List of Abbreviations Used BMP bone morphogenetic protein; EGF endothelial growth factor; FGF fibroblast growth factor; HLA human leukocyte antigen; IGF insulinlike growth factor; IL interleukin; LMP LIM-mineralizing protein; OP-1 osteogenic protein-1; PDGF platelet derived growth factor; TGF transforming growth factor; TNF tumor necrosis factor
A significant proportion of clinical orthopaedic surgery involves attempts to stimulate bone healing. Although the results of fracture repair and reconstructive surgery usually are very good, complications of skeletal repair occur frequently enough that surgeons often need bone graft or a skeletal substitute material. Bone grafts and synthetic skeletal materials are being used with increased frequency worldwide, but there are many unanswered questions concerning the basic processes of graft immunology, incorporation, and remodeling. Clinical and basic science data do not yet provide sufficient information to chose the most appropriate synthetic materials to be used as skeletal substitutes, alone or in combination with autogenous bone grafts. Experience to date suggests that grafting materials and composites will become progressively specialized for use in specific applications. Optimum clinical application of these materials and progressive improvement on current constructs requires a detailed understanding of their biologic effects. Thorough reviews of different aspects of bone graft biology have been published previously.5,24-27,39,48,49,63,64 The purpose of the current study is to summarize selected basic science aspects of the incorporation and remodeling of bone graft materials.
The definitions of bone grafts and skeletal substitute materials can be confusing.25,57 Physicians usually think of a graft or a transplant as a tissue or organ containing donor cells that are intended to survive in the recipient. An implant generally is considered to be nonviable material.48 Bone allografts are different from most solid organ transplants, however, in that cells are removed intentionally as thoroughly as possible to minimize immunologic rejection. Strictly speaking, a bone allograft in which cells have been removed efficiently might be better described as an alloimplant, but this does not reflect the common vocabulary used by surgeons today. As orthopaedic surgeons now commonly are using composite materials for skeletal reconstruction, the authors of this review will adapt the definition proposed by Muschler and Lane:48 a bone graft material is any implanted material that, alone or in combination with other materials, promotes a bone healing response by providing osteogenic, osteoconductive, or osteoinductive activity to a local site. An osteogenic material can be defined as one which contains living cells that are capable of differentiation into bone. An osteoconductive material promotes bone apposition to its surface, functioning in part as a receptive scaffold to facilitate enhanced bone formation. An osteoinductive material provides a biologic stimulus that induces local or transplanted cells to enter a pathway of differentiation leading to mature osteoblasts.
The terms osteogenic, osteoconductive, and osteoinductive are not absolute, and are best understood when used in the context of a comparative study in which variables of the substrate material, porosity, surface geometry, and surface chemistry are highly controlled and defined. For example, when matched by size, shape and surface texture, hydroxyapatite is more osteoconductive than Ti, and Ti is more osteoconductive than a similar segment of CoCr alloy or stainless steel. However, changes in surface texture will change the relative performance of these materials. Similarly, one should expect to find differences in relative osteoinductivity between allograft products that have been processed with the use of different methods, and also among batches of allograft bone from different sites or donors, even if processed identically.
Bone graft materials can be divided broadly into autograft, allograft, xenograft, synthetic materials, and combinations thereof (Table 1). Autograft (autogenous graft) refers to bone tissue harvested from and implanted in the same individual. Autograft preparations include aspirated bone marrow, cancellous or cortical bone, or vascularized grafts. Vascularized bone autografts and cancellous graft inserted into a healthy host site may be simultaneously osteogenic (because of the viable cells), osteoinductive (because of the matrix proteins), and osteoconductive (because of the bony matrix). Although only a small fraction of the cells transplanted within devascularized segments of autograft bone survive, they may contribute to an improved healing response.
An allograft is defined as tissue that has been harvested from one individual and implanted into another individual of the same species. In this setting, the host is expected to mount an immune reaction against cells of a fresh allograft, so human bone allograft preparations are cleaned and processed to remove cells and reduce the host immune reaction. Removal of cell debris also reduces the risk of transplantation of viral particles, which may be intracellular. Allografts can be classified based on the graft anatomy, methods of processing and sterilization, and handling properties (Table 1). Lacking viable cells, allografts do not provide the osteogenic properties of viable autograft. The extent of osteoinductive and osteoconductive properties, and the mechanical strength of allograft vary depending in part on methods of graft processing.
