Method for preparing cell matrix nerve grafts to repair peripheral nerve damage

Abstract

The objective of the present invention is to provide a cell-substrate nerve graft for repairing peripheral nerve damage and a method for producing the same. [Solution] The present invention discloses a cell-matrix nerve graft for repairing peripheral nerve injury and a method for preparing the same. The cell-matrix nerve graft includes an acellular matrix and a scaffold, the acellular matrix is ​​obtained by decellularization after secretion and formation of stem cells, and is wrapped around the scaffold, which overcomes the disadvantages of autologous nerve grafts and avoids the immunogenicity of allogeneic cell grafts, provides a cell-matrix nerve graft favorable for the proliferation and migration of nerve cells, and achieves the goal of nerve regeneration and functional recovery by constructing a microenvironment favorable for axon growth, providing a feasible means for clinical treatment.

Description

Technical Field

[0001] The present invention relates to a cell matrix nerve graft for repairing peripheral nerve injury and a method for producing the same, and particularly relates to the technical field of medical biomaterials that can be implanted into the human body.

Background Art

[0002] Peripheral nerve injury is frequently seen clinically, which not only significantly affects the quality of life of patients but also increases the economic burden on patients. In the prior art, for short-distance peripheral nerve defects, a repair method of alignment suture by microsurgery is usually used. The repair of long-distance large peripheral nerve defects is achieved by autologous nerve transplantation or scaffold material transplantation. Autologous nerve transplantation is the gold standard for nerve transplantation and repair. That is, a part of the autologous nerve such as the medial or lateral antebrachial cutaneous nerve, ulnar nerve, femoral nerve, or radial nerve branch is surgically excised and then transplanted to repair the damaged nerve. The Schwann cells of the graft are activated and secrete a large amount of nerve growth factors, enabling rapid regeneration of axons. However, problems such as limitations of donor sources, immune rejection, mismatch of nerve diameters, and permanent denervation of donor sites restrict the clinical application of autologous nerve transplantation. The application of biological scaffold materials is to grow nerves in a certain direction to prevent the occurrence of neuromas, but the repair and regeneration process takes a long time, the nerve regeneration rate is slow, and the effector and receptor atrophy soon.

[0003] Tissue-engineered nerve grafts are organic integrations composed of scaffold materials, supporting cells, extracellular matrix, nerve growth factors, etc., and can be used as a bridge to guide severed nerve fibers from the proximal end of the injury to the distal end. However, the translation to clinical practice faces many problems such as the type and amount of cells or factors, cell viability or factor activity, stability of cell phenotypes, treatment time, regulatory intervention, and high cost.

[0004] Cell-free matrix (ACM) is increasingly favored by researchers as a viable tissue engineering graft and is used for repairing tissue damage. Decellularization of allogeneic or heterogeneous nerve (or non-nerve) tissue using various physical, chemical, and enzymatic methods can produce tissue-derived cell-free matrix. Tissue-derived cell-free matrix is ​​a suitable non-cellular biomaterial for creating nerve scaffolds and can be used as an alternative to autologous nerve grafts for repairing peripheral nerve defects. Compared to scaffolds made from individual extracellular matrix components, tissue-derived cell-free matrix scaffolds (also known as cell-free nerve grafts) have a superior ability to preserve the basic structure of natural tissue and promote peripheral nerve regeneration. While tissue or organ-derived cell-free matrix scaffolds possess biochemical and physical factors that support tissue or cell growth, obtaining donor tissue of consistent quality remains a significant limitation. Furthermore, there are several drawbacks, including potential immunogenicity, pathogen transfer, uncontrolled degradation, and high manufacturing costs. Therefore, repair and regeneration after clinical peripheral nerve injury remain significant challenges. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to overcome the shortcomings of the prior art and to provide a cell matrix nerve graft and a method for producing the same for repairing peripheral nerve damage, which is advantageous for cell adhesion and migration and promotes nerve regeneration and functional recovery. [Means for solving the problem]

[0006] To achieve the above objective, the present invention employs the following technical means.

