Rapid and reliable healing of critical size bone defects with genetically modified sheep muscle.

Large segmental defects in bone fail to heal and remain a clinical problem. Muscle is highly osteogenic, and preliminary data suggest that autologous muscle tissue expressing bone morphogenetic protein-2 (BMP-2) efficiently heals critical size defects in rats. Translation into possible human clinical trials requires, inter alia, demonstration of efficacy in a large animal, such as the sheep. Scale-up is fraught with numerous biological, anatomical, mechanical and structural variables, which cannot be addressed systematically because of cost and other practical issues. For this reason, we developed a translational model enabling us to isolate the biological question of whether sheep muscle, transduced with adenovirus expressing BMP-2, could heal critical size defects in vivo. Initial experiments in athymic rats noted strong healing in only about one-third of animals because of unexpected immune responses to sheep antigens. For this reason, subsequent experiments were performed with Fischer rats under transient immunosuppression. Such experiments confirmed remarkably rapid and reliable healing of the defects in all rats, with bridging by 2 weeks and remodelling as early as 3-4 weeks, despite BMP-2 production only in nanogram quantities and persisting for only 1-3 weeks. By 8 weeks the healed defects contained well-organised new bone with advanced neo-cortication and abundant marrow. Bone mineral content and mechanical strength were close to normal values. These data demonstrate the utility of this model when adapting this technology for bone healing in sheep, as a prelude to human clinical trials.

Equine model for soft-tissue regeneration.

Soft-tissue regeneration methods currently yield suboptimal clinical outcomes due to loss of tissue volume and a lack of functional tissue regeneration. Grafted tissues and natural biomaterials often degrade or resorb too quickly, while most synthetic materials do not degrade. In previous research we demonstrated that soft-tissue regeneration can be supported using silk porous biomaterials for at least 18 months in vivo in a rodent model. In the present study, we scaled the system to a survival study using a large animal model and demonstrated the feasibility of these biomaterials for soft-tissue regeneration in adult horses. Both slow and rapidly degrading silk matrices were evaluated in subcutaneous pocket and intramuscular defect depots. We showed that we can effectively employ an equine model over 6 months to simultaneously evaluate many different implants, reducing the number of animals needed. Furthermore, we were able to tailor matrix degradation by varying the initial format of the implanted silk. Finally, we demonstrate ultrasound imaging of implants to be an effective means for tracking tissue regeneration and implant degradation.

Comparison of two methods for presurgical disinfection of the equine hoof.

OBJECTIVES: To determine for equine hooves the normal resident aerobic bacterial population and the efficacy of 2 methods of disinfection. Study Design-Measurement of total bacterial, gram-positive bacterial, and gram-negative bacterial surface populations from the frog, sole, and hoof wall after each step of 2 different preoperative surgical disinfection techniques. ANIMALS: Six adult horses. METHODS: Hoof wall, sole, and frog samples were collected for quantitative bacteriology before, during, and after 2 multistep antiseptic preparation techniques: Method A-6-minute scrub with povidone-iodine soap, followed by 24-hour submersion in povidone-iodine solution-soaked cotton; and Method B-initial removal of superficial layer of hoof capsule before completing Method A disinfection procedures. RESULTS: Removal of the superficial hoof layer, application of the povidone iodine scrub, and completion of the povidone-iodine soak all significantly (P < .0008) decreased total bacterial numbers. Method B had significantly lower bacterial counts than method A at each consecutive step. Final total bacterial counts remained greater than 10(5) bacteria per gram of tissue regardless of preparation method. CONCLUSIONS: The hoof surface hosts a broad spectrum of aerobic gram-positive and -negative bacteria, many of which are potential pathogens. Bacterial numbers can be significantly reduced by removal of the superficial hoof surface, by application of a povidone-iodine scrub, and by use of a 24-hour povidone-iodine soak. However, bacterial populations >10(5) g per tissue persist after these disinfection procedures. CLINICAL RELEVANCE: Regardless of the preparation methods used in this study, bacterial populations capable of inducing wound infection remain on the hoof capsule.

Safety of an oral chondroprotective agent in horses.

