Thoroughbreds are particularly susceptible to joint diseases1 that result in substantial wastage in the horse racing industry.1–3 Repetitive impact of the carpus during high-speed galloping generates higher amounts of stress in the dorsal and medial regions than in other regions of opposing joint surfaces on the radial facet of the third carpal bone and the radial carpal bone.4–7 High amounts of stress in these regions are associated with the forces of weight-bearing and muscle activity passing through a forelimb during part of the stance phase at a gallop when the carpus is in maximum extension or even hyperextension.7 Repetitive stresses, when applied over a prolonged period, can lead to localized degeneration of joint cartilage and bone4,7,8 and may be an important factor in the initiation and progression of osteoarthritis with associated SCB sclerosis and necrosis and increased risk of osteochondral fracture.9,10
Musculoskeletal tissues respond readily to training exercise through adaptive changes that can increase the physiologic threshold of safe function.11,12 Thus, exercise during early development in horses has been proposed as a means of increasing resistance to the forces that will be sustained during athletic training for racing, with a possible associated resistance to the onset of joint injury and disease.13
Intensity, duration, frequency, and other features of loading associated with exercise programs are known to be important determinants for adaptive tissue responses14–16 but may also be related to pathological changes, although details of exercise regimens associated with joint compromise remain unclear.17–19 Better understanding of the safe thresholds for exercise intensity may aid in raising athletic horses with increased musculoskeletal strength and enhanced resistance to exercise-related tissue injury, degeneration, or both.
The study reported here includes histologic characterization and analysis of defects found in the mid-carpal joints of a subset of Thoroughbreds enrolled in a large study20 in which musculoskeletal characteristics of horses that did or did not undergo a track-based conditioning exercise program from shortly after birth until 18 months of age were compared. In a previous study,13 we identified 6 common sites of defects on the ASs of these same midcarpal joints and reported types and prevalence of gross cartilage defects found therein. In one of those sites (the dorsal aspect of the radial facet of the third carpal bone), we also reported grossly visible focal whitening in the articular cartilage associated with greater cartilage matrix swelling, compared with that of samples from joint surfaces that did not have this abnormality.
The purpose of the study reported here was to determine the histologic characteristics of grossly detected abnormalities in common midcarpal joint defect sites and to quantify the osteochondral and vascular features of the dorsal aspect of the radial facet of the third carpal bone in Thoroughbreds that did or did not undergo an early conditioning exercise regimen. We hypothesized that the histologic appearance of the third and radial carpal bones and histomorphometric values determined for the third carpal bone would be significantly different between bones that had evidence of CC abnormalities and those that did not. We further hypothesized that early conditioning exercise would not influence the prevalence of these defects.
Materials and Methods
Sample—The midcarpal joints of 12 of 33 Thoroughbreds that were allocated to 1 of 2 groups (spontaneous exercise at pasture only [PASTEX] or with additional conditioning exercise [CONDEX] beginning shortly after birth21) were used in the present study. The subset of 12 horses (n = 6/group) had been euthanized at 18 months of age, and various tissues were used in numerous studies,13,21–26 including the evaluation of gross defects in the same carpal bones used in the present study. The study and its procedures were approved by the Massey University Animal Ethics Committee.
Exercise program—For approximately 18 months beginning at a mean age of 3 weeks, both groups of horses were kept on pasture, but horses in the CONDEX group were additionally exercised (1,030 m/d and 5 d/wk) on an oval track under controlled conditions described elsewhere.21 From 3 weeks of age to weaning (approx 120 days), the mean target speed for exercise was 5.4 m/s; from weaning to first sprint (approx 100 days), the mean target speed was 7.5 m/s; and for the remaining phase of exercise (approx 300 days), the mean target speed was 9.6 m/s with a brief 129-m sprint at 12.5 m/s. During the 18-month exercise period, lameness was not detected, but similar prevalences of mild (CONDEX, 29%; PASTEX, 25%) and moderate (CONDEX, 23%; PASTEX, 20%) effusion of the midcarpal joints were found during 1,132 evaluations in 33 horses.21
Sample collection and processing—Midcarpal joints (n = 24) were collected from 12 horses (6 CONDEX and 6 PASTEX) at the time of euthanasia (mean ± SD age, 535 ± 30 days); dissection, storage, visual examination, photography, and description of macroscopic cartilage defects on the midcarpal joint surfaces have been previously described.13 Detailed gross examination of cartilage defects on the midcarpal cartilage surfaces was performed and was enhanced by use of macroscopic photographs. In 6 common cartilage defect sites, the size and location of defects such as undulations, focal whitening, softening, fissures, and tissue loss were recorded.13
Defect sites—Cartilage defect sites selected for histologic analysis were a modification of those described in our previous study of gross lesions13 and were identified as follows: sites C3r_dorsal and Crr_dorsal were regions (size, approx 10 × 20 mm) centered midway between the most lateral and medial aspects of the dorsal rim of the radial facet of the third carpal bone and the corresponding opposing surface of the radial carpal bone, respectively (Figure 1). Site C3r_palmar (approx 10 × 20 mm) included portions of the radial and intermediate facets of the proximal surface of the third carpal bone close to the notch at the bone's palmar aspect. Site C3l_ridge (approx 5 × 20 mm) was located on the proximal surface of the lateral facet of the third carpal bone, palmar to the ridge adjoining the radial and intermediate facets. Site C2ridge (approx 10 × 20 mm) was located on the ridge between the lateral and medial facets of the proximal surface of the second carpal bone. Site Crridge (approx 10 × 20 mm) was located on the ridge between the lateral and medial facets of the distal surface of the radial carpal bone.