[0007] In a first aspect, the present invention provides a cell matrix nerve graft for repairing peripheral nerve injury, comprising a cell-free matrix and a scaffold. The cell-free matrix is ​​obtained by the secretion and subsequent decellularization of stem cells and retains various important components and frameworks of the extracellular matrix, thereby favoring the induction of nerve cell adhesion and axonal regeneration, promoting peripheral nerve regeneration and functional recovery, and the cell-free matrix is ​​wrapped around the scaffold.

[0008] When combined with the first embodiment, the stem cells are further human bone marrow-derived mesenchymal stem cells, which facilitates the clinical application of tissue-engineered nerve grafts.

[0009] Furthermore, the aforementioned scaffold is a biodegradable scaffold containing chitosan and recombinant human collagen, characterized by good biocompatibility, degradability, and the ability to guide the growth direction of nerve axon regeneration.

[0010] In a second embodiment, the present invention provides a method for preparing a cell matrix nerve graft for repairing peripheral nerve damage as described in any one of the above descriptions. This method comprises the following steps: Steps to prepare a cell-free matrix, The process of wrapping a cell-free matrix around the scaffold to construct the initial morphology of the cell matrix nerve graft. A step of placing the initial morphology of the cell matrix nerve graft at 2-6°C for at least 24 hours to allow it to self-organize and form a tubular structure, and The process of freeze-drying the tubular structure to construct it as a cell matrix nerve graft. Includes.

[0011] The cell matrix nerve grafts produced by the above method do not contain toxic exogenous substances introduced during the production process and possess good biocompatibility, biodegradability, and good mechanical properties.

[0012] When combined with the second embodiment, the cell-free matrix can be wound in multiple layers around the scaffold, wrapped around the biodegradable scaffold, and form a tubular structure through self-organization, providing not only pathways necessary for nerve cell growth but also inducible and oriented growth.

[0013] Furthermore, the cell-free matrix is ​​rolled up in 6, 9, or 12 layers around the biodegradable scaffold and wrapped around the biodegradable scaffold.

[0014] Furthermore, the freeze-drying temperature is -80°C.

[0015] Furthermore, the process for preparing the cell-free matrix is ​​as follows: Human bone marrow-derived mesenchymal stem cells are diluted and seeded in a large culture dish. When the cell saturation density reaches 90% or higher, serum-free medium containing ascorbic acid is added and the cells are stimulated for 7 to 14 days to promote the secretion of extracellular matrix from the cells. The matrix is ​​washed with phosphate buffer, hypotonic treatment is performed by adding sterile ultrapure water, the sterile ultrapure water is removed, cell lysate is added for extraction, and then the matrix is ​​washed again with phosphate buffer to obtain a cell-free matrix. [Effects of the Invention]

[0016] Compared to conventional technology, the advantageous effects of the present invention include: This invention provides a natural extracellular matrix by decellularizing cells after culture, retaining various important components and frameworks of the extracellular matrix, which is advantageous for inducing nerve cell adhesion and axonal regeneration, and promotes peripheral nerve regeneration and functional recovery. Cell matrix nerve grafts do not contain toxic exogenous substances introduced during the fabrication process, and possess good biocompatibility, biodegradability, and good mechanical properties. By selecting human bone marrow-derived mesenchymal stem cells and using serum-free media, the clinical application of tissue-engineered nerve grafts is facilitated. The acellular matrix can be wrapped around the biodegradable scaffold and can form a tubular structure by self-organization, not only providing a path necessary for the growth of nerve cells, but also providing induced and oriented growth. The cell matrix nerve graft prepared by the method of the present invention avoids the immunogenicity of allogeneic cell transplantation and is suitable for use by a large population.

