Current vascular implant materials poorly interact with medial smooth muscle cells (SMCs) allowing the permanent loss of vascular elastin, eliminated by trauma or disease, a crucial element in maintaining the natural biomechanics of the blood vessel and overall vascular homeostasis. In addition, these materials insufficiently recruit vascular endothelial cells (ECs) to form a normally functional, confluent endothelium that acts as the interface between the blood and vascular tissue and regulates numerous vital vascular processes. As a result, the restenosis, or re-occlusion, rate within these devices has remained fairly high stimulating the investigation of numerous new materials capable of providing the necessary stimuli for the regeneration of vascular tissue. Our lab has reported extensively on hyaluronic acid (HA) as a vascular regenerative agent. We found it beneficially impact two key aspects of vascular regeneration, namely functional endothelialization and elastin matrix repair/ regeneration, and that the biologic impact is dependant on HA molecular weight. This project was initiated to investigate the size-specific ability of exogenous HA to promote endothelialization, and then attempted to utilize this, and previously acquired information on the elastogenesis of vascular SMCs, for the development of HA biomaterials, in the form of surface coatings and hydrogels, for vascular tissue engineering.
Exogenous supplementation of a HA digest (D2) containing a concentrated mixture of HA oligomers (0.75-10 kDa) promoted EC proliferation and tube formation, but also enhanced platelet attachment, CAM expression, and cytokine release. Conversely, the exogenous addition of HMW HA (HA 1500) inhibited platelet deposition on cultured ECs and limited their CAM expression and cytokine release. Upon immobilization onto a substrate through derivatization of HA without crosslinking, ECs were able to attach on D2, however HA 1500 deterred attachment. As observed with exogenous D2, ECs were able to proliferate on D2 substrates similar to cultures on fibronectin but also elicited CAM expression. Another HA digest (D1) containing a broad mixture of HA oligomers, shown to promote elastogenesis of vascular SMCs as an exogenous supplement, was also immobilized and stimulated SMCs to upregulate synthesis of soluble tropoelastin, and crosslinked matrix elastin similar to the levels previously observed with exogenous D1. These surfaces also enhanced elastin organization into fibrils and desmosine crosslinking to a greater degree than exogenous D1. These HA digests (D2, D1) were then separately embedded within divinyl sulfone (DVS) and glycidyl methacrylate (GM) crosslinked HA 1500 hydrogels (DVS-HA, GM-HA), respectively, and we found the mechanical and physical properties of these hydrogels can be modulated by varying the crosslinker and oligomer concentration within them. However, the mechanical properties of these gels were too weak to act as standalone vascular scaffolding biomaterials. DVS-HA gels were cytotoxic stimulating and inflammatory reaction in vivo and EC CAM expression in vitro. Yet, the highest amount of EC attachment and proliferation was observed on DVS-HA that contained the highest amount of D2. SMCs survived encapsulation into the more biocompatible GM-HA gels and D1 incorporation elevated elastin levels to 2x the amount within GM-HA without D1.
Overall, we showed that HA oligomers are useful to incorporate within vascular biomaterial scaffold, either as surface-immobilized coatings or as hydrogels to promote endothelialization and elastic tissue regeneration. Our studies also indicate that incorporation of HMW HA is also necessary to temper adverse cell responses to HA oligomers (e.g., CAM expression) and provide mechanical stability/ ease of handling to the gels.