Supraglenoid tubercle fractures repair with transverse locking compression plates in 4 horses.

OBJECTIVE: To report on a series of 4 horses with supraglenoid tubercle fractures repaired with locking compression plates. STUDY DESIGN: Case series. ANIMAL: Four horses ranging in age from 6 weeks to 20 months and weighing from 121 to 425 kg with supraglenoid tubercle fractures of 1 day to 6 weeks in duration. METHODS: Supraglenoid tubercle fractures were reduced and stabilized with transversely positioned locking compression plate(s) with and without additional tension band wiring. RESULTS: All fractures reached bony union. Two postoperative surgical site infections were managed with drainage and antibiotherapy. Three of the 4 horses continued onto athletic careers including flat racing, dressage, and hunter/jumper competition. The remaining horse was lame for a prolonged period, but was sound at 4 years. CONCLUSIONS: The application of one or two, transversely positioned LCPs should be considered for the repair of SGT fractures because of the relative ease of the technique, and its elimination of a biceps brachii tenotomy. All screws can be inserted in a lateral to medial direction without transection or drill penetration of the biceps brachii tendon. CLINICAL RELEVANCE: SGT fractures of various durations can be repaired in a wide range of horses with transversely positioned LCPs, and allow return to athletic function.

Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.

REASONS FOR PERFORMING STUDY: Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. OBJECTIVES: The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. STUDY DESIGN: Randomised in vivo experiment. METHODS: We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. RESULTS: Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken ß-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. CONCLUSIONS: The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the distribution pattern of transduction.

Microdialysis measurements of lamellar perfusion and energy metabolism during the development of laminitis in the oligofructose model.

REASONS FOR PERFORMING STUDY: Failure of lamellar energy metabolism, with or without ischaemia, may be important in the pathophysiology of sepsis-associated laminitis. OBJECTIVES: To examine lamellar perfusion and energy balance during laminitis development in the oligofructose model using tissue microdialysis. STUDY DESIGN: In vivo experiment. METHODS: Six Standardbred horses underwent laminitis induction using the oligofructose model (OFT group) and 6 horses were untreated controls (CON group). Microdialysis probes were placed in the lamellar tissue of one forelimb (all horses) as well as the skin dermis of the tail in OFT horses. Dialysate and plasma samples were collected every 2 h for 24 h and concentrations of energy metabolites (glucose, lactate, pyruvate) and standard indices of energy metabolism (lactate to glucose ratio [L:G] and lactate to pyruvate ratio [L:P]) determined. Microdialysis urea clearance was used to estimate changes in tissue perfusion. Data were analysed nonparametrically. RESULTS: Median glucose concentration decreased to <30% of baseline by 8 h in OFT lamellar (P = <0.01) and skin (P<0.01) dialysate. Lactate increased mildly in skin dialysate (P = 0.04) and plasma (P = 0.05) but not lamellar dialysate in OFT horses. Median pyruvate concentration decreased to <50% of baseline in OFT lamellar dialysate (P = 0.03). A >5-fold increase in median L:G compared with baseline occurred in OFT lamellar and skin dialysate (P<0.03). From a baseline of <20, median L:P increased to a peak of 80 in OFT skin and 38.7 in OFT lamellar dialysates (P<0.02); however, OFT lamellar dialysate L:P was not significantly different from CON. Urea concentration decreased significantly in OFT lamellar dialysate (increased urea clearance) but not in OFT skin or CON lamellar dialysate. CONCLUSIONS: Increased lamellar perfusion occurred during the development of sepsis-associated laminitis in the oligofructose model. Glucose concentrations in the lamellar interstitium decreased, suggesting increased glucose consumption but there was no definitive evidence of lamellar energy failure.

The effect of weightbearing and limb load cycling on equine lamellar perfusion and energy metabolism measured using tissue microdialysis.

