Neural intimal basement membrane extracellular matrix repair material and preparation method thereof

Abstract

The invention discloses an endometrial and basilar membrane extracellular matrix repair material which is a porous patch obtained by treating endometrial and basilar membrane tissues of peripheral nerves of mammals. The endometrial and basilar membrane extracellular matrix repair patch only retains the endometrial and basilar membrane matrix of peripheral nervous tissues, is not doped with non-endometrial and basilar membrane tissues, and has a remarkable effect of specifically promoting regeneration of injured nerves. The invention also discloses a preparation method of the endometrial-basilar membrane extracellular matrix repair material, and the endometrial-basilar membrane extracellular matrix repair material is prepared by selecting mammal nervous tissues, separating nerve tracts, decellularizing and tabletting.

Description

Technical Field

[0001] This invention relates to the field of nerve repair technology, and in particular to a nerve endometrial basement membrane extracellular matrix repair material for repairing peripheral nerve injuries and its preparation method. Background Technology

[0002] Peripheral nerve injury is a severe and potentially disabling trauma, requiring surgical repair via microsurgery after nerve rupture. Despite significant advancements in microsurgical suturing techniques, the regenerative capacity of peripheral nerves remains limited. The injured area is prone to persistent fibrosis, scarring, and disordered axonal tangles, hindering patient recovery and leading to poor prognosis. The medical community typically utilizes biological repair patches to act as a biological barrier after nerve injury, effectively reducing the negative impact of the adverse post-traumatic microenvironment on regeneration and repair.

[0003] Patent (CN201510364908.5) discloses a peripheral nerve repair membrane and its preparation method, made from non-extracellular matrix repair materials such as chitosan, collagen, hyaluronic acid, chitin, trehalose, and gelatin. The nerve repair membrane prepared by this method does not have the function of specifically guiding nerve regeneration. Patent (CN 107737374 A) discloses an extracellular matrix nerve repair membrane material, which removes cellular components that can trigger immune responses by decellularizing animal peripheral nerve tissue and then processes it to form a porous membrane material. This matrix material is derived from peripheral nerve tissue and has tissue specificity. However, the content of basement membrane components in this repair membrane material is relatively low, and the effect of promoting nerve regeneration is not ideal. Summary of the Invention

[0004] In view of this, the present invention provides an extracellular matrix repair material for the neuroendometrial basement membrane for repairing peripheral nerve injuries and a method for preparing the same, aiming to solve or at least improve the above-mentioned problems to a certain extent.

[0005] To address the above problems, this invention provides a method for preparing an extracellular matrix repair material for the neuronal basement membrane, comprising the following steps:

[0006] (1) Select mammalian nerve tissue, remove attached connective tissue, and freeze-dry; (2) Vacuum dry the peripheral nerve tissue and separate the freeze-dried nerve bundles from it; (3) Decellularize the freeze-dried nerve bundles separated in step (2) to obtain a decellularized nerve bundle scaffold; (4) Perform acid hydrolysis, enzymatic digestion and alkali neutralization reaction on the decellularized nerve bundle scaffold in step (3) in sequence, and freeze-dry to obtain the extracellular matrix repair material of the nerve endometrium basement membrane.

[0007] In some embodiments, the method further includes pressing the neuronal basement membrane extracellular matrix repair material from step (4) into a neuronal basement membrane extracellular matrix repair patch of the desired size.

[0008] In some embodiments, the nerve tissue in step (1) is taken from the peripheral nerve tissue of a human, dog or pig, and the nerve tissue is frozen at -20°C for 1 hour.

[0009] In some embodiments, after step (1) and before step (2), frozen nerve tissue slices are soaked and dispersed in distilled water and then placed at -80°C for 3 hours.

[0010] In some embodiments, the temperature of the vacuum drying process in step (2) is -20℃ to -80℃, and the processing time is 2 to 6 hours.

[0011] In some embodiments, in step (2), the anatomical structures of nerve bundles and non-nerve bundles of the freeze-dried peripheral nerve tissue are identified under a microscope, and the freeze-dried nerve bundles are removed using micro-forceps.

