Recombinant human bone morphogenetic protein-2 in absorbable collagen sponge enhances bone healing of tibial osteotomies in dogs

OBJECTIVE: To compare the efficacy of 2 doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) on tibial osteotomy healing in dogs. STUDY DESIGN: Experimental, randomized complete block (n=7). ANIMALS: Adult female dogs (n=21). METHODS: Right midshaft tibial osteotomies were created and stabilized with a 1-mm gap using type I external fixators. Seven dogs were untreated controls and 14 with osteotomies were treated with either 0.05 or 0.2 mg/mL rhBMP-2 delivered in an absorbable collagen sponge (ACS). At 8 weeks, dogs were euthanatized and bones were mechanically tested and examined by microscopy. RESULTS: Bone healing based on radiographic scoring, was significantly improved in dogs treated with 0.2 mg/mL of rhBMP-2 compared with the other groups; these tibiae were also significantly stronger and stiffer than 0.05 mg/mL rhBMP-2 and control osteotomized tibiae. Histologic scores were significantly better for 0.2 mg/mL rhBMP-2 group than 0.05 mg/mL rhBMP-2 group, but neither was significantly different from control. CONCLUSIONS: rhBMP-2 in ACS at a concentration of 0.2 mg/mL improves healing of tibial osteotomies in dogs compared with untreated controls and 0.05 mg/mL rhBMP-2 based on force plate analysis and radiographic evaluation. This was not confirmed histologically but treated bones had improved mechanical properties at 8 weeks. CLINICAL RELEVANCE: After a long bone fracture, dogs may face a long recovery period before full return of limb function. rhBMP-2, in association with good fracture fixation principles, may enhance bone healing in dogs with diaphyseal fractures.     

Clinical application of recombinant human bone morphogenetic protein-2 in 4 dogs

OBJECTIVE: To describe outcome in dogs with insufficient bone healing treated with recombinant human bone morphogenetic protein-2 (rhBMP-2). STUDY DESIGN: Retrospective study. ANIMALS: Four dogs clinically affected with delayed union or nonunion bone healing. METHODS: Medical records were reviewed for signalment, clinical problem, treatment, and outcome. RESULTS: Four dogs that had delayed- or nonunion of bone fracture, osteotomy, or arthrodesis were treated with either minimally invasive, fluoroscopically guided, percutaneous administration or direct surgical application of rhBMP-2. Doses used ranged from 0.2 to 1.6 mg of rhBMP-2. In 3 dogs, a calcium phosphate matrix (CPM) carrier was used whereas in 1 dog commercially prepared rhBMP-2 impregnated in an absorbable collagen sponge (INFUSE Bone Graft) was used. This latter dog had osteomyelitis associated with implant infection before rhBMP-2 administration. Rapid radiographic union was noted in all dogs with excellent long-term outcome. Adverse effects were minimal and included transient worsening of lameness after percutaneous administration of rhBMP-2 in 2 dogs. CONCLUSIONS: rhBMP-2 stimulated rapid bone formation at delayed- or nonunion sites resulting in radiographic bone union with minimal adverse effects and excellent long-term outcome in 4 dogs. CLINICAL RELEVANCE: Direct intraoperative administration or fluoroscopically guided, minimally invasive delivery of rhBMP-2 may be an effective treatment modality for bone delayed- or nonunions and could potentially be used to stimulate new bone production in a variety of orthopedic surgical conditions in dogs.     

Clinical application of recombinant human bone morphogenetic protein in cats and dogs: A review of 13 cases

Databases (2001–2008) for cases in which recombinant bone morphogenetic protein (rhBMP) was used to aid in management of orthopedic disease were reviewed and cases were categorized as non-unions, delayed unions, and cases expected to heal with difficulty. If follow-up in the medical record was < 6 mo for live animals, owners were surveyed by telephone. Thirteen cases (11 dogs, 2 cats) were identified; OP-1 (rhBMP-7) was used in 3 cases and INFUSE (rhBMP-2) in 10. Mean time from injury to rhBMP use for non- and delayed union cases was 156 d; mean time from rhBMP use to radiographic healing was 101 d. No systemic side effects were reported. All patients achieved clinical and radiographic bone union following rhBMP administration. Recombinant human BMP was used in 13 veterinary patients to successfully achieve bone union without serious deleterious effects in a variety of clinical applications.     