REASONS FOR PERFORMING STUDY: Lamellar perfusion is thought to be affected by weightbearing and limb load cycling; this may be critical in the development of supporting limb laminitis. OBJECTIVES: To document the effects of unilateral weightbearing and altered limb load cycling on lamellar energy metabolism and perfusion. STUDY DESIGN: Randomised, controlled (within subject), experimental trial. METHODS: Nine Standardbred horses were instrumented with microdialysis probes in the foot lamellar tissue and skin (over the tail base). Urea (20 mmol/l) was added to the perfusate. Samples were collected every 15 min for a 1 h control period, then during periods of unilateral weightbearing (opposite limb held off the ground for 1 h); enhanced static limb load cycling (instrumented limb lifted every 10 s for 30 min); reduced limb load cycling activity (i.v. detomidine sedation) and continuous walking (30 min). Dialysate concentrations of glucose, lactate, pyruvate and urea were measured and lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios were calculated. For each intervention, values were compared with baseline using nonparametric statistical testing. RESULTS: Lamellar dialysate glucose increased and L:G decreased significantly during enhanced static limb load cycling. Glucose and pyruvate increased, and L:G, L:P and urea decreased significantly during walking. Simultaneous skin dialysate values did not change significantly. There were no significant dialysate changes during unilateral weightbearing or after detomidine administration, but only the latter resulted in a significant decrease in limb load cycling frequency. CONCLUSIONS: Increases in limb load cycling frequency (particularly walking) caused dialysate changes consistent with increased lamellar perfusion. Unilateral weightbearing (1 h) and a sedation-induced reduction in limb load cycling frequency did not have a detectable effect on lamellar perfusion. More research is needed to confirm the role of hypoperfusion in supporting limb laminitis, but strategies to increase limb load cycling may be important for prevention.

Microdialysis measurements of equine lamellar perfusion and energy metabolism in response to physical and pharmacological manipulations of blood flow.

REASONS FOR PERFORMING STUDY: A suitable method for evaluating lamellar perfusion changes and their metabolic consequences is currently lacking. OBJECTIVES: To examine perfusion changes in lamellar tissue using serial microdialysis measurements of urea clearance and energy metabolites. STUDY DESIGN: Randomised, controlled (within subject) experimental trial. METHODS: Nine Standardbred horses were instrumented with microdialysis probes in the foot lamellar tissue and skin (over the tail base). Urea (20 mmol/l) was added to the perfusate and its clearance was used to estimate local perfusion. Samples were collected every 15 min for a 1 h control period, then during application of a distal limb tourniquet, during periods when norepinephrine or potassium chloride (KCl) were included in both skin and lamellar perfusates, and after systemic (intravenous) acetylpromazine. Dialysate concentrations of glucose, lactate, pyruvate and urea were measured and lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios calculated. Values were compared with pre-intervention baseline and also between simultaneous skin and lamellar samples using nonparametric statistical methods. RESULTS: Lamellar glucose decreased and lactate, urea, L:G and L:P increased significantly with tourniquet application, without significant changes in skin dialysate values. Lamellar and skin glucose decreased and L:G increased significantly during norepinephrine infusion, but mild increases in urea were not significant at either site. KCl caused significant decreases in lamellar and skin L:G, and an increase in skin glucose, but did not affect urea clearance. Acetylpromazine caused profound decreases in lamellar glucose and L:P, with increased L:G and pyruvate, but did not affect urea clearance or any skin dialysate values. CONCLUSIONS: Significant changes in microdialysis urea clearance only occurred with severe lamellar hypoperfusion. However, changes in dialysate metabolite concentrations reflected less profound fluctuations in perfusion. This method may be useful for examining lamellar perfusion and energy balance during laminitis development and for the evaluation of vasoactive therapeutics.

Equine lamellar energy metabolism studied using tissue microdialysis.

Failure of lamellar energy metabolism may contribute to the pathophysiology of equine laminitis. Tissue microdialysis has the potential to dynamically monitor lamellar energy balance over time. The objectives of this study were to develop a minimally invasive lamellar microdialysis technique and use it to measure normal lamellar energy metabolite concentrations over 24 h. Microdialysis probes were placed (through the white line) into either the lamellar dermis (LAM) (n = 6) or the sublamellar dermis (SUBLAM) (n = 6) and perfused continuously over a 24 h study period. Probes were placed in the skin dermis (SKIN) for simultaneous comparison to LAM (n = 6). Samples were collected every 2 h and analysed for glucose, lactate, pyruvate, urea and glycerol concentrations. LAM was further compared with SUBLAM by simultaneous placement and sampling in four feet from two horses over 4 h. Horses were monitored for lameness, and either clinically evaluated for 1 month after probe removal (n = 4) or subjected to histological evaluation of the probe site (n = 10). There were no deleterious clinical effects of probe placement and the histological response was mild. Sample fluid recovery and metabolite concentrations were stable for 24 h. Glucose was lower (and lactate:glucose ratio higher) in LAM compared with SUBLAM and SKIN (P < 0.05). Pyruvate was lower in SUBLAM than SKIN and urea was lower in LAM than SKIN (P < 0.05). These differences suggest lower perfusion and increased glucose consumption in LAM compared with SUBLAM and SKIN. In conclusion, lamellar tissue microdialysis was well tolerated and may be useful for determining the contribution of energy failure in laminitis pathogenesis.

