Preliminary study of ethylene oxide sterilization of full-thickness cortical allografts used in segmental femoral fracture repair

Full-thickness canine cortical allografts were cleanly harvested, sterilized with ethylene oxide, and stored at room temperature. The allografts were incorporated into canine segmental femoral fracture repairs and compared clinically, radiographically, and morphologically with control femoral cortical autografts for function of the limb, graft acceptance, and bone union. Sterility was maintained and the cortical allografts were well accepted by the host animals, resulting in full use of the limb which was subjected to surgical operation. The allografts showed healing patterns similar to those of the autografts, as determined by radiographic, gross, and histologic evaluation of the proximal and distal host-graft interfaces. Evaluations were made monthly. The host-graft interfaces of the allografts and autograft were filled with woven bone with adjacent vascular invasion and remodeling of the graft at the final 4th-month evaluation.     

Evaluation of Canine Cortical Bone Graft Remodeling

A stable cortical bone fracture model was developed to evaluate the remodeling rate of cortical bone grafts. Samples of cortical bone were harvested with a trephine and press fit into predrilled holes in the femoral diaphyses of four live dogs. The percentages of new bone, unremodeled graft bone, porosity, forming bone surface area, and resorbing bone surface area were determined morphometrically and compared in cortical autografts, cortical allografts sterilized with 84% ethylene oxide (EO), and allografts sterilized with 12% EO. The host-graft interfaces healed without formation of fibrous tissue or cartilage, indicating a stable fracture surface. The amount of new bone formed in cortical autografts and allografts sterilized with 84% EO was significantly greater than the amount of new bone in allografts sterilized with 12% EO. There was no significant difference between the amounts of new bone formed in the allografts sterilized with 84% EO and the cortical autografts. No significant differences were detected in percentages of porosity or bone surface areas.     

Effects of tissue antigen matching on the healing of fresh cancellous bone allografts in dogs

The radiographic and histologic healing patterns of fresh cancellous bone allografts were compared with those of fresh cancellous autografts in dogs. Two groups of allografts were studied: 1 group of littermate pairs with minor histocompatibility mismatches, and 1 group of unrelated pairs with major histocompatibility mismatches. Pairs were chosen on the basis of preoperative serologic typing and mixed lymphocyte cultural assay of lymphocyte-defined compatibility. Generally, grafts with minor mismatches and autografts achieved bony union in 6 weeks with grafted bone serving as a scaffold for new bone formation. Grafts with major mismatches were resorbed and replaced by fibrocartilage. Healing of osteotomy sites grafted with mismatched bone occurred by ingrowth of host periosteal and endosteal new bone.     

Combination of a Corticai Allograft With a Cancellous Autograft in the Canine Tibia

This study was undertaken to determine what difference, if any, occurred when autogenous cancellous bone was placed in the medullary canal of a fresh, 4-cm cortical allograft compared to a fresh cortical allograft alone. Twelve mature dogs were used. A tubular cortical graft was placed in the midshaft of both tibias. The right tibia received the combination graft while the left tibia received a fresh cortical allograft only. Study periods ranged from one week to 17 weeks. The progress of the grafts was assessed by technetium-99m radionuclide bone scans, radiographs, gross and histological examination, and tetracycline labeling. Results indicate that autogenous cancellous bone placed in the medullary canal of a fresh cortical allograft was beneficial to the ingrowth of new blood vessels and incorporation of the cortical graft.     

Antigenicity of cortical bone allografts in dogs and effect of ethylene oxide-sterilization.

