Heparan sulfate co-immobilized with cRGD ligands and BMP2 on biomimetic platforms promotes BMP2 mediated osteogenic differentiation. Figure 4

- The p-SMAD 1/5/9 signaling of C2C12 cells plated on biomimetic platforms presenting immobilized heparan sulfate (iHS) with adsorbed BMP2 (aBMP2) was analysed at different cRGD surface concentrations and compared to soluble BMP2 (sBMP2) and immobilised BMP2 (iBMP2). The condition with only iHS is the negative control. The SMAD phosphorylation decreases with the cRGD surface concentration in the case of sBMP2 and iBMP2 whereas on the surfaces presenting iHS + aBMP2 the levels of p-SMAD 1/5/9 do not change with the cRGD surface concentration. The presence of iHS maintained high levels of p-SMAD 1/5/9 independently of cRGD surface concentration. In human periosteum derived stem cells the p-SMAD 1/5/9 levels are higher in iHS+aBMP2 with respect to the other type of BMP2 presentation showing that iHS enhanced BMP2-SMAD signaling. Figure 4c: Western blot of p-SMAD 1/5/9 in C2C12 cells plated on platforms functionalized with different cRGD surface concentrations and with sBMP2, iHS, iHS and aBMP2 and iBMP2. Cells were lysed one hour after plating and the phosphorylation of SMAD 1/5/9 has been normalised to GAPDH. The loading of the samples has been modified in two over the four experiments. The quantification of the four experiments is shown in Figure 4c of the paper Sefkow-Werner et al. 2020, (submitted) Figure 4d: Western blot of p-SMAD 1/5/9 levels obtained from hPDSCs plated 2 hours on different platforms presenting sBMP2, iHS, iHS a BMP2 and IBMP2 and cRGD surface concentration of 0.5 ng/cm2. The quantification of the western blots is shown in Figure 4d of the paper Sefkow-Werner et al. 2020, (submitted).