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.
Highlights
Bone is a complex multicellular organ that possesses the ability to functionally adapt its structure to meet the biochemical and biophysical demands placed upon it
To determine the effect of substrate composition upon osteocyte viability and metabolic activity, MLO-Y4 cells were seeded on collagen-based 2D films consisting of: (i) collagen, (ii) collagen-GAG, (iii) collagen-HA, and (iv) collagen-GAG-HA
Bone formation is a complex process regulated in part by the osteocyte specific protein Bone formation is aline complex process part by thethe osteocyte specific prosclerostin
Summary
Bone is a complex multicellular organ that possesses the ability to functionally adapt its structure to meet the biochemical and biophysical demands placed upon it. Evidence suggests that the master orchestrator of bone mechanoadaptation is the osteocyte [3,4,5], which accounts for over 90% of the cell population in mature bone [3]. These stellate cells reside within a calcified matrix, in spaces termed lacunae, and extend long processes that interconnect to coordinate secretion of biological signalling factors, such as sclerostin, regulating bone anabolism [6,7,8,9,10]. Bone extracellular matrix (ECM), formed by osteoblasts, is a composite material comprised of both organic and inorganic components.
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