartic acid (RGD), which mediates cell attachment to several
matrix proteins including brinogen, bronectin, vitronectin,
and osteopontin. Several groups have successfully incorporated or coated RGD, or RGD-containing oligopeptides into
synthetic materials and shown a modest improvement in
osteoblast and osteoprogenitor cell functionality.87,88,159 One
explanation for the only modest improvement in cell adhesion, spreading, and mineralization is that the RGD peptide
lacks integrin binding selectivity and triggers non-discriminatory cell attachment. Research is ongoing to discover more
selective peptide sequences which trigger osteoblast adhesion. Recently, Garcia et al. synthesized a collagen-mimetic

1547

peptide (glycine-phenlalanine-hydroxyproline-glycine-glutamate-arganine, GFOGER), which substantially enhanced

osteoblast functionality and osseointegration in vivo compared with control surfaces.160 This approach is relatively
new and holds tremendous promise for all tissue
engineering.
Growth and Transcription Factors. Another popular
strategy for enhancing osteoprogenitor and osteoblast functionality is the incorporation of growth, adhesion, and transcription factors into a synthetic scaffold. Methods for
molecular encapsulation and release from synthetic scaffolds
will be reviewed in a later section. Here, a brief overview of
the importance of these factors in bone regeneration through
scaffold technologies is provided. Growth and transcription
factors are large polypeptides that are synthesized and
expressed in very low physiological concentrations to act as
local regulators of cell behavior. These large molecules
direct cellular activity by binding to specic transmembrane
cell receptors which subsequently triggers the intracellular
domain and activates transcription of a gene into mRNA and
consequently protein production. An excellent review of
physiological roles and in vitro/in vivo analysis of growth
factors is provided by Solheim et al.161 The motivation for
utilizing these factors in synthetic bone scaffolds is established by the fact that in normal bone physiology, osteoprogenitor proliferation, migration into osseous tissue, and
differentiation into osteoblasts (osteoconduction) occur under
the inuence of growth and transcription factors. For example, the osteoblast secreted transcription factor Cbfa-1/Runx2 plays key roles in bone remodeling and is required for the
expression of osteoblast-specic genes and proteins, such as
osteocalcin.52,162 There have been a variety of growth factors
identied and isolated for in vitro and in vivo analysis
including: PDGFs, IGF-Is and IIs, TGFs, and acidic or basic
broblast growth factors (FGSs).163,164 The general principle
of growth factor encapsulation and release is the same for all
of them. A key subset of growth factors (BMPs) is reviewed
here. BMPs are multifunctional growth factors belonging to
the TGF-b superfamily. Since their discovery in 1965, over
20 BMPs have been investigated for cell signaling and bone
forming ability.165 Of the 20 discovered, only three (BMP-2,
-4, and -7) have been able to stimulate the osteoprogenitor
differentiation into mature osteoblasts in vitro. One common
method of BMPs delivery is via biodegradable polymeric
scaffolds or hydrogel.166168 BMPs have been tested in preclinical and clinical studies, showing their denite potential
in osteoinduction and have been FDA-approved for clinical
use in open fracture of long bones, non-unions, and vertebral
arthrodesis.169,170 However, the transition into clinical studies has had only mild success and relies on large doses of
BMPs for bone formation.171,172 When BMPs are released
naturally by cells, mere nanogram quantities of the proteins
per gram of bone matrix are enough to trigger the bone
repair cascade. Yet microgram quantities of BMP per gram
of matrix material (over six orders of magnitude higher)
seem to be needed to produce the same effect with an articial matrix.27 Additionally, their clinical usefulness as regenerative agents may be cost prohibitive and also limited by a
short in vivo half-life and low specic activity. Therefore,
gene therapy is an alternative route for exploiting the boneinductive activity of this class of molecules.173
In gene therapy, genes coding for the production of specic proteins are delivered to cells via a virus vector (i.e.,
retrovirus, adenovirus, adeno-associated virus, herpesvirus),

1548

or via non-viral delivery (i.e., naked DNA, oligonucleotides,

lipoplexes). The most effective delivery system depends on
the application. Gene therapy aims to eliminate problems
with direct protein therapy by genetically modifying cells to
produce an increased amount of a selected protein in a sustained manner in vivo or ex vivo. Two basic gene therapy
approaches have been taken in bone regeneration. In one
approach, genetically modied cells are injected directly into
the fracture site. In the other approach, genetically modied
cells are seeded and cultured on a scaffold in vitro before
implantation. Several groups have shown favorable results
for this therapy in bone regeneration, a comprehensive
review is provided in the literature.174 Gene therapy is an
exciting new prospect in bone repair and regeneration. There
are still a number of considerations to be made, however,
before widespread clinical studies can begin such as: (1)
safety of administration of viral vectors, (2) identication of
the optimal combination of gene transfer vector, regenerative
molecules, and cell source, (3) characterization of protein
release rate and therapeutic dose requirements, and (4) characterization of cell fate after implantation.

