A method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs

Abstract

This invention discloses a method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs, belonging to the field of biomedical technology. The method includes the following steps: S1: synthesizing GGA; S2: preparing a PEG-PLL aqueous solution; S3: preparing a PLGA electrospun nerve conduit; S4: synthesizing a GGA-PEG-PLL hydrogel sheath and assembling the PLGA@GPP nerve conduit. This invention uses a PLGA electrospun nerve conduit as a mechanical support framework, providing a stable, directional axonal extension channel for nerve defects. Its controllable degradation rate is highly matched to the nerve regeneration cycle. The inner composite GGA-PEG-PLL hydrogel coating can biomimeticly simulate the endoneurotic microenvironment, effectively improving the high-oxidative-stress inflammatory microenvironment at the site of nerve injury. This solves the problems of existing nerve conduits lacking a stable lumen structure and failing to provide a three-dimensional microenvironment for cell adhesion and growth.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs. Background Technology

[0002] Peripheral nerve injury (PNI) is a common disabling disease in clinical practice, mainly caused by factors such as mechanical trauma, iatrogenic injury, or metabolic diseases. This type of injury leads to sensory, motor, and autonomic dysfunction, severely impacting patients' quality of life and placing continuous pressure on the social healthcare system. Statistics show that the global incidence of PNI is approximately 13 to 23 cases per 100,000 people per year; while in China, the number of cases is as high as 300,000 to 500,000 per year, highlighting a huge unmet clinical need in this field. Although peripheral nerves possess a certain intrinsic regenerative capacity compared to the central nervous system, their spontaneous repair process has significant limitations. First, axonal regeneration is extremely slow, growing an average of only 1 to 2 millimeters per day. For long-segment nerve defects, regenerating axons often fail to successfully reach and re-establish innervation connections before irreversible atrophy and fibrosis occur in effector organs (such as muscles and skin receptors), leading to permanent functional loss.

[0003] Secondly, the precision of nerve regeneration is a key challenge. Without proper physical and chemical guidance, regenerated axons rely on the chemotaxis of the severed ends to grow, leading to a mismatch in sensory and motor signal transmission. This mismatch results in incomplete functional recovery; even if neural continuity is restored, patients may still suffer from long-term sequelae such as decreased motor coordination, sensory abnormalities, and intractable neuropathic pain.

[0004] Currently, autologous nerve transplantation is considered the "gold standard" for repairing nerve defects. However, this method has many inherent drawbacks, such as limited donor availability, loss of donor site function, neuroma formation, and size mismatch. Therefore, developing tissue-engineered neural conduits that can effectively guide and promote precise nerve regeneration has become a research focus and urgent need in this field.

[0005] With the development of tissue engineering, nerve conduits have provided new treatment ideas for peripheral nerve regeneration. The core of this approach lies in constructing a biomimetic structure that can provide directional physical guidance and create a favorable microenvironment for regeneration.

[0006] However, existing neural conduit materials often suffer from limited functionality: while synthetic conduits provide sufficient mechanical strength, they often lack the necessary bioactivity; and while natural materials have good cell compatibility, they are prone to collapse after implantation due to insufficient mechanical properties and lack effective axonal guidance capabilities. In particular, there is a lack of ideal conduits that can simultaneously meet the following key requirements: a stable luminal structure, a three-dimensional microenvironment for cell adhesion and growth, and a composite functional system that can continuously provide neurotrophic support. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs. This PLGA@GPP composite biomimetic nerve conduit can not only serve as a physical channel for nerve regeneration, but also continuously maintain the activity and function of bone marrow mesenchymal stem cells. Through its paracrine and differentiation-promoting effects, it creates a favorable regenerative microenvironment, providing a novel comprehensive solution for the repair of peripheral nerve injuries and solving the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs includes the following steps:

[0010] S1, Synthesize GGA:

[0011] Gelatin was dissolved in deionized water by stirring to obtain a gelatin aqueous solution. Gallic acid was dissolved in a mixed solution of deionized water and dimethylformamide. After complete dissolution, EDC and NHS were added, and the mixture was stirred at room temperature. The gallic acid reaction solution was slowly added to the gelatin aqueous solution and stirred overnight. The mixture was then placed in a dialysis bag and dialyzed with deionized water for 3-5 days. The dialysate was aliquoted into petri dishes, frozen, and then freeze-dried to obtain GGA powder.

[0012] S2. Preparation of PEG-PLL aqueous solution:

[0013] PEG-NH2 was weighed and dissolved in DMF, and PLL monomer was weighed and dissolved in DMF. The mixture was heated and stirred under N2 environment. The product was added dropwise to ice-cold ether, filtered and collected, and dried under vacuum to obtain MPEG5K-PLL7K. PEG-PLL was weighed and completely dissolved in trifluoroacetic acid solvent. An acetic acid solution containing hydrogen bromide was added under ice bath stirring. After the reaction, the crude product was precipitated in ice-cold ether. The crude product was then dissolved in water to adjust the pH value, placed in a dialysis bag and dialyzed. After dialyzing in deionized water, the purified PEG-PLL block copolymer was obtained as a white flocculent solid by freeze drying.

[0014] S3. Preparation of PLGA electrospun nerve conduit:

[0015] PLGA polymer is dissolved in hexafluoroisopropanol or trifluoroethanol and continuously stirred magnetically at room temperature until a homogeneous and transparent spinning solution is formed. The spinning solution is injected into an injection pump equipped with a flat-headed metal needle for spinning. Spinning continues until a uniform tubular fiber membrane is deposited on the surface of the columnar mandrel. The tubular fiber membrane is carefully peeled off from the mandrel to obtain a PLGA electrospun nerve conduit with a through lumen.

[0016] S4. Synthesis of GGA-PEG-PLL hydrogel cannulas and assembly of PLGA@GPP nerve condyloma acuminatum:

[0017] PEG-PLL and GGA were dissolved in deionized water to obtain a mixed precursor solution containing PEG-PLL and GGA. TG enzyme was added to the mixed precursor solution, and after shaking and mixing, the liquid was injected into the inner layer of the PLGA electrospun nerve conduit. Kirschner wires were then inserted and fixed in the center of the lumen. After cross-linking at room temperature, the Kirschner wires were slowly removed to obtain the PLGA@GPP nerve conduit, which was then sterilely sealed and stored for later use.

