Healing of Cortical Bone Grafts in the Dog The Role of Azathioprine

Sixteen unrelated beagles, randomly divided into groups of four, were used in evaluating the role of therapeutic immunosuppression in the healing of fresh cortical bone allografts over a 16-week period. The four groups included: nontreated allograft, treated allograft, nontreated autograft, and treated autograft. A 2.7-cm tibial cortical graft was fixed orthotopically using a dynamic compression plate. Healing was evaluated by radiography and by gross and histologic studies at 2, 4, 8, and 16 weeks after surgery. The treated dogs were immunosuppressed with azathioprine for eight weeks postsurgery. The conclusions were that: temporary immunosuppression did not significantly alter healing of fresh cortical bone autografts; healing of fresh cortical bone allografts in immunosuppressed dogs was similar to healing of fresh cortical bone autografts; slight differences were observed in the healing of bone grafts in all groups after eight weeks; and cellular reaction typical of graft rejection was found in nontreated allografts, but healing still occurred.     

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.     

Ultrasonographic and radiographic assessment of uncomplicated secondary fracture healing of long bones in dogs and cats

OBJECTIVES: To evaluate the use of ultrasonography (US) to detect bone healing in uncomplicated diaphyseal fractures of dogs and cats, and to compare these observations with detection of healing by radiography (RG). STUDY DESIGN: Clinical study. ANIMALS: Dogs (33) and cats (11). METHODS: RG and brightness mode US were used to follow uncomplicated secondary fracture healing. Fractures were examined at admission and then every 2-4 weeks until healed or implant removal. Temporal differences in definitive detection of healed fracture by imaging technique were examined by species, patient age, bone, and fracture type. RESULTS: US images obtained during uncomplicated secondary fracture healing were consistent with images of fracture healing described in humans. Mean time to US diagnosis of a healed fracture (mean 46 days) was significantly shorter than by RG (mean 66 days). Mean time until diagnosis of a healed fracture (US and RG) did not differ significantly between open and closed treatment. Patients 36 months (n=11), but there was no significant difference between the latter 2 groups. Diagnosis of a healed simple fracture by US was significantly quicker than for a comminuted fracture (P<.05), but no difference was noted when using RG. CONCLUSIONS: US can be used to evaluate secondary fracture healing in biologically treated fractures in dogs and cats. US permits detection of a healed fracture earlier than RG. CLINICAL RELEVANCE: Earlier diagnosis of a healed fracture by US can prevent unnecessarily long limb immobilization and allow earlier dynamization.     

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.     

Renal transplantation for treatment of end-stage renal failure in cats.

Renal transplantation was performed as treatment of end-stage renal failure in 23 cats. Twenty-two cats had chronic renal disease and 1 cat had acute renal disease associated with ethylene glycol-induced toxicosis. Sixteen cats were discharged from the hospital. Nine survived a mean of 8.4 +/- 6.5 months, and 7 cats continue to survive at the time of this report (mean 12.6 months). Seven cats died within 2 weeks of surgery. All renal allografts were obtained from unrelated blood-crossmatch-compatible donors. No deaths were attributable to acute renal allograft rejection, demonstrating the successful maintenance of renal allografts by use of cyclosporine and prednisolone immunosuppression in cats.     

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.     

Retrograde placement of a novel 3.5 mm titanium interlocking nail for supracondylar and diaphyseal femoral fractures in cats.

Twenty-four simple or comminuted supracondylar and diaphyseal femoral fractures in cats, which had been treated by retrograde insertion of a new 3.5 mm titanium interlocking nail (IN) from the intercondylar notch, were evaluated between June 2000 and October 2004 at the Ecole Nationale Vétérinaire d'Alfort. Patient data (species, breed, weight, and age), fracture characteristics, details of the surgery, post-operative complications and radiographic follow-up were recorded. The mean body weight was 3.75 kg and the cats ranged in age between four and 66 months (mean 29 months). The IN had a diameter of 3.5 mm and a length of 100, 109 or 119 mm, and were all fixed in a static position (two screws in seven cats, three screws in 10 cats and four screws in seven cats). Cerclage wires were used in seven cats and an autogenous bone graft was used in two cats. Screw or nail breakage were not recorded. Nineteen fractures healed without any complications, three cats died during the post-operative period from unknown causes, and two cats showed delayed bone healing. Twenty cats were considered to have an excellent limb function at one month. One cat with a sciatic injury was non-weight bearing for several months. Radiographic signs of degenerative joint disease of the stifle joint were not observed except in the cat with the sciatic nerve injury. The results of this study suggest that this new 3.5 mm titanium IN can be introduced from the intercondylar notch and be used in static fixation mode to stabilize supracondylar and diaphyseal femoral fractures in cats.     

