Authors: Yunhao Zhai, Xiangheng Guan, Caorui Lu, Ruixuan Sun, Yun Qian, Yi Li, Kaihang Zhang, Xu Wang, Linbin Xu, Xinghao Yin, Shang Guo, Jinglei Wu, Cunyi Fan
Categories: Full Length Article, Peripheral nerve injury, Chitosan-ibuprofen conjugate, Minimally invasive, Injectable hydrogel
Source: Materials Today Bio
Authors: Yunhao Zhai, Xiangheng Guan, Caorui Lu, Ruixuan Sun, Yun Qian, Yi Li, Kaihang Zhang, Xu Wang, Linbin Xu, Xinghao Yin, Shang Guo, Jinglei Wu, Cunyi Fan
Non-transecting peripheral nerve injuries require effective inflammation control, support of axon regeneration, and strategies that minimize additional surgical trauma. Injectable hydrogels are attractive carriers for local therapy, and chitosan is a widely used biocompatible matrix; however, conventional chitosan systems often depend on acidic dissolution and crosslinking or cytotoxic crosslinkers, which may aggravate nerve injury and hinder clinical translation. Here, we develop an injectable chitosan-based hydrogel (IBU-CS-GP) in which ibuprofen is complexed with chitosan for solubility range expansion toward near-neutral pH, thereby permitting genipin-mediated crosslinking under near-physiological pH and resolving the mismatch between chitosan solubility and the optimal pH for genipin. The resulting hydrogel forms a stable depot after perineural injection, enabling minimally invasive in situ gelation and localized drug delivery. We characterize its physicochemical properties, ibuprofen release profile, and biosafety, and evaluate its immunomodulatory and pro-regenerative effects in vitro and in a rat sciatic nerve crush model. In vitro, the IBU-CS-GP hydrogel suppresses macrophage inflammatory activation and reduces pro-inflammatory mediator production, thereby promoting a repair-supportive phenotype; in parallel, it indirectly enhances endothelial and stromal cell activities involved in angiogenesis and matrix remodeling. In vivo, perineural injection results in sustained ibuprofen release, accompanied by accelerated recovery of gait and nerve conduction, better preservation of gastrocnemius muscle mass and architecture, and more organized axon regeneration. These data suggest that the IBU-CS-GP hydrogel is a promising minimally invasive local therapy for non-transecting peripheral nerve injuries, as it enables near-neutral-pH in situ gelation and modulates the post-injury microenvironment.
Peripheral nerve injuries are common sequelae of traffic accidents, occupational trauma, and iatrogenic injuries, affecting about 18 % of patients with limb trauma [1,2]. Epidemiologic data indicate that 551,612 U S. patients sustained activity-related peripheral nerve injuries between 2009 and 2018; among these, traumatic brachial plexus injuries carry long-term indirect costs exceeding US$0.8 million per patient [3,4]. These injuries impose substantial, long-term functional and quality-of-life burdens, and despite advances in microsurgical repair, many patients retain sensory and motor deficits that necessitate prolonged rehabilitation and, in some cases, secondary operations [5,6]. In clinical practice, entrapment neuropathies are especially common yet frequently underdiagnosed or misattributed to radiculopathies, because of overlapping symptom patterns and the limited sensitivity of current electrodiagnostic and imaging techniques [7]. Experimentally, heterogeneity in model design and outcome measures impedes cross-study comparison, while Wallerian degeneration, neuroinflammation, Schwann-cell plasticity, and target-organ denervation together constitute an intricate, incompletely mapped cascade [8,9].
At present, the main clinical treatments for peripheral nerve injury are direct neurorrhaphy, autologous nerve grafting, and artificial nerve conduits. Autologous nerve grafting is still considered as the “gold standard” for repairing peripheral nerve defects. However, surgeons often lack suitable donor nerves, donor-site complications are common, the diameters of donor and recipient nerves may not match, and painful neuromas can form. Artificial nerve conduits have shown progress in the repair of short peripheral nerve defects. However, most nerve conduits still act mainly as passive physical bridges, with limited control over the inflammatory, oxidative, and angiogenic microenvironment, and with mechanical properties that often mismatch those of native nerve, thereby undermining long-term regeneration [10]. Clinical studies suggest that implant-associated infections at the injury or repair site markedly increase antibiotic consumption, scar formation, reoperation rates, and even the risk of amputation, with potentially catastrophic consequences for nerve repair [11]. Moreover, infection-driven chronic inflammation and bacterial toxins can substantially amplify the inflammatory cascade triggered by peripheral nerve injury and are strongly associated with impaired vascular regeneration and delayed electrophysiological recovery of the injured nerve. In addition, epineurial scarring is one of the major negative determinants of peripheral nerve regeneration; excessive hemorrhage promotes scar formation, and the resulting mechanical compression and perineural adhesions slow axonal regrowth and may ultimately culminate in compression neuropathy [12]. In peripheral nerve crush injuries, the nerve trunk usually remains continuous. In these cases, traditional extensive open surgery is often not the first choice. There is an urgent need for a minimally invasive and convenient local treatment, which can support nerve regeneration in situ [13].
Hydrogels are widely used as three-dimensional scaffolds that mimic the extracellular matrix for tissue engineering applications [14]. In recent years, many tissue engineering studies on peripheral nerve injury have used hydrogels as intraluminal fillers in nerve conduits, as wrapping materials around nerves, and as local sustained-release drug depots. By precisely tuning the material composition and microstructure, these hydrogels can recruit Schwann cells, support axon regeneration, and promote angiogenesis [10]. Injectable hydrogels can be delivered as liquids through a syringe, then undergo in situ gelation in response to temperature, pH, or chemical crosslinking. This minimally invasive approach can fill irregular defects and provide sustained local drug release. Because of these advantages, injectable hydrogels have become an important strategy for local drug delivery in nerve regeneration [15]. Clinically approved or investigational injectable hydrogels are largely confined to a limited number of material platforms, predominantly systems based on hyaluronic acid and polyethylene glycol, whose clinical indications are mainly restricted to soft-tissue augmentation and local sustained drug delivery [16]. In contrast, mature products specifically designed for peripheral nerve regeneration remain scarce and are insufficient to meet current clinical needs in peripheral nerve repair.
Chitosan (CS), a cationic polysaccharide derived from the deacetylation of chitin, has been extensively employed in peripheral nerve conduits and related scaffolds owing to its favorable biocompatibility, biodegradability, and intrinsic hemostatic and antibacterial activities [17]. At the molecular level, chitosan is composed of repeating units of β-(1 → 4)-linked D-glucosamine and N-acetyl-D-glucosamine, with the primary amine at C2 (C2–NH2) and the hydroxyl groups at C3 and C6 (C3/C6–OH) serving as its main reactive sites [18]. This structure enables protonation of the amino groups under acidic conditions, rendering the polymer positively charged, but also entails practical limitations. As a weakly basic polysaccharide with a pKa value of 6.5 for the C2–NH2 groups, chitosan becomes largely deprotonated at or above physiological pH (7.4), which strengthens interchain hydrogen bonding and increases crystallinity, thereby making it almost insoluble in neutral and alkaline aqueous media, while remaining soluble only in dilute acidic solutions [19]. These solubility characteristics markedly restrict the application of chitosan in near-neutral pH environments, such as injectable systems and tissue-engineered hydrogels for in vivo use. To address these structural limitations, current mainstream strategies focus on chemical modification of the C2–NH2 and C3/C6–OH groups of chitosan, in combination with crosslinking and composite design, to tailor its solubility, charge properties, and network architecture [20]. Acylation is one of the most widely used chemical modification strategies for chitosan-based materials, as it disrupts intra- and intermolecular hydrogen bonds within the polymer chains, reduces crystallinity, and thereby improves the aqueous solubility of chitosan. Among these derivatives, O-acylation increases the lipophilicity of chitosan, whereas N-acylation of the C2–NH2 groups enhances its water solubility. Ibuprofen (IBU) is a classic small-molecule nonsteroidal anti-inflammatory drug (NSAID) that, in addition to exerting cyclooxygenase (COX) pathway–mediated anti-inflammatory effects, can form N-acyl linkages to the C2–NH2 groups of chitosan via its carboxyl group to form an amide-linked chitosan–ibuprofen conjugate (IBU-CS), which remains soluble under near-neutral pH conditions [21]. Recent studies have shown that the natural crosslinker genipin (GP) achieves the highest degree of crosslinking with chitosan under neutral pH conditions [22]. This implies that ibuprofen-mediated modification of chitosan may enable the fabrication of genipin-crosslinked, chitosan-based injectable hydrogels under near-neutral pH conditions.
