Oncostatin-M enhances osteoinduction in a rabbit critical calvarial defect model
Appendix I
TABLE OF CONTENTS
Introduction
page 3
Bone
page 5
Bone remodeling and regulation
page 7
Cellular communication in bone and elsewhere
page 9
Tissue engineering
page 11
Bone growth factors (cytokines)
page 14
Oncostatin-M
page 20
Osteoconductive materials and carriers
page 24
Conclusion
page 29
Appendix I references
page 30
2
APPENDIX I
INTRODUCTION
Otolaryngologists, especially those who specialize in reconstructive surgery, are often
faced with the difficult task of rebuilding bony defects. In the head and neck,
vascularized and nonvascularized grafts are used for reconstructing congenital, traumatic,
or post-oncologic surgery defects. Grafts range from nonvascularized split calvarium for
cranioplasty or maxillofacial trauma to free tissue transfer of vascularized bone, such as
fibula or iliac crest for mandibular reconstruction.
Since Macewen pioneered the clinical interest in bone grafting, in 1881, the longstanding
clinical and research question of how bone formed has had increasing light shed on itA1.
A considerable body of literature now exists both on bone homeostasis and fracture
repair. Specifically, since UristA2 discovered the ability of bone matrix to create new
bone in non-bony sites, the area of bone growth factor research has exploded. With the
discovery of the inherent activity of bone matrix came the ability to purify the factors or
cytokines contained within this matrix. Eventually, through more advanced technology
over the last 2 decades, extensive basic science research has lead to the clinical
applications of bone growth factors in commercially available formulations. Although
great progress has been made, a major combined clinical and basic science research
undertaking is still ongoing and required to formulate the proper carriers for and
3
combinations of bone active cytokines to replicate the processes of fracture repair or bone
growth. The promise this technology holds is the ability to control and model bone
growth in situations where it would otherwise not be possible.
4
BONE
Bone is a biphasic material made up of an organic matrix of collagen fibrils embedded in
an amorphous substance, with mineral crystals precipitated within this matrix. Bone is
derived from mesenchyme through either intramembranous or endochondral
ossificationA3. In endochondral ossification, a cartilaginous model is replaced by bone
through vascular invasion (angiogenesis) which recruits undifferentiated mesenchymal
stem cells (MSC) which differentiate to become osteoblasts. Osteoid is then produced
and mineralized. The formation of membranous bone is similar but lacks the
cartilaginous phase. Endochondral bones include long bones, ribs, vertebrae, and skull
base. Membranous bones include the cranium, facial skeleton, and clavicles.
Bone may also be generally divided into two morphological categories: Compact bone
(Haversian or cortical bone) and trabecular bone (cancellous or spongious bone). About
80% of the skeletal mass is compact bone with the remainder trabecular. Cortical bone is
characterized by the Osteon as its structural unit. In an osteon, an osteocyte is centrally
located and surrounded by concentric layers of bone and blood vessels and a Haversian
canalA4.
Bone is a composite structure which also consists of organic and inorganic compounds.
Two-thirds of bone by weight is inorganic, largely calcium-based salts with the
remainder fibrous protein matrix collagenA1. This inorganic component is biocompatible
5
and non-immunogenic. The major constituent is in the form of a poorly crystalline,
calcium phosphate compound, called hydroxyapatiteA5. The characteristic toughness and
strength of bone result from the nature of the bone matrix, the mineralized extracellular
component produced by osteoblastsA6.
Bone is a living substance and contains four main cell types within itA7:
(1) Osteoblasts are the cells which lay down bone. This process can occur during
embryogenesis, during routine remodeling, or during fracture healing. As the process of
laying down bone slows, osteoblasts are incorporated into the matrix and become
osteocytes, others will continue to line the surface.
(2) Osteocytes have cell bodies which are found in lacunae and their cytoplasmic
processes extend through canaliculi to communicate with each other and the surface cells.
(3) Lining cells are remnants of osteoblasts that laid down matrix but were not
incorporated as osteocytes.
(4) Osteoclasts are multinucleated giant cells which resorb bone. They are found
in shallow pits on flat bone surfaces or in the edge of cutting cones in Haversian bone.
6
BONE REMODELING & REGULATION
Friedlaender has termed the balance between bone formation and remodeling as the
remodeling cycleA8. When formation and remodeling are occurring at the same rate, the
process is described as coupled or in homeostasis. Alterations in either direction result in
pathologic changes to bone – an uncoupling of the balance.
ReckerA7 discussed the regulation and control of coordinated cellular events which occur
in living bone. He described four discrete functional subdivisions:
(1) Growth to affect elongation of the skeleton and creation of trabeculae.
(2) Modeling to shape and sculpt the skeleton during growth in response to
mechanical forces.
(3) Remodeling to remove aged bone coupled with formation to lay down new
bone in its place. This ensures that damaged bone from mechanical usage is not allowed
to deteriorate to the point of fracture, but, rather is replaced with new strong bone.
(4) Fracture healing consists of clot formation, MSC proliferation, and callus
creation. The appearance and formation of a large volume of cartilage and woven bone
around the fracture represents the genesis of a callus. This callus will then be removed
and replaced with normal lamellar bone.
The healing of fractures has some features which are similar to the process of soft tissue
repair but bone, itself, has distinct properties which contribute to its capacity for
7
regeneration. In contrast to soft tissue, bone undergoes true regeneration rather than
simple repair through fibrosis and, eventually, scar formation. Osseous tissue has the true
ability, after injury, to be restored to its characteristic organizational structureA9.
8
CELLULAR COMMUNICATION IN BONE & ELSEWHERE
Communication among individual cell types which populate bone is of critical
importance to determining the properties of this tissue. Cytokines, which may be broadly
defined as soluble products released from one cell that can modulate the activity of other
cells, play an important role in this process. Individual cytokines may have multiple
biological activities and multiple cytokines may share similar or common functional
properties. The term cytokine has been used to refer to a variety of substances including:
growth factors, monokines, lymphokines, interleukins, and differentiation factors.
Cytokines may act by 3 main mechanisms:
(1) Autocrine: by modifying the action of the cell of origin to produce feedback
regulation.