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.     

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.     

Evaluation of the Clinical and Histologic Features of Renal Allograft Rejection in Cats

OBJECTIVES: To describe the clinical signs and histopathologic features of renal allograft rejection in cats, and to provide a historical, untreated control group for use in future studies of feline renal allograft rejection. ANIMALS: Fourteen adult research cats. METHODS: Renal transplantation and bilateral nephrectomy were performed in pairs of immunogenically mismatched cats. A physical examination was performed, and packed cell volume, total protein, and plasma creatinine concentrations were measured each day after surgery. The cats were euthanatized when plasma creatinine concentration exceeded 7 mg/dL or when weight loss exceeded 20%. Renal histopathology was scored according to the Banff 97 criteria by 3 pathologists. RESULTS: Nine cats completed the study. Plasma creatinine exceeded 7 mg/dL in 5 cats, weight loss exceeded 20% in 3 cats, and 1 cat was found dead. Clinical signs in cats with rejection were nonspecific or absent. Rectal temperature decreased by 0.8 +/- 0.5 degrees C in the 24 hours before euthanasia. The pathologists agreed on the allograft histopathologic category in 6 of 9 cats. The histologic consensus was acute/active rejection in 8 cats and normal in 1 cat. Median survival time of the 8 cats with histologically confirmed allograft rejection was 23 days (range, 8-34 days). CONCLUSIONS AND CLINICAL RELEVANCE: Renal allograft rejection is associated with minimal clinical signs. Therefore, plasma creatinine concentration should be measured routinely in patients with a functioning allograft. An increase in plasma creatinine concentration is highly suspicious for allograft rejection, although a biopsy of the renal allograft is needed for definitive diagnosis.     

Comparison of healing of the osteotomy gap after tibial tuberosity advancement with and without use of an autogenous cancellous bone graft

Objective: To evaluate and compare healing, with and without the use of bone graft, of the gap created during tibial tuberosity advancement (TTA). Study Design: Prospective study and case series. Animals: Dogs treated with TTA (n=67). Methods: Prospective study: Mediolateral radiographic projections (6 weeks and 4 months) after TTA without use of bone graft (group I, n=14) were compared with radiographs of consecutive TTA in which the gap was filled with autologous cancellous bone graft (group II, n=14). Two scoring techniques (A, B) were used. Score A was used to grade the overall osteotomy healing (0=no healing, 4=healed osteotomy). Score B evaluated, independently of each other, healing in 3 sites: proximal to the cage (B1), between cage and plate (B2), and distal to the plate (B3). Case series: nongrafted TTA (4–25 weeks, n=39) were evaluated for healing (Score A). Data was analyzed using t‐tests and ANOVA. Significance was set at P≤.05. Results: Prospective study: Score A, B2, and B3 showed no difference in healing between groups at 6.8 weeks and 4.2 months. Score B1 revealed, in both rechecks, a significantly higher density in group II. Case series: Radiographs at 11.59±5.99 weeks scored 3.3 (2–4). No healing related complications were observed. Conclusion: The osteotomy gap created during TTA healed within expected time regardless of bone graft use.     

Cutaneous Wound Healing in Ehlers-Danlos Syndrome

Wound healing in five dogs and five cats affected with a connective tissue dysplasia resembling Ehlers-Danlos syndrome of humans was compared with wound healing in 10 nonaffected animals. Six skin incisions on the lateral aspects of the thorax and abdomen of each animal were sutured and assessed daily for 75 days for evidence of healing. All wounds in nonaffected dogs, affected cats, and nonaffected cats healed by first intention. Three incisions in affected dogs had dehiscence of all or part of the incision line and healed by granulation, contraction, and epithelialization. Biopsies taken at 3, 6, 9, 12, 15, and 75 days were compared histologically to determine if there were any differences in rates of healing between affected and nonaffected animals. Epidermal thickening and scab formation were noted at days 3 and 6 in both affected and nonaffected animals. Infiltration with mononuclear cells and fibroplasia steadily increased from day 6 to day 15 in all groups. Collagen fibril formation was evident by day 9. At day 75, incision sites were recognized by fine, more compact collagen bundles and lack of adnexal structures, as compared with the adjacent dermis in both affected and nonaffected animals. Although delayed wound healing has been reported to be a complication of Ehlers-Danlos syndrome in humans, using clinical and histologic criteria, wound healing in dogs and cats with Ehlers-Danlos syndrome appears to be similar to nonaffected animals.     