The aims of the present study were to determine the antigenicity of cortical bone allografts and the effect of ethylene oxide-sterilization (EO-sterilization). Cortical bone allografts from one donor dog were implanted in a muscle pouch in four groups of four dogs each. The grafts were either fresh, EO-sterilized, demineralized or demineralized and EO-sterilized. The immune response against the grafts was determined by measuring the antibody response against surface antigens of donor cells and by the mixed lymphocyte reaction. Dogs receiving EO-sterilized grafts or bone matrix did not demonstrate an immune response. Only two of the four dogs with fresh cortical bone grafts showed a very weak immune response. This suggests a priming of the host by the fresh bone grafts. However, implanting skin grafts from the donor dog subdermally, in one dog of each of the groups, four months after implanting the bone grafts did not induce a secondary immune response. Macroscopic and histologic examination of the bone grafts five months after their implantation consistently revealed graft resorption (activity of osteoclasts) and vascularization of the fresh bone grafts, but not of EO-sterilized fresh grafts. For most EO-sterilized grafts, a strong inflammatory reaction was present in the tissues surrounding the graft and this was not apparent around the non-sterilized grafts. The absence of resorption and the presence of the inflammation seemed to be unwanted effects of the EO-sterilization. The EO-sterilisation did not affect osteoinduction since osteocytes were observed in the EO-sterilized demineralized grafts. Results indicate that cortical bone allografts used in the present study are very weak antigens and that the EO-sterilization procedure used has no effect on osteoinduction, but decreases bone resorption.     

Feline model for the study of frozen osteoarticular hemijoint transplantation: qualitative and quantitative assessment of bone healing

Twenty-four outbred cats underwent massive osteoarticular allograft and control autograft transplantation, using the right distal femur with its articular cartilage, capsule, and medial collateral ligament intact. The cats were monitored clinically and radiographically for 1 year. Groups of cats (4 allografts and 2 control autografts) were euthanatized at 3-, 6-, 9-, and 12-month intervals. At necropsy, the grafts were photographed and assessed for bone healing and replacement by standard radiography, quantitative 99mTc bone scans, microradiography, and histologic examination of decalcified and nondecalcified specimens. The osteosynthesis site of the allografts usually healed by 5 months, compared with the autografts that healed by 3 months. As illustrated by quantitative bone scans, creeping appositional new bone slowly invaded and replaced the allograft bone. Seemingly, the cat can be used as an acceptable and clinically comparable model for the massive osteoarticular allografts currently being used for the reconstruction of joints damaged or destroyed by neoplasm surgery in limb-sparing procedures in human beings. This model may also be used to assess the rate and method of bone healing.     

The effect of cancellous autograft and novel plate design on radiographic healing and postoperative complications in tibial tuberosity advancement for cranial cruciate-deficient canine stifles

Objective: To assess the effect of autogenous cancellous bone graft (autograft) and novel plate use on radiographic healing and complications in tibial tuberosity advancement (TTA) for treatment of cranial cruciate ligament (CrCL)‐deficient stifles in dogs. Study Design: Prospective clinical study. Animals: Consecutive dogs (n=125) with unilateral CrCL‐deficient stifles. Methods: Four treatment groups: CPG, conventional plate with autograft; CPNG, conventional plate without autograft; NPG, novel plate with autograft; NPNG, novel plate without autograft were studied. Radiographs from 60 dogs were scored for healing at 6 and 10 weeks postoperatively; all 125 dogs were assessed for radiographic complications. Variables evaluated for relationship with healing scores and radiographic complications were age, weight, sex, cage and plate size, implant type, and graft use. Results: Dogs with autograft had overall higher healing scores at 6 and 10 weeks. Radiographic complications occurred in 13 dogs (12 minor, 1 major), and were not influenced by graft or novel plate use. Conclusion: Autograft increases healing scores, but was not found to have a significant impact on the rate of complications in TTA. The novel plate was not found to have healing scores or radiographic complication rates significantly different from the conventional plate design.     

