BOOK CHAPTER

Biology and Enhancement of Skeletal

Repair  Download PDF
 J. Tracy Watson

Skeletal Trauma: Basic Science, Management, and Reconstruction, 5, 79-128

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Introduction
The rapid development of contemporary fracture fixation devices has occurred as the
concepts of minimally invasive surgical techniques have advanced. Percutaneous
instrumentation, the refinement of insertion portals, and the development of blocking
screw techniques have expanded the range of intramedullary (IM) nailing from midshaft
injuries to the articular margins. Locking plates have extended the role for periarticular
fracture fixation using minimalistic exposures. Fully implantable limb lengthening nails
and IM devices with bone transport capabilities are coming on line, and these devices
will further define the ability to perform complex deformity correction, limb lengthening
procedures, and bone transport, all without the need for cumbersome and patient-
demanding external fixation devices.

Similarly, the understanding and use of growth factors and other biologic adjuvants for
the treatment of long bone fracture healing, subchondral defects, and nonunion repair
continue to evolve. It is increasingly important that the orthopaedic surgeon be familiar
with the biologic characteristics and clinical application of these therapies. Multiple
materials have become available to enhance fracture healing through a myriad of
biologic pathways. The purpose of this chapter is to review the basic fund of knowledge
on the topic of orthobiologic surgical adjuvants and the newer arena of orthobiologic
manipulation by pharmacologic agents, and to provide a comprehensive review of the
use of these currently available adjuvants for approved clinical use. The current levels of
evidence supporting the use of these various materials will be reviewed if available.

Biology of Graft Substitutes

The biology of bone grafts and their substitutes is appreciated from an understanding of
the bone formation processes of osteogenesis, osteoinduction, and
osteoconduction. 12345

Graft Osteogenesis
Graft osteogenesis is the ability of cellular elements within a donor graft, which survive
transplantation, to synthesize new bone at the recipient site. Osteogenesis can occur in
two ways. Surface osteoblasts can survive the transplantation by receiving nutrition
through diffusion at the recipient site, and then proliferate to form more living bone
tissue. There is more surface area in cancellous graft, and thus it has more potential for
surviving cells than a cortical graft. Likewise the transplantation of marrow elements
alone has demonstrated this ability to survive and form bone. 6
The ability to provide a threshold concentration of viable bone-forming elements
presents a viable mode of intervention for the treatment of fractures or nonunions
provided the recipient site provides the appropriate biologic environment to allow these
cells to produce their osteogenic stimulus.

These transplanted cells or circulating pluripotential cells must have the appropriate
substrate to attach to, or become attached to, once the cells have localized to the site of
defect or injury. This substrate site for cellular attachment has to have the appropriate
three-dimensional architecture to allow for these cells to proliferate. Subsequently,
these cells then use this substrate as a scaffolding through which to build bone. This
three-dimensional process involves vascular proliferation and ingrowth of capillaries
along the open spaces in the substrate and thus the porosity of these materials is critical.
Attachment is then followed by the differentiation of cells into bone-forming cell types
and the production and remodeling of bone then proceeds. 789

Providing a competent three-dimensional architecture for cellular attachment is the

next area of intervention in a process termed graft osteoconduction.

Graft Osteoconduction
A major category of available graft substitutes for defect management intervention is to
provide a lattice-like substrate material that will facilitate the attachment and
proliferation of osteoblastic cellular elements. This is followed closely by vascular
proliferation and ingrowth of capillaries along the open spaces in the substrate. The
porosity of these materials is critical to facilitate the revascularization of these grafts.
Attachment is then followed by the differentiation of cells into bone-forming cell types
and the production and remodeling of bone then proceeds. 78 The conductive substrate
then undergoes incorporation into the developing bone or degradates by a chemical
dissolution process. The incorporation kinetics and timing are material dependent as
these materials have a host of properties that are unique to each individual substrate.

Graft Osteoinduction
As all skeletal tissues evolve from mesenchyme, undifferentiated mesenchymal cells
make a genetic commitment to a particular cellular lineage early in the developmental
or repair process. In the case of repair, some stimulus must signal the undifferentiated
mesenchymal cells to differentiate along a chondro-osteogenic pathway. Before this
differentiation into an osteogenic lineage, these cells are affected by multiple growth
factors that provide chemotactic and mitogenic stimuli to these cells. They influence
these cells to migrate, attach, and multiply at the locale, which provides a competent
osteoconductive substrate as a site of cellular attachment. 6 This phenomenon, known as
osteoinduction, is defined as “a process that supports the mitogenesis of
undifferentiated mesenchymal cells leading to the formation of osteoprogenitor cells
with the capacity to form new bone.” 789 Thus any material that induces this process
could be considered to be an osteoinductive growth factor.

Growth Factors
Proteins secreted by cells that bind to receptors on target cells to carry out a specific
action are known as growth factors. This action may be autocrine, paracrine, or
endocrine. Autocrine signaling occurs when a cell secretes a growth factor that binds to
that same cell, resulting in self-activation. Paracrine signaling refers to the secretion of
growth factors to neighboring cells, where the signal only travels a short distance to act
in a local fashion on other cells. Endocrine signaling occurs when a growth factor is
released into the circulation and acts on a cell at a distant site. Once a growth factor
binds to a receptor, the receptor is activated and undergoes a change in its
conformation. This change in conformation activates an intracellular protein known as a
transcription factor, which travels to the nucleus by various messenger factors and
induces the expression of a new gene or set of genes. 1011

This cascade of activity is jump-started by graft-derived growth factors such as

transforming growth factor–β (TGF-β), insulin-like growth factors (IGFs) 1 and 2,
platelet-derived growth factor (PDGF), and others. They influence these cells to migrate,
attach, and multiply at the locale that provides a competent osteoconductive substrate
as a site of cellular attachment. 12 This phenomenon, known as osteoinduction, is defined
as “a process that supports the mitogenesis of undifferentiated mesenchymal cells
leading to the formation of osteoprogenitor cells with the capacity to form new
bone.” 131415 Thus any material that induces this process could be considered
osteoinductive.

Several aspects of this cascade are important to understand when considering the
therapeutic potential of each individual growth factor. First, two different growth factors
may have the same effect on a cell. Thus it is simplistic to assume that each growth
factor works alone. These proteins work in concert with one another, and the
homeostatic and regulatory mechanisms that serve to modulate the activity of these
factors is just now beginning to be understood. Although each growth factor family has
its own corresponding family of receptors, many of these receptors activate the same set
of genes. Therefore different growth factors may lead to the same cellular response.
Many growth factors display pleiotropic activity in that a single growth factor may elicit
a variety of different effects in the same cell, in the same cell at different stages of
development, or in different cells. This is due to the fact that the signals transmitted to
the nucleus frequently activate several genes at once. This has important clinical
implications, as growth factors may also activate deleterious cellular effects in addition
to their advantageous ones.

The temporal relationship of growth factor action is also dependent on the timing of
application of the particular adjuvant, as well as the dose response that is variable
depending on the timing of the factor application. Many of these factors act within a
specific stage of fracture healing. The concept of osteoinductive new bone formation is
realized through the active recruitment of host mesenchymal stem cells (MSCs) from the
surrounding tissue, which differentiate into bone-forming osteoblasts. This process is
facilitated by the presence of growth factors within the graft, principally bone
morphogenetic proteins (BMPs). However, many factors take part in the fracture
healing cascade before the potent action that the BMPs impart on the pluripotential
cells.
It has been shown that the growth factors expressed during fracture healing include
BMPs, TGF–β, fibroblast growth factor (FGF), and PDGF. 1617181920212223 On the
basis of these findings, these growth factors have been most extensively studied in the
context of fracture healing. The use of each of these growth factors in the treatment of
fracture healing is discussed indivually.

Growth Factor Effects on the Phases of Fracture Healing

Fracture healing is reviewed in Chapter 4 ; the particular growth factors that affect each
stage of fracture healing are reviewed here. Many of these factors are now available for
clinical application, and it is important to understand their locale of intervention and
overall mode of action.

Platelets found in the hematoma degranulate, releasing various signaling molecules that
are contained within each platelet. These include factors such as TGF-β and PDGF.
These cytokines and signaling molecules are involved in the processes of chemotaxis and
angiogenesis and also regulate cell proliferation and differentiation of the cells that have
migrated to the site of the fracture. 24 The TGF-β family of growth factors, which includes
all of the TGF-βs, BMPs, and growth and differentiation factors (GDFs), controls a
number of processes during this initial phase of skeletal repair. BMP-2 most likely
induces both chondrogenesis and periosteal osteogenesis, leading to the initiation of the
endochondral healing response and intramembranous ossification.

The inflammatory phase of fracture healing is characterized by neovascularization and

ingrowth of proliferative blood vessels. In addition to this oxygen-rich environment, it is
vitally important that the mesenchymal cells that have recently migrated to the site of
injury have a suitable osteoconductive surface for their implantation.

Cellular attachment to the extracellular matrix (ECM) and conductive substrate is a

basic requirement for fracture healing. This is accomplished with integrins, which are
transmembrane receptors that are the bridges for cell-cell and cell-ECM interactions.
Essentially, they act as “glue” to allow the attachment of cells to the conductive matrix.

Plasma fibronectin has been found in the fracture hematoma within the first 3 days after
fracture. Its production by cells associated with the callus appears to be greatest in the
earliest stages of fracture healing. This is consistent with its function to establish
provisional fibers and attachments in the developing cartilaginous matrices.

When activated, integrins in turn trigger chemical pathways to the interior (signal
transduction), such as the chemical composition and mechanical status of the ECM,
which results in a response (activation of transcription) such as regulation of the cell
cycle, cell shape, and/or motility; or new receptors being added to the cell membrane.
Integrins give the cell critical signals about the nature of its surroundings. Together with
signals arising from receptors for soluble growth factors such as vascular endothelial
growth factor (VEGF), epidermal growth factor (EGF), PDGF, and many others, they
enforce a cellular decision on what biologic action to take, be it attachment, movement,
death, or differentiation. Thus integrins lie at the heart of the early inflammatory
process. The actual attachment of the cells takes place through formation of cell
adhesion complexes, which consist of integrins and many cytoplasmic proteins, such as
α-actinin. 25 This cellular binding to a competent conductive substrate is necessary for
the circulating inductive factors (BMPs) to then differentiate these cells into an
osteoblastic lineage.

Factors Influencing the Inflammatory Phase

Arachidonic Acid Metabolism and the Action of Nonsteroidal
Antiinflammatory Drugs
Arachidonic acid metabolism exerts complex control over many bodily systems; it is
mainly involved in inflammation and immunity. Two families of enzymes catalyze fatty
acid oxygenation of arachidonic acid and produce either prostaglandin or leukotriene
factors, both of which can affect the inflammatory phase of healing with their actions.
These actions include a direct role in the stimulation of platelet aggregation and
vasoconstriction, both important for the activation and degranulation of platelets. As
leukocyte chemotaxis is activated these factors may directly activate gene transcription
of the attached MSCs.

The enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) metabolize

arachidonic acid to prostaglandin G2 and prostaglandin H2, which in turn may be
converted to various prostaglandins. The classical cyclooxygenase (COX) inhibitors
prevent the production of prostaglandin products. They are not selective and inhibit all
phases of the inflammatory process. The main COX inhibitors are the nonsteroidal
antiinflammatory drugs (NSAIDs). However, the selectivity for COX-2 inhibition was
the main feature of this newest member of this drug class. Because COX-2 is usually
specific to inflamed tissue, there is much less gastric irritation associated with COX-2
inhibitors, with a decreased risk of peptic ulceration. The selectivity of COX-2 does not
seem to negate other fracture healing side effects of NSAIDs. Specifically, COX-2
inhibitors and other NSAIDs in general should be considered a potential risk factor for
fracture healing and therefore be avoided in patients at risk for delayed fracture
healing. 26 It appears as though the temporal time for their action is during the
inflammatory phase of fracture healing and thus this has been firmly established within
the first 3 to 4 weeks of injury. Thus if possible these medications should be avoided
during this time frame. 2627

Wnt Pathway
Wnt signaling pathways are a group of signal transduction pathways made of proteins
that pass signals from outside a cell through cell surface receptors to the inside of the
cell. Wnt proteins form a family of highly conserved secreted signaling molecules that
regulate cell-to-cell interactions during development and adult tissue homeostasis.
Three Wnt signaling pathways have been characterized. All three Wnt signaling
pathways are activated by the binding of a Wnt-protein ligand to a Frizzled family
receptor, which passes the biologic signal to the protein Disheveled inside the cell. The
canonical Wnt pathway leads to regulation of gene transcription and thus is important
for the conversion of undifferentiated MSCs into an osteoblastic lineage that takes place
under the influence of inductive factors during the inflammatory phase of fracture
healing. 28 Induction of the Wnt signaling pathway promotes bone formation, whereas
inactivation of the pathway leads to osteopenic states. Potential therapeutic approaches
attempt to stimulate the Wnt signaling pathway by upregulating the intracellular
mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of
the pathway.

Sclerostin is produced by the osteocyte and has antianabolic effects on bone formation.
It binds to the LRP5/LRP6/Frizzled coreceptor group inhibiting the Wnt signaling
pathway. 29 The inhibition of the Wnt pathway leads to decreased bone formation.
Although the underlying mechanisms are unclear, it is believed that the antagonism of
BMP-induced bone formation by sclerostin is mediated by Wnt signaling, but not BMP
signaling pathways. Antibodies against endogenous antagonists, such as antisclerostin,
have demonstrated promising results in promoting bone formation and increased callus
size. Improved fracture healing rates are equivocal and more studies are needed in this
regard. Sclerostin production by osteocytes is inhibited by parathyroid hormone
(PTH) 30 and cytokines, including prostaglandin E2. Sclerostin production is increased
by calcitonin 31 ( Fig. 5.1 ).

Open full size image

Fig. 5.1
(A) Platelets found in the hematoma degranulate, releasing various signaling molecules (TGF-β,
PDGF, and FGF), which are involved in the processes of chemotaxis and angiogenesis, as well as
regulate cell proliferation and differentiation of the undifferentiated marrow cells that have migrated
to the fracture site. (B) The Wnt pathway leads to regulation of gene transcription and thus is critical
for the conversion of undifferentiated mesenchymal stem cells into an osteoblastic lineage. Induction
of the Wnt signaling pathway promotes bone formation, whereas inactivation of the pathway leads to
osteopenic states. (C) Sclerostin is produced by the osteocyte and has antianabolic effects on bone
formation. It binds to receptor groups inhibiting the Wnt signaling pathway, thus inhibiting further
formation of osteoblasts. Sclerostin production by osteocytes is inhibited by parathyroid hormone.
(D) PTH binds to the osteoblasts. Binding stimulates osteoblasts, and continual exposure to PTH
leads to an increase in osteoclast activity and density; intermittent exposure stimulates osteoblasts
more than osteoclasts and results in an increase in bone formation. PTH/teriparatide inhibits
osteoblast apoptosis, thus prolonging the synthetic life span of this bone-forming cell. (E) BMP-2,
BMP-4, and BMP-7 have been found to play a critical role in bone healing, as they are able to
stimulate differentiation of undifferentiated mesenchymal cells directly into an osteochondroblastic
lineage.
An antibody for sclerostin is available in clinical trials (antisclerostin ab). Its use has
increased bone growth in preclinical trials in osteoporotic rats and monkeys. 32 In a
phase 1 study, a single dose of antisclerostin antibody (romosozumab) increased bone
density in the hip and spine in healthy men and postmenopausal women and the drug
was well tolerated. 33 In a phase 2 trial, 1 year of the antibody treatment in osteoporotic
women increased bone density more than bisphosphonate and teriparatide. Monthly
treatments of the antibody for 1 year increased the bone mineral density of the spine and
hip by 18% and 6%, respectively, compared with the placebo group. 34 A recent study
reported that postmenopausal women with osteoporosis who received 12 months of
treatment with romosozumab followed by alendronate had a significantly lower risk of
fracture than did those who received alendronate alone. 35 However, the study also
demonstrated an increased risk of cardiovascular adverse events and has not obtained
full US Food and Drug Administration (FDA) approval. These results demonstrate the
interplay that these signaling pathways have on bone formation and fracture healing,
and in the future will likely be available for fracture augmentation.

Factors Affecting the Early Callus Phase

Platelet concentrate contains alpha granules, which contain more than 30 bioactive
proteins, many of which have a fundamental role in hemostasis and/or tissue healing.
These proteins include PDGF (including aa, bb, ab isomers), TGF-β (including β1 and β2
isomers), platelet factor 4, interleukin-1, platelet-derived angiogenesis factor, VEGF,
epidermal growth factor, platelet-derived endothelial growth factor, epithelial growth
factor, IGF, osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin, and
thrombospondin-1. 3 Of these, PDGF and TGF-β appear to have the most potent effect
on the soft callus stage of healing 36 (see Fig. 5.1 ).

TGF-β activates fibroblasts to induce collagen formation, endothelial cells for

angiogenesis, chondroprogenitor cells for cartilage, and mesenchymal cells in an effort
to increase the population of factors crucial to the propagation of the soft callus phase of
healing.

The main function of PDGF is stimulating cellular replication (mitogenesis). This

growth factor increases populations of healing cells, including MSCs and
osteoprogenitor cells. PDGF also activates macrophages, resulting in débridement of the
surgical or traumatic site. The macrophage activation then triggers a second source of
growth factors released from the host tissues under the influence of the macrophage
action. 19 Recombinant PDGF has been approved by the FDA for use as an alternative to
autograft for ankle and/or hindfoot fusion indications (Augment). This recombinant
growth factor is combined with an osteoconductive beta–tricalcium phosphate (β-
TCP)–collagen matrix, which is also injectable. Studies have shown that this material
was an effective alternative to autograft for ankle and hindfoot fusions. 373839 These
results from multiple studies demonstrated comparable fusion rates in these patients
when using this material compared with autograft. Additionally, these patients had a
marked decrease in pain and fewer side effects compared with the treatment groups
where autograft was used for the fusion procedure.

Remodeling Phase
As osteoblastic formation of the woven bone takes place, subsequently the formation of
more organized secondary bone also begins. The newly formed woven bone remodels to
recapitulate the mature lamellar bone and restore the original cortical end plates and
cortical structure. 40 Although the major cell type responsible for fracture callus
remodeling is the osteoclast, it is the interaction between osteoblastic and osteoclastic
function that leads to successful remodeling.

Recent evidence suggests that cells in the osteoblastic lineage, including mesenchymal
stromal cells, secrete factors that induce fully differentiated osteoblasts to express
ligands that regulate the activity of osteoclasts. One of these, the receptor activator of
nuclear factor kappa B (NF-κB) ligand (RANKL), has been shown to be essential in the
development of osteoclast precursors and thus modulates this remodeling activity. This
surface-bound molecule RANKL found on osteoblasts serves to activate osteoclasts,
which are critically involved in bone resorption. Osteoclastic activity is triggered via the
osteoblasts’ surface-bound RANKL activating the osteoclasts’ surface-bound receptor
activator of NF-κB (RANK). 40 A molecule that can block the binding of RANKL to RANK
would successfully block the formation of osteoclasts.

When the hemopoietic mononuclear osteoclast precursor binds the osteoblast through
the interaction of RANKL and RANK, these mononuclear cells are induced to fuse and
form multinucleated osteoclasts with bone-resorbing capacity. Through the binding of
RANKL, osteoclasts and osteoblasts play a vital role in normal bone remodeling. The
osteocyte produces RANKL and sclerostin to control bone turnover and production in
response to the mechanosensors in the osteocytes.

Studies show that RANKL is expressed at very low levels in unfractured intact bones but
is strongly induced by a fracture to increase its activity. 41 The coregulation of this
process takes place through the action of macrophage colony-stimulating factor, which
shows a peak in expression just before calcified cartilage removal begins (remodeling
phase). Overproduction of RANKL is implicated in a variety of degenerative resorptive
bone diseases, such as rheumatoid arthritis and psoriatic arthritis.

Factors Affecting the Remodeling Phase: New Venues for Effective

Pharmacologic Intervention
Activation of RANK by RANKL promotes the maturation of preosteoclasts into
osteoclasts. Denosumab is the first monoclonal antibody that binds to be approved by
the FDA. Denosumab inhibits maturation of osteoclasts by binding to and inhibiting
RANKL. This mimics the natural action of osteoprotegerin, an endogenous RANKL
inhibitor, that presents with decreasing concentrations (and perhaps decreased avidity)
in patients who are suffering from osteoporosis. This protects bone from degradation
and helps counter disease progression. 4243 This has the effect of inhibiting the
differentiation of the osteoclast precursor into a mature osteoclast. The first human
monoclonal antibody for the treatment of osteoporosis, denosumab is approved for use
by the FDA for postmenopausal women with risk of osteoporosis and fractures.
Emerging data indicate that denosumab may also reduce the risk of osteoporotic
fracture in patients with steroid-induced osteoporosis. 35444546

PTH is an 84-amino-acid polypeptide involved in the regulation of calcium and

phosphate metabolism. Bone resorption is the normal destruction of bone by
osteoclasts, which are indirectly stimulated by PTH. Stimulation is indirect because
osteoclasts do not have a receptor for PTH; rather, PTH binds to the osteoblasts and
indirectly stimulates these precursors to fuse, forming new osteoclasts, which ultimately
enhances bone resorption. 47 Although continual exposure to PTH leads to an increase in
osteoclast activity and density, intermittent exposure stimulates osteoblasts more than
osteoclasts and results in an increase in bone formation. PTH given in intermittent
doses inhibits osteoblast apoptosis, thus prolonging the synthetic life span of this bone-
forming cell (see Fig. 5.1 ).

Teriparatide is a recombinant form of PTH. Teriparatide is identical to a portion of

human PTH, and intermittent use activates osteoblasts more than osteoclasts, which
leads to an overall increase in bone. It is an effective anabolic (i.e., bone-growing)
agent 48 used in the treatment of some forms of osteoporosis.

