Incorporation of perforated and demineralized cortical bone allografts. Part I: radiographic and histologic evaluation.

Massive cortical bone allografts have been found to incorporate slowly into host bone. They are subject to complications such as nonunion, fatigue fracture and infection. In an attempt to improve osteoinduction in cortical bone allografts, laser perforated and partially demineralized cortical bone allografts were orthotopically transplanted into the sheep tibia. In this model, mid-shaft tibial bone allografts from out-bred sheep donor animals were prepared by partial demineralization and drilling of 0.33-mm diameter holes with a pulsed, 2.94-microm wavelength Erbium:Yttrium-Aluminum-Garnet laser. Recipient animals of the same out-bred strain were divided into three groups of eight according to the type of cortical allograft used: group 1, fresh-frozen, no treatment; group 2, laser hole grid; and group 3, laser hole grid and partial demineralization. Plain films were taken in two standard views at monthly intervals. Incorporation was evaluated at nine months postoperatively. Longitudinal radiographic data was correlated to a histologic and morphometric evaluation of each bone graft. Computer tomography was used for the latter analysis. Results showed that untreated allografts, although surrounded by a periosteal bone cuff, were poorly incorporated. Partial demineralization lead to excessive resorption of allografts, but little new bone formation. Laser perforation and partial demineralization induced complete incorporation of allografts into the host bone. Based on the results of the radiographic, histologic and morphometric evaluation, the development of laser-perforated and partially demineralized bone allografts was proposed for clinical use.

Incorporation of perforated and demineralized cortical bone allografts. Part II: A mechanical and histologic evaluation.

Laser perforated and partially demineralized cortical bone allografts were orthotopically transplanted into sheep tibiae. This paper reports results of the mechanical testing of the transplanted bones, which was done at nine months postoperatively. Animals were divided into three groups of eight according to the type of cortical allograft used: group 1, no treatment; group 2, laser hole grid; and group 3, laser hole grid and partial demineralization. Thus, changes in flexural rigidity of 24 transplanted whole tibiae were investigated. Starting in the anterior direction at the tibial tuberosity, the flexural rigidity was determined using a nondestructive 4-point bending test. The elliptical distribution of the flexural rigidity was compared to the untreated contralateral control bone of each animal. Mechanical parameters were defined as percentage rates for comparative analysis between groups. Flexural rigidity measurement showed that bones transplanted with untreated allografts were stiffer than contralateral control bones. Partial demineralization of allografts reduced the flexural rigidity of transplanted bones below the level of contralateral control bones. Flexural rigidities of test bones transplanted with laser perforated and partially demineralized allografts were higher than those seen in bones transplanted with partially demineralized allografts. These results were corroborated by the histologic analysis which showed that untreated allografts, although surrounded by a periosteal bone cuff that effectively increased their outer diameter. In contrast, excessive bone resorption was observed in partially demineralized allografts. Laser-perforated and partially demineralized allografts showed histologic evidence of complete incorporation into the host bone. Based on this mechanical evaluation, it was concluded that processing of cortical bone allografts by the combination of perforation and partial demineralization resulted in improved mechanical strength of the transplanted bones as compared to processing by partial demineralization alone.

Allograft fractures revisited.

A retrospective review of patients with allograft fractures was done at the authors' institution. Between 1974 and 1998, 185 of 1046 (17.7%) structural allografts fractured in 183 patients at a mean of 3.2 years after transplantation. Initial allograft fixation included internal fixation with plates and screws in 181 patients. Patients with grafts that were longer than the average length (15.5 cm) tended to have worse results. Adjuvant therapy had no effect on fracture rate. Seventy-three patients with fractures had other allograft complications. Infection and nonunion with allograft fracture significantly worsened the outcome. The incidence of fracture in the patients with osteoarticular and arthrodesis transplants was significantly higher than those patients who had intercalary and composite reconstructions. Treatment of the allograft fractures included open reduction and internal fixation in 41 patients, reconstruction with a new allograft in 38, allograft-prosthesis composite in five, oncologic prosthesis in 19, amputation in 15, arthroscopic removal of loose bodies in three, resurfacing of fractured osteoarticular allograft surfaces in 39, allograft removal and cement spacer placement in 15. Twenty patients did not receive treatment. Eight of the fractures in patients who were not treated healed spontaneously. Outcomes were judged as excellent in nine patients (4.9%), good in 72 patients (38.9%), fair in 17 patients (9.2%), and in 85 patients (45.9%) the allograft reconstruction failed.

