Deep-Frozen Allogeneic Cancellous Bone Grafts in 10 Dogs: A Case Series

Deep-frozen, aseptically collected and processed allogeneic cancellous bone was implanted in eight dogs during the surgical repair of diaphyseal long bone fractures and in two dogs during arthrodeses. A combined allogeneic and autogeneic cancellous bone graft was used in two fractures with a segmental bone loss of more than 5 cm. Bone union occurred in five fractures and in both arthrodeses. Failure of fixation occurred in two dogs with nonunion fractures and in a third dog with an open, infected fracture. Biopsies from the fracture sites were obtained from these dogs following failure of their fracture fixation. The cancellous bone graft appeared to be in the process of normal incorporation in each case. Failure of fixation was attributed to technical or case management errors or both, in each of the three fractures that failed to achieve bony union. Frozen allogeneic cancellous bone grafts were effectively incorporated when used in the primary repair of fractures and arthrodeses. Combined autogenous and allogeneic cancellous bone grafts may be particularly useful in the repair of fractures with large segmental diaphyseal bone defects. The use of allogeneic cancellous bone grafts in nonunion fractures requires further investigation before it can be recommended.     

Effects of autogenous cancellous bone on healing of homogenous cortical bone grafts

A comparative clinical and histological study was made of the effects of autogenous cancellous grafts on the rate of healing of homogenous cortical bone grafts in dogs: their right foreleg received homogenous bone grafts and their left foreleg received pre-drilled homogenous bone grafts filled with autogenous cancellous bone. Histologically, the use of homogenous cortical grafts with pre-drilled holes and autogenous cancellous bone appeared to accelerate reorganization and development of these grafts. This was seen particularly 1–2 months post-operatively and at nine months when the graft in the left leg was made up of spongy bone marrow; whereas the right leg graft still contained compact dead bone and the marrow was filled with fibrous tissue.     

Comparison of bone healing,as assessed by computed tomography,following tibial tuberosity advancement in dogs with and without autogenous cancellous bone grafts

Abstract

AIMS: To objectively compare measures of bone healing, using computed tomography (CT) in dogs following bilateral tibial tuberosity advancement (TTA), between tibiae treated with and without autogenous cancellous bone grafts.

METHODS: Ten dogs with bilateral cranial cruciate ligament disease requiring surgical stabilisation were prospectively recruited to undergo single-session bilateral TTA, with only one, randomly assigned, tibia receiving bone graft in the osteotomy deficit. Bone healing at the osteotomy site was assessed using CT performed 38–70 days post-operatively. CT images were evaluated using both objective measurements of osseous bridging and subjective evaluation by six radiologists. Repeated measures ANOVA was used to compare the objective outcomes between the grafted and non-grafted tibiae.

RESULTS: The mean percentage of the osteotomy deficit bridged at the lateral cortex was greater in grafted (77.6, SD 35.2%) compared to non-grafted (63.0, SD 36.5%) tibiae (p=0.001), but did not differ at the medial cortex (p=0.1). The mean minimum callus width was greater in grafted (7.2, SD 3.3 mm) compared to non-grafted (3.6, SD 2.9 mm) tibiae (p<0.001). There was no difference in mean attenuation (measured in Hounsfield units) of the callus between grafted and non-grafted tibiae (p=0.5). The grafted tibia was deemed to have superior bone healing in 50/60 subjective assessments made by radiologists.

CONCLUSIONS: Superior osseous bridging was detected by CT analysis following TTA using autogenous cancellous bone grafts compared with no graft. This was shown by greater bridging percentage at the lateral cortex and formation of a broader callus. Qualitative assessments made by six radiologists also supported the conclusion that bone healing was improved by use of autogenous cancellous bone graft. CT was a useful method for assessing evidence of bone healing following TTA.

CLINICAL RELEVANCE: These findings justify the application of autogenous cancellous bone graft to augment healing following TTA in dogs.     

Vascularization of cancellous chip bone grafts

Fresh autogenous, frozen allogeneic, and commercially prepared xenogeneic cancellous chip bone grafts were placed into plug holes in the tibia and into surgically simulated nonunion defects in the ulna of dogs. The microvascular and correlated histologic reactions were studied at given times up to 12 weeks after the graft implantation. Marked and characteristic differences were noted among the 3 graft materials. Fresh autogenous chips were extensively vascularized by 1 week, and subsequent microscopic healing was rapid. Allogeneic chips were incorporated into all defects, but vascularization and histologic healing rates were slower than those with the fresh autogenous graft material. The xenogeneic graft was poorly incorporated. A foreign body reaction was seen histologically, and corresponding vascularization of the graft was slow and incomplete.     

