Guided bone regeneration with beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone membrane in partial defects of canine humerus

This study was performed to evaluate the effect of beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone (TCP/PLGC) membrane in the repair of partial bone defects in canine proximal humerus. Three adult mixed-breed dogs were used during the experimental period. The length of the defect was quarter of the full length of humerus, and width of the defect was quarter of middle diameter of the lateral aspect of humerus. The humeri of each dog were divided into treatment (TCP/PLGC) and control groups. The defect was covered with TCP/PLGC membrane in treatment group. To evaluate regeneration of the bone, computerized tomography (CT) and histopathologic examination were performed. The radiopaque lines were appeared at the original defect sites in TCP/PLGC group but below the original site in control at 4th week. Radiopacity and thickness of the defect sites, and radiopaque lines were more increased at 8th week than those of 4th week. Histopathologic findings revealed fibrous connective tissue migration into the defect and the migration inhibited the structure of new cortex to be placed in the original level in control whereas new cortex growth was found in the level of original line in TCP/PLGC group. However, the new cortical bone in the TCP/PLGC group was thinner and less organized than the adjacent intact cortex, and the amount of new cancellous bones were also scanty. The result suggested that TCP/PLGC membrane is a good guided bone regeneration material to restore the original morphology of humerus in partial defect.     

Comparative study of beta-tricalcium phosphate mixed with platelet-rich plasma versus beta-tricalcium phosphate, a bone substitute material in dentistry

Animal experiments were carried out with osteoconductive bone substitute beta-tricalcium phosphate (beta-TCP), with the aim of assessing the effects of the growth factors synthesised by thrombocytes on the speed of beta-TCP incorporation and on the quality of newly formed bone. The question to be answered was the extent to which platelet-rich plasma (PRP) accelerated the resorption of beta-TCP and the formation of new bone. Two teeth were removed symmetrically from each side of the mandible of 12 Beagle dogs; the resulting cavities were filled on one side with beta-TCP alone, and on the other side with a mixture of beta-TCP + PRP (obtained from autologous blood). The quality of the newly formed bone and the effects of this PRP were studied by histological and histomorphometric methods. In week 6, bone formation was already more effective when PRP was applied in comparison with beta-TCP alone, and in week 12 the growth was significantly greater. The results demonstrate that the use of PRP accelerates the remodelling of new bone created by beta-TCP.     

Effect of subchondral drilling on repair of partial-thickness cartilage defects of third carpal bones in horses

Arthrotomies of middle carpal joints were done on 13 horses, and a 1-cm partial thickness, round defect was made on the radial facet of both third carpal bones. In one joint, 1-mm diameter 1-cm deep holes were drilled within the defect, and one joint was used as a control. horses were assigned to 2 groups--group 1 (n = 6 horses), 5 drill holes; group 2 (n = 7 horses), 11 drill holes. At 1 and 3 weeks after surgery, differences between joints in synovial fluid total protein values, WBC counts, or results of mucin precipitate tests were not significant (P = 0.005). Physically and radiographically, horses were the same during the 12 initial weeks they were housed in stalls and the 9 weeks they were kept in paddocks. Twenty-one weeks after surgery, horses were euthanatized. Joints with drill holes had a significantly greater area (P less than 0.05) of healthy fibrocartilage new tissue: group 1-33 to 68% new tissue, compared with 0 to 23% new tissue in controls; and group 2-22 to 64% new tissue, compared with 0 to 37% new tissue in controls. Differences between healing of defects with drill holes in groups 1 and 2 were not significant. Thickness of new tissue over drill holes was 33 to 61% of thickness of cartilage adjacent to the defect, and thickness of tissue between drill holes was 11 to 43% (group 1) and 8 to 79% (group 2) of the thickness of cartilage adjacent to the defect.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of compacted cancellous bone grafting on the healing of subchondral bone defects of the medial femoral condyle in horses

