Introduction and Aims: Fibrin-sealant has been recommended as a tissue glue for autologous chondrocyte implantation. It is known that the active compound of fibrin-sealant is thrombin, but its effect on chondrocytes is still unclear. The aims of this study are to examine if fibrin-sealant stimulates proliferation and survival of human chondrocytes.

Method: To determine if human chondrocytes express thrombin receptors, we have conducted immunoconfocal analyses and RT-PCR for the detection of PAR type I, II, III and IV. To examine if thrombin activates intracellular signalling of chondrocytes, we have examined the intracellular calcium signalling by thrombin. Proliferation of chondrocytes was also tested with various concentrations of thrombin. The migration of chondrocytes was monitored by co-culturing of the cells with fibrin-sealant for up to 15 days.

Results: Primary human chondrocytes express thrombin receptor PAR types I, II, II and IV as evidenced by immunohistochemistry and RT-PCR. Induction of intracellular calcium signals was evidenced in majority of chondrocytes at 100 seconds after addition of thrombin. To confirm if evaluation of calcium signal activation is by a specific PAR receptor, we have examined the effect of specific peptides, which mimic the receptor activation on calcium signalling. The result showed that expression of PAR I and II receptor in chondrocytes is responsible for the activation of intracellular calcium. When human chondrocytes were co-cultured with thrombin at a dose between 1u/mL to 10u/mL, there was no effect on cellular proliferation at 24 hours. However at 48 hours, thrombin stimulated proliferation and survival of chondrocytes in a dose-dependent manner. A maximum of threefolds induction was evidenced at a dose of 10u/mL (p< 0001). Co-culture of chondrocytes with fibrin-sealant showed that after 12 hours only a few cells had migrated from the membrane to the fibrin-sealant, but after 36 hours many cells had formed a layer on the surface of the fibrin-sealant. By 15 days of co-culture, it was evidenced that the majority of chondrocytes were migrating into the fibrin-sealant.

Conclusion: The results of this study show that human chondrocytes express thrombin receptor and fibrin-sealant is capable of inducing chondrocyte proliferation and migration.

Introduction Fibrin-sealant has been widely used clinically for the protection of haemorrhage, wounds and tissue fluid leakage. Recently fibrin-sealant has been recommended as a tissue glue for autologous chondrocyte implantation. It is known that the active compound of fibrin-sealant is thrombin but its effect on chondro-cyte is still unclear. The aims of this study are to examine if fibrin-sealant stimulates proliferation and survival of human chondrocytes.

Methods Primary human chondrocytes derived from articular cartilage were used for the detection of thrombin receptors RAR type I, II, III and IV by immunohistochemistry and RT-PCR. To examine the effect of thrombin on chondrocytes, the changes in free intra-cellular calcium were monitored after the addition of thrombin. Proliferation of chondrocytes were also tested with various concentrations of thrombin. The survival of chondrocytes was monitored by co-culturing of the cells with fibrin-sealant for up to 15 days. Primary human chondrocytes express thrombin receptor RAR types I, II, III and IV as evidenced by immunohistochemistry and RT-PCR. However, the level of expression appears to be varied between cells. This has been reflected by the measurement of intracellular calcium signal in chondrocytes.

Results Induction of intracellular calcium signals was evidenced in the majority of chondrocytes at 100 seconds after addition of thrombin. When human chondrocytes were co-cultured with thrombin at a dose between 1u/ml to 10u/ml, there was no effect on cellular proliferation at 24 hours. However, at 48 hours thrombin stimulated proliferation and survival of chondrocytes in a dose dependent manner. A maximum of three folds induction was evidenced at a dose of 10u/ml (p< 0001). Co-culture of chondrocytes with fibrin-sealant showed that after 12 hours only a few cells had migrated from the membrane to the fibrin-sealant, but after 36 hours many cells had formed a layer on the surface of fibrin-sealant. By 15 days of co-culture, it was evidenced that majority of chondrocytes were migrating into the fibrin-sealant. Immunohistology study showed that these cells express type II collagen, suggesting that they maintain the phenotype of chondrocytes.

Conclusions The results of this study show that human chondrocytes express thrombin receptor and fibrin-sealant is capable of inducing chondrocyte proliferation and maintain the survival of chondrocytes.

In relation to the conduct of this study, one or more of the authors is in receipt of a research grant from a non-commercial source.