Advancing Peripheral Nerve Regeneration: 3D Bioprinting of GelMA-Based Cell-Laden Electroactive Bioinks for Nerve Conduits

Abstract

Peripheral nerve injuries often result in substantial impairment of the neurostimulatory organs. While the autograft is still largely used as the "gold standard" clinical treatment option, nerve guidance conduits (NGCs) are currently considered a promising approach for promoting peripheral nerve regeneration. While several attempts have been made to construct NGCs using various biomaterial combinations, a comprehensive exploration of the process science associated with three-dimensional (3D) extrusion printing of NGCs with clinically relevant sizes (length: 20 mm; diameter: 2−8 mm), while focusing on tunable buildability using electroactive biomaterial inks, remains unexplored. In addressing this gap, we present here the results of the viscoelastic properties of a range of a multifunctional gelatin methacrylate (GelMA)/poly(ethylene glycol) diacrylate (PEGDA)/carbon nanofiber (CNF)/gellan gum (GG) hydrogel bioink formulations and printability assessment using experiments and quantitative models. Our results clearly established the positive impact of the gellan gum on the enhancement of the rheological properties. Interestingly, the strategic incorporation of PEGDA as a secondary crosslinker led to a remarkable enhancement in the strength and modulus by 3 and 8-fold, respectively. Moreover, conductive CNF addition resulted in a 4-fold improvement in measured electrical conductivity. The use of four-component electroactive biomaterial ink allowed us to obtain high neural cell viability in 3D bioprinted constructs. While the conventionally cast scaffolds can support the differentiation of neuro-2a cells, the most important result has been the excellent cell viability of neural cells in 3D encapsulated structures. Taken together, our findings demonstrate the potential of 3D bioprinting and multimodal biophysical cues in developing functional yet critical-sized nerve conduits for peripheral nerve tissue regeneration.

Low-Concentration Gelatin Methacryloyl Hydrogel with Tunable 3D Extrusion Printability and Cytocompatibility: Exploring Quantitative Process Science and Biophysical Properties

“…The as-cured samples were freeze-dried for 72 h to obtain a dry scaffold for further experiments. To determine the swelling ratio, the freeze-dried scaffolds ( n = 4) were submerged in 1× PBS at 37 °C, and swelled weight was measured at different time intervals for 24 h. The percentage swelling ratio ( Q t ) was calculated using the following equation Q t = W t − W 0 W t × 100 % where W 0 and W t are freeze-dried and swelled weights at time t , respectively.…”

“…At various time intervals, the samples ( n = 4) were retrieved, and their masses were measured before ( M 0 ) and after ( M t ) exposure to the enzymatic solutions. Using the following equation, these masses were then used to determine the percentage of remaining mass ratio (RM) RM = M t M 0 × 100 % …”

“…As previously described, a standardized in-house synthesis protocol was followed to prepare GelMA by chemically modifying gelatin. , Briefly, 10% (w/v) of gelatin was dissolved in 1× phosphate-buffered saline (PBS) medium under continuous stirring at 60 °C for 20–30 min. After complete dissolution, the solution temperature was reduced to 50 °C, followed by adding 8% (v/v) methacrylic anhydride (MA) dropwise.…”

Gelatin Methacryloyl (GelMA)-Based Biomaterial Inks: Process Science for 3D/4D Printing and Current Status

“…The hydrogel-inorganic phase systems (composite bioinks) are the multimaterial bioinks consisting of various nanoparticles or anisotropic fillers, such as graphene, graphene oxide (GO), carbon nanotubes (CNTs), carbon nanofibers, hydroxyapatite (HAp), β-tricalcium phosphate (β-TCP), bioactive glasses (BG), silica nanoparticles, and nano clays into the primary biopolymer matrix component to enhance the mechanical and biological properties of biomaterials inks. ,,− In this section, the applications of several inorganic biomaterials, which are utilized as the macro- or nanofiller in GelMA hydrogel matrix to formulate printable biomaterial inks will be summarized. Moreover, nanoscale biomaterials have a macroscale impact on mimicking natural ECM environments on artificially constructed cell-embedded scaffolds, which profoundly influence cellular responses such as cell adhesion, proliferation, and differentiation.…”