Three-layer nerve conduit based on cell-loaded near-field direct writing printing as well as preparation method and application of three-layer nerve conduit

Abstract

The invention discloses a three-layer nerve conduit based on cell-loaded near-field direct-writing printing and a preparation method and application thereof, and is characterized in that the inner layer of the three-layer nerve conduit is a cell-loaded ALG / GelMA / PEO hydrogel stent, the middle layer is a PVA / GelMA electrostatic spinning membrane, and the outer layer is a fused near-field direct-writing PCL stent; the preparation method comprises the following steps: preparing an ordered fiber membrane PVA / GelMA from a spinning solution through electrostatic spinning, adding the ordered fiber membrane PVA / GelMA into an acetone / glutaraldehyde solution to obtain a PVA / GelMA electrostatic spinning membrane, and printing a direct-writing PCL support on any single surface of the electrostatic spinning membrane through fusion near-field direct-writing. The preparation method comprises the following steps: preparing an ordered cell-loaded hydrogel scaffold from cell-containing bio-ink through cell-loaded near-field direct writing printing, and finally preparing the three-layer nerve conduit, and the three-layer nerve conduit has the advantages of accelerating axon growth, promoting myelin sheath regeneration and improving nerve function recovery.

Description

Technical Field

[0001] The present invention relates to the technical field of medical supplies, and in particular to a three-layer nerve conduit based on cell-carrying near-field direct writing printing, and a preparation method and application thereof. Background Art

[0002] Peripheral nerve injury is a common clinical condition with complex and diverse causes, including metabolic disorders, trauma, and infection. This significantly reduces patients' quality of life and places a heavy medical and economic burden on society. Compared to the central nervous system, neurons in the peripheral nervous system possess a certain ability to regenerate and grow after injury. However, axonal extension is slow and lacks directionality, which restricts the recovery of peripheral nerve function. Autologous nerve transplantation remains the preferred option for repairing large peripheral nerve defects. However, issues such as a shortage of donor nerves, dysfunction of the donor site, mismatched nerve fiber types, potential postoperative nerve distortion or displacement, neuroma formation, and immune rejection severely limit its clinical application. Nerve conduits have emerged as an alternative to autologous nerve transplantation. They can connect the proximal and distal ends of the nerve injury site, providing physical and biochemical guidance for axonal regeneration.

[0003] The directional migration and functional activation of Schwann cells play a critical role in neural regeneration. These cells not only secrete neurotrophic factors but also form oriented Büngner bands to guide axonal extension. However, the limited proliferation capacity of adult Schwann cells has prompted researchers to explore the differentiation potential of mesenchymal stem cells. Adipose-derived stem cells (ADSCs) have attracted considerable attention due to their accessible and multidirectional differentiation potential. However, traditional transplantation systems face bottlenecks such as low cell survival and insufficient directional differentiation efficiency, hindering their clinical application.

[0004] Existing single-layer or double-layer catheters have limitations in mechanical gradient design and cell microenvironment simulation. Modern biomanufacturing technology provides a new approach to the structural optimization of nerve catheters. Electrospinning technology can produce oriented fibers with a diameter of 50-500 nm, which effectively simulates the ordered structure of the neural matrix. Polycaprolactone (PCL) is often used as the outer layer of the stent due to its excellent mechanical properties, but its inherent hydrophobicity affects cell adhesion and requires surface modification to enhance bioactivity. The selection of the inner layer material needs to take into account both cell loading and three-dimensional microenvironment construction. How to achieve high-precision cell encapsulation remains a challenge. The design of the middle layer structure of the catheter needs to balance the coordination of mechanical support and biodegradation, but the interface fusion quality of the multi-layer composite catheter directly affects the overall structural stability. How to achieve seamless integration of functional layers prepared by different processes remains a technical difficulty. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a three-layer nerve conduit based on cell-loaded near-field direct writing printing, which can accelerate axonal growth, promote myelin regeneration and improve nerve function recovery, as well as its preparation method and application, for the treatment of peripheral nerve injury.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a three-layer nerve conduit based on cell-laden near-field direct writing printing, wherein the inner layer of the three-layer nerve conduit is a cell-laden ALG / GelMA / PEO hydrogel scaffold, the middle layer is a PVA / GelMA electrospinning membrane, and the outer layer is a melt near-field direct writing PCL scaffold.

