Biology and Enhancement of Skeletal Repair

C h a p t e r 4
Biology and Enhancement of

Skeletal Repair
J. TRACY WATSON
INTRODUCTION substrate and thus the porosity of these materials is critical.

Attachment is then followed by the differentiation of cells into
The last 10 years has seen the rapid advancement of fracture bone-forming cells types, and the production and remodeling
fixation devices and surgical techniques. The development of of bone then proceeds.2,3,5
preshaped locking plates in concert with minimally invasive Graft osteoconduction: A major category of available graft
surgical exposures has revolutionized the trauma surgeon’s substitutes for defect management intervention is to provide
ability to treat complex periarticular injuries. Percutaneous a lattice-like substrate material that will facilitate the attach-
instrumentation, the refinement of insertion portals, and the ment and proliferation of osteoblastic cellular elements.
development of blocking screw techniques has expanded the Graft osteoinduction: As all skeletal tissues evolve from
range of intramedullary (IM) nailing from midshaft injuries mesenchyme, undifferentiated mesenchymal cells make a
to the articular margins. Likewise, the arena of orthobiologics genetic commitment to a particular cellular lineage early in
has seen tremendous technological advancements in bioma- the developmental or repair process.6 In the case of repair,
terials and biologics in an attempt to avoid the perceived some stimulus must signal the undifferentiated mesenchymal
morbidity associated with the harvest of autogenous iliac cells to differentiate along a chondro-osteogenic pathway.
crest bone graft (AICBG). Multiple materials have become Prior to this differentiation into an osteogenic lineage, these
available to enhance fracture healing through a myriad of cells are affected by multiple factors that provide chemotactic
biologic pathways. The purpose of this chapter is to review and mitogenic stimuli to these cells. This process is jump-
the basic fund of knowledge on the topic of bone graft substi- started by graft-derived growth factors, such as transforming
tutes. Currently available adjuvants for approved clinical growth factor-ß, insulin-like growth factors 1 and 2, platelet-
use will be discussed. As well, the current levels of evidence derived growth factor, and others. They influence these cells
in supporting the use of these various materials will be to migrate, attach, and multiply at the locale that provides a
reviewed. competent osteoconductive substrate as a site of cellular
attachment.7 This phenomenon, known as osteoinduction, is
defined as “a process that supports the mitogenesis of undif-
BIOLOGY OF GRAFT SUBSTITUTES ferentiated mesenchymal cells leading to the formation of
osteoprogenitor cells with the capacity to form new bone.”8-10
The biology of bone grafts and their substitutes is appreciated Thus, any material that induces this process could be consid-
from an understanding of the bone formation processes of ered to be osteoinductive.
osteogenesis, osteoinduction, and osteoconduction.1-5 The concept of osteoinductive new bone formation is real-
Graft osteogenesis: This is the ability of cellular elements ized through the active recruitment of host mesenchymal stem
within a donor graft, which survive transplantation, to synthe- cells (MSCs) from the surrounding tissue, which differentiate
size new bone at the recipient site. Osteogenesis can occur in into bone-forming osteoblasts. This process is facilitated by
two ways. Surface osteoblasts can survive the transplantation the presence of growth factors within the graft, principally
by receiving nutrition through diffusion at the recipient site, bone morphogenic proteins.
and then proliferate to form more living bone tissue. There is Marshall R. Urist described the induction principle in the
more surface area in cancellous graft, and thus it has more 1960s.8 The concept of bone matrix—demineralized bone
potential for surviving cells than a cortical graft. Likewise the matrix (DBM)—is that this material contains properties that
transplantation of marrow elements alone has demonstrated can induce de novo new bone formation when implanted into
this ability to survive and form bone. an extraskeletal site. It should be noted, however, this response
These transplanted cells or circulating pluripotential cells is seen in animal models but has not been demonstrated in
must have the appropriate substrate to attach to, or become humans.
attached to, once the cells have localized to the site of defect Urist and his colleagues soon identified a protein that they
or injury. This substrate site for cellular attachment has to named bone morphogenetic protein (BMP).8-10 Other mole-
have the appropriate three-dimensional architecture to allow cules were soon identified and helped to characterize an
for these cells to proliferate. Subsequently, these cells then use entire family of osteoinductive molecules.11 This family of spe-
this substrate as a scaffolding through which to build bone. cific factors now contains at least 15 BMPs and is part of the
This three-dimensional process involves vascular proliferation larger transforming growth factor-β (TGF-β) superfamily of
and ingrowth of capillaries along the open spaces in the molecules.
69
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70 SECTION ONE — General Principles
TABLE 4-1 AVAILABLE FRACTURE HEALING ADJUVANTS WITH THEIR INHERENT PROPERTIES
Osteoconductive Osteogenic
Scaffold Osteoinductive Growth Factors Living Cells
Synthetic Ca+ ceramics •
Marrow concentrates •
Bone morphogenic protein (BMP) •
Autograft • • •
Platelet-rich concentrates Osteopromotive indirect cellular effect
No intracellular transcription
Banked demineralized bone matrix (DBM) • •
Protein extracts derived from bone can initiate the process STAGES OF BONE GRAFT INCORPORATION
that begins with cartilage formation and ends in de novo bone
formation. The critical components of this extract, BMPs, Bone grafts and bone graft substitutes incorporate via a well-
which direct cartilage and bone formation as well as the con- defined pathway that can be divided into five distinct stages
stitutive elements supplied by the animal during this process, of host response, with the duration of each phase depending
have long remained unclear. Amino acid sequence has been on the type of graft or adjuvant material used.2,3,14,15
derived from a highly purified preparation of BMP from Stage 1: Initial injury and hemorrhage: Initiates the pathway
bovine bone. Now, human complementary DNA clones cor- with the degranulation of platelets at the site of injury.
responding to three polypeptides present in this BMP prepa- Stage 2: Inflammation: Under the influence of many active
ration have been isolated, and expressions of the recombinant cytokines that are produced at the site of injury. These cause
human proteins have been obtained.9,10 migration of cells to the site of injury.
Each of the three (BMP-1, BMP-2A, and BMP-3) appears Stage 3: Vascular proliferation and ingrowth. Under the
to be independently capable of inducing the formation of car- influence of many cytokines invading capillaries bring
tilage in vivo. Two of the encoded proteins (BMP-2A and perivascular tissue with mesenchymal cells that can differenti-
BMP-3) are new members of the TGF-β supergene family, ate into osteoprogenitor cell lines. A competent conductive
while the third, BMP-1, appears to be a novel regulatory mol- substrate is required for this process to occur. This vascular
ecule. BMP factors can be synthesized by recombinant gene invasion can be significantly inhibited by nonsteroidal antiin-
technology or derived from autologous bone, allogeneic bone, flammatory medications, which will inhibit this process and
or from DBM.9,10,12 thus alter the fracture healing pathway.
DBM is allogenic bone that has undergone the acid extrac- Stage 4: The fourth stage consists of osteoclastic resorption
tion of the mineralized extracellular matrix of the allograft of the avascular (dead) bone graft lamellae and simultaneous
bone. In theory, the noncollagenous proteins, including osteo- production of new bone matrix by osteoblasts.
inductive proteins such as the BMPs, remain viable while the Stage 5: In the final stage, the newly formed bone is remod-
structural portion of the allograft has been removed.13 eled and reoriented based on the mechanical environment of
All bone graft and bone-graft-substitute materials can be the host site.
described through these three processes. While fresh autolo- Orthobiologic interventions have been specifically designed
gous graft has the capability of supporting new bone growth to target these stages to achieve a specific effect. Each broad
by all three mechanisms, it may not be necessary for a bone category of intervention will be discussed in terms of the
graft replacement material to have all three properties inher- desired effect these material have on the specific stage of graft
ent in that material in order to be clinically efficacious (Table incorporation. The gold standard and prime material that all
4-1). When inductive molecules are locally delivered on a others are compared to is AICBG.
scaffold, ultimately, MSCs are attracted to that site and are
capable of reproducibly inducing new bone formation pro-
vided minimal concentration and dose thresholds are met. In AUTOGENOUS ILIAC CREST BONE GRAFT
some clinical studies, osteoinductive agents have been shown
to potentially perform superiorly compared to conductive Fresh cancellous autograft provides the quickest and most reli-
materials alone. able type of bone graft. Its open structure allows rapid revas-
When bone marrow aspirate (viable stem cells) is applied cularization; a 5-mm graft may be totally revascularized in 20
to an osteoconductive scaffold, these cells are still reliant on to 25 days. These grafts depend on ingrowth of host vessels
the local mechanical and biological inductive signals to ulti- and perform best in well-vascularized beds. The large surface
mately form bone. Similarly, osteoconductive materials work area of harvested autograft allows for survival of numerous
well when filling noncritical size defects that would normally graft cells. It is estimated that approximately 30 mL of graft
heal easily without any additional adjuvants added. However, can be reliably harvested from an anterior iliac crest.16
in more challenging critical-size defects, all three types of However, many other sites of harvest have been described,
these materials are necessary as an adjunct in order to achieve differing only in the amount of graft obtained from each
efficacy equivalent to autograft.2,3,14 harvest site (see Table 4-1). Recent literature has demonstrated
Chapter 4 — Biology and Enhancement of Skeletal Repair 71
histologic differences between iliac crest and tibial bone graft, and osteogenic. Cell viability and osteogenic potential were
suggesting superiority of iliac crest in terms of osteogenic and similar between bone grafts obtained from both the RIA
hematopoietic progenitor cell content.17 Studies document system and the iliac crest.36
success rates approaching 100% for subcritical-size defects (1- Elevated levels of fibroblast growth factor-α (FGF-α),
to 2-cm defects) requiring 20 mL or less of autograft.9,18 platelet-derived growth factor (PDGF), insulinlike growth
There are many issues regarding AICBG because of limited factor-1 (IGF-1), TGF-β1, and BMP-2 were measured in the
quantity available, and the reported rates of postoperative pain reaming debris as compared to iliac crest curetting’s. However,
from the graft harvest site.19,20 Substantial rates of complica- vascular endothelial growth factor (VEGF) and FGF-β were
tions related to the harvest site have been reported.17,19 It has significantly lower in the reaming debris than from iliac crest
been thought that this technique is restricted to short defects, samples. In comparing platelet-rich plasma (PRP) and platelet-
in the range of 4 to 6 cm. Numerous studies report favorable poor plasma (PPP), all detectable growth factors, except
union results for critical-size defects up to 4 cm. However, in IGF-1, were enhanced in the PRP. In the reaming irrigation
many of these studies, multiple graft procedures were required FGF-α (no measurable value in the PRP) and FGF-β were
to achieve solid union.18,21-23 higher, but VEGF, PDGF, IGF-1, TGF-β1, and BMP-2 were
The ability to obtain substantial amounts of autogenous lower compared to PRP. BMP-4 was not measurable in any
graft material would appear as an advantage for the treatment sample. The bony reaming debris is a rich source of growth
of critical-sized defects. The reamer-irrigator-aspirator (RIA; factors with a content comparable to that from iliac crest. The
Synthes, Paoli, PA) offers a technique to achieve substantial irrigation fluid from the reaming also contains growth factors.
amounts of graft volumes for the treatment of larger segmental Although the early evidence regarding RIA bone grafting is
defects. The medullary canal of the femur or tibia is reamed encouraging, there is currently a lack of high-level compara-
with a device designed to collect the reamings and deliver tive evidence.
them for potential grafting procedures.24,25 Variable amounts
of harvested graft with this technique have been reported in
the literature and range from 30 to 90 mL. A recent compari- OSTEOCONDUCTIVE GRAFT SUBSTITUTES
son between a historical control group using anterior iliac
harvesting (40 patients) versus a study group using femoral There is considerable interest in creating osteoconductive
shaft RIA harvesting (41 patients) documented on average 25 matrices using nonbiological porous structures implanted into
to 75 mL of harvested RIA graft (average = 40.3 mL).24 The or adjacent to bone. The host substrate must mimic the cancel-
authors reported a favorable union rate with RIA bone graft- lous bony architecture and have very specific surface kinetics
ing (37 of 41 patients) versus AICBG (32 of 40 patients), to facilitate the migration, attachment, and proliferation of
although not statistically significant. There were significantly MSCs, which then differentiate into osteoprogenitor cells
lower postoperative harvest site pain scores from the RIA (augmentation of stages 2 and 3 of graft incorporation). Broad
group versus the AICBG group at 48 hours, 48 hours to 3 categories of these materials are available and, in general, are
months, and greater than 3 months (P = 0.001, 0.001, and classified as calcium ceramics. These include the specific mate-
0.004, respectively). There were a total of two complications rials of calcium sulfate, calcium phosphate, synthetic trical-
related to the graft harvest site in the RIA group (one perfora- cium phosphate as well as β-tricalcium phosphate, and
tion of the distal anterior femoral cortex treated conservatively coralline hydroxyapatite (HA).
and one excessive reaming of the femoral neck treated with The history of bioceramics dates back to 1892 with the use
prophylactic cannulated screws) versus 12 harvest site compli- of calcium sulfate for space-occupying lesions. Calcium sulfate
cations in the AICBG group (3 infections, 1 hematoma, and 8 has the distinction of being the alternative that is both one of
patients with numbness). the simplest as well as that which has the longest clinical
This study has several limitations including the concurrent history as a synthetic bone graft material—spanning more
use of BMP-2 in most cases. This somewhat limits the ability than 100 years.37
to draw strong conclusions regarding the relative efficacy of The original material was plaster of Paris, which is non-
RIA bone graft versus AICBG from this study.24 inflammatory and nonreactive and encouraged bone healing
A recent study reported on the treatment of 20 bone defects in a contained lesion. Peltier took commercial-grade plaster
ranging from 2 to 14.5 cm (average = 6.6 cm) using RIA bone of Paris, which was then mixed with water, poured into
graft. Eighteen of the 20 patients were initially treated with an molds made of wax paper or aluminum foil, and then allowed
antibiotic cement spacer using the Masquelet technique26-29 to set, forming small pellets or columns. He performed a
(Fig. 4-1). The average graft volume obtained using the RIA series of bone defect studies in dogs to determine the role
was 64 mL. Seventeen of 20 bone defects ultimately healed, these materials had in the ability to heal these defects.38 From
although 7 of these required repeat surgery. The authors his experiments, he determined that the plaster of Paris itself
reported no significant complications related to the bone graft does not stimulate osteogenesis. Its chief effect was found to
harvest site19,30 (see Fig. 4-1). be a mechanical one of preventing the collapse of the peri-
Numerous basic science studies have demonstrated the osteal tube and favoring regeneration. In this way the mate-
biologic potential of RIA bone graft. Investigators have docu- rial provided a supportive scaffolding.38-40 There is no doubt,
mented elevated amounts of osteoinductive growth factors31-35 however, that subperiosteal resections in which plaster of
and osteoprogenitor and endothelial progenitor cell types Paris columns were inserted regenerated, in whole or in part,
compared to AICBG. The RIA filtrate contains large numbers more frequently than was the case in subperiosteal resections
of MSCs that could potentially be extracted without enzymatic alone.
digestion and used for bone repair without prior cell expan- Subsequently, coralline HA was reported for similar lesions
sion. Medullary autograft cells harvested using RIA are viable followed by other ceramics. All of these bone graft substitutes
A B
C D
Figure 4-1. 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
to form (Masquelet technique), 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 grafting.
have the advantage of being nonimmunogenic, noninflamma- into sulfate and calcium ions and is then absorbed into the
tory, in an unlimited supply, and packaged sterile. synovium. This resorption mechanism involves a fluid
exchange mechanism and may promote excessive drainage if
Calcium Sulfate Substitutes used in wounds with questionable soft tissue coverage or
Calcium sulfate was one of the first orthobiologic materials to integrity. A low but consistent complication rate, specifically
be used commercially as a bone graft substitute. Calcium serous drainage from the wound as the calcium sulfate absorbs,
sulfate has a crystalline-independent rate of incorporation and has been reported. This complication is higher when the mate-
is very consistent in terms of dissolution and incorporation. rial is used in higher volumes (greater than 20 mL) or in
The crystalline structure is consistent throughout a whole subcutaneous bones (tibia, ulna).41
range of materials, with a constant rate of osteointegration. In This may cause a potential increase in osmotic load at the
contrast to calcium phosphate, calcium sulfate behaves as a site of implantation. Therefore, to avoid subsequent drainage
true salt, that is, if it egresses into the joint, it quickly dissolves at the graft site, the calcium sulfate product should be reserved
for situations with adequate blood supply and competent soft Hydroxyapatite
tissue coverage. Additionally, this material should be used in Synthetic HA is a crystalline calcium phosphate osteoconduc-
a contained defect only.42 Studies document that implanted tive bone substitute that is also manufactured as a ceramic
calcium sulfate pellets in contact with joint synovial fluid through a sintering process. This material was some of the
are at risk for resorption without any significant bony first available to be used clinically as a bone graft substitute.
healing response.43 If calcium sulfate pellets are to be implanted Porous HA (Interpore 500) was formed by conversion of the
in periarticular locations, complete bony containment is Porites goniopora coral exoskeleton, with pores averaging
necessary.44 600 µm and pore interconnections averaging 260 µm in
Calcium sulfate hemihydrate has been used for many diameter.1,21
years as a self-setting biomaterial due to its good setting Clinical studies with this material used for augmentation
properties. The fairly rapid degradation rate of these materials, of tibial plateau fractures were successful when used with
which occurs in 3 to 4 months, was once viewed as an internal fixation. No significant radiographic or clinical differ-
advantage.45 However, as these materials began to be used ences were appreciated between those patients that were ran-
to support articular subchondral surfaces in cases of periar- domized to have the defect filled with either autogenous bone
ticular plateau and pilon fractures, this rapid degradation or porous HA.51
becomes a distinct disadvantage.46 Transition to full weight The investigators felt that a major drawback of the material
bearing occurs normally at 3 to 4 months after surgery, and was that the rate of incorporation was very slow. The apposi-
many cases of late articular collapse have been subsequently tional process of incorporation of the implant was confirmed
reported due to this rapid incorporation with the simultane- by the finding that only 66.5% of the surface of the Interpore
ous loss of articular support.21,47 Additionally, this material 500 was covered with bone ingrowth at 12 months. Thus, a
demonstrates rapid loss in its mechanical compressive strength relatively long incorporation time was documented.51-53
following implantation, when compared to the phosphate Other investigators studied the ability of this material to act
ceramics. as a conductive substrate and determine the effectiveness of
This combination of rapid degradation rate, speedy loss of coralline HA as a bone graft substitute for lumbar spine fusion
compressive strength, and lack of bioactivity have currently when used in combination with bone marrow, or when mixed
limited its application for bone defect management.37 Three with autogenous bone graft (ABG), or combined with an
case series examining the use of calcium sulfate for the treat- osteoinductive material (BMP).54 The data indicated that cor-
ment of bone nonunion revealed a significant failure rate, alline HA with bone marrow was not an acceptable bone graft
suggesting that this material, used in isolation, is not optimal substitute used alone for posterolateral spine fusion when
to promote union in that setting.41,48 compared to autograft.
