FEATURE ARTICLE

Limitations of Autograft and Allograft: New Synthetic Solutions

Randal R. Betz, MD

Abstract
Autogenous cancellous bone is widely regarded as an ideal construct for graft procedures, supplying osteoinductive growth factors, osteogenic cells, and a structural scaffold. However, procurement morbidity and constraints on obtainable quantities limit its use. Allograft is the next best alternative at present; however, minor immunogenic rejection and risk of disease transmission are unresolved issues. Although synthetic grafting materials eliminate these risks, these materials do not transfer osteoinductive or osteogenic elements to the host site. To offer the advantages of autograft and allograft, a composite graft may be considered. Such a graft can combine a synthetic scaffold with biologic elements to stimulate cell infiltration and new bone formation.

Bone is the most common tissue for transplant procedures, second only to blood. More than 2.2 million bone grafts worldwide and 450,000 in the United States are performed each year.1 Autogenous bone has been the implant of choice for most of these procedures; in the United States, autogenous bone was used in approximately 60% of bone grafts in 1996, compared with 34% allograft and 7% other materials.2 Autologous bone supplies the scaffold (bone mineral and collagen) for osteoconduction, growth factors (noncollagenous bone matrix proteins) for osteoinduction, and progenitor stem cells for osteogenesis. The iliac crest is chosen most frequently as the donor tissue site because of the quality and volume of bone available. However, procurement difficulties arise as a result of lengthened surgery with a second

From Shriners Hospital for Children, Philadelphia, Pa.

operative incision, limited supply of bone, and iatrogenic complications secondary to graft procurement.3 Allograft, the most common alternative to autograft, carries a finite risk of transferring contaminants, toxins, or infection from the donor. The processing of allograft tissue lowers this risk but, unfortunately, can substantially degrade the biologic and mechanical properties initially present in the donated tissue. An ample supply is not assured; processed and banked donor bone is not always available at the time of surgery.4,5 Great strides have been made with synthetic alternatives over the past decade. These materials may soon provide results comparable to or even superior to autograft (Table). Until recently, synthetics had been viewed less favorably than autograft and allograft; only 10% of bone graft procedures worldwide have relied on synthetic materials, including ceramics and polymers.1 Specific disadvantages encountered

with such materials in clinical settings have included low or unpredictable resorbability, difficulty in handling (coral-derived hydroxyapatite [HA]),6 and poor clinical results with occasional inflammatory foreign body reaction (degradable polymers).7,8 These complications can be surmounted with newer, modified materials, such as specialty ceramics. One of the most promising emerging surgical options may be the use of a composite graft that contains osteogenic cells and osteoinductive growth factors along with a synthetic osteoconductive matrix. Composite materials being tested in preclinical and clinical trials may demonstrate equivalent or superior functionality to autograft and allograft. Autograft Using autologous bone graft bypasses potential complications of host rejection or disease transfer. The autograft brings with it an osteoconductive s561
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GRAFT MATERIALS

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TABLE

Comparison of Bone Graft Materials

Graft Autograft Allograft Properties Osteogenic, osteoinductive, osteoconductive Osteoconductive, weakly osteoinductive Advantages No host rejection; no disease transmission; retains viable osteoblasts Greater availability of banked bone than autograft; customized forms available; no additional surgical procedure needed Limitless availability; no additional surgical procedure needed Limitless supply, injectable, biocompatible, some initial structural support Limitless supply, biocompatible; can be manipulated during placement Ease of use; no additional surgical procedure needed Structure provides a favorable milieu to bone regeneration Disadvantages Procurement morbidity, limited availability; high cost Immunogenic, disease transfer risk, not osteogenic; variable clinical results; expensive Not osteogenic or osteoinductive, little initial structural support Not osteogenic or osteoinductive, expensive, lack of placement control Expense reflects R&D, commercialization costs No structural support, not osteogenic Minimal structural support, potentially immunogenic

Ceramics (TCP, Osteoconductive hydroxyapatite) Injectable cerOsteoconductive amic cements -TCP/BMA Osteogenic, osteoinductive, composite osteoconductive DBM Osteoconductive, osteoinductive Collagen Good delivery vehicle for other synthetic graft materials

Abbreviations: -TCP5 -tricalcium phosphate, BMA5 bone marrow aspirate, DBM5 demineralized bone matrix, and R&D5 research and development.