Schematic illustration of the locations of 6 common defect sites (C3r_dorsal, Crr_dorsal, C3r_palmar, C3l_ridge, C2ridge, and Crridge) on the AS of midcarpal joints in twelve 18-month-old Thoroughbreds that did (CONDEX group) or did not (PASTEX group) undergo a conditioning exercise regimen that began shortly after birth. Descriptive histologic assessment was conducted at all 6 sites; histomorphometric analysis was confined to site C3r_dorsal (asterisk). Boxes bordered by a dashed line indicate the sample collection site for tissues used to evaluate cartilage swelling in a previous study.13 C2 = Second carpal bone. C3 = Third carpal bone. C4 = Fourth carpal bone. Ci = Intermediate carpal bone. Cr = Radial carpal bone. Cu = Ulnar carpal bone.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Schematic illustration of the locations of 6 common defect sites (C3r_dorsal, Crr_dorsal, C3r_palmar, C3l_ridge, C2ridge, and Crridge) on the AS of midcarpal joints in twelve 18-month-old Thoroughbreds that did (CONDEX group) or did not (PASTEX group) undergo a conditioning exercise regimen that began shortly after birth. Descriptive histologic assessment was conducted at all 6 sites; histomorphometric analysis was confined to site C3r_dorsal (asterisk). Boxes bordered by a dashed line indicate the sample collection site for tissues used to evaluate cartilage swelling in a previous study.13 C2 = Second carpal bone. C3 = Third carpal bone. C4 = Fourth carpal bone. Ci = Intermediate carpal bone. Cr = Radial carpal bone. Cu = Ulnar carpal bone.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Schematic illustration of the locations of 6 common defect sites (C3r_dorsal, Crr_dorsal, C3r_palmar, C3l_ridge, C2ridge, and Crridge) on the AS of midcarpal joints in twelve 18-month-old Thoroughbreds that did (CONDEX group) or did not (PASTEX group) undergo a conditioning exercise regimen that began shortly after birth. Descriptive histologic assessment was conducted at all 6 sites; histomorphometric analysis was confined to site C3r_dorsal (asterisk). Boxes bordered by a dashed line indicate the sample collection site for tissues used to evaluate cartilage swelling in a previous study.13 C2 = Second carpal bone. C3 = Third carpal bone. C4 = Fourth carpal bone. Ci = Intermediate carpal bone. Cr = Radial carpal bone. Cu = Ulnar carpal bone.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Sample preparation—A 4-mm-thick osteochondral slab was cut from the opposing surfaces of the third carpal and radial carpal bones and used for cartilage swelling analysis in a previous study,13 and the remaining portion of each bone was stored frozen (–20°C) for approximately 6 months prior to histologic analysis (Figure 1). Osteochondral blocks were then cut from the 6 identified defect sites to include the defects and adjacent unaffected tissue; this resulted in various block sizes (approx 5 × 10 to 5 × 30 mm). The C3r_dorsal block (approx 5 × 20 mm) was immediately adjacent to the location of the slab cut for the previously described cartilage swelling analysis.13
The osteochondral blocks were fixed in 10% formaldehyde solution for 1 to 3 days, decalcified for 3 to 5 days in 10% formic acid solution buffered with sodium formate,27,28 rinsed in water for 1 hour, and mounted on a sledge microtome with a medium for frozen tissue specimensa and liquid nitrogen for cryosectioning. From each block, osteochondral sections (n = 10 to 30; thickness, 30 μm) were obtained in the sagittal plane over a span of approximately 3 mm; 3 to 5 sections were then selected for histologic examination and photo-micrographic (bright-field microscopy)b imaging.
The selected sections were gently washed with water at room temperature, wet-mounted on glass slides, examined at 20× magnification (unstained), and digitally photographed; 5 to 10 images of each section were obtained and then digitally merged to produce a high-resolution composite image of the entire cartilage-bone thick section.
Histologic assessment—Prepared osteochondral block sections were assessed histologically. Previously defined histopathologic features29 were used to evaluate alterations in the AS (discontinuities, surface undulations, and fibrillation), chondrocyte number and morphology (clustering, orientation, and hypertrophy), HC (fissures, erosion, delamination, and matrix texture or fibrosity), CC (tidemark continuity, duplication, and texture), OCJ integrity, SCB (microfractures, fibrocartilaginous repair, and sclerosis), and VC area and numbers. Defects of the C3l_ridge, C2ridge, and Crridge were classified subjectively as mild, moderate, or severe.
Histologic assessment of osteochondral sections of sites C3r_dorsal and Crr_dorsal was used to determine the presence or absence of CC abnormalities. Tissues without a CC abnormality were characterized by the following features: homogenous CC matrix texture; normal-appearing chondrocytes (1 to 5 cells radially arranged in a unit)30; absence of microcracks or fissures; consistency of CC layer thickness across the site; a uniformly undulating and well-defined OCJ; and VCs advancing up to, but not through, the OCJ. Histologic features of tissues with a CC abnormality included the following: heterogeneous appearance of the CC matrix, locally thickened CC, presence of microcracks or fissures in the CC, hypertrophic chondrocytes with obvious cell clustering, disruption of the OCJ, and widened VCs at the OCJ and in the SCB > 2 to 3 mm from the OCJ. A bone was categorized as having a CC abnormality if it had > 3 of these features.
Histomorphometric assessment—Each high-resolution composite image was overlaid with a 3,000 × 3,000-μm matrix grid that was prescaled to the microscope magnification (Figure 2). Within the grid, a pen-tablet digitizerc with image-editing softwared was used to hand-trace the boundaries of tissues of interest, which were then colorized (blue for HC, green for CC, and red for VC). Because VCs may participate in remodeling of the OCJ by advancement up to and into the CC,31–34 VCs were included only if they were observed to contact the OCJ or were within approximately 50 μm of the OCJ. The area of any VC intruding into the CC was also included. Data from the 5 previously selected osteochondral sections from each C3r_dorsal site were analyzed by use of image analysis softwaree to calculate mean thickness of the HC and CC and mean number and area of VCs over the length of the matrix grid (3,000 μm).
Images depicting the third carpal bone region selected for histomorphometric analysis. A—Composite photomicrograph of a dorsal-to-palmar sagittal section of the proximal aspect of the third carpal bone showing the location of the C3r_dorsal site. B—Colorized histomorphometric image of affected tissue in a bone with CC abnormalities at the C3r_dorsal site (inset of panel A; grid size, 3,000 × 3,000 μm). Analysis included measurement of HC (blue) and CC (green) thickness and area, VC area (red), and number of VCs in contact with the OCJ or located within approximately 50 μm of the OCJ (arrowheads).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Images depicting the third carpal bone region selected for histomorphometric analysis. A—Composite photomicrograph of a dorsal-to-palmar sagittal section of the proximal aspect of the third carpal bone showing the location of the C3r_dorsal site. B—Colorized histomorphometric image of affected tissue in a bone with CC abnormalities at the C3r_dorsal site (inset of panel A; grid size, 3,000 × 3,000 μm). Analysis included measurement of HC (blue) and CC (green) thickness and area, VC area (red), and number of VCs in contact with the OCJ or located within approximately 50 μm of the OCJ (arrowheads).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Images depicting the third carpal bone region selected for histomorphometric analysis. A—Composite photomicrograph of a dorsal-to-palmar sagittal section of the proximal aspect of the third carpal bone showing the location of the C3r_dorsal site. B—Colorized histomorphometric image of affected tissue in a bone with CC abnormalities at the C3r_dorsal site (inset of panel A; grid size, 3,000 × 3,000 μm). Analysis included measurement of HC (blue) and CC (green) thickness and area, VC area (red), and number of VCs in contact with the OCJ or located within approximately 50 μm of the OCJ (arrowheads).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Statistical analysis—Statistical analyses were performed by use of linear mixed modelingf to determine whether there were significant differences between bones grouped according to the presence or absence of CC abnormalities with respect to the following histomorphometric data: HC thickness, CC thickness, VC area, and VC number. Variables analyzed in the statistical model included limb (left or right) and section number (1 to 5), and the horses were considered to be randomly selected.