The current best use of this material appears to be that of When combined with AICBG, coralline HA served as a
a carrier for adjuvant antibiotics as a treatment for osteomy- graft extender yielding results comparable to those obtained
elitis. The characteristics regarding rapid resorption and deg- with autograft alone.51 In addition, coralline HA served as an
radation are now advantageous for delivering high-dose excellent carrier for the bovine osteoinductive bone protein
antibiotics. McKee and colleagues demonstrated results for the extract yielding superior results to those obtained with auto-
treatment of chronic osteomyelitis and infected nonunions, graft or bone marrow.54 As with other characteristics of these
using an antibiotic-impregnated calcium sulfate pellet. He felt materials, the rates of osteoinduction were greatest when a
that this was equivalent to standard surgical therapy in eradi- porous architecture was maintained.55,56 Animal studies have
cating infection and reducing the number of subsequent surgi- suggested that HA may have some osteoinductive properties
cal procedures necessary.49,50 in addition to its osteoconductive capabilities.1-3,57,58
There is level I and II evidence (one randomized trial, The crystalline structure dictates the rate of osteointegra-
one case-control study, one prospective cohort study) that tion. These materials integrate via a cell-mediated response,
antibiotic-impregnated bioabsorbable calcium sulphate has and the pore structure found in these materials serves as sites
the potential to reduce the number of procedures and surgical for cellular attachment. This porosity makes these materials
morbidity associated with the surgical treatment of chronic very brittle with minimal tensile strength.59 Because of these
osteomyelitis and infected nonunion while maintaining a high material properties, and concerns regarding very slow bone
rate of infection eradication. Calcium sulphate remains an formation, HA used alone is not commonly used as an osteo-
inexpensive, safe, reliable bone-void filler that can also serve conductive bone substitute at this time.
as an absorbable delivery vehicle for antibiotics or other The porosity of these materials is the primary factor in
compounds.49,50 determining the ability to foster ingrowth and osteointegra-
tion. No osseous ingrowth occurs with pore sizes of 15 to
Calcium Phospate Substitutes 40 µm. Osteoid formation requires minimum pore sizes of
Calcium phosphate substitutes are osteoconductive, but they 100 µm, with pore sizes of 300 to 500 µm reported to be ideal
are not osteoinductive unless growth factors, BMPs, or other for osseous ingrowth.60 Some authors, however, have reported
osteoinductive substances are added to create a composite that pore size may be less critical than the presence of inter-
graft. Calcium phosphate is available in a variety of forms and connecting pores for osseous ingrowth. Interconnecting pores
products, including ceramics, powders, and cements. Ceram- prevent the formation of blind alleys, which are associated
ics are highly crystalline structures created by heating nonme- with low oxygen tension; low oxygen tension prevents osteo-
tallic mineral salts at temperatures greater than 1000° C, a progenitor cells from differentiating into osteoblasts.1,61
process known as sintering. These phosphate materials have Highly porous interconnected materials have abundant
variable rates of osteointegration based on their crystalline sites available for cellular interactions, which help these
size and stoichiometry.1 materials osteointegrate faster. This is accompanied by a
corresponding decrease in the compressive strength afforded. and may serve to increase the immediate weight-bearing capa-
If the material is designed with minimal porosity, the rate of bilities of the repaired plateaus in clinical practice.67
osteointegration will be prolonged because of the paucity of A large clinical retrospective case series reviewed 43
cellular interactions. The corresponding compressive strength patients with traumatic bone defects or nonunions treated.
will also be very high. As noted, their ability to provide struc- Defects of the femur, tibia, calcaneus, humerus, ulna, or radius
tural support is dependent on the degree of porosity inherent had treatment augmented with TCP. Ninety percent of the
in each unique material that can be highly manipulated.1,21 fractures and 85% of the nonunions had united at the time of
These materials have the advantage of incorporating at a follow-up, average of 12 months. The authors concluded that
slower rate than calcium sulfate materials. They increase bone TCP was a useful substitute for cancellous68 bone if the local
formation by providing an osteoconductive matrix for host biology provided a suitable blood supply. This was deemed
osteogenic cells to create bone under the influence of host necessary to provide competent cells and circulating inductive
osteoinductive factors shores. factors.
The use of injectable bone calcium phosphate cements
Tricalcium Phosphate offers the opportunity to support the reduced joint surface
Tricalcium phosphate (TCP) is a commonly available resorb- without open bone grafting. This is a valuable adjuvant, as less
able ceramic. It can be obtained in block, granular, powder, or invasive fixation approaches are becoming widely accepted
putty form. Coralline ceramics are formed by thermochemi- and the ability to limit the exposures for grafting and subchon-
cally treating coral with ammonium phosphate, leaving TCP dral defect augmentation would also be a valuable tool. Jubel
with a structure and porosity that is similar to that of cancel- and colleagues evaluated the clinical and radiologic outcomes
lous bone. of an injectable calcium phosphate material (Norian SRS) used
TCP is less brittle and has a faster resorption rate than HA for tibial plateau fractures. The time interval from surgery to
because of the increased porosity. Animal studies demon- partial weight bearing was 3.7 weeks. The results demon-
strated that 95% of calcium phosphate is resorbed in 26 to 86 strated that this material can be successfully used to fill
weeks.62,63 This is much faster when compared to the previous metaphyseal bone defects in tibial plateau fractures. The clini-
HA reported rates of osteointegration. cal and radiologic results were comparable to those of frac-
TCP and HA have been combined into a biphasic calcium tures treated with autologous bone graft. The high compression
phosphate composite that has a faster resorption rate than strength allows early full weight bearing without the risk of
pure HA. In a clinical study using a composite graft, a mixture secondary loss of reduction. Soft tissue reactions due to the
of porous beads composed of 60% HA and 40% TCP ceramic cement were not observed.69 However, on all radiographs
and fibrillar collagen was used as a graft substitute and com- taken 36 months after the operation, the phosphate cement
bined with autogenous bone marrow. Collagraft (Zimmer and block was still visible indicating a very slow rate of osteointe-
Collagen Corporation) was randomized against cancellous gration, which may be of some concern to some clinicians.
iliac crest autografts in the treatment of long bone fractures. Once the transition to full weight bearing has been achieved,
This material appeared to function as well as autogenous graft it would be desirable to have the material almost completely
when used in the treatment of acute long bone fractures.64 integrated. There are newer phosphate materials now available
Calcium phosphate can also be manufactured as cement, that have shorter times to complete osteointegration as these
by adding an aqueous solution to dissolve the calcium, which crystalline structures can be manipulated accordingly.
is followed by a precipitation reaction in which the calcium In a critical study by Russell and colleagues, the efficacy of
phosphate crystals grow and the cement hardens. The primary a bioresorbable calcium phosphate cement was compared with
advantage of cements over blocks, granules, or powders is the standard autogenous iliac bone grafting in a multicenter, pro-
ability to custom-fill defects21,47 and produce increased com- spective, randomized study for the treatment of these osseous
pressive strength. However, cement can be extruded beyond defects.70 Randomization to treatment with calcium phos-
the boundaries of the fracture, potentially damaging the sur- phate cement (82 fractures) or autogenous iliac bone graft (38
rounding tissue. This is especially problematic if these materi- fractures) occurred at the time of surgery. There was a signifi-
als extrude into a joint cavity following repair of a subchondral cantly (P = 0.009) higher rate of articular subsidence during
defect such as a tibial plateau. This presents a potential disad- the 3- to 12-month follow-up period in the bone graft group.
vantage of these phosphate materials, as they will not dissolve The bioresorbable calcium phosphate cement used in this
if they happen to migrate into the joint.65 The ability of calcium study appeared to be a better choice, at least in terms of the
phosphate bone substitutes to act as a bone-void filler has been prevention of subsidence, than autogenous iliac bone graft for
documented in multiple preclinical animal studies and biome- the treatment of subarticular defects associated with unstable
chanical and human case series.66 tibial plateau fractures.
One such representative biomechanical study evaluated the Many studies have specifically evaluated these materials
fatigue strength of calcium phosphate–augmented repairs as bone graft substitutes in the management of subchondral
versus ABG repairs for lateral tibia plateau fractures. Repro- bone defects associated with tibial plateau fractures. A meta-
ducible split-depression fractures were simulated and repaired analysis study compared calcium phosphate cement substi-
with each specimen randomly assigned to either calcium tutes directly to these other conductive substrate materials
phosphate or ABG as augmentation. Calcium phosphate– used for plateau augmentation: HA granules, calcium sul-
augmented repairs subsided less and were more stiff during phate, bioactive glass, TCP, DBM, allografts, autografts, and
the fatigue loading than were ABG repairs at the 70,000, xenografts.71 Fracture healing was uneventful in more than
140,000, and 210,000 cycle intervals (P < 0.03). The authors 90% of the cases over the variable time period of the meta-
concluded that the calcium phosphate repairs had significantly analysis. Secondary collapse of the knee joint surface ≥2 mm
higher fatigue strength and ultimate load than ABG repairs was highest in the biological substitutes group, 8.6% (allograft,
DBM, autograft, and xenograft). This is similar to the results fractures underwent this method of treatment. Full weight
noted by Russell in his randomized study comparing autolo- bearing was started at 1 month postoperatively, and no
gous iliac bone graft (AIBG) to calcium phosphate cement. cases demonstrated loss of reduction. The authors felt that
Late collapse was less in the HA-treated group, 5.4%, and the the early results with stabilization and augmentation were
material that had the least subsidence was the calcium phos- encouraging.76
phate cement group. 3.7%. The group that experienced the In a similar study Eisner and colleagues compared patients
highest rate of subsidence was the calcium sulfate cases, treated surgically with and without injectable carbonated
11.1%.72 This is consistent with the rapid dissolution time and apatite cement. In the cement group, full weight bearing on
relative biomechanical properties of this material discussed the affected extremity was regained at an average of 4 weeks
previously in this chapter. postoperatively. During the study period of 3 years, only a
The recorded incidence of primary surgical site and donor slight decrease in the density of the peripheral zones of the
site infection (3.6%) was not statistically significantly different cement block was observed (again referring to the relatively
among all the grafting groups. However, donor site-related long integration time with these first- and second-generation
pain was reported up to 12 months following AIBG harvest. materials). Complete resorption and remodeling of the bone
Shorter total operative time, greater tolerance of early weight cement were not complete at 3 years.77
bearing, and improved early functional outcomes within the These findings should be temporized by the results of an
first year after surgery were noted in the studies reporting on earlier study by Schildhauer and colleagues, who reported on
the use of injectable calcium phosphate cements. Despite a lack a series of 36 joint depression–type calcaneal fractures treated
of good quality randomized controlled trials, there is arguably by internal fixation augmented with calcium phosphate
sufficient evidence supporting the use of bone graft substitutes cement.78 Patients began to bear weight as early as 3 weeks
in the clinical setting of depressed plateau fractures.69,71,72 after the surgery without loss of reduction, and no significant
These materials have also been extensively used for the difference in functional outcome scores between patients with
augmentation of distal radius fractures. Zimmermann and early versus late weight bearing. Of concern, however, was a
colleagues evaluated patients treated with cement augmenta- finding of an 11% infection rate. The majority of infections
tion 2 years after the surgery. The authors concluded that the occurred in smokers. In spite of the infection rate, the authors
use of injectable calcium phosphate cement (Norian SRS) to concluded that cement augmentation of the fixation for joint-
supplement pin and screw fixation was effective in maintain- depression calcaneal fractures allowed earlier weight bearing
ing the reduction of unstable intraarticular distal radius frac- with no change in postoperative outcomes.
tures in osteoporotic patients and provided superior functional Proximal humeral fracture augmentation has also been
outcomes at 2 years after the surgery compared to percutane- reported. Reinforcement with calcium phosphate cement in
ous pinning alone.73 With the widespread use of locked plating the treatment of proximal humeral fractures with locked
for these injuries, the efficacy of these materials for use in plates decreased fracture settling and significantly decreased
this situation must be questioned. A recent randomized study intraarticular screw penetration and helped to prevent varus
sought to determine whether augmentation of volar locking- deformation in clinical series. Augmentation was able to
plate fixation with calcium phosphate bone cement had any decrease the tendency for subsequent superior screw cutout
benefit over volar locking-plate fixation alone in an elderly from the humeral in biomechanical studies.79,80
patient population with unstable distal radial fractures.74 The Civinini and colleagues reported on the results of the treat-
two groups were comparable with regard to age, sex, fracture ment in patients with early-stage osteonecrosis of the femoral
type, injury mechanism, and bone mineral density. No signifi- head (ONFH) that underwent core decompression, injection
cant differences were observed between the groups with regard of autologous bone marrow concentrate, and the use of a com-
to the clinical outcomes at the 3- or 12-month follow-up posite injectable bone substitute calcium sulfate and trical-
examination. No significant intergroup differences in radio- cium phosphate as a mechanical supplementation associated
graphic outcomes were observed at 1-year follow-up. with decompression.81
The authors concluded that augmentation of metaphyseal At final follow-up, the mean hip scores increased from 68
defects with calcium phosphate bone cement after volar points preoperatively to 86 points postoperatively, and radio-
locking-plate fixation offered no benefit over volar locking- graphic improvement occurred in 29 hips (78.4%). The overall
plate fixation alone in elderly patients with an unstable clinical success rate of the procedure was 86.5%. The authors
distal radial fracture. This highlights the point that with the felt that backfilling the defect with an injectable bioceramic
improved biomechanics that locked plating provides, prospec- for the treatment of early stages of ONFH helped to relieve hip
tive studies are required to determine the role of these conduc- pain and prevent the progression of ONFH in the majority of
tive substrates when they are combined with locked plating the cases.81
techniques.75 Rouvillain reported on the clinical, radiologic, and
The treatment of intraarticular calcaneal fractures is com- histologic findings following high tibial valgus osteotomy
monplace in busy trauma practices. Although open reduction (HTVO) augmenting the distraction osteotomy with a micro-
and fixation are favored by many authors, increased risk of soft macroporous biphasic calcium phosphate wedge. This is one
tissue complications makes this method of treatment a chal- of the first studies to examine the results by histologic analysis
lenge. Again the value of an injectable, minimally invasive of the graft incorporation.82
material to augment posterior facet elevation would be valu- Forty-three knees underwent clinical and radiologic
able. Wee and colleagues documented in a recent clinical study follow-up at days 1, 90, and 365 to evaluate consolidation and
the safe use of an injectable calcium phosphate cement to bone substitute interfaces. Biopsies were obtained at least 1
augment standard plate fixation of calcaneal fractures. A total year after implantation from 10 patients who requested plate
of 10 patients with 12 displaced intraarticular calcaneal removal. Radiographic consolidation was observed in 98% of
patients with no late collapse noted. Histology confirmed (BM) aspirate (TCP/BM). While significant improvements in
normal bony architecture with trabecular and/or dense lamel- radiographic parameters were observed in both TCP groups
lar bone growth throughout the wedge implants. Radiographs over 2 years of follow-up, the addition of BM was not found
and microcomputed tomography (CT) scan revealed a well- to provide any significant benefit.90 This highlights the need
organized, mineralized structure in the newly formed bone. for comparative data to determine the effectiveness of just
This study confirmed that using medial biphasic calcium randomly combining these adjuvants.
phosphate (MBCP) wedges in combination with locked plates In an effort to improve the performance of calcium phos-
offered a simple, safe, and fast surgical technique for HTVO.82 phate materials, many investigators are manipulating the
Early results have demonstrated that augmentation of materials themselves in an attempt to improve the incorpora-
femoral neck and intertrochanteric hip fractures with calcium tion characteristics or the osteoinductive capabilities of these
phosphate cement is feasible, with no substantial increase in materials. The inherent osteoinductivity of silicate-substituted
complications.83,84 calcium phosphate materials was investigated in a sheep
A recent meta-analysis was undertaken to evaluate this muscle pouch study.
concept of hip fracture augmentation and came to differing Silicate substitution had a significant effect on the forma-
conclusions: 411 studies were identified, of which 22 met the tion of bone both within the implant and on the implant
rigorous inclusion criteria, comprising 12 experimental and surface during the study period. The formation of bone within
10 clinical reports. The clinical studies were evaluated with muscle during the 12-week period showed both silicate-
regard to their levels of evidence. Only four were prospective substituted calcium phosphate and stoichiometric calcium
and randomized. Polymethylmethacrylate (PMMA) and phosphate to be osteoinductive in this model. Silicate substitu-
calcium phosphate cements increased the primary stability of tion significantly increased the amount of bone that formed
the implant-bone construct in all experimental and clinical and the amount of bone attached to the implant surface. New
studies. In randomized, controlled studies, augmentation of bone formation occurred through an intramembranous
intracapsular fractures of the neck of the femur with calcium- process within the implant structure.91 This material manipu-
phosphate cement was associated with poor long-term results. lation continues to be the focus of great research interest at
There was a lack of data on the long-term outcome for tro- this time in attempt to develop materials that have both induc-
chanteric fractures. Because there were only a few, random- tive and conductive properties within the same material.
ized, controlled studies, there is currently poor evidence for
the use of any orthobiologic bone cement in the treatment of
fractures of the hip and it should not be undertaken.85 DEMINERALIZED BONE MATRIX
Investigators continue to manipulate these calcium phos-
phate substrates in an attempt to find the optimal resorption/ DBM is formed by acid extraction of the mineralized extracel-
incorporation rate with the optimal strength ratios main- lular matrix of allograft bone. It contains type I collagen, non-
tained. No authors of human studies have been able to clearly collagenous proteins, and osteoinductive growth factors,92
demonstrate the actual resorption rate of calcium phosphate including BMPs and other inductive factors found in the
cement (Figs. 4-2 and 4-3). However, the creation of macro- TGF-β group of proteins. As noted earlier, the TGF-β super-
pores can significantly improve the resorption rate of these family includes a number of factors in addition to BMPs. The
materials. This increased degradation is associated with almost factors that are known to be osteoinductive are BMPs, growth
complete bone replacement. Animal studies have shown that differentiation factors (GDFs), and possibly TGF-β1, TGF-β2,
up to 80% of the cement is resorbed at 10 weeks, with resorp- and TGF-β3.93 DBM is highly osteoconductive because of its
tion and replacement with bone continuing for as long as 30 particulate nature and presents a large surface area and three-
weeks.86,87 This process occurs by dissolution as well as by dimensional architecture to serve as a site of cellular attach-
osteoclast resorption. Biomaterials depicted a significant ment.94,95 Thus, when DBM is implanted in an animal, all of
increase in bone content, when compared to ABG, concerning these factors potentially work in combination to produce the
bone regeneration at the same time period.88 observed osteogenic response.
The lack of osteoprogenitor cells and osteoinductive poten- DBM is, strictly speaking, allogeneic bone tissue. In theory,
tial of calcium-based bone substitutes has led to the develop- the noncollagenous proteins, including osteoinductive pro-
ment of composite grafts in an attempt to accelerate bone teins such as the BMPs remain viable. However, because the
formation. A composite graft is created by adding an osteoin- true test of osteoinductivity is whether a material that has been
ductive factor to an osteoconductive calcium phosphate implanted in a nonosseous site forms bone, the inability of
matrix to theoretically increase bone formation. Many clinical allograft bone to do this in human patients argues against
series use composite ceramic grafts combining the scaffolding allograft bone having substantial osteoinductive activity. DBM
properties of TCP materials with biological elements to stimu- has been shown to produce this effect in animal studies,96-98
late cell proliferation and differentiation.89 but it too has never demonstrated this effect in human patients.
