Cloning and expression of type III collagen in normal and injured tendons of horses

OBJECTIVE: To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses. ANIMALS: 14 healthy adult horses. PROCEDURE: The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides were assessedby gel electrophresis. RESULTS: Type III collagen mRNA expression and protein content increased immediately after injury and remained increased. Type III collagen was localized to the endotenon in normal tendon and in injured tendon at 1 week. At 8 and 24 weeks, expression became more widely distributed throughout the tendon parenchyma. Injured tendon contained 6 times more type I than type III collagen mRNA. Quantities of type III collagen protein were maximal in the first 4 weeks after injury (approx 33%) and then began to decrease. CONCLUSIONS AND CLINICAL RELEVANCE: Type III collagen expression is increased initially in endotenon and subsequently in parenchyma of healing tendon; however, type III remains the minor collagen throughout the healing process. The role of type III collagen in tendon healing is not fully elucidated.     

Healing of surgically created defects in the equine superficial digital flexor tendon: effects of pulsing electromagnetic field therapy on collagen-type transformation and tissue morphologic reorganization

The effect of pulsing electromagnetic field (PEMF) therapy on the healing of surgically created defects in equine superficial digital flexor tendons was evaluated. Defects were created in both front superficial digital flexor tendons of 20 horses. The defect in 1 limb was exposed to a PEMF for 2 hours daily. The other limb served as a control. Histologic and immunofluorescent evaluations were done in horse killed at postsurgical weeks 2, 4, 8, 12, and 24. Therapy with the PEMF significantly (P less than 0.05) delayed the maturation of the tissue formed within the defect at postsurgical weeks 8 and 12, as determined by histologic examination. The collagen-type transformation was also delayed by the PEMF therapy, but to a degree that was not significant.     

Arthroscopic Subchondral Bone Plate Microfracture Technique Augments Healing of Large Chondral Defects in the Radial Carpal Bone and Medial Femoral Condyle of Horses

OBJECTIVE: To evaluate the effect of arthroscopic subchondral bone microfracture on healing of large chondral defects in horses. STUDY DESIGN: Short- (4 months) and long-term (12 months) in vivo experimental chondral defect model. ANIMALS: 10 horses, aged 2 to 5 years. METHODS: Each horse had a 1 cm2 full-thickness chondral defect created in both radial carpal bones and both medial femoral condyles. One carpus and one femoral condyle of each horse had the subchondral bone plate under the defect perforated using an orthopedic awl. All horses were exercised, five horses were evaluated after 4 months and five horses after 12 months. Gross, histologic, and histomorphometric examination of defect sites and repair tissues was performed, as was collagen typing of the repair tissue. RESULTS: On gross observation a greater volume of repair tissue filled treated defects (74%) compared with control defects (45%). Histomorphometry confirmed more repair tissue filling treated defects, but no difference in the relative amounts of different tissue types was observed. There was an increased percentage of type II collagen in treated defects compared with control defects and evidence of earlier bone remodeling as documented by changes in porosity. CONCLUSIONS: In full-thickness chondral defects in exercised horses, treatment with subchondral bone microfracture increased the tissue volume in the defects and the percentage of type II collagen in the tissue filling the defects when compared to nontreated defects. CLINICAL RELEVANCE: No negative effects of the microfracture technique were observed and some of the beneficial effects are the basis for recommending its use in patients cases with exposed subchondral bone.     

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)     

Effect of adipose-derived nucleated cell fractions on tendon repair in horses with collagenase-induced tendinitis

OBJECTIVE: To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis. ANIMALS: 8 horses. PROCEDURES: Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed. RESULTS: Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons. CONCLUSIONS AND CLINICAL RELEVANCE: ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.     

Morphologic study of repair of induced osteochondral defects of the distal portion of the radial carpal bone in horses by use of glued periosteal autografts [corrected