Xenograft is bone tissue harvested from one species and implanted into a different species. A vigorous immune response precludes the use of most xenograft preparations. Deproteinated and defatted xenograft bone (Kiel bone or Oswestry bone) shows reduced immune response,17 but this process also destroys osteoinductive matrix proteins. One study reported osteogenesis in animals and humans when processed xenograft bone was supplemented with autologous marrow,55 but human allograft materials are considered more effective and more widely available at this time. Processed bovine collagen derived from bone or skin seems to be biocompatible and is a component of several evolving bone graft preparations. Collagen is a flexible substrate material, which can be prepared as a gel powder, sponge, paper, or feltlike mesh, depending on methods of preparation and cross-linking.
Synthetic materials have greatly expanded the available tools for bone grafting. Various extracted or synthesized protein growth factors and adhesion molecules, and synthetic osteoconductive materials are becoming available for use in orthopaedic surgery. These materials vary greatly in osteoconductivity and osteoinductivity, and in mechanical strength, handling properties, and cost. Several of these materials could be considered bone graft materials, but are beyond the scope of the current review.
Overview of Incorporation
The events leading to healing and mechanical stability of bone grafts have been reviewed many times.14,19,32,37,48,63,65 and will only be summarized in the current study. The term incorporation is used to describe the biologic interactions between graft material and host site that result in bone formation leading to adequate mechanical properties. This healing response includes the host inflammatory reaction to the trauma of surgical preparation of the graft site, the host inflammatory and/or immune reaction to the graft material itself, and the processes of cell proliferation, migration, differentiation and revascularization resulting in new bone formation and union between graft and host. The rate and extent of incorporation are dependent on the type of graft material, the tissues at the margin of the graft site, and the systemic physiologic state of the host. The biologic events occurring in the graft and graft site during the process of incorporation include: (1) hematoma formation with release of cytokines and growth factors; (2) inflammation, migration, and proliferation of mesenchymal cells and development of fibrovascular tissue in and around the graft; (3) invasion of vessels into the graft, often via existing Haversian and Volkmann canals; (4) focal osteoclastic resorption of graft surfaces; and (5) intramembranous and/or endochondral bone formation on graft surfaces.
Of these factors, the inflammatory reaction that occurs within the graft site may be especially important. As in any wound healing response, platelets adhere quickly to the wound surfaces and degranulate, releasing a large number of peptide growth factors, including FGF-2, PDGF, TGF-β and others into the fibrin network formed as blood and extracellular fluid coagulate within the graft site. Neutrophies, lymphocytes, and monocytes are attracted to the site and migrate into the organizing hematoma. Neutrophies in particular release kinins and prostaglandins that are angiogenic. The resulting granulation tissue is composed of relatively porous small blood vessels and edematous fibrous tissue rich in cytokines and growth factors. This sequence of changes is controlled by numerous cytokines, including prostaglandins, nitric oxide, vasoactive amines, complement factors, and interleukins. Collagen also is produced by fibroblasts, in part under stimulation by growth factors (such as TGF-β) and interleukins, and necrotic bone that is undergoing remodeling commonly shows fibrosis. Net collagen accumulation, however, also depends on the extent of collagen degradation, achieved primarily by metalloproteinases that also are released by fibroblasts, macrophages, and other cells.11,12 This process, through which an inflammatory response initiates vascular proliferation is necessary to provide the graft with nutrients and cells. It follows that modifiers of the inflammatory response might alter the process of bone graft incorporation. For example, indomethacin has been shown to delay the onset of mineralization if administered in the first 6 days of healing.54
The mechanical environment in the graft site also will have a profound effect on revascularization and cell differentiation. If the bone graft material does not achieve adequate mechanical stability, then granulation tissue and fibrosis are likely to develop at the interface between graft and host, precluding graft incorporation.
The rate and extent of incorporation also are influenced by the quality of the tissues in the host site. Of particular importance is the vascularity in the graft bed, and the abundance and competence of progenitors of endothelial cells and of connective tissue progenitors. A site deficient in one or both of these cell types will be less able to respond to an osteoinductive, osteoconductive, or angiogenic stimulus at the site. Settings in which stem cell deficiency may limit a graft site include areas of extensive scar, poor vascularity, large bone defects, previous infection, immunosuppressed hosts, and previous radiation therapy. Patients taking some pharmacologic agents, such as nicotine, also may have a compromised pool of local progenitor cells. Selected experimental and clinical studies of the incorporation of different types of graft materials will be summarized below.