As previously mentioned, the incorporation times of some The relative quantities of BMPs in DBMs are low, in the order
of these materials can be prolonged because of a number of of 1 × 10-9 g of BMP per gram of DBM. The osteoinductive
factors. In an attempt to improve the incorporation and inte- variability has been found not only across different DBM
gration characteristics, investigators combined ultraporous products but also among production lots from the same DBM
β-TCP synthetic graft material (Vitoss Bone Graft Substitute, formulation.99 Bae and colleagues demonstrated that there
Orthovita) with bone marrow aspirates, with the hypothesis is higher variability in the concentration of BMPs among
that bone marrow aspirate speeds incorporation of the bone different lots of the same DBM formulation than among dif-
graft substitute. The study prospectively examined healing of ferent DBM formulations. Each individual lot has a different
cavitary defects filled with TCP versus TCP and bone marrow osteoinductive capacity that can vary considerably among
A B
C D
E
Figure 4-2. A, Split depression tibial plateau fracture. Computed tomography (CT) scans demonstrate significant lateral compartment articular
impaction and comminution. B, Lateral articular surface was elevated and the subchondral defect grafted with a particulate calcium-phosphate-
sulfate composite osteoconductive bone-void filler to help maintain articular reconstruction (left). To fill all cancellous voids, an injectable form
of the same material was injected, and following a short set period, the plate and screw construct was applied, drilling directly through the
material (center). This particulate 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 a small particulate piece of material has egressed into joint. Because of the particulate material properties, this small amount
dissolved in the synovial fluid environment and was absorbed without any harmful articular sequela (right). C, 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. D, Three-month postoperative images. Articular surface reduction remains intact. Calcium
ceramic is noted to be much smaller with enveloping bone around the material noted. E, Six-month postoperative images. 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.
different donors. This variability questions the reliability of DBM has also been evaluated.102 Wildemann and colleagues
DBM products and, possibly, their efficacy in providing con- attempted to quantify the activity and presence of eight growth
sistent osteoinduction.100 factors important for bone healing in multiple different “off-
Prior investigations have shown that the osteoinductive the-shelf ” DBM formulations, which are already in human
potential can vary widely, with influence from both donor and use. Differences between the products were seen in total
processing sources. There appears to be sex-related differences protein content and the absolute growth factor values. The type
in donor DBM. DBM derived from female donors appears to of the growth factors found in these preparations was almost
have significantly greater concentrations of BMP-2 and BMP-7 comparable between the materials. FGF and BMP-4 were not
than that derived from male donors (P = 0.0257 and 0.0245, detectable in any analyzed sample. BMP-2 revealed the highest
respectively). There was no significant correlation between concentration extractable from the samples without a signifi-
donor age and the levels of any of the measured BMPs in a cant difference between the three DBM formulations.
study evaluating BMP levels in commercially available DBM In general, all the materials had variable concentrations
preparations.101 The presence of other growth factors found in of TGF-β1, FGF-α, IGF-1, and PDGF. No differences were
A B
Figure 4-3. A, Computed tomography (CT) images demonstrating significant metaphyseal defect following a crushing injury to the medial
tibial plateau. B, At the time of definitive fixation, a percutaneous injection of a calcium ceramic was completed, completely filling the metaphy-
seal defect and supplying articular support in conjunction with the fixation hardware. Plain radiograph at 3 months demonstrates minimal
incorporation of the material. This material was selected for its longer integration time period to help maintain the metaphyseal region past the
transition to full weight bearing.
accessed for VEGF. This and other similar studies highlight limitations imposed by radiation, it seems most practical to
the differences in the growth factor concentrations between handle DBM aseptically throughout the procedures of com-
the individual materials, independent of the product formula- positing pastes, putties, or suspensions, and only if necessary,
tion. As well, there is evidence of differential potencies of each exposing bone components to radiation sterilization prior to
available growth factor within individual DBM preparations, mixing.103,104
based on the individual manufacturer and manufacturing In addition, many of the proteins responsible for “turning
process.13 on” the differentiation process for stem cells will not function
There are numerous DBM formulations based on refine- if damaged, and protein preservation is therefore important in
ments of the manufacturing process. They are available as the delivery of DBM and BMPs. Some DBM products are
freeze-dried powder, granules, gel, putty, or strips. They have lyophilized and mixed with carrier at the time of implantation,
also been developed as combination products with other while others are prehydrated or premixed and could poten-
materials such as allogeneic bone chips and calcium sulfate tially remain on the shelf for a long period of time.105 If precise
granules. There is now evidence of differential potencies of control of conditions is not maintained, many proteins might
DBM preparations based on the manufacturer and manufac- be susceptible to chemical and physical degradation.106
turing process.13 Sterile processing of the bone may also affect Ethylene oxide as a means for sterilization of DBM is used
the protein effectiveness of these materials. Some tissue banks by some manufacturers and has been shown to attenuate its
harvest and process the DBM under “sterile” conditions osteoinductive potential.107 Exposure of DBM to ethylene
whereas other manufacturers sterilize the DBM after pro oxide for the duration required to kill most common bacterial
cessing. Gamma irradiation is frequently used to sterilize pathogens results in a marked reduction of its osteoinductivity
implanted devices but has limitations when used on biologi- most likely due to destruction of BMPs and other inductive
cally active materials and composites. factors.108 The effect of ethylene oxide has been shown to be
Studies indicate that if the DBM is irradiated prior to the dose dependent.108
addition of any aqueous carrier, the activity of DBM in the dry It is clear that processing and sterilization techniques have
state remains relatively stable with only a small loss of activity. significant effects on DBM viability. With this in mind, clini-
Composites of DBM with a carrier such as lecithin, to which cians should be aware of how these factors influence the effi-
no water has been added, lose activity at approximately the cacy of each particular DBM product that they may choose to
same rate as DBM in the dry form. In composites that contain use in each clinical application.
water, the loss of activity occurs even at much lower levels of The shelf life of the product may also vary with the specific
radiation exposure. Osteoinductivity of DBM decreased with carrier, which may or may not affect the overall activity of the
the increase of gamma-irradiation dose at ambient tempera- product. All available in vitro assays evaluate the DBM without
ture, whereas no decrease occurred when treated with gamma the carrier added by the manufacturers. Carriers include glyc-
irradiation at low temperature. However, the hydrated DBM erol (Grafton DBM, Osteotech, Inc., Eatontown, NJ), synthetic
showed diminishing osteoinductivity after 6-month storage at polymer (Dynagraft, GenSci OrthoBiologics, Inc., Irvine,
ambient conditions, whereas the DBMs in dry form retained CA), porcine gel (Osteofil, Regeneration Technologies, Inc.,
their osteoinductivity after the 6-month storage. The findings Alachua, FL), and carboxymethylcellulose (Allomatrix Inject-
in this study indicate that DBM and demineralized bone able Putty, Wright Medical Technology, Inc., Arlington, TN).
matrix/acellular dermal matrix (DBM/AM) composites could DBM is also available in a particulate powder form that has
retain their osteoinductivity when they are in dry configura- no carrier material present. This form is useful to admix to
tion and are irradiated at low temperature (−40° C to −70° C).103 other materials such as iliac aspirate or autograft and platelet
Gamma irradiation does not change cell attachment to gels (Fig. 4-4).
the DBM matrix but has an influence on both stem cell Bioactivity can be measured by in vivo assays (which
and osteoprecursor cell proliferation rates. Because of the measure de novo bone formation and alkaline phosphatase
C
Figure 4-4. A, Demineralized bone matrix (DBM) can be delivered in many forms. Particulate DBM is a powder without any carrier (top left).
A DBM composite putty is infiltrated with a carrier that allows the material to be applied directly or injected (bottom left). Demineralized cancel-
lous chips can be infiltrated with concentrated marrow or platelet concentrate as seen here (right). 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 with
interconnecting pores and presenting a tremendous surface area (DBM) as a site for potential marrow cellular attachment.
levels) and in vitro assays (which measure stimulation of these studies suggest that DBM putty enriched with BM may
human osteoblast culture).109-111 be comparable to autograft for treating long bone fractures
Only a few tissue banks use an in vivo model to measure and nonunions. This option offers the distinct advantages of
the bioactivity of the final DBM product with the carrier. A decreased morbidity, reduced costs, and shorter hospital stay
study by Han and colleagues evaluated the effects of moisture compared to AICBG.117
from water-based carriers and the storage temperatures on There is only one level 1 randomized prospective study in
osteoinductivity of known DBM products. This was done to humans available, evaluating the use of DBM as a solitary graft
evaluate these materials with regard to shelf life in an operat- material. This study used DBM alone to treat a standardized
ing room. In a dry state, without a carrier, DBM can preserve critical-sized fibular defect, grafted using only DBM (positive
its osteoinductive activity when temperatures reached 65° C control) compared to a similar defect with no graft applied
(149° F), but in the presence of moisture, (carrier substances) negative (untreated) controls.119
the activity decreases with incubation time. Nearly 90% of the This was the first phase of a study done in conjunction with
DBM activity is lost when maintained for 5 weeks at 65° C a prospective, randomized double-blind study in 24 patients
(149° F).105 undergoing high tibial osteotomy to evaluate the effectiveness
While the preclinical data are impressive for DBM forming of human recombinant osteogenic protein (OP-1) on a type I
de novo bone in lesser animal models,98 the human clinical collagen carrier in a critically sized fibular defect (phase II).
data are deficient with only isolated case reports and uncon- The results of phase I established the critically sized nature of
trolled retrospective reviews. These level 3 and 4 studies the defect. In the defect group with no graft augmentation, no
suggest the potential therapeutic effects of DBM.112 The bony changes were observed while nonunion resulted in all of
maxilla-craniofacial field published the bulk of the literature these defects. In the group grafted with DBM, formation of
documenting successful reconstruction of complex deformi- new bone was visible from 6 weeks onward with many defects
ties using DBM alone as a bone graft substitute.113-115 Reported undergoing complete healing. The results of the second phase
success rates for mandibular and maxillary reconstructions showed no significant formation of new bone in the presence
are consistently more than 90%. of collagen graft alone, while in the OP-1 group, all patients
One of the first clinical series was published by Tiedeman except one showed formation of new bone from 6 weeks
and colleagues. They reported on an uncontrolled case series onward. This study demonstrated that both DBM and OP-1
of 48 patients in whom DBM had been used in conjunction exhibited osteogenic activity in a validated critically sized
with BM for the treatment of skeletal injuries. Thirty-nine human defect.
patients were available for follow-up, and 30 of them demon- In a similar study, Hierholzer and colleagues attempted to
strated healing.116 These results were encouraging; however, evaluate the healing of humeral shaft nonunions using a con-
because there was no control group, the role of DBM in sistent surgical protocol but compared the use of two different
patients who demonstrated healing remains unknown. Unfor- types of bone graft: autologous iliac crest bone graft and DBM
tunately, most clinical series combined DBM with other adju- in a consecutive retrospective cohort series.120 Forty-five
vants and, as noted earlier, the singular effectiveness of DBM patients were treated with AICBG and 33 grafted with DBM
alone is difficult to elucidate. only. Union was noted clinically and radiographically in 100%
Two separate investigators evaluated the effectiveness of of the 45 patients treated with autologous bone graft and 97%
combining DBM with BM aspirate for the treatment of acute (32) of the 33 patients treated with DBM (not significant evi-
long bone fractures and nonunions. Wilkins and colleagues dence). The overall functional outcome did not differ between
treated 66 patients with 69 “stiff ” nonunions with a prospec- the groups; however, 20 (44%) of the autologous bone graft
tive protocol. The only therapeutic intervention was the per- recipients had donor site morbidity, including prolonged pain
cutaneous administration of a mixture of autologous BM and in the majority and a superficial infection requiring irrigation
allograft DBM on an outpatient basis. Sixty-one of the percu- and débridement in 1 patient. These results demonstrated that
taneous treatments (88%) resulted in union (level III evi- consistent healing could be achieved with commercially avail-
dence), however, there was no comparative control group able DBM alone (level III evidence).
other than historic controls with which to compare the effec- Augmentation of spinal fusion appears to be the most fre-
tiveness of this composite graft.117 quent application of DBM. In the more challenging environ-
Lindsey studied the effectiveness of a composite graft con- ment, such as posterolateral spine fusion, multiple formulations
sisting of DBM putty (Grafton DBM) and aspirated BM for and combinations have been used.121 Significant differences
treating long bone acute fractures.118 Patients were random- exist between the various forms of DBM in their ability to
ized to treatment with either the DBM putty composite or iliac generate spine fusion in animal models. This must be consid-
crest autograft, with a minimum of 12 months of radiographic ered when analyzing the data from human studies.
follow-up. Ninety percent of DBM patients achieved full bone In a side-by-side comparative study, a total of 120 patients
formation compared to 75% of autograft patients (P = 0.41). underwent posterolateral spine fusion with pedicle screw fixa-
Additionally, all DBM patients were healed compared with tion and bone grafting. Iliac crest autograft was implanted on
63% of autograft patients (P = 0.07). Both of these studies one side of the spine and a Grafton DBM/autograft composite
suggest that DBM putty enriched with BM may be comparable was implanted on the contralateral side in the same patient.
to autograft for treating long bone fractures and nonunions. The bone graft mass was fused in 42 cases (52%) on the
However, in the case of acute fractures, the number of Grafton DBM side and in 44 cases (54%) on the autograft side.
patients in each group was 10 or less, and although this was a The authors concluded from this level III study that DBM can
randomized prospective trial (level I evidence), the numbers extend a smaller quantity of autograft than is normally
are too small to draw definitive conclusions regarding this required to achieve a solid spinal arthrodesis. Consequently, a
composite graft option. Despite these limitations, both of reduced amount of harvested autograft may be required,
potentially diminishing the risk and severity of donor site aspirate has a high concentration of connective tissue progeni-
complications.122 tors. One milliliter of iliac aspirate contains approximately 40
Thus, the use of DBM as a bone graft extender appears to million nucleated cells, 1500 of which are connective tissue
be a viable indication for its use. progenitors.126
In a larger 2-year prospective study, a randomized clinical
trial compared the outcomes of Grafton DBM combined with Historic Perspective
local bone, with that of iliac crest bone graft (ICBG) in a Connelly’s work with unfractionated BM aspirate was instru-
single-level instrumented posterior lumbar fusion. Patients mental in stimulating clinical interest for using marrow as an
were randomly assigned (2 : 1) to receive Grafton DBM with adjunct graft material for fracture and nonunion healing. His
local bone or autologous ICBG. initial clinical series used autologous marrow injection to
At 2-year follow-up, subjects who were randomized to stimulate healing in 20 ununited tibial fractures over a 5-year
Grafton DBM and local bone achieved an 86% overall fusion period. His injections were combined with either the use of a
rate versus 92% (ICBG) (P = 1.0, not significant evidence) and cast (10 patients) or a Lottes nail (10 patients).128 The two
improvements in clinical outcomes that were comparable with failures were in the cast treatment group. BM injection was as
those in the ICBG group.123 There was a statistically significant effective as past open autologous grafting but with consider-
greater mean intraoperative blood loss in the ICBG group ably fewer disadvantages. These early results were not dupli-
than in the Grafton group (P < 0.0031). cated by others, but did stimulate researchers to document
A recent comprehensive meta-analysis reviewed the use of enhanced bone healing through the use of marrow cell–based
DBM for spinal fusion series. Articles were critically examined strategies in vitro and in animal studies.
and compared according to study design, DBM type, out- Connolly continued to investigate the effects of concentrat-
comes, and results. The primary outcome of interest was ing marrow by centrifugation in a rabbit nonunion model. The
fusion rate. Secondary outcomes included the Oswestry Dis- results with centrifugation were superior to those with unpro-
ability Index, SF-36 survey, Odom’s criteria, visual analog scale cessed marrow. And thus the concept of a threshold level of
(VAS), neurologic pain score, Japanese Orthopedic Associa- cells necessary for osteogenesis to occur in ectopic sites was
tion myelopathy score, Neck Disability and Ishihara Curvature promulgated.128-130
indices, and pseudarthrosis and surgical failure rates. The
majority of human clinical trials report high fusion rates when Current Methodology
DBM is employed as a graft extender or a graft enhancer. Few The failures and low rates of healing associated with the use
prospective randomized controlled trials have been performed of unfractionated BM may reflect the paucity of osteoprogeni-
comparing DBM to autologous ICBG in spine fusion.124 tor cells present in the mature marrow aspirate. Even in a
Although many animal and human studies demonstrate normal adult only 36 to 55 of every million nucleated BM cells
comparable efficacy of DBM when combined with autograft will undergo osteogenic differentiation.127 Because osseous
or compared to autograft alone, additional high level of evi- regeneration is dependent on the number of cells available to
dence studies are required to clearly define the indications for participate in bone synthesis, patients with fewer local cellular
its use in spine fusion surgeries and the appropriate patient precursors will typically have a poorer healing response
population that will benefit from DBM. leading to potential nonunion. As greater volumes of BM are
harvested from the iliac crest, the concentration of osteopro-
genitor cells decreases because the sample undergoes consid-
PATIENT-DERIVED CELLULAR THERAPIES erable dilution with peripheral blood.
The aspiration technique is very specific in order to maxi-
Current orthobiologic strategies have progressed from an mize the number of effective progenitor cells per unit
approach based primarily on biomaterials to a cell- and tissue- volume.131 Muschler and colleagues studied this issue and
based approach that includes understanding of cell sourcing determined that no more than 2 mL of blood should be aspi-
and bioactive stimuli. New options include methods for rated from any given area in the iliac crest to avoid dilution
harvest and transplantation of tissue-forming cells, combined with peripheral blood127 (Fig. 4-5). On the basis of these data,
with bioactive scaffold matrix materials, and delivery of bioac- a selective retention (filtration) system was developed that has
tive molecules that allow these stem and progenitor cells to the ability to concentrate progenitor cells three to four times
differentiate into the appropriate cell lineage for specific tissue and load them onto an allograft substrate for delivery.132
repair.125,126 Available cell-based strategies include targeting Other sites of harvest for marrow aspirate have been
local cells with use of scaffolds or bioactive factors, transplan- assessed to determine if there is variability of the quality of
tation of autogenous connective tissue progenitor cells derived cells obtained compared to the results of aspirate from the iliac
from BM or other tissues, and the use of autologous growth crest.133 A comparative study harvested BM aspirates from the
factors obtained from the patient’s own platelets. ipsilateral anterior iliac crest, distal tibial metaphysis, and cal-
caneal body all from the same patients. The samples were then
Marrow Aspirate centrifuged to obtain a concentrate of nucleated cells, which
The critical component necessary to all bone formation is the were plated and grown in cell culture. The anatomic locations
ability to provide viable osteoprogenitor cells. BM is a plentiful were compared between the 40 patients enrolled in the study.
source of musculoskeletal stem cells, but the cells can also be Clinical parameters (including sex, age, tobacco use, body
found in periosteum, cartilage, muscle, fat, and vascular peri- mass index, and diabetes) were assessed as possible predictors
cytes.127 Connective tissue progenitors describe the population of osteoblastic progenitor cell yield. BM aspirate collected
of stem cells and progenitors that are actively engaging in from the iliac crest had a higher mean concentration of osteo-
proliferation and differentiation into connective tissue. A BM blastic progenitor cells compared with the distal tibia or the
Figure 4-5. 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; 3 to 4 cc 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. Following needle repositioning,
the plunger can be unlocked and additional aspirate obtained.
calcaneus (P = 0.0001). There was no significant difference in Preclinical Substantiation

concentration between the tibia and the calcaneus (P = 0.063). Many investigators reported enhanced bone healing through
Age, sex, tobacco use, and diabetes were not predictive of the use of cell-based strategies in vitro and in multiple pre-
osteoblastic progenitor cell yield.133 clinical animal studies. In addition to the rabbit model that
Similar comparisons evaluated aspirates of vertebral body Connolly used to evaluate the beneficial effects of cellular
marrow compared to matched controls from the iliac crest of concentration, a similar study was performed using a canine
the same patient. Vertebral body aspirates demonstrated com- tibial nonunion model. Distraction gaps were maintained
parable or greater concentrations of progenitor cells compared with external fixators and were treated with either a bone graft
with matched controls from the iliac crest. The concentration of concentrated marrow aspirate, or DBM, or a composite
of osteogenic progenitor cells was, on the average, 71% higher graft of both materials.137 A separate group treated with auto-
in the vertebral aspirates than in the paired iliac crest samples graft was used as the control group. Use of the combination
(P = 0.05).133,134 of DBM and marrow concentrate (composite graft) yielded
However, the concentration and yield of colony-forming results that were superior to DBM or aspirate groups alone.
connective-tissue progenitors is greater when aspirate is The results of the composite graft were similar to those in the
obtained from the posterior crest compared with the anterior autograft group.
iliac crest. It has also been found that the biologic potential of The concept of composite grafts combining marrow ele-
the cells derived from both of these sites appears to be ments with other conductive and or inductive substrates has
comparable.135 become a major area of interest based on the early results
Cellularity of BM has been found to decrease with age and documenting the superiority of composite grafts compared to
for women.126,136 Surprisingly, there appears to be no relation- grafting with marrow aspirates alone. The ability to load cells
ship between smoking status and marrow cellularity, cellular into an osteoconductive substrate with a microporous struc-
prevalence, or cellular numbers. Studies have found that ture provides the cells with a potentially stable and well-
tobacco use is not associated with a change in prevalence of vascularized environment.138 In this situation, most of these
osteogenic progenitor cells in BM, or their intrinsic biologic cells will differentiate into osteoblasts, whereas in sites that are
capacity to undergo early osteoblastic differentiation.126 mechanically unstable and less well vascularized, the cells tend
to become chondrocytes.139 If conditions are not right for the Clinical Application of Marrow Elements
optimal differentiation of these MSCs into bone or cartilage The use of marrow elements has been used clinically to
cells, they will differentiate along a default pathway and augment bone healing with variable results. Most of the early
become fibroblasts140; when this occurs, nonunion results. series involved the simple aspiration and injection of iliac
Thus, the emphasis on providing the optimal conditions for crest aspirates as noted previously in this chapter document-
these composite grafts. ing the work of Connelly.128-130,147 He was one of first surgeons
Bruder and colleagues evaluated BM combined with a to report on the use of BM aspirate as a clinical alternative to
porous TCP cylinder in a canine nonunion model stabilized autograft (level IV evidence). Other early case studies or series
with plates. Use of the composite graft demonstrated results document results for simple marrow injections. These studies
that were superior to those of treatment with the ceramic were primarily single or multiple surgeons with no historic or
cylinders alone, which resulted in only modest bone case-matched controls (level IV evidence). These studies
formation.141 include Garg et al., who reported good results in his series of
Bruder continued to investigate marrow elements in a 20 patients treated with BM injection148 for long bone non-
composite graft using a selective retention filtration strategy union. Healey et al. successfully treated nonunions in a group
to concentrate marrow elements. In a canine model, selective of eight children with cancer by simply injecting BM aspi-
retention cellular aspirate concentrates were mixed with DBM rate.149 Wientroub et al. reported on the use of autologous
and were found to be equivalent to autograft in their ability marrow to improve the effectiveness of allografts in 23 chil-
to repair critical-size defects.142 Additional studies using the dren.150 And finally, Goel et al. sequentially injected 3 to 5 mL
concept of cellular concentrates enriching a DBM carrier of aspirated BM directly into the nonunion site. Multiple iliac
matrix in a canine spine fusion model demonstrated similar crest aspirations were performed until a maximum of 15 mL
results. The selective cellular concentration process allows the of marrow was injected. Clinical and radiologic bone union
number of connective tissue progenitors to be increased three- occurred in 15 out of 20 patients (75%).151
to fourfold. These studies were performed without the benefit of our
In a canine study by Muschler and colleagues, enriched current knowledge regarding appropriate aspiration tech-
aspirate composite DBM grafts delivered a mean of 2.3 times niques, the advantage of cellular concentrates, or the use of
more cells and approximately 5.6 times more progenitors than composite grafting methods, yet meaningful results were
DBM mixed with simple unconcentrated BM. A union score, achieved.
