The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

Abstract

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model.

Bone on-a-chip: a 3D dendritic network in a screening platform for osteocyte-targeted drugs

“…We observed a trend towards increased expression of Cx43 in 3D Oc also by the analysis of mRNA, isolated from cells recovered from the microfluidic channel, compared to cells cultured under standard conditions (figure 4(c)). Remarkably, Sost, which is usually not expressed in 2D cultured of MLO-Y4 cells [50], as also observed in our cultures, was significantly induced in 3D (figure 4(c), * * p = 0.0043). Perfusion and inclusion in the matrix also strongly promoted the expression of alkaline phosphatase, an additional marker of the Oc phenotype, as was clearly revealed by the comparison with the 2D perfused condition (channel A) or with the 2D standard culture condition, for both cell lines (figure 4(d)).…”

In vitrodevelopment and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale

“…This protein is related to bone metabolism regulation, activating the Wnt/βcatenin signaling pathway [33]. Stiffness (mineralized matrix) has been demonstrated to influence the expression of SOST [34], and although this mechanism is not entirely understood, osteocytes have been proposed to have mechanosensors such as fibrils, integrin-based focal adhesion, ion channels, and connexin gap junctions, which could regulate sclerostin activation [32,35]. All of these mechanoregulatory factors point to an important participation of calcium, phosphate and their transformation into rigid minerals in the bone matrix as important determinants of osteocyte response.…”