A hydrogel material for nerve repair after liver resection and a preparation method thereof

Abstract

A hydrogel material for nerve repair after liver resection, characterized in that the hydrogel material is a 3-aminophenylboronic acid modified hyaluronic acid hydrogel, which is synthesized by 3-aminophenylboronic acid (3-APBA) and hyaluronic acid (HA) through amidation reaction, and then cross-linked to form a three-dimensional porous hydrogel structure. The HAB hydrogel proposed in the application has a degradation performance suitable for the postoperative repair microenvironment of the liver, good biocompatibility and slight inflammatory reaction; the hydrogel proposed in the application has a three-dimensional porous structure, which efficiently promotes the proliferation and differentiation of dorsal root ganglion stem cells and accelerates the functional regeneration of perihilar nerves. The hydrogel proposed in the application has adjustable physicochemical properties and has the advantages of minimally invasive injection and convenient clinical operation; the hydrogel proposed in the application fills the blank in the field of special nerve repair materials after liver resection, and is a targeted solution to the pain point of clinical repair; the raw materials and preparation process of the hydrogel proposed in the application are suitable for industrialization and landing, and have a broad clinical conversion prospect.

Description

Technical Field

[0001] This invention relates to the field of neuroregenerative medicine, and in particular to a hydrogel material for nerve repair after liver resection and its preparation method. Background Technology

[0002] Hepatectomy is a core surgical procedure for treating liver diseases such as liver cancer, hepatic hemangioma, and liver abscess. However, the procedure is prone to damaging the nerves around the liver and in the porta hepatis, leading to postoperative complications such as small liver syndrome, persistent pain in the liver area, gastrointestinal dysfunction, and abnormal bile secretion, which seriously affect the patient's postoperative recovery and quality of life. Peripheral nerve injury (PNI) suffers from limited nerve regeneration capacity and a lack of suitable repair scaffold materials that are compatible with the postoperative liver microenvironment and possess both biocompatibility and neurogenic activity, resulting in poor clinical repair outcomes.

[0003] Dorsal root ganglion stem cells (DRGSCs) are multipotent stem cells in the peripheral nervous system, capable of differentiating into neurons and glial cells, participating in nerve regeneration and injury repair, and possessing significant application value in peripheral nerve injury repair. Traditional two-dimensional culture systems struggle to maintain the activity, proliferation capacity, and neural differentiation efficiency of DRGSCs. Furthermore, while high molecular weight hyaluronic acid (HA) hydrogels exhibit good biocompatibility, they easily form physical barriers, hindering cell migration, nutrient diffusion, and neural differentiation, thus limiting their application in nerve repair.

[0004] Partial hepatectomy inevitably damages afferent and regulatory nerves distributed around the liver. Insufficient repair of these nerve defects will continue to induce adverse consequences such as postoperative chronic pain and disordered liver regeneration regulation. Current research and development of nerve repair hydrogels focuses on the repair of central nervous system injuries such as those to the brain and spinal cord, as well as peripheral nerve injuries to the limbs, lacking a functionalized hydrogel system specifically for perihepatic nerve repair after hepatectomy. Existing materials of this type exhibit several shortcomings when applied to perihepatic wounds: First, the material degradation rate cannot be matched with the regeneration cycle of perihepatic nerves; Secondly, the implanted material can easily stimulate a severe inflammatory response in the local area, further inhibiting nerve repair; Third, the material has a weak effect on the adhesion and migration of dorsal root ganglion stem cells (DRGSCs), and it is difficult to efficiently induce stem cells to differentiate into functional neurons.

[0005] In summary, developing specialized hydrogels with excellent biocompatibility, precisely regulated degradation behavior, efficient promotion of DRGSCs adhesion and migration and neuronal differentiation, and adaptability to the complex wound microenvironment after liver resection is the core approach to overcoming the technical obstacles of perihepatic nerve repair after liver resection. Summary of the Invention

[0006] In view of the above problems, the present invention provides a hydrogel material for nerve repair after liver resection, which has excellent biocompatibility, is tunable and degradable, can efficiently promote the adhesion, migration and differentiation of DRGSCs and neurons, and is adapted to the microenvironment after liver resection. It is the key to solving the problem of nerve repair after liver resection.

[0007] A hydrogel material for nerve repair after liver resection is a 3-aminophenylboronic acid modified hyaluronic acid hydrogel, abbreviated as HAB hydrogel. It is synthesized by an amidation reaction of 3-aminophenylboronic acid (3-APBA) and hyaluronic acid (HA), and then cross-linked to form a three-dimensional porous hydrogel structure. The hyaluronic acid has a molecular weight of 1.0–1.8 MDa; The degree of substitution of the 3-aminophenylboronic acid on hyaluronic acid is 2.09 ± 0.02 mmol / g; The hydrogel concentrations are 1.25%, 2.5%, and 5.0% (w / w).

[0008] The yield stress of the hydrogel is 18.6, 165.9, and 711.5 Pa.

[0009] The hydrogel has a three-dimensional porous structure.

[0010] The pore size of the hydrogel decreases with increasing concentration, and its degradation period is 28 days, which matches the nerve regeneration cycle after liver surgery.

[0011] The hydrogel can promote the adhesion rate of dorsal root ganglion stem cells (DRGSCs) to 78.5%±3.2%, the migration speed to 21.4μm / h, and the neuronal differentiation efficiency to 72.6%±4.1%, and upregulate the expression of synapse-related genes Gnao1 and Grm7, as well as neurotrophic factors BDNF and NGF.

