Despite the well-known effect of type-I collagen in promoting cartilage repair, the mechanism still remains unknown. In this study we investigated this mechanism using a rabbit model of cartilage defects. 5-mm-diameter full-thickness defects were created on both patellar grooves of 53 Japanese white rabbits (approximately 13 weeks old). The left defect was filled with collagen gel and the right defect was left empty. The rabbits were killed and examined morphometrically until the twenty-fourth postoperative week, by (1) evaluation of matrix production, (2) enumeration of the total number of cells engaged in cartilage repair, (3) enumeration of the proliferating cells, (4) localization of mesenchymal stem cells, and (v) localization of apoptotic cells. We found that type-I collagen enhances cell recruitment, and thereby increases the number of proliferating cells. A considerable proportion of the proliferating cells were identified as bone marrow-derived mesenchymal stem cells. However, type-I collagen does not prevent the chondrocyte precursors from undergoing apoptotic disengagement from the chondrogenic lineage. Type-I collagen promotes cartilage repair by enhancing recruitment of bone marrow-derived mesenchymal stem cells. Additional use of agent(s) that sustain mesenchymal stem cells along the chondrogenic path of differentiation may constitute an appropriate environment for cartilage repair.

Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA). Recently, we reported that activation of volume-sensitive Cl⁻ current (I_Cl,vol) mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX) blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of I_Cl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl⁻ conductance. The TNFα-evoked Cl⁻ current exhibited electrophysiological and pharmacological properties similar to those of I_Cl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited I_Cl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

To investigate the mutational effect for the stability of thermolysin (TLN) in conserved regions. Mutational effects for stability at autodegradation sites of TLN in conserved region were studied. The bands of mutant TLN (34 kDa) on SDS-PAGE were decreased. However, those of mutant TLN cultivated with CaCl2 recovered to the same level as WT TLN. Dialysis study shows that these mutant TLN require more calcium ions than WT TLN. From these results, calcium affinity of mutant TLN in the conserved regions seem to become weak, subsequently mutant TLN were easily autodegraded in the case of low concentration of CaCl2. The autodegradation sites located in conserved regions of bacilli neutral proteases are important for the tertiary structure formation concerning the stability of the protein.