Because the variance in CC thickness was high in the CC abnormality group, a log transformation was applied initially. However, this failed to overcome unequal variances between the groups. A reciprocal transformation was then applied, which did balance the degrees of variance. Values of P < 0.05 were considered significant.
Results
The bones of 24 midcarpal joints from 12 horses were examined grossly. Histologic evaluation was performed for 18 of these joints from 5 PASTEX and 4 CONDEX horses (samples from 3 horses were unavailable).
Sites C3r_dorsal and Crr_dorsal—Gross examination of third and radial carpal bones revealed distinct focal whitening of the cartilage with a smooth AS in 7 bones and focal whitening with AS undulations at the affected site in 1 bone. Of 18 histologically examined bones, 9 (which included the 8 with focal whitening) had macroscopically visible and abruptly thickened CC layers (Figure 3). Three of these bones (1 third carpal and 2 radial carpal) were from 1 CONDEX horse, and 6 bones (3 third carpal and 3 radial carpal) were from 2 PASTEX horses.
Images depicting CC abnormalities at sites C3r_dorsal and Crr_dorsal. A—Macroscopic photograph of a sagittal section of the third and radial carpal bones in articular contact showing corresponding CC thickening at sites C3r_dorsal and Crr_dorsal (arrowheads). Bar = 5 mm. B—A composite photomicrograph of sections of the bones in panel A reveals details of the CC abnormality (arrowheads). Thickening of the CC (approx 6 mm long and 1 mm deep) is evident near the dorsoproximal aspect of the third carpal bone, and a similar but slightly smaller abnormality can be seen near the dorsodistal aspect of the radial carpal bone. Bar = 2 mm. C—Higher magnification of the transition from unaffected to affected CC (inset in panel B). Notice microcracks (arrowheads) and disruption of the OCJ in the region of the CC abnormality, compared with the unaffected adjacent tissue. Bar = 200 μm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Images depicting CC abnormalities at sites C3r_dorsal and Crr_dorsal. A—Macroscopic photograph of a sagittal section of the third and radial carpal bones in articular contact showing corresponding CC thickening at sites C3r_dorsal and Crr_dorsal (arrowheads). Bar = 5 mm. B—A composite photomicrograph of sections of the bones in panel A reveals details of the CC abnormality (arrowheads). Thickening of the CC (approx 6 mm long and 1 mm deep) is evident near the dorsoproximal aspect of the third carpal bone, and a similar but slightly smaller abnormality can be seen near the dorsodistal aspect of the radial carpal bone. Bar = 2 mm. C—Higher magnification of the transition from unaffected to affected CC (inset in panel B). Notice microcracks (arrowheads) and disruption of the OCJ in the region of the CC abnormality, compared with the unaffected adjacent tissue. Bar = 200 μm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Images depicting CC abnormalities at sites C3r_dorsal and Crr_dorsal. A—Macroscopic photograph of a sagittal section of the third and radial carpal bones in articular contact showing corresponding CC thickening at sites C3r_dorsal and Crr_dorsal (arrowheads). Bar = 5 mm. B—A composite photomicrograph of sections of the bones in panel A reveals details of the CC abnormality (arrowheads). Thickening of the CC (approx 6 mm long and 1 mm deep) is evident near the dorsoproximal aspect of the third carpal bone, and a similar but slightly smaller abnormality can be seen near the dorsodistal aspect of the radial carpal bone. Bar = 2 mm. C—Higher magnification of the transition from unaffected to affected CC (inset in panel B). Notice microcracks (arrowheads) and disruption of the OCJ in the region of the CC abnormality, compared with the unaffected adjacent tissue. Bar = 200 μm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Histologically, bones with and without CC abnormalities had intact ASs, and the texture of HC matrices was homogeneous with normal-appearing chondrocytes evenly spaced but obliquely aligned toward the dorsoproximal aspect of the third carpal bone or the dorsodistal aspect of the radial carpal bone (Figure 4). However, CC in bones with CC abnormalities was visibly thickened and had a substantially modified texture, compared with the smooth and amorphous appearance in bones without this defect (Figure 5).