Scapulohumeral osteochondrosis. A retrospective study of 32 horses.

OBJECTIVES: To review the outcome of equine shoulder osteochondrosis (OC) with surgical or conservative treatment. METHODS: Retrospective study of 32 horses, less than two years of age, with scapulohumeral joint (SHJ) OC. The lesion severity was graded based on measurements taken from lateromedial radiographs. Follow-up information was obtained from racing records or telephone conversations with owners. Successful outcome was defined as the ability of the horse to perform its intended use. RESULTS: Sixteen of 32 horses were affected bilaterally (48 joints). Eleven of 16 horses with bilateral OC underwent arthroscopic surgery; five had bilateral arthroscopy, six had unilateral arthroscopy of the most severely affected joint. Eight of 16 horses with unilateral OC underwent arthroscopy. The overall outcome was 'poor'. Only 15.4% (4/26) of potential race horses started a race, whereas 67% (4/6) non-racehorses were 'sound' for the intended use. Statistical analysis evaluating the effect of breed on outcome showed a statistically significant difference. There were no significant interactions between outcome and gender, affected limb, unilateral versus bilateral involvement, treatment or severity of the radiographic lesion. However, radiographic lesion severity on the humerus and glenoid showed significant positive correlation. CLINICAL SIGNIFICANCE: The overall poor prognosis for shoulder OC in young horses appears to be, in part, dependent on breed and intended use. There was not any difference in outcome between surgically and conservatively treated horses.

Clinical use of the locking compression plate (LCP) in horses: a retrospective study of 31 cases (2004-2006).

REASONS FOR PERFORMING STUDY: Fracture repair and arthrodesis in horses require rigid fixation in order to achieve early post operative comfort. The locking compression plate (LCP) was designed to allow greater stability and less periosteal disruption than conventional plates. OBJECTIVE: To describe a series of clinical cases in which the LCP was used for fracture stabilisation or arthrodesis. METHODS: Medical records, radiographs and follow-up for 31 horses having fracture stabilisation or arthrodesis with the LCP were reviewed. Reasons for treatment included fracture of the olecranon (n = 2), metatarsal III (n = 2), ilial shaft (n = 1), femur (n = 2), radius (n = 2), medial condylar (n = 5) and complex lateral condylar (n = 1); or arthrodesis of the metacarpophalangeal joint (n = 8), proximal interphalangeal joint (n = 8) and carpus (n = 2). RESULTS: Of the 31 horses, 27 were discharged from the hospital. Complications included incisional infection (32%), implant infection (19%), implant loosening/ breakage (22%), contralateral limb laminitis (16%), colic (3%) and diarrhoea (3%). Follow-up was available on all horses with at least 6 weeks of follow-up ranging up to 25 months. Of the 31 horses, 25 are sound for intended purpose, one is lame and 5 were subjected to euthanasia for complications associated with the original injury. CONCLUSIONS: The LCP is an acceptable means of fracture stabilisation and arthrodesis in the horse. POTENTIAL RELEVANCE: The LCP should be considered for difficult fracture configurations and arthrodeses, especially when increased stability of the fixation is needed.

Minimally invasive plate fixation of lower limb injury in horses: 32 cases (1999-2003).

REASONS FOR PERFORMING STUDY: Complications of osteosynthesis including superficial and deep infections continue to affect adversely outcome in equine surgery of the lower limb. OBJECTIVE: To describe a technique for minimally invasive plate fixation in horses and evaluate clinical results of the technique. HYPOTHESIS: Lower complication rates would occur in horses treated with a minimally invasive approach compared with conventional techniques. METHODS: Cases of minimally invasive plate fixation from January 1999 to December 2003 were evaluated. Case records, radiographs and race records for horses that sustained distal third metacarpal/metatarsal (Mc/tIII) condylar fractures were assessed. Follow-up information was obtained and results analysed. In addition, results for horses that received arthrodesis by a conventional, open approach during the same period were evaluated and statistical analysis was performed. RESULTS: Thirty-two cases of minimally invasive plate fixation were identified (22 incomplete distal Mc/tIII condylar fractures with a spiralling or diaphyseal component, 10 metacarpophalangeal arthrodeses and 4 pastern arthrodeses). For both types of arthrodesis, surgery times and infection rates were lower and survival rates higher in the minimally invasive group, but no statistical difference was found. Spiralling condylar fractures were successfully managed, without catastrophic fracture. CONCLUSIONS AND POTENTIAL RELEVANCE: Minimally invasive percutaneous plate osteosynthesis (MIPPO) can be successfully used for select applications in horses, and may reduce morbidity and mortality in equine fracture repair.

