Page 225 - IJB-10-3

International Journal of Bioprinting Preparation and characterization of branched NGCs

branched NGCs group, and sham group. Anesthesia 2.8. Histological analysis of the regenerated nerves
was induced in all rats via intraperitoneal injection of Following the electrophysiology assessment, the
10% chloral hydrate. Subsequently, the sciatic nerve was regenerated sciatic nerves were harvested. The nerve
exposed, and a 6 mm nerve defect was created by surgically tissues were fixed in 10% formalin, dehydrated in ethanol,
excising a segment of the nerve for conduit implantation. and embedded in paraffin to prepare paraffin sections
For dual-branched NGC groups, the 8-0 absorbable Vicryl (7 μm). Subsequently, the tissue sections were stained with
sutures were used to suture the proximal nerve ends, H&E and luxol fast blue (LFB) and observed using a digital
which were then connected with a soft silicone guider. slide scanner (Pannoramic MIDI).
The sutures were guided to the contralateral side through
the inner channel and sutured with the distal stumps at 2.9. Statistical analysis
the connector of branched NGCs. For the multi-branched Statistical analysis was performed using GraphPad Prism
NGCs group, the proximal and distal stumps were placed 7 software. The data were expressed as mean values ±
into the two connectors of the NGCs, and the nerve standard deviation (SD). Significance was determined
stumps within the conduits were stabilized by coaptating utilizing the one-way ANOVA method, with p < 0.05
8-0 absorbable surgical sutures (Vicryl) around the considered statistically significant.
outsides of conduits. For the sham group, no treatment 3. Results
was administered to the sciatic nerve. Finally, the muscle
and skin layers were sutured with 2-0 nylon sutures. 3.1. Design and fabrication of the branched NGCs
The scaffold material is the basic component of conduits,
2.7. Functionality assessment of regenerated nerves and an ideal scaffold for nerve repair should possess
Sixteen weeks post-surgery, SD rats were anesthetized, characteristics of biodegradability, biocompatibility, and
and the injured sites were exposed to observe mechanical properties aligned with the injured sites. In this
nerve regeneration within branched NGCs. An study, GelMA and PEGDA were combined as a composite
electrophysiology assessment was performed to evaluate prepolymer for NGC fabrication. GelMA, containing cell-
the functional recovery of regenerated nerves. Bipolar binding motifs, fosters cell adhesion and proliferation,
electrodes were used to stimulate the proximal and distal albeit hindered by its poor mechanical properties. PEGDA,
ends of the regenerated sciatic nerves, with a monopolar a synthetic polymer, has received FDA approval for various
recording electrode placed in the gastrocnemius muscle. human applications. The mechanical properties of PEGDA
After surgically removing the dual-branched NGCs, hydrogel can be fine-tuned to provide favorable mechanical
the regenerated nerve dual branches, denoted Branch 1 strength for NGC. Thus, we integrated GelMA and
(B1) and Branch 2 (B2), were exposed. Meanwhile, the PEGDA to formulate a composite prepolymer that is both
electrophysiological conduction of each nerve branch was biocompatible and mechanically strong for nerve repair. 47-49
tested by attaching the bipolar stimulating electrodes to Notably, hydrogels containing GelMA have been reported
the proximal and distal ends of the branched nerves. Nerve to be beneficial for cell adhesion, a benefit not dependent on
conduction velocity (NCV), the latency of compound GelMA concentration in a linear relationship. Therefore,
50
muscle action potential (CMAP), and the peak amplitude our optimization of the composite prepolymer mainly
of CMAP were measured using an electromyography focused on hydrogel mechanical properties. We fabricated
machine (Nuocheng, Shanghai, China). various scaffolds using a series of composite prepolymers,
Following the electrophysiology assessment, varying the concentrations of GelMA and PEGDA. As
gastrocnemius muscles on both sides of the rats were shown in Figure S1 (Supplementary File), the composition
harvested. The wet weight of the gastrocnemius muscle of 4% GelMA: 12% PEGDA yielded Young’s modulus of
was immediately measured, and the wet weight ratio was 0.318 MPa, a value relatively optimal for matching the
calculated based on the mass ratio of the injured side to the elastic modulus of peripheral nerves (0.5–13 MPa).
normal side. Subsequently, the muscles were preserved in To investigate the biocompatibility of GelMA/PEGDA
10% formalin overnight and dehydrated with an automated hydrogel and elucidate the effects of scaffold structures on
tissue processer (ASP300s, Leica), followed by embedding neuron growth in vitro, we employed a DLP 3D printing
in paraffin. Tissue sections of 7 μm were prepared using process to fabricate a micropatterned scaffold (Figure S2a
a rotary microtome (RM2125 RTS, Leica). Finally, in Supplementary File). The centrosymmetric scaffold was
hematoxylin and eosin (H&E) staining was performed, designed, featuring circular microchannels at the center,
and images were acquired via a digital slide scanner along with several central strip channels and two side
(Pannoramic MIDI). The diameter of muscle fibers was loops (Figure S2b in Supplementary File). Rhodamine-
measured using ImageJ software. phalloidin staining revealed that PC12 cells exhibited

Volume 10 Issue 3 (2024) 217 doi: 10.36922/ijb.1750