Recent reviews have further highlighted the critical role of microenvironmental modulation and advanced material design in promoting peripheral nerve regeneration [23,24]. These works have significantly advanced our understanding of inflammation control and functional recovery mechanisms. However, a gap remains in developing minimally invasive interventions that can be delivered precisely to non-transecting injury sites while providing localized, sustained immunomodulation without the need for open surgery. To address this gap, we designed an injectable chitosan-based hydrogel (IBU-CS-GP) by covalently coupling ibuprofen to chitosan and using genipin as a natural crosslinker (Scheme 1). In this study, we utilized the standardized rat sciatic nerve crush model as a representative paradigm of non-transecting injury. The resulting in situ–forming hydrogel can be administered as a minimally invasive therapy for peripheral nerve crush injury, rapidly gelling at the injection site and promoting nerve regeneration by modulating the local inflammatory microenvironment. It is also expected to exhibit antibacterial activity and good biocompatibility. In parallel, the material may modulate macrophage polarization, attenuate oxidative stress, and support angiogenesis [25,26]. By acting on these targets in concert, IBU-CS-GP may enhance axon regeneration and improve functional recovery after sciatic nerve crush injury in rats. To test this hypothesis, we systematically evaluated the physicochemical properties, biosafety, in vitro anti-inflammatory and antioxidant effects, and in vivo nerve repair performance of this hydrogel. Our goal is to develop a candidate injectable immunomodulatory hydrogel and provide preclinical experimental basis for treating non-transecting peripheral nerve injuries.Scheme 1The IBU-CS-GP hydrogel was synthesized by chemically modifying chitosan with ibuprofen and subsequently using genipin as a natural crosslinker to obtain a neutral, chitosan-based injectable hydrogel. Upon minimally invasive local injection, the precursor solution undergoes in situ gelation at the injury site, modulates the local post-injury inflammatory microenvironment, and thereby promotes peripheral nerve repair. Created in https://BioRender.com. IBU, ibuprofen; CS; chitosan; GP, genipin; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide.Scheme 1
Chitosan (degree of deacetylation 90 %, 200 kDa), ibuprofen (IBU), and genipin (GP) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and sodium hydroxide (NaOH) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). N-hydroxysuccinimide (NHS) was purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Luria–Bertani (LB) medium and agar were supplied by Sangon Biotech Co., Ltd. (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin antibiotics were obtained from Gibco (USA). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (USA).
IBU-CS-GP hydrogel was prepared in two steps. First, ibuprofen (IBU) was grafted onto chitosan (CS) in an ethanol/water system using EDC/NHS carbodiimide chemistry. In brief, 1.61 g of CS (10 mM repeating units) was dissolved in 100 mL of 0.1 M HCl and stirred at room temperature until the solution became clear. At the same time, 0.206 g of IBU (1 mM) was dissolved in 50 mL of ethanol. EDC and NHS were added to the IBU solution to activate the carboxyl groups. The activated IBU solution was then added dropwise into the CS solution under vigorous stirring to obtain a clear and uniform reaction mixture. The reaction was kept at 25 °C, protected from light, and stirred slowly for 6 h. After the reaction, the mixture was dried at 37 °C and washed several times with ethanol to remove unreacted small molecules, and the CS-IBU conjugate was collected. In our previous studies, we established that IBU can be successfully grafted onto CS via EDC/NHS activation [21]. The reaction was verified by FTIR, which indicated the formation of amide linkages between the carboxyl group of IBU and the amino group of the CS backbone, evidenced by the characteristic Amide I and Amide II bands. To ensure the hydrophilicity of the hydrogel, the degree of substitution of IBU is typically controlled at < 30 %.
In the second step, the CS-IBU conjugate was crosslinked with genipin (GP) to form the IBU-CS-GP hydrogel. Briefly, 80 mg of CS-IBU conjugate was dissolved in 4 mL of phosphate-buffered saline (PBS) under vigorous stirring at room temperature to prepare a 2 % (w/v) CS-IBU solution. Then, 5 mg of GP was dissolved in 1 mL of PBS under vigorous stirring to obtain a 0.5 % (w/v) GP solution. The CS-IBU solution (2 % w/v) and GP solution (0.5 % w/v) were mixed at a volume ratio of 1 under vigorous stirring, and the mixture was poured into molds to form the IBU-CS-GP hydrogel. For the CS-GP control hydrogel, 80 mg of CS was dissolved in 4 mL of 0.1 M HCl under vigorous stirring at room temperature to prepare a 2 % (w/v) CS solution. The CS solution and GP solution were then mixed at a volume ratio of 1 under vigorous stirring and poured into molds to obtain the CS-GP hydrogel. A previously reported CS-MA photo-crosslinked hydrogel was used as an additional control [21].
To ensure sterility, the CS-IBU conjugate and genipin powders were subjected to ultraviolet irradiation for 30 min in a sterile laminar flow cabinet prior to dissolution. The precursor solutions were prepared using sterile PBS. All preparation steps were conducted in a sterile laminar flow cabinet. For both cell culture and animal experiments, the hydrogel precursor solution was prepared freshly and maintained on ice (4 °C) to inhibit gelation until immediate use.
The microstructure of the hydrogels was observed by scanning electron microscopy (SEM). CS-MA, CS-GP, and IBU-CS-GP hydrogels were freeze-dried and then sputter-coated with a thin layer of gold. The samples were fixed on conductive tape and imaged using a desktop SEM (XL desktop, Phenom, Netherlands) at an accelerating voltage of 5 kV. For pore size analysis, 27 pores were randomly selected from independent hydrogel samples. The pore diameters were measured using ImageJ software and then subjected to statistical analysis.
Pre-gel solutions were added into a 48-well plate at a volume of 600 μL (V0) per well to form hydrogels. After gelation, the hydrogels were removed and frozen at −80 °C overnight. The frozen hydrogels were then freeze-dried and weighed to obtain the dry weight (Wd). After that, the samples were immersed in water for a defined period, taken out, and weighed again to obtain the wet weight (Wt). The porosity (P) was calculated according to the following equation.p=(Wd−Wt)/ρH2OV0×100%
The mechanical properties of the hydrogels were tested according to a reported method [21]. Cylindrical hydrogel samples with a diameter of 10 mm and a height of 5 mm were prepared. The samples were soaked in normal saline for 12 h before testing to reach equilibrium and to mimic the in vivo environment. Before the test, excess surface water was gently removed with a Kimwipe. Unconfined compression was then performed on a universal testing machine (Instron 5567, USA) equipped with a 200 N load cell. The crosshead speed was set at 3 mm/min. The compressive modulus was calculated from the linear region of the stress-strain curve within 5 % strain.
The water uptake capacity and equilibrium water content (EWR) of the hydrogels were measured as described in our previous study [21]. For EWR testing, freeze-dried hydrogel samples with the same volume were immersed in deionized water until water uptake reached equilibrium. In brief, hydrogels were first freeze-dried and weighed to obtain the dry weight (Wd). The samples were then immersed in deionized water. At predetermined time points, the hydrogels were taken out, gently blotted to remove surface water, and weighed to obtain the wet weight (Ww). The water uptake (w) of the hydrogels was calculated using the following equation.w=Ww−WdWd×100%
In vitro degradation of the hydrogels was evaluated by incubating the samples in phosphate-buffered saline (PBS) in centrifuge tubes at 37 °C on a shaker. Pre-gel solutions (600 μL per well) were first added into a 48-well plate and allowed to crosslink. The crosslinked hydrogels were then freeze-dried, collected, and weighed to obtain the initial dry weight (W0). At predefined time points, the samples were taken out, freeze-dried again, and weighed to obtain the remaining dry weight (Wt). The degradation rate of the hydrogels was calculated using the following equation.Massremaining=WtW0×100%
The release of IBU from the IBU-CS-GP hydrogel was evaluated by UV–visible spectrophotometry. Cylindrical hydrogels (10 mm in diameter, 5 mm in height) were placed in centrifuge tubes containing 4 mL of PBS and incubated at 37 °C. At predetermined time points, 1 mL of the release medium was withdrawn for measurement, and 1 mL of fresh PBS was added back to keep the total volume constant. The collected samples were transferred to quartz cuvettes, and the absorbance was recorded at 220 nm using a TU1810 spectrophotometer (Thermo Fisher Scientific). The IBU concentration in each sample was calculated from a standard calibration curve prepared with known IBU concentrations.