matrix of cartilage, minerals and matrix proteins, osteoinductive proteins, and osteogenic bone and marrow cells.9 As a result, high success rates are achieved with certain graft procedures (Table). In spinal segment fusion, for example, autogenous graft in posterior cervical and lumbar procedures produces union in > 90% of cases.10 Successful incorporation and union, however, depend highly on the graft location and technique. For example, posterolateral lumbar fusion, the most common spinal arthrodesis procedure, shows failure (nonunion) in 5%-44% of surgeries in which autografts are used.10 The theoretical potential of autograft does not always translate into successful outcomes in clinical practice. Although bone harvested from the patient's iliac crest or rib transfers osteogenic precursor cells, its osteogenic capability is rapidly depleted; most of the cellular (osteogenic) components do not survive the transplant.10 Often, the supply of autogenous bone is not sufficient given the patient's overall physical condition or the indication for surgery. Autogenous bone graft may not be feasible in the elderly, in small pediatric patients, or in patients with metastatic disease. The treatment of spinal deformity in adults

or neuromuscular scoliosis in children can require a relatively large autograft.11 This may be impractical if it leads to iliac crest weakening to the point of precluding its later use for fixation points in spinal instrumentation. The volume of iliac crest is usually inadequate in cases of congenital scoliosis and neurofibromatosis. In cases of paralytic scoliosis and pelvic obliquity, posterior spinal fusion commonly involves additional instrumentation to the pelvis, thus precluding harvest from the posterior ilium.11,12 Additionally compromised pulmonary function, as is frequently seen in these patients, would bar retrieval of bone from the posterior rib cage.12,13 Atrophic osteoporotic pelvic bone, as accompanies paralytic scoliosis or progressive scoliosis secondary to cerebral palsy, compounds the difficulty in retrieving bone stock.14,15 Insufficient autogenous bone in a graft procedure may be a cause of subsequent failure to fuse.14 When long, multisegmental spinal fusions or revisions for fracture repair are required, retrieving adequate bone subsequent to previous harvests may not be possible. When the quality and quantity of a patient's bone stock make harvesting of an autograft possible, the surgical procedure presents risks of significant

complications. These risks include hernia formation, blood loss, nerve injury, postoperative infection, hematoma formation, and chronic pain at the donor site.9,16 In a study of 214 children, the incidence of perioperative complications was 2%. However, when interviewed, 24% of patients reported pain at the donor site and 15% had complications that affected daily living activities.17 Seiler and Johnson18 identified the reported risks of bone donor site surgeries: arterial injury, ureteral injury, herniation, chronic pain, nerve injury, infection, fracture, pelvic instability, cosmetic defects, hematoma, and tumor transplantation. Chronic pain has been attributed to heterotopic bone formation.19 Improved surgical techniques, such as limiting subcutaneous dissection and providing layered tension-free incision closures, have been suggested as a means of reducing some complications.20 Allograft Bone obtained from a donor is the most frequently chosen substitute for autogenous bone and is used to either replace or extend its volume. Procedures with allograft increased 14-fold between 1985 and 1996 and recently account for approximately one-third of bone grafts performed in the United States.2 s562
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The increased availability of banked, donated bone tissue relative to the supply of the patient's own bone has made possible the manufacture and fabrication of customized forms, such as dowels, strips, and chips, made from allograft tissue. Some of these machine-structured allograft forms, such as threaded bone dowels, can serve dually as fixation devices and delivery systems for bioactive agents.10 Including allograft as a bone filler can provide early structural support without procurement site morbidity and is associated with a 60%-90% overall success rate for large segmental replacements (Table).21 The best allograft results may be obtained in the adolescent patient undergoing scoliosis correction and fusion.10 Dodd et al22 assessed results in 40 patients with idiopathic scoliosis treated with grafts obtained from either femoral head allograft or iliac crest autograft. Rates of successful union were equivalent (100%) for the 2 groups. The allograft group demonstrated a low rate of infection, less blood loss at surgery, and reduced postoperative pain. The primary drawbacks of allograft include highly variable, less satisfactory clinical results compared with autograft, especially for posterior lumbar spinal fusion,23 and the finite risk of disease transfer and immunogenic reaction.24 These outcomes may be attributed in part to the preservation, storage, and sterilization procedures for banked allograft to lower the risk of disease transmission and rejection but which eliminate osteogenic cells and weaken osteoinductive potential. Processing and radiation also degrade structural integrity, reducing mechanical strength up to 50%.10,25 Allograft is more slowly incorporated than autograft and may require more hardware protection.23,25 Other drawbacks related to procurement are expense, donor-to-donor variation in quality, and limited availability. The cost of allogenic bone for a spine deformity procedure requiring 6 bottles of cancellous chips (30 cc per

