Preparation method of decellularized tissue nerve graft stent with electric conductivity

Abstract

The invention discloses a preparation method of a decellularized tissue nerve graft stent with electric conductivity, and belongs to the field of biological materials. The method comprises the following steps: separating umbilical cord tissues in an embryonic period, removing cells and lipids, and then freeze-drying; the preparation method comprises the following steps: preparing a titanium carbide single-layer nanosheet solution by adopting a chemical etching method, preparing a GelMA solution by adopting a phosphate buffer salt solution of a photoinitiator, and mixing the GelMA solution with the titanium carbide single-layer nanosheet solution to obtain conductive hydrogel; the conductive hydrogel is used for wrapping the umbilical cord tissue in the embryonic period, and ultraviolet light curing is carried out, so that the decellularized tissue nerve graft stent with the conductive capability is obtained. Compared with the prior art, the nerve graft stent provided by the invention has good conductivity, mechanical property and degradability.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a method for preparing a decellularized nerve graft scaffold with electrical conductivity. Background Art

[0002] Nerve rupture caused by severe trauma may lead to complete disconnection of nerve bundles or even separation of the entire nerve trunk, which can easily lead to permanent damage to peripheral nerve function, seriously affecting the patient's daily life and possibly increasing the country's healthcare burden. In order to restore functional connection, the severed nerve endings must be rearranged to ensure that the axons can re-extend to the distal end. However, in actual operation, due to tension limitations, end-to-end suturing techniques are only applicable to cases where the nerve gap does not exceed a few millimeters. For breaks with gaps exceeding 1 to 2 centimeters, bridging techniques must be used for repair. Although autologous nerve grafts are regarded as the "gold standard" for this type of surgery, due to the limitations of donor tissue and potential pathogenic risks, the actual functional benefits they bring are often less than half of the expected.

[0003] As an implant for treating severe peripheral nerve injury, conductive hydrogel can not only promote the conduction of bioelectrical signals in the body while isolating injured nerves from surrounding tissues, but can also be used to repair extensive nerve defects. By loading a variety of cells and bioactive factors, conductive hydrogel can provide an optimized microenvironment for nerve regeneration, provide guidance for the migration of Schwann cells and the extension of axons, thereby achieving the repair of nerve damage. However, the process of loading cells and bioactive factors is relatively complicated, which limits the convenience of preparing such catheters. Decellularized tissue can retain the components of extracellular matrix (ECM) and growth factors, provide a good regenerative microenvironment for damaged nerves, and is a promising tissue engineering material for nerve repair. It is expected to provide a promising simple-to-prepare tissue engineering nerve graft scaffold for clinical treatment, which can be used to bridge long-distance peripheral nerve injuries and promote nerve and functional recovery. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for preparing a decellularized nerve graft scaffold with electrical conductivity.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention relates to a method for preparing a decellularized nerve graft scaffold having electrical conductivity, comprising the following steps:

[0007] Isolation of embryonic umbilical cord tissue, removal of cells and lipids, and subsequent freeze-drying;

[0008] Titanium carbide single-layer nanosheet solution was prepared by chemical etching.

[0009] A GelMA solution is prepared by using a phosphate buffered saline solution of a photoinitiator, and the solution is mixed with the titanium carbide single-layer nanosheet solution to obtain a conductive hydrogel;

[0010] The conductive hydrogel is used to wrap the embryonic umbilical cord tissue, and is cured by ultraviolet light to obtain the decellularized tissue nerve graft scaffold with conductive ability.

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

[0012] Optionally, the method for removing cells and lipids comprises a chemical extraction method, and the solvent used is a 2 wt % sodium deoxycholate solution.

[0013] Optionally, the method for removing cells and lipids comprises treatment with phosphate buffered saline using deoxyribonucleases and ribonucleases.

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

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

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

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

[0018] The second aspect of the present invention relates to a nerve graft scaffold prepared by the above-mentioned method for preparing the decellularized tissue nerve graft scaffold with electrical conductivity.