Brief Description of Drawings

[0017] [Figure 1] Optical microscope images before and after the preparation of the acellular matrix provided by the examples of the present invention (A is the optical microscope image of stem cells before decellularization, and B is the optical microscope image of the matrix after decellularization). [Figure 2] Immunochemical photographs of CollagenIV and Fibronectin of the acellular matrix provided by the examples of the present invention (A shows CollagenIV staining, and B shows Fibronectin staining). [Figure 3] Scanning electron microscope images of the acellular matrix provided by the examples of the present invention (A is an electron microscope image at 2500 times magnification, and B is an electron microscope image at 5000 times magnification). [Figure 4] Scanning electron microscope images of the cell matrix nerve graft provided by the examples of the present invention (A is an electron microscope image at 800 times magnification, and B is an electron microscope image at 2500 times magnification). [Figure 5] Schematic diagram showing the electrophysiological examination results of the regenerated nerves provided by the examples of the present invention (the left figure is the composite muscle action potential waveform diagram of each group, where A represents the cell matrix group, B represents the autologous group, and C represents the sham operation group. The right figure is the comparison diagram of the composite muscle action potential amplitudes of the regenerated nerves of different samples in each group, ****p < 0.0001). [Figure 6] Transmission electron microscope image of the cross-section of the middle part of the regenerated nerve provided by the examples of the present invention (the upper part is a transmission electron microscope image at 20000 times magnification of the myelin sheath part expansion of each group, where A represents the cell matrix group, B represents the autologous group, and C represents the sham operation group. The lower part is the comparison diagram of the number of myelin sheath lamellae in each group, ****p < 0.0001). [Figure 7]A schematic diagram showing the results of the wet muscle weight ratio of the target muscle provided by an embodiment of the present invention (the upper part is a schematic diagram of the external morphology of the gastrocnemius muscle in each group, where A represents the cell matrix group, B represents the autologous group, and C represents the sham operation group. The lower part is the analysis result of the wet muscle weight ratio of the gastrocnemius muscle in each group, ***p < 0.001, ****p < 0.0001). [Figure 8] A diagram showing the results of the reconstruction of the motor endplate provided by an embodiment of the present invention (the upper part is a schematic diagram of the maturity of the motor endplate, the middle part is an observation diagram of the motor endplate in each group, where A represents the cell matrix group, B represents the autologous group, and C represents the sham operation group. The lower part is a histogram of the statistical analysis of the ratio of the motor endplate at different times). [Figure 9] A diagram showing the results of the cross-sectional area of the target muscle fibers provided by an embodiment of the present invention (the upper part is an observation diagram of the cross-section of the gastrocnemius muscle fibers in each group, where A represents the cell matrix group, B represents the autologous group, and C represents the sham operation group. The lower part is a statistical comparison diagram of the cross-sectional area of the gastrocnemius muscle fibers in each group, **p < 0.01, ****p < 0.0001).

Embodiment for Implementing the Invention

[0018] Hereinafter, the present invention will be further described with reference to the accompanying drawings. The following examples are only used to more clearly illustrate the technical means of the present invention and cannot be used to limit the protection scope of the present invention.

[0019] (Example 1) Preparation of acellular matrix: hBMSC cells of passage P3 (purchased from the ATCC cell bank) were seeded in a large culture dish at 10 5 cells / ml. When the cell saturation density reached 90% or more, a serum-free medium containing ascorbic acid was added for stimulation to promote the secretion of extracellular matrix from the cells. In this example, a stimulation of 7 - 14 days was selected, washed three times with phosphate buffer, then sterile ultrapure water was added, and hypotonic treatment was performed at 37°C ± 0.5°C for at least 10 minutes, After removing the sterile ultrapure water, a cell lysis solution consisting of 3% Triton X-100 and 2% SDS was added, and the mixture was extracted at 37°C ± 0.5°C for no more than 5 minutes. After washing three times with phosphate buffer, a cell-free matrix was obtained.

[0020] In this example, when human bone marrow-derived mesenchymal stem cells were stimulated with ascorbic acid for 10 days, the secreted extracellular matrix gradually thickened over time. In the later stages, the cells aged, their adhesion decreased, and the extracellular matrix appeared "rolled up," resulting in the sample being deemed unsuitable.

[0021] The cell-free matrix obtained in this example is immersed in phosphate buffer and stored in a low-temperature range of 2-6°C. In this example, a low-temperature environment of 4°C was selected, allowing for storage for approximately two weeks. Figure 1B shows an optical microscope image of the obtained cell-free matrix, while Figure 1A shows an optical microscope image of stem cells before decellularization.

