Functional hydrogel biomaterials for peripheral nerve regeneration

Abstract

Peripheral nerve injury (PNI) causes sensory or motor function deficits, numbness, muscle weakness, and pain, leading to serious psychosocial health issues for patients. Autografts have been the gold standard, however, donor site morbidity and size mismatch remain unsolved issues. To address these limitations, various nerve guidance conduits (NGC) has been extensively explored. Among them, hydrogel NGCs have been considered to provide biomimetic environments, such as softness, high-hydration, and permeability; however, mechanical weakness hinders their practical applications and effectiveness. Accordingly, hydrogel NGCs exhibiting improved mechanical strength of hollow NGCs and additional biological and electrical cues are desired to sufficiently protect regenerating nerves from external shocks and enhance nerve regeneration. This thesis aims at fabrication of advanced hydrogel-based NGCs and luminal fillers integrating structural, cellular, and electrical functions for efficient PNI regeneration. Accordingly, this thesis topic includes (i) development of a chemical-free double network (DN) hydrogel NGC for simple fabrication and enhanced mechanical properties, (ii) direct encapsulation of human umbilical-cord mesenchymal stem cells (ucMSCs) into DN NGC for therapeutic cellular activity, and (iii) development of an injectable conductive luminal filler for electroactive intraluminal environment. First, I fabricated a DN NGC through the crosslinking of gelatin and alginate in a chemical-free method. The primary gelatin network was crosslinked via gamma irradiation, while the secondary alginate network was subsequently formed through ionic crosslinking. Gamma irradiation does not use toxic chemical crosslinkers, avoids chemical residues, simplifies manufacturing process, and sterilizes the NGC products simultaneously. This Ph.D./MS 20214040 conduit shows improved mechanical properties and enhanced sciatic nerve regeneration compared to single network NGC controls. Second, I directly encapsulated ucMSCs within a gelatin methacrylate (GelMA)/alginate DN NGC to provide biological cues. This ucMSC laden DN NGC exhibited enhanced mechanical properties, maintained MSC viability (>80%), and enabled paracrine factor release (e.g. GM-CSF, HGF etc.). In the rat sciatic nerve defect model, functional and histological improvement was demonstrated in ucMSC NGC group compared with non-cellular and medical grade silicone group. To our knowledge, this is the first study to treat PNI by encapsulating ucMSCs into DN hydrogel NGC. Third, I developed an injectable, adhesive and in situ-gelling conductive luminal filler in conjunction with electrical stimulation (ES). The conductive luminal filler is synthesized via thiol–maleimide Michael addition reaction between gelatin-maleimide (GelMal) and gelatin-thiol (GelSH) with dispersible-reduced graphene oxide (rGO). Conductive filler and ES treatment led to significantly enhanced neurite outgrowth of dorsal root ganglia (DRG) explants in vitro. In vivo animal studies revealed that the electrically conductive filler and ES group exhibited promoted axonal regeneration, myelination, and functional recovery compared to control groups. Collectively, this thesis demonstrates comprehensive and progressive design for advanced hydrogel-based NGCs and luminal fillers for PNI repair. These results provide a practical blueprint for hydrogel-based tissue engineering toward clinically meaningful PNI repair.