The use of periosteal autografts to resurface osteochondral defects was investigated in 10 horses (2 to 3 years old), and the repair tissue was characterized morphologically. Middle carpal joint arthrotomies were made, and osteochondral defects were induced bilaterally on the distal articular surface of each radial carpal bone. Each defect measured approximately 1 cm2 and extended 3 mm into the subchondral bone plate. Residual subchondral bone plate of control and principal defects was perforated by drilling. A sterile fibrin adhesive was made by mixing a fibrinogen component and a thrombin component. A periosteal autograft was harvested from the proximal portion of the tibia and was glued onto the recipient osseous surface, with its cambium facing the joint cavity. Control defects were glued, but not grafted. Horses were walked 1 hour daily on a walker, starting at postoperative week 7 and continuing for 9 weeks. Sixteen weeks after the grafting procedure was done, carpal radiography was performed, after which horses were euthanatized. Quality of repair tissue of control and grafted defects was evaluated and compared grossly, histologically, and histochemically. Using a reticule, the proportions of various repair tissue types filling each defect were quantitated. Seven weeks after the grafting procedure was done, bilateral arthroscopy revealed synovial adhesions and marginal pannus formation in control and grafted defects. None of the autografts was found floating unattached within the respective middle carpal joints. At 16 weeks, the gross appearance of most grafted and nongrafted defects was similar, and repair was dominated by a fibrous pannus. In 4 grafted defects, bone had formed either concentrically within the defect or eccentrically in the fibrous adhesions between the defect and the joint margin. Histologically, all grafted and nongrafted defects were repaired similarly by infiltration of a mixture of fibrous tissue, fibrocartilage, and bone. Fibrous tissue was the predominant tissue in most defects and its mean proportion was 56 and 59% in the grafted and nongrafted defects, respectively. Fibrocartilaginous tissue in the deeper layers approximated 20%, and woven bone at the base of the defect was 20% in all defects. Histochemically, difference in staining for proteoglycans was not observed between grafted and nongrafted defects. Little remaining original periosteal graft tissue was evident at the defect sites. The only distinguishing feature of grafted defects was the presence of islands of bone formation either at the defect site (n = 2 horses), or in somewhat dorsally displaced tissue that was incorporated in fibrous adhesions (n = 2 horses).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of platelet-derived growth factor-BB on the metabolic function and morphologic features of equine tendon in explant culture

OBJECTIVE: To evaluate the effects of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) on the metabolic function and morphologic features of equine superficial digital flexor tendon (SDFT) in explant culture. Animals-6 euthanized horses (2 to 5 years old). METHODS: Forelimb SDFT explants were cultured for 6 days as untreated control specimens or treated with rhPDGF-BB (1, 10, 50, or 100 ng/mL of medium). Treatment effects on explant gene expression were evaluated via real-time PCR analysis of collagen type I, collagen type III, PDGF-A, and PDGF-B mRNA. Explants were assayed for total collagen, glycosaminoglycan, and DNA content; histologic changes were assessed via H and E staining and immunohistochemical localization of collagen types I and III. RESULTS: No morphologic or proliferative changes were detected in tendon explant sections. After high-dose rhPDGF-BB treatment, gene expression of collagen types I and III was increased and decreased, respectively. Expression of PDGF-A and PDGF-B mRNA was significantly increased at 24 hours, but later decreased to have few or negative autoinductive effects. Although PDGF gene expression waned after 48 hours of culture, collagen type I gene expression was significantly increased at 48 hours and reached peak value on day 6. Glycosaminoglycan and DNA content of explants were unchanged with rhPDGF-BB treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that rhPDGF-BB use may be of benefit in the repair of equine tendon, particularly through induction of collagen type I mRNA. Positive autoinductive effects of PDGF-BB in equine SDFT explants were detected early following culture medium supplementation, but these diminished with time.     

Comparison of carbon fibre and nylon suture for repair of transected flexor tendons in the horse

Carbon fibre-polylactic acid composites and monofilament non-absorbable suture material were compared for the repair of surgically transected superficial digital flexor tendons in 10 horses. All surgical wounds healed by first intention. The repaired tendons were enlarged, the carbon implanted tendons being larger than those sutured. The horses were killed at six, eight, 12,20 or 24 weeks. Greater fibrous thickening occurred in tendons repaired with carbon fibre, especially at 12 weeks postoperatively. Carbon fibre incited a greater histological response with macrophages, lymphocytes, plasma cells, eosinophils and fibroblasts. The fibrous tissue in the repair sites appeared to mature and the collagen to align at a similar rate irrespective of the method of repair. Only that tissue within and immediately surrounding the carbon bundles was immature at six months. There was minimal tendency for carbon filaments to separate and those that did were often surrounded by epithelioid macrophages forming a granuloma. Massive eosinophil concentrations were present between each granuloma. With each sequential test period the sutured tendons became increasingly stronger than the carbon implanted tendons. This may have been because of the immature core of tissue associated with the carbon bundles. No carbon particles were detected in draining lymph nodes.     

