Quantitative Evaluation of the Factors Affecting the Process of Fibroblast-Mediated Collagen Gel Contraction by Separating the Process into Three Phases

Abstract

Kinetics of collagen gel contraction by fibroblasts cultured in vitro was examined in detail for quantitative analysis. The process of collagen gel contraction was not expressed by a simple function of time. It appeared to consist of three distinct phases; a lag phase before the initiation of contraction, a rapid contraction phase and a slow contraction phase. Factors affecting the gel contraction can be classified into four groups. The first group includes increase in cell number, in culture temperature or in serum concentration, which strengthened the contraction in all the three phases, suggesting that they affected cellular activity particularly in interacting with collagen. The second group repressed the later two phases of contraction but not the first lag phase, typically increase in collagen concentration and a low dose of nocodazole or colcemid. Increasing population doubling levels of fibroblasts belongs to the third group which caused a reduced lag time but no change in the later two phases. Cytochalasin D at a low dose (0.03–0.1 µg/ml) is another example of the third group which shortened the lag time. The last group did not change the contraction curves. Donor age of fibroblasts isolated from the skin is an example of this group. The rate of rapid contraction in the second phase was always found to be closely correlated with the degree of contraction at the end of the third phase, in a whole set of the factors above mentioned. The results suggest that the extent of the later two phases might be a reflection of the same cellular activity, particularly cytokinetical one. The lag time is directly related to the time for cells to become elongate in shape as observed by using the video-microscopy, suggesting that the lag phase is also governed by cytokinetical activity. The two cytokinetical activities are closely related, but may be distinct, since the factors affecting the collagen gel contraction can be differentiated into four groups.