Sericin-akermanite composite nerve conduit and manufacturing method and application thereof

Abstract

The invention relates to a sericin-akermanite composite nerve conduit. A preparation method of the sericin-akermanite composite nerve conduit comprises the following steps: dissolving silkworm cocoons with a lithium bromide solution to obtain a sericin solution; the sericin solution and the Tris-Hcl aqueous solution are mixed and added into a dialysis bag; adding the dialysis bag into the dialysate for dialysis; centrifuging, filtering and collecting the dialyzed sericin solution; adding the sericin solution into a polyethylene glycol supersaturated solution, and concentrating; adding akermanite into the genipin solution to prepare a cross-linking solution; mixing the sericin solution and the cross-linking solution, upside down and uniformly mixing, and injecting into a mold; and incubating to obtain the sericin hydrogel. And freezing the sericin hydrogel, demolding and freeze-drying to obtain the sericin-akermanite composite nerve conduit. According to the present invention, the cell compatibility, the degradability and the immunogenicity are good; proliferation and migration of nerve regeneration key cells-Schwann cells and secretion of various nutritional factors can be promoted; and the structural regeneration and functional recovery of long-distance peripheral nerve dissection are promoted.

Description

Technical Field

[0001] This invention relates to a sericin-magnesium feldspar composite nerve conduit for repairing peripheral nerve transection injuries, its manufacturing method, and its application. Background Technology

[0002] Peripheral nerve injury (PNI) is commonly caused by traumatic injuries such as traffic accidents, workplace injuries, natural disasters, surgical trauma, or war injuries, with over 5 million new cases each year. PNI causes loss of sensory and motor function at the site of injury, seriously endangering the patient's life and health. Autologous nerve transplantation is the "gold standard" of clinical treatment, but it is severely limited by problems such as difficulty in nerve matching and secondary damage to the donor site. Nerve conduits are currently the main research direction for the treatment of PNI and hold promise as an ideal alternative to autologous nerve transplantation.

[0003] Peripheral nerve regeneration depends on the proliferation, migration, and cytokine secretion of endogenous Schwann cells to construct a pro-regenerative microenvironment. Therefore, intraductal delivery of Schwann cells or cytokines (such as NGF, BDNF, and VEGF) has become a common pro-regenerative strategy. While these methods can replace the role of endogenous Schwann cells to some extent, the limited availability and poor survival rate of exogenous Schwann cells, coupled with the limited functionality, short half-life, and poor stability of cytokines, lead to insufficient nerve function recovery. Therefore, developing a long-acting and stable repair strategy to reshape the function of endogenous Schwann cells and rebuild the pro-regenerative microenvironment holds promise for achieving efficient regeneration of peripheral nerve infarction (PNI).

[0004] The important role of bioactive ions in nerve tissue regeneration and repair is becoming increasingly prominent. Studies have shown that magnesium ions can promote the secretion of neurotrophic factors from Schwann cells and repair peripheral nerve compression injuries; calcium ions can promote Schwann cell migration and maintain the function of various nerve cells. Therefore, combining the effects of multiple bioactive ions can be a new method to reshape endogenous Schwann cell function and promote nerve regeneration. Magnesia feldspar is a bioceramic material rich in magnesium, silicon, and calcium, with advantages such as wide availability, good degradability, and high stability. It has already demonstrated good repair effects in bone tissue regeneration and repair, but its role in nerve regeneration and repair has not yet been explored.

[0005] Sericin, a major component of silkworm cocoons, possesses excellent biocompatibility, biodegradability, and low immunogenicity, and has been extensively studied in fields such as skin repair and bone tissue repair. Previous research by the applicant has revealed that sericin also exhibits neurotrophic and neuroprotective functions, promoting neuronal adhesion and enhancing axonal elongation, making it an ideal matrix material for ductal construction.