[0012] A method for preparing a hydrogel material for nerve repair after liver resection includes the following steps: (1) Preparation of raw material pretreatment and proportioning limitation The raw materials include: hyaluronic acid (HA, molecular weight 1.0–1.8 MDa), 3-aminophenylboronic acid (3-APBA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sodium hydroxide, hydrochloric acid, phosphate buffered saline (PBS, pH 7.4), and deionized water. All chemical raw materials and biochemical reagents are of analytical grade and can be used directly after purchase without secondary purification or refining; the pH of the physiological phosphate buffer is kept constant at 7.4. (2) Synthesis of phenylboronic acid functionalized hyaluronic acid intermediate HAB S201. Homogeneous dissolution of hyaluronic acid: Weigh 3g of hyaluronic acid raw material, add it to 500mL of deionized water, and stir at a constant temperature until the solute is completely homogeneously dissolved to prepare a homogeneous hyaluronic acid aqueous solution. S202, Activation of carboxyl groups in hyaluronic acid: Add 1.504g EDC and 0.911g NHS to the above aqueous solution of hyaluronic acid at one time, and stir the reaction under closed conditions at room temperature for 30 minutes. Use the dual activation reagent to activate the carboxyl groups on the hyaluronic acid molecular chain to provide active sites for the amide grafting reaction. S203, Controllable amide grafting modification: After activation with hydrochloric acid and sodium hydroxide, the pH value of the system was stabilized to 5.5. 1.084g of 3-aminophenylboronic acid was added to the system, and the reaction temperature was kept constant at 25℃. The system was stirred at a constant temperature for 6h. Through amide covalent bonding reaction, the functional group of 3-aminophenylboronic acid was covalently grafted and fixed to the main chain of hyaluronic acid polymer to obtain the crude modified reaction solution. S204. Dialysis purification: The modified crude reaction solution was transferred into a regenerated cellulose dialysis bag with a molecular weight cutoff of 14000 Da and purified by continuous dialysis with deionized water at room temperature for 3 days to completely remove unreacted monomers, free activators, small molecule byproducts and soluble impurities from the system. S205, Powder solidification preparation: The clarified modified liquid after dialysis purification is subjected to vacuum freeze-drying to obtain pure white phenylboronic acid modified hyaluronic acid solid powder. The obtained HAB powder is sealed and stored at room temperature in the dark for later use. (3) Preparation of medical nerve repair HAB hydrogel by in-situ crosslinking S301. Preparation of modified polymer precursor solution: Take the spare HAB powder, add it to sterile physiological phosphate buffer with pH=7.4, and stir at a constant temperature of 50℃ until the HAB powder is completely dissolved and homogenized. Prepare a sterile HAB precursor aqueous solution with a mass fraction w / w of 1.25%~5.0%. S302, Thermosensitive in-situ crosslinking molding: The prepared HAB precursor aqueous solution is naturally cooled to 25°C, sealed and left to stand for 1 hour. The medical HAB hydrogel with an internal three-dimensional porous structure is obtained by relying on the dynamic phenylboronic acid ester bonds between polymer chains for autonomous crosslinking. Among them, the HAB precursor aqueous solution has the characteristics of rapid prototyping in response to body temperature: when the system temperature drops to 37°C or below, it can achieve rapid gel solidification, the material flowability is controllable, it can be used for non-invasive injection with 18G medical injection needles, and it is suitable for intraoperative minimally invasive injection after liver resection and local implantation of wounds after surgery.

[0013] The application of hydrogel materials for nerve repair after liver resection in drugs for nerve repair after liver resection: The HAB hydrogel can upregulate the expression of neurotrophic factors BDNF and NGF, as well as synapse-related genes Gnao1 and Grm7, induce functional synapse formation, enhance neurophysiological activity, and accelerate the regeneration of damaged perihepatic nerves.

[0014] The HAB hydrogel has a degradation cycle of 28 days in vivo, which matches the nerve regeneration cycle after liver surgery; the HAB hydrogel has a three-dimensional porous biomimetic extracellular matrix structure; the HAB hydrogel is adapted to the low-inflammatory repair microenvironment after liver surgery and is used for the regeneration and repair of perihepatic nerve damage after liver resection.

[0015] The HAB hydrogel can significantly downregulate the expression of inflammatory factors IL-1β and TNF-α during in vivo degradation, with no obvious immune rejection, excellent biocompatibility, and low degree of inflammatory response.

[0016] The three-dimensional porous structure can improve the adhesion rate, migration rate and neuronal differentiation efficiency of DRGSCs, and can significantly improve the cell activity and directed differentiation ability of DRGSCs compared with the two-dimensional culture system.

[0017] The HAB hydrogel can upregulate the expression of neurotrophic factors BDNF and NGF, as well as synapse-related genes Gnao1 and Grm7, induce functional synapse formation, enhance neurophysiological activity, and accelerate the regeneration of damaged perihepatic nerves.

[0018] The HAB hydrogel is specifically designed to adapt to the microenvironment of the perihepatic wound after liver resection, overcoming the shortcomings of existing nerve repair materials which are only suitable for the central nervous system and peripheral nerves of the limbs and cannot be adapted to the perihepatic nerve repair scenario.

[0019] The beneficial effects of this invention are as follows: I. The HAB hydrogel proposed in this invention has excellent degradation properties that are suitable for the postoperative liver repair microenvironment, exhibits good biocompatibility, and causes mild inflammatory response. The HAB hydrogel of this invention has an in vivo degradation cycle of 28 days, which can be precisely matched with the regeneration cycle of perihepatic nerves after liver surgery. The material degradation process can significantly downregulate the expression levels of inflammatory factors IL-1β and TNF-α. There is no obvious immune rejection reaction after implantation. It can construct a low-inflammatory repair microenvironment after liver surgery, and has high overall safety and excellent biocompatibility.

[0020] II. The hydrogel proposed in this invention has a three-dimensional porous structure. This three-dimensional porous structure efficiently promotes the proliferation and differentiation of dorsal root ganglion stem cells and accelerates the functional regeneration of perihepatic nerves. This invention utilizes a hydrogel to construct a three-dimensional porous biomimetic structure that can simulate the body's natural extracellular matrix microenvironment. Compared to traditional two-dimensional culture systems, this structure can significantly improve the adhesion rate, migration rate, and neuronal differentiation efficiency of DRGSCs, effectively maintaining and enhancing DRGSC cell activity. Simultaneously, it can upregulate the expression of neurotrophic factors BDNF and NGF, as well as synapse-related genes Gnao1 and Grm7, inducing functional synapse formation, improving neurophysiological activity, and thus significantly accelerating the regeneration process of damaged perihepatic nerves.

[0021] Third, the hydrogel proposed in this invention has adjustable physicochemical properties and offers the advantage of minimally invasive injection application, making it convenient for clinical operation. This invention can flexibly adjust the internal pore size, mechanical stiffness and overall degradation rate of the hydrogel by controlling the concentration of HAB raw materials, thus adapting to different needs for repairing perihepatic nerve injuries. The material is injectable and can be directly injected into the perihepatic nerve injury lesion, resulting in minimal surgical trauma and simple clinical application.

[0022] Fourth, the hydrogel proposed in this invention fills the gap in the field of specialized nerve repair materials after liver resection, and is a targeted solution to the pain points of clinical repair. Existing hydrogel materials for nerve repair are mostly developed for injuries to the central nervous system and peripheral nerves of the limbs, and cannot adapt to the special microenvironment of the perihepatic wound after liver surgery, making it difficult to achieve efficient repair of perihepatic nerves. This invention is specifically designed for the repair needs of perihepatic nerve injuries after liver resection, making up for the shortcomings of existing materials and providing a new, efficient and safe scaffold repair material for perihepatic nerve repair after liver resection.