quantitative CT, and mechanical testing results all demon- In spite of the encouraging preclinical and rudimentary
strated the use of the selective-retention-enriched bone DBM clinical studies, the use of these grafts as a substitute have not
to be superior to the use of bone DBM alone and DBM plus enjoyed widespread use. This may be due to a host of reasons,
unconcentrated marrow.132 such as (1) the variability resulting from inconsistent and
Composite grafts have also been devised using cellular incorrect aspiration techniques and faulty instrumentation
elements in addition to osteoinductive proteins. Lane and necessary to achieve consistent aspirates; (2) low osteopro-
colleagues investigated the potential of combining BM cells genitor content of these insufficient aspirates; and (3) the com-
with recombinant human BMP (rhBMP-2) in a rat femoral bination of these aspirates with suboptimal scaffolding
defect model.143 This combination was superior to either materials.152
rhBMP-2 or marrow cells by themselves as well as to treatment Contemporary clinical series have demonstrated excellent
with syngeneic bone grafting. The authors believed that results when the above factors have been attended to. Studies
this represented a synergistic effect of the two materials that use correct aspiration and concentration methodologies
and emphasized the importance of growth factors being as well as adhere to using appropriate documented composite
present. grafting techniques do document the value of marrow aspi-
Animal models have been used to evaluate composite rates for graft substitution.
grafts that incorporate all three components, that is, cellular The use of bone marrow aspirate (BMAC) in conjunction
concentrate, a conductive substrate, and inductive proteins in with specific osteoconductive substrates appears to be a suit-
varying combinations. A preclinical defect study evaluated a able substitution for AICBG when used in specific situations.
sheep tibial defect treated with HA combined with either Multiple level IV studies have documented the effectiveness of
rhBMP-7 or BM compared to autograft application.134 Treat- this modality for a variety of indications. It has been used
ment with the composite grafts yielded results that were primarily for defect management found in posttraumatic long
as good as those in an autograft control group and were supe- bone defects and acetabular bone defects discovered during
rior to those in either a void group or a group treated with revision total hip arthroplasty.153-155
HA alone. A new area of clinical use for BMAC is for the treatment
With the vast improvements in limb salvage techniques, the of avascular necrosis (AVN) of the femoral head, as results
reconstruction of large defects in humans has become more have been seen following grafting using BMAC with a variety
commonplace and has historically relied on autograft bone of carrier matrices.156,157
grafts. Limiting factors include availability of graft material, Hernigou and colleagues reported on 60 patients with non-
comorbidity, and insufficient integration into the damaged infected nonunion who had undergone BM aspiration from
bone. Complex animal models now routinely evaluate hard both iliac crests. These aspirates were then concentrated using
and soft tissue scaffoldings in combination with a multitude a centrifuge technique to achieve a baseline number of colony-
of inductive materials as well as applied cellular concentrates forming units (CFUs) present in the composite graft to be
in an attempt to determine the optimum cocktail that can injected into the nonunion site.
compare to the gold standard of AICBG for the treatment of He demonstrated complete healing in 53 of the 60 patients.
segmental defects.144-146 Those patients contained >1500 progenitors/cm3 and an
average total of 54,962 ± 17,431 progenitors. The concentra- designed to encourage rapid vascular ingrowth with
tion (634 ± 187 progenitors/cm3) and the total number (19,324 maximal cellular viability.
± 6843) of progenitors injected into the nonunion sites of 3. The graft has osteogenic, osteoinductive, and osteoconduc-
the seven patients in whom union was not obtained were tive properties, and is capable of directing new bone forma-
both significantly lower (P = 0.001 and P < 0.01, respectively) tion at the site of implantation.164
than those in the patients who healed. There was a high cor- The material appears promising; however, there is a paucity
relation between the volume of mineralized callus at 4 months of clinical data available regarding its use. Most of the informa-
and the number and concentration of CFUs available at the tion available is limited to “commercial white papers” (level V),
time of implantation. The seven patients in whom the fracture case reports (level V), and uncontrolled case series (level IV).165
did not unite had lower numbers and concentrations of CFUs. The largest uncontrolled retrospective study reviewed the
The efficacy of this technique appears to be related to the clinical effectiveness of a MSC allograft to achieve radiologic
number of progenitors available in the graft and highlights the arthrodesis in adult patients undergoing lumbar interbody
need to concentrate the aspirates and achieve the baseline fusion surgery for a variety of different indications. Fifty-two
number of cells necessary to achieve osteogenesis (level III consecutive patients received lumbar interbody fusion at one
evidence).158,159 However, there is a lack of prospective ran- (69%) or two contiguous (31%) levels of lumbar spine for
domized studies including controls for cell therapy for bone various indications.166 Solid arthrodesis was achieved in 92.3%
defects. of patients at median follow-up time of 5 months and the
A randomized prospective spine fusion study evaluated authors felt this was a safe and effective graft alternative for
posterolateral fusion patients (PLF). Following PLF instru- adult patients undergoing lumbar interbody spinal fusion pro-
mentation, approximately half the patients were randomized cedures. This study has significant limitations with multiple
to be grafted with allograft alone, and the other group ran- levels being treated, no control group, limited follow-up
domized to BMAC together with allograft (level I evidence). numbers, and follow-up time of only 5 months.
Statistically significant differences between both groups was Twenty-three patients were treated and results reported in
proven for CT evidence of ongoing fusion at 12 months (P = a retrospective review of foot and ankle patients using MSC
0.041) and at 24 months (P = 0.011) with the BMAC group, allografts for the treatment of various nonunion conditions
which was a significantly better alternative for the PLF than (level IV). This uncontrolled case review found radiographic
the allograft alone. The authors state that the use of autologous new bone formation was observed at the area of implantation
MSCs in form of the BMAC in combination with allograft was and a 91.3% union rate was observed, and no evidence of graft
an effective option to enhance the PLF healing.160 rejection or complications associated with implantation was
Another randomized study using contemporary BMAC recorded. No evidence of graft rejection or complications
concepts was completed for the treatment of posttraumatic associated with implantation was noted.164 With no level I
bone defects. Thirty-nine patients with critical bone deficien- evidence currently available, and minimal level IV and V data
cies were treated with concentrated BMAC in a prospective presented, the routine use of this material cannot be recom-
clinical trial. A collagen sponge served as scaffold in 12 patients mended at this time.
and a bovine HA substrate was applied in the other 27 indi-
viduals for the composite graft applications. All patients Platelet-Rich Plasma
showed new bone formation in radiographs during follow-up. Following an acute fracture or an operative intervention, plate-
Complete bone healing was achieved in the HA group after lets are activated by thrombin and subendothelial collagen.
17.3 weeks compared to 22.4 weeks in the collagen sponge During activation, the α granules within platelets fuse with
group. The average concentration factor of BMAC was 5.2 the platelet plasma membrane and release some of their protein
with a standard deviation of 1.3. Flow cytometry confirmed contents to the surroundings (degranulation). The α granules
the mesenchymal nature of the cells161 (level II evidence). This in platelets contain more than 30 bioactive proteins, many of
excellent study documented the value of cellular concentra- which have a fundamental role in hemostasis and/or tissue
tion (BMAC) as well as the importance of a specific conduc- healing.3 These proteins include PDGF (including aa, bb, ab
tive substrate used as a competent cellular carrier.162,163 isomers), TGF-β (including β1 and β2 isomers), platelet factor
In an effort to avoid any marrow harvest and intraoperative 4, interleukin-1, platelet-derived angiogenesis factor, VEGF,
graft manipulation, as well as provide a consistent graft mate- epidermal growth factor, platelet-derived endothelial growth
rial, allogeneic human stem cells harvested from cadaver factor, epithelial growth factor, IGF, osteocalcin, osteonectin,
donors is now available as a “stem cell” graft. The majority of fibrinogen, vitronectin, fibronectin, and thrombospondin-1.3
technical information is proprietary and not readily available Platelets begin actively secreting these proteins within 10
with regard to graft specifics in terms of harvesting, immuno- minutes after clotting, with more than 95% of the presynthe-
genicity, and cell morphology. The grafts are composed of sized growth factors secreted within 1 hour.167,168
viable MSCs derived from cadaveric donor tissue and are
delivered on an osteoconductive substrate (DBM). This graft Platelet-Derived Growth Factor
has many theoretical advantages over BMAC: PDGF is the first growth factor to jump-start nearly all
1. The graft is “preconcentrated,” such that a threshold con- wound healing.169 When activated, this growth factor attaches
centration of cells is present on arrival, without having to to transmembrane receptors on target cells, including osteo-
manipulate the aspiration prior to implantation, as is done blasts and fibroblasts. The main function of PDGF is stimulat-
with an autogenous BMAC harvest. ing cellular replication (mitogenesis). This growth factor
2. The grafts are delivered preadmixed with a competent increases cell populations of healing cells, including MSCs
osteoconductive substrate with a matrix designed for and osteoprogenitor cells. PDGF also activates macrophages,
optimal cellular attachment and porosity. This matrix was resulting in débridement of the surgical or traumatic site. The
macrophage activation then triggers a second source of growth The data obtained from these in vitro investigations dem-
factors released from the host tissues under the influence of onstrate that the cellular responses induced by PRC and bone
the macrophage action.170 graft materials in human bone marrow stromal cell (hBMSC)
can be significantly (positively or negatively) modified by
Transforming Growth Factor-β adding the agents in combination. These in vitro data high-
TGF-β stimulates the proliferation of osteoblast precursor cells, light the need to consider the potential interaction between
and it has direct stimulatory effects on bone collagen synthesis. biologic agents when added in combination.174 PRC is not a
Therefore, TGF-β modulates bone matrix synthesis by increas- true osteogenic agent but rather is osteopromotive, with cell
ing the number of cells capable of expressing the osteoblast fate determination being dependent on additional signals
genotype, as well as direct upregulation of osteoblasts. TGF- derived from the microenvironment.
β also decreases bone resorption by inducing apoptosis of
osteoclasts.169 TGF-β activates fibroblasts to induce collagen Preclinical Studies
formation, endothelial cells for angiogenesis, chondroprogeni- The majority of early preclinical and basic science work
tor cells for cartilage, and mesenchymal cells in an effort to regarding the use of PRC for potential clinical applications has
increase the population of wound-healing cells. Thus, platelet been encouraging and was primarily found in the periodontal,
gels provide a rich source of growth factors that serve a critical oral surgery, and maxillofacial surgery literature. PRP has
function in wound and fracture healing and can stimulate the been suggested for use to increase the rate of bone deposition
formation of blood vessels; the invasion of pluripotential MSCs, and quality of bone regeneration when augmenting sites prior
monocytes, and macrophages; and the further aggregation of to or in conjunction with dental implant placement.175 The
platelets. These factors have direct chemotactic and mitogenic orthopaedic focus has primarily been on the ability of PRP to
effects on osteoblasts and osteoblast precursors and act syner- augment bone formation and healing in diabetic fracture
gistically to help stimulate bone remodeling and healing.21,170 healing models, wound healing, and defect augmentation.
In a diabetic fracture model study, a significant reduction
Platelet-Rich Plasma Augmentation in PDGF, TGF-β1, IGF, and VEGF expression was demon-
of Graft Materials strated in the diabetic fracture callus compared to the nondia-
By providing a variety of concentrated growth factors, PRP betic fracture callus.176 The application of PRP restored early
can play an important role in enhancing the bone healing cell proliferation during healing to levels comparable to non-
pathways. It has been demonstrated that the treatment of diabetic controls. Biomechanical testing revealed improved
human mesenchymal stem cells (hMSCs) with clotted PRP, in fracture healing in platelet-rich treated diabetic fractures com-
an osteoinductive environment, enhances osteoblastic compared to those in nontreated diabetic controls.164 PRP delivery
mitment and bone formation. At the molecular level, PRP at the fracture site normalized the early (cellular proliferation
treatment stimulates a transient enhancement of BMP-2 mes- and chondrogenesis) parameters while improving the late
senger RNA and induces an earlier and a sustained increase (mechanical strength) parameters of diabetic fracture healing.
in the key osteogenic transcription factor, RUNX2. PRP can The biomechanical properties were only partially restored
enhance alkaline phosphatase activity more than twofold, and in late diabetic fracture callus. These data are consistent with
the noted increase in osteoblastic commitment translates to that seen clinically in diabetic patients having improved
enhanced calcified matrix deposition.171 healing and decreased complications after ankle fusion when
Currently, it is common to combine the platelet-rich mate- treated with PRP.177 These results suggest a role for PRP in
rial with autograft, allograft, DBM, or other graft material. In mediating diabetic fracture healing and potentially other
fact, PDGF release from platelet-rich concentrate was mark- high-risk fractures.164
edly reduced in the presence of DBM. When PRP was applied Siebrecht and colleagues demonstrated that PRP, prepared
in conjunction with ABG, the rate of bone formation doubled from human blood using a commercially available platelet
and bone density increased by 25% when compared to con- concentrate system, significantly increased bone and total
trols.168 However, there has been some data presented stating tissue ingrowth distance compared with untreated controls
that PRP has limited or even negative efficacy when combined (porous HA) in an athymic rat bone chamber model. The
with certain delivery vehicles.172,173 investigators theorized that porous HA lacks the cells and
Yet, more recent data has shown the improved efficacy of growth factors present in bone graft, but that PRC could
platelet-rich concentrate (PRC) and bone graft materials on potentially reverse this phenomenon.178
human BM stromal cell activity, with bone formation being Soft tissue effects of PRP were evaluated in a rabbit Achil-
significantly modified by adding the agents in combination. les tendon injury model where PRP was compared against
PRP was combined with various bone graft materials such as saline injections to determine augmentation of healing that
DBM and autograft to evaluate the effect of these factors on the materials may facilitate. PRP seems to enhance neovascu-
the hMSCs themselves. larization, which may accelerate the healing process and
Combining PRP with the graft materials increased cellular promote scar tissue of better histologic quality compared
proliferation above that seen with the graft materials alone; to the degree of healing (nonhealing) and scar quality found
however, only DBM and allograft were capable of increasing in the saline injection group.179 The authors felt that these
cellular proliferation above that seen with PRP alone. PRP results indicated that PRP may promote tendon healing
increased mineralization compared with DBM, collagen, or acceleration.
β-TCP alone. When compared with PRP alone, addition of
DBM or allograft decreased mineralization. Collagen gave rise Platelet-Rich Plasma Preparation
to a small increase in mineralization, whereas β-TCP yielded In terms of processing, platelet collection should commence
the same level of mineralization as PRP alone. before surgery because activity at the surgical site will initiate
nonconcentrated autologous blood.183 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
Platelet-poor, concentrated platelets, the relative concentration of white
fibrinogen-rich portion blood cells (WBCs) and/or red blood cells in the suspension
and the concomitant use of thrombin all affect the final
product.184,185
The absence of a validated classification system that
Platelet-rich portion
identifies crucial differences between PRP formulations
makes it difficult to compare acquisition systems or compara-
tive studies.185 The caveat “buyer beware” should be strictly
RBCs and WBCs with serum adhered to when considering the PRP in terms of the device
to be used.21,170
Figure 4-6. Platelet concentrate centrifuge cell, demonstrating the
multiple layers of materials obtained following the platelet separation Clinical Evidence
centrifugation process. This particular processing device now requires The use of PRP has been reported for a wide variety of clinical
this container to leave the sterile field in order to have the platelet-rich applications, most predominately for the problematic wound,
portion aspirated from this container prior to its delivery to the
patient. RBC, Red blood cell; WBC, white blood cell. maxillofacial applications, and spine. Collectively, these
studies provide variable support for the clinical use of PRP.
However, many reports are anecdotal, and few level 1 studies
clotting, thereby reducing the systemic platelet concentration. with control group comparison are available to definitively
It is felt by some that even the initiation of an inhalation anes- determine the role of PRP. There is little consensus regarding
thetic agent will initiate the activation of platelets. Until the production and characterization of PRP, which can impede
recently, centrifugation systems were the primary method of the establishment of standards that are necessary to integrate
producing a platelet-rich fraction. One of the disadvantages of the enormous amount of literature from various studies on the
centrifugation is that it can lead to fragmentation and lysis subject.
of the platelets, which triggers early release of growth factors BONE APPLICATIONS. A spinal fusion study performed by
and cytokines compromising bioactivity.180 Fragmentation and Wiener and colleagues highlights the problems with PRP
early activation have also been shown with previous filtration- studies, and the issues that an inconsistent PRP product can
based systems that were either too vigorous or used pediatric have on study results.186 A retrospective, consecutive series
dialysis filters in combination with cell saver, resulting in sig- was performed to evaluate two groups undergoing lumbar
nificant platelet degranulation and decreased efficacy.181 fusion: one with and one without autologous growth factors
Most systems in clinical use today use platelet-specific (AGFs). AGF is a product name given to a system of filtration
centrifugation devices or cell-sensitive filtration devices to devices to acquire and concentrate autologous platelets. Two
produce a platelet concentrate of sufficient concentration to groups were studied. The control group consisted of 27 con-
produce a physiologic effect. A PRP count of 1,000,000/mL as secutive patients who underwent a single-level intertransverse
measured in a standard 6-mL aliquot has become the bench- lumbar fusion using ICBG. The AGF group consisted of 32
mark for therapeutic PRC.167 Both of these separation tech- consecutive patients undergoing an identical procedure for
nologies deliver intact platelets that then require some the same indications with ICBG augmented with AGF (graft
activation at the time of delivery to the patient (Fig. 4-6). extender). The fusion rate for the iliac crest–only control
The regenerative potential of PRP depends on the amount group was 91%. However, the fusion rate for the AGF group
and viability of the inherent growth factors present in the was only 62%, which was significantly inferior.
delivered materials. Platelet activation and growth factor Later evaluation of the AGF technique (which is no longer
release can be accomplished by the addition of calcium or used) revealed that this device shredded platelets during the
thrombin to the platelet concentrate. filtration process and activated the platelets prior to use, essen-
The exact role of thrombin in PRP has been debated. tially delivering a substrate with limited biologic activity.181
Thrombin and/or calcium chloride is necessary to catalyze the The Weiner study used this PRP as an augmentation material
conversion of fibrinogen to fibrin, but it also induces platelets combined with a smaller amount of AICBG. It is conceivable
to secrete growth factors. Demineralized bone matrices sup- that a lesser amount of iliac crest graft was combined with an
plemented with PRP, with or without thrombin activation, inferior PRP, and thus the results were significantly inferior to
were implanted intramuscularly in athymic rats and were what was found using AICBG alone. This study was widely
examined. The results of this study suggested that exogenous seen as a condemnation of PRP and was thought that PRP
thrombin activation of PRP may actually diminish its ability was actually detrimental to bone healing. In actuality, the par-
to induce bone formation compared with non–thrombin- ticular formulation of PRP was at fault and not the concept of
activated PRP.182 PRP itself.