[0012] In some embodiments, in step (3), the lyophilized nerve bundle scaffold is decellularized by sequentially using distilled water, 3%–5% Triton X-100 solution and 3%–5% sodium deoxycholate solution.

[0013] In some embodiments, step (4) involves acid hydrolysis using an HCl solution with a concentration of 0.01N to 0.1N, enzymatic digestion using a pepsin solution with a mass concentration of 5% to 30%, digestion time of 12 to 24 hours, neutralization treatment using a NaOH solution with a pH of 7.4, and freeze-drying at -20°C.

[0014] In another aspect, the present invention provides a neuroendometrial basement membrane extracellular matrix repair material, which is prepared by the preparation method of the neuroendometrial basement membrane extracellular matrix repair material described in any one of the above claims.

[0015] In some embodiments, the extracellular matrix repair material of the neuroendometrial basement membrane is a porous patch with a thickness of 0.1 mm to 0.5 mm.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) Before the decellularization treatment of the peripheral nerves of mammals, the non-nerve bundle tissues of the repair material prepared in this invention are removed, and only the freeze-dried nerve bundle scaffold of the peripheral nerve tissue is retained to obtain a high-purity neuronal basement membrane matrix material. It is completely free of interfascicular connective tissue (fat, fascia) and non-nerve matrix such as the epineurium, which can give full play to the specific advantages of the peripheral nerves and improve the nerve repair effect.

[0018] (2) The extracellular matrix repair material of the neuroendometrial basement membrane prepared by the present invention is derived from the complete neuroendometrial basement membrane matrix. Therefore, it has high performance stability and will not be affected by non-neural tissue matrix, so it will not have large performance fluctuations due to different anatomical positions of surrounding nerve tissue.

[0019] (3) The extracellular matrix repair material of the nerve endometrium basement membrane prepared by the present invention is rich in microporous structure. When it is attached to the anastomosis of the severed nerve, it can effectively play the role of a temporary biological barrier before the autologous nerve barrier is fully restored, reduce the adverse stimulation of the trauma microenvironment, facilitate integration with autologous tissue during the autologous nerve barrier repair process, and accelerate nerve repair.

[0020] (4) The chemical reagents used in the process of decellularizing and extracting nerve tissue in this invention are highly water-soluble and do not react with the extracellular matrix of the nerve endometrium and basement membrane. They can be completely removed after washing with water, which can effectively avoid adverse reactions to the human body during use. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0022] Figure 1 This image shows a section of Panamanian pig sciatic nerve tissue stained with Laminin immunohistochemistry.

[0023] Figure 2 A-2F shows a scanning electron microscope image of the sciatic nerve of a Panamanian pig stained with laminin immunohistochemistry.

[0024] Figure 3 The image shows a photograph of the Panamanian pig ischius, taken at the marked location.

[0025] Figure 4 A-4B shows cross-sectional photographs of the sciatic nerve of a Panamanian pig before and after freeze-drying, taken under a microscope.

[0026] Figure 5 The repair materials shown in the examples and comparative examples demonstrate the promoting effect of nerve regeneration in in vitro cell culture experiments.

[0027] Figure 6 The diagram shows the statistical results of the maximum neural axon assay in in vitro cell culture experiments for the repair materials of the embodiments and comparative examples.

[0028] Figure 7A-7B shows a comparison of axon regeneration in in vivo repair experiments using the repair materials from the examples and comparative examples.

[0029] Figure 8 The diagram shows the regenerated axons in in vivo repair experiments of the repair materials used in the examples and comparative examples. Detailed Implementation

[0030] 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 a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Laminin immunohistochemical staining experiment

[0032] Complete sciatic nerve tissue was harvested from anesthetized live Panama pigs and subjected to Laminin immunohistochemical staining. The tissue section photographs are shown below. Figure 1 As shown below. The microstructure was observed under a scanning electron microscope, as follows. Figure 2 As shown in A-2F.

