Ascorbic acid is essential for significant collagen deposition by human tenocytes in vitro

Abstract

Osnat Hakimi, Raewyn Poulson, Dipti Thakkar, Clarence Yapp, Andrew Carr

Objective: We sought to compare L-ascorbic acid (AA), the natural form of vitamin C, and magnesium ascorbyl phosphate (MAP), a stable derivative, for their effect on cell proliferation, culture collagen content and collagen expression in tendon-derived cells. Methods: Tenocytes received these two ascorbate formulations at concentrations between 0.01 mM and 2 M. The effect of supplementation was evaluated in terms of cell growth, collagen expression, collagen secretion, cell morphology and collagen localization. Results: MAP supplementation resulted in dose-dependent increase in growth rates, whilst AA supplementation was toxic to tendon-derived cells from as low as 1 mM. Collagen expression was only mildly modulated by ascorbic acid, but collagen accumulation was significantly increased, supporting the hypothesis of its possible role in secretion. Immunofluorescence staining revealed differential localization of collagen I in vitro with collagen localized outside cells in the presence of ascorbic acid. Multi-photon microscopy further demonstrated that collagen fibrils were only formed in the presence of ascorbic acid. Conclusions: Taken together, these results suggest that ascorbic acid, preferably the magnesium ascorbyl phosphate formulation, should be routinely used as a supplement to tendon cell culture when collagen fibrils are required, especially in the context of tissue engineering and scaffold development.