Objective— To compare the quality of second-intention healing and that of compacting sternally harvested cancellous bone into subchondral bone defects of the medial femoral condyle in horses.
Study Design— A controlled experiment using a surgical technique that minimizes soft tissue trauma, customized for consistency among horses.
Animals or Sample Population— Ten horses, aged 2 to 5 years, free of hindlimb lameness and with radiographically normal stifles.
Methods— After a 12.7-mm-diameter × 19-mm-deep defect was created into randomly selected medial femoral condyles, bone and cartilage healing was evaluated over a 6-month period in control horses (  n = 5  ) and horses receiving a compacted cancellous bone graft (  n = 5  ). Healing was evaluated using lameness assessment, radiographic and microradiographic interpretation, arthroscopic appearance, percent bone fill, proteoglycan content, and histology.
Results— Six months after surgery, there was no significant difference between grafted and ungrafted defects with respect to lameness, radiographic score, or percent bone fill. Histologically, grafted defects were characterized by the presence of dead graft and secondary cyst formation in four defects. Ungrafted defects filled with fibrous tissue and no cyst formation were identified.
Conclusions— Grafted defects do not heal better than ungrafted defects, and lameness was not affected by surgical technique.
Clinical Significance— Cartilage healing is similar in grafted and ungrafted defects in the equine medial femoral condyle at 6 months, suggesting that surgical debridement alone of cystic structures remains the treatment of choice.     

Alteration of distal tarsal subchondral bone thickness pattern in horses with tarsal pain

REASONS FOR PERFORMING STUDY: Understanding of the development of pathology and source of pain in distal tarsal osteoarthritis is poorly understood. Magnetic resonance imaging is often used in the analysis of human osteoarthritis (OA) because it is sensitive to early changes. HYPOTHESIS: In association with distal tarsal joint (DTJ) pain, there will be an alteration in the characteristic subchondral bone (SCB) thickness pattern of horses with no history of pain when subjected to low-level exercise. METHODS: Sixteen cadaver tarsal joints were collected from 9 mature horses with a history of tarsal pain and radiographic evidence of OA; 3 cadaver tarsi were collected from 2 mature horses with a history of tarsal pain and no radiographic abnormality. Magnetic resonance images were acquired using high-resolution sagittal 3D T1 weighted spoiled gradient echo sequence. Subchondral bone thickness was measured on sagittal images at dorsal and plantar locations on the proximal and distal aspects of the central (CT) and third (T3) tarsal bones and proximal aspect of the third metatarsal bone (MT3). RESULTS: In tarsi with radiographic evidence of OA medial and lateral SCB thicknesses were greater than midline on the proximal and distal aspects of CT and T3. Lateral SCB thickness was greater than medial on the proximal aspect of MT3. There was an increase in SCB thickness at the majority of sites compared with normal horses. There were too few joints in the group without radiographic changes to analyse statistically. In painful tarsi SCB thickness was greater medially than laterally at all sites. In horses without tarsal pain all lateral sites had greater SCB thickness, except the proximal aspect of CT. CONCLUSIONS: There is alteration of normal SCB thickness patterns in painful tarsi. Different thickness patterns could represent different types of pathological processes. Potential clinical relevance: Further work is required to elucidate the pathological processes leading to OA of the DTJs.     

Clinical effects of exercise on subchondral bone of carpal and metacarpophalangeal joints in horses

OBJECTIVE: To determine effects of treadmill exercise on subchondral bone of carpal and metacarpophalangeal joints of 2-year-old horses. ANIMALS: 12 healthy 2-year-old horses. PROCEDURE: Horses were randomly assigned to the control (n = 6) or exercised (6) groups. Horses in the exercised group ran on a high-speed treadmill 5 d/wk for 6 months. Horses in the control group were hand walked for the same amount of time. Results of clinical, radiographic, nuclear scintigraphic, and computed tomographic examinations, and serum and synovial concentrations of biochemical markers of bone metabolism were compared between groups. RESULTS: Exercised horses were significantly lamer at the end of the study than control horses. Radionuclide uptake in the metacarpal condyles, but not in the carpal joints, was greater in exercised horses, compared with control horses. Exercised horses also had a higher subchondral bone density in the metacarpal condyles than control horses, but such differences were not detected in the carpal bones. CONCLUSIONS AND CLINICAL RELEVANCE: None of the diagnostic techniques evaluated was sufficiently sensitive to detect all osteochondral damage. Computed tomography and computed tomographic osteoabsorptiometry were superior to conventional radiography for detecting small osteochondral fragments. Nuclear scintigraphy was a sensitive indicator of subchondral bone change but lacked specificity for describing lesions and discerning normal bone remodeling from damage. Newer techniques such as computed tomography may help clinicians better diagnose early and subtle joint lesions in horses prior to development of gross joint damage.     

