Objective—To determine effects of various concentrations of retinoic acid (RA) or a synthetic RA receptor antagonist (LE135) on equine chondrocytes or bone marrow—derived equine mesenchymal stem cells (BMDMSCs) in monolayer cultures.
Sample—Articular cartilage and BMDMSCs from 5 clinically normal horses.
Procedures—Monolayers of chondrocytes cultured in standard media and of BMDMSCs cultured in chondrogenic media were treated with RA at concentrations of 0, 0.1, 1, or 10μM or LE135 at concentrations of 0, 0.1, 1, or 10μM on day 0. On days 7 and 14, samples were analyzed for DNA concentration, chondrocyte morphology or features consistent with chondrogenesis (ie, chondral morphology [scored from 0 to 4]), and gene expression of collagen type Ia (CI), collagen type II (CII), and aggrecan.
Results—Chondrocytes treated with RA had more mature chondral morphology (range of median scores, 3.0 to 4.0) than did untreated controls (range of median scores, 0.5 to 0.5). Chondrocytes treated with LE135 did not sustain chondrocyte morphology. All BMDMSCs had evidence of chondral morphology or high CII:CI ratio. Retinoic acid (1 or 10μM) or LE135 (10μM) treatment decreased DNA content of BMDMSC cultures. At 0.1 and 1μM concentrations, LE135 weakly but significantly increased chondral morphology scores, compared with untreated controls, but lack of aggrecan expression and lack of increased CII:CI ratio, compared with that of controls, did not affect chondrogenesis.
Conclusions and Clinical Relevance—RA promoted maturation and hypertrophy in chondrocytes but not BMDMSCs in monolayer cultures. Deficiency or blockade of RA may prevent hypertrophy and maturation of differentiated chondrocytes.
Objective—To evaluate effects of extracorporeal shock wave therapy (ESWT) and polysulfated glycosaminoglycan treatment (PSGAGT) on subchondral bone (SCB), serum biomarkers, and synovial fluid biomarkers in horses with induced osteoarthritis.
Animals—24 healthy 2- to 3-year-old horses.
Procedures—An osteochondral fragment was created on the distal aspect of the radial carpal bone in 1 middle carpal joint of each horse. Horses were randomly allocated to receive local application of ESWT (days 14 and 28; n = 8), PSGAGT (IM, q 4 d for 28 days; 8), or a sham ESWT probe (placebo; days 14 and 28; 8). Serum biomarkers were measured every 7 days, and synovial fluid biomarkers were measured every 14 days. Bone density was measured by use of computed tomography on days 0 and 70, and microdamage and bone formation variables were compared among groups at the end of the study (day 70).
Results—There was no significant effect of ESWT or PSGAGT on any bone variable. Serum osteocalcin concentration was significantly greater in horses that received ESWT, compared with placebo-treated horses, and serum concentration of the C-terminal telopeptide of type I collagen was significantly higher in horses that received ESWT, compared with placebo- and PSGAG-treated horses. Concentrations of the synovial fluid epitope CS846 were significantly higher in joints with osteoarthritis treated with ESWT
Conclusions and Clinical Relevance—Treatment of osteoarthritis with ESWT had no effect on SCB but did induce increases in serum biomarkers indicative of bone remodeling. Treatment of osteoarthritis with PSGAG had no effect on SCB or biomarkers.
Objective—To evaluate effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs.
Animals—12 healthy female Beagles.
Procedures—A mid-diaphyseal transverse osteotomy (stabilized with an intramedullary pin) of the right tibia was performed in each dog. The carprofen group (n = 6 dogs) received carprofen (2.2 mg/kg, PO, q 12 h) for 120 days; the control group (6) received no treatment. Bone healing and change in callus area were assessed radiographically over time. Dogs were euthanized 120 days after surgery, and tibiae were evaluated biomechanically and histologically.
Results—The osteotomy line was not evident in the control group on radiographs obtained 120 days after surgery. In contrast, the osteotomy line was still evident in the carprofen group. Callus area was significantly less in the carprofen group, compared with the area in the control group, at 20, 30, and 60 days after surgery. At 120 days after surgery, stiffness, elastic modulus, and flexural rigidity in the carprofen group were significantly lower than corresponding values in the control group. Furthermore, histologic evaluation revealed that the cartilage area within the callus in the carprofen group was significantly greater than that in the control group.
Conclusions and Clinical Relevance—Long-term administration of carprofen appeared to inhibit bone healing in dogs that underwent tibial osteotomy. We recommend caution for carprofen administration when treating fractures that have delays in healing associated with a reduction in osteogenesis as well as fractures associated with diseases that predispose animals to delays of osseous repair.
Objective—To determine the in vitro effects of selected growth factors on fibrochondrogenesis by synovial membrane cells from nonosteoarthritic (normal) and osteoarthritic joints of dogs.
Animals—5 dogs with secondary osteoarthritis of shoulder or stifle joints and 6 dogs with normal joints.