Factors affecting nonunion of the allograft-host junction.

Nonunion of allograft-host junction after bone transplantation is not uncommon, and its treatment frequently is problematic. To improve the understanding of these nonunions, a retrospective review was performed of 163 nonunions in 945 patients who underwent allograft transplantation (17.3%) for various benign and malignant tumors at the authors' institution between 1974 and 1997. Of these 945 patients, 558 did not receive adjuvant therapy. Chemotherapy was administered to 354 patients and only 33 patients received radiation therapy alone. Seventy-one patients had radiation treatment and chemotherapy. Of the 163 patients who had nonunion develop at the allograft-host junction, there were 269 reoperations performed on the involved extremity. In 108 patients, treatment was successful resulting in union of the allograft-host junction. Forty-nine patients did not respond to multiple surgical treatment attempts. The greater the number of surgical procedures, the worse the outcome. The rate of nonunions increased to 27% for the patients who received chemotherapy as compared with 11% for the patients who did not receive chemotherapy. The order of allografts from highest rate of nonunion to lowest was as follows: alloarthrodesis, intercalary, osteoarticular, and alloprosthesis. Infection and fracture rates were higher in the patients with nonunions as compared with the patients without nonunions.

Immune response to perforated and partially demineralized bone allografts.

Immune responses have been shown to be involved in the pathogenesis of clinical complications of cortical bone allografts. In an attempt to reduce the immunogenicity of these allografts, we evaluated cortical bone allografts modified by laser perforation and partial demineralization transplanted orthotopically into sheep tibiae. The recipient animals were divided into three groups, of eight animals each, according to the type of cortical allograft that was transplanted: group 1, no treatment (control); group 2, demineralization only; and group 3, laser perforation and partial demineralization. All animals were tissue-typed by biochemical definition of MHC class I molecules, using unidimensional isoelectric focusing and Western blotting. Mismatches of donors and recipients were assessed by testing samples of each donor and recipient pair in parallel and by comparing their individual bands. Donor-specific alloantibodies were detected by a similar technique, using an enzyme-linked immunosorbent assay (ELISA) format. Negative controls were included in all tests. All grafts were poorly immunogenic, whether they were untreated, processed by partial demineralization, or processed by both laser perforation and partial demineralization. Only two recipient animals showed a transient, antibody-mediated donor-specific immune response. One of these animals had received a control allograft, whereas the other animal had received a laser-perforated and partially demineralized bone allograft. All of the grafts in this study, including control grafts, were stripped of soft tissues and their bone marrow was removed; cellular sources of alloantibody stimulation may have been eliminated by these processes. The results of this study suggest that immune responses to bone allografts may be reduced by removing the bone marrow and adjacent soft tissues. The processing of cortical bone allografts by laser perforation and partial demineralization appeared to have little effect on immune responses.

Biodegradable foam coating of cortical allografts.