Incorporation of Autografts
Incorporation is achieved most predictably using vascularized autograft, in which the inflammatory reaction that accompanies implantation is complicated by neither extensive ischemic necrosis of cells in the graft, nor immunologic reaction to the graft by the host. It has been suggested that if adequate vascular anastomosis and graft stability are achieved, more than 90% of the osteocytes may survive the transplantation.15 New bone formation by graft and host can lead to rapid graft incorporation (Fig 1); the amount of graft persisting with time then is determined by the remodeling process, influenced mostly by mechanical load (discussed below).
Even in a suitable host site the majority of transplanted cells die as a result of ischemia or are induced to undergo apoptosis. Among the cells that are most resistant to ischemia after transplantation are primitive mesenchymal cells present in the bone marrow and progenitors of endothelial cells. These cells may survive and even be stimulated to proliferate by changes in oxygen tension, pH, and the cytokine environments resulting from transplantation. Survival of these cells is thought to account in large part for the greater efficacy of autogenous cancellous bone compared with allograft. The graft matrix is invaded by granulation tissue relatively quickly. In an experimental study, osteoclastic bone resorption and new bone formation were seen after only a few weeks.5 In a pattern similar to that seen at the edge of a bone infarct, the formation of new bone on a scaffold of graft trabeculae may impart a slightly increased radiodensity to the graft area. As the graft matrix subsequently is resorbed by the process of remodeling, overall radiodensity returns to normal.5 The rate of autograft incorporation is influenced by many factors, including the host site, size of the grafted area, and the species of the host. A controlled study in rats showed maximum expression of the genes for Type I and Type III collagen at 1 week after cancellous autograft, and 2 weeks after cancellous allograft.72 This correlated with invasion of the graft by mesenchymal cells and early bone formation. Most of the autograft had been remodeled by 8 weeks.72
Nonvascularized Cortical Autograft
Segments of autograft (cortex or vascularized autografts in which the vascular anastomosis has been compromised) are composed of necrotic bone that does not induce immunologic rejection by the host. The matrix of cortical bone does not allow sufficient diffusion to support the survival of any useful fraction of the osteocytes after transplantation. Therefore, these segments are not appreciably osteogenic, but provide an osteoconductive substrate for bone formation by the host (Fig 2). Revascularization of cortical allografts is slow, in part because the density and low surface area of cortical bone physically bars vascular ingrowth.
Nonvascularized autograft cortex has the advantage of providing mechanical support at the graft site. Use of autograft also eliminates the risk of immunologic reaction, but overall, autograft cortical bone offers little advantage over allograft cortical bone, particularly when donor site morbidity is considered.
Autologous Bone Marrow
Bone marrow is a source of osteogenic cells that may be underused in contemporary orthopaedic surgery.49 Several studies have shown bone formation induced by marrow cells implanted in extraskeletal sites.1,52 Friedenstein et al23 showed that fibroblasts and stromal elements proliferate and are associated with bone production after transplantation of bone marrow cells. This suggested the presence of an undifferentiated precursor cell in postnatal bone marrow that could be induced to form osteoblasts. Many investigators have continued work to characterize the progenitor populations present in bone marrow. In vitro studies of bone marrow-derived osteoblastic progenitors have helped to define the potential role of many growth factors involved in regulating osteoblast differentiation.45 A few uncontrolled clinical studies suggest that aspirated bone marrow autograft may be of value in treating nonunions,28,35 but no prospective trials have studied isolated marrow grafting. A recent clinical study has shown that bone marrow-derived progenitors can be harvested by aspiration in essentially all patients, but that the concentration of progenitors is diluted with peripheral blood.46 This dilution can be limited, if the volume of aspiration from an individual site is held to 2 cc or less.46 Increasing the number of progenitors available in a graft site by manipulation of bone marrow to concentrate the progenitor population can increase the biologic result of bone grafting.8,9 The number of progenitors also can be increased by in vitro expansion using cell culture techniques. For example, Bruder and coworkers4 recently have reported healing of critical sized canine femoral defects using calcium phosphate cylinders loaded with cultured autologous mesenchymal stem cells. Implants containing cells developed more abundant periosteal new bone around the implants than untreated controls or defects treated with the implants alone.