The effect of intralesional injection of bone marrow derived mesenchymal stem cells and bone marrow supernatant on collagen fibril size in a surgical model of equine superficial digital flexor tendonitis

Reasons for performing study: Collagen fibril size is decreased in repair tissue following tendon injury compared to normal tendon matrix in horses. Mesenchymal stem cells have been suggested to promote regeneration of tendon matrix rather than fibrotic repair following injury, although this concept remains unproven. Objectives: To explore the hypothesis that implantation of autologous mesenchymal stem cells derived from bone marrow into a surgically created central core defect in the superficial digital flexor tendon (SDFT) of horses would induce the formation of a matrix with greater ultrastructural similarities to tendon matrix than the fibrotic scar tissue formed in control defects. Methods: Tissue was collected 16 weeks after induction of injury and 12 weeks after treatment from normal and injured regions of control and treated limbs of 6 horses and examined using transmission electron microscopy. Collagen fibril diameters were measured manually with image analysis software and surface areas calculated. Three parameters assessed for normal and injured tissue were mass average diameter (MAD), collagen fibril index (CFI) and the area dependent diameter (ADD). Results: Normal regions from both treated and control limbs displayed higher MAD and CFI values, as well as a characteristic bimodal distribution in fibril size. Injured regions from both treated and control limbs displayed significantly lower MAD and CFI values, as well as a unimodal distribution in fibril size. There were no significant differences between treated and control limbs for any of the parameters assessed. Conclusions: Intralesional injection of autologous bone marrow derived mesenchymal stem cells had no measurable effect on the fibril diameter of collagen in healing tissue in the SDFT of this experimental model 16 weeks after injury. Potential relevance: Favouring matrix regeneration over fibrotic repair may not be the mechanism by which autologous mesenchymal stem cells assist healing of tendon injury.     

Effects of extracorporeal shock wave therapy on desmitis of the accessory ligament of the deep digital flexor tendon in the horse

Objective: To evaluate the effects of extracorporeal shock wave therapy (ESWT) on collagenase‐induced lesions in the accessory ligament of the deep digital flexor tendon (ALDDFT) of horses. Study Design: Paired, blinded controlled study. Animals: Eight Thoroughbred horses (3 mares, 5 geldings; mean±SD weight, 464±26 kg, mean age, 8±1.7 years). Methods: Lesions were created in both ALDDFTs of all horses by injection of 2 × 10³ IU of collagenase type I. Percent lesion and structure (fiber alignment and echogenicity) were quantified with ultrasonographic imaging 3, 6, and 9 weeks after collagenase injection. After ultrasound examinations, ESWT (1000 shocks at 0.15 mJ/mm²) was applied to 1 ALDDFT in each horse. ALDDFT were harvested 15 weeks after collagenase injection and the microstructure, mRNA levels of collagen types I and III, and collagen and glycosaminoglycan content were evaluated. Results: There were no differences in percent lesion, echogenicity, or fiber alignment between control‐ and ESWT‐treated ligaments at each evaluation time; however, compared with 3‐week values, there was a significant increase in percent lesion and echogenicity for EWST treated ligaments at 6 weeks and significant decrease in both variables for treated and control ligaments at 12 weeks. Fiber alignment improved significantly at 9 weeks in controls and at 12 weeks in treated and control ligaments. Collagen type I mRNA levels were significantly higher in the ESWT treatment group compared with the control group 15 weeks after collagenase injection though differences in other mRNA levels, microstructure, and composition were not significant. Conclusions: Our results do not support an effect of ESWT on collagenase‐induced lesions in the equine ALDDFT.     

Tissue strength and wound morphology of the equine linea alba after ventral median celiotomy

