The interactions between cells and their surrounding extra-cellular matrix (ECM) affects  cell behaviour during various cellular processes, like attachment, proliferation, differentiation, and has a determining role in the final cell fate. Guiding cellular differentiation through modulation of the substrate stiffness was proven to be an effective method to change the stem cell fate towards osteogenesis. Furthermore, mechanical loading is known to be a strong catalyst for osteogenic differentiation while tissue is being structured under physiological conditions.                  

We aimed to design a multifactorial system that will use mechanical loading, by substrate deformation, together with altered substrate elasticity properties.We would like to understand whether the combination of an appropriate substrate elasticity with the presence of mechanical loading could have a synergistic effect on osteoblast differentiation, or otherwise what is the over-ruling mechanism that determines the in vitro cell differentiation for hard tissue regeneration.   

Stretchable poly-dimethylsiloxane (PDMS) elastomer dishes were fabricated by mixing the monomer and catalyst components in 10:1 ratio and cured overnight at RT. Poly-acrylamide (PAAm) (123 kPa) hydrogels were synthesized according to previous protocol. Following the Ar-plasma treatment and silanization of PDMS surface, PAAm hydrogel films were attached on half of the PDMS dishes and incubated overnight at 37°C (Figure 1). Both types of dishes were coated with collagen (50µg/ml) monomer to obtain cell attachment. Rat bone marrow-derived stem cells (BMSCs ) of early passage numbers (<5) were used for all experiments. 1x104 cells/cm² were seeded on each dish. Following 8 hours rest after the medium change uniaxial stretch was applied with the magnitude of 8% and a constant frequency of 1 Hz for 16 hours intermittently (Figure 2). Samples were collected 3 days after the mechanical loading protocol was stopped and cell nuclei and actin filaments of the cytoskeleton were visualized by DAPI and Phalloidin, respectively and imaged by CLSM. The expression of the genes of interest such as COL-1, RUNX-2, OCN, BSP, and OPN was analyzed by RT-PCR. 

Our findings show that cells were perpendicularly aligned to the stretch direction (Figure 3). We observed higher number of cells on PDMS surface than PAAm coated substrates. According to the RT-PCR results COL-1 and OCN expression was upregulated for PAAm coated samples with mechanical loading (Figure 4). In conclusion, the mechanical loading overruled the stiffness sensing during in vitro osteogenic differentiation of mesenchymal stem cells.