Six healthy, adult female horses were administered five times the minimum maintenance dose of an oral low-molecular-weight chondroitin sulfate, glucosamine HCl, and manganese ascorbate chondroprotective agent (Cosequin; Nutramax Laboratories, Inc., Edgewood, MD) daily for 35 days. Hematology, serum biochemistry, and synovial fluid parameters were assessed twice prior to administering the product and again at the end of the treatment period. Physical examinations performed daily throughout the study showed no abnormal clinical changes attributable to the product. All hematologic parameters measured were within normal reference ranges; however, hematocrit, hemoglobin, and white blood cell counts were significantly (P < .05) increased after treatment, as compared with values on Day 0. Mean serum urea nitrogen was mildly elevated above the reference range before and after treatments, and mean serum creatinine was significantly (P < .05) decreased after treatment. Several other biochemical parameters (calcium, phosphorus, potassium, total and indirect bilirubin, alkaline phosphatase, gamma-glutamyltransferase, lactic dehydrogenase, and sodium:potassium ratio) were significantly (P < .05) altered following administration of the chondroprotectant, but all remained within normal reference ranges. Mean creatine kinase levels were significantly higher after treatment than on Day 0 (429 U/L versus 310 U/L), but this represented only a mild elevation relative to the reference range (10 to 350 U/L). Synovial fluid total protein and specific gravity were unaffected. The minor shifts encountered in hematology and serum biochemistry parameters are not considered to have clinical significance. The results of this study suggest that the oral chondroprotective agent tested is safe for administration to horses at recommended dose rates.

Potential applications and delivery strategies for bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines which are members of the Transforming Growth Factor-beta superfamily. They are the only signaling molecules which can singly induce de novo bone formation at orthotopic and heterotopic sites and their osteoinductive potency makes them clinically valuable as alternatives to bone graft. Several means of delivering BMPs to patients are undergoing evaluation including systemic administration, gene transfer and local matrix delivery vehicle implantation. The latter methodology is in advanced stages of development for application in humans in the treatment of selected spinal fusions, fracture repairs, craniomaxillofacial surgery and periodontal injury and disease. The BMPs are also widely distributed in non-skeletal tissues such as nerve, gastrointestinal tract, kidney, heart and lungs and they have a central role in vertebrate and non-vertebrate organogenesis. Initial studies indicate that the BMPs have neuro, cardio and reno-protective actions and it is likely that therapeutic indications for their use will extend well beyond skeletal disease and injury.

Concentrations of substance P and prostaglandin E2 in synovial fluid of normal and abnormal joints of horses.

OBJECTIVE: To correlate substance P content of synovial fluid with prostaglandin E2 content, radiographic evidence of joint abnormality, and anatomic location of the joint for normal and osteoarthritic joints of horses. SAMPLE POPULATION: Synovial fluid from 46 normal joints in 21 horses and 16 osteoarthritic joints in 10 horses. PROCEDURE: Normal and osteoarthritic joints were identified by clinical and radiographic examination, by response to nerve blocks, during scintigraphy or surgery, or by clinicopathologic evaluation. Substance P and prostaglandin E2 contents of synovial fluid were determined by radioimmunoassay. Radio-graphs of joints were assigned a numeric score reflecting severity of lesions. Joints were assigned a numeric score reflecting anatomic location. RESULTS: Median concentrations of substance P and prostaglandin E2 were significantly increased in osteoarthritic joints, compared with normal joints. A significant correlation was found between concentrations of substance P and prostaglandin E2 in synovial fluid, but a correlation was not detected between substance P concentration in synovial fluid and anatomic location of the joint or between radiographic scores of osteoarthritic joints and concentrations of substance P or prostaglandin E2. CONCLUSIONS AND CLINICAL RELEVANCE: A correlation existed between concentrations of substance P and prostaglandin E2 in synovial fluid obtained from normal and osteoarthritic joints. However, content of substance P in synovial fluid cannot be predicted by the radiographic appearance of the joint or its anatomic location. Substance P and prostaglandin E2 may share an important and related role in the etiopathogenesis of osteoarthritis, lending credence to the importance of neurogenic inflammation in horses.

