Characterization of Photo-Crosslinking ECM-Mimetic Gallol Modified Hyaluronic Acid-Protein Composite Hydrogels

Abstract

The need for multifunctional advanced hydrogel materials for biomedical research and biomedicine has never been greater to find better alternatives to animal modelling and to make more physiologically relevant in vitro models. A novel natural photo-crosslinking hy-drogel system has been created with modified hyaluronic acid (HA) to help fill this demand as an all-natural advanced hydrogel. This hydrogel system can also be coupled with various extracellular matrix (ECM) proteins and other proteoglycan components to more closely imitate the in vivo microenvironment with all non-toxic components. The use of naturally occurring and medicinal phenolic acids such as polyphenols, natural neurotransmitters, and other phenolic compounds as components in hydrogels and hydrogel systems has gained traction in recent years. Previously, hyaluronic acid hydrogels have been covalently crosslinked with integrated dopamine, but no success has been made with photo-crosslinking approaches using phenolic compounds with HA. Gallic acid (GA), a common phenolic acid and natural metabolite found in fruits, nuts, and vegetables, was chemically attached via EDC chemistry to the HA backbone and Riboflavin, vitamin B2, was incorporated into the system as the photo-sensitizer and photo-initiator. HA-GA crosslinks through an irreversible photo-mediated oxidation reaction after exposure to UV or Blue light due to Riboflavin’s dual absorption peaks and ensuing release of oxidative free radicals that strip electron’s away from Gallol residues on the HA-backbone and forms strong covalent bonds. The HA-GA material components and parameters were rheologically tested and optimized to give a range of mechanical properties from very soft to stiff, matching a variety of tissue types from brain to tendons or cartilage. The mechanical properties of HA-GA can be tuned solely by changing light exposure time or light power without altering the solid content of the hydrogel. Moreover, HA-GA alone shows promise with self-healing capabilities after crosslinking, and further proteins and ECM constituents can be added to enhance the material’s capabilities as a human ECM mimicking material. The gelation kinetics of photo-crosslinking HA-GA was also assessed and proved much faster than previously reported HA-GA systems on the order of seconds rather than minutes to hours. Different types of proteins including Collagen I, Fibronectin, Vitronectin, Gelatin, and Albumin were all successfully incorporated into the HA-GA system to form Protein-Polysaccharide composite gels to serve a variety of purposes and match many ECM compositions from different tissues. The degradation and stability of HA-GA and HA-GA composite gels was also assessed in PBS, Hyaluronidase solution, and DMEM cell culture media. HA-GA sufficiently maintains structure for more than three weeks before degrading depending on protein and polysaccharide composition. HA-GA with riboflavin also shows promise as a 3D printable and moldable material with excellent tissue adhesive properties, and can be modified to fit many different purposes. Biocompatibility and proliferation studies were conducted with HA-GA and HA-GA composite gels with UV and blue light exposure with multiple cell types. HA-GA with Riboflavin and HA-GA composite gels prove promising as an advanced hydrogel system for a wide variety of applications.