Studies on primary osteocytes, which compose >90-95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 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. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk 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. Histological analysis showed, after only 1 wk, 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 plasticcultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. 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 cross-talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.

Prostate and mammary cancer are among the leading cancers diagnosed and the second leading cause of cancer death in men and women, respectively.1 Both cancers show a high propensity to metastasize to bone. Whereas prostate cancer (CaP) elicits predominantly an osteoblast response resulting in osteosclerotic lesions, mammary cancer (CaM) triggers preferentially an osteoclast reaction with bone resorption and consequent osteolytic lesions.2 Osteolytic and osteosclerotic lesions are prone to pathological fractures. A better understanding of the mechanism(s) determining the osteoclast and osteoblast response to cancer metastases is essential for the identification of therapeutic strategies for prevention of pathological bone fractures in cancer patients.Several factors stimulating osteoblast proliferation and differentiation in a paracrine manner have been shown to be released by CaP and CaM cells in the bone microenvironment and have been postulated to mediate osteoblast response in bone metastasis. 3,4 Factors that modulate proliferation and differentiation can act directly on the osteoblast progenitors or indirectly by activation of factors involved in their generation. 4 Paradigmatic molecules regulating directly osteoblast generation are the bone morphogenetic proteins (BMPs).5 BMPs were first identified by their ability to induce ectopic chondro-osteogenesis in vivo. 6 They play a crucial role in skeletal and joint morphogenesis, bone

Protein kinase CK2, a vital, pleiotropic and highly conserved serine/threonine phosphotransferase is involved in transcription-directed signaling, gene control and cell cycle regulation and is suspected to play a role in global processes. Searching for these global roles, we analyzed the involvement of CK2 in gene expression at cell cycle entry by using genome-wide screens. Comparing expression profiles of Saccharomyces cerevisiae wild-type strains with strains with regulatory or catalytic subunits of CK2 deleted, we found significant alterations in the expression of genes at all cell cycle phases and often in a subunit-and isoform-specific manner. Roughly a quarter of the genes known to be regulated by the cell cycle are affected. Functionally, the genes are involved with cell cycle entry, progression and exit, including spindle pole body formation and dynamics. Strikingly, most CK2-affected genes exhibit no common transcriptional control features, and a considerable proportion of temporarily altered genes encodes proteins involved in chromatin remodeling and modification, including chromatin assembly, (anti-)silencing and histone (de-)acetylation. In addition, various metabolic pathway and nutritional supply genes are affected. Our data are compatible with the idea that CK2 acts at different levels of cellular organization and that CK2 has a global role in transcription-related chromatin remodeling.