[0006] Based on the above, the applicant prepared a composite nerve conduit by mixing magnesium feldspar with sericin to repair PNI damage. This method can promote cell proliferation, migration, and secretion of trophic factors, remodel endogenous Schwann cell regeneration function, accelerate the construction of axons and Bünger bands, and achieve effective restoration of neural structure and function. This method has advantages such as abundant material sources, simple preparation method, good biocompatibility, and stable regeneration effect, and has important application value and translational prospects in the field of PNI repair. Summary of the Invention

[0007] To achieve effective structural and functional recovery of peripheral nerve injuries, this invention provides a method for preparing nerve conduits that remodels endogenous Schwann cell regeneration activity. Specifically, a novel conduit mold is designed, and a mixture of magnesium feldspar and sericin solution is injected into the mold to prepare a sericin-magnesium feldspar composite nerve conduit, providing a new conduit repair method for peripheral nerve regeneration.

[0008] The technical solution of this invention is as follows: A method for manufacturing a sericin-magnesia feldspar composite nerve conduit, comprising the following steps: 1) Dissolve the genetically mutated silkworm cocoons in a lithium bromide solution to obtain a sericin solution; 2) Add the sericin solution to a Tris-HCl aqueous solution with pH=9, stir and mix well, and then add it to a dialysis bag to obtain a dialysis bag containing the sericin solution; 3) Add 2 mL of Tris-HCl to every 2 L of ultrapure water to obtain the dialysis solution; 4) Add the dialysis bag from step 2) to the dialysis solution, stir at room temperature for 24 hours, replace with new dialysis solution every 2 hours, and perform dialysis with ultrapure water for the last time; 5) Centrifuge the dialyzed sericin solution at 3000 rpm for 10 minutes, discard the upper insoluble matter, filter it through a 70-micron pore size cell sieve, collect the sericin solution and put it into a dialysis bag; 6) Dissolve polyethylene glycol 6000 in ultrapure water to prepare a supersaturated solution, add the dialysis bag from step 5) into it, stir and concentrate at room temperature to obtain a concentrated sericin solution; 7) Dissolve genipin in ultrapure water to prepare a genipin solution of 10 mg / mL. Add magnesia feldspar powder to the genipin solution at a ratio of 70-140 mg: 1 ml, mix by inverting the mixture to obtain a crosslinking solution. 8) Mix the concentrated sericin solution from step 6) and the crosslinking solution from step 7) at a volume ratio of 6:1, invert the mixture to mix thoroughly, and inject it into the mold using a syringe. 9) Under humid conditions, incubation at 37 ℃ for 24 hours yielded a deep blue sericin-magnesia feldspar hydrogel; 10) Place the hydrogel conduit in a -20 ℃ freezer for 5 hours, remove it from the mold, freeze dry it, and obtain the sericin-magnesium feldspar composite nerve conduit.

[0009] The lithium bromide solution was prepared by dissolving 33.55 g of lithium bromide in 40 mL of ultrapure water.

[0010] The sericin solution in step 1) above is obtained by cutting 1 g of silkworm cocoon into small pieces with scissors, adding it to a lithium bromide solution, stirring and mixing, and then dissolving it in a water bath at 38 ℃ for 48 hours.

[0011] Furthermore, the present invention provides a sericin-magnesium feldspar composite nerve conduit prepared by the above-described manufacturing method.

[0012] Furthermore, the present invention also provides the application of the aforementioned sericin-magnesium feldspar composite nerve conduit for repairing peripheral nerve transection injuries. The present invention has the following advantages and effects:

[0013] 1. This invention, from the perspective of the influence of inorganic elements on cell regeneration behavior, first discovered that magnesium feldspar can promote the proliferation of Schwann cells, a key cell in nerve regeneration, as well as the secretion of various nutritional factors and cell migration, and elucidated the molecular mechanism affecting this remodeling function—the activation of the PI3K / AKT and MAPK signaling pathways. 2. The sericin-magnesium feldspar nerve conduit prepared by this invention has good cell compatibility and biodegradability; 3. The sericin-magnesium feldspar nerve conduit prepared in this invention can accelerate the extension speed of nerve axons and promote the formation of Bing-Bünger's band after in vivo transplantation for the treatment of long-distance peripheral nerve transection injuries. 4. This invention can promote tissue regeneration in cases of long-distance peripheral nerve transection injuries; 5. This invention can promote the recovery of motor function and nerve conduction function in cases of long-distance peripheral nerve transection injuries. Attached Figure Description

