Immobilized sonic hedgehog N-terminal signaling domain enhances differentiation of bone marrow-derived mesenchymal stem cells.

The signaling domain of Sonic hedgehog (Shh), a potent upstream regulator of cell fate that has been implicated in osteoblast differentiation from undifferentiated mesenchymal cells in its endogenous form, was investigated in an immobilized form as a means for accelerating differentiation of uncommitted cells to the osteoblast phenotype. A recombinant cysteine-modified N-terminal Shh (mShh) was synthesized, purified, and immobilized onto interpenetrating polymer network (IPN) surfaces also grafted with a bone sialoprotein-derived peptide containing the Arg-Gly-Asp (RGD) sequence (bsp-RGD (15)), at calculated densities of 2.42 and 10 pmol/cm2, respectively. The mitogenic effect of mShh was dependent on the mode of presentation, as surfaces with immobilized mShh and bsp-RGD (15) had no effect on the growth rate of rat bone marrow-derived mesenchymal stem cells (BMSCs), while soluble mShh enhanced cell growth compared to similar surface without mShh supplementation. In conjunction with media supplemented with bone morphogenetic protein-2 and -4, mShh and bsp-RGD (15)-grafted IPN surfaces enhanced the alkaline phosphatase activity of BMSCs compared with tissue culture polystyrene and bsp-RGD (15)-grafted IPN surfaces supplemented with soluble mShh, indicating enhanced osteoblast differentiation. The adhesive peptide bsp-RGD (15) was necessary for cell attachment and proliferation, as well as differentiation in response to immobilized mShh. The addition of immobilized Shh substantially improved the differentiation of uncommitted BMSCs to the osteoblast lineage, and therefore warrants further testing in vivo to examine the effect of the stated biomimetic system on peri-implant bone formation and implant fixation.