Healing of full-thickness cartilage compared with full-thickness cartilage and subchondral bone defects in the equine third carpal bone.

The effect of lesion depth on the quality of third carpal bone cartilage repair was examined. A 1-cm diameter articular defect penetrating the calcified cartilage in one limb and the subchondral bone plate in the opposite limb was created in the radial facet of the third carpal bones. Clinical and xeroradiographic examinations were performed every 4 weeks until 4 months (3 horses) and 6 months (3 horses) after surgery. The synovial membrane, non-opposing articular surfaces and articular defects were examined grossly, histologically and histochemically. Grossly, deeper defects contained thicker, whiter tissue, but both joints contained generalised degenerative changes. Defects extending through calcified cartilage were filled deeply by fibrocartilage and superficially by fibrous connective tissue. Defects extending through subchondral bone were consistently filled with hyaline-like cartilage in the depths of the lesion, fibrocartilage in the intermediate layer and fibrous connective tissue superficially. The results indicate that subchondral bone is the source of hyaline-like cartilage repair tissue and suggest that quality of healing of cartilage defects may be improved by penetrating the subchondral bone plate. It also appears that the synovitis associated with the procedure must be controlled before the procedure can be advocated for treatment of clinical cases.     

Healing of surgically created defects in the equine superficial digital flexor tendon: collagen-type transformation and tissue morphologic reorganization

Full-thickness defects were surgically created in the superficial digital flexor tendons of the front limbs of 20 horses. Tissues formed within the defect were evaluated histologically, and the collagen composition of the tissue was determined by immunofluorescence. Transformation occurred from loose fibrillar areas of types I and III collagen and pericellular types IV and V collagen to dense bundles of type I collagen fibers. Loose fibrillar areas of types I and III collagen were present after 24 weeks. Histologically, in horses killed after 2 weeks, the tissue within the defect was a randomly oriented mass of fibrovascular tissue. In horses killed after 24 weeks the tissue within the defect resembled normal tendon tissue.     

Evaluation of increased subchondral bone density in areas of contact in the metacarpophalangeal joint during joint loading in horses

OBJECTIVE: To quantitatively evaluate contact area under 2 loads and subjectively compare contact areas with subchondral bone (SCB) density patterns in intact metacarpophalangeal joints of horses. SAMPLE POPULATION: 6 forelimbs from horses without musculoskeletal disease. PROCEDURES: Computed tomographic scans of intact metacarpophalangeal joints were analyzed to obtain SCB density measurements. Each limb was loaded on a materials testing system to 150 degrees and 120 degrees extension in the metacarpophalangeal joint, and the joint was stained via intra-articular injection with safranin-O or toluidine blue, respectively. Each joint was disarticulated, and the surface area was digitized. Total articular surface area, contact area, and percentage contact area at each angle were calculated for the distal third metacarpal condyles, the proximal phalanx, and the proximal sesamoid bones. RESULTS: Contact area on the third metacarpal condyles, proximal sesamoid bones, and the proximal phalanx significantly increased with increased load. Areas of contact subjectively appeared to have a higher density on computed tomographic scans. CONCLUSIONS AND CLINICAL RELEVANCE: Areas consistently in contact under higher load were associated with increased SCB density. This supports the idea that the SCB adapts to the load applied to it. As load increased, contact area also increased, suggesting that areas not normally loaded may have a high degree of stress during impact loading. Quantifying how contact in the joint changes under different loading conditions and the adaptation of the bone to this change in normal and abnormal joints may provide insight into the pathogenesis of osteochondral disease.     

Evaluation of the healing of surgically created small colon serosal lesions in horses, treated by homologous pericardium implantation: An experimental study

The healing of serosal lesions created in the small colon of horses and treated by homologous pericardium implantation was evaluated. A standing left flank laparotomy was performed in 6 horses. The small colon was partially exposed, and a defect measuring 2 × 2 cm was created in the serosa between the mesenteric border and the anti-mesenteric taenia. A square piece of homologous pericardium preserved in a 98% glycerin solution was rehydrated, dried, and sutured over the defect. Recovery of the animals was uneventful, with no signs of abdominal discomfort and no significant changes in physiologic variables, intestinal motility, behavior, appetite, or defecation pattern. The same was true for the erythrogram (packed cell volume, red cell count, and hemoglobin concentration), leukogram, and plasma fibrinogen values. The animals were reoperated on through the ventral midline approach after 4, 5, 6, 7, 8, and 35 weeks, respectively, and the implants were inspected visually and collected for microscopic study. No adhesions were found. Initially, there was a rim of fibrotic tissue covering the suture line, which regressed over time and was no longer evident after 7 weeks. From that moment on, progressive integration of the implant to the intestinal serosa was observed. Histopathologic examination revealed a late healing process, with infiltration of mononuclear cells, fibroblasts, neovascularization, and mature collagen fibers, which increased in intensity up to 6 weeks and then progressively regressed. At 5 weeks, it was already difficult to distinguish between the implant and the intestinal serosa. Our conclusion was that homologous pericardium implants have good acceptance in intestinal serosal lesions in horses and offer a good perspective for clinical application.     