bottle) is $2100. Families of potential donors harbor a fear of gross disfigurement that substantially curbs donation of bone relative to tenders of solid organs; tissue banks, therefore, experience difficulty with procurement.5,26 Processing of allogenic bone reduces, but does not fully eliminate, the risk of disease transfer and immunogenicity. Contaminants or pathologies that may be transferred include viral (especially human immunodeficiency virus [HIV] and hepatitis B and C) and bacterial infection, malignancy, systemic disorders (rheumatoid arthritis, autoimmune disease), or toxins. In a study of 1146 femoral heads removed from patients with osteoarthritis who elected hip arthroplasty, 8% of the femoral heads showed evidence of diseases that were unreported and undiagnosed at the time of hip surgery and were not pathologically associated with osteoarthritis, including occult malignant tumors. All 1146 bone specimens would have been deemed suitable for allograft by existing criteria apart from their analysis for this study.27 Even so, the risk of disease transmission with bone is low, even lower than that of blood.2 Risk of HIV in properly screened allograft is estimated to be 1 in 1.6 million, compared with 1 in 450,000 in blood. Nevertheless, at least 4 cases of HIV transmission by fresh-frozen allograft have been documented.23 The US Food and Drug Administration (FDA) has recently expressed concern that musculoskeletal tissue can contain prions implicated in degenerative neurologic disease, and that these prions may not be inactivated by approved processing techniques.28 The potential for any allograft to trigger an immune response is attributed to the major histocompatibilitycomplex antigens expressed in cellular material composing the allograft and, to a lesser extent, proteoglycans in the ground substance. In addition to HLA class I antigens expressed by the stromal cells of the graft, HLA class II antigens are expressed by the

parenchymal osteoblasts.29 Immunogenic responses delay, in a dose-dependent manner, the development of a new blood supply to the graft site, which, in turn, retards the rate of new bone growth and limits the long-term extent of incorporation and bone remodeling.23 The ability to promote an immune response is a function of allograft processing; more aggressive processing has been associated with less frequent and less intense immunologic responses. Fresh allografts are no longer used clinically for this reason. Frozen allografts induce stronger immune responses than frozen, freezedried allografts.23 In a multicenter study of 84 patients receiving massive frozen allografts, 49 (58%) patients were sensitized to class I antigens and 46 (55%) were sensitized to class II antigens.30 Friedlaender et al21 concur that long-term clinical outcome of allografts is improved when class II antigens are closely matched between donors and recipients, but they comment that biologic consequences of immunologic events remain speculative. In their series of 33 patients with large segmental deficits of long bones repaired with massive frozen allograft, 23 of 29 (79%) patients available for long-term follow-up exhibited satisfactory outcomes.21 While noting encouraging results in this study of unmatched grafts, Friedlaender et al21 also acknowledge that an understanding of immunologic responses associated with bone allografts provides opportunities to improve the predictability of clinical outcomes. Allografts are processed by 2 primary means, freezing and demineralizing. Both processes ensure the death of host cells. As reviewed by Burwell,31 death occurs primarily at the time of harvesting bone, although some peripheral cells may survive depending on the extent of the blood supply. Freezing, freeze-drying, and demineralizing processes are severe enough to kill any remaining cells. Allografts may be fresh-frozen or freeze-dried after at least one washing s563
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with an antibiotic solution. Fresh allografts are no longer used because they engender inflammatory responses,23 but freezing fresh allografts at 70C after an antibiotic wash reduces the immunogenicity. Freeze-dried (lyophilized) allografts are washed in antibiotic twice, frozen at 70C, and dried to 5 weight-percent water. These freshfrozen allografts are more osteoinductive and stronger than freeze-dried grafts. Human immunodeficiency virus has been transmitted in fresh-frozen allograft, but not in freeze-dried bone.23 Freeze-dried allograft is the least immunogenic but has inferior osteoinductive and mechanical properties and mechanical strength. Further sterilization of freeze-dried bone by ethylene oxide or gamma irradiation may also diminish osteoinductive properties.10 Rehydration of freeze-dried bone can result in longitudinal microscopic and macroscopic cracks, which may account for decreased graft strength of up to 50%.10,25 The shelf-life of freshfrozen bone stored at 20C is 1 year,10 at 70C, the shelf-life is 5 years23; the shelf-life of freeze-dried bone is indefinite.23 No amount of allograft processing will fully eliminate the possibility of an immune response or disease transfer. The enhanced safety of processing is offset by reductions in the biologic and mechanical function of grafts with more unpredictable outcomes than the autograft gold standard. Demineralized Bone Matrix Demineralized bone matrix (DBM) is an osteoconductive scaffold that is produced by acid extraction of banked allograft bone. It provides no structural strength and contains noncollagenous proteins, osteoinductive growth factors, and type I collagen.32 Demineralized bone matrix has greater osteoinductive potential than allograft due to enhanced bioavailability of growth factors secondary to the demineralization process.10,24 The efficacy of DBM in promoting new bone formation

