Effects of dermal-fibroblast-derived ECM and dextran sulfate supplementation on osteoblast differentiation - results of a preliminary in vitro study.

Abstract

Critical size bone defects represent a clinical challenge, associated with considerable morbidity, and frequently trigger the requirement of secondary procedure. To fill osseous gaps, multiple steps are required, such as proliferation and differentiation on the cellular level and the building of extracellular matrix. In addition, the osteogenic potential of cell-derived extracellular matrices (CD-ECM) is known to enhance bone healing. We therefore examined the osteogenic potential of fibroblast-derived ECM (Fibro-ECM) and assessed the influence of Dextran-sulfate (Dx-S) addition regarding the production of extracellular matrix (ECM). ECMs were generated by culturing human dermal fibroblasts, adipose-derived stromal cells (ASCs), and osteoblasts derived from ASCs (Osteo-ECM) for four days, with or without Dx-S supplementation. After decellularization, skeletal stem cells (SSCs) isolated from femoral head aspirations were seeded onto the ECMs and differentiated under osteogenic conditions for 17 days. Osteogenesis was assessed by Alizarin Red S staining for calcium deposition and RT-qPCR analysis of osteogenic marker genes. SSCs cultured on Fibro-ECM exhibited enhanced osteogenesis compared to Osteo-ECM and ASC-derived ECM, as evidenced by increased calcium deposition. Notably, Dx-S supplementation further improved the osteoinductive capacity of Fibro-ECM, leading to an upregulation of osteocalcin (OCN) and bone morphogenetic protein 2 (BMP2). In contrast, Dx-S had no significant effect on Osteo-ECM. The addition of Dx-S in autologous fibroblast-derived ECM induces an improvement in osteoinductivity. Addition of Dx-S may therefore be a useful adjunct in the in vitro bone generation models. Whether these results may represent a piece in the puzzle for difficult healing situations in patients with nonunions and bone defects should be subject to further study.