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.

Substrate topography: A valuable in vitro tool, but a clinical red herring for in vivo tenogenesis

“…We attribute this indicative trans-differentiation to the far from physiological substrate stiffness of the PLGA material employed in this study. Indeed, substrate stiffness has been shown to strongly regulate protein expression, cell phenotype maintenance and stem cell differentiation, with soft substrates observed to be neurogenically conductive and rigid substrates to be chondrogenic / osteogenic [73][74][75][76]. This implies that multifactorial, rather than mono-domain, approaches should be assessed in future studies, in accordance with previous observations, where it was suggested that topography should be combined with mechanical loading for physiological bovine tenocyte morphology maintenance [27].…”

Osteocyte differentiation and the formation of an interconnected cellular network in vitro

“…Osteocytes are formed when cuboidal-like osteoblasts become embedded within soft secreted osteoid and start to change morphologically to a dendritic shape characteristic of an osteocyte. This transition is accompanied by a loss of cell volume (reduced organelle content) (Knothe Tate et al, 2004;Palumbo et al, 2004) and an increase in the formation and elongation of thin cytoplasmic projections that interconnect with neighbouring osteocytes within the bone ECM and osteoblasts on the surface of the bone (Palazzini et al, 1998;Palumbo, 1986;Mullen et al, 2013;Prideaux et al, 2012;Palumbo et al, 2004). Furthermore, as an osteoblast differentiates to an osteocyte, expression of the osteoblast marker enzyme alkaline phosphatase (ALP) is greatly reduced (Jee, 2001;Mikuni-Takagaki et al, 1995;Nakano et al, 2004) along with an upregulation in dentin matrix protein-1 (DMP-1) (Narayanan et al, 2003;Rios et al, 2005).…”

“…Through these methods important cellular responses have been identified, in particular regulatory mechanisms for cell proliferation, Formation of an interconnected osteocyte network migration, and matrix production, which provide information that is driving the development of approaches for regenerating bone tissue in vitro as a treatment for large bone defects. It has been shown that substrate (2D) (Engler et al, 2004;Mullen et al, 2013) and matrix (3D) (Tan et al, 2014) stiffnesses are important physical factors that induce a phenotypic shift towards osteogenic differentiation. Indeed, extracellular mechanical cues are recognised as regulators of a variety of cell behaviours such as migration (Zaman and Trapani, 2006), proliferation (Hadjipanayi et al, 2009), and differentiation (Lo et al, 2000;Tan et al, 2014).…”

“…Moreover, the extracellular mechanical environment can partially direct osteogenic differentiation and mineralisation of a variety of cells including myoblasts (Tan et al, 2014), osteoblasts (Chatterjee et al, 2010), MSCs (Engler et al, 2006;Huebsch et al, 2010;Wang et al, 2012) and embryonic stem cells (Evans et al, 2009). In particular, the effect of varying 2D substrate stiffness on cultured osteoblastic cells has been shown to favour osteocyte differentiation on relatively soft ECM substrates (0.286 kPa) (Mullen et al, 2013), whereas culture on a more rigid substrate (20-40 kPa) led to osteogenic differentiation (Engler et al, 2004).…”

“…Cell seeding density has also been shown to be a critical parameter controlling cell proliferation, ECM synthesis and osteogenic signal expression, as it dictates the paracrine signalling distance between cells (Kim et al, 2009;Mullen et al, 2013;Zhou et al, 2011). Using a 2D culture of preosteoblastic cells seeded on collagen substrates, we have recently shown that a low cell seeding density was important for dendrite formation and osteogenic differentiation, as indicated by reduced alkaline phosphatase (ALP) activity and increased mineral production (Mullen et al, 2013).…”