A micron fiber membrane beneficial to cartilage tissue repair and its preparation method

Abstract

The present invention relates to a micron fiber membrane that is beneficial for cartilage tissue repair and a preparation method thereof. The micron fiber comprises a cross-linked network structure formed by several micron fibers, wherein the micron fibers have a diameter of 1-6 μm and an inter-fiber pore size of 10-30 μm. The micron fibers are made of a natural, degradable polymer material. The preparation method comprises the following steps: uniformly mixing the natural, degradable polymer material and a mixed solvent to obtain a spinning solution, then electrospinning the solution to obtain a micron spinning membrane. The micron spinning membrane is chemically cross-linked and sterilized to obtain the micron fiber membrane that is beneficial for cartilage tissue repair. The spinning apparatus used for the electrospinning is disposed in a sealed cabin, and the internal environmental conditions of the cabin are: a negative pressure of 100-500 Pa, a humidity of 45-65% RH, and a temperature of 35-45°C. The micron fiber membrane of the present invention promotes the adhesion, capture, growth, and secretion of extracellular matrix of mesenchymal stem cells, thereby facilitating the repair of articular cartilage tissue.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a micron fiber membrane that is beneficial to cartilage tissue repair and a preparation method thereof. Background Art

[0002] Articular cartilage damage is a persistent condition affecting people of all ages, and has been on the rise in recent years. Due to the lack of blood vessels, lymphatics, and nerve tissue in articular cartilage, damaged articular cartilage is unable to repair itself through the bloodstream, transporting mesenchymal stem cells and nutrients, making repair difficult.

[0003] Microfracture surgery combined with bioscaffolds is an advanced method for tissue regeneration and repair. It has become an effective treatment for full-thickness cartilage defects measuring 2-6 cm² worldwide, typically completing cartilage repair within 4-8 weeks. The bioscaffold provides sealing, support, and chondrogenesis during the cartilage repair process. The biofilm's material composition and macro / microstructure can influence the effectiveness of cartilage repair.

[0004] Existing bioscaffolds for cartilage tissue repair are primarily categorized as hydrogels, decellularized extracellular matrices (ECMs), and electrospun nanofiber membranes. For example, the hydrogel described in patent CN 114073791 A is easy to prepare and easily drug-loaded, but its mechanical properties are relatively low, making it insufficient for supporting and sealing damaged areas. For example, the decellularized ECM described in patent CN111840642A possesses an extracellular fiber structure, but its fiber diameters range from 40 to 640 nm, and the interfiber pores are small, making it difficult for cells to penetrate and grow rapidly.

[0005] Among existing patented technologies, there is no application of natural biodegradable materials in the preparation of micron fiber membranes with a fiber diameter of 1 to 6 μm for cartilage tissue repair. This is mainly because the composition of the spinning solution, spinning temperature, and humidity during the electrospinning process for preparing micron-sized fibers using natural biodegradable materials as raw materials affect the microstructure of the produced fiber membranes. Furthermore, the continuous stability of the spinning process is difficult to ensure, ultimately resulting in the inability to stably produce micron fiber membranes that meet the requirements and are beneficial for cartilage tissue repair. Summary of the Invention

[0006] To stably produce micron-fiber membranes that are beneficial for cartilage tissue repair, a method for preparing a micron-fiber membrane and its preparation are provided. Using natural, degradable polymer materials, the present invention employs electrospinning to stably produce a mesh-like micron-fiber membrane with a fiber diameter of 1-6 μm and an inter-fiber pore size of 10-30 μm. This membrane promotes the adhesion, capture, growth, and extracellular matrix secretion of mesenchymal stem cells in the environment, thereby facilitating the repair of articular cartilage tissue.

[0007] In order to achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] A micron fiber membrane that is beneficial to cartilage tissue repair, comprising a cross-linked network structure formed by several micron fibers, wherein the diameter of the micron fibers is 1-6 μm and the pore size between the fibers is 10-30 μm; the material of the micron fibers is a natural degradable polymer material; the natural degradable polymer material is one or more of gelatin, silk fibroin, and collagen.