A hemolysis assay was performed using a 2 % (v/v) red blood cell (RBC) suspension. RBCs were obtained from fresh anticoagulated whole blood collected from healthy rats. The blood was diluted with normal saline and centrifuged at 3000 rpm for 10 min. The supernatant was discarded. The RBCs were then washed three times with normal saline and finally adjusted to a 2 % (v/v) suspension. For the assay, 200 μL of pre-warmed hydrogel sample solution (37 °C, 30 min) was mixed with 500 μL of the RBC suspension. The mixtures were incubated at 37 °C for 1 h. After incubation, the samples were centrifuged at 3000 rpm, and the supernatants were collected. The absorbance of each supernatant was measured at 540 nm to evaluate hemoglobin release. Normal saline and deionized water were used as the negative and positive controls, respectively. The hemolysis rate was calculated according to the following equation.Hemolysisrate=As−AnAp−An
As is the absorbance of the supernatant in the test group, An is the absorbance in the negative control, and Ap is the absorbance in the positive control.
To assess the hemostatic ability of the hydrogels, a dynamic whole-blood clotting assay was performed. Hydrogels were incubated at 37 °C for 5 min. Then 100 μL of fresh anticoagulated rabbit whole blood (supplemented with 10 % sodium citrate) was added onto each sample, followed by 10 μL of CaCl2 solution (0.2 M). The samples were incubated at 37 °C for 10 min to allow blood clotting. After incubation, 10 mL of deionized (DI) water was added to each sample and shaken on an orbital shaker at 37 °C for 5 min to remove any non-adherent blood. The absorbance of the supernatant was measured at 540 nm (Dt). DI water and normal saline were used as the positive (Dm) and negative (Dn) controls. The blood clotting index (BCI) was calculated using the following equation.BCI=Dt−DnDm−Dn×100%
Whole blood clotting time was also measured. In brief, 400 μL of hydrogel was placed in a centrifuge tube, and 2 mL of whole blood containing 10 % sodium citrate was added. Then 60 μL of CaCl2 solution (0.25 M) was added to start coagulation. For the gauze control, rectangular pieces of gauze (10 mm long and 5 mm wide) were prepared. The tubes were inverted, and clot formation was checked each time. The time required for visible clot formation in each tube was recorded.
The in vitro antibacterial activity of the hydrogels was evaluated against Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923). Bacteria were grown overnight in LB broth at 37 °C on a shaker. Then, 400 μL of hydrogel was mixed with 200 μL of bacterial suspension (10^8^ CFU/mL) and incubated at 37 °C for 12 h. After incubation, 100 μL of each diluted sample was spread onto LB agar plates and cultured at 37 °C for another 12 h. Bacterial colonies were imaged using an automatic colony counter (Shineso Science & Technology Co., Ltd., Hangzhou, China). The number of colonies was counted in ImageJ, and the normalized survival rate was calculated based on the colony counts.
Hydrogels were incubated in complete culture medium for 24 h to prepare hydrogel-conditioned medium. The complete medium consisted of high-glucose DMEM supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin. After incubation, the supernatant was collected and used as hydrogel-conditioned medium.
Mouse fibroblasts (L929, ATCC), human umbilical vein endothelial cells (HUVECs, ATCC), and mouse macrophages (RAW 264.7, ATCC) were provided by the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM supplemented with 10 % FBS and 1 % penicillin/streptomycin at 37 °C in a humidified incubator with 5 % CO2. Cells were seeded in 48-well plates at a density of 0.8 × 10^4^ cells per well and allowed to attach at 37 °C. After firm attachment, the medium was replaced with different hydrogel-conditioned media. Cell viability and proliferation were assessed by live/dead staining and CCK-8 assays. Cells cultured in complete medium without hydrogel-conditioned medium served as the control group.
LPS-stimulated macrophages were used to evaluate the anti-inflammatory and antioxidant effects of the hydrogels. RAW264.7 cells (3 × 10^6^ cells/well) were seeded in six-well plates and cultured for 12 h. The cells were then incubated in serum-free medium containing 100 ng/mL LPS for 8 h. After that, the LPS-containing medium was removed, and the cells were cultured in hydrogel-conditioned medium for 24 h.
For immunophenotyping, cell pellets were collected and resuspended in 1 mL PBS containing 1 μg PE-conjugated anti-mouse CD206 antibody and 1 μg FITC-conjugated anti-mouse CD86 antibody (BioLegend, USA). The cell suspensions were incubated on ice for 30 min to maintain cell viability and reduce non-specific binding. The cells were then washed twice with PBS to remove unbound antibodies. Expression of CD86 and CD206 was analyzed using a CytoFLEX flow cytometer (Becton Dickinson, USA), and data were processed with FlowJo software (FlowJo v10).
Intracellular reactive oxygen species (ROS) levels were measured using a 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay kit (Beyotime, China), following the manufacturer's instructions. ROS levels were observed with an inverted fluorescence microscope and quantified by flow cytometry. Nitric oxide (NO) levels in the culture supernatants were determined using an NO detection kit (Beyotime, China) according to the manufacturer's protocol.
RAW264.7 cells were also used for RNA extraction as described above. Total RNA was isolated using an EZ-press RNA purification kit (EZBioscience, Roseville, MN, USA). The isolated RNA was reverse transcribed into complementary DNA (cDNA) using a Color reverse transcription kit (EZBioscience). Quantitative RT-PCR was then performed with 2 × Color SYBR Green qPCR Master Mix (EZBioscience). Relative expression of target genes was calculated using the 2-ΔΔCt method. Primer sequences are listed in Table S1 (Supplementary Table), and GAPDH was used as the internal reference gene.
RAW264.7 cells were seeded into six-well plates at a density of 3 × 10^5^ cells per well and cultured until they adhered. The medium was then replaced with fresh medium containing LPS (100 ng/mL), and the cells were stimulated for 12 h. After stimulation, the cells were gently washed with PBS. Then 1.5 mL of complete medium and 200 μL of hydrogel were added to each well, and the cells were cultured for another 24 h. The culture supernatants were collected and filtered through a 0.22 μm syringe filter (Millipore). The filtered supernatants were mixed with fresh medium for other cell types at a 2 (v/v) ratio to obtain macrophage-conditioned medium, which was used for subsequent experiments.
Tube formation in Matrigel (Cat. No. 356234, BD Matrigel, USA) was thawed at 4 °C and added to 48-well plates (100 μL per well). The plates were then incubated at 37 °C for 20 min to allow gelation. HUVECs were seeded onto the Matrigel-coated wells (2 × 10^4^ cells per well), and 200 μL of different macrophage-conditioned media was added to each well. The cells were cultured for 6 h. During this period, the cells migrated, extended, and formed capillary-like networks. Tube-like structures were observed under an optical microscope. ImageJ was used to analyze angiogenesis-related parameters, including the number of junctions, meshes, and main vessel segments.
Scratch wound healing L929 fibroblasts and HUVECs (both from ATCC) were seeded in 24-well plates (3 × 10^4^ cells per well) and grown to about 90 % confluence. A straight scratch was made across the cell monolayer using a sterile 200 μL pipette tip. The cells were washed with PBS to remove debris, and 1 mL of the corresponding macrophage-conditioned medium was added to each well. Cell migration was observed under an optical microscope at 0 h (t = 0) and 24 h (t = 24). The migration rate was calculated from the wound area at 0 h (P0) and 24 h (Pt) according to the following equation.Migrationrate=PtP0×100%
Transwell migration The migratory capacity of L929 fibroblasts and HUVECs was evaluated using a Transwell assay. Transwell inserts were placed in 24-well plates. A 200pl200 μL cell suspension (2 × 10^5^ cells/well) was added to the upper chamber, and 800 μL of different macrophage-conditioned media was added to the lower chamber. The plates were incubated at 37 °C for 12 h. After incubation, the cells were fixed with 4 % paraformaldehyde (PFA) for 30 min and stained with 100 μL of 1 % crystal violet solution for 5 min. Cells on the upper surface of the membrane were carefully removed with a wet cotton swab. Cells that had migrated to the lower surface were observed under an optical microscope (Eclipse, Nikon, Japan).