[0022] Identification of partial components of the cell-free matrix by CollagenIV and Fibronectin immunochemistry: The obtained cell-free matrix was fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed three times with phosphate buffer, and then incubated overnight at 4°C with primary antibodies: rabbit anti-collagenIV (1:100) and sheep anti-fibronectin (1:100). After washing three times with phosphate buffer, add secondary antibodies: Cy3-Goat anti-rabbit IgG (H+L) (1:600) and donkey anti-sheep (AF647) (1:500) and incubate overnight at 4°C. The cells were washed three times with phosphate buffer, mounted with DAPI staining, and detected using a fluorescence confocal microscope (DMR, Leica). Figure 2 shows the immunochemical diagrams of Collagen IV and Fibronectin in the cell-free matrix provided by the embodiment of the present invention. A shows Collagen IV staining, and B shows Fibronectin staining.

[0023] This invention utilizes a cell-free matrix derived from cultured cells, and compared to a tissue-derived cell-free matrix, it eliminates the transfer of pathogens during culture and proliferation, overcomes the inherent structural limitations of extracellular matrix derived from tissues or organs, and, when combined with tissue-engineered biomaterials, provides a controllable biodegradation rate and effective mechanical properties while maintaining the necessary geometric shapes and flexibility for scaffolding.

[0024] The cell matrix in the tissue-engineered nerve graft used in this invention is obtained by secreting cells to be cultured and decellularizing them after formation. A novel cell matrix nerve graft was constructed by combining a cell-free matrix derived from hBMSCs with a biodegradable scaffold. Since immunogenicity decreases or disappears after transplantation, it is suitable for use in large populations. The BMSCs of this invention are human-derived, and serum-free culture was performed between them, establishing a strong foundation for bridging the gap between tissue-engineered nerve grafts and clinical applications.

[0025] (Example 2) Electron microscopy detection of cell-free matrix: Figure 3 shows scanning electron microscope images of a cell-free matrix provided according to an embodiment of the present invention, where A is a 2500x electron microscope image and B is a 5000x electron microscope image. Scanning electron microscope observation revealed that the cell-free matrix exhibits a dense fibrous network structure, with spherical substances of varying sizes distributed between the network fibers, and that the fiber arrangement has a certain directionality.

[0026] Specific method for electron microscopy detection of cell-free matrix in this embodiment: Place the cell-free matrix on a 14 mm circular glass slide, fix it with 4% glutaraldehyde in a refrigerator at 4°C for 2-4 hours, and rinse three times with phosphate buffer every 10 minutes. Fix in 1% osmium acid in the dark at room temperature for 2 hours, then rinse three times with redistilled water for 10 minutes each time. Subsequently, dehydration treatment with gradient ethanol for at least 10 minutes each time (ethanol concentrations of 30%, 50%, 70%, 80%, and 95%) was performed. Finally, the samples were immersed overnight in anhydrous ethanol, replaced with fresh anhydrous ethanol the next day, and then substituted with isoamyl acetate (ratios of 1:1, 1:2, and pure isoamyl acetate). After drying in a critical point dryer, the samples were gold plated and observed with a Hitachi S-3400II scanning electron microscope.

[0027] (Example 3) Construction of cell matrix nerve grafts: Using calipers, a jelly-like, membrane-like cell-free matrix approximately 0.12 mm thick is wrapped around a biodegradable scaffold in three configurations: 6, 9, or 12 layers, to construct the initial morphology of the cell matrix nerve graft. The initial morphology of the cell matrix nerve graft is placed at 2-6°C for at least 24 hours to form a tubular structure through self-organization. In this example, self-organization at 4°C for 24 hours was selected, and then the cell matrix nerve graft was constructed by freeze-drying at -80°C.

[0028] Figure 4 shows scanning electron microscope images of a cell matrix nerve graft provided according to an embodiment of the present invention, where A is an 800x electron microscope image and B is a 2500x electron microscope image. Scanning electron microscope results showed that the graft exhibited a certain directionality and that spherical substances of different sizes were distributed between the fibers.

[0029] Specific methods of scanning electron microscopy: The cell matrix nerve graft was cut longitudinally down the middle, fixed with 4% glutaraldehyde in a refrigerator at 4°C for 2-4 hours, and rinsed three times with phosphate buffer every 10 minutes. Fix in 1% osmium acid in the dark at room temperature for 2 hours, then rinse three times with redistilled water for 10 minutes each time. Subsequently, dehydration treatment with gradient ethanol for at least 10 minutes each time (ethanol concentrations of 30%, 50%, 70%, 80%, and 95%) was performed. Next, the samples were immersed overnight in anhydrous ethanol, and the following day the ethanol was replaced with fresh anhydrous ethanol. After substituting the anhydrous ethanol with isoamyl acetate (ratios of 1:1, 1:2, and pure isoamyl acetate), the samples were dried in a critical point dryer, gold plated, and then observed with a Hitachi S-3400II scanning electron microscope.