[0023] V. The hydrogel raw materials and preparation process proposed in this invention are well-suited for industrialization and have broad prospects for clinical translation. The raw materials used in this invention are readily available and have low production costs; the entire synthesis and molding process is mild and simple to operate, requiring no large and complex processing equipment, making it easy to achieve large-scale batch preparation and possessing extremely high potential for clinical translational applications. Attached Figure Description

[0024] Figure 1 af is a schematic diagram of the characterization of the chemical structure of HAB using infrared spectroscopy; in: Figure 1 a represents the FTIR spectra of HA, BA, and HAB.

[0025] Figure 1 b shows the proton NMR spectra of HA, BA, and HAB.

[0026] Figure 1 c is a plot of the degree of substitution of HA for BA calculated using elemental analysis data (%w / w).

[0027] Figure 1 d represents the molecular weight distribution of HA and HAB.

[0028] Figure 1 e is a strain scanning analysis graph of HAB powder-derived hydrogel.

[0029] Figure 1 The left figure shows the formation of HAB powder-derived hydrogels at different concentrations (1.25%, 2.5%, and 5.0% (w / w)) in DMEM / F12. Scale bar = 1 cm.

[0030] Figure 1 The right image shows a scanning electron microscope image of the HAB powder-derived hydrogel, revealing its porous structure (n=3). Scale bar = 20 μm.

[0031] Figure 2 a, Figure 2 b is a schematic diagram comparing HAB hydrogel with traditional 2D culture;

[0032] Figure 2 Image a shows bright-field images and immunofluorescence staining of DRGSCs cultured in plastic culture dishes and HAB hydrogels. Red fluorescence corresponds to the NSC label Nestin, green fluorescence represents the peripheral NSC label p75NTR, and blue fluorescence represents the nuclear label Dapi. Black circles highlight the observed cell morphology.

[0033] Figure 2 b is a real-time quantitative polymerase chain reaction (qPCR) assay showing the μ expression level of DRGSCs during fetal bovine serum-induced differentiation. The blue bar represents the neuronal marker Tuj1, and the brown bar represents the satellite glial cell marker GS.

[0034] Data are presented as mean ± SD, n=3; **P<0.01.

[0035] Figure 3 Figure a shows a simulation of the release of neurotrophic factors in HAB hydrogel.

[0036] Figure 3 b shows the enhanced electrophysiological activity of DRGSCs cultured in HAB hydrogel compared to plastic culture dishes, exhibiting a higher excitation frequency.

[0037] Figure 3 cd represents the mRNA expression levels of neurotrophic factors BDNF and NGF in DRGSCs cultured in plastic petri dishes and HAB hydrogels.

[0038] Figure 3ef represents the expression levels of Wnt pathway-related genes (Wnt and β-catenin) in DRGSCs. Green bars represent plastic culture dishes, and yellow bars represent HAB hydrogels.

[0039] Data are presented as mean ± SD, n=3; *P<0.05, **P<0.01.

[0040] Figure 4 Image a shows a bright-field image and live / dead staining of DRGSCs cultured in plastic culture dishes and HAB hydrogels. Green fluorescence indicates live cells (calcein staining), red fluorescence indicates dead cells (PI staining), and blue fluorescence indicates the nuclear marker DAPI. Black circles highlight cell morphology. Scale bar = 100 μm.

[0041] Figure 4 b shows the cell cycle analysis of DRGSCs cultured in plastic culture dishes and HAB hydrogels. Blue, green, and pink represent the proportions of cells in G0-G1, S, and G2-M phases, respectively. Data are presented as mean ± SD, n=3.

[0042] Figure 4 c represents the OD values ​​of DRGSCs cultured in plastic culture dishes and HAB hydrogels at 450 nm, as shown by the CCK-8 assay. The green bars represent the OD values ​​in the plastic culture dishes, and the brown bars represent the OD values ​​in the HAB hydrogels.

[0043] Data are presented as mean ± SD, n=3; **P<0.01.

[0044] Figure 5 Image a is a bright-field image showing the degradation process of HAB hydrogel implanted under the skin one month later. Scale bar = 1 cm.

[0045] Figure 5 b shows the expression of tumor necrosis factor-α and interleukin-1β in the surrounding tissue as revealed by immunohistochemistry. The implanted HAB hydrogel (n=6) is marked by black circles.

[0046] Figure 5 c represents the protein levels of tumor necrosis factor-α and interleukin-1β in subcutaneous tissue as shown by immunoblotting analysis (n=3).

[0047] Data are presented as mean ± SD; *P<0.05, **P<0.01.

[0048] Figure 6 a shows a volcano plot of DEG in DRGSCs co-cultured in HAB hydrogel and plastic culture dishes. Red dots represent upregulated DEGs, and blue dots represent downregulated DEGs.

[0049] Figure 6b shows a Pearson correlation clustering heatmap displaying different gene expression profiles. Blue clusters represent the HAB hydrogel group, and red clusters represent the plastic culture dish group.

[0050] Figure 6 c represents the KEGG pathway enrichment analysis, highlighting the top-level pathways associated with DEGS.

[0051] The data is presented as mean ± standard deviation, n=3.

[0052] Figure 7 The expression results of synapse-related genes Gnao1 and Grm7 in plastic plate culture and HAB hydrogel system are shown, n=3;

[0053] Figure 7 a represents the relative mRNA expression levels of Gnao1 and Grm7 genes obtained by qRT-PCR detection;

[0054] Figure 7 b represents the expression levels of GNAO1 and GRM7 proteins obtained by Western blotting.

[0055] Data are presented as mean ± SD; *P<0.05, **P<0.01, *P<0.001. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0057] Example 1

[0058] Characterization of HAB hydrogel raw materials and physicochemical structure of HAB hydrogel

[0059] 1. Characterization of the chemical structure of HAB using Fourier transform infrared spectroscopy (FT-IR).

[0060] Fourier transform infrared spectroscopy was performed on the raw material HA and the modified product HAB powder to analyze the changes in molecular structure. The test results are shown in [Figure number missing]. Figure 1 a. By Figure 1 As can be seen from the infrared spectrum, the HAB sample shows a new characteristic absorption peak at the position of 1350 cm⁻¹, which is attributed to the stretching vibration of the BO bond.

[0061] Comparing the infrared spectrum of HA, HAB showed a characteristic absorption peak in the range of 1408–1384 cm⁻¹. This absorption peak is a characteristic peak of CN stretching vibration generated by the amidation reaction between HA and BA, which preliminarily proves that BA was successfully grafted onto the HA molecular chain.

