The Interrelationship Between Human Gingival Fibroblast Differentiation and Cultivating Time

Gingival fibroblasts (GF) are the most abundant cell types in periodontal connective tissues, and have distinct functional activities in the repair of periodontal tissues as well as in inflammatory periodontal diseases. Human gingival fibroblasts (hGF) can be used as stem cells for periodontal tissue engineering. This study examined whether the in vitro differentiation of hGF into chondroblast (or osteoblasts) is possible depending on the cultivating time. hGF were obtained from the excised gingival tissue of an implant patient undergoing 2(nd) surgery. The tissue was incubated at 37A degrees C in 5% CO2 and 95% humidity, and the cultivating media was changed every 2 days. The 5(th) passage hGF cells were cultured in a medium containing Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and a 1 x antibiotic antimycotic solution. The differentiation pattern of hGF was examined using ALP staining, H & E, Alcian blue, von-Kossa and Alizarin-red staining. For ALP assay, the hGF were cultured for 1, 6 and 15 days. The control group (C) was cultured for 1 day. The experimental group (E1) was cultured 6 days. The experimental group 2 (E2) was cultured 15 days. For histologic assay using the cell block, the hGF were cultured for 8 and 57 days. The control hGF were cultured for 8 days. The experimental hGF were cultured for 57 days. The cultured cells were collected. The cell sections were stained with hematoxylin and eosin, Alcian blue, von-Kossa and Alizarin red for general histology observation. With increasing culture time, the experiment groups showed a clear spindle shape more compared to the control group as well as an increase in the number of cells. In the experimental groups, the hGF exhibited enhanced positive ALP and Alcian blue staining with increasing cultivating time. No cells in the control group showed positive staining. After cultivating for 57 days, the hGF showed positive cells for Alcian blue staining. Overall, the differentiation pattern of hGF can be changed by increasing the cultivating time.