Neural repair material with high-orientation tube bundle structure and preparation method thereof

Abstract

The invention relates to a neural restoration material with a high-orientation tube bundle structure and a preparation method of the neural restoration material. Natural wood is taken as a raw material, and the lignocellulose scaffold material with an anisotropic pore structure of original wood is prepared by combining normal-pressure delignification treatment with freeze drying; and pouring the methacrylated gelatin solution into the lignocellulose scaffold material, and carrying out photo-crosslinking curing to obtain the nerve repair material with the high-orientation tube bundle structure. According to the invention, the materials are environment-friendly, the preparation process is simple, the controllability is good, and the prepared nerve repair material has a directional tube bundle structure matched with the natural nerve tissue form and excellent biocompatibility, also has good mechanical support, can promote the directional arrangement and growth of cells and nerve protrusions along the nerve fiber direction in the nerve tissue, and has a good application prospect. The maturation of nerve structures and functions is realized, and the nerve injury repairing effect is finally achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering and bionic structure, and in particular relates to a nerve repair material with a highly oriented tube bundle structure and a preparation method thereof. Background Art

[0002] The demand for nerve injury repair and nerve regeneration caused by trauma, disease, aging population and other factors is increasing. However, autologous nerve transplantation is difficult to meet the growing clinical needs due to its limited sources and donor site complications. Allogeneic nerve transplantation is limited by immune response and disease transmission risks and is difficult to promote on a large scale in clinical practice. Therefore, neural tissue engineering scaffolds have become one of the most commonly used methods for clinical treatment of nerve injury repair. Nerve tissue is a special tissue with a complex anatomical structure. It is composed of nerve bundles with a highly oriented structure (anisotropy). Functional structures such as microvessels and collagen-rich connective tissue are also distributed around the nerve bundles. In addition, the microenvironment is complex when the damaged nerve tissue is repaired. Therefore, the ideal neural tissue engineering scaffold must have good biocompatibility, mechanical properties that adapt to the growth of neural tissue, provide an environment that facilitates material exchange, and directional guidance of cell migration and growth.

[0003] Studies in recent years have shown that three-dimensionally connected highly oriented structural neural repair materials can better simulate the growth environment of natural neural tissues, providing space and direction for the growth of new cells and neurites. At present, the common designs of highly oriented structural neural repair materials mainly include two types: one is the "bottom-up" design method based on the development of technologies such as biological 3D printing or self-assembly. This type of method includes biological 3D printing, self-assembly, and directional freezing, which have a large degree of freedom and can realize the active regulation of the porous structure and mechanical properties of neural repair materials. However, current research reports that highly oriented structural neural repair materials based on such design methods are difficult to take into account the cell scale and appearance size that match natural neural tissues. Another type of highly oriented structural neural repair material design is mostly prepared by simple homogeneous materials such as natural materials and decellularized matrix materials. This method also carries out bionic design of natural neural tissues, but at present, this type of neural repair material has a relatively simple composition and structure, cumbersome preparation process, and high price, making it difficult to achieve active regulation of its porous structure and mechanical properties, and animal-derived materials also have the risk of pathogenic bacteria / virus transmission. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the present invention provides a nerve repair material with a highly oriented tube bundle structure, which has a cell-scale three-dimensional oriented porous structure matching natural nerve tissue, excellent biocompatibility, and takes into account both adjustable appearance size and mechanical support. The radial overall compressive strength is 50-2000KPa, and the compression modulus is 120-10000kPa. The compression modulus of the hydrogel matrix filled inside is 1.25-500kPa. It can promote the oriented arrangement of cells and neurites along the direction of nerve fibers in the nerve tissue, realize the maturation of nerve structure and function, and ultimately achieve the effect of nerve damage repair.

[0005] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0006] The first aspect of the present invention provides a nerve repair material with a highly oriented tubular bundle structure, comprising a lignocellulose scaffold with a natural porous structure and a hydrogel matrix, wherein the hydrogel matrix is ​​filled in the pores of the lignocellulose scaffold with a natural porous structure.

[0007] Preferably, the lignocellulose scaffold with a natural porous structure is a lignocellulose scaffold with a natural porous structure that has been subjected to delignification treatment.

[0008] Preferably, the delignification treatment is as follows: the natural wood is placed in a NaOH and Na2SO3 solution for normal pressure cooking, and then placed in a H2O2 solution for further normal pressure cooking.

[0009] Preferably, the residual lignin content in the lignocellulose scaffold with a natural porous structure does not exceed 3%.

[0010] Preferably, the lignocellulose scaffold with a natural porous structure has a tubular array structure formed by oriented arrangement of plant fiber cells in the original wood.

[0011] Preferably, the diameter of the lignocellulose support with a natural porous structure is the same as the diameter of wood fibers in natural wood and the size of the ducts and sieve tubes.