Effect of age at presentation on outcome following arthroscopic debridement of subchondral cystic lesions of the medial femoral condyle: 85 horses (1993--2003).

REASONS FOR PERFORMING STUDY: Subchondral cystic lesions of the medial femoral condyle (SCMFC) are well documented in horses < or =3 years; arthroscopic debridement or enucleation of the cyst is currently the surgical treatment of choice. However, studies of occurence and outcome following surgery in older horses are lacking. OBJECTIVE: To identify factors important in outcome for horses with SCMFC treated by arthroscopic debridement. HYPOTHESIS: Age of horse at presentation has a significant influence on return to work following arthroscopic treatment for SCMFC. Clinical and diagnostic findings are also significant with respect to prognosis. METHODS: A retrospective review of medical records from 6 equine referral centres identified 85 horses that underwent arthroscopic debridement of SCMFC. Clinical examination, radiographic and arthroscopic findings were analysed together with follow-up data. Univariable analysis and multivariable logistic regression models were used to determine factors affecting return to soundness. Event-time analysis was performed to evaluate return to work. RESULTS: Older horses (>3 years) were less likely to return to soundness (P = 0.02) or to work (P = 0.04) than younger horses (< or = 3 years). Of 39 horses age 0-3 years, 25 (64%, 95% CI 49-79%) returned to soundness. Of 46 horses age >3 years, 16 (35%,95% CI 21-49%) returned to soundness. In addition, cartilage damage at sites other than the SCMFC negatively affected prognosis (P = 0.05). The hospital where treatment was performed had no influence on return to soundness. CONCLUSIONS: Older horses carry a worse prognosis for both return to soundness and return to work. POTENTIAL RELEVANCE: It is important for clients to be made aware of the difference in outcome between age groups.

Mechanical effects of high density polyethylene dynamic compression plate hole inserts on bone-plate constructs.

This study was conducted in order to investigate the mechanical effects of high density polyethylene screw hole inserts in 4.5 mm Dynamic Compression Plate (DCP)--synthetic bone constructs. A mid-shaft 'osteotomy' was created in synthetic bone cylinders. The bisecting 'osteotomy' was reduced using six-hole broad DCPs and 4.5 mm cortical bone screws. The screws adjacent to the 'osteotomy' were placed using a load-guide. The remaining screws were placed in neutral position. High density polyethylene DCP screw hole inserts were incorporated with each screw in neutral position, in the experimental group. The bone plate constructs were tested in four point cyclical bending with the plates loaded at 2,000 Newtons, for a total of 6,000 cycles. Osteotomy gap was measured at 3,000 and 6,000 cycles. Screw head deflection adjacent to the osteotomy was measured. Kruskal-Wallis non-parametric testing was used for statistical comparisons. There was significantly less gapping at the osteotomy site in the treatment group after 3,000 cycles (0.49 +/- 0.18 mm [control] vs. 0.06 +/- 0.14 mm [treated], P=0.02) and 6,000 cycles (0.6 +/- 0.18 mm [control] vs. 0.1 +/- 0.22 mm [treated], P=0.02). The screws adjacent to the gap were significantly more deformed in the control group than those in the treated constructs (3.63 +/- 1.81 [control] vs. 1.06 +/- 1.55 [treated], P=0.0002). The polyethylene inserts improved the interface between bone plate and screw head, resulting in decreased relative movement of the implant and bone. The polyethylene inserts also resulted in less bending of the loaded screws.

Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes.

