Heparan sulfate co-immobilized with cRGD ligands and BMP2 on biomimetic platforms promotes BMP2 mediated osteogenic differentiation. Figure 5
We knocked down beta1 or beta3 integrins on C2C12 cells and we plated these cells on biomimetic platforms presenting immobilized heparan sulfate (iHS) with adsorbed BMP2 (aBMP2) compared to the conditions with soluble BMP2 (sBMP2) and BMP2 directly immobilized (iBMP2). The knocking down reduced p-SMAD 1/5/9 levels at short time points (1.5 hour) and ALP expression 3 days after plating. p-SMAD 1/5/9 remained low on cRAD platforms, however the expression of ALP was up-regulated on cRAD platforms presenting iHS and aBMP2. Figure 5a: Cells were silenced for either beta1 or beta3 integrins and plated on cRGD and cRAD platforms presenting sBMP2, iHS, iHS + aBMP2 and iBMP2, and fixed after 1.5 h for p-SMAD1/5/9 staining. Representative immunofluorescence images shown the intensity of p-SMAD1/5/9 translocated into cell nuclei on all different conditions and cell-type. The quantification of the triplicate expriment is shown in figure 5b of Sefkow-Werner et al. 2020 (submitted). Figure 5c: Representative ALP staining of C2C12 cells scrambled, silenced for beta1 (si beta1) and beta3 (si beta3) integrins, plated on cRGD or cRAD platforms co-presenting sBMP2, iHS, iHS+aBMP2 and iBMP2 and fixed after 3 days. The quantification of the triplicate expriment is shown in figure 5d of Sefkow-Werner et al. 2020 (submitted).