The migration of cells from the ruptured human anterior cruciate ligament into collagen-glycosaminoglycan regeneration templates in vitro.

Guided tissue regeneration of the ruptured anterior cruciate ligament (ACL) offers the potential benefits of retaining the complex footprints of the ACL and the proprioceptive nerve fibers of the tissue. For this approach to be successful, ACL cells must retain the ability to migrate into an adjacent regeneration template, or scaffold, after ligament rupture. Ruptured ACLs were obtained from the knees of four men, ages 25-35, at the time of ACL reconstruction. Explants of ACL tissue were taken from three locations along the longitudinal axis of the remnant: the rupture site, the middle of the remnant, and far from the rupture site. These three areas have been found to be distinct histologically, with the region far from the rupture site having a histologic appearance similar to the intact ligament. Explants from each area were cultured in conventional tissue culture dishes (2-D culture) and on porous collagen-glycosaminoglycan (CG) scaffolds. Two-dimensional outgrowth was measured 3 times a week, and the 3-D explant/scaffold constructs were examined at 1, 2, 3 and 4 weeks to assess outgrowth of cells into the scaffold. The cell number density and expression of a-smooth muscle actin (SMA) were determined at each time point. The decrease in the diameter of the scaffolds and non-seeded controls were determined as a function of time in culture. The outgrowth of cells onto the tissue culture dishes was observed to begin as early as 3 days and as late as 21 days, with outgrowth first detected at an average of 6.8 +/- 2.0 days after explantation. In general, there was a larger area of outgrowth at the 2-week time point from explants with higher cell number density and higher blood vessel density. The 2-week area of outgrowth also correlated with the percentage of SMA-positive cells in the explant. In the experimental constructs with CG scaffolds, fibroblasts were noted to migrate from the human ACL explants into the templates at the earliest time point recorded (I week). The migration and proliferation of cells from the explants in the CG matrices resulted in an increase in the cell density in the scaffolds with time. There was a significant effect of the location from which the explant was taken on cell density in the scaffold, with a higher density of cells migrating from the explants from the rupture site of the ACL specimens. The percentage of cells staining positive for the SMA isoform varied from 0 to 50% of cells in the scaffold. Scaffolds co-cultured with explants showed a reduction in diameter that was significantly affected by time in culture and the location in the ACL from which the explant was taken. The percentage contraction attributed to the cells was 15% at 2 weeks, and increased to 27% for the injury-zone explant at 4 weeks. There was a significant correlation of the cell-mediated contraction of the matrices at 4 weeks with the cell density in the scaffolds, but not with the number of SMA-positive cells in the scaffolds. These data demonstrate that cells in the human ACL retain their ability to migrate into an adjacent CG scaffold in vitro, weeks after complete rupture. Moreover, the ACL-derived cells can express a contractile actin isoform and can contract a CG analog of extracellular matrix.