All animal experiments were designed and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 8th Edition, revised 2011) and all surgical procedures were reviewed and approved by the Ethics Committee of Donghua University (DWSY202510280225). Male adult Sprague–Dawley rats (200–250 g, 6–8 weeks) were used to establish the sciatic nerve crush injury model. Rats were randomly allocated to the four groups (a control group and three hydrogel groups) using a random number generator. Anesthesia was induced with isoflurane using a small-animal anesthesia system. After the rats reached a stable anesthetic state, the isoflurane flow was reduced. The rats were placed in the prone position, and the limbs were fixed. The hair around the hip region was removed with an electric shaver. The surgical field was disinfected thoroughly with povidone–iodine. A longitudinal skin incision was made parallel to the body axis and crossing the medial third of the femur. The skin and muscle were bluntly dissected layer by layer to expose the main trunk of the sciatic nerve. A single vascular clamp (Jinzhong Surgical Instrument, J31050) was used for all procedures. The tip of the clamp was applied to the sciatic nerve trunk and locked at the second ratchet position for 20 s. This crush procedure was repeated three times. Compound muscle action potentials (CMAPs) were recorded before and after the crush to confirm successful axonotmesis. A 6-0 non-absorbable suture was tied to the muscle tissue adjacent to the crush site to mark the injury level.
After model establishment, 10 μL of sterile normal saline (Control group) or sterilized hydrogel precursor solution (Hydrogel CS-MA photo-crosslinked hydrogel, CS-GP hydrogel, or IBU-CS-GP hydrogel) was injected around the sciatic nerve crush site using a 26-gauge needle. The needle was inserted parallel to the long axis of the nerve to ensure precise delivery around the epineurium without damaging the nerve fascicles. For the CS-MA photo-crosslinked hydrogel, visible light from an LED source (405 nm) was applied for 2 min at a distance of 1.5 cm after injection. For the other two hydrogels, the injected material was left in place for 5 min to allow spontaneous crosslinking at body temperature. After confirmation of in situ gelation, the muscle and skin were closed layer by layer. Anesthesia was discontinued, and the rats were returned to their cages after full recovery from anesthesia. A priori exclusion criteria included severe wound infection, autophagy of the operated limb, or death prior to the endpoint. No animals were excluded from the final analysis in this study.
On postoperative days 7, 14, and 28, rats were placed on a small-animal gait analysis system (XR-FP101, Shanghai Xinruan) and allowed to walk freely. Hind paw prints were recorded during normal walking. The footprints of the hind limbs were highlighted with a blue fluorescent marker to improve contrast. For each rat, gait parameters were measured on the experimental side (E) and the normal side (N). The following footprint parameters were print length (PL; distance from the heel to the tip of the third toe), toe spread (TS; distance between the first and fifth toes), and intermediary toe spread (IT; distance between the second and fourth toes). The sciatic functional index (SFI) was calculated using the following formula.SFI=−38.3×EPL−NPLNPL+109.5×ETS−NTSNTS+13.3×EIT−NITNIT−8.8
EPL/NPL, ETS/NTS, and EIT/NIT represent the ratios of the experimental side to the normal side for each parameter. The SFI ranges from −100 to 0, where 0 indicates normal sciatic nerve function and −100 indicates complete loss of function.
On postoperative day 28, rats were re-anesthetized using the same protocol as in the initial surgery. The sciatic nerve on the operated side was re-exposed. Bipolar hook stimulating electrodes were placed sequentially at the distal and proximal sites relative to the crush lesion. Latency and compound muscle action potentials (CMAPs) were recorded from the belly of the ipsilateral tibialis anterior (TA) muscle using an electromyography system (Medtronic LeadPoint 4 EEG/EMG System). Nerve conduction velocity (NCV) and CMAP amplitude of the regenerated sciatic nerve were then calculated.
On postoperative days 7, 14, and 28, three rats from each group were randomly selected and anesthetized. The gastrocnemius muscles on the experimental side and the normal side were carefully dissected. Each muscle was weighed three times using an electronic analytical balance (FA124C, Shanghai Lichen), and the mean value was recorded. The gastrocnemius wet weight ratio (Pw) was calculated using the following formula.Pw=Wewn×100%
We is the wet weight of the gastrocnemius muscle on the experimental side, and Wn is the wet weight on the normal side. On postoperative day 28, gastrocnemius samples collected after weighing were fixed in 4 % paraformaldehyde for subsequent histological staining.
Sciatic nerve tissue at the crush site was harvested and fixed in 2.5 % glutaraldehyde solution for 48 h. Ultrathin transverse sections with a thickness of 50 nm were then cut using an ultramicrotome (EM UC6, Leica). The sections were placed on grids coated with 0.5 % formvar and stained with uranyl acetate and lead citrate. Transmission electron microscopy (TEM) imaging was performed at an accelerating voltage of 100 kV using a HITACHI HT7700 Exalens microscope. In each TEM image, random fields containing intact myelinated fibers were selected. Axon inner diameter, fiber outer diameter, myelin sheath thickness, and g-ratio (ratio of axon inner diameter to fiber outer diameter) were measured in these fields.
At postoperative day 28, sciatic nerve tissue at the crush site and gastrocnemius muscles were harvested for H&E, toluidine blue (TB), immunohistochemical, and immunofluorescence staining. The mid-portion of each muscle or nerve sample was fixed, dehydrated, embedded in paraffin, and sectioned at 5 μm thickness. For general morphology, nerve sections were stained with H&E or 1 % toluidine blue (TB), and images were acquired under a light microscope. Muscle sections were stained with Masson's trichrome. The proportion of collagen fiber area in random fields was quantified. For immunohistochemistry, paraffin-embedded nerve sections were deparaffinized and rehydrated through a graded ethanol series. Sections were then incubated in 0.1 mol/L sodium citrate for 20 min for antigen retrieval and blocked with 10 % bovine serum albumin (BSA). The sections were incubated with anti-NF200 primary antibody at 4 °C overnight. After washing, an HRP-conjugated secondary antibody was applied at room temperature for 1 h. DAB was used as the chromogen, and images were captured with a light microscope. For immunofluorescence, paraffin-embedded nerve sections were deparaffinized, rehydrated, and subjected to antigen retrieval. The tissue was then fixed with 4 % paraformaldehyde and permeabilized with 0.2 % Triton X-100, followed by blocking with 10 % BSA. Sections were incubated with specific primary antibodies. To identify regenerating sciatic axons, anti-NF200 or anti-TUBB3 antibodies were used. To evaluate Schwann cells and myelin, anti-S100β and anti-MBP antibodies were applied. After incubation with appropriate fluorescent secondary antibodies, nuclei were counterstained with DAPI. Immunofluorescence images were observed and recorded using a fluorescence microscope.
To minimize bias, data collection and analysis for gait, electrophysiology, and histology were performed by investigators blinded to the experimental grouping. For histological quantification, at least 3 random non-overlapping fields of view (FOVs) were captured per section (or per animal for TEM). Quantitative parameters from these FOVs were averaged to generate a single representative value for each biological replicate for subsequent statistical comparison. Unless otherwise specified, the sample size (n) reported in the figure legends refers to the number of independent biological replicates rather than technical replicates. Statistical differences between two groups were evaluated using a two-tailed Student's t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey's post hoc test. All statistical analyses were carried out using GraphPad Prism 10.0. A p value < 0.05 was considered statistically significant. Significance levels are shown as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Detailed statistical results are provided in the corresponding figure legends.
