Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model.

This study evaluated healing of rabbit bilateral ulnar osteotomies 6 and 8 weeks after surgery in response to percutaneous injection of transgenic adenoviral (Ad) bone morphogenetic protein-6 (BMP-6) vector or green fluorescent protein vector control (Ad-GFP) administered 7 days after surgery compared to untreated osteotomy controls. The amount, composition and biomechanical properties of the healing bone repair tissue were compared among groups and to historical data for intact rabbit ulnae obtained from similar studies at the same institution. Quantitative computed tomography was used to determine area, density and mineral content of the mineralized callus in the harvested ulnae. Maximum torque, torsional stiffness, and energy absorbed to failure were determined at 1.5 degrees /s. Calcified sections of excised ulnae (5 microm) were stained with Goldner's Trichrome and Von Kossa, and evaluated for callus composition, maturity, cortical continuity, and osteotomy bridging. Radiographic assessment of bone formation indicated greater mineralized callus in the ulnae injected with Ad-hBMP-6 as early as 1 week after treatment (2 weeks after surgery) compared to untreated osteotomy ulnae (p < 0.006) and Ad-GFP treated osteotomy ulnae (p < 0.002). Quantitative computed tomography confirmed greater bone area and bone mineral content at the osteotomy at 6 weeks in Ad-BMP-6 treated osteotomy as compared to untreated osteotomy ulnae (p < 0.001) and Ad-GFP treated osteotomy ulnae (p < 0.01). Ad-BMP-6 treated osteotomy ulnae were stronger (p < 0.001 and 0.003) and stiffer (p < 0.004 and 0.003) in torsion at 6 weeks than untreated osteotomy ulnae or Ad-GFP treated osteotomy ulnae, respectively. Maximum torque, torsional stiffness, and energy absorbed to failure were greater in Ad-BMP-6 treated osteotomy ulnae compared to their respective untreated contralateral osteotomy ulnae at 8 weeks [p < 0.03]. Maximum torque and torsional stiffness in the Ad-BMP-6 treated osteotomy ulnae were not different to intact ulnae values at 6 and 8 weeks. These experiments confirm that BMP-6 can be potently osteoinductive in vivo resulting in acceleration of bone repair.

Recombinant human bone morphogenetic protein-2 accelerates healing in a rabbit ulnar osteotomy model.

BACKGROUND: Approximately 5% to 20% of fractures have delayed or impaired healing. Therefore, it is desirable to develop new therapies to enhance fracture-healing that can be used in conjunction with traditional treatment methods. The purpose of this study was to evaluate the ability of a single application of recombinant human bone morphogenetic protein-2 to accelerate fracture-healing in a rabbit ulnar osteotomy that heals spontaneously. METHODS: Bilateral mid-ulnar osteotomies (approximately 0.5 to 1.0 mm wide) were created in seventy-two skeletally mature male rabbits. The limbs were assigned to one of three groups: those treated with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2, those treated with an absorbable collagen sponge containing buffer, and those left untreated. In the first two groups, an 8 20-mm strip of absorbable collagen sponge containing either 40 g of recombinant human bone morphogenetic protein-2 or buffer only was wrapped around the osteotomy site. The rabbits were killed at two, three, four, or six weeks after surgery. In addition, twenty-four age-matched rabbits were used to provide data on the properties of intact limbs. The retention of recombinant human bone morphogenetic protein-2 at the osteotomy site was determined with scintigraphic imaging of (125)I-labeled recombinant human bone morphogenetic protein-2. After the rabbits were killed, the limbs were scanned with peripheral quantitative computed tomography to assess the area and mineral content of the mineralized callus. The limbs were then tested to failure in torsion, and undecalcified specimens were evaluated histologically. RESULTS: Gamma scintigraphy of (125)I-recombinant human bone morphogenetic protein-2 showed that 73% +/- 6% (mean and standard deviation) of the administered dose was initially retained at the fracture site. Approximately 37% +/- 10% of the initial dose remained at the site one week after surgery, and 8% +/- 7% remained after two weeks. The mineralized callus area was similar in all groups at two weeks, but it was 20% to 60% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 than in either the ulnae treated with buffer or the untreated ulnae at three, four, and six weeks (p < 0.05). Biomechanical properties were similar in all groups at two weeks, but they were at least 80% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 at three and four weeks than in either the ulnae treated with buffer (p < 0.005) or the untreated ulnae (p < 0.01). By four weeks, the biomechanical properties of the ulnae treated with recombinant human bone morphogenetic protein-2 were equivalent to those of the intact ulnae, whereas the biomechanical properties of both the ulnae treated with buffer and the untreated ulnae had reached only approximately 45% of those of the intact ulnae. At six weeks, the biomechanical properties were similar in all groups and were equivalent to those of the intact ulnae. The callus geometry and biomechanical properties of the ulnae treated with buffer were equivalent to those of the untreated ulnae at all time-points. CONCLUSIONS AND CLINICAL RELEVANCE: These findings indicate that treatment with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2 enhances healing of a long-bone osteotomy that heals spontaneously. Specifically, osteotomies treated with recombinant human bone morphogenetic protein-2 healed 33% faster than osteotomies left untreated. The results of this study provide a rationale for testing the ability of recombinant human bone morphogenetic protein-2 to accelerate healing in patients with fractures requiring open surgical management.