Critically sized osteo-periosteal femoral defects: a dog model.

A 21-mm defect was created in 1 femoral diaphysis each of 15 dogs. Periosteum as well as a cylinder of bone was removed, and the defect was stabilized with a bone plate. Twelve of the defects were filled with an equal volume of autogenous cancellous bone harvested from the ipsilateral ilium. Three defects were left untreated. Cranial to caudal radiographs were taken postoperatively and every 4 weeks for 16 weeks. The radiographs were evaluated for healing using two ordinal scales. At 16 weeks, the dogs were euthanized and the femurs harvested for biomechanical testing and histologic evaluation. Both operated and contralateral not operated femurs were mechanically tested to failure in torsion, and load at failure and stiffness were calculated. All dogs tolerated the procedure well, and were using the operated limb within 1 or 2 days postoperatively. There were no complications noted during the 16 weeks of the study. Unfilled defects did not heal and became atrophic nonunions. The defects filled with autogenous cancellous bone healed in a consistent pattern of consolidation, incorporation, and remodeling, with uniform increases of both ordinal scales used. The femoral cortex opposite the bone plate demonstrated most mature remodeling, evident both radiographically as well as histologically. Unoperated femurs failed at 13.61 +/- 3.88 N-m and grafted femurs failed at 2.96 +/- 1.3 N-m, which was 23% of the measurement of the unoperated femur. Relative stiffness of the unoperated femurs was 5974 +/- 4316 N-m2/radian, and grafted femurs had a relative stiffness of 642 +/- 561 N-m2/radian, which was 10.4% of the measurement of unoperated femur. This model proved to be a critically sized defect, which when left unfilled resulted in an atrophic nonunion, and when filled with cancellous bone resulted in a consistent healing pattern.

A comparison of the Synthes 4.5-mm cannulated screw and the Synthes 4.5-mm standard cortex screw systems in equine bone.

OBJECTIVE: To determine risk of failure of the Synthes 4.5-mm cannulated screw system instrumentation in equine bone and to compare its application with the Synthes 4.5-mm standard cortex screw system. STUDY DESIGN: The maximum insertion torque of the cannulated and standard cortex screw systems were compared with the ultimate torsional strengths of the equipment. Pullout strength and ultimate tensile load of cannulated and standard cortex screws were also determined. SAMPLE POPULATION: Paired equine cadaver third metacarpal and third carpal bones. METHODS: Maximum insertion torque and ultimate torsional strengths were determined by using an axial-torsional, servohydraulic materials testing system and a hand-held torquometer. Pullout tests were performed by using a servohydraulic materials testing system. RESULTS: Maximum insertion torque of all cannulated instrumentation was less than ultimate torsional strength at all locations (P < .05). Maximum insertion torques of cannulated taps and screws were greater than for standard taps and screws in the third carpal bone (P < .002). Pullout strength of the cannulated screws was less than the standard cortex screws at all sites (P < .001). Cannulated screws broke before bone failure in all but one bone specimen. CONCLUSIONS: The risk of cannulated instrument or screw failure during insertion into bone is theoretically low. The relatively low pullout strength of the cannulated screws implies that the interfragmentary compression achievable is likely to be less than with standard cortex screws. CLINICAL RELEVANCE: The relatively low pullout strength of the cannulated screw suggests that its risk of failure during fracture repair is greater than with the standard cortex screw.

Healing bone using recombinant human bone morphogenetic protein 2 and copolymer.