[0030] (Example 4) Repair of sciatic nerve defects in rats using cell matrix nerve grafts: We repaired sciatic nerve defects in rats using cell matrix nerve grafts, and detected nerve regeneration rate and sciatic nerve function recovery using electrophysiological, transmission electron microscopy, and immunohistochemical methods. The specific steps were as follows: First, a 10mm rat sciatic nerve defect model was established and the rats were randomly divided into three groups: A, B, and C. Group A, which used cell matrix nerve grafts to repair the rat sciatic nerve defect, was called the cell matrix group. Group B, which used autologous nerves to repair the rat sciatic nerve defect, was called the autologous group. Group C, in which the sciatic nerve was exposed without repair of the defect, was called the sham surgery group. Twelve weeks after surgery, the sciatic nerve on the surgical side was exposed under moderate anesthesia, and neuroelectrophysiological testing was performed.

[0031] Figure 5 is a schematic diagram showing the electrophysiological examination results of regenerated nerves provided by embodiments of the present invention. The left figure shows the composite muscle action potential waveforms of each group, with A representing the cell matrix group, B representing the autologous group, and C representing the sham surgery group. The right figure is a comparative diagram of the composite muscle action potential amplitudes of regenerated nerves in each group (****p<0.0001). Different shaped symbols in the figure represent different groups, and the number represents the number of samples. From the figure, it was found that the mean CMAP amplitudes of the cell matrix group, autologous group, and sham surgery group were 15.49±1.82mV, 15.33±2.98mV, and 21.56±1.67mV, respectively. There was no statistically significant difference between the cell matrix group and the autologous group (P>0.05).

[0032] Sections of the middle portion of regenerated nerves were taken and examined using transmission electron microscopy. Figure 6 shows transmission electron microscopy images of cross-sections of the middle portion of regenerated nerves provided according to the embodiment of the present invention. The upper part shows 20,000x magnification transmission electron microscopy images of the myelin sheath portion of each group, with A representing the cell matrix group, B representing the autologous group, and C representing the sham surgery group. The lower part is a comparison chart of the number of myelin sheath layers in each group (****p<0.0001). From the figure, it was found that the number of myelin sheath layers in the cell matrix group, autologous group, and sham surgery group were 41±5, 46±6, and 90±17, respectively. There was no statistically significant difference between the cell matrix group and the autologous group (P>0.05).

[0033] Measurement of the wet muscle weight ratio and motor endplate of the target muscle after surgery is an important indicator for evaluating the reconstruction of target muscle weight innervation function by regenerating nerves. Denervation of the muscle occurs early, and over time, regenerating nerves re-innervate the target muscle. Figure 7 is a schematic diagram showing the results of the wet muscle weight ratio of the target muscle provided by an embodiment of the present invention. The upper part shows the external morphology of the gastrocnemius muscle of each group, with A being the cell matrix group, B being the autologous group, and C being the sham surgery group. The lower part shows the analysis of the wet muscle weight ratio of the gastrocnemius muscle of each group (***p<0.001, ****p<0.0001). As can be seen from the figure, at 12 weeks post-surgery, the wet muscle weight ratios of the gastrocnemius muscle of the cell matrix group, autologous group, and sham surgery group were 0.48±0.22, 0.58±0.16, and 0.88±0.06, respectively. There was no statistically significant difference between the cell matrix group and the autologous group (P>0.05).

[0034] The motor endplate is the neuromuscular junction between the nerve terminal and the target muscle it innervates, and it has a synaptic structure. The snake venom α-bungarotoxin specifically labels acetylcholine receptors on the postsynaptic membrane. Gastrocnemius muscle tissue from the surgical side was collected from rats 12 weeks after bridging surgery in cell matrix, autologous, and sham surgery groups. After fixation, dehydration, and freezing, the tissue was cut longitudinally, and the motor endplate was stained to analyze its maturity.