Studies have typically shown a three- to fourfold increase Currently, there is no level I evidence to indicate using PRP
in growth factor concentrations in PRP as compared to alone or in combination with other materials has a substantial
effect on bone healing. The available evidence (levels III and to saline with regard to pain reduction for lateral epicondylitis
IV) indicates that PRP may have a positive effect as an adjunct at the primary end point at 3 months. However, injection
to local bone graft and has been suggested for use to increase of glucocorticoid had a short-term pain-reducing effect at
the rate of bone deposition and quality of bone regeneration 1 month in contrast to the other therapies. Injection of
in fusion and nonunion situations. Overall, there is clearly a glucocorticoid in lateral epicondylitis reduces both color
lack of scientific evidence to support the use of PRP in com- Doppler activity and tendon thickness compared with PRP
bination with bone grafts during augmentation procedures.187 and saline.193 Despite these results, the clinical evidence sug-
Well-controlled randomized clinical studies are needed to gests that local injection of PRP-containing WBCs may be
assess the ideal concentration of the different factors and to beneficial to patients with chronic elbow epicondylitis refrac-
determine which materials provide the greatest effect. At tory to standard nonsurgical treatment. While there have
present, platelet gel appears to function best as a physiologic been some promising results documented for the treatment
carrier for other graft materials. of lateral epicondylitis, the results have not been as encourag-
SOFT TISSUE APPLICATIONS. Platelet-rich therapies are ing for the treatment of other tendinopathies including
being used increasingly in the treatment of musculoskeletal jumper’s knee.
soft tissue injuries such as ligament, muscle and tendon tears, Two similar level I studies in patients with chronic Achilles
and tendinopathies. These therapies can be used as the prin- tendinopathy were randomized to receive either a blinded
cipal treatment or as an augmentation procedure (application injection containing PRP or saline (placebo group) in addition
after surgical repair or reconstruction). Reported series to eccentric training programs. Functional scores improved in
have been published for clinical trials covering eight clinical both the PRP groups and the placebo groups after 1 year. There
conditions: rotator cuff tears (arthroscopic repair); shoulder was no significant difference in the increase in the improve-
impingement syndrome surgery, elbow epicondylitis; anterior ment between both groups in both studies. Ultrasonographic
cruciate ligament (ACL) reconstruction (donor graft site tendon structure improved significantly in both groups in one
application); patellar tendinopathy (one trial), Achilles tendi- study. Overall the results demonstrated no clinical and ultra-
nopathy, and acute Achilles rupture surgical repair.188 In sonographic superiority of PRP injections over a placebo
reviewing these multiple studies, one factor again becomes injection in chronic Achilles tendinopathy at 1 and 2 years
problematic: The methods of preparing PRP were highly when combined with an eccentric training program.194-196
varied and lacked standardization. The quantification of the Substantial research has gone into evaluating the effect of
PRP amounts applied to the patient were also not universally PRP on ACL surgery. In vitro studies document the positive
documented. effects on the graft tissue itself. Isolated ACL cells were cul-
Another confounding variable making comparison tured in the presence of differing concentrations of PRP. Cell
between trials difficult is that the primary treatments were viability, collagen synthesis, and collagen typing was ana-
either open surgery for repair with PRP application versus lyzed.197 PRP-treated cells resulted in a significant increase in
trials that simply injected PRP into tendinopathies where cell number compared with platelet-poor clot. Total collagen
platelet-rich therapy injections were the main treatment. production by the PRP-treated cells was significantly higher
Five level I and II controlled studies have compared results than that of the platelet-poor treated cells only because of
after surgical repair of rotator cuff injuries with and without enhanced cell proliferation. Expression of type III collagen
the adjunctive use of PRP. Subacromial decompression aug- was significantly enhanced by the treatment with PRP.
mented with PRP led to significantly decreased pain scores Multiple studies have been carried out evaluating specific
and improved shoulder range of motion.189 A double-blind factors related to ACL reconstruction. A systematic review
randomized controlled trial (RCT) of 53 patients, intraopera- of eight controlled clinical trials (level I, II and III studies)
tive application of PRP during arthroscopic rotator cuff repair concluded that the addition of platelet concentrates to ACL
led to significantly higher constant and University of Califor- reconstruction may have a 20% to 30% beneficial effect on
nia, Los Angeles (UCLA) scores and strength in external rota- graft maturation, as well as improved healing of the femoral
tion 3 months after surgery but not at long-term follow-up tunnels.198
when compared with control subjects.190 PRP was found to have a positive effect with regard to
Despite some encouraging results that PRP does have a improved graft remodeling and enveloping tissue when
positive benefit for rotator cuff repair for pain relief and patients were treated with PRP at the time of ACL reconstruc-
improved early motion, overall, the evidence indicates that tion. Many studies documented this enhanced ligamentization
PRP does not have an effect on re-tear rates or clinical out- process in tendon grafts.199
comes after arthroscopic repair.191 Further study is required While studies documented improved graft incorporation,
before the routine use of adjunctive PRP during shoulder this has not translated to improved functional outcomes.
surgery can be recommended.192 Many separate RCTs (level I) document no difference in radio-
The treatment of lateral epicondylitis has received signifi- graphic, International Knee Documentation Committee
cant attention by treating this condition with PRP. Most of the scores, KT-1000 arthrometer (MEDmetric), plain radiogra-
studies are RCTs with reasonable control groups and long- phy, and magnetic resonance imaging (MRI) findings in those
term follow-up. Most of the studies document some improve- ACL patients treated with or without PRP. The authors con-
ment in pain and VAS scores following the injection of PRP. cluded that there were no significant differences in any param-
The longevity of pain relief was variable in most of these eter related to functional outcome status.200,201 When treating
studies, and many of these studies used glucocorticoid injec- the tibial harvest sites with PRP, studies documented a positive
tion as the control group. However, contrary results were effect on patellar tendon harvest site healing on MRI after 6
found in a recent study. Krough and colleagues demonstrated months and also reduced pain in the immediate postoperative
that neither injection of PRP nor glucocorticoid was superior period.202,203
A similar study attempted to evaluate whether PRP, because mesenchymal cells (stem cells), changing them directly into
of its antiinflammatory properties and capacity to stimulate osteoprogenitor cells.
tissue regeneration, was able to reduce the anterior knee pain, The early use of this material began with multiple clinical
the kneeling pain, and donor-site morbidity at the tibial series and case reports by Johnson and colleagues.12,209-212 Urist
harvest site.191 The authors determined outcomes by evalua- purified the human protein in his laboratory at UCLA and his
tion of Victorian Institute of Sports Assessment (VISA) and clinical colleagues were able to use this material. Because
VAS scoring scales and MRI analysis of the tendon and bone extraction of purified human BMP (hBMP) from cadaver
defect. The PRP-treated group was successful in reducing sub- bone provided small yields, the ability to produce it in large
jective pain at the donor site. The tibial and patellar bone quantities was limited, and thus limited clinical application
defects were satisfactorily filled by new bony tissue in 85% of followed. However, early results were promising, treating
PRP group patients. complex femoral nonunions,210 posttraumatic shortened atro-
Although no significant difference in clinical outcomes phic femoral nonunions,209 and resistant nonunions with
has been found, preliminary clinical evidence suggests that partial or complete segmental defects including tibial defects
PRP may be beneficial during the ligamentization and matura- with bone loss ranging up to 17 cm.12 These were all treated
tion processes of graft healing as well as that of the patellar with a composite alloimplant of hBMP and autolyzed antigen-
tendon graft harvest sites in ACL reconstruction. For rotator free allogeneic (AAA) bone.12,209-212
cuff and Achilles tendon repairs, the results of clinical studies Purification of DBM by Sampath and Reddi in 1981 led to
are equivocal. the isolation and identification of several members of the BMP
Overall, and for the individual clinical conditions, there is family.213 Due to the time and expense of formulating these
currently insufficient evidence to support the routine use of materials in limited quantities, recombinant technologies
PRP for treating musculoskeletal soft tissue injuries.204 Further were developed to deliver BMPs in large amounts to proceed
study is needed before definitive conclusions can be drawn with significant research efforts. BMPs such as rhBMP-2,
and recommendations made.185 rhBMP-4, and rhBMP-6 (Genetic Institute, Cambridge, MA)
and rhBMP-7 (OP-1) (Olympus Corporation, Tokyo, Japan)
Recombinant Human Platelet-Derived were synthesized. When these factors are loaded on a collagen
Growth Factor sponge or particulate collagen, these carriers act as a reservoir
In an effort to take advantage of the beneficial effects of autog- for delivery to the site of involvement. In an overwhelming
enous PDGF while avoiding the perceived limitations (as out- amount of preclinical animal data, these factors have been
lined in the previous section) a recombinant human PDGF found to induce enchondral bone formation in segmental
was developed (rhPDGF). The clinical success and safety that bone defects and achieve spinal fusion in a number of animal
have been demonstrated with use of rhPDGF in the repair of models.214 Substantial preclinical and preapproval data are
periodontal defects has led to US Food and Drug Administra- required because the use of this technology is viewed by the
tion (FDA) approval of rhPDGF for this indication. The potent FDA as being associated with risk. Recombinant BMPs were
stimulatory effects of rhPDGF have been determined to be classified as Class III devices and face a much more rigorous
similar to the properties of autogenous PDGF.205 approval pathway.
Augment Bone Graft, a fully synthetic bone graft material Faced with demanding premarket FDA approval, from
composed of rhPDGF and a TCP matrix (rhPDGF/TCP), 1994 through 2000, multiple rigorously controlled preclinical
has been considered as a possible alternative to ABG and has studies were performed primarily evaluating the efficacy of
been used in many preclinical studies. The data have consis- BMPs in their ability to achieve spine fusion compared to
tently indicated that rhPDGF-BB treatment ameliorates the AICBG in various animal models. More than 25 studies com-
effects of diabetes on fracture healing by promoting early cel- pared BMP-2 versus autogenous graft, and 12 studies evalu-
lular proliferation that ultimately leads to more bone forma- ated BMP-7 versus autogenous graft, all demonstrating
tion. It was felt that the local application of rhPDGF-BB may equivalency or superiority of the BMP implants.
be efficacious for diabetic fracture treatment and for defect A representative study was performed by Boden and col-
management.206 leagues, where they used a primate lumbar intertransverse
Two RCTs (level I) have demonstrated efficacy in patients process arthrodesis model to evaluate rhBMP-2 in a HA-TCP
requiring hind-foot or ankle arthrodesis. Treatment with carrier as a complete bone graft substitute. The rhBMP com-
rhPDGF-BB/β-TCP resulted in comparable fusion rates, less posite sites were compared to AICBG sites with radiography
pain, and fewer side effects as compared with treatment with and histology to assess fusion and to detect any bony growth
autograft.207,208 This material is currently awaiting FDA approval into the laminectomy defect. One hundred percent of the
for clinical use in long bone and foot and ankle indications. rhBMP composite fusions healed, whereas fusion was not
achieved in any of the monkeys treated with AICBG. 215
These studies set the stage for the crucial pivotal clinical
INDUCTIVE SUBSTRATES (BONE trials for which there were at least 11 randomized prospective
MORPHOGENIC PROTEIN) studies evaluating BMP-2 and 15 level I studies investigating
BMP-7. These preapproval level I studies evaluated both spine
The BMPs belong to the TGF-β superfamily of growth and and long bone applications.
differentiation factors. Unlike DBM, which is a mixture of One of the most prominent of these was a multicenter,
BMPs and immunogenic noninductive proteins, the pure prospective, randomized, nonblinded, 2-year study performed
form of BMP is nonimmunogenic and nonspecies specific. by Burkus and colleagues.216 Nearly 300 patients with degen-
The BMPs are true “osteoinducers.” As they are released, erative lumbar disc disease were randomly divided into two
they feed back onto circulating undifferentiated perivascular groups that underwent interbody fusion using two tapered
threaded fusion cages. The investigational group (143 patients) Danek, Memphis, TN) in the treatment of open tibial frac-
received rhBMP-2 on an absorbable collagen sponge, and a tures.219 Four hundred and fifty patients with such an injury
control group (136 patients) received AICBG. Fusion rates in were initially managed with irrigation, débridement, and IM
the BMP-treated group was 94.5% compared with 88.7% in nail fixation. At the time of definitive wound closure, the
ABG-treated group. The ABG-treated group had eight adverse patients were randomized to one of three groups: standard
events related to bone graft harvest procedure, and 32% of closure, standard closure and the addition of 6 mg of rhBMP-2
patients reported graft site discomfort. This pivotal trial led to to the fracture site, or standard closure and the addition of
the approval of BMP-2 for spinal surgery for the following 12 mg of rhBMP-2 to the fracture site. The primary outcome
indication: the INFUSE Bone Graft in conjunction with an measure in this study was the rates of secondary interventions
interbody fusion device, that is, either the LT-Cage Lumbar (returns to the operating rooms for additional treatment).
Tapered Fusion Device or the Inter Fix RP Threaded Fusion The group treated with the higher dose of rhBMP-2 (1.5 mg/
Device, implanted via an anterior open or a laparoscopic kg) had fewer secondary interventions. Interestingly, although
approach. not used as primary outcome measures, an accelerated time
In a large prospective, randomized, controlled, partially to union, improved wound healing, and a reduced infection
blinded, multicenter study, Friedlaender and colleagues rate were also found in the patients treated with the high
assessed the efficacy of the OP-1 device (3.5 mg of rhBMP-7 dose of rhBMP-2. The FDA subsequently granted approval
in a bovine bone–derived type I collagen-particle delivery for the treatment of acute, open fractures of the tibial shaft
vehicle; Olympus Corporation, Tokyo, Japan) in comparison (Fig. 4-8).
with that of autografting in the treatment of 122 patients with A recent study has brought into question the value of
a total of 124 tibial nonunions. All of the nonunions were at rhBMP-2 for the treatment of open tibial shaft fractures. Aro
least 9 months old and had shown no progress toward healing and colleagues evaluated the use of rhBMP-2 in the treatment
for the 3 months prior to the patients’ enrollment in the study. of acute open tibial fractures treated with reamed IM nail
All patients were treated with reamed IM nailing of the non- fixation. This study was very similar to the aforementioned
union and were then randomized to have either autograft bone BESTT study. Patients were randomly assigned (1 : 1) to receive
or OP-1 implanted at the nonunion site. Nine months after the IM fixation and routine soft tissue management (the standard
surgery, 81% of the OP-1 group and 85% of the autograft of care group) or an IM nail plus an absorbable collagen sponge
group had clinical evidence of union. Radiographic assess- implant placed at the time of wound closure. The implant con-
ments suggested healing of 75% and 84% of these nonunions, tained 1.5 mg/mL of rhBMP-2 (total, 12.0 mg) (the rhBMP-2/
respectively. At 2 year follow-up, these results continued at American College of Surgeons [ACS] group). Healing and
similar levels.217 other outcome measures were assessed. By week 20, the pro-
OP-1 statistically proved to be a safe and effective alterna- portions of patients with fracture healing were 68% and 67% in
tive to bone graft in the treatment of tibial nonunions. A limi- the rhBMP-2/ACS and standard of care groups, respectively
tation of the study was that the investigators could not control (not significant evidence). All other outcome parameters were
for the potential healing effects produced by reamed IM similar between the two groups and the authors concluded that
nailing of the tibial nonunions. This, among other study criti- the healing of open tibial fractures treated with reamed IM nail
cisms, prevented the full FDA approval of this material. fixation was not significantly accelerated by the addition of an
Limited approval of OP-1 by the FDA for use in the treat- absorbable collagen sponge containing rhBMP-2.220
ment of tibial nonunions and other long bone nonunions Following the approval of these two BMPs for specific trau-
was designated as a humanitarian use device (HUD) for matic conditions (acute open tibial shaft fracture, recalcitrant
this particular indication.218 An HUD is a device that is nonunions), very limited data have been published using these
intended to benefit patients by treating or diagnosing a disease devices with their strict on-label indications. Many investiga-
or condition that affects or is manifested in fewer than 4000 tors have sought to combine these materials with other bio-
individuals in the United States per year and the manufacturer logic adjuvants. Just as we discussed previously in this chapter,
is not required to demonstrate unequivocal benefit, but only the ability to combine multiple inductive, conductive, and or
“probable” benefit. OP-1 may only be used in facilities that osteogenic factors continues with the BMPs as well. This has
have established a local institutional review board (IRB) to been done for a variety of clinical issues regarding the clinical
supervise clinical testing of devices and after an IRB has handling and application of these materials, as well as to
approved the use of the OP-1 to treat recalcitrant nonunions attempt to augment the healing potential of these specific
(Fig. 4-7). BMPs for their trauma application.
OP-1 putty also has FDA approval through the humanitar- Jones and colleagues investigated the implantation of
ian device exemption process and is indicated for use as an BMP-2 combined with allograft compared to autograft for the
alternative to autograft in compromised patients requiring treatment of acute segmental defects in a prospective RCT of
revision posterolateral (intertransverse) lumbar spinal fusion, 30 patients with tibial diaphyseal bone defects.221 The average
for whom autologous bone and BM harvest are not feasible or defect size was 4 cm (range, 1–7 cm). At 12-month follow-up,
are not expected to promote fusion. 80% of patients (12 of 15 patients) in the AICBG group and
In addition to the approved spinal indications for BMP-2, 87% of patients (13 of 15 patients) in the allograft/rhBMP-2
a prospective randomized study evaluated rhBMP-2 for the group had healed without reintervention (P = 0.2). There were
treatment of open tibial shaft fractures. The BMP-2 Evaluation no significant differences in complication rates or functional
in Surgery for Tibial Trauma (BESTT) Study Group reported outcomes between the two groups. This study suggests that
the results of a large multinational, prospective, randomized, rhBMP-2/allograft is safe and as effective as traditional autog-
controlled study of the effects of INFUSE Bone Graft (rhBMP-2 enous bone grafting for the treatment of tibial fractures associ-
on an absorbable type I collagen sponge; Medtronic Sofamor ated with extensive traumatic diaphyseal bone loss.221
A B
C D
E
Figure 4-7. A, Recalcitrant atrophic humeral nonunion in a 67-year-old woman who had failed a previous autogenous iliac crest bone graft
(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
osteogenic protein (OP-1) (recombinant human bone morphogenic protein-7 [rhBMP-7]). An off-label application was used combining the OP-1
with concentrated marrow aspirate and particulate demineralized bone matrix (DBM), to help provide a carrier for the particulate OP-1 graft
material. C, At 10-month postgraft follow-up, the nonunion has healed with excellent bridging of the nonunion site, as well as developing
massive heterotopic bone. The heterotopic ossification restricted the patient’s elbow and shoulder motion, requiring eventual resection. D, At
excision surgery, a large longitudinal mass of heterotopic 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 sequela.
A B
C D
Figure 4-8. A, A motorcycle crash resulted in a grade 3b open distal tibia fracture with limited intraarticular extension and 3 to 4 cm of seg-
mental 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 intramedullary (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), recombinant human bone morphogenic protein-2
(rhBMP-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 x-rays. The external fixator allowed full weight
bearing following maturation of the free flap. Near-complete healing of the segmental defect occurred over a 5-month time period following
graft application. The external fixator was removed at 21 weeks postinjury.