Physical effectors in synthetic bone scaffolds

The elaborate hierarchical geometric structure of bone
(Figure 5) is critical not only for the macroscopic mechanical properties but also for progenitor and bone cell survival
and functionality at the micro and nanoscale. Because of the
direct apposition and binding of the ECM proteins and cell
cytoskeleton through cell receptors, cells sense and respond
to the physical properties of the matrix by converting mechanical cues into intracellular chemical signals which drive
activities such as gene expression, protein production, and
general phenotypic behavior.73,175178 Therefore, a primary
goal in bone scaffold design is to mimic the unique micro
and nanoscale characteristics of bone. In recent years, it has
been well established in the literature that bone cells are profoundly inuenced by topography in vitro.11,107,179182
The microscale features of bone provide a conduit for vascularization, nutrient delivery, and cell migration. Several
researchers have suggested that for a bone cell and nutrients
to migrate into a scaffold, the pores should be at least the
size of the cell.183 Highly porous microscale scaffolds also
allow for higher levels of nutrient diffusion, vascularization,
and better spatial organization for cell growth and ECM production.184,185 There is still some ambiguity surrounding the
optimal porosity and pore size for a 3D bone scaffold. A literature review indicates that a pore size in the range of 10
400 lm may provide enough nutrient and osteoblast cellular
infusion, while maintaining structural integrity.185190 A
wide variety of fabrication techniques have been investigated
to recreate the microscale porosity and special organization
of native bone. Some examples of well developed techniques
include: micromachining, photolithography, calciumphosphate sintering, rapid prototyping, melt extrusion, salt leaching, emulsion templating, phase separation, ber bonding,
membrane lamination, and polymer demixing.179 Arguably,
the most successful fabrication techniques for macro and
microporous synthetic bone scaffolds have been emulsion
and sintering of calciumphosphate materials. Using these
techniques, synthetic bone scaffolds have been prepared with
porosities of up to 70%. These scaffolds have demonstrated
excellent bioactivity in vitro and bone ingrowth in
vivo.191,192