Clinical outcomes of bone allograft procedures may be improved by modifying the surface of the graft with an osteoconductive biopolymeric coating. In this comparative in vitro study, we evaluated the dimensional stability, mechanical strength, hydrophilicity, and water uptake of biodegradable foams of poly(propylene fumarate) (PPF) and poly(d,l-lactic-co glycolic acid) (PLGA) when applied as surface coatings to cortical bone. Cortical bone samples were divided into four groups: Type I, untreated bone; Type II, laser-perforated bone; Type III, partially demineralized bone; and Type IV, laser-perforated and partially demineralized bone. Results show that PPF wets easily, achieving 12.5% wt/wt in 30 min. Compressive tests on the PPF foam material showed that the compressive strength was 6.8 MPa prior to in vitro incubation but then gradually reduced to 1.9 MPa at 8 weeks. Push-out and pulloff strength tests showed that initially both PPF and PLGA foam coatings had comparable adherence strengths to the cortical bone samples (100-150 N). When additional geometrical surface alteration by perforation and demineralization of the bony substrate was employed, in vitro adherence of the PPF foam coating was further increased to 120 N, demonstrating a statistically significant improvement of push-out strength throughout the entire 8-week observation period (p<0.0002 for all four data points). The pore geometry of PPF-foam coatings changed little over the 2-month evaluation period. In comparison, PLGA foam coating around the cortical bone samples rapidly lost structure with a decrease of 67% in strength seen after 1-week in vitro incubation. These new types of bone allografts may be particularly useful where the use of other replacement materials is not feasible or practical.

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.

Retrograde nailing of femur fractures in patients with myelopathy and who are nonambulatory.

The authors studied 10 consecutive patients with closed femoral shaft or supracondylar fractures who were nonambulatory and who were treated by reamed retrograde intramedullary nailing via an intercondylar notch approach. The study consisted of five women and five men with an average age of 60.7 years (range, 40-89 years). Six patients had spinal cord lesions, one had a brain injury, one had cerebral palsy, one had multiple sclerosis, and one had progressive myelopathy. Three fractures were supracondylar, and seven fractures involved the mid-distal diaphysis. The average time of surgery was 110 minutes (range, 70-225 minutes) with an average estimated blood loss of 288 mL (range, 150-400 mL). There were two postoperative deaths (at 15 days and 2 months, respectively) after the procedure that were attributable to pneumonia. The remaining eight patients were observed for an average of 13 months (range, 6-20 months) after surgery. All fractures healed as evaluated radiographically. Retrograde intramedullary nailing is a simple, safe, and effective alternative to nonoperative treatment for femoral shaft or supracondylar fractures in patients who are nonambulatory. Stabilization by this method allows fracture healing and rapid return of patients to their previous level of function. There were no nonunions, malunions, significant shortening, implant failure, or wound infections.

Bone allografts: past, present and future.

Bone allograft transplantation has been performed in humans for more than one hundred and twenty years. During the first one hundred years (1880-1980), the major problem in bone allograft transplantation was availability. Most of the bone grafts used during this time were autografts. Allografts were not available due to a lack of legislation protecting procurers and processers. In addition, surgical procedures requiring allografts were not being performed. During the next twenty years (1980-2000), as allografis began to be used, the major issue was safety. Diseases transmitted during this period included AIDS and hepatitis. Avoidance of disease transmission became paramount. Sensitive blood tests and extensive efforts by bone banks to develop ways to clean. bone and clear it of infectious agents helped provide safe transplants. With concerns of availability and safety receding, the major issue in the future (2000-? ) will be the efficacy of the transplant. How allograft bone remodels in the host, how it incorporates and heals to host bone and how it integrates with the host skeleton will be the most important concerns of bone bankers and tissue transplant surgeons. Future research efforts will be applied to bone allograft transplantation to ensure that bone transplants heal quickly and sufficiently to be able to function as part of the weight-bearing skeletal system.

Bone banking. Update on methods and materials.

Bone allografts are being used in increasing numbers by orthopedic surgeons, yet many surgeons are unfamiliar with their preparation and processing, as well as their use as safe and effective transplants. This article reviews current sources of bone allografts, new methods of processing to achieve optimal results, the biology of incorporation of allografts, and new bone substitutes.