Photomicrographs of sections of the third carpal bones from 2 horses revealing structural differences in the CC and OCJ of bones without (A) and with (B) CC abnormalities at site C3r_dorsal. Although HC morphology is similar, CC and OCJ morphologies differ substantially between these samples. A—Notice the intact CC (double-headed arrow) and intact OCJ (arrowhead). B—The affected CC (double-headed arrow) is greatly thickened and contains multiple parallel cracks (arrow); the OCJ is fragmented (arrowhead). Bars = 400 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of sections of the third carpal bones from 2 horses revealing structural differences in the CC and OCJ of bones without (A) and with (B) CC abnormalities at site C3r_dorsal. Although HC morphology is similar, CC and OCJ morphologies differ substantially between these samples. A—Notice the intact CC (double-headed arrow) and intact OCJ (arrowhead). B—The affected CC (double-headed arrow) is greatly thickened and contains multiple parallel cracks (arrow); the OCJ is fragmented (arrowhead). Bars = 400 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of sections of the third carpal bones from 2 horses revealing structural differences in the CC and OCJ of bones without (A) and with (B) CC abnormalities at site C3r_dorsal. Although HC morphology is similar, CC and OCJ morphologies differ substantially between these samples. A—Notice the intact CC (double-headed arrow) and intact OCJ (arrowhead). B—The affected CC (double-headed arrow) is greatly thickened and contains multiple parallel cracks (arrow); the OCJ is fragmented (arrowhead). Bars = 400 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of sections of the third carpal bone showing morphological differences in the CC matrix, OCJ, and chondrocytes at site C3r_dorsal between bones from horses without (A) and with CC abnormalities (B and C). The bottom row shows corresponding magnified views of insets in the top row. A—The unaffected CC has a smooth and amorphous matrix with columns of chondrocytes and a clearly defined OCJ with a VC (arrowhead). B—The affected CC matrix is markedly striated and heterogeneous in appearance (white and black arrowheads), and chondrocytes (asterisk) are difficult to detect; the OCJ is severely disrupted. C—In another type of affected CC, the matrix texture appears uneven and clusters of chondrocytes are present, with a severely fragmented OCJ. Bars = 50 μm (top row).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of sections of the third carpal bone showing morphological differences in the CC matrix, OCJ, and chondrocytes at site C3r_dorsal between bones from horses without (A) and with CC abnormalities (B and C). The bottom row shows corresponding magnified views of insets in the top row. A—The unaffected CC has a smooth and amorphous matrix with columns of chondrocytes and a clearly defined OCJ with a VC (arrowhead). B—The affected CC matrix is markedly striated and heterogeneous in appearance (white and black arrowheads), and chondrocytes (asterisk) are difficult to detect; the OCJ is severely disrupted. C—In another type of affected CC, the matrix texture appears uneven and clusters of chondrocytes are present, with a severely fragmented OCJ. Bars = 50 μm (top row).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of sections of the third carpal bone showing morphological differences in the CC matrix, OCJ, and chondrocytes at site C3r_dorsal between bones from horses without (A) and with CC abnormalities (B and C). The bottom row shows corresponding magnified views of insets in the top row. A—The unaffected CC has a smooth and amorphous matrix with columns of chondrocytes and a clearly defined OCJ with a VC (arrowhead). B—The affected CC matrix is markedly striated and heterogeneous in appearance (white and black arrowheads), and chondrocytes (asterisk) are difficult to detect; the OCJ is severely disrupted. C—In another type of affected CC, the matrix texture appears uneven and clusters of chondrocytes are present, with a severely fragmented OCJ. Bars = 50 μm (top row).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Within the CC matrix of bones with CC abnormalities, there were numerous microcracks, fissures, or both. The matrices were populated by mostly normal-appearing chondrocytes, with occasional cell clustering and hypertrophy (2/9 bones, from 1 CONDEX horse and 1 PASTEX horse; Figures 4 and 5). The severely disrupted OCJ in these bones contrasted with the undulating form of OCJ (associated with VC advancement) in bones without CC abnormalities. Whereas most VCs did not penetrate into the CC layer of bones with CC abnormalities, a small number did so through the damaged OCJ (Figure 6). Furthermore, adjacent to the thickened CC, there were wide curvilinear spaces deep within the SCB, interpeted as enlarged VCs (Figures 7 and 8).
Images of the OCJ at site Crr_dorsal in a radial carpal bone with a CC abnormality. A—Photomicrograph of a section at the affected site. The OCJ is indistinct with focal disruptions (asterisk), and the CC matrix is heterogeneous. A VC extends into the CC (arrowhead). B—Line tracings of the VC (solid line) and the OCJ (dashed line) in panel A. Bar = 100 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Images of the OCJ at site Crr_dorsal in a radial carpal bone with a CC abnormality. A—Photomicrograph of a section at the affected site. The OCJ is indistinct with focal disruptions (asterisk), and the CC matrix is heterogeneous. A VC extends into the CC (arrowhead). B—Line tracings of the VC (solid line) and the OCJ (dashed line) in panel A. Bar = 100 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Images of the OCJ at site Crr_dorsal in a radial carpal bone with a CC abnormality. A—Photomicrograph of a section at the affected site. The OCJ is indistinct with focal disruptions (asterisk), and the CC matrix is heterogeneous. A VC extends into the CC (arrowhead). B—Line tracings of the VC (solid line) and the OCJ (dashed line) in panel A. Bar = 100 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Composite photomicrograph of a radial carpal bone section with a CC abnormality at the Crr_dorsal site (arrowhead). Trabecular bone adjacent to the CC abnormality contains enlarged VCs (arrow). Bar = 1 mm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Composite photomicrograph of a radial carpal bone section with a CC abnormality at the Crr_dorsal site (arrowhead). Trabecular bone adjacent to the CC abnormality contains enlarged VCs (arrow). Bar = 1 mm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Composite photomicrograph of a radial carpal bone section with a CC abnormality at the Crr_dorsal site (arrowhead). Trabecular bone adjacent to the CC abnormality contains enlarged VCs (arrow). Bar = 1 mm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of third and radial carpal bone sections from the left (A) and right (B) midcarpal joints of 1 PASTEX horse. A—Severely widened VCs (arrowheads) are present in bones with CC abnormalities at the C3r_dorsal and Crr_dorsal sites. B—The CC of bones without these defects appears normal. Bars = 1 mm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of third and radial carpal bone sections from the left (A) and right (B) midcarpal joints of 1 PASTEX horse. A—Severely widened VCs (arrowheads) are present in bones with CC abnormalities at the C3r_dorsal and Crr_dorsal sites. B—The CC of bones without these defects appears normal. Bars = 1 mm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of third and radial carpal bone sections from the left (A) and right (B) midcarpal joints of 1 PASTEX horse. A—Severely widened VCs (arrowheads) are present in bones with CC abnormalities at the C3r_dorsal and Crr_dorsal sites. B—The CC of bones without these defects appears normal. Bars = 1 mm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Site C3r_palmar—Two bones from 1 CONDEX horse had 2 to 3 parallel cartilage fissures (2 to 3 mm long) and an elongated, raised bulla-type lesion was detected in 1 of these bones (Figure 9). In 2 bones from 1 PASTEX horse, regions of focal softening (1 mild and 1 severe) were detected at this site by means of gentle probing.