In an effort to improve fracture healing in closed tibial shaft of either concentration of rhBMP-2/CPM compared with IM
fractures and develop an injectable form of BMP-2, investiga- nailing alone without adjuvant injection.
tors evaluated rhBMP combined with a new, injectable calcium
phosphate matrix (CPM). Patients were randomized (1 : 2 : 2 : 1) Off-Label Use and Complications
to receive standard of care, with a locked reamed IM nail, As is highlighted by these two recent studies, investigators are
versus IM nailing with injection with 1.0 mg/mL of rhBMP-2/ evaluating off-label indications. Most of the use of rhBMPs in
CPM; or an increased concentration 2.0 mg/mL of rhBMP-2/ orthopaedics is off label, which means that the product has
CPM.222 The results were very similar to the Aro study where not been approved for such use by the FDA. Physician-directed
no significant difference with or without BMP was noted.220 It use of such products for uses not approved by the FDA is
appears that the time to fracture union and pain-free full allowable because the FDA does not regulate physician prac-
weight bearing were not significantly reduced by the injection tice; its statutory authority extends only to the regulation of
the manufacturers of drugs and medical devices and listing of nonunion repair.237 All the patents in this series required sur-
known complications. Physician-directed (off-label) use of gical removal of this extra bone. It has been proposed that
devices or drugs has been approved by some state legislatures, BMP-4 is involved in the extensive endochondral ossification
and it is generally acknowledged that physicians should be seen in fibrodysplasia ossificans progressiva.238,239 The transfer
able to apply their best medical judgment in the use of off-label of the gene for BMP-2 into skeletal muscle has been shown to
devices or drugs. However with an increase of reported com- promote both endochondral and intramembranous ossifica-
plications of BMP usage, the unregulated nature of BMP has tion.240 Expression of BMP-2 and BMP-7 has also been shown
come under intense scrutiny both from third-party payers and to induce BMP-4 expression.240 The osteoinductive stimulus
the federal government. for chondro-osteogenic differentiation leading to the develop-
Isolated complications with the use of rhBMP in spine ment of HO is the proposed mechanism in these cases.
fusions, such as retrograde ejaculation in males, have Another proposed mechanism could be the inability of the
occurred in clinical studies, but was initially delayed in their BMP carrier to contain the material to the site of involvement
reporting. In some instances, it was felt that undue bias from only and, thus, the additional bone formation found in areas
industry-sponsored studies had intentionally underreported of soft tissue injury as well as the primary application site.241
complications.223-225 Subsequent studies cited the following
adverse events that occurred when used in spinal applica- Bone Morphogenic Protein Interactions
tions. These consist of heterotopic ossification (HO), graft As noted earlier, it has been shown that the presence of one
osteolysis, increased infection, arachnoiditis, increased neu- BMP can enhance the expression of other BMPs. For example,
rologic deficits, and retrograde ejaculation.226-229 The use of the combinations of BMP-2 with BMP-7 have been reported
rhBMPs in cervical spine fusion has resulted in significant to be 5 to 10 times more potent than BMP-2 alone in inducing
edema with resultant compromise of tracheal air flow.230-232 cartilage and bone formation,242 perhaps by the induction of
Some of these issues resulted in devastating consequences for BMP-4 as mentioned previously.
the patients who incurred these complications. BMPs are regulated at multiple levels by various BMP
Surgeons have noted several safety issues associated with antagonists as well as affected by other circulating BMPs.243,244
the use of BMPs, including cancer risk, stating both BMP and Thus, the current use of single-agent BMPs is very simplistic,
their receptors have been isolated from human tumors. In and begs the question as to why supraphysiologic dosages of
addition, data presented to the FDA on the product AMPLIFY a single BMP is required to have an osteoinductive effect. The
rhBMP-2 Matrix (40 mg) revealed a higher number of cancers effective doses are orders of magnitude greater than the
in the investigational group compared with the control. An endogenous amounts of BMPs expressed during normal bone
independent review of the cancer risk of rhBMP use in spine repair or in normal bone remodeling. Presumably the com-
fusions as published in peer-reviewed literature and in the bined action of other factors may be required for maximal
publicly available FDA data summaries was evaluated. They efficacy of BMP-mediated osteoinduction. It has been shown
concluded that cancer risk with BMPs may be dose dependent, that BMPs and induction are regulated at multiple levels by
illustrating the need to continue to study this technology and various BMP antagonists and are also affected by other circu-
obtain longer follow-up on patients currently enrolled in the lating BMPs.243,244
FDA trials.233 The current application of giving one factor and expecting
However, another large population-based analysis of Medi- a complex cascade of bone-forming events to occur in an
care beneficiaries found no evidence that administration of unencumbered fashion is quite simplistic. However, the exper-
rhBMP at the time of lumbar fusion surgery was associated imental complexities of manipulating multiple factors makes
with an elevated cancer risk.234 Propensity-matched studies investigational clarification of this hypothetical issue nearly
suggest that the use of BMP in lumbar fusions is associated impossible to carry out.
with a significantly higher rate of benign neoplasms but not
malignancies.235 Economic Impact
Complications also occurred when BMPs were used in While the efficacy and possible complications caused by
acute traumatic or posttraumatic situations. HO appears to be rhBMPs continue to be debated, the economic impact of using
the most common and consistent complication when used for these devices off label is relatively unknown. A recent study
trauma-related conditions (see Fig. 4-7).236 Boraiah and col- evaluated the economic impact of “on-label” versus “off-label”
leagues noted a significant incidence of HO occurred when use of BMPs for spinal fusions.245 The authors determined that
plateau fractures were treated with BMP. The BMP was used in 2010 (at the authors’ institution), 96% of the spinal fusions
to augment the structural support provided by the bone-void that used BMP were used in an off-label capacity (cost
filler used to support the elevated defect. Even though this was $4,547,822), while only 4% were performed on-label at a cost
an off-label indication, the authors sought to determine if of $296,419.
BMP enhanced the ability to maintain the surgically elevated An interesting cost analysis study was undertaken using the
subchondral bone; 60% (10 of 17) of the patients developed data from the BESTT trials to evaluate the potential cost
heterotopic bone, of which 40% (4) required additional surgery savings associated with the use of BMP-2 for open fractures
to have this excised. Additionally, the BMP did not appear to care. Data from the United Kingdom, Germany, and France
have any positive effect on the maintenance of the subchon- was analyzed. The cost of BMP-2 was offset by the ability of
dral repair.236 injured patients to return to work, which was assumed to cor-
A second series also reported the phenomenon of hetero- respond with fracture healing time. Reduced productivity
topic bone. This bone occurred in the humerus, of which losses were realized by the significantly faster fracture healing
50% were related to acute application of BMP for traumatic and earlier return to work compared to the non–BMP-2
bone loss, and 50% had BMP applied at the time of humeral treated patients. Total net savings were estimated to be 9.6
million euros for the United Kingdom, 14.5 million euros for osteoinductive, structurally similar to bone, easy to use, and
Germany, and 11.4 million euros for France. The authors felt cost-effective and at minimal risk for material-based compli-
that despite the apparent high direct cost of rhBMP-2 for use cations. Within these parameters, a growing number of bone
in grade III A and B open tibial fractures, at a national level, alternatives are commercially available for orthopaedic appli-
there are net cost savings from a societal perspective for all cations. Future comprehensive strategies for therapeutic appli-
three countries.246 cation will combine concepts of tissue engineering with a
simple delivery mechanism and biologic scaffolding.
Summary
This review highlights the paucity of current data present for KEY REFERENCES
the use of rhBMPs for traumatic indications. There is limited
The level of evidence (LOE) is determined according to the criteria provided
evidence to suggest that BMP may be more effective than in the preface.
controls for acute tibial fracture healing; however, the use of 1. De Long WG, Jr, Einhorn TA, Koval K, et al: Bone grafts and bone graft
BMP for treating nonunion remains unclear. The limited avail- substitutes in orthopaedic trauma surgery. A critical analysis. J Bone
able economic evidence indicates that BMP treatment for Joint Surg Am 89(3):649–658, 2007.
33. Sagi HC, Young ML, Gerstenfeld L, et al: Qualitative and quantitative
acute open tibial fractures may be more favorable economi- differences between bone graft obtained from the medullary canal (with
cally when used in patients with the most severe fractures.247 a Reamer/Irrigator/Aspirator) and the iliac crest of the same patient.
Well-designed and executed studies are necessary to com- J Bone Joint Surg Am 94(23):2128–2135, 2012.
pletely define the indications for the use of BMPs. Studies need 70. Russell TA, Leighton RK: Comparison of autogenous bone graft and
to be designed to account for the specific BMP to be used, and endothermic calcium phosphate cement for defect augmentation in
tibial plateau fractures. A multicenter, prospective, randomized study.
the definitive pathology and region of application. Standard- J Bone Joint Surg Am 90(10):2057–2061, 2008.
ized surgical technique is required, as well as control of the 100. Bae H, Zhao L, Zhu D, et al: Variability across ten production lots of a
dosage applied and the specific carrier substances used to single demineralized bone matrix product. J Bone Joint Surg Am
deliver the materials. Most importantly, investigators should 92(2):427–435, 2010.
strive to define the natural history and management of any 127. Muschler GF, Boehm C, Easley K: Aspiration to obtain osteoblast pro-
genitor cells from human bone marrow: the influence of aspiration
associated complications. volume. J Bone Joint Surg Am 79(11):1699–1709, 1997.
159. Hernigou P, Poignard A, Beaujean F, et al: Percutaneous autologous
bone-marrow grafting for nonunions. Influence of the number and con-
CONCLUSIONS centration of progenitor cells. J Bone Joint Surg Am 87(7):1430–1437,
2005.
185. Hsu WK, Mishra A, Rodeo SR, et al: Platelet-rich plasma in orthopaedic
Although current regulations on informed consent do not applications: evidence-based recommendations for treatment. J Am
require a physician to note that he or she is using a product Acad Orthop Surg 21(12):739–748, 2013.
such as rhBMP off label, the planned use of allografts, bone 217. Friedlaender GE, Perry CR, Cole JD, et al: Osteogenic protein-1 (bone
morphogenetic protein-7) in the treatment of tibial nonunions. J Bone
substitutes, and bone enhancers in surgery should be disclosed Joint Surg Am 83(Suppl 1 Pt 2):S151–S158, 2001. LOE I
in informed consent. 219. Govender S, Csimma C, Genant HK, et al: Recombinant human bone
This is an exciting time in the development of orthobio- morphogenetic protein-2 for treatment of open tibial fractures: a pro-
logic- and factor-based therapies for enhancement of bone spective, controlled, randomized study of four hundred and fifty
healing for traumatic conditions. Areas of application for patients. J Bone Joint Surg Am 84(12):2123–2134, 2002, LOE I
221. Jones AL, Bucholz RW, Bosse MJ, et al: Recombinant human BMP-2 and
new technologies include the acceleration of fracture healing, allograft compared with autogenous bone graft for reconstruction of
treatment of nonunions, enhancement of fusion mass, and diaphyseal tibial fractures with cortical defects. A randomized, con-
the treatment of massive segmental bone loss, including trolled trial. J Bone Joint Surg Am 88(7):1431–1441, 2006. LOE II
reconstruction of cavitary bone deficiency in situations of
segmental bone loss and spine fusion. The ideal bone-graft The complete References list is available online at https://
substitute is biocompatible, bioresorbable, osteoconductive, expertconsult.inkling.com.
Chapter 4 — Biology and Enhancement of Skeletal Repair 93.e1
REFERENCES 28. Pelissier P, Masquelet AC, Bareille R, et al: Induced membranes secrete
growth factors including vascular and osteoinductive factors and could
The level of evidence (LOE) is determined according to the criteria provided stimulate bone regeneration. J Orthop Res 22(1):73–79, 2004.
in the preface. 29. Stafford PR, Norris BL: Reamer-irrigator-aspirator bone graft and Mas-
1. De Long WG Jr, Einhorn TA, Koval K, et al: Bone grafts and bone graft quelet technique for segmental bone defect nonunions: a review of 25
substitutes in orthopaedic trauma surgery. A critical analysis. J Bone cases. Injury 41(Suppl 2):S72–S77, 2010.
Joint Surg Am 89(3):649–658, 2007. 30. McCall TA, Brokaw DS, Jelen BA, et al: Treatment of large segmental
2. Goldberg VM: The biology of bone grafts. Orthopedics 26(9):923–924, bone defects with reamer-irrigator-aspirator bone graft: technique
2003. and case series. Orthop Clin North Am 41(1):63–73, 2010.
3. Goldberg VM, Stevenson S: The biology of bone grafts. Semin Arthro- 31. Schmidmaier G, Herrmann S, Green J, et al: Quantitative assessment of
plasty 4(2):58–63, 1993. growth factors in reaming aspirate, iliac crest, and platelet preparation.
4. Li XQ, Stevenson S, Klein L, et al: Differential patterns of incorporation Bone 39(5):1156–1163, 2006.
and remodeling among various types of bone grafts. Acta Anat (Basel) 32. Cox G, McGonagle D, Boxall SA, et al: The use of the reamer-irrigator-
140(3):236–244, 1991. aspirator to harvest mesenchymal stem cells. J Bone Joint Surg Br 93(4):
5. Heiple KG, Goldberg VM, Powell AE, et al: Biology of cancellous bone 517–524, 2011.
grafts. Orthop Clin North Am 18(2):179–185, 1987. 33. Sagi HC, Young ML, Gerstenfeld L, et al: Qualitative and quantitative
6. Burchardt H: The biology of bone graft repair. Clin Orthop Relat Res differences between bone graft obtained from the medullary canal (with
(174):28–42, 1983. a Reamer/Irrigator/Aspirator) and the iliac crest of the same patient.
7. Bishop JA, Palanca AA, Bellino MJ, et al: Assessment of compromised J Bone Joint Surg Am 94(23):2128–2135, 2012.
fracture healing. J Am Acad Orthop Surg 20(5):273–282, 2012. 34. Stannard JP, Sathy AK, Moeinpour F, et al: Quantitative analysis of
8. Urist MR: Bone: formation by autoinduction. Science 150(3698):893– growth factors from a second filter using the reamer-irrigator-aspirator
899, 1965. system: description of a novel technique. Orthop Clin North Am 41(1):
9. Urist MR, Dowell TA: Inductive substratum for osteogenesis in pellets 95–98, 2010.
of particulate bone matrix. Clin Orthop Relat Res 61:61–78, 1968. 35. Hak DJ, Pittman JL: Biological rationale for the intramedullary canal
10. Urist MR, Strates BS: The classic: bone morphogenetic protein. Clin as a source of autograft material. Orthop Clin North Am 41(1):57–61,
Orthop Relat Res 467(12):3051–3062, 2009. 2010.
11. Wozney JM, Rosen V, Celeste AJ, et al: Novel regulators of bone forma- 36. Uppal HS, Peterson BE, Misfeldt ML, et al: The viability of cells obtained
tion: molecular clones and activities. Science 242(4885):1528–1534, using the reamer-irrigator-aspirator system and in bone graft from the
1988. iliac crest. Bone Joint J 95-b(9):1269–1274, 2013.
12. Johnson EE, Urist MR, Finerman GA: Repair of segmental defects of the 37. Beuerlein MJ, McKee MD: Calcium sulfates: what is the evidence?
tibia with cancellous bone grafts augmented with human bone morpho- J Orthop Trauma 24(Suppl 1):S46–S51, 2010.
genetic protein. A preliminary report. Clin Orthop Relat Res (236):249– 38. Peltier LF, Bickel EY, Lillo R, et al: The use of plaster of paris to fill defects
257, 1988. in bone. Ann Surg 146(1):61–69, 1957.
13. Peterson B, Whang PG, Iglesias R, et al: Osteoinductivity of commer- 39. Peltier LF: The use of plaster of paris to fill large defects in bone. Am J
cially available demineralized bone matrix. Preparations in a spine Surg 97(3):311–315, 1959.
fusion model. J Bone Joint Surg Am 86(10):2243–2250, 2004. 40. Peltier LF: The use of plaster of Paris to fill defects in bone. Clin Orthop
14. Nauth A, McKee MD, Einhorn TA, et al: Managing bone defects. 21:1–31, 1961.
J Orthop Trauma 25(8):462–466, 2011. 41. Ziran BH, Smith WR, Morgan SJ: Use of calcium-based demineralized
15. Bhandari M, Schemitsch EH: Stimulation of fracture healing: osteobio- bone matrix/allograft for nonunions and posttraumatic reconstruction
logics, bone stimulators, and beyond. J Orthop Trauma 24(Suppl 1):S1, of the appendicular skeleton: preliminary results and complications.
2010. J Trauma 63(6):1324–1328, 2007.
16. Sen MK, Miclau T: Autologous iliac crest bone graft: should it still be 42. Kelly CM, Wilkins RM, Gitelis S, et al: The use of a surgical grade
the gold standard for treating nonunions? Injury 38(Suppl 1):S75–S80, calcium sulfate as a bone graft substitute: results of a multicenter trial.
2007. Clin Orthop Relat Res (382):42–50, 2001.
17. Chiodo CP, Hahne J, Wilson MG, et al: Histological differences in iliac 43. Moed BR, Willson Carr SE, Craig JG, et al: Calcium sulfate used as bone
and tibial bone graft. Foot Ankle Int 31(5):418–422, 2010. graft substitute in acetabular fracture fixation. Clin Orthop Relat Res
18. Christian EP, Bosse MJ, Robb G: Reconstruction of large diaphyseal (410):303–309, 2003.
defects, without free fibular transfer, in Grade-IIIB tibial fractures. 44. Walsh WR, Morberg P, Yu Y, et al: Response of a calcium sulfate bone
J Bone Joint Surg Am 71(7):994–1004, 1989. graft substitute in a confined cancellous defect. Clin Orthop Relat Res
19. Goulet JA, Senunas LE, DeSilva GL, et al: Autogenous iliac crest bone (406):228–236, 2003.
graft. Complications and functional assessment. Clin Orthop Relat Res 45. Peters CL, Hines JL, Bachus KN, et al: Biological effects of calcium
339:76–81, 1997. sulfate as a bone graft substitute in ovine metaphyseal defects. J Biomed
20. Loeffler BJ, Kellam JF, Sims SH, et al: Prospective observational study Mater Res A 76(3):456–462, 2006.
of donor-site morbidity following anterior iliac crest bone-grafting 46. Yu B, Han K, Ma H, et al: Treatment of tibial plateau fractures with
in orthopaedic trauma reconstruction patients. J Bone Joint Surg Am high strength injectable calcium sulphate. Int Orthop 33(4):1127–1133,
94(18):1649–1654, 2012. 2009.
21. Watson JT: Overview of biologics. J Orthop Trauma 19(Suppl 10):S14– 47. Watson JT: The use of an injectable bone graft substitute in tibial
S16, 2005. metaphyseal fractures. Orthopedics 27(Suppl 1):s103–s107, 2004.
22. Watson JT, Anders M, Moed BR: Management strategies for bone 48. Borrelli J Jr, Prickett WD, Ricci WM: Treatment of nonunions and
loss in tibial shaft fractures. Clin Orthop Relat Res (315):138–152, osseous defects with bone graft and calcium sulfate. Clin Orthop Relat
1995. Res (411):245–254, 2003.
23. Reckling FW, Waters CH 3rd: Treatment of non-unions of fractures 49. McKee MD, Li-Bland EA, Wild LM, et al: A prospective, randomized
of the tibial diaphysis by posterolateral cortical cancellous bone-grafting. clinical trial comparing an antibiotic-impregnated bioabsorbable bone
J Bone Joint Surg Am 62(6):936–941, 1980. substitute with standard antibiotic-impregnated cement beads in the
24. Belthur MV, Conway JD, Jindal G, et al: Bone graft harvest using a new treatment of chronic osteomyelitis and infected nonunion. J Orthop
intramedullary system. Clin Orthop Relat Res 466(12):2973–2980, 2008. Trauma 24(8):483–490, 2010.
25. Kobbe P, Tarkin IS, Frink M, et al: Voluminous bone graft harvesting of 50. McKee MD, Wild LM, Schemitsch EH, et al: The use of an antibiotic-
the femoral marrow cavity for autologous transplantation. An indication impregnated, osteoconductive, bioabsorbable bone substitute in the
for the “reamer-irrigator aspirator (RIA)” technique. Unfallchirurg treatment of infected long bone defects: early results of a prospective
111(6):469–472, 2008. trial. J Orthop Trauma 16(9):622–627, 2002.
26. Masquelet AC: Muscle reconstruction in reconstructive surgery: soft 51. Bucholz RW, Carlton A, Holmes RE: Hydroxyapatite and tricalcium
tissue repair and long bone reconstruction. Langenbecks Arch Surg phosphate bone graft substitutes. Orthop Clin North Am 18(2):323–334,
388(5):344–346, 2003. 1987.
27. Pelissier P, Martin D, Baudet J, et al: Behaviour of cancellous bone 52. Holmes R, Mooney V, Bucholz R, et al: A coralline hydroxyapatite bone
graft placed in induced membranes. Br J Plast Surg 55(7):596–598, graft substitute. Preliminary report. Clin Orthop Relat Res (188):252–
2002. 262, 1984.
93.e2 SECTION ONE — General Principles
53. Holmes RE, Bucholz RW, Mooney V: Porous hydroxyapatite as a bone- densitometry, histology, and functional outcome of 18 patients. J Foot
graft substitute in metaphyseal defects. A histometric study. J Bone Joint Ankle Surg 44(5):390–395, 2005.
Surg Am 68(6):904–911, 1986. 78. Schildhauer TA, Bauer TW, Josten C, et al: Open reduction and aug-
54. Boden SD, Martin GJ Jr, Morone M, et al: The use of coralline hydroxy- mentation of internal fixation with an injectable skeletal cement for the
apatite with bone marrow, autogenous bone graft, or osteoinductive treatment of complex calcaneal fractures. J Orthop Trauma 14(5):309–
bone protein extract for posterolateral lumbar spine fusion. Spine 317, 2000.
24(4):320–327, 1999. 79. Egol KA, Sugi MT, Ong CC, et al: Fracture site augmentation with
55. Gosain AK, Song L, Riordan P, et al: A 1-year study of osteoinduction calcium phosphate cement reduces screw penetration after open
in hydroxyapatite-derived biomaterials in an adult sheep model: part I. reduction-internal fixation of proximal humeral fractures. J Shoulder
Plast Reconstr Surg 109(2):619–630, 2002. Elbow Surg 21(6):741–748, 2012.