Incorporation of Allografts
Bone allografts have been in clinical use for decades, and the histologic events in allograft incorporation have been reviewed many times.26,32,33,37,63-65 In general, the processes involved in allograft incorporation are qualitatively similar to those of nonvascularized autograft incorporation, but occur more slowly, and are accompanied by a variable amount of inflammation that can be attributed to an immunologic host response to the allograft.
Immunology of Transplant Rejection in General
Rejection of most solid organ transplants involves cell-mediated and humoral (antibody) immunity, and requires recognition of the graft by the host. The antigens most responsible for that recognition are those of the major histocompatibility complex, or HLA complex. The HLA molecules most important in transplant rejection are those of Class I and Class II. Class I antigens are expressed on essentially all nucleated cells and usually are expressed with proteins that were synthesized within the cell, especially viral proteins. Briefly, a foreign protein within a cell is bound to a newly synthesized subunit of the Class I molecule and then is transported to the cell surface. The foreign antigen is oriented in the Class I molecule on the cell membrane so that the receptor on a CD8+ T-lymphocyte can recognize it. Class II HLA molecules have a similar structure, but more commonly present antigens of extracellular origin to CD4+ helper T-lymphocytes. Unlike B-lymphocytes, T-lymphocytes recognize antigens only when presented by antigen-presenting cells, commonly called dendritic cells (which are similar to macrophages). Dendritic cells of either graft or host origin present antigens of graft cells to host T-lymphocytes. Once activated, CD4+ helper cells secrete cytokines that influence many other cells in the immune system, including B-cells, macrophages, natural killer cells, and other T-cells. Activated CD8+ cells also can secrete cytokines, but function primarily as cytotoxic cells. These populations of CD8+ and CD4+ T-lymphocytes are key to the inflammatory reaction directed at destroying the epithelial and endothelial cells of the transplant.11,12
CD4+ T cells also can stimulate B-lymphocytes to produce antibodies directed against the transplant. These antibodies can cause lysis of graft cells, and also are directed against the blood vessels of the graft. One histologic feature of rejection of any epithelial organ transplant is lymphocyte infiltration of the parenchymal cells of the transplant and the small blood vessels. Intimal hyperplasia of vessels, the consequence of antibody-mediated damage and lymphocytic vasculitis eventually can cause ischemic graft necrosis. Progressive chronic rejection causes fibrosis, more prominent vascular changes, and loss of tissue parenchyma.
Immunologic Reaction to Bone Allografts
As described above, the antigens most commonly associated with organ transplantation are Class I and Class II HLA antigens associated with the cells of the graft. It is not surprising that transplantation of fresh bone allograft also is associated with an immunologic reaction from the host. Many experimental studies have shown that in general, bone graft materials show optimum incorporation with the host when histocompatibility differences are minimized by either matching tissue types, or treating the allografts with techniques that reduce immunogenicity.2,3,18,30,31,34,39,64 For example, Gotfried and coworkers33 compared the histologic appearance of vascularized and nonvascularized bone grafts in genetically defined rats. Although lymphocytic infiltration was not described, the authors found evidence of damage to small blood vessels in mismatched bone grafts, especially vascularized allografts. Vascular damage was associated with extravasation of red cells with fibrin deposition, necrosis of marrow cells and osteoblasts, and later features suggestive of ischemic damage.
There is also ample evidence from animal studies that allografts induce the production of graft-specific antibodies.34,66,67 For example, using a canine fibular model to compare matched and mismatched, fresh and frozen osteochondral allografts, Stevenson66 found evidence of cell-mediated immunity and specific antibody production in mismatched, fresh grafts. An additional study using the same model suggested that the humoral immunity was directed primarily at Class I antigens.67 Antidonor antibody response was transient and less frequent in animals that received frozen allografts.67 Similarly, Horowitz and Friedlaender40 incubated allogenic bone with isolated T-cells in vitro to characterize the nature of T-cell activation. The authors showed activation of CD8+ T-cells with reactivity specific for antigens of the murine major histocompatibility complex, primarily Class I and Class II.39 Although several studies suggest that bone marrow cells are the major source of immunogenicity,12,21 Horowitz and Friedlander40 found that removing bone marrow cells did not eliminate the T cell activation. This suggests that cells within the diaphyseal cortex are capable of activating allogenic T cells.