Photomicrographs of third carpal bone sections from 2 horses showing histopathologic features of 2 defects at the C3r_palmar site. A—A bulla detected in 1 CONDEX horse may have been induced by a small intercarpal gap between the apposing intermediate carpal and radial carpal bones; these appeared to articulate with the third carpal bone on either side of the bulla (arrowheads). B—Higher magnification of the HC (inset 1 in panel A) reveals small numbers of chondrocytes and a fibrous appearance (arrowhead) suggestive of fibrillar network destructuring. C—Higher magnification of the tidemark (inset 2 in panel A) shows a vague, almost feather-like appearance of this feature. D—Unaffected CC adjacent to the affected region in panel C. Notice the clearly delineated tidemark, more homogeneous appearance of the matrix, and distinct chondrocytes, compared with the affected tissue in panel C. E—A mild focally softened region detected in 1 CONDEX horse is shown. The AS is intact, but localized thickening of the HC and chondrocyte proliferation in both the HC and CC are evident (inset). The dashed line indicates the tidemark. F—A severe focally softened region detected in the contralateral third carpal bone of the same CONDEX horse in panel E has locally thinned HC with fibrillation (arrowhead) and chondrocyte proliferation (inset). Notice that tidemark and OCJ levels appear to be unaffected. Bars = 200 μm (panel A), 50 μm (B, C, and D), and 1 mm (E and F).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of third carpal bone sections from 2 horses showing histopathologic features of 2 defects at the C3r_palmar site. A—A bulla detected in 1 CONDEX horse may have been induced by a small intercarpal gap between the apposing intermediate carpal and radial carpal bones; these appeared to articulate with the third carpal bone on either side of the bulla (arrowheads). B—Higher magnification of the HC (inset 1 in panel A) reveals small numbers of chondrocytes and a fibrous appearance (arrowhead) suggestive of fibrillar network destructuring. C—Higher magnification of the tidemark (inset 2 in panel A) shows a vague, almost feather-like appearance of this feature. D—Unaffected CC adjacent to the affected region in panel C. Notice the clearly delineated tidemark, more homogeneous appearance of the matrix, and distinct chondrocytes, compared with the affected tissue in panel C. E—A mild focally softened region detected in 1 CONDEX horse is shown. The AS is intact, but localized thickening of the HC and chondrocyte proliferation in both the HC and CC are evident (inset). The dashed line indicates the tidemark. F—A severe focally softened region detected in the contralateral third carpal bone of the same CONDEX horse in panel E has locally thinned HC with fibrillation (arrowhead) and chondrocyte proliferation (inset). Notice that tidemark and OCJ levels appear to be unaffected. Bars = 200 μm (panel A), 50 μm (B, C, and D), and 1 mm (E and F).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of third carpal bone sections from 2 horses showing histopathologic features of 2 defects at the C3r_palmar site. A—A bulla detected in 1 CONDEX horse may have been induced by a small intercarpal gap between the apposing intermediate carpal and radial carpal bones; these appeared to articulate with the third carpal bone on either side of the bulla (arrowheads). B—Higher magnification of the HC (inset 1 in panel A) reveals small numbers of chondrocytes and a fibrous appearance (arrowhead) suggestive of fibrillar network destructuring. C—Higher magnification of the tidemark (inset 2 in panel A) shows a vague, almost feather-like appearance of this feature. D—Unaffected CC adjacent to the affected region in panel C. Notice the clearly delineated tidemark, more homogeneous appearance of the matrix, and distinct chondrocytes, compared with the affected tissue in panel C. E—A mild focally softened region detected in 1 CONDEX horse is shown. The AS is intact, but localized thickening of the HC and chondrocyte proliferation in both the HC and CC are evident (inset). The dashed line indicates the tidemark. F—A severe focally softened region detected in the contralateral third carpal bone of the same CONDEX horse in panel E has locally thinned HC with fibrillation (arrowhead) and chondrocyte proliferation (inset). Notice that tidemark and OCJ levels appear to be unaffected. Bars = 200 μm (panel A), 50 μm (B, C, and D), and 1 mm (E and F).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Histologically, the focally thickened HC bulla contained a substantially destructured middle zone matrix with thick aggregated bundles of optically resolvable collagen fibers approximately 0.2 mm below the AS; the matrix also lacked chondrocytes (Figure 9). The tidemark had an ill-defined, almost feathery appearance, compared with that of unaffected CC in the same section.
In the bone with mild focal softening, the AS was intact but the CC was thinner and the OCJ protruded slightly into the SCB in the affected region; there were few chondrocytes in the HC and CC, and these, where present, were in clusters (Figure 9). In the bone with severe focal softening, fibrillation was evident and the HC had local thinning at the defect site, creating an indentation in the AS. However, the tidemark and OCJ were at the same level as that in adjacent areas with normal-appearing cartilage. The tidemark at the defect site was also indistinct and feathery, and throughout the HC, chondrocytes were distributed irregularly and cluster formation was evident.
Site C3l_ridge—Grossly, defects of similar severity were found bilaterally at this site in 14 carpal bones of 3 CONDEX and 4 PASTEX horses. Nine of these 14 bones (from 3 CONDEX and 2 PASTEX horses) were histologically examined. The remaining 5 bones underwent gross examination only.
Eight bones (5 from 3 CONDEX horses and 3 from 3 PASTEX horses) had mild cartilage disruption, characterized by focal fibrillation, superficial fissures, and focal concavity or flattening of the AS and CC, with or without cell loss in the HC matrix in those that underwent histologic evaluation (Figure 10). No obviously abnormal features were observed in the tidemarks or CC.
Composite (A, C, and F) and individual (B, D, E, and G) photomicrographs of sections of the third carpal bones from horses with site C3l_ridge defects of various severities. A—Focal flattening or concavity of the HC is evident in the mild form of defects at this site. B—Higher magnification of the AS (inset of panel A) reveals local fibrillation (arrowhead). C—In the moderate form of the defect, a single deep fissure (arrowhead) is evident, with thickening of HC and focal concavity of the CC. D and E—Higher magnifications of the fissure (insets of panels C and D, respectively) reveal localized chondrocyte loss near the AS. F—In the severe form of the defect, a deep fissure penetrates into the SCB and substantial cartilage thickness loss is apparent. G—Higher magnification of the fissure (inset of panel F) and the adjacent region of cartilage loss (inset) reveals proliferation of chondrocyte clusters in the remaining HC and CC. Bars = 2 mm (panels A, C, and F), 250 μm (B, D, and G), and 60 μm (E).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Composite (A, C, and F) and individual (B, D, E, and G) photomicrographs of sections of the third carpal bones from horses with site C3l_ridge defects of various severities. A—Focal flattening or concavity of the HC is evident in the mild form of defects at this site. B—Higher magnification of the AS (inset of panel A) reveals local fibrillation (arrowhead). C—In the moderate form of the defect, a single deep fissure (arrowhead) is evident, with thickening of HC and focal concavity of the CC. D and E—Higher magnifications of the fissure (insets of panels C and D, respectively) reveal localized chondrocyte loss near the AS. F—In the severe form of the defect, a deep fissure penetrates into the SCB and substantial cartilage thickness loss is apparent. G—Higher magnification of the fissure (inset of panel F) and the adjacent region of cartilage loss (inset) reveals proliferation of chondrocyte clusters in the remaining HC and CC. Bars = 2 mm (panels A, C, and F), 250 μm (B, D, and G), and 60 μm (E).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Composite (A, C, and F) and individual (B, D, E, and G) photomicrographs of sections of the third carpal bones from horses with site C3l_ridge defects of various severities. A—Focal flattening or concavity of the HC is evident in the mild form of defects at this site. B—Higher magnification of the AS (inset of panel A) reveals local fibrillation (arrowhead). C—In the moderate form of the defect, a single deep fissure (arrowhead) is evident, with thickening of HC and focal concavity of the CC. D and E—Higher magnifications of the fissure (insets of panels C and D, respectively) reveal localized chondrocyte loss near the AS. F—In the severe form of the defect, a deep fissure penetrates into the SCB and substantial cartilage thickness loss is apparent. G—Higher magnification of the fissure (inset of panel F) and the adjacent region of cartilage loss (inset) reveals proliferation of chondrocyte clusters in the remaining HC and CC. Bars = 2 mm (panels A, C, and F), 250 μm (B, D, and G), and 60 μm (E).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Moderate cartilage disruption was detected in 4 bones (1 from 1 CONDEX horse and 3 from 3 PASTEX horses) and was characterized in histologic samples by the presence of a single fissure penetrating into the HC middle zone together with histologic features similar to those found in bones with mild cartilage disruption (Figure 10).