[0018] Preferably, in the preparation process of the PLGA electrospun nerve conduit, the PLGA has a molecular weight of 10 kDa, a LA:GA molar ratio of 75:25, a viscosity of 1.21 dL / g (CHCl3 / 25℃), and is prepared using trichloroethanol. A 10% (w / v) PLGA electrospun solution is prepared, and the initial inner diameter of the PLGA electrospun nerve conduit is 1.5 mm, with a wall thickness of 0.25 mm.

[0019] Preferably, in the GGA synthesis process, the mass ratio of gallic acid to gelatin is 1:3-1:2.

[0020] Preferably, during the assembly of the PLGA@GPP nerve conduit, the mass ratio of GGA to PEG-PLL is 2:1.

[0021] Preferably, the amount of TG enzyme used is 2-4 U / ml.

[0022] Preferably, in the mixed solution of deionized water and dimethylformamide, the volume ratio of deionized water to dimethylformamide is 3:2.

[0023] Preferably, when the spinning solution is injected into an injection pump equipped with a flat-headed metal needle for spinning, the applied voltage is 17kV, the collection distance between the needle and the receiving device is 12cm, and the spinning solution injection rate is 2.0mL / h.

[0024] Preferably, the receiving device uses a high-speed rotating cylindrical spindle with a rotation speed of 800 r / min.

[0025] Preferably, the mass ratio of EDC to NHS is 1:2 to 1:1.

[0026] Preferably, the PLGA@GPP nerve conduit has an inner diameter of 1 mm, an outer diameter of 2.0 mm, and a length of 12 mm.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. This invention uses PLGA electrospun nerve conduit as a mechanical support framework, which can provide a stable and directional axon extension channel for long-segment nerve defects, avoiding the collapse of the lumen and compression of regenerating nerves. Its controllable degradation rate is highly matched with the nerve regeneration cycle. The inner composite GGA-PEG-PLL hydrogel coating can biomimize the endometrial microenvironment and has excellent ROS scavenging ability. It can effectively improve the high oxidative stress inflammatory microenvironment at the nerve injury site, significantly improve the survival rate of subsequently injected BMSCs, and its controllable degradation rate is highly matched with the rat sciatic nerve regeneration cycle. It can provide a stable adhesion interface and nutrient exchange channel for cells in the early stage of regeneration, and gradually degrade into space for axon extension in the later stage.

[0029] 2. The accompanying intraoperative in situ injection protocol for BMSCs, which involves injecting the BMSCs suspension after catheter bridging and fixation, completely avoids the damage to cell viability caused by cross-linking reactions and temperature changes during pre-encapsulation preparation. The cell dosage can be flexibly adjusted according to the actual situation of the nerve defect during surgery, resulting in stronger clinical adaptability. BMSCs can maintain high survival rate and bioactivity in the biomimetic microenvironment of the catheter, and continuously secrete neurotrophic factors through paracrine effects, synergistically promoting Schwann cell proliferation, axonal regeneration and myelination, significantly improving the repair effect and motor function reconstruction of long-segment peripheral nerve defects. Its preparation process is simple, the conditions are mild, and the biosafety is excellent, with no obvious immune rejection or systemic toxicity, providing a new approach and a transferable solution for the clinical treatment of peripheral nerve defects. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the Fourier transform infrared spectrum of the cross-linked GA and Gel of the present invention;

[0031] Figure 2 This is a schematic diagram of the hydrogen NMR spectrum of GGA cross-linked with Gel according to the present invention;

[0032] Figure 3 This is a schematic diagram of the cross-linking of GGA and PEG-PLL at room temperature under the catalysis of TG enzyme according to the present invention;

[0033] Figure 4 This is a schematic diagram of the swelling curve of the hydrogel of the present invention;

[0034] Figure 5This is a schematic diagram of the hydrogel degradation curve of the present invention;

[0035] Figure 6 This is a schematic diagram of the inner and outer diameters of the lumen of the PLGA@GPP nerve conduit of the present invention;

[0036] Figure 7 This is a schematic diagram showing the length of the PLGA@GPP composite nerve conduit of the present invention;

[0037] Figure 8 This is a schematic diagram of the morphology of the PLGA electrospun fibers on the outer layer of the conduit under SEM images of the present invention.

[0038] Figure 9 This is a schematic diagram of the morphology of the GGA-PEG-PLL hydrogel in the inner layer of the catheter under SEM images according to the present invention.

[0039] Figure 10 This is a schematic diagram of the cross-sectional morphology of the PLGA@GPP composite nerve conduit under SEM according to the present invention;

[0040] Figure 11 This is a schematic diagram illustrating the activity of PLGA@GPP neural conduit CCK-8 cells according to the present invention;

[0041] Figure 12 This is a schematic diagram of the PLGA@GPP live / dead staining of nerve conduits according to the present invention;

[0042] Figure 13 This is a schematic diagram of the mitochondrial membrane potential detection in the PLGA@GPP neural conduit according to the present invention;

[0043] Figure 14 This is a schematic diagram of ROS detection in the PLGA@GPP nerve conduit according to the present invention;

[0044] Figure 15 This is a schematic diagram of HE staining of the heart, liver, spleen, lungs, and kidneys 12 weeks after PLGA@GPP nerve conduit suturing according to the present invention;

[0045] Figure 16 This is a schematic diagram of the gait of the right paw in rats 12 weeks after surgery according to the present invention;

[0046] Figure 17 This is a schematic diagram of the 3D pressure map of the right paw of a rat 12 weeks after surgery according to the present invention;

[0047] Figure 18 This is a schematic diagram of the gross morphology of the right gastrocnemius muscle in rats 12 weeks post-surgery according to the present invention.

[0048] Figure 19 This is a schematic diagram of Masson staining of the right gastrocnemius muscle of a rat 12 weeks post-surgery according to the present invention.

[0049] Figure 20 This is a schematic diagram of the wet weight of the right gastrocnemius muscle in rats 12 weeks post-surgery according to the present invention.