Despite experimental evidence from animal studies that allografts can induce an immune reaction in the host,34,66,67 the clinical significance of this reaction in humans is unclear. Biopsy specimens of sites containing allograft sometimes show chronic inflammatory cells (Fig 3), but the histologic appearance is nonspecific and it is difficult to attribute the inflammation to an immune reaction with certainty. Similarly, Friedlaender24 identified graft-specific antiHLA antibodies in the sera of nine of 44 patients who had received freeze-dried allografts, but none of the nine patients had a poor clinical result.
Besides the potential consequences of local graft rejection, the implications of sensitization of the host to an expanded pool of HLA antigens also should be considered. Lee and coworkers43 have reported on a woman with kidney and pancreatic disease who was being evaluated as a potential kidney and pancreas transplant recipient. She was found to have a chondrosarcoma of the distal femur and later received a total knee prosthesis and frozen allograft. There was good incorporation of the graft to the host, but within weeks of surgery, the patient had broadly reactive HLA antibodies develop, which precluded her receiving organs from many cadaver donors. At least one animal study has shown that sensitization is less likely with deep-frozen allogenic bone when compared with fresh bone,7 but orthopaedic surgeons should be aware that bone allografts still have the potential to sensitize a patient, thereby limiting options for subsequent organ transplants. Although most patients receive allograft from only one donor, sensitization would be even more likely to occur in patients who receive allograft from several different donors.
Several studies have evaluated the histologic features of retrieved human bone grafts or bone grafts in which a biosy was done.20,36,38,51 For example, Muscolo and coworkers51 obtained 23 biopsy specimens from 16 patients, 9 to 78 months after transplanting large frozen allografts to the distal femur. The HLA types of all patients and all graft donors were determined before transplantation, but no attempt was made to match graft with recipient. Allografts from six patients showed infiltration by lymphocytes, and some of these cases had a poor HLA match, but overall there was no clear relationship between the extent of graft incorporation (revascularization and new bone formation) and the degree of histocompatibility match between graft and host.51
The histologic features of allograft incorporation in humans also was documented by Enneking and Mindell,20 who evaluated 16 retrieved massive human allografts. Their frozen or freeze-dried allografts had been implanted after tumor resections and had been in situ from 4 to 65 months. The authors of this review agree with numerous observations of Enneking and Mindell, including the following:
Union. Most allografts fuse to host bone at cortical to cortical or medullary to medullary junctions. At cortical junctions union occurs by intramembranous bone formation from reconstituted periosteum, not by extension of bone from the cut cortical ends. Gaps between host and graft cortex may be filled by bone extending in from the periosteum. Thus, the end of the graft usually can be recognized by a persisting cement (reversal) line (Fig 4). The osteons of the new bone are perpendicular to the axis of the cortical graft. In the study of Enneking and Mindell,20 the perpendicular orientation of Haversion systems did not remodel and remained distinguishable from the allograft, even after 5 years in vivo. Enneking and Mindell found that junctions between cancellous bone of graft and host formed more rapidly than between segments of cortical bone, in a pattern of repair reminiscent of the pattern of tissue healing around a bone infarct. The inner zone of the allograft remained necrotic and essentially acellular (Fig 5). A zone several millimeters thick showed evidence of creeping substitution, imparting a band of increased radiodensity to the interface (Fig 6). Enneking and Mindell showed that the extent of bony repair was correlated with revascularization. Although reparative tissue in most allografts penetrated only a few millimeters, a specimen retrieved at 65 months showed 25 mm of repair at the graft to host junction.
Graft Surface. Enneking and Mindell20 found that only a 1- to 2-mm thick layer of bone had been deposited by intramembranous bone formation onto the external surface of most grafts (Fig 7). Distinct cement lines indicated the peripheral edge of the allograft, although buds of remodeling had extended into Volkmann canals. In other areas of the graft surface, unrepaired erosions were present, lined by osteoclasts and filled with loose fibrous tissue and occasional inflammatory cells (Fig 8).
Rejection. Two of the specimens studied by Enneking and Mindell20 had been retrieved for presumed rejection. Superficial areas of resorption with rare lymphocytes were present, but neither specimen showed other histologic features thought to be typical of rejection.
Type of Bone Formation. In mechanically stable constructs of mineralized allografts, bone formation occurs by intramembranous deposition rather than by endochondral ossification. Cartilage is seen only if there is motion at the graft site. Based on these observations, frozen and freeze-dried human allografts seem to offer mostly osteoconductive surfaces rather than osteogenesis or significant osteoinductivity.