As shown in Fig. 1A, the pre-gel solutions of CS-MA, CS-GP, and IBU-CS-GP were clear before gelation. All three systems formed stable hydrogels after crosslinking. CS-MA remained as a transparent hydrogel, whereas the clear CS-GP and IBU-CS-GP solutions turned into dark green hydrogels. SEM images revealed the internal microstructure of the hydrogels (Fig. 1B). All groups exhibited a typical porous architecture. Quantitative pore size analysis (Fig. 1C) showed that the average pore sizes of CS-MA, CS-GP, and IBU-CS-GP hydrogels were 248 ± 92 μm, 216 ± 56 μm, and 192 ± 80 μm, respectively. The IBU-CS-GP hydrogel had a significantly smaller pore size than the CS-MA hydrogel (p < 0.05), while no significant difference was observed between the CS-GP and IBU-CS-GP hydrogels. The porosity results are shown in Fig. 1D. The porosity of the CS-MA, CS-GP, and IBU-CS-GP hydrogels was 43.9 ± 4.4 %, 52.6 ± 2.0 %, and 52.6 ± 3.6 %, respectively. The CS-MA hydrogel showed a significantly lower porosity than both the CS-GP and IBU-CS-GP hydrogels (p < 0.05).Fig. 1Physicochemical properties of hydrogels. Macroscopic photos of CS–MA, CS-GP and IBU-CS-GP injectable hydrogel (A). SEM images of the cross sections of injectable hydrogels (B). Pore size (C), and porosity (D), typical compressive stress–strain curves (E) with peak compression strength (F), compressive modulus (G), and breaking strains (H), water absorption (I), in vitro degradation (J), and drug release profile (K). Unpaired two-tailed student's t-test n = 27 for C, n = 3 for D and F-K; ∗p < 0.05, n.s. CS, chitosan; IBU, ibuprofen; GP, genipin; MA, methacrylic anhydride; n.s., not significant; SEM, scanning electron microscopy.Fig. 1
Fig. 1E–H shows the mechanical properties of the hydrogels in the wet state. All three groups had similar stress–strain curves; in the early stage of compression, stress increased linearly with strain, indicating an elastic response at low strains (Fig. 1E). The peak compressive strength of IBU-CS-GP reached 18.7 ± 6.4 kPa and was significantly higher than that of CS-GP (12.4 ± 2.3 kPa; p < 0.05), but was similar to that of CS-MA (14.6 ± 2.3 kPa) (Fig. 1F). The fracture strain of IBU-CS-GP was 50.1 ± 4.1 %, which was significantly higher than that of CS-GP (36.4 ± 5.9 %; p < 0.05) and close to that of CS-MA (44.5 ± 5.4 %) (Fig. 1G). However, the compressive modulus of IBU-CS-GP (3.1 ± 0.4 kPa) did not differ from that of CS-GP or CS-MA (Fig. 1H).
Fig. 1I shows the water uptake behavior of CS-MA, CS-GP, and IBU-CS-GP. The IBU-CS-GP hydrogel took up water quickly during the first 30 min; then the swelling rate slowed and the system reached a steady state. The three groups showed similar swelling curves over time, but the CS-GP hydrogel consistently exhibited greater water uptake than IBU-CS-GP and CS-MA.
The degradation profiles of the hydrogels are shown in Fig. 1J. After 28 days, IBU-CS-GP retained 35.9 ± 6.2 % of its initial dry mass, and CS-GP retained 26.0 ± 4.0 %. In contrast, CS-MA was completely degraded by day 21, indicating that CS-MA degraded more rapidly than CS-GP and IBU-CS-GP.
IBU release from the IBU-CS-GP hydrogel was measured for up to 28 days (Fig. 1K). In the first 7 days, the hydrogel released about 60 % of the loaded IBU—this phase exhibited relatively rapid release kinetics. The release rate then became slower, and the cumulative release was nearly complete by day 28 and subsequently plateaued. Taken together, these data suggest that IBU-CS-GP provides a slow and sustained drug release profile in vitro.
Chitosan can bind to the negatively charged surfaces of bacterial cell membranes and exhibits strong antibacterial activity [27]. Based on this property, we examined the antibacterial performance of the hydrogels. As shown in Supplementary Fig. 1A, we assessed their antibacterial activity in vitro against two common bacterial strains, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Compared with the phosphate-buffered saline (PBS) control group, all three hydrogels (CS-MA, CS-GP, and IBU-CS-GP) resulted in a marked decrease in bacterial colony counts. Quantitative analysis showed that the normalized survival of E. coli and S. aureus in the IBU-CS-GP group was 33.9 ± 2.2 % for E. coli (Supplementary Fig. 1B) and 50.7 ± 2.3 % for S. aureus (Supplementary Fig. 1C). These values were not significantly different from those observed for the CS-MA and CS-GP groups. Together, these data indicate that the modified chitosan-based hydrogels retain appreciable antibacterial activity, supporting their potential to reduce infection risk associated with minimally invasive clinical procedures.
Hemocompatibility assessments showed that hydrogels promoted clot formation with significantly lower BCI values than gauze (Supplementary Fig. 2A–D). Furthermore, hemolysis rates were all ≤5 %, adhering to ISO 10993-4 standards (Supplementary Fig. 2E and F). HUVECs, L929 cells, and RAW264.7 cells maintained high viability in all hydrogel-conditioned media. Live/dead staining and CCK-8 assays confirmed sustained metabolic activity, with no significant differences among the groups (Supplementary Fig. 3). Histological examination of major organs revealed no overt damage, indicating good in vivo biocompatibility (Supplementary Fig. 4).
Inflammation plays a key role in nerve repair by initiating the healing process [8]. Macrophages are key effector cells in this response [28]. They adopt different phenotypes, such as the pro-inflammatory M1 and the anti-inflammatory M2 phenotype [29]. M1 macrophages drive inflammatory responses, whereas M2 macrophages support tissue repair and exert anti-inflammatory effects. A shift in macrophages from the M1 to the M2 phenotype is therefore considered a promising anti-inflammatory strategy. We next examined how the hydrogels modulated macrophage polarization and ROS scavenging after LPS stimulation. Flow cytometry revealed distinct changes in macrophage subsets. After treatment with IBU-CS-GP hydrogel–conditioned medium, the percentages of CD206+ M2 macrophages and CD86^+^ M1 macrophages both increased compared with the Control, CS-MA, and CS-GP groups (Fig. 2A and B). We also measured the mRNA levels of TNF-α and IL-4 by qPCR. As shown in Fig. 2C and D, cells treated with IBU-CS-GP hydrogel–conditioned medium showed a marked decrease in the pro-inflammatory cytokine TNF-α compared with the CS-MA and CS-GP groups. At the same time, the anti-inflammatory cytokine IL-4 was further upregulated in cells treated with IBU-CS-GP hydrogel–conditioned medium.Fig. 2Anti-inflammatory and antioxidative capacity of hydrogels. Flow cytometry (A) reveals that IBU-CS-GP hydrogel significantly promote M2 polarization of LPS-stimulated RAW264.7 macrophages (B) RT-PCR analysis suggests that IBU-CS-GP downregulates pro-inflammatory gene TNF-α (C) and upregulates anti-inflammatory genes of IL-4 (D) in comparison to CS-MA and CS-GP. IBU-CS-GP markedly attenuates ROS and NO production in LPS-stimulated macrophages, as indicated by DCFH staining (H), flow cytometry analysis (G), and ROS & NO quantification (E–F). One-way ANOVA with Tukey's post hoc test and n = 3 for B-F; ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. ANOVA, analysis of variance; CS, chitosan; IBU, ibuprofen; GP, genipin; MA, methacrylic anhydride; LPS, lipopolysaccharides; n.s., not significant; NO, nitric oxide; PCR, polymerase chain reaction; ROS, reactive oxygen species; DCFH, 2′,7′-Dichlorodihydrofluorescein; DAPI, 4′,6-diamidino-2-phenylindole.Fig. 2
Previous studies have shown that NO production plays an important role in inflammation [30]. High NO levels are cytotoxic and are linked to many inflammatory diseases [31]. In our study, the IBU-CS-GP hydrogel group showed a significant reduction in NO levels, which suggests that this treatment alleviated oxidative stress–related damage (Fig. 2E). We then used the fluorescent probe DCFH-DA to evaluate intracellular reactive oxygen species (ROS) in macrophages under oxidative stress. The fluorescence signal was recorded and quantified by flow cytometry and fluorescence imaging. As shown in Fig. 2F and G, LPS treatment resulted in markedly elevated ROS levels, whereas the percentage of DCFH-positive cells in the IBU-CS-GP group was significantly lower than in the other groups. Consistent with this, DCFH fluorescence staining showed a clear decrease in green fluorescence intensity in the IBU-CS-GP group (Fig. 2H), indicating a stronger ROS-scavenging capacity.