[0050] Figure 21 This is a schematic diagram of the cross-sectional area of ​​the right gastrocnemius muscle in rats 12 weeks post-surgery according to the present invention. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] To address the issues of existing neural conduits lacking stable luminal structures, failing to provide a three-dimensional microenvironment for cell adhesion and growth, and being unable to continuously provide neurotrophic support, please refer to [link to relevant documentation]. Figures 1-21 This embodiment provides the following technical solution:

[0053] Example 1

[0054] A method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs includes the following steps:

[0055] S1. Synthetic gallic acid-modified gelatin (GGA):

[0056] Add 5g of gelatin to 150mL of deionized water and stir at 40℃ for 1h to dissolve, thus obtaining a gelatin aqueous solution;

[0057] 1.7 g gallic acid was dissolved in 125 mL of a mixed solution of deionized water and dimethylformamide (volume ratio 3:2). After complete dissolution, 1.91 g EDC and 1.6 g NHS were added, and the mixture was stirred at room temperature for 1 h.

[0058] Slowly add the gallic acid reaction solution to the gelatin aqueous solution and stir overnight at 40°C;

[0059] The mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water at 40°C for 3 days, with the deionized water changed twice a day.

[0060] The dialysate was dispensed into culture dishes, frozen at -80°C for 4 hours, and then freeze-dried for 2 days to obtain GGA powder.

[0061] S2. Preparation of polyethylene glycol-polylysine (PEG-PLL) aqueous solution:

[0062] Weigh 1 g of PEG-NH2 and dissolve it in 5 mL of dry DMF. Weigh 3.88 g of PLL monomer and dissolve it in 20 mL of DMF. Heat and stir in N2 environment, react at 40 °C for 48 h. After the reaction is completed, add the product dropwise to a large amount of ice-cold ether, filter to collect the product, and dry under vacuum to obtain MPEG5K-PLL7K.

[0063] 3.5g of PEG-PLL was weighed and completely dissolved in trifluoroacetic acid solvent. An acetic acid solution containing 33% hydrogen bromide was added under stirring in an ice bath. After reacting for 4 hours, the crude product was precipitated in a large amount of ice-cold ether. The crude product was then dissolved in water, and the pH was adjusted to 6.0-7.0. The product was then placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in deionized water for 24 hours. The purified PEG-PLL block copolymer was obtained as a white flocculent solid by freeze drying.

[0064] S3. Preparation of PLGA electrospun nerve conduit:

[0065] Dissolve 1g of PLGA polymer in 10mL of trifluoroethanol and stir magnetically for 24 hours at room temperature until a homogeneous and transparent solution is formed, with the solution concentration controlled at 10% (w / v).

[0066] The above spinning solution was injected into an injection pump equipped with a flat-headed metal needle, and spinning was carried out within the following process parameters: the applied voltage was 17kV, the collection distance between the needle and the receiving device was 12cm, the spinning solution injection rate was 2.0mL / h, and the receiving device adopted a high-speed rotating cylindrical mandrel with its rotation speed controlled at 800 r / min.

[0067] Continue spinning until a uniform tubular fiber membrane with a wall thickness of 0.25 mm is deposited on the surface of the columnar mandrel. Carefully peel the obtained tubular fiber membrane off the mandrel (1.5 mm in diameter) to obtain a PLGA electrospun nerve conduit with a through lumen. Cut the PLGA electrospun nerve conduit to a specific length (e.g., 10-15 mm) as needed.

[0068] S4. Synthesis of GGA-PEG-PLL hydrogel cannulas and assembly of PLGA@GPP nerve condyloma acuminatum:

[0069] 50 mg of PEG-PLL and 100 mg of GGA were dissolved in 1 mL of deionized water to obtain a mixed precursor solution containing 5% PEG-PLL and 10% GGA (GGA to PEG-PLL mass ratio 2:1). 2 U / ml of TG enzyme (glutamin transferase) was added to the precursor solution, and after shaking to mix, the liquid was injected into the inner layer of the PLGA electrospun nerve conduit. A 1 mm diameter Kirschner wire was then inserted and fixed in the center of the lumen. After incubation and cross-linking at room temperature for 20 min, the Kirschner wire was slowly removed to obtain a PLGA@GPP nerve conduit with an inner diameter of 1 mm, an outer diameter of 2.0 mm, and a length of 12 mm. The conduit was then sterilely sealed and stored for later use.

[0070] Example 2

[0071] The difference from Example 1 is that the amount of TG enzyme used during hydrogel crosslinking is different. In this example, the amount of TG enzyme used is... 3U / mL ;

[0072] The method for preparing PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs includes the following steps:

[0073] S1. Synthetic gallic acid-modified gelatin (GGA):

[0074] Add 5g of gelatin to 150mL of deionized water and stir at 40℃ for 1h to dissolve, thus obtaining a gelatin aqueous solution;

[0075] 1.7 g gallic acid was dissolved in 125 mL of a mixed solution of deionized water and dimethylformamide (volume ratio 3:2). After complete dissolution, 1.91 g EDC and 1.6 g NHS were added, and the mixture was stirred at room temperature for 1 h.

[0076] Slowly add the gallic acid reaction solution to the gelatin aqueous solution and stir overnight at 40°C;

[0077] The mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water at 40°C for 3 days, with the deionized water changed twice a day.

[0078] The dialysate was dispensed into culture dishes, frozen at -80°C for 4 hours, and then freeze-dried for 2 days to obtain GGA powder.

[0079] S2. Preparation of polyethylene glycol-polylysine (PEG-PLL) aqueous solution:

[0080] Weigh 1 g of PEG-NH2 and dissolve it in 5 mL of dry DMF. Weigh 3.88 g of PLL monomer and dissolve it in 20 mL of DMF. Heat and stir in N2 environment, react at 40℃ for 48 h. After the reaction is completed, add the product dropwise to a large amount of ice-cold diethyl ether, filter to collect the product, and dry under vacuum to obtain crude PEG-PLL product.

[0081] 3.5 g of crude PEG-PLL product was weighed and completely dissolved in trifluoroacetic acid solvent. An acetic acid solution containing 33% hydrogen bromide was added under stirring in an ice bath. After reacting for 4 h, the crude product was precipitated in a large amount of ice-cold ether. The product was then dissolved in water, and the pH was adjusted to 6.0-7.0. The product was then placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in deionized water for 24 h. After freeze-drying, the purified PEG-PLL block copolymer was obtained as a white flocculent solid.

[0082] S3. Preparation of PLGA electrospun nerve conduit:

[0083] Dissolve 1g of PLGA polymer in 10mL of trifluoroethanol and stir magnetically for 24 hours at room temperature until a homogeneous and transparent solution is formed, with the solution concentration controlled at 10% (w / v).