[0014] Figure 1The images show the biological activity assays for argentite. (AB) High-resolution TEM results and elemental composition analysis of Ca, Mg, and Si from argentite. (CD) Cell viability was assessed by culturing Schwann cells with argentite extract (AKTDEs) for 7 days, followed by live / dead staining and CCK-8 assays. (E) Cell proliferation was assessed using the CCK-8 assay after culturing Schwann cells with AKTDEs. (F) qPCR was used to detect the expression of multiple nutrient factor genes after culturing Schwann cells with AKTDEs at different dilutions. (GH) Cell migration was assessed using the Trans-well assay after culturing Schwann cells with AKTDEs at different dilutions. (IJ) Cell migration was assessed using the scratch assay after culturing Schwann cells with AKTDEs at different dilutions. (K) Western blot analysis was used to detect the activation of the PI3K / AKT and MAPK signaling pathways.

[0015] Figure 2 This is a design drawing of the catheter mold for the present invention. Figure 2 The parameters of the catheter male mold are displayed in the image, showing the front view, top view, side view, and 3D model in sequence.

[0016] Figure 3 This is a schematic diagram illustrating the catheter preparation process and in vivo repair of the present invention.

[0017] Figure 4 This diagram shows the regeneration-promoting activity of different concentrations of magnesia feldspar-serice ducts according to the present invention. (A) shows the Transwell experimental results of different concentrations of magnesia feldspar-serice ducts. (B) is a statistical graph of (A). (C) shows the detection results of different concentrations of magnesia feldspar-serice ducts promoting axonal elongation. (D) is a statistical graph of (C).

[0018] Figure 5 Characterization of the sericin-magnesia feldspar composite conduit of the present invention, where SC is a sericin conduit and ASC is a sericin-magnesia feldspar composite conduit. (A) Macroscopic view of the conduit: Figures 1-3 are cross-sectional views of the conduit after 1000 compressions, with the dashed line representing the outer diameter before compression. (B) SEM results of the conduit. (CD) Results of water absorption swelling rate and porosity of the conduit. (E) Results of FTIR test of the conduit. (FG) Experimental diagram and results of elastic modulus test of the conduit. (HI) Experimental diagram and results of structural compression hardness test of the conduit.

[0019] Figure 6This invention's composite catheter promotes axonal extension and Bünger band regeneration. (A) Timeline of catheter in vivo transplantation and histological and functional testing. (BD) Ten days after catheter in vivo transplantation, Tuj-1 immunofluorescence staining was used to detect axonal extension; Figure C shows the statistical results of axonal extension distance, and Figure D shows the statistical results of intra-catheter fluorescence ratio. (EF) S100 immunofluorescence staining was used to detect Schwann cells; fluorescence showed that the cells exhibited cord-like Bünger band structures; Figure F shows the fluorescence intensity of Schwann cells in different regions.

[0020] Figure 7 This invention demonstrates the promotion of axonal and Schwann cell regeneration 14 weeks after composite catheter transplantation. (A) Macroscopic view of the regenerated nerve. Autograft shows the location of fluorescent staining of the regenerated nerve in autograft transplantation. (B) Schematic diagram of the regenerated nerve fluorescence staining location. (CE) Staining results and statistical graphs of axons and Schwann cells at the proximal end of the regenerated nerve. (FH) Staining results and statistical graphs of axons and Schwann cells at the distal end of the regenerated nerve. (IK) Staining results and statistical graphs of axons and Schwann cells longitudinally along the middle segment of the regenerated nerve.

[0021] Figure 8 This invention utilizes a composite nerve conduit to promote the recovery of nerve function in rats. (AC) Behavioral evaluation 14 weeks after conduit transplantation. Figure A is a schematic diagram, Figure B shows characteristic footprints, and Figure C shows the sciatic nerve index (SFI) test results. (DF) Electrophysiological function test results of regenerated nerves. Figure D is a schematic diagram, Figure E shows the statistical results of nerve conduction time, and Figure F shows the test results of nerve conduction velocity (NCV). (GI) Masson staining results of gastrocnemius muscle (G), Figure H shows the statistical results of gastrocnemius muscle fiber diameter, and Figure I shows the statistical results of collagen fiber proportion. Detailed Implementation