[0062] 2. Characterization by proton nuclear magnetic resonance (¹H NMR) spectroscopy

[0063] HAB powder was subjected to ¹H nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 As shown in b, the HAB NMR spectrum shows a new set of characteristic peaks in the chemical shift range of δ7.70ppm to 7.46ppm, which correspond to the benzene ring protons in the BA structure. Compared with the NMR peaks of pure BA, the benzene ring proton peaks in HAB show a low-field shift, proving that intermolecular / intramolecular hydrogen bonds are formed in the system, further confirming that BA and HA are covalently bonded.

[0064] 3. Elemental analysis to quantify the degree of graft substitution

[0065] The mass percentage (%w / w) of N, C, and H elements in HA and HAB samples were determined using an elemental analysis instrument. The grafting substitution degree (SD) of BA on the HA molecule was calculated by the increase in nitrogen content. The results are shown in [Figure number missing]. Figure 1 c. Test results showed that the nitrogen content of the modified HAB was significantly higher than that of the raw material HA, proving that HA and BA underwent stable covalent modification; the calculated degree of BA substitution of HAB was SD = 2.09 ± 0.02 mmol / g.

[0066] 4. Molecular weight and polydispersity index test

[0067] The relative molecular masses and polydispersity index (PDI) of HA and HAB were determined by gel permeation chromatography (GPC), and the results are shown in the figure. Figure 1 d. The relative molecular mass of the raw material HA was 1.02 MDa, and the PDI was 1.32; after modification, the relative molecular mass of HAB increased to 1.19 MDa, and the PDI was 1.27. The increase in molecular weight and slight optimization of dispersibility confirmed the successful preparation of the HAB graft-modified product at the molecular scale.

[0068] 5. HAB hydrogel formation and microporous structure observation

[0069] HAB powder was mixed with DMEM / F12 culture medium, and the system rapidly formed a structurally complete HAB hydrogel. The internal microstructure of the hydrogel at different HAB concentrations was observed using scanning electron microscopy (SEM), and the results are shown in the figure. Figure 1 e Figure 1 f. SEM images show that all HAB hydrogels form interconnected three-dimensional porous structures; as the HAB concentration increases, the average pore size inside the hydrogel gradually decreases, and the pore structure of the scaffold can be precisely controlled by adjusting the HAB concentration.

[0070] 6. Rheological property testing

[0071] HAB hydrogels with mass concentrations of 1.25%, 2.5%, and 5.0% (w / w) were prepared, and their rheological properties were tested. The yield stress of each group of hydrogels was determined: the yield stress of 1.25% (w / w) HAB hydrogel was 18.6 Pa; the yield stress of 2.5% (w / w) HAB hydrogel was 165.9 Pa; and the yield stress of 5.0% (w / w) HAB hydrogel was 711.5 Pa.

[0072] Rheological test results show that the HAB hydrogel constructed by borate crosslinking and multiple physical interactions in this invention has excellent gel mechanical properties. Its mechanical strength can be adjusted with the concentration gradient of raw materials. It has both injectability and mechanical stability, which can meet the needs of in vivo cell delivery and minimally invasive injection, and can also be used as a stable three-dimensional scaffold for in vitro cell culture research.

[0073] Example 2

[0074] Three-dimensional co-culture and performance verification experiment of dorsal root neural stem cells with HAB hydrogel

[0075] I. Preparation of Experimental Materials and Instruments

[0076] 1. Experimental cells

[0077] Primary rat dorsal root neural stem cells (DRGSCs) were isolated and purified. After primary culture identification, the cell purity was ≥95%. DRGSCs in the logarithmic growth phase of the third generation were selected for this co-culture experiment.

[0078] 2. Experimental reagents and materials

[0079] Self-developed modified HAB composite functional hydrogel, complete cell culture medium, fetal bovine serum (FBS), nestin primary antibody, p75NTR primary antibody, corresponding fluorescent secondary antibody, DAPI cell nuclear staining solution, Trizol cell lysis buffer, reverse transcription kit, real-time quantitative PCR kit, BDNF, NGF, Wnt, β-catenin, GS, Tuj1 gene-specific primers, and conventional cell culture-grade plastic culture dishes.

[0080] 3. Experimental instruments

[0081] Inverted bright-field optical microscope, laser confocal immunofluorescence microscope, Maestro high-throughput microelectrode array (MEA) detection system, real-time quantitative PCR instrument, high-speed low-temperature centrifuge, and constant temperature cell culture incubator.

[0082] II. Experimental Group Setup

[0083] This experiment set up two parallel control systems. All cell culture environments were uniformly set at 37℃ in a 5% CO2 constant temperature and humidity incubator. The amount of culture medium and the cell seeding density were exactly the same in all culture systems.

[0084] Control group: DRGSCs were cultured in conventional 2D plastic culture dishes;

[0085] Experimental group: DRGSCs were co-cultured on a three-dimensional HAB hydrogel system.

[0086] The seeding density for both groups of cells was uniformly set at 1×10⁻⁶. 5 The cells / mL was set up with 6 replicates per group, and all detection experiments were repeated 3 times biologically to ensure the validity of the experimental data.

[0087] Co-cultivation operation method

[0088] The solidified HAB hydrogel was pre-treated with sterile substrate, sterilized by UV irradiation for 30 min, and then incubated with basal culture medium for 12 h. The incubation waste liquid was then removed. The prepared DRGSCs cell suspension was evenly seeded into the three-dimensional pores of the HAB hydrogel for cell loading co-culture. The control group had an equal amount of DRGSCs directly seeded into sterile 2D plastic culture dishes. After the cells adhered stably, both groups were simultaneously infused with 10% fetal bovine serum (FBS) for directed differentiation induction culture. Various indicators were measured after 7 and 14 days of induction culture, respectively.

[0089] III. Detection Methods and Experimental Results

[0090] 1. Identification of cell morphology and stem cell phenotype

[0091] The morphology of two groups of DRGSCs was observed using an inverted bright-field microscope. The results are shown in [Figure number missing]. Figure 2 a. Compared to 2D plastic culture dish systems, the HAB three-dimensional hydrogel co-culture system can maintain the complete three-dimensional clonal morphology of DRGSCs, with good cell spreading and no apoptosis, shrinkage, or morphological fragmentation.