OBJECTIVE AND DESIGN: To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture. MATERIAL OR SUBJECTS: Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses. TREATMENT: Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10(-10) to 10(-4) M. METHODS: Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Northern blots of total RNA from the treated cells were probed with equine specific cDNA probes for a number of cartilage matrix-related genes. Zymography, Western blotting, and fluorography were also performed to study the effects on protein synthesis. RESULTS: The glucocorticoids, dexamethasone, triamcinolone, and prednisolone, markedly decreased MMP1, MMP3, MMP13, TIMPI, and ferritin steady-state mRNA levels. There were no qualitative differences seen among the tested corticosteroids although dexamethasone and triamcinolone appeared to be slightly more potent than prednisolone. The effects of the glucocorticoids on MMP transcription occurred consistently at lower doses than those required to similarly downregulate type II collagen and aggrecan. Link protein and fibronectin mRNA were increased by the glucocorticoids, and biglycan and decorin were minimally affected. Fluorography of [14-C] proline-labeled media demonstrated that the decrease in type II collagen transcription (mRNA levels) was paralleled at the protein level. Zymography and Western blotting confirmed the decrease in functional metalloproteinases found in chondrocyte cultures following glucocorticoid treatment. CONCLUSIONS: The effects of glucocorticoids are complex inasmuch as they differentially affect numerous genes involved in the composition of cartilage matrix and the degradation of that matrix. This study provides new insight into the effects of glucocorticoids on the regulation of extra-cellular matrix and matrix-related genes by demonstrating that low doses of glucocorticoids can inhibit the degradative metalloproteinases with minimal negative effects on the transcription of extracellular matrix genes.

Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes.

Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.

Characterization of a biomaterial with cartilage-like properties expressing type X collagen generated in vitro using neonatal porcine articular and growth plate chondrocytes.

OBJECTIVE: The availability of cartilage with or without the potential to ossify and suitable for surgical restoration and resurfacing of joints is an important clinical problem in arthritis-related pathology, trauma and reconstructive surgery. Here, we designed experiments to generate a biomaterial with cartilage-like properties by culturing neonatal porcine articular and growth plate chondrocytes on a hydrogel substrate and to examine the biochemical and histological characteristics of the resulting tissue. DESIGN: Neonatal porcine epiphyseal and growth plate chondrocytes were cultured on poly(2-hydroxyethyl methacrylate) (polyHEMA)-coated dishes to prevent their adherence to plastic. We previously described that this procedure allows the maintenance of the chondrocyte-specific phenotype for > or = 8 months. Chondrocytes were isolated by successive enzymatic digestions and cultured at high density (>2.0 x 10(7) cells/ml) in DMEM with 10% FBS, 50 microg/ml ascorbic acid, glutamine, vitamins, and antibiotics for up to 10 weeks on 60 mm plastic culture dishes coated with polyHEMA. The tissues produced during culture were studied histologically and biochemically and were examined for cellular proliferation employing(3)H-thymidine incorporation and for their collagen production employing biosynthetic labeling with(14)C-proline and Western blot with specific antibodies. The expression of relevant collagen genes was examined employing RT-PCR. RESULTS: Within 24 h of culture, isolated chondrocytes organized into well-formed clusters and in 2 weeks formed structures with gross appearance and consistency similar to those of natural cartilage. The wet weight of the tissue formed in vitro increased six-fold during the 10-week period of study. Cell proliferation measured by the incorporation of(3)H-thymidine increased during the first 3 weeks and reached a plateau in subsequent weeks. Histological examination showed that the cultures contained rounded chondrocytes embedded in an abundant cartilaginous extracellular matrix. The cartilage formed contained large amounts of collagen and sulfated proteoglycans as examined by staining with Masson's Trichrome and Alcian blue, respectively. Deposition of calcium in the deeper layers of the tissue was demonstrated with the von Kossa stain. Western analyses with specific antibodies showed that type II collagen was present from the first week and progressively increased in the cultures, whereas type X collagen was first detected at 4 weeks and increased with length of culture. When chondrocytes isolated from the growth plate were included, small amounts of type I collagen were detected in the medium of cultured biomaterial as expected. Type III collagen was not detected by Western blot over the 10-week period. High levels of type II and type X collagen gene expression were demonstrated by RT-PCR. CONCLUSION: These studies demonstrate the production in vitro of cartilage-like tissue with similar morphological, histochemical and biochemical characteristics to those of natural growth plate cartilage. The cartilage generated in vitro has the potential to be used in reconstructive surgery and in joint resurfacing and restoration of skeletal defects.

Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage.

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes.

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.

