The objective of this study was to investigate the changes in synovial fluid concentration of collagen type II cleavage site (C2C) and pro collagen II C-propeptide (CPII), markers of joint cartilage degeneration and synthesis, respectively, in horses with intraarticular fracture or osteochondrosis dissecans (OCD), and to examine the relationship between arthroscopic findings and these biomarker levels. Synovial fluid was collected from 36 joints in 18 horses (6 fractures and 12 OCDs). Samples from contralateral normal joints, when available, served as controls (n=12). Concentrations of C2C and CPII were measured using enzyme-linked immunosorbant assays. Moreover, the severity of the cartilage degradation was graded arthroscopically in 16 horses, and the correlation between the C2C and CPII levels and the arthroscopic scores were investigated. Compared to the control, the concentration of C2C was increased in OCD joints but not in fracture joints, whereas the concentration of CPII was increased in fracture joints but not in OCD joints. Within each disease group there was no correlation between biomarker levels and arthroscopic findings. Therefore, although C2C and CPII have diagnostic potential further knowledge is required to provide accurate analysis.

Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-Rassociated factor 2 (TRAF2) and nuclear factor-kappa B (NE-KappaB) , and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTS of thoroughbred horses. The tendinocytes were treated with 10ng/ ml equine TNFAlpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF-R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFAlpha-treated cells (inflamed condition). Intense TRAF2 and NF-KappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in uitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.

Our previous study established protein gene product 9.5 (PGP 9.5), a ubiquitin C-terminal hydrolase, as a specific cytochemical marker of synovial lining cells (type B synoviocytes) in the horse joint. The present study aimed to detect PGP 9.5 in the synovial fluid and shows that PGP 9.5 is a valuable marker of osteoarthritis in the horse. Immunohistochemical staining confirmed rich and consistent localization of PGP 9.5 immunoreactivity in the cytoplasm of synovial lining cells in the normal horse joint. Western blot analysis of synovial fluid from normal joints could detect a significant band corresponding to that contained in the brain and synovial membrane extracts. When 60 synovial fluid samples from normal and abnormal joints were assayed with an enzyme-linked immunosorbent assay (ELISA) system, the concentration of PGP 9.5 tended to be elevated in osteochondrosis dissecance, inflammatory arthropathy and intra-articular fracture, among which a statistiture and the control. Thus, this study demonstrated the possibility that PGP 9.5, derived from synovial lining cells, may be a new biochemical marker for arthritic disorders of the horse.

This study aimed to evaluate a system that identifies cartilage turn over and/or degradation through measurement of a new keratan sulfate (KS) epitope concentration in equine sera. Blood samples were collected from 30 horses, 1 (n=15) and 2 year-olds (n=15). Serum samples were analyzed for an epitope of keratan sulfate by 1/20/5D4 (KS5D4) and new epitopes of keratan sulfate using high sensitive keratan sulfate (HSKS), measured by two respective enzyme-linked immunosorbant assays (ELISAs). There was no correlation in serum concentration of KS evaluated using 5D4 and HSKS. Age had no significant effect on concentrations of KS measured with KS5D4 while 1 year-old horses showed significantly higher amounts than 2 year-olds with HSKS. Results suggest that HSKS could detect early signs of cartilage metabolic changes.

Bone marrow derived mesenchymal stem cells (MSCs) can be used to repair articular cartilage defects, these cells should be properly stimulated so that they could differentiate morphologically and hold cellular synthetic features closer to maturely differentiated chondrocytes. It is well known that tissue specific environment plays an important role in cell fate determination. Once improved isolation, proliferation and differentiation protocols have been developed, the likelihood of spontaneous differentiation of MSCs into divergent lineages will be reduced, thus increasing their value for cartilage repair. The purpose of this study was to improve chondrogenic differentiation of equine MSCs using coculture with mature equine articular chondrocytes (ACs), along with the determination of the effect of adding transforming growth factor (TGF) beta1 in the pellet culture system. Following confirmation of multilineage (adipogenic, osteogenic and chondrogenic) differentiation, isolated MSCs, ACs and coculture of both cell types were transferred into pellet culture system in a DMEM-based medium supplemented with or without TGFbetal. Chondrogenic differentiation was evaluated histologically and the relative mRNA expressions of collagen type 1 alpha1 (COL1A1), collagen type 2 alpha1 (COL2A1), aggrecan (ACAN) and SRY-box 9 (SOX9) were estimated by quantitative RT-PCR. Cocultured cells showed diffuse distribution of extracellular matrix (ECM), whereas in chondrocyte pellets it was more localized to central regions. Expression of COL2A1, ACAN and SOX9 genes were higher in cocultured pellets when compared to MSCs and ACs-composed pellets. Addition of TGFbeta1 in chondrogenic differentiating medium did not consistently amplify expression of the above mentioned genes. Differentiation of equine MSCs was enhanced by coculturing in association with mature ACs, improving expression of cartilage-specific genes and producing a more homogeneous production of ECM within the newly formed cocultured cartilage. The use of the coculture system could possibly enhance the capacity of MSC-derived chondrocytes to build up stable articular cartilage-like constructs, which could play an important role in articular cartilage repair and regeneration.