The anterior cruciate ligament has little capacity to heal after injury, in contrast to the medial collateral ligament. Cell migration and cell proliferation are essential steps in the wound healing of connective tissue, and we have developed an in vitro assay to study cell outgrowth (i.e., both cell migration and cell proliferation) from tissue explants. Using this assay, we have previously shown a synergistic effect of four growth factors on the early stages of cell outgrowth (i.e., at days 3 and 6) from these explants. Hyaluronan, a linear polysaccharide with a molecular weight of 3 x 10(6) daltons, has been shown to enhance the anterior cruciate ligament healing capacity in vivo by over 50%. The purpose of this study was to assess in vitro whether hyaluronan and the combination of growth factors (transforming growth factor-beta1 25 ng/ml, platelet-derived growth factor-BB 100 ng/ml, basic fibroblast growth factor 100 ng/ml, and insulin 25 microg/ml) would enhance cell outgrowth from explants at 3 and 6 days. At 3 days the mean cell outgrowth from the explants treated with hyaluronan and the combination of growth factors exceeded the growth in the explants treated with growth factors alone for the anterior cruciate ligament (mean cell count +/- standard error: 19 +/- 4 and 2 +/- 1 cells, respectively) and for the medial collateral ligament (297 +/- 40 and 87 +/- 18 cells, respectively). At 6 days, the mean cell outgrowth in the presence of growth factors was enhanced sixfold by hyaluronan for the anterior cruciate ligament (160 +/- 27 compared with 26 +/- 15 cells) and fourfold for the medial collateral ligament (1363 +/- 219 compared with 330 +/- 74 cells). As previously shown, little cell outgrowth occurred from the anterior cruciate or medial collateral ligament explants when no growth factors were added to the modified Eagle's media at 3 and 6 days (<20 cells). For the medial collateral ligament only, hyaluronan had a small stimulatory effect on cell outgrowth in minimal essential media in the absence of growth factors. At 3 days the stimulation by hyaluronan was fourfold (31 +/- 8 cells compared with 7 +/- 4 cells), and at 6 days the stimulation by hyaluronan was nearly fourfold (73 +/- 17 compared with 20 +/- 12 cells). These results suggest that hyaluronan could be used together with growth factors in efforts to enhance the healing of ligamentous tissue injuries.