[0022] To enable a better understanding of the present invention, the principles and features of the present invention are described below with reference to examples and accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] The present invention provides a method for preparing a neural conduit that reshapes the endogenous Schwann cell regeneration activity, comprising: Step 1: Conduit mold preparation: The conduit is designed using modeling software, and a positive mold is prepared using a 3D printer; a casting is performed using polydimethylsiloxane (PDMS); a 36 mm long and 1.5 mm diameter iron rod is added to the center of the positive mold to prepare the conduit mold. Step 2: Combining sericin and magnesium feldspar to construct a composite neural conduit, the preparation steps of which are as follows: weighing... 140NDsAfter washing and cutting the genetically mutated silkworm cocoons, lithium bromide solution was added to dissolve them. After dialysis, filtration, and concentration, a sericin solution was obtained. Magnesia feldspar powder (70-140 mg / m) was added to the genipin solution to prepare a crosslinking solution. The sericin solution and the crosslinking solution were mixed and injected into a mold to prepare a sericin-magnesia feldspar hydrogel. After freezing and lyophilization, a sericin-magnesia feldspar composite nerve conduit was obtained.

[0024] Magnesia feldspar remodels Schwann cell function: (1) such as Figure 1 As shown, the elemental composition of magnesia feldspar was detected, and the effect of magnesia feldspar on the regeneration behavior of Schwann cells was investigated to explore the potential mechanism. (2) The structure of magnesia was determined using TEM and EDS to clarify its calcium, magnesium, silicon, and oxygen composition. Figure 1 (A and B) (3) Magnesia feldspar was prepared into an extract (AKTDEs), and RSC96 Schwann cells were continuously cultured in the extract for 7 days to assess the cell compatibility of magnesia feldspar. Cell viability was detected by CCK-8 assay every 2 days, and cell survival was assessed by Live / Dead staining on days 0 and 7. Figure 1 (C and D). The results demonstrate that magnesium feldspar has good cellular compatibility; (4) The extract was diluted at a ratio of 1 / 64 and 1 / 32, and then 10% fetal bovine serum (FBS) was added for cell culture. RSC96 cells were cultured for 48 hours. CCK-8 assay showed that cell proliferation was significantly increased. Figure 1 E); After culturing RSC96 cells for 24 hours, the expression of integrin, GDNF, VEGF, Ncam, BDNF, NGF, and FGF-2 was detected by qRT-PCR. The results showed that the expression of these cytokines was upregulated. Figure 1 F); Transwell and scratch assays showed that the extract promoted cell migration (F); Figure 1 GJ). After culturing RSC96 cells with the above extract for 24 hours, Western blot analysis was performed to detect the activation of the key signaling pathways PI3K / AKT and MAPK, which are crucial for cell proliferation and secretion. The results showed that these two signaling pathways were significantly activated. Figure 1 The above results demonstrate that magnesia can promote the proliferation, secretion, and migration of Schwann cells by activating the PI3K / AKT and MAPK signaling pathways, revealing the ability of magnesia to remodel the pro-regenerative behavior of Schwann cells. Preparation of catheter mold:

[0025] (1) such as Figure 2 As shown, the catheter was designed using modeling software, and the positive mold of the catheter was prepared using a 3D printer; (2) Place the positive mold into a petri dish, add polydimethylsiloxane (PDMS) until it is submerged, and let it stand at room temperature for 24 hours. (3) Remove the male mold from the solidified PDMS and add an iron rod with a length of 36 mm and a diameter of 1.5 mm in the middle to obtain the conduit mold.

[0026] By combining sericin and magnesium feldspar, composite nerve conduits can be constructed, such as... Figure 3 As shown, the preparation process and in vivo repair of the conduit: 140 Nd-s silkworm cocoons were shredded, dissolved, dialyzed, and concentrated to obtain a sericin solution. Magnesia feldspar and genipin crosslinking agent were then added, mixed thoroughly, and injected into a mold to prepare a sericin-magnesia feldspar composite nerve conduit for repairing sciatic nerve injury in rats. The specific preparation steps of this conduit are as follows: (1) 1g 140NDs After washing and cutting the genetically mutated silkworm cocoons, add 40 mL of lithium bromide solution (6 mol / L), mix well, and dissolve in a water bath at 38°C for 48 hours; (2) Add 10 mL of Tris-HCl (1 mol / L, pH=9), stir and mix well, then add to the dialysis bag (3000 MW); (3) Add 2 ml of Tris-HCl to every 2 L of ultrapure water to prepare dialysis solution.