[0035] The maturation stages of the motor endplate are divided into the following stages: (1) Early: small in morphology, non-porous structure "plaque", (2) Mature: large in morphology, reticular, porous structure "pretzel", and (3) Mid-stage: "intermediate" (between mature and early). Figure 8 shows the reconstruction results of the motor endplate provided by the embodiment of the present invention. The upper part is a schematic diagram of the motor endplate maturation stages, with Pretzel being mature, Intermediate being transitional, and Plaque being immature. The middle section shows observations of the motor endplates of each group, with A representing the cell matrix group, B representing the autologous group, and C representing the sham surgery group. The lower part is a histogram of the statistical analysis of the proportion of motor endplates at different stages.

[0036] In the sham surgery group, the majority of motor endplates were mature, with large, butterfly-wing-shaped plaques and a reticular porous structure. In the cell matrix and autologous groups, the area of ​​motor endplates innervated by regenerated nerves was small and elongated. Quantitative statistics of motor endplates at 12 weeks post-surgery showed that, compared to the other two groups, the majority of motor endplates in the sham surgery group were mature (52%), while the proportion of mature motor endplates in the cell matrix group (44%) was not statistically significant compared to the autologous group (46%) (P>0.05).

[0037] Figure 9 shows the results of target muscle fiber cross-sectional area provided by the embodiment of the present invention. Gastrocnemius muscle tissue was collected from rats 12 weeks after bridging surgery in the cell matrix group, autologous group, and sham surgery group. The tissue was fixed, dehydrated, frozen, transversely dissected, and the extracellular matrix of the muscle fibers was stained with laminin. The upper part of Figure 9 shows cross-sectional observations of gastrocnemius muscle fibers in each group, with A representing the cell matrix group, B representing the autologous group, and C representing the sham surgery group. The lower part is a statistical comparison of the gastrocnemius muscle fiber cross-sectional area in each group (**p<0.01, ****p<0.0001). Statistically, the muscle fiber cross-sectional area of ​​the cell matrix group, autologous group, and sham surgery group was 923.4±98.85 μm², respectively. 2 , 1131±92.23μm 2 , 1606±229.9μm 2 There was no statistically significant difference between the cell substrate group and the autologous group (P>0.05).

[0038] The cell matrix nerve grafts used in this invention do not contain toxic exogenous substances introduced during the fabrication process and possess good biocompatibility, biodegradability, and excellent mechanical properties. By using a biodegradable scaffold and forming a tubular structure, it not only provides the pathways necessary for nerve cell growth but also offers inducible and oriented growth, and the cell-free matrix used can effectively promote nerve regeneration and functional recovery.

[0039] This invention addresses shortcomings in the prior art and provides a cell matrix nerve graft and a method for producing the same that are advantageous for cell adhesion and migration and can promote nerve regeneration. Stem cells are removed using in vitro decellularization technology to obtain a cell-free matrix, which is then wrapped around a biodegradable scaffold to form a cell matrix nerve graft, thereby repairing peripheral nerve defects. This invention overcomes the drawbacks of autologous nerve grafting and avoids the immunogenicity of allogeneic cell transplantation, providing a cell-free matrix that is advantageous for the proliferation and migration of nerve cells and has a certain orientation. By constructing a local microenvironment that facilitates the growth of regenerated axons, this invention achieves the ideal goal of rapid nerve growth and functional recovery, and provides a means that can be realized in clinical treatment.

[0040] It should be noted that the above describes only preferred embodiments of the present invention, and that those skilled in the art may make several improvements and modifications without departing from the technical principles of the present invention, and such improvements and modifications should be considered to fall within the scope of protection of the present invention.

[0041] (Note) (Note 1) A cell matrix nerve graft for repairing peripheral nerve damage, comprising a cell-free matrix and a scaffold, wherein the cell-free matrix is ​​obtained by the secretion and subsequent decellularization of stem cells and is wrapped around the scaffold.

[0042] (Note 2) The cell matrix nerve graft for repairing peripheral nerve damage, as described in Appendix 1, is characterized in that the stem cells are human bone marrow-derived mesenchymal stem cells.

[0043] (Note 3) The scaffold is a biodegradable scaffold containing chitosan and recombinant human collagen, and is characterized by being a cell matrix nerve graft for repairing peripheral nerve damage as described in Appendix 1.