Angiogenesis is widely regarded as a fundamental process in the regeneration and restoration of diverse tissues. The induction of de novo blood vessels is considered essential for the successful performance of scaffolds in peripheral nerve regeneration [32]. To test whether macrophages activated by the IBU-CS-GP hydrogel could support tissue repair, we used macrophage-conditioned medium (CM) to culture HUVECs and performed a tube formation assay. After 6 h, HUVECs treated with CM from the IBU-CS-GP group formed more prominent capillary-like networks than those in the Control, CS-MA, and CS-GP groups (Fig. 3A). Quantitative analysis showed that the number of branch points was highest in the IBU-CS-GP group (Control, CS-MA, CS-GP, and IBU-CS-GP: 6.7 ± 2.1, 11.7 ± 1.5, 13.3 ± 1.5, and 32.0 ± 6.6, respectively) (Fig. 3D). Consistently, the IBU-CS-GP group also induced more branches (Control, CS-MA, CS-GP, and IBU-CS-GP: 13.7 ± 3.8, 15.3 ± 3.1, 17.7 ± 2.3, and 31.3 ± 3.1, respectively) (Fig. 3E).Fig. 3Pro-angiogenic and pro-migratory effects of hydrogels. Immunofluorescence staining shows blood vessel formation of HUVECs in LPS-macrophage condition medium (A). Scratch assays and Transwell migration assays of HUVECs (B) and L929 cells (C). Quantification of junctions (D), branches (E), wound closure percentage (F–G) and migrated cells (H–I). One-way ANOVA with Tukey's post hoc test and n = 3 for D-I; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. ANOVA, analysis of variance; CS, chitosan; IBU, ibuprofen; GP, genipin; MA, methacrylic anhydride; LPS, lipopolysaccharides; HUVEC, human umbilical vein endothelial cell; n.s., not significant.Fig. 3
We then used scratch assays and Transwell migration assays to examine how different CM samples affected HUVECs and L929 fibroblasts. After 24 h of co-culture with CM from the IBU-CS-GP group, both HUVECs and L929 cells exhibited markedly increased migratory capacity compared with the other groups (HUVEC migration IBU-CS-GP = 82.3 % ± 2.9 % vs. Control = 27.2 % ± 1.8 %, CS-MA = 23.6 % ± 2.3 %, CS-GP = 36.5 % ± 8.1 %; L929 migration IBU-CS-GP = 83.2 % ± 1.0 % vs. Control = 25.2 % ± 3.7 %, CS-MA = 25.5 % ± 3.5 %, CS-GP = 40.6 % ± 2.6 %) (Fig. 3B, C, F, G). Transwell assays for both cell types showed a similar trend. For HUVECs, the number of migrated cells per high-power field (HPF) in the Control, CS-MA, CS-GP, and IBU-CS-GP groups was 139.0 ± 42.2, 231.0 ± 23.4, 232.3 ± 19.7, and 396.7 ± 20.6, respectively (Fig. 3B–H). For L929 cells, the corresponding values were 219.3 ± 10.2, 206.3 ± 22.9, 270.3 ± 17.6, and 436.0 ± 34.7 cells/HPF (Fig. 3C–I). These results suggest that CM from the IBU-CS-GP hydrogel not only enhances cell migration in vitro but also supports the formation of well-defined tubular networks, giving the strongest pro-angiogenic effect among all groups. These findings further indicate that this chitosan-based hydrogel exhibits ECM-like properties that are conducive to cell adhesion and growth.
To further verify the in vivo anti-inflammatory and nerve-repair effects of the IBU-CS-GP co-loaded hydrogel, we established a rat sciatic nerve crush injury model. The IBU-CS-GP hydrogel, photo-crosslinked CS-MA hydrogel, and drug-free CS-GP hydrogel were each injected around the sciatic nerve crush site, while rats in the control group received an equal volume of sterile saline. After confirming in situ gelation, the muscle and skin were closed in layers, and the rats were returned to their home cages.
Gait footprint analysis (Fig. 4A) and SFI evaluation (Fig. 4B) suggested that rats in the IBU-CS-GP group exhibited better hindlimb functional recovery than those in the other groups. After sciatic nerve crush, all groups showed severe motor deficits, and SFI values were close to −100, indicating almost complete loss of function. Over time, SFI scores gradually improved in all groups. This trend suggests that regenerating nerve fibers gradually reinnervated the target muscles and that motor function partly recovered. However, the IBU-CS-GP group showed faster and greater improvement in SFI than the other groups. By day 28, rats in the IBU-CS-GP group already exhibited a more normal gait pattern. Their footprints displayed more physiological toe spread and stepping pattern. In contrast, the control group and the CS-MA group still showed abnormal gait with paw contracture (Fig. 4A). These gait data indicate that treatment with the IBU-CS-GP co-loaded hydrogel can accelerate motor function recovery in rats after sciatic nerve crush injury.Fig. 4Functional assessment in rats after sciatic nerve crush injury. Representative hind paw footprints at different postoperative time points (A), with SFI calculated from footprint parameters (B). Electrophysiological evaluation of the sciatic nerve was performed at post-operation day 28 (C), NCV and recovery indices of CMAP were quantified (D–E). Bilateral gastrocnemius muscles were harvested and weighed at three postoperative time points (F), and the gastrocnemius wet weight ratio was calculated (G). One-way ANOVA with Tukey's post hoc test and n = 3 for B, D, E and G; ∗p < 0.05, ∗∗p < 0.01. ANOVA, analysis of variance; CS, chitosan; IBU, ibuprofen; GP, genipin; MA, methacrylic anhydride; SFI, sciatic functional index; PL, print length; TS, toe spread; IT, intermediary toe spread; NCV, nerve conduction velocity; CMAP, compound muscle action potential.Fig. 4
Electrophysiological recordings of the regenerated sciatic nerve showed that the IBU-CS-GP hydrogel performed best in restoring nerve conduction (Fig. 4C). At day 28, the NCV in the IBU-CS-GP group was the highest among all groups (Fig. 4D) and was significantly higher than in both the untreated control and CS-MA groups (p < 0.05). This suggests more complete remyelination of regenerated axons and a more effective recovery of conduction in the IBU-CS-GP group. We then used the recovery index of CMAP amplitude to evaluate how well the injured nerve could drive the target muscle. This index was calculated as the percentage of CMAP amplitude on the injured side relative to the normal side (Fig. 4E). Rats treated with IBU-CS-GP showed a much higher CMAP recovery index than control rats (p < 0.01), which means that muscle function was better restored after reinnervation in this group. Overall, these electrophysiological data indicate that local ibuprofen delivery via the IBU-CS-GP hydrogel enhances nerve regeneration and leads to a more complete recovery of neuromuscular function than the other treatments.
It is well known that prolonged denervation after peripheral nerve injury leads to marked atrophy of target muscles [33,34]. On postoperative day 28, the gastrocnemius muscle on the injured side in the Control group was clearly smaller than the muscle on the contralateral side, which points to marked atrophy due to poor reinnervation. In the IBU-CS-GP group, the situation was different. The injured gastrocnemius muscle kept much more of its volume, and its size was closer to that of the opposite limb. The CS-MA and CS-GP groups showed changes between these two extremes, with visible atrophy, but less severe than in the Control group (Fig. 4F). To quantify this, we measured the gastrocnemius wet weight ratio (injured/contralateral side) on days 7, 14, and 28 (Fig. 4G). A higher ratio means less muscle loss, and a value near 1 would suggest almost no atrophy. On day 7, the IBU-CS-GP group had a slightly higher ratio than the other three groups, but the differences were not significant. By day 14, the wet weight ratio in the IBU-CS-GP group was already significantly higher than in the Control group. By day 28, this ratio in the IBU-CS-GP group was significantly higher than in both the Control and CS-MA groups (p < 0.01), indicating better protection of muscle mass over time.
When we compared the three time points within each group, we found that the decline in gastrocnemius wet weight ratio over time was relatively slow in the IBU-CS-GP group, whereas the other groups showed a sharper decrease, with the largest drop in the Control group. Together with the gait and electrophysiological data, this pattern suggests that the IBU-CS-GP hydrogel supports earlier and more effective reinnervation of muscle fibers, helps to preserve muscle mass, and slows the development of gastrocnemius atrophy after denervation. Overall, these findings show that the IBU-CS-GP hydrogel has a clear advantage in promoting nerve repair and limiting muscle loss. Compared with the Control group and the hydrogel groups without ibuprofen, rats treated with IBU-CS-GP showed faster and more complete recovery of motor function, as seen in better gait patterns and higher SFI scores. Electrophysiological outcomes were also better in this group, with higher NCV and a greater CMAP recovery index. At the same time, gastrocnemius atrophy was less pronounced, and muscle mass on the injured side was better maintained in the IBU-CS-GP group.