Two bones from 2 PASTEX horses had severe HC loss (approx 20% to 30% of the adjacent HC thickness), each with a single fissure penetrating into the SCB surrounded by apparently retained HC and CC. The HC and CC matrices had obvious chondrocyte loss and clustering (Figure 10).
Site C2ridge—Grossly, defects of similar severity were found bilaterally at this site in 2 CONDEX and 2 PASTEX horses. Of 8 C2 bones with these defects, 3 (from 1 CONDEX and 1 PASTEX horse) were histologically examined (Figure 11). The remaining 5 bones underwent gross examination only.
Photomicrographs of sections of the second carpal bones from horses with defects of various severities at site C2ridge. A—In the mild form of this defect, the HC appears normal. B—Higher magnification of the AS (inset of panel A) reveals only microscopic undulations at the ridge. C—The more severe form of defect includes fibrillation of the AS, HC loss, and a cyst-like defect in the SCB (inset). D—Higher magnification of the defect (arrow; inset of panel C) shows severe chondrocyte loss in the middle zone of the remaining HC and chondrocyte clustering in the deep zone HC and CC; CC is not detectable in the immediate area of the defect (arrowhead). Bars = 250 μm (panel A), 100 μm (B and D), and 500 μm (C).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of sections of the second carpal bones from horses with defects of various severities at site C2ridge. A—In the mild form of this defect, the HC appears normal. B—Higher magnification of the AS (inset of panel A) reveals only microscopic undulations at the ridge. C—The more severe form of defect includes fibrillation of the AS, HC loss, and a cyst-like defect in the SCB (inset). D—Higher magnification of the defect (arrow; inset of panel C) shows severe chondrocyte loss in the middle zone of the remaining HC and chondrocyte clustering in the deep zone HC and CC; CC is not detectable in the immediate area of the defect (arrowhead). Bars = 250 μm (panel A), 100 μm (B and D), and 500 μm (C).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs of sections of the second carpal bones from horses with defects of various severities at site C2ridge. A—In the mild form of this defect, the HC appears normal. B—Higher magnification of the AS (inset of panel A) reveals only microscopic undulations at the ridge. C—The more severe form of defect includes fibrillation of the AS, HC loss, and a cyst-like defect in the SCB (inset). D—Higher magnification of the defect (arrow; inset of panel C) shows severe chondrocyte loss in the middle zone of the remaining HC and chondrocyte clustering in the deep zone HC and CC; CC is not detectable in the immediate area of the defect (arrowhead). Bars = 250 μm (panel A), 100 μm (B and D), and 500 μm (C).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Six bones (4 from 2 CONDEX horses and 2 from 1 PASTEX horse) had mild defects that were characterized by minor AS undulations without apparent changes in the HC in those samples evaluated histologically (Figure 11). Two bones from 1 PASTEX horse had severe defects characterized by HC loss (approx 50% of HC thickness), fissure formation, cell loss, chondrocyte clustering, and a discontinuous CC layer. In 1 bone, a cyst-like lesion was present with no apparent CC in the immediate vicinity.
Site Crridge—Grossly, defects at this site were found bilaterally in 2 CONDEX and 2 PASTEX horses. A cartilage flap was also present in 1 bone from 1 of the 2 CONDEX horses. Of 8 radial carpal bones with defects, 3 (2 from 1 CONDEX horse and 1 from 1 PASTEX horse) were histologically examined, and the remaining 5 underwent gross examination only.
Histologically, all HC or CC defects were present at the apex of the ridge and extended to the HC or CC. The defects were characterized as a focal concavity at the ridge with an adjacent destructured matrix, a single HC fissure extending to the tidemark, or an intact AS (observed grossly and in the histologic section) with a fissure in the deep zone extending into the CC layer (Figure 12). The deep-zone fissure was associated with an HC matrix that had obvious cell loss and with discontinuity in the CC layer.
The bone with the cartilage flap also had a fragmented OCJ, allowing cartilage in the region to be partially detached from underlying SCB (Figure 12). The CC layer in the region of the defect was 2 to 3 times thicker than that in the adjacent unaffected tissue regions, although neither region had any obvious cell loss or clustering.