Biotechnol. Prog., 2009, Vol. 25, No. 6

Although microscale porosity plays a key role in the

osteoconductivity of a scaffold, the nanoscale architecture of
the material is the primary physical inuence on the osteoinductivity and osseointegrativity of the scaffold. Until
recently, most synthetic bone scaffold fabrication techniques
were limited to the macro and microscale which are unable
to recreate the sophisticated nanoarchitecture in bone. In the
past decade, however, there have been several advances in
nanofabrication and hence control over cell behavior through
nanomanipulation. Bone cells in native tissue interact with
nanoscale proteins and minerals. It is well established that
all living systems are governed by molecular interactions at
the nanometer scale. The unique properties of all the molecular building blocks of life, such as proteins, lipids, carbohydrates, and nucleic acids are all governed by their nanoscale
sizes, patterns, and folding.91 Consequently, bone cells are
predisposed to adhere, grow, proliferate, differentiate, and
produce ECM based on these nanoscale interactions.91,193,194
In addition to enhancing cellular activities, nanoscale materials may have tunable surface area and energy which has
been shown to effect adhesion protein adsorption.86 Similar
to microscale fabrication, a wide variety of nanofabrication
techniques have emerged in recent years. Examples of
successful 2D and 3D nanofabrication techniques include
electrospinning,195199 nanotemplating,179,200 electrostatic
spray deposition,201,202 RF plasma spray/deposition,203 molecular self assembly,204206 and metal oxide anodization.104,182,207209 Of the fabrication techniques listed above,
electrospinning has emerged as a very promising strategy for
fabricating bone-mimetic nanober scaffolds. The popularity
of electrospinning is likely due to the simplicity of the experimental setup, the ability to incorporate bioactive molecules, and the versatility is provides. A literature review
revealed that over 100 biodegradable polymers have been
successfully electrospun, more than 30 of which were used
in tissue engineering applications.91 Fiber diameter, and
hence porosity, can be nely tuned through simple changes
made in the spinning setup. In electrospinning, the bulk material
of interest is dissolved in an organic solvent and subsequently
pulled under the inuence of an electric eld through a needle
and nally deposited in a non-woven pattern on a collector plate
(Figure 6).210 Both in vitro and in vivo studies have demonstrated that osteoprogenitor cells differentiate, proliferate, and
adhere in synthetic nanobrous matrices.211,212
Another emerging fabrication technique for nanoscale
manipulation for tissue engineering applications is called
nanotemplating or template synthesis. Template synthesis is
a simple procedure which provides a controlled approach for
developing nanoscale polymer constructs for tissue engineering applications.182,213 In template synthesis, a polymer is
heated above its melting temperature and gravimetrically
extruded into a nanopore membrane which is subsequently
removed leaving the negative template of nanowires (Figure
7). Recently, substrate-bound arrays of poly(caprolactone)
(PCL) nanowires were synthesized using this technique.
MSCs seeded on PCL nanowire surfaces displayed enhanced
functionality in terms of accelerated differentiation and ECM
production over those seeded on smooth control surfaces.200
Considering the important and distinct roles which microscale and nanoscale architectures play in native bone physiology, researchers are aggressively pursuing technologies
which lend themselves to multiscale hierarchical manipulation. Electrospinning combined with ber bonding is one
technique which has been used to accomplish this.195,214,215

Biotechnol. Prog., 2009, Vol. 25, No. 6

1549

Figure 6. Typical electrospinning setup consisting of a high-voltage supply, syringe and syringe pump, and blunt-tip catheter. Fiber
mat (right) SEM shows ber morphology.

Figure 7. PCL nanowire fabrication.At 658C, PCL is gravimetrically extruded in to an alumina membrane (AC) followed by removal
of the membrane in NaOH (D). SEM images of the PCL nanowire surfaces indicates that uniform distinct nanowires were
developed using this technique (F,G).

Templating is another fabrication technique which is capable

of developing multidimensional structures.104,182,200,213 Other
techniques such as modied rapid prototyping,216 electrochemical anodization, template synthesis, and polymer-mineral emulsion have also demonstrated the ability to construct
multiscale synthetic scaffolds.

Localized Bioactive Molecule and

Drug Delivery in Bone Repair
As bone biology, natural bone healing cascades, and bone
pathogenesis is more clearly understood, new therapeutics
and therapeutic strategies are emerging. The challenge to tissue engineers is to design and develop temporary bone scaffolds which deliver these bioactive molecules and drugs to
the injury site and hence extend its biological functionality
(i.e., direct and accelerate healing and tissue regeneration
while simultaneously preventing pathology). Although mimicking the geometric architecture of bone in a synthetic scaffold has been shown to promote favorable cellular activity,
the overall capacity for a scaffold to direct cell behavior can

be substantially improved through the controlled delivery of

biospecic cues.49,217221 Administration of growth factors
and other bioactive molecules to promote bone formation
and repair has achieved promising results in several preclinical and clinical models.222227 A variety of administration
methods have been investigated including: bolus injection,
surface adsorbed protein release, osmotic pumps, and controlled release from biodegradable scaffolds. The efcacy of
the delivery vehicle relies on its ability to provide the appropriate dose over the appropriate therapeutic time frame.
Therefore, many of the strategies investigated (such as bolus
administration) is not effective due to rapid diffusion from
the target site and deactivation/digestion by ubiquitous
enzymes.228 Ideally, the presentation of bioactive molecules
or drugs must be nely tuned to dynamically match the
physiological needs of the tissue as it regenerates. Because
of the hydrolytically unstable linkages in their backbone and
tunable biodegradation rate, polymers have demonstrated the
most success in controlled delivery of bioactive molecules.
Ceramic materials have also demonstrated the ability to biodegrade and release bioactive molecules at a controlled

1550

rate.123,129,130,140,147 However, these materials hold little clinical utility due to their poor mechanical properties discussed
above. Natural polymers such as collagen, brin, alginate,
gelatin, and GAGs have also been extensively investigated
as drug delivery vehicles in bone tissue engineering. These
natural polymers have distinct advantages due to their inherent biocompatibility and bioactivity but lack the mechanical
properties required for load bearing applications, may have
inappropriate (xed) degradation rates, are difcult to harvest and sterilize, and may induce an immunogenic response.
Therefore, the focus of this section is on the use of biodegradable polymer materials as drug delivery systems in bone
tissue engineering. Bioactive molecules can be covalently
bound to polymers or physically entrapped inside a polymer
matrix.88,229 In either case, the molecule release as the polymer degrades in the physiological environment.