The effect of recombinant human bone morphogenetic protein-2 on femoral reconstruction with an intercalary allograft in a dog model.

This study compared the effect of augmentation of allograft host bone junctions with recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge (ACS), autogenous cancellous bone graft (CBG), and a collagen sponge alone in a canine intercalary femoral defect model repaired with a frozen allograft. Outcome assessment included serial radiographs, dual energy X-ray absorptiometry scans, and gait analyses, and mechanical testing and histology of post-mortem specimens. The distal junction healed more quickly and completely with rhBMP-2 than ACS alone based on qualitative radiography and histologic evaluations. The primary tissue in the unhealed gaps in the ACS group was fibrous connective tissue. The proximal allograft host bone junction had complete bone union in the three treatment groups. There was significantly greater new bone callus formation at both junctions with rhBMP-2 than with CBG or ACS alone that resulted in increased bone density around the allograft host bone junctions. All dogs shifted their weight from the treated leg to the contralateral pelvic limb immediately after surgery. Weight bearing forces were redistributed equally between the pelvic limbs at 12 weeks after surgery with rhBMP-2, at 16 weeks after surgery with CBG, and at 24 weeks after surgery with ACS alone. Bending and compressive stiffnesses of the whole treated femora were equal to the contralateral control femora in all treatment groups, whereas torsional rigidities of the whole treated femora for the CBG and ACS groups were significantly less than the control. Both the proximal and distal junctions the treated with rhBMP-2 had torsional stiffnesses and strengths equal to intact control bones. Ultimate failure torques of the proximal junctions of the CBG group and of both junctions of the ACS group were significantly less than the BMP-treated bones. Augmentation of the allograft host bone junctions with rhBMP-2 on an ACS gave results for all parameters measured that equaled or exceeded autogenous graft in this canine intercalary femoral defect model.

The role of computed tomography in evaluation of subchondral osseous lesions in seven horses with chronic synovitis.

Seven horses with severe, persistent lameness of sudden onset were evaluated with scintigraphy and/or computed tomography. The lameness was localised to the front fetlock joint in 2 horses and to the tibiotarsal joint in 5 horses. Five of the horses had a history of intra-articular injections of the involved joint prior to presentation. All horses had effusion of the affected joint and were positive to flexion tests. Intraarticular anaesthesia eliminated or improved the lameness in 4 cases and a nerve conduction block proximal to the affected joint improved the lameness in another. Cytology examination of fluid from affected joints identified normal joint fluid (one horse) or elevations in nucleated cell counts of 0.9 x 10(9)/l-36.8 x 10(9)/l and total protein 20-42 g/l (6 horses). The joint fluid of 2 of these horses cultured positive for bacteria. Initial radiographs were either normal (4 cases) or the changes seen were not sufficient to explain the degree of lameness. In the 6 cases where scintigraphy was performed, intense focal isotope uptake was found in the suspected region, which corresponded to the proximal portion of the first phalanx (2 cases), distal tibia (2 cases), or talus (3 cases). Computed tomography (CT) was performed because occult fracture or osteomyelitis was suspected; and knowledge of the precise anatomical location of the lesion was considered necessary to assess the need for surgery and to plan the surgical approach. Hypodense focal lesions with hyperdense haloes were found in the subchondral bone deep to the sagittal groove of the first phalanx (P1) (2 cases) in the cochlea of the distal tibia (2 cases), and in the intertrochlear portion of the talus (3 cases). Communication between the lesion and the joint space was demonstrated by CT in 5 cases. Post mortem examination of one case revealed synovitis and a chronic bone abscess (Brodie's abscess) communicating with the joint space.