[0084] The above spinning solution was injected into an injection pump equipped with a flat-tipped metal needle, and spinning was carried out within the following process parameters: applied voltage of 17kV, collection distance between the needle and the receiving device of 12cm, and spinning solution injection rate of 2.0mL / h. The receiving device adopted a high-speed rotating cylindrical mandrel, and its rotation speed was controlled at 800 r / min;

[0085] Continue spinning until a uniform tubular fibrous membrane with a wall thickness of 0.25 mm is deposited on the surface of the columnar mandrel. The resulting tubular fibrous membrane is then carefully peeled off from the mandrel (1.5 mm in diameter) to obtain a PLGA electrospun nerve conduit with a penetrating lumen. The conduit is then cut to specific lengths (e.g., 10-15 mm) for later use, as needed.

[0086] S4. Synthesis of GGA-PEG-PLL hydrogel cannulas and assembly of PLGA@GPP nerve condyloma acuminatum:

[0087] 50 mg of PEG-PLL and 100 mg of GGA were dissolved in 1 mL of deionized water to obtain a mixed precursor solution containing 5% PEG-PLL and 10% GGA (GGA to PEG-PLL mass ratio 2:1). 3 U / mL of TG enzyme (glutamin transferase) was added to the precursor solution, and after shaking to mix, the liquid was injected into the inner layer of the PLGA electrospun nerve conduit. A 1 mm diameter Kirschner wire was then inserted and fixed in the center of the lumen. After incubation and cross-linking at room temperature for 10 min, the Kirschner wire was slowly removed to obtain a PLGA@GPP nerve conduit with an inner diameter of 1 mm, an outer diameter of 2.0 mm, and a length of 12 mm. The conduit was then sterilely sealed and stored for later use.

[0088] Specifically, PLGA (polylactic acid-glycolic acid copolymer) provides a supportive material for the repair of peripheral nerve injuries, offering durable and robust mechanical support and a clear physical guiding channel, effectively preventing lumen collapse and providing space for directional axonal extension; GGA endows the hydrogel with antioxidant and anti-inflammatory capabilities, clearing excessive ROS locally after nerve injury, helping to reduce oxidative stress damage in the early stages of transplantation, inhibiting stem cell apoptosis, and optimizing the regenerative microenvironment; PEG-PLL (polyethylene glycol-polylysine) enhances the mechanical strength and stability of the hydrogel, and its good hydrophilicity facilitates the exchange of nutrients while providing sufficient active sites for cell adhesion; TG enzyme, as a biological cross-linking agent, has mild cross-linking conditions, avoiding the potential toxicity of chemical cross-linking agents and ensuring high cell survival rates.

[0089] It should be noted that when GGA and PEG-PLL are mixed in a 2:1 ratio to obtain a GGA-PEG-PLL composite hydrogel, and different concentrations of TG enzyme are added for cross-linking, it is found that GGA and PEG-PLL in a 2:1 ratio can complete gelation within 2-10 minutes at room temperature. With 2 U / ml TG enzyme, the gelation time is shortened to within 2 minutes. Therefore, the GGA-PEG-PLL hydrogel prepared with 2 U / ml TG enzyme was selected for subsequent experiments.

[0090] The physicochemical characterization of the PLGA@GPP neural conduit is as follows:

[0091] 1) Structural verification of GGA synthesis

[0092] Fourier transform infrared spectroscopy (FTIR) analysis: Total reflectance absorption (ATR) mode was used. The freeze-dried gelatin, gallic acid and GGA samples were ground into uniform fine powder and detected by infrared spectrometer. The scanning range was 4000 cm⁻¹ - 400 cm⁻¹ and the resolution was 4 cm⁻¹. The successful synthesis of GGA was verified by observing the changes in characteristic absorption peaks before and after grafting.

[0093] ¹H NMR analysis: 5 mg of each sample was dissolved in 0.5 mL of deuterated water (D2O). A 400 MHz superconducting NMR spectrometer was used with tetramethylsilane as an internal standard to record and analyze the proton chemical shifts and to quantitatively calculate the grafting efficiency of gallic acid.

[0094] 2) Evaluation of the gelation properties of GGA-PEG-PLL hydrogel

[0095] To verify the effectiveness of cross-linking between GGA and PEG-PLL catalyzed by TG enzyme: the premixed solution was added to a test tube and TG enzyme was added. After standing at room temperature for 20 minutes, the test tube was tilted at 45° or inverted. The flow of the liquid surface was observed and recorded. The formation of gel was defined as when the liquid surface no longer moved.

[0096] 3) Evaluation of swelling properties and in vitro degradation

[0097] Swelling rate determination: Weigh the dry weight (W0) of the lyophilized hydrogel, immerse it in PBS at 37℃, remove the sample at preset time points (1-24h), absorb the surface moisture, and weigh it again (W0). t The swelling ratio is calculated using the following formula:

[0098] Swelling rate = (W t - W0) / W0 × 100%;

[0099] In vitro degradation rate determination: Hydrogels with a known initial dry weight (W0) were placed in PBS solution in a 37°C constant temperature shaker. Samples were taken out on days 1, 3, 7, 14, and 28, lyophilized again, and weighed (W0). t The mass residue rate is calculated to evaluate degradation kinetics, where the formula for the mass residue rate is as follows:

[0100] Quality Residual Rate = W t / W0 × 100%;

[0101] The in vitro biocompatibility evaluation of the PLGA@GPP neural conduit is as follows:

[0102] 1) Cell viability (Live / Dead staining)

[0103] In the Transwell co-culture model, rat bone marrow mesenchymal stem cells (BMSCs) were co-cultured with PLGA@GPP catheters for 1 day and 3 days. Fluorescent staining was performed using Calcein-AM / PI working solution, and the cells were observed under a laser confocal microscope: live cells appeared green and dead cells appeared red, thus assessing the contact toxicity of the material to cells.

[0104] 2) Cell proliferation activity assay (CCK-8 assay)

[0105] BMSCs were cultured using the extract of the duct material, and CCK-8 working solution was added at 0, 24, 48, and 72 h. The OD value at 450 nm was measured to evaluate the effect of the material on cell proliferation rate.