[0027] (4) Add the dialysis bag containing sericin solution in (2) to the dialysis solution in (3), stir at room temperature for 24 hours, replace the dialysis solution every 2 hours, and finally dialyze with ultrapure water.

[0028] (5) Centrifuge the sericin solution after dialysis at 3000 rpm for 10 minutes, discard the upper insoluble matter, filter it with a 70 μm pore size cell sieve, and put the collected sericin solution back into the dialysis bag.

[0029] (6) Dissolve PEG6000 in ultrapure water to prepare a supersaturated solution, add the dialysis bag containing the sericin solution in (5) into it, stir slowly at room temperature to concentrate, and collect the concentrated sericin solution for later use.

[0030] (7) Dissolve genipin in ultrapure water to prepare a genipin solution of 10 mg / mL. Add magnesium feldspar powder (provided by Shanghai Institute of Ceramics, Chinese Academy of Sciences) to the genipin solution at a ratio of 70 or 140 mg: 1 ml. Mix well by inverting the solution to prepare a crosslinking solution.

[0031] (8) Mix the sericin solution from step (6) and the crosslinking liquid from step (7) at a volume ratio of 6:1, mix them thoroughly by inverting the mixture, and inject them into the mold using a syringe.

[0032] (9) Seal the mold with transparent tape and incubate at 37°C for 24 hours in a humid environment to prepare sericin / magnesia feldspar composite hydrogel.

[0033] (10) The composite conduit was placed in a -20℃ freezer and frozen for 5 hours. It was then removed from the mold and freeze-dried to obtain a sericin-magnesium feldspar composite nerve conduit with a magnesium feldspar concentration of 10 mg / mL or 20 mg / mL.

[0034] (11) The composite catheter is disinfected by immersing it in 75% ethanol for 1 hour, and then washed with sterile PBS to remove residual ethanol before use. Comparison of in vitro regeneration activity of catheters:

[0035] (1) Sesaccharide-magnesium feldspar composite nerve conduits of 10 mg / mL and 20 mg / mL were placed into the lower chamber of a Transwelll plate, and RSC96 cells were seeded in the upper chamber. (2) Add cell culture medium, incubate for 12 hours, remove the upper chamber, stain with crystal violet, take a picture, and record the migration of Schwann cells. Figure 4 (A and B) (3) Collect the cell culture medium and continue to culture the activated PC12 cells; (4) After 24 hours and 48 hours of culture, phalloidin staining was performed, microscopic photography was conducted, and the axonal extension distance was counted. Figure 4 (C and D) (5) Based on the above comparative experiments, it was confirmed that 20 mg / mL has a stronger regeneration effect and will be used in subsequent experiments. Catheter characterization:

[0036] (1) The prepared catheter is dark blue ( Figure 5 A-①, ②), and the structure remains stable even after 1000 repeated compressions ( Figure 5 A-③); (2) Scanning electron microscopy (SEM) results showed that the catheter had a porous structure. Figure 5 B); (3) The water absorption and swelling experiment showed that the conduit reached a steady state after being soaked for about 24 hours. Figure 5 C), porosity testing showed that the duct porosity was approximately 62% ( Figure 5 D), Fourier transform infrared (FTIR) spectroscopy results proved that magnesium feldspar did not significantly alter the structure of sericin. Figure 5 E); (4) Tensile testing was performed to measure the elastic modulus of the conduit. The results showed that the elastic modulus of the composite nerve conduit decreased ( Figure 5 FG), while the compression test results showed that the structural stiffness of the catheter did not change significantly (FG). Figure 5 HI). Treatment of 13 mm sciatic nerve transection injury in rats: (1) A rat model of 13 mm sciatic nerve transection injury was constructed, and a catheter was transplanted for treatment;