[0092] Immunofluorescence staining was used to identify the expression of stem cell-specific markers: After fixing two groups of cells, they were permeabilized and sealed, and then incubated sequentially with nestin primary antibody, p75NTR primary antibody and corresponding fluorescent secondary antibody. The cell nuclei were labeled with DAPI reagent. Laser confocal microscopy imaging results showed that DRGSCs in the experimental group could specifically express the neural stem cell marker nestin (red fluorescence) and the peripheral neural stem cell marker p75NTR (green fluorescence) at high levels in HAB hydrogel. The cell nuclei were completely stained with blue fluorescence, and the cell stem cell phenotype was stable.

[0093] 2. Detection of mRNA expression of genes related to neural differentiation

[0094] After 7 and 14 days of FBS-directed differentiation, total RNA was extracted from two groups of cells using the Trizol method. cDNA was obtained through reverse transcription, and the relative mRNA expression levels of neuronal marker Tuj1 and glial cell marker GS were detected by real-time fluorescence polymerase chain reaction (qRT-PCR). The results are as follows: Figure 2 As shown in b: Under the same induction culture period, the mRNA expression level of Tuj1, a neuronal marker, in DRGSCs of the HAB hydrogel experimental group was significantly higher than that of glial cell marker GS, and the difference within the group was statistically significant (P<0.01), confirming that HAB hydrogel can efficiently promote the directional differentiation of DRGSCs into neuronal lineages.

[0095] 3. Release and electrophysiological function detection of neurotrophic factors from hydrogels

[0096] The in vitro neurotrophic factor sustained-release performance of HAB hydrogel was tested, and the results are shown in [the table below]. Figure 3 a. The self-developed HAB composite hydrogel has the advantage of long-term sustained release of neurotrophic factors, which can stably provide a nutrient microenvironment for co-cultured DRGSCs.

[0097] The electrophysiological excitation frequencies of the two groups of cells were detected using the Maestro high-throughput MEA system. The results are shown in [Figure number missing]. Figure 3 b: Compared with the 2D plastic culture dish control group, the stability of the electrophysiological excitation frequency of DRGSCs in the HAB hydrogel experimental group was significantly improved, and the difference between the groups was statistically significant (P<0.05).

[0098] 4. Detection of expression of nutritional factors and Wnt pathway-related genes

[0099] qRT-PCR was used to quantitatively detect the mRNA expression levels of neurotrophic factors and key factors in the Wnt signaling pathway. The results are as follows: Figure 3 As shown in c-3f:

[0100] (1) The mRNA expression levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the HAB hydrogel experimental group were significantly higher than those in the 2D plastic culture dish control group, and the difference between the groups was statistically significant (P<0.05).

[0101] (2) There was no significant difference in the expression level of classical Wnt factor mRNA between the control group and the experimental group, and there was no statistical difference between the groups (P>0.05).

[0102] (3) β-catenin, the core activator of the Wnt signaling pathway, was highly expressed in the HAB hydrogel experimental group, and the difference between the groups was statistically significant (P<0.01).

[0103] This embodiment demonstrates through co-culture experiments that, compared to the traditional 2D planar culture mode, HAB three-dimensional hydrogel can effectively maintain the complete clonal morphology of DRGSCs, optimize the cell growth microenvironment, and enhance cell survival. By activating the downstream β-catenin-mediated Wnt signaling pathway, it upregulates the expression of neurotrophic factors such as BDNF and NGF, ultimately significantly improving the directed differentiation efficiency of dorsal root nerve stem cells into functional neurons. It can be used to construct stem cell vectors for peripheral nerve injury repair.

[0104] Example 3

[0105] HAB hydrogel in vitro cytotoxicity assessment experiment

[0106] 1. Experimental conditions

[0107] A control group was set up: DRGSCs were cultured in conventional 2D plastic culture dishes.

[0108] Experimental group: DRGSCs were cultured in a three-dimensional HAB hydrogel system;

[0109] The culture period lasted 28 days, with 6 replicates per group and 3 biological replicates of the experiment.

[0110] 2. Testing Indicators and Methods

[0111] Cell morphology and viability were observed using an inverted bright-field microscope and a live / dead cell staining kit, respectively; cell cycle distribution was detected by flow cytometry; and cell proliferation activity was detected by a CCK-8 kit. The entire process was performed in accordance with the instructions of the corresponding kits.

[0112] 3. Experimental Results

[0113] Cell morphology and long-term survival activity observation

[0114] After 28 days of continuous culture, the morphology and cell viability of the two groups of DRGSCs were observed using bright-field microscopy and live / dead staining reagents. The results are shown in the figure. Figure 4 a.

[0115] In the 2D plastic culture dish group, the DRGSCs cells were mostly spherical, and in the later stage of culture, the cells showed fragmentation and apoptosis.

[0116] In the HAB hydrogel experimental group, DRGSCs exhibited a normal, elongated growth morphology, and their cell growth status was excellent.

[0117] Live / dead staining results confirmed that DRGSCs in the HAB hydrogel group maintained stable cell viability during the 28-day culture period, while cell viability in the 2D plastic culture dish group decreased significantly by day 28.

[0118] 4. Cell cycle distribution detection

[0119] Cell cycle distribution was analyzed by flow cytometry after 28 days of culture. The results are shown in [Figure number missing]. Figure 4 b: Compared to the 2D planar culture group, the HAB hydrogel group showed a higher proportion of DRGSCs in the G0-G1 phase and a lower proportion in the S phase, with no statistically significant difference in the proportion of each cell cycle phase between the two groups (P>0.05). This suggests that the conventional 2D plastic culture environment can inhibit the G0-G1 cell cycle progression of DRGSCs and interfere with DNA synthesis metabolism in the S phase.

[0120] 5. Cell proliferation activity detection

[0121] Cell proliferation was assessed using the CCK-8 assay after 28 days of culture. Results are shown below. Figure 4 c: Compared with the 2D plastic culture system, HAB hydrogel can significantly promote the proliferation and growth of DRGSCs, and the difference between groups is statistically significant (P<0.01).

[0122] in conclusion:

[0123] HAB hydrogel has no in vitro cytotoxicity and excellent biocompatibility; it can optimize the growth morphology of DRGSCs, maintain long-term cell viability, avoid the cell apoptosis and fragmentation problems caused by 2D culture, and enhance the proliferation capacity of DRGSCs, making it suitable for the long-term three-dimensional culture of dorsal root nerve stem cells.

[0124] Example 4

[0125] HAB hydrogel in vivo biocompatibility evaluation experiment

[0126] 1. Laboratory animals and experimental conditions

[0127] Healthy SPF-grade SD rats aged 6-8 weeks were selected, with half males and half females, weighing 200±20g. All rats were housed in an environment with a temperature of 22~25℃, a 12h day-night cycle, and free access to food and water. After 7 days of acclimatization, animal modeling experiments were conducted. The animal experimental procedures complied with the approval standards of the Laboratory Animal Ethics Committee.