[0019] The third aspect of the present invention relates to the use of the above-mentioned nerve graft scaffold in the preparation of nerve damage repair products.

[0020] Beneficial effects of the present invention:

[0021] The present invention provides a method for preparing a decellularized tissue nerve graft scaffold with electrical conductivity. The raw materials are cheap and readily available, the preparation process is simple, and high-throughput preparation can be achieved. The nerve graft scaffold obtained by the preparation method of the present invention has the advantages of excellent biocompatibility, degradability, reduced inflammatory response and promotion of axon growth, so that it can be used for bridging long-distance peripheral nerve injuries and promoting nerve and function recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below in conjunction with the accompanying drawings.

[0023] Figure 1 Schematic diagram of the preparation process and application of acellular tissue nerve graft scaffold with conductive ability.

[0024] Figure 2 The figure shows the mechanical properties and conductivity of the decellularized nerve graft scaffold with electrical conductivity, where AC is the compression cycle diagram after adding different components to the nerve graft scaffold, and D is the conductivity performance diagram after adding different components to the nerve graft scaffold.

[0025] Figure 3 The figures show the repair effect of acellular tissue nerve graft scaffolds with conductive ability implanted in rats with sciatic nerve defect, wherein A is a statistical graph of inflammatory cells in the proximal tissue of the sciatic nerve regeneration 14 days after the nerve graft scaffolds were implanted in rats with sciatic nerve defect, B is a statistical graph of the length of regenerated axons of the sciatic nerve 14 days after the nerve graft scaffolds were implanted in rats with sciatic nerve defect, and C is a statistical graph of the sciatic nerve function index at the first, second and third months after the nerve graft scaffolds were implanted in rats with sciatic nerve defect.

[0026] Figure 4 Bright field image showing the in vivo degradation characterization of the acellular tissue nerve graft scaffold with electrical conductivity after implantation into the sciatic nerve defect in rats. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1

[0029] This embodiment provides a method for preparing a decellularized nerve graft scaffold with electrical conductivity. The preparation process 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 embryonic days 18-21 was isolated and repeatedly frozen and thawed three times with liquid nitrogen and ultrapure water at 37°C, with the treatment time in each medium being 3 minutes, and then placed in ultrapure water and incubated at room temperature for 2 hours, then placed in 2wt% sodium deoxycholate and incubated at room temperature for 2.5 hours, then placed in ultrapure water and phosphate buffer in turn and washed at room temperature for 30 minutes, then placed in phosphate buffer containing deoxyribonuclease (100U / mL) and ribonuclease (75μg / mL) and incubated at 37°C for 4 hours, 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 lithium fluoride and hydrogen fluoride synthesized in situ with concentrated hydrochloric acid at a mass volume ratio of 1:10 as an etchant. The etching temperature was 40°C and the etching speed was 400 rpm. The reaction product was separated by centrifugation, washed and resuspended in deionized water. The centrifugal speed was gradually increased from 3000 rpm to 7500 rpm to ensure the complete separation of the titanium carbide monolayer nanosheet solution. After the concentration of the product was determined, it was packaged and stored at 4°C for later use.

[0034] Step S3, preparation of GelMA:

[0035] A gelatin solution with a concentration of 0.1 g / mL was prepared using Dulbecco's phosphate buffer, and then methacrylic acid (final concentration of 6 wt%) was added dropwise to the gelatin solution and stirred at 50°C for 3 hours (speed 400 rpm), followed by placement in a 12-14 kDa dialysis bag and dialyzed in ultrapure water at 40°C for 7 days and adjusting the pH to 7.2, and 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, and then MXene (100 μg / mL) was added to the GelMA solution to obtain a conductive hydrogel.