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.     

Evaluation of Collagen as a Retainer for Autogenous Cancellous Bone Used in Repair of Full Thickness Cortical Bone Defects

A commercially available collagen sponge acting as a cylindrical retainer containing autogenous cancellous bone graft was proposed for treatment of full thickness cortical defects in dogs. Three groups of three dogs each were subjected to removal of at least 1.5 cm of the tibial diaphyses. All tibias were stabilized with bilateral external fixators. Group 1 received collagen surrounding an autogenous cancellous bone graft. Group 2 received collagen and group 3 received no treatment of the ostectomy site. The collagen sponge appeared to allow vascularization of the bone graft as evidenced by new bone formation and was resorbed by 3 months after implantation. Radiographic, histomorphologic, and histomorphometric evaluation at 3 months after surgery showed a greater per cent of bone in the ostectomy site in group 1.     

Mesenchymal stem cells and bone regeneration

OBJECTIVE: To review the role of mesenchymal stem cells (MSC) in bone formation and regeneration, and outline the development of strategies that use MSC in bone healing and regeneration. STUDY DESIGN: Literature review. METHODS: Medline review, synopses of authors' published research. RESULTS: The MSC is the basic cellular unit of embryologic bone formation. Secondary bone healing mimics bone formation with proliferation of MSC then their differentiation into components of fracture callus. Bone regeneration, where large amounts of bone must form, mimics bone healing and can be achieved with MSC combined with strategies of osteogenesis, osteoinduction, osteoconduction, and osteopromotion. MSC based strategies first employed isolated and culture expanded stem cells in an osteoconductive carrier to successfully regenerate a critical segmental defect in the femur of dogs, which was as effective as autogenous cancellous bone. Because MSC appeared to be immunologically privileged, a study using mismatched allogeneic stem cells demonstrated that these cells would regenerate bone without inciting an immunologic response, documenting the possibility of banked allogeneic MSC for bone regeneration. A technique was developed for selectively retaining MSC from large bone marrow aspirates at surgery for bone regeneration. These techniques utilized osteoconductive and osteoinductive carriers and resulted in bone regeneration that was similar to autogenous cancellous bone. CONCLUSION: MSC can be manipulated and combined with carriers that will result in bone regeneration of critically sized bone defects. CLINICAL RELEVANCE: These techniques can be employed clinically to regenerate bone and serve as an alternative to autogenous cancellous 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.     

Use of cancellous bone grafting to promote granulation tissue in a distal limb wound in a dog

A 5‐year‐old female spayed Shetland sheepdog presented for evaluation of bandage sores on the left pelvic limb after a splint was placed to stabilise a digit fracture incurred approximately 2 weeks previously. Multiple areas of necrosis were identified upon bandage removal. After debridement of devitalised tissue, a 4 cm wound on the plantar surface of the metatarsal bones (with exposed bone and tendons) remained. The Papineau technique (which involves removal of infected or necrotic tissue, placement of autogenous cancellous bone graft within a wound, and delayed skin closure) was used to treat this wound. Use of bone graft in this manner aims to promote early granulation tissue formation and reduce the time until wound reconstruction can be performed. Autogenous cancellous bone graft was harvested from the proximal humerus and placed within the metatarsal wound. Once a healthy granulation tissue bed was noted to cover the bone graft, a meshed full thickness skin graft was placed. To our knowledge, this is the first report of the Papineau technique being used for this purpose in a dog.     

Qualitative aspects of the incorporation of equine cancellous bone grafts

The incorporation of autogenous cancellous bone graft was studied in eight yearling ponies. The site for the defect to be grafted was chosen so that the effect on the graft, of both the host cortical and trabecular bone, could be assessed. To obtain information concerning the vitality of the graft and the dynamic aspects of the modelling and remodelling processes of graft incorporation, a double and treble tetracycline intravital labelling technique was used. Radiographs of the graft and host tissues of all ponies were obtained regularly, but were of little assistance in assessing graft incorporation. The ponies were destroyed humanely at regular intervals between nine and 241 days after installation of the graft, followed by histological examination of undecalcified sections. The study revealed that all installed graft trabeculae showed signs of non-vitality at nine days after installation and gradually disintegrated. Two processes of new bone formation were observed. First, finger-like projections of immature new trabeculae were found to originate from the graft/host interfaces. Second, a gradual process of accretion of osteoid and woven bone upon disintegrating graft trabeculae occurred uniformly throughout the graft. The graft adapted to the structure of opposing host bone by corticalisation and trabecularisation. The present study confirmed clinical observations relating to convalescence time following grafting of large osseous defects in horses and indicated that equine bone reacts to autogenous bone grafts in a similar manner to other mammals.     

