The osteoinductive capacity of differently HCl-decalcified bone alloimplants

Three procedures to obtain bone inductive implants were tested heterotopically in 3-month-old allogeneic rats: 1) antigen-extracted HCl-decalcified at 4 degrees C, autolysed implant (AAA bone); 2) HCl-decalcified implant at 4 degrees C; 3) HCl-decalcified implant at room temperature. Each type of implant was either deep-frozen at -35 degrees C for at least 2 months or immediately freeze-dried. The bone inductive capacity of the differently HCl-decalcified cortical bone implant was evaluated at 2 months by isotopic strontium incorporation and by ash-weight measurements. Bone HCl-decalcification alone, either at 4 degrees C or at room temperature, gave a higher new bone yield than the freeze-dried AAA bone. The type or short-term preservation technique had no effect on the osteoinductive capacity of either of the differently treated implants, AAA bone expected.     

Comparative in vivo study of six hydroxyapatite‐based bone graft substitutes

Improvement of synthetic bone graft substitutes as suitable alternatives to a patient's own bone graft remains a challenge in biomaterials research. Our goal was to answer the question of whether improved osteoinductivity of a material would also translate to better bone‐healing orthotopically. Three porous biphasic calcium phosphate (BCP) ceramics (BCPA, BCPB, and BCPC), consisting of hydroxyapatite and β‐tricalcium phosphate, a porous biphasic calcium phosphate ceramic reinforced with a bioresorbable polylactic acid to improve its mechanical properties (BCPC+), a pure hydroxyapatite ceramic (HA), and a carbonated apatite ceramic (CA) were implanted intramuscularly and orthotopically by using a transverse process model in 11 goats for 12 weeks. BCPA and BCPB had similar chemical composition but differed in their microstructure. BCPB was not osteoinductive at all, but BCPA induced ectopic bone formation in 9 of 11 animals. Orthotopically, BCPA performed better than BCPB in both the amount and rate of bone formation. BCPC, similar to BCPA structurally and physicochemically, showed comparable results ectopically and orthotopically. Addition of resorbable polymer to BCPC made the material less osteoinductive (4 of 11 animals) and delayed bone formation orthotopically. Neither HA nor CA were osteoinductive, and their orthotopic performance was inferior to the osteoinductive ceramics. The results of the present study showed that material‐derived osteoinduction significantly enhanced bone healing orthotopically, and that this material property appeared more sensitive for predicting orthotopic performance than physicochemical and structural characteristics. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1363–1370, 2008     

Ossification of decalcified and nondecalcified homologous bone grafts

This two-year study examined the process of reossification and survival of decalcified and nondecalcified bone grafts implanted under the scalp tissue of 15 rabbits. Both radiologic and histologic examination showed that the nondecalcified fresh-frozen bone specimens became totally viable and recalcified while largely retaining their initial size. The decalcified bones, however, were totally absorbed within a period of four months. Possible reasons for failure of the decalcified bone to reossify are discussed.     

The osteoinductive capacity of differently HCI-decalcified bone alloimplants

Three procedures to obtain bone inductive implants were tested hetero-topically in 3-month-old allogeneic rats: 1) antigen-extracted HCl-decalci-fied at 4°C, autolysed implant (AAA bone); 2) HCl-decalcified implant at 4°C; 3) HCl-decalcified implant at room temperature. Each type of implant was either deep-frozen at-35°C for at least 2 months or immediately freeze-dried. The bone inductive capacity of the differently HCl-decalcified cortical bone implant was evaluated at 2 months by isotopic strontium incorporation and by ash-weight measurementsx. Bone HCl-decalcification alone, either at 4°C or at room temperature, gave a higher new bone yield than the freeze-dried AAA bone. The type or short-term preservation technique had no effect on the osteoinductive capacity of either of the differently treated implants, AAA bone expected.     