[0128] The experimental group was sterilely injected and cured into HAB hydrogel, while a blank control group was implanted subcutaneously. There were 6 rats in each group. The in vivo implantation observation period was 28 days. All biochemical tests were performed in 3 replicates. The statistical test standards were the same as those in Example 1 and Example 2.

[0129] 2. Implantation Procedures and Sampling Methods

[0130] After intraperitoneal anesthesia, the subcutaneous area of ​​the rat's back was prepared and disinfected. Sterile pretreated HAB hydrogel was implanted in equal amounts into the subcutaneous cavities on both sides of the rat's back. The gross morphological degradation of the hydrogel at the implantation site was observed by the naked eye on days 3, 14, and 28 after implantation. Subcutaneous tissue around the implantation site was collected on schedule, fixed and preserved, and used for immunohistochemistry and Western blotting protein detection.

[0131] 3. Detection Method

[0132] General visual observation:

[0133] The morphology, texture, and residual volume of subcutaneous HAB hydrogel implants in rats were observed periodically, and the in vivo degradation rate was assessed.

[0134] Immunohistochemical staining:

[0135] The tissue expression levels of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in subcutaneously implanted tissue were detected.

[0136] Western blotting:

[0137] Quantitatively detect the expression levels of TNF-α and IL-1β inflammatory factor proteins in the tissue surrounding the implant.

[0138] 4. Experimental Results

[0139] Observation of HAB hydrogel degradation behavior in vivo

[0140] The 28-day in vivo subcutaneous implantation degradation observation results showed that: on the 3rd day after HAB hydrogel was implanted subcutaneously in rats, a well-defined subcutaneous solid mass was formed at the implantation site; on the 14th day after implantation, the solid gel mass gradually dissolved and liquefied, transforming into a liquid water-like substance; on the 28th day after implantation, the liquefied degradation products were metabolized and absorbed by the body, and there was no gel residue at the implantation site. The material can complete complete in vivo degradation and metabolism within 1 month.

[0141] Detection of expression levels of inflammatory factors in vivo

[0142] Immunohistochemical results are shown in Figure 5 a: As HAB hydrogel gradually degrades in vivo, the expression levels of inflammatory factors TNF-α and IL-1β in the surrounding tissues show a continuous decreasing trend, and the differences in inflammatory factor expression at different time points are extremely significant (P<0.01).

[0143] Western blotting protein quantification results are shown below. Figure 5b: Consistent with the trend of immunohistochemical results, as the HAB hydrogel degrades in vivo, the expression levels of TNF-α and IL-1β inflammatory proteins in the body tissues are gradually downregulated, and the local inflammatory response in the body gradually subsides.

[0144] in conclusion:

[0145] HAB hydrogel can be controlled to degrade and be completely metabolized and absorbed under the skin of rats within one month. After implantation, it does not induce a persistent local inflammatory response. The inflammatory response of the body gradually weakens as the material degrades. The material as a whole has no obvious in vivo irritation and has excellent in vivo biocompatibility, meeting the requirements for in vivo application of neurorepair implantable biomaterials.

[0146] Example 5

[0147] Differential gene expression profile analysis of DRGSCs co-cultured with HAB hydrogel

[0148] 1. Basic experimental conditions

[0149] Using the same third-generation logarithmic growth phase rat DRGSCs as described in the above examples, the experimental groups, cell seeding density, culture environment, and culture period were completely consistent with the aforementioned examples:

[0150] The control group consisted of DRGSCs cultured in conventional 2D plastic culture dishes.

[0151] The experimental group consisted of DRGSCs co-cultured in a three-dimensional HAB hydrogel system;

[0152] Cell samples were collected 14 days after differentiation. Three biological replicates were set up for each group. Outliers were removed, and whole transcriptome gene expression profile sequencing analysis was performed. A uniform screening threshold was set for sequencing and bioinformatics analysis.

[0153] 2. Sample processing and detection analysis methods

[0154] Two groups of DRGSCs cell pellets were collected, and total RNA was extracted from the cells using the Trizol method. The purity and integrity of the RNA were detected, and a cDNA library was constructed. Gene sequencing was performed using a high-throughput transcriptome sequencing platform. Differentially expressed genes (DEGs) were screened, and Pearson correlation clustering analysis, GO functional enrichment analysis, and KEGG pathway enrichment analysis were conducted. Bioinformatics data analysis and plotting were standardized.

[0155] 3. Sequencing and bioinformatics analysis results

[0156] Overall distribution of differentially expressed genes

[0157] Compared to the 2D plastic culture dish control group, 990 differentially expressed genes were screened in the HAB hydrogel co-culture system, of which 627 were upregulated and 363 were downregulated. The distribution of differentially expressed genes is shown in the figure. Figure 6 a.

[0158] Sample Pearson correlation cluster analysis

[0159] The results of the Pearson correlation cluster analysis of gene expression are shown below. Figure 6 b. Sequencing samples can be precisely clustered and divided according to the culture system, clearly divided into two major clusters: HAB hydrogel culture group and 2D plastic culture dish culture group. There are significant differences in the gene expression patterns between the two groups, and the samples within the groups have good repeatability.

[0160] GO functional enrichment analysis results

[0161] GO enrichment analysis was performed on the differentially expressed genes identified through screening, and the results are shown in [the table below]. Figure 6 c: ① Biological process level: Differentially expressed genes are significantly enriched in biological processes such as neurotransmitter transport, synaptic assembly, and membrane potential regulation; ② Cell component level: Differentially expressed genes are mainly enriched in cellular components that regulate synaptic transmission, postsynaptic membrane, and intrinsic synapse-related functions; ③ Molecular function level: Differentially expressed genes are concentrated in metal ion transporter functions and glutamate receptor activity functions.

[0162] KEGG pathway enrichment analysis results

[0163] The results of KEGG pathway enrichment analysis are shown in Table 1: Differentially expressed genes of DRGSCs mainly participate in the regulation of three core signaling pathways: synaptic vesicle circulation, glutamatergic signal transduction, and cell adhesion molecules, mediating the regulation of cellular neural function.

[0164] in conclusion:

[0165] HAB hydrogels can significantly regulate the differential expression of genes related to the neuronal function of DRGSCs. By enriching functional genes related to synapse construction, ion transport, and neural signal transduction, HAB hydrogels can activate neural functional pathways such as glutamatergic signaling and synaptic vesicle circulation, thereby regulating and enhancing the neuronal function of DRGSCs at the molecular transcriptional level. This elucidates the molecular mechanism by which HAB hydrogels promote neural differentiation and optimize neuronal function.

