Objective—To investigate the density of the primary epidermal lamellae (PEL) around the solar circumference of the forefeet of near-term fetal feral and nonferal (ie, domesticated) horses. Sample—Left forefeet from near-term Australian feral (n = 14) and domesticated (4) horse fetuses. Procedures—Near-term feral horse fetuses were obtained from culled mares within 10 minutes of death; fetuses that had died in utero 2 weeks prior to anticipated birth date and were delivered from live Thoroughbred mares were also obtained. Following disarticulation at the carpus, the left forefoot of each fetus was frozen during dissection and data collection. In a standard section of each hoof, the stratum internum PEL density was calculated at the midline center (12 o'clock) and the medial and lateral break-over points (11 and 1 o'clock), toe quarters (10 and 2 o'clock), and quarters (4 and 6 o'clock). Values for matching lateral and medial zones were averaged and expressed as 1 density. Density differences at the 4 locations between the feral and domesticated horse feet were assessed by use of imaging software analysis. Results—In fetal domesticated horse feet, PEL density did not differ among the 4 locations. In fetal feral horse feet, PEL density differed significantly among locations, with a pattern of gradual reduction from the dorsal to the palmar aspect of the foot. The PEL density distribution differed significantly between fetal domesticated and feral horse feet. Conclusions and Clinical Relevance—Results indicated that PEL density distribution differs between fetal feral and domesticated horse feet, suggestive of an adaptation of feral horses to environment challenges.

Bilateral osteochondral defects (10 mm2 X 3 mm deep) were created on the distal articular surface of the radial carpal bone of ten, 2- to 3-year-old horses. One defect of each horse was repaired, using a sternal cartilage autograft (treated), and the other was left untreated (control). The horses were exercised on a high-speed treadmill at incrementally increased speed and duration over the course of 12 months. Horses were evaluated arthroscopically at 6 to 7 weeks, and clinical examinations were conducted weekly at exercise. Twelve months after surgery, carpuses of each horse were radiographed and clinically examined prior to euthanasia. A gross pathologic evaluation of each joint was conducted, and samples were collected for histologic, histochemical, histomorphometric, and biochemical evaluation. Radiographically, the grafted joints had more extensive evidence of arthropathy, and clinically, 8 of the 10 horses were more lame in the grafted limb. On the basis of histomorphometry, the repair tissue of the grafted defects contained a greater median percentage of hyaline cartilage (45%) than that of control defects 4.5%), and the control defects contained a greater percentage of fibrocartilage (82%) than did grafted defects (28.5%). A greater median percentage of repair tissue stained with safranin-O in the grafted defects (24.5%) than in the control defects (3.5%). On gross pathologic and histologic evaluation, repair tissue of the control defects had better continuity and was more firmly attached to the subchondral bone than was repair tissue of the grafted defects. Repair tissue of the grafted defects had extensive fissure and flap formation. Histologically, subchondral bone reactivity and fibroplasia was extensive in grafted joints. Repair tissue of grafted defects had a greater percentage of type II collagen (mean sem, 83.5 +/- 2.95%) than did controls (mean, 79.4 3.87%) that was not statistically significant. Hexosamine content was significantly higher (P < 0.05) in repair tissue of the grafted defect (mean, 28.9 +/- 3.00 mg/g of dry weight) vs control (mean, 20.6 +/- 1.85 mg/g of dry weight). On the basis of this experimental model, sternal cartilage autografts cannot be recommended at this time for repair of osteochondral defects in athletic horses.