Post operative performance of racing Standardbreds treated arthroscopically for carpal chip fractures: 176 cases (1986-1993).

A retrospective study of 176 Standardbred horses that had arthroscopic surgery for carpal chip fractures over a 7 year period was done in order to determine fracture location and post operative performance. Chip fractures of the proximal third carpal bone and the distal radiocarpal bone occurred with equal frequency (49.2 and 49.6% respectively), and chip fractures involving the antebrachiocarpal joint were rare. Trotters had significantly more third carpal bone lesions than radiocarpal bone lesions when compared to pacers (P = 0.0304). Seventy-four per cent of horses made at least one start following surgery. Pacers were significantly more likely to have at least one start after surgery than were trotters (P = 0.0009). Median earnings per start significantly decreased after surgery (P = 0.0081), while median race mark significantly increased after surgery (P = 0.0100). It is concluded that the majority of Standardbreds will be useful racehorses following carpal arthroscopy; however, most earn less money per start and many race at a lower class.

Cecocolic and cecocecal intussusception in horses: 30 cases (1976-1996).

OBJECTIVE: To determine the prognosis in horses with cecocolic or cecocecal intussusception. DESIGN: Retrospective study. ANIMALS: 30 horses with cecocolic intussusception or cecocecal intussusception. PROCEDURE: Information on history, physical examination findings, and laboratory values was summarized from the medical records. Laboratory data included results of hematologic examination, serum biochemical analysis, and peritoneal fluid color, total nucleated cell count, and total protein concentration. A one-year follow-up via the telephone was used to determine long-term survival. RESULTS: Horses ranged from 7 months to 30 years old, but 63% were < or = 3 years. Standardbred horses were significantly overrepresented. Twenty-six horses had acute-to-subacute disease, and 4 had a chronic wasting disease. Cecal intussusceptions were suspected on the basis of finding a mass on abdominal palpation per rectum (14 of 24 horses) and positive ultrasonographic findings (2 of 3 horses). Thirteen horses with colic for > 1 day had scant, soft feces. Six horses died or were euthanatized without undergoing surgery, and 24 were treated surgically. Six of the latter horses were euthanatized during surgery because of peritonitis, rupture of the cecum, and irreducible intussusception. All 4 horses with a chronic disease were euthanatized because of irreversible changes in the cecum. Of the 18 horses allowed to recover from surgery, 15 survived long-term. Surgical treatments were reduction, with or without partial typhlectomy (6 horses), partial typhlectomy through a colotomy and reduction (6), reduction through a colotomy and partial typhlectomy (3), partial typhlectomy for a cecocecal intussusception (1) and an ileocolostomy (2). CLINICAL IMPLICATIONS: Cecal intussusception has a good prognosis with surgical correction without delay. Reduction through colotomy has a high success rate. Bypass by ileocolostomy should be used as a last resort.

Molecular characteristics of equine stromelysin and the tissue inhibitor of metalloproteinase 1.

OBJECTIVE: To clone the entire coding sequence of equine matrix metalloproteinase-3 (MMP-3, stromelysin) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and compare their nucleotide and amino acid sequences with those of MMP-3 and TIMP-1 from other species. SAMPLES: Articular cartilage harvested from the joints of 4 foals, 2 yearlings, and 3 adult horses. PROCEDURE: A cDNA library was constructed from mRNA extracted from equine chondrocytes. The library was screened and clones selected that contained the cDNA for MMP-3 and TIMP-1. The cDNA was sequenced and the nucleotide and deduced amino acid sequences compared with known sequences in other species. Northern blot analysis was performed, using the resulting cDNA clones. RESULTS: An 1803-bp cDNA for MMP-3 including the entire coding sequence of 1434 bases was cloned and sequenced. A 744-bp cDNA for TIMP-1 including the entire coding sequence of 624 bases was cloned and sequenced. Northern analysis revealed MMP-3 to hybridize to a single mRNA species at approximately 2.1 kb. TIMP-1 hybridized to a single mRNA species at approximately 0.8 kb. CONCLUSIONS: MMP-3 and TIMP-1 were highly homologous to that of other species at the nucleotide and amino acid level although each had unique residues in part of the peptide that is generally conserved. CLINICAL RELEVANCE: Understanding the molecular structure of MMP-3 and TIMP-1 and the availability of their cDNA should allow a more detailed understanding of their balance in cartilage and the degradative processes in joint disease.