Mechanisms of polymer biodegradation

Biodegradation of polymers can be generally categorized
as bulk or surface degradation.230,231 In bulk degradation,
the rate of water penetration exceeds the degradation and
solubilization rate of surface molecules resulting in a bulk
material degradation and consequently the loss of macroscopic mechanical properties of the scaffold. In surface degradation, the surface molecules degrade and solubilize faster
than the water penetration rate resulting in surface erosion
while bulk material maintains its structural integrity. Polyesters, polyetheresters, and polyesteramides are classied as
bulk-degraders and typically yield rst order release proles,
while polyanhydrides and polyorthoesters are classied as
surface eroders which typically yield zero-order proles.
Most polymers utilized for synthetic bone scaffolds are
semi-crystalline polyesters, in which case there are two primary mechanisms of degradation. First, the polymer is
degraded through random chain scission characterized by a
reduction in molecular weight.232 Random chain scission
means that any ester bond in the chain has equal probability
of cleavage via hydrolysis. This is in contrast to chain-end
scission reaction which, as the name implies, occurs at the
terminus of the polymer chain. No weight loss is observed in
this initial phase which covers a molecular weight of
200
kDa to 5 kDa. Second, when the molecular weight is
reduced to a sufciently low number (5 kDa has been suggested233), polymer fragments diffuse out of the bulk polymer matrix characterized by a measurable weight loss. A
decrease in chain scission is likely in the second phase due
to an increase in polymer crystallinity since chain scission
preferentially occurs on amorphous regions of the polymer.233235 In practice, however, biodegradation of a polymer scaffold cannot be characterized this simply as the
degradation rate and mechanisms will also depend on (1) the
micro and nanoscale structure of the scaffold, (2) the polymerization of multiple polymers/polymer types, and (3) the
presence of hydrolytic accelerators or suppressors. The in
vivo degradation rate of polyesters is signicantly enhanced
compared with in vitro degradation rates.236238 This is
assigned in part to the optimum concentration of esterdegrading enzymes such as lipases in the human
body.232,237,238 To mimic the in vivo environment, many
researchers utilize bacterial enzymes such as Pseudomonas
cepacia lipase at higher than physiologic concentrations.232,239,240 Selection of a polymer for synthetic bone
scaffold development pivots on some key design criteria

Biotechnol. Prog., 2009, Vol. 25, No. 6

such as: (1) degradation rate is comparable to tissue development/ingrowth rate, (2) biodegradation products are biocompatible, and (3) polymer processability (i.e., ease of
copolymerization, covalent attachment of key molecules). In
the following paragraphs, a brief introduction to several of
the most commonly utilized polymers and copolymers for
synthetic bone scaffolds is provided. A more detailed comprehensive review on the application of biodegradable polymers to bone regeneration and drug delivery is available in
the literature.49

Polyesters
Aliphatic polyesters such as poly(lactic-acid)(PLA), poly
(glycolic-acid)(PGA), and poly(caprolactone) (PCL), and
their copolymers are the most commonly utilized polymers
in bone tissue engineering.11,49,133,241 These polymers have
been FDA-approved and utilized in a wide variety of clinical
applications such as sutures, systemic drug delivery, spinal
fusion cages, coronary stents, xation screws, and nerve conduits.233,242245 PLA is the cyclic dimer of lactic acid, which
exists as two isomers: D- and L- Poly(lactic aid) (PLLA) is