Photomicrographs (A through C, E) and a photograph (D) depicting cartilage defects at Crridge sites in the radial carpal bones of horses. A—A focal concavity is present at the surface of the ridge (top); at higher magnification (bottom [inset of top image]), destructured matrix (arrowhead) is evident in the middle zone HC. B—An HC fissure (top) extends through the ridge; at higher magnification (bottom [inset of top image]), the defect is seen disrupting the tidemark. C—The AS at the site is intact, but an HC fissure is evident in the deep zone (top) with CC damage and cell loss (arrowhead; bottom [inset of top image]). D—A cartilage flap disrupts the AS; inset shows the same defect at higher magnification. E—Section through the ridge site indicated by the dashed line in panel D shows partial detachment of the HC and CC and a fragmented OCJ (arrowhead). Bars = 300 μm (A, B, C, and E) and 3 mm (D).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs (A through C, E) and a photograph (D) depicting cartilage defects at Crridge sites in the radial carpal bones of horses. A—A focal concavity is present at the surface of the ridge (top); at higher magnification (bottom [inset of top image]), destructured matrix (arrowhead) is evident in the middle zone HC. B—An HC fissure (top) extends through the ridge; at higher magnification (bottom [inset of top image]), the defect is seen disrupting the tidemark. C—The AS at the site is intact, but an HC fissure is evident in the deep zone (top) with CC damage and cell loss (arrowhead; bottom [inset of top image]). D—A cartilage flap disrupts the AS; inset shows the same defect at higher magnification. E—Section through the ridge site indicated by the dashed line in panel D shows partial detachment of the HC and CC and a fragmented OCJ (arrowhead). Bars = 300 μm (A, B, C, and E) and 3 mm (D).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Photomicrographs (A through C, E) and a photograph (D) depicting cartilage defects at Crridge sites in the radial carpal bones of horses. A—A focal concavity is present at the surface of the ridge (top); at higher magnification (bottom [inset of top image]), destructured matrix (arrowhead) is evident in the middle zone HC. B—An HC fissure (top) extends through the ridge; at higher magnification (bottom [inset of top image]), the defect is seen disrupting the tidemark. C—The AS at the site is intact, but an HC fissure is evident in the deep zone (top) with CC damage and cell loss (arrowhead; bottom [inset of top image]). D—A cartilage flap disrupts the AS; inset shows the same defect at higher magnification. E—Section through the ridge site indicated by the dashed line in panel D shows partial detachment of the HC and CC and a fragmented OCJ (arrowhead). Bars = 300 μm (A, B, C, and E) and 3 mm (D).
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Histomorphometric assessment—Histomorphometry was performed on sections acquired from the C3r_dorsal sites of 18 bones from 5 CONDEX and 4 PASTEX horses. Sixty-five sections were from 13 bones without CC abnormalities, and 25 sections were from 5 bones with CC abnormalities; the latter had thicker CC and appeared to have more prominent VCs in these sections than did the former (Figure 13). The VCs in bones with CC abnormalities were aligned approximately perpendicular to the AS in this region. In contrast, the VCs in bones without these defects appeared to be more randomly oriented and occupied a smaller area.
Colorized histomorphometric images of sections of equine third carpal bones showing differences in CC thickness and VC morphology between C3r_dorsal sites in which a CC abnormality is absent (A) or present (B). Notice the HC (blue), differences in thickness and cellularity of the CC (green), and prominence and orientation of the VCs (red). Grid width = 3,000 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Colorized histomorphometric images of sections of equine third carpal bones showing differences in CC thickness and VC morphology between C3r_dorsal sites in which a CC abnormality is absent (A) or present (B). Notice the HC (blue), differences in thickness and cellularity of the CC (green), and prominence and orientation of the VCs (red). Grid width = 3,000 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
Colorized histomorphometric images of sections of equine third carpal bones showing differences in CC thickness and VC morphology between C3r_dorsal sites in which a CC abnormality is absent (A) or present (B). Notice the HC (blue), differences in thickness and cellularity of the CC (green), and prominence and orientation of the VCs (red). Grid width = 3,000 μm.
Citation: American Journal of Veterinary Research 73, 4; 10.2460/ajvr.73.4.498
The mean CC thickness was 98% greater (P = 0.019) in bones with (452 μm [range, 224 to 770 μm]) than in bones without (228 μm [range, 115 to 307 μm]) CC abnormalities. However, the VC area, VC number, and HC thickness were not significantly different between the 2 groups (P = 0.237, P = 0.184, and P = 0.145, respectively; Table 1).
Results of histomorphometric analysis of the C3r_dorsal sites of third carpal bones from 9 Thoroughbred horses with (5 bones) and without (13) CC abnormalities.
| CC abnormality* | |||||
|---|---|---|---|---|---|
| Absent (n = 65) | Present (n = 25) | ||||
| Variable | Mean | SE | Mean | SE | P value |
| HC thickness (μm) | 603 | 68.2 | 681 | 44.5 | 0.145 |
| CC thickness (μm) | 228 | 94.4 | 452 | 12.5 | 0.019 |
| VC area (μm2) | 490,320 | 64,542 | 624,894 | 39,979 | 0.237 |
| VC number | 18.0 | 0.826 | 15.9 | 0.721 | 0.184 |
Five sections were obtained from each bone for analysis.
Distribution of midcarpal joint defects in CONDEX and PASTEX horses—The distribution of defects, as well as frequency and severity, appeared to be similar between CONDEX and PASTEX horses; however, these variables could not be evaluated statistically. Two CONDEX horses and 1 PASTEX horse had no cartilage defects on any bones examined.
One CONDEX horse had midcarpal joint defects at multiple sites including C3r_dorsal (unilaterally), Crr_dorsal (bilaterally), C3r_palmar (unilaterally [raised bulla] and bilaterally [cartilage fissures]), and C3l_ridge, C2ridge, and Crridge (mild defects at each site bilaterally, in addition to the described Crridge cartilage flap). One CONDEX horse had C3l_ridge defects bilaterally (1 mild and 1 moderate), another had mild C2ridge defects bilaterally, and 1 had C3l_ridge defects (both mild) and Crridge defects (1 mild and 1 severe) bilaterally.
Similarly, 1 PASTEX horse had cartilage defects at multiple sites including C3r_dorsal (bilaterally), Crr_dorsal (bilaterally), C3r_palmar (bilaterally [focal softening]), C3l_ridge (bilaterally [1 moderate and 1 severe]), and C2ridge (bilaterally [both severe]). One PASTEX horse had unilateral defects at the C3r_dorsal and Crr_dorsal sites with mild C2ridge defects bilaterally, 1 had C3l_ridge defects (1 mild and 1 moderate) and Crridge defects (both mild) bilaterally, 1 had C3l_ridge defects (1 mild and 1 moderate) and Crridge defects (1 mild and 1 moderate) bilaterally, and 1 had bilateral C3l_ridge defects (1 mild and 1 severe).
