| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
|
|---|
| Research Abstract |
Objective. We have shown for the first time that it is possible to enhance the proliferation of cultured chondrocytes and promote the production of type-IX collagen in primary chondrocytes grown on a three-dimensional (3-D) culture system through low-intensity pulsed ultrasound (LIPUS) stimulation. Moreover, one of the underlying mechanisms of intracellular signaling pathways such as MAPK/MAPKK and Integrin/PI3K/Akt involved in mechano-transduction during the proliferation of chondrocytes was identified.
Methods. Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly slaughtered porcines. Isolated chondrocytes in collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. The final cell density was adjusted to 2×10^6 cells/well/ml. LIPUS stimulation was applied to the chondrocyte cultures for 20 minutes daily. Anti-type-II and anti-type-IX collagen antibodies were used to investigate the phenotype of the chondrocytes and t
… More
o detect type-II and type-IX collagen production at post-culture week 2. For immuno-staining and Western blot analysis, the following primary antibodies were used: type-II and type-IX collagen, Akt, p-Akt, Cyclin B1 and D1, MAPK, p-MAPK, FAK, p-FAK, Paxillin, p-Paxillin, β-catenin, β-1 Integrin, and α-1, 2 and v Integrins.
Results. Chondrocytes proliferated and a collagenous matrix formed on the surface of the collagen sponge. By exposing chondrocytes to LIPUS, thicker layers of matrix containing type-II collagen and proteoglycan accumulated. The rate of proliferation of chondrocytes cultured under LIPUS stimulation was significantly higher than in the control group during the two-week culture period. In Western blot analysis, type-IX collagen, Cyclin B1 and D1, p-FAK, and p-Akt were found to be more strongly expressed in the LIPUS stimulation group than in the control. However, there was no difference between the two groups in MAPK, p-MAPK and type-II collagen expression.
Conclusion. LIPUS stimulation promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a 3-D culture system using collagen sponge as a vehicle to create an environment similar to cartilaginous tissue. Less
|