[0038] Step S5: Preparation of a decellularized nerve graft scaffold with electrical conductivity:

[0039] A glass capillary with an inner diameter of 3 mm was cut into short tubes with a length of 1.5 cm. The cut capillary was placed in a solution of octadecyltriethoxysilane diluted in anhydrous ethanol with a concentration of 10 mg / mL, and the capillary was immersed in the solution for 1 hour. After the treatment, the capillary was transferred to a drying oven at 45°C for 2 hours to remove the residual solvent and solidify the silanization layer. Thereafter, the capillary was thoroughly washed with deionized water and dried again. The decellularized umbilical cord was coaxially nested and fixed with the capillary, and approximately 80 μL of conductive hydrogel was injected into the gap, followed by ultraviolet irradiation for 30 seconds to obtain a decellularized tissue nerve graft scaffold with conductive ability. The prepared catheter was immersed in physiological saline containing antibiotics and stored at a temperature of 2-5°C for subsequent experimental use.

[0040] Example 2

[0041] Evaluation of the mechanical properties and electrical conductivity of acellular tissue-based nerve graft scaffolds with electrical conductivity:

[0042] The decellularized nerve graft scaffold with electrical conductivity (abbreviated as graft scaffold) was trimmed into 1 cm length and placed on the compressor and LED circuit respectively to measure the mechanical properties and electrical conductivity of the graft scaffold. Figure 2 As shown in Figures A, B, and C, the incorporation of GelMA and MXene significantly improves the rigidity of the nerve graft scaffold, and after 20 compression cycles, it still shows a relatively consistent compression force, confirming its outstanding structural integrity and mechanical durability, which is of great significance for the in vivo application of the graft scaffold. Figure 2 As shown in D, the incorporation of MXene enables the nerve graft scaffold to connect to the LED circuit, confirming its outstanding conductive properties, which is of great significance for the graft scaffold to promote bioelectrical conduction in vivo.

[0043] Example 3

[0044] The repair effect of the acellular nerve graft scaffold with conductive ability implanted in rats with sciatic nerve defect was evaluated:

[0045] A rat sciatic nerve defect model (1 cm) was constructed, and a conductive acellular nerve graft scaffold (abbreviated as 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 A, 14 days after bridging, the proximal regeneration tissue was obtained and fluorescent immunohistochemically stained with CD68 antibody. The results showed that compared with the autologous nerve transplantation group, the CD68 +The number of cells was significantly reduced, confirming that the graft scaffold can inhibit the inflammatory response after injury. Figure 3 As shown in Figure B, 14 days after bridging, the tissue at the bridging site was taken and fluorescent immunohistochemically stained with NF200 antibody. The results showed that there was no significant difference in the length of regenerated axons in the graft scaffold group compared with the autologous nerve transplant group, confirming the potential of graft scaffolds to replace autologous nerves for bridging long-distance nerve defects. Figure 3 As shown in Figure 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 once again confirmed the potential of graft scaffolds to replace autologous nerves for bridging long-distance nerve defects.

[0046] Example 4

[0047] Evaluation of the in vivo degradation of the acellular tissue nerve graft scaffold with electrical conductivity implanted into rats with sciatic nerve defects:

[0048] A rat sciatic nerve defect model (1 cm) was constructed, and a conductive acellular nerve graft scaffold (abbreviated as graft scaffold) was used to bridge the defect site. The in vivo degradation of the graft scaffold was evaluated at different time points. Figure 4 As shown, the bridging site was exposed at the time of bridging, 14 days and 3 months after bridging, respectively. The results showed that the graft scaffold showed partial degradation 14 days after bridging, and there was no graft scaffold visible to the naked eye after 3 months of bridging. In addition, the appearance of the regenerated tissue was continuous and there was no neuroma growth at the bridging site. There was no significant difference in appearance from the autologous nerve transplantation group, confirming the potential of the graft scaffold to replace autologous nerves for bridging long-distance nerve defects.

[0049] In summary, the embodiments of the present application verify that the prepared nerve graft scaffold has good electrical conductivity and good mechanical properties, and can provide reliable support. In addition, the embodiments of the present application also verify that the nerve graft scaffold has achieved good results in the repair of sciatic nerve defect in rats, and can inhibit the inflammatory response after injury. Finally, the embodiments of the present application also verify that the nerve graft scaffold has good degradability in rats.