Demineralized Bone Allograft
The observation that demineralized bone matrix could induce bone formation when transplanted to soft tissue,69 initiated a search for the factors responsible for this osteoinduction. Urist et al70 later developed a chemosterilized, autolyzed, antigen-extracted allogenic bone (AAA bone), and described its use in posterolateral lumbar spinal fusion.71 Demineralized bone allograft has been used effectively alone or in combination with autograft as reported in an experimental study16 and has been used in several different clinical applications.56,73 Numerous different demineralized bone allograft preparations now are available. As described above, conventional mineralized allograft preparations are mostly osteoconductive, with minimal osteoinductive activity. Demineralized preparations, however, show clear osteoinductive activity, characterized by endochondral bone formation when placed into soft tissue (Fig 9).
Incorporation of Synthetic Materials
Many surgeons now are using composite preparations for skeletal reconstruction, including mixtures of autograft (bone marrow or cancellous bone), allograft (demineralized allograft bone), and synthetic materials (osteoconductive calcium phosphates, calcium sulfate, or a recombinant osteoinductive protein)48-50,61,64,75(Fig 10). It therefore is reasonable to consider the biologic events that may lead to mechanical stability (incorporation) of these synthetic materials or composites.
Most currently available biomaterials have undergone extensive preclinical testing materials that would induce a clinically significant immune reaction. Therefore, the inflammatory reaction that occurs at implantation is not complicated by the immune response seen with fresh bone allograft. Instead, the nature of the inflammatory reaction depends largely on the composition, size, and surface properties of the synthetic material. A discussion of the attributes of all currently available synthetic bone graft materials is beyond the scope of this discussion, but most currently available materials are osteoconductive and do not provoke a clinically important inflammatory reaction at the time of insertion. Experience with total joint prostheses, however, has heightened concern about potential bone resorption as a consequence of an inflammatory reaction to particles of wear debris,75 and concern has been expressed that wear debris from osteoconductive biomaterials also could lead to bone resorption. The cellular processes involved in debris-associated osteolysis now are thought to revolve around cytokines released from macrophages in response to contact with (or phagocytosis of) particles of debris smaller than approximately 2 um. Debris that is not readily dissolved by macrophages seems to be associated with the release of many cytokines, including TNF-alpha and IL-1β among others. These cytokines in turn directly, or indirectly promote osteoclast chemotaxis, maturation, attachment, and activation leading to bone resorption.41 Most skeletal substitute materials are available as relatively large granules, blocks, or cements, but it is possible that motion in vivo or the incorporation process could degrade these materials into particulate form. The clinical significance of these particles still is unknown, but because most of these materials are soluble in acid pH, it is anticipated that macrophages will digest the particles without initiating a sustained inflammatory reaction. At this time, bone resorption caused by particles of currently available skeletal substitute materials does not seem to be a clinically significant problem.
Synthetic Osteoconductive Bone Graft Materials
The specific events responsible for the osteoconductive properties seen with some skeletal substitute materials are unclear, and may involve extracellular mineral precipitation with crystal growth, and osteoblast-mediated bone formation. Regardless of the specific mechanisms involved, when placed into a suitable host site some osteoconductive materials show extensive bone apposition (Fig 11). However, if these materials are placed into an unsuitable host site, bone apposition will not occur. Once a bone graft material has become incorporated into the host bone, other factors, including the composition of the material and mechanical load influence long-term remodeling (see below).
Bone Morphogenetic Proteins
As described above, recognition of the osteoinductive properties of demineralized bone led to a search for the factors responsible for bone induction. A group of BMPs has been identified, most of which now are classified within the TGF superfamily. The classification, biologic properties, mechanisms of action, and receptor sites have been reviewed recently,49,61 and are beyond the scope of this discussion. Very briefly, BMPs play a critical role in the growth and differentiation of many different cell types during embryogenesis and homeostasis. They are important in the differentiation of stem cells into osteoblasts44 and also may influence other steps in fracture healing and bone remodeling. Among the BMPs most active in bone are BMP-2, BMP-4 and BMP-7 (OP-1). Several preclinical studies have shown bone formation to be induced by these synthesized proteins.10,47 Clinical trials are under way to test the use of these materials in various human applications. In the first report of a prospective randomized multicenter clinical trial of a human recombinant growth factor for bone healing, Muschler et al50 reported that BMP-7 (OP-1) delivered in a collagen carrier had comparable efficacy to autogenous cancellous bone for treatment of established tibial nonunions. Applications for other osteotrophic cytokines, specifically FGF-2, EGF, PDGF, TGF-β, parathyroid hormone and parathyroid hormone analogs, IGF-1, IGF-11, Indian Hedgehog (IHH), and LMP, also are being investigated and may play an important role in optimizing the bone healing response.
