Human Gingival Fibroblast: Single Colonies vs. Heterogeneous Culture, Characterization and Osteogenic Differentiation

Abstract

Cells with mesenchymal stem cell (MSC) properties were successfully isolated and characterized from different dental tissues. Amongst these cells are the human gingival fibroblast cells (GFs). Their use in tissue engineering is promising. However, choosing the right cellular population is one of several factors that are necessary for a successful tissue engineering approach. In order to better choose which population of cells to use, we isolated the GFs single colonies. We identified them, osteogenically induced them and compared them to the heterogeneous culture of these cells. Materials and methods: GFs cells were extracted from human gingival tissue; incubated to confluency. After which they were counted, serially diluted and seeded in 6 well plates. The cells were observed daily to locate the first formed colonies. Borosilicate cylinders were used to pick up the colonies. Flow cytometry was used to identify Stem cells surface markers to compare single colonies and heterogeneous cultures. The cells were then osteogenically induced for 21 days. The following assays were performed to compare the osteogenic potential between single colonies and heterogeneous cultures; Calcium assay, ALP/DNA specific activity, RT-qPCR for osteogenic related genes (OPN, OCN, ALP) and western blot analysis. Results: All the assays results were consistent in revealing an increased osteogenic differentiation potential of the heterogeneous culture of the GFs over the single colonies cultures. These results indicate that the heterogeneous cultures of GFs have a higher stem cell population and subsequent osteogenic differentiation potential than the single cell colonies’ cultures.