Pasteurized tumoral autograft and adjuvant chemotherapy for the treatment of canine distal radial osteosarcoma: 13 cases

OBJECTIVE: To report outcome in 13 dogs with distal radial osteosarcoma, without evidence of metastasis, treated by a combination of adjuvant chemotherapy and a pasteurized autograft limb-sparing procedure. STUDY DESIGN: Prospective clinical study. ANIMALS: Thirteen dogs with distal radial osteosarcoma. METHODS: Limb-sparing procedure was performed using an autograft from the excised tumoral segment, pasteurized at 65 degrees C for 40 minutes. Adjuvant chemotherapy (cisplatin or cisplatin and doxorubicin) was administered in all dogs. RESULTS: Mean and median survival times were 531 and 324 days, respectively (range, 180 to 1,868 days). Overall survival was 100% at 6 months, 50% at 12 months, 44% at 18 months, and 22% at 24 months. Lung metastasis occurred in 5 (38%) dogs. Observed complications were local recurrence (2 dogs, 15%), allograft infection (4 dogs, 31%), and implant failure (3 dogs, 23%). Limb function was good in 12 dogs (92%) and fair in 1 dog. CONCLUSIONS: Pasteurized bone autograft derived from the tumoral bone segment was an effective alternative to cortical bone allograft for limb sparing in canine distal radial osteosarcoma, in terms of feasibility, pattern of healing, complications, and survival. CLINICAL RELEVANCE: Use of a pasteurized bone autograft eliminates the need for a canine bone allograft bank and has the added advantage of good fit to the recipient site.     

Enhancement of bone healing using non-glycosylated rhBMP-2 released from a fibrin matrix in dogs and cats

OBJECTIVES: To test a non-glycosylated recombinant human bone morphogenetic protein-2 (ngly-rhBMP-2)/fibrin composite, which has been shown experimentally to enhance healing of bone defects in rodents, in a clinical case series of dogs and cats undergoing treatment for fracture non-unions and arthrodesis. METHODS: A ngly-rhBMP-2/fibrin composite was applied in 41 sites in 38 dogs and cats for which a cancellous bone autograft was indicated, replacing the graft. RESULTS: Bridging of the bone defect with functional bone healing was achieved in 90 per cent of the arthrodesis and fracture nonunions treated in this manner. CLINICAL SIGNIFICANCE: This prospective clinical study demonstrates the beneficial effects of ngly-rhBMP-2 in a specially designed fibrin matrix on the treatment of bone defects, and validates the use of this composite as an alternative to bone autografts in dogs and cats.     

Treatment of severely comminuted diaphyseal fractures in the dog, using standard bone plates and autogenous cancellous bone graft to span fracture gaps: 11 cases (1979-1983)

Severely comminuted diaphyseal fractures in 11 dogs were repaired with standard bone plates that spanned a fracture gap filled with autogenous cancellous bone graft. Five dogs had closed injuries, 4 dogs had open fractures, and 2 dogs had infected nonunion fractures for which previous attempts at internal pin fixation had failed. A second autogenous cancellous bone graft was performed in 3 of the dogs during the healing period. The technique was successful in all dogs. The technique was considered a versatile and relatively simple alternative, compared with meticulous small fragment reconstruction and cortical bone allografts.     

Healing of transverse humeral fractures in pigeons treated with ethylene oxide-sterilized, dry-stored, onlay cortical xenografts and allografts

Transverse humeral fractures were created in adult male pigeons (Columbia livia). They were bridged with an allograft or a xenograft, or they were allowed to heal without treatment. Tissues were examined for wound healing, bony union, infection, and sequestration of the graft at 28, 43, 85, 106, and 168 days after surgery. Neither allografts nor xenografts contributed to nor interfered with bone healing. In nontreated pigeons (controls), bony union with marked callus formation developed between misaligned fragment ends. When onlay grafts were used, bone fragments remained aligned, with minor callus formation. There was a significant (P = 0.0001) increase in the number of wounds opening after surgery and infections of the surgical site (xenograft, P = 0.0026; allograft, P = 0.0021) associated with graft use. A significant (P = 0.0001) frequency of graft sequestration was observed, regardless of graft type. A significant (P = 0.0001) frequency of foreign body reaction also was observed. Under these circumstances, the application of a graft should be considered as an alignment device, rather than a stimulus to healing.     