Clinically, PTH has been approved by the FDA for its use in the treatment of
osteoporosis in postmenopausal women and men. Several recent clinical trials have
demonstrated that daily systemic treatment with PTH increases bone mineral density
and reduces fracture risk in osteoporotic patients. It is also occasionally used off-label to
speed fracture healing and treat nonunions. Numerous animal studies using PTH to
augment fracture healing have consistently demonstrated increased fracture callus
volume and enhanced mechanical properties as well as increased bone mineral density,
bone mineral content, and total osseous tissue volume. 49505152 These findings have
supported the initiation of clinical trials to study the role of systemic administration of
PTH in fracture patients. 5253

Recent data using this as an adjuvant for fracture healing in patients with atypical
femoral shaft fractures when given immediately after surgical repair suggest a trend for
superior healing of these fractures and improved bone mineral density compared with
those who received teriparatide in a delayed fashion. The number of patients was too
small to achieve statistical significance. 54 In the context of a patient who has
experienced an atypical femoral fracture after receiving bisphosphonate treatment,
therapy with teriparatide for 24 months increased bone mineral density and reduced the
risk of additional fractures resulting from osteoporosis. 55 Again, patient cohort size was
too small to achieve significance, but these results do lend support for the adjuvant use
of teriparatide after surgical repair of atypical femoral shaft fractures.

Atypical femoral shaft fractures have been shown to have a high association with chronic
bisphosphonate therapy for osteoporosis. Nitrogenous bisphosphonates act on bone
metabolism by binding and blocking multiple enzymatic pathways that are essential for
connecting some small proteins to the cell membrane. Inhibition of these specific
proteins can affect osteoclastogenesis, cell survival, and cytoskeletal dynamics. In
particular, the cytoskeleton is vital for maintaining the “ruffled border” that is required
for contact between a resorbing osteoclast and a bone surface. Thus inhibition of the
ruffled border with subsequent dysfunction of resorption is the main osteoclast effect
seen with this class of bisphosphonates. Unfortunately the biphosphonate inhibition of
the ruffled border has long-lasting effects and thus simple discontinuation of this drug
does not immediately reverse the osteoclast inhibition effects. 56 Several animal models
have shown increased callus volume, trabecular bone volume, and bone mineral content
with bisphosphonate treatment but have also shown delayed maturation and
remodeling of the callus. There are concerns that long-term bisphosphonate use can
result in oversuppression of bone turnover. It is hypothesized that microcracks in the
bone are unable to heal and eventually unite and propagate, resulting in atypical
fractures.

Such fractures tend to heal poorly and often require some form of bone stimulation, for
example, bone grafting as a secondary procedure. This complication is not common, and
the benefit of overall fracture reduction still holds. 5758 However, with these uncertain
effects on bone repair, their role in fracture healing is unclear. In cases where there is
concern of such fractures occurring, teriparatide therapy is potentially a good adjuvant
to assist in healing of these fractures due to its ability to reduce damage caused by
suppression of bone turnover. 59

Graft Incorporation and Healing of Fractures

Fracture healing is a complex process in which a series of molecular and cellular events
leads to the formation of new bone. This process requires osteogenic cells, osteogenic
factors, and an osteoconductive scaffold under the proper mechanical stimuli and with
adequate vascularization. The majority of fractures heal uneventfully, but it has been
reported that 5% to 10% of fractures either fail to unite or demonstrate a delay in
healing. 3 Therefore alternative strategies designed to enhance fracture healing and to
improve the treatment of delayed unions and nonunions are required.

Therapies aimed at enhancing fracture repair act through one of the following
mechanisms: (1) directly increase the conductive substrate and available sites of
competent cellular attachment, (2) increase osteoblastic activity, (3) indirectly recruit
progenitor cells that will mature to osteoblasts, (4) inhibit osteoclastic activity, or (5)
stimulate tissue revascularization. Growth factors, by virtue of their ability to regulate
cell behavior, may influence a number of these processes.

All bone graft and bone-graft-substitute materials’ mechanism of action can be

described through these processes. Although fresh autologous graft has the capability of
supporting new bone growth by many of these mechanisms, it may not be necessary for
a bone graft replacement material to have all the properties inherent in that material to
be clinically efficacious ( Table 5.1 ). When inductive molecules are locally delivered on a
scaffold, ultimately MSCs are attracted to that site and are capable of reproducibly
inducing new bone formation provided minimal concentration and dose thresholds are
 met. In some clinical studies, osteoinductive agents have been shown to potentially
perform superiorly compared with conductive materials alone. 60

Table 5.1
Available Fracture Healing Adjuvants With Their Inherent Properties
Osteoconductive Osteoinductive Growth Osteogenic
Scaffold Factors Living Cells
Synthetic Ca + ceramics •
Marrow concentrates •
Bone morphogenetic •
proteins (BMPs)
Autograft • • •
Platelet-rich Osteopromotive
concentrates indirect cellular effect
No intracellular
transcription
Banked demineralized • •
bone matrix (DBM)
 View full size
When bone marrow aspirate (viable stem cells) is applied to an osteoconductive
scaffold, these cells are still reliant on the local mechanical and biologic inductive signals
to ultimately form bone. Similarly, osteoconductive materials work well when filling
noncritical-sized defects that would normally heal easily without any additional
adjuvants added. However, in more challenging critical-sized defects, all three types of
these materials are necessary to achieve efficacy equivalent to autograft. 616263

Stages of Bone Graft Incorporation

Bone grafts and bone graft substitutes incorporate via a well-defined pathway that can
be divided into five distinct stages of host response, with the duration of each phase
depending on the type of graft or adjuvant material used. 23616263

 Stage 1: Initial injury and hemorrhage. Initiates the pathway with the degranulation of
platelets at the site of injury. (Platelet-derived factors.)

 Stage 2: Inflammation. Under the influence of many active cytokines that are produced
at the site of injury. These cause migration of cells to the site of injury. (Growth factor
intervention.)

 Stage 3: Vascular proliferation and ingrowth. Under the influence of many cytokines,
invading capillaries bring perivascular tissue with mesenchymal cells that can
differentiate into osteoprogenitor cell lines. (Autograft, marrow cellular therapies.)  64 A
competent conductive substrate is required for this process to occur. (Allograft and
alloplastic conductive substrates.) This vascular invasion can be significantly inhibited by
 nonsteroidal antiinflammatory medications, which will inhibit this process and thus
alter the fracture healing pathway. (NSAIDs.)

 Stage 4: The fourth stage consists of osteoclastic resorption of the avascular (dead) bone
graft lamellae and simultaneous production of new bone matrix by
osteoblasts. (Pharmacologic manipulation.)

 Stage 5: In the final stage, the newly formed bone is remodeled and reoriented based on
the mechanical environment of the host site. (Pharmacologic manipulation.)

Orthobiologic interventions have been specifically designed to target these stages to

achieve a specific effect. Each broad category of intervention will be discussed in terms
of the desired effect these materials have on the specific stage of graft incorporation. The
gold standard and prime material that all others are compared with is autogenous iliac
crest bone graft (AICBG). 6465

Urist and colleagues identified a protein that they termed bone morphogenetic

protein (BMP). 131415 Other molecules were soon identified and helped to characterize
an entire family of osteoinductive molecules. 66 This family of specific factors now
contains at least 15 BMPs and is part of the larger TGF-β superfamily of molecules.

Protein extracts derived from bone can initiate the process that begins with cartilage
formation and ends in de novo bone formation. The critical components of this extract
(BMP) that direct cartilage and bone formation, as well as the constitutive elements
supplied by the animal during this process, have long remained unclear. Amino acid
sequence has been derived from a highly purified preparation of BMP from bovine bone.
Now, human complementary DNA clones corresponding to three polypeptides present
in this BMP preparation have been isolated, and expressions of the recombinant human
proteins have been obtained. 967

Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of
inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and
BMP-3) are new members of the TGF-β supergene family, and the third, BMP-1,
appears to be a novel regulatory molecule. BMP factors can be synthesized by
recombinant gene technology or derived from autologous bone, allogeneic bone, or
demineralized bone matrix (DBM). 141516

DBM is allogeneic bone that has undergone the acid extraction of the mineralized ECM
of the allograft bone. In theory, the noncollagenous proteins, including osteoinductive
proteins such as the BMPs, remain viable while the structural portion of the allograft
has been removed. 68

All bone graft and bone-graft-substitute materials can be described through these three
processes. Although fresh autologous graft has the capability of supporting new bone
growth by all three mechanisms, it may not be necessary for a bone graft replacement
material to have all three properties inherent in that material to be clinically efficacious
(see Table 5.1 ). When inductive molecules are locally delivered on a scaffold, ultimately
MSCs are attracted to that site and are capable of reproducibly inducing new bone
formation provided minimal concentration and dose thresholds are met. In some
clinical studies, osteoinductive agents have been shown to potentially perform
superiorly compared with conductive materials alone.

Autogenous Iliac Crest Bone Graft

Fresh cancellous autograft provides the quickest and most reliable type of bone graft. Its
open structure allows rapid revascularization; a 5-mm graft may be totally
revascularized in 20 to 25 days. These grafts depend on ingrowth of host vessels and
perform best in well-vascularized beds. The large surface area of harvested autograft
allows for survival of numerous graft cells. It is estimated that approximately 25 to
30 mL of graft can reliably be harvested from an anterior iliac crest. 6569 Cadaver
studies also indicate that on average 35 mL is achieved from a posterior crest harvest
and 18 mL on average is harvested from a proximal tibial site. However, many other
sites of harvest have been described, differing only in the amount of graft obtained from
each harvest site (see Table 5.1 ). Using volumetric computed tomography (CT) scans on
healthy volunteers the amount of potential graft harvested from the pelvis has been
estimated. It has been shown that more cancellous and total corticocancellous bone can
be harvested from the posterior than the anterior iliac crest, together with similar or
smaller volumes of cortical bone. Sex of the donor (not age) was an important factor in
terms of the amount of bone than can be harvested, with a wide range of volumes
individually from both iliac crests. 70 Literature has demonstrated histologic differences
between iliac crest and tibial bone graft, suggesting superiority of iliac crest in terms of
osteogenic and hematopoietic progenitor cell content. 71 Studies document success rates
approaching 100% for subcritical-sized defects (1- to 2-cm defects) requiring 20 mL or
less of autograft. 147172

Many issues exist regarding AICBG due to the limited quantity available and the
reported rates of postoperative pain from the graft harvest site. 7374 Substantial
complication rates related to the harvest site have been reported. 1315 It has been
thought that this technique is restricted to short defects in the range of 4 to 6 cm.
Numerous studies report favorable union results for critical-sized defects up to 4 cm.
However, in many of these studies multiple graft procedures were required to achieve
solid union. 72757677

The ability to obtain substantial amounts of autogenous graft material would appear to
be an advantage for the treatment of critical-sized defects. The reamer irrigator
aspirator (RIA, Synthes; Paoli, PA) offers a technique to achieve substantial amounts of
graft volumes for the treatment of larger segmental defects. The medullary canal of the
femur or tibia is reamed with a device designed to collect the reamings and deliver them
for potential grafting procedures. 7879 Variable amounts of harvested graft with this
technique have been reported in the literature and range from 30 to 90 mL. A
comparison between a historical control group using anterior iliac harvesting (40
patients) versus a study group using femoral shaft RIA harvesting (41 patients)
documented on average 25 to 75 mL of harvested RIA graft (average, 40.3 mL). 78 The
authors reported a favorable union rate with RIA bone grafting (37 of 41 patients) versus
AICBG (32 of 40 patients), although not statistically significant. There were significantly
lower postoperative harvest site pain scores from the RIA group versus the AICBG group
at 48 hours, 48 hours to 3 months, and greater than 3 months ( P = 0.001, 0.001, and
0.004, respectively). There were a total of two complications related to the graft harvest
site in the RIA group (one perforation of the distal anterior femoral cortex treated
conservatively and one excessive reaming of the femoral neck treated with prophylactic
cannulated screws) versus 12 harvest site complications in the AICBG group (3
infections, 1 hematoma, and 8 patients with numbness).

This study has several limitations, including the concurrent use of bone morphogenetic
protein-2 (BMP-2) in most cases. This somewhat limits the ability to draw strong
conclusions regarding the relative efficacy of RIA bone graft versus AICBG from this
study. 7879

A study reported on the treatment of 20 bone defects ranging from 2 to 14.5 cm (average
= 6.6 cm) using RIA bone graft. Eighteen of the 20 patients were initially treated with an
antibiotic cement spacer using the Masquelet technique 80818283 (see Fig. 5.1 ). The
average graft volume obtained using the RIA was 64 mL. Seventeen of 20 bone defects
ultimately healed, although 7 of these required repeat surgery. The authors reported no
significant complications related to the bone graft harvest site 7384 ( Fig. 5.2 ).
Open full size image
Fig. 5.2
(A) Open distal femoral shaft fracture with 13-cm segmental defect. (B) Fracture underwent multiple
débridements with eventual placement of a large antibiotic spacer into the skeletal defect with
stabilization using a plate and spanning external fixator. (C) After complete soft tissue healing had
been accomplished, and the development of an enveloping psuedomembrane surrounding the
antibiotic cement spacer (Masquelet), massive autografting was carried out. The reamer irrigator
aspirator (RIA) was used to harvest a substantial amount of graft from the contralateral femur. The
spacer was carefully removed leaving the psuedomembrane intact grafting into the well-defined
space spanning the defect. (D) Complete healing of the defect at approximately 6 months after graft.
Numerous basic science studies have demonstrated the biologic potential of RIA bone
graft. Investigators have documented elevated amounts of osteoinductive growth
factors 85868788 and osteoprogenitor/endothelial progenitor cell types compared with
AICBG. 89 The RIA filtrate contains large numbers of MSCs that could potentially be
extracted without enzymatic digestion and used for bone repair without prior cell
expansion. Medullary autograft cells harvested using RIA are viable and osteogenic. Cell
viability and osteogenic potential were similar between bone grafts obtained from both
the RIA system and the iliac crest. 90

Elevated levels of FGFa, PDGF, IGF-I, TGF-β1, and BMP-2 were measured in the
reaming debris compared with iliac crest curettings. However, VEGF and FGFb were
significantly lower in the reaming debris than from iliac crest samples. In comparing
platelet-rich plasma (PRP) and platelet-poor plasma (PPP), all detectable growth
factors, except IGF-I, were enhanced in the PRP. In the reaming irrigation FGFa (no
measurable value in the PRP) and FGFb were higher, but VEGF, PDGF, IGF-I, TGF-β1,
and BMP-2 were lower compared with PRP. BMP-4 was not measurable in any sample.
The bony reaming debris is a rich source of growth factors with a content comparable to
that from iliac crest.

The irrigation fluid from the reaming also contains growth factors. Proteins present in
the RIA effluent water indicate the potential for clinical use of this filtrate as an adjunct
for enhancing bone production, healing, and remodeling. This finding has encouraged
the development of processing protocols for viable use of the effluent RIA waste water.
Similarly cells cultured from RIA compared favorably to those from iliac crest bone
grafts (ICBGs) with respect to their potential bone formation. 91

The use of the device has been associated with pathologic fractures, due to the eccentric
reaming caused by malposition of the reaming guide wire. An eccentric canal can
significantly compromise a long bone's torsional strength, more than if reamed
concentric to a larger diameter having the same minimum wall thickness. This puts the
shaft at risk for pathologic fracture with trivial forces. 9293 In addition significant
perioperative blood loss remains a prevalent complication during RIA reaming, as well
as persistent postoperative pain in the reamed extremity. 94 A recent series found that
44% of patients undergoing RIA graft harvest required transfusion with a mean
hematocrit drop of 113.7 across all subjects, which is significantly greater than that
associated with ICBG. 95

Although the early evidence regarding RIA bone grafting is encouraging, there is
currently a lack of high-level comparative evidence; however, a randomized controlled
trial (RCT) comparing RIA grafting to ICBG revealed similar rates of union of the
grafted bone, with significantly less donor site pain in the RIA patients (Level I). RIA
also demonstrated a greater volume of graft compared with anterior ICBG and a shorter
harvest time compared with posterior ICBG. For larger volume grafts, cost analysis
favored using RIA, primarily related to the more invasive crest harvest with attendant
longer operating room (OR) times. 96

A study published in 2010 compared the osteoprogenitor and endothelial progenitor

cells obtained from RIA versus hip aspirate. 97 Patients in both groups did not
significantly differ regarding their age, gender, or preexisting health conditions. In
comparison to aspirates obtained from iliac crest, the RIA aspirates from the femur
contained a significantly higher percentage of CD34+ progenitor cells, a significantly
higher concentration of MSCs, and a significantly higher concentration of early
endothelial progenitor cells (EPCs). The results demonstrated that RIA aspirate is a rich
source for different types of autologous progenitor cells, which can be used to accelerate
healing of bone and other musculoskeletal tissues. The major flaw of this study was that
for the clinical use of marrow aspsirates to be effective, a threshold concentration needs
to be obtained. This study only evaluated the cellular types and found comparative
numbers of active cells. 97

Osteoconductive Graft Substitutes

There is considerable interest in creating osteoconductive matrices using nonbiologic
porous structures implanted into or adjacent to bone. The host substrate must mimic
the cancellous bony architecture and have very specific surface kinetics to facilitate the
migration, attachment, and proliferation of MSCs, which then differentiate into
osteoprogenitor cells (augmentation of stages 2 and 3 of graft incorporation). Broad
categories of these materials are available and in general are classified as calcium
ceramics. These include the specific materials of calcium sulfate (CS), calcium
phosphate, synthetic tricalcium phosphate and beta tricalcium phosphate, and coralline
hydroxyapatite. All of these materials have been used as carriers for bone marrow
aspirate concentrate (BMAC), all with comparable results. 9899100

The history of bioceramics dates back to 1892 with the use of CS for space-occupying
lesions. CS has the distinction of being the alternative that is both one of the simplest
and the one that has the longest clinical history as a synthetic bone graft material—
spanning more than 100 years. 99

The original material was plaster of Paris, which is noninflammatory and nonreactive
and encouraged bone healing in a contained lesion. Peltier took commercial-grade
plaster of Paris, which was then mixed with water, poured into molds made of wax
paper or aluminum foil, and then allowed to set forming small pellets or columns. He
performed a series of bone defect studies in dogs to determine the role these materials
had in the ability to heal these defects. 100 From his experiments he determined that the
plaster of Paris itself does not stimulate osteogenesis. Its chief effect was found to be a
mechanical one of preventing the collapse of the periosteal tube and favoring
regeneration. In this way the material provided a supportive
scaffolding. 101102103104 There is no doubt, however, that subperiosteal resections in
which plaster of Paris columns were inserted regenerated, in whole or in part, more
frequently than was the case in subperiosteal resections alone.

Subsequently, coralline hydroxyapatite was reported for similar lesions followed by

other ceramics. All of these bone graft substitutes have the advantage of being
nonimmunogenic, noninflammatory, in an unlimited supply, and packaged sterile.

Calcium Sulfate Substitutes

CS was one of the first orthobiologic materials to be used commercially as a bone graft
substitute. CS has a crystalline-independent rate of incorporation and is very consistent
in terms of dissolution/incorporation. The crystalline structure is very consistent
throughout a whole range of materials, with a very constant rate of osteointegration. In
contrast to calcium phosphate, CS behaves as a true salt; that is, if it egresses into the
joint it quickly dissolves into sulfate and calcium ions and is then absorbed into the
synovium. This resorption mechanism involves a fluid exchange mechanism and may
promote excessive drainage if used in wounds with questionable soft tissue coverage or
integrity. A low but consistent complication rate, specifically serous drainage from the
wound as the calcium sulfate absorbs, has been reported. This complication is higher
when the material is used in higher volumes (greater than 20 mL) or in subcutaneous
bones (tibia, ulna). 105

This may cause a potential increase in osmotic load at the site of implantation.
Therefore to avoid subsequent drainage at the graft site, the CS product should be
reserved for situations with adequate blood supply and competent soft tissue coverage.
Additionally, this material should be used in contained defects only. 106 Studies
document that implanted CS pellets in contact with joint synovial fluid are at risk for
resorption without any significant bony healing response. 107 If CS pellets are to be
implanted in periarticular locations, complete bony containment is necessary. 108

CS hemihydrate has been used for many years as a self-setting biomaterial due to its
good setting properties. The fairly rapid degradation rate of these materials that occurs
in 3 to 4 months was once viewed as an advantage. 109 However, as these materials began
to be used to support articular subchondral surfaces in cases of periarticular plateau and
pilon fractures, this rapid degradation became a distinct disadvantage. 110 Transition to
full weight bearing occurs normally at 3 to 4 months postsurgery, and many cases of late
articular collapse have been subsequently reported due to this rapid incorporation with
simultaneous loss of articular support. 75111 Additionally, this material demonstrates
rapid loss in its mechanical compressive strength after implantation, compared with the
phosphate ceramics.

