Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects. Articular cartilage was isolated from donors undergoing total hip or knee replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase, and expanded in DMEM-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1,
Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Human ACL cells were harvested from ligament samples donated by patients who underwent total knee arthroplasties with informed consents. Interface cells were isolated from the 5-mm-end of ACL. Midsubstance cells were cultured from the middle part of ACL. The cells were seeded onto stretch chambers (2Ä−2 cm, 50,000 cells/chamber) and uni-axial cyclic mechanical stretch (0.5 Hz, 7%) was applied for 2 h using a ST140. RNA samples were reverse-transcripted and quantitative real-time RT-PCR analysis were performed. To inhibit the function of integrin alphaVbeta3 subunit or alpha5 in stretching experiments, anti-human integrin alphaVbeta3 and alpha5 functional blocking antibodies (alphaVbeta3: 20 mg/ml, alpha5: 4 mg/ml) were used. To investigate the cellular attachments responding to mechanical stretch, we observed the distribution of integrins and stress fibers in both ACL cells. The shape of midsubstance cells showed spindle and fibroblastic cellular morphologies. On the other hand, the interface cells displayed chondroblastic appearances such as small and triangular morphologies. The expressions of COL1A1,