[0009] The method for preparing the micron fiber membrane that is beneficial to cartilage tissue repair comprises the following steps:

[0010] A natural degradable polymer material and a mixed solvent are uniformly mixed to obtain a spinning solution, which is then electrospun to obtain a micron spinning membrane. The micron spinning membrane is chemically cross-linked and sterilized to obtain a micron fiber membrane that is beneficial to cartilage tissue repair.

[0011] The spinning device used in the electrospinning is placed in a sealed cabin, and the internal environmental conditions of the cabin are: negative pressure 100-500 Pa, humidity 45-65% RH, and temperature 35-45°C;

[0012] The mixed solvent consists of an organic solvent and water, wherein the volume percentage of the organic solvent in the mixed solvent is 20-80%, and the mass percentage of the natural degradable polymer material in the spinning solution is 30-40%; the natural degradable polymer material is one or more of gelatin, silk fibroin, and collagen.

[0013] Furthermore, the cabin has an air pumping port for forming a negative pressure in the internal environment of the cabin, and the air pumping port is arranged right behind the receiver of the spinning device.

[0014] Furthermore, the process conditions of the electrospinning are as follows: the temperature of the spinning solution is set to the same as the internal ambient temperature of the cabin, the spinning voltage is 10-30kv, the spinning distance is 10-18cm, the liquid supply speed of the spinning solution is 1-5mL / h, and the drum speed of the receiver of the spinning device is 100-300rpm.

[0015] Furthermore, the organic solvent is one or more of formic acid, acetic acid, ethyl acetate, and N,N-dimethylformamide.

[0016] Preferably, the mixed solvent is composed of the following materials in 100% by volume: 5-10% formic acid, 15-30% acetic acid, 2-30% ethyl acetate, 0-10% N,N-dimethylformamide, and the balance is water.

[0017] Furthermore, the crosslinking agent used in the chemical crosslinking is one or more of formaldehyde, acetaldehyde, and glutaraldehyde.

[0018] Furthermore, the chemical cross-linking process is: placing the micron spinning membrane in cross-linking agent vapor for cross-linking for 8-32 hours, and the concentration of the cross-linking agent vapor in the spinning device is 5-10 mL / m3.

[0019] Furthermore, the sterilization treatment is one or more of irradiation sterilization, ethylene oxide sterilization, and ultraviolet sterilization.

[0020] Beneficial technical effects: The micron fiber membrane of the present invention is made of natural degradable materials, has high biocompatibility, and the fiber surface has the ability to promote the adhesion of mesenchymal stem cells; the fiber diameter of the micron fiber membrane is 1-6μm, the pore size between fibers is 10-30μm, and the fiber membrane is in a cross-linked network, which can three-dimensionally capture free mesenchymal stem cells in the environment; the fiber membrane degrades synchronously with cell proliferation, and the degradation time is controllable within 4-8 weeks, providing nutrients for the growth of mesenchymal stem cells; the fibers in the micron fiber membrane are tightly combined through crossing, stacking, and interlocking, and its mechanical properties, swelling properties, and water absorption properties can ensure the three-dimensional growth of cells on the surface and inside the membrane; the micron fiber membrane has high biocompatibility, and the fiber surface has the ability to promote the adhesion of mesenchymal stem cells. The spinning materials used in the present invention are all natural degradable polymer materials, and the solvent raw materials used are easy to obtain and easy to remove from the material. The material composition, micro / macro structure and mechanical properties of the micron fiber membrane of the present invention can promote its effects on promoting adhesion, capture, growth and extracellular matrix secretion of mesenchymal stem cells in the environment. The micron fiber membrane of the present invention can be used as a biological scaffold required in the process of cartilage repair to facilitate articular cartilage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are SEM morphologies of the cross-linked micron fiber membranes of Examples 1-3 and the cross-linked nanofiber membrane of Comparative Example 1. The scale lengths in the figures are: Example 1—50.0 μm, Example 2—100.0 μm, Example 3—10.0 μm, and Comparative Example 1—5.0 μm. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention and the accompanying drawings 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, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. Technology and methods known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology and methods should be considered as a part of the specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments can have different values.