56. Klar RM, Duarte R, Dix-Peek T, et al: Calcium ions and osteoclastogen- 80. Gradl G, Knobe M, Stoffel M, et al: Biomechanical evaluation of locking
esis initiate the induction of bone formation by coral-derived macropo- plate fixation of proximal humeral fractures augmented with calcium
rous constructs. J Cell Mol Med 17(11):1444–1457, 2013. doi: 10.1111/ phosphate cement. J Orthop Trauma 27(7):399–404, 2013.
jcmm.12125, [Epub 2013 Sep 23]. 81. Civinini R, De Biase P, Carulli C, et al: The use of an injectable calcium
57. Ripamonti U: Osteoinduction in porous hydroxyapatite implanted in sulphate/calcium phosphate bioceramic in the treatment of osteonecro-
heterotopic sites of different animal models. Biomaterials 17(1):31–35, sis of the femoral head. Int Orthop 36(8):1583–1588, 2012.
1996. 82. Rouvillain JL, Lavalle F, Pascal-Mousselard H, et al: Clinical, radiologi-
58. Ripamonti U, Crooks J, Khoali L, et al: The induction of bone formation cal and histological evaluation of biphasic calcium phosphate bio
by coral-derived calcium carbonate/hydroxyapatite constructs. Bioma- ceramic wedges filling medial high tibial valgisation osteotomies. Knee
terials 30(7):1428–1439, 2009. 16(5):392–397, 2009.
59. Shors EC: Coralline bone graft substitutes. Orthop Clin North Am 83. Mattsson P, Larsson S: Stability of internally fixed femoral neck fractures
30(4):599–613, 1999. augmented with resorbable cement. A prospective randomized study
60. Kuhne JH, Bartl R, Frisch B, et al: Bone formation in coralline hydroxy- using radiostereometry. Scand J Surg 92(3):215–219, 2003.
apatite. Effects of pore size studied in rabbits. Acta Orthop Scand 84. Mattsson P, Larsson S: Unstable trochanteric fractures augmented with
65(3):246–252, 1994. calcium phosphate cement. A prospective randomized study using
61. Nakahara H, Goldberg VM, Caplan AI: Culture-expanded periosteal- radiostereometry to measure fracture stability. Scand J Surg 93(3):223–
derived cells exhibit osteochondrogenic potential in porous calcium 228, 2004.
phosphate ceramics in vivo. Clin Orthop Relat Res (276):291–298, 1992. 85. Lindner T, Kanakaris NK, Marx B, et al: Fractures of the hip and osteo-
62. Knaack D, Goad ME, Aiolova M, et al: Resorbable calcium phosphate porosis: the role of bone substitutes. J Bone Joint Surg Br 91(3):294–303,
bone substitute. J Biomed Mater Res 43(4):399–409, 1998. 2009.
63. Wiltfang J, Merten HA, Schlegel KA, et al: Degradation characteristics 86. del Real RP, Ooms E, Wolke JG, et al: In vivo bone response to
of alpha and beta tri-calcium-phosphate (TCP) in minipigs. J Biomed porous calcium phosphate cement. J Biomed Mater Res A 65(1):30–36,
Mater Res 63(2):115–121, 2002. 2003.
64. Cornell CN, Lane JM, Chapman M, et al: Multicenter trial of Collagraft 87. Herron S, Thordarson DB, Winet H, et al: Ingrowth of bone into absorb-
as bone graft substitute. J Orthop Trauma 5(1):1–8, 1991. able bone cement: an in vivo microscopic evaluation. Am J Orthop (Belle
65. Mauffrey C, Fader R, Hammerberg EM, et al: Incidence and pattern of Mead NJ) 32(12):581–584, 2003.
technical complications in balloon-guided osteoplasty for depressed 88. Guimaraes KB, Vasconcelos BC, Limeira Junior Fde A, et al: Histomor-
tibial plateau fractures: a pilot study in 20 consecutive patients. Patient phometric evaluation of calcium phosphate bone grafts on bone repair.
Saf Surg 7(1):8, 2013. Braz J Otorhinolaryngol 77(4):447–454, 2011.
66. Szpalski M, Gunzburg R: Applications of calcium phosphate-based can- 89. Faour O, Dimitriou R, Cousins CA, et al: The use of bone graft substi-
cellous bone void fillers in trauma surgery. Orthopedics 25(Suppl 5): tutes in large cancellous voids: any specific needs? Injury 42(Suppl 2):
s601–s609, 2002. S87–S90, 2011.
67. McDonald E, Chu T, Tufaga M, et al: Tibial plateau fracture repairs 90. Damron TA, Lisle J, Craig T, et al: Ultraporous beta-tricalcium phos-
augmented with calcium phosphate cement have higher in situ fatigue phate alone or combined with bone marrow aspirate for benign cavitary
strength than those with autograft. J Orthop Trauma 25(2):90–95, 2011. lesions: comparison in a prospective randomized clinical trial. J Bone
68. McAndrew MP, Gorman PW, Lange TA: Tricalcium phosphate as a bone Joint Surg Am 95(2):158–166, 2013.
graft substitute in trauma: preliminary report. J Orthop Trauma 2(4): 91. Coathup MJ, Samizadeh S, Fang YS, et al: The osteoinductivity of
333–339, 1988. silicate-substituted calcium phosphate. J Bone Joint Surg Am 93(23):2219–
69. Jubel A, Andermahr J, Mairhofer J, et al: Use of the injectable bone 2226, 2011.
cement Norian SRS for tibial plateau fractures. Results of a prospective 92. Friedlaender GE: Immune responses to osteochondral allografts.
30-month follow-up study. Orthopade 33(8):919–927, 2004. Current knowledge and future directions. Clin Orthop Relat Res (174):
70. Russell TA, Leighton RK: Comparison of autogenous bone graft and 58–68, 1983.
endothermic calcium phosphate cement for defect augmentation in 93. Ramoshebi LN, Matsaba TN, Teare J, et al: Tissue engineering: TGF-
tibial plateau fractures. A multicenter, prospective, randomized study. beta superfamily members and delivery systems in bone regeneration.
J Bone Joint Surg Am 90(10):2057–2061, 2008. Expert Rev Mol Med 4(20):1–11, 2002.
71. Goff T, Kanakaris NK, Giannoudis PV: Use of bone graft substitutes in 94. Kirk JF, Ritter G, Waters C, et al: Osteoconductivity and osteoinductivity
the management of tibial plateau fractures. Injury 44(Suppl 1):S86–S94, of NanoFUSE DBM. Cell Tissue Bank 14(1):33–44, 2013.
2013. 95. Moore ST, Katz JM, Zhukauskas RM, et al: Osteoconductivity and
72. Bajammal SS, Zlowodzki M, Lelwica A, et al: The use of calcium phos- osteoinductivity of Purost® DBM putty. J Biomater Appl 26(2):151–171,
phate bone cement in fracture treatment. A meta-analysis of random- 2011.
ized trials. J Bone Joint Surg Am 90(6):1186–1196, 2008. 96. Bolander ME, Balian G: The use of demineralized bone matrix in the
73. Zimmermann R, Gabl M, Lutz M, et al: Injectable calcium phosphate repair of segmental defects. Augmentation with extracted matrix pro-
bone cement Norian SRS for the treatment of intra-articular compres- teins and a comparison with autologous grafts. J Bone Joint Surg Am
sion fractures of the distal radius in osteoporotic women. Arch Orthop 68(8):1264–1274, 1986.
Trauma Surg 123(1):22–27, 2003. 97. Honsawek S, Powers RM, Wolfinbarger L: Extractable bone morphoge-
74. Kim JK, Koh YD, Kook SH: Effect of calcium phosphate bone cement netic protein and correlation with induced new bone formation in an in
augmentation on volar plate fixation of unstable distal radial fractures vivo assay in the athymic mouse model. Cell Tissue Bank 6(1):13–23,
in the elderly. J Bone Joint Surg Am 93(7):609–614, 2011. 2005.
75. Suhm N: Gisep A. Injectable bone cement augmentation for the treat- 98. Einhorn TA, Lane JM, Burstein AH, et al: The healing of segmental
ment of distal radius fractures: a review. J Orthop Trauma 22(8 Suppl): bone defects induced by demineralized bone matrix. A radiographic
S121–S125, 2008. and biomechanical study. J Bone Joint Surg Am 66(2):274–279,
76. Wee AT, Wong YS: Percutaneous reduction and injection of Norian bone 1984.
cement for the treatment of displaced intra-articular calcaneal fractures. 99. Bae HW, Zhao L, Kanim LE, et al: Intervariability and intravariability of
Foot Ankle Spec 2(2):98–106, 2009. bone morphogenetic proteins in commercially available demineralized
77. Elsner A, Jubel A, Prokop A, et al: Augmentation of intraarticular bone matrix products. Spine 31(12):1299–1306, discussion 1307–1298,
calcaneal fractures with injectable calcium phosphate cement: 2006.
100. Bae H, Zhao L, Zhu D, et al: Variability across ten production lots 125. Patterson TE, Kumagai K, Griffith L, et al: Cellular strategies for
of a single demineralized bone matrix product. J Bone Joint Surg Am enhancement of fracture repair. J Bone Joint Surg Am 90(Suppl 1):111–
92(2):427–435, 2010. 119, 2008.
101. Pietrzak WS, Woodell-May J, McDonald N: Assay of bone morphoge- 126. Muschler GF, Midura RJ: Connective tissue progenitors: practical con-
netic protein-2, -4, and -7 in human demineralized bone matrix. J Cra- cepts for clinical applications. Clin Orthop Relat Res (395):66–80, 2002.
niofac Surg 17(1):84–90, 2006. 127. Muschler GF, Boehm C, Easley K: Aspiration to obtain osteoblast pro-
102. Wildemann B, Kadow-Romacker A, Haas NP, et al: Quantification of genitor cells from human bone marrow: the influence of aspiration
various growth factors in different demineralized bone matrix prepara- volume. J Bone Joint Surg Am 79(11):1699–1709, 1997.
tions. J Biomed Mater Res A 81(2):437–442, 2007. 128. Connolly JF, Guse R, Tiedeman J, et al: Autologous marrow injection as
103. Qiu QQ, Connor J: Effects of gamma-irradiation, storage and hydration a substitute for operative grafting of tibial nonunions. Clin Orthop Relat
on osteoinductivity of DBM and DBM/AM composite. J Biomed Mater Res (266):259–270, 1991.
Res A 87(2):373–379, 2008. 129. Connolly J, Guse R, Lippiello L, et al: Development of an osteogenic
104. Han B, Yang Z, Nimni M: Effects of gamma irradiation on osteoinduc- bone-marrow preparation. J Bone Joint Surg Am 71(5):684–691, 1989.
tion associated with demineralized bone matrix. J Orthop Res 26(1):75– 130. Connolly JF: Clinical use of marrow osteoprogenitor cells to stimulate
82, 2008. osteogenesis. Clin Orthop Relat Res (Suppl 355):S257–S266, 1998.
105. Han B, Yang Z, Nimni M: Effects of moisture and temperature on the 131. Hernigou P, Homma Y, Flouzat Lachaniette CH, et al: Benefits of small
osteoinductivity of demineralized bone matrix. J Orthop Res 23(4):855– volume and small syringe for bone marrow aspirations of mesenchymal
861, 2005. stem cells. Int Orthop 37(11):2279–2287, 2013.
106. Carpenter JF, Pikal MJ, Chang BS, et al: Rational design of stable lyophi- 132. Muschler GF, Matsukura Y, Nitto H, et al: Selective retention of bone
lized protein formulations: some practical advice. Pharm Res 14(8):969– marrow-derived cells to enhance spinal fusion. Clin Orthop Relat Res
975, 1997. (432):242–251, 2005.
107. Boyce T, Edwards J, Scarborough N: Allograft bone. The influence of 133. Hyer CF, Berlet GC, Bussewitz BW, et al: Quantitative assessment of the
processing on safety and performance. Orthop Clin North Am 30(4):571– yield of osteoblastic connective tissue progenitors in bone marrow aspi-
581, 1999. rate from the iliac crest, tibia, and calcaneus. J Bone Joint Surg Am
108. Doherty MJ, Mollan RA, Wilson DJ: Effect of ethylene oxide steriliza- 95(14):1312–1316, 2013.
tion on human demineralized bone. Biomaterials 14(13):994–998, 134. McLain RF, Fleming JE, Boehm CA, et al: Aspiration of osteoprogenitor
1993. cells for augmenting spinal fusion: comparison of progenitor cell con-
109. Han B, Tang B, Nimni ME: Quantitative and sensitive in vitro assay for centrations from the vertebral body and iliac crest. J Bone Joint Surg Am
osteoinductive activity of demineralized bone matrix. J Orthop Res 87(12):2655–2661, 2005.
21(4):648–654, 2003. 135. Pierini M, Di Bella C, Dozza B, et al: The posterior iliac crest
110. Zhang M, Powers RM Jr, Wolfinbarger L Jr: A quantitative assessment outperforms the anterior iliac crest when obtaining mesenchymal
of osteoinductivity of human demineralized bone matrix. J Periodontol stem cells from bone marrow. J Bone Joint Surg Am 95(12):1101–1107,
68(11):1076–1084, 1997. 2013.
111. Bormann N, Pruss A, Schmidmaier G, et al: In vitro testing of the 136. Bidula J, Boehm C, Powell K, et al: Osteogenic progenitors in bone
osteoinductive potential of different bony allograft preparations. Arch marrow aspirates from smokers and nonsmokers. Clin Orthop Relat Res
Orthop Trauma Surg 130(1):143–149, 2010. 442:252–259, 2006.
112. Upton J, Boyajian M, Mulliken JB, et al: The use of demineralized xeno- 137. Tiedeman JJ, Connolly JF, Strates BS, et al: Treatment of nonunion by
geneic bone implants to correct phalangeal defects: a case report. J Hand percutaneous injection of bone marrow and demineralized bone matrix.
Surg [Am] 9(3):388–391, 1984. An experimental study in dogs. Clin Orthop Relat Res (268):294–302,
113. Mulliken JB, Glowacki J, Kaban LB, et al: Use of demineralized alloge- 1991.
neic bone implants for the correction of maxillocraniofacial deformities. 138. Schwartz Z, Doukarsky-Marx T, Nasatzky E, et al: Differential effects of
Ann Surg 194(3):366–372, 1981. bone graft substitutes on regeneration of bone marrow. Clin Oral
114. Smartt JM, Lin IC, Kim E, et al: Hybrid constructs for craniofacial Implants Res 19(12):1233–1245, 2008.
reconstruction: sustained gene delivery using demineralized bone 139. Olivares-Navarrete R, Hyzy SL, Hutton DL, et al: Direct and indirect
matrix putty. Ann Plast Surg 52(6):592–596, discussion 597, 2004. effects of microstructured titanium substrates on the induction of mes-
115. Neigel JM, Ruzicka PO: Use of demineralized bone implants in orbital enchymal stem cell differentiation towards the osteoblast lineage. Bio-
and craniofacial reconstruction and a review of the literature. Ophthal materials 31(10):2728–2735, 2010.
Plast Reconstr Surg 12(2):108–120, 1996. 140. Schwartz Z, Kieswetter K, Dean DD, et al: Underlying mechanisms
116. Tiedeman JJ, Garvin KL, Kile TA, et al: The role of a composite, demin- at the bone-surface interface during regeneration. J Periodontal Res
eralized bone matrix and bone marrow in the treatment of osseous 32(1 Pt 2):166–171, 1997.
defects. Orthopedics 18(12):1153–1158, 1995. 141. Bruder SP, Kraus KH, Goldberg VM, et al: The effect of implants loaded
117. Wilkins RM, Chimenti BT, Rifkin RM: Percutaneous treatment of long with autologous mesenchymal stem cells on the healing of canine seg-
bone nonunions: the use of autologous bone marrow and allograft bone mental bone defects. J Bone Joint Surg Am 80(7):985–996, 1998.
matrix. Orthopedics 26(5 Suppl):s549–s554, 2003. 142. Brodke D, Pedrozo HA, Kapur TA, et al: Bone grafts prepared with
118. Lindsey RW, Wood GW, Sadasivian KK, et al: Grafting long bone frac- selective cell retention technology heal canine segmental defects as
tures with demineralized bone matrix putty enriched with bone marrow: effectively as autograft. J Orthop Res 24(5):857–866, 2006.
pilot findings. Orthopedics 29(10):939–941, 2006. 143. Lane JM, Yasko AW, Tomin E, et al: Bone marrow and recombinant
119. Geesink RG, Hoefnagels NH, Bulstra SK: Osteogenic activity of OP-1 human bone morphogenetic protein-2 in osseous repair. Clin Orthop
bone morphogenetic protein (BMP-7) in a human fibular defect. J Bone Relat Res (361):216–227, 1999.
Joint Surg Br 81(4):710–718, 1999. 144. Janicki P, Schmidmaier G: What should be the characteristics of the ideal
120. Hierholzer C, Sama D, Toro JB, et al: Plate fixation of ununited humeral bone graft substitute? Combining scaffolds with growth factors and/or
shaft fractures: effect of type of bone graft on healing. J Bone Joint Surg stem cells. Injury 42(Suppl 2):S77–S81, 2011.
Am 88(7):1442–1447, 2006. 145. Reichert JC, Cipitria A, Epari DR, et al: A tissue engineering solution
121. Vaccaro AR, Stubbs HA, Block JE: Demineralized bone matrix compos- for segmental defect regeneration in load-bearing long bones. Sci Transl
ite grafting for posterolateral spinal fusion. Orthopedics 30(7):567–570, Med 4(141):141–193, 2012.
2007. 146. Qi Y, Wang Y, Yan W, et al: Combined mesenchymal stem cell sheets
122. Cammisa FP Jr, Lowery G, Garfin SR, et al: Two-year fusion rate equiva- and rhBMP-2-releasing calcium sulfate-rhBMP-2 scaffolds for segmen-
lency between Grafton DBM gel and autograft in posterolateral spine tal bone tissue engineering. Cell Transplant 21(4):693–705, 2012.
fusion: a prospective controlled trial employing a side-by-side compari- 147. Connolly JF: Injectable bone marrow preparations to stimulate osteo-
son in the same patient. Spine 29(6):660–666, 2004. genic repair. Clin Orthop Relat Res (313):8–18, 1995.
123. Kang J, An H, Hilibrand A, et al: Grafton and local bone have compa- 148. Garg NK, Gaur S, Sharma S: Percutaneous autogenous bone marrow
rable outcomes to iliac crest bone in instrumented single-level lumbar grafting in 20 cases of ununited fracture. Acta Orthop Scand 64(6):671–
fusions. Spine 37(12):1083–1091, 2012. 672, 1993.
124. Aghdasi B, Montgomery SR, Daubs MD, et al: A review of demineral- 149. Healey JH, Zimmerman PA, McDonnell JM, et al: Percutaneous bone
ized bone matrices for spinal fusion: the evidence for efficacy. Surgeon marrow grafting of delayed union and nonunion in cancer patients. Clin
11(1):39–48, 2013. Orthop Relat Res (256):280–285, 1990.
150. Wientroub S, Goodwin D, Khermosh O, et al: The clinical use of autolo- 174. Butcher A, Milner R, Ellis K, et al: Interaction of platelet-rich concen-
gous marrow to improve osteogenic potential of bone grafts in pediatric trate with bone graft materials: an in vitro study. J Orthop Trauma
orthopedics. J Pediatr Orthop 9(2):186–190, 1989. 23(3):195–200, discussion 201–192, 2009.
151. Goel A, Sangwan SS, Siwach RC, et al: Percutaneous bone marrow graft- 175. Slater M, Patava J, Kingham K, et al: Involvement of platelets in stimulat-
ing for the treatment of tibial non-union. Injury 36(1):203–206, 2005. ing osteogenic activity. J Orthop Res 13(5):655–663, 1995.
152. Jaiswal N, Haynesworth SE, Caplan AI, et al: Osteogenic differentiation 176. Gandhi A, Doumas C, O’Connor JP, et al: The effects of local platelet
of purified, culture-expanded human mesenchymal stem cells in vitro. rich plasma delivery on diabetic fracture healing. Bone 38(4):540–546,
J Cell Biochem 64(2):295–312, 1997. 2006.