Clinical investigation of local implantation of gentamicin-impregnated collagen sponges in dogs

The purpose of this study was to evaluate the effects on health and kidney function of local implantation of commercial gentamicin-impregnated collagen sponges. Four healthy dogs were submitted to local surgical implantation of collagen impregnated sponges. Follow-up with serial physical examinations and measurements of serum creatinine, urea nitrogen, and gentamicin were performed for 7 d. There were no adverse reactions, or changes in measurements of kidney function.     

Comparison of two doses of aqueous bovine thyrotropin for thyroid function testing in dogs

Thyroid function was evaluated in 20 healthy dogs by thyrotropin (TSH) response testing. Two dose regimens were used: 5 IU of TSH given IV and 1 IU of TSH given IV. Blood samples were collected prior to and at 4 and 6 hours after TSH administration. Serum was obtained and analyzed for total 3,5,3'-tri-iodothyronine and thyroxine (T4) concentrations by radioimmunoassay. All dogs were classified as euthyroid on the basis of response to 5 IU of TSH at 4 and 6 hours. The 1-IU dose of TSH failed to induce adequate increase in T4 concentration in 7 dogs at 4 and 6 hours when the criteria for normal response were post-TSH serum concentration T4 greater than or equal to 3.0 micrograms/dl and serum T4 increase by greater than or equal to 100% over baseline serum T4 concentration. One IU of TSH induced increase in serum T4 concentration over baseline; however, the increase was significantly (P less than 0.05) less than that in response to a 5-IU dose at 6 hours after administration of TSH.     

Comparison of three closure methods and two absorbable suture materials for closure of jejunal enterotomy incisions in healthy dogs

The macroscopic and histological appearance of jejunal antimesenteric incisions approximated with two different absorbable suture materials (monofilament versus multifilament) and three closure techniques (appositional single layer, crushing single layer, and double layer) were compared in healthy dogs at 14 or 28 days, postoperatively. No significant differences between the two suture materials were observed for most of the macroscopic or histological variables. However, the monofilament suture material caused significantly more fibrous tissue reaction in the muscular layer of the jejunum than did the multifilament suture material. Of the three enterotomy closure techniques used in this study, the appositional single-layer method proved to be the best. The double-layer closure method caused a significant decrease in the incisional circumference, the relative circumference, and volume of the jejunum, and a significant increase in jejunal wall thickness. Our findings suggest that canine jejunal enterotomy incisions can be closed using an appositional suture pattern with relatively rapidly absorbable monofilament suture material. The use of double-layer suture patterns for closure of jejunal enterotomy incisions should be avoided because the size of the intestinal lumen may be reduced.     

Surgery: Comparison of three closure methods and two absorbable suture materials for closure of jejunal enterotomy incisions in healthy dogs

Summary

The macroscopic and histological appearance of jejunal antimesenteric incisions approximated with two different absorbable suture materials (monofilament versus multifilament) and three closure techniques (appositional single layer, crushing single layer, and double layer) were compared in healthy dogs at 14 or 28 days, postoperatively. No significant differences between the two suture materials were observed for most of the macroscopic or histological variables. However, the monofilament suture material caused significantly more fibrous tissue reaction in the muscular layer of the jejunum than did the multifilament suture material. Of the three enterotomy closure techniques used in this study, the appositional single‐layer method proved to be the best. The double‐layer closure method caused a significant decrease in the incisional circumference, the relative circumference, and volume of the jejunum, and a significant increase in jejunal wall thickness. Our findings suggest that canine jejunal enterotomy incisions can be closed using an appositional suture pattern with relatively rapidly absorbable monofilament suture material. The use of double‐layer suture patterns for closure of jejunal enterotomy incisions should be avoided because the size of the intestinal lumen may be reduced.     