[0106] The anti-inflammatory and oxidative stress protective functions of PLGA@GPP nerve conduits were evaluated as follows:

[0107] 1) Detection of intracellular reactive oxygen species (ROS) levels

[0108] A BMSCs oxidative stress model was established using TBHP. The experiment was divided into a control group, a model group, a PLGA group, and a PLGA@GPP group. The DCFH-DA probe was used for labeling, and fluorescence images were acquired by laser confocal microscopy to evaluate the ability of the ducts (GGA components) to clear excess intracellular ROS.

[0109] 2) Evaluation of mitochondrial membrane potential (MMP)

[0110] The JC-1 staining method was used to observe the change in intracellular fluorescence from red to green in different treatment groups. The protective effect of PLGA@GPP ducts on mitochondrial function of BMSCs under oxidative stress was evaluated by calculating the red / green fluorescence ratio.

[0111] The in vivo biocompatibility of the PLGA@GPP neural conduit was verified as follows:

[0112] The PLGA@GPP nerve conduit was implanted into a 10mm sciatic nerve defect in rats. Twelve weeks post-surgery, major organs such as the heart, liver, spleen, lungs, and kidneys were collected. After fixation with 4% paraformaldehyde, paraffin embedding, and 4μm sectioning, hematoxylin-eosin (HE) staining was performed. The presence of inflammatory cell infiltration, tissue damage, or pathological fibrosis in each organ was assessed under an optical microscope to confirm the long-term biocompatibility of the material in vivo.

[0113] The establishment of the peripheral nerve injury model and the grouping of animals are as follows:

[0114] 1) Before the operation, the rats were transferred to the operating room and allowed to rest for 30 minutes to confirm that the rats were in good mental condition, had normal limb movement, and no limb deformities or skin damage. The gas anesthesia machine was connected and isoflurane gas was used for anesthesia. After the rats’ corneal reflex and toe pinch pain reflex were completely lost and the depth of anesthesia was confirmed to be up to standard, the rats were fixed in a prone position on a constant temperature operating board, and the limbs and head were fixed with tape. The right hind limb was kept naturally extended to fully expose the surgical area.

[0115] 2) Thoroughly shave the hair from the posterolateral thigh to the buttock of the rat's right hind leg with an electric shaver. Clean the skin debris in the surgical area with a damp gauze. Disinfect the surgical area three times in a spiral motion from the inside out with iodine-soaked cotton balls, covering a 5cm area around the surgical area. Then remove the iodine twice with 75% alcohol cotton balls. Strictly follow the aseptic operation principle throughout the process. After disinfection, cover with a sterile drape, exposing only the skin in the surgical area.

[0116] 3) Make a transverse skin incision of about 2 cm long on the posterolateral aspect of the right hind leg of the rat, along the course of the muscle fibers. Use ophthalmic forceps to bluntly separate the subcutaneous fascia tissue to fully expose the gluteus maximus. Use a retractor to gently pull the gluteus maximus to both sides to fully expose the main trunk of the sciatic nerve. The exposure range extends from the piriformis foramen to the bifurcation of the tibial and common peroneal nerves. Take care to avoid mechanical damage to the nerve trunk by pulling or clamping it.

[0117] 4) At the midpoint of the sciatic nerve trunk, about 5 mm from the piriformis foramen, the proximal cut point is located. The distal cut point is precisely measured 10 mm away from the distal cut point. The nerve is then sharply cut along the marked point using microscopic ophthalmic scissors. A 10 mm long sciatic nerve segment is completely cut to construct a standardized 10 mm rat sciatic nerve defect model. The nerve ends are moistened with physiological saline.

[0118] 5) According to the pre-set 5 experimental protocols, rats were subjected to corresponding interventions, and the epineurium was sutured using 6-0 medical nylon sutures: Group 1: Sciatic nerve defect (SND) group, after 10 mm of sciatic nerve was removed, the proximal and distal ends were rotated 180° respectively, and the epineurium of the severed ends was sutured and fixed to the surrounding myofascia with 6-0 nylon sutures to block the contact and regeneration of the two severed ends and prevent spontaneous nerve anastomosis; Group 2: Autograft group, The 10mm autologous nerve was inverted, and the proximal and distal ends were aligned with the epineurium at both ends. End-to-end epineurium sutures were performed, with 3 sutures evenly placed at each end. Group 3: Simple PLGA@GPP catheter group. A pre-treated PLGA@GPP composite nerve catheter was used. The proximal and distal ends of the nerve were gently inserted into the lumen of the catheter at both ends, with an insertion depth of 1mm, ensuring a precise 10mm gap in the nerve defect within the catheter. The epineurium was then sutured to the catheter port edge with nylon sutures, with 3 sutures placed at each end. Group 4: BMSCs / PLGA@GPP group. The procedure was the same as the catheter group above. After anastomosis and fixation of the catheter and nerve ends, 10ul of cells at a density of 1×10⁻⁶ were injected using a microsyringe. 7 The BMSCs cell suspension was slowly and evenly injected into the catheter lumen to avoid leakage.

[0119] 6) After the intervention was completed, the surgical area was rinsed with saline to remove residual blood and tissue debris; the gluteus maximus muscle layer was intermittently sutured with 4-0 absorbable surgical sutures. After confirming that there was no active bleeding, the skin incision was intermittently sutured with 4-0 medical silk sutures, and the skin around the incision was disinfected again with povidone-iodine. After the rats recovered, they were transferred to their rearing cages.

[0120] The gait analysis is as follows:

[0121] Sciatic nerve injury directly leads to lower limb motor dysfunction in rats, causing characteristic abnormal changes in gait patterns, plantar pressure distribution, and footprint morphology. Using a small animal plantar pressure gait analysis system, plantar pressure distribution and footprint morphology parameters of both hind limbs of rats are collected non-invasively through a high-sensitivity pressure sensing track. The system accurately calculates quantitative indicators such as peak plantar pressure and foot-ground contact area, and objectively evaluates the overall recovery of motor function after sciatic nerve injury in rats.

[0122] Specific experimental procedures: The day before the experiment, rats in each group were placed in the walking channel of the gait analysis system for adaptive walking training, 10 minutes per rat, to ensure that the rats could walk through the walking channel at a constant speed without stopping, so as to eliminate the interference of environmental stress on gait data; before data collection, it was confirmed that the system pressure sensor module was calibrated and that the ambient light and temperature were kept constant; the rats were placed at the beginning of the walking channel and induced to walk in a straight line at a constant speed to the end, and the footprint information was collected by the system and the data was analyzed.