[0007] The present invention also provides a method for preparing the above-mentioned three-layer nerve conduit based on cell-carrying near-field direct writing printing, comprising the following steps: Step 1: Preparation of the middle layer: Powdered polyvinyl alcohol (PVA) and block methacrylated gelatin (GelMA) are added to distilled water and ultrasonically prepared into a spinning solution; the spinning solution is electrospun into an ordered fiber membrane PVA / GelMA; the ordered fiber membrane PVA / GelMA is added to an acetone / glutaraldehyde solution and incubated at room temperature to obtain a cross-linked ordered fiber membrane, namely the PVA / GelMA electrospun membrane; Step 2, outer layer preparation: using molten polycaprolactone (PCL) as the material, a direct writing scaffold is printed on any single side of the PVA / GelMA electrospinning membrane obtained in step 1 by melt near-field direct writing, thereby obtaining a melt near-field direct writing PCL scaffold; Step 3, inner layer preparation: Sodium alginate (ALG), polyethylene oxide (PEO), and GelMA are added to a photoinitiator solution and dissolved to obtain a uniform ALG / GelMA / PEO solution, and cells are mixed into it to obtain a cell-containing bio-ink; the cell-containing bio-ink is prepared into an ordered cell-laden ALG / GelMA / PEO hydrogel scaffold by cell-laden near-field direct writing printing, and printed on the other side of the PVA / GelMA electrospinning membrane obtained in step 1. The ordered cell-laden ALG / GelMA / PEO hydrogel scaffold is photocured and incubated in a CaCl2 solution at room temperature to obtain a cross-linked ordered hydrogel scaffold, namely the cell-laden ALG / GelMA / PEO hydrogel scaffold; Step 4, preparation of three-layer nerve conduit: The three-layer membrane obtained in step 3 was cut into rectangles, rolled up and bonded with a stainless steel stick to prepare a three-layer nerve conduit, named CEDW / ES / MEW.

[0008] Furthermore, in step 1, the mass concentration of PVA in the spinning solution is 8-12%, the mass concentration of GelMA is 2-8%, and the mass concentration of acetone in the acetone / glutaraldehyde solution is 1-3%.

[0009] Furthermore, in step 1, the electrospinning liquid flow rate is 0.2-0.6 mL / h, the spinning voltage is 10-15 kV, the printing speed is 300-1500 mm / min, and the spinning height is 10-15 cm.

[0010] Furthermore, in step 2, the molecular weight of the polycaprolactone is 80,000 Da, the heating temperature is 70-120° C., the feed pressure of the melt near-field direct writing is 0.02-0.1 MPa, the printing speed is 500-2000 mm / min, the direct writing voltage is 2-6 kV, and the distance from the nozzle to the collection plate is 2-5 mm.

[0011] Furthermore, in step 3, the ALG / GelMA / PEO solution has an ALG mass concentration of 0.5-3%, a PEO mass concentration of 0.5-3%, a GelMA mass concentration of 2-10%, and a photoinitiator mass concentration of 0.2-0.8%.

[0012] Furthermore, in step 3, the feed air pressure of the cell-carrying near-field direct writing printing is 0.02-0.1 MPa, the printing speed is 500-2000 mm / min, the direct writing voltage is 2-6 kV, and the distance from the nozzle to the collecting plate is 50-200 μm.

[0013] Furthermore, in step 3, the light curing is carried out under ultraviolet light, and the energy of the ultraviolet light is 15 to 20 mW / cm 2 The cross-linking time of light curing is 20 to 120 seconds; the mass concentration of the CaCl2 solution is 1 to 5%, and the incubation time at room temperature is 1 to 5 minutes.

[0014] Furthermore, in step 4, the product is cut into a rectangle with a length of 6 to 24 mm and a width of 2.5 to 10 mm, and rolled up with a stainless steel rod with a diameter of 0.5 to 2.5 mm.

[0015] The present invention also provides the use of a three-layer nerve conduit prepared by the above preparation method based on cell-loaded near-field direct writing printing in the preparation of a nerve conduit for treating peripheral nerve damage.

[0016] Compared with the prior art, the advantages of the present invention are: 1. The present invention provides a three-layer nerve conduit CEDW / ES / MEW, in which the inner layer adopts the cell-loaded near-field direct writing printing technology to prepare the cell-loaded ALG / GelMA / PEO hydrogel scaffold, the middle layer adopts the electrospinning technology to prepare the PVA / GelMA electrospinning membrane, and the outer layer adopts the melt near-field direct writing technology to prepare the melt near-field direct writing PCL scaffold, realizing the complementary advantages of the multi-layer structure and more effectively simulating the structure and function of natural neural tissue.

[0017] 2. The inner layer of the present invention adopts cell-loaded near-field direct writing printing technology, which can evenly wrap the cells inside the hydrogel scaffold, thereby improving the cell survival rate and biological activity; the middle layer PVA / GelMA electrospinning membrane has good biocompatibility and degradability. By introducing GelMA for blending modification, the cell affinity of the material can be improved and the degradation cycle can be regulated, which can provide a good microenvironment for the growth of nerve cells; the outer layer melt near-field direct writing PCL scaffold has good mechanical properties and can provide structural support and protection for the nerve conduit.

