Method for preparing a decellularized tissue nerve graft scaffold with electrical conductivity

Abstract

The application discloses a preparation method of a decellularized tissue nerve graft scaffold with electric conductivity, and belongs to the field of biomaterials. The method comprises the following steps: separating embryonic period umbilical cord tissue, removing cells and lipids, and then performing freeze-drying; a titanium carbide monolayer nanosheet solution is prepared by using a chemical etching method, a GelMA solution is prepared by using a photo initiator phosphate buffer salt solution, and the GelMA solution is mixed with the titanium carbide monolayer nanosheet solution to obtain a conductive hydrogel; the embryonic period umbilical cord tissue is wrapped with the conductive hydrogel, and is cured by ultraviolet light to obtain the decellularized tissue nerve graft scaffold with electric conductivity. Compared with the prior art, the nerve graft scaffold has good electric conductivity, mechanical properties and degradability.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and more specifically to a method for preparing a decellularized tissue neural graft scaffold with electrical conductivity. Background Technology

[0002] Severe trauma-induced nerve rupture can lead to complete separation of nerve bundles or even the entire nerve trunk, which can easily result in permanent damage to peripheral nerve function, severely impacting patients' daily lives and potentially increasing the national healthcare burden. To restore functional connectivity, the severed nerve endings must be rearranged to ensure the axons can extend distally again. However, in practice, due to tension limitations, end-to-end suturing techniques are only suitable for nerve gaps no larger than a few millimeters. For ruptures with gaps exceeding 1 to 2 centimeters, bridging techniques must be used for repair. Although autologous nerve grafts are considered the "gold standard" for this type of surgery, due to limitations in donor tissue and potential pathogenic risks, the actual functional benefits are often less than half of the expected.

[0003] As an implant for treating severe peripheral nerve injuries, conductive hydrogels not only isolate injured nerves from surrounding tissues while promoting the conduction of bioelectrical signals in vivo, but also repair extensive nerve defects. By loading various cells and bioactive factors, conductive hydrogels can provide an optimized microenvironment for nerve regeneration, guiding Schwann cell migration and axonal extension, thereby achieving nerve repair. However, the complexity of loading cells and bioactive factors limits the ease of fabrication of such conduits. Decellularized tissue, which retains extracellular matrix (ECM) and growth factor components, provides a favorable regenerative microenvironment for damaged nerves and is a promising tissue engineering material for nerve repair. It holds promise for providing a simple tissue-engineered nerve graft scaffold for bridging long-distance peripheral nerve injuries and promoting nerve and functional recovery. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a method for preparing a decellularized tissue neural graft scaffold with electrical conductivity.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A first aspect of the present invention relates to a method for preparing a conductive decellularized tissue neural graft scaffold, comprising the following steps:

[0007] Embryonic umbilical cord tissue was isolated, and cells and lipids were removed, followed by freeze-drying;

[0008] Titanium carbide monolayer nanosheet solution was prepared using a chemical etching method.

[0009] A GelMA solution was prepared using a phosphate buffer solution of a photoinitiator, and then mixed with the titanium carbide monolayer nanosheet solution to obtain a conductive hydrogel.

[0010] The conductive hydrogel was used to encapsulate the embryonic umbilical cord tissue, and then cured by ultraviolet light to obtain the conductive decellularized tissue neural graft scaffold.

[0011] Optionally, the method for removing cells and lipids includes a freeze-thaw process, using liquid nitrogen and 37°C ultrapure water to repeatedly freeze and thaw several times.

[0012] Optionally, the method for removing cells and lipids includes chemical extraction using a 2 wt% sodium deoxycholate solution as the solvent.

[0013] Optionally, the method for removing cells and lipids includes treatment with a phosphate-buffered saline solution containing deoxyribonuclease and ribonuclease.

[0014] Optionally, the method for preparing the titanium carbide monolayer nanosheets includes: chemically etching Ti3AlC2 using lithium fluoride and hydrogen fluoride synthesized in situ with hydrochloric acid at a mass-to-volume ratio of 1:10 as an etchant.

[0015] Optionally, the preparation method of the GelMA includes the following steps: preparing a gelatin solution using Durbeco phosphate buffer, adding methacrylic acid dropwise to the gelatin solution; placing it in a dialysis bag and dialyzing in ultrapure water and adjusting the pH to 7.2; obtaining GelMA by centrifugation, filtration and freeze-drying.

[0016] Optionally, the photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone at a concentration of 1 wt%.

[0017] Optionally, the concentration of the GelMA solution is 15 wt%, and the concentration of the titanium carbide monolayer nanosheet solution is 100 μg / mL.