[0123] The wet weight of the gastrocnemius muscle is as follows:

[0124] After sciatic nerve injury, the gastrocnemius muscle, the target organ, undergoes denervation and atrophy due to loss of nerve innervation. Changes in muscle wet weight can directly reflect the degree of muscle atrophy and are the core indicator for evaluating the effect of nerve re-innervation of the target organ after nerve regeneration.

[0125] Experimental methods: Rats were euthanized by over-anesthesia, and the bilateral hind limbs were quickly dissected. The gastrocnemius muscle was completely dissected, and the muscle tissue was quickly rinsed with pre-cooled physiological saline. Excess moisture on the surface was gently absorbed with sterile filter paper. The wet weight of the bilateral gastrocnemius muscles was weighed using an electronic analytical balance and the value was recorded.

[0126] Masson staining of the gastrocnemius muscle is as follows:

[0127] Denervated gastrocnemius muscles are accompanied by muscle fiber atrophy and collagen fiber hyperplasia. Masson trichrome staining can specifically stain muscle fibers red and collagen fibers blue. By quantitatively analyzing the area ratio of collagen fibers, the degree of muscle fibrosis can be accurately evaluated, and further reflect the functional recovery of target organs after nerve repair.

[0128] Experimental Procedure: Gastrocnemius muscle tissue fixed in 4% paraformaldehyde for 24 h was dehydrated with a gradient of alcohols, cleared with xylene, and embedded in paraffin. Continuous paraffin sections of 4 μm thickness were prepared, and three discontinuous sections were taken from each tissue block for staining. Xylene dewaxing was performed twice, 10 min each time. Gradient alcohols (100%, 95%, 80%, 70%) were used for hydration sequentially, 5 min each time, followed by two rinsings with distilled water, 3 min each time. Weigert iron hematoxylin staining was performed for 5 min, followed by rinsing with running water for 10 min to achieve blue reversion. Ponceau S and Acid Fuchsin staining was performed for 8 min, followed by three rapid rinsings with distilled water. Differentiation was performed with phosphomolybdic acid solution for 5 min, without rinsing, and then directly stained with aniline blue for 5 min. Differentiation was performed with 1% glacial acetic acid solution for 1 min. Gradient alcohol dehydration and xylene clearing were performed, followed by mounting with neutral resin. Images were acquired under an optical microscope, and the muscle cross-sectional area was calculated.

[0129] In this experiment, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0130] Depend on Figure 1 The results show that Gel exhibits typical amide characteristic peaks of collagen at 3280 cm⁻¹, 1630 cm⁻¹, and 1526 cm⁻¹, confirming the integrity of its protein backbone structure. GA shows a phenolic hydroxyl characteristic peak at 3280 cm⁻¹ and a benzene ring backbone characteristic peak at 1450 cm⁻¹. The grafted product GGA completely retains the amide characteristic peaks of Gel, while a benzene ring characteristic peak of GA appears at 1450 cm⁻¹. The free carboxyl characteristic peak of GA completely disappears, and the amide peak shape undergoes characteristic changes consistent with the amidation reaction. Infrared spectroscopy results confirm that through the EDC / NHS-catalyzed amidation reaction, the carboxyl group of GA successfully combines with the amino group on the Gel molecular chain to form a new amide bond. GA is successfully grafted onto the Gel molecular chain, the GGA grafted product is successfully synthesized, and the main protein structure of Gel is not destroyed during the reaction.

[0131] Depend on Figure 2 The results show that the GGA spectrum completely retains all the characteristic peaks of the polypeptide backbone of gelatin in the δ 0.5~4.5 ppm range, with no obvious distortion in peak shape and position; at 7.0 ppm, a specific characteristic peak of the aryl hydrogen (Ha) of the GA benzene ring appears; the characteristic peak of the ε-CH2- of the lysine residue at 3.0 ppm in the Gel spectrum shows a significant chemical shift at the corresponding position in the GGA spectrum, and the peak intensity is significantly reduced, proving that the carboxyl group of GA undergoes a specific amidation reaction with the free amino group of the lysine residue in Gel, and the active site is covalently modified. The experiment confirms that the amidation reaction catalyzed by EDC / NHS successfully synthesizes GGA.

[0132] Depend on Figure 3The results show that the mixed precursor solution of GGA and PEG-PLL is a free-flowing liquid. Under the catalysis of TG enzyme, after 20 min of reaction at room temperature, the system undergoes a transformation from a mixed solution to a gel and completely loses its fluidity. After inversion, there is no liquid flow or dripping, and a stable hydrogel is formed, which confirms that GGA and PEG-PLL can be crosslinked under mild conditions.

[0133] Depend on Figure 4 The results show that: 0-3h is the rapid swelling stage, and the swelling rate of both hydrogels increases sharply with time. The initial swelling rate is fast. 3-18h is the swelling plateau period, during which the swelling rate of the hydrogel slows down significantly, and the swelling rate increases slowly. At 18h, both hydrogels reach swelling equilibrium. From 18-24h, the hydrogels swell slowly. After reaching swelling equilibrium, the swelling rate of both hydrogels does not decrease significantly, and they maintain good swelling stability, confirming that the material can maintain the stability of the network structure in physiological environment.

[0134] Depend on Figure 5 The results show that in the early stage, there was no significant difference in the degradation trend of the two groups of hydrogels, both showing a slow decline in quality; in the middle and late stages of degradation, the difference in degradation rate between the two groups of hydrogels gradually became apparent, with the hydrolysis rate of GGA hydrogel accelerating significantly, while GGA-PEG-PLL hydrogel still maintained a slow degradation trend.

[0135] Depend on Figure 6-7 The results showed that the PLGA@GPP nerve conduit had a uniform milky white hollow tubular structure, with an intact appearance and no damage or cracks in the tube wall. It was flexible and could meet the surgical requirements for implantation in rats. The total length of the PLGA@GPP nerve conduit was 12 mm, the outer diameter was 2 mm, and the inner diameter was 1 mm, which matched the physiological diameter of the rat sciatic nerve.

[0136] Depend on Figure 8 The results show that the PLGA scaffold has a disordered, interwoven three-dimensional fiber network structure with uniform diameter of individual fibers and a large number of interconnected micron-level gaps between the fibers.