(2) Paracrine: by modulating the activities of other cell types.
(3) Endocrine: by traveling through the circulation to affect cells in a target
organA10.
Some authorsA11 also consider a fourth method to be incorporation of the cytokines into
the bone matrix where they may be released later during remodeling or fracture. This
may facilitate coupling of resorption with regeneration in bone. The trapped cytokines
may function as local regulators of bone cell activity by release during various stages of
9
the remodeling sequenceA12. At time of fracture, cytokines released by platelets and from
local inflammatory cells would also interact with this liberated pool.
Cytokines may act at a variety of stages in development where they influence cell
activities such as recruitment, migration, differentiation, and proliferationA13. Skeletal
embryogenesis is the product of a defined sequence of cellular differentiation from basic
pluripotent stem cells to individual differentiated cell types. These cellular changes are
also accompanied by extracellular matrix changesA10.
10
TISSUE ENGINEERING
In 1993, Langer and VacantiA14 described tissue engineering as a branch of medicine and
engineering that seeks to develop biological substitutes for repair or regeneration of
tissues. In terms of osseus tissue regeneration, in particular, focus in tissue engineering
was to shift reconstruction from inert cements to biologic, cell-based tissue and organ
replacementA15. The basic premise of bone tissue engineering is that the repair and
regeneration of bone can be guided through control of cells, materials, and the
microenvironment into which these cells are deliveredA16. The classic bone tissue
engineering paradigm involves 3 components: (1) A scaffold or matrix, (2) Bone active
cytokines, and (3) MSCA17.
Broadly speaking, there are 2 current pathways in tissue engineering: ex vivo and in vivo.
The former relies on the creation of an implantable product through cell culture based
techniques – cultured osteoblasts creating bone in the lab. The latter represents the use of
growth factors to recruit MSC to differentiate into osteoblasts to begin the process of
bone creation within the host. Although another avenue to in vivo bone production exists
in the form of transplantation of genetically modified cells or expansion of stem cells –
this technology is currently in a more primitive state and is well reviewed
elsewhereA18,A19.
When discussing bone grafts or bone replacement strategies, one usually uses 3 terms:
11
(1) Osteogenesis – represents new bone formation with no indication of cellular
originA20. This new bone can be formed around the graft either from recipient cells or
from cells transferred alive in the graftA21. In many respects, cancellous bone, because of
its large surface area covered by lining cells, has greater potential for graft-origin
osteogenesis than cortical boneA8.
(2) Osteoinduction – refers to recruitment from the surrounding host bed
(periosteum, adjacent bone) of undifferentiated MSC that can become osteoblasts. This
process is mediated through graft-derived or graft-carried (if added exogenously) growth
factors. Demineralized bone matrix is potently osteoinductive and will be discussed in
more detail later.
(3) Osteoconduction – means acting as a scaffold or trellis for the in-growth of all
components of bone. It is observed when porous structures are placed adjacent to living
bone. This is a 3-dimensional process beginning with angiogenesis and angioinvasion of
sprouting capillaries, perivascular tissue, and then osteoprogenitor cells from the host bed
into the scaffoldA5. Eventually the scaffold is replaced by remodeled new bone. It can
occur secondary to osteoinduction (as in demineralized bone matrix) or can occur
passively (as in cortical grafts) without any participation of the graft. Osteoconduction is
not a random process and follows an orderly sequence of events partly dictated by the
structure of the graft, the vascular supply, and the mechanical environmentA22. The
osteoinductive substrate is not usually viable – it is a passive scaffold which provides a
route to active in-growth. ShorsA23 has described a triad of osteoconduction, requiring
proximity between the graft and the surrounding bone, viability of the surrounding bone,
12
and stability of the graft to prevent shearing of the attachments which develop between it
and the host bone.
Bone grafts, themselves, can be termed autografts when moved from one site to another
within the same individual – autologous or autogenous would be synonyms. An allograft
is defined as tissue transferred between 2 genetically different individuals of the same
species. A xenograft would represent transfer between differing species.
In the field of in vivo based bone replacement research, osteoinduction has been a
common route to examine. As early as 1965, UristA2 was able to show bone formation by
purified growth factors in a non-bony site. Further research by several labs in the 1980’s
and early 1990’s was essential to identifying members of the Transforming growth
factor-beta (TGF-β) superfamily as osteoinductive agentsA24,A25. In particular, the bone
morphogenetic proteins (BMP) gained popularity in researchA26. They were isolated
from extensive purification of the bone-inductive activity found in bovine bone. Further
cDNA cloning resulted in identification of multiple BMPs as well as other osteoinductive
proteins. Currently there are many cytokines with known osteoinductive activities,
including: BMP, TGF-β1, Insulin-like growth factor-I (IGF-I), and platelet-derived
growth factor (PDGF) A27 to name but a few.
13
BONE GROWTH FACTORS (CYTOKINES)
Osteoclastic bone resorption and osteoblastic bone formation dictate the balance of bone
homeostasis. Although systemic regulation of this process by hormones is important
(calcitonin and parathyroid hormone serving as examples), regulation by local bone
growth factors is probably more essential. These cytokines are ultimately responsible for
the maintenance of constant bone volume during physiologic and pathologic
conditionsA28. It is believed that these cytokines act in an autocrine or paracrine manner
on regional osteoblasts and osteoclasts, affecting cellular proliferation and activityA29.
Most growth factors exert their biologic functions by binding to cell surface receptors’
extracellular domain. Binding then triggers the intracellular component which actives
protein kinases, usually of the serine/threonine variety. The ultimate result is mRNA
production and development of specific proteins. These proteins may be intracellular or
extracellularA30. Proteins produced by these growth factors can then influence cell
proliferation, differentiation, and chemotaxisA29.
Bone active cytokines would appear to play critical roles in fracture healing as well as
bone homeostasis. Like many cascades involving tissue healing, fracture repair is
orchestrated by chemical mediators. The process is complex and probably redundant in
some aspects. It is also incompletely understood on a cytokine level.