This combination of rapid degradation rate, speedy loss of compressive strength, and
lack of bioactivity has limited its application for bone defect management. 101 Three case
series examining the use of calcium sulfate for the treatment of bone nonunion revealed
a significant failure rate, suggesting that this material, used in isolation, is not optimal
to promote union in that setting. 105112

The current best use of this material appears to be that of a carrier for adjuvant
antibiotics as a treatment for osteomyelitis. The characteristics regarding rapid
resorption and degradation are now advantageous for delivering high dose antibiotics.
McKee et al. demonstrated results for the treatment of chronic osteomyelitis and
infected nonunions, using an antibiotic-impregnated CS pellet. 113114 Recent studies
have evaluated the strategy of débridement, with removal of any implants, and excision
back to bleeding bone. 115 A composite of CaSO 4 plus tobramycin preformed pellets
(OSTEOSET T) was packed into a contained defect or the intramedullary canal with
further bony stabilization and soft tissue reconstruction ( n = 7) undertaken as required.
The average follow-up was 15 months. Union rate after tibial reconstruction was 100%.
Wound complications were encountered in 52%: a wound discharge in the early
postoperative period was noted in seven patients (33%) independent of site of pellet
placement. This is thought to be due to an osmotic load as CS dissolves by chemical
dissolution and acts like a salt in a fluid dynamic, as previously discussed.
In cases where the soft tissues may be incompetent, this material then leaks onto the
surface and can be mistaken for infection. This is a sterile exudate from local wound
fluid overload. Surgeons must be familiar with this common phenomenon if they use
this material. The authors concluded that this material was an effective adjunct in the
treatment of chronic tibial osteomyelitis after trauma. This was based on the low
incidence of residual infection; however, wound drainage is an issue that must be
recognized (Level III). 115

There is Level I and II evidence (one randomized trial, one case-control study, one
prospective cohort study) that antibiotic-impregnated bioabsorbable calcium sulfate has
the potential to reduce the number of procedures and surgical morbidity associated with
the surgical treatment of chronic osteomyelitis and infected nonunion while
maintaining a high rate of infection eradication. Calcium sulfate remains an
inexpensive, safe, reliable bone void filler that can also serve as an absorbable delivery
vehicle for antibiotics or other compounds. 113114

In addition to antibiotic delivery, these materials have also been used recently for the
management of dead space. This is a fundamental aspect of surgery. Residual dead
space after surgery can fill with hematoma and provide an environment for bacterial
growth, increasing the incidence of postoperative infection. Materials for managing
dead space have historically included polymethylmethacrylate (PMMA), which is
nonresorbing and usually requires removal in a second surgical procedure. The use of
CS offers the advantage of being fully absorbed and does not require subsequent surgical
removal. As calcium phosphate has historically been used as a bone void filler, there are
some concerns for the risk of heterotopic ossification (HO) when implanted adjacent to
soft tissue, and thus it may not be an ideal material for dead space management.

A recent study assessed the osteoinductive potential of CS and identified and

characterized residual material present in muscle tissue using histology, energy-
dispersive radiograph spectroscopy analysis, and scanning electron microscopy. CS
beads with and without antibiotic were implanted in intramuscular sites in both athymic
rats and New Zealand white rabbits. At 28 days after implantation no signs of
osteoinduction or HO were observed. At 3 weeks after implantation near-complete bead
absorption was confirmed and thus this material appears to be an excellent alternative
to bone cement and may help avoid additional surgery for removal 116 ( Fig. 5.3 ).
Open full size image
Fig. 5.3
Calcium sulfate beads are visible after débridement procedures and provide a local depot of high-
dose antibiotics for delivery to these previously infected regions. In addition to the local antibiotic
delivery, they are an excellent means of filling the dead space that occurs after débridement. They
have the added benefit of dissolving over the course of 6 to 8 weeks and prevent the need for a
secondary removal procedure that is common when using PMMA beads for similar purposes.
Calcium Phosphate Substitutes
Calcium phosphate substitutes are osteoconductive, but they are not osteoinductive
unless growth factors, BMPs, or other osteoinductive substances are added to create a
composite graft. Calcium phosphate is available in a variety of forms and products,
including ceramics, powders, and cements. Ceramics are highly crystalline structures
created by heating nonmetallic mineral salts at temperatures greater than 1000°C, a
process known as sintering. These phosphate materials have variable rates of
osteointegration based on their crystalline size and stoichiometry. 1

Hydroxyapatite
Synthetic hydroxyapatite is a crystalline calcium phosphate osteoconductive bone
substitute that is also manufactured as a ceramic through a sintering process. This
material was some of the first available to be used clinically as a bone graft substitute.
Porous hydroxyapatite (Interpore 500) was formed by conversion of the Porites
goniopora coral exoskeleton, with pores averaging 600 µm and pore interconnections
averaging 260 µm in diameter. 175

Clinical studies with this material used for augmentation of tibial plateau fractures were
successful when used with internal fixation. No significant radiographic or clinical
differences were appreciated between those patients who were randomized to have the
defect filled with either autogenous bone or porous hydroxyapatite. 117

The investigators thought that a major drawback of the material was the slow rate of
incorporation. The appositional process of incorporation of the implant was confirmed
by the finding that only 66.5% of the surface of the Interpore 500 was covered with bone
ingrowth at 12 months. Thus a relatively long incorporation time was
documented. 117118119

Other investigators studied the ability of this material to act as a conductive substrate
and determine the effectiveness of coralline hydroxyapatite as a bone graft substitute for
lumbar spine fusion when used in combination with bone marrow, when mixed with
autogenous bone graft (ABG), or when combined with an osteoinductive material
(BMP). 120 The data indicated that coralline hydroxyapatite with bone marrow was not an
acceptable bone graft substitute used alone for posterolateral spine fusion compared
with autograft.

When combined with AICBG, coralline hydroxyapatite served as a graft extender

yielding results comparable to those obtained with autograft alone. 117 Additionally,
coralline hydroxyapatite served as an excellent carrier for the bovine osteoinductive
bone protein extract yielding superior results to those obtained with autograft or bone
marrow. 120 As with other characteristics of these materials, the rates of osteoinduction
were greatest when a porous architecture was maintained. 121122 Animal studies have
suggested that hydroxyapatite may have some osteoinductive properties in addition to
its osteoconductive capabilities. 123123124

The crystalline structure dictates the rate of osteointegration. These materials integrate
via a cell-mediated response and the pore structure found in these materials serves as
sites for cellular attachment. This porosity makes these materials very brittle with
minimal tensile strength. 125 Because of these material properties and concerns regarding
very slow bone formation, hydroxyapatite, used alone, is not commonly used as an
osteoconductive bone substitute at this time.

The porosity of these materials is the primary factor in determining the ability to foster
ingrowth and osteointegration. No osseous ingrowth occurs with pore sizes of 15 to
40 µm. Osteoid formation requires minimum pore sizes of 100 µm, with pore sizes of
300 to 500 µm reported to be ideal for osseous ingrowth. 126 Some authors, however,
have reported that pore size may be less critical than the presence of interconnecting
pores for osseous ingrowth. Interconnecting pores prevent the formation of blind alleys,
which are associated with low oxygen tension; low oxygen tension prevents
osteoprogenitor cells from differentiating into osteoblasts 1127 ( Fig. 5.4 ).
Open full size image
Fig. 5.4
Left, Calcium ceramic materials present a three-dimensional architecture to the defect that serves as
a site for potential cellular attachment. This porosity provides a matrix for osteointegration and
degradation of the implanted material at a rate dependent on the porosity of the material. Center,  A
combination of particulate and injectable CaPO 4 has been applied to the defect after articular
elevation and the creation of a large subchondral void. Right, The ability to place fixation hardware
through the material is material specific. The “setting” properties of these injectable materials if
variable and can be disrupted by the process of hardware manipulation. Thus not all can be “drilled
and filled.”
Highly porous interconnected materials have abundant sites available for cellular
interactions, which help these materials osteointegrate faster. This is accompanied by a
corresponding decrease in the compressive strength afforded. If the material is designed
with minimal porosity, the rate of osteointegration will be very prolonged because of the
paucity of cellular interactions. The corresponding compressive strength will also be
very high. As noted, their ability to provide structural support is dependent on the
degree of porosity inherent in each unique material, which can be highly
manipulated. 175 These materials have the advantage of incorporating at a slower rate
than CS materials. They increase bone formation by providing an osteoconductive
matrix for host osteogenic cells to create bone under the influence of host osteoinductive
factors.

Tricalcium Phosphate
Tricalcium phosphate is a commonly available resorbable ceramic. It can be obtained in
block, granular, powder, or putty form. Coralline ceramics are formed by
thermochemically treating coral with ammonium phosphate, leaving tricalcium
phosphate with a structure and porosity that are similar to those of cancellous bone.

Tricalcium phosphate is less brittle and has a faster resorption rate than hydroxyapatite
due to the increased porosity. Animal studies have demonstrated that 95% of calcium
phosphate is resorbed in 26 to 86 weeks. 128129 This is much faster compared with the
previous hydroxyapatite reported rates of osteointegration.
Tricalcium phosphate and hydroxyapatite have been combined into a biphasic calcium
phosphate (BCP) composite that has a faster resorption rate than pure hydroxyapatite.
In a clinical study using a composite graft, a mixture of porous beads composed of 60%
hydroxyapatite and 40% tricalcium phosphate ceramic and fibrillar collagen was used as
a graft substitute and combined with autogenous bone marrow. Collagraft (Zimmer and
Collagen Corporation) was randomized against cancellous iliac crest autografts in the
treatment of long bone fractures. This material appeared to function as well as
autogenous graft when used in the treatment of acute long bone fractures. 130

Calcium phosphate can also be manufactured as cement by adding an aqueous solution

to dissolve the calcium, which is followed by a precipitation reaction in which the
calcium phosphate crystals grow and the cement hardens. The primary advantage of
cements over blocks, granules, or powders is the ability to custom-fill defects 75111 and
produce increased compressive strength. However, cement can be extruded beyond the
boundaries of the fracture, potentially damaging the surrounding tissue. This is
especially problematic if these materials extrude into a joint cavity after repair of a
subchondral defect such as a tibial plateau. This presents a potential disadvantage of
these phosphate materials, as they will not dissolve if they happen to migrate into the
joint. 131 The ability of calcium phosphate bone substitutes to act as a bone void filler has
been documented in multiple preclinical animal studies, biomechanical studies, and
human case series. 132

One such representative biomechanical study evaluated the fatigue strength of calcium
phosphate–augmented repairs versus ABG repairs for lateral tibia plateau fractures.
Reproducible split-depression fractures were simulated and repaired with each
specimen randomly assigned to either calcium phosphate or ABG as augmentation.
Calcium phosphate–augmented repairs subsided less and were more stiff during the
fatigue loading than were ABG repairs at the 70,000-, 140,000-, and 210,000-cycle
intervals ( P < 0.03) The authors concluded that the calcium phosphate repairs had
significantly higher fatigue strength and ultimate load than ABG repairs and may serve
to increase the immediate weight-bearing capabilities of the repaired plateaus in clinical
practice. 133

A large clinical retrospective case series reviewed 43 patients with traumatic bone
defects or nonunions treated. Defects of the femur, tibia, calcaneus, humerus, ulna, or
radius had treatment augmented with tricalcium phosphate. Ninety percent of the
fractures and 85% of the nonunions had united at the time of follow-up (average of 12
months). The authors concluded that tricalcium phosphate was a useful substitute for
cancellous 134 bone if the local biology provided a suitable blood supply. This was deemed
necessary to provide competent cells and circulating inductive factors.

The use of injectable bone calcium phosphate cements offers the opportunity to support
the reduced joint surface without open bone grafting. This is a valuable adjuvant, as less
invasive fixation approaches are becoming widely accepted and the ability to limit the
exposures for grafting and subchondral defect augmentation would also be a valuable
tool. Jubel and colleagues 135 evaluated the clinical and radiologic outcomes of an
injectable calcium phosphate material (Norian SRS) used for tibial plateau fractures.
The time interval from surgery to partial weight bearing was 3.7 weeks. The results
demonstrated that this material can be successfully used to fill metaphyseal bone defects
in tibial plateau fractures. The clinical and radiologic results were comparable to those
of fractures treated with autologous bone graft. The high compression strength allows
early full weight bearing without the risk of secondary loss of reduction. Soft tissue
reactions due to the cement were not observed. 135

However, on all radiographs taken 36 months after the operation, the phosphate cement
bloc was still visible, indicating a very slow rate of osteointegration, which may be of
some concern to some clinicians. Once the transition to full weight bearing has been
achieved, it would be desirable to have the material almost completely integrated.
Newer phosphate materials are now available that have shorter times to complete
osteointegration as these crystalline structures can be manipulated accordingly.

In a critical study by Russell and Leighton 136 the efficacy of a bioresorbable calcium

phosphate cement was compared with standard autogenous iliac bone grafting in a
multicenter, prospective, randomized study for the treatment of these osseous defects.
Randomization to treatment with calcium phosphate cement (82 fractures) or
autogenous iliac bone graft (38 fractures) occurred at the time of surgery. There was a
significantly ( P = 0.009) higher rate of articular subsidence during the 3- to 12-month
follow-up period in the bone graft group. The bioresorbable calcium phosphate cement
used in this study appeared to be a better choice, at least in terms of the prevention of
subsidence, than autogenous iliac bone graft for the treatment of subarticular defects
associated with unstable tibial plateau fractures.

A recent matched cohort study evaluated the value of an injectable calcium phosphate
(CaPO) cement for the augmentation of tibial plateau fracture defects matched to
patients with identical fracture patterns treated with similar fixation but augmented
with routine autogemous bone grafting. Articular step-off and variation of articular step-
off were significantly lower in the CaPO groups compared with the control group using
autograft. At last follow-up, patients of the control group presented a higher rate of step-
off greater than 2 mm and step-off Δ greater than 2 mm (respectively, 56% and 35%)
than patients of the CaPO group (26% and 9%). At mean follow-up of 29 months, pain
scores were significantly better in patients of the CaPO group than in control
patients 137 ( Fig. 5.5 ).
Open full size image
Fig. 5.5
(A and B) A large subchondral defect is produced after the elevation of the articular surface using a
tibial-plasty balloon reduction technique. (C) The large defect is then filled with an injectable
CaPO 4 material that allows percutaneous placement. (D and E) The subchondral void is filled
completely, and fixation hardware is then applied to augment the joint reduction and elevation. (F) At
4 postoperative months there is gradual incorporation of the material with a halo of new bone
surrounding the material and no loss of articular reduction.
Open full size image
Leucht et al. 138 compared the clinical and radiologic outcomes between acetabular
fractures with marginal impaction that were treated with either cancellous bone graft
(CBG) or tricalcium phosphate cement (TPC) as bone void filler. Forty-three patients
with acetabular fractures with marginal impaction were treated. Eighteen patients
received cancellous bone graft and 25 patients received tricalcium phosphate cement as
bone void filler.

Radiographs were evaluated for postoperative reduction, arthritis grade, and

development of HO. At final follow-up, a significantly higher number of patients in the
cancellous bone graft group exhibited signs of moderate to severe posttraumatic
arthritis and required a total hip arthroplasty (THA). There was no significant difference
between the two groups regarding functional outcome. The authors concluded that
patients with acetabular fractures with marginal impaction treated with tricalcium
phosphate cement exhibit a significantly lower incidence of posttraumatic arthritis
compared with patients treated with cancellous bone graft (Level III). 138

At this time there is ample Level I evidence to suggest that calcium phosphate materials
are more reliable and maintain the articular reduction of plateau fractures through the
transition of fracture healing to full weight bearing. These are primarily radiographic
superiority studies. 138 This evidence suggests that phosphate cements can now be used
routinely without the complication of late subsidence that is commonly seen with
allograft and autograft augmentation of subchondral defects associated with plateau
fractures. There are, however, other considerations that must be taken into account.

The application systems available for the consistent delivery of the material into the
subchondral void are not as sophisticated as the materials themselves and warrant
improvements. As well, there are costs associated with the use of these materials that
can be quite expensive. A cost-benefit analysis has yet to be performed that will help
determine whether the extra costs associated with these materials are worth the
perceived benefits of avoiding late subsidence. The big question yet to answer is, Does a
few millimeters of subsidence seen on radiograph have any detrimental effects on the
long-term clinical outcomes of these injuries, regardless of the material used to augment
the fracture reduction?

Many studies have specifically evaluated these materials as bone graft substitutes in the
management of subchondral bone defects associated with tibial plateau fractures. A
meta-analysis compared CaPO cement substitutes directly to these other conductive
substrate materials used for plateau augmentation: hydroxyapatite granules, calcium
sulfate, bioactive glass, tricalcium phosphate, DBM, allografts, autografts, and
xenografts. 139 Fracture healing was uneventful in over 90% of the cases over the variable
time period of the meta-analysis. Secondary collapse of the knee joint surface ≥2 mm
was highest in the biologic substitutes group, 8.6% (allograft, DBM, autograft, and
xenograft).

This is similar to the results noted by Russell in his randomized study comparing
autologous iliac bone graft (AIBG) to calcium phosphate cement. 136 Late collapse was
less in the hydroxyapatite-treated group, 5.4%, and the material that had the least
subsidence was the CaPO cement group, 3.7% ( Fig. 5.6 ). The group that experienced the
highest rate of subsidence was the calcium sulfate cases, 11.1%. 140 This is consistent with
the rapid dissolution time and relative biomechanical properties of this material,
discussed previously in this chapter.

Open full size image

Fig. 5.6
Split depression fracture with articular elevation and subchondral support using CaPO 4 void filler and
a lateral raft plate. The fracture healed uneventfully, and the patient required hardware removal at 6
months after implantation. The articular surface reduction has been maintained and the material
continued to osteointegrate.
The recorded incidence of primary surgical site and donor site infection (3.6%) was not
statistically significant different among all the grafting groups. However, donor site–
related pain was reported up to 12 months after AIBG harvest. Shorter total operative
time, greater tolerance of early weight bearing, and improved early functional outcomes
within the first year after surgery were noted in the studies reporting on the use of
injectable CaPO cements. Despite a lack of good-quality RCTs, there is arguably
sufficient evidence supporting the use of bone graft substitutes at the clinical setting of
depressed plateau fractures. 135139140

These materials have also been extensively used for the augmentation of distal radius
fractures. Zimmermann et al. 141 evaluated patients treated with cement augmentation 2
years after the surgery. The authors concluded that the use of injectable CaPO cement
(Norian SRS) to supplement pin-and-screw fixation was effective in maintaining the
reduction of unstable intraarticular distal radius fractures in osteoporotic patients and
provided superior functional outcomes at 2 years after the surgery compared with
percutaneous pinning alone. 141 With the widespread use of locked plating for these
injuries, the efficacy of these materials for use in this situation must be questioned. A
randomized study sought to determine whether augmentation of volar locking plate
fixation with CaPO bone cement had any benefit over volar locking plate fixation alone
in an elderly patient population with unstable distal radial fractures. 142 The two groups
were comparable with regard to age, sex, fracture type, injury mechanism, and bone
mineral density. No significant differences were observed between the groups regarding
the clinical outcomes at the 3- and 12-month follow-up examinations. No significant
intergroup differences in radiographic outcomes were observed at 1-year follow-up.

The authors concluded that augmentation of metaphyseal defects with CaPO bone
cement after volar locking plate fixation offered no benefit over volar locking plate
fixation alone in elderly patients with an unstable distal radial fracture. This highlights
the point that, with the improved biomechanics that locked plating provides,
prospective studies are required to determine the role of these conductive substrates
when they are combined with locked plating techniques. 143

The treatment of intraarticular calcaneal fractures is commonplace in busy trauma

practices. Although open reduction and fixation is favored by many authors, increased
risk of soft tissue complications makes this method of treatment a challenge. Again the
value of an injectable minimally invasive material to augment posterior facet elevation
would be valuable. Wee and Wong 144 documented the safe use of an injectable CaPO
cement to augment standard plate fixation of calcaneal fractures. A total of 10 patients
with 12 displaced intraarticular calcaneal fractures underwent this method of treatment.
Full weight bearing was started at 1 postoperative month, and no cases demonstrated
loss of reduction. The authors felt that the early results with stabilization and
augmentation were encouraging. 144

In a similar study Elsner et al. 145 compared patients treated surgically with and without
injectable carbonated apatite cement. In the cement group, full weight bearing on the
affected extremity was regained at an average of 4 weeks postoperatively. During the
study period of 3 years, only a slight decrease in the density of the peripheral zones of
the cement block was observed (again referring to the relatively long integration time
with these first- and second-generation materials). Complete resorption and remodeling
of the bone cement were not complete at 3 years.

A more recent study using minimally invasive balloon-assisted fracture augmentation

was undertaken using defect management with CaPO to fill the subchondral void after
balloon-assisted postfacet elevation (minimally invasive percutaneous calcaneoplasty).
The results were encouraging as all cases obtained bony union in 2 to 3 months, with
average Bohler's angle maintained and no subsidence. No skin complications or adverse
reactions were observed secondary to the use of the phosphate bone void filler. 146

These techniques have also been utilized for tibial plateau fractures injecting CaPO after
balloon reduction of depressed articular plateau surfaces. The authors found that the
articular reductions were maintained throughout the transition to full weight bearing,
even when the material was placed via very minimally invasive injection
methodologies. 147
These new findings are consistent with the results of an earlier study by Schildhauer
et al., 148 who reported on a series of 36 joint-depression–type calcaneal fractures treated
by standard internal fixation techniques augmented with CaPO cement. Patients began
to bear weight as early as 3 weeks after the surgery without loss of reduction and no
significant difference in functional outcome scores between patients with early versus
late weight bearing. Of concern, however, was a finding of an 11% infection rate. Most
infections occurred in smokers. Despite the infection rate the authors concluded that
cement augmentation of the fixation for joint-depression calcaneal fractures allowed
earlier weight bearing with no change in postoperative outcomes.