[0137] Depend on Figure 9 The results show that the GGA-PEG-PLL hydrogel exhibits a three-dimensional interconnected porous network structure with uniform pore distribution and continuous interconnected channels between pores, without any obvious closed-pore structure.

[0138] Depend on Figure 10The results show that the PLGA@GPP nerve conduit has a complete concentric hollow tubular structure. The inner lumen is smooth and continuous, without collapse or cracks, and the inner diameter of the lumen is highly consistent with the design value. The outer layer has a multi-layered convolutional structure. This gradient structure can significantly improve the radial mechanical support performance of the PLGA@GPP nerve conduit, resist the compression of surrounding soft tissues in the body, and avoid lumen occlusion.

[0139] Depend on Figure 11 The results showed that after co-culturing PLGA and PLGA@GPP neural conduits with BMSCs, the viability of CCK-8 cells was as follows: at 0 h after cell seeding, there was no significant difference in cell viability among the BMSCs blank control group, the BMSCs+PLGA@GGA co-culture group, and the BMSCs+PLGA@GPP co-culture group. The initial cell seeding density was consistent across the three groups, and the experimental baseline was uniform. As the co-culture time increased from 24 h to 72 h, the cell viability of all three groups increased significantly in a time-dependent manner, and there was no statistically significant difference in the co-culture results among the groups from 0 to 72 hours (P>0.05). The results indicate that PLGA and PLGA@GPP neural conduits have no significant cytotoxicity and do not affect the normal survival and proliferation of BMSCs, demonstrating excellent in vitro cell compatibility and biosafety.

[0140] Depend on Figure 12 The results showed that after 24 hours of co-culture, a large number of uniformly distributed green fluorescent live cells were observed in the BMSCs alone, BMSCs+PLGA@GGA, and BMSCs+PLGA@GPP groups, with only a very small number of scattered red fluorescent dead cells. No obvious abnormal morphology such as shrinkage or death was observed in any of the three groups. After 72 hours of co-culture, the density of green fluorescent live cells in all three groups was significantly higher than that after 24 hours, and the cells were well-spread. Only a very small number of red fluorescent dead cells were still observed, and there was no mass cell death. Combining the 24-hour and 72-hour co-culture results, it is confirmed that both materials possess excellent long-term cell compatibility and can support the normal proliferation and growth of BMSCs.

[0141] Depend on Figure 13The results showed that: in the Control group, BMSCs exhibited abundant JC-1 red fluorescence but no obvious green fluorescence, indicating that the mitochondrial structure was intact and the membrane potential was relatively stable under normal physiological conditions; after TBHP intervention, the red fluorescence of the TBHP model group cells almost completely disappeared, and the cytoplasm was filled with green fluorescence signals, proving that TBHP induced mitochondrial damage and significantly depolarized and decreased the mitochondrial membrane potential; compared with the TBHP model group, the red fluorescence signal of the TBHP / PLGA group cells did not recover significantly and was still dominated by strong green fluorescence, suggesting that pure PLGA ducts could not alleviate TBHP-induced mitochondrial membrane potential damage; while the red fluorescence signal of the TBHP / PLGA@GPP group cells was significantly increased compared with the TBHP model group and the TBHP / PLGA group, and the intensity of the green fluorescence signal was significantly weakened, indicating that the degree of mitochondrial membrane damage was smaller, confirming that the introduction of GGA-PEG-PLL enabled the PLGA@GPP neural ducts to alleviate mitochondrial damage under oxidative stress.

[0142] Depend on Figure 14 The results showed that after TBHP intervention, a large amount of green fluorescence signal was visible in the cytoplasm of cells in the TBHP model group, and the fluorescence intensity was significantly higher than that in the control group. This confirmed that TBHP induced severe oxidative stress damage in BMSCs, resulting in a large accumulation of intracellular ROS. Furthermore, the green fluorescence intensity of cells in the TBHP / PLGA group was not significantly reduced, indicating that PLGA ducts did not have significant ROS scavenging ability and could not effectively enhance the cells' antioxidant stress capacity. In contrast, the green fluorescence signal intensity of cells in the TBHP / PLGA@GPP group was significantly downregulated compared to both the TBHP model group and the TBHP / PLGA group, with only a small amount of weak scattered fluorescence. The intracellular ROS level was close to that of the control group. This confirmed that the introduction of GGA-PEG-PLL enabled PLGA@GPP neural ducts to have the ability to scavenge ROS.

[0143] Depend on Figure 15 The results showed that: 12 weeks after the sciatic nerve in situ suture in rats, the heart, liver, spleen, lungs, and kidneys of rats were stained with hematoxylin and eosin (HE) to observe histopathological changes; the experimental results showed that: in the sciatic nerve transection group (Control group), the tissue structures of all organs of rats were intact and the morphology was normal, with no obvious pathological changes; compared with the control group, no obvious tissue morphological abnormalities, cell degeneration and necrosis, or inflammatory damage were observed in the heart organs of rats in the PLGA@GPP nerve conduit implantation group.

[0144] Depend on Figure 16-17The results showed that at 12 weeks post-surgery, the sciatic nerve function of rats in each group was assessed using a plantar pressure gait analysis system. The results indicated that in the SND group where nerve defects were not repaired, the affected side of the rats exhibited severely shrunken and deformed paw prints, with only a very small amount of point pressure contact observed at the heel. The forefoot toes were completely curled up, with no effective toe contact points. (3D...) The pressure topography showed only a single, wide, and low heel peak, with no toe-related pressure peaks, indicating that the affected side lacked effective weight-bearing capacity and that sciatic nerve motor function was completely lost. In the PLGA@GPP simple catheter group, the affected side of the rats retained only basic heel weight-bearing capacity, the footprint morphology was abnormal, the toe contact points did not effectively extend, and the 3D pressure map showed only two low pressure peaks with uneven pressure distribution, indicating only limited functional recovery and that the fine nerve innervation function of the toes was not effectively repaired. In the BMSCs / PLGA@GPP group, the integrity of the footprint on the affected side of the rats was significantly improved, the contact between the heel and forefoot became clearer, and the 3D pressure map showed a clear heel peak and the tips of the three toes, indicating that the nerve function recovery was better than that of the simple catheter group. The fine nerve innervation capacity of the toes and the overall weight-bearing function were close to the gold standard group of Autograft autologous nerve transplantation. As the gold standard for clinical repair of peripheral nerve injury, the Autograft autologous nerve transplantation group showed complete footprints on the affected side of the rats, high pressure peaks, and good recovery of basic weight-bearing capacity.