14
At the time of fracture, damaged cells and other local inflammatory cells release multiple
mediators, including IL-1 and tumor necrosis factor (TNF). These two cytokines cause
attraction of polymorphonuclear leukocytes (PMN). Basic fibroblast growth factor
(bFGF) is released from macrophages. Increased vascular permeability allows leaking of
platelets, red blood cells, and clotting cascade factors. TGF-β, PDGF, and bFGF released
from platelets and surrounding injured bone help create the fracture hematoma. The
initial hypoxic environment is rapidly vascularized by angiogenesis from bFGF and TGFβ. Recruitment of BMPs, TGF-β, and bFGF then leads to the necessary conditions to
begin deposition of matrix proteins and osteogenic cell interactions to lay down bone.
The osteogenic precursor cells or MSC are recruited from the surrounding normal bone,
periosteum, or possibly bloodstream. Eventually, the process of laying down new bone
and removing old bone remodels the fracture such that it is indistinguishable from the
surrounding normal boneA29.
As noted previously, there are several important bone growth factors which play roles in
the orchestration of fracture healing, including:
Bone Morphogenetic Protein (BMP): BMPs are low molecular weight peptides that
initiate endochondral bone formation by recruiting and stimulating local MSC to become
osteoblasts. They are members of the TGF-β superfamily. As such, they are the most
extensively studied of all bone growth factors. Like all members of the TGF-β
superfamily, they conserve 7 cysteine residues in their mature domain. Each mature,
active BMP is a heterodimer or homodimer. Heterodimers appear to be more potent at
15
bone formation. BMPs bind to transmembrane serine/threonine receptor complexes
which mediate their intracellular interactions. The most studied and seemingly potent of
the group are BMP-2 and BMP-7A31. Compared to all other known cytokines, BMPs are
unique in their ability to generate bone in non-bony sites. BMPs have been shown
effective in enhancing osteoinduction in both animal and human trialsA29.
Tranforming Growth Factor-β (TGF-β): TGF-β has also been extensively studied in bone
growth and regulation. TGF-β1 is a polyfunctional regulatory cytokine. It is a 25 kDa,
homodimeric, disulfide-linked polypeptide which was first identified from human blood
platelets and placentaA32. It is the prototypical cytokine of the TGF-β superfamilyA33 and
its structure is highly conserved phylogenetically, being identical in primate, bovine,
avian, and porcine systemsA34. Its first demonstration as a potential bone-forming
compound was its activity in culture medium conditioned with intact fetal rat
calvariaeA35. TGF-β1 is one of the most abundant cytokines in bone matrix and has
potent effects on osteoblastsA36. Studies have shown TGF-β1 to be a potent
osteoinductive agent in both critical-sized weight-bearing defectA37 and calvarial
defectA38 models. TGF-β1 may potentiate the activity of BMP which may relate it to the
cascade of fracture healingA39. It may be a prime regulator of extracellular matrixA6. The
most concentrated form of TGF-β is TGF-β1 which is stored in platelet alpha granules,
again, presumably to aid in fracture repair. TGF-β1 and the other isotypes of TGF-β
form homo- or heterodimers, like the BMPs. TGF-β1 also interacts by one of 3
transmembrane receptor proteins which act as serine/threonine kinases to unleash the
intracellular mRNA transcription and protein regulation.
16
Platelet Derived Growth Factor (PDGF): PDGF is a dimer which consists of 2
polypeptide chains termed A and B. PDGF-AA homodimer acts locally on bone growth.
PDGF-BB appears to demonstrate the greatest bone-inducing activity, while PDGF-AB
heterodimer is slightly less potent but retains some systemic actionA29. It is synthesized
by platelets, monocytes, and endothelial cells. It strongly acts on MSC and their progeny,
such as osteoblasts. Due to its role in bone formation, platelet rich plasma has been used
to enhance osteoinduction in bone experimentationA40. Animal experimentsA41,A42 using
PDGF to enhance fracture healing have shown improvement over controls but it would
appear not to the degree of the BMPs or TGF- β1.
Insulin-Like Growth Factor (IGF-I): IGF-I and its relative, growth hormone, play critical
roles in skeletal growth and development but a key role in fracture healing is less
certainA43. Both IGF-I and IGF-II will promote matrix synthesis and proliferation of
bone-related cells. Studies have shown massive recruitment of MSC by administration of
growth hormone but most have failed to show an ability of IGF alone to enhance
osteoinductionA31.
Fibroblast Growth Factor (FGF): FGFs are present in fracture healing and are both
strongly angiogenic and mitogenic. Although there are multiple members of the FGF
family, acidic (aFGF) and basic (bFGF) remain the best studied. FGFs bind to their
receptors which exhibit dimerization and tyrosine kinase activityA44. FGFs have a high
affinity for heparin and most extracellular FGF is bound in heparin sulfate proteoglycan,
17
which is thought to act as a mobile reservoirA45. FGFs have been shown to have mixed
effects on bone repair which are dose dependent. They have also been shown to mediate
at least some of their effects through modulation of TGF- βA46,A47. It is possible that IGF,
PDGF, and FGF are all principally mitogens as opposed to TGF- β1 and BMPs which
appear to be osteoinductive agentsA29.
Interleukin (IL)-6 family cytokines: IL-6 family cytokines tend not to appear on any
major list of bone related growth factors, but a body of literature does exist to support
their inclusionA48. This role may not imply true osteoinductive ability but, rather, a role
in maintenance of homeostasis. The cytokine member of the IL-6 family of particular
interest to this thesis is Oncostatin-M (OSM) which is more thoroughly reviewed in the
next section of this appendix. Other members of the IL-6 family include: leukemia
inhibitory factor (LIF), granulocyte colony stimulating factor, cardiotrophin 1, ciliary
neurotrophic factor, IL-11 and IL-6. IL-6, itself, has been linked to osteoclastogenesisA49
and postmenopausal osteoporosisA50 suggesting negative roles in bone homeostasis. LIF
has been shown to have conflicting roles depending on dosage and model with respect to
bone formation or resorptionA51.
