A Bioinspired Self‐Healing Conductive Hydrogel Promoting Peripheral Nerve Regeneration

Abstract The development of self‐healing conductive hydrogels is critical in electroactive nerve tissue engineering. Typical conductive materials such as polypyrrole (PPy) are commonly used to fabricate artificial nerve conduits. Moreover, the field of tissue engineering has advanced toward the use of products such as hyaluronic acid (HA) hydrogels. Although HA‐modified PPy films are prepared for various biological applications, the cell–matrix interaction mechanisms remain poorly understood; furthermore, there are no reports on HA‐modified PPy‐injectable self‐healing hydrogels for peripheral nerve repair. Therefore, in this study, a self‐healing electroconductive hydrogel (HASPy) from HA, cystamine (Cys), and pyrrole‐1‐propionic acid (Py‐COOH), with injectability, biodegradability, biocompatibility, and nerve‐regenerative capacity is constructed. The hydrogel directly targets interleukin 17 receptor A (IL‐17RA) and promotes the expression of genes and proteins relevant to Schwann cell myelination mainly by activating the interleukin 17 (IL‐17) signaling pathway. The hydrogel is injected directly into the rat sciatic nerve‐crush injury sites to investigate its capacity for nerve regeneration in vivo and is found to promote functional recovery and remyelination. This study may help in understanding the mechanism of cell–matrix interactions and provide new insights into the potential use of HASPy hydrogel as an advanced scaffold for neural regeneration.

Draw the curve of sodium hydroxide consumption (volume, ml) vs. conductivity according to the method mentioned above (Fig.S3), and calculate the carboxyl group content according to the formula: CC = 0.04 mol /L (V B -V A ) mL/0.3g (1) Where V A and V B are respectively the x-coordinate values corresponding to the intersection of the left and right tangent lines of the conductivity curve and the horizontal line of the lowest point of the curve.
Graft ratio= (CC HA -CC HA-Cys-Py )/ CC HA 100 (2) Electroactivity measurement The conductivity of the hydrogels was measured using four probes and an electrochemical workstation. In 0.1 mol/L PBS electrolyte solution, a traditional three-electrode system with Pt as the counter electrode, an Ag/AgCl electrode as the reference electrode, and a hydrogel sample as the working electrode was used for electroactivity analysis at room temperature using an electrochemical workstation (CHI660D, China). Based on cyclic voltammetry (CV) measurements, the potential range was -0.8-0.2 V and the scanning rate 100 mV/s. The conductivity of the hydrogels is calculated according to the following formulas: V = I×R (3) σ = L/RS (4) R = ρL/S (5) Where σ (S·cm −1 ) is the desired conductivity, L (cm) is the distance between the reference electrode and the working electrode, S (cm 2 ) is the cross-sectional area of the measured hydrogel, R (Ω) is the ohmic resistance, and ρ (Ω·cm) is the resistivity.

All-Atom Molecular Dynamics simulations method
Materials Studio software was used to simulate the interactions between HASPy chains with water molecules and iron ions under the Compass force field. A simulation crystal Cell was constructed by Amophous Cell calculation. For HASPy chains with water molecules (HASPy-W), two HASPy chains and 300 water molecules were placed in the box. For HASPy chains with iron ions, two HASPy chains, 300 water molecules, 40 Fe 3+ and 120 Cl − ions were added in the box. The initial size of the simulation unit cell is 30.03 Å  30.03 Å 30.03 Å, with the lattice parameters of α = β = γ = 90°. The models were equilibrated at 298 K for 25 ps in the NPT ensemble.

Self-healing experiment
The self-healing behavior of hydrogels was directly observed. The original sample was usually made into rectangular strips (10 × 5 × 3 mm), and then the hydrogel was cut into two equal parts from the middle. The cut surfaces were then placed in full contact and the hydrogel was wrapped in a petri dish for self-healing. After 30 min of self-healing, the hydrogel completely returned to its original state, and tensile and tensile tests were conducted to evaluate the tensile strength before and after self-healing.

In vitro degradation test
First, 10 mg of hydrogel was placed in 1 mL PBS, and 10 mM DTT was added for incubation at 37 °C. PBS without DTT was used as the control group. Then images were obtained at regular intervals to record the morphologies of the hydrogels.

In vivo biocompatibility and degradation test
The hydrogel was disinfected overnight under a UV lamp and the surgical instruments were sterilized by autoclaving. Nine adult female Sprague-Dawley (SD) rats (200-230 g) were randomly divided into three groups. After anesthesia, the rats were placed on the surgical plate, and 10-mm incisions were made on the backs of the SD rats and 30 mg of hydrogel implanted. Skin tissue samples were collected on the 7th 14th, and 28th postoperative days (PODs). Tissue samples were fixed in paraformaldehyde for 24 h and dehydrated in sucrose for 48 h. The sample was then embedded in OCT at the optimum cutting temperature and cut into slices that were 8 μm thick. Finally, in vivo biocompatibility and hydrogel degradation were assessed using H&E staining.

Hemolysis assay
Hemolysis testing of the samples was performed according to the ISO 10993-4 procedure. An anticoagulant agent (3 wv% sodium citrate + 0.1 wv % citrate + 2.5 wv % glucose) was added to the red blood cells from SD rats at a ratio of 7:1. Then 2 mL of whole blood was diluted to 4.5 ml, and a 10 mg sample was placed in 10 mL PBS, and 0.2 mL blood was added, and the solution was incubated at 37 °C for 1 h. Then, the mixed solution was centrifuged at 3000 rpm for 5 min. The absorbance at a wavelength of 545 nm was measured using a UV spectrometer, and the hemolysis rate was calculated as follows: Hemolysis percentage= (absorbance experimental group − absorbance negative group) / (absorbance positive group − absorbance negative group) ×100% (6)         (B) Statistics of fluorescence intensity of S100-β in control, injury, HA, HASPy. Scale bar: 200 µm. Table S1. The interaction energy (E int ), van der Waals energy (E vdw ) and electrostatic interaction energy (E ele ) between HASPy and IL-17A compound protein (IL-17(A, RAH)). Table S2. The distance and angle DHY of the hydrogen bonds between HASPy and IL-17A compound protein (IL-17(A, RAH)). Table S3. The interaction energy (Eint), van der Waals energy (Evdw), electrostatic interaction energy (Eele) and total hydrophohic interaction energy (Etotal)between HASPy and individual residues in IL-17(A, RAH). Table S4. The interaction energy (Eint), van der Waals energy (Evdw) and electrostatic interaction energy (Eele) between HASPy and IL-17A compound protein (IL-17RA (H, L)). Table S5. The distance and angle DHY of the hydrogen bonds between HASPy and IL-17A compound protein (IL-17RA (H, L)). Table S6. The interaction energy (Eint), van der Waals energy (Evdw), electrostatic interaction energy (Eele) and total hydrophohic interaction energy (Etotal)between HASPy and individual residues in IL-17RA (H, L).