The specific objective of this study was to determine the chronology of the appearance of the myofibroblast in the healing ligament. The overall goal of our work is to elucidate the cellular mechanism of contraction in this tissue. The myofibroblast has been found to be responsible for wound contraction in many tissues and to be the cause of the contracture in several pathological conditions. This cell type contains the actin isoform previously thought to be unique to smooth muscle cells and displays certain characteristic features at the ultrastructural level. In 26 New Zealand White male rabbits, the right medial collateral ligament was transected, whereas the left medial collateral ligament received a sham operation. The central third of the ligament (ligament scar tissue) was evaluated at 2, 3, 6, 8, 10, and 12 weeks postoperatively by immunohistochemical techniques, transmission electron microscopy, and Western blot analyses. Three other rabbits served as anatomic controls. During the early reparative phase (2 and 3 weeks after transection), there was an increase in the number of cells containing alpha-smooth muscle actin as well as augmentation of the alpha-smooth muscle actin content within each cell--a finding attributed to smooth muscle cells and pericytes associated with neovascularity. No myofibroblasts were detected at this stage, immediately postoperatively, or in the sham-operation controls. Ligaments in the remodeling phase of healing (6, 8, 10, and 12 weeks) exhibited alpha-smooth muscle actin in fibroblasts (myofibroblasts) as well as in vascular pericytes and smooth muscle cells. During this stage of healing, transmission electron microscopy demonstrated an increase in the number of cells displaying myofibroblastic features. It was estimated that at 12 weeks of healing 10% of the cells at the site of injury were myofibroblasts. This is the first definitive finding of myofibroblasts in the injury site of the healing ligament, to our knowledge. The appearance of myofibroblasts in the 6-12 week healing period, the interval during which the ligament has been shown to contract in studies by other investigators, is a rationale for a hypothesis that a cellular contractile apparatus comprising alpha-smooth muscle actin (i.e., the myofibroblast) may contribute to the recovery of original ligament length (and normal in situ strain).

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The significantly higher incidence of anterior cruciate ligament (ACL) injuries in collegiate women compared with men may result from relative ligament laxity. Differences in estrogen and relaxin activity, similar to that seen in pregnancy, may account for this. We quantified estrogen receptors by flow cytometry and relaxin receptors by radioligand binding assay in human ACL cells and compared the presence of these receptors in males and females. ACL stumps were harvested from seven males and eight females with acute ACL injuries. The tissue was placed in M199 cell culture medium. Outgrowth cultures were obtained, and passage 2 cells were used for all studies. Estrogen receptor determination was performed using flow cytometry. Relaxin binding was performed in ACL cells derived from five female and male patients using I(125)-labeled relaxin. Estrogen receptors were identified by flow cytometry in 4 to 10% of ACL cells. Mean fluorescence of cells expressing estrogen receptors was approximately twice that of controls, with no significant differences between males and females. Relaxin studies showed low-level binding of I(125)-relaxin-labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

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