Proximal humeral fracture augmentation has also been reported. Reinforcement with
CaPO cement in the treatment of proximal humeral fractures with locked plates
decreased fracture settling and significantly decreased intraarticular screw penetration
and helped to prevent varus deformation in clinical series. Augmentation was able to
decrease the tendency for subsequent superior screw cutout from the humerus in
biomechanical studies. 149150

This technique has fallen out of favor recently as the reports of screw cutout and
increased failure rates for proximal humeral locking plates have vastly increased.
Failure appears to be the result of varus reductions and inability to appropriately place
calcar support screws properly rather than the lack of bony support that cement
augmentation could help remedy. 151

Civinini et al. 152 reported on the treatment of patients with early-stage osteonecrosis of

the femoral head (ONFH) that underwent core decompression (CD), injection of
autologous bone marrow concentrate, and the use of a composite injectable bone
substitute CS and tricalcium phosphate as a mechanical supplementation associated
with decompression. At final follow-up the mean hip scores increased from 68 points
preoperatively to 86 points postoperatively and radiographic improvement occurred in
29 hips (78.4%). The overall clinical success rate of the procedure was 86.5%. The
authors thought that backfilling the defect with an injectable bioceramic for the
treatment of early stages of ONFH helped to relieve hip pain and prevent the
progression of ONFH in the majority of the cases. 152

Rouvillain and colleagues 153 reported on the clinical, radiologic, and histologic findings

after high tibial valgus osteotomy (HTVO) augmenting the distraction osteotomy with a
micromacroporous biphasic calcium phosphate (MBCP) wedge. This is one of the first
studies to examine the results by histologic analysis of the graft incorporation. 153 Forty-
three knees underwent clinical and radiologic follow-up at days 1, 90, and 365 to
evaluate consolidation and bone substitute interfaces. Biopsies were obtained at least 1
year after implantation from 10 patients who requested plate removal. Radiographic
consolidation was observed in 98% of patients with no late collapse noted. Histology
confirmed normal bony architecture with trabecular and/or dense lamellar bone growth
throughout the wedge implants. Radiograph and micro-CT scan revealed a well-
organized, mineralized structure in the newly formed bone. This study confirmed that
using MBCP wedges in combination locked plates offered a simple, safe, and fast
surgical technique for HTVO. 153
Early results have demonstrated that augmentation of femoral neck and
intertrochanteric hip fractures with CaPO cement is feasible, with no substantial
increase in complications. 154155

A prospective multicenter study evaluated clinical and radiologic results using an IM

implant with a perforated cephalomedullary device that offers an option for
standardized CaPO augmentation to provide more stability to the fracture fixation. The
mean follow-up time was 15 months and radiographs were analyzed for the cortical
thickness index, changes of the trabecular structure around the material, and the hip
joint space. There were no complications related to the cement augmentation, and no
complication was reported due to this procedure. The mean hip joint space did not
change significantly until follow-up and there were no signs of osteonecrosis in the
follow-up radiographs. Furthermore, no blade migration was seen or cutout visualized.

The authors concluded that standardized biologic augmentation of a cephalomedullary

device with a perforated blade was a safe method to treat trochanteric femoral fractures.
It leads to good functional results and was not associated with cartilage or bone
necrosis; however, it is only surmised that this type of technique will decrease the rates
of intertrochanteric fracture screw cutout and ultimately reduce the rates of failure for
unstable intertrochanteric fractures. 156

A recent meta-analysis was undertaken to evaluate this concept of hip fracture

augmentation and came to differing conclusions. In the meta-analysis 411 studies were
identified, of which 22 met the rigorous inclusion criteria, comprising 12 experimental
and 10 clinical reports. The clinical studies were evaluated with regard to their levels of
evidence. Only four were prospective and randomized. PMMA and CaPO cements
increased the primary stability of the implant-bone construct in all experimental and
clinical studies. In randomized, controlled studies, augmentation of intracapsular
fractures of the neck of the femur with CaPO cement was associated with poor long-term
results.

One study demonstrated that augmentation of the proximal femur did not improve
patients’ walking ability compared with the use of a nonaugmented device but might
have the potential to prevent reoperations by strengthening the osteosynthesis
construct. 157 There was a lack of data on the long-term outcome for trochanteric
fractures. Because there were only a few, randomized, controlled studies, there is
currently poor evidence for the use of any orthobiologic bone cement in the treatment of
fractures of the hip and should not be undertaken. 158

Investigators continue to manipulate these CaPO substrates to find the optimal

resorption/incorporation rate with the optimal strength ratios maintained. No authors
of human studies have been able to clearly demonstrate the actual resorption rate of
CaPO cement ( Fig. 5.7 ). However, the creation of macropores can significantly improve
the resorption rate of these materials. This increased degradation is associated with
almost complete bone replacement. Animal studies have shown that up to 80% of the
cement is resorbed at 10 weeks, with resorption and replacement with bone continuing
for as long as 30 weeks. 159160 This process occurs by dissolution as well as by
osteoclast resorption. Biomaterials depicted a significant increase in bone content,
compared with ABG, concerning bone regeneration over the same time frame. 161

Open full size image

Fig. 5.7
(A) Left, Split depression tibial plateau fracture with significant lateral compartment articular
impaction and comminution. Lateral articular surface was elevated and the subchondral defect
grafted with a particulate Ca phosphate/sulfate composite osteoconductive bone void filler to help
maintain articular reconstruction. Center,  To fill all cancellous voids, an injectable form of the same
material was injected, and after a short set period the plate-and-screw construct was applied drilling
directly through the material. Right,  This particular material allows instrumentation immediately after
the initial set time without degradation of the resulting crystalline structure of the material. Final
fixation radiograph demonstrating reconstructed surface with large void filled with the conductive
substrate. Note that a small particulate piece of material has egressed into joint. Because of the
particular material properties, this small amount dissolved in the synovial fluid environment and was
absorbed without any harmful articular sequelae. (B) Sequential images and CT scan to document
osseous incorporation of material over time. Initial postoperative radiograph and CT scan showing
material in place with articular reduction maintained and supported by the material. (C)
Postoperative images at 3 months. Articular surface reduction remains intact. Ca ceramic is noted to
be much smaller with enveloping bone around the material noted. No cyst formation is noted with
complete apposition of developing bone in direct contact with the material. (D) Postoperative images
at 6 months. With most of the material osteointegrated, the articular reduction has been maintained
and the patient has advanced to full weight bearing at approximately 2.5 months.
The lack of osteoprogenitor cells and osteoinductive potential of calcium-based bone
substitutes has led to the development of composite grafts in an attempt to accelerate
bone formation. A composite graft is created by adding an osteoinductive factor to an
osteoconductive CaPO matrix to theoretically increase bone formation. Many clinical
series use composite ceramic grafts combining the scaffolding properties of tricalcium
phosphate materials with biologic elements to stimulate cell proliferation and
differentiation. 162

As mentioned, the incorporation times of some of these materials can be prolonged due
to a number of factors. In an attempt to improve the incorporation and integration
characteristics, investigators combined ultraporous beta-tricalcium phosphate (TCP)
synthetic graft material (Vitoss; Orthovita) with bone marrow aspirates, with the
hypothesis that bone marrow aspirate speeds incorporation of the bone graft substitute.
The study prospectively examined healing of cavitary defects filled with TCP versus TCP
and bone marrow aspirate (TCP/BM). Although significant improvements in
radiographic parameters were observed in both TCP groups over 2 years of follow-up,
the addition of BM was not found to provide any significant benefit. 163 This highlights
the need for comparative data to determine the effectiveness of randomly combining
these adjuvants.

To improve the performance of CaPO materials, many investigators are manipulating

the materials themselves to improve the incorporation characteristics or the
osteoinductive capabilities of these materials. The inherent osteoinductivity of silicate-
substituted CaPO materials was investigated in a sheep muscle pouch study. Silicate
substitution had a significant effect on the formation of bone both within the implant
and on the implant surface during the study period. The formation of bone within
muscle during the 12-week period showed both silicate-substituted CaPO and
stoichiometric CaPO to be osteoinductive in this model. Silicate substitution
significantly increased the amount of bone that formed and the amount of bone attached
to the implant surface. New bone formation occurred through an intramembranous
process within the implant structure. 164

Similar findings were reported using a novel ceramic bone substitute that resembles the
autologous bone behavior when used as graft material. This silicated material was
composed of β-tricalcium phosphate (β-TCP) and a BCP with silicocarnotite (CaPO
silicate mineral). A rabbit bone defect model was used to assay the bone regenerative
capabilities and degradation times of the material. The novel BCP induced the filling of
about 73% of the bone defect with a newly formed bone tissue and an almost complete
degradation after 12 weeks of healing. This material underwent cytocompatibility testing
with human osteoblast cell lines and revealed significantly enhanced cell proliferation
on the silicated ceramic. 165

This novel ceramic with silicate is also known as bioglass and appears to induce an
osteo inductive response, as well as demonstrate enhanced conductive substrate
characteristics. This is an area of current investigation and the exact mechanism for this
material's enhanced bone-forming properties is not fully known. However, it resembles
the autologous bone properties of complete degradation and efficient enhancement of
bone formation, making it promising as bone graft material.

Recent research has found that these bioactive bioglass composites may be an ideal
delivery vehicle for antibiotics and/or growth factors. These materials demonstrate a
sophisticated interconnected pore structure, to which human bone marrow–derived
MSCs can attach, spread, and proliferate, promoting upregulation of the expression of
osteogenic markers. Thus these bioglass scaffolds exhibit better cytocompatibility and
osteoblastic differentiation properties compared with pure CaPO scaffolds.

Vancomycin-loaded silicone scaffolds have been found to yield sustained release that
lasts for more than 8 weeks in vitro. In vitro experiments demonstrated that loading
vancomycin onto the scaffold promoted antibacterial activity and inhibited biofilm
formation without deleterious effects on surrounding cells. These composite bioglass
materials may have a myriad of potential applications as these materials continue to be
developed. 166

Demineralized Bone Matrix

Marshall R. Urist described the induction principle in the 1960s. 13 The concept of bone
matrix (DBM) is that this material contains properties that can induce de novo new
bone formation when implanted into an extraskeletal site. It should be noted that this
response is seen in animal models but has not been demonstrated in humans.

As noted, Urist and colleagues soon identified a protein that they named bone
morphogenetic protein (BMP). 13141520212223 Protein extracts derived from bone can
initiate the process that begins with cartilage formation and ends in de novo bone
formation. The critical components of this extract (BMP) that direct cartilage and bone
formation, as well as the constitutive elements supplied by the animal during this
process, have long remained unclear. Amino acid sequence has been derived from a
highly purified preparation of BMP from bovine bone. Now, human complementary
DNA clones corresponding to three polypeptides present in this BMP preparation have
been isolated, and expressions of the recombinant human proteins have been
obtained. 1415

Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of
inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and
BMP-3) are new members of the TGF-β supergene family, and the third, BMP-1,
appears to be a novel regulatory molecule. BMP factors can be synthesized by
recombinant gene technology or derived from autologous bone, allogeneic bone, or
DBM. 141516172223

DBM is allogeneic bone that has undergone the acid extraction of the mineralized ECM
of the allograft bone. In theory, the noncollagenous proteins, including osteoinductive
proteins such as the BMPs, remain viable while the structural portion of the allograft
has been removed. 167 It contains type-1 collagen, noncollagenous proteins, and
osteoinductive growth factors, 168 including the BMPs and other inductive factors found
in the TGF-β group of proteins. As noted, the TGF-β superfamily includes a number of
factors in addition to the BMPs. The factors that are known to be osteoinductive are the
BMPs, GDFs, and possibly TGF-βs 1, 2, and 3. 169 DBM is highly osteoconductive due to
its particulate nature and presents a large surface area and three-dimensional
architecture to serve as a site of cellular attachment. 170171 Thus when DBM is
implanted in an animal, all of these factors potentially work in combination to produce
the observed osteogenic response.

However, because the true test of osteoinductivity is whether a material that has been
implanted in a nonosseous site forms bone, the inability of allograft bone to do this in
human patients argues against allograft bone having substantial osteoinductive activity.
DBM has been shown to produce this effect in animal studies, 172173174 but it too has
never demonstrated this effect in human patients. The relative quantities of BMPs in
DBMs are low, on the order of 1 × 10 −9 g of BMP per gram of DBM. The osteoinductive
variability has been found not only across different DBM products but also among
production lots from the same DBM formulation. 175

Bae et al. 176 demonstrated that there is higher variability in concentration of BMPs

among different lots of the same DBM formulation than among different DBM
formulations. Each individual lot has a different osteoinductive capacity that can vary
considerably among different donors. This variability questions DBM products’
reliability and, possibly, efficacy in providing consistent osteoinduction. 176

Prior investigations have shown that the osteoinductive potential can vary widely, with
influence from both donor and processing sources. There appears to be a sex-related
difference in donor DBM. DBM derived from female donors appears to have
significantly greater concentrations of BMP-2 and BMP-7 than that derived from male
donors ( P = 0.0257 and 0.0245, respectively). There was no significant correlation
between donor age and the levels of any of the measured BMPs in a study evaluating
BMP levels in commercially available DBM preparations. 177

The presence of other growth factors found in DBM has also been evaluated. Wildemann
et al. 178 attempted to quantify the activity and presence of eight growth factors important
for bone healing in multiple different “off the shelf” DBM formulations, which are
already in human use. Differences between the products were seen in total protein
content and the absolute growth factor values. The type of growth factors found in these
preparations was almost comparable between the materials. FGF and BMP-4 were not
detectable in any analyzed sample. BMP-2 revealed the highest concentration
extractable from the samples without a significant difference between the three DBM
formulations.

In general all the materials had variable concentrations of TGF-β1, FGFa, IGF-I, and
PDGF. No differences were accessed for VEGF. This and other similar studies highlight
the differences in the growth factor concentrations between the individual materials,
independent of the product formulation. There is evidence of differential potencies of
each available growth factor within individual DBM preparations, based on the
individual manufacturer and manufacturing process. 62
There are numerous DBM formulations based on refinements of the manufacturing
process. They are available as freeze-dried powder, granules, gel, putty, or strips. They
have also been developed as combination products with other materials such as
allogeneic bone chips and CS granules. Sterile processing of the bone may also affect the
protein effectiveness of these materials. Some tissue banks harvest and process the DBM
under “sterile” conditions, whereas other manufacturers sterilize the DBM after
processing. Gamma irradiation is frequently used to sterilize implanted devices but has
limitations when used on biologically active materials and composites.

Studies indicate that if the DBM is irradiated before the addition of any aqueous carrier,
the activity of DBM in the dry state remains relatively stable with only a small loss of
activity. Composites of DBM with a carrier such as lecithin, to which no water has been
added, lose activity at approximately the same rate as DBM in the dry form. In
composites that contain water, the loss of activity occurs even at much lower levels of
radiation exposure. Osteoinductivity of DBM decreased with the increase of gamma
irradiation dose at ambient temperature, whereas no decrease occurred when treated
with gamma irradiation at low temperature. However, the hydrated DBM showed
diminishing osteoinductivity after 6-month storage at ambient condition, whereas the
DBM in dry form retained its osteoinductivity after the 6-month storage. The findings in
this study indicate that DBM and DBM/AM composites could retain their
osteoinductivity when they are in dry configuration and are irradiated at low
temperature (−40°C [approximately −70°C]). 179

Gamma irradiation does not change cell attachment to the DBM but has an influence on
both stem cell and osteoprecursor cell proliferation rates. Because of the limitations
imposed by radiation, it seems most practical to handle DBM aseptically throughout the
procedures of compositing pastes, putties, or suspensions, and only if necessary,
exposing bone components to radiation sterilization before mixing. 179180

In addition, many of the proteins responsible for “turning on” the differentiation
process for stem cells will not function if damaged, and protein preservation is therefore
important in the delivery of DBM and BMPs. Some DBM products are lyophilized and
mixed with carrier at the time of implantation, whereas others are prehydrated or
premixed and could potentially remain on the shelf for a long period of time. 181 If precise
control of conditions is not maintained, many proteins might be susceptible to chemical
and physical degradation. 182

Ethylene oxide as a means for sterilization of DBM is used by some manufacturers and
has been shown to attenuate its osteoinductive potential. 183 Exposure of DBM to
ethylene oxide for the duration required to kill most common bacterial pathogens
results in a marked reduction of its osteoinductivity, most likely due to destruction of
BMPs and other inductive factors. 184 The effect of ethylene oxide has been shown to be
dose dependent. 185

Processing and sterilization techniques have significant effects on DBM viability. With
this in mind clinicians should be aware of how these factors influence the efficacy of
each particular DBM product that they may choose to use in each clinical application.
The shelf life of the product may also vary with the specific carrier, which may or may
not affect the overall activity of the product. All available in vitro assays evaluate the
DBM without the carrier added by the manufacturers. Carriers include glycerol
(Grafton; Medtronic, Minneapolis, MN), synthetic polymer (Dynagraft; GenSci
OrthoBiologics, Inc., Irvine, CA), porcine gel (Osteofil; Regeneration Technologies, Inc.,
Alachua, FL), and carboxymethylcellulose (Allomatrix; Wright Medical Technology,
Inc., Arlington, TN), as well as many other carrier technologies. DBM is also available in
a particulate powder form that has no carrier material present. This form is useful to
admix to other materials such as iliac aspirate, autograft, and platelet gels ( Fig. 5.8 ).
Bioactivity can be measured by in vivo assays (which measure de novo bone formation
and alkaline phosphatase levels) and in vitro assays (which measure stimulation of
human osteoblast culture). 185186187
Open full size image
Fig. 5.8
(A) Demineralized bone matrix (DBM) can be delivered in many forms. Top left, Particulate DBM is a
powder without any carrier. Bottom left,  A DBM composite putty is infiltrated with a carrier that allows
the material to be applied directly or injected . Right, Demineralized cancellous chips can be
infiltrated with concentrated marrow or platelet concentrate as seen here. The BMAC is admixed
with DBM plus chips to form a composite bone graft material. This graft has a putty-like consistency
and is implanted into the nonunion or fracture site through an open surgical procedure. (B) A graft
chamber is filled with particulate DBM as well as demineralized cancellous chips. This chamber is
then loaded with other adjuvants to facilitate delivery of the DBM graft. (C) This graft chamber was
injected with concentrated marrow elements. This allowed delivery of a graft with significant
conductive properties replete with interconnecting pores and presents a tremendous surface area
(DBM) as a site for potential marrow cellular attachment.
Only a few tissue banks use an in vivo model to measure the bioactivity of the final DBM
product with the carrier. A study by Han et al. 181 evaluated the effects of moisture from
water-based carriers and the storage temperatures on osteoinductivity of known DBM
products. This was done to evaluate these materials with regard to shelf life in an
operating room. In a dry state, without a carrier, DBM can preserve its osteoinductive
activity when temperatures reached 65°C, but in the presence of moisture (carrier
substances) the activity decreases with incubation time. Nearly 90% of the DBM activity
is lost when maintained for 5 weeks at 65°C. 181

Although the preclinical data are impressive for DBM forming de novo bone in lesser
animal models, 174 the human clinical data are deficient with only isolated case reports
and uncontrolled retrospective reviews. These Level III and IV studies have suggested
potential therapeutic effects of DBM. 188 The maxilla-craniofacial literature has published
the bulk of the literature documenting successful reconstruction of complex deformities
using DBM alone as a bone graft substitute. 189190191 Reported success rates for
mandibular and maxillary reconstructions are consistently more than 90%.

One of the first clinical series was published by Tiedeman and colleagues. They reported
on an uncontrolled case series of 48 patients in whom DBM had been used in
conjunction with bone marrow for the treatment of skeletal injuries. Thirty-nine
patients were available for follow-up, and 30 of them demonstrated healing. 192 These
results were encouraging; however, because there was no control group, the role of DBM
in patients who demonstrated healing remains unknown. Unfortunately, most clinical
series combined DBM with other adjuvants and, as noted, the singular effectiveness of
DBM alone is difficult to elucidate.

Two separate investigators evaluated the effectiveness of combining DBM with bone
marrow aspirate for the treatment of acute long bone fractures and nonunions. Wilkins
et al. 193 treated 66 patients with 69 “stiff” nonunions with a prospective protocol. The
only therapeutic intervention was the percutaneous administration of a mixture of
autologous bone marrow and allograft DBM on an outpatient basis. Sixty-one of the
percutaneous treatments (88%) resulted in union (Level III evidence). However, there
was no comparative control group other than historical controls with which to compare
the effectiveness of this composite graft. 193
Lindsey et al. 194 studied the effectiveness of a composite graft consisting of DBM putty
(Grafton DBM) and aspirated bone marrow for treating long bone acute fractures.
Patients were randomized to treatment with either the DBM putty composite or iliac
crest autograft, with a minimum of 12 months of radiographic follow-up. Ninety percent
of DBM patients achieved full bone formation compared with 75% of autograft patients
( P = 0.41). Additionally, all DBM patients were healed compared with 63% of autograft
patients ( P = 0.07). Both studies suggest that DBM putty enriched with bone marrow
may be comparable to autograft for treating long bone fractures and nonunions.

However, in the case of acute fractures the number of patients in each group was 10 or
less, and although this was a randomized prospective trial (Level I), the numbers are too
small to draw any definitive conclusions regarding this composite graft option.

The use of DBM for fractures and/or nonunions is best served as an adjuvant to expand
autograft or to use as a conductive substrate for BMAC (composite grafts are discussed
later).

Despite these study limitations, all suggest that DBM putty enriched with bone marrow
may be comparable to autograft for treating long bone fractures and nonunions. This
option offers the distinct advantages of decreased morbidity, reduced costs, and shorter
hospital stay compared with AICBG. 193

There is only one Level I randomized prospective study in humans available, evaluating
the use of DBM as a solitary graft material. This study used DBM alone to treat a
standardized critical-sized fibular defect, grafted using only DBM (positive control)
compared with a similar defect with no graft applied to negative (untreated) controls. 195

This was phase one of a study done in conjunction with a prospective, randomized,
double-blind study in 24 patients undergoing high tibial osteotomy to evaluate the
effectiveness of human recombinant osteogenic protein (OP-1) on a collagen type-I
carrier in a critically sized fibular defect (phase two). The results of phase one
established the critically sized nature of the defect. In the defect group with no graft
augmentation, no bony changes were observed while nonunion resulted in all of these
defects. In the group grafted with DBM, formation of new bone was visible from 6 weeks
onward with many defects undergoing complete healing. The results of the second phase
showed no significant formation of new bone in the presence of collagen graft alone,
whereas in the OP-1 group all patients except one showed formation of new bone from 6
weeks onward. This study demonstrated that both DBM and OP-1 exhibited osteogenic
activity in a validated critically sized human defect.

In a similar study, Hierholzer and colleagues 196 attempted to evaluate the healing of

humeral shaft nonunions using a consistent surgical protocol but compared the use of
two different types of bone graft—autologous ICBG and DBM—in a consecutive
retrospective cohort series. Forty-five patients were treated with AICBG and 33 grafted
with DBM only. Union was noted clinically and radiographically in 100% of the 45
patients treated with autologous bone graft and 97% (32) of the 33 patients treated with
DBM (not significant). The overall functional outcome did not differ between the
groups; however, 20 (44%) of the autologous bone graft recipients had donor site
morbidity, including prolonged pain in the majority and a superficial infection requiring
irrigation and débridement in one patient. These results demonstrated that consistent
healing could be achieved with commercially available DBM alone (Level III).