[0033] Figure 1 This shows that in peripheral nerve tissue, laminin, an extracellular matrix component of the basement membrane, is enriched in nerve bundles. Figure 2 A and Figure 2 B shows that, under scanning electron microscopy, nerves are composed of numerous nerve bundles and non-nerve bundle connective tissue. Figure 2 A, 2B). Figure 2 B is Figure 2 A magnified view of the area within the square in A. Figure 2 C-2F are magnified views of point CF in Figure B. Figure 2 C and 2D show that the non-nerve bundle connective tissue is the epineurium. Figure 2 C) and dense longitudinal fibers ( Figure 2 D). Figure 2 E indicates that the nerve bundle is located within the perineal membrane (marked with &). Figure 2 F shows that the nerve bundle contains numerous endoneurotic basement membrane tubes, with the basement membrane as the inner wall (marked with *) and the endoneurium as the outer wall (marked with #). The nerve bundle is clearly rich in laminin, the extracellular matrix of the basement membrane.

[0034] Examination of the proportion of nerve bundles and non-nerve bundles

[0035] Different sites of the sciatic nerve were harvested from live Panamanian pigs under anesthesia. The sampling diagram is shown below. Figure 3The area of ​​nerve bundles (Laminin-positive areas) and the area of ​​intact nerves at different sampling sites were measured and calculated using image processing software, and the proportion of nerve bundles was calculated. The results are shown in Table 1 below.

[0036] Table 1. Ratio of nerve tracts to non-nerve tracts

[0037]

[0038]

[0039] The results showed that the proportion of nerve bundles and intact peripheral nerve tissue freeze-dried scaffolds was 15.2%-33.6% (average 24.6%), while the proportion of non-nerve bundles was 66.4%-84.8% (average 75.4%). The ratio of the cross-sectional area of ​​nerve bundles to non-nerve bundles was 33.4% ± 11.8%, indicating that nerve bundles accounted for a lower proportion of intact nerves. Furthermore, the content of neuromatrix in peripheral nerve tissue varied across different anatomical levels.

[0040] Example: Preparation of an extracellular matrix repair patch for the endoneurium basement membrane (ECM group)

[0041] Step (1): Take a number of sciatic nerve tissues from Panamanian pigs, quickly remove excess connective tissues such as fascia, fat and blood clots attached to the nerve epineurium, shake twice with sterile distilled water and soak in sterile distilled water for 30 minutes, then place the nerve tissue on a plate and wipe off the liquid on its surface.

[0042] Nervous tissue of Panamanian pigs under a microscope. Figure 4 A shows a cross-section of the peripheral nerve tissue in its fresh state, where nerve bundles appear light yellow (red arrows) and non-nerve bundles appear dark yellow (white arrows). Nerve bundles (red arrows) and non-nerve bundles (white arrows) can be vaguely distinguished with the naked eye, but it is difficult to separate the nerve bundles and non-nerve bundles at this time.

[0043] The nerve tissue was then placed at -20°C for 1 hour to freeze-harden it in order to maintain structural stability.

[0044] Step (2): The frozen and hardened nerve tissue from step (1) was rapidly cut to obtain several tissue slices with a flat cut surface and a thickness of 3 mm. These slices were then immersed in a petri dish containing distilled water to fully disperse them until they floated on the surface. The petri dish was then placed at -80°C for 3 hours. Subsequently, the tissue slices were removed and subjected to rapid vacuum drying. The vacuum drying temperature was -20°C, the vacuum degree was 0.1 kPa, and the treatment time was 5 hours to obtain freeze-dried peripheral nerve tissue.

[0045] Observe the cross-section of freeze-dried porcine sciatic nerve tissue under a microscope. For example... Figure 4As shown in Figure B, nerve bundles (red arrows at the top) and non-nerve bundles (white arrows at the bottom) can be clearly distinguished with the naked eye, demonstrating that nerve bundles and non-nerve bundles in peripheral nerve tissue can be differentiated through microsurgical manipulation. Subsequently, the anatomical structures of nerve bundles and non-nerve bundles in the freeze-dried peripheral nerve tissue were identified under a microscope. The freeze-dried nerve bundle scaffolds were removed one by one using micro-forceps, and the remaining non-nerve bundle tissue was discarded.