Procedures—Synovial membrane cells were harvested from normal and osteoarthritic joints and cultured in monolayer with or without (control) basic fibroblast growth factor, transforming growth factor-β1, and insulin-like growth factor-1. In the cultured cells, fibrochondrogenesis was measured by use of a real-time reverse transcriptase PCR assay to determine relative expressions of collagen I, collagen II, and aggrecan genes and of 3 genes involved in embryonic chondrogenesis: Sry-type homeobox protein-9 (SOX-9), frizzled-motif associated with bone development (Frzb), and regulator of G-protein signaling-10 (RGS-10). Tissue collagen content was measured via a hydroxyproline assay, and sulfated glycosaminoglycan content was measured via a 1,9-dimethylmethylene blue assay. Cellularity was determined via a double-stranded DNA assay. Immunohistochemical analysis for collagens I and II was also performed.
Results—In vitro collagen synthesis was enhanced by growth factor stimulation. Although osteoarthritic-joint synoviocytes could undergo a fibrocartilage-like phenotypic shift, their production of collagenous extracellular matrix was less than that of normal-joint synoviocytes. Gene expressions of SOX-9 and RGS-10 were highest in the osteoarthritic-joint cells; Frzb expression was highest in growth factor treated cells.
Conclusions and Clinical Relevance—Autogenous synovium may be a viable cell source for meniscal tissue engineering. Gene expressions of SOX-9 and RGS-10 may be potential future targets for in vitro enhancement of chondrogenesis.
Objective—To investigate topographic and age-dependent adaptation of subchondral bone density in the elbow joints of healthy dogs by means of computed tomographic osteoabsorptiometry (CTOAM).
Animals—42 elbow joints of 29 clinically normal dogs of various breeds and ages.
Procedures—Subchondral bone densities of the humeral, radial, and ulnar joint surfaces of the elbow relative to a water-hydroxyapatite phantom were assessed by means of CTOAM. Distribution patterns in juvenile, adult, and geriatric dogs (age, < 1 year, 1 to 8 years, and > 8 years, respectively) were determined and compared within and among groups.
Results—An area of increased subchondral bone density was detected in the humerus distomedially and cranially on the trochlea and in the olecranon fossa. The ulna had maximum bone densities on the anconeal and medial coronoid processes. Increased bone density was detected in the craniomedial region of the joint surface of the radius. A significant age-dependent increase in subchondral bone density was revealed in elbow joint surfaces of the radius, ulna, and humerus. Mean subchondral bone density of the radius was significantly less than that of the ulna in paired comparisons for all dogs combined and in adult and geriatric, but not juvenile, dog groups.
Conclusions and Clinical Relevance—An age-dependent increase in subchondral bone density at the elbow joint was revealed. Maximal relative subchondral bone densities were detected consistently at the medial coronoid process and central aspect of the humeral trochlea, regions that are commonly affected in dogs with elbow dysplasia.
Objective—To determine whether histopathologic characteristics of the osteochondral units of equine distal tarsal joints were associated with exercise history in horses without lameness.
Sample Population—30 cadaver tarsi from horses without lameness and with known exercise history were separated into 3 groups: nonridden, pasture exercise (group P); low-intensity, ridden exercise (group L); and high-intensity, elite competition exercise (group E).
Procedures—Standardized sites from the centrodistal and tarsometatarsal joints under went histologic preparation. A grading system was adapted to describe location, depth, and shape of lesions; cellular arrangement; organization at cartilage and subchondral bone (SCB) junctions; and organization of SCB. A high score signified a more severe pathological change than a low score. Exercise groups were compared by calculation of Spearman rank correlations.
Results—In the centrodistal joint, lesions were present in groups L and E but only medially. Cellular arrangement scores were higher at the dorsomedial location in group P than in groups L and E. Groups L and E had higher scores than group P for the organization of the cartilage, SCB junctions, and SCB, with higher scores at the dorsomedial location. In the tarsometatarsal joint, lesions were evident across the whole joint surface, with more severe lesions located laterally in all 3 groups. Overall, group E had higher scores for cellular arrangement and SCB organization than groups P and L.
Conclusions and Clinical Relevance—Ridden exercise may increase the risk of osteochondral lesions at distal tarsal sites predisposed to osteoarthritis relative to the risk with nonridden exercise.
Objective—To establish the dose-dependent effects of high-molecular-weight hyaluronic acid (HA) supplementation on chondrogenesis by mesenchymal stem cells (MSCs) cultured on chitosan sponges and to determine the extent to which MSC matrix production (chondrogenesis) can be influenced by incorporation of high-molecular-weight HA into chitosan scaffolds.
Sample Population—Murine MSCs derived from a multipotent bone marrow stromal precursor.
Procedures—MSCs were seeded on chitosan and chitosan-HA scaffolds in chondrogenic medium with various HA concentrations. Scanning electron microscopy, fluorescence microscopy (viability assay), and DNA quantification were used to assess cell attachment, distribution, and viability 48 hours after seeding. Constructs were cultured for 3 weeks prior to evaluation of cell distribution and chondrogenic differentiation via histologic evaluation and quantification of DNA, glycosaminoglycan, and collagen II.
