Differentiation of osteoblasts to osteocytes in 3D type I collagen gels - a novel tool to study osteocyte responses to mechanical loading
Year: 2015
Type: dissertation
Abstract: Osteocytes are currently regarded as being pivotal in maintaining bone homeostasis. \nThey differentiate from osteoblasts, are embedded in mineralised matrix and difficult \nto isolate. Current models for in vitro osteocyte studies are limited. Others have \nsuggested that osteoblasts in 3-dimensional (3D) cultures differentiate to osteocytes. \nThis study aimed to develop 3D cultures enabling differentiation of osteoblasts to \nosteocytes, which could be used for studies of osteocyte differentiation and \nresponses to mechanical loading. Furthermore, the effects of external compounds on \nosteoblast differentiation in 3D were assessed. \nMouse (MC-3T3, IDG-SW3) and human primary osteoblasts (hOBs) were \nmaintained in type I collagen gels in either non-osteogenic or osteogenic media, and \n+/- compounds such as insulin-like growth factor-1 (IGF-1). Furthermore, \nmechanical loading (5 mins, 10 Hz, 2.5 N) was applied to 3D cultures and responses \ncharacterised. \nCells were viable in collagen gels for 25 days, and expressed mRNA for mature \nosteocyte markers e.g. sclerostin in osteogenic medium. Furthermore, IGF-1 upregulated \nmRNA expression of osteocyte markers and other molecules (e.g. receptor \nactivator of the nuclear factor kappa-β ligand - RANKL - 43-fold) in MC-3T3 cells, \nindicating modulation of cell differentiation and function. Osteocyte markers were \nexpressed earlier in IDG-SW3 cells in 3D compared to published marker expression \nprofiles in 2D monolayer cultures. Following mechanical loading, known mechanosensitive \nmarkers were modulated in IDG-SW3 cells in 3D, for example, RANKL \nand vascular endothelial growth factor (VEGF) up-regulated and sclerostin downregulated \npost-loading. \nThis 3D model enables differentiation of osteoblasts to osteocytes in an environment \nakin to osteocytes in vivo. External compounds accelerated cell differentiation, and \nthis was also accelerated in 3D compared to monolayer. Furthermore, the 3D model \nenabled osteocyte mechanical loading. This model can be used with human cells, \nwill further our understanding of osteocyte differentiation, and inform on osteocyte \nfunction including their responses to mechanical loading.
Author: Nicole Scully
Language: English
Topic: Bone Metabolism and Diseases
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1879392941