Middiaphyseal 2.5-cm segmental defects in the right femurs of 12 sheep were stabilized with stainless steel plates and implanted with (1) 2 mg recombinant human bone morphogenetic protein 2 and poly[D,L-(lactide-co-glycolide)] bioerodible polymer with autologous blood (n = 7), (2) 4 mg recombinant human bone morphogenetic protein 2 and poly[D,L-(lactide-co-glycolide)] and blood (n = 3), or (3) poly[D,L-(lactide-co-glycolide)] and blood only (n = 2). Bone healing was evaluated for 1 year using clinical, radiographic, gross pathologic, and histologic techniques. Union occurred in three sheep in Group 1, two in Group 2, and none in Group 3. In the animals that healed, new bone first was visible radiographically between Weeks 2 and 6 after implantation; new bone mineral content equaled that of the intact femur not surgically treated by Week 16; recanalization of the medullary cavity approached completion at Week 52; and at necropsy the surgical treated femurs were rigidly healed, the poly[D,L-(lactide-co-glycolide)] was resorbed completely, and woven and lamellar bone bridged the defect site. In two Group 1 sheep euthanized at Weeks 2 and 6, polymer particles were permeated by occasional multinucleated giant cells. Some plasma cells, lymphocytes, and neutrophils were present locally. The poly[D,L-(lactide-co-glycolide)] tended to fragment during surgical implantation. Despite these observations, the recombinant human bone morphogenetic protein 2/poly[D,L-(lactide-co-glycolide)] implant was able to heal large segmental bone defects in this demanding model.

A new animal model for maxillary sinus floor augmentation: evaluation parameters.

This study describes a novel animal model of the maxillary sinus floor augmentation procedure used to assess bone formation during 12 weeks in response to a recombinant human bone morphogenetic protein-2 (rhBMP-2)/absorbable collagen sponge (ACS) sinus implant. A buffer-ACS implant was used as a control. Animal response was monitored using computerized tomography and physical, hematologic, gross pathologic, and histologic evaluations. The rhBMP-2/ACS implants maintained a relatively constant size postsurgery and showed a time-dependent increase in mineralization. The buffer/ACS control implants failed to mineralize and were resorbed by 4 weeks. The model served effectively and without complication. Results indicate rhBMP-2/ACS implants deserve consideration as alternatives to traditional grafting procedures.

Endodontic therapy and surgical excision of a chronic suppurative osteomyelitic lesion in a horse: a case report.

A 22-year-old thoroughbred gelding was presented for evaluation and treatment of chronic dental disease. The horse had a history of quidding and abnormal bite behavior. Intraoral examination revealed signs of chronic generalized gingival inflammation and severe dental caries affecting the maxillary and mandibular incisor teeth. Treatment was provided on two separate visits over an interval of four months. The first visit consisted of the surgical extraction of three unrestorable incisor teeth and restoration of six carious maxillary incisor teeth. The second visit consisted of conventional endodontic therapy on the remaining mandibular incisor teeth and the surgical removal of a chronic suppurative osteomyelitic lesion. Immediate and long term improvements in eating habits were noted. Three month follow-up examinations following completion of treatment have shown the teeth to be in functional position, the restorations intact, and the surgical site well healed.

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.

Long-term healing of bone using recombinant human bone morphogenetic protein 2.

A 2.5-cm-long middiaphyseal plate-stabilized segmental defect in the right femora of 5 adult sheep was implanted with 1.5 mg of recombinant human bone morphogenetic protein 2 mixed with inactivated demineralized ovine bone matrix. Bone healing was evaluated for 12 months using clinical, radiographic, gross pathologic, and histologic techniques. Bone formation within the defect was first visible radiographically between Weeks 2 and 4 after surgery; bone union was apparent between Weeks 12 and 16, at which time the plates were removed. Recanalization of the medullary cavity with neocortex formation was near completion at Week 52. Bone mineral content at the defect sites equaled that of the nonsurgically treated intact femora by Week 16. Perifemoral soft tissue mineralization did not occur, and callus size was not greater than that formed with autograft. By Week 52, the sheep were not lame, and at necropsy the surgically treated femora were rigidly healed. Woven and lamellar bone bridged the defect site. An apparently normal sequence of ossification, modeling, and remodeling events had occurred. Recombinant human bone morphogenetic protein 2 mixed with a suitable carrier could provide an alternative to autograft for use in a variety of orthopaedic procedures.

Recombinant bone morphogenetic proteins: novel substances for enhancing bone healing.