153. Vulcano E, Murena L, Cherubino P, et al: Treatment of severe post- 177. Grant WP, Jerlin EA, Pietrzak WS, et al: The utilization of autologous
traumatic bone defects with autologous stem cells loaded on allogeneic growth factors for the facilitation of fusion in complex neuropathic
scaffolds. Surg Technol Int 22:291–301, 2012. fractures in the diabetic population. Clin Podiatr Med Surg 22(4):561–
154. Vulcano E, Murena L, Falvo DA, et al: Bone marrow aspirate and bone 584, vi, 2005.
allograft to treat acetabular bone defects in revision total hip arthro- 178. Siebrecht MA, De Rooij PP, Arm DM, et al: Platelet concentrate increases
plasty: preliminary report. Eur Rev Med Pharmacol Sci 17(16):2240– bone ingrowth into porous hydroxyapatite. Orthopedics 25(2):169–172,
2249, 2013. 2002.
155. den Boer FC, Wippermann BW, Blokhuis TJ, et al: Healing of segmental 179. Lyras DN, Kazakos K, Verettas D, et al: The influence of platelet-rich
bone defects with granular porous hydroxyapatite augmented with plasma on angiogenesis during the early phase of tendon healing. Foot
recombinant human osteogenic protein-1 or autologous bone marrow. Ankle Int 30(11):1101–1106, 2009.
J Orthop Res 21(3):521–528, 2003. 180. Gonshor A: Technique for producing platelet-rich plasma and platelet
156. Cuervas-Mons M, Narbona J, Laguna R, et al: Autologous concentrated concentrate: background and process. Int J Periodontics Restorative Dent
bone marrow graft in the treatment of femoral head avascular necrosis: 22(6):547–557, 2002.
clinical outcome after two years of follow up in a non-controlled pro- 181. Kevy SV, Jacobson MS: Comparison of methods for point of care prepa-
spective study. Rev Esp Cir Orthop Traumatol 57(2):106–110, 2013. ration of autologous platelet gel. J Extra Corpor Technol 36(1):28–35,
157. Yan ZQ, Chen YS, Li WJ, et al: Treatment of osteonecrosis of the femoral 2004.
head by percutaneous decompression and autologous bone marrow 182. Han B, Woodell-May J, Ponticiello M, et al: The effect of thrombin
mononuclear cell infusion. Chin J Traumatol 9(1):3–7, 2006. activation of platelet-rich plasma on demineralized bone matrix osteo-
158. Hernigou P, Mathieu G, Poignard A, et al: Percutaneous autologous inductivity. J Bone Joint Surg Am 91(6):1459–1470, 2009.
bone-marrow grafting for nonunions. Surgical technique. J Bone Joint 183. Zimmermann R, Arnold D, Strasser E, et al: Sample preparation tech-
Surg Am 88(Suppl 1 Pt 2):322–327, 2006. nique and white cell content influence the detectable levels of growth
159. Hernigou P, Poignard A, Beaujean F, et al: Percutaneous autologous factors in platelet concentrates. Vox Sang 85(4):283–289, 2003.
bone-marrow grafting for nonunions. Influence of the number and con- 184. Mazzucco L, Balbo V, Cattana E, et al: Not every PRP-gel is born equal.
centration of progenitor cells. J Bone Joint Surg Am 87(7):1430–1437, Evaluation of growth factor availability for tissues through four PRP-gel
2005. preparations: Fibrinet, Regen PRP-Kit, Plateltex and one manual proce-
160. Hart R, Komzak M, Okal F, et al: Allograft alone versus allograft with dure. Vox Sang 97(2):110–118, 2009.
bone marrow concentrate for the healing of the instrumented postero- 185. Hsu WK, Mishra A, Rodeo SR, et al: Platelet-rich plasma in orthopaedic
lateral lumbar fusion. Spine J 2013 Dec 20 (Epub ahead of print). applications: evidence-based recommendations for treatment. J Am
161. Jager M, Herten M, Fochtmann U, et al: Bridging the gap: bone marrow Acad Orthop Surg 21(12):739–748, 2013.
aspiration concentrate reduces autologous bone grafting in osseous 186. Weiner BK, Walker M: Efficacy of autologous growth factors in lumbar
defects. J Orthop Res 29(2):173–180, 2011. intertransverse fusions. Spine 28(17):1968–1970, discussion 1971,
162. Jager M, Hernigou P, Zilkens C, et al: Cell therapy in bone healing 2003.
disorders. Orthop Rev (Pavia) 2(2):e20, 2010. 187. Sanchez AR, Sheridan PJ, Kupp LI: Is platelet-rich plasma the perfect
163. Jager M, Jelinek EM, Wess KM, et al: Bone marrow concentrate: a novel enhancement factor? A current review. Int J Oral Maxillofac Implants
strategy for bone defect treatment. Curr Stem Cell Res Ther 4(1):34–43, 18(1):93–103, 2003.
2009. 188. Moraes VY, Lenza M, Tamaoki MJ, et al: Platelet-rich therapies for
164. Rush SM, Hamilton GA, Ackerson LM: Mesenchymal stem cell allograft musculoskeletal soft tissue injuries. Cochrane Database Syst Rev (12):
in revision foot and ankle surgery: a clinical and radiographic analysis. CD010071, 2013.
J Foot Ankle Surg 48(2):163–169, 2009. 189. Everts PA, Devilee RJ, Brown Mahoney C, et al: Exogenous application
165. Guyton GP, Miller SD: Stem cells in bone grafting: Trinity allograft with of platelet-leukocyte gel during open subacromial decompression con-
stem cells and collagen/beta-tricalcium phosphate with concentrated tributes to improved patient outcome. A prospective randomized
bone marrow aspirate. Foot Ankle Clin 15(4):611–619, 2010. double-blind study. Eur Surg Res 40(2):203–210, 2008.
166. Kerr EJ 3rd, Jawahar A, Wooten T, et al: The use of osteo-conductive 190. Randelli P, Arrigoni P, Ragone V, et al: Platelet rich plasma in arthroscopic
stem-cells allograft in lumbar interbody fusion procedures: an alterna- rotator cuff repair: a prospective RCT study, 2-year follow-up. J Shoulder
tive to recombinant human bone morphogenetic protein. J Surg Orthop Elbow Surg 20(4):518–528, 2011.
Adv 20(3):193–197, 2011. 191. Castricini R, Longo UG, De Benedetto M, et al: Platelet-rich plasma
167. Marx RE: Platelet-rich plasma: evidence to support its use. J Oral Maxil- augmentation for arthroscopic rotator cuff repair: a randomized con-
lofac Surg 62(4):489–496, 2004. trolled trial. Am J Sports Med 39(2):258–265, 2011.
168. Marx RE, Carlson ER, Eichstaedt RM, et al: Platelet-rich plasma: growth 192. Chahal J, Van Thiel GS, Mall N, et al: The role of platelet-rich plasma in
factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral arthroscopic rotator cuff repair: a systematic review with quantitative
Radiol Endod 85(6):638–646, 1998. synthesis. Arthroscopy 28(11):1718–1727, 2012.
169. Grageda E: Platelet-rich plasma and bone graft materials: a review 193. Krogh TP, Fredberg U, Stengaard-Pedersen K, et al: Treatment of lateral
and a standardized research protocol. Implant Dent 13(4):301–309, epicondylitis with platelet-rich plasma, glucocorticoid, or saline: a ran-
2004. domized, double-blind, placebo-controlled trial. Am J Sports Med
170. Mehta S, Watson JT: Platelet rich concentrate: basic science and current 41(3):625–635, 2013.
clinical applications. J Orthop Trauma 22(6):432–438, 2008. 194. de Jonge S, de Vos RJ, Weir A, et al: One-year follow-up of platelet-rich
171. Parsons P, Butcher A, Hesselden K, et al: Platelet-rich concentrate sup- plasma treatment in chronic Achilles tendinopathy: a double-blind ran-
ports human mesenchymal stem cell proliferation, bone morphogenetic domized placebo-controlled trial. Am J Sports Med 39(8):1623–1629,
protein-2 messenger RNA expression, alkaline phosphatase activity, and 2011.
bone formation in vitro: a mode of action to enhance bone repair. 195. de Vos RJ, van Veldhoven PL, Moen MH, et al: Autologous growth factor
J Orthop Trauma 22(9):595–604, 2008. injections in chronic tendinopathy: a systematic review. Br Med Bull
172. Ranly DM, Lohmann CH, Andreacchio D, et al: Platelet-rich plasma 95:63–77, 2010.
inhibits demineralized bone matrix-induced bone formation in nude 196. de Vos RJ, Weir A, van Schie HT, et al: Platelet-rich plasma injection for
mice. J Bone Joint Surg Am 89(1):139–147, 2007. chronic Achilles tendinopathy: a randomized controlled trial. JAMA
173. Ranly DM, McMillan J, Keller T, et al: Platelet-derived growth factor 303(2):144–149, 2010.
inhibits demineralized bone matrix-induced intramuscular cartilage 197. Fallouh L, Nakagawa K, Sasho T, et al: Effects of autologous platelet-rich
and bone formation. A study of immunocompromised mice. J Bone Joint plasma on cell viability and collagen synthesis in injured human anterior
Surg Am 87(9):2052–2064, 2005. cruciate ligament. J Bone Joint Surg Am 92(18):2909–2916, 2010.
198. Vavken P, Sadoghi P, Murray MM: The effect of platelet concentrates on 220. Aro HT, Govender S, Patel AD, et al: Recombinant human bone mor-
graft maturation and graft-bone interface healing in anterior cruciate phogenetic protein-2: a randomized trial in open tibial fractures treated
ligament reconstruction in human patients: a systematic review of con- with reamed nail fixation. J Bone Joint Surg Am 93(9):801–808, 2011.
trolled trials. Arthroscopy 27(11):1573–1583, 2011. 221. Jones AL, Bucholz RW, Bosse MJ, et al: Recombinant human BMP-2 and
199. Sanchez M, Anitua E, Azofra J, et al: Ligamentization of tendon grafts allograft compared with autogenous bone graft for reconstruction of
treated with an endogenous preparation rich in growth factors: gross diaphyseal tibial fractures with cortical defects. A randomized, con-
morphology and histology. Arthroscopy 26(4):470–480, 2010. trolled trial. J Bone Joint Surg Am 88(7):1431–1441, 2006. LOE II
200. Magnussen RA, Flanigan DC, Pedroza AD, et al: Platelet rich plasma 222. Lyon T, Scheele W, Bhandari M, et al: Efficacy and safety of recombinant
use in allograft ACL reconstructions: two-year clinical results of a human bone morphogenetic protein-2/calcium phosphate matrix for
MOON cohort study. Knee 20(4):277–280, 2013. closed tibial diaphyseal fracture: a double-blind, randomized, controlled
201. Nin JR, Gasque GM, Azcarate AV, et al: Has platelet-rich plasma any Phase-II/III trial. J Bone Joint Surg Am 95(23):2088–2096, 2013.
role in anterior cruciate ligament allograft healing? Arthroscopy 223. Carragee EJ, Hurwitz EL, Weiner BK: A critical review of recombinant
25(11):1206–1213, 2009. human bone morphogenetic protein-2 trials in spinal surgery: emerging
202. de Almeida AM, Demange MK, Sobrado MF, et al: Patellar tendon safety concerns and lessons learned. Spine J 11(6):471–491, 2011.
healing with platelet-rich plasma: a prospective randomized controlled 224. United States Senate Finance Committee: Staff report on Medtronic’s
trial. Am J Sports Med 40(6):1282–1288, 2012. influence on INFUSE clinical studies. Int J Occup Environ Health
203. Cervellin M, de Girolamo L, Bait C, et al: Autologous platelet-rich 19(2):67–76, 2013.
plasma gel to reduce donor-site morbidity after patellar tendon graft 225. Epstein NE: Complications due to the use of BMP/INFUSE in spine
harvesting for anterior cruciate ligament reconstruction: a randomized, surgery: the evidence continues to mount. Surg Neurol Int 4(Suppl 5):
controlled clinical study. Knee Surg Sports Traumatol Arthrosc 20(1):114– S343–S352, 2013.
120, 2012. 226. Chrastil J, Patel AA: Complications associated with posterior and trans-
204. Sheth U, Simunovic N, Klein G, et al: Efficacy of autologous platelet-rich foraminal lumbar interbody fusion. J Am Acad Orthop Surg 20(5):283–
plasma use for orthopaedic indications: a meta-analysis. J Bone Joint 291, 2012.
Surg Am 94(4):298–307, 2012. 227. Joseph V, Rampersaud YR: Heterotopic bone formation with the use of
205. Hollinger JO, Hart CE, Hirsch SN, et al: Recombinant human platelet- rhBMP2 in posterior minimal access interbody fusion: a CT analysis.
derived growth factor: biology and clinical applications. J Bone Joint Surg Spine 32(25):2885–2890, 2007.
Am 90(Suppl 1):48–54, 2008. 228. Burkus JK, Dryer RF, Peloza JH: Retrograde ejaculation following single-
206. Al-Zube L, Breitbart EA, O’Connor JP, et al: Recombinant human level anterior lumbar surgery with or without recombinant human bone
platelet-derived growth factor BB (rhPDGF-BB) and beta-tricalcium morphogenetic protein-2 in 5 randomized controlled trials: clinical
phosphate/collagen matrix enhance fracture healing in a diabetic rat article. J Neurosurg Spine 18(2):112–121, 2013.
model. J Orthop Res 27(8):1074–1081, 2009. 229. Comer GC, Smith MW, Hurwitz EL, et al: Retrograde ejaculation after
207. Daniels T, DiGiovanni C, Lau JT, et al: Prospective clinical pilot trial in anterior lumbar interbody fusion with and without bone morphogenetic
a single cohort group of rhPDGF in foot arthrodeses. Foot Ankle Int protein-2 augmentation: a 10-year cohort controlled study. Spine J
31(6):473–479, 2010. 12(10):881–890, 2012.
208. DiGiovanni CW, Lin SS, Baumhauer JF, et al: Recombinant human 230. Cahill KS, Chi JH, Day A, et al: Prevalence, complications, and hospital
platelet-derived growth factor-BB and beta-tricalcium phosphate charges associated with use of bone-morphogenetic proteins in spinal
(rhPDGF-BB/beta-TCP): an alternative to autogenous bone graft. J Bone fusion procedures. JAMA 302(1):58–66, 2009.
Joint Surg Am 95(13):1184–1192, 2013. 231. Goode AP, Richardson WJ, Schectman RM, et al: Complications, revi-
209. Johnson EE, Urist MR: One-stage lengthening of femoral nonunion sion fusions, re-admissions and utilization over a one-year period fol-
augmented with human bone morphogenetic protein. Clin Orthop Relat lowing bone morphogenetic protein use during primary cervical spine
Res (347):105–116, 1998. fusions. Spine J 2013 Dec 7 (Epub ahead of print).
210. Johnson EE, Urist MR: Human bone morphogenetic protein allografting 232. Mroz TE, Wang JC, Hashimoto R, et al: Complications related to osteo-
for reconstruction of femoral nonunion. Clin Orthop Relat Res (371):61– biologics use in spine surgery: a systematic review. Spine 35(Suppl
74, 2000. 9):S86–S104, 2010.
211. Johnson EE, Urist MR, Finerman GA: Distal metaphyseal tibial non- 233. Devine JG, Dettori JR, France JC, et al: The use of rhBMP in spine
union. Deformity and bone loss treated by open reduction, internal fixa- surgery: is there a cancer risk? Evid Based Spine Care J 3(2):35–41, 2012.
tion, and human bone morphogenetic protein (hBMP). Clin Orthop 234. Cooper GS, Kou TD: Risk of cancer after lumbar fusion surgery with
Relat Res (250):234–240, 1990. recombinant human bone morphogenic protein-2 (rh-BMP-2). Spine
212. Johnson EE, Urist MR, Finerman GA: Resistant nonunions and partial 38(21):1862–1868, 2013.
or complete segmental defects of long bones. Treatment with implants 235. Lad SP, Bagley JH, Karikari IO, et al: Cancer after spinal fusion: the role
of a composite of human bone morphogenetic protein (BMP) and auto- of bone morphogenetic protein. Neurosurgery 73(3):440–449, 2013.
lyzed, antigen-extracted, allogeneic (AAA) bone. Clin Orthop Relat Res 236. Boraiah S, Paul O, Hawkes D, et al: Complications of recombinant
(277):229–237, 1992. human BMP-2 for treating complex tibial plateau fractures: a prelimi-
213. Sampath TK, Reddi AH: Dissociative extraction and reconstitution of nary report. Clin Orthop Relat Res 467(12):3257–3262, 2009.
extracellular matrix components involved in local bone differentiation. 237. Axelrad TW, Steen B, Lowenberg DW, et al: Heterotopic ossification
Proc Natl Acad Sci U S A 78(12):7599–7603, 1981. after the use of commercially available recombinant human bone mor-
214. Friedlaender GE: OP-1 clinical studies. J Bone Joint Surg Am 83(Suppl phogenetic proteins in four patients. J Bone Joint Surg Br 90(12):1617–
1 Pt 2):S160–S161, 2001. 1622, 2008.
215. Boden SD, Martin GJ Jr, Morone MA, et al: Posterolateral lumbar inter- 238. Kaplan FS, Glaser DL, Hebela N, et al: Heterotopic ossification. J Am
transverse process spine arthrodesis with recombinant human bone Acad Orthop Surg 12(2):116–125, 2004.
morphogenetic protein 2/hydroxyapatite-tricalcium phosphate after 239. Olmsted EA, Kaplan FS, Shore EM: Bone morphogenetic protein-4
laminectomy in the nonhuman primate. Spine 24(12):1179–1185, 1999. regulation in fibrodysplasia ossificans progressiva. Clin Orthop Relat Res
216. Burkus JK, Gornet MF, Dickman CA, et al: Anterior lumbar interbody (408):331–343, 2003.
fusion using rhBMP-2 with tapered interbody cages. J Spinal Disord Tech 240. Kawai M, Bessho K, Maruyama H, et al: Simultaneous gene transfer of
15(5):337–349, 2002. bone morphogenetic protein (BMP)-2 and BMP-7 by in vivo electro-
217. Friedlaender GE, Perry CR, Cole JD, et al: Osteogenic protein-1 (bone poration induces rapid bone formation and BMP-4 expression. BMC
morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Musculoskelet Disord 7:62, 2006.
Joint Surg Am 83(Suppl 1 Pt 2):S151–S158, 2001. LOE I 241. King GN: The importance of drug delivery to optimize the effects of
218. Blokhuis TJ, Calori GM, Schmidmaier G: Autograft versus BMPs for the bone morphogenetic proteins during periodontal regeneration. Cur
treatment of non-unions: what is the evidence? Injury 44(Suppl 1):S40– Pharm Biotechnol 2(2):131–142, 2001.
S42, 2013. 242. Israel DI, Nove J, Kerns KM, et al: Expression and characterization of
219. Govender S, Csimma C, Genant HK, et al: Recombinant human bone morphogenetic protein-2 in Chinese hamster ovary cells. Growth
bone morphogenetic protein-2 for treatment of open tibial fractures: Factors 7(2):139–150, 1992.
a prospective, controlled, randomized study of four hundred and 243. Dean DB, Watson JT, Jin W, et al: Distinct functionalities of bone mor-
fifty patients. J Bone Joint Surg Am 84(12):2123–2134, 2002, phogenetic protein antagonists during fracture healing in mice. J Anat
LOE I 216(5):625–630, 2010.
244. Dean DB, Watson JT, Moed BR, et al: Role of bone morphogenetic 246. Alt V, Donell ST, Chhabra A, et al: A health economic analysis of the
proteins and their antagonists in healing of bone fracture. Front Biosci use of rhBMP-2 in Gustilo-Anderson grade III open tibial fractures for
(Landmark Ed) 14:2878–2888, 2009. the UK, Germany, and France. Injury 40(12):1269–1275, 2009.
245. Epstein NE, Schwall GS: Costs and frequency of “off-label” use of 247. Garrison KR, Shemilt I, Donell S, et al: Bone morphogenetic protein
INFUSE for spinal fusions at one institution in 2010. Surg Neurol Int (BMP) for fracture healing in adults. Cochrane Database Syst Rev
2:115, 2011. (6):CD006950, 2010.

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Biology and Enhancement of

INTRODUCTION substrate and thus the porosity of these materials is critical.

calcaneus (P = 0.0001). There was no significant difference in Preclinical Substantiation

nonconcentrated autologous blood.183 Large variations exist

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