[0050] It should be additionally explained that the nerve graft scaffold in the embodiment of the present application can be implanted into the animal body by surgical methods known in the art, bridging between damaged nerves. The specific operation method can be selected by those skilled in the art from the known technologies, and this application will not make any specific repetitions and limitations. In addition to the preparation raw materials disclosed in the present invention, those skilled in the art can use other known auxiliary raw materials that can be implanted into the animal body, such as adjuvants that promote nerve growth or raw materials for further improving conductivity, which will not be repeated in this application. One of the raw materials used in the above embodiments of the present application is rat umbilical cord tissue, which can be replaced with human umbilical cord tissue when used in the preparation of human nerve grafts. Umbilical cord tissue is relatively simple to collect as a raw material, the cost is relatively low, and the expected effect can be achieved in the above embodiments.

[0051] The degradation time period measured in the examples of the present application is about three months, but there may be different requirements for this period in actual use. Those skilled in the art can adjust the degradation time by adding other promoting or inhibiting adjuvants. 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 stent of the present application according to the situation of nerve damage to obtain the shortest possible degradation time under the premise of completing the repair.

[0052] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0053] The above shows and describes 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, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a decellularized nerve graft scaffold having electrical conductivity, characterized in that: The following steps are involved: Isolation of embryonic umbilical cord tissue, removal of cells and lipids, and subsequent freeze-drying; Titanium carbide single-layer nanosheet solution was prepared by chemical etching. A GelMA solution is prepared by using a phosphate buffered saline solution of a photoinitiator, and the solution is mixed with the titanium carbide single-layer nanosheet solution to obtain a conductive hydrogel; The conductive hydrogel is used to wrap the embryonic umbilical cord tissue, and is cured by ultraviolet light to obtain the decellularized tissue nerve graft scaffold with conductive ability.

2. The method for preparing the acellular tissue nerve graft scaffold with electrical conductivity according to claim 1, characterized in that: The method for removing cells and lipids includes a freeze-thaw method, using liquid nitrogen and 37° C. ultrapure water to repeatedly freeze and thaw several times.

3. The method for preparing the acellular tissue nerve graft scaffold with electrical conductivity according to claim 1, characterized in that: The method for removing cells and lipids comprises a chemical extraction method, and the solvent used is a 2wt% sodium deoxycholate solution.

4. The method for preparing the acellular tissue nerve graft scaffold with electrical conductivity according to claim 1, characterized in that: The method for removing cells and lipids includes treatment with phosphate buffered saline using deoxyribonucleases and ribonucleases.

5. The method for preparing the acellular tissue nerve graft scaffold with electrical conductivity according to claim 1, characterized in that: The method for preparing the titanium carbide single-layer nanosheet comprises: using lithium fluoride and hydrogen fluoride synthesized in situ with hydrochloric acid in a mass-to-volume ratio of 1:10 as an etchant to chemically etch Ti3AlC2.

6. The method for preparing the acellular tissue nerve graft scaffold with electrical conductivity according to claim 1, characterized in that: The preparation method of GelMA comprises the following steps: preparing a gelatin solution using Dulbecco's phosphate buffer, and adding methacrylic acid dropwise into the gelatin solution; placing the gelatin solution in a dialysis bag and dialyzing it in ultrapure water and adjusting the pH to 7.2; and obtaining GelMA by centrifugation, filtration and freeze drying.

7. The method for preparing the acellular tissue nerve graft scaffold with electrical conductivity according to claim 1, characterized in that: The photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone, and the concentration is 1 wt %.

8. The method for preparing the acellular tissue nerve graft scaffold with electrical conductivity according to claim 1, characterized in that: The concentration of the GelMA solution is 15 wt %, and the concentration of the titanium carbide single-layer nanosheet solution is 100 μg / mL.

9. A nerve graft scaffold prepared by the method for preparing a decellularized tissue nerve graft scaffold with electrical conductivity according to any one of claims 1 to 8.

10. Use of the nerve graft scaffold according to claim 9 in the preparation of nerve damage repair products.

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