[0012] Preferably, the average tube diameter of the lignocellulose scaffold with a natural porous structure is 10-500 μm.

[0013] Preferably, the nerve repair material with a highly oriented tube bundle structure has a radial overall compressive strength of 50-2000 kPa and a compression modulus of 120-10000 kPa.

[0014] Preferably, the compression modulus of the hydrogel matrix is ​​1.25-500 kPa, so as to take into account both the mechanical property requirements of nerve tissue growth and external mechanical support.

[0015] Preferably, the hydrogel matrix is ​​selected from methacrylated hydrogel (GelMA); it is suitable for Schwann cells, neuronal cells or neural stem cells to grow therein.

[0016] Preferably, the appearance shape of the nerve repair material with a highly oriented tube bundle structure is selected from one or more of a catheter, a cuboid, a film, a cylinder, an ellipsoid or other irregular shapes.

[0017] The second aspect of the present invention provides a method for preparing a nerve repair material having a highly oriented tube bundle structure, comprising the following steps:

[0018] (1) Cutting natural wood and immersing it in a hot solution containing NaOH and Na2SO3 for normal pressure cooking; then rinsing it and transferring it to a hot H2O2 solution for further normal pressure cooking until a white sample is obtained; placing the obtained white sample in a dialysis bag for dialyzing with distilled water, and then freeze-drying it to obtain a lignocellulose scaffold;

[0019] (2) immersing the lignocellulose scaffold in a hydrogel matrix solution containing a photoinitiator, and treating under negative pressure until the internal spaces of the lignocellulose scaffold are filled with the hydrogel matrix solution;

[0020] (3) Irradiating with ultraviolet light to fully gel the hydrogel matrix.

[0021] Preferably, the concentration of NaOH in the hot solution in step (1) is 2.5 mol / L, and the concentration of Na2SO3 is 0.4 mol / L.

[0022] Preferably, the normal pressure cooking time in step (1) is 1-12 hours.

[0023] Preferably, the concentration of the H2O2 hot solution in step (1) is 2.5 mol / L.

[0024] Preferably, the temperature of the hydrogel matrix solution in step (2) is 37° C. and the concentration is 5-30% (w / v).

[0025] Preferably, the photoinitiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0026] Preferably, the hydrogel matrix is ​​selected from GelMA.

[0027] Preferably, the wavelength of the ultraviolet light in step (3) is 405 nm, and the light intensity is 0.1-3 mW / cm 2 .

[0028] Preferably, the irradiation time in step (3) is 10-120s.

[0029] Compared with the prior art, the present invention has the following beneficial technical effects:

[0030] (1) The nerve repair material of the present invention has a cell-scale three-dimensional directional porous structure that matches natural nerve tissue, providing an environment that facilitates material exchange for nerve repair and conditions that directional guide cell migration and growth.

[0031] (2) The lignocellulose scaffold of the present invention is made of original wood with good bio-tissue compatibility through delignification treatment, and the scaffold is filled with methacryloyl gelatin suitable for the growth of Schwann cells, neurons, neural stem cells, and vascular endothelial cells, without additional material modification.

[0032] (3) The present invention accurately regulates the mechanical properties of nerve repair materials by adjusting the composition and concentration of the filler inside the scaffold, thereby providing appropriate mechanical support for nerve repair.

[0033] (4) The overall appearance of the lignocellulose scaffold of the present invention can be molded into different shapes according to needs and used to repair different types of nerve damage.

[0034] (5) The materials used in the present invention are environmentally friendly, the preparation process is simple, the controllability is good, and it is easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The figures are scanning electron micrographs of the nerve repair material with a highly oriented tubular bundle structure of Example 1; wherein 1(a) is a scanning electron micrograph of the porous structure inside natural wood; 1(b) is a scanning electron micrograph of the lignocellulose scaffold prepared in Example 1 that retains the highly oriented porous structure of natural wood; 1(c) is a scanning electron micrograph of the nerve repair material with a highly oriented tubular bundle structure obtained after filling the porous structure of the lignocellulose scaffold with a hydrogel matrix.

[0036] Figure 2 This is an optical microscope photograph of the nerve repair material prepared in Example 2.

[0037] Figure 3 This is the CCK-8 test result of the biocompatibility test of the nerve repair material in Example 3. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the examples below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents used in the present invention are all commercially available, wherein GelMa is purchased from Engineering For Life, and the cell proliferation / toxicity detection kit (CCK-8 kit) is purchased from Donjido. The experimental methods used in the present invention are all conventional methods and techniques in the art. Representative results are selected from the biological experiments repeated and presented in the context figures, and the data are displayed as mean±SD and mean±SEM as specified in the figure. All experiments were repeated at least three times. The data were processed using GraphPad Prism 8.0.