As fracture healing, embryologic bone development, or the process of bone homeostasis
obviously require all of the above growth factors and many others to occur, more
research is required to adequately understand both the cascade of events that develop
bone under normal circumstances, as well as elucidate mechanisms to augment these
responses and apply them to appropriate clinical scenarios (ie, malunion, unbridgeable
18
defect, etc). Temporal expression of cytokines in fracture healing has been shown to be
differential in animal modelsA52, whether the same sequence applies in humans will also
be a question requiring further study.
In terms of clinical relevance of growth factor research, at present there are commercially
available formulations of BMP which can be applied to a variety of carriers to enhance
osteoinduction. A recent phase II study using BMP-2 to augment maxillary sinus floors
was published in December 2005A53. Reviews of BMP-7 and BMP-2 clinical trials
suggested improved results in tibial nonunion, long bone nonunion, and open tibial shaft
fracturesA54,A55. However, one studyA56 suggested a higher rate of complications when
BMP-2 delivered in an absorbable collagen sponge was used in cervical spine fusion,
while anotherA57 failed to show any improvement in hip surgery outcomes. While these
negative outcome studies suggest caution, it is important to note that they were both
retrospective reviews, while the positive studies listed before were prospective
randomized clinical trials. In the cervical spine fusion paperA56, a high dose of BMP-2
was chosen and the authors postulated that it may have induced too great an
inflammatory effect, which spread beyond the confines of the surgical space into adjacent
normal tissue. The authors recommended further appropriate dosage investigation.
Other recent research is attempting to identify synergistic combinations of growth factors
to more closely mimic the natural environment. Some of these have had positiveA58 and
some have had negativeA59 outcomes.
19
ONCOSTATIN-M
Oncostatin M (OSM) is a 28 kDa glycoprotein originally isolated from the conditioned
medium of U937 human histiocytic leukemia cells that had been induced to differentiate
into macrophage-like cells. OSM was identified by its ability to inhibit growth of human
A375 melanoma cells but not normal human fibroblastsA60. OSM shows similarities
between amino acid sequences and predicted secondary structures with leukemia
inhibitory factor (LIF), granulocyte colony stimulating factor, and interleukin-6A60. OSM
is now considered part of the IL-6 family of cytokines which all signal via complexes
containing glycoprotein gp130A61 and includes IL-6, IL-11, OSM, LIF, cardiotrophin 1,
and ciliary neurotrophic factor. OSM and LIF appear to have evolved from a common
ancestral gene (the genes for OSM and LIF are both located on chromosome 22) and both
proteins adopt a four-alpha-helical bundle structure due to intramolecular disulphide
bondsA62. The receptor complex for OSM always contains gp130 coupled with either LIF
receptor-beta or OSM receptor-betaA63. Signaling occurs via activation of members of
the Janus (JAK) family of tyrosine kinases, which leads to phosphorylation of signal
transducers and activators of transcription (STATs). These STATs then translocate to the
nucleus where they bind specific DNA sequences to initiate transcriptionA64.
Reports have suggested a role for OSM in bone homeostasis although the exact role
remains unclear. Confusion has often existed because the primary cytokine of the IL-6
family, IL-6, itself, is an erosive cytokine which promotes osteoclasts. OSM has often
20
been implicated by association through sequence homology and receptor sharing. More
recent evidence has suggested that different members of the IL-6 family play different
roles during bone-related inflammation. In fact, different members of the IL-6 family can
influence osteoblasts and/or osteoclasts thus influencing overall bone homeostasisA48.
Chondrocyte regulation appears to also be influenced in differing ways by OSM and IL6A65.
The first evidence of a role for OSM in bone homeostasis appeared in the mid 1990’s. A
transgenic mouse modelA62 produced mice with excessive ossification, especially of the
hind limbs and phalanges. Marrow cavities were filled with bone and the mice exhibited
new periosteal bone deposition. A second paperA66 showed that OSM had both positive
and negative effects. It had modest effects on osteoblastic proliferation and synthesis of
matrix proteins and inhibited alkaline phosphatase activity and bone resorption. By the
end of the 20th century, the scientific literature was developing a collective picture of
OSM as a regulatory cytokine involved in wound healing and attenuation of
inflammatory responses to restore tissue homeostasisA67.
Further supporting evidence has come from research documenting osteoblasts in bone
marrow as target cells for OSMA68. As osteoblasts are the cell types associated with
laying down bone, this supports a role in bone homeostasis for OSM. When comparing a
variety of gp130 receptor based cytokines, it was found that only OSM increased bone
colony numbers in a calvarial progenitor modelA69 – again, suggesting differential effects
of different IL-6 family members, with a pro-bone formation role for OSM. As noted
21
previously, the recruitment of MSC to differentiate into osteoblasts is essential in
osteoinduction and OSM has also been shown to stimulate the proliferation of MSCA70.
Matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMP, or
TIMPs) are important regulators of the extracellular matrix. Extracellular matrix is of
particular importance in bone. The TIMPs regulate the enzymatic function of the MMPs
by complexation and maintenance of an enzyme-inhibitor balance. An imbalance of the
interactions between MMPs and TIMPs may be involved in pathologic turnover of
extracellular matrix as seen in bone regulation disorders such as osteoarthritis. OSM
appears to play a role in the regulation of this extracellular matrix balanceA71.
In addition to promoting bone proliferation, OSM has also been implicated in the
formation of osteoclast-like cells, relating it to bone erosionA72. These dual qualities of
IL-6 family cytokines have been described and appear to be dose, time, and environment
dependentA51, A73.
Another potential property of OSM which is of interest in bone replacement strategies is
its angiogenic abilities. Angiogenesis is an important event in fracture healing and bony
defect bridging as it supplies the oxygen and nutrients to the area. OSM would appear to
induce angiogenesis both in vitro and in vivoA73. It has also been shown to induce basic
fibroblast growth factor (bFGF) expression in endothelial cellsA74. bFGF is a potent
angiogenic agent in its own right. Several lines of evidence suggest that endothelial cells
are a primary target of OSM reinforcing an impression of angiogenic potentialA75.
22
Endothelial cells have 10-20 fold greater expression of high and low affinity OSM
receptors than they do for other growth factorsA76.
