Modification of bioactive hyaluronic acid for stereolithography 3D printing of hydrogel conduits for peripheral nerve regeneration

Abstract

Peripheral nerve injuries occur as a result of illness or injury, and present a significant healthcare and economic burden. Many of these cases occur in otherwise healthy individuals, in the age range 20-40 years, due to trauma in the work environment. Despite all that is known of this condition, complete functional recovery remains difficult to obtain through current surgical methods. The aim of this research was to modify hyaluronic acid (HA) to enable 3D printing of hydrogel nerve conduits which could enable full functional recovery of a peripheral nerve injury. An array of compounds were screened in conjunction with HA to assess any potential neurotrophic benefits to their inclusion in the final formulation. As HA is not conducive to cell attachment, neuronal and glial cell lines were initially used to characterise HA in order to design a testing procedure and acquire a baseline response. HA was then successfully modified with cysteamine HCl and methacrylic anhydride, to produce thiolated HA (HA-SH) and methacrylated HA (HA-MA) respectively, as confirmed by colorimetric and spectroscopic methods. The modification degree was approximately 20% so as not to interfere with the receptor interaction of HA. Modified HA was characterised chemically and biologically to ensure cytocompatibility in neuronal and glial cell lines. UV photo-polymerised hydrogels were created via click chemistry reactions by combining thiolated HA and methacrylated HA in various ratios and their properties were evaluated. Due to the poor mechanical properties of the HA hydrogels, a synthetic polymer, polyethylene glycol dimethacrylate (PEGDMA), was introduced to the matrix. An optimal formulation was discovered and prints were created via stereolithography 3D printing. Direct contact and elution extract testing revealed no significant cytotoxicity over a 24 h period, in contrast to the hydrogels of each of the four polymeric matrices tested (PEGDMA, 50 %wv HA-MA: PEGDMA, 50 %wv HA-SH: PEGDMA and hybrid blend). This would indicate that 3D printing yielded a sample which is representative of the conduit formulation, which is capable of providing biological and physical support to enhance peripheral nerve regeneration. Through extensive testing of potential neurotrophic compounds, the lead compound Tyrosol failed to produce significant proliferative effects from elution extract testing of 3D printed samples using the resazurin assay. Given its antioxidant status and the significant proliferative effects observed with this compound in direct cell assays, future studies should vi refine the test methods in order to evaluate the effective elution concentration of Tyrosol from the final conduit, rather than from a representative sample. This research could have a significant impact on the future of not only nerve regeneration, but also bioengineering as a whole. This thesis has elucidated the use of click-chemistry reactions to enable highly reliable cross-linking reactions in biological polymers to enable processing which would otherwise be difficult. Further work would involve the ex vivo testing of this formulation in dorsal root ganglion cells before in vivo testing could take place. This formulation could also be tested in other formats such as injectable hydrogels and foams for a number of pathologies