Evaluation of the repair process of cartilage defects of the equine third carpal bone with and without subchondral bone perforation

To determine the effect of subchondral bone drilling (forage) on the cartilage repair process after injury has occurred, a cartilage defect (1 cm in diameter) was created on the radial facet of the proximal surface of each third carpal bone in 6 adult horses. In one of the third carpal bones (right or left thoracic limb) of each horse, a 1-cm cartilage defect was created, and 5 holes (1 mm in diameter and 10 mm deep) were drilled through the subchondral bone into the cancellous bone. In the other thoracic limb, an identical defect was created, but not drilled. Analyses of cell numbers and types in the synovia and the mucin precipitate quality were done before, at 1 week after, and 3 weeks after surgical manipulation was done and showed no significant difference between the joint environment of drilled carpi and those of nondrilled carpi. At 21 weeks after surgical manipulation was done, each joint was examined radiographically, macroscopically, and microscopically to compare the condition of the joints and the state of repair of the cartilage in each defect. The amount of surface of the defect covered by the dense fibrous and fibrocartilagenous repair tissue and the thickness of the repair tissue were significantly greater (P less than 0.05 and P less than 0.01, respectively) in the drilled carpal bones. In addition, the attachment of the repair tissue to underlying chondro-osseous tissue was better in the drilled carpal bones. Fibrocartilage was resurfacing the drilled defects, whereas only fibrous tissue was present in the nondrilled defects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrous tissue of subchondral cystic lesions in horses produce local mediators and neutral metalloproteinases and cause bone resorption in vitro

OBJECTIVES: To define the release of nitric oxide (NO), prostaglandin E2 (PGE2), and the neutral metalloproteinases (NMPs) in horses with subchondral cystic lesions (SCL) and to study bone resorption triggered by conditioned media of fibrous tissue of SCL in vitro. STUDY DESIGN: Equine explant cultures of fibrous tissue of SCL, and synovial membrane and articular cartilage of normal horses and horses affected with moderate and severe osteoarthritis were performed. NO, PGE2, and NMP concentrations of media samples were measured, and osteoclast formation and activation was studied in vitro. ANIMALS: Experiment 1: 32 horses with SCL (n = 8), normal joints (7), and joints with moderate (7) and severe (10) osteoarthritis (OA). Experiment 2: 22 horses with SCL (n = 3), normal joints (7), and chip fractures (12). Experiment 3: Conditioned media of fibrous tissue from 3 horses with SCL of the medial femoral condyle (n = 1), distal metacarpal bone (1), and tarsal bone (1). METHODS: Determinations of local mediator concentrations were made with the Griess assay for NO and an enzyme immunoassay kit for PGE2 concentrations in biological fluids. Enzyme activities were assessed with radiolabeled substrates indicating collagenolytic, gelatinolytic, and caseinolytic activities. The resorption pit assay was used to assess osteoclast recruitment and activity. RESULTS: Fibrous tissue of SCL produced NO, PGE2, and NMPs. Of all the variables measured, PGE2 concentrations were the highest in cystic tissue of SCL compared with synovial membrane and articular cartilage from normal joints and joints with chip fractures, indicating that this mediator may play an important role in pathological bone resorption associated with SCL. These findings were supported by the observation that conditioned media of SCL tissue were capable of recruiting osteoclasts and increasing their activity. CONCLUSION: Fibrous tissue of SCL released NO, PGE2, and NMPs into the culture media. It is suspected that intralesional fibrous tissue may play an active role in the pathological process of bone resorption occurring in SCL in horses and may be partly responsible for the maintenance, slow healing rate, and expansion of these lesions. CLINICAL RELEVANCE: Understanding the pathogenesis of SCL will help to establish successful therapy in horses affected with SCL.     