[0039] Example 1

[0040] A nerve repair material with a highly oriented tube bundle structure, the preparation method of which comprises the following steps:

[0041] (1) Natural wood is cut and immersed in a hot solution containing NaOH (2.5 mol / L) and Na2SO3 (0.4 mol / L) and steamed at normal pressure for 12 h. The steamed material is then rinsed with hot distilled water and transferred to a hot H2O2 solution (2.5 mol / L) and steamed at normal pressure until the material changes color to white. The obtained white sample is placed in a dialysis bag and dialyzed with distilled water to remove residual chemical reagents, and then freeze-dried to obtain a cellulose scaffold.

[0042] (2) The cellulose scaffold was immersed in a 5% (w / v) GelMA solution containing lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 37° C. and treated under negative pressure until the internal spaces of the cellulose scaffold were completely filled with the GelMA solution.

[0043] (3) At 405nm, light intensity is 0.1mW / cm 2 The hydrogel matrix was fully gelled by irradiating the hydrogel matrix under ultraviolet light for 30 seconds, thereby obtaining a nerve repair material with a highly oriented bundle structure suitable for the adhesion of neurons.

[0044] The above-mentioned nerve repair material with highly oriented bundle structure was tested by scanning electron microscopy. The results are as follows: Figure 11(a) is a scanning electron micrograph of the porous structure inside natural wood; 1(b) is a scanning electron micrograph of the lignocellulose scaffold prepared in this example that retains the highly oriented porous structure of natural wood; 1(c) is a scanning electron micrograph of the nerve repair material with a highly oriented tubular bundle structure obtained after the porous structure of the lignocellulose scaffold is filled with a hydrogel matrix.

[0045] Furthermore, the physical and chemical properties were tested, and the results showed that the radial overall compressive strength of the nerve repair material with a highly oriented bundle structure prepared in this embodiment was 50 kPa, the compression modulus was 120 kPa, and the compression modulus of the hydrogel matrix filled inside was 1.25 kPa, which could promote the directional arrangement of cells and neurites along the direction of nerve fibers in nerve tissue, realize the maturation of nerve structure and function, and ultimately achieve the effect of nerve damage repair.

[0046] Example 2

[0047] A nerve repair material with a highly oriented tube bundle structure, the preparation method of which comprises the following steps:

[0048] (1) Natural wood is cut and immersed in a hot solution containing NaOH (2.5 mol / L) and Na2SO3 (0.4 mol / L) and steamed at normal pressure for 12 h. The steamed material is then rinsed with hot distilled water and transferred to a hot H2O2 solution (2.5 mol / L) and steamed at normal pressure until the material changes color to white. The obtained white sample is placed in a dialysis bag and dialyzed with distilled water to remove residual chemical reagents, and then freeze-dried to obtain a cellulose scaffold.

[0049] (2) The cellulose scaffold was immersed in a 25% (w / v) GelMA solution containing lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 37° C. and treated under negative pressure until the internal spaces of the cellulose scaffold were completely filled with the GelMA solution.

[0050] (3) At 405nm, light intensity is 3mW / cm 2 The hydrogel matrix was fully gelled by irradiating the hydrogel matrix under ultraviolet light for 20 seconds, thereby obtaining a neural repair material with a highly oriented bundle structure suitable for the adhesion of neurons (see Figure 1 ).

[0051] The physical and chemical properties of the nerve repair material with a highly oriented bundle structure prepared in this embodiment were tested. The results showed that the radial overall compressive strength was 2000 KPa, the compression modulus was 9600 kPa, and the compression modulus of the hydrogel matrix filled inside was 500 kPa. The nerve repair material with a highly oriented bundle structure was tested using an optical microscope. The results are as follows: Figure 2 The results showed that the pores of the obtained material were observed to be arranged in parallel along the longitudinal direction of the scaffold, which was suitable for the growth of neurons along the directional pores and had the structural basis of a neural scaffold.

[0052] Example 3

[0053] A nerve repair material with a highly oriented tube bundle structure, the preparation method of which comprises the following steps:

[0054] (1) Natural wood was cut and immersed in a hot solution containing NaOH (2.5 mol / L) and Na2SO3 (0.4 mol / L) and then steamed at normal pressure for 6 h. The steamed material was then rinsed with hot distilled water and transferred to a hot H2O2 solution (2.5 mol / L) and continued to be steamed at normal pressure until the color of the material turned white. The obtained white sample was placed in a dialysis bag and dialyzed with distilled water to remove residual chemical reagents, and then freeze-dried to obtain a cellulose scaffold.

[0055] (2) The cellulose scaffold was immersed in a 10% (w / v) GelMA solution containing lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 37° C. and treated under negative pressure until the internal spaces of the cellulose scaffold were completely filled with the GelMA solution.