Sustained release carriers used to delivery bone morphogenetic proteins in the bone healing process

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor beta superfamily, especially BMP-2, induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to delivery BMPs for practical use need to be developed with the objective to heal cartilage and bone-related diseases in medical, dental and veterinary practice. Thus, the aim of this article was to present an overview of the principals carriers used to delivery BMPs and alternative delivery systems for these proteins.     

Treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 delivered from a fibrin matrix

OBJECTIVE: To report the results of the treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 (nglBMP-2) delivered from a designed fibrin matrix. STUDY DESIGN: Experimental trial in rodents and prospective clinical study in dogs and cats with nonunion fractures. ANIMALS: Twenty adult female, albino, Sprague-Dawley rats; 8 client-owned cats and dogs. METHODS: After development of a fibrin matrix and evaluation of nglBMP-2 in a rodent femoral defect model, 8 consecutive long bone nonunion fractures (no progression in healing in > or = 3 months), were treated using 300 microg nglBMP-2 in a liquid fibrin precursor, injected into the defect gap after fracture revision and stabilization, or through a stab incision into the fracture site. The fibrin matrix was designed to clot in the wound after 60 seconds and to release the nglBMP-2 continuously over several days. RESULTS: Using only fibrin gel, 7% of the rat femoral defect was filled with new formed bone compared with 79% defect filling using 2 microg nglBMP-2 (P=.006). Five and 10 microg nglBMP in fibrin resulted in union of all femoral defects with complete filling of the gap with new bone. Bony bridging and clinical healing was achieved in 7 patients within 24 weeks of administration of nglBMP-2. CONCLUSIONS: Application of nglBMP-2 in a functional matrix can induce bone healing. Controlled release of nglBMP-2 from a fibrin matrix mimics the natural fracture hematoma. CLINICAL RELEVANCE: nglBMP-2/fibrin can successfully replace a cancellous bone autograft in fracture treatment with an associated reduction in graft donor site morbidity and surgical time.     

Expression of bone morphogenetic protein-6 and bone morphogenetic protein receptors in myoepithelial cells of canine mammary gland tumors

To study the ectopic chondrogenesis in canine mammary mixed tumors, the expression of bone morphogenetic protein-6 (BMP-6) and specific BMP receptors (BMPRs), BMPR-IA, BMPR-IB, and BMPR-II, was examined using immunohistochemical and immunoblot analysis in 39 canine mammary gland tumors. Immunohistochemically, BMP-6 and all three types of BMPRs were coexpressed in the myoepithelial cells and chondrocytes in six of eight benign mixed tumors. In complex adenomas, myoepithelial cells showed an expression pattern of BMP-6, BMPR-IA, and BMPR-II similar to those in benign mixed tumors, whereas immunoreactivity for BMPR-IB was very mild. The myoepithelial cells proliferating within the basement membrane showed more intense immunoreactivity for BMP-6 and all BMPRs as compared with those proliferating in the interstitial areas. Western blotting analysis revealed immunopositive bands at 40-45 kDa for BMP-6 in the samples from simple and complex adenomas and benign mixed tumors. The BMPR-IB-specific bands at 45 kDa were most detected in benign mixed tumors. Because among BMPRs, BMPR-IB is thought to be the major receptor for BMP-6 for primary chondrogenesis, these findings suggest that the expression of BMP and its receptors on the myoepithelial cells might play a role in the ectopic cartilage formation in canine mammary gland tumors, especially in benign mixed tumors.     

Comparison of arrhythmogenic doses of epinephrine in heartworm-infected and noninfected dogs

The arrhythmogenic dose of epinephrine (ADE) was determined in heartworm-infected and noninfected (control) dogs during thiamylal-induced and halothane-maintained anesthesia to assess the myocardial sensitization. The ADE in heartworm-infected dogs (2.42 +/- 0.26 micrograms/kg of body weight) was significantly lower than that for the controls (3.36 +/- 0.29 micrograms/kg). After 2 weeks, ADE was determined again in these dogs after atropine treatment. Atropine treatment lowered the ADE to 1.76 +/- 0.33 micrograms/kg and 1.77 +/- 0.19 micrograms/kg in heartworm-positive and -negative dogs, respectively. After 2 weeks more, the ADE was determined after administration of prazosin, an alpha 1-antagonist. Only 2 of 6 controls and 3 of 6 heartworm-positive dogs had arrhythmias after a threefold increase of ADE. The mean ADE in the dogs that responded to treatment were 7.4 micrograms/kg and 7.2 micrograms/kg for heartworm-positive and -negative dogs, respectively. The finding of this study indicated that ADE in heartworm-infected dogs were lower than those in the control dogs, which makes the heartworm-infected dogs more vulnerable to arrhythmia during anesthesia. Atropine did not protect the dogs of either group. However, prazosin protected the dogs of both groups by significantly increasing the threshold of the ADE. On the basis of our findings, to reduce the risk of arrhythmia, we suggest that routine screening of dogs for heartworm infection be done before anesthetics are used.     

