Epithelial-derived Microvesicles Regulate Osteogenic Differentiation in Fibroblasts

Objectives: Microvesicles (MVs) are membrane-bound vesicles shed from a variety of cells, ranging 100–1000 nm in diameter. We have reported previously that epithelial-derived MVs regulate the fibroblast phenotype. In the present study, we hypothesized that MVs from gingival epithelial cells (GEC) could regulate osteogenic differentiation in human gingival fibroblasts (HGFs).

Methods: MVs were isolated by centrifugation from the GEC conditioned medium. HGFs were cultured in basic (DMEM+FBS) or osteogenic medium (basic medium, 50 mg/mL of ascorbic acid, 100 nM dexamethasone, 100 nM vitamin D3, and 10 mM β-glycerophosphate) with or without MVs for up to 4 weeks. Von Kossa staining and energy-dispersive x-ray spectroscopy (EDX) techniques were used to determine the endpoint mineralization and calcium accumulation in HGFs, respectively. Mineralization of the HGF cultures was monitored in real-time by using calcein green staining and the IncuCyte Zoom system. RNA expression profiling by RNAseq and real-time quantitative RT-PCR was used to analyze gene expression in HGFs undergoing osteogenic differentiation. Statistical significance was set at p<0.05.
Results: Incubation of HGFs with MVs in osteogenic medium induced a cumulative osteogenic differentiation in HGFs, reaching statistical significance after one week, as detected by Von Kossa staining. Calcium and phosphorus were detected accumulating in cultures of MV-treated HGFs after 4 weeks by EDX. Mineralization in HGFs treated with MVs in osteogenic medium measured by calcein green fluorescence reached significance at 15 days. Multitude of genes showed differential gene expression between MV-treated and non-treated HGFs in osteogenic medium in early time points.
Conclusions: Human GEC-derived MVs promote fast osteogenic differentiation of HGFs in vitro and may provide a novel approach to induce bone formation in vivo.