Primary closure of equine laryngotomy incisions: a review of 42 cases.

Laryngotomy incisions for either staphylectomy, ventriculectomy, cordectomy, resection of the palatopharyngeal arch, or subepiglottal cyst removal, were closed primarily in 42 horses. Incisional complications were subcutaneous emphysema (11 horses, 26%), incisional discharge (4 horses, 10%), postoperative fever (4 horses, 10%), incisional abscessation (3 horses, 7%), incisional seroma (2 horses, 5%), and subcutaneous edema (2 horses, 5%). Incisional complications were identified in 22 horses, but only 8 horses (19%) required intervention for incisional healing to occur. Factors such as preoperative and postoperative administration of antibiotics or nonsteroid anti-inflammatory drugs, use of antibiotic lavage or drains, type of suture material and suture pattern, were not significantly associated with incisional complications. Horses with incisional complications had significantly shorter mean surgical time (P = .011) than horses without incisional complications. Surgical experience was associated with fewer complications (P = .018), but had no significant effect on the frequency of complications requiring intervention. Results of this study indicate that equine laryngotomy incisions can be closed primarily and that most will heal without need for further surgical intervention.

The effect of reversible left recurrent laryngeal neuropathy on the metabolic cost of locomotion and peak aerobic power in thoroughbred racehorses.

The effect of left recurrent laryngeal neuropathy (LRLN) on the metabolic cost of locomotion (MCL) and peak aerobic power (VO2peak) was evaluated in four trained Thoroughbred racehorses. Oxygen consumption (VO2), carbon dioxide production (VCO2), venous lactate concentrations (LAC), and heart rate (HR) were measured during a treadmill exercise test (TET). Each horse performed the exercise test four times, alternating between normal upper airway function and reversibly induced LRLN. Subcutaneous infusion of 2% mepivicaine, a local anesthetic, into the region were the left recurrent laryngeal nerve passes caudal to the cricoid cartilage was used to induce LRLN. The induction of LRLN did not alter the relationship between VO2 and treadmill speed at exercise intensities where VO2 was less than VO2peak (< 9 m/sec). However, a 15.3% reduction in VO2peak (Normal = 165.3 +/- 3.4, LRLN = 140.0 +/- 3.2 mL/kg/min +/- SE, P < .001) occurred at higher treadmill speeds in horses with induced LRLN. A significant group (Normal v LRLN) by treadmill speed effect was found for LAC and R only at treadmill speeds where VO2 = VO2peak. Peak lactate (LACpeak) did not change after the induction of LRLN. The relationship between HR and treadmill speed increased in horses with induced LRLN at exercise intensities where VO2 < VO2peak. Peak heart rate (HRpeak) remained unchanged. Performance as indicated by the maximum number of speed intervals completed (STEPmax) decreased 7% in horses with induced LRLN (Normal = 9.1 +/- 0.04, LRLN = 8.5 +/- 0.2 minutes +/- SE, P < .04). A comparison of paired exercise test measurements showed no evidence of a training effect, or decreased performance caused by a learned response, over the course of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Scintigraphic identification of skeletal muscle damage in horses 24 hours after strenuous exercise.

The uptake of the bone-seeking radiopharmaceutical 99mTc-MDP by damaged skeletal muscle in horses is evaluated. Twenty-four hours following strenuous exercise, 109 racehorses with a history of inadequate athletic performance and subtle lameness were imaged using scintigraphic techniques. Ten horses (9.2 per cent) demonstrated abnormal uptake of the radioisotope within skeletal muscles. A muscle biopsy from one of these horses confirmed that the muscles with increased scintigraphic activity had histologic evidence of rhabdomyolysis. This technique allows localisation and relative quantification of muscle damage and is a valuable aid in the evaluation of the athletic horse.