Effects of a hydrolyzed collagen dressing on the healing of open wounds in dogs

OBJECTIVE: To determine the effects of a hydrolyzed bovine collagen dressing (HBCD) on healing of open wounds in healthy dogs. ANIMALS: 9 female Beagles. PROCEDURES: 2 full-thickness skin wounds were made bilaterally on the trunk of each dog. Wounds on 1 side were treated with powdered HBCD covered with a semiocclusive nonadherent bandage. Wounds on the other side (control wounds) were covered with a semiocclusive nonadherent bandage only. Wound healing was subjectively assessed, and percentage increase in tissue perfusion was assessed by use of laser Doppler perfusion imaging (LDPI). Planimetry was performed to determine the percentages of contraction, epithelialization, and total wound healing. Biopsy specimens were examined microscopically to evaluate histologic changes. RESULTS: The HBCD did not induce a strong inflammatory reaction, as reflected by results of LDPI and histologic examination. Moreover, HBCD appeared hydrophilic and provided an environment to keep wounds clean and enhance early epithelialization. After treatment for 7 days, treated wounds had a significantly greater percentage of epithelialization than control wounds (12.13 vs. 7.03%). CONCLUSION AND CLINICAL RELEVANCE: The hydrophilic property of HBCD may cleanse contaminated wounds with the body's homeostatic fluids and enhance early wound epithelialization.     

Comparison of bone healing by demineralized bone matrix and autogenous cancellous bone in horses

OBJECTIVE: The purpose of this study was to compare bone healing induced by equine demineralized bone matrix (DBM) to autogenous cancellous bone graft (ACB) or no graft (control) in a rib-defect model in horses. STUDY DESIGN: The osteogenic properties of ACB and DBM were evaluated in bilateral 19-mm circular defects created in the outer cortex of the 6th and 8th ribs of each horse. ANIMALS OR SAMPLE POPULATION: Eight mature horses. METHODS: Three rib defects in each horse were randomly treated with each of the 3 treatment groups, and the fourth rib defect received a random treatment. Rib sections, including the defects, were harvested 56 days after implantation and examined for bone mineral density, percent ash and calcium and graded for signs of radiographic and histological healing. RESULTS: All ribs were fractured at the defect site and were classified as nonunion fractures 56 days after implantation. There were no significant differences among groups in bone mineral density and signs of radiographic or histological healing. There was an increased volume of bone in control and ACB-treated sites compared with DBM-treated sites. Rib defects treated with ACB were significantly higher in percent ash and calcium than those treated with DBM. DBM elicited no inflammatory reaction, and remodeling occurred around the periphery and within vascular channels of the decalcified particles. CONCLUSION: DBM particles remodel from the periphery, which may explain the significantly lower percent ash, calcium, and bone when compared with ACB, because 2- to 4-microL pieces of DBM may act as space-occupying masses until completely mineralized. There was no evidence of enhanced healing associated with the use of DBM in this model. CLINICAL RELEVANCE: Particles of 2 to 4 mm DBM should not be used as an aid to fracture repair because particles of this size interfere with normal mineralization. However, our model of nonunion fracture healing may be useful in future studies.     

Accessory carpal bone displacement in two dogs.