[0166] rno04721 Synaptic vesicle cycle 6.92481E-15 Tcirg1 / Atp6v0d2 / Slc17a6 / Stx1b / Slc1a1 / Slc6a5 / Slc1a3 / Slc6a11 / Slc6a1 / Dnm1 / Snap25 / Slc32 a1 / Stxbp1 / Slc6a9 / Syt1 / Slc1a6 / Rims1 / Cplx1 / Nsf / Atp6v0a1 / Unc13a / Slc6a7 / Cacna1a / Atp6v1g2 rno04724 Glutamate synapse 5.81494E-11 Shank1 / Slc17a6 / Grm1 / Slc1a1 / Grin2d / Grm5 / Prkcb / Grik5 / Gria2 / Slc1a3 / Grm8 / Grm7 / Grin1 / Gria3 / Adcy8 / Slc1a6 / Gng7 / Gria4 / Grin2c / Gng4 / Gnao1 / Cacna1a / Grm4 / Grik2 rno04721 GABAergic synapse 1.07229E-09 Gabra5 / Prkcb / Slc6a11 / Slc6a1 / Slc12a5 / Slc32a1 / Gad1 / Gabbr2 / Gabre / Gphn / Adcy8 / Gng7 / Hap1 / Gabrg2 / Nsf / Gad2 / Gng4 / Gnao1 / Cacna1a / Gabbr1 rno05033 Nicotine addiction 5.67552E-09 Slc17a6 / Gabra5 / Grin2d / Gria2 / Chrna4 / Slc32a1 / Grin1 / Gria3 / Gabre / Gria4 / Grin2c / Gabrg2 / Cacna1a rno04514 Cell adhesion molecules 1.75702E-07 Sdc1 / Icam1 / Selp / F11r / RT1-CE5 / RT1-A2 / RT1-CE10 / Vsir / Nrxn2 / Lrrc4b / Nlgn1 / Ntng1 / Lrrc4 / Lrrc4c / L1cam / Nrxn3 / Nrcam / Cntn1 / Nectin1 / Nfasc / Cadm3 / Slitrk5 / Slitrk1 / RT1-DMb / RT1-N2 rno04974 Protein digestion and absorption 3.10605E-07 Col5a1 / Col16a1 / Col8a2 / Col22a1 / Col12a1 / Col5a2 / Col4a1 / Col4a2 / Col8a1 / Col13a1 / Col6a2 / Col18a1 / Eln / Slc1a1 / Atp1a3 / Slc3a1 / Atp1b1 / Atp1b2 rno04080 Neuroactive ligand receptor interactions 5.90217E-07 Aplnr / Vipr2 / Tspo / Pth1r / Kng1 / Gabra5 / Grm1 / Grin2d / Grm5 / Grik5 / Glrb / Gria2 / Npy2r / Grm8 / Adcyap1r1 / Grm7 / Chrna4 / Lypd6b / Lypd6 / Gri n1 / Cnr1 / Gabbr2 / Glra2 / Htr2c / Gria3 / Gabre / Sstr1 / Lynx1 / Kiss1r / Gria4 / Adcyap1 / Grin2c / Gabrg2 / Grid1 / Sst / Grm4 / Grik2 / Gabbr1 / Glp1r rno04723 Retrograde endocannabinoid signaling 2.29134E-05 LOC679739 / Slc17a6 / Gabra5 / Grm1 / Grm5 / Prkcb / Gria2 / Slc32a1 / Cnr1 / Gria3 / Gabre / Adcy8 / Gng7 / Gria4 / Rims1 / Gabrg2 / Gng4 / Gnao1 / Cacna1a rno04713 Biological clock calibration 6.82966E-05 Grin2d / Prkcb / Camk2d / Gria2 / Adcyap1r1 / Grin1 / Gria3 / Adcy8 / Gng7 / Gria4 / Adcyap1 / Grin2c / Gng4 / Gnao1 rno04390 Hippo signal path 0.00013931 Tgfb1 / Ccn2 / Fzd1 / Frmd6 / Bmp5 / Afp / Tcf7 / Wnt9a / Snai2 / Tead3 / Sox2 / Ctnna2 / Ywhab / Prkcz / Ywhah / Wnt3 / Ppp2r2b / Ywhag

[0167] Table 1. KEGG pathway enrichment analysis

[0168] Example 6

[0169] 1. Basic experimental conditions

[0170] Using the same third-generation logarithmic growth phase rat DRGSCs as described in the above examples, the experimental groups, cell seeding density, culture environment, and differentiation culture period were completely identical: The control group consisted of DRGSCs cultured in conventional 2D plastic culture dishes. The experimental group consisted of DRGSCs co-cultured in a three-dimensional HAB hydrogel system; The core differentially expressed genes Gnao1 and Grm7, which are highly associated with mutation development and neural stem cell differentiation and obtained from the transcriptome screening in the above examples, were selected. The transcriptome results were verified by qRT-PCR and Western blotting. Each group had 3 biological replicates and 3 technical replicates.

[0171] 2. Detection Method

[0172] Real-time quantitative PCR (qRT-PCR): Two groups of cells were collected on schedule, total RNA was extracted and reverse transcribed into cDNA, an amplification system was prepared, and the target genes Gnao1 and Grm7 were specifically amplified. The relative expression levels of the two gene mRNAs were detected.

[0173] Western blotting: Total protein was extracted from two groups of cells, quantified, electrophoresed, transferred to a membrane, blocked, and incubated with primary antibodies Gnao1 and Grm7 and fluorescent secondary antibodies. The relative expression level of the target protein was then detected by imaging and quantification.

[0174] The qRT-PCR test results are shown below. Figure 7 a: Compared to the 2D plastic culture dish control group, the expression levels of synapse- and stem cell differentiation-related genes Gnao1 and Grm7 mRNA were significantly upregulated in the HAB hydrogel experimental group; among them, the difference in Gnao1 among the groups was extremely significant (P<0.01), and the difference in Grm7 among the groups was significant (P<0.05).

[0175] Determination of the reliability of transcriptome results

[0176] The intergroup expression trends of Gnao1 and Grm7 genes at the mRNA and protein levels are completely consistent with the differential expression trends of high-throughput transcriptome sequencing in Example 4, confirming that the aforementioned transcriptome gene expression profile detection data are authentic and reliable and the experimental results are credible.

