Combined Effects Of Microenvironment Factors Of Joint Cavity On MMP-2 Production After ACL Injury

Injury to the anterior cruciate ligament (ACL) causes knee instability, pain, and may result in progressive degeneration and premature osteoarthritis. An injured ACL does not heal satisfactorily as compared to other joint tissues such as medial collateral ligament (MCL), making it as one of the most challenging and intractable clinical problem today. Apart from these intrinsic differences, microenvironment in knee joint cavity also regulates the ligament's healing ability. Therefore, in this study, we focus on microenvironment of joint cavity to explore the cause of ACL repair failure. The main experiments and results are as follows:1) Contributions of different intraarticular tissues to the acute phase elevation of synovial fluid MMP-2 following rat ACL ruptureWith a rat ACL rotating injury model, we found that levels of IL-1β, IL-6, and TNF-αwere significantly higher in synovial fluids after ACL injury. MMP-2 activity and global MMP activity in synovial fluids also increased significantly in a time-dependent manner. Ex vivo studies showed that all tissues contributed to the elevation of MMP-2 in synovial fluids especially synovium and the injuried ACL. We concluded that although the regular wound healing mechanism also accurs after ACL injury, accumulation of MMP activity in the synovial fluids, due to all of the intraarticular tissues, may be at least one of the important reasons why an injured ACL cannot be repaired.2) The effects of microenvironment factors on MMP-2 production①Differential MMP-2 activity induce by mechanical compression and inflammatory factors in synoviocytesSynovium may be the major regulator of MMPs in joint cavity after injury, in vitro study to determine whether mechanical injury and inflammatory factors will induce MMP-2 production in synoviocytes. We found mechanical compression increased the MMP-2 production. In addition, TNF-αcan also elevate the activity of MMP-2 in a dose dependent manner, while IL-1αdoes not. However, mixture of these two factors dramatically increased MMP-2 production. In addition, mechanical injury had a strong synergistic effect on MMP-2 production with IL-1α, TNF-αand their mixture. The generic MMP activity assay revealed that mechanical compression increased the generic activity by only , APMA treatment increased the generic activity of MMPs induced by compression but not inflammatory factors, which indicated that compression would induced MMPs in pro-form while inflammatory factors induced MMPs mostly in active-form.②Combined effects of TNF-αand IL-1βon MMP-2 production in ACL fibroblasts under mechanical stretchThe dynamics between inflammatory factors, mechanical stress and healing factors, in an intra-articular joint, are very complex after injury. Injury to intra-articular tissue (anterior cruciate ligament, ACL, synovium) results in accumulation of various pro-inflammatory factors, cytokines and metallo-proteases. Although the presence of increased amounts of MMPs in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP-2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury and inflammation on induction of MMP-2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF-αand IL-1βelevate the activity of MMP-2 in a dose and time dependent manner. In addition, mechanical stretch further enhances the MMP-2 protein levels with TNF-α, IL-1β, and their mixture. Our results conclude that mechanical injury and inflammatory factors collectively induce increased MMP-2 production in ACL fibroblasts.③Mechanical stretch and HIF-1αaffects vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) in ACL fibroblastsHypoxia plays important role in regulating microenvironment of joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we investigated the influence of hypoxia on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and hypoxia inducible factor-1 alpha (HIF-1α) expression in ACL fibroblasts after mechanical injury. The real-time PCR results show that mechanical stretch or hypoxia increases the expression of MMP-2, VEGF, CTGF and HIF-1α; however, the synergistic effects of mechanical stretch and hypoxia increased MMP-2 production but decreased the VEGF, CTGF and HIF-1αexpression. Western blot analysis and ELISA also confirmed these results. Our results demonstrate that increased levels of MMP-2 and decreased levels of HIF-1α, VEGF and CTGF are detrimental to ACL fibroblasts hence affecting their healing ability and may be one of the prime reason's of tissue degeneration under hypoxia resulted after injury. 3) NF-κB pathway is one of critical pathway of the MMP-2 release in the ACL fibroblasts.In these studies, the production and underlying signaling pathway for MMP-2 were investigated. Further studies showed that the induced MMP-2 was inhibited by Bay11-7082 (NF-ΚB inhibitor), but was not inhibited by PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB203850 (p38 inhibitor).In summary, our results suggested that the differentially effect of microenvironmental factors on ACL might alter the balance in tissue healing, which might in turn be responsible for the ACL poor healing ability. We believe that improving the microenvironment and intervention of MMPs production in the joint fluid after knee injury would be very important in the tissue injury/remodeling processes of human knee.