Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration.

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades. However, efficient harvest and handling of cell sheets remain challenging, including insufficient extracellular matrix content and poor mechanical strength. Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types. However, currently, there are no effective ways to apply mechanical loading to cell sheets. In this study, we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide) (PNIPAAm) to poly(dimethylsiloxane) (PDMS) surfaces. The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting. Subsequently, MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates. Upon maturation, the cell sheets were harvested by lowering the temperature. We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning. Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated. After implantation into the critical-sized calvarial defects of mice, the mechanically conditioned cell sheets significantly promoted new bone formation. Findings from this study reveal that thermo-responsive elastomer, together with mechanical conditioning, can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.