[0024] In the following examples, experimental methods without specific conditions are generally measured according to national standards. If there are no corresponding national standards, they are measured according to the general international standards or the standards proposed by relevant companies. Unless otherwise stated, all parts are by weight and all percentages are by weight.

[0025] Example 1

[0026] A method for preparing a micron fiber membrane that is beneficial for cartilage tissue repair comprises the following steps:

[0027] Preparation of mixed solvent: Mix 5% formic acid, 15% acetic acid, 2% ethyl acetate, 3% N,N-dimethylformamide and the balance water in a volume ratio of 100%;

[0028] Evenly mixing gelatin with the mixed solvent to prepare a spinning solution, wherein the mass percentage of gelatin in the mixed solvent accounts for 30%;

[0029] Then, electrospinning is performed, wherein the spinning device used for the electrospinning is placed in a sealed cabin, the cabin has a pump air port for connecting a vacuum device to form a negative pressure in the internal environment of the cabin, the pump air port is placed directly behind the receiver of the spinning device, the internal environmental conditions of the cabin are: negative pressure 100 Pa, humidity 45% RH, and temperature 35°C, and the process conditions of the electrospinning are: the temperature of the spinning solution is set to be the same as the internal environmental temperature of the cabin, the spinning voltage is 10 kV, the spinning distance is 10 cm, the liquid supply rate of the spinning solution is 1 mL / h, and the drum speed of the receiver of the spinning device is 100 rpm, thereby obtaining a micron spun membrane;

[0030] Then the micron spinning membrane is placed in formaldehyde vapor, and the concentration of formaldehyde vapor in the spinning device is 5mL / m 3 The chemical cross-linking time is 8 hours. After the chemical cross-linking is completed, irradiation sterilization treatment is performed to obtain a micron fiber membrane that is beneficial to cartilage tissue repair.

[0031] The SEM image of the micron fiber membrane obtained in this example is beneficial to cartilage tissue repair. Figure 1As shown in , it can be seen that the fiber diameter of the fiber membrane is about 1-3 μm and the pore size between fibers is about 10-30 μm.

[0032] Example 2

[0033] A method for preparing a micron fiber membrane that is beneficial for cartilage tissue repair comprises the following steps:

[0034] Preparation of mixed solvent: Mix 5% formic acid, 20% acetic acid, 15% ethyl acetate and the balance 60% water in a volume ratio of 100%;

[0035] Evenly mixing gelatin and collagen with the mixed solvent to prepare a spinning solution, wherein the mass percentage of gelatin in the mixed solvent accounts for 20%, and the mass percentage of collagen in the mixed solvent accounts for 15%;

[0036] Then, electrospinning is performed, wherein the spinning device used for the electrospinning is placed in a sealed cabin, the cabin has a pump air port for connecting a vacuum device to form a negative pressure in the internal environment of the cabin, the pump air port is arranged directly behind the receiver of the spinning device, and the internal environmental conditions of the cabin are: negative pressure 300 Pa, humidity 50% RH, and temperature 40°C. The process conditions for electrospinning are: the temperature of the spinning solution is set to the same as the internal environmental temperature of the cabin, the spinning voltage is 20 kV, the spinning distance is 15 cm, the liquid supply rate of the spinning solution is 3 mL / h, and the drum speed of the receiver of the spinning device is 200 rpm, thereby obtaining a micron spun membrane;

[0037] Then the micron spinning membrane is placed in a mixed steam of formaldehyde and glutaraldehyde, wherein the concentration of formaldehyde vapor in the mixed steam in the spinning device is 5 mL / m 3 The concentration of glutaraldehyde vapor in the spinning device is 3 mL / m 3 The chemical cross-linking time is 8 hours. After the chemical cross-linking is completed, ethylene oxide sterilization treatment is performed to obtain a micron fiber membrane that is beneficial to cartilage tissue repair.