NONGRAFTED AND GRAFTED OSTEOTOMIES OF PROXIMAL SESAMOID BONES OF THE HORSE

A radiographic study of healing patterns of nongrafted and grafted basilar osteotomies of proximal sesamoid bones in 14 horses was performed. Osteotomies were created in one proximal sesamoid bone of each fromt leg. One was treated by an autogenous rib graft, and the other was left nongrafted. Wedge-shaped cortical bone, corticocancellous bone with multiple drill holes, and chips of cancellous hbone were used as autogenous grafts. Presurgical, surgical, and postsurgical radiographic examinations were performed. The longest follow-up period was 40 weeks. High detail radiography of 3-mm bone sections and microangiography were also perofromed. Radiographic interpretation of lack of bone healing was erroneous in approximately one-half of the cases. Lack of an external bridging callus was incorrectly interpreted as lack of bony union. If noted, periosteal new bone formation failed to develop into a pattern of bridging callus. Radiographs did not permiit detection of the osteotomy line entering the articular surface or displacement of distal fragments. From angiography obtained ten weeks after surgery, the pattern of blood supply was similar in nontreated and grafted sesamoid bones. Microangiography showed rich vascularization of the cancellous graft and callus, reflecting good healing activity, whitle vascularization of the osteotomy site was absent in the nontreated osteotomies.     

Vascularized ulnar bone grafts for limb-sparing surgery for the treatment of distal radial osteosarcoma

The objective of this retrospective study was to compare vascularized free or roll-in ulnar bone grafts for limb-sparing surgery in dogs with radial osteosarcoma with the cortical allograft, metal endoprosthesis, or distraction osteogenesis techniques. Overall, the ulnar graft techniques used in this study demonstrated excellent healing properties. Complications included recurrence of the tumor in 25% (2/8) of the dogs, metastasis in 50% (4/8) of the dogs, implant loosening in 37.5% (3/8) of the dogs, implant failure in 12.5% (1/8) of the dogs, and infection in 62.5% (5/8) of the dogs. Mean survival time was 29.3 mo (range, 9 to 61 mo). The mean metastasis-free interval was 33.67 mo (range, 8 to 54 mo). Tumors recurred locally in two dogs at 10 mo and 20 mo postoperatively. This study yielded similar long-term complications as other limb-sparing options (such as cortical allografts and metal endoprostheses) and allowed dogs to bear weight on the operated limb with acceptable limb function. More research is needed regarding specific healing times for ulnar vascularized grafts, time until implant removal, and the extent of radial bone that could ultimately be replaced by the ulna.     

Evaluation of the proximal portion of the femur as an autogenous cancellous bone donor site in dogs.

The proximal portion of the femur was evaluated as a source of autogenous cancellous bone in dogs. Bilateral oval cortical defects were created in the lateral subtrochanteric area of the femur in 16 dogs. Cancellous bone was removed and the weight recorded. Cancellous bone was similarly harvested from the proximal portion of the humerus in 7 of these dogs. Subtrochanteric femoral defects in 11 dogs were randomly assigned to receive cancellous bone graft obtained from the femur (n = 4) or the humerus (n = 7). Subtrochanteric defects in 5 dogs were not grafted. Radiographic assessment of subtrochanteric defects was performed at 4-week intervals, and histologic assessment at 4, 8, 16, and 24 weeks after surgery. Nongrafted donor sites healed by ingrowth of trabecular bone during the first 12 weeks after surgery. By week 24, the lateral cortical wall had reformed, but remodeling was incomplete. Donor sites grafted with cancellous bone healed similarly, but with more rapid healing and more complete remodeling evident by week 24. Although the mean weight of cancellous bone harvested from the proximal portion of the femur (0.82 +/- 0.22 g) was significantly (P less than 0.05) less than that harvested from the proximal portion of the humerus (1.38 +/- 0.29 g), there was no qualitative histologic or radiographic difference in bony healing of grafted defects. We determined that the proximal portion of the femur can be safely used to provide moderate amounts of cancellous bone, and that a second bone graft can be collected from the same subtrochanteric donor site after 12 weeks.     

