Enhanced Production of Mineralized Nodules and Collagenous Proteins In Vitro by Calcium Ascorbate Supplemented With Vitamin C Metabolites

Background: Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite‐supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate). Methods: Cells derived from ePTFE membranes were cultured with β‐glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite‐supplemented ascorbate. Incorporation of radioactive praline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase‐digestible protein (radioactivity released by collagenase digestion.) Co‐localization of collagen and fibronectin was examined by immunofluorescence. Results: In vitro treatment of these cells with metabolite‐supplemented ascorbate increased the area of The cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite‐supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell‐associated fraction containing the extracellular matrix. The additional collagen exhibited normal co‐distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate‐treated cultures, but was less than the observed in cultures treated with metabolite‐supplemented ascorbate. Conclusions: In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of the mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitates the formation of