Long-term follow-up of patients with osteochondral allografts. A correlation between immunologic responses and clinical outcome.

This article confirms immunologic responses in humans to histocompatibility antigens (Class I and II) presented by frozen osteochondral allografts. These observations include a correlation of immune responses with long-term clinical outcome. As found in animal models, matching of histocompatibility antigens, particularly to Class II, improves clinical and, presumably, biologic success following implantation of massive frozen bone allografts in humans. The presence of sensitization clearly does not preclude a satisfactory outcome, nor have other reconstructive alternatives (e.g., metallic implants) been shown to be superior in their long-term results.

Improved osteoconduction of cortical bone grafts by biodegradable foam coating.

Alteration of the geometrical surface configuration of cortical bone allografts may improve incorporation into host bone. A porous biodegradable coating that would maintain immediate structural recovery and subsequently allow normal graft healing and remodeling by promoting bony ingrowth could provide an osteoconductive surface scaffold. We investigated the feasibility of augmenting cortical bone grafts with osteoconductive biodegradable polymeric scaffold coatings. Three types of bone grafts were prepared: Type I--cortical bone without coating (control), Type II--cortical bone coated with PLGA-foam, Type III--cortical bone coated with PPF-foam. The grafts were implanted into the rat tibial metaphysis (16 animals for each type of bone graft). Post-operatively the animals were sacrificed at 2 weeks and 4 weeks (8 animals for each type of bone graft at each time point). Histologic and histomorphometric analysis of grafts showed that the amount of new bone forming around the foam-coated grafts was significantly higher than in the control group (uncoated; p < 0.02). Although both foam formulations were initially equally osteoconductive, PLGA-based foam coatings appeared to have degraded at two weeks postoperatively, whereas PPF-based foam coatings were still present at 4 weeks postoperatively. While significant resorption was present in control allografts with little accompanying reactive new bone formation, PLGA-coated bone grafts showed evidence of bone resorption and subsequent bony ingrowth earlier than those coated with PPF-based foams suggesting that PPF-coated cortical bone grafts were longer protected against host reactions resulting in bone resorption.

An electron microscopic study on the process of acid demineralization of cortical bone.

Demineralization has been shown to foster osteoinductive properties of cortical bone grafts, yet little is known about the process of demineralization and how to control it. The purpose of this study was to investigate the process of cortical bone demineralization by using scanning electron microscopy to evaluate how hydrochloric acid demineralizes cortical bone. Results showed that in the demineralization of diaphyseal cortical bone specimens using hydrochloric acid, a uniformly thick circumferential band of demineralized bone matrix surrounds an inner undecalcified bone core as the process of demineralization occurs. The interface between the demineralized and mineralized section of the bone specimens was extremely sharp. This interface between demineralized and undemineralized bone was noted to advance as a reaction front with increasing demineralization which resulted in continuous shrinkage of the inner cortical bone core. This study suggests that cortical bone demineralization can be best described using an advancing reaction front theory, and this explanation can be used for implementation of the concept of controlled demineralization.

Improved osteoinduction of cortical bone allografts: a study of the effects of laser perforation and partial demineralization.