depends on a number of factors, such as processing solutions, demineralization time, temperature extremes, DBM particle size, and method of terminal sterilization.2 Since the early 1990s, DBM products have been widely used in orthopedic surgery.10 The first commercially available preparation was Grafton gel (Osteotech Inc, Eatontown, NJ), consisting of DBM with a glycerol carrier; Grafton is also currently supplied as a malleable putty and as flexible strips. A moldable bone paste (Opteform, Medtronic Sofamor Danek, Memphis, Tenn), containing cortical bone chips, and an injectable bone paste (Osteofil, Medtronic Sofamor Danek), formed from demineralized powder, are recent, commercially marketed alternatives. Although prospective clinical studies are underway, available data suggest a role for DBM as a bone graft extender rather than as a bone graft substitute.10,33 Collagen Collagen is the most abundant protein in the extracellular bone matrix. The structure of collagen is conducive to mineral deposition, vascular ingrowth, and growth-factor binding, providing both a physical and chemical milieu favorable to bone regeneration.8 However, it provides little or no initial structural support and has potential immunogenicity. So far, studies have demonstrated its primary use as a delivery system for other osteoconductive, osteoinductive, or osteogenic factors with mixed clinical results.24 A composite of collagen gel with granules of a biphasic ceramic of HA and tricalcium phosphate (TCP) was studied in a posterior segmental canine spinal fusion model.34 The addition of collagen-ceramic composite to autograft was actually inferior to using a smaller volume of autograft alone. However, Zerwekh et al35 used the same collagen-ceramic composite in a canine anterior spinal fusion model and found it to be an effective autograft extender. A prospective, randomized

clinical trial comparing autograft with a composite of TCP and autogenous marrow in the treatment of long bone fractures found no significant difference with regard to rates of union or functional measures.36 Ceramics Advantages. Synthetic ceramics offer potentially limitless availability. Alone, synthetic ceramics impart no osteogenic or osteoinductive properties. Synthetic implants do not require a second operative site, and there is no risk of disease transmission or immunogenic response. The 3-dimensional (3-D) structure is the critical determinant of the speed of incorporation and remodeling; a more porous, lower density construct provides greater surface area exposure for supply of nutrients, vascularization, and bony ingrowth. The osteoconductive scaffold provides an appropriate environment in which bone cells and bone morphogenetic proteins (BMPs) can adhere and proliferate. Historically, the materials used have been hydroxyapatite (Ca10[PO4]6[OH]2), one of the calcium phosphate stoichiometries, or combinations of the two. Initially, the newly placed ceramic lacks compressive and tensile strength. Subsequent to incorporation and healing, the area of the implant attains mechanical strength similar to cancellous bone.37 Success with specific formulations for treatment of spinal misalignment or fracture repair has been reported. In a prospective study of posterior spinal fusion in adolescents with idiopathic scoliosis, 341 patients were randomized to either autograft or synthetic porous ceramic blocks (macroporous biphasic calcium phosphate [MBCP]).38 Overall results were comparable between the 2 groups at 18 months postoperatively except for increased wound complications in the autograft group. In a prospective study of 106 patients with degenerative spine or spondylolisthesis undergoing lumbar spinal fusion, MBCP granules were s564
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SYNTHETIC OPTIONS