[0018] 3. The preparation method of the three-layer nerve conduit provided by the present invention has a reasonable process flow, adjustable parameters, and is easy to achieve large-scale production, providing an effective tool for the clinical treatment of peripheral nerve injury.

[0019] 4. The three-layer nerve conduit of the present invention has excellent biocompatibility and achieves seamless integration: when the outer layer polycaprolactone material is deposited on the electrospun film, it gradually cools and solidifies from a molten state. The molten PCL fibers penetrate into the gaps of the middle layer fiber network during the electrospinning deposition process. When cooled and solidified, they achieve physical bonding between the outer layer and the middle layer through mechanical intercalation and intermolecular adhesion with the electrospun fibers. The inner layer hydrogel scaffold and the middle layer electrospun membrane both contain GelMA. The same GelMA component enables the two layers of material to form a continuous molecular cross-linked network at the interface, avoiding the weak points of bonding between heterogeneous materials and achieving seamless integrated bonding. The organic combination of the above three processes to construct a composite conduit with an inner layer of cell-laden hydrogel, a middle layer of functional nanofibers, and an outer layer of structured polymer scaffold can produce complementary advantages in mechanical support, biochemical guidance, and cell synergy, providing a new solution for the repair of peripheral nerve defects.

[0020] 5. The three-layer nerve conduit of the present invention was successfully implanted into the 10 mm defect of the sciatic nerve of SD rats, which can accelerate axonal growth, promote myelin regeneration and improve nerve function recovery.

[0021] In summary, the present invention provides a three-layer nerve conduit based on cell-loaded near-field direct writing printing, its preparation method and application. By combining three processes of cell-loaded near-field direct writing printing, electrospinning and melt near-field direct writing, a three-layer nerve conduit CEDW / ES / MEW with specific functions is prepared and applied to the treatment of peripheral nerve injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart for preparing a three-layer nerve conduit based on cell-loaded near-field direct writing printing prepared in Example 1; Figure 2Macroscopic and microscopic images of the three-layer nerve conduit prepared in Example 1 based on cell-loaded near-field direct writing printing, including (a) overall morphology of the composite nerve conduit, (b) cross-sectional morphology of the composite nerve conduit, (c) inner layer Schwann cell-loaded hydrogel scaffold, (d) electron micrograph of the outer layer melt near-field direct writing scaffold, (e) electron micrograph of the middle layer electrospun film, and (f) confocal image of the three-layer composite conduit. Green fluorescence indicates living cells. Figure 3 This is a diagram showing the toxicity test of the electrostatic fiber membrane of the three-layer nerve conduit based on cell-laden near-field direct writing printing in Example 2; Figure 4 This is a diagram showing an in vitro cell activity experiment of a three-layer nerve conduit with a cell-laden hydrogel scaffold based on cell-laden near-field direct writing printing in Example 3; Figure 5 Figure 4 shows the 10 mm sciatic nerve defect of rats after nerve conduits were implanted in different groups. Figure 6 Representative images of the footprints of the rats in each group walking on the normal side (left) and experimental side (right) in Example 4; Figure 7 The figure is a statistical graph of the sciatic nerve function index (SFI) of rats in each group in Example 4, NS indicates no significant difference, * indicates a P value less than 0.05, and ** indicates a P value less than 0.01; Figure 8 The waveforms of compound motor action potentials at the distal (upper) and proximal (lower) ends of the stimulation defect on the experimental side of each group of rats in Example 4 are shown; Figure 9 This is a statistical graph of compound motor action potential amplitudes in Example 4, * indicates a P value less than 0.05, and ** indicates a P value less than 0.01; Figure 10 The statistical graph of compound motor action potential latency in Example 4, NS indicates no significant difference, * indicates a P value less than 0.05, and ** indicates a P value less than 0.01; Figure 11 This is a morphological observation diagram of the gastrocnemius muscle in Example 4, where the left side is the experimental side and the right side is the normal side. Scale bar: 1 cm; Figure 12 Graph showing the weight ratio of the gastrocnemius muscle in Example 4. NS indicates no significant difference, and ** indicates a P value less than 0.01. DETAILED DESCRIPTION

[0023] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0024] The present invention provides a three-layer nerve conduit based on cell-loaded near-field direct writing printing. The three-layer nerve conduit is named CEDW / ES / MEW, that is, a composite nerve conduit consisting of an inner layer of cell-loaded near-field direct writing hydrogel scaffold, a middle layer of electrospun membrane, and an outer layer of melt near-field direct writing scaffold. The preparation method thereof comprises the following steps: Step 1. Preparation of the middle layer: Powdered polyvinyl alcohol (PVA) and block gelatin methacryloyl (GelMA) are added to distilled water, and the materials are dissolved evenly through ultrasonic treatment to obtain a spinning solution for electrospinning; the above spinning solution is prepared into an ordered fiber membrane PVA / GelMA using an electrospinning device; then, the prepared ordered fiber membrane PVA / GelMA is immersed in an acetone / glutaraldehyde solution and incubated at room temperature. After a cross-linking reaction, a cross-linked ordered fiber membrane is obtained, and finally a middle layer PVA / GelMA electrospinning membrane is obtained.