REMODELING OF BONE GRAFT MATERIALS
Once a bone graft material has achieved sufficient healing to become mechanically functional, factors besides the initial immune and inflammatory response begin to regulate its homeostasis. The overall rate of skeletal remodeling is one factor likely to influence long-term graft turnover. For example, cancellous grafts become integrated with the surrounding skeleton more rapidly in young patients who still are undergoing skeletal growth than elderly patients. Although little clinical data are available, bone grafts in patients with metabolic bone disease (renal osteodystrophy, hyperparathyroidism, or Paget's disease) would be expected to show different patterns of incorporation and remodeling than grafts in patients with normal skeletal homeostasis.
Perhaps the most important influence on long-term graft integrity is mechanical load. It has been recognized from before the time of Wolff74 that bone adapts to mechanical loads. The mechanism whereby mechanical load (strain) influences bone remodeling is unknown. There is experimental evidence that strain induces changes in anabolic activity of osteocytes and other cells present on the surface of bone42 and that the absence of strain leads to bone resorption. Strain may affect osteocytes directly by changing the shape of processes within osseous cannaliculi, or indirectly by, for example, hydrostatic pressure. Intermittent hydrostatic pressures have been shown to cause anabolic and catabolic effects on bone organ cultures6 and on osteoblasts in vitro.58 Therefore, it is not surprising that viable bone graft, such as vascularized autografts show architectural features consistent with adaptation to mechanical loads (Fig 1). Radiographic studies of patients with structural allografts occasionally have shown evidence of graft remodeling in response to mechanical load. For example, in a study of allografts used for pelvic reconstructive at revision arthroplasty, Trancik and coworkers68 showed resorption of a portion of allograft overhanging the edge of the acetabulum in an area not expected to transmit mechanical load. Bone was preserved in areas subjected to load, and the authors suggested that there had been resorption of the nonstressed portion of the bone graft (Fig 12). Biopsy specimens from other patients with similar grafts suggest that the bulk of the allograft is necrotic, with only a few millimeters of the graft surface replaced by viable bone. This observation supports the concept that the local signal transduction mechanism whereby mechanical load influences the activity of bone remodeling units on the surface of bone can occur in the absence of osteocytes. The authors of this review speculate that hydrostatic pressures or streaming potentials of ions associated with strain are transmitted to graft surface where these signals are transduced into cytokine signals by osteoblasts, osteocytes, or other surface lining cells that in turn influence activation of bone remodeling units.
Not only is incorporated but partially necrotic structural allograft remodeled under the influence of mechanical load, but some acellular biomaterials also may be remodeled. For example, Norian Skeletal Repair System (SRS®, Cupertino, CA) is an injectable, osteoconductive calcium phosphate cement that cures in vivo to form a mineral nearly identical to bone mineral. Frankenburg and coworkers22 inserted the cement into a transmural defect in the canine proximal tibia so that it occupied space within cortex and medulla. The material showed early bone apposition, followed by focal osteoclastic resorption, vascular ingrowth, and the formation of haversian systems histologically indistinguishable from those of remodeling bone (Fig 13). Serial evaluations showed that the cement within the cortex, presumably subjected to relatively high compressive loads, was resorbed and replaced by host cortical bone (remodeled) much more rapidly than bone present in the medulla (Fig 14). This suggests that mechanical load also can influence the rate of remodeling of skeletal substitute materials. The Norian Skeletal Repair System has a relatively high extent of microporosity and would be capable of transmitting changes in hydrostatic pressures that might influence bone remodeling in a manner similar to that of conventional bone graft. The authors of this review suggest that for a synthetic material to respond to Wolff's law, it must meet several requirements: (1) it must be osteoconductive so that it can become physically and mechanically incorporated with the host such that mechanical loads are transmitted through the graft site; (2) it must have mechanical properties that prevent mechanical failure (fracture, deformation, or particulate wear) under the loads experienced in vivo; and (3) it must be composed of a material that permits or facilitates osteoclastic resorption. These material properties might include porosity that allows transmission of changes in hydrodynamic pressure or streaming potentials throughout the bulk of the material. Under these circumstances, it seems likely that at least some acellular skeletal substitute materials may undergo remodeling in response to load. Exploiting this concept could lead to the development of composite biomaterials with properties that are better suited to specific skeletal applications.