[0038] The SEM image of the micron fiber membrane obtained in this example is beneficial to cartilage tissue repair. Figure 1 As shown in , it can be seen that the fiber diameter of the fiber membrane is about 1-4 μm and the pore size between fibers is about 10-30 μm.

[0039] Example 3

[0040] A method for preparing a micron fiber membrane that is beneficial for cartilage tissue repair comprises the following steps:

[0041] Preparation of mixed solvent: Mix 10% formic acid, 30% acetic acid, 30% ethyl acetate, 10% N,N-dimethylformamide and the balance 20% water in a volume ratio of 100%;

[0042] Evenly mixing gelatin, silk fibroin and collagen with the mixed solvent to prepare a spinning solution, wherein the mass percentage of gelatin in the mixed solvent accounts for 20%, the mass percentage of silk fibroin in the mixed solvent accounts for 5%, and the mass percentage of collagen in the mixed solvent accounts for 15%;

[0043] Then, electrospinning is performed, wherein the spinning device used for the electrospinning is placed in a sealed cabin, the cabin has a pump air port for connecting a vacuum device to form a negative pressure in the internal environment of the cabin, the pump air port is arranged directly behind the receiver of the spinning device, and the internal environmental conditions of the cabin are: negative pressure 500 Pa, humidity 65% RH, and temperature 35°C. The process conditions for electrospinning are: the temperature of the spinning solution is set to the same as the internal environmental temperature of the cabin, the spinning voltage is 30 kV, the spinning distance is 18 cm, the liquid supply rate of the spinning solution is 5 mL / h, and the drum speed of the receiver of the spinning device is 300 rpm, thereby obtaining a micron spun membrane;

[0044] Then the micron spinning membrane is placed in a mixed steam of formaldehyde, acetaldehyde and glutaraldehyde, wherein the concentration of formaldehyde vapor in the mixed steam in the spinning device is 5 mL / m 3 The concentration of acetaldehyde vapor in the spinning device is 2 mL / m 3 The concentration of glutaraldehyde vapor in the spinning device is 3 mL / m 3 The chemical cross-linking time is 32 hours. After the chemical cross-linking is completed, ultraviolet sterilization treatment is performed to obtain a micron fiber membrane that is beneficial to cartilage tissue repair.

[0045] The SEM image of the micron fiber membrane obtained in this example is beneficial to cartilage tissue repair. Figure 1 As shown in , it can be seen that the fiber diameter of the fiber membrane is about 1-6 μm and the pore size between fibers is about 10-30 μm.

[0046] Comparative Example 1

[0047] This comparative example is the preparation of nano-scale gelatin fiber membrane: its preparation method adopts a common stable preparation process, and the specific preparation method is as follows:

[0048] Trifluoroethanol is used as a solvent and is evenly mixed with gelatin to prepare a spinning solution, wherein the mass concentration of gelatin in the spinning solution is 6%; an electrospinning process is used for spinning: the spinning device used for the electrospinning is arranged in a sealed cabin, and the cabin is not set as a negative pressure environment, the liquid supply rate of the spinning solution is 1 mL / h, the spinning voltage is 10 kV, the spinning distance is 10 cm, the spinning temperature is 25°C, the spinning humidity is 55% RH, and the rotation speed of the spinning receiving device roller is 100 rpm, thereby preparing a nanofiber membrane; then, formaldehyde vapor is used for chemical crosslinking, and the concentration of formaldehyde vapor in the spinning device during steam crosslinking is 5 mL / m 3 , the cross-linking time is 8h; after the cross-linking treatment is completed, the fiber membrane is irradiated and sterilized.

[0049] Test Case

[0050] The cross-linked fiber membranes prepared in the above examples and comparative examples were subjected to performance tests, and the test items were as follows:

[0051] The fiber membrane size test was performed using a measuring tool based on the scanning electron microscope image.

[0052] The degradation time was obtained by in vitro degradation with sterile PBS.