23
OSTEOCONDUCTIVE MATERIALS & CARRIERS
Most of the research occurring around the world with osteogenic proteins has involved
linking them with a carrier substance. Choosing the proper carrier or scaffolding for the
growth factor formulation is equally if not more important than the growth factor, itself.
The proper carrier will provide the necessary strength, shape, and functional space for
delivery of growth factors. Scaffolds provide osteoconduction to facilitate in-growth of
microvasculature and provide a highway for MSC recruitment. Ideal scaffolds must have
the correct stability to prevent shearing of vasculature as well as the ideal ‘porosity’ –
they must be the correct size to allow this in-growth. General consensus is that the most
suitable pore size is 100-500µmA23. Other factors considered important in a carrier
scaffold include: biocompatibility, surface properties including pH, mechanical
properties such as rigidity, and potential for biodegradabilityA77,A78,A79.
Early strategies for carriers were based on the concept that tissue form and function could
be restored by using biocompatible materials which would bear load. Materials were
fabricated that resembled bone in its chemical composition. As the field has become
more sophisticated, this has been shown to be incompatible with a long-term solution
because of issues of tissue integrationA80. The problems confronting bone material
engineers, even today, are rooted in the limits of nonviable materials – excessive
brittleness, poor handling characteristics, and easy to fracture. They can be difficult to
24
place into the correct position in the body and may, in themselves, represent unfavorable
microenvironments for cells to occupyA17.
Several varieties of carriers are available and include biocompatible materials such as
calcium based ceramics and polymers made of fibrinogen, hyaluronan, collagen, and the
like. Synthetic carriers are also available and are often made of suture-like biodegradable
materials. In general, calcium or collagen based biomaterials are preferred to synthetics
as the latter can sometimes lead to toxic degradation productsA81.
Autogenous bone arguably remains the ‘gold standard’ for most reconstructions. The
disadvantages of autografts include donor site morbidity, increased operative time, and
lack of adequate volume to fill large defects. Autogenous bone grafts will also resorb
over time, especially if not vascularizedA82.
Methyl methacrylate is an acrylic based resin that was first used to fill bony defects
during World War II. Unlike autogenous bone, it was found to be resistant to any
resorption. Methyl methacrylate has a large body of literature evaluating its usage which
includes its several disadvantages. It may have a significant infection rate, especially in
the face of any communication between it and the nasal cavity. It is also fixed and inert
and will not adapt to the changing craniofacial skeleton or growth of limbs. It does not
allow any bony incorporation and, as such, is susceptible to dislodgementA82.
25
Hydroxyapatite is the underlying compound giving strength to teeth and bones. It can be
produced as a ceramic by a process known as sintering. It is a porous material which will
allow and promote osteointegration and is biocompatibleA83. When prepared properly
hydroxyapatite should allow bony ingrowth and eventual replacementA84. The cement
paste version of hydroxyapatite can be easily sculpted or shaped to fit defects and has
extensive use in adult craniofacial reconstruction.
Porous polyethylene (Medpor, Porex Surgical, College Park, GA) has been used for
craniofacial reconstruction. It is a resin of polyethylene which is a straight chain
aliphatic hydrocarbon. It is inert and causes little tissue reactivity. It also shows little
degradation. Its porous structure does facilitate some in-growth of bone and soft tissue.
YaremchukA85 has described an extensive clinical experience with several hundred
implants. They were used for many craniofacial clinical scenarios with good effect.
Polylactic and polyglycolic acid polymers have vast surgical experience as suture
materials. They are slowly biodegradable through hydrolysis. Although well tolerated,
they incite an inflammatory response when implanted in a bulky formA86. These
compounds do have the advantage of being able to be assembled in a variety of forms and
can be used as a platform for the delivery of growth factors or other compounds to create
multiphase delivery systems. Although pores can be created to meet optimal size criteria,
it has been shown that they do not function well as osteoconductive agentsA5.
26
In the 1970s it was recognized that naturally occurring marine corals exhibited a structure
similar to bone. Corals of the genus Porites or Gonipora have appropriately sized
channels for osteoconduction with interconnecting fenestrations to allow good 3dimensional in-growthA5. Extensive experimentation in animal models followed by
human trials has shown implantation of hydroxyapatite porous materials is followed by
osteoconduction and conversion to mature bone and that these coralline-based implants
are superior to ceramics because of this increased invasionA87,A88,A89.
In terms of degradation of calcium-based bone substitutes or carriers there are 2 main
processes: dissolution and resorption. Dissolution is a chemical phenomenon which is
controlled by many factors, including surface area, volume, pH, and temperature.
Resorption is a cell-based phenomenon and generally mediated by osteoclasts whose
enzyme carbonic anhydrase will resorb bone and coralline implantsA23. This resorption
allows for replacement of the carrier by normal bone over time.
Type I collagen bonded into its cross-linked fibrillar structure is the most abundant
protein in the extracellular matrix. The surface of collagen is osteoconductive and has
multiple sites for binding of proteins, including growth factorsA90. Soluble collagen
products tend to degrade quickly when placed in surgical defects. Chemical cross-linking
can provide more stability and decrease the rate of dissolution. Collagen alone provides
no structural support and is thus unsuitable for weight-bearing defects. Commercial
formulations of collagen (often bovine) either mixed with more solid substances (such as
27
hydroxyapatite or tricalcium phosphate) or alone have been in use with varied resultsA90.
As a medium for the carriage of growth factors, however, collagen is very successful.
A common experimental agent used in animal models has been demineralized bone
matrix (DBM) derived from the animal being studied. DBM continues to be used in
humans both inside and outside of clinical trials with good results. DBM represents a
combination of type I collagen and a variety of endogenous growth factors which would
normally be resident in bone (BMP, TGF-β1, IGF-I, interleukins, granulocyte colony
stimulating factor, and PDGF) as well as proteins including osteocalcin, bone
sialoprotein, osteopontin, and thrombospondinA91. DBM is quickly vascularized,
provides no structural support, but may be both osteoinductive (if the endogenous growth
factors have not been removed) and osteoconductive. When DBM is implanted there is
initial platelet aggregation, hematoma formation, and inflammatory response by PMNs.