[0044] (Note 4) A method for preparing a cell matrix nerve graft for repairing peripheral nerve damage as described in any one of the appendices 1 to 3, comprising the following steps: Steps to prepare a cell-free matrix, The process of wrapping a cell-free matrix around the scaffold to construct the initial morphology of the cell matrix nerve graft. A step of placing the initial morphology of the cell matrix nerve graft at 2-6°C for at least 24 hours to allow it to self-organize and form a tubular structure, and The process of freeze-drying the tubular structure to construct it as a cell matrix nerve graft. A method for preparing a cell matrix nerve graft for repairing peripheral nerve damage, characterized by including the following:

[0045] (Note 5) The method for preparing a cell matrix nerve graft for repairing peripheral nerve damage, as described in Appendix 4, characterized in that the freeze-drying temperature is -80°C.

[0046] (Note 6) The method for preparing a cell matrix nerve graft for repairing peripheral nerve damage, as described in Appendix 4, is characterized in that the cell-free matrix is ​​wound in multiple layers around a scaffold and wrapped around a biodegradable scaffold.

[0047] (Note 7) The method for producing a cell matrix nerve graft for repairing peripheral nerve damage, as described in Appendix 4, is characterized in that the cell-free matrix is ​​wound up in 6, 9, or 12 layers around a biodegradable scaffold and wrapped around the biodegradable scaffold.

[0048] (Note 8) The process for preparing the cell-free matrix is ​​as follows: Human bone marrow-derived mesenchymal stem cells are diluted and seeded in a large culture dish. Once the cell saturation density reaches 90% or higher, serum-free medium containing ascorbic acid is added and the cells are stimulated for 7 to 14 days. After washing with phosphate buffer and hypotonic treatment with sterile ultrapure water, the sterile ultrapure water is removed, cell lysate is added for extraction, and then the matrix is ​​washed again with phosphate buffer to obtain a cell-free matrix. A method for preparing a cell matrix nerve graft for repairing peripheral nerve damage as described in Appendix 4, characterized by the above.

Claims

1. A method for preparing a cell matrix nerve graft for repairing peripheral nerve damage, The cell matrix nerve graft comprises a membranous cell-free matrix and a biodegradable scaffold. The aforementioned membranous cell-free matrix contains extracellular matrix secreted by human bone marrow-derived mesenchymal stem cells, and is wound in multiple layers around the biodegradable scaffold, and is wrapped around the biodegradable scaffold. The biodegradable scaffold is a biodegradable scaffold containing chitosan and recombinant human collagen, The method for preparing the cell matrix nerve graft is as follows: Steps to prepare a membrane-like cell-free matrix, The process involves wrapping the aforementioned membranous cell-free matrix around a biodegradable scaffold to construct the initial morphology of the cell matrix nerve graft. A step of placing the initial morphology of the cell matrix nerve graft at 2-6°C for at least 24 hours to allow it to self-organize and form a tubular structure, and The process of freeze-drying the tubular structure to construct it as a cell matrix nerve graft. A method for preparing a cell matrix nerve graft for repairing peripheral nerve damage, characterized by including the following:

2. The method for preparing a cell matrix nerve graft for repairing peripheral nerve damage according to claim 1, characterized in that the freeze-drying temperature is -80°C.

3. The method for producing a cell matrix nerve graft for repairing peripheral nerve damage according to claim 1, characterized in that the membranous cell-free matrix is ​​wound up in 6, 9, or 12 layers around the biodegradable scaffold and wrapped around the biodegradable scaffold.

4. The process for preparing the aforementioned membranous cell-free matrix is ​​as follows: Human bone marrow-derived mesenchymal stem cells are diluted and seeded in a large culture dish. Once the cell saturation density reaches 90% or higher, serum-free medium containing ascorbic acid is added and the cells are stimulated for 7 to 14 days. After washing with phosphate buffer and hypotonic treatment with sterile ultrapure water, the sterile ultrapure water is removed, cell lysate is added for extraction, and then the mixture is washed again with phosphate buffer to obtain a membrane-like cell-free matrix. A method for producing a cell matrix nerve graft for repairing peripheral nerve damage, as described in claim 1.

Images