Hyaluronic acid stimulates human fibroblast proliferation within a collagen matrix

Human dermal fibroblasts suspended in a collagen matrix exhibit a 4-day delay in cell division, while the same cells in monolayer divided by day 1. The initial rates of 3H-thymidine incorporation by cells in monolayer or suspended in collagen were not significantly different. When suspended in collagen, there was a threefold increase in the proportion of cells in a tetraploidal (4N) DNA state compared to the same cells in monolayer. Flow cytometry analysis and 3H-thymidine incorporation studies identified the delay of cell division as a consequence of a block in the G2/M of the cell cycle and not an inhibition of DNA synthesis. The inclusion of 150 microg/ml of hyaluronic acid (HA) in the manufacture of fibroblast populated collagen lattices (FPCL) caused a stimulation of cell division, as determined by cell counting; increased the expression of tubulin, as determined by Western blot analysis; and reduced the proportion of cells in a 4N state, as determined by flow cytometry. HA added to the same cells growing in monolayer produced a minimal increase in the rate of cell division or DNA synthesis. HA supplementation of FPCLs stimulated cell division as well as tubulin concentrations, but it did not enhance lattice contraction. The introduction of tubulin isolated from pig brain or purchased tubulin into fibroblasts by electroporation prior to their transfer into collagen lattices promoted cell division in the first 24 hours and enhanced FPCL contraction. It is proposed that tubulin protein, the building blocks of microtubules, is limited in human fibroblasts residing within a collagen matrix. When human fibroblasts are suspended in collagen, one effect of added HA may be to stimulate the synthesis of tubulin which assists cells through the cell cycle.