The Negative Impact of High Molecular Weight Hyaluronan on Anterior Cruciate Ligament Wound Repair

Surgical reconstruction of the anterior cruciate ligament (ACL) has historically been the only method by which knee stability is restored following ACL rupture. Following ACL rupture, the torn ends of the ligament are exposed to the synovial environment, containing hyaluronan (HA), which has been implicated in the poor migratory function of ACL fibroblasts (ACLF). We hypothesize that the HA in synovial fluid attenuates the wound healing response of the ACL by inhibition of new focal adhesions between ACLF and the surrounding environment. Juvenile bovine ACLF were isolated and cultured in the presence of endogenous and exogenous high molecular weight HA (HMWHA) to monitor in vitro wound closure. Concurrently, cells were assayed for focal adhesion formation and adhesion strength. Next, human ACLF were cast into tissue-engineered constructs to assess their ability to contract within a 3D matrix after treatment with HA. A cellular viability assay was used to determine the cytotoxicity of HMWHA. Co-culture of synoviocytes with ACLF wounds demonstrated that HMWHA was the primary cause for attenuated wound healing. When exogenous HMWHA was cultured with ACLF, a dose-dependent negative correlation (r = -0.65, p < 0.001) in cell migration was observed. A significant decrease in the number and strength of focal adhesions was found to mirror the dose-dependent pattern. Collagen gel contraction was inhibited in the presence of HMWHA. Direct exposure of ACLF to HMWHA was shown to inhibit ACLF wound healing and contraction. As cytotoxicity remained unchanged, this decreased healing capacity is attributed to reduced focal adhesion formation and weakened adhesion strength of ACLF in the presence of HMWHA. This study identifies HMWHA exclusion as a potential therapeutic strategy and provides insight into the mechanism by which traditional primary repair of the ACL, as well as graft reconstructions, may fail.