Efficacy of Azathioprine Versus Cyclosporine on Kidney Graft Survival in Transfused and Nontransfused Ummatched Mongrel Dogs

Sixteen mongrel dogs had bilateral nephrectomy and received a renal allograft from an unmatched mongrel. One group of eight dogs was treated orally with azathioprine and prednisone; another group of eight dogs was treated orally with cyclosporine and prednisone. Four dogs of each group received four blood transfusions each prior to surgery. Mean survival time was nearly the same in the azathioprine-treated and the cyclosporine-treated dogs. Transfusions prolonged survival in the azathioprine-treated group but not in the cyclosporine-treated group. Retrospective measurement of whole blood trough cyclosporine concentrations indicated marked variation between dogs and in the same dog at different times. This variation may have influenced graft survival. Only one dog survived the 9-month period of observation, indicating that refinements of the techniques used in this study will be required for long-term survival of renal allografts in unrelated mongrel dogs.     

Orthotopic Osteochondral Transplantation of the Canine Proximal Femur

An 18-week study was conducted to evaluate orthotopic osteochondral transplantation of the proximal femur in the dog. Eighteen dogs were divided into 3 groups of 6 each. The first group received autografts, the second received fresh allografts, and the third received grafts that had been frozen in a bone bank for 24–28 days. The grafts were fixed in position using dynamic compression plates. The grafted limbs were maintained in a sling and thus were nonfunctional and non-weight bearing throughout the 18-week study. Postoperatively the dogs were given oral tetracycline to assess osteo- cyte viability. The dogs were radiographed at 2–week intervals and 1 dog in each group was euthanatized every 3 weeks. The femurs were examined using standard histopathologic and fluorescent labeling techniques. All femoral heads were luxated by the 2nd postoperative week. The bones of all the femoral heads underwent avascular necrosis and degenerative changes were present in the transplanted cartilage by the 6th postoperative week. During the first 18 weeks following transplantation there was little radiographic and histologic difference among the 3 types of grafts regarding the nature and rate of bone healing. Based on the data obtained from techniques utilized in this study, the femoral head, neck, and articular cartilage did not survive, while the femoral diaphysis did survive the transplantation process. Different techniques may alter to some degree the results obtained.     

Pasteurized tumoral autograft as a novel procedure for limb sparing in the dog: A clinical report

OBJECTIVE: To evaluate use of a pasteurized tumoral autograft prepared from the resected primary bone neoplasm for limb sparing in a dog with distal radial osteosarcoma (OSA). STUDY DESIGN: Clinical case report. ANIMALS: A 9-year-old male Maremma shepherd dog. METHODS: After right distal radial OSA removal, the tumoral autograft was pasteurized. The excised bone segment was placed in a sterile watertight box containing sterile saline solution preheated to 65 degrees C in a water bath. The box was kept immersed in the water bath at 65 degrees C for 40 minutes to kill the tumor cells. The autograft was then fixed in the host with a plate and screws based on standard AO/ASIF technique for carpal arthrodesis. Three doses of cisplatin (70 mg/m(2) intravenously) were administered, 3 weeks apart; the initial dose was administered the day after surgery. RESULTS: The autograft was incorporated in a manner comparable to an allograft, and after 708 days, the metallic implants were removed. A 1-month activity restriction as well as spoon splint to protect the leg from a full loading were used thereafter. Limb function was fair to good, and the dog remains disease free after 56 months. CONCLUSIONS: A pasteurized autograft consisting of the resected primary bone neoplasm is a valid alternative to a cortical bone allograft for limb sparing in dogs with appendicular OSA in terms of feasibility and pattern of healing. CLINICAL RELEVANCE: This procedure can be an alternative method of limb sparing when difficulties are encountered in establishing and maintaining a canine bone allograft bank.     

