Human Primary Osteocyte Differentiation in a Three-Dimensional Culture System

Abstract Introduction: Investigations on primary osteocytes, which compose over 90-95% of bone cells, embedded throughout the mineralized matrix, is a major challenge due to their difficult accessibility and the very rare models available in vitro. We engineered a three-dimensional (3D) culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Materials and Methods: Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for four weeks in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Results: Histological analysis revealed, after only one week, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real time PCR expression of a set of bone-related genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. Conclusions: This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the crosstalk of osteocytes with tumor cells in bone metastasis since it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could be also a very efficient tool for drug testing and bone tissue engineering applications.