[0025] Step 2, outer layer preparation: Select polycaprolactone (PCL) as the material of the outer layer bracket. After melting it, use near-field direct writing technology to print the direct writing bracket structure on any surface of the cross-linked nanofiber membrane prepared in step 1, thereby obtaining the outer layer melt near-field direct writing PCL bracket.

[0026] Step 3, inner layer preparation: sodium alginate (ALG), polyethylene oxide (PEO) and gelatin methacryloyl (GelMA) are added to a pre-prepared photoinitiator solution, stirred and dissolved to obtain a uniform ALG / GelMA / PEO mixed solution; cells are uniformly mixed with the mixed solution to prepare a cell-containing bio-ink; using cell-laden near-field direct writing printing technology, the above-mentioned cell-containing bio-ink is printed into an ordered cell-laden ALG / GelMA / PEO hydrogel scaffold, and printed on the other side surface of the cross-linked nanofiber membrane obtained in step 1); the printed ordered cell-laden ALG / GelMA / PEO hydrogel scaffold is subjected to UV light curing treatment, and calcium chloride (CaCl2) solution is added to incubate at room temperature, and cross-linking reaction is carried out again to finally obtain a cross-linked ordered hydrogel scaffold, which is the inner layer cell-laden ALG / GelMA / PEO hydrogel scaffold.

[0027] Step 4, preparation of three-layer nerve conduit: The three-layer composite film prepared in step 3 is cut into a rectangular shape, and then a stainless steel rod is used as a curling mold to roll up the cut rectangular film and bond it with a wound adhesive to finally prepare a three-layer nerve conduit CEDW / ES / MEW.

[0028] The above-mentioned PCL (polycaprolactone) and PEI (polyethyleneimine) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); ALG (sodium alginate), PEO (polyethylene oxide), and anhydrous calcium chloride were purchased from Sigma Reagent Company (USA); GelMA (methacrylated gelatin) and photoinitiator were purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. (Suzhou, China); PVA (polyvinyl alcohol) 1799 was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China); and Calcein-AM / PI live / dead cell double staining kit was purchased from Beijing Solebao Technology Co., Ltd. (Beijing, China).

[0029] Example 1: A method for preparing a three-layer nerve conduit based on cell-laden near-field direct writing printing, comprising the following steps: Step 1. Accurately weigh 0.1g of polyvinyl alcohol (PVA) powder and 0.05g of gelatin methacryloyl (GelMA) blocks and add them to 10mL of distilled water. Ultrasonicate the solution for 60 minutes to ensure thorough dispersion and dissolution of the materials, thereby obtaining a uniform electrospinning solution. An electrospinning apparatus was used to prepare an ordered PVA / GelMA fibrous membrane. The electrospinning process parameters were set as follows: a liquid flow rate of 0.4mL / h, an applied voltage of 12kV, a distance of 12cm between the printhead tip and the receiving plate, and a printing speed of 800mm / min. Spinning was continued for a specified time to produce an ordered PVA / GelMA fibrous membrane of the desired thickness. The prepared ordered PVA / GelMA membrane was immersed in a mixed solution of acetone and glutaraldehyde (1% acetone concentration) and incubated at room temperature for 8 hours to induce crosslinking. This resulted in a crosslinked ordered fibrous membrane, the middle-layer PVA / GelMA electrospun membrane, which was set aside.

[0030] Step 2: Heat polycaprolactone (PCL) particles to 90°C to melt them, producing a molten PCL material. A melt near-field direct writing device was used to prepare the outer layer scaffold. The melt near-field direct writing parameters were set as follows: feed pressure 0.06 MPa, print speed 1000 mm / min, direct writing voltage 4 kV, and a distance of 3 mm between the nozzle and the collecting plate. The molten PCL material was printed using near-field direct writing onto a single surface of the cross-linked nanofiber membrane prepared above, producing a melt near-field direct writing PCL scaffold, which served as the outer layer scaffold.

