oa Matrix-controlled In-growth Selectivity as a Principle of Heart Valve Regeneration

Spontaneous scaffold based tissue regeneration may be a promising alternative to in vitro tissue engineered heart valve prostheses. Selective promotion of ingrowth of desired cells versus suppression of potentially harmful cell invasion would be a key principle of such an approach. We have begun to demonstrate the utility of hydrogels engineered with specific adhesive and degradation peptide sites towards achieving this long-term goal. Polyethylene glycol hydrogels that through Michaels addition chemistry allow for both polymerization via enzymatically (matrix metalloproteinase (MMP)) degradable peptides and appendage of adhesive peptides were developed. Hydrogels were crosslinked with either a relatively enzymatically promiscuous peptide (MMP-pep) or sequences specifically degradable by either MMP-14 or MMP-9 (MMP-14pep, MMP-9pep). MMPs are of interest as there is evidence of cell specific expression. For example in vascular cells under physiological conditions MMP-9 is expressed at low levels except in macrophages. Adhesive peptides utilised were RGD, YIGSR and/or PHRSN. A range of primary vascular cells (smooth muscle cells (SMC), endothelial cells (EC), valvular intestitial cells (VIC) and fibroblasts (FB)) were cultured individually or as spheroids in 2 or 3-D. Migration and invasion were then assessed by time-lapse phase and confocal microscopy. ECs were shown to migrate significantly more rapidly on a surface containing YIGSR/RGD relative to other peptide combinations whilst the migration of SMC was unaffected. Hydrogels polymerized with MMP-14pep or MMP-9pep were shown to be highly preferentially cleaved by their relative enzymes. VICs, Fb and SMC primarily utilised MMPs to invade the hydrogels as demonstrated by inhibition of sprout formation by the MMP inhibitor, GM6001. VICs and FB invaded the gels in the following order, MMP-pep>>MMP-14>MMP-9. However, SMC showed a marked preference for invasion into MMP-14pep crosslinked hydrogels above that observed in their MMP-pep and MMP-9pep counterparts. Confocal microscopy analysis also showed a significantly more branched and interconnected pattern of invasion for SMC in MMP-14pep hydrogels. Thus we have begun to show that through engineering of extracellular matrix mimics with precise enzymatic and adhesive recognition sites it is possible to selectively influence specific cell types invasive behaviour suggesting that cell-specific ingrowth scaffolds may be achievable.