[0177] in conclusion: HAB hydrogels can significantly upregulate the transcription and protein expression levels of Gnao1 and Grm7, key genes for DRGSC synapse construction and stem cell differentiation, thereby regulating neuronal synapse maturation and directed stem cell differentiation at the molecular level. This validation experiment confirms the accuracy and effectiveness of the aforementioned transcriptome sequencing results, further clarifying that HAB hydrogels can optimize DRGSC synaptic function and promote neural differentiation and maturation by upregulating the expression of Gnao1 and Grm7.

[0178] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. The various components mentioned in this invention are common technologies in the existing field. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A hydrogel material for nerve repair after hepatectomy, characterized in that The hydrogel material is a 3-aminophenylboronic acid modified hyaluronic acid hydrogel, which is synthesized by an amidation reaction of 3-aminophenylboronic acid (3-APBA) and hyaluronic acid (HA), and then cross-linked to form a three-dimensional porous hydrogel structure.

2. A hydrogel material for nerve repair after liver resection according to claim 1, characterized in that, The hyaluronic acid has a molecular weight of 1.0-1.8 MDa.

3. A hydrogel material for nerve repair after liver resection according to claim 1, characterized in that, The degree of substitution of the 3-aminophenylboronic acid on hyaluronic acid is 2.09 ± 0.02 mmol / g.

4. A hydrogel material for nerve repair after liver resection according to claim 1, characterized in that, The hydrogel concentrations are 1.25%, 2.5%, and 5.0% (w / w); the hydrogel yield stresses are 18.6, 165.9, and 711.5 Pa.

5. A hydrogel material for nerve repair after liver resection according to claim 1, characterized in that, The hydrogel has a three-dimensional porous structure; the pore size of the hydrogel decreases with increasing concentration, and the degradation period is 28 days, which matches the nerve regeneration period after liver surgery. The hydrogel can promote the adhesion rate of dorsal root ganglion stem cells (DRGSCs) to 78.5%±3.2%, the migration speed to 21.4μm / h, and the neuronal differentiation efficiency to 72.6%±4.1%, and upregulate the expression of synapse-related genes Gnao1 and Grm7, as well as neurotrophic factors BDNF and NGF.

6. A method for preparing a hydrogel material for nerve repair after liver resection, comprising the following steps: (1) Preparation of raw material pretreatment and proportioning; (2) Synthesis of phenylboronic acid functionalized hyaluronic acid intermediate HAB; (3) Preparation of medical nerve repair HAB hydrogel by in-situ cross-linking.

7. A method for preparing a hydrogel material for nerve repair after liver resection according to claim 6, characterized in that, The specific steps for the pretreatment of the raw materials are as follows: The raw materials include: hyaluronic acid (HA, molecular weight 1.0–1.8 MDa), 3-aminophenylboronic acid (3-APBA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sodium hydroxide, hydrochloric acid, phosphate buffered saline (PBS, pH 7.4), and deionized water. All chemical raw materials and biochemical reagents are of analytical grade and can be used directly after purchase without secondary purification or refining. The pH of the physiological phosphate buffer is kept constant at 7.

4.

8. A method for preparing a hydrogel material for nerve repair after liver resection according to claim 6, characterized in that, The specific steps for synthesizing the phenylboronic acid functionalized hyaluronic acid intermediate HAB are as follows: S201. Homogeneous dissolution of hyaluronic acid: Weigh 3g of hyaluronic acid raw material, add it to 500mL of deionized water, and stir at a constant temperature until the solute is completely homogeneously dissolved to prepare a homogeneous hyaluronic acid aqueous solution. S202, Activation of carboxyl groups in hyaluronic acid: Add 1.504g EDC and 0.911g NHS to the above aqueous solution of hyaluronic acid at one time, and stir the reaction under closed conditions at room temperature for 30 minutes. Use the dual activation reagent to activate the carboxyl groups on the hyaluronic acid molecular chain to provide active sites for the amide grafting reaction. S203, Controllable amide grafting modification: After activation with hydrochloric acid and sodium hydroxide, the pH value of the system was stabilized to 5.

5. 1.084g of 3-aminophenylboronic acid was added to the system, and the reaction temperature was kept constant at 25℃. The system was stirred at a constant temperature for 6h. Through amide covalent bonding reaction, the functional group of 3-aminophenylboronic acid was covalently grafted and fixed to the main chain of hyaluronic acid polymer to obtain the crude modified reaction solution. S204. Dialysis purification: The modified crude reaction solution was transferred into a regenerated cellulose dialysis bag with a molecular weight cutoff of 14000 Da and purified by continuous dialysis with deionized water at room temperature for 3 days to completely remove unreacted monomers, free activators, small molecule byproducts and soluble impurities from the system. S205, Powder solidification preparation: The clarified modified liquid after dialysis purification is subjected to vacuum freeze-drying to obtain pure white phenylboronic acid modified hyaluronic acid solid powder. The obtained HAB powder is sealed and stored at room temperature in the dark for later use.

9. A method for preparing a hydrogel material for nerve repair after liver resection according to claim 6, characterized in that, The specific steps for preparing the medical nerve repair HAB hydrogel by in-situ crosslinking are as follows: S301. Preparation of modified polymer precursor solution: Take the spare HAB powder, add it to sterile physiological phosphate buffer with pH=7.4, and stir at a constant temperature of 50℃ until the HAB powder is completely dissolved and homogenized. Prepare a sterile HAB precursor aqueous solution with a mass fraction w / w of 1.25%~5.0%. S302, Thermosensitive in-situ crosslinking molding: The prepared HAB precursor aqueous solution is naturally cooled to 25°C, sealed and left to stand for 1 hour. The medical HAB hydrogel with an internal three-dimensional porous structure is obtained by relying on the dynamic phenylboronic acid ester bonds between polymer chains for autonomous crosslinking. Among them, the HAB precursor aqueous solution has the characteristics of rapid prototyping in response to body temperature: when the system temperature drops to 37°C or below, it can achieve rapid gel solidification, the material flowability is controllable, it can be used for non-invasive injection with 18G medical injection needles, and it is suitable for intraoperative minimally invasive injection after liver resection and local implantation of wounds after surgery.

10. The application of a hydrogel material for nerve repair after liver resection according to any one of claims 1-9 in a nerve repair drug after liver resection, wherein the HAB hydrogel can upregulate the expression of neurotrophic factors BDNF and NGF and synapse-related genes Gnao1 and Grm7, induce functional synapse formation, enhance neurophysiological activity, and accelerate the regeneration of damaged perihepatic nerves.

Images