[0031] Step 3: Accurately weigh 0.015g of sodium alginate (ALG), 0.015g of polyethylene oxide (PEO), and 0.06g of gelatin methacryloyl (GelMA), and add them to the pre-prepared solution containing 0.5wt% of photoinitiator. Stir thoroughly to dissolve them evenly to prepare an ALG / GelMA / PEO mixed solution. Neural stem cells (NSCs) were cultured at a rate of 5×10 6The concentration of cells / mL was added to the above mixed solution, mixed evenly, and prepared into cell-containing bio-ink. The inner layer of cell-loaded hydrogel scaffold was prepared using a cell-loaded near-field direct writing printing device. The cell-loaded near-field direct writing printing parameters were set as follows: feed air pressure 0.06MPa, printing speed 1000mm / min, direct writing voltage 4kV, and distance from nozzle to collection plate 100μm. The cell-containing bio-ink was printed on the other side surface of the cross-linked nanofiber membrane prepared in step

[0052] . After printing was completed, light curing was immediately performed. When the ultraviolet light energy was 18mW / cm 2 The cells were then irradiated under a UV lamp for 60 seconds for photocrosslinking. Subsequently, a 1% calcium chloride (CaCl2) solution was added and incubated at room temperature for 5 minutes for ionic crosslinking, resulting in a crosslinked, ordered hydrogel scaffold, the inner layer of which was the cell-laden ALG / GelMA / PEO hydrogel scaffold.

[0032] Step 4: Cut the prepared three-layer composite film into rectangles measuring 15 mm x 5 mm. A 1.5 mm diameter stainless steel rod was used as a rolling die. The cut rectangular film was wound around the rod and the edges were bonded with medical wound adhesive to form a tubular structure. This resulted in a three-layer nerve conduit, designated CEDW / ES / MEW.

[0033] like Figure 1 As shown in the figure, during the preparation of a three-layer nerve conduit made of CEDW / ES / MEW, the parameters of each step can be adjusted according to specific needs, such as material concentration, spinning parameters, printing parameters, cross-linking conditions, and conduit dimensions. By optimizing these parameters, three-layer nerve conduits can be prepared to meet different application requirements.

[0034] like Figure 2 (a) Overall morphology of the composite nerve conduit. Figure 2 Middle (b) Cross-sectional morphology of the composite nerve conduit. Figure 2 Middle (c) inner layer of hydrogel scaffold loaded with Schwann cells, Figure 2 Middle (d) Electron microscope image of the outer layer molten near-field direct writing stent, Figure 2 (e) Electron microscopy image of the middle layer electrospun film. Figure 2 (f) The confocal image of the three-layer composite conduit shows the fabricated three-layer CEDW / ES / MEW nerve conduit. The inner layer is a cell-laden ALG / GelMA / PEO hydrogel scaffold for cell loading and delivery; the middle layer is a PVA / GelMA electrospun membrane, providing structural support and guiding nerve regeneration; and the outer layer is a melt-near-field direct-written PCL scaffold, providing additional mechanical strength and protection. These three layers work synergistically to promote the repair of peripheral nerve injuries.

[0035] Example 2: Toxicity test of electrostatic fiber membrane of three-layer nerve conduit based on cell-laden near-field direct writing printing.

[0036] Take the PVA / GelMA electrospun fiber membrane prepared in step 1 of Example 1, cut the obtained fiber membrane into 24mm×24mm rectangular sheets, perform liquid crosslinking treatment, and then immerse it in sufficient deionized water for more than 12 hours to remove residual glutaraldehyde crosslinking agent.

[0037] This experiment involved glycine-treated and untreated groups, with fiber membrane samples prepared in each group. One day before cell inoculation, one group immersed each sample in 5 mL of a 5% glycine solution overnight to fully block any remaining aldehyde groups. Prior to the experiment, the glycine solution was removed and the membranes were disinfected by soaking in 75% ethanol for one hour. The membranes were then sterilized in a UV sterilizer for at least two hours. The other group underwent only sterilization.

[0038] During the experiment, the two sterilized fiber membranes were transferred to 12-well plates, rinsed with PBS, and flattened to adhere to the bottom of the wells. 1 mL of culture medium was pre-added to each well. Rat pheochromocytoma PC12 cells were seeded at a cell density of 150,000 cells / mL, and 1 mL of cell suspension was added to each well (approximately 150,000 cells per well). The cells were incubated at 37°C in a 5% CO2 incubator, with the culture medium replaced regularly. On the third day of culture, each sample was stained with Calcein-AM / PI live-dead staining. Three replicates were performed for each experiment. After staining, the cells were observed using an inverted biological microscope (Nikon Eclipse Ti-S). Green fluorescence indicated live cells, and red fluorescence indicated dead cells. The live and dead cell channels were superimposed to create a merged image.

[0039] like Figure 3 As shown in the figure, by comparing the cell survival of the glycine-treated group and the untreated group, the glycine-treated group had more green fluorescence and almost no red fluorescence, indicating that there were more living cells and basically no dead cells, indicating that the glycine treatment step can effectively reduce the cytotoxicity that may be caused by glutaraldehyde cross-linking and improve the biocompatibility of PVA / GelMA fiber membrane.