Postmortem degradation of demineralized bone matrix osteoinductive potential. Effect of time and storage temperature

The osteoinductive growth factors present in demineralized bone are degraded by tissue enzymes. Storage of rat limbs at low temperature (4 degrees C) before harvesting of bones preserves the osteoinductive potential of such factors. Storage at room temperature for more than 24 hours causes the recovered bone matrix to be biologically inactive, presumably as the result of biodegradation.     

Morphometric and physical investigations of segmental cortical bone autografts and allografts in canine ulnar defects.

Cortical bone grafts were implanted for six months in mature dogs using an osteoperiosteal 3-cm defect in the ulna to evaluate their respective morphometric and physical values compared with autografts. The bone-grafting material included fresh auto- and allografts, frozen and thimerosal preserved allografts, and partially demineralized bone allografts. The grafts were evaluated by roentgenograms, microradiograms, photon absorptiometry, porosity, fluorescence labeling measurements, and torsional loading at failure. Autografts achieved a better union score than the allografts, but intracortical bone porosity, percentage of cumulative new bone, and mineral apposition rate were not variables with statistical significance. Lamellar bone was found earlier and in greater quantity in autografts. Within the graft, new bone was deposited at a slower rate than in the recipient bone. Autografts showed less peripheral resorption and a greater torsional resistance than allografts. Photon absorptiometry demonstrated that nondemineralized allografts underwent a substantial loss of peripheral bone. This marked reduction in the outer diameter of the graft had more influence on torsional resistance than did the intracortical porosity of the graft. Demineralized allografts were osteoinductive in only 28% of the cases and appeared to respond in an all-or-nothing pattern. Frozen and thimerosal preserved allografts were the most acceptable substitutes to autografts.     

Influence of irradiation on the osteoinductive potential of demineralized bone matrix

Summary Samples of demineralized bone matrix (DBM) were exposed to graduated doses of radiation (1–15 Megarad) (Mrad) utilizing a linear accelerator and then implanted into the thoracic region of Long-Evans rats. Subcutaneous implantation of DBM into allogenic rats induces endochondral bone. In response to matrix implantation, a cascade of events ensues; mesenchymal cell proliferation on day 3 postimplantation, chondrogenesis on day 7, calcification of the cartilagenous matrix and chondrolysis on day 9, and osteogenesis on day 11 resulting in formation of an ossicle containing active hemopoietic tissue. Bone formation was assessed by measuring alkaline phosphatase activity, the rate of mineralization was determined by measuring⁴⁵Ca incorporation to bone mineral, and⁴⁰Ca content measured the extent of mineralization; acid phosphatase activity was used as a parameter for bone resorption. The dose of radiation (2.5 Mrad) currently used by bone banks for sterilization of bone tissue did not destroy the bone induction properties of DBM. Furthermore, radiation of 3–5 Mrad even enhanced bone induction, insofar as it produced more bone at the same interval of time than was obtained from unirradiated control samples. None of the radiation doses used in these experiments abolished bone induction, although the response induced by matrix irradiated with doses higher than 5 Mrad was delayed.     

Incorporation of impacted morselized bone allografts in rabbits

Morselized bone allografts have been used for the treatment of bone stock loss in orthopedic revision surgery with encouraging results. However, several parameters can influence the graft incorporation including the processing treatments. This experimental work used a cavitary bone defect in 90 rabbits to evaluate the sequence of incorporation of three different kinds of morselized bone allografts: uncryopreserved cancellous bone, freeze-dried cancellous bone, and totally demineralized cortical bone each of which were prepared in accordance with our rigid protocol. Revascularization and remodeling of the transplanted bone grafts were evident upon histological evaluation. Bone apposition and bone resorption resulted in a mixture of graft and new bone. Mineralized cancellous grafts showed great osteoconductive capacity, whereas demineralized cortical grafts showed an intense osteoinductive capacity and a weak osteoconductive capacity. In a general evaluation, cryopreserved cancellous bone grafts showed superior biological efficacy for reconstruction of experimental bone defects, closely followed by freeze-dried cancellous bone grafts, and, finally, by demineralized cortical bone grafts. These results validate our protocol for the processing and preservation of these three kinds of bone grafts.     

