Enzymatic, physicochemical and biological properties of MMP-sensitive alginate hydrogels

Protease-sensitive hydrogels that recapitulate the mechanisms of cell-driven enzymatic remodelling of the natural extracellular matrix (ECM) have been gaining popularity as artificial 3D cell-microenvironments. Here, the matrix metalloproteinase (MMP)-sensitive peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG) was double-end grafted to alginate forming water-soluble PVGLIG-alginate conjugates. The PVGLIG peptide was synthesized as a Fluorescence Resonance Energy Transfer (FRET) sensor and showed to be a good substrate for MMP-2, MMP-9, MMP-13 and MMP-14. After demonstrating that human MSC (hMSC) expressed both MMP-2 and MMP-14 under basal and osteogenic in vitro conditions, the effect of 3D-culture within MMP-sensitive alginate hydrogels on hMSC behaviour was addressed. In situ-forming alginate hydrogels containing only cell-adhesive RGD peptides (RGD-alginate, MMP-insensitive) or both peptides (PVGLIG/RGD-alginate, MMP-sensitive) were used. Cell-matrix and cell-cell interactions were enhanced in hMSC-laden MMP-sensitive alginate hydrogels, as compared to MMP-insensitive hydrogels with identical viscoelastic and microstructural properties. hMSC underwent osteogenic differentiation in both types of matrices. However, the presence of PVGLIG stimulated the secretion of proteases (most likely MMP-2) by hMSC, in both undifferentiated and differentiated cultures. By using the FRET sensor, it was possible to demonstrate that the cocktail of hMSC-secreted MMPs was effectively active in cleaving the PVGLIG motif. Protease-sensitive alginates can be used to creat(e) cell-responsive 3D microenvironments and offer promise as injectable carriers for therapeutic hMSC-delivery.