OBJECTIVE: To evaluate the tensile strength and wound morphology of the equine linea alba at intervals over 6 months after ventral median celiotomy. STUDY DESIGN: Linea alba tensile strength and wound morphology were determined at 2, 4, 8, 16, and 24 weeks postoperatively. Linea alba samples from 3 unoperated horses were used as controls. ANIMALS: Eighteen adult horses, weighing 400 to 500 kg, 4 to 15 years old. METHODS: Tensile strength and thickness of incised linea alba samples collected at 2, 4, 8, 16, and 24 weeks after ventral median celiotomy were compared with control linea alba samples. Additional samples were subjectively evaluated for wound morphology, notably collagen morphology. RESULTS: Control linea alba had a mean (+/-SEM) tensile strength of 484.9 +/- 58.3 N and was significantly (P < or = .05) stronger than at 2 weeks (87.7 +/- 61.4 N) after surgery. The tensile strength of 4-week (305.8 +/- 61.7 N), 8-week (465.4 +/- 56.5 N), and 16-week (477.8 +/- 57.2 N) samples were not significantly different from control linea alba. At 24 weeks, the tensile strength (721.0 +/- 57.9 N) was significantly stronger than control. The 2-, 4-, and 8-week samples were significantly thicker than controls, whereas the 16- and 24-week samples were not different from controls. On microscopy, control samples were characterized by dense mature collagen bundles. At 2 weeks, samples consisted primarily of granulation tissue, whereas at 4 weeks, samples had immature collagen fibers that were not formed into bundles, and at 8, 16, and 24 weeks, all samples had abundant mature collagen fibers formed into bundles. CONCLUSIONS: At 8 weeks, incised and sutured equine linea alba had a tensile strength comparable with non-incised linea alba and was characterized by mature collagen. CLINICAL RELEVANCE: Based on tensile strength and wound morphology, horses that have had an uncomplicated recovery after ventral median celiotomy should be able to return to controlled exercise as early as 60 days after surgery.     

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.     

The effect of age on serum antibody titers after rabies and influenza vaccination in healthy horses

BACKGROUND: The proportion of geriatric horses within the equine population has increased in the past decade, but there is limited information on the immune function of these animals. HYPOTHESIS: Aged horses will have a lesser increase in serum antibody response to vaccination. ANIMALS: Thirty-four aged healthy horses (> or = 20 years) and 29 younger adult horses (4-12 years) of various breeds. METHODS: All horses were vaccinated with vaccines of killed rabies and influenza virus. Horses in each age group were allocated to receive either rabies or influenza booster vaccine 4 weeks after the initial vaccination. Serum samples were taken at 0, 4, 8, and 24 weeks. Rabies serum neutralization titers and equine influenza virus specific antibody sub-isotypes (IgGa, IgGb, IgG(T), and IgA) as well as single radial hemolysis (SRH) titers were determined. RESULTS: Rabies antibody titers were similar in the 2 age groups at all sampling times. Aged horses had higher IgGa and IgGb influenza antibody titers before vaccination than younger horses but similar titers after vaccination (P= .004 and P= .0027, respectively). Younger horses had significantly greater increases in titer than aged horses at all sampling times for IgGa (P= .001) and at 8 and 24 weeks for IgGb (P= .041 and .01, respectively). There was no detectable serum IgG(T) at any time point. A significant booster vaccine effect was seen for both antirabies and anti-influenza titers. Anti-influenza titer before vaccination also had a significant effect on subsequent antibody response. CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy aged horses generated a primary immune response to a killed rabies vaccine similar to that of younger adult horses. Aged horses had a significantly reduced anamnestic response to influenza vaccine.     

Proportion of types I and III collagen in longissimus collagen from bulls and steers

The proportion of types I and III intramuscular collagen in longissimus muscles of Simmental bulls (n = 8) and steers (n = 8) 17 mo of age was studied. Longissimus samples taken 7 d after slaughter were evaluated for total collagen, types I and III collagen, heat-soluble collagen, sensory panel traits and Warner-Bratzler shear force. Intramuscular collagen (IMC) was isolated and digested with cyanogen bromide, and peptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Percentage of type III IMC was calculated from the total of types I and III collagen as determined from the peak area of densitometric scans of the cyanogen bromide peptides alpha 1(I)CB8 and alpha 1(III)CB8. Longissimus muscles from steers had lower (P less than .05) Warner-Bratzler shear values, less (P less than .05) sensory panel-detectable connective tissue and more (P less than .05) tender panel ratings for muscle fiber tenderness and overall tenderness. Muscles from steers had more (P less than .05) heat-soluble collagen than those from bulls, but no differences (P greater than .05) were found for total collagen and percentage of type III collagen. Some intramuscular-collagen characteristics may have contributed to the less tender muscle of bulls. However, the proportion of types I and III collagen did not account entirely for the tenderness difference between steer and bull muscles. Because there were differences in collagen solubility in muscles from steers and bulls, other collagen characteristics such as crosslinking or fiber size may have been more important than collagen type.     