Effects of gelatin sponge implantation in cancellous bone defects in dogs

Reports from oral surgeons suggested that packing bone defects with cellulose or collagen stimulated cancellous bone replacement. The study reported here was designed to evaluate a similar method for augmenting the remodeling of cancellous bone defects and graft donor sites. Gelatin sponge was implanted into bony defects created in the proximal humeral metaphysis of 5 adult Beagles after collection of cancellous bone. A similar defect created in the contralateral humerus was left unpacked. Four months after creation of the defect, the dogs were euthanatized, and the surgical sites were evaluated by use of correlated microradiographic and histologic examination. Unpacked sites had filled completely with loosely woven trabecular bone. Remodeling had not taken place in sites packed with gelatin sponge. Gelatin sponge did not elicit any reaction in the surrounding bone, but remained in situ and obstructed new bone formation. Gelatin sponge blocked rather than augmented cancellous bone replacement.     

Distracted cervical spinal fusion for management of caudal cervical spondylomyelopathy in large-breed dogs

Using an autogenous bone graft (obtained from the iliac crest), 4-mm cancellous bone screws, and polymethylmethacrylate, a distracted cervical spinal fusion technique was performed on 10 dogs with myelographic evidence of caudal cervical spondylomyelopathy. All dogs had evidence of dynamic soft tissue spinal cord compression, as indicated by flexion, extension, and traction myelographic views. Of the 10 dogs, 4 previously had undergone surgery by use of ventral slot or cervical disk fenestration techniques, and their neurologic status had deteriorated after the original surgery. Preoperative neurologic status of the 10 dogs included nonambulatory tetraparesis (n = 5), severe ataxia with conscious proprioceptive deficits (n = 2), and mild ambulatory ataxia with conscious proprioceptive deficits (n = 3). Five dogs had signs of various degrees of cervical pain. Clinical improvement was observed in 8 of 10 dogs--either improved neurologic status or elimination of cervical pain. Implant loosening developed in 3 dogs; 2 of them were euthanatized because of lack of neurologic improvement. Radiographic evidence of bony cervical fusion was observed during a 9- to 24-week period in 6 of the 8 surviving dogs. The distracted cervical fusion technique appears to be a valid surgical procedure to manage cervical spondylomyelopathy in those dogs in which the lesions are limited to one cervical intervertebral disk space.     

Use of an external skeletal fixation device and bone graft for arthrodesis of the metacarpophalangeal joint in horses

Arthrodesis of the metacarpophalangeal joint was attempted in 9 adult horses, using a new external skeletal fixation device and autogenous cancellous bone graft. Four horses had excellent results, 2 horses had fair results, arthrodesis failed in 2 horses, and 1 horse fractured the third metacarpal bone. The advantages of the technique included minimal surgical trauma to the area of injury, immediate rigid fixation of the metacarpophalangeal joint, and access for treatment of soft-tissue lesions at the site of injury. Although pin loosening and pin-track infection were complications when the device was in place, all pin tracks healed without complications after device removal.     

Effects of a fixed compression load on the osteogenic effect of autogenous cancellous bone grafts in dogs.

A standardized cortical defect was created on the caudal cortex of the proximal portion of each ulna in 5 adult mixed-breed dogs. One gram of autogenous cancellous bone graft (ACBG) was obtained from the greater tubercle of the ipsilateral humerus. The cortical defect in the ulna of 1 limb was filled with 1 g of ACBG that had been compressed with 2-MPa pressure for 30 seconds. One gram of noncompressed ACBG was placed into the contralateral ulnar cortical defect. The compressed and noncompressed ACBG recipient sites were radiographed at weekly intervals. Dogs were euthanatized 8 weeks after surgery, and the ACBG recipient sites were harvested for histomorphometric analysis. Optical densitometry was performed on all radiographs. There was no significant difference between compressed and noncompressed ACBG with optical densitometry or histomorphometric analysis for total bone area. We concluded that there was no difference in osteogenic capability between compressed and noncompressed ACBG of equal mass.     

Bone grafts.

