Introduction: The role of pro-angiogenic materials is to support the formation of new vasculature, restoring tissue perfusion and function. The material could enable rapid integration of cells and microvessel networks by mimicking the native biochemical and mechanical tissue environment. However, the development of compliant and degradable scaffolds for soft vascularized tissue regeneration has remained difficult, leading to the use of overly stiff materials that fail to recapitulate endogenous tissue mechanics. Here we have created enzymatically degradable biomimetic poly(ethylene glycol) (PEG) hydrogels which utilize competitive crosslinking sites to alter network connectivity and control the hydrogel mechanics independent of changes to the polymer density or chain length. This advancement allows the creation of highly compliant hydrogels with enhanced lifetime to investigate endothelial cell (EC) behaviors in vitro as well as micrvessel formation in vivo.
Materials and Methods: Peptides containing an enzymatically degradable site (PQ) that incorporated or omitted a competitive allyloxycarbonyl (alloc) crosslinking site were synthesized via Fmoc chemistry and PEGylated at terminal amines, creating a photopolymerizable acrylate-PEG-peptide-PEG-acrylate macromer. Compression testing was used to quantify the effects of the competitive crosslinking site on hydrogel mechanics. For in vitro studies, ECs were encapsulated alone or with pericytes in the presence of PEG-RGDS then evaluated via confocal microscopy[1]. For in vivo studies, hydrogels were photopolymerized acellularly and implanted subcutaneously in the dorsum of Lewis rats. Gels were excised after 3, 7, 14, or 28 days and analyzed via histology to evaluate cellular integration and microvessel density.
Results and Discussion: Compression testing revealed our ability to tune the hydrogels’ mechanical properties from < 1 to ~20 kPa by copolymerizing PEG macromers that contained (PEG-PQ) or omitted [PEG-PQ(alloc)] the competitive alloc crosslinking site (Figure 1A). This range of mechanical properties was able to modulate EC interactions with their local matrix in 3D via enhanced cell spreading after just 24 hours (Figure 1B). Similarly, when ECs were co-cultured with pericytes in compliant gels, networks formed in less than 3 days whereas stiffer hydrogels took at least 6 days (Figure 1C).
Compliant hydrogels exhibit significant quantifiable enhancements in tubule length and branching. Furthermore, cell networks were stable for at least 4 weeks in the highly compliant hydrogel environment and were capable of depositing collagen IV and laminin perivacsularly. Compliant subcutaneous hydrogel implants exhibited enhanced cellular integration and overall increased microvessel density as assessed by H&E and CD31 staining (Figure 2).
Conclusions: Our ability to control the hydrogel mechanical properties independent of polymer density permits investigations into the long and short term effects of material compliance on vascular integration. Here, material compliance enhances the ability of ECs to assemble into vessel-like networks in vitro as well as infiltrate and form vascular networks in vivo, suggesting that compliant materials may improve therapeutic outcomes for many vascular tissue engineering efforts.
References:
[1] Schweller, RM; West, JL; Encoding Hydrogel Mechanics via Network Cross-Linking Structure. ACS Biomater Sci Eng. 2015 1 (5), 335-344.