Results—48 hours after MSC seeding, cell viability and DNA content were similar among groups. Three weeks after seeding, HA supplementation of the culture medium improved matrix production in a dose-dependent manner, as indicated by matrix glycosaminoglycan and collagen II concentrations. The scaffold composition, however, had no significant effect on matrix production.
Conclusions and Clinical Relevance—High-molecular-weight HA supplementation in culture medium had a dose-dependent effect on matrix production and thus chondrogenic differentiation of MSCs cultured on chitosan sponges. The addition of HA in the surrounding fluid during chondrogenesis should improve cartilage production and may be useful for producing engineered cartilage tissues.
Objective—To determine whether camel articular chondrocytes can be maintained in tissue culture without phenotype loss and whether the response to cytokine stimulation can be modulated.
Sample Population—Cartilage from 4 carpal joints of healthy adult dromedary camels (Camelus dromedarius).
Procedures—Chondrocytes were evaluated for type II collagen and aggrecan production They were incubated with control media or with 2 test mixtures (alone and then in combination) that have anti-inflammatory activity (avocado-soybean unsaponifiables, glucosamine, and chondroitin sulfate [ie, ASU + GLU + CS] and pentosan polysulfate and N-acetyl glucosamine [ie, PPS + NG]). Cells were then stimulated with interleukin-1β and tumor necrosis factor-α to determine prostaglandin (PG) E2 production and nuclear factor (NF)-κB activation.
Results—Chondrocytes proliferated in media used for propagating equine chondrocytes; they produced type II collagen and aggrecan. Cytokine stimulation induced PGE2 production and translocation of NF-κB. Incubation with each test mixture significantly inhibited PGE2 production. The combination of ASU + GLU + CS and PPS + NG significantly potentiated PGE2 inhibition and disrupted NF-κB translocation, compared with effects for either mixture alone.
Conclusions and Clinical Relevance—Chondrocytes proliferated without loss of the cartilage phenotype. Responses to cytokines were significantly inhibited by the mixtures of ASU + GLU + CS and PPS + NG, which indicated that this response can be modulated. This culture technique can be used to study the functional properties of camel chondrocytes and identify agents that may potentially be used to treat and manage joint inflammation.
Objective—To determine the items (question topics) for a subjective instrument to assess degenerative joint disease (DJD)–associated chronic pain in cats and determine the instrument design most appropriate for use by cat owners.
Animals—100 randomly selected client-owned cats from 6 months to 20 years old.
Procedures—Cats were evaluated to determine degree of radiographic DJD and signs of pain throughout the skeletal system. Two groups were identified: high DJD pain and low DJD pain. Owner-answered questions about activity and signs of pain were compared between the 2 groups to define items relating to chronic DJD pain. Interviews with 45 cat owners were performed to generate items. Fifty-three cat owners who had not been involved in any other part of the study, 19 veterinarians, and 2 statisticians assessed 6 preliminary instrument designs.
Results—22 cats were selected for each group; 19 important items were identified, resulting in 12 potential items for the instrument; and 3 additional items were identified from owner interviews. Owners and veterinarians selected a 5-point descriptive instrument design over 11-point or visual analogue scale formats.
Conclusions and Clinical Relevance—Behaviors relating to activity were substantially different between healthy cats and cats with signs of DJD-associated pain. Fifteen items were identified as being potentially useful, and the preferred instrument design was identified. This information could be used to construct an owner-based questionnaire to assess feline DJD-associated pain. Once validated, such a questionnaire would assist in evaluating potential analgesic treatments for these patients.
Objective—To determine the prevalence and severity of osteoarthritis in the metacarpophalangeal joints of Thoroughbred racehorses via development and validation of a quantitative macroscopic evaluation system.
Sample Population—Metacarpophalangeal joints from 50 Thoroughbred racehorses.
Procedures—Joints were collected from horses that died or were euthanized within 60 days of racing. Metacarpophalangeal joints were assessed for osteoarthritic degeneration by use of macroscopic and histologic scoring systems, polarized light microscopy, and cartilage biochemical analysis. The global macroscopic score for the entire metacarpophalangeal joint was based on factors that reflected the size and severity of lesions as well as the involvement of weight-bearing surfaces.
Results—One-third of all 2- and 3-year-old horses had partial-or full-thickness cartilage lesions and osteoarthritis. Osteoarthritis severity increased until age 6 in this population. Significant correlations were found between macroscopic grade and age, cause of death, glycosaminoglycan depletion, and loss of superficial cartilage zone polarized light intensity.
Conclusions and Clinical Relevance—The macroscopic system devised for this study had good correlations with quantitative methods. Two-and 3-year-old horses had full-thickness cartilage lesions that may have been career limiting. Year-to-year attrition and a small population of older horses may have led to underestimation of the prevalence of osteoarthritis in older horses. The macroscopic scoring system was reliable when used by nonexpert and expert users. (Am J Vet Res 2010;71:1284–1293)