Changes in allograft bone irradiated at different temperatures

Secondary sterilisation of allograft bone by gamma irradiation is common, but the conditions under which it is performed vary between tissue banks. Some do so at room temperature, others while the bone is frozen. Bone is made brittle by irradiation because of the destruction of collagen alpha chains, probably mediated by free radicals generated from water molecules. Freezing reduces the mobility of water molecules and may therefore decrease the production of free radicals. We found that bone irradiated at -78 degrees C was less brittle and had less collagen damage than when irradiated at room temperature. These findings may have implications for bone-banking.     

Extracorporeal irradiation and incorporation of bone grafts: Autogeneic cortical grafts studied in rats

The incorporation of resected, extracorporeally irradiated (1, 5, 25 and 50 kGy) and orthotopically reim-planted autogeneic cortical bone was investigated in 116 adult Wistar rats. 7 mm-long diaphyseal segments of the tibia were resected, irradiated and re-implanted using K-wire osteosynthesis. Autogeneic fresh grafts served as controls. Graft healing was evaluated by radiography and histomorphometric study at 3, 6, 9, and 12 weeks. At 3 weeks, two thirds of the 50 kGy irradiated grafts were fractured and therefore the series with this dose was interrupted because of mechanical graft insufficiency. After 3 and 6 weeks there were no statistically significant differences among the control group and 1 or 5 kGy irradiated grafts. The healing of 25 kGy irradiated grafts was delayed from the sixth week onwards and continued until the end of the experiment at 12 weeks (50% reduction of incorporation). The incorporation of 1 and 5 kGy irradiated grafts showed a 16% (1 kGy) to 24% (5 kGy) delay at 12 weeks, compared to autogeneic fresh grafts. 1 and 5 kGy irradiated autogeneic bone grafts retain most of their biological potential. Resection, extracorporeal irradiation and reimplantation of bone tumors may therefore be a possible alternative to allografting.     

Changes in subchondral bone in cartilage resurfacing--an experimental study in sheep using different types of osteochondral grafts

OBJECTIVE: This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. DESIGN: Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. RESULTS: Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. CONCLUSION: Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.     

Femoral and acetabular revision using impacted nondemineralized freeze-dried bone allografts

Background  Favorable results have been obtained by the use of deep-frozen bone allografts in total hip arthroplasty. However, owing to the shortage of deep-frozen allografts and the risk of infectious disease, other materials have been studied, such as sterile nondemineralized freeze-dried allografts. The aim of this study was to describe midterm clinical outcomes and radiographic bone incorporation of human freeze-dried bone grafts in 42 revision total hip arthroplasty procedures using cancellous impacted bone grafting. Methods  This report presented clinical and radiographic evidence of allograft incorporation in 42 hip reconstructions performed between 1996 and 2002. The patient group included 13 (31%) men and 29 (69%) women with mean ± SD age of 63 ± 14 years (range 28–80 years). Meanfollow-upwas 82 months (range 63–127) months. Clinical analysis was based on the D’Aubigné-Postel score. Radiographic incorporation was defined according to specific criteria. Results  The D’Aubigné and Postel criteria showed adequate outcome in 38 (90%) of the patients. The radiographic evaluation revealed that allograft remodeling and incorporation were found in 39 (93.0%) and 36 (86.5%) of acetabular and femoral cases, respectively. The overall graft survival rate at an average follow-up of 8 years (range 5–10 years) was 90%. Conclusions  Bone grafts obtained by the lyophilization process developed and carried out in our tissue bank provide suitable grafts for revision total hip arthroplasty. Clinical and radiographic midterm results were excellent, indicating that nondemineralized freeze-dried bone allografts are suitable for replacing deep-frozen grafts.     

Irradiation has no effect on the incorporation of impacted morselized bone: a bone chamber study in goats

Background Gamma irradiation has been widely used for sterilization of bone allografts. However, gamma irradiation alters proteins. This is favorable when it reduces immunogenicity, but is undesirable when osteoinductive proteins are damaged. Although the effect of gamma irradiation on BMPs has been studied, the effect of irradiation on the process of incorporation of morselized bone chips remains unclear. We studied the effects of sterilization by gamma irradiation on the incorporation of impacted morselized allografts.