[0056] (3) At 405nm, light intensity is 1mW / cm 2 The hydrogel matrix was fully gelled by irradiating the hydrogel matrix under ultraviolet light for 30 seconds, thereby obtaining a nerve repair material with a highly oriented tubular structure.

[0057] The physical and chemical properties thereof were tested, and the results showed that the nerve repair material with a highly oriented tube bundle structure prepared in this embodiment had an overall radial compressive strength of 1000 KPa and a compression modulus of 2000 kPa, and the compression modulus of the hydrogel matrix filled therein was 200 kPa.

[0058] Example 4

[0059] The nerve repair material with a highly oriented bundle structure prepared in Example 3 was tested for its tissue compatibility, and the specific steps are as follows:

[0060] (1) After SD rats were killed, the dorsal skin was peeled off to separate the spinal cord. The dorsal root ganglion (DRG) was carefully separated with forceps. The central and peripheral axons were cut off, and the DRG neurons were obtained after digestion and centrifugation, which were then purified and identified.

[0061] (2) The DRG neurons were divided into 4 groups, designated as Group 1 to Group 4, where Group 1 was cultured with a simple culture medium (DRG+M group), Group 2 was cultured with a culture medium + natural wood (DRG+WOOD group), Group 3 was cultured with a culture medium + GelMA (DRG+GelMA group), and Group 4 was cultured with a culture medium + the neural repair material with a highly oriented tubular structure obtained in Example 3 (DRG+WOOD+GelMA group).

[0062] (3) The proliferation activity of DRG neurons was detected by CCK-8 on the 1st, 3rd, and 7th days of culture: 10% of the total volume of CCK-8 solution was added to the culture medium and cultured in a cell culture incubator at 37°C and 5% CO2 for 2 h; the absorbance was measured at 620 nm using an enzyme reader, and the results were processed and analyzed using Graphpad Prism.

[0063] Test results such as Figure 3 The results showed that there was no significant difference in the proliferation and activity of DRG neurons in the four culture systems, indicating that the nerve repair material with a highly oriented bundle structure prepared according to this embodiment has good tissue compatibility.

[0064] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. A nerve repair material with a highly oriented tube bundle structure, characterized in that: The invention comprises a wood cellulose support with a natural porous structure and a hydrogel matrix, wherein the hydrogel matrix is ​​filled in the pores of the wood cellulose support with a natural porous structure.

2. The nerve repair material with a highly oriented tube bundle structure according to claim 1, characterized in that: The lignocellulose scaffold with a natural porous structure is a lignocellulose scaffold with a natural porous structure that has been subjected to delignification treatment.

3. The nerve repair material with a highly oriented tube bundle structure according to claim 1, characterized in that: The nerve repair material with a highly oriented tube bundle structure has a radial overall compressive strength of 50-2000 kPa and a compression modulus of 120-10000 kPa.

4. The nerve repair material with a highly oriented tube bundle structure according to claim 1, characterized in that: The compression modulus of the hydrogel matrix is ​​1.25-500 kPa.

5. The nerve repair material with a highly oriented tube bundle structure according to claim 1, characterized in that: The hydrogel matrix is ​​selected from methacrylated hydrogels.

6. The nerve repair material with a highly oriented tube bundle structure according to any one of claims 1 to 5, characterized in that: The appearance shape of the nerve repair material with a highly oriented tube bundle structure is selected from one or more of a catheter, a cuboid, a film, a cylinder, an ellipsoid or other irregular shapes.

7. A method for preparing a nerve repair material having a highly oriented tube bundle structure, characterized in that: The steps include: (1) Cutting natural wood and immersing it in a hot solution containing NaOH and Na2SO3 for normal pressure cooking; then rinsing it and transferring it to a hot H2O2 solution for further normal pressure cooking until a white sample is obtained; placing the obtained white sample in a dialysis bag for dialyzing with distilled water, and then freeze-drying it to obtain a lignocellulose scaffold; (2) immersing the lignocellulose scaffold in a hydrogel matrix solution containing a photoinitiator, and treating under negative pressure until the internal spaces of the lignocellulose scaffold are filled with the hydrogel matrix solution; (3) Irradiating with ultraviolet light to fully gel the hydrogel matrix.

8. The preparation method according to claim 7, characterized in that: In step (1), the concentration of NaOH in the hot solution is 2.5 mol / L, the concentration of Na2SO3 is 0.4 mol / L, and the concentration of H2O2 in the hot solution is 2.5 mol / L.

9. The preparation method according to claim 7, characterized in that: The wavelength of the ultraviolet light in step (3) is 405nm, and the light intensity is 0.1-3mW / cm 2 .

10. The preparation method according to claim 7, characterized in that: The irradiation time in step (3) is 10-120s.

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