37% crystalline with a melting temperature of 6065 C,
and a degradation time of up to several years.246,247 Both
PGA and PLA scaffolds has been investigated as a slowdelivery carrier for growth factors in several in vitro and in
vivo studies, and demonstrated the ability to promote healing
and osseointegration compared with control scaffolds.248250
However, due to the low modulus of PLA, it must be either
copolymerized with a higher modulus polymer, or made into
a composite with a different material. PGA, the simplest linear aliphatic polyester is highly crystalline (4555%), has a
high melting point (220 C), and a glass transition temperature of
35 C. PGA alone has a high modulus (7 GPa), and
completely degrades in vivo within 46 months.251 Like
PLA, PGA has also been used in a bone tissue engineering
applications. However, most researchers copolymerize PLA
and PGA to increase control over degradation rates and
mechanisms for a specic application. A description of common copolymers utilized in drug delivery for bone tissue
engineering applications is provided below. Poly(e-caprolactone) is another semi-crystalline polyester with a glass transition and melting temperature of approximately 60 C and
60 C, respectively. The degradation time for PCL is similar
to PLA (2 years in vivo). Because of it relatively slow
degradation rate, and high modulus compared with other
FDA approved biodegradable polyesters, it is well suited for
orthopedic and drug delivery applications.233,252 Additionally, PCL degradation products are easily resorbed through
metabolic pathways and do not produce local acidic environments as opposed to polylactides and glycolides. The local
acidic environment produced by polylactides and glycolides
may effect the stability of a protein or other bioactive molecule in the preparation and delivery stage.233,253,254 One
group has recently demonstrated enhanced osteoblast functionality in vitro and bone formation in vivo as a result of
controlled delivery of calciumphosphates and growth factors from PCL scaffolds.255258 In another novel approach,
PCL scaffolds were functionalized with laminin-derived peptide sequences known to promote adhesion and proliferation
(i.e., RGD, YIGSR). In vitro analysis with adipose-derived
stem cells indicated that peptide grafting to PCL materials
enhanced cellular adhesion and proliferation.259 PEG is a
hydrophilic polyether with an extensive clinical history. On

Biotechnol. Prog., 2009, Vol. 25, No. 6

the basis of the synthesis technique, PEG can have a wide

range in % crystallinity, molecular weight, melting temp,
glass transition temperature, and degradation rate. Many
researchers have co-polymerized PEG with other hydrophobic polymers (such as PLA, PGA, PCL) to enhance the degradation rate and neutralize acidic degradation products.260

1551

successfully loaded with recombinant human BMPs and

demonstrated accelerated healing and osteogenesis in
vivo.285,286 In a novel in vitro approach, another group systematically varied the ratio of PLA/PEG and measured the
release of a growth factor cocktail to determine to ideal ratio
for osteoblast functionality indicated by matrix mineralization and ALP activity.229

Common copolymers in bone engineering

To gain more control over the degradation rate, hydrophobicity, % crystallinity, and subsequent biological functionality, researchers combine multiple polymers in a chemical
process called copolymerization. Copolymerization is analogous to the design of composite materials where multiple
constituents are combined resulting in a new material with
desirable properties from each constituent. Undoubtedly, the
most commonly utilized copolymer for bioactive molecule
encapsulation and release for bone tissue engineering is the
copolymer poly(lactic acid-co-glycolic acid) (PLGA). Several researchers have utilized this well-characterized copolymer for encapsulation and release of a wide variety of
bioactive molecules and drugs including TGF-b, BMPs,
IGFs, VEGF, NGF, DNA, vancomycin, gentamysin, cisplatin, and others.261268 To successfully fabricate and encapsulate PLGA scaffold with these molecules, a wide variety of
creative approaches have been taken such as emulsion
freeze-drying,269 nano and microparticle encapsulation,270272
double emulsion solvent extraction,273 electrospinning,274276
compression molding,277 and others. As mentioned earlier, a
fundamental problem with utilizing growth factors is their
characteristic short half life in physiological environments.
Long-term delivery of a set quantity of growth factors substantially enhances MSC response in vitro and bone formation in vivo compared with bolus administration. In an effort
to prolong the release of viable growth factors, several
groups have loaded variable sized PLGA microspheres with
growth factors and subsequently imbedded them within other
polymer matrices with variable degradation rates. For
example, one group recently demonstrated that growth factor-loaded PLGA microspheres imbedded within a poly(propylene fumarate) (PPF) matrix displayed prolonged release
of active growth factor in vivo for a period of 12 weeks.278
This resulted in a substantially better bone formation
compared with controls. Other groups have investigated similar approached by entrapping growth factors within PLGA
scaffolds which have been mineralized through soaking in a
simulated body uid. Another novel strategy to prolonging
viable growth factor release from PLGA scaffolds is to
conjugate the copolymer with heparin, a highly sulfated glycosaminoglycan. Since heparin is known to bind growth factors, several groups have incorporate it into PLGA scaffolds
to immobilize growth factors and prevent diffusion thereby
prolonging their viable release.279283 This strategy has proven to be very successful and holds promise for the future of
growth factor delivery systems for bone regeneration. However, although PLGA has shown promise in bone scaffold
applications, its clinical utility is limited due to its relatively
poor mechanical properties (specically Youngs Modulus)
compared with cancellous bone, and therefore must be combined with other materials to enhance its mechanical
properties.284
PLA-PEG is another example of a common copolymer
investigated for bone tissue engineering applications. Several
groups have fabricated PLA-PEG scaffolds which have been

