Mullen C.A.,Center for Biomechanics Research |
Mullen C.A.,National University of Ireland |
Haugh M.G.,Center for Biomechanics Research |
Haugh M.G.,National University of Ireland |
And 4 more authors.
Journal of the Mechanical Behavior of Biomedical Materials
Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for the regulation of bone formation and resorption. ECM stiffness and cell seeding density have been shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast-osteocyte differentiation are not yet understood. In this study, we cultured MC3T3-E1 cells on (A) substrates of different chemical compositions and stiffnesses, as well as, (B) substrates of identical chemical composition but different stiffnesses. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP-1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP-1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro. © 2013 Elsevier Ltd. Source
Birmingham E.,Center for Biomechanics Research |
Birmingham E.,National Center for Biomedical Engineering Science |
Birmingham E.,Regenerative Medicine Institute REMEDI |
Niebur G.L.,National Center for Biomedical Engineering Science |
And 14 more authors.
European Cells and Materials
Mesenchymal stem cells (MSCs) within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely infl uence how MSCs differentiate to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro; however, the role of the natural cues from bone's regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP) activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more infl uential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel coculture system confi rmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications. Source