Cell therapy for tendinitis, experimental and clinical report

To compare cultured bone marrow mesenchymal cells (cBMSC), bone marrow mononucleated cells (BMMNCs), and placebo to repair collagenase-induced tissue damage in an equine model of experimental tendonitis, 6 Standardbred horses with no signs of previous SDF tendon injury have been recruited. Three weeks after collagenase treatment an average of either 5.5 x 10(6) cBMSCs or 122.3 x 10(6) BMMNCs, saline solution (placebo) or fibrin glue were injected intralesionally in random order. Horses were stall rested for 21 weeks, and tendon ultrasound scans performed before and during this period. Horses were euthanized and tendons harvested for histology and immunohistochemistry. Data observed in this study showed effectiveness of cBMSC and BMMNC in regenerating tendon tissue after collagenase -induced tendonitis. Both cBMSC and BMMNC transplantation resulted in qualitatively similar regeneration of tendon extracellular matrix in terms of type I/III collagen ratio, fiber orientation, and COMP expression. After this favourable results, 20 horses were recruited referred for spontaneous lesions of the flexor tendons or the suspensory ligament. Horses were treated with autologous graft of BMMNCs.After treatment the. the exercise program allowed was 8 weeks stall rest, 4 weeks hand walking, 4 weeks trotting, 4 weeks of gradually raising of exercise level then horses were gone back to race. US characteristics of lesions started to improve at T3. CSA-l, FPS and TLS were better in all patients, with an appreciable filling of lesions indicated by a decreasing of CSA-l and increasing of TLS. When horses started the exercise program T8 tendon architecture improved, demonstrated by their longitudinal alignment and length. At T6, and persistently in later follow-up, no lameness was evident by clinical examination. At time of writing 12 patients (60%) were go back to races, while other 8 (40%) are under controlled exercise program. Re-injury rate was assessed at 25%. All the owners judged good to excellent the outcome in term of athletic success.     

Biochemical study of repair of induced osteochondral defects of the distal portion of the radial carpal bone in horses by use of periosteal autografts

Periosteal autografts were used for repair of large osteochondral defects in 10 horses aged 2 to 3 years old. In each horse, osteochondral defects measuring 1.0 x 1.0 cm2 were induced bilaterally on the distal articular surface of each radial carpal bone. Control and experimental defects were drilled. Periosteum was harvested from the proximal portion of the tibia and was glued into the principal defects, using a fibrin adhesive. Control defects were glued, but were not grafted. Sixteen weeks after the grafting procedure, the quality of the repair tissue of control and grafted defects was assessed biochemically. Total collagen content and the proportion of type-II collagen were determined. Galactosamine and glucosamine contents also were determined. From these measurements, contents of chondroitin and keratan sulfate and total glycosaminoglycan, and galactosamine-to-glucosamine ratio were calculated. All biochemical variables were compared with those of normal equine articular cartilage taken from the same site in another group of clinically normal horses. Total collagen content was determined on the basis of 4-hydroxyproline content, using a colorimetric method. The proportions of collagen types I and II in the repair tissue were assessed by electrophoresis of their cyanogen bromide-cleaved peptides on sodium dodecyl sulfate slab gels. Peptide ratios were computed and compared with those of standard mixtures of type-I and type-II collagens. Galactosamine and glucosamine contents were determined by use of ion chromatography. In general, the biochemical composition of repair tissue of grafted and nongrafted defects was similar, but clearly differed from that of normal articular cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphologic and biochemical study of sternal cartilage autografts for resurfacing induced osteochondral defects in horses.

Using biodegradable pins, sternal cartilage autografts were fixed into osteochondral defects of the distal radial carpal bone in ten 2 to 3-year-old horses. The defects measured 1 cm2 at the surface and were 4 mm deep. Control osteochondral defects of contralateral carpi were not grafted. After confinement for 7 weeks, horses were walked 1 hour daily on a walker for an additional 9 weeks. Horses were euthanatized at 16 weeks. Half of the repair tissue was processed for histologic and histochemical (H&E and safranin-O fast green) examinations. The other half was used for the following biochemical analyses: type-I and type-II collagen contents, total glycosaminoglycan content, and galactosamine-to-glucosamine ratio. On histologic examination, the repair tissue in the grafted defects consisted of hyaline-like cartilage. Repair tissue in the nongrafted defects consisted of fibrocartilaginous tissue, with fibrous tissue in surface layers. On biochemical analysis, repair tissue of grafted defects was composed predominantly of type-II collagen; repair tissue of non-grafted defects was composed of type-I collagen. Total glycosaminoglycan content of repair tissue of grafted defects was similar to that of normal articular cartilage. Total glycosaminoglycan content of nongrafted defects was 62% of that of normal articular cartilage (P less than 0.05). Repair tissue of all defects was characterized by galactosamine-to-glucosamine ratio significantly (P less than 0.05) higher than that of normal articular cartilage. These results at 16 weeks after grafting indicate that sternal cartilage may potentially constitute a suitable substitute for articular cartilage in large osteochondral defects of horses.     