[0046] Step (3): The lyophilized nerve bundle scaffold obtained in step (2) was washed twice with distilled water and then subjected to decellularization extraction treatment with double distilled water at 4°C, Triton X-100 solution and sodium deoxycholate solution in sequence to remove the cellular components in the nerve bundle scaffold and obtain the decellularized nerve bundle scaffold.

[0047] The Triton X-100 solution has a mass concentration of 4%, and the sodium deoxycholate solution has a mass concentration of 4%.

[0048] Step (4): The decellularized nerve bundle scaffold was cut into pieces at high speed and ground into powder. Hydrochloric acid solution was added to dissolve it, and then pepsin was added to digest it. After stirring at room temperature for 1 hour, NaOH solution was added to neutralize the reaction and the resulting gel solution was poured into a culture dish. The gel solution was vacuum dried at -20℃ to obtain sheet-like nerve endometrial basement membrane matrix material.

[0049] The concentration of the HCl solution was 0.01N, the mass ratio of pepsin to decellularized matrix was 1:10, and the pH of the NaOH solution was 7.4.

[0050] The endometrial basement membrane matrix material obtained in step (4) was then flattened, compacted, and its edges trimmed with a smooth stick to obtain a porcine endometrial basement membrane extracellular matrix repair patch with a thickness of 0.02 mm.

[0051] Preparation of comparative decellularized peripheral nerve matrix repair patches (decellularized nerve ECM group)

[0052] Step (1): Take the complete sciatic nerve tissue of a live Panamanian pig under anesthesia, quickly remove the fascia, fat and blood clots attached to the nerve epineurium, shake it twice with sterile distilled water and soak it in sterile distilled water for 30 minutes, then take the nerve tissue, place it on a plate and wipe off the liquid on its surface, and then place it in a -20℃ environment for 1 hour to freeze harden.

[0053] Step (2): After washing the sciatic nerve tissue from step (1) twice with distilled water, it was then subjected to decellularization extraction treatment with double distilled water at 4°C, Triton X-100 solution, and sodium deoxycholate solution in sequence to remove the cellular components in the nerve bundle and obtain the peripheral nerve matrix scaffold.

[0054] The Triton X-100 solution has a mass concentration of 4%, and the sodium deoxycholate solution has a mass concentration of 4%.

[0055] Step (3): The peripheral nerve matrix scaffold was sheared and ground into powder at high speed, dissolved in 0.01N HCl solution, digested with pepsin, stirred at room temperature for 1 hour, and then neutralized with NaOH solution at pH=7.4 to obtain a gel solution. The gel solution was poured into a culture dish and vacuum dried at -20℃ to obtain decellularized peripheral nerve matrix material.

[0056] Subsequently, the decellularized peripheral nerve matrix material from step (3) was flattened, compacted, and its edges trimmed with a smooth stick to obtain a decellularized peripheral nerve matrix repair patch with a thickness of 0.02 mm.

[0057] In vitro cell culture experiment

[0058] The extracellular matrix repair patch of the nerve endometrium basement membrane in the embodiment and the decellularized peripheral nerve matrix repair patch in the comparative example were used in an in vitro dorsal root ganglion cell culture experiment to compare the differences in their nerve regeneration promoting effects. The results are as follows: Figure 5 and 6 As shown, compared to the comparative decellularized peripheral nerve matrix culture system, the maximum diameter of nerve fibers in the extracellular matrix culture system of the neuronal endometrial basement membrane in this embodiment is significantly increased. Statistical graphs of the two sets of data are shown below. Figure 6 This indicates that the difference was statistically significant (P < 0.05).