Thereafter, fibroblast-like MSC attach to the edges and differentiate into chondrocytes.
A cartilaginous matrix is created which is later mineralized. Eventually, osteoblast
precursors arrive, differentiate into bone forming cells, which begin the process of laying
down new bone. The final pathway is the complete remodeling of the DBM into new
boneA22.
28
CONCLUSION
The ultimate desire of osteoinduction research is to develop a variety of formulations of
carriers to deliver a mixture of cytokines to exactly reproduce and augment the process of
bone formation. Solid, weight bearing carriers as well as softer, moldable carriers would
be required depending on the defect – long bone versus craniofacial on-lay, as examples.
In either case, the appropriate combination of cytokines delivered within the carrier
would activate the cascade of bone formation – potentially speeding it up and allowing
bridging of previously unbridgeable defects. If tooled properly, augmented angiogenesis
could be of great value for hypoxic sites (post-irradiation or traumatic).
Intuitively, one cytokine alone does not hold the promise to completely create new bone.
To this end, eventual experiments and models must examine the synergistic application
of cytokines to better mimic the natural environment. Some early results would favor a
positive role for OSM in conjunction with bone morphogenetic proteinsA92. Further
research is required, perhaps in a similar model to that described in the body of this
thesis, to more fully exploit this potential synergy.
As knowledge of the physiologic processes and interactions of different bone cytokines
grows, surgeons’ longstanding goal of healing presently unbridgeable defects by
accelerating the repair and regeneration of bone inches closer to realityA93.
29
APPENDIX REFERENCES
A1
Manson PN: Bone Grafts: Clinical review of factors affecting survival. In: Portland Bone Symposium
1993, Oregeon Health Sciences University, Portland, 1993:355-380
A2
Urist MR. Bone: Formation by autoinduction. Science 1965;150:893-899
A3
Bassett C: Current concepts of bone formation. J Bone Joint Surg 1962;44A:1217-1244
A4
Motoki DS and Mulliken JB: The healing of bone and cartilage. Clin Plast Surg 1990;17:527-544
A5
Cornell CN. Osteoconductive materials and their role as substitutes for autogenous bone grafts. Orthop
Clin NA 1999;30(4):591-598
A6
Balooch G, Balooch M, Nalla RK, et al. TGF-beta regulates the mechanical properties and composition
of bone matrix. Proc Natl Acad Sci USA 2005;102(52):18813-18818
A7
Recker RR: Bone remodeling and metabolic bone disease. In: Portland Bone Symposium 1993, Oregon
Health Sciences University, Portland 1993:133-146
A8
Friedlaender GE: Current concepts review: Bone Grafts. J Bone Joint Surg 1987;69:786-790
A9
Glowacki, J: Influence of inflammation and vascularization on bone repair. In: Portland Bone
Symposium 1993, Oregon Health Sciences University, Portland 1993:33-38.
A10
Goldring MB and Goldring SR: Skeletal tissue response to cytokines. In: Portland Bone Symposium
1993, Oregon Health Sciences University, Portland 1993:61-133
A11
Canalis E, McCarthy T, and Centrella M: Growth factors and the regulation of bone remodeling. J Clin
Invest 1988;81:277-281
A12
Hauschka PV, Mavrakos AE, Iafrati MD, Doleman SE, and Klagsburn M: Growth factors in bone
matrix. Isolation of multiple types by affinity chromatography on heparin-sepharose. J Biol Chem
1986;261:12665-12674
A13
Mercola M and Stiles CD: Growth factor superfamilies and mammalian embryogenesis. Development
1988;102:451-460
A14
Langer R, Vacanti J. Tissue engineering. Science 1993;260:920-926
A15
Muschler GF, Nakamoto C, Griffith LG. Engineering principles of clinical cell-based tissue
engineering. J Bone Joint Surg Am 2004;86:1541-1558
A16
Vacanti CA, Vacanti JP. The science of tissue engineering. Orthop Clin North Am 2000;31:351-355
A17
Calvert JW, Weiss LE, Sundine MJ. New frontiers in bone tissue engineering. Clin Plast Surg
2003;30:641-648
A18
Blum JS, Barry MA, Mikos AG. Bone regeneration through transplantation of genetically modified
cells. Clin Plast Surg 2003;30:611-620
A19
Greenberger JS, Goff JP, Bush J, et al. Expansion of hematopoietic stems cells in vitro as a model
system for human tissue engineering. Orthop Clin NA 2000;31:499-509
30
A20
Buckwalter JA, Glimscher MJ, Cooper RR, et al. Bone biology. J Bone Joint Surg Am 1995;77:12561289
A21
Burchardt H. The biology of bone graft repair. Clin Orthop 1983;42:28-42
A22
Stevenson S. Biology of bone grafts. Orthop Clin NA 1999;30(4):543-551
A23
Shors EC. Coralline bone graft substitutes. Orthop Clin NA 1999;30(4):599-611
A24
Wang EA, Rosen V, Cordes P, et al. Purification and characterization of other distinct bone-inducing
factors. Proc Natl Acad Sci USA 1988;85:9484-9488.
A25
Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: Molecular clones and
activities. Science 1988;242:1528-1534
A26
Celeste AJ, Iannazzi JA, Taylor RC, et al. Identification of transforming growth factor beta family
members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci USA
1990;87:9843-9847
A27
Rose FRAJ, Hou Q, Oreffo ROC. Delivery systems for bone growth factors: the new players in skeletal
regeneration. J Pharm Pharmacol 2004;56:415-427.