The use of sports medicine techniques in evaluating the problem equine athlete.

Discovering the cause of poor performance in racehorses can often represent a considerable challenge eluding the more common diagnostic techniques available at the racetrack. Application of sports medicine techniques to these problem cases can aid in the diagnosis of poor performance. Central to the development of this capability has been the use of highspeed treadmills, allowing the racehorse to be evaluated in the controlled laboratory setting, at exercise intensities equivalent to those of racing. Video and cinematographic gait analysis can be used in the diagnosis of subtle lameness conditions. Evaluating hoof balance at high speed has also become an important technique for both lameness diagnosis and prevention. Correcting hoof imbalance normalizes the applied stresses on joints, ligaments, and tendons. Abnormal upper airway function resulting in increased resistance to airflow is major cause of poor racing performance. Often this cause of upper airway dysfunction is difficult to evaluate at rest or after exercise. A definitive diagnosis can be made in these cases using treadmill endoscopy to visualize upper airway function during peak exercise. Lower airway function can also affect performance capability. Radiographic and scintigraphic imaging modalities can be used to evaluate both global and regional lung function in cases of suspected pneumonia, EIPH, COPD, or emphysema. Reduced metabolic fitness can be a primary cause of poor performance due to inherent differences in capability, pathologic changes in the major body systems involved in exercise, or inadequate training. Metabolic stress testing can be used to evaluate the level of fitness in these cases. Orthopedic imaging has also become a valuable diagnostic technique for evaluating musculoskeletal injuries. Scintigraphic evaluation of soft tissue and bone and CT scanning are used to localize the source of lameness and to grade the severity of various orthopedic conditions such as arthritis and stress-induced bone disease that are often difficult to categorize with conventional radiography.

Evaluation of upper respiratory tract function during strenuous exercise in racehorses.

Forty-six racehorses with a history of poor performance underwent endoscopic evaluation of laryngeal and pharyngeal function while exercising on a high-speed treadmill. This evaluation allowed the definitive diagnosis of intermittent or continual upper respiratory tract obstruction as a cause of poor performance, as well as the documentation of the dynamic functional anatomy of the obstruction. Ten of the horses (22%) were determined to have a functional abnormality of the upper respiratory tract. These abnormalities included epiglottic entrapment (1 horse), persistent dorsal displacement of the soft palate during exercise (4 horses), and left laryngeal hemiplegia (5 horses). Thirty-two horses were observed to have signs of left laryngeal hemiparesis (asynchronous arytenoid movement) at rest that did not impair full laryngeal abduction during strenuous exercise.

Distribution of fiber types in locomotory muscles of dogs.

The distribution of Type I and Type II fibers, as determined from histochemical estimation of myofibrillar ATPase activity, was studied within and among the locomotory muscles of the forelimb, trunk, and hindlimb of three mongrel dogs. All Type II fibers had high oxidative capacities as estimated from the histochemical assay for reduced nicotinamide adenine dinucleotide tetrazolium reductase, so they were not further divided into subpopulations. Furthermore, Type I and Type II fibers had similar oxidative potentials as indicated by both histochemistry and biochemistry. Type I fiber populations ranged between 14% and 100% in the muscles sampled. The highest percentages of Type I fibers were found in deep muscles of physiological extensor groups in the arm and thigh that serve to resist gravity (antigravity muscles) when the dog is in the quadrupedal standing position. More superficial muscles in these same groups had fewer Type I fibers. The patterns of Type I fiber distribution among muscles in the antigravity groups of the forearm and leg were the opposite of those in the arm and thigh, with the more superficial muscles of the distal limb segments having more Type I fibers than the deeper muscles. In all limb segments, muscle groups that do not serve to resist gravity did not show as much intermuscular variation. Type I fiber populations in these muscles did not exceed 50%. A stratification of fiber types also existed within muscles, both in extensor and flexor groups, with the deeper portions of the muscles having more Type I fibers than the more superficial portions.