| BackgroundPeripheral nerve injury（PNI）is a severe clinical condition that predominantly affects young adults.It is characterized by a high incidence rate,limited treatment options,and poor clinical prognosis.Severe PNI is one of the major causes of permanent functional impairments and disabilities,leading to significant medical and economic burdens on society.When PNI occurs,a series of cellular and molecular events known as Wallerian degeneration occurs.Subsequently,proliferating macrophages are activated and recruited to the injury site to remove myelin debris and necrotic tissue,ultimately initiating the nerve regeneration process.However,excessive inflammatory response can result in elevated levels of reactive oxygen species（reactive oxygen species ROS）at the local tissue,and macrophages are one of the primary sources of ROS production while engulfing the inflammatory necrotic tissue.Excessive accumulation of ROS can induce oxidative stress,exerting neurotoxic effects and causing damage to peripheral nerve cells,thereby inhibiting peripheral nerve growth.Therefore,effectively modulating the inflammatory response and oxidative stress at the site of injury is crucial for promoting peripheral nerve regeneration and functional recovery.Recently,there has been an increasing focus among researchers on promoting neural tissue regeneration and facilitate the recovery of damaged nerve function,yielding promising results.Due to their unique physical and chemical properties,hydrogels are frequently utilized as materials for the culture and differentiation of neural progenitor cells.However,conventional hydrogels lack the ability to regulate the inflammatory microenvironment,which limits their application in PNI therapy.To this end,we have developed a hyaluronic acid-based hydrogel crosslinked with tannic acid and explore its therapeutic effect and mechanism of promoting peripheral nerve repair（PNR）by eliminating ROS at the injury site and regulating the inflammatory microenvironment.Purpose:This series of experiments primarily focused on the application of hyaluronic-based dynamic multi-functional hydrogels with tannic acid .Firstly,the Hyaluronic acid HA-based multifunctional hydrogel（HPTA）with anti-oxidative stress and anti-inflammatory ability was constructed by using TA as a crosslinking agent.The precursors that make up the hydrogel were fully characterised along with the physicochemical properties of the hydrogel and its effects on cell behaviour were explored in cellular experiments before the hydrogel was injected into rat sciatic nerve crush injuries for treatment to assess the effects of HPTA hydrogel on nerve repair.Subsequently,We developed a new injectable HPTA@MeCbl hydrogel based on the successful construction of HPTA hydrogels loaded with Methylcobalamin MeCbl as an alternative to the traditional injection therapy of MeCbl for the continuous treatment of localised nerve injuries.The physicochemical properties of the HPTA@MeCbl hydrogel were fully characterised to verify whether the addition of MeCbl would alter the properties of the HPTA gel itself.The results of in vitro cellular assays and in vivo treatment of rats with sciatic nerve transection injuries were used to evaluate the effect of HPTA@MeCbl hydrogel on nerve regeneration.Research Methods:In this study,HPTA hydrogel with antioxidant and anti-inflammatory functions was constructed.Its physical and chemical properties were fully characterized,and the effect of hydrogel on cell behavior was investigated through cell experiments in vitro.Finally,hydrogel was applied to the model of sciatic nerve crush injury in rats to verify its effect on sciatic nerve regeneration and functional recovery in rats.The specific research methodology are as follows:（1）This study initially involved the grafting of phenylboronic acid onto hyaluronic acid（HA）through an amide reaction between 3-aminophenylboronic acid（PBA,Bidepharm）and the carboxyl groups on HA,resulting in the formation of HA-PBA conjugates.The HA-PBA conjugates were characterized by nuclear magnetic resonance（NMR）and Fourier transform infrared spectroscopy（FTIR）.Subsequently,tannic acid （TA）was used as a crosslinking agent to blend with the HA-PBA conjugates,forming a hyaluronic acid-based hydrogel with dynamic boronic ester bonds,named HPTA.The HPTA hydrogel was explored for its material properties through morphology,rheological testing,adhesion,degradation rate,and release performance.The optimal gelation ratio was selected for further experiments.The antioxidative capacity of the hydrogel was evaluated through DPPH·and PTIO·radical scavenging assays.（2）The effect of HPTA hydrogel on the cell behavior of pheochromocytoma（PC12）cells and Schwann cells（SCs）was characterized in vitro.The protective effects of HPTA hydrogel on cells under reactive oxygen species（ROS）conditions were investigated.（3）In an in vivo animal experiment,a rat sciatic nerve crush injury model was utilized to evaluate the nerve repair efficacy of the hydrogel.The rats were randomly divided into groups,and after modeling,functional recovery of the lower limbs was evaluated at 2 and 4 weeks post-surgery through footprint analysis,nerve electrophysiology testing,muscle histology analysis,nerve histology evaluation,immunofluorescence staining,immunoblotting,and assessment of inflammatory factor expression in nerve tissue.The study aimed to assess the effects of the dynamic HPTA hydrogel on nerve repair after sciatic nerve injury,without the use of any other therapeutic agents.Subsequently,we added mecobalamine（MeCbl）on the basis of the successful construction of HPTA hydrogel to develop a new HPTA@MeCbl hydrogel.（1）HPTA@MeCbl hydrogel was extensively characterized through scanning electron microscopy,rheological testing,adhesion,in vitro and in vivo degradation,and MeCbl release experiments.The obtained results were compared with a blank HPTA hydrogel for comparison.（2）In vitro cell experiments using PC12 cells were performed to characterize the cell viability,proliferation,and cell viability staining of HPTA@MeCblhydrogel.