A28
Mohan S, Baylink D. Bone growth factors Clin Orthop 1991;263:30-48
A29
Khan SN, Bostrom MPG, Lane JM. Bone growth factors. Orthop Clin NA 2000;31(3):375-387
A30
Solheim E. Growth factors in bone. Int Orthop 1998;22:410-416
A31
Bostrum MPG, Saleh K, Einhorn TA. Osteoinductive growth factors in preclinical fracture and long
bone defects models. Orthop Clin NA 1999;30(4):647-658
A32
Sporn MB, Roberts AB. TGF-β: problems and prospects. Cell Regulation 1990;1:875-882
A33
Chang H, Brown CW, and Matzuk MM. Genetic analysis of the mammalian transforming growth
factor-β superfamily. Endocrine Reviews 2002;23(6):787-823
A34
Roberts AB, Sporn MB. The transforming growth factor betas. In: Peptide growth factors and their
receptors. Berlin: Springer-Verlag, 1990:417-472
A35
Centrella M, Canalis E. Transforming and nontransforming growth factors are present in medium
conditioned by fetal rat calvariae. Proc Natl Acad Sci USA 1985;82:7335
Maeda S, Hayashi M, Komiya S, Imamura T, Miyazono K. Endogenous TGF-β signaling suppresses
maturation of osteoblastic mesenchymal cells. EMBO J 2004;23:552-563
A36
A37
Moxham JP, Kibblewhite DJ, Dvorak M, et al. TGF-β 1 forms functionally normal bone in a segmental
sheep tibial diaphyseal defect. J Otolaryngol. 1996;25(6):388-92.
A38
Moxham JP, Kibblewhite DJ, Bruce AG, Rigley T, Gillespy T 3rd, Lane J. Transforming growth
factor-β 1 in a guanidine-extracted demineralized bone matrix carrier rapidly closes a rabbit critical
calvarial defect. J Otolaryngol. 1996;25(2):82-7
A39
Carrington JL, Roberts AB, Flanders KC, et al. Accumulation, localization, and compartmentation of
transforming growth factor-beta during endochondral bone development. J Cell Biol 1988;107:1969-1975
31
A40
Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet rich plasma: Growth factor enhancement for bone
grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endo 1998;85:638-646
A41
Nash TJ, Howlett CR, Martin C, et al. Effect of platelet-derived growth factor on tibial osteotomies in
rabbits. Bone 1994;15:203-208
A42
Canalis E. Effect of platelet-derived growth factor on DNA and protein synthesis in cultured rat
calvariae. Metabolism 1981;30:970-978
A43
Trippel SB. Potential role of insulinlike growth factors in fracture healing. Clin Orthop 1998;355S:301
A44
Jaye M, Schlessinger J, Dionne CA. Fibroblast growth factor receptor tyrosine kinase-molecular
analysis and signal transduction. Biochem Biophys Acta 1992;1135:185-199
A45
Galzie Z, Kinsella AR, Smith JA. Fibroblast growth factors and their receptors. Biochem Cell Biol
1997;75:669-685
A46
Nakamura T, Hanada K, Tamura M, et al. Stimulation of endosteal bone formation by systemic
injections of recombinant basic fibroblast growth factor in rats. Endocrinology 1995;136:1276-1284
A47
Noda M, Vogel R. Fibroblast growth factor enhances type beta 1 transforming growth factor gene
expression in osteoblast-like cells. J Cell Biol 1989;109:2529-2535
A48
Heymann D, Rousselle AV. Gp130 cytokine family and bone cells. Cytokine 2000;12(10):1455-1468
A49
Tamura T, Udagawa N, Takahashi N, et al. Soluble interleukin-6 receptor triggers osteoclast formation
by interleukin-6. Proc Natl Acad Sci USA 1993;90:11924-11928
A50
Jilka RL, Hangoc G, Girasole G, et al. Increased osteoclast development after estrogen loss: mediation
by interleukin-6. Science 1992;257:88-91
A51
Metcalf D. The unsolved enigmas of leukemia inhibitory factor. Stem Cells 2003;21:5-14
A52
Cho TJ, Gerstendfeld LC, Einhorn TA. Differential expression of members of the transforming growth
factor beta superfamily during murine fracture healing. J Bone Miner Res 2002;17(3):513-520
A53
Boyne PJ, Lilly LC, Marx RE, et al. De novo bone induction by recombinant human bone
morphogenetic protein-2 (rhBMP-2) in maxillary sinus floor augmentation. J Oral Maxillofac Surg
2005;63(12):1693-1707
A54
McKee MD. Recombinant human bone morphogeneic protein-7: applications for clinical trauma. J
Orthop Trauma. 2005;19(10 Suppl):S26-28.
A55
Jones AL. Recombinant human bone morphogenic protein-2 in fracture care. J Orthop Trauma.
2005;19(10 Suppl):S23-25
A56
Shields LB, Raque GH, Glassman SD, et al. Adverse effects associated with high-dose recombinant
human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine 2006;31(5):542-547
A57
Karrholm J, Hourigan P, Timperley J, Razaznejad R. Mixing bone graft with OP-1 does not improve
cup or stem fixation in revision surgery of the hip: 5-year follow-up of 10 acetabular and 11 femoral study
cases and 40 control cases. Acta Orthop 2006;77(1):39-48
32
A58
Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cellexpressed VEGF and bone morphogenetic protein-4. J Clin Invest 2002;110:751-759
A59
Vonau RL, Bostrum MP, Aspenberg P, et al. Combination of growth factors inhibits bone ingrowth in
the bone harvest chamber. Clin Orthop 2001;386:243-251
A60
Rose TM, Bruce AG. Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory
factor, granulocyte colony-stimulating factor, and interleukin 6. Proc Natl Acad Sci USA 991;88:86418645
A61
Anhuf D, Weissenbach M, Schmitz J, Sobota R, et al. Signal transduction of IL-6, Leukemia-inhibitory
factor, and Oncostatin M: Structural receptor requirements for signal attenuation. J Immunol 2000;165:
2535-2543
A62
Malik N, Haugen HS, Modrell B, Shoyab M, Clegg CH. Developmental abnormalities in mice
transgenic for bovine Oncostatin M. Mol Cell Biol 1995;15(5):2349-2358
A63
Thoma B, Bird TA, Friend DJ, Gearing DP, Dower SK. Oncostatin M and leukemia inhibitory factor
trigger overlapping and different signals through partially shared receptor complexes. J Biol Chem
1994;269:6215-6222
A64
Levy JB, Schindler C, Raz R, Levy DE, Baron R, Horowitz MC. Activation of the JAK-STAT signal
transduction pathway by Oncostatin-M in cultured human and mouse osteoblastic cells. Endocrinology
1996;137:1159-1165
A65
Sanchez C, Deberg MA, Burton S, Devel P, Reginster JY, Henrotin YE. Differential regulation of
chondrocyte metabolism by Oncostatin M and interleukin-6. Osteoarthritis & Cartilage 2004;12(10):801810
A66
Jay PR, Centrella M, Lorenzo J, Bruce AG, Horowitz MC. Oncostatin-M: A new bone active cytokine
that activates osteoblasts and inhibits bone resorption. Endocrinology 1996;137:1151-1158
A67
Wallace PM, MacMaster JF, Rouleau KA, et al. Regulation of inflammatory responses by Oncostatin
M. J Immunol 1999;162:5547-5555
A68
Bellido T, Stahl N, Farruggella TJ, Borba V, Yancopoulos GD, Manolagas SC. Detection of receptors
for interleukin-6, interleukin-11, leukemia inhibitory factor, Oncostatin M, and ciliary neurotrophic factor
in bone marrow stromal/osteoblastic cells. J Clin Invest 1996;97(2):431-437.