[0040] Example 3: In vitro cell activity experiment of a three-layer nerve conduit cell-laden hydrogel scaffold based on cell-laden near-field direct writing printing; To fully evaluate the effectiveness and biocompatibility of the cell-loaded near-field direct writing printing technology used in the present invention in cell printing applications, this example used Schwann cells (SCs) to construct a 3D printed structure and systematically evaluated the survival rate of the cells after printing.

[0041] 1.0% (w / v) sodium alginate, 5.0% (w / v) gelatin methacryloyl and 1.0% (w / v) polyethylene oxide were prepared in PBS, and 0.5% (w / v) photoinitiator was added and ultrasonically dispersed to obtain a uniform solution. Subsequently, enzyme digestion and counting were adjusted to 5×10 6 cells / mL Schwann cell suspension was gently mixed in to prepare cell-containing printing ink.

[0042] At 25°C, CEDW printing was performed using a 160 μm inner diameter needle, 0.04 MPa pressure, 1.0 kV voltage, and a 100 μm gap between the needle and the base plate. Ten layers of grid-like supports were constructed by stacking them up 15 μm each time. After printing, the scaffold was first illuminated with 400–500 nm visible light (15 mW / cm 2 ) for 30 s for initial solidification, then immersed in 1 wt% CaCl2 solution for solidification for 5 min, and finally washed three times with TBS buffer and cultured in DMEM medium containing 10% fetal bovine serum.

[0043] Live / dead cell staining was performed using a Calcein-AM / PI kit on days 1, 4, and 7 of culture. Green (live cells) and red (dead cells) fluorescence images were acquired using a laser confocal microscope. Live and dead cell counts were counted in each field of view using ImageJ, and survival rates were calculated. Three replicates were performed at each time point to ensure data reliability.

[0044] The fluorescence images at each time point showed regular grid pores, which were consistent with the designed size. Figure 4 As shown, the survival rate was approximately 80% after staining (day 1), increasing to approximately 88% by day 4 and reaching approximately 92% by day 7. Concurrently, the green signal intensity and cell spreading range increased, while the red signal significantly decreased, indicating that SCs maintained high activity and continued proliferation within the scaffold. This example demonstrates that the selected CEDW parameters and two-step cross-linking protocol achieve high-fidelity microstructure fabrication while maximizing cell viability and exhibiting excellent biocompatibility.

[0045] Example 4: Animal experiment on a three-layer nerve conduit based on cell-laden near-field direct writing printing.

[0046] 1. Experimental setup: The catheter had an inner diameter of 1.5 mm, an outer diameter of 2.0 mm, and a length of 12 mm. This three-layer catheter was used to bridge a 10 mm sciatic nerve gap in SD rats. The experiment was divided into four groups: autologous nerve transplantation group (Autograft group), ES / MEW group (implantation of a double-layer catheter containing only the middle electrospun membrane and the outer melt near-field direct writing scaffold), EDW / ES / MEW (cell-free group) (implantation of a complete three-layer catheter, but without cells in the inner hydrogel scaffold), and CEDW / ES / MEW (Schwann cell-containing group) (implantation of a complete three-layer cell-loaded nerve conduit with Schwann cells in the inner hydrogel scaffold).

[0047] 2. Experimental method: 40 SD male rats were selected and randomly divided into four groups, with 10 rats in each group. The rats were fasted for 12 hours before the operation but not water, and anesthetized with 3% sodium pentobarbital solution (30 mg / kg) by intraperitoneal injection. The skin of the right hind limb was prepared and disinfected, and a sterile surgical sheet was laid. Under a stereomicroscope, the 8mm sciatic nerve in the middle of the right thigh of the rat was removed (near the obturator tendon), and a 10mm defect was formed after the nerve was retracted. Each group used the corresponding nerve conduit for bridging suture, using 11 / 0 absorbable nylon suture. The autologous nerve transplantation group reversed the severed nerve 180° and sutured it end to end. The muscle and skin were sutured layer by layer with 5 / 0 absorbable sutures. Penicillin was injected intramuscularly seven days after the operation to prevent infection and ensure the smooth progress of the experiment.

[0048] Gait analysis was performed 16 weeks after nerve conduit implantation to assess motor function recovery. A treadmill was set up, and rats were acclimated and fasted for 12 hours before testing. The rats were guided to run across the treadmill, and their movement trajectories were recorded. The sciatic nerve function index (SFI) was measured and calculated using the following formula: The sciatic nerve function index (SFI) = -38.3 × (EPL - NPL) / NPL + 109.5 × (ETS - NTS) / NTS + 13.3 × (EIT - NIT) / NIT - 8.8, where N is the normal side (left leg) and E is the experimental side (right leg); PL is the footprint length, IT is the distance between toes 2 and 4, and TS is the distance between toes 1 and 5. The SFI ranges from 0 to -100, with higher values ​​indicating better function and lower values ​​indicating worse function. Each rat was assessed at least three times.