Regenerated sciatic nerve tissue at the crush site was harvested on postoperative day 28. Representative cross-sections from the mid-portion of the lesion were processed for histological staining. Hematoxylin and eosin (H&E) staining revealed no overt tissue necrosis or scar-like fibrosis in any group (Fig. 5C). In the IBU-CS-GP group, the overall nerve architecture was better preserved. Nerve fascicles were more compact and regularly arranged, whereas the regenerated nerve tissue in the control group appeared more disorganized. Toluidine blue (TB) staining was used to visualize myelin (Fig. 5C). Sections from the IBU-CS-GP group showed intense dark-blue staining, indicating a higher content of mature myelinated fibers than in the other groups.Fig. 5Histological evaluation of sciatic nerve and gastrocnemius muscle sections at post-operation day 28. Double immunofluorescence staining for S100β and NF200 in sciatic nerve sections (A). Double immunofluorescence staining for MBP and TUBB3 in sciatic nerve sections (B). H&E staining and TB staining of the sciatic nerve (C). Immunohistochemical staining for NF200 in the injured sciatic nerve (D). Masson's trichrome staining of gastrocnemius muscle sections(E). Quantitative analysis of the collagen fiber area fraction in the gastrocnemius muscle (F). One-way ANOVA with Tukey's post hoc test and n = 3 for F; ∗p < 0.05, ∗∗p < 0.01. ANOVA, analysis of variance; CS, chitosan; IBU, ibuprofen; GP, genipin; MA, methacrylic anhydride; S100β, a Schwann cell marker; NF200, neurofilament protein NF200; MBP, myelin basic protein; TUBB3, β3-Tubulin; H&E, hematoxylin and eosin; TB, toluidine blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 5
In parallel, dual immunofluorescence staining was performed on regenerated sciatic nerves on postoperative day 28. S100β and MBP were used to evaluate regeneration of Schwann cells and myelin, and NF200 and TUBB3 were used to assess axon regeneration (Fig. 5A and B). The immunofluorescence images implied a clear advantage in the IBU-CS-GP group. Regenerating nerve fibers in this group were dense and well aligned. The number of regenerating axons was higher, and Schwann cells were distributed around the axonal bundles, indicating close interactions between regenerating axons and Schwann cells. In contrast, the other groups exhibited weaker and more scattered positive signals for these markers, suggesting slower axonal regrowth and less efficient Schwann cell recruitment.
Consistent with the immunofluorescence findings, NF200 immunohistochemical staining further confirmed the pro-regenerative effect of IBU-CS-GP (Fig. 5D). Regenerated nerves in the IBU-CS-GP group contained a substantially higher axonal density and numerous dark-brown NF200-positive fibers than the control groups. Overall, these histological data indicate that, by postoperative day 28, the IBU-CS-GP hydrogel markedly enhances axon regeneration and promotes Schwann cell recruitment to support peripheral nerve repair.
On postoperative day 28, samples from the midportion of the gastrocnemius muscle were stained with Masson's trichrome to quantify muscle fibrosis and to further assess how early reinnervation affected muscle quality (Fig. 5E). In the Control group, there was marked deposition of blue-stained collagen fibers, which almost completely filled the spaces between muscle fibers and indicated severe muscle atrophy and fibrosis. In the CS-MA and CS-GP groups, the proportion of collagen fibers was lower than in the Control group, with CS-GP performing slightly better than CS-MA. The IBU-CS-GP group showed the lowest collagen content; only a small amount of collagen was observed between muscle bundles, and the overall structure and staining pattern of the muscle more closely resembled those of normal tissue (Fig. 5F). These findings suggest that the IBU-CS-GP hydrogel effectively reduces denervation-induced muscle fibrosis and helps preserve muscle quality.
To examine how each hydrogel influenced sciatic nerve repair at the microscopic level, we performed TEM of four representative transverse sections from each group at day 28 (Fig. 6A). Compared with the Control group, all three hydrogel groups showed markedly thicker myelin sheaths. Among them, the IBU-CS-GP group had visibly thicker myelin than the CS-MA group, while myelin thickness was comparable between the IBU-CS-GP and CS-GP groups (Fig. 6B). We then analyzed the g-ratio, defined as the ratio of axon inner diameter to fiber outer diameter, which reflects the maturation of myelinated fibers; within an appropriate range, a lower g-ratio indicates more mature myelin. The IBU-CS-GP group exhibited a significantly lower g-ratio than each of the other three groups (Fig. 6C), supporting the notion that this hydrogel speeds up the maturation of regenerating axons. When we plotted g-ratio against axon diameter, the fitted line for the IBU-CS-GP group lay below those of the other groups over the range of smaller axon diameters; as axon size increased, these lines gradually converged and ultimately almost overlapped (Fig. 6D). This pattern indicates that IBU-CS-GP mainly promotes the maturation of small-diameter myelinated fibers.Fig. 6Transmission electron micrographs of regenerated sciatic nerves at post-operation day 28 (A). Quantitative analysis of myelin sheath thickness of regenerated myelinated fibers in each group (B). Calculated g-ratio of regenerated myelinated fibers based on morphometric measurements (C). Linear regression between g-ratio and axon diameter in each group (D). One-way ANOVA with Tukey's post hoc test and n = 9 for B-D; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ANOVA, analysis of variance; CS, chitosan; IBU, ibuprofen; GP, genipin; MA, methacrylic anhydride.Fig. 6
In summary, the histological findings at day 28 indicate that the IBU-CS-GP hydrogel enhances axonal regrowth after peripheral nerve injury, recruits more Schwann cells, supports remyelination of newly regenerated axons, increases myelin thickness, and improves fiber maturity. Consistent with these findings, earlier and more effective reinnervation in the IBU-CS-GP group is associated with reduced collagen deposition and less muscle fibrosis.
Chitosan-based hydrogels have emerged as versatile platforms for peripheral nerve repair, leveraging chitosan's cationic, biodegradable nature to support Schwann cell proliferation, limit scarring, and enhance axonal regrowth compared with untreated nerve injury [35]. As integral components of nerve guidance conduits, chitosan hydrogels have been engineered to provide ECM-like architecture, tune elasticity and topography, and deliver growth factors or therapeutic cells, thereby improving structural and functional recovery in sciatic nerve defect models [36]. Recent reviews further underscore that functionalized chitosan hydrogels represent a major class within broader hydrogel-based strategies for peripheral nerve repair, yet their performance remains highly dependent on precise control of crosslinking chemistry, degradation, and microenvironmental modulation [10]. In this study, we developed an injectable chitosan-based hydrogel (IBU-CS-GP) that undergoes spontaneous crosslinking with genipin at physiological temperature and near-neutral pH. Administered via minimally invasive local injection for the treatment of peripheral nerve crush injury, this hydrogel enables in situ gelation at the lesion site, affords good moldability at the lesion site and adequate mechanical strength, and exhibits a favorable biosafety profile and good biocompatibility. The IBU-CS-GP hydrogel showed better overall performance than the drug-free CS-GP hydrogel and our previously reported CS-MA photocrosslinked hydrogel in terms of physicochemical properties, immunomodulatory and antioxidant functions, and neuromuscular recovery. These findings support the IBU-CS-GP hydrogel as a promising platform for repairing peripheral nerve crush injuries.
From a materials design perspective, grafting ibuprofen onto chitosan addresses a critical pH mismatch between chitosan solubility and genipin crosslinking while simultaneously introducing a bioactive anti-inflammatory cue. Native chitosan displays poor solubility at neutral pH because its primary amines (pKa = 6.5) are deprotonated and extensive inter- and intramolecular hydrogen bonding confers a highly crystalline, water-insoluble structure. At the same time, genipin–chitosan chemistry is strongly pH-dependent: Mi et al., as summarized in a recent comprehensive review, reported that the highest genipin–chitosan degree of crosslinking (∼96 %) and the lowest swelling and enzymatic degradation were achieved at pH 7.4, whereas crosslinking dropped to ∼39.9 % at pH 5 and to 1.4 % at pH 13.6 [37]. Chemical substitution on the amino groups is known to disrupt the hydrogen-bond network of chitosan, reduce crystallinity, and broaden the solubility window toward neutral and even alkaline conditions [38]. By enabling chitosan–ibuprofen complexes to remain soluble and amenable to crosslinking near physiological pH, our design effectively aligns the polymer's dissolution window with the optimal genipin reaction range, allowing a densely crosslinked, mechanically stable network to form under near-neutral conditions. This is particularly relevant for peripheral nerve repair, where pH values below 6.5 exacerbate conduction failure and impair the recovery of nerve action potentials, and where acidic microenvironments after neurotrauma have been shown to impede functional regeneration while pH neutralization improves outcomes [39]. These findings imply the rationale that ibuprofen-grafted, genipin-crosslinked chitosan hydrogels uniquely integrate optimized neutral-pH network formation with synergistic anti-inflammatory and pro-regenerative signaling in the peripheral nerve injury microenvironment.