Methods Bone chambers with impacted allografts, rinsed impacted allografts, allografts that were rinsed and subsequently irradiated, and an empty control were implanted in proximal medial tibiae of goats. Incorporation was evaluated using histology and histomorphometry.

Results Histology revealed evidence of bone graft incorporation, which proceeded in a similar way in unprocessed, rinsed, and both rinsed and irradiated bone grafts. After 12 weeks, no difference in bone and tissue ingrowth was found between the unprocessed, the rinsed, and the rinsed and subsequently irradiated allografts. The amount of unresorbed graft remnant was highest in the unprocessed bone grafts.

Interpretation We conclude that sterilization with gamma irradiation does not influence the incorporation of impacted rinsed bone allografts.     

Fate of vascularized and nonvascularized autografts

Controlled laboratory data demonstrate biologic and mechanical characteristics of orthotopically placed canine ulnar autografts. The pattern of bone repair was similar in vascular and nonvascular ulnar grafts beginning with resorption followed by appositional reactive bone formation. The time intervals in the sequence were accelerated in the vascularized grafts. Both the strength and stiffness of the vascularized grafts were found to be significantly greater in Groups studied from six weeks to six months postoperation. There was no statistical difference between the grafts for mechanical testing performed on dogs either one week or one year postoperation. The vascularized ulnar bone grafts fared better than comparably sized nonvascularized grafts and were more rapidly repaired. The mechanical testing demonstrated superior strength and stiffness of the vascularized grafts throughout the repair process.     

Natural history of autografts and allografts

The clinical outcome of bone grafting procedures depends on many factors, including type and fixation of the bone graft as well as the site and status of the host bed. Bone grafts serve one or both of two main functions, as a source of osteogenetic cells and as a mechanical support. Autografts, both cancellous and cortical, are usually implanted fresh and are often osteogenetic, whether by providing a source of osteoprogenitor cells or by being osteoinductive. The latter is a process whereby the transplanted tissue induces mesenchymal cells of the recipient to differentiate into osteoblastic cells. Cortical grafts, whether autogeneic or allogeneic, at least initially act as weight-bearing space fillers or struts. All bone grafts are initially resorbed, but cancellous grafts are completely replaced in time by creeping substitution, while cortical grafts remain an admixture of necrotic and viable bone for a prolonged period of time. The three-dimensional framework, which supports invasion of the bone grafts by capillaries and osteoprogenitor cells, termed "osteoconduction", is another important function of both autografts and allografts. Fresh allografts are more slowly and less completely replaced by host bones because they invoke both local and systemic immune responses that diminish or destroy the osteoinductive and conductive processes. Although freezing or freeze-drying of allografts improves acceptance, their failure rate is still too high. These processes are also influenced by the vascularity and composition of the host bed. Thus, the interaction of the host and the bone graft determines the success of these procedures, which ultimately is to provide a mechanically efficient support structure.     

Intact articular cartilage cryopreservation. In vivo evaluation.

By varying the parameters that determine the cryopreservation process (freezing rate, storage temperature, cryoprotective agents, storage time), various protocols have been designed for preservation of osteochondral allografts of rabbit femoral condyles. The grafts were implanted orthotopically for three months. After harvesting, the results were evaluated by optical microscopy, histochemistry, and electron microscopy (scanning and transmission). A quantitative study and statistical analysis was performed using a gradation scale of chondral degeneration. Synovial reaction was higher in the fresh allograft than in the cryopreservation groups. Glycerol yielded better results than DMSO as a cryoprotective agent. No cryopreservation protocols produced the results equal to the control groups of either fresh auto- or allografts. Optimal preservation was achieved with either the group with hypothermic storage at 4 degrees for no more than 48 hours or the group with slow freezing to -80 degrees and preparative exposure to 15% glycerol at 4 degrees for 60 minutes. No statistically significant differences were found in these two groups.     