（3）In the in vivo animal experiment,HPTA@MeCbl hydrogel was applied for the treatment of rat sciatic nerve transection injury.At 2 months post-surgery,a series of experiments,including footprint analysis,nerve electrophysiology testing,muscle histology analysis,nerve histology evaluation,and immunostaining,were conducted to evaluate lower limb functional recovery and sciatic nerve regeneration.Result and discussion:（1）Firstly,information was collected through nuclear magnetic resonance（NMR）to analyze the HA-PBA conjugate,and these characteristic peaks indicated the successful modification of the phenylboronic acid group onto the hyaluronic acid（HA）molecular chain,with a calculated grafting rate of 23.4%.After synthesizing the HPTA hydrogel,FT-IR analysis revealed that the characteristic peak at 1326 cm-1 was mainly attributed to the stretching vibration of the boronic ester bond（B-OH）,confirming TA and HA-PBA conjugate were successfully crosslinked to form gel.Subsequently,HPTA-3 was determined as the optimal gel formulation through screening using scanning electron microscopy（SEM）,storage modulus,adhesion,degradation rate,and release performance,and further experiments were conducted.At a concentration of 20mg/m L,HPTA-3 hydrogel exhibited a free radical scavenging rate of（76.23±0.7522%）for DPPH·and（66.95±0.1305%）for PTIO·,confirming the excellent antioxidant capacity of HPTA-3.（2）Furthermore,in cell experiments,PC-12 cells incubated with 20 mg/m L HPTA-3 hydrogel extract and 0.5 m M H2O2solution showed cell viability and vitality greater than 80%,indicating the effective elimination of intracellular reactive oxygen species（ROS）by HPTA-3 hydrogel and its protective role against ROS-induced cell damage.Cell viability of both PC-12 and SCs neuronal cells tested against HPTA-3hydrogel showed values above 80%.In vivo degradation experiments did not show significant inflammatory reactions four weeks after surgery.This demonstrated that HPTA-3 hydrogel exhibited good biocompatibility in vivo.（3）HPTA-3 hydrogel was then applied for the treatment and study of a rat sciatic nerve crush injury model.Results from gait analysis,sciatic function index（SFI）,nerve electrophysiology test,muscle histology,nerve histology,transmission electron microscopy（TEM）for observing axon diameter and myelin sheath thickness,as well as staining for glial fibrillary acidic protein（GFAP）,myelin basic protein（MBP）,neurofilament-200（NF200）,andβ3-tubulin（Tuj-1）as specific protein markers,along with ELISA analysis of inflammatory factor expression and immunoblotting,indicated that although the HPTA-3 treatment group still showed differences compared to the Sham group that did not undergo surgery,it achieved better outcomes compared to the Crush group without hydrogel treatment.ELISA measurement of chemotactic factors in nerve tissue demonstrated that the application of HPTA-3 hydrogel upregulated anti-inflammatory cytokines and suppressed pro-inflammatory factor expression,helping macrophages transition from the M1 phenotype to the M2 phenotype after neural tissue injury,confirming the anti-inflammatory properties of HPTA-3.The Western blot expression results of MBP and NF200 were corresponding to immunoprotein fluorescence staining,which again proved that HPTA-3 hydrogel can effectively promote the re-myelination and regeneration of axons after injury.We then further synthesised injectable HPTA@MeCbl hydrogels based on HPTA hydrogels loaded with MeCbl.（1）Primarily,the microstructure of HPTA@MeCbl hydrogel was observed by SEM,and it was found that MeCbl crystals were deposited in the network structure of hydrogel.Rheological properties,adhesion,and degradation of HPTA@MeCbl hydrogel were compared with blank HPTA-3（HPTA）without MeCbl,and no significant differences were observed in the material characterization results between the two hydrogel groups.This indicates that the addition of MeCbl did not interfere significantly with the crosslinking and boronic ester bond formation,thereby not causing substantial changes in the physicochemical properties of the hydrogel.The release rate of MeCbl was calculated using the standard curve regression equation:y=181.94x-0.1471.The release rate increased rapidly during the first six days and then slowed down noticeably between days 6 and 10.This release pattern of HPTA@MeCbl hydrogel is expected to reach the required concentration of treatment in the early stage,which can help repair damaged nerves earlier.（2）We have also demonstrated through cellular assays that HPTA@MeCbl hydrogels have good cytocompatibility and a significant cell proliferation promoting effect can be observed after 48 hours of co-incubation with cells.（3）Finally,HPTA@MeCbl hydrogel was used for the treatment of sciatic nerve transection injury.Two months later,the sciatic function index（SFI）,nerve electrophysiology test,muscle histology test,nerve histology test,transmission electron microscopy（TEM）for axon diameter and myelin sheath thickness,as well as the results of 4 specific neuroprotein staining showed that the treatment effect of HPTA@MeCbl group was good,and the results were closer to those of Sham group compared with blank HPTA group and Transection group.Notably,semi-quantitative analysis of specific protein staining for Neurofilament-200（NF200）andβ3-tubulin（Tuj-1）after two months post-surgery showed no statistically significant differences between the HPTA@MeCbl group and the Sham group.This further indicates that HPTA@MeCbl hydrogel significantly enhances axonal regeneration,achieving results similar to normal nerve tissue.In summary,this series of studies has constructed HA-based hydrogels using TA as a crosslinking agent.Without the combination of other therapeutic agents,HPTA hydrogel effectively promotes nerve repair after PNI through its intrinsic antioxidant and anti-inflammatory effects.Moreover,as a drug delivery vehicle,it achieves sustained local therapy of MeCbl in neural tissue damage,validating the feasibility of this"new application of a classic drug."The research findings provide new insights and directions for the design and development of high-performance materials for nerve injury repair in the future. |
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