BIOMATERIALS

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used with autogenous bone marrow and bone chips.39 Fusion was achieved in 100 of the 106 patients, suggesting that MBCP provides a suitable alternative to autografting. Disadvantages. The main disadvantage of using pure ceramic as a bone graft substitute is the minimal immediate structural support. However, purely cancellous (as opposed to corticocancellous) autograft or allograft also delivers little in the way of early structural support. A second disadvantage is the absence of osteoinductive or osteogenic properties. Both of these disadvantages may be addressed in future composites of biologic and synthetic elements. A third disadvantage is the potential expense reflecting research and development costs for synthetics; this, however, is offset by the reduction of procurement costs associated with autograft and allograft. Rapidly Resorbing Ceramics. Tricalcium phosphate constructs generally contain approximately 39% calcium and 20% phosphorus by weight, similar to natural bone.40 Such constructs have been used safely as synthetic bone void fillers in dentistry and orthopedics for decades.41,42 The construct's calcium phosphaterich surface layers seem to enhance bonding with adjacent host bone. This stimulates osteoclastic resorption and osteoblastic new bone formation within the resorbed implant.8 Slowly Resorbing Ceramics. Hydroxyapatite exhibits brittleness and resorbs slowly; implants of this material can become a focus of mechanical stress. It is often modified and combined with other materials for improved functionality and faster resorption. A pediatric study assessed results for 12 patients with severe scoliosis who underwent internal fixation and fusion with HA/TCP alone or combined with autogenous cancellous bone.43 Successful fusion was achieved for all patients treated with HA/TCP alone or combined with autograft, based on clinical and radiologic examination at follow-up averaging

15 months. Another study by Bucholz et al44 used either interporous HA or cancellous autograft to fill metaphyseal defects in 40 patients with displaced tibial plateau fractures.44 They found no radiologic or clinical difference between the 2 groups at follow-up. Injectable Ceramic Cements. Injectable calcium phosphate (Norian SRS, Norian Corp, Cupertino, Calif) represents a class of ceramics that combines some of the qualities of a cement with those of a bone void filler. Norian cement contains -TCP mixed with calcium carbonate and monocalcium phosphate monohydrate. In its initial liquid state, it can be injected by syringe into a fracture site or bone defect via closed or open techniques; it then hardens in vivo without generating significant heat. The initial compressive strength of the hardened material is similar to that of cancellous bone. The calcium phosphate implant undergoes long-term remodeling, and is ultimately completely replaced by host tissue.8 Vascular channel invasion, osteoclastic activity, and new bone formed in direct contact with the implant become visible within a few weeks of implantation, with minimal foreign body reaction.24 Injectable calcium phosphate shows promise for treating distal radius fractures and as an adjunct to treating femoral neck fractures, vertebral body compression fractures, and reinforcement of pedicle screw fixation.24 In a prospective, randomized study of 40 patients with redisplaced distal radial fractures, Kopylov et al45 compared treatment with injectable calcium phosphate bone cement (and 2 weeks of cast immobilization) with external fixation for 5 weeks. Functional outcome was identical except for more rapid recovery of grip strength and wrist mobility in the injected group, presumably due to the shorter immobilization time. Another prospective, randomized study compared conservative treatment (traction, 6 weeks of cast immobilization) with calcium phosphate bone cement injection (and 2 weeks of cast immobilization) in

110 patients with distal radius fractures.46 At the 1-year follow-up, the union rate was only 58% in patients treated by immobilization alone, compared with 82% in those injected with cement before immobilization. Patients treated with injectable cement experienced less pain and earlier restoration of movement and grip strength. The material has also been successfully used to augment hip fracture treatment (52 femoral neck fractures and 39 intertrochanteric fractures).47 A potential drawback of the liquid injectable cement is accidental extraosseous extrusion. Difficulties in controlling final placement may result in soft-tissue or intra-articular deposits.46 Ultraporous -TCP. A highly porous -TCP bone void filler that is composed of 90% interconnected void space with a broad range of pore sizes (1-1000 m) has been developed to mimic the trabecular structure of natural cancellous bone (Vitoss, Orthovita, Malvern, Pa). Small pores allow the wicking of phagocytic cells for resorption and bone-forming cells, nutrients, and growth factors for bone recovery through capillary refill. The larger size pores encourage vascularization and bone ingrowth.48 This ceramic construct includes pore sizes within the optimum range for osteoconductivity, 150-500 m, as cited by Gazdag et al.37 The -TCP particles have an average diameter of 100 nm, small enough for osteoclastic digestion in the remodeling phase. The material can be manipulated during placement, sculpted as blocks, or packed as morsels; after it is packed to conform to the shape of the bone defect, its high porosity remains intact. Like all ceramic scaffolds, -TCP does not possess intrinsic osteogenic or osteoinductive properties. A canine study was undertaken to determine the rate of new bone ingrowth into defect sites repaired with -TCP and to measure the rate of resorption of the -TCP scaffold.49,50 Cylindrical metaphyseal defects measuring 10 X 25 mm in canine humeri s565
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metals, such as porous tantalum) offer the potential for absolute control of the final structure, no immunogenicity, and excellent biocompatibility.8

Rationale A composite graft combines an osteoconductive matrix with bioactive agents that provide osteoinductive and osteogenic properties, potentially replicating autograft functionality. The osteoconductive substrate becomes a delivery system for bioactive agents,10 requiring less chemotaxis and less migration of osteoblast progenitor cells to the graft site. The direct infusion of progenitor cells should lead to more rapid and consistent bone recovery. When an osteoconductive scaffold is seeded with bone marrow aspirate (BMA) or BMP, for example, the composite graft may become both osteogenic and osteoinductive, providing a competitive alternative to autograft.
Figure: Radiograph of -tricalcium phosphate ( -TCP) implants from 3-52 weeks.