Immunologic responses to West Nile virus in vaccinated and clinically affected horses

OBJECTIVE: To compare neutralizing antibody response between horses vaccinated against West Nile virus (WNV) and horses that survived naturally occurring infection. DESIGN: Cross-sectional observational study. ANIMALS: 187 horses vaccinated with a killed WNV vaccine and 37 horses with confirmed clinical WNV infection. PROCEDURE: Serum was collected from vaccinated horses prior to and 4 to 6 weeks after completion of an initial vaccination series (2 doses) and 5 to 7 months later. Serum was collected from affected horses 4 to 6 weeks after laboratory diagnosis of infection and 5 to 7 months after the first sample was obtained. The IgM capture ELISA, plaque reduction neutralization test (PRNT), and microtiter virus neutralization test were used. RESULTS: All affected horses had PRNT titers > or = 1:100 at 4 to 6 weeks after onset of disease, and 90% (18/20) maintained this titer for 5 to 7 months. After the second vaccination, 67% of vaccinated horses had PRNT titers > or = 1:100 and 14% had titers < 1:10. Five to 7 months later, 33% (28/84) of vaccinated horses had PRNT titers > or = 1:100, whereas 29% (24/84) had titers < 1:10. Vaccinated and clinically affected horses' end point titers had decreased by 5 to 7 months after vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: A portion of horses vaccinated against WNV may respond poorly. Vaccination every 6 months may be indicated in certain horses and in areas of high vector activity. Other preventative methods such as mosquito control are warranted to prevent WNV infection in horses.     

Changes in molecular expression of aggrecan and collagen types I, II, and X, insulin-like growth factor-I, and transforming growth factor-beta1 in articular cartilage obtained from horses with naturally acquired osteochondrosis.

OBJECTIVE: To determine molecular changes in the expression of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) in horses with osteochondrosis, and to characterize expression of matrix aggrecan and collagen types I, II, and X in articular cartilage of affected joints. SAMPLE POPULATION: Articular cartilage from affected stifle or shoulder joints of 11 horses with naturally acquired osteochondrosis and corresponding joints of 11 clinically normal horses. PROCEDURE: Harvested specimens were snap frozen in liquid nitrogen, and total RNA was isolated. Specimens were fixed in 4% paraformaldehyde for histologic examinations. Expression of matrix molecules was assessed by analysis of northern blots and in situ hybridization, using equine-specific cDNA probes and riboprobes, respectively. Expression of IGF-I and TGF-beta1 was assessed by use of noncompetitive quantitative polymerase chain reaction, in situ hybridization, and immunohistochemical analysis. RESULTS: Cartilage obtained from osteochondrosis lesions had significantly greater expression of IGF-I, compared with normal cartilage. Expression of TGF-beta1 and collagen type I were higher, but not significantly so, in affected tissues. Expression of aggrecan or collagen types II and X did not differ between affected and clinically normal cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of growth factors and collagen type I was found in cartilage from osteochondrosis lesions. However, this probably reflects a healing response to injured tissue rather than a primary alteration. Therefore, methods aimed at altering concentrations of growth factors in cartilage of growing horses would be unlikely to alter the incidence or progress of the disease.     

Gunshot Fractures of Extremities: Classification, Management, and Complications

Fifteen clinical cases of gunshot fractures were studied. A radiographic classification of the fractures was developed to aid in fracture evaluation. Type I fractures involved a simple transverse or oblique fracture with minimal soft tissue damage. Type II fractures were severely comminuted with no cortical bone defect and minimal soft tissue damage. Type III fractures were "shatter" fractures, characterized by severe comminution, cortical bone defects, and extensive soft tissue damage. Type I and Type II fractures (combined 26.7%) healed in 8 weeks or less. Eleven of the 15 cases evaluated (73.3%) were found to be Type III fractures, requiring more than 14 weeks for complete cortical healing. Osteomyelitis was associated with 3 cases of Type III tibial fractures, 2 of which developed after a second surgical intervention. Management, complications, and prognosis of gunshot fractures are discussed.