Impaction Bone Grafting
Another controversial clinical application of allograft is impaction grafting at the time of revision arthroplasty in the presence of severe proximal femoral bone loss. Originally described for use in the acetabulum by Sloof et al,62 and later adapted for use in the femur,29 impaction grafting involves cementing a femoral component into compressed bone graft, usually morselized allograft. Although many variations of the technique are in current practice, impaction grafting as promoted by Fowler et al,29 uses a polished, tapered femoral component that is not designed to bond to the cement. It has been suggested that the absence of cement bonding allows the femoral component to convert shear loads into radial compression, thereby subjecting the impacted graft to compressive loads and at the same time preserving the sometimes relatively thin mantle of bone cement. There are numerous controversial aspects to this procedure, a complete discussion of which is beyond the scope of the current study. One of the controversies pertains to the extent to which impacted bone graft can be incorporated and remodeled, especially when the graft is adjacent to a cemented implant. Biopsy specimens from the proximal femur obtained at the time of removal of trochanteric wires in a small group of patients provided evidence of graft remodeling in some cases.53 More recently the authors have studied multiple histologic sections of the proximal femur from a man who died of a myocardial infarction 2 years after rerevision arthroplasty with the use of impaction grafting. These sections show zones of differentiation similar to those described by Enneking and Mindell20 in retrieved massive allografts. An inner zone of necrotic trabecular bone (unincorporated impacted allograft) lies next to the cement, surrounded by a zone showing evidence of creeping substitution and relative sclerosis, and by a peripheral zone of histologically normal cancellous bone and cortex (Fig 15). Interestingly, sections from the inner zone immediately adjacent to the cement were found to contain small blood vessels with red blood cells, suggesting that the complete thickness of the impacted graft layer may have been neovascularized, even though there was not yet evidence of incorporation or remodeling of the adjacent bone (Fig 16). Anectodal findings from autopsy retrievals should be interpreted with caution, but histologic evidence of extensive vascular ingrowth and at least focal graft incorporation provide some support to the general concept of impaction grafting in the proximal femur.
The influence of hardware fixation in conjunction with bone graft materials is complex, and beyond the scope of this discussion. For many applications, hardware is necessary to provide sufficient stability to allow for graft incorporation. The extent to which the bone graft will be shielded from load by the hardware depends on many factors, not the least of which are the size, shape, and composition of the hardware. Of particular interest is the use of cages in conjunction with autograft, allograft, and other bone graft materials to help achieve spinal fusion. Although few controlled trials have been completed, early clinical experience using cages of several different designs generally has been good. The authors' evaluation of cages retrieved for persistent nonunion or malposition generally has shown histologically viable bone within the cages, sometimes with features consistent with remodeling bone graft (Fig 17). Additional studies using standardized graft procedures and quantification of viable bone after loading in vivo will be required to determine the influence of cage design on incorporation and remodeling of bone graft materials used for spinal fusion.
Bone graft preparations and synthetic skeletal substitute materials will continue to play an important role in reconstructive orthopaedic surgery. The mechanical properties, osteoconductive and/or osteoinductive properties, and the rate and extent of remodeling vary among graft materials. These properties are influenced by many factors, including the nature of the graft itself, the quality of the host site and the local mechanical environment (strain). As application-specific graft products and bioactive composite materials become available, understanding the advantages and disadvantages of each material will be necessary for optimum use.
The authors would like to express their appreciation to the following surgeons who contributed specimens that were illustrated in this manuscript: William Head, MD; W.M. Michael Mikhail, MD; Michael Joyce, MD; Bernard Stulberg, MD; Ken Marks, MD; P.P. Castelyan, MD; Maarten Persenaire, MD; and Arthur Malkani, MD. The authors also thank Nelson Scarborough, PhD for contributing an experimental case, Alan Wilde, MD for contributing radiographs, Sharon Stevenson DVM, for helpful suggestions, and Jessica Ancker for help editing the manuscript.
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