Cellular repopulation of deep-frozen meniscal autografts: An experimental study in the dog

This study evaluated the cellular repopulation of deep-frozen meniscal autografts. Medial menisci of adult dogs were excised, deep-frozen in liquid nitrogen (-196 degrees C) for 10 min, and orthotopically reimplanted into the joint. Deep-freezing was found to effectively kill all the cells within the meniscus as determined by the absence of Na(2)35SO4 incorporation. Following orthotopic replacement within the knee joint, menisci were repopulated with cells that seemed to originate from the adjacent synovium. These cells migrated over the surface of the meniscus and began to invade the deeper layers of the tissue. However, even after 6 months, the central core of the meniscus remained acellular. While the new cells appeared to modulate into cells that are similar in appearance to meniscal fibrochondrocytes, the exact phenotypic expression of these newly differentiated cells has yet to be determined. Histological alterations, as manifested by a loss of normal orientation of the collagen architecture of the superficial layers of the meniscus; was evident at 6 months and suggests that a remodeling phenomenon may be associated with the cellular repopulation. While biomaterial studies have not been carried out on these specimens, the morphologic alterations observed in the collagen orientation would suggest a possible alteration in the material properties of the repopulated meniscus. The clinical implication of this study is that the structural remodeling associated with the cellular repopulation of deep-frozen meniscal allografts may make the transplanted meniscus more susceptible to injury.     

Allograft reconstruction in total knee arthroplasty.

We reviewed 32 deep-frozen irradiated allografts used for the reconstruction of bone defects in 20 knees. They were subdivided into bulk grafts, cortical strut grafts, and morsellised bone. The average follow-up was 4.2 years (2 to 7.2). Radiographs showed union of the allograft to the host in all cases. Two allografts later fractured and three knees required further surgery because of infection. The allografts effectively filled large bone defects around the knee, lessening the need for custom-made and constrained prostheses.     

The Clinical Use of Allografts,Demineralized Bone Matrices,Synthetic Bone Graft Substitutes and Osteoinductive Growth Factors: A Survey Study

The emergence of new bone grafting options and alternatives has led to significant uncertainty when determining the most appropriate product for surgical procedures requiring bone graft in orthopedics. Allografts, demineralized bone matrices, synthetic bone graft substitutes, and osteoinductive growth factors are all viable options, yet there is a lack of data reporting clinical usage of these products. This correspondence reports on the use of bone grafting products at the Hospital for Special Surgery for a 27-month period and makes recommendations based on surgical usage, safety, and cost. Approximately half (48.6%) of all bone graft substitutes were implanted during spinal surgery. Arthroplasty, trauma, and foot/hand cases all used considerable amounts of bone grafting products as well (20.1%, 19.0%, 12.1%, respectively). Considerable differences were noticed in usage of bone grafting products among each orthopedic discipline. Of all bone graft substitutes used in arthroplasty, 14.4% were demineralized bone matrices, whereas 56.8% were allografts. Demineralized bone matrix grafts were used in 82% of trauma surgery and 89% of foot/hand cases. An increase in synthetic bone graft alternatives was noticed near the end of our investigation period.     

Complications of irradiated allografts in orthopaedic tumor surgery

Massive structural allografts used for replacement of bone defects after removal of bone tumors have several complications, including fracture, infection, and nonunion. To decrease the rate of infection, irradiation of selected allografts before their implantation was performed. This study evaluated the complications in patients with these irradiated grafts. Twenty-four patients were identified who had received allografts from 1987 through 1991 that were irradiated before implantation. The dosage of radiation was between 10 kGy and 30 kGy. The mean length of followup of the patients was 5 years (range, 2-9 years). These grafts were compared with a control group of grafts that were not irradiated but were implanted during the same time and used for similar diagnostic problems with defects of similar size. The outcomes of the groups differed significantly only in the incidence of allograft fracture. These findings indicate that high-dose irradiation to bone allografts is associated with a higher rate of fracture than are similar reconstructions using nonirradiated allografts.