Keratan sulfate (KS) is a glycosaminoglycan, distribution of which is confined mostly to hyaline cartilage. As such, it is a putative marker of hyaline cartilage catabolism. In experiment 1, a focal osteochondral defect was made arthroscopically in 1 radial carpal bone of 2 ponies, and in 2 other ponies, chymopapain was injected into the radiocarpal joint to induce cartilage catabolism. Sequential and concurrent plasma and synovial fluid concentrations of KS were measured, up to 13 months after induction of cartilage injury, to determine whether changes in KS concentrations reflected cartilage catabolism. In experiment 2, a large, bilateral osteochondral defect was made in the radial carpal bones of 18 ponies, which were subsequently given postoperative exercise and/or injected intra-articularly with 250 mg of polysulfated glycosaminoglycan (PSGAG). Medication was given at surgery, then weekly for 4 weeks. Blood samples were collected and synovial fluid was aspirated before surgery, when medication was given, and at postmortem examination (postoperative week 17). The KS concentration was measured in these fluids to determine whether changes in KS concentration indicated an effect of joint treatment. In experiment 1, the concentration of KS in synovial fluid was highest 1 day after joint injury, and the concentration in plasma peaked 2 days after joint injury. For ponies receiving chymopapain intra-articularly (generalized cartilage catabolism), a fivefold increase over baseline was observed in the concentration of KS in plasma (peak mean, 1.2 microgram/ml), and a tenfold increase over baseline in synovial fluid (peak mean, 2.0 mg/ml) was observed. On average, these maxima were threefold higher than values in fluids of ponies with osteochondral defects (focal cartilage disease). In experiment 2, nonexercised ponies had lower KS concentration (as a percentage of the preoperative concentration) in synovial fluid than did exercised ponies at all postoperative times, and at postoperative week 17, this effect was significant (P < 0.05). This may be related to decreased turnover of KS in articular cartilage attributable to stall confinement and late increase in turnover related to exercise. Seventeen weeks after surgery, synovial fluid from exercised, medicated ponies had significantly (P < 0.05) higher KS content than did fluid from exercised, nonmedicated ponies. This indicated that exercise, when combined with medication, may increase KS release from articular cartilage. Synovial fluid from medicated joints of nonexercised ponies had significantly (P < 0.05) lower KS concentration than did synovial fluid from nonmedicated joints of nonexercised ponies. This indicated that, in nonexercised joints, medication with PSGAG may have decreased either release of KS from the articular cartilage into the synovial fluid or inhibited synthesis of KS. Concentration of KS in synovial fluid was not related clearly to the development of osteoarthritis in these ponies. Exercise or medication did not affect plasma KS concentration, and synovial fluid and plasma KS concentrations were not correlated. Data indicated that KS concentration in plasma and synovial fluid may be increased in acute, marked, generalized articular cartilage catabolism and that KS turnover in cartilage of joints with large osteochondral defects was affected by intra-articular PSGAG and postoperative exercise.

Periosteal autograft 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. Total glycosaminoglycan content, galactosamine and glucosamine contents, and galactosamine-to-glucosamine ratio of grafted and nongrafted defects were all significantly (P < 0.05) less than corresponding values in normal equine articular cartilage. By contrast, total collagen content of neocartilaginous tissues of grafted and nongrafted defects was greater than that of normal articular cartilage, although the difference was not significant. The proportion of type-I and type-II collagens in repair tissue in grafted and nongrafted defects was 70 and 30%, respectively. The fibrous nature of the repair tissue reported in a companion morphologic and histochemical study was substantiated by the biochemical results. We concluded that use of periosteal autograft did not improve the healing of osteochondral defects.

The third carpal bone (C3) was collected from both forelimbs of 27 Thoroughbreds. On the basis of age, training, and history, specimens were assigned to 1 of 5 groups: yearling, untrained horses (group 1, n = 4); 2- to 3-year-old, untrained horses (group 2, n = 7); trained 2-year-old horses (group 3, n = 6); trained 3-year-old horses (group 4, n = 6); and 3-year-old, trained horses with carpal pathologic features (group 5, n = 4). A transverse section of subchondral bone 5-mm thick was cut in a precise fashion 10 mm below the proximal articular surface of all specimens. After high-detail radiography was done, indentation testing was performed on the proximal surface of the section at points 5 mm apart. The stiffness of the subchondral cancellous bone was determined from the slope of the load vs displacement curve. Topographic plots of stiffness measurements were compared with radiographs of each specimen. Point determinations were averaged to derive measures for the radial and intermediate facets, and for regions 5, 10, 15, and 20 mm from the dorsal margin of C3. Area fraction (1-p; p = porosity) was measured for the radial and intermediate facets, using an automated image analysis system. Significant (P < 0.05) increases in stiffness and area fraction were found in the C3 from trained horses (groups 3 to 5), compared with untrained horses (groups 1 to 2). Stiffness and area fraction of the radial facet of pathologic C3 were significantly higher than the same variables measured in C. from any other group. A typical profile of regional subchondral stiffness was identified in C3 from normal horses, with maximal stiffness measured 10 mm from the dorsal articular margin. A different pattern was found in pathologic C3, with significantly greater stiffness 15 and 20 mm from the dorsal articular margin when compared with normal horses. A highly significant (P < 0.0001) direct linear correlation between stiffness and area fraction at the radial facet was found. Topographic and radiographic analysis demonstrated good correlation between stiffness and radiographic density of the bone sections. The observed patterns of normal and pathologic C3 were contrasted. In particular, a large gradient in sub-chondral stiffness was identified in pathologic C3 at the dorsomedial aspect of the bone.

