title: Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation.
abstract: 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.
authors.affiliation: Centre for Biomechanics Research (BMEC), Mechanical and Biomedical Engineering, NUI Galway, Ireland; National Centre for Biomedical Engineering Science (NCBES), NUI Galway, Ireland.
authors.forename: C A
authors.initials: CA
authors.lastname: Mullen
authors.forename: M G
authors.initials: MG
authors.lastname: Haugh
authors.forename: M B
authors.initials: MB
authors.lastname: Schaffler
authors.forename: R J
authors.initials: RJ
authors.lastname: Majeska
authors.forename: L M
authors.initials: LM
authors.lastname: McNamara
mesh_terms: D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D002454:Cell Differentiation; D005109:Extracellular Matrix / Q000378:metabolism*; D005786:Gene Expression Regulation; D055595:Mechanical Phenomena; D051379:Mice; D008903:Minerals / Q000378:metabolism; D010011:Osteocytes / Q000166:cytology* / Q000378:metabolism
chemical_list: D008903:Minerals; D000469:Alkaline Phosphatase
keywords: Bone; Cellular differentiation; Extracellular mechanical environment; Osteoblast; Osteocyte