Dental Pulp Stem Cell Conditioned Medium Enhancing Gingival Fibroblast Properties

Objectives: To investigate the biological effects of dental pulp stem cell-derived conditioned medium (DPSC-CM) on human gingival fibroblasts (HGFs).
Methods: DPSC was isolated and characterized. DPSC-CM were obtained from cell culture supernatants after 2 days in serum-free medium. The total protein in DPSC-CM was determined using Bradford assay. For the cell viability assay using MTT, HGFs were treated with various concentrations of DPSC-CM (1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µg/ml) in serum-free DMEM for 24 hours. For the proliferation assay using CCK-8 assay, HGFs were treated with 3.12 and 6.25 µg/ml of DPSC-CM in DMEM with serum. The proliferation of HGFs after treatment on days 1, 3, 5, and 7 was measured. For collagen production using picrosirius red staining, HGFs were treated with 3.12 and 6.25 µg/ml of DPSC-CM in DMEM with serum. The qualitative and quantitative analyses of collagen amount were conducted on days 7 and 14. The COL1A1 and COL3A1 gene expression was examined at days 7, 14 and 21 by real-time PCR.
Results: HGFs treated with DPSC-CM at 3.12 and 6.25 µg/ml showed significantly higher cell viability than that of the control group without DPSC-CM. DPSC-CM at concentrations of 3.12 and 6.25 µg/ml significantly increased proliferation on day 7. Collagen amount was significantly higher in HGFs treated with 3.12 and 6.25 µg/ml of DPSC-CM at days 7 and 14, compared to the control group. Relative gene expression of COL3A1 was upregulated at days 7 and 14, compared to the control. The 6.25 µg/ml showed the highest expression of COL3A1 at both days, and of COL1A1 at day 14. At day 21, both COL3A1 and COL1A1 were down-regulated.
Conclusions: DPSC-CM has the potential to promote the viability, proliferation, and collagen production in HGFs, indicating its promising properties to apply for enhancing oral wound healing.