New 3D system for evaluating the response of osteocyte to mechanical loading

Osteocytes play a vital role in the adaptation of bone to mechanical loads since they translate mechanical stimuli into a biological response which regulate bone resorption and bone formation. The molecular mechanisms governing the dynamics of osteocyte response to mechanical loads are not completely understood.

Here we present a novel 3D osteocyte culture under shear force to characterize the kinetics of their response to different load regimes. In our system osteocytes are cultured on collagen scaffolds in a unique bioreactor as a perfusion vessel, the system is a closed system. The flow pressure could be controlled and the shear stress applied to the cells could be calculated. Up to 40 scaffolds could be cultured in a single experiment providing an optimal source for mRNAs, proteins and metabolites expressed and secreted by osteocytes under dynamic loading.

Our preliminary data shows that MLO-Y4 and IDG-SW3 osteocytic cell lines could adhere to the 3D scaffolds in a unified manner. IDG-SW3 cells cultured on the collagen scaffolds were induced to differentiate to mature osteocytes and expressed DMP-1, a marker for osteocyte maturation was significantly upregulated as compared to earlier time points. In addition alizarin red staining of scaffolds after two weeks of differentiation was significantly increased suggesting osteocyte could mature when cultured on the collagen scaffolds. Cells cultured on the scaffold showed typical osteocyte morphology with numerous membrane projections. Finally measurements of NO secretion, a marker for osteocyte response to mechanical loading showed increased NO levels after fluid flow shear force was applied by the bioreactor.