Pharmacokinetics of tacrolimus after multidose oral administration and efficacy in the prevention of allograft rejection in cats with renal transplants

OBJECTIVE: To describe pharmacokinetics of multi-dose oral administration of tacrolimus in healthy cats and evaluate the efficacy of tacrolimus in the prevention of allograft rejection in cats with renal transplants. ANIMALS: 6 healthy research cats. PROCEDURE: Cats received tacrolimus (0.375 mg/kg, PO, q 12 h) for 14 days. Blood tacrolimus concentrations were measured by a high performance liquid chromatography-mass spectrometry assay. Each cat received an immunogenically mismatched renal allograft and native kidney nephrectomy. Tacrolimus dosage was modified to maintain a target blood concentration of 5 to 10 ng/mL. Cats were euthanatized if plasma creatinine concentration exceeded 7 mg/dL, body weight loss exceeded 20%, or on day 50 after surgery. Kaplan-Meier survival curves were plotted for 6 cats treated with tacrolimus and for 8 cats with renal transplants that did not receive immunosuppressive treatment. RESULTS: Mean (+/- SD) values of elimination half-life, time to maximum concentration, maximum blood concentration, and area under the concentration versus time curve from the last dose of tacrolimus to 12 hours later were 20.5 +/- 9.8 hours, 0.77 +/- 0.37 hours, 27.5 +/- 31.8 ng/mL, and 161 +/- 168 hours x ng/mL, respectively. Tacrolimus treated cats survived longer (median, 44 days; range, 24 to 52 days) than untreated cats (median, 23 days; range, 8 to 34 days). On histologic evaluation, 3 cats had evidence of acute-active rejection, 1 cat had necrotizing vasculitis, and 2 cats euthanatized at study termination had normal appearing allografts. CONCLUSIONS AND CLINICAL RELEVANCE: Tacrolimus may be an effective immunosuppressive agent for renal transplantation in cats.     

Miniplate Fixation for Repair of Mandibular and Maxillary Fractures in 15 Dogs and 3 Cats

Maxillofacial miniplates and screws were used for skeletal fixation in 15 dogs and 3 cats that sustained a variety of mandibular and maxillary fractures. These implants were used as neutralization or buttress fixation in 11 caudal (junction of the ramus with the mandibular body) and 2 rostral mandibular fractures, 4 maxillary fractures, and 2 zygomatic arch fractures. All but one of the fractures healed with appropriate occlusion and excellent function. In one case of a rostral mandibular fracture, soft tissue dehiscence occurred accompanied by a loss of the fixation and subsequent distraction of the bone fragments; reasonable function was obtained by performing a rostral mandibulectomy. Plate contouring and application of the miniplates along the appropriate biomechanical lines of stress was easily performed and permitted the biomechanical principles of tension band fixation to be applied in most cases. Miniplate fixation, either used alone or in combination with other fracture fixation techniques, achieved sufficiently rigid skeletal fixation to provide uncomplicated healing and good to excellent functional and cosmetic results in 14 dogs and 3 cats.     

Evaluation of autogeneic and allogeneic cortical chip grafting in a feline tibial nonunion model

A feline tibial nonunion model was used to evaluate the efficacy of fresh autogeneic and deep-frozen allogeneic cortical chip grafts for bone repair. Rigidly fixated tibial ostectomies with a segmental defect length of 1 cm were grafted with 3 mm X 2 mm chips of cortical bone in 12 skeletally mature cats. Six cats were given fresh autogeneic bone (group A) and 6 were given deep-frozen allogeneic bone (group B). Bone healing was evaluated radiographically and histologically over a 12-week period. Consolidating bony callus and palpable stability of the tibia after bone plate removal were evident in all group A cats and 4 of the 6 group B cats by 12 weeks after surgery. Bone remodeling had progressed to intermediate restoration of diaphyseal structure in 2 cats of each group. Fibrous replacement of graft chips, absence of bony callus, and instability of the tibia after bone plate removal were evident in 1 group B cat at 12 weeks. The remaining group B cat was withdrawn from the study at 6 weeks due to loosening of the bone plate and screws.     

Use of veterinary interlocking nails for diaphyseal fractures in dogs and cats: 121 cases