Delayed union and nonunion are common complications in atypical femoral fractures
despite having good fracture fixation. A prospective study was conducted on patients
with atypical femoral fractures using DBM compared with a retrospective consecutive
case series of similar patients treated without DBM. All patients were treated with the
same standard guidelines and followed up until fractures completely united.
Postoperative outcomes were then compared.

The DBM group demonstrated a significantly shorter healing time compared with the
controls. Delayed union was found in 4 patients (44%) in the DBM group compared with
7 patients (78%) in the control group ( P > 0.05). No statistical difference of nonunion
was demonstrated between both. The authors concluded that DBM is safe and effective
for accelerating fracture healing in patients with atypical femoral shaft fractures and
possibly reduces nonunion after fracture fixation. They also recommend this method for
enhancing fracture healing and in the treatment of atypical femoral shaft fractures
(Level IV). 197

Although AIBG remains the gold standard for spine fusion, harvesting morbidity has
prompted the search for alternatives, especially for multisegment fusion. Augmentation
of spinal fusion appears to be the most frequent application of DBM. In the more
challenging environment such as posterolateral spine fusion multiple formulations and
combinations have been utilized. 198 Significant differences exist between the various
forms of DBM in their ability to generate spine fusion in animal models. This must be
considered when analyzing the data from human studies.

To evaluate the efficacy of using DBM as a substitute of AIBG for long instrumented
posterolateral fusion (PLF; three-level fusion or greater), a total of 47 consecutive
patients underwent laminectomy decompression, and multilevel instrumented PLFs
were reviewed. Group 1 comprised 26 patients having DBM with autologous
laminectomy bone (ALB). Group 2 consisted of 21 patients having AIBG with ALB. The
fusion success was evaluated radiographically at 1 year.

Patients in group 1 (80.8%) and patients in group 2 (85.7%) were observed to achieve
solid bony fusion. There was no statistical difference in the fusion success ( P = 0.72).
Blood loss was significantly more in the autograft group 2 ( P = 0.02). The duration of
the hospital stays and operative times were also longer, but the difference was not
significant. The authors concluded that DBM combined with local laminectomy bone is
as effective as an AIBG with respect to long multisegment PLF success. They concluded
that DBM can be used as an effective bone graft substitute and may decrease morbidities
associated with iliac bone graft harvest (Level II). 199

In a side-by-side comparative study, a total of 120 patients underwent posterolateral

spine fusion with pedicle screw fixation and bone grafting. Iliac crest autograft was
implanted on one side of the spine and a Grafton DBM/autograft composite was
implanted on the contralateral side in the same patient. The bone graft mass was fused
in 42 cases (52%) on the Grafton DBM side and in 44 cases (54%) on the autograft side
(Level III). The authors concluded that DBM can extend a smaller quantity of autograft
than is normally required to achieve a solid spinal arthrodesis. Consequently, a reduced
amount of harvested autograft may be required, potentially diminishing the risk and
severity of donor site complications. 200 Thus the use of DBM as a bone graft extender
appears to be a viable indication for its use.

In a larger 2-year prospective study, a randomized clinical trial compared the outcomes
of Grafton DBM combined with local bone with that of ICBG in a single-level
instrumented posterior lumbar fusion. Patients were randomly assigned (2 : 1) to receive
Grafton DBM with local bone or autologous ICBG. At 2-year follow-up, subjects who
were randomized to Grafton Matrix and local bone achieved an 86% overall fusion rate
versus 92% (ICBG) ( P = 1.0; not significant) and improvements in clinical outcomes
that were comparable with those in the ICBG group. 201 There was a statistically
significant greater mean intraoperative blood loss in the ICBG group than in the Grafton
group ( P < 0.0031).

Anterior cervical discectomy and fusion (ACDF) is one of the most widely used
procedures in cervical spine. A systematic search of the literature was conducted
evaluating patient-directed outcomes specifically evaluating the use of DBM as the sole
agent for fusion augmentation. Secondary outcome measures were fusion rate,
nonunion, subsidence, collapse, displacement, spinal alignment, and reoperation.

Patient-reported outcomes were similar for DBM compared with the autograft and other
bone substitute materials. The DBM had a fusion rate comparable with other graft
materials, particularly in the long term (88.8% to 100%, after 18-month follow-up). The
majority of studies reported no collapse, subsidence, or displacement with DBM. The
revision surgery was mainly due to the symptomatic nonunion in 4.1% to 8.3% of the
DBM cases. Most of the studies reported similar results for DBM compared with
autograft and other graft substitute materials in terms of patient-reported outcomes,
fusion rate, and safety. However, the quantity and quality of evidence are limited, and
isolated use for fusion in the area must be cautiously approached. 202

A comprehensive meta-analysis reviewed the use of DBM for all spinal fusion series,
including cervical and lumber. Articles were critically examined and compared
according to study design, DBM type, outcomes, and results. Primary outcome of
interest was fusion rate. Secondary outcomes included Oswestry Disability Index; Short
Form-36 survey; Odom's criteria; Visual Analog Scale neurologic pain score; Japanese
Orthopedic Association myelopathy score; Neck Disability and Ishihara Curvature
Indices; and pseudoarthrosis and surgical failure rates. The majority of human clinical
trials report high fusion rates when DBM is employed as a graft extender or a graft
enhancer. Few prospective RCTs have been performed comparing DBM to autologous
ICBG in spine fusion. 203

Conclusion
Most of the studies reported noninferior results for DBM compared with autograft and
other graft substitute materials in terms of patient-reported outcomes, fusion rate, and
safety. However, the quantity and quality of evidence are limited, and use of DBM alone
cannot be recommended. The best evidence demonstrates its use as an autograft
extender or as a carrier for cellular therapies. It is not recommended that DBM be used
as a stand-alone graft.

Although many animal and human studies demonstrate comparable efficacy of DBM
when combined with autograft or compared with autograft alone, additional high-level
evidence studies are required to define the indications for its use in spine fusion
surgeries, acute fractures and nonunion management, and the appropriate patient
population that will benefit from DBM.

Patient-Derived Cellular Therapies

Current orthobiologic strategies have progressed from an approach based primarily on
biomaterials to a cell- and tissue-based approach that includes understanding of cell-
sourcing and bioactive stimuli. The need for suitable bone grafts is high; however, there
are limitations to all current graft sources, such as limited availability, the invasive
harvest procedure, insufficient osteoinductive properties, poor biocompatibility, ethical
problems, and degradation properties. The lack of osteoinductive properties is a
common problem.

As an allogeneic bone graft, DBM can overcome issues such as limited sources and
comorbidities caused by invasive harvest; however, DBM is not sufficiently
osteoinductive. Bone marrow has been known to magnify osteoinductive components
for bone reconstruction because it contains osteogenic cells and factors. MSCs derived
from bone marrow are the gold standard for cell seeding in tissue-engineered
biomaterials for bone repair, and these cells have demonstrated beneficial effects.

By providing a significant surface area and optimal pore size DBM/cellular concentrate
scaffolds possess a much higher enriching yield for osteogenic cells and factors
compared with DBM-alone scaffolds. At the same time, composites can build a cellular
microenvironment for cell adhesion, proliferation, and differentiation that promotes
bone reconstruction. As a result, a suitable bone graft fabricated using DBM/cellular
scaffolds and bone marrow represents a new strategy and product for bone
transplantation in the clinical setting. 204

New options include methods for harvest and transplantation of tissue-forming cells,
combined with bioactive scaffold matrix materials, and delivery of bioactive molecules
that allow these stem and progenitor cells to differentiate into the appropriate cell
lineage for specific tissue repair. 205206 Available cell-based strategies include
targeting local cells with use of scaffolds or bioactive factors, transplantation of
autogenous connective tissue progenitor (CTP) cells derived from bone marrow or other
tissues, and the use of autologous growth factors obtained from the patient's own
platelets.

Marrow Aspirate
The critical component necessary to all bone formation is the ability to provide viable
osteoprogenitor cells. Bone marrow is a plentiful source of musculoskeletal stem cells,
but the cells can also be found in periosteum, cartilage, muscle, fat, and vascular
pericytes. 207 CTPs describe the population of stem cells and progenitors that are actively
engaging in proliferation and differentiation into connective tissue. A bone marrow
aspirate has a high concentration of CTPs. One milliliter of iliac aspirate contains
approximately 40 million nucleated cells, 1500 of which are CTPs. 206207

Historical Perspective
Connolly's work with unfractionated bone marrow aspirate was instrumental in
stimulating clinical interest for using marrow as an adjunct graft material for fracture
and nonunion healing. His initial clinical series used autologous marrow injection to
stimulate healing in 20 ununited tibial fractures over a 5-year period. His injections
were combined with either the use of a cast (10 patients) or a Lottes nail (10
patients). 208 The two failures were in the cast treatment group. Bone marrow injection
was as effective as past open autologous grafting but with considerably fewer
disadvantages. These early results were not duplicated by others, but did stimulate
researchers to document enhanced bone healing through the use of marrow cell-based
strategies in vitro and in animal studies.

Connolly continued to investigate the effects of concentrating marrow by centrifugation

in a rabbit nonunion model. The results with centrifugation were superior to those with
unprocessed marrow, and thus the concept of a threshold. 208209210

Current Methodology
The failures and low rates of healing associated with the use of unfractionated bone
marrow may reflect the paucity of osteoprogenitor cells present in the mature marrow
aspirate. Even in a normal adult only 36 to 55 of every million nucleated bone marrow
cells will undergo osteogenic differentiation. 207 Because osseous regeneration is
dependent on the number of cells available to participate in bone synthesis, patients
with fewer local cellular precursors will typically have a poorer healing response leading
to potential nonunion. As greater volumes of bone marrow are harvested from the iliac
crest, the concentration of osteoprogenitor cells decreases because the sample
undergoes considerable dilution with peripheral blood.

The aspiration technique is very specific to maximize the number of effective progenitor
cells per unit volume. 211 Muschler et al. 207 studied this issue and determined that no
more than 2 mL of blood should be aspirated from any given area in the iliac crest to
avoid dilution with peripheral blood ( Fig. 5.9 ). On the basis of these data, a selective
retention (filtration) system was developed that has the ability to concentrate progenitor
cells three to four times and load them onto an allograft substrate for delivery. 212
Open full size image
Fig. 5.9
Many devices are available for marrow harvest. These all have large-bore needles with multiple side
ports and differing handle types to facilitate correct aspiration technique. From 3 to 4 mL should be
aspirated at any one location and as a single aspirate. The needle should be slowly withdrawn,
rotated, and reoriented to aspirate from a different location. Many aspiration syringes available can
be locked into position after small quantities are aspirated. This permits needle repositioning without
losing the suction of the plunger. After needle repositioning, the plunger can be unlocked and
additional aspirate obtained.
Other sites of harvest for marrow aspirate have been assessed to determine whether
there is variability of the quality of cells obtained compared with the results of aspirate
from the iliac crest. 213 A comparative study harvested bone marrow aspirates from the
ipsilateral anterior iliac crest, distal tibial metaphysis, and calcaneal body all from the
same patients. The samples were then centrifuged to obtain a concentrate of nucleated
cells, which were plated and grown in cell culture. The anatomic locations were
compared among the 40 patients enrolled in the study. Clinical parameters (including
sex, age, tobacco use, body mass index, and diabetes) were assessed as possible
predictors of osteoblastic progenitor cell yield. Bone marrow aspirate collected from the
iliac crest had a higher mean concentration of osteoblastic progenitor cells compared
with the distal tibia or the calcaneus ( P = 0.0001). There was no significant difference
in concentration between the tibia and the calcaneus ( P = 0.063). Age, sex, tobacco use,
and diabetes were not predictive of osteoblastic progenitor cell yield. 213

Similar comparisons evaluated aspirates of vertebral body marrow compared with

matched controls from the iliac crest of the same patient. Vertebral body aspirates
demonstrated comparable or greater concentrations of progenitor cells compared with
matched controls from the iliac crest. The concentration of osteogenic progenitor cells
was, on average, 71% higher in the vertebral aspirates than in the paired iliac crest
samples ( P = 0.05). 213214

However, the concentration and yield of colony-forming connective-tissue progenitors

are greater when aspirate is obtained from the posterior crest compared with the
anterior iliac crest. It has also been found that the biologic potential of the cells derived
from both of these sites appears to be comparable. 215

Cellularity of bone marrow has been found to decrease with age and for
women. 206216 Surprisingly, there appears to be no relationship between smoking
status and marrow cellularity, cellular prevalence, or cellular numbers. Studies have
found that tobacco use is not associated with a change in prevalence of osteogenic
progenitor cells in bone marrow or their intrinsic biologic capacity to undergo early
osteoblastic differentiation. 206216

Preclinical Substantiation
Many investigators reported enhanced bone healing through the use of cell-based
strategies in vitro and in multiple preclinical animal studies. In addition to the rabbit
model that Connolly used to evaluated the beneficial effects of cellular concentration, a
similar study was performed using a canine tibial nonunion model. Distraction gaps
were maintained with external fixators and were treated with either a bone graft of
concentrated marrow aspirate, or DBM, or a composite graft of both materials. 217 A
separate group treated with autograft was used as the control group. Use of the
combination of DBM and marrow concentrate (composite graft) yielded results that
were superior to DBM or aspirate groups alone. The results of the composite graft were
similar to those in the autograft group.

The concept of composite grafts combining marrow elements with other conductive
and/or inductive substrates has become a major area of interest based on the early
results documenting the superiority of composite grafts compared with grafting with
marrow aspirates alone. The ability to load cells into an osteoconductive substrate with
a microporous structure provides the cells with a potentially stable and well-
vascularized environment. 217218 In this situation most of these cells will differentiate
into osteoblasts, whereas in sites that are mechanically unstable and less well
vascularized, the cells tend to become chondrocytes. 219 If conditions are not right for the
optimal differentiation of these MSCs into bone or cartilage cells, they will differentiate
along a default pathway and become fibroblasts, 220 and when this occurs, nonunion
results. Thus the emphasis is on providing the optimal conditions for these composite
grafts.
Bruder et al. 221 evaluated bone marrow combined with a porous tricalcium phosphate
cylinder in a canine nonunion model stabilized with plates. Use of the composite graft
demonstrated results that were superior to those of treatment with the ceramic cylinders
alone, which resulted in only modest bone formation.

Bruder continued to investigate marrow elements in a composite graft using a selective

retention filtration strategy to concentrate marrow elements. In a canine model selective
retention cellular aspirate concentrates were mixed with DBM and were found to be
equivalent to autograft in their ability to repair critical-sized defects. 222 Additional
studies using the concept of cellular concentrates enriching a DBM carrier matrix in a
canine spine fusion model demonstrated similar results. The selective cellular
concentration process allows the number of CTPs to be increased threefold to fourfold.

In a canine study by Muschler et al., 212 enriched aspirate composite DBM grafts

delivered a mean of 2.3 times more cells and approximately 5.6 times more progenitors
than DBM mixed with simple unconcentrated bone marrow. A union score, quantitative
CT, and mechanical testing results all demonstrated the use of the selective-retention–
enriched bone DBM to be superior to the use of DBM alone and DBM plus
unconcentrated marrow. 212

Composite grafts have also been devised using cellular elements in addition to
osteoinductive proteins. Lane et al. 223 investigated the potential of combining bone
marrow cells with recombinant human BMP (rh-BMP-2) in a rat femoral defect model.
This combination was superior to either rh-BMP-2 or marrow cells by themselves, as
well as to treatment with syngeneic bone grafting. The authors believed that this
represented a synergistic effect of the two materials and emphasized the importance of
growth factors being present.

Animal models have been used to evaluate composite grafts that incorporate all three
components (i.e., cellular concentrate, a conductive substrate, and inductive proteins) in
varying combinations. A preclinical defect study evaluated a sheep tibial defect treated
with hydroxyapatite combined with either rh-BMP-7 or bone marrow compared with
autograft application. 214 Treatment with the composite grafts yielded results that were as
good as those in an autograft control group and were superior to those in either a void
group or a group treated with hydroxyapatite alone.

With the vast improvements in limb salvage techniques the reconstruction of large
defects in humans has become more commonplace and has historically relied on
autograft bone grafts. Limiting factors include availability of graft material, comorbidity,
and insufficient integration into the damaged bone. Complex animal models now
routinely evaluate hard and soft tissue scaffoldings in combination with a multitude of
inductive materials, as well as applied cellular concentrates, in an attempt to determine
the optimum cocktail that can compare to the gold standard of AICBG for the treatment
of segmental defects. 224225226

Clinical Application of Marrow Elements

 Marrow elements have been used clinically to augment bone healing with variable
results. Most of the early series involved the simple aspiration and injection of iliac crest
aspirates as noted previously in this chapter documenting the work of
Connolly, 208209210227 one of the first surgeons to report on the use of bone marrow
aspirate as a clinical alternative to autograft (Level IV). Other early case studies or series
document results for simple marrow injections. These studies were primarily single or
multiple surgeon with no historic or case-matched controls (Level IV). These studies
include Garg et al., 228 who reported good results in a series of 20 patients treated with
bone marrow injection for long bone nonunion.

Healey et al. 229 successfully treated nonunions in a group of eight children with cancer

by simply injecting bone marrow aspirate. Wientroub et al. 230 reported on the use of
autologous marrow to improve the effectiveness of allografts in 23 children. Goel
et al. 231 sequentially injected 3 to 5 mL of aspirated bone marrow directly into the
nonunion site. Multiple iliac crest aspirations were performed until a maximum of
15 mL of marrow was injected. Clinical and radiologic bone union occurred in 15 of 20
patients (75%).

These studies were performed without the benefit of our current knowledge regarding
appropriate aspiration techniques, the advantage of cellular concentrates, or the use of
composite grafting methods, yet meaningful results were achieved.

In spite of the encouraging preclinical and rudimentary clinical studies, the use of these
grafts as a substitute have not enjoyed widespread use. This may be the result of a host
of reasons:

 1. 

The variability resulting from inconsistent and incorrect aspiration techniques and
faulty instrumentation necessary to achieve consistent aspirates

 2. 

Low osteoprogenitor content of these insufficient aspirates

 3. 

The combination of these aspirates’ with suboptimal scaffolding materials 232

The correct aspiration technique is described next and requires multiple limited-
quantity aspirates through multiple trajectories and needle reorientation.

Iliac Crest Marrow Aspiration Technique

The aspiration of 80 to 100 mL of marrow from the iliac crest is performed, using a
sequential technique. A small incision is made over the iliac crest. The superior table is
identified and a 2-mm drill used to broach the hard-dorsal cortex. A multiport large-
bore aspiration trochar/needle is inserted between the inner and outer iliac tables
(see Fig. 5.9 ). Aspiration is initiated obtaining a small amount of aspirate (per the
technique of Muschler).

The needle is then withdrawn and rotated to aspirate a “new” region of cells. This is
repeated multiple times until the needle is withdrawn. A second or third drill hole may
be required to obtain the volume of aspirate necessary ( Fig. 5.10 ). The quantity of
aspirate harvested is dependent on the desired concentration and amount required for
each specific indication. This may also vary with the particular concentrating device
used to produce the concentrate. Repeat aspiration via different trajectories is done to
obtain the highest possible cell counts without venus blood dilution before processing.

Open full size image

Fig. 5.10
The orientation of the aspiration needle is directed down between the two iliac tables. The trajectory
is changed frequently to harvest from a large area, all through single drill holes in the superior table
of the iliac crest.
This material is then transferred to the concentrating device, which then removes the
red blood cell fractions and plasma. A substantial volume reduction occurs with most
devices and achieves typical total nucleated cells (TNCs) concentration factors that are
three to six times the baseline TNC concentration in bone marrow aspirate. 233

Contemporary clinical series have demonstrated excellent results when these factors
have been attended to. Studies that use correct aspiration and concentration
methodologies and adhere to appropriate documented composite grafting techniques
document the value of marrow aspirates for graft substitution. Thus the efficacy of this
technique appears to be related to the number of progenitors present in the graft and
highlights the need to concentrate the aspirates and achieve the baseline number of cells
necessary to achieve osteogenesis (Level III) 234235236237238 ( Fig. 5.11 ).
Open full size image
Fig. 5.11
(A) At 10 months post-IM nailing for grade 3 open tibia. Large fracture gap with no evidence of
healing. (B) At revision surgery the nail was removed with takedown of the nonunion site application
of BMAC directly into the compressed nonunion gap through the trocar, and the plate was applied
and augmented with composite grafting as well (BMAC + DBM). (C) Complete healing of the
nonunion achieved by 5 months. Follow-up radiographs demonstrate excellent remodeling and
absence of the fracture site at 17 months after surgery.
Open full size image

Open full size image

The concept of composite grafts combining marrow elements with other conductive
and/or inductive substrates has become a major area of interest based on the results
documenting the superiority of composite grafts compared with grafting with marrow
aspirates alone. 239240241 The ability to load cells onto an osteoconductive substrate
with a microporous structure provides the cells with a potentially stable and well-
vascularized environment. In this situation most of these cells will differentiate into
osteoblasts. If conditions are not right for the optimal differentiation of these MSCs into
bone or cartilage cells, they will differentiate along a default pathway and become
fibroblasts, 239 and nonunion may result.
The use of BMAC in conjunction with specific osteoconductive substrates appears to be
a suitable substitution for AICBG when used in specific situations. Many materials have
been used as conductive carriers for concentrated bone marrow aspirate materials. Once
the marrow elements have been concentrated and removed from the specific device,
they are admixed to the carrier of choice and then implanted much as one would
perform an AICBG procedure. In some case series these materials are delivered via a
percutaneous injection.