[0053] The tensile strength test refers to the film tensile property test method in GB / T 1040.3-2006, and the tensile strength of the sample is tested using a universal tensile testing machine.

[0054] The tear strength test refers to the film tear resistance test method in GB / T 16578.1-2008, and the tear strength test of the sample is carried out using a universal tensile testing machine.

[0055] The swelling rate test refers to the test method in GB / T 14797.3-2008.

[0056] The water absorption test refers to the test method in GB / T 3903.33-2008.

[0057] The cell adhesion rate test method is as follows: the fiber membrane is cut into 3mm×3mm size, moistened with cell culture medium, and placed in a microplate. 5×10 bone marrow mesenchymal stem cells (BM-MSCs) that have been screened and passaged twice are inoculated into each microplate. 5An appropriate volume of culture medium and serum was added to each well. The negative control group consisted of the microplate bottom material. After culturing in a cell culture incubator for 8 hours, the culture medium in the microwells was removed, and the microwells and fiber membrane surface were gently rinsed three times with sterile PBS to remove cells that were not adhered or not firmly adhered. Ten fields of view of the control and experimental groups were photographed using a microscope, and the cells in the images were counted. The number of cells in the experimental group was designated N1, and the number of cells in the negative control group was designated N0. The cell adhesion promotion rate (%) = N1 / N0.

[0058] The cell proliferation rate test method is as follows: refer to GB / T 16886.1-2001 Biological Evaluation of Medical Devices Part 1, use cell culture medium to extract the fiber membrane extract, and inoculate 5×10 bone marrow mesenchymal stem cells (BM-MSCs) that have been passaged twice into each microwell. 5 The extract was used as the cell culture medium and cultured in a carbon dioxide incubator for 1 day, 3 days, 7 days, and 14 days. After trypsin digestion, the cells were counted, which was A1. The cells were cultured in cell culture medium and counted, which was A0. The cell proliferation rate (%) = A1 / A0.

[0059] The above test results are shown in Table 1.

[0060] Table 1 Comparison of fiber membrane performance

[0061]

[0062]

[0063] As shown in Table 1, the microfiber membrane of Example 1 is larger in fiber size and interfiber pore size than that of Comparative Example 1. It also exhibits comparable mechanical, swelling, and water absorption properties, and significantly outperforms Comparative Example 1 in terms of cell adhesion and cell proliferation. This is due to the presence of multiple functional groups on the gelatin surface, such as amino, hydroxyl, and carboxyl groups, which have a unique physical adsorption effect on cells. Furthermore, the microfiber membrane of the present invention has a fiber size of 1-3 μm, which provides a more suitable surface density than nanofibers. The pore size of the microfiber membrane is controlled at 10-30 μm, matching the size of mesenchymal stem cells. This allows mesenchymal stem cells in the environment to adhere to the microfiber surface and directly enter the microfiber membrane, achieving rapid three-dimensional growth. The nanofiber membrane of Comparative Example 1, with its smaller interfiber pore size (1-3.5 μm), lacks this performance. While the tensile strength and tear strength of the microfiber membrane of Example 1 are slightly lower than those of Comparative Example 1, they are sufficient to facilitate operations such as folding and unfolding the microfiber membrane and provide good support for stable cell growth. The swelling properties of the micron fiber membrane ensure that a slight interference fit can be achieved during wound patching, which can better seal the wound. Its excellent water absorption performance can ensure that the membrane provides a good wetting environment for the mesenchymal stem cells inside and on the surface during the entire repair process, and provides an ideal growth environment for the proliferation, differentiation and extracellular matrix secretion of mesenchymal stem cells. The present invention uses cross-linking of aldehyde vapor to produce moderate cross-linking of the amino groups of the gelatin molecules in the membrane fibers, and the time for complete degradation can be stably controlled at 4 weeks. During this period, as the mesenchymal stem cells grow, they are gradually degraded into essential amino acids and other nutrients required for cell life activities, ensuring their good proliferation rate. The micron fiber membrane of Example 1 has better cell proliferation promoting properties than that of Comparative Example 1. This is because the mixed solvent used to prepare the micron fiber membrane of the present invention is better than that of Comparative Example 1 in biocompatibility.