The diagnosis and treatment of luxation of the accessory carpal bone in a racing greyhound, and subluxation of the same bone in a lurcher, are described. The injury in the lurcher occurred in both carpi, but on separate occasions. Both dogs had severe thoracic limb lameness with marked carpal swelling. The diagnosis of luxation was obvious from carpal radiographs. The subluxations were difficult to detect on palpation, but were suspected and confirmed on exploratory surgery, which showed an avulsion of the lateral support structures of the accessory carpal bone from the distal ulna. Pancarpal arthrodesis with accessory carpal bone excision undertaken in the greyhound was successful. Following repair of the torn ligaments, the lurcher returned to full activity without lameness before sustaining the same injury to the other carpus. The anatomy of the accessory carpal support structures and the aetiology of the injuries are discussed.     

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.     

Nasal adenocarcinoma metastatic to bone in two dogs

Diagnosis of nasal adenocarcinoma was made in a 6-year-old 35-kg neutered Golden Retriever and a 6-year-old 8-kg spayed mixed-breed dog with chronic bilateral nasal discharge unresponsive to antibiotics. Treatment for the Golden Retriever consisted of bilateral rhinotomy, curettage, and postoperative fractionated 48-Gy orthovoltage irradiation. The mixed-breed dog was treated with cisplatin. After complete remission of the primary neoplasm, the dogs were reevaluated because of acute lameness. Radiography of the right stifle of the Golden Retriever revealed soft tissue swelling, extensive bony destruction of the distal femoral metaphysis and epiphysis, and pathologic fracture involving the medial condyle. Radiography of the left scapula of the mixed-breed dog revealed lysis of the glenoid cavity and subchondral scapular bone. Diagnosis of metastatic carcinoma was made in both dogs. With treatment improvements and longer survival time of affected dogs, sinonasal neoplasia may be observed to develop in similar life-threatening metastatic sites.     

Comparison of biological activity and safety of recombinant canine erythropoietin with that of recombinant human erythropoietin in clinically normal dogs.

OBJECTIVE: To determine whether recombinant canine erythropoietin (rcEPO) stimulates erythropoiesis in dogs without causing the immunogenicity problem (ie, erythroid hypoplasia) associated with recombinant human erythropoietin (rhEPO). ANIMALS: 13 clinically normal dogs. PROCEDURE: Dogs were randomly assigned to 2 groups; 1 group (n = 6) received rhEPO, whereas the other group (7) received rcEPO. Both groups received SC injections of diluent for 4 weeks before initiating treatment with erythropoietin (100 U/kg of body weight, SC, 3 times/wk). Hematocrit and absolute reticulocyte count were monitored weekly, CBC were done monthly, and bone marrow aspirates for cytologic evaluation were obtained before and at 4, 8, 16, and 24 weeks during treatment. RESULTS: Weekly mean Hct and absolute reticulocyte count increased in both groups of dogs during the first 2 weeks of treatment. For dogs receiving rhEPO, precipitous decreases in reticulocyte number and more gradual decreases in Hct were associated with development of erythroid hypoplasia. Dogs receiving rhEPO developed erythroid hypoplasia by week 4 (n = 4), 8 (1), or 16 (1). With cessation of rhEPO treatment after diagnosis of erythroid hypoplasia, RBC production recovered 5 to 11 weeks (median, 7 weeks) later. In contrast, rcEPO treatment caused sustained increases in Hct and reticulocytosis. None of the dogs receiving rcEPO developed erythroid hypoplasia. CONCLUSIONS: rcEPO stimulated erythrocyte production in clinically normal dogs during a 24-week period without causing the erythroid hypoplasia encountered in rhEPO-treated dogs. CLINICAL RELEVANCE: Because rcEPO did not cause erythroid hypoplasia, rcEPO may represent an improved option, compared with rhEPO, for treatment of erythropoietin-dependent anemia in dogs.     

1,25-Dihydroxyvitamin D3, recombinant human transforming growth factor-beta 1, and recombinant human bone morphogenetic protein-2 induce in vitro differentiation of canine osteosarcoma cells.

Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), recombinant human transforming growth factor (rhTGF)-beta 1 and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1,25(OH)2D3 increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-beta 1 increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1,25(OH)2D3 or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1,25(OH)2D3 seemed to be more effective than rhTGF-beta 1. These agents are potential inducers of apoptosis.