Massive cortical bone allografts have been found to incorporate slowly into host bone and thus are subject to complications such as nonunion, fatigue fracture, and infection. To better understand and improve the process of osteoinduction in these types of bone grafts, a new experimental model was developed with use of diaphyseal cortical bone grafts from rat tibiae that were prepared by partial demineralization and drilling of 0.33 mm diameter holes with a pulsed, 2.94 microns wavelength, erbium:yttrium-aluminum-garnet laser. Six types of grafts were analyzed: untreated (Type I), demineralized 25 microns deep (Type II), demineralized 150 microns deep (Type III), laser perforated (Type V), laser perforated and then demineralized 25 microns deep (Type V), and laser perforated and then demineralized 150 microns deep (Type VI). The graft was orthotopically transplanted in the tibia of an adult Sprague-Dawley rat and followed for as long as 4 months. Histologic evaluation at 1 and 4 months postoperatively with use of hematoxylin and eosin staining confirmed that there was new bone growth in Types II, III, V, and VI grafts. The amount of growth was estimated by comparing bone mineral density before implantation with values obtained after retrieval of the graft. These measurements were correlated to histomorphometric analysis of graft incorporation. The results show that the processes of partial demineralization (p < 0.000001) and laser perforation with partial demineralization (p < 0.000001) were both significant in enhancing bone growth in this model. New bone growth was significantly increased when the grafts were prepared with extensive demineralization (p < 0.015). This study demonstrates that osteogenesis in cortical bone grafts can be fostered through the process of partial demineralization and laser perforation. To the extent that minimal partial demineralization and laser perforation allow maintenance of structural integrity while altering the osteoinductive properties in such a way as to promote ingrowth of new bone, this experimental model represents an advance in understanding how osteogenesis in cortical bone grafts may be improved.

Kinetics of cortical bone demineralization: controlled demineralization--a new method for modifying cortical bone allografts.

We investigated the kinetics of hydrochloric acid demineralization of human cortical bone with the objective of developing a method of controlled demineralization for structural bone allografts. It is known that the demineralization of cortical bone is a diffusion rate limited process with a sharp advancing reaction front. The demineralization kinetics of human cortical bone, described as the advance of the reaction front versus immersion time, were determined by measuring extraction of bone mineral in both planar and cylindrical geometries. Mathematical models based on diffusional mass transfer were developed to predict this process. The experimental data fit well with the behavior predicted by the model. The model for planar geometry is applicable to controlled demineralization of cortical bone allografts of irregular shapes such as cortical struts. The model for cylindrical geometry is appropriate when curved surfaces are involved such as in diaphyseal bone allografts. This method of demineralization has direct application to clinical modification of cortical bone allografts to potentially enhance their osteoinductive properties.

Immunologic responses in human recipients of osseous and osteochondral allografts.

A multiinstitutional study was carried out to evaluate immunologic responses for human recipients of massive frozen (-80 degrees C) osseous and osteochondral allografts. Allografts were used to reconstruct skeletal defects associated with a variety of traumatic degenerative and neoplastic disorders. Serum samples were obtained before surgery and from 1 month to 4 years after surgery. Sera were tested by microcytotoxicity against T cells from 60 donors for human leukocyte antigen Class I antibodies and against beta 2-microglobulin treated B cells from 40 donors for human leukocyte antigen Class II antibodies. Panels were selected to represent the majority of known human leukocyte antigen specificities. Of the 84 cases evaluated, 62 (74%) received blood transfusions and 28 of 44 (64%) female recipients had been previously pregnant. Sensitization before transplant was shown in 33 of 84 (39%) patients. After grafting, 49 of 84 (58%) recipients showed evidence of sensitization to Class I antigens and 46 of 84 (55%) recipients showed evidence to sensitization to Class II antigens. Overall sensitization was 67%.

Chondrocyte survival in cryopreserved osteochondral articular cartilage.

Fluorescent staining techniques were used in combination with confocal and conventional fluorescent microscopy to determine the location of viable chondrocytes in frozen and thawed osteochondral articular cartilage. The results showed that cell survival was confined to the superficial layer of the cartilage matrix. The addition of a cryopreservative agent (dimethyl sulfoxide) increased chondrocytes survival but only in the same layer. There was no evidence of cell survival in the middle or deep layers of the cartilage with or without the use of dimethyl sulfoxide. Under the conditions employed in this study, chondrocyte survival in a cryopreserved osteochondral allograft appears t be limited to the superficial layer of the articular cartilage.

Long-term results of allograft replacement in the management of bone tumors.