[0059] In vivo repair experiment

[0060] A hydrogel was prepared by dissolving a comparative endoneurotic basement membrane extracellular matrix repair patch (n=5, i.e., 5 parallel experiments) and the decellularized peripheral nerve matrix repair patch of this embodiment (n=5). The hydrogel was then infused into a silicone catheter with an inner diameter of 1.2 mm and a length of 1 cm, and used to repair sciatic nerve defects in SD rats. At week 10 post-surgery, the regenerated nerve tissue was harvested for histological examination, and the regenerated axons were compared. The results are shown in Figure 7.

[0061] Figure 7 Example A shows that the extracellular matrix repair patch group of the neuronal endometrial basement membrane was characterized by a large number of regenerated myelinated axons. Figure 7 B shows that the decellularized peripheral nerve matrix repair patch group had fewer regenerated myelinated axons. Statistical graphs of the two groups are shown below. Figure 8 The difference was statistically significant (P < 0.05).

[0062] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for preparing an extracellular matrix repair material for the neuronal basement membrane, characterized in that, Includes the following steps: (1) Select peripheral nerve tissue from mammals, remove the attached connective tissue, and freeze it. (2) Vacuum drying of peripheral nerve tissue to obtain freeze-dried peripheral nerve tissue, from which freeze-dried nerve bundles are separated. (3) The lyophilized nerve bundles separated in step (2) are decellularized to obtain a decellularized nerve bundle scaffold; (4) The decellularized nerve bundle scaffold from step (3) is subjected to acid hydrolysis, enzymatic digestion and alkali neutralization reaction in sequence, and then freeze-dried to obtain the extracellular matrix repair material of the nerve endometrium basement membrane.

2. The method for preparing the neuroendometrial basement membrane extracellular matrix repair material according to claim 1 further includes pressing and processing the neuroendometrial basement membrane extracellular matrix repair material of step (4) into a neuroendometrial basement membrane extracellular matrix repair patch of the required size.

3. The method for preparing the extracellular matrix repair material for the neuronal basement membrane according to claim 1, characterized in that, The peripheral nerve tissue mentioned in step (1) is obtained from the peripheral nerve tissue of humans, dogs or pigs, and the peripheral nerve tissue is frozen at -20°C for 1 hour.

4. The method for preparing the extracellular matrix repair material for the neuroendometrial basement membrane according to claim 1, characterized in that, It also includes slicing frozen peripheral nerve tissue after step (1) and before step (2), dispersing it by soaking it in distilled water, and then placing it at -80°C for 3 hours.

5. The method for preparing the extracellular matrix repair material for the neuronal basement membrane according to claim 1, characterized in that, The temperature of the vacuum drying process in step (2) is -20℃ to -80℃, the processing time is 2 to 6 hours, and the vacuum degree is 0.01 to 0.1 kPa.

6. The method for preparing the extracellular matrix repair material for the neuronal basement membrane according to claim 1, characterized in that, In step (2), the anatomical structures of nerve bundles and non-nerve bundles of the freeze-dried peripheral nerve tissue are identified under a microscope, and the freeze-dried nerve bundles are removed using micro-forceps.

7. The method for preparing the extracellular matrix repair material for the neuronal basement membrane according to claim 1, characterized in that, In step (3), the lyophilized nerve bundle scaffold is decellularized in sequence using distilled water, 3%–5% Triton X-100 solution and 3%–5% sodium deoxycholate solution.

8. The method for preparing the extracellular matrix repair material for the neuronal basement membrane according to claim 1, characterized in that, In step (4), acid hydrolysis is performed using an HCl solution with a concentration of 0.01N to 0.1N, enzymatic digestion is performed using a pepsin solution with a mass concentration of 5% to 30%, the digestion time is 12 to 24 hours, alkali neutralization is performed using a NaOH solution with a pH of 7.4, and the mixture is freeze-dried at -20°C.

9. A neuroendometrial basement membrane extracellular matrix repair material, characterized in that, Prepared by the method for preparing extracellular matrix repair material of the neuroendometrial basement membrane as described in any one of claims 1-8.

10. The extracellular matrix repair material for the neuronal basement membrane according to claim 9, characterized in that, It is a porous patch with a thickness of 0.1mm to 0.5mm.

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