Discussion
In the study reported here, we performed a histologic evaluation of osteochondral defects found at 6 sites in the midcarpal joints of horses that did or did not undergo a conditioning exercise regimen from shortly after birth until 18 months of age. We also conducted a histomorphometric analysis of tissues at 1 of these 6 sites, C3r_dorsal, in bones with or without CC abnormalities. Details of the exercise program and a previous evaluation of gross cartilage defects in the midcarpal joints of these horses were described elsewhere.13 Close examination of the previously described gross defects revealed that 1 site was not associated with histologic changes, and 2 sites were in very close proximity with each other, suggesting they should be combined; a previously unidentified defect site was also detected. Thus, a modified site-labeling system was used in the present study.
There was little apparent difference between CONDEX and PASTEX horses in the distribution, frequency, or severity of defects in the 6 midcarpal joint sites evaluated in the present study, although statistical comparisons could not be made. This suggests that the early conditioning exercise regimen may have had little influence on the development of defects and that even the spontaneous exercise undertaken at pasture by the PASTEX horses was sufficient to lead to easily detectable changes in the AS, HC, CC, OCJ, VCs, or SCB, similar to those in the CONDEX group. In other published studies13,21–26 involving the same cohort of horses, it was concluded that early conditioning exercise was not associated with tissue damage.13,22,24
One CONDEX and 2 PASTEX horses in the present study had CC thickening without HC damage at sites C3r_dorsal and Crr_dorsal. These are sites where osteochondral fractures commonly occur.35 The thickening and associated changes in or near the CC are thought to represent an early form of osteochondral disease,36–39 a common joint disease in Thoroughbreds40 that may arise from repetitive overloading during high-intensity exercise.41 The CC changes detected in the present study might also be associated with the development of an early secondary osteoarthrosis.42,43 This is a condition that may precede more clinically relevant lesions such as cartilage furrowing and collapse, SCB softening, and osteochondral fragmentation, all of which have been commonly reported in racehorses at these sites.41,44–46,g
With regard to site C3r_dorsal, histologic assessment indicated that the morphology and cellularity of HC was similar in bones with and without CC abnormalities. However, the HC in contiguous sections adjacent to sites with CC abnormalities was shown in our previous study13 to have a 4.2% higher mean swelling strain (P = 0.04) than that adjacent to sites without these defects. This suggested that subtle changes in the HC47,48 could have already taken place, and because HC matrix swelling is dependent on factors such as fibrillar network integrity47,49,50 and proteoglycan concentration and type,51,52 the mechanical properties of the affected region may have already been compromised without structural changes detectable by use of the optical microscope.
The fact that VCs were occasionally seen entering the abnormal CC through the fragmented OCJ suggested that a vascular response might have occurred, thus leading to partial healing of the damaged CC; confirmation of such a process would, however, require further study. Microcracks, fissures, or both were commonly observed in the CC of all 9 bones with CC abnormalities. This consistently detected morphological feature may possibly have resulted from repeated loading associated with spontaneous or imposed activity in the horses; it is also possible that the thickened CC may be more vulnerable to failure under even low loads, as is apparently the case in foals with retained HC.53
In histomorphometric assessment of site C3r_dorsal, the mean VC area fraction in the SCB appeared to be greater in bones that had CC abnormalities than in those that did not, although the difference was not significant. Also, there were unusually large curvilinear VCs in the cancellous bone deep in the SCB adjacent to the OCJ of all bones with, but none without, CC abnormalities. In our opinion, expansion of VC area fractions could be associated with repetitive high stresses or with a vascular or secondary remodeling response caused by thickening of the CC, and VC area fraction expansion. These changes, together with microcracks in the CC matrix and fragmentation of the OCJ, might be sufficient to weaken the bone by reducing the fracture toughness index54 and thus render it more susceptible to osteochondral fracture.
The elongated bulla-type cartilage defect found in 1 bone at site C3r_palmar may have resulted from compression of the margins of the ASs of the opposing intermediate and radial carpal bones against the third carpal bone, such that the cartilage was extruded into the small intercarpal bone gap. Interestingly, this type of localized extrusion of cartilage has been successfully duplicated by use of an indenter with a small central relief channel,55,56 thus confirming, at least in principle, that discontinuous contact conditions can generate such deformities. The complex shear field shown to be associated with this unusual type of extrusion55,56 may constitute a mechanism that could induce rapid HC disruption consistent with the considerable degree of localized destructuring and fibrosity detected in the region of the bulla in the present study.
The study reported here was limited by the small number of midcarpal joints available for analysis and the small number of osteochondral defects identified in the bones. Furthermore, the osteochondral sections examined, although considered thick (approx 30 μm), were not serially obtained, and thus the fissures observed in the HC may have penetrated more deeply through the cartilage and into the SCB. Despite these limitations, results of the study provide a histologic characterization of a spectrum of osteochondral defects in damage-prone sites in the dorsal aspects of the third and radial carpal bones, and these defects are unlikely to be reliably imaged in a clinical setting.
Results of histomorphometric assessment of relevant features revealed a significant difference in CC thickness of bones that had CC abnormalities, compared with those that did not. We hypothesize that the described changes in CC (eg, localized thickening, matrix texture changes, and development of microcracks) and OCJ disruption represent the earliest morphological changes associated with at least 1 pathway of osteochondral disease in the dorsal aspects of the third and radial carpal bones. Osteochondral disease requiring diagnostic or therapeutic arthroscopic intervention is commonly detected at these same sites in young racehorses. We postulate that the CC abnormalities detected in the present study could progress toward clinically important lesions; whether healing of such lesions might occur and what might be the conditions and exercise regimen that would best encourage such healing are unknown. However, results of the present study indicate that CC abnormalities can develop without obvious histologic changes of the HC in young horses with or without early conditioning exercise. The insights gained into the early stages of development of CC abnormalities in regions of clinical interest should provide a stimulus for further research into this problem.
ABBREVIATIONS
| AS | Articular surface |
| CC | Calcified cartilage |
| HC | Hyaline cartilage |
| OCJ | Osteochondral junction |
| SCB | Subchondral bone |
| VC | Vascular channel |
Tissue-Tek OCT Compound, Sakura Finetek USA Inc, Torrance, Calif.
Nikon AZ100, Nikon Instruments Inc, Melville, NY.
Model Graphire CTE-630, Wacom Co Ltd, Saitama, Japan.
Adobe Photoshop, version CS, Adobe Systems Inc, San Jose, Calif.
Image J, version 1.39d, National Institutes of Health, Bethesda, Md.
PROC MIXED, SAS, version 9.1, SAS Institute Inc, Cary, NC.
Sippel W. Equine degenerative arthritis. MS thesis, Department of Pathology and Bacteriology, Cornell University, Ithaca, NY, 1942.
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