Collagen sponges, hydroxyapatite substrates, and other porous ceramics, as well as

particulate DBM, have all been used in combination with BMAC to fabricate “composite
grafts.” The use of BMAC in conjunction with specific osteoconductive substrates
appears to be a suitable substitution for AICBG when used in specific situations.
Multiple Level IV studies have documented the effectiveness of this modality for a
variety of indications. It has been used primarily for defect management found in
posttraumatic long bone defects and nonunion management. 242243244245

A new area of clinical use for BMAC is for the treatment of avascular necrosis (AVN) of
the femoral head, as promising results have been seen after grafting using BMAC with a
variety of carrier matrices. 237238244245

Studies that use correct aspiration and concentration methodologies and adhere to
appropriate documented composite grafting techniques document the value of marrow
aspirates for graft substitution. Thus the efficacy of this technique appears to be related
to the number of progenitors present in the graft and highlights the need to concentrate
the aspirates and achieve the baseline number of cells necessary to achieve osteogenesis.

Hernigou and colleagues 244 reported on 60 patients with a noninfected nonunion who

had undergone bone marrow aspiration from both iliac crests. These aspirates were then
concentrated using a centrifuge technique to achieve a baseline number of colony-
forming units present in the composite graft to be injected into the nonunion site.
Complete healing occurred in 53 of the 60 patients. Those patients contained more than
1500 progenitors/cm 3 and an average total of 54,962 ± 17,431 progenitors. The
concentration (634 ± 187 progenitors/cm 3 ) and the total number (19,324 ± 6843) of
progenitors injected into the nonunion sites of the seven patients in whom union was
not obtained were both significantly lower ( P = 0.001 and P < 0.01, respectively) than
those in the patients who healed. There was a high correlation between the volume of
mineralized callus at 4 months and the number and concentration of colony-forming
units available at the time of implantation. The seven patients in whom the fracture did
not unite had lower numbers and concentrations of colony-forming units. The efficacy of this
technique appears to be related to the number of progenitors available in the graft and
highlights the need to concentrate the aspirates and achieve the baseline number of cells
necessary to achieve osteogenesis (Level III). 244245246 However, there is a lack of
prospective randomized studies including controls for cell therapy for bone defects.
Additionally the ultimate threshold concentration of cells necessary to promote
osteogenesis is not known at this time. 206207212214 Based on current concepts,
concentrations of three to four times baseline are necessary for competent graft
materials. 246
Clinical Results
A randomized prospective spine fusion study evaluated PLF patients. After PLF
instrumentation, approximately half the patients were randomized to be grafted with
allograft alone, and the other group randomized to BMAC together with allograft (Level
I). Statistically significant difference between both groups was proven for CT evidence of
ongoing fusion at 12 months ( P = 0.041) and at 24 months ( P = 0.011), with the BMAC
group a significantly better alternative for the PLF than the allograft alone. The authors
state that the use of autologous MSCs in the form of the BMAC in combination with
allograft was an effective option to enhance the PLF healing. 247

A separate randomized study using contemporary BMAC concepts was completed for
the treatment of posttraumatic bone defects. Thirty-nine patients with critical bone
deficiencies were treated with BMAC in a prospective clinical trial. A collagen sponge
(Col) served as a scaffold in 12 patients and a bovine hydroxyapatite substrate was
applied in the other 27 individuals for the composite graft applications. All patients
showed new bone formation in radiographs during follow-up. Complete bone healing
was achieved in the hydroxyapatite group after 17.3 weeks compared with 22.4 weeks in
the Col group. The average concentration factor of BMAC was 5.2 (SD 1.3). Flow
cytometry confirmed the mesenchymal nature of the cells 248 (Level II). This excellent
study documented the value of cellular concentration (BMAC), as well as the importance
of a specific conductive substrate used as a competent cellular carrier. 249250

The application of BMAC has increasingly been used as a regenerative therapy for
musculoskeletal pathologic conditions despite limited evidence-based support. BMAC
has demonstrated promising results in the clinical application for repair of chondral
defects as an adjuvant procedure or as an independent management technique. A
subcomponent of BMAC, bone marrow–derived MSCs possess the ability to
differentiate into cells important for osteogenesis and chondrogenesis. Modulation of
paracrine signaling is perhaps the most important function of BM-MSCs in this setting.

Many current studies are lacking in numbers, but early data suggest that this concept
may have value. In a recent prospective, single-blind, placebo-controlled trial, 251 patients
with bilateral knee pain from bilateral osteoarthritis were randomized to receive BMAC
into one knee and saline placebo into the other. Fifty-two milliliters of bone marrow was
aspirated from the iliac crests and concentrated in an automated centrifuge. The
resulting BMAC was combined with platelet-poor plasma for an injection into the
arthritic knee and was compared with a saline injection into the contralateral knee as a
control. Pain relief and function as measured by Osteoarthritis Research Society
International (OARSI) measures and the visual analog scale (VAS) score were tracked
initially at 1 week, 3 months, and 6 months after the procedure. There were no serious
adverse events from the BMAC procedure. OARSI, Intermittent and Constant
Osteoarthritis Pain, and VAS pain scores in both knees decreased significantly from
baseline at 1 week, 3 months, and 6 months ( P ≤ 0.019 for all). Pain relief, although
dramatic, did not differ significantly between treated knees ( P > 0.09 for all).
Early results show that BMAC is safe to use and is a reliable and viable cellular product.
No adverse events resulted from the injections. However, both BMAC- and saline-
treated arthritic knees experienced similar degrees of pain relief. 251

Another study evaluated the safety of autologous bone marrow mesenchymal stem cells
(BM-MSCs) expanded in vitro and given intraarticularly to patients with stage II and III
knee osteoarthritis. As a secondary end point, the study evaluated the ability of these
cells to relieve symptoms and restore the knee cartilage as judged by normalized Knee
Injury and Osteoarthritis Outcome Score (KOOS) and by magnetic resonance imaging
(MRI).

Patients were given two doses of BM-MSCs 1 month apart by intraarticular injection in a
prospective clinical trial. Each patient was followed for a minimum of 24 months for any
adverse events and for clinical outcome using normalized KOOS. Cartilage thickness
was assessed by quantitative MRI T2 at 12 months of follow-up.

Results were much more refined than the previous study reviewed. At 24 months, no
serio us adverse events had been reported. Normalized KOOS improved significantly.
Most impressively, mean knee cartilage thickness measured by MRI improved
significantly.

The authors of this prospective protocol determined that BM-MSCs given

intraarticularly were safe for knee osteoarthrosis. The procedure described significantly
improved the KOOS and knee cartilage thickness, indicating that they may enhance the
functional outcome as well as the structural component. 252

The equivocal results noted previously are also confirmed in well-controlled meta-
analyses. A systematic review of the literature was performed in 2016 using the
Cochrane Database of Systematic Reviews, the Cochrane Central Register of Controlled
Trials, PubMed, and MEDLINE from 1980 to present. Three studies investigated the
clinical efficacy of BMAC in the treatment of osteoarthritis, and eight studies evaluated
the efficacy of BMAC on focal cartilage injuries. All three studies regarding osteoarthritis
and all eight studies regarding focal chondral defects reported good to excellent overall
outcomes with the use of BMAC.

The conclusion was that there remains a paucity of high-quality studies. The studies
included in this systematic review reported varying degrees of beneficial results with the
use of BMAC with and without an additional procedure for the treatment of chondral
defects and early stages of osteoarthritis. Most articles present the use of BMAC as a safe
procedure and report good results, 253 but no definitive recommendations could be made
for their routine use.

As more information has been published on the use of BMAC for knee arthritis, a follow-
up meta-analysis in 2017 reported on five RCTs and one non-RCT. Bone marrow–
derived stem cells, adipose-derived MSCs, and peripheral blood stem cells were used. In
these studies, all trials were at high risk of bias, resulting in Level III evidence. All five
RCTs reported superior efficacy for patient-reported outcomes compared with controls
at final follow-up (range 24 to 48 months). Superior radiologic outcomes were found
 favoring stem cell injection. Superior histologic outcomes and/or improved
arthroscopically scored healing rates were reported in two trials. No serious adverse
events were reported.

Most of the studies were heavily biased and demonstrated only Level III or IV evidence
supporting the use of stem cell injections for knee arthritis. The authors concluded that
in the absence of high-level evidence, they did not recommend stem cell therapy for
knee arthritis. 254

Conclusion
The use of biologic agents in the treatment of knee pathologies has emerged as a
potential option. Despite the increasing use of biologic agents for knee pathology, there
are conflicting results on the efficacy of these products. Furthermore, strong data
supporting the optimal preparation methods and composition for widely used biologic
agents, such as PRP and BMAC, largely remain absent from the literature. 255 Further
study is required to determine the mechanisms of action, duration of efficacy, optimal
frequency of treatments, and regenerative potential.

Allograft Stem Cell Preparations

In an effort to avoid any marrow harvest and intraoperative graft manipulation, as well
as provide a consistent graft material, allogeneic human stem cells harvested from
cadaver donors are now available as a stem cell graft. The majority of technical
information is proprietary and not readily available with regard to graft specifics in
terms of harvesting, immunogenicity, and cell morphology. These “live” MSC allograft-
containing allogeneic bone grafts have recently gained popularity and currently account
for greater than 17% of all bone grafts and bone graft substitutes used in spinal surgery.
Although the claim of cellular bone matrices containing osteogenic cells with
osteoinductive properties is attractive, little is known about their clinical success. 256 No
supporting data have been published on their use for acute bone loss or nonunion
treatment.

The grafts are composed of viable MSCs derived from cadaveric donor tissue and are
delivered on an osteoconductive substrate (DBM). This graft has many theoretic
advantages over BMAC:

 1. 

The graft is “preconcentrated,” such that a threshold concentration of cells is present on

arrival, without having to manipulate the aspiration before implantation, as is done with
an autogenous BMAC harvest.

 2. 

The grafts are delivered preadmixed with a competent osteoconductive substrate with a
matrix designed for optimal cellular attachment and porosity. This matrix was designed
to encourage rapid vascular ingrowth with maximal cellular viability.
 3. 

The graft has osteogenic, osteoinductive, and osteoconductive properties and is capable
of directing new bone formation at the site of implantation. 257

The material appears promising; however, a paucity of clinical data are available
regarding its use. Most of the information available is limited to “commercial white
papers” (Level V), case reports (Level V), and uncontrolled case series (Level IV). 258
One of the largest uncontrolled retrospective studies reviewed the clinical effectiveness
of an MSC allograft to achieve radiologic arthrodesis in adult patients undergoing
lumbar interbody fusion surgery for a variety of different indications. Fifty-two
consecutive patients received lumbar interbody fusion at one (69%) or two contiguous
(31%) levels of lumbar spine for various indications. 259 Solid arthrodesis was achieved in
92.3% of patients at median follow-up time of 5 months, and the authors thought this
was a safe and effective graft alternative for adult patients undergoing lumbar interbody
spinal fusion procedures. This study has significant limitations with multiple levels
being treated, no control group, limited follow-up numbers, and a follow-up time of only
5 months.

Twenty-three patients were treated and results reported in a retrospective review of foot
and ankle patients using MSC allografts for the treatment of various nonunion
conditions (Level IV). This uncontrolled case review found that radiographic new bone
formation was observed at the area of implantation and a 91.3% union rate was
observed, and no evidence of graft rejection or complications associated with
implantation was noted. 257

More recent data have come to light evaluating these materials. A consecutive series of
57 patients who underwent a one- or two-level instrumented ACDF were retrospectively
analyzed. All fusion constructs comprised an interbody allograft, an anterior plate, and
an allograft stem cell preparation. These patients were matched to a control group of 57
patients.

At the 1-year follow-up, 87.7% patients in the allograft stem cell cohort demonstrated a
solid fusion compared with 94.7% in the control group. Among patients in the allograft
cohort, 12.3% were reported as having a failed fusion at 1 year. This was the first non–
industry-sponsored study to analyze a matched cohort assessing the 1-year arthrodesis
rates associated with a nonstructural MSC allograft in one- and two-level ACDF
procedures. Although not statistically significant, patients treated with MSC allografts
demonstrated lower fusion rates compared with a matched non-MSC cohort. 256

A similar retrospective review from 2017 evaluated instrumented ACDF using an

autograft substitute with instrumentation. There was no control comparative group,
however. Results were stratified as their subgroup analysis of subjects with risk factors
for pseudoarthrosis (current or former smokers, diabetic, or obese/extremely obese)
compared with those without risk factors demonstrated no significant differences in
fusion rates. The authors concluded that patients undergoing two-level ACDF with
Trinity Evolution® (TE) in combination with a polyetheretherketone plastic like
material (PEEK) interbody spacer and supplemental anterior fixation had a high rate of
fusion success without any serious adverse events related to the graft material. 260

With no Level I evidence currently available, and equivocal Level IV and V studies using
these materials, the routine use of this material cannot be recommended at this time.

A new area of clinical use for BMAC is for the treatment of AVN of the femoral head, as
results have been seen after grafting using BMAC with a variety of carrier
matrices. 237238

In the precollapse phase, CD, with or without the addition of bone marrow (e.g., BMAC)
or bone graft, is a common treatment alternative. A recent matched pair analysis was
carried out comparing patients treated with CD. All patients were divided into group A
(CD + BMAC) and group B (CD alone). The primary endpoint was a THA. In group A
two patients needed THA and in group B four patients were treated with THA. This
study demonstrates that CD in combination with the application of autologous BMAC
into the femoral head seems to be a safe and efficient treatment alternative in the early
stages of AVN of the femoral head compared with CD alone. 261

Platelet-Rich Plasma
After an acute fracture or an operative intervention, platelets are activated by thrombin
and subendothelial collagen. During activation, the alpha granules within platelets fuse
with the platelet plasma membrane and release some of their protein contents to the
surroundings (degranulation). The alpha granules in platelets contain more than 30
bioactive proteins, many of which have a fundamental role in hemostasis and/or tissue
healing. 3 These proteins include PDGF (including aa, bb, and ab isomers), TGF-β
(including β1 and β2 isomers), platelet factor 4, interleukin-1, platelet-derived
angiogenesis factor, VEGF, epidermal growth factor, platelet-derived endothelial growth
factor, epithelial growth factor, IGF, osteocalcin, osteonectin, fibrinogen, vitronectin,
fibronectin, and thrombospondin-1. 3 Platelets begin actively secreting these proteins
within 10 minutes after clotting, with more than 95% of the presynthesized growth
factors secreted within 1 hour. 262263264

Platelet-Derived Growth Factor

PDGF is the first growth factor to jump start nearly all wound healing. 264 When
activated, this growth factor attaches to transmembrane receptors on target cells,
including osteoblasts and fibroblasts. The main function of PDGF is stimulating cellular
replication (mitogenesis). This growth factor increases cell populations of healing cells,
including MSCs and osteoprogenitor cells. PDGF also activates macrophages, resulting
in débridement of the surgical or traumatic site. The macrophage activation then
triggers a second source of growth factors released from the host tissues under the
influence of the macrophage action. 265

Transforming Growth Factor–β

TGF-β stimulates the proliferation of osteoblast precursor cells, and it has direct
stimulatory effects on bone collagen synthesis. Therefore TGF-β modulates bone matrix
synthesis by increasing the number of cells capable of expressing the osteoblast
genotype, as well as direct upregulation of osteoblasts. TGF-β also decreases bone
resorption by inducing apoptosis of osteoclasts. 264 TGF-β activates fibroblasts to induce
collagen formation, endothelial cells for angiogenesis, chondroprogenitor cells for
cartilage, and mesenchymal cells in an effort to increase the population of wound
healing cells.

Thus platelet gels provide a rich source of growth factors that serve a critical function in
wound and fracture healing and can stimulate the formation of blood vessels; the
invasion of pluripotential MSCs, monocytes, and macrophages; and the further
aggregation of platelets. These factors have direct chemotactic and mitogenic effects on
osteoblasts and osteoblast precursors and act synergistically to help stimulate bone
remodeling and healing. 265266

Platelet-Rich Plasma Augmentation of Graft Materials

By providing a variety of concentrated growth factors, PRP can play an important role in
enhancing the bone healing pathways. It has been demonstrated that the treatment of
human mesenchymal stem cells (hMSCs) with clotted PRP, in an osteoinductive
environment, enhances osteoblastic commitment and bone formation. At the molecular
level, PRP treatment stimulates a transient enhancement of BMP-2 messenger RNA and
induces an earlier and a sustained increase in the key osteogenic transcription factor,
Runx2. PRP can enhance alkaline phosphatase activity more than twofold, and the
noted increase in osteoblastic commitment translates to enhanced calcified matrix
deposition. 266

Currently, it is common to combine the platelet-rich material with autograft, allograft,

DBM, or other graft material. In fact, PDGF release from platelet-rich concentrate was
markedly reduced in the presence of DBM. When PRP was applied in conjunction with
ABG, the rate of bone formation doubled and bone density increased by 25% compared
with controls. 263 However, data have been presented stating that PRP has limited or
even negative efficacy when combined with certain delivery vehicles. 267268

More recent data have shown the improved efficacy of platelet rich concentrate (PRC)
and bone graft materials on human bone marrow stromal cell activity, with bone
formation being significantly modified by adding the agents in combination. PRP was
combined with various bone graft materials such as DBM and autograft to evaluate the
effect of these factors on the hMSCs themselves.

Combining PRP with the graft materials increased cellular proliferation above that seen
with the graft materials alone; however, only DBM and allograft were capable of
increasing cellular proliferation above that seen with PRP alone. PRP increased
mineralization compared with DBM, collagen, or β-TCP alone. Compared with PRP
alone, addition of DBM or allograft decreased mineralization. Collagen gave rise to a
small increase in mineralization, whereas β-TCP yielded the same level of mineralization
as PRP alone.
The data obtained from these in vitro investigations demonstrate that the cellular
responses induced by PRC and bone graft materials in hMSCs can be significantly
(positively or negatively) modified by adding the agents in combination. These in vitro
data highlight the need to consider the potential interaction between biologic agents
when added in combination. 269 PRC is not a true osteogenic agent but rather is
osteopromotive, with cell fate determination being dependent on additional signals
derived from the microenvironment.

Preclinical Studies
The majority of early preclinical and basic science work regarding the use of PRC for
potential clinical applications has been encouraging and was primarily found in the
periodontal, oral surgery, and maxillofacial surgery literature. PRP has been suggested
for use to increase the rate of bone deposition and quality of bone regeneration when
augmenting sites before or in conjunction with dental implant placement. 270 The
orthopaedic focus has primarily been on the ability of PRP to augment bone formation
and healing in diabetic fracture healing models, wound healing, and defect
augmentation.

In a diabetic fracture model study, a significant reduction in PDGF, TGF-β1, IGF, and
VEGF expression was demonstrated in the diabetic fracture callus compared with the
nondiabetic fracture callus. 271 The application of PRP restored early cell proliferation
during healing to levels comparable to nondiabetic controls. Biomechanical testing
revealed improved fracture healing in platelet-rich treated diabetic fractures compared
with those in nontreated diabetic controls. 257

PRP delivery at the fracture site normalized the early (cellular proliferation and
chondrogenesis) parameters while improving the late (mechanical strength) parameters
of diabetic fracture healing. The biomechanical properties were only partially restored in
late diabetic fracture callus. These data are consistent with that seen clinically in
diabetic patients having improved healing and decreased complications after ankle
fusion when treated with PRP. 272 These results suggest a role for PRP in mediating
diabetic fracture healing and potentially other high-risk fractures. 257271272

Siebrecht and colleagues 273 demonstrated that PRP, prepared from human blood using a
commercially available platelet concentrate system, significantly increased bone and
total tissue ingrowth distance compared with untreated controls (porous
hydroxyapatite) in an athymic rat bone chamber model. The investigators theorized that
porous hydroxyapatite lacks the cells and growth factors present in bone graft, but that
PRC could potentially reverse this phenomenon. 273

Soft tissue effects of PRP were evaluated in a rabbit Achilles tendon injury model where
PRP was compared against saline injections to determine augmentation of healing that
the materials may facilitate. PRP seems to enhance neovascularization, which may
accelerate the healing process and promote scar tissue of better histologic quality
compared with the degree of healing (nonhealing) and scar quality found in the saline
injection group. 274 The authors thought that these results indicated that PRP may
promote tendon healing acceleration.
Platelet-Rich Plasma Preparation
In terms of processing, platelet collection should commence before surgery because
activity at the surgical site will initiate clotting, thereby reducing the systemic platelet
concentration. It is thought by some that even the initiation of an inhalation anesthetic
agent will initiate the activation of platelets. Until recently, centrifugation systems were
the primary method of producing a platelet-rich fraction. One of the disadvantages of
centrifugation is that it can lead to fragmentation and lysis of the platelets, which
triggers early release of growth factors and cytokines compromising
bioactivity. 275 Fragmentation and early activation have also been shown with previous
filtration-based systems that were either too vigorous or used pediatric dialysis filters in
combination with cell saver, resulting in significant platelet degranulation and
decreased efficacy. 276

Most systems in clinical use today use platelet-specific centrifugation devices or cell-
sensitive filtration devices to produce a platelet concentrate of sufficient concentration
to produce a physiologic effect. A PRP count of 1,000,000/mL as measured in a
standard 6-mL aliquot has become the benchmark for therapeutic PRC. 262 Both of these
separation technologies deliver intact platelets that then require some activation at the
time of delivery to the patient ( Fig. 5.12 ).
Open full size image
Fig. 5.12
Platelet concentrate centrifuge cell, demonstrating the multiple layers of materials obtained after the
platelet separation centrifugation process. This particular processing device now requires this
container to leave the sterile field to have the platelet-rich portion aspirated from this container
before its delivery to the patient.
The regenerative potential of PRP depends on the amount and viability of the inherent
growth factors present in the delivered materials. Platelet activation and growth factor
release can be accomplished by the addition of calcium or thrombin to the platelet
concentrate.