[0064] During the preparation process of the invented product, the matching of spinning solution viscosity with the spinning temperature and humidity is crucial to the structural stability of the entire fiber. Furthermore, the negative pressure applied to the chamber balances the uncontrollable fluctuations during the spinning process, improving the continuity and stability of micron fiber production. By controlling the concentration of the naturally degradable polymer in the spinning solution to 30-40%, the viscosity, surface tension, and conductivity of the spinning solution meet the requirements for preparing micron fiber membranes. By matching the spinning temperature to 35-45°C and the humidity to 45-65% RH, the viscosity, surface tension, conductivity, and solvent evaporation rate of the spinning solution are optimized to ensure stable micron fiber membrane production. By setting the negative pressure in the spinning chamber to 100-500 Pa, the driving force for the spinning solution supply is increased to ensure continuous liquid supply during the spinning process. The negative pressure environment and the specific airflow increase the solvent evaporation rate and ensure uniform fiber diameter distribution. The specific airflow generated by the negative pressure assists the electric field force in pulling the spun fibers, ensuring the stability of the fibers' flight to the receiving device during spinning, ultimately producing a structurally stable micron fiber membrane.

[0065] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a micron fiber membrane that is beneficial to cartilage tissue repair, characterized in that: The steps include: A natural degradable polymer material and a mixed solvent are uniformly mixed to obtain a spinning solution, which is then electrospun to obtain a micron spinning membrane. The micron spinning membrane is chemically cross-linked and sterilized to obtain a micron fiber membrane that is beneficial for cartilage tissue repair. The obtained micron fiber membrane is composed of a cross-linked network structure composed of several micron fibers, the diameter of the micron fibers is 1-6 μm, and the pore size between fibers is 10-30 μm; The mixed solvent consists of an organic solvent and water, wherein the volume percentage of the organic solvent in the mixed solvent is 22-80%, and the mass percentage of the natural degradable polymer material in the spinning solution is 30-40%; the natural degradable polymer material is one or more of gelatin, silk fibroin, and collagen; The mixed solvent consists of the following 100% by volume materials: Composition: formic acid 5-10%, acetic acid 15-30%, ethyl acetate 2-30%, N,N-dimethylformamide 0-10%, the balance is water; The spinning device used in the electrospinning is placed in a sealed cabin, and the internal environmental conditions of the cabin are: negative pressure 100-500 Pa, humidity 45-65% RH, and temperature 35-45°C; The chemical crosslinking process is as follows: placing the micron spinning membrane in crosslinking agent steam for crosslinking for 8-32 hours, and the concentration of the crosslinking agent steam in the spinning device is 5-10 mL / m 3 .

2. The method for preparing a micron fiber membrane that is beneficial to cartilage tissue repair according to claim 1, characterized in that: The cabin body has an air pumping port for forming a negative pressure in the internal environment of the cabin body. The air pumping port is arranged right behind the receiver of the spinning device.

3. The method for preparing a micron fiber membrane that is beneficial to cartilage tissue repair according to claim 1, characterized in that: The process conditions of the electrospinning are as follows: the temperature of the spinning solution is the same as the internal ambient temperature of the cabin, the spinning voltage is 10-30 kV, the spinning distance is 10-18 cm, the liquid supply rate of the spinning solution is 1-5 mL / h, and the drum speed of the receiver of the spinning device is 100-300 rpm.

4. The method for preparing a micron fiber membrane that is beneficial to cartilage tissue repair according to claim 1, characterized in that: The crosslinking agent used in the chemical crosslinking is one or more of formaldehyde, acetaldehyde and glutaraldehyde.

5. The method for preparing a micron fiber membrane that is beneficial to cartilage tissue repair according to claim 1, characterized in that: The sterilization treatment is one or more of radiation sterilization, ethylene oxide sterilization, and ultraviolet sterilization.

Images