[0145] Depend on Figure 18-21 The results showed that: in the SND group, the volume of the gastrocnemius muscle on the operated side was significantly reduced, the color was pale, and the texture was shriveled, forming a stark contrast with the normal muscle on the healthy side, exhibiting typical denervation atrophy characteristics. The muscle fibers were extremely atrophied, with small diameters, disordered arrangement, widened intermuscular spaces, and a large amount of blue collagen fibers infiltrated, indicating severe fibrosis. In the PLGA@GPP catheter group, the muscle volume on the operated side was significantly larger than that of the unrepaired group, but still smaller than that of the healthy side. The muscle fibers were atrophied and larger in diameter than those in the defect group, with reduced collagen deposition. In the BMSCs / PLGA@GPP group, the muscle volume was further restored, the morphology was closer to normal, the muscle fibers were further thickened, the size tended to be uniform, the arrangement was more regular, and the morphology was close to that of the autologous transplant group.

[0146] In summary, this invention utilizes TG enzyme-catalyzed crosslinking to composite gallic acid-functionalized gelatin (GGA) with polyethylene glycol-polylysine (PEG-PLL), and encapsulates this composite within a polylactic acid-glycolic acid copolymer (PLGA) electrospun sheath, thus constructing a novel composite nerve conduit. In this system, GGA alleviates oxidative stress at the transplantation site through its antioxidant properties; PEG-PLL significantly enhances the mechanical properties of the hydrogel; and the PLGA shell ensures the conduit maintains structural integrity during regeneration. This composite conduit not only serves as a physical channel for nerve regeneration but also continuously maintains the activity and function of bone marrow mesenchymal stem cells, creating a favorable regenerative microenvironment through its paracrine and differentiation-promoting effects, providing a novel and comprehensive solution for the repair of peripheral nerve injuries.

[0147] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0148] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs, characterized in that, Includes the following steps: S1, Synthesize GGA: Gelatin was dissolved in deionized water by stirring to obtain a gelatin aqueous solution. Gallic acid was dissolved in a mixed solution of deionized water and dimethylformamide. After complete dissolution, EDC and NHS were added, and the mixture was stirred at room temperature. The gallic acid reaction solution was slowly added to the gelatin aqueous solution and stirred overnight. The mixture was then placed in a dialysis bag and dialyzed with deionized water for 3-5 days. The dialysate was aliquoted into petri dishes, frozen, and then freeze-dried to obtain GGA powder. S2. Preparation of PEG-PLL aqueous solution: PEG-NH2 was weighed and dissolved in DMF, and PLL monomer was weighed and dissolved in DMF. The mixture was heated and stirred under N2 environment. The product was added dropwise to ice-cold ether, filtered and collected, and dried under vacuum to obtain MPEG5K-PLL7K. PEG-PLL was weighed and completely dissolved in trifluoroacetic acid solvent. An acetic acid solution containing hydrogen bromide was added under ice bath stirring. After the reaction, the crude product was precipitated in ice-cold ether. The crude product was then dissolved in water to adjust the pH value, placed in a dialysis bag and dialyzed. After dialyzing in deionized water, the purified PEG-PLL block copolymer was obtained as a white flocculent solid by freeze drying. S3. Preparation of PLGA electrospun nerve conduit: PLGA polymer is dissolved in hexafluoroisopropanol or trifluoroethanol and continuously stirred magnetically at room temperature until a homogeneous and transparent spinning solution is formed. The spinning solution is injected into an injection pump equipped with a flat-headed metal needle for spinning. Spinning continues until a uniform tubular fiber membrane is deposited on the surface of the columnar mandrel. The tubular fiber membrane is carefully peeled off from the mandrel to obtain a PLGA electrospun nerve conduit with a through lumen. S4. Synthesis of GGA-PEG-PLL hydrogel cannulas and assembly of PLGA@GPP nerve condyloma acuminatum: PEG-PLL and GGA were dissolved in deionized water to obtain a mixed precursor solution containing PEG-PLL and GGA. TG enzyme was added to the mixed precursor solution, and after shaking and mixing, the liquid was injected into the inner layer of the PLGA electrospun nerve conduit. Kirschner wires were then inserted and fixed in the center of the lumen. After cross-linking at room temperature, the Kirschner wires were slowly removed to obtain the PLGA@GPP nerve conduit, which was then sterilely sealed and stored for later use.

2. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 1, characterized in that, In the preparation of the PLGA electrospun nerve conduit, the PLGA has a molecular weight of 10 kDa, a LA:GA molar ratio of 75:25, a viscosity of 1.21 dL / g (CHCl3 / 25℃), and is prepared using trichloroethanol. A 10% (w / v) PLGA electrospun solution is prepared, and the initial inner diameter of the PLGA electrospun nerve conduit is 1.5 mm, with a wall thickness of 0.25 mm.

3. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 2, characterized in that, In the GGA synthesis process, the mass ratio of gallic acid to gelatin is 1:3-1:

2.

4. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 3, characterized in that, During the assembly of the PLGA@GPP nerve conduit, the mass ratio of GGA to PEG-PLL is 2:

1.

5. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 4, characterized in that, The dosage of the TG enzyme is 2-4 U / ml.

6. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 5, characterized in that, In the mixed solution of deionized water and dimethylformamide, the volume ratio of deionized water to dimethylformamide is 3:

2.

7. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 6, characterized in that, When the spinning solution is injected into an injection pump equipped with a flat-headed metal needle for spinning, the applied voltage is 17kV, the collection distance between the needle and the receiving device is 12cm, and the injection rate of the spinning solution is 2.0mL / h.

8. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 7, characterized in that, The receiving device uses a high-speed rotating cylindrical spindle with a rotation speed of 800 r / min.

9. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 8, characterized in that, The mass ratio of EDC to NHS is 1:2 to 1:

1.

10. The method for preparing a PLGA@GPP composite biomimetic nerve conduit loaded with BMSCs according to claim 9, characterized in that, The PLGA@GPP nerve conduit has an inner diameter of 1 mm, an outer diameter of 2.0 mm, and a length of 12 mm.

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