Passaging of gingival fibroblasts from periodontally healthy and diseased sites upregulates osteogenesis-related genes

Abstract

Abstract In order to investigate biological processes of the periodontium, in vitro primary cell models have been established. To study the biology of the gingiva, primary gingival fibroblast cell models are widely used. For such experiments, cells need to be expanded and passaged. A key assumption is that primary cells maintain most of their original characteristics they have in situ. The aim of this research is to explore the impact of early passaging on selected gene expression of human gingival fibroblast cells. For this purpose, gene expression from outgrowth of the resected tissues until the fourth passage was followed for 9 tissue samples, from both healthy and diseased sites. Micrographs were taken from the cultures, RNA was extracted from the samples of each passage and quantitative PCR was performed for selected genes representing various biological processes. Epithelial cells were present during the first outgrowth, but were no longer present in the second passage. Our results indicate that the morphology of the gingival fibroblast cells does not change with passaging and that passages 2 to 4 contain only gingival fibroblasts. Gene expression of IL-1β, M-CSF, TNF-α, TLR4, POSTN, and FAPα was unchanged by passaging, expression of IL-6 and TLR2 decreased due to passaging and expression of in particular the selected osteogenesis genes (ALP, RUNX2, Osteonectin, COL1A), OPG and MKI67 increased with passaging. Our results emphasize the importance of using the same passage in experiments that use primary gingival fibroblasts from various donors.