Effects of resveratrol on enrichment of adipose-derived stem cells and their differentiation to osteoblasts in two-and three-dimensional cultures

Abstract

The goal of this study was to develop a method for increasing the yield of multipotent adipose-derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271(+), CD73(+) and CD45(-)) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11(+) and OCN(+). Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µM) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two-dimensional (2D) cultures as a function of alkaline phosphatase-specific activity and osteocalcin production. In addition, cells were seeded onto three-dimensional (3D) poly-ε-caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro-CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol-treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy.