Glycoprotein Hydrogel-Based Implantable Nerve Guidance Conduits for Peripheral Nerve Regeneration

Peripheral nerve injuries (PNIs) beyond the critical gap length pose a serious problem in neural tissue engineering and regenerative medicine. Research on effective therapeutic strategies has centered primarily on implantable nerve guidance conduits (INGCs) over the past several decades.<br/>A significant amount of progress has been made in INGC development. Still, the field has remained stagnant due to the lack of an optimal solution for ensuring target reinnervation accuracy, which requires a delicate balance of biochemical cues, including growth factors that govern tissue formation and regeneration throughout the regeneration process. In recent years, researchers have utilized heparin-based materials for sustained growth factor delivery owing to the intrinsic characteristic of heparin to bind and stabilize the growth factors through electrostatic interactions to afford more efficient regenerative outcomes. However, several shortcomings of heparin, such as immunogenicity, could hinder the successful clinical translation of heparin-based INGCs. For this reason, structurally well-defined synthetic mimics fulfilling the same function without triggering immune response are in demand.<br/>In this work, we introduce a silk fibroin-modified gelatin tyramine hydrogel matrix fabricated through enzymatic crosslinking. Both 2D-shaped (planar film) and 3D-shaped (tubular INGC) protein hydrogel scaffolds with the same composition were produced for <i>in vitro</i> and <i>in vivo</i> investigations, respectively. At the macroscale, silk fibroin modification facilitated enhanced mechanical strength to provide the native nerve tissue-like mechanical properties and prolonged the degradation time of the hydrogel implant. It acted as a biocompatible scaffold for Sprague Dawley rat Schwann cells (SCs).<br/>Then, we directed our efforts toward creating a small library of synthetic small-molecule heparin glycomimetics to address the limitations of native heparin. We synthesized monosaccharides and disaccharides with different sulfation patterns to conjugate to the hydrogel matrix covalently via a PEG linker for cellular-level nerve regeneration. The binding affinities of these synthetic heparin glycomimetics to nerve regeneration-promoting biochemical cues, namely, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and fibroblast growth factor 2 (FGF2), were screened using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) techniques and our findings showed sulfation pattern-dependent binding affinity. The best-performing molecule was able to bind to NGF and FGF2 with acceptable affinity. We covalently conjugated this compound to the hydrogel matrix. Fluorescence assays and gel permeation chromatography (GPC) confirmed small molecule sugar conjugation to the hydrogel. Hydrogel scaffold, which bears small molecule heparin glycomimetic complexed with NGF, referred to as glycoprotein-based hydrogel, accelerated the neurite growth of Sprague Dawley rat dorsal root ganglion (DRG) neurons and extended retention time of NGF.<br/>Inspired by <i>in vitro</i> evaluations, we currently fabricated 3D tubular glycoprotein hydrogel-based INGCs (heavily grafted with our small molecule heparin glycomimetic and with native heparin having the same degree of grafting for comparison) to assess <i>in vivo</i> nerve regeneration in Sprague Dawley rat sciatic nerve defect model. Our final goal is long-term monitoring of INGC to unveil how heparin glycomimetics affect the cumulative release of NGF <i>in</i> <i>vivo</i> compared to native heparin and as well as to analyze the immunogenicity of both INGCs at the same degree of grafting.<br/>To the best of our knowledge, it is the first report systematically investigating the structure-activity relationship of small molecule heparin glycomimetics for <i>in vivo</i> nerve regeneration applications. This approach could be further extended to related regenerative medicine and tissue engineering disciplines.