Evaluation of bone healing in canine tibial defects filled with cortical autograft,commercial-DBM,calf fetal DBM,omentum and omentum-calf fetal DBM

The present study was conducted to compare the effects of xenogenic bovine fetal demineralized bone matrix (DBM), commercial DBM, omentum, omentum-calf fetal DBM, cortical autograft and xenogenic cartilage powder on the healing of tibial defects in a dog model to determine the best material for bone healing. Seven male adult mongrel dogs, weighing 26.2 ± 2.5 kg, were used in this study. Seven holes with a diameter of 4-mm were created and then filled with several biomaterials. Radiographs were taken postoperatively on day 1 and weeks 2, 4, 6, 8. The operated tibias were removed on the 56th postoperative day and histopathologically evaluated. On postoperative days 14, 42 and 56, the lesions of the control group were significantly inferior to those in the other group (p < 0.05). On the 28th postoperative day, the autograft group was significantly superior to the control and omentum groups (p < 0.05). Moreover, calf fetal DBM was significantly superior to the control group. There was no significant difference between the histopathological sections of all groups. Overall, the omentum and omentum-DBM groups were superior to the control group, but inferior to the autograft, commercial-DBM, calf fetal DBM and calf fetal cartilage groups.     

Epiglottic Augmentation in the Horse

Epiglottic augmentation with injectable bovine collagen or an autogenous or allogenous auricular cartilage graft was performed in 12 horses with endoscopically and radiographically normal epiglottises. The grafting procedures were easy to perform and did not cause apparent discomfort. Cartilage graft extrusion or resorption may have occurred, but was not seen by endoscopy and lateral laryngeal radiography. Only collagen implants remained evident endoscopically, as smooth round submucosal bulges ventral to the epiglottic cartilage. Two horses with collagen implants, and all horses with cartilage autografts and allografts, were euthanatized at week 16. One horse with a collagen implant was euthanatized at week 4 and one at week 6. The epiglottis appeared thickened in three horses with collagen implants, two horses with autogenous grafts, and three horses with allogenous grafts. Pharyngeal lymphoid tissue was hyperplastic in two horses with autografts and three horses with allografts, but not in horses with collagen implants. Collagen grafts persisted as one or two smooth bulges 8 mm in diameter. Collagen incited a brisk foreign body reaction that was surrounded by a fibrous connective tissue capsule. Epiglottises of the horses with collagen implants were significantly thicker 20 mm from the tip than those of normal horses and horses with allografts. Cartilage graft incorporation was not evident grossly and was seen on microscopic examination in only one autograft. Thickening was caused by submucosal fibrosis.     

Effects of a 98% solution of glycerol or sterilization with ethylene oxide on FeLV in bone allografts and effects on bone incorporation of allografts in cats

OBJECTIVES: To compare virucidal effects and bone incorporation properties of cortical bone allografts transplanted into specific-pathogen-free (SPF) cats. Allografts consisted of untreated bone from a SPF cat (negative-control group) and bone from 5 FeLV-infected cats that was subjected to sterilization with ethylene oxide (ETO), preservation with glycerol, or no treatment (positive-control group). SAMPLE POPULATION: Bones from the aforementioned groups and twenty 8-week-old SPF cats (5 cats/group) implanted with an allograft from 1 of the aforementioned groups. PROCEDURE: After implantation, blood samples were collected weekly to monitor FeLV p27 antigen and antibody titers. Quantification of FeLV provirus was performed on blood samples at weeks 0, 4, and 8 and donor bone samples at time of implantation. Cats were euthanatized 8 weeks after transplantation, and graft sites were evaluated. RESULTS: All results for negative-control cats were negative. All ETO group cats had negative results for antigen and provirus in blood, whereas 1 cat had a low antibody titer. Although 3 ETO-treated allografts were positive for provirus, the DNA appeared denatured. One cat in the glycerol group had positive results for all tests in blood samples. All glycerol-preserved allografts were positive when tested for provirus. All results for positive-control group cats were positive. Differences in incorporation of bone grafts were not observed. CONCLUSIONS AND CLINICAL RELEVANCE: Glycerol preservation of FeLV-infected bone allografts did not eliminate transmission of retrovirus to recipients. In contrast, ETO sterilization appeared to denature DNA and prevent infection. Treatments did not affect incorporation of bone grafts in young cats.