[0049] After motor function assessment, rats were anesthetized with sodium pentobarbital (30 mg / kg), and the sciatic nerve was exposed for electrophysiological testing. A recording electrode was inserted into the gastrocnemius muscle belly, and a ground electrode was inserted into the tail. The distal and proximal sciatic nerve trunks were stimulated sequentially (0.5 mA, 1 Hz) through the nerve guide, and the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were recorded. The test was repeated six times per group.

[0050] Sixteen weeks after surgery, the gastrocnemius muscles of the experimental side (GM(E)) and the normal side (GM(N)) of the rats were weighed. Gastrocnemius muscle atrophy was compared between the groups, and the gastrocnemius muscle wet weight ratio (GMRatio) was calculated. Gastrocnemius muscle weight ratio (%) = (GM(E) / GM(N)) × 100%.

[0051] 3. Analysis of experimental results like Figure 5 As shown, to verify the effect of CEDW / ES / MEW three-layer nerve conduits in promoting peripheral nerve regeneration in vivo, ES / MEW, EDW / ES / MEW (cell-free) and CEDW / ES / MEW (containing Schwann cells) nerve conduits were implanted into 10 mm sciatic nerve defects in SD rats.

[0052] like Figure 6 As shown, the footprints of ES / MEW and EDW / ES / MEW (cell-free) displayed typical features of sciatic nerve dysfunction, including severe foot contracture, inadequate toe extension, and abnormally extended heel-toe spacing, suggesting incomplete reinnervation. In contrast, the footprints of CEDW / ES / MEW (containing Schwann cells) were relatively uniform and well-defined, with significantly improved toe extension, indicating significantly better motor function recovery than the first two groups.

[0053] Figure 7 Gait analysis images and the sciatic nerve function index (SFI) are shown 16 weeks after surgery to assess functional recovery of nerve deficits. A more negative SFI value indicates more severe functional impairment, while values ​​close to zero indicate normal functional recovery. The SFI in rats in the autologous nerve transplantation group and the CEDW / ES / MEW (Schwann cell-containing) group was significantly better than that in the ES / MEW and EDW / ES / MEW (cell-free) groups.

[0054] Figure 6 and Figure 7 The results showed that the SFI of rats in the autologous nerve transplantation group and the CEDW / ES / MEW (Schwann cell-containing group) was superior to that in the ES / MEW group and the EDW / ES / MEW (cell-free) group. Peripheral nerves serve as information bridges connecting the central nervous system and sensory / motor endings, enabling bidirectional transmission of bioelectrical signals.

[0055] To evaluate the reinnervation of the gastrocnemius muscle distal to the injured nerve, rats underwent electrophysiological testing of compound motor action potentials and latency. Increased CMAP amplitude indicated increased density of regenerated nerve fibers and improved nerve function.

[0056] like Figure 8As shown, both the autologous transplantation group and the CEDW / ES / MEW (Schwann cell-containing group) recorded strong, rapid compound muscle action potentials with regular waveforms close to normal physiological states, indicating that nerve conduction function has been effectively restored and the neuromuscular junction has been well reconstructed. In contrast, although some potential signals were detected in the EDW / ES / MEW (cell-free group), the signal intensity was significantly weakened, suggesting that functional recovery was still incomplete. The ES / MEW group showed almost no effective electrical signal response, with only extremely weak background noise being recorded, confirming that its functional repair effect was minimal and nerve conduction function was essentially lost.

[0057] like Figure 9 As shown, except for the autologous nerve transplantation group, the CMAP amplitude of CEDW / ES / MEW (containing Schwann cells group) (9.80±2.65 mV) was higher than that of ES / MEW group (1.87±0.33 mV) and EDW / ES / MEW (no cell group) (5.50±1.27 mV).

[0058] like Figure 10 As shown in the data, the latency of CEDW / ES / MEW (containing Schwann cells) (4.99±1.09 ms) was also significantly lower than that of ES / MEW group (11.39±2.59 ms) and EDW / ES / MEW (cell-free group) (7.34±0.87 ms), and was close to the level of autologous nerve transplantation group (5.22±0.66 ms).

[0059] like Figure 11 As shown, the bilateral gastrocnemius muscles of each group of rats were isolated, photographed, and weighed. The gastrocnemius muscle photos showed that the right side was healthy, while the left side had varying degrees of atrophy after modeling.

[0060] Figure 12 The gastrocnemius muscle wet weight ratio showed that the wet weight ratios of the experimental side and the healthy side in the CEDW / ES / MEW (containing Schwann cells group) and autologous nerve transplantation group were 49.42±9.65% and 61.53±4.64%, respectively, which were significantly higher than those in the ES / MEW group (23.79±8.67)% and EDW / ES / MEW (cell-free group) (37.70±4.83)%.