BIOLOGIC/SYNTHETIC COMPOSITE GRAFTS

were repaired with blocks of -TCP. Specimens were examined up to 1 year postimplantation, and bone ingrowth and scaffold resorption were quantified using standard histomorphometric techniques. The -TCP scaffold showed bone formation and resorption as early as 3 weeks postimplantation. By 6 weeks, the volume of new bone formed throughout the implants exceeded the volume of bone in areas adjacent to the defects by approximately 20%. Bone density was in the normal range and the scaffold was almost completely resorbed by the end of 12 weeks. By 24 weeks, the implant's trabecular orientation and thickness approximated that of adjacent bone. The bone within the defects was virtually indistinguishable histologically and radiographically from the surrounding bone at the 12-, 24-, and 52-week follow-ups (Figure).49,50 These findings were more encouraging than earlier histomorphometric

studies with traditional ceramics.51-53 Bruder et al52 found that a porous ceramic composed of HA and conventional -TCP showed no change in its structural geometry and essentially no resorption over the first 16 weeks after implantation. Delayed bone formation was evident mostly at the periphery of defects containing the ceramic implants,51,52 in contrast to the pattern of bone formation and resorption found throughout the implants examined in the canine ultraporous -TCP study discussed in the previous paragraph. The more uniform deposition of bone in the ultraporous -TCP study may have resulted from the greater interconnected microporosity of ultraporous -TCP, which may accelerate remodeling by facilitating in-migration and retention of osteogenic cells and nutrients. Nonbiologic Osteoconductive Substrates. Nonbiologic substrates (eg, degradable polymers and fabricated

Potential Composites Bone Marrow/Synthetic Composites. Clinical experience with bone marrow transplantation has established bone marrow as a highly reliable source of osteoblast progenitor cells.54 It is the only source that does not require an open surgical procedure or the additional time and cost of in vitro cell growth.24 In addition, marrow cells can be delivered percutaneously.55 Uncontrolled clinical studies suggest that aspirated bone marrow autograft may be of value in treating nonunions,56,57 but no prospective trials have studied isolated marrow grafts.58 Studies have looked at composites of bone marrow and synthetics for bone grafting with generally positive results. In athymic mice, HA loaded with human bone marrow cells placed subcutaneously showed an early bone response.59 In rabbits, coralline HA plus bone marrow was found to be an unacceptable combination to achieve lumbar spinal fusion.60 Solchaga et al61 s566
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attribute variable results with bone marrow to the interindividual variability of rabbit specimens. In sheep, a composite of a ceramic mixture of calcium phosphates and collagen mixed with bone marrow achieved spinal fusions of similar mechanical properties to autogenous corticocancellous bone after 6 months.62 Hydroxyapatite with bone marrow was comparable to autograft in repairing 3-cm gaps in the tibia of sheep,63 but composites of resorbable calcium phosphates and bone marrow were inadequate alternatives for autografts in the same model, although the stimulatory effect of bone marrow was noted.64 Some of these materials have been investigated clinically. A composite of a ceramic mixture of calcium phosphates and collagen mixed with bone marrow and autogenous bone chips available from the spine was used to fuse lumbar vertebrae in a series of 106 patients, most (95/106) with degenerative spine etiologies. After a minimum follow-up period of 2 years, 100 of 106 patients achieved spinal fusion. As degenerating spines are difficult to obtain arthrodesis, these results were considered especially encouraging.39 Ultraporous -TCP/BMA Composite. The newer ultraporous version of TCP, Vitoss synthetic cancellous bone void filler, may be well suited for use with BMA in a composite graft. The broad pore size range and interconnected microporosity endow the scaffold with favorable wicking and hydrophilic properties, encourage tissue ingrowth, and allow percolation of body fluids that facilitates the delivery of nutrients and physical dissolution of the scaffold. These attributes encourage retention of both seeded marrow cells and growth factors, as well as potentially greater penetration of bone-forming cells, phagocytic cells, growth factors, and nutrients from adjacent bone.65 Osteoinductive Growth Factors and Synthetic Composites. Bone morphogenetic proteins consist of a family of at least 15 structurally related osteoin-