Bone grafting provides a method of enhancing bone healing in veterinary orthopedic patients. Specifically, autogenous cancellous bone graft provides the cellular components and matrix proteins that can accelerate bone healing, dramatically. Allografts provide immediate mechanical support for fracture repair and patient function, but these grafts do not create the osteogenic environment seen with the use of autogenous cancellous bone graft. Xenograft bone implants may also hold a place for use in fracture management. With the advent of recombinant bone-derived tissue growth factor technology, bone grafting may some day become a practiced technique of the past. For now, however, bone grafting still holds a strong place in orthopedic surgery when dealing with bone defects in animals.     

Clinical Applications of Demineralized Bone Matrix: A Retrospective and Case‐Matched Study of Seventy‐Five Dogs

Objectives— To evaluate the outcome in dogs treated with demineralized bone matrix (DBM) as an adjunct to orthopedic procedures. Study Design— Retrospective and case‐match study. Animals— Dogs (n=75). Methods— Medical records (1999–2006) and radiographs of dogs that had orthopedic procedures (comminuted fractures, tibial plateau leveling osteotomy [TPLO] where correction for tibial rotation created an osteotomy gap, arthrodeses, open corrective osteotomies) where DBM was used were reviewed for signalment, quantity of DBM implanted, duration of exercise restriction, radiographic healing, and complications. Dogs that had TPLO and correction of tibial torsion (n=15), or arthrodesis (n=16) were compared with case‐matched controls. Data were analyzed using Kruskal–Wallis test, ANOVA, Tukey's HSD test, and logistic regression analysis. Results— Mean (±SD) healing time for orthopedic surgeries with DBM augmentation were 15±6.97 (weeks) and complication rate was 19% (14 dogs). Dogs with a TPLO gap filled with DBM were allowed to return to normal exercise 2 weeks earlier than dogs with a well‐apposed TPLO site. Radiographic healing, duration of exercise restriction, and timing of destabilization were similar in dogs undergoing carpal and tarsal arthrodesis whether they received DBM, autogenous graft, or both. Conclusions— DBM can be used to treat uncomplicated bone defects associated with comminuted fracture repairs, open osteotomies, and arthrodeses in dogs. Under these circumstances, clinicians might expect similar clinical outcomes without the possibility of side effects associated with the harvest of autogenous cancellous bone. Clinical Relevance— DBM is safe for use in dogs.     

Use of an autogenous cancellous bone graft in the treatment of subchondral bone cysts in the medial femoral condyle of the horse

The clinical features, diagnosis and radiological findings of eight cases of a subchondral bone cyst involving the medial condyle of the femur are described. Surgical treatment, which comprised performing an arthrotomy of the femorotibial joint, curetting and packing the cystic cavity with an autogenous cancellous bone graft, is described.     

Cancellous Bone Graft Collection from the Tibia in Dogs

Autologous cancellous bone was collected from the metaphysis of the proximal end of the tibia in 30 medium and large dogs. Access to the metaphyseal cancellous bone was gained by making a medial opening in the cortex with a 5.6, 7.9, or 10 mm bone trephine. Sufficient bone was obtained from this site for grafting in each case. Four dogs had bilateral carpal arthrodesis that necessitated graft collection from both tibias. Complications arising from graft collection included wound disruption 5 days postoperatively (3/34 graft sites) and incomplete fracture of the tibial cortex created during the surgical procedure (1/34 graft sites). Collection of autologous cancellous bone from this sita in mature, medium and large dogs was found to be a safe procedure with a low incidence of donor site morbidity.     

Antebrachial deformities in the dog: Treatment with external fixation

The results of 12 dogs with antebrachial deformity treated by ulnar ostectomy, radial osteotomy and external skeletal fixation are presented. Postoperative complications were seen in only one dog; a delayed union requiring placement of autogenous cancellous bone graft at a second surgery to achieve healing. Postoperatively, owners reported limb function was good in almost all cases, although they were less pleased with the cosmetic appearance.     

Histologic features of the healing of bone graft donor sites in dogs

Healing of cancellous bone graft donor sites in the proximal tibial metaphysis of 12 healthy adult dogs was studied histologically. Cancellous bone was curetted from the metaphysis of the proximal end of the tibia, via a 1-cm diameter circular opening in the medial cortex. A hematoma and fibrovascular tissue filled the bone defect at 2 weeks. At 4 and 8 weeks, endosteal callus, composed initially of cartilage and woven bone and later of lamellar bone, filled the marrow cavity. At 12 weeks, the normal structural arrangement of lamellar bone and hematopoietic marrow was reestablished in the marrow cavity. The medial cortex defect was filled only with lamellar trabecular bone. It was concluded that, in adult dogs, a second cancellous bone graft could be collected from the proximal portion of the tibial metaphysis 12 weeks or more after an initial collection.