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

Abstract

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 3Dculture 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 create cell-responsive 3D microenvironments and offer promise as injectable carriers for therapeutic hMSC-delivery.

Matrix metalloproteinase-13 mediated degradation of hyaluronic acid-based matrices orchestrates stem cell engraftment through vascular integration

“…In another study, conditioned medium from MMP-13-overexpressing cells stimulated capillary formation of immortalized human umbilical vein endothelial cells (HUVECs), while treatment of HUVECs with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo [51]. Furthermore, production of MMP13 induces the secretion of VEGF from fibroblasts and endothelial cells, and promotes angiogenesis by activation of FAK and ERK pathways [20, 50–52]. Consistent with these previous reports, we observed that the high amount of MMP-13 retained in the QPQGLAK-linked hydrogel induced significantly higher amounts of VEGF 165 compared to rapidly degradable GPLGMHGK- and GPLGLSLGK-linked hydrogels (Fig.…”

“…Isolated GFP + /Sca-1 + /CD105 + /CD45 − CPCs were cultured in Iscove’s Modified Dulbecco’s Medium(IMDM) basal media containing 10% Fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) as previously described [12, 20, 21]. Cells were encapsulated in the hydrogels at the density of 5x10 6 cells/mL as described in our previous report [7].…”

Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells

“…Different OGP analogs were designed and synthesized by Fmoc/tBu SPPS, where the target bioactive fragment YGFGG was either flanked by a poly-G sequence (in OGP1), the MMP-cleavable substrate PVGLIG (in OGP2) [24], or the PVGLIG-scrambled sequence GIVGPL (in OGP3). Their molecular weight and purity were assessed by LC-MS and HPLC, respectively (Table 1).…”

“…Both were chemically grafted to alginate, a natural polysaccharide with the ability to form hydrogels in situ, which has been extensively used as a delivery vehicle for entrapped cells [21][22][23]. We hypothesized that in the presence of specific MMPs, in particular MMP-2 [20,24], the bioactive YGFGG fragment would be released from the hydrogel upon enzymatic cleavage of the PVGLIG sequence, while it would remain mostly immobilized when using the scrambled linker. Once implanted, such systems may, thus, provide localized OGP delivery for variable time periods, remaining in close proximity to the targeted host cells at the injury site.…”

Modular protein domains: an engineering approach toward functional biomaterials

“…For example, Fonseca and coworkers profiled the in vitro gene expression of hMSCs grown in basal or osteogenic medium and found that in osteogenic medium there was an increase in MMP-14 gene expression levels and alkaline phosphatase (ALP) activity at one week [43]. Thus, it is anticipated that by selecting specific MMP-sensitive sequences it is possible to design materials that are selectively degraded by desired cells at specific stages of differentiation.…”