Delivery of multiple bioactive molecules

In vivo, bone healing and regeneration is driven by the
action of a number of growth factors. Several researchers
have reported on the sequential cellular production and
expression of multiple growth factors in preclinical fracture
healing experiments.287,288 Multiple researchers have also
observed a synergistic effect (i.e., enhance bone formation)
from local sequential administration of multiple growth factors on osteoprogenitor and osteoblast cells in vitro and in
vivo.289292 Despite this, many synthetic bone scaffolds rely
on the delivery of single factors, which may partially explain
the limited clinical utility of many current approaches.262
Therefore, researchers have been investigating techniques to
encapsulate and release multiple bioactive molecules in a
highly controlled spatial and temporal manner. Research has
shown that this method signicantly enhances tissue regeneration compared with the controlled release of single biological cues.293295 For example, one group recently reported
enhanced pluripotent cellular differentiation, ALP activity,
and matrix mineralization through sequential delivering
BMP-2 and IGF-I through a novel layered biodegradable
polymer scaffold.222 Another group demonstrated enhanced
osteogenic differentiation in vitro through sequential administration of BMP-2 and -7 via polymer degradation.296 The
technology of incorporating multiple chemical effectors and
controlling their spatial and temporal release is a very promising strategy, but is still in its infancy and has not yet demonstrated widespread preclinical or clinical utility to date.
Antibiotics and chemotherapeutics
Most of the discussion above is focused on the controlled
delivery of growth factors to promote healing and tissue integration. It is equally important, however, to encapsulate and
release drugs which prevent pathologies that can occur post
implantation of a synthetic scaffold. A wide variety of drugs
have been encapsulated and released from biodegradable
polymer scaffolds including antibiotics, DNA, RNA, cathepsin inhibitors, chitin, chemotherapeutics, bisphosphonates,
statins, sodium uoride, dihydropyridine, and many
others.48,297300 The scope of this section is limited to basic
motivation and strategies in encapsulation and local controlled release of antibiotics and chemotherapeutics for the
purpose of sepsis and cancer recurrence prevention. Infection
is dened as the homeostatic imbalance between the host tissue and the presence of microorganisms at a concentration
exceeding 105 per gram of tissue.301 Infection seriously limits the healing and regenerative capacity of a tissue and
remains a major limitation in the long-term utility of orthopedic implants. Bacteria are ubiquitous, as are the potential
contamination sources in a clean operating room despite
painstaking efforts to maintain an aseptic environment.
Infection (sepsis) and subsequent patient morbidity and huge
associated costs has been reported to occur as frequently as
5% in the case of total joint replacement surgeries.302304