[0061] In conclusion, CEDW / ES / MEW (containing Schwann cells) can increase action potential amplitude and accelerate nerve conduction, thus promoting the recovery of nerve function.

[0062] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A three-layer nerve conduit based on cell-laden near-field direct writing printing, characterized by: The inner layer of the three-layer nerve conduit is a cell-laden ALG / GelMA / PEO hydrogel scaffold, the middle layer is a PVA / GelMA electrospinning membrane, and the outer layer is a melt near-field direct writing PCL scaffold.

2. A method for preparing a three-layer nerve conduit based on cell-carrying near-field direct writing printing according to claim 1, characterized in that The following steps are involved: Step 1, middle layer preparation: adding powdered polyvinyl alcohol and block methacrylated gelatin into distilled water and ultrasonically obtaining a spinning solution; The spinning solution is electrospun to prepare an ordered fiber membrane PVA / GelMA, and the ordered fiber membrane PVA / GelMA is added to an acetone / glutaraldehyde solution and incubated at room temperature to obtain a cross-linked ordered fiber membrane, namely the PVA / GelMA electrospun membrane; Step 2, outer layer preparation: using molten polycaprolactone as the material, a direct writing scaffold is printed on either side of the PVA / GelMA electrospinning membrane obtained in step 1 by melt near-field direct writing, thereby obtaining a melt near-field direct writing PCL scaffold; Step 3: Preparation of the inner layer: Sodium alginate, polyethylene oxide, and GelMA are added to the photoinitiator solution and dissolved to obtain a uniform ALG / GelMA / PEO solution, into which cells are mixed to obtain a cell-containing bio-ink; The cell-containing bio-ink was prepared into an ordered cell-laden ALG / GelMA / PEO hydrogel scaffold by cell-laden near-field direct writing printing, and printed on the other side of the PVA / GelMA electrospinning membrane obtained in step 1. The ordered cell-laden ALG / GelMA / PEO hydrogel scaffold was photocured and incubated in CaCl2 solution at room temperature to obtain a cross-linked ordered hydrogel scaffold, namely the cell-laden ALG / GelMA / PEO hydrogel scaffold; Step 4, preparation of three-layer nerve conduit: The three-layer membrane obtained in step 3 was cut into rectangles, rolled up and bonded with a stainless steel stick to prepare a three-layer nerve conduit, named CEDW / ES / MEW.

3. The preparation method according to claim 2, wherein: In step 1, the mass concentration of PVA in the spinning solution is 8-12%, the mass concentration of GelMA is 2-8%, and the mass concentration of acetone in the acetone / glutaraldehyde solution is 1-3%.

4. The preparation method according to claim 2, wherein: In step 1, the electrospinning liquid flow rate is 0.2-0.6 mL / h, the spinning voltage is 10-15 kV, the printing speed is 300-1500 mm / min, and the spinning height is 10-15 cm.

5. The preparation method according to claim 2, wherein: In step 2, the molecular weight of the polycaprolactone is 80,000 Da, the heating temperature is 70-120° C., the feed pressure of the melt near-field direct writing is 0.02-0.1 MPa, the printing speed is 500-2000 mm / min, the direct writing voltage is 2-6 kV, and the distance from the nozzle to the collection plate is 2-5 mm.

6. The preparation method according to claim 2, wherein: In step 3, the ALG / GelMA / PEO solution has an ALG mass concentration of 0.5-3%, a PEO mass concentration of 0.5-3%, a GelMA mass concentration of 2-10%, and a photoinitiator mass concentration of 0.2-0.8%.

7. The preparation method according to claim 2, characterized in that: In step 3, the feed gas pressure of the cell-carrying near-field direct writing printing is 0.02-0.1 MPa, the printing speed is 500-2000 mm / min, the direct writing voltage is 2-6 kV, and the distance from the nozzle to the collecting plate is 50-200 μm.

8. The preparation method according to claim 2, wherein: In step 3, the light curing is carried out under ultraviolet light, and the energy of the ultraviolet light is 15 to 20 mW / cm 2 The cross-linking time of light curing is 20 to 120 seconds; the mass concentration of the CaCl2 solution is 1 to 5%, and the incubation time at room temperature is 1 to 5 minutes.

9. The preparation method according to claim 2, wherein: In step 4, the film is cut into rectangles with a length of 6 to 24 mm and a width of 2.5 to 10 mm, and rolled up using a stainless steel rod with a diameter of 0.5 to 2.5 mm.

10. Use of a three-layer nerve conduit based on cell-loaded near-field direct writing printing prepared by the preparation method according to any one of claims 2 to 8 in preparing a nerve conduit for treating peripheral nerve injury.

Images