The exact role of thrombin in PRP has been debated. Thrombin and/or calcium chloride
is necessary to catalyze the conversion of fibrinogen to fibrin, but it also induces
platelets to secrete growth factors. Demineralized bone matrices supplemented with
PRP, with or without thrombin activation, were implanted intramuscularly in athymic
rats and were examined. The results of this study suggested that exogenous thrombin
activation of PRP may actually diminish its ability to induce bone formation compared
with non–thrombin-activated PRP. 277

Studies have typically shown a threefold to fourfold increase in growth factor

concentrations in PRP compared with nonconcentrated autologous blood. 278 Large
variations exist between the 40-plus commercially available systems; therefore each
system needs to be tested individually for growth factor and cytokine expression. The
final platelet concentration of any PRP product depends on a host of factors, including
the initial volume of whole blood to be processed and the platelet recovery efficiency of
the chosen technique. Dilution variables regarding the final volume of plasma used to
suspend the concentrated platelets, the relative concentration of white blood cells
(WBCs) and/or red blood cells in the suspension, and the concomitant use of thrombin
all affect the final product. 279280

The absence of a validated classification system that identifies crucial differences

between PRP formulations makes it difficult to compare acquisition systems or
comparative studies. 280 The caveat “buyer beware” should be strictly adhered to when
considering the PRP in terms of the device to be used. 240265

Clinical Evidence
The clinical use of PRP has been reported for a wide variety of clinical applications, most
predominantly for the problematic wound, maxillofacial applications, and spine.
Collectively, these studies provide variable support for the clinical use of PRP. However,
many reports are anecdotal, and few Level I studies with control group comparison are
available to definitively determine the role of PRP. There is little consensus regarding
the production and characterization of PRP, which can impede the establishment of
standards that are necessary to integrate the enormous amount of literature from
various studies on the subject.

Bone Applications
A spinal fusion study performed by Weiner and Walker 281 highlights the problems with
PRP studies and the issues that an inconsistent PRP product can have on study results.
A retrospective, consecutive series was performed to evaluate two groups undergoing
lumbar fusion: one with and one without autologous growth factors (AGF). AGF is a
product name given to a system of filtration devices to acquire and concentrate
autologous platelets. Two groups were studied. The control group consisted of 27
consecutive patients who underwent a single-level intertransverse lumbar fusion using
ICBG. The AGF group consisted of 32 consecutive patients undergoing an identical
procedure for the same indications with ICBG augmented with AGF (graft extender).
The fusion rate for the iliac crest–only control group was 91%. However, the fusion rate
for the AGF group was only 62%, which was significantly inferior.
Later evaluation of the AGF technique (which is no longer used) revealed that this
device shredded platelets during the filtration process and activated the platelets before
use, essentially delivering a substrate with limited biologic activity. 277 The Weiner and
Walker study used this PRP as an augmentation material combined with a smaller
amount of AICBG. It is conceivable that a lesser amount of iliac crest graft was
combined with an inferior PRP, and thus the results were significantly inferior than
what was found using AICBG alone. This study was widely seen as a condemnation of
PRP and it was thought that PRP was actually detrimental to bone healing. In actuality,
the particular formulation of PRP was at fault and not the concept of PRP itself.

Currently, there is no Level I evidence to indicate that using PRP alone or in

combination with other materials has a substantial effect on bone healing. The available
evidence (Levels III and IV) indicates that PRP may have a positive effect as an adjunct
to local bone graft and has been suggested for use to increase the rate of bone deposition
and quality of bone regeneration in fusion and nonunion situations. Overall, however,
there is clearly a lack of scientific evidence to support the use of PRP in combination
with bone grafts during augmentation procedures. 282 Well-controlled randomized
clinical studies are needed to assess the ideal concentration of the different factors and
to determine which materials provide the greatest effect. At present, platelet gel appears
to function best as a physiologic carrier for other graft materials.

Soft Tissue Applications

Platelet-rich therapies are being used increasingly in the treatment of musculoskeletal
soft tissue injuries such as ligament, muscle, and tendon tears and tendinopathies.
These therapies can be used as the principal treatment or as an augmentation procedure
(application after surgical repair or reconstruction). Platelet-rich therapies are produced
by centrifuging a quantity of the patient's own blood and extracting the active, platelet-
rich, fraction. The platelet-rich fraction is applied to the injured tissue, for example, by
injection.

Reported series have been published for clinical trials covering eight clinical conditions:
rotator cuff tears (arthroscopic repair); shoulder impingement syndrome surgery, elbow
epicondylitis; anterior cruciate ligament (ACL) reconstruction, ACL reconstruction
(donor graft site application); patellar tendinopathy (one trial), Achilles tendinopathy,
and acute Achilles rupture surgical repair. 283 In reviewing these multiple studies one
factor again becomes problematic: The methods of preparing PRP were highly varied
and lacked standardization. The quantification of the PRP amounts applied to the
patient were also not universally documented. 284

Another confounding variable making comparison between trials difficult is that the
primary treatments were either open surgery for repair with PRP application versus
trials that simply injected into “tendinopathies” where platelet-rich therapy injections
were the main treatment.

Five Level I and II controlled studies have compared results after surgical repair of
rotator cuff injuries with and without the adjunctive use of PRP. Subacromial
decompression augmented with PRP led to significantly decreased pain scores and
improved shoulder range of motion. 285 In a double-blind RCT of 53 patients,
intraoperative application of PRP during arthroscopic rotator cuff repair led to
significantly higher constant and University of California, Los Angeles, scores and
strength in external rotation 3 months after surgery but not at long-term follow-up
compared with control subjects. 286

Despite some encouraging results that PRP does have a positive benefit for rotator cuff
repair for pain relief and improved early motion, overall the evidence indicates that PRP
does not have an effect on retear rates or clinical outcomes after arthroscopic
repair. 287 Further study is required before the routine use of adjunctive PRP during
shoulder surgery can be recommended. 288

The treatment of lateral epicondylitis has received significant attention by treating this
condition with PRP. Most of the studies are RCTs with reasonable control groups and
long-term follow-up. Most of the studies document some improvement in pain and VAS
scores after the injection of PRP. The longevity of pain relief was variable in most of
these studies and many used glucocorticoid injection as the control group. However,
contrary results were found in a recent study. Krough et al. 289 demonstrated that neither
injection of PRP nor glucocorticoid was superior to saline with regard to pain reduction
for lateral epicondylitis at the primary end-point at 3 months. However, injection of
glucocorticoid had a short-term pain-reducing effect at 1 month in contrast to the other
therapies. Injection of glucocorticoid in lateral epicondylitis reduces both color Doppler
activity and tendon thickness compared with PRP and saline. 289

Despite these results, the clinical evidence suggests that local injection of PRP
containing WBCs may be beneficial to patients with chronic elbow epicondylitis
refractory to standard nonsurgical treatment. A major limitation in the evaluation of the
efficacy of PRP in treatment of lateral epicondylitis and other musculoskeletal diseases
is the heterogeneity in the way with which PRP is prepared and administered. This
heterogeneity of PRP preparations may account for the variable outcomes noted in the
prior studies evaluating efficacy of PRP for lateral epicondylitis. Several groups are now
working to find a method of standardizing the preparation of PRP, which will be greatly
beneficial for future research and application of this treatment. 290

Overall, autologous blood preparations including PRP have shown variable results in
RCTs but have shown some promise in the treatment of refractory lateral epicondylitis.
Future studies with long-term follow-up, larger patient groups, and a standardized
method for preparation of the injection are needed to better define the efficacy of these
treatments for lateral epicondylitis. 291

There have been some promising results documented for the treatment of lateral
epicondylitis, but the results have not been as encouraging for the treatment of other
tendonopathies, including jumper's knee. Two similar Level I studies in patients with
chronic Achilles tendinopathy were randomized to receive either a blinded injection
containing PRP or saline (placebo group) in addition to eccentric training programs.
Functional scores improved in both the PRP and the placebo groups after 1 year. There
was no significant difference in the increase in the improvement between both groups in
both studies. Ultrasonographic tendon structure improved significantly in both groups
in one study. Overall the results demonstrated no clinical and ultrasonographic
superiority of PRP injections over a placebo injection in chronic Achilles tendinopathy
at 1 and 2 years when combined with an eccentric training program. 292293294

There is insufficient evidence from RCTs to draw conclusions on the use, or to support
the routine use, of injection therapies for treating Achilles tendinopathy. Reviews have
highlighted a need for definitive research in the area of injection therapies for Achilles
tendinopathy, including in older nonathletic populations. 295

Substantial research has gone into evaluating the effect of PRP on ACL surgery. In vitro
studies document the positive effects on the graft tissue itself. Isolated ACL cells were
cultured in the presence of differing concentrations of PRP. Cell viability, collagen
synthesis, and collagen typing were analyzed. 296 PRP-treated cells resulted in a
significant increase in cell number compared with platelet-poor clot. Total collagen
production by the PRP-treated cells was significantly higher than that of the platelet-
poor treated cells only because of enhanced cell proliferation. Expression of type-III
collagen was significantly enhanced by the treatment with PRP.

Multiple studies have been carried out evaluating specific factors related to ACL
reconstruction. A systematic review of eight controlled clinical trials (Level I, II, and III
studies) concluded that the addition of platelet concentrates to ACL reconstruction may
have a 20% to 30% beneficial effect on graft maturation, as well as improved healing of
the femoral tunnels. 297 PRP was found to have a positive effect regarding improved graft
remodeling and enveloping tissue when patients were treated with PRP at the time of
ACL reconstruction. Many studies documented this enhanced ligamentization process in
tendon grafts. 298

Although studies documented improved graft incorporation, this has not translated to
improved functional outcomes. Many separate RCTs (Level I) document no difference in
radiographic, International Knee Documentation Committee scores, KT-1000
arthrometer (MEDmetric), plain radiography, and MRI findings in those ACL patients
treated with or without PRP. The authors concluded that there were no significant
differences in any parameter related to functional outcome status. 299300 When
treating the tibial harvest sites with PRP, studies have documented a positive effect on
patellar tendon harvest site healing on MRI after 6 months and also reduced pain in the
immediate postoperative period. 301302

A similar study attempted to evaluate whether PRP, due to its antiinflammatory

properties and capacity to stimulate tissue regeneration, was able to reduce the anterior
knee pain, the kneeling pain, and donor-site morbidity at the tibial harvest site. 287 The
authors determined outcomes by evaluation of VISA and VAS scoring scales and MRI
analysis of the tendon and bone defect. The PRP-treated group was successful in
reducing subjective pain at the donor site. The tibial and patellar bone defects were
satisfactorily filled by new bony tissue in 85% of PRP-group patients.

Although no significant difference in clinical outcomes has been found, preliminary

clinical evidence suggests that PRP may be beneficial during the ligamentization and
maturation processes of graft healing as well as that of the patellar tendon graft harvest
sites in ACL reconstruction. For rotator cuff and Achilles tendon repairs, the results of
clinical studies are equivocal.

There is a need to assess whether this translates into clinical benefit. Overall, and for the
individual clinical conditions, there is currently insufficient evidence to support the
routine use of PRP for treating musculoskeletal soft tissue injuries. 303 Further study is
needed before definitive conclusions can be drawn and recommendations
made. 280284

Recombinant Human Platelet-Derived Growth Factor

In an effort to take advantage of the beneficial effects of autogenous PDGF while
avoiding the perceived limitations (as outlined in the preceding section), a recombinant
PDGF was developed. The clinical success and safety that have been demonstrated with
use of recombinant human platelet-derived growth factor (rh-PDGF) in the repair of
periodontal defects has led to FDA approval of rh-PDGF for this indication. The potent
stimulatory effects of rh-PDGF have been determined to be similar to the properties of
autogenous PDGF. 304

Augment Bone Graft, a fully synthetic bone graft material composed of recombinant
human PDGF and a calcium phosphate matrix (rh-PDGF/TCP), has been considered as
a possible alternative to ABG and has been used in many preclinical studies. The data
have consistently indicated that rh-PDGF-BB treatment ameliorates the effects of
diabetes on fracture healing by promoting early cellular proliferation that ultimately
leads to more bone formation. It was felt that the local application of rh-PDGF-BB may
be efficacious for diabetic fracture treatment and for defect management. 305

Level I studies (RCTs) have demonstrated efficacy in patients requiring hindfoot or

ankle arthrodesis. Treatment with rh-PDGF-BB/β-TCP resulted in comparable fusion
rates, less pain, and fewer side effects compared with treatment with
autograft. 3738306 This material is currently FDA approved for clinical use to treat foot
and ankle indications, as noted in the previous section.

Inductive Substrates (Bone Morphogenetic Proteins)

The bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily of GDFs.
Unlike DBM, which is a mixture of BMPs and immunogenic noninductive proteins, the
pure form of BMP is nonimmunogenic and non–species specific. The BMPs are true
“osteoinducers.” As they are released, they feed back onto circulating undifferentiated
perivascular mesenchymal cells (stem cells), changing them directly into
osteoprogenitor cells. The pivotal role of BMPs in the growth and differentiation of
various tissues and organs during embryonic development has been noted. 307 BMPs
have also been found to be potent osteoinductive proteins, inducing a sequential cascade
of events leading to chondrogenesis, osteogenesis, angiogenesis, and the synthesis of
ECM. 308 BMPs exert their action by binding to the extracellular domain of the BMP
receptor, which leads to the activation of SMAD proteins (intracellular messengers that
are protein dependent and act as signaling pathways to increase transcription of
osteoconductive genes). 307309 BMP-2, BMP-4, and BMP-7 have been found to play a
critical role in bone healing, as they are able to stimulate mesenchymal cells to develop
into an osteochondroblastic lineage. 310

The early use of this material began with multiple clinical series and case reports by
Johnson et al. 61311312313314 Urist purified the human protein in his laboratory at
UCLA and his clinical colleagues were able to utilize this material. Because extraction of
purified human BMP from cadaver bone provided small yields, the ability to produce it
in large quantities was limited, and thus limited clinical application followed. However,
early results were promising, treating complex femoral nonunions, 312 posttraumatic
shortened atrophic femoral nonunions, 311 and resistant nonunions with partial or
complete segmental defects including tibial defects with bone loss ranging up to
17 cm. 61 These were all treated with a composite alloimplant of human bone
morphogenetic protein (h-BMP) and autolyzed, antigen-free, allogeneic bone
(AAA). 311312313314315

Purification of DBM by Sampath and Reddi 316 in 1981 led to the isolation and
identification of several members of the BMP family. Because of the time and expense of
formulating these material in limited quantities, recombinant technologies were
developed to deliver BMPs in large amounts to proceed with significant research efforts.
BMPs such as rh-BMP-2, rh-BMP-4, and rh-BMP-6 (Genetic Institue, Cambridge, MA)
and rh-BMP-7 (OP-1) (Olympus Corp., Tokyo, Japan) were synthesized. When these
factors are loaded on a collagen sponge or particulate collagen, these carriers act as a
reservoir for delivery to the site of involvement. In an overwhelming amount of
preclinical animal data, these factors have been found to induce enchondral bone
formation in segmental bone defects and achieve spinal fusion in a number of animal
models. 317 Substantial preclinical and preapproval data are required because the use of
this technology is viewed by the FDA as being associated with risk. Recombinant BMPs
were classified as Class-III devices and face a much more rigorous approval pathway.

Faced with demanding premarket FDA approval, from 1994 to 2000 multiple rigorously
controlled preclinical studies were performed primarily evaluating the efficacy of BMPs
in their ability to achieve spine fusion compared with AICBG in various animal models.
More than 25 studies compared BMP-2 versus autogenous graft, and 12 studies
evaluated BMP-7 versus autogenous graft, all demonstrating equivalency or superiority
of the BMP implants.

A representative study was performed by Boden and colleagues 318 in which they used a
primate lumbar intertransverse process arthrodesis model to evaluate rh-BMP-2 in a
hydroxyapatite-tricalcium phosphate (HA-TCP) carrier as a complete bone graft
substitute. The rh-BMP composite sites were compared with AICBG sites with
radiography and histology to assess fusion and to detect any bony growth into the
laminectomy defect. All of the rh-BMP composite fusions healed, whereas fusion was
not achieved in any of the monkeys treated with AICBG. 318

These studies set the stage for the crucial pivotal clinical trials for which there were at
least 11 randomized prospective studies evaluating BMP-2 and 15 Level I studies
investigating BMP-7. These preapproval Level I studies evaluated both spine and long
bone applications.
One the most prominent of these was a multicenter, prospective, randomized,
nonblinded, 2-year study performed by Burkus and colleagues. 319 Nearly 300 patients
with degenerative lumbar disc disease were randomly divided into two groups that
underwent interbody fusion using two tapered threaded fusion cages. The
investigational group (143 patients) received rh-BMP-2 on an absorbable collagen
sponge (ACS), and a control group (136 patients) received AICBG. Fusion rate in the
BMP-treated group was 94.5% compared with 88.7% in ABG-treated group. The ABG-
treated group had eight adverse events related to bone graft harvest procedure and 32%
of patients reported graft site discomfort. This pivotal trial lead to the approval of BMP-
2 for spinal surgery for the following indication: the InFUSE Bone Graft in conjunction
with an interbody fusion devices (i.e., either the LT-CAGE Lumbar Tapered Fusion
Device or the Inter Fix RP Threaded Fusion device), implanted via an anterior open or a
laparoscopic approach.

BMP-7
BMP-7 is a potent osteoinductive agent that has been found to play a critical role in
fracture healing. 12 Despite similarities, the testing and regulation of BMP-7 have been
different from that of BMP-2. In a large prospective, randomized, controlled, partially
blinded, multicenter study, Friedlaender and colleagues 320 assessed the efficacy of the
OP-1 Device (3.5 mg of rh-BMP-7 in a bovine bone derived type-1 collagen-particle
delivery vehicle; Olympus Corp., Tokyo, Japan) in comparison with that of autografting
in the treatment of 122 patients with a total of 124 tibial nonunions. All of the nonunions
were at least 9 months old and had shown no progress toward healing for the 3 months
before the patients' enrollment in the study. All patients were treated with reamed IM
nailing of the nonunion and were then randomized to have either autograft bone or OP-1
implanted at the nonunion site. Nine months after the surgery, 81% of the OP-1 group
and 85% of the autograft group had clinical evidence of union. Radiographic
assessments suggested healing of 75% and 84% of these nonunions, respectively. At 2-
year follow-up these results continued at similar levels. 320

OP-1 statistically proved to be a safe and effective alternative to bone graft in the
treatment of tibial nonunions. A limitation of the study was that the investigators could
not control for the potential healing effects produced by reamed IM nailing of the tibial
nonunions. This, among other study criticisms, prevented the full FDA approval of this
material.

Limited approval of OP-1 by the FDA for use in the treatment of tibial nonunions and
other long bone nonunions was designated as a humanitarian use device (HUD) for this
particular indication. 321 An HUD is intended to benefit patients by treating or diagnosing
a disease or condition that affects or is manifested in fewer than 4000 individuals in the
United States per year and the manufacturer is not required to demonstrate unequivocal
benefit, but only “probable” benefit. OP-1 was only used in facilities that had established
a local institutional review board (IRB) to supervise clinical testing of devices and after
an IRB has approved the use of the OP-1 to treat recalcitrant nonunions ( Fig. 5.13 ).
Open full size image
Fig. 5.13
(A) Recalcitrant atrophic humeral nonunion in a 67-year-old woman who had failed a previous
AICBG and revision humeral nailing. The patient has many comorbidities, including diabetes and
hypertension. Note the distraction gap at the site of the atrophic nonunion. (B) Revision
osteosynthesis was carried out using a compression locking plate and graft augmentation using rh-
BMP. An off-label application was used combining the OP-1 with concentrated marrow aspirate and
particulate DBM to help provide a carrier for the particulate rh-BMP graft material. (C) At 10 months
postgraft, the nonunion has healed with excellent bridging of the nonunion site, as well as
developing massive heterotopic bone. The heterotopic ossification (HO) followed the natural tissue
planes and probably resulted from the inability of the carrier material to contain the protein
exclusively to the site of the nonunion. The HO restricted the patient's elbow and shoulder motion,
requiring eventual resection. (D) At excision surgery, a large longitudinal mass of heteroptopic bone
running the entire length of the interval between the biceps and brachialis muscle was exposed. This
was interconnected to the bone found at the nonunion site, which was united (white arrows).  (E) A
large quantity of heterotopic bone was resected from the humerus. This restored the patient's
shoulder and elbow function. The fracture completely healed with no other residual sequelae.
There are few high-quality studies on the use of rh-BMP-7 in treatment of acute
fractures and nonunions. A 2010 Cochrane systematic review identified eight RCTs of
the use of rh-BMP-7 in fracture healing, but these studies varied substantially in their
methodology and setting. 322 Overall, the quality of the studies was graded as “poor,” as
most studies were small, did not report methods of randomization or allocation, or were
industry sponsored. 37 Further studies are needed to assess the efficacy of BMP-7 in the
treatment of acute fractures and nonunions.

OP-1 putty was also FDA approval through the humanitarian device exemption (HDE)
process and was indicated for use as an alternative to autograft in compromised patients
requiring revision posterolateral (intertransverse) lumbar spinal fusion, for whom
autologous bone and bone marrow harvest are not feasible or are not expected to
promote fusion. Both materials have subsequently been removed from the market for
commercial use and are no longer available.