1552

Naturally, prophylactic administration of antibiotics has been

shown to reduce the risk of sepsis by 81%.303 However, conventional systemic delivery of antibiotics has major drawbacks such as systemic toxicity, renal complications, liver
complications, poor penetration into necrotic tissue, and the
need for hospitalization.305 In an effort to prevent these
adverse systemic side effects, researchers are aggressively
pursuing strategies to deliver antibiotics locally to the site of
injury/surgery. The most common biodegradable polymer/antibiotic combination is PLGA scaffolds loaded with antibiotics such as ciprooxin, gentamysin, and vancomysin. PLGA
scaffolds have demonstrated successful sustained local delivery of these antibiotics for up to 20 or more days in vitro
and in preclinical animal models.263,264,306308 The focus in
the majority of these experiments is on characterizing polymer degradation, encapsulation efciency, and drug release
rates and not on clinical outcome. In fact, there is very limited literature available on preclinical and clinical outcomes
of treatments using this strategy. Although local delivery of
antibiotics has a very promising outlook, there remains a
number of challenges (such as antibiotic stability within the
scaffold and antibiotic deactivation during fabrication),
which need to be addressed before clinical trials can begin.
Under the similar motivation for local delivery of antibiotics,
inter-institutional multidiscipline groups of researchers, scientists, and oncologists are working towards the development
of biodegradable bone scaffolds which locally deliver physiologically relevant doses of chemotherapeutics to prevent
cancer recurrence in osteosarcoma patients. Similar to local
antibiotic delivery, the primary advantage of local chemotherapeutics delivery is that the patient does not experience
the serious side effects from systemic administration. In an
effort to improve patient functionality and quality of life, the
primary treatment strategy in osteosarcoma patients has transitioned from amputation to limb sparing techniques in
recent years.309 However, unlike amputation, marginal resection of the primary tumor in limb sparing techniques can
result in the rate of local recurrence to be as high as 28%.310
Therefore, biodegradable bone scaffolds capable of sustained
and controlled drug elution are ideal candidates for limb
sparing treatments in osteosarcoma patients. One group
recently investigated an open cell polylactic acid scaffold
containing 8 wt % cisplatin (platinum-based chemotherapeutic) in an animal model. The rate of local recurrence was
signicantly reduced for mice with marginally resected
mammary carcinomas, and dog with incompletely resected
soft tissue and bone tissue sarcomas.311,312

Biotechnol. Prog., 2009, Vol. 25, No. 6

cation techniques include: sintering/compression followed by

ultrasonic welding,264,285 hot melt encapsulation,313,315,319
high pressure CO2,314 and mechanical mixing. In hot melt
encapsulation, the processing temperature must be below the
denaturation or deactivation temperature of the bioactive
molecule or drug encapsulated. Another practical challenge
in polymeric scaffold fabrication is the prevention of
high loading conditions during processing which may cause
protein aggregation and denaturation.220 Sterilization techniques must also be considered from the onset of scaffold
design.

Concluding Remarks
Limitations with utilizing autogenous bone grafts as the
gold standard treatment for critical-sized defects in bone
have motivated this dynamic eld of bone tissue engineering
whose nal goal is the design of synthetic biodegradable
replacements for bone in cases where critical-sized defects
have been induced by tumor resection, orthopedic surgery,
or trauma. Although technologies and strategies reviewed in
this article are progressive towards the development of a
synthetic component capable of mimicking the physiochemical attributes of bone, no single tissue engineering scaffold
to date has demonstrated the ability to meet the comprehensive requirements outlined here. Bone tissue engineers are
aggressively pursuing solutions to remaining fundamental
challenges in this eld such as the: (1) development of a
scaffold which presents appropriate mechanical properties
throughout the course of biodegradation, (2) effective, nontoxic strategies for drug and bioactive molecule encapsulation resulting in tightly controlled temporal and spatial longterm release proles, and (3) directing local pluripotent cell
population behavior through physically mimicking the multiscale hierarchical architecture of native bone.
The technological advances in synthetic bone scaffolds
discussed herein demonstrate several obstacles that have already been overcome, and also that there is still a substantial
gap to bridge before bone scaffolds are able to address the
aforementioned challenges. Arguably, an elegant union of
solutions to each of those individual challenges will likely
result in the most positive patient outcomes. In the near
term, such technological unions are unlikely to be able to
match the close spatial and temporal control over the bone
environment that has been observed in healthy bone. The
best strategies to developing bone scaffolds will be to incorporate cross-functional interdisciplinary approaches aimed at
enhancing key events rather than exerting start-to-nish control over the bone development process.

Other critical design considerations

In addition to the challenges associated with controlling
polymer degradation and delivery of bioactive molecules and
drugs, a number of practical fabrication challenges exist. The
predominate method for particle or molecular encapsulation
in polymeric scaffolds entails the use of organic solvents.
Although this technique has advanced the overall science of
controlled drug delivery in tissue engineering, unintended
release of residual solvent trapped in the polymer after processing is harmful to adherent cells, local growth factors, and
neighboring tissue.221,313318 Residual organic solvents in a
polymer scaffold limit or prevent the encapsulation of active
growth factors and hence functionality of the scaffold.
Therefore, novel solvent-free techniques are being aggressively pursed. Some examples of solvent-free scaffold fabri-

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