The effects of substrate stiffness on mesenchymal stem cell proliferation and differentiation

The aim of the project was to test the hypothesis that substrate stiffness will affect MSC proliferation and differentiation. In order to achieve this two systems were developed; a natural fibrin substrate, whereby altering the concentration of fibrinogen changes the stiffness of the resultant gel and an artificial PDMS substrate where the stiffness is controlled by altering the degree of crosslinking. To determine the effect of various fibrin matrices, providing more physiological growth conditions, on the MSC phenotype; cells were cultured on the gels and then analysed or re-plated onto TCP before analysis. It was found that cells, that had an initial 7-day culture period on the fibrin, proliferated. and maintained their osteogenic differential potential better when compared to cells pre-cultured on TCP. Similarly, a concentration relationship between colony number and fibrin concentration was seen with a decrease in colony number as the fibrin number increased suggesting that' progenitor cell numbers are better maintained on low-stiffness gels. Furthermore, direct culture on the gels demonstrated a stiffness related increase in colony number. PDMS is easily produced with a large range of mechanical properties. Uncoated PDMS does not support MSC attachment and growth in vitro and therefore an acrylic acid coating was applied. Although XPS analysis was unable to establish that a complete coating was deposited on all of the substrates, once coated the PDMS supported MSC
attachment and growth. CFU-f efficiency was not directly altered by the mechanical properties of the underlying substrate, however, the differentiation of the cells showed a trend; with an increase in osteoblastic differentiation as the stiffness increased. This trend was also seen under high-density culture conditions with no correlation to the rate of proliferation. Although the exact mechanism is unknown the data presented here supports the concept that substrate signals influence MSC growth and differentiation.