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 approximatively 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 autograft 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). It was concluded that use of periosteal autograft did not improve the healing of osteochondral defects of the distal portion of the radial carpal bone. The repair tissue produced in grafted and nongrafted defects was similar and was principally fibrous in nature.

Using arthroscopic technique, identical diameter defects were created in the proximal articular surface of both intermediate carpal bones of 6 horses. One of each pair of defects was deepened to penetrate the subchondral plate. Removed cartilage was assayed for [35S] sulfate incorporation, total hexosamine content, and DNA content. Six weeks later, cartilage was harvested and similarly analyzed from the distolateral portion of the radius directly opposite the created lesions and the distomedial portion of the radius distant from the lesion. The repair tissue filling the full-thickness defect and the cartilage at the periphery of the partial-thickness lesion also were analyzed. There was a marked increase in synthetic activity (35S sulfate incorporation) opposite the full-thickness defect, compared with the cartilage opposite the partial-thickness defect. A marked decrease in glycosaminoglycan content in the cartilage opposite the full-thickness defect was found as compared with that opposite the partial-thickness defect. The repair tissue filling the full-thickness defect was highly cellular, high in synthetic activity, but low in glycosaminoglycan content. Insignificant changes occurred in the cartilage adjacent to the partial-thickness defect. On the basis of these results, we suggest that full-thickness defects at 6 weeks result in more detrimental change to the cartilage opposite it than do partial-thickness lesions of the same diameter.

Objective- To evaluate the feasibility and repeatability of in vivo measurement of stiffness gradients by means of acoustoelastography in the superficial digital flexor tendons (SDFTs) of clinically normal horses. Animals- 15 clinically normal horses. Procedures- For each horse, stiffness gradient index and dispersion values for SDFTs in both forelimbs were evaluated in longitudinal orientation by use of acoustoelastography at 3 sites (5, 10, and 15 cm distal to the accessory carpal bone) by 2 observers; for each observer, data were acquired twice per site. The left forelimb was always scanned before the right forelimb. Lifting of the contralateral forelimb with the carpus flexed during image acquisition resulted in the required SDFT deformation in the evaluated limb. Interobserver repeatability, intraobserver repeatability, and right-to-left limb symmetry for stiffness gradient index and dispersion values were evaluated. Results- Stiffness gradient index and dispersion values for SDFTs at different locations as well as effects of age or sex did not differ significantly among the 15 horses. Interclass correlation coefficients for interobserver repeatability, intraobserver repeatability, and limb symmetry revealed good to excellent agreement (intraclass correlation coefficients, > 0.74). Conclusions and Clinical Relevance- Results indicated that acoustoelastography is a feasible and repeatable technique for measuring stiffness gradients in SDFTs in clinically normal horses, and could potentially be used to compare healthy and diseased tendon states.

Three doses of sodium monoiodoacetate (MIA) were used to induce degenerative changes in articular cartilage in middle carpal joints of horses. Twelve young (2- to 5-year-old) horses, free of lameness, were randomly allotted to 3 groups. One middle carpal joint of each horse was injected with 0.9% NaCl solution (control joint). The contralateral middle carpal joint was injected with 0.09 mg of MIA/kg of body weight (group 1); 0.12 mg(kg (group 2); or 0.16 mg(kg (group 3). After MIA administration, horses were allowed ad libitum exercise in a 2-acre paddock for 12 weeks. At the end of the study, gross and microscopic tissue changes were evaluated and biochemical analyses of articular cartilage were done. Grossly, diffuse partial-thickness articular cartilage lesions were observed in group-2 (n = 2) and group-3 (n = 4) horses, but not in group-1 horses. Articular cartilage uronic acid content was significantly (P < 0.03) decreased in all MIA-injected joints, compared with controls. Articular cartilage matrix staining with safranin-O was decreased in 3 of 4 MIA-injected joints of group-1 horses and in all MIA-injected joints of group-2 and group-3 horses, compared with controls (P < 0.06). Microscopic degenerative changes in articular cartilage were not significantly different between MIA-injected and control joints in group-1 horses, but were increased (P < 0.06) in all MIA-injected joints of group-2 and group-3 horses, compared with controls. Qualitatively, decreased matrix staining and degenerative changes were more severe in group-3 horses. On the basis of articular cartilage gross and microscopic changes, as well as biochemical changes, 0.12 mg of MIA/kg injected intra-articularly was determined to induce moderate degrees of articular cartilage degeneration. This model of chemically induced articular cartilage injury could be useful for evaluating treatment effects of anti-arthritic drugs 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 nongrafted 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 < 0.05). Repair tissue of all defects was characterized by galactosamine-to-glucosamine ratio significantly (P < 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.