Metformin and PRF enhaced the osteogenic differentiation of hPLSCs

Abstract

This study compares the regenerative power of Met and PRF and the ability of both materials alone and in combination to induce osteogenesis in-vitro using human periodontal ligament stem cells (hPLSCs). PRF was prepared using 10 ml venous blood and centrifuged at 1200 rpm for 12 minutes. The hPLSCs were divided into 4 groups; cells cultured in normal culture medium served as the control group, cells cultured in osteogenic medium served as Osteo group, cells cultured in osteogenic medium conditioned with PRF served as PRF group, and the combination group which consisted of cells cultured in osteogenic medium conditioned with PRF and Met. After osteogenic induction, osteogenic differentiation was assessed by measuring mineralization degree, alkaline phosphatase enzyme (ALP) activity, and the expression of osteoprotegerin (OPG), RUNX Family Transcription Factor 2 (RUNX2), and osteocalcin (OC) by RT-PCR. The Met concentration of 250 uM showed 100% cells viability and was used in the all the experiments of this study. The combination group showed the highest mineralized nodules formation among all study groups with mean value of 0.7 ± 0.02. Same results were observed with ALP assay, the combination group showed the highest p-NP accumulation denoting the highest ALP activity (p<0.0001). The combination group showed the highest expression of osteogenic markers including RUNX2, OC and OPG with mean value of 5.83 ±0.08 (p< 0.05), 2.88 ±0.03 (p<0.0001), 3.32 ±0.16 (p<0.001), respectively. The combination of Met and PRF showed better enhancement of osteogenic differentiation than using each material alone.