[0018] A second aspect of the present invention relates to a neural graft scaffold prepared by the above-described method for preparing acellular tissue neural graft scaffolds with conductive properties.

[0019] A third aspect of the present invention relates to the application of the above-described neural graft scaffold in the preparation of nerve injury repair products.

[0020] The beneficial effects of this invention are:

[0021] This invention provides a method for preparing a decellularized tissue neural graft scaffold with electrical conductivity. The raw materials are inexpensive and readily available, the preparation process is simple, and high-throughput preparation can be achieved. The neural graft scaffold obtained by the preparation method of this invention has the advantages of excellent biocompatibility, biodegradability, reduced inflammatory response, and promotion of axonal growth, thus it can be used for bridging long-distance peripheral nerve injuries and promoting nerve and functional recovery. Attached Figure Description

[0022] The invention will now be further described with reference to the accompanying drawings.

[0023] Figure 1 A schematic diagram illustrating the fabrication process and application of a conductive decellularized tissue neural graft scaffold.

[0024] Figure 2 The figures show the mechanical properties and conductivity of acellular tissue neural graft scaffolds with electrical conductivity. AC represents the compression cycle diagrams after adding different components to the neural graft scaffold, and D represents the conductivity diagram after adding different components to the neural graft scaffold.

[0025] Figure 3 The images show the repair effect of a decellularized tissue neural graft scaffold with electrical conductivity implanted in rats with sciatic nerve defects. In the images, A is a statistical diagram of inflammatory cells in the proximal tissue of sciatic nerve regeneration 14 days after implantation of the neural graft scaffold in rats with sciatic nerve defects; B is a statistical diagram of the axon length of sciatic nerve regeneration 14 days after implantation of the neural graft scaffold in rats with sciatic nerve defects; and C is a statistical diagram of sciatic nerve function index at the first, second, and third months after implantation of the neural graft scaffold in rats with sciatic nerve defects.

[0026] Figure 4 Bright-field image of the degradation characterization of a decellularized tissue nerve graft scaffold with electrical conductivity implanted in rats with sciatic nerve defects. Detailed Implementation

[0027] 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.

[0028] Example 1

[0029] This embodiment provides a method for preparing a conductive decellularized tissue neural graft scaffold, the preparation process of which is as follows: Figure 1 As shown, the specific steps are as follows:

[0030] Step S1: Preparation of decellularized umbilical cord tissue:

[0031] Umbilical cord tissue from rat embryos aged 18-21 days was isolated and subjected to three freeze-thaw cycles in liquid nitrogen and 37°C ultrapure water, with each treatment lasting 3 minutes. The tissue was then placed in ultrapure water and incubated at room temperature for 2 hours. Next, it was placed in 2 wt% sodium deoxycholate and incubated at room temperature for 2.5 hours. Then, it was sequentially placed in ultrapure water and phosphate buffer and washed at room temperature for 30 minutes. Finally, it was placed in phosphate buffer containing deoxyribonuclease (100 U / mL) and ribonuclease (75 μg / mL) and incubated at 37°C for 4 hours. The tissue was washed again with phosphate buffer for 30 minutes and finally freeze-dried to obtain the main scaffold.

[0032] Step S2, Preparation of MXene solution:

[0033] Ti3AlC2 was chemically etched for 30 hours using a mixture of lithium fluoride and concentrated hydrochloric acid synthesized in situ at a mass-to-volume ratio of 1:10 as the etchant. The etching temperature was 40℃, and the etching speed was 400 rpm. The reaction products were separated by centrifugation, washed, and resuspended in deionized water. The centrifugation speed was gradually increased from 3000 rpm to 7500 rpm to ensure complete separation of the titanium carbide monolayer nanosheet solution. After the product concentration was determined, it was aliquoted and stored at 4℃ for later use.

[0034] Step S3, Preparation of GelMA:

[0035] A gelatin solution with a concentration of 0.1 g / mL was prepared using Durbeco phosphate buffer. Then, dry methacrylic acid (final concentration 6 wt%) was added dropwise to the gelatin solution and stirred at 50°C for 3 hours (400 rpm). The solution was then placed in a 12-14 kDa dialysis bag and dialyzed in ultrapure water at 40°C for 7 days, with the pH adjusted to 7.2. Finally, GelMA was obtained by centrifugation, filtration, and freeze-drying.

[0036] Step S4, Preparation of conductive hydrogel:

[0037] A GelMA solution (15 wt%) was prepared using a phosphate buffer solution containing 2-hydroxy-2-methyl-1-phenylpropanone (1 wt%) as a photoinitiator. MXene (100 μg / mL) was then added to the GelMA solution to obtain a conductive hydrogel.