[0037] (2) 10 days after transplantation ( Figure 6 A), the catheter was removed and longitudinally sectioned. Axon staining results showed that the composite catheter transplantation significantly promoted axonal elongation. Figure 6 BD), Schwann cell staining results from different sites showed that the cells in the composite catheter treatment group were arranged in cords and the density was significantly increased, proving that the composite catheter promoted the establishment of the Bünger band (BD). Figure 6 EF); (3) The regenerated nerve was removed 14 weeks after transplantation. Figure 7 A), the results showed that the duct had been completely degraded, and the absence of obvious connective tissue attachment around it proved good biocompatibility; transverse sections were made at the proximal and distal ends of the nerve, and transverse sections were made in the middle segment ( Figure 7 B), immunofluorescence staining of axons and Schwann cells was performed to evaluate axonal and Schwann cell regeneration. The results showed a significant increase in axonal and Schwann cell density at both the proximal and distal ends, comparable to autologous transplantation. Figure 7 CH); the intermediate segment axons and Schwann cells are arranged in a distinct parallel longitudinal pattern, and the Schwann cells and axons are co-localized ( Figure 7 IK). The above results indicate that the composite catheter can significantly promote neural structure regeneration; (4) Fourteen weeks after transplantation, a footprint analysis was performed to assess the recovery of motor function. The results showed that the sciatic nerve index (SFI) of the composite catheter group was significantly improved. Figure 8 AC; Electrophysiological testing of nerve conduction capacity was performed, and the results showed that the nerve conduction time and rate were significantly restored in the composite catheter treatment group (AC). Figure 8 DF); Masson staining of the gastrocnemius muscle showed that the treatment group had increased muscle fiber diameter and decreased collagen accumulation. Figure 8 (GI), demonstrating that the composite catheter treatment group can alleviate target organ atrophy. These results indicate that the composite nerve catheter can promote the recovery of damaged nerve function.

Claims

1. A method for fabricating a sericin-magnesia feldspar composite nerve conduit, comprising the following steps: 1) Dissolve the genetically mutated silkworm cocoons in a lithium bromide solution to obtain a sericin solution; 2) Add the sericin solution to a Tris-HCl aqueous solution with pH=9, stir and mix well, and then add it to a dialysis bag to obtain a dialysis bag containing the sericin solution; 3) Add 2 mL of Tris-HCl to every 2 L of ultrapure water to obtain the dialysis solution; 4) Add the dialysis bag from step 2) to the dialysis solution, stir at room temperature for 24 hours, replace with new dialysis solution every 2 hours, and perform dialysis with ultrapure water for the last time; 5) Centrifuge the dialyzed sericin solution at 3000 rpm for 10 minutes, discard the upper insoluble matter, filter it through a 70-micron pore size cell sieve, collect the sericin solution and put it into a dialysis bag; 6) Dissolve polyethylene glycol 6000 in ultrapure water to prepare a supersaturated solution, add the dialysis bag from step 5) into it, stir and concentrate at room temperature to obtain a concentrated sericin solution; 7) Dissolve genipin in ultrapure water to prepare a genipin solution of 10 mg / mL. Add magnesia feldspar powder to the genipin solution at a ratio of 70-140 mg: 1 ml, mix by inverting the mixture to obtain a crosslinking solution. 8) Mix the concentrated sericin solution from step 6) and the crosslinking solution from step 7) at a volume ratio of 6:1, invert the mixture to mix thoroughly, and inject it into the mold using a syringe. 9) Under humid conditions, incubation at 37 ℃ for 24 hours yielded a deep blue sericin-magnesia feldspar hydrogel; 10) Place the hydrogel conduit in a -20 ℃ freezer for 5 hours, remove it from the mold, freeze dry it, and obtain the sericin-magnesium feldspar composite nerve conduit.

2. The manufacturing method according to claim 1, characterized in that: The lithium bromide solution was prepared by dissolving 33.55 g of lithium bromide in 40 mL of ultrapure water.

3. The manufacturing method according to claim 1 or 2, characterized in that: The sericin solution in step 1) is obtained by cutting 1 g of silkworm cocoon into small pieces with scissors, adding it to a lithium bromide solution, stirring and mixing, and then dissolving it in a water bath at 38 ℃ for 48 hours.

4. A sericin-magnesium feldspar composite nerve conduit prepared by the method described in claim 1, 2 or 3.

5. The application of the sericin-magnesium feldspar composite nerve conduit according to claim 4 in the preparation of materials for repairing long-distance peripheral nerve transection.

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