ductive growth factors first identified in 1976 as the active osteoinductive fraction of DBM and characterized via molecular cloning in 1988.32 Of the BMPs, recombinant human bone morphogenetic protein-2 (rhBMP-2) (Genetics Institute, Cambridge, Mass) is closest to clinical application. Osteoinductive growth factors other than BMPs include transforming growth factor (TGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and fibroblast growth factor (FGF).32 The role of these other growth factors in fracture repair is not yet fully defined.66 Histologic analyses of tissue obtained from dental studies have confirmed new bone growing into defects repaired with composites of absorbable collagen sponge (ACS) and rhBMP-2.67 The safety and feasibility of the composite to augment local osseous defects and preserve alveolar ridges after extractions were shown in a 24-month trial of 12 patients in 2 centers, although some implant volume was lost from week 4 to week 8.68 Biopsies taken 3 years posttreatment in a 12-patient study showed normal bone formation in extraction sites and alveolar ridges augmented with rhBMP-2/ACS.69 Feasibility studies and prospective clinical trials have also begun to investigate orthopedic applications of BMPs. BMP-7, also called recombinant human osteogenic protein-1 (rhOP-1), was used in a prospective, randomized clinical trial of tibial nonunions initiated in 1994.70 Multicenter clinical trials have begun in the United States and abroad, and an interim report on the use of rhBMP2/ACS in open tibial fractures became available in 1999.71 In a prospective, randomized, double-blind 24-patient trial, 5 of the 6 patients receiving rhOP-1 in a collagen carrier demonstrated new bone formation from 6 weeks onward in a critically sized fibular defect.72 In a 2-year prospective, randomized, controlled trial, 14 patients with lumbar degenerative disk disease received threaded

interbody fusion cages filled with either rhBMP-2/collagen sponge or autograft.73 At 6, 12, and 24 months, fusions occurred in all 11 of the rhBMP-2 patients compared to only 2 of 3 autograft patients. In addition, patients in the rhBMP-2 group had shorter hospital stays, most likely due to the iliac crest procurement in the autograft group. Because previous animal studies had shown that bone would not spontaneously grow through collagen-filled cages alone, bone formation was attributed to the presence of rhBMP-2. In a prospective, randomized, controlled, partially blinded, multicenter 24-month trial, 122 patients with 124 tibial nonunions were treated with rhOP-1 in collagen or with autograft.74 Judging by clinical criteria 9 months after surgery, 81% of nonunions treated with rhOP-1/collagen fused versus 85% of nonunions treated with autograft (P= .52); by radiographic criteria, corresponding rates of fusion were 75% and 84%, respectively (P= .22). These initial results were continued after 2 years with no difference in outcomes (P= .94). The incidence of donor site pain was 20% in patients receiving autogenous bone. Luque compared the use of rhBMP2/biphasic-ceramic block composite with autograft. During posterior transverse process spinal fusion in 7 patients, the BMP/ceramic composite was implanted unilaterally, and the contralateral side was implanted with autograft. Preliminary results are similar between the 2 groups.10 The implantation of rhBMP-2/ceramic bilaterally, which would eliminate the need for autograft, is being examined in the second phase of this study. Another ongoing study will involve 45 patients undergoing anterior cervical diskectomy and fusion with autograft.10 An rhBMP-2/collagen sponge construct in various concentrations will be placed to fill the iliac crest procurement defect site. The future role of BMPs in orthopedic surgery should be better defined by the results of these studies. s567
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Growth factor enhancements can now be prepared intraoperatively from patients' blood. Proteins involved in healing and reparative processes are initially delivered to injury sites via platelets and white blood cells, where they are localized and concentrated by clotting. One surgical aid, Autogenous Growth Factors (Interpore Cross, Irvine, Calif), collects and processes the platelet- and leukocyte-rich buffy coat at the time of need to form a gel that stabilizes bone and graft fragments and assists in their fixation. The gel serves a dual function: it delivers and localizes higher levels of proteins needed for repair and constrains graft elements to achieve incorporation. The use of this type of gel to deliver exogenous osteoinductive growth factors also appears attractive.75 A method has been developed to separate platelet-rich plasma from patients' blood (Symphony, DePuy AcroMed, Raynham, Mass), which can then be used to enhance allograft. BMP/Polyglycolic Acid Polymer Composites. Two of the broader categories of polymeric membranes that have been investigated for use in bone grafting procedures are polytetrafluoroethylene (PTFE) and the degradable poly--hydroxy acids (PHAs), polylactic acid (PLA), and polyglycolic acid (PGA). Periodontal applications of PTFE and rhBMP were reported and reviewed in 199576; the combination of PHAs and BMP were reviewed in 199677 with a more general and current review of polymers and BMP following in 1999.78 Although degradable polymers have little osteoconductive potential and are associated with adverse foreign body reactions,24 they are potentially versatile as growth factor delivery vehicles with customized degradation rates and 3-D structure. Research in this area remains active. The addition of bovine-derived BMP to both PTFE and degradable membranes did not increase the rate of healing of critical-sized defects in the mandibles of rats, but rhBMP-2 accel-