[0038] Step S5: Preparation of a conductive decellularized tissue neural graft scaffold:

[0039] Glass capillaries with an inner diameter of 3 mm were cut into 1.5 cm lengths. The cut capillaries were then immersed in a 10 mg / mL solution of octadecyltriethoxysilane diluted in anhydrous ethanol for 1 hour. After treatment, the capillaries were transferred to a 45°C drying oven for 2 hours to remove residual solvent and solidify the silanized layer. Subsequently, the capillaries were thoroughly rinsed with deionized water and dried again. Acellular umbilical cord was coaxially nested and fixed to the capillaries, and approximately 80 μL of conductive hydrogel was injected into the gap. The grafts were then irradiated with ultraviolet light for 30 seconds to obtain conductive acellular tissue nerve graft scaffolds. The prepared catheters were immersed in antibiotic-containing saline and stored at 2–5°C for subsequent experimental use.

[0040] Example 2

[0041] The mechanical properties and electrical conductivity of conductive acellular tissue neural graft scaffolds were evaluated:

[0042] Acellular neural graft scaffolds with electrical conductivity were cut into 1 cm lengths and placed on a compressor and an LED circuit, respectively, to measure their mechanical properties and conductivity. Figure 2 As shown in A, B, and C, the incorporation of GelMA and MXene significantly improved the rigidity of the neural graft scaffold, and after 20 compression cycles, it still exhibited relatively consistent compressive force, confirming its outstanding structural integrity and mechanical durability, which is of great significance for the in vivo application of graft scaffolds. Figure 2 As shown in D, the incorporation of MXene enables the neural graft scaffold to connect to LED circuits, demonstrating its outstanding conductivity, which is of great significance for promoting bioelectrical conduction in vivo.

[0043] Example 3

[0044] The efficacy of electrically conductive decellularized tissue nerve graft scaffolds implanted in rats with sciatic nerve defects for in vivo repair was evaluated.

[0045] A rat model of sciatic nerve defect (1 cm) was constructed. A decellularized tissue nerve graft scaffold with electrical conductivity (hereinafter referred to as the graft scaffold) was used to bridge the defect site. The in vivo repair effect of the graft scaffold was evaluated at different time points. Figure 3 As shown in Figure A, 14 days after bridging, proximal regenerated tissue was harvested and subjected to fluorescent immunohistochemical staining with CD68 antibody. The results showed that, compared with the autologous nerve transplantation group, the CD68 antibody in the graft scaffold group was significantly higher. +The significant reduction in cells confirms that the graft scaffold can suppress the inflammatory response following injury. Figure 3 As shown in Figure B, 14 days after bridging, tissue from the bridging site was taken for fluorescent immunohistochemical staining with NF200 antibody. The results showed no significant difference in the length of regenerated axons between the graft scaffold group and the autologous nerve transplantation group, confirming the potential of graft scaffolds to replace autologous nerves for bridging long-distance nerve defects. Figure 3 As shown in C, the sciatic nerve function index of rats was measured at the first, second, and third months after bridging. The results showed that there was no significant difference in the sciatic nerve function index between the graft scaffold group and the autologous nerve transplant group, which further confirmed the potential of graft scaffolds to replace autologous nerves for bridging long-distance nerve defects.

[0046] Example 4

[0047] The in vivo degradability of conductive decellularized tissue nerve graft scaffolds implanted in rats with sciatic nerve defects was evaluated.

[0048] A rat model of sciatic nerve defect (1 cm) was established. A conductive decellularized tissue nerve graft scaffold (hereinafter referred to as the graft scaffold) was used to bridge the defect site. The in vivo degradability of the graft scaffold was evaluated at different time points. Figure 4 As shown, the bridging site was exposed at the time of bridging and 14 days and 3 months after bridging. The results showed that the graft scaffold showed partial degradation 14 days after bridging, and no graft scaffold was visible to the naked eye 3 months after bridging. Furthermore, the regenerated tissue showed no neuroma growth at the continuous bridging site, and its appearance was not significantly different from that of the autologous nerve transplantation group, confirming the potential of graft scaffolds to replace autologous nerves for bridging long-distance nerve defects.

[0049] In summary, the embodiments of this application verify that the prepared neural graft scaffold possesses good electrical conductivity and excellent mechanical properties, thus providing reliable support. Furthermore, the embodiments of this application also verify that the neural graft scaffold achieves good results in repairing sciatic nerve defects in rats, while simultaneously inhibiting the inflammatory response following injury. Finally, the embodiments of this application also verify that the neural graft scaffold has good biodegradability in rats.

