Effects Of Mechanical Injury And PGE₂on MMP-1,2,3Expressions In ACL Fibroblasts By Exposing Cells To Conditioned Medium From Injure Synovial Cells

The incidence of the injury to the knee ligaments has been steadily increased as a result of growing popularity of sport activities. Treatments of the cruciate ligaments (anterior and posterior cruciate ligaments or ACL and ACL) are the most important, which have poor healing abilities that cannot be healed after primary surgery. The current standard treatment for ACL injuries in active adults with functional instability is ligament reconstruction but it does not restore the normal ACL biomechanics. It is revealed that there was high amount of matrix metalloproteinases (MMPs) accumulated in the joint fluids after ACL injury that may disturb the delicate balance between the removal and deposition of the extracellular matrix components, and interrupt the ACL remodeling processes.In our study wound healing assay was performed to determine the migration rate of PCL fibroblasts in absence or presence of PGE2under mono-culture or co-culture. Cultured ACL fibroblasts exposed to conditioned medium from synovial cells were treated with mechanical injure and/or combination of PGE2, and Quantitative real-time PCR assay was performed to detect MMP-1,2,3gene expression at1,3,6h and12h after treatment; gelatin zymography was performed to detect MMP-2activity at12,24,48h and72h after treatment. The results shown that the co-culture or/and PGE2also stimulated ACL fibroblasts to grow and migrate. With time passing, the mRNA expression of MMP-1,3increase significantly but MMP-2decrease in the condition of mechanical injure. On the other hand, combination of PGE2up-regulated both mRNA expression of MMP-1,2and MMP-3. The gelatin zymography shown that mechanical injure and/or combination of PGE2promoted MMP-2activity in ACL cells exposing to conditioned medium from synovial cells in time dependent manner.6%and1.0HZ stretch ACL cell is generated by Flex cell4000tension systems under different co-culture systems for4h. The activity of ACL fibroblast was detected with MTS. Gelatin zymography was applied to detect the MMP-2activity in culture supernatants which were collected at12h after incubation. The results shown that The morphology of ACL fibroblasts showed that the cell grew well without any physical damage. The longitude direction of ACL fibroblasts under dynamic and co-culture dynamic groups have been oriented to perpendicular direction of the stretched force direction. The cellular activity of static co-culture group and dynamic co-culture group are improved significantly than the static group. However, ACL-synovial cells interaction markedly increase the expression of MMP-2in ACL.It is hypothesized Synovium and PGE2from knee joint cavity play an important role for the ACL repair. And The knee cavity micro-environment can be simulated well by the ACL and FLS dynamic co-culture system.