Effect of ranitidine on gastric acid secretion in young male horses

Gastric cannulas were placed surgically in 5 young male horses. After a 2-week recovery period, horses were studied once a week. Horses were fasted for 24 hours, and gastric fluid output was collected for 5 continuous hours. Volumes were recorded every 15 minutes, and pH and hydrogen ion concentration were determined in an aliquot from each period. In 10 basal experiments, using 5 horses, volume, pH, and hydrogen ion concentration were continuously variable. Mean acid output was 45.1 +/- 2.02 microEq/15 min/kg (mean +/- SEM). In 6 experiments, using 3 horses, 0.5 mg of ranitidine/kg of body weight, given as an IV bolus after a 1-hour basal collection, significantly (P less than 0.02) inhibited hourly total acid output for 4 hours, but did not significantly change pH. The cannulation technique was done without complications, and horses tolerated the cannula for several months. Seemingly, the horse has a continuously variable gastric acid secretion, and histamine type-2 receptors have a role in this process.     

Implantation of canine umbilical cord blood-derived mesenchymal stem cells mixed with beta-tricalcium phosphate enhances osteogenesis in bone defect model dogs

This study was performed to evaluate the osteogenic effect of allogenic canine umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) mixed with beta-tricalcium phosphate (β-TCP) in orthotopic implantation. Seven hundred milligrams of β-TCP mixed with 1 × 10⁶ UCB-MSCs diluted with 0.5 ml of saline (group CM) and mixed with the same volume of saline as control (group C) were implanted into a 1.5 cm diaphyseal defect and wrapped with PLGC membrane in the radius of Beagle dogs. Radiographs of the antebrachium were made after surgery. The implants were harvested 12 weeks after implantation and specimens were stained with H&E, toluidine blue and Villanueva-Goldner stains for histological examination and histomorphometric analysis of new bone formation. Additionally, UCB-MSCs were applied to a dog with non-union fracture. Radiographically, continuity between implant and host bone was evident at only one of six interfaces in group C by 12 weeks, but in three of six interfaces in group CM. Radiolucency was found only near the bone end in group C at 12 weeks after implantation, but in the entire graft in group CM. Histologically, bone formation was observed around β-TCP in longitudinal sections of implant in both groups. Histomorphometric analysis revealed significantly increased new bone formation in group CM at 12 weeks after implantation (p < 0.05). When applied to the non-union fracture, fracture healing was identified by 6 weeks after injection of UCB-MSCs. The present study indicates that a mixture of UCB-MSCs and β-TCP is a promising osteogenic material for repairing bone defects.     

Use of magnetic resonance imaging for identifying subchondral bone damage in horses: 11 cases (1999-2003)

OBJECTIVE: To assess the use of magnetic resonance (MR) imaging for identifying subchondral bone damage in the distal limbs of horses. DESIGN: Retrospective study. ANIMALS: 11 horses. PROCEDURE: Medical records of horses with lameness and subsequent evidence of subchondral bone damage as determined by MR imaging were reviewed. Severity and duration of lameness, results of diagnostic local anesthesia and diagnostic testing, surgical and necropsy findings, and treatment were recorded. Outcome was determined by follow-up information obtained from the owner or referring veterinarian. RESULTS: Lameness was localized by physical examination and diagnostic local anesthesia. Lameness was localized to the metacarpophalangeal or metatarsophalangeal joint in 4 horses, distal interphalangeal joint in 5 horses, and tarsocrural joint in 2 horses. The duration of lameness ranged from 2 weeks to 20 months. Magnetic resonance imaging of the affected joints revealed abnormal fluid accumulation within the subchondral bone. None of the abnormalities observed by MR imaging were detected by radiography. Subchondral bone damage was diagnosed in all horses. Arthroscopy of the affected joint was performed in 4 horses. Communication with the articular surface of the affected bone was suspected on the basis of results of MR imaging in 4 horses and was confirmed by arthroscopy in 1 horse and by necropsy in 1 horse. CONCLUSIONS AND CLINICAL RELEVANCE: Magnetic resonance imaging was useful for providing a diagnosis when other imaging techniques did not definitively identify the cause of lameness. Subchondral bone damage was clearly identified by MR imaging and should be considered as a cause of lameness in horses in which radiographic findings are unremarkable.