A69
Malaval L, Liu F, Vernallis AB, Aubin JE. GP130/OSMR is the only LIF/IL-6 family receptor complex
to promote osteoblast differentiation of calvaria progenitors. J Cell Physiol 2005;204(2):585-93
A70
Song HY, Jeon ES, Jung JS, Kim JH. Oncostatin M induces proliferation of human adipose tissuederived mesenchymal stem cells. Int J Biochem Cell Biol 2005;37(11):2357-2365
A71
Li WQ, Zafarullah M. Oncostatin M up-regulates tissue inhibitor of metalloproteinases-3 gene
expression in articular chondrocytes via de novo transcription, protein synthesis, and tyrosine kinase- and
mitogen-activated protein kinase-dependent mechanisms. J Immunol 1998;161:5000-5007
A72
Richards CD, Langdon C, Deschamps P, Pennica D, and Shaughnessy SG. Stimulation of osteoclast
differentiation in vitro by mouse Oncostatin M, leukaemia inhibitory factor, cardiotrophin-1, and
interleukin 6: synergy with dexamethasone. Cytokine 2000;12:613-621
A73
Vasse M, Pourtau J, Trochon V, et al. Oncostatin M induces angiogenesis in vitro and in vivo
Arterioscler Thromb Vasc Biol 1999;19:1835-1842.
33
A74
Wijelath ES, Carlsen B, Cole T, Chen J, Kothari S, Hammond WP. Oncostatin M induces basic
fibroblast growth factor expression in endothelial cells and promotes endothelial cell proliferation,
migration and spindle morphology. J Cell Sci 1997;110(7):871-879
A75
Brown TJ, Rowe JM, Jingwen L, Mohammed S. Regulatin of IL-6 expression by oncostatin M. J
Immunol 1991:147;2175-2180
A76
Linsley PS, Bolton-Hanson M, Horn D, et al. Identification and characterization of cellular receptors for
the growth regulator, Oncostatin M. J Biol Chem 1989:264;4282-4289
A77
Muschler GF, Nakamoto C, Griffith LG. Engineering principles of clinical cell-based tissue
engineering. J Bone Joing Surg Am 2004;86:1541-1558
A78
Wiesmann HP, Joos U, Meyer U. Biological and biophysical principles in extracorporeal bone tissue
engineering: part I. Int J Oral Maxillofac Surg 2004;33:325-332
A79
Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the art and future trends.
Macromol Biosci 2004;4:743-765
A80
Boyan BD, Lohmann CH, Romero J, Schwartz Z. Bone and cartilage tissue engineering. Clin Plast Surg
1999;26(4):629-635
A81
Hunter AC, Moghimi SM. Therapeutic synthetic polymers: a game of Russian roulette? Drug Discov
Today 2002;7:998-1001
A82
Cho YR, Gosain AK. Biomaterials in craniofacial reconstruction. Clin Plast Surg 2004;31:377-385
A83
Jackson IT, Yavuzer R. Hydroxyapatite cement: an alternative for craniofacial skeletal contour
refinements. Br J Plast Surg 2000;53:24-29
A84
Constantino PD, Friedman CD, Jones K, Chow LC, Sisson GA. Experimental hydroxyapatite cement
cranioplasty. Plast Reconstr Surg 1992;90:174-191
A85
Yaremchuk MJ. Facial skeletal reconstruction using porous polyethylene implants. Plast Reconstr Surg
2003;111:1818-1827
A86
Hollinger JO, Buttistone GC. Biodegradable bone repair materials: Synthetic polymers and ceramics.
Clin Orthop 1986;207:290-305
A87
Chapman MW, Bucholz R, Cornell CN. Treatment of acute fractures with a collagen-calcium phosphate
graft material: A randomized clinical trial. J Bone Joint Surg Am 1997;79:495-502
A88
Holmes RE, Bucholz RW, Mooney V. Porous hydroxyapatite as a bone graft substitute in metahyseal
defects. J Bone Joint Surg Am 1986;68:904-911
A89
Holmes RE, Mooney V, Bucholz R, et al. A Coralline hydroxyapatite bone graft substitute. Clin
Orthop 1984;188:252-262
A90
Fleming JE, Cornell CN, Muschler GF. Bone cells and matrices in orthopedic tissue engineering.
Orthop Clin NA 2000;31(3):357-374
A91
Heinegard D, Oldberg A. Structure and biology of cartilage and bone matrix noncollagenous
macromolecules. FASEB J 1989;3:2042-2050
34
A92
deHooge ASK, van de Loo FAJ, Bennink MB, et al. Adenoviral transfer of murine Oncostatin M elicits
periosteal bone apposition in knee joints of mice, despite synovial inflammation and up-regulated
expression of interleukin-6 and receptor activator of nuclear factor-kappaB ligand. Am J Pathol
2002;160:1773-1743
A93
Habal MB. Bone tissue engineering applications in craniofacial reconstructive surgery. Clin Plast Surg
2004;31:387-392
35