Functional change of osteoblasts subjected to different mechanical stimulation

Objectives: Mechanical stress plays a crucial role in bone homeostasis, and several studies have been performed to understand the effect of mechanical stimulation on osteoblast functions by applying mechanical stimulation. However, control cells in these studies are still subjected to continuous strain induced by cell adhesion on the tissue culture plate. The use of a floating cell sheet might be effective means of eliminating mechanical strain from the substrate while maintaining adhesion of the cell to the matrix. We created a technique to obtain cell sheets, and evaluated osteoblasts functional change. Methods: Poly-N-isopropylacrylamide (PNIPAAm) gel (30×30×1mm) was prepared by the mixture of PNIPAAm solution (7.7wt%) with polyethylene glycol dimethacrylate. Osteoblast-like cells (MC3T3-E1) were seeded on the gel, and were detached in the form of a cell sheet by decreasing the temperature below the lower critical solution temperature (LCST;32°C) after 24h. Expression of osteopontin (OPN) and osteocalcin (OC) genes was investigated by real-time PCR. To compare the effect of cyclic strain, mRNA expression of osteoblasts subjected to cyclic tensile strain was also investigated. Results: When the temperature was decreased below LCST, the surface area of the gel increased by approximately 43% and the cells detached from the gel surface in the form of a cell sheet. This detachment was caused by alterations in the surface area and hydrophobicity of the polymer surface. In the stress-free condition, OPN gene expression increased 121±13% and OC gene expression decreased 52±6%. In contrast, mechanical stress induced a 56±3% decrease in OPN expression and an 84±3% increase in OC expression. Conclusion: An osteoblast sheet-mechanical stress originating from the cell culture substrate-was fabricated using PNIPAAm gel. The levels of expression of OPN and OC genes in the absence of mechanical stress were negatively correlated with those in the presence of mechanical stimulation.