erated healing when used with PLA/PGA membranes.79 A porous biodegradable copolymer, poly(D,L [lactide-glycide]) (PLGA) was evaluated as a carrier for rhBMP-2 using a canine posterior segmental spinal fusion model.80 The rhBMP-2/PLGA composite was compared with autogenous cancellous bone and with PLGA alone. Union score and mechanical testing showed no difference between autogenous cancellous bone and rhBMP-2/PLGA; both were superior to PLGA alone. BMA/BMP/Polyglycolic Acid Polymer Composite. Lane et al81 argues that studies examining the use of either osteoconductive matrices or osteogenic BMA alone, or even in combination, will be inferior to studies that include an osteoinductive stimulant such as BMP. A rat defect model showed the apparent synergistic effect of osteoinductive rhBMP-2 combined with bone marrow and a polylactic-glycolic acid carrier, with results superior to that of autogenous cancellous bone.

Several genes applicable to bone repair have been successfully transferred. The gene coding for the expression of TGF- 1 has been transferred directly into the nucleus pulposus of vertebral disks of rabbits, resulting in a doubling of the synthesis of proteoglycans relative to control disks.83 Genes from bovine intervertebral endplates have been transferred indirectly, thus suggesting novel ways of synthesizing pharmacotherapies in situ.84 In addition, potential clinical treatments to achieve spinal arthrodesis have been attempted by transferring genes that express the osteoinductive proteins LMP-1 and BMP-2. A mixture of bone marrow cells transfected with the cDNA for an osteoinductive protein, LMP-1, and devitalized bone matrix has been used to fuse vertebrae in nude rats,85 and an adenovirus containing the gene for BMP-2 has been injected percutaneously along the spine at the lumbosacral junction in nude mice to produce ectopic new bone formation.

ADVANCEMENTS
The early concept of gene therapy was described as the treatment of congenital diseases by transferring copies of healthy DNA into cells with defective genes. The broader view now includes the treatment and prevention of congenital and acquired diseases by transferring nucleic acid material (DNA or RNA). This transfer can be accomplished by direct (in vivo) application of DNA or RNA in vectors (generally, bacterial viruses are used) or by an indirect (ex vivo) means whereby nucleic acids are incorporated into cultured host cells out of the body and then put back into the body.82 The use of gene therapy to repair bone defects may have advantages over the injection of an exogenous bolus of osteoinductive BMPs. For example, the use of a cellular delivery vehicle containing modified genetic material may be more physiologic than an osteoconductive matrix, both in terms of timing and concentration.

SUMMARY
Bone grafting is one of the most common orthopedic procedures.86 Although autograft and banked allograft are used most frequently, synthetic composite bone substitutes that combine an osteoconductive matrix with osteoinductive growth factors and osteogenic cells may ultimately replace them.

1. Lewandrowski K, Gresser JD, Wise DL, Trantolo DJ. Bioresorbable bone graft substitutes of different osteoconductivities: a histologic evaluation of osteointegration of poly(propylene glycol-co-fumaric acid)-based cement implants in rats. Biomaterials. 2000; 21:757-764. 2. Boyce T, Edwards J, Scarborough N. Allograft bone: the influence of processing on safety and performance. Orthop Clin North Am. 1999; 30:571-581. 3. Hu RW, Bohlman HH. Fracture at the iliac bone graft harvest site after fusion of the spine. Clin Orthop. 1994; 309:208-213. 4. Broom MJ, Banta JV, Renshaw TS. Spinal fusion augmented by Luque-rod segmental instrumentation for neuromuscular scoliosis. J Bone Joint Surg Am. 1989; 71:32-44. 5. Henman P, Finlayson D. Ordering allograft by weight: suggestions for the efficient use of frozen bone-graft for impaction grafting. J

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