[0050] It should be further noted that the neural graft scaffold in this application embodiment can be implanted into an animal body using surgical methods known in the art, bridging the damaged nerves. The specific operation method can be selected by those skilled in the art from known techniques, and this application will not elaborate further or limit it. In addition to the preparation materials disclosed in this invention, those skilled in the art can use other known auxiliary materials that can be implanted into an animal body, such as adjuvants to promote nerve growth or materials to further improve conductivity, which will also not be elaborated upon in this application. One of the raw materials used in the above embodiments of this application is rat umbilical cord tissue. When applied to the preparation of human neural grafts, it can be replaced with human umbilical cord tissue accordingly. Umbilical cord tissue, as a raw material, is relatively simple to collect, has relatively low cost, and can achieve the expected results in the above embodiments.

[0051] The degradation time period measured in this embodiment is approximately three months, but in actual use, this period may have different requirements. Those skilled in the art can adjust the degradation time by adding other adjuvants that promote activity or inhibit inhibition. Of course, the degradation time may also be affected by the size and implantation site. Those skilled in the art can adjust the material or size of the graft scaffold of this application according to the condition of nerve damage to obtain the shortest possible degradation time while completing the repair.

[0052] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0053] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing a conductive decellularized tissue neural graft scaffold, characterized in that, The method comprises the following steps: Embryonic umbilical cord tissue is separated and cells and lipids are removed, followed by freeze-drying; A titanium carbide monolayer nanosheet solution is prepared by chemical etching, A GelMA solution is prepared using a phosphate buffer solution of a photoinitiator, mixed with the titanium carbide monolayer nanosheet solution to obtain a conductive hydrogel; A glass capillary tube with an inner diameter of 3 mm is cut into a short tube with a length of 1.5 cm. The cut capillary tube is immersed in an octadecyl triethoxysilane solution diluted in anhydrous ethanol with a concentration of 10 mg / mL for 1 hour. After treatment, the capillary tube is transferred to a drying oven at 45°C for 2 hours to remove residual solvents and solidify the silanized layer. Then the capillary tube is thoroughly washed with deionized water and dried again. The decellularized umbilical cord is coaxially nested and fixed with the capillary tube, and 80 μL of conductive hydrogel is injected into the gap. Then it is irradiated with ultraviolet light for 30 seconds to obtain a decellularized tissue nerve graft scaffold with conductive ability.

2. The method for preparing a decellularized tissue neural graft scaffold with conductive properties according to claim 1, characterized in that, The method for removing cells and lipids includes freeze-thaw method, using liquid nitrogen and 37°C ultrapure water to freeze-thaw repeatedly for several times.

3. The method for preparing a decellularized tissue neural graft scaffold with conductive properties according to claim 1, characterized in that, The method for removing cells and lipids includes chemical extraction method, using 2 wt% sodium deoxycholate solution as solvent.

4. The method for preparing a decellularized tissue neural graft scaffold with conductive properties according to claim 1, characterized in that, The method for removing cells and lipids includes using deoxyribonuclease and ribonuclease phosphate buffer.

5. The method for preparing a decellularized tissue neural graft scaffold with conductive properties according to claim 1, characterized in that, The preparation method of the titanium carbide monolayer nanosheet includes: using lithium fluoride and in-situ synthesized hydrogen fluoride as etchant to chemically etch Ti3AlC2 with a mass-volume ratio of 1:

10.

6. The method for preparing a decellularized tissue neural graft scaffold with conductive properties according to claim 1, characterized in that, The preparation method of the GelMA includes the following steps: using Dulbecco's phosphate buffer to prepare a gelatin solution, adding methyl acrylate dropwise to the gelatin solution; placing it in a dialysis bag and dialyzing in ultrapure water and adjusting the pH to 7.2; centrifuging, filtering and freeze-drying to obtain GelMA.

7. The method for preparing a decellularized tissue neural graft scaffold with conductive properties according to claim 1, characterized in that, The photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone with a concentration of 1 wt%.

8. The method for preparing a decellularized tissue neural graft scaffold with conductive properties according to claim 1, characterized in that, The concentration of the GelMA solution is 15 wt%, and the concentration of the titanium carbide monolayer nanosheet solution is 100 μg / mL.

9. A nerve graft scaffold prepared by the method of any one of claims 1-8.

10. The use of the nerve graft scaffold of claim 9 in the preparation of a nerve injury repair product.

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