For non-transecting peripheral nerve injuries such as crush lesions, the key challenge is to control inflammation and fibrosis without converting a potentially reversible conduction block into a chronic scar neuropathy. Open exploration and neurolysis require wide dissection of the nerve and surrounding soft tissues; post-surgical perineural scarring and adhesions are well-recognized complications that impair nerve gliding and cause recurrent compression neuropathies and pain, with symptom recurrence reported in 7–20 % of patients after primary median nerve release largely due to scar tethering [40]. Experimental data further show that injury confined to the soft-tissue bed, even without direct nerve transection, is sufficient to induce intraneural collagen deposition, extraneural adhesions and myelin disruption, thereby underscoring that additional surgical trauma may worsen long-term nerve structure and function [41]. In contrast, injectable hydrogels are delivered through narrow needles and undergo in situ gelation under physiological conditions, enabling minimally invasive placement of a scaffold that conforms to irregular lesion geometries and sustains local release of bioactive materials. For small-molecule non-opioid analgesics such as NSAIDs, hydrogel depots provide controlled and sustained release, improving anti-inflammatory and analgesic efficacy while reducing systemic exposure and gastrointestinal toxicity [42]. Building on this evidence, an injectable ibuprofen-modified chitosan-based hydrogel that gels at near-neutral pH has the potential to avoid iatrogenic soft-tissue damage, limit perineural scar formation and provide prolonged local immunomodulation in the setting of nerve crush injury, thereby offering a mechanistically rational, minimally invasive alternative or adjunct to traditional open surgical management.
This study shows that IBU-CS-GP can reshape the immune microenvironment in vitro by acting on both macrophage polarization and oxidative stress. In LPS-stimulated macrophages, IBU-CS-GP reduced TNF-α expression and lowered levels of reactive oxygen and nitrogen species, while simultaneously increasing IL-4 expression and the proportion of CD206^+^ macrophages. These changes suggest a shift toward an M2-like phenotype. Moreover, IBU-CS-GP hydrogel helps to control both inflammation and oxidative stress in a coordinated way. Previous work has shown that chitosan and its degradation products can improve the microenvironment for peripheral nerve regeneration by modulating macrophage polarization and limiting scar formation [43]. IBU-CS-GP hydrogel enhanced tube formation and increased cell migration, suggesting that it may alter the secretome of polarized macrophages in a way that indirectly promotes angiogenesis and cell recruitment. Intraneural vascularization, orchestrated by the crosstalk between macrophages and endothelial cells, is a prerequisite for successful nerve repair [32,44]. After injury, macrophages sense inflammatory and hypoxic cues in the local microenvironment and can polarize toward an M2 phenotype that secretes pro-angiogenic factors and initiates vessel growth [45]. The pro-angiogenic and pro-migratory effects observed in this study, together with the M2-skewing pattern induced by IBU-CS-GP, help to build a permissive microenvironment for subsequent remyelination and axon regeneration.
In the rat sciatic nerve crush model, IBU-CS-GP hydrogel yielded superior outcomes in motor function, electrophysiology, and muscle preservation. Earlier studies have shown that local delivery of anti-inflammatory drugs can markedly improve neuromuscular recovery after peripheral nerve injury [26,46]. In this study, both the SFI recovery curves and the electrophysiological results showed that IBU-CS-GP restored hindlimb motor function more efficiently. When these data were considered together with the gastrocnemius wet weight ratio and Masson staining, IBU-CS-GP clearly reduced denervation-induced collagen deposition in muscle fibers. This finding indicates that earlier reinnervation of the target muscle contributes to attenuating pathological structural alterations. Histological staining and ultrastructural analysis provided microscopic support for these functional improvements. In the IBU-CS-GP group, axonal markers and Schwann cell markers increased in parallel, which reflects the coordinated timing and localization of axonal regrowth, Schwann cell recruitment, and remyelination. Previous reviews have emphasized that fiber diameter, myelin thickness, and g-ratio are closely related to axon maturity and conduction capacity and thus serve as key indicators of the quality of peripheral nerve regeneration [47]. Thicker myelin and a lower g-ratio indicate that IBU-CS-GP hydrogel can accelerate nerve regeneration and help form conduction units that are closer to normal function. The improvement in g-ratio was most evident in small-diameter myelinated fibers, which suggests that IBU-CS-GP may preferentially promote the early maturation of sensory fiber populations.
To comprehensively evaluate the positioning of this study, we compared the IBU-CS-GP hydrogel with state-of-the-art hydrogel repair materials reported recently. Current research on nerve repair hydrogels primarily focuses on imparting specific physical properties to the materials. For instance, Park et al. developed a reduced graphene oxide (rGO)/gelatin conductive hydrogel, highlighting the significance of electrical signal conduction for peripheral nerve regeneration [48]. Meanwhile, Feng et al. utilized PEDOT:PSS to endow PVA hydrogels with an anisotropic topology to guide axonal extension, while simultaneously achieving superior mechanical stability [49]. In contrast to these design strategies that prioritize physical cues, our work emphasizes the modulation of the microenvironment associated with peripheral nerve injury. By chemically grafting ibuprofen, we not only addressed the limitation of chitosan solubility at neutral pH, but also enabled the drug to directly target the post-injury inflammatory cascade. This immunomodulatory effect targeting the early inflammatory microenvironment suggests a critical pathway for promoting peripheral nerve regeneration, acting as a vital complement to physical factors. Our results indicate the hydrogel's efficacy in promoting regeneration following acute peripheral nerve crush injury, suggesting its potential translational value for broader non-transecting nerve injuries.
Despite the comprehensive evaluation, several limitations remain. First, the 28-day observation period, while sufficient to capture early regeneration and functional recovery trends, limits the assessment of long-term target organ reinnervation. Second, a single drug loading concentration was employed, and the dose-response relationship warrants further optimization. Third, while distinct macrophage polarization was observed, the precise downstream signaling pathways require further elucidation. Looking forward, our future work will focus on optimizing release kinetics and conducting long-term evaluations in large animal models to verify the safety and efficacy of the IBU-CS-GP hydrogel in clinically relevant scenarios, thereby paving the way for potential translation.
Overall, this study used a chitosan–ibuprofen conjugate and genipin crosslinking strategy to design an injectable, in situ gelling hydrogel with combined immunomodulatory and sustained anti-inflammatory functions. In a peripheral nerve crush model, IBU-CS-GP promoted axon regeneration, supported remyelination, and improved neuromuscular functional recovery. At the same time, it reduced fibrosis in the target muscle and showed good hemocompatibility and antibacterial activity. These findings suggest that IBU-CS-GP hydrogel is a minimally invasive local treatment candidate with translational potential for peripheral nerve crush injury. Taken together, these data provide experimental support and a more systematic evaluation to facilitate the clinical translation of injectable hydrogels designed for peripheral nerve repair.
Yunhao Zhai: Writing – original draft, Validation, Software, Methodology, Investigation, Data curation. Xiangheng Guan: Writing – original draft, Methodology, Investigation, Data curation. Caorui Lu: Writing – original draft, Software, Methodology, Data curation. Ruixuan Sun: Methodology, Investigation, Data curation. Yun Qian: Writing – review & editing, Supervision, Project administration, Investigation, Conceptualization. Yi Li: Methodology, Investigation, Data curation. Kaihang Zhang: Methodology, Investigation, Data curation. Xu Wang: Methodology, Investigation, Data curation. Linbin Xu: Methodology, Investigation. Xinghao Yin: Methodology, Investigation, Data curation. Shang Guo: Writing – review & editing, Supervision, Project administration, Data curation. Jinglei Wu: Writing – review & editing, Supervision, Project administration, Investigation, Formal analysis, Data curation. Cunyi Fan: Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.