Bone morphogenetic proteins (BMPs) are differentiative factors whose principal function is to induce transformation of undifferentiated mesenchymal cells into chondroblasts and osteoblasts in a dose-dependent manner. Bone morphogenetic proteins have been isolated postnatally in mammals from bone matrix, periosteal cells, mesenchymal cells of marrow stroma, tooth analagen, and cells of osteosarcoma and chondrosarcoma. Distribution in additional embryonic tissues implies a broader organogenic function. Bone morphogenetic proteins are the only differentiative factors able to singularly induce de novo bone formation in vitro and in vivo. Recombinant DNA technology allows their production in large and highly purified quantities. The BMPs' osteoinductive ability has been shown with a variety of carriers including collagens and polymers at heterotopic and orthotopic sites in a wide range of species. They are presently being readied for clinical use as alternatives to bone grafts. Other potential applications include use as pulp capping agents, promoters of implant osteointegration and soft tissue reunion with bone, treatments for nonadaptive bone disease, and implants for use with mitotically expanded skeletal stem cell populations. Errors in the genetic coding of BMPs may manifest as clinical disease entities.

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.

Vascular supply of the tendon of the equine deep digital flexor muscle within the digital sheath.

The vascular and microvascular anatomy of the equine deep digital flexor tendon (DDFT) within the digital sheath was studied by injecting the vasculature with either colored latex or barium sulphate for radiographic, microangiographic, histologic, and computed tomographic (CT) evaluation. Consecutive 4-mm thick two-dimensional CT slice data were reconstructed to 3-dimensional volumetric images to enhance spatial evaluation of the blood supply. Gross dissection and angiographic studies identified three major vascular sources. Above the fetlock, the DDFT was supplied by either a branch of the medial palmar artery (Arteriae digitalis palmaris communis II) or a branch of the medial palmar digital artery (A. digitalis [palmaris propria III] medialis). Below the fetlock, the DDFT was supplied by branches from the lateral and medial palmar branches to the proximal phalanx (Ramus palmaris phalangis proximalis). The most distal aspect of the tendon received small branches from the medial and lateral palmar digital arteries. Using histology and microangigraphy we observed an extensive and uniform intratendinous vascular network above and below the fetlock, with a relatively avascular region of tendon palmar to the fetlock. The most distal 2.0 to 2.5 cm of the tendon within the sheath was heavily infiltrated with fibrocartilage along its dorsal aspect.

Bilateral hind limb hypoplasia in a foal.

Hypoplasia affecting both hind limbs was observed in a neonatal foal. Scintigraphy aided in establishing diagnosis and prognosis. Radiography revealed an abnormally shaped middle phalanx and lack of the distal phalanx and navicular bone in the right hind limb, with an abnormally shaped distal phalanx and navicular bone in the left hind limb. Scintigraphic findings were closely correlated with the radiographic findings. Radiographic and scintigraphic findings were confirmed at postmortem examination.

Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein.

A middiaphyseal, 2.5-cm osteoperiosteal segmental defect stabilized by plate fixation was created in the right femur of 17 sheep. Four treatment groups were included: Group I, no implant; Group II, inactive bone matrix; Group III, recombinant human bone morphogenetic protein (rhBMP-2) mixed with inactive bone matrix; and Group IV, autogeneic bone graft. Three animals had early failure of fixation, and the remaining 14 were evaluated at three months after implantation. Radiographs showed bony union of all defects treated with rhBMP-2 (six) and a lack of bony union in the negative-control groups treated with no implant (three) and inactive bone matrix without BMP (three). Both defects treated with autograft healed. New bone formation in the defect sites treated with rhBMP-2 first appeared one month after implantation and had a mean bending strength (expressed as a percentage of the contralateral femur) of 91% +/- 59% (mean +/- standard deviation) for defects treated with BMP-2, 77% +/- 34% for autograft, 9% +/- 8% for no implant, and 11% +/- 7% for inactive matrix without BMP. Three sheep treated with rhBMP-2 had their fixation plates removed at four months and were followed for one year. Their bone defect sites remained solidly healed one year after the initial operation.

Surgical repair of ruptured trachea in a horse.

An Arabian mare was referred with traumatically induced complete transection of the trachea. A temporary airway was established through the open wound at the site of the rupture. Resection of devitalized tissue including 3 tracheal ring segments, and anastomosis of the tracheal ends was subsequently performed under general anesthesia. Wound healing was complicated by a peritracheal abscess, which responded successfully to drainage and antibiotic administration.