BMP-2
In addition to the approved spinal indications for BMP-2, a prospective randomized
study evaluated rh-BMP-2 for the treatment of open tibial shaft fractures. The BMP-2
Evaluation in Surgery for Tibial Trauma (BESTT) Study Group reported the results of a
large multinational, prospective, randomized, controlled study of the effects of INFUSE
(rh-BMP-2 on an absorbable type-1 collagen sponge; Medtronic Sofamor Danek,
Memphis, TN) in the treatment of open tibial fractures. 323

Four hundred and fifty patients with such an injury were initially managed with
irrigation, débridement, and IM nail fixation. At the time of definitive wound closure,
the patients were randomized to one of three groups: standard closure, standard closure
and the addition of 6 mg of rh-BMP-2 to the fracture site, or standard closure and the
addition of 12 mg of rh-BMP-2 to the fracture site. The primary outcome measure in this
study was the rate of secondary interventions (returns to the operating rooms for
additional treatment). The group treated with the higher dose of rh-BMP-2 (1.5 mg/kg)
had fewer secondary interventions. Interestingly, although not used as primary outcome
measures, an accelerated time to union, improved wound healing, and a reduced
infection rate were also found in the patients treated with the high dose of rh-BMP-2.
The FDA subsequently granted approval for the treatment of acute, open fractures of the
tibial shaft ( Fig. 5.14 ).
Open full size image
Fig. 5.14
(A) A motorcycle crash resulted in a grade 3b open distal tibia fracture with limited intraarticular
extension and 3 to 4 cm of segmental bone loss. The fracture was initially stabilized with a temporary
spanning external fixator. (B) The injury presented with a severe soft tissue injury in addition to the
major bone loss. The fracture was also severely contaminated, which prevented emergent IM
nailing, and was therefore converted to a more stable external fixator and ultimately to a ring fixator
for definitive treatment. (C) The soft tissue injury required free flap coverage, as shown here. At the
time of flap coverage (wound closure) rh-BMP-2 was applied with a particulate ceramic to act as a
conductive substrate and facilitate cellular ingrowth into this relatively avascular diaphyseal
region (black arrows). (D) Healing of the segmental defect is noted on these postframe radiographs.
The external fixator allowed full weight bearing after maturation of the free flap. Near-complete
healing of the segmental defect occurred over a 5-month period after graft application. The external
fixator was removed at 21 weeks after injury.
A 2011 study has brought into question the value of rh-BMP-2 for the treatment of open
tibial shaft fractures. Aro and colleagues 324 evaluated the use of rh-BMP-2 in the
treatment of acute open tibial fractures treated with reamed IM nail fixation. This study
was very similar to the aforementioned BESST study. Patients were randomly assigned
(1 : 1) to receive IM fixation and routine soft tissue management (the SOC group) or an
IM nail plus an ACS implant placed at the time of wound closure. The implant contained
1.5 mg/mL of rh-BMP-2 (total, 12.0 mg) (the rh-BMP-2/ACS group). Healing and other
outcome measures were assessed. By week 20, the proportions of patients with fracture-
healing were 68% and 67% in the rh-BMP-2/ACS and SOC groups, respectively (not
significant). All other outcome parameters were similar between the two groups and the
authors concluded that the healing of open tibial fractures treated with reamed IM nail
fixation was not significantly accelerated by the addition of an ACS containing rh-BMP-
2. 324

After the approval of these two BMPs for specific traumatic conditions (acute open tibal
shaft fracture, recalcitrant nonunions), limited data have been published using these
devices with their strict on-label indications. Many investigators have sought to combine
these materials with other biologic adjuvants. Just as we have discussed previously in
this chapter, the ability to combine multiple inductive, conductive, and/or osteogenic
factors continues with the BMPs as well. This has been done for a variety of issues
regarding the clinical handling and application of these materials, as well as to attempt
to augment the healing potential of these specific BMPs for their trauma application.

Jones and colleagues 325 investigated the implantation of BMP-2 combined with allograft

compared with autograft for the treatment of acute segmental defects in a prospective
RCT of 30 patients with tibial diaphyseal bone defects. The average defect size was 4 cm
(range, 1 to 7 cm). At 12-month follow-up 80% of patients (12 of 15 patients) in the
AICBG group and 87% of patients (13 of 15 patients) in the allograft/rh-BMP-2 group
had healed without reintervention ( P = 0.2). There were no significant differences in
complication rates or functional outcomes between the two groups. This study suggests
that rh-BMP-2/allograft is safe and as effective as traditional autogenous bone grafting
for the treatment of tibial fractures associated with extensive traumatic diaphyseal bone
loss. 325

In an effort to improve fracture healing in closed tibial shaft fractures and to develop an
injectable form of BMP-2, investigators evaluated rh-BMP combined with a new,
injectable calcium phosphate matrix (CPM). Patients were randomized (1 : 2 : 2 : 1) to
receive standard of care, with a locked reamed IM nail, versus IM nailing with injection
with 1.0 mg/mL of rh-BMP-2/CPM; or an increased concentration 2.0 mg/mL of rh-
BMP-2/CPM. 326 The results were similar to the Aro study where no significant difference
with or without BMP was noted. 324 It appears that the times to fracture union and pain-
free full weight bearing were not significantly reduced by the injection of either
concentration of rh-BMP-2/CPM compared with IM nailing alone without adjuvant
injection.

In addition to questions regarding the clinical efficacy of BMP-2, several studies have
highlighted potential safety concerns regarding this treatment. In a clinical trial of 287
patients with open tibial shaft fractures, patients were randomized to receive either
standard of care (IM nail and soft tissue management) or standard of care in addition to
rh-BMP-2 (1.5 mg/mL; n = 139). 324 The study was halted before completion due to the
higher rate of infection in the treatment group (19%) compared with controls (11%).
Although the reason for an increased rate of infection is unclear, it raises concerns over
the safety of this treatment. In addition to infection, there are a number of other
complications associated with rh-BMPs that are related to either the initial
inflammatory response induced by the protein (seroma, neuritis, neck swelling) or to
their osteoinductive properties (HO, transient osteopenia) 327328329 (see Fig. 5.13 ).
This is probably due to the nonspecific nature of the BMP-2 active molecule itself having
a variety of effects in addition to the desired response to facilitate bone formation.

Because of safety concerns, rh-BMP-2 is not approved for use in children, pregnant
women, and cancer patients. rh-BMP-2 is also contraindicated for use in the cervical
spine as a result of severe complications, including infection and dysphagia. 330331

As the complications related to BMP-2 may be due to the supraphysiologic doses

currently used in clinical trials, recent studies have focused on optimizing delivery,
enhancing BMP expression, or finding synergistic substances to reduce the required
dose. The development of a more specific molecule is ongoing. In this way, it is hoped
that the secondary side effects will be minimized with a definitive mode of action
molecule. The optimal carrier matrix for rh-BMP-2 is unknown. Currently, an ACS is
used, but kinetic studies have shown that up to two-thirds of the total drug is released
within the first 4 days. 332 In animal studies, the delivery of BMP-2 has been improved
using alternate carrier systems such as synthetic hydrogel scaffolds, polyelectrolyte
complexes, bare colloidal gelatin gels, and microspheres. 333334335336337 Strategies
to reduce the required dose of BMP-2 to enhance fracture healing have also been
studied, such as amplifying BMP-2 with biomimetic supramolecular nanofibers within
collagen scaffolds, which was able to significantly reduce the dose of BMP-2 required to
heal a rat femoral defect. 338 Although these methods have shown promise in animal
models, they have yet to be translated to the clinical setting.

In addition to safety concerns, another consideration with regard to the use of BMP-2 is
cost. The use of rh-BMP-2 may add $5000 to $15,000 to the cost of treatment,
depending on the amount of protein needed. 339 Proponents argue that these high costs
are offset by savings due to shorter surgery time, lack of a bone grafting procedure,
faster hospital discharge, and faster return to work. There is limited evidence to support
these claims, and a high risk of bias as the authors received financial support from the
BMP manufacturing company. 340341 A 2007 systematic review did find that BMPs
were associated with a reduced operating room time, improvement in clinical outcomes,
and a shorter hospital stay compared with autograft. 342 This study evaluated both BMP-
2 and BMP-7, and included acute tibial fractures, nonunions, and spinal procedures.
The true cost-efficacy of BMP is probably not known at this time. There is a need for
more rigorous cost-effectiveness analysis related to the use of BMP. 343

The high cost and safety concerns for the use of BMP-2 currently limit its utility as a
therapy in the treatment of fractures and nonunions. Despite these concerns, BMP-2 is
one of the most potent osteoinductive agents available and holds promise for future use
as the molecule undergoes further refinement. 343
BMP-2 has also found widespread off-label use augmenting fracture fixation for the
treatment of osteoporotic fractures ( Fig. 5.15 ). There are only limited data on the
clinical relevance and optimal indications for the use of bone graft substitute materials
and BMPs on the treatment of osteoporotic fractures despite the clinical benefits of
these materials in other clinical indications. Given the general compromised outcome in
osteoporotic fractures and limited alternatives for enhancement of fracture healing,
clinicians and researchers should focus on this important topic and provide more data
in this field to enable a sound clinical use of these materials in osteoporotic fractures. 344

Open full size image

Fig. 5.15
(A) A 75-year-old woman sustained IIIb distal humerus fracture with 2 to 3 cm of acute bone loss.
Additionally, she had a history of osteoporosis and malabsorption, and was taking oral agents to
treat her metabolic bone disease. (B) To treat her bone loss and osteoporosis, her fixation construct
required an off-label application of rh-BMP-2 in combination with DBM + cancellous allograft +
BMAC, as a “composite graft.” Two plates were also used to achieve construct stability. (C) Six
weeks after implantation there is early consolidation of the composite graft. (D) Complete
consolidation of this defect in a patient with metabolic bone disease at 14 weeks after implantation.
No loss of reduction or hardware loosening is noted. Composite grafting provides all factors a
competent bone graft substitute requires: conductive surface area (DBM + cancellous allograft);
growth factors and inductive proteins for chemotaxis, mitogenesis, and differentiation (DBM + rh-
BMP 2); viable undifferentiated and pluripotential cells for transformation to an osteoblastic lineage
(BMAC).
Open full size image
Fibroblast Growth Factor
The FGFs are a family of 22 structurally related polypeptides that are characterized by
their high affinity for heparin sulfate proteoglycans. 345 This growth factor family requires
heparin sulfate to activate their cell-surface receptors. 12345 FGFs have a wide range of
cellular activities, including the regulation of cell proliferation, differentiation, and
apoptosis. 345 FGF-1 and FGF-2 are the most abundant in normal adult tissues and can be
detected in the early stages of fracture repair at the fracture site, facilitating vascular
ingrowth and other hallmarks of the inflammatory stage of fracture healing. 12346 FGF-
1 exerts its mitogenic effect primarily in chondrocyte proliferation. 347348 FGF-2 is
released primarily by osteoblasts and is considered to be a more potent growth factor
than FGF-1. 349

Preclinical studies in both small and large animal models have demonstrated that
recombinant human fibroblast growth factor (rh-FGF) is able to enhance fracture
healing and increase bone volume after a single injection. 350351 Kawaguchi
et al. 352 examined the effect of rh-FGF-2 in an RCT of 70 patients with transverse or
short-oblique closed tibial shaft fractures. The authors randomized patients to one of
three groups to receive placebo, low-dose, or high-dose rh-FGF-2 hydrogel injected into
the fracture site at the time of surgery with IM nail. Results noted that radiographic
union was higher in the rh-FGF-2 treated groups compared with controls. Although this
study demonstrated a beneficial effect of rh-FGF-2 on radiographic bone healing, there
was no demonstrable clinical benefit compared with controls. Future clinical trials
examining the effect of this growth factor in the enhancement of fracture healing are
required.

Off-Label Use and Complications

As is highlighted by these two recent studies, investigators are evaluating off-label
indications. Most of the use of rh-BMPs in orthopaedics is off-label, which means that
the product has not been approved for such use by the FDA. Physician-directed use of
such products for uses not approved by the FDA is allowable because the FDA does not
regulate physician practice; its statutory authority extends only to the regulation of the
manufacturers of drugs and medical devices and listing of known complications.
Physician-directed (off-label) use of devices or drugs has been approved by some state
legislatures, and it is generally acknowledged that physicians should be able to apply
their best medical judgment in the use of off-label devices or drugs. However, with an
increase of reported complications of BMP usage the unregulated nature of BMP has
come under intense scrutiny both from third-party payers and the federal government.

Isolated complications with the use of rh-BMP in spine fusions, such as retrograde
ejaculation in males, have occurred in clinical studies, but was initially delayed in their
reporting. In some instances it was felt that undue bias from industry-sponsored studies
had intentionally underreported complications. 353354355 Subsequent studies have
cited the following adverse events that have occurred when used in spinal applications:
HO, graft osteolysis, increased infection, arachnoiditis, increased neurologic deficits,
and retrograde ejaculation. 356357358359 The use of rh-BMPs in cervical spine fusion
has resulted in significant edema with resultant compromise of tracheal air
flow. 360361362 Some of these issues resulted in devastating consequences for the
patients who incurred these complications.

Surgeons have noted several safety issues associated with the use of BMPs, including
cancer risk, stating that both BMP and their receptors have been isolated from human
tumors. In addition, data presented to the FDA on the product AMPLIFY (rh-BMP-2,
40 mg) revealed a higher number of cancers in the investigational group compared with
the control. An independent review of the cancer risk of rh-BMP use in spine fusions as
published in peer-reviewed literature and in the publicly available FDA data summaries
was evaluated. They concluded that cancer risk with BMPs may be dose dependent,
illustrating the need to continue to study this technology and obtain longer follow-up on
patients currently enrolled in the FDA trials. 363

However, a large population-based analysis of Medicare beneficiaries found no evidence

that administration of rh-BMP at the time of lumbar fusion surgery was associated with
an elevated cancer risk. 364 Propensity-matched studies suggest that the use of BMP in
lumbar fusions is associated with a significantly higher rate of benign neoplasms but not
malignancies. 365

Complications also occurred when BMPs were used in acute traumatic or posttraumatic
situations. HO appears to be the most common and consistent complication when used
for trauma-related conditions (see Fig. 5.13 ). Boraiah and colleagues 366 noted a
significant incidence of HO when plateau fractures were treated with BMP. The BMP
was used to augment the structural support provided by the bone void filler used to
support the elevated defect. Even though this was an off-label indication, the authors
sought to determine whether BMP enhanced the ability to maintain the surgically
elevated subchondral bone. Sixty percent (10 of 17) of the patients developed heterotopic
bone of which 4 (40%) required additional surgery to have this excised. Additionally, the
BMP did not appear to have any positive effect on the maintenance of the subchondral
repair. 324

A second series also reported the phenomenon of heterotopic bone. This bone occurred
in the humerus of which 50% were related to acute application of BMP for traumatic
bone loss, and 50% had BMP applied at the time of humeral nonunion repair 367 (see Fig.
5.13 ). All of the patients in this series required surgical removal of this extra bone. It has
been proposed that BMP-4 is involved in the extensive endochondral ossification seen in
fibrodysplasia occificans progressive. 368369 The transfer of the gene for BMP-2 into
skeletal muscle has been shown to promote both endochondral and intramembranous
ossification. 370 Expression of BMP-2 and BMP-7 has also been shown to induce BMP-4
expression. 370 The osteoinductive stimulus for chondro-osteogenic differentiation
leading to the development of HO is the proposed mechanism in these cases. Another
proposed mechanism could be the inability of the BMP carrier to contain the material to
the site of involvement only and thus the additional bone formation found in areas of
soft tissue injury, as well as the primary application site. 371

Bone Morphogenetic Protein Interactions

As noted, it has been shown that the presence of one BMP can enhance the expression of
other BMPs. For example, the combinations of BMP-2 with BMP-7 have been reported
to be 5 to 10 times more potent than BMP-2 alone in inducing cartilage and bone
formation, 372 perhaps by the induction of BMP-4 as mentioned previously.

BMPs are regulated at multiple levels by various BMP antagonists, as well as being
affected by other circulating BMPs. 373374 Thus the current use of single-agent BMPs is
simplistic and raises the question as to why supraphysiologic doses of a single BMP are
required to have an osteoinductive effect. The effective doses are orders of magnitude
greater than the endogenous amounts of BMPs expressed during normal bone repair or
in normal bone remodeling. Presumably the combined action of other factors may be
required for maximal efficacy of BMP-mediated osteoinduction. It has been shown that
BMPs and induction are regulated at multiple levels by various BMP antagonists, as well
as affected by other circulating BMPs. 373374

The current application of giving one factor and expecting a complex cascade of bone
forming events to occur in an unencumbered fashion is simplistic. However, the
experimental complexities of manipulating multiple factors make investigational
clarification of this hypothetical issue nearly impossible to carry out. However, other
factors regarding the timing and mode of delivery may also be crucial to the appropriate
mechanism of action that these molecules are capable of stimulating.

Future Directions: Timing of Delivery

After fracture, the fracture site progresses through the stages of healing, and the types of
cells that are present vary substantially. 375376 In addition, the growth factor receptor
expression on each of these cells changes over time. 377378 Therefore a growth factor
delivered to the fracture site may be completely ineffective at a certain time point during
healing, while being very potent during a different phase of repair. The temporal
relationship of growth factor delivery is unknown at this time and future studies will
seek to determine the optimal timing of growth factor delivery, which may differ by
growth factor and fracture type. 374

Future Directions: Mode of Delivery

There are a number of potential methods of growth factor delivery, including open
insertion versus percutaneous injection, suspension in a carrier or buffer vehicle, and
local application versus systemic treatment. The most popular carrier used in clinical
trials is collagen, which has been shown to be biocompatible, degradable, and
osteoconductive. 379380381382 The major disadvantages of collagen are that it
requires an open procedure for insertion and it releases up to 30% of the total protein it
is carrying immediately after implantation. 383384 More recent studies have focused on
addressing these issues. Systemic administration of PTH and PTH-rP obviates the need
for open insertion of growth factor delivery to the fracture site and has been shown to
accelerate bone healing. 385386 To improve the sustained release of growth factor at the
fracture site, various biodegradable polymers have been created and tested, including
hyaluronic-based gels, gelatin gels, and calcium phosphate pastes. 386387388389 These
polymers have shown promise in animal studies, but have not been tested clinically at
this point.

Despite advancements in local delivery methods, many have suggested that a single dose
of exogenous protein administered at the fracture site is inadequate to enhance fracture
healing. To address this limitation, a popular strategy under study is gene therapy. Gene
therapy refers to the delivery of a complementary DNA sequence of a particular growth
factor to the cells surrounding the site of fracture. With successful incorporation of this
DNA, the cells at the fracture site can produce the desired growth factor. In gene
therapy, DNA is typically delivered in either viral (adenovirus or retrovirus) or nonviral
(liposomes or polypeptides) vectors. It may be delivered directly at the fracture site or
transferred to cells ex vivo and then transferred to the fracture site. The major
theoretical advantage of gene therapy for fracture healing is prolonged expression of
growth factor at the fracture site. Potential disadvantages of this therapy include a host
immune response, recombination of the defective virus with viruses in the host cell, and
excess bone growth. Preclinical studies have had promising results for the use of gene
therapy in the enhancement of fracture healing, but no clinical studies have been
performed to date. 390

Economic Impact
While the efficacy and possible complications due to rh-BMPs continue to be debated,
the economic impact of using these devices off-label is relatively unknown. A recent
study evaluated the economic impact of “on-label” versus “off-label” use of BMPs for
spinal fusions. 391 The authors determined that in 2010 (at the authors’ institution), 96%
of the spinal fusions that used BMP were used in an off-label capacity (cost $4,547,822),
whereas only 4% were performed on-label at a cost of $296,419. 392
An interesting cost analysis study was undertaken using the data from the BESTT trials
to evaluate the potential cost savings associated with the use of BMP-2 for open fracture
care. Data from the UK, Germany, and France were analyzed. The cost of BMP-2 was
offset by the ability of injured patients to return to work, which was assumed to
correspond with fracture healing time. Reduced productivity losses were realized by the
significantly faster fracture healing and earlier return to work compared with the non–
BMP-2–treated patients. Total net savings were estimated to be 9.6 million euros for the
UK, 14.5 million euros for Germany, and 11.4 million euros for France. The authors felt
that despite the apparent high direct cost of rh-BMP-2 for use in grade III A and B open
tibial fractures, at a national level there are net cost savings from a societal perspective
for all three countries. 393

Summary
This chapter highlights the paucity of current data present for the use of rh-BMPs for
traumatic indications. There is limited evidence to suggest that BMP may be more
effective than controls for acute tibial fracture healing; however, the use of BMP for
treating nonunion remains unclear. The limited available economic evidence indicates
that BMP treatment for acute open tibial fractures may be more favorable economically
when used in patients with the most severe fractures. 394

Well-designed and well-executed studies are necessary to completely define the

indications for the use of BMPs. Studies need to be designed to account for the specific
BMP to be used and the definitive pathology and region of application. Standardized
surgical technique is required, as well as control of the dosage applied and the specific
carrier substances used to deliver the materials. Most important, investigators should
strive to define the natural history and management of any associated complications.

Conclusion
Although current regulations on informed consent do not require physicians to note that
they are using a product such as rh-BMP off-label, the planned use of allografts, bone
substitutes, and bone enhancers in surgery should be disclosed in informed consent.
Currently, many organizations are formulating and establishing guidelines for the
correct disclosure of these materials and their approved indications for their use based
on the best available evidence.

The ideal bone graft substitute is biocompatible, bioresorbable, osteoconductive,

osteoinductive, structurally similar to bone, easy to use, cost-effective, and at minimal
risk for material-based complications. Within these parameters a growing number of
bone alternatives are commercially available for orthopaedic applications.

This is an exciting time in the development of orthobiologic/factor-based therapies for

enhancement of bone healing for traumatic conditions. Areas of application for new
technologies include the acceleration of fracture healing, treatment of nonunions,
enhancement of fusion mass, and the treatment of massive segmental bone loss. These
adjuvants also have been valuable for the reconstruction of cavitary bone deficiency in
situations of segmental bone loss and spine fusion. Additionally, the technologies for the
 use of cell-based adjuvants are constantly being improved. Early results are encouraging
for cell-based therapies, and it is hoped that they will have a positive effect on the
clinical outcomes for the treatment of tendinopathies, as well as for symptomatic and
radiographic improvement in the early treatment of arthritic joints.

As the understanding and use of growth factors in the treatment of fracture healing
continue to evolve, it is increasingly important that the orthopaedic surgeon be familiar
with the biologic characteristics and clinical application of these therapies. Given the
enormous consequences of delayed fracture healing from both a patient-centered and
societal perspective, further research is needed to bring these strategies from the
laboratory bench to the bedside.

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