Mikko Lammi

This study was undertaken to evaluate the influences of loading on chondrocyte extracellular matrix (ECM) and cytoskeletal elements. The capacity of the articular cartilage matrix of young beagle dogs to recover during 50 weeks' remobilization period after an atrophy caused by 11 weeks of immobilization of the knee (stifle) joint was studied. The effects of hydrostatic pressure on the stress fibers, microtubules and Golgi apparatus were examined in bovine chondrocyte cultures. In addition, the role of dynamic microtubules in hydrostatic pressure induced alterations in proteoglycan (PG) biosynthesis in chondrocytes was investigated A sensitive assay was developed for quantification of glycosaminoglycans (GAGs) and PGs.

   The concentration and synthesis of articular cartilage PGs were studied at the onset of 50 weeks' immobilization and immediately after it. Cartilage PGs were characterized by chemical analyses, gel filtration and Western blotting. The PG concentrations, reduced in all sample sites after immobilization, remained 14-28% lower than controls in certain joint surfaces after remobilization. In the contralateral joint, a 49% increase of PG in the inferior femoropatellar area (FPI) appeared following remobilization, while in the intermediate medial femoral condyle (FMI) a 34% decrease was noticed. Total PG synthesis was not significantly changed after immobilization or remobilization. The chondroitin 6- to  4-sulphate was reduced after immobilization both in the newly synthesized and the total tissue PGs. In the remobilized joint, this ratio was restored in FPI, while in tibia it remained at a level lower than controls. Neither immobilization nor remobilization induced the appearance of any of the immunologically detectable epitopes reported to be associated with early degenerative changed in native PGs of articular cartilage. These results show that the depletion of PGs observed after 11 weeks of immobilization was not completely restored after 50 weeks of remobilization. The changes that developed in the contralateral joint during the remobilization provide support for the concept that a permanent alteration of matrix metabolism can result from even a temporary modification of laoding patter in immature joints.

   Changes in the stress fiber organization was studied in vitro by exposing the cultures to 5, 15 or 30 MPa hydrostatic pressures for two hours continuously or at frequencies of 0.125 Hz or 0.05 Hz. A 30 MPa continuous pressure caused a nearly complete disappearance of stress fibers. At 15 MPa, or cyclic pressures, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in controls. The pressure effects were reversible after two hours. These results indicate that cytoskeletal changes may be an integral part of the chondrocyte response to hydrostatic pressure.

   The role of microtubules in the regulation of PG synthesis in pressurized chondrocyte cultures was examined using drugs affecting microtubular organization. Disruption of the microtubular array by nocodazole inhibited [35S]sulphate incorporation, and a further decrease was noticed when nocodazole cultures were exposed to 30 MPa continuous hydrostatic pressure, suggesting that high pressure exerts its inhibition through mechanisms independent of microtubules. Accordingly, stabilization of microtubules with taxol did not rescue the suppressed [35S]sulphate incorporation by cyclic 0.5 Hz 5 MPa hydrostatic pressure, suggesting the the stimulatory effect was microtubule dependent.

   This study showed that immobilization-induced alterations in immature articular cartilage matrix in vivo may well be permanent. Hydrostatic pressure was found to affect the organization of cytoskeletal components in chondrocytes, and to modulate chondrocyte PG synthesis in vitro, with this modulation being mediated partly by mechanisms which are dependent on dynamic microtubules.