OBJECTIVE: To report clinical outcome after use of an interlocking nail (veterinary interlocking nail [VIN]) for stabilization of diaphyseal fractures in dogs and cats. STUDY DESIGN: Retrospective study. Animals: Seventy-eight dogs and 43 cats with diaphyseal fractures of the femur (n = 96), tibia (n = 14), or humerus (n = 11). METHODS: Interlocking nails (4 mm diameter [n = 72], 6 mm [n = 25] or 8 mm [n = 24]), were used in static (n = 106) or dynamic (n = 15) fixation mode. Cerclage wires also were used in 63 (52%) cases. Data about the patient (species, breed, weight, age), characteristics of the fracture, and details of the surgery and perioperative complications were recorded. The surgeon evaluated functional outcome, and fracture healing was quantified 6 weeks (W6) and 3 months (M3) after surgery with a radiographic index. RESULTS: Twelve cases had been unsuccessfully treated by another technique. Of 106 comminuted fractures, 60 were classified as unstable. Only 112 animals were evaluated at W6; 86 (77%) healed without complication and had a functional outcome considered excellent (n = 80, 93%), good (n = 5, 4%), or fair (n = 1). Twenty-six complications were noted: 16 (14%) patients did not require additional surgery and had a good or excellent outcome, whereas 10 (8%) patients needed surgical intervention to CONCLUSIONS: VINs can be used to repair diaphyseal fractures of the femur, tibia, and humerus in dogs and cats provided the implants are appropriately sized for the fractured bone. The high healing rate (even with unstable fractures), associated with a functional outcome, and low complication rate support the use of VINs for these fracture types. However, a period of training and the application of basic principles are necessary to ensure successful results. CLINICAL RELEVANCE: VINs should be considered as alternative technique for management of selected diaphyseal fractures of the femur, tibia, and humerus in dogs and cats.     

Heterotopic Transfer of Fresh and Cryopreserved Autogenous Articular Cartilage in the Horse

Two 10 mm thick osteochondral grafts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. The grafts were frozen in a 7.5% DMSO solution and stored in liquid nitrogen. The horses were anesthetized again on day 14 and the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifle. A fresh graft was transferred from the right hock to the left stifle. To control for the effects of surgery, another fresh graft was transferred from the right stifle to the left stifle. The result was two grafts in each femoropatellar joint. Fresh and frozen osteoarticular autografts appeared to maintain a durable weight-bearing surface for 3 months; however, the fresh grafts were clearly superior. Frozen grafts had fewer living chondrocytes, decreased safranin-O staining, and decreased SO435 uptake. Graft stability and articular surface congruency were determining factors in the outcome of all grafts. Since the availability of osteochondral autografts is limited, further work on the use of preserved allogeneic osteochondral tissue is warranted.     

Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects: an ovine model

OBJECTIVES: To report an ovine model that can be used to evaluate the efficacy of bone substitutes for repair of segmental diaphyseal bone defects. STUDY DESIGN: Experimental study. ANIMALS: Eleven 2-year-old Pré-Alpes Sheep. METHODS: Mid-diaphyseal metatarsal bone defects (25 mm long) were stabilized by a dynamic compression plate over a polymethylmethacrylate (PMMA) cement spacer, and by external coaptation. The PMMA spacer was removed at 6 weeks by incising the encapsulating membrane. The defect remained unfilled (Group 1; n=5) or was filled with morselized autologous corticocancellous graft (Group 2; n=6), the membrane sutured closed, and external coaptation applied for 6 months, when healing was evaluated. RESULTS: Radiographic, computed tomographic, and histologic examinations at 6 months after the 2nd surgery revealed non-union in ungrafted defects whereas grafted defects showed bone healing. The induced membrane had blood vessels, CBFA1+ cells, and very few macrophages entrapped in a collagenous tissue positive for type I collagen. CONCLUSION: This ovine metatarsal defect model resulted in a critical-size defect (non-union) that healed when grafted. The PMMA-induced membrane constrained the graft, was well vascularized, and may have osteogenic properties. CLINICAL RELEVANCE: This model may be useful to evaluate new strategies in bone tissue engineering because the PMMA-induced membrane may help confine bone morphogenetic proteins, skeletal stem cells, or other agents to the defect cavity where they could be useful to enhance bone formation.     

Treatment of tibial fractures with plates using minimally invasive percutaneous osteosynthesis in dogs and cats

OBJECTIVES: The aim of the here described case series was to develop and evaluate the minimally invasive percutaneous osteosynthesis for the plate fixation of tibial fractures in dogs and cats. METHODS: Six dogs and four cats with shaft fractures of the tibia were treated using minimally invasive percutaneous osteosynthesis. Follow-up radiographs four to six weeks after fracture fixation were evaluated for fracture healing. For the long-term follow-up (minimum 2.4 years), owners were contacted by phone to complete a questionnaire. RESULTS: All fractures healed without the need for a second procedure. Follow-up radiographs obtained after four to six weeks in seven cases showed advanced bony healing with callus formation and filling of the fracture gaps with calcified tissue in all seven. All the patients had a good to excellent long-term result with full limb function. The time needed for regaining full limb use was two to three months. CLINICAL SIGNIFICANCE: Minimally invasive percutaneous osteosynthesis seems to be a useful technique for the treatment of tibial shaft fractures in dogs and cats.