AN IN-VITRO MODEL OF CALCIFICATION FOR THE STUDY OF THE OSTEOGENIC POTENTIAL OF ADULT HUMAN DERMAL FIBROBLASTS.

In order to investigate the calcification process in both physiological or pathological conditions, in vitro osteogenic assays are generally performed using bone-derived cells, bone-marrow-derived mesenchymal stromal cells or vascular smooth muscle cells. In normal healthy individuals, mineral formation is limited to specialized tissues as skeletal bone and teeth, however, there are many disorders ( i.e. diabetes, kidney diseases,atherosclerosis as well as genetic conditions) in which soft connective tissues undergo mineralization. In the present study a calcification assay has been established by isolating dermal fibroblasts from adult individuals and by growing these cells in a calcifying medium in which DMEM has been supplemented with 10mM β-glycerophosphate, 50μg/ml ascorbic acid and 10 nM dexametasone. After different periods of culture, up to 40 days, fibroblast cell cultures were stained with the Von Kossa method and the activity of alkaline phosphatase (ALP) measured by a spectrophometric assay. Results indicate that in-vitro human dermal fibroblasts, which are characterized by a limited life span in culture, are capable to mineralize their secreted extracellular matrix, when grown in the presence of an osteogenic medium. Moreover, the process of mineralization appeared to progresses with time, since areas of calcifications become visible after two weeks of culture. Consistently with the activation of the osteogenic phenotype, fibroblasts exhibited also an upregulation of ALP activity. However, we have observed a remarkable heterogeneity among cells from different individuals, supporting the hypothesis that ALP is not a unique marker of calcification and that the mineralization process is the result of a fine regulation of many inhibitors and stimulatory factors.