Over the past 24 years, the authors have implanted >870 massive frozen cadaveric allografts mostly for the treatment of defects created by the resection of a bone tumor. Most of the grafts were obtained from the authors' institutional bone bank. The results show that only stage and type of graft affected outcome predictably. Specifically, grafts for a Stage 2 or Stage 3 tumor had a poorer outcome than those for Stages 0 and 1. The results for allograft arthrodeses were considerably poorer than osteoarticular, intercalary, and allograft plus prosthesis. The other major factors in results were complications--recurrence, infection, fracture, and nonunion--with the former 2 having a profound negative effect on outcome. After the first year of susceptibility to infection (10%) and the third year of increased risk of fracture (19%), the grafts become stable, and approximately 75% are retained by patients and are considered to be successful for >20 years after implantation. Osteoarthritis becomes a problem at approximately 6 years for osteoarticular grafts, and so far, 16% of the patients with distal femoral, proximal tibial, or proximal femoral grafts have required total joint replacements. Although the current results are adequate, they are imperfect, and research should be directed at improving the results.

Repopulation of laser-perforated chondroepiphyseal matrix with xenogeneic chondrocytes. An experimental model.

Growth of chondrocytes into a xenogeneic chondroepiphyseal matrix was investigated in an in vitro experimental model by combining viable calf chondrocytes with chick epiphyseal matrix devoid of viable chondrocytes. The chondrocytes were harvested from the wrist joints of newborn calves and cultured for 2 days. The epiphyses were harvested from the distal femurs and the proximal tibias of fetal chicks after development was arrested at 17 days by freezing. The epiphyseal specimens were prepared in four ways. These included femoral and tibial epiphyses without holes and femoral and tibial epiphyses with holes made by a laser. These epiphyseal specimens were co-cultured with calf chondrocytes for various periods. After digestion of the epiphyseal matrix, viable chondrocytes were counted in suspension. Chondrocyte division in the matrix was assessed by [3H]thymidine incorporation. The growth of calf chondrocytes into the xenogeneic chick matrix was evaluated by fluorescence microscopy on fresh thick epiphyseal sections. The percentage of viable chondrocytes in the xenogeneic epiphyseal matrix increased with culture time to a maximum at day 21. The addition of laser-drilled holes was found to extend a plateau of chondrocyte viability until day 29. A decrease in cell viability was detected at later observation points. This study demonstrates that xenogeneic matrix may serve as a morphogenetic scaffold for chondrocytic growth.

Effect of Er:YAG laser holes on osteoinduction in demineralized rat calvarial allografts.

Massive cortical autografts and allografts have been found to incorporate into host bone very slowly and thus are subject to complications such as fatigue fracture and infection. In order to understand and improve the process of osteogenesis in these types of bone grafts, a new experimental model was developed using bone discs from rat calvaria prepared by demineralization and drilling of 0.5 mm diameter holes with a pulsed, 2.94 microns wavelength Erbium:Yttrium-Aluminum-Garnet laser. Four types of bone discs were analyzed: untreated (Type I), demineralized (Type II), laser-ablated (Type III), and laser-ablated then demineralized (Type IV). The discs were transplanted into a subcutaneous site in adult Sprague-Dawley rats and followed for as long as 6 weeks. Histologic analysis of the discs at weekly intervals with use of hematoxylin and eosin staining confirmed the presence of new bone growth in Type-II and Type-IV discs. The amount of new bone growth in each disc was estimated by determining the mineral x-ray attenuation coefficient, which is proportional to mineral density, from digitized radiographs of the discs. The results showed that the processes of demineralization (P < 0.001) and laser ablation with demineralization (p < 0.05) were both significant in enhancing new bone growth in this model. This study demonstrated that osteoinduction can be fostered in cortical bone through the processes of demineralization and laser ablation. To the extent that laser ablation may allow maintenance of structural integrity while altering the surface geometry in such a way as to promote ingrowth of new bone, this experimental model represents an advance in understanding how osteogenesis in cortical bone grafts might be improved.