Oriented collagen-based biological material as well as preparation method and application thereof
Through the self-assembly and multi-step preparation process of collagen fibers, combined with airflow-assisted orientation and mechanical stretching, the problem of difficult to manufacture large-scale and biologically active oriented collagen-based biomaterials in the prior art is solved, and materials with good biocompatibility and biological activity are prepared, which is suitable for tissue regeneration and repair and tissue engineering applications.
Patent Information
- Application Number
- CN202411880415.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to manufacture large-scale, biologically active oriented collagen-based biomaterials, and common methods will change or fail to form the characteristic micro-nano structure of collagen fibers, affecting biological activity.
Through the preparation processes of collagen fibers such as self-assembly, concentration, pre-cooling, pre-freezing, airflow-assisted orientation and mechanical stretching, combined with freeze-drying and chemical crosslinking, collagen-based sponge or membrane material with fiber orientation arrangement structure characteristics are constructed.
The preparation of oriented collagen-based materials with good biocompatibility and biological activity is achieved, which is suitable for the regeneration and repair of disease-deficient tissues and the construction of tissue-engineered artificial organs.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomedical engineering and biomaterials, and in particular relates to an oriented collagen-based biomaterial and a preparation method and application thereof. Background Art
[0002] Tissue defects caused by trauma, infection, tumors and degenerative diseases are the main hazards affecting human health. Currently, the repair and reconstruction of diseased and defective tissues still face many challenges. The construction of biomaterials with biomimetic composition and structure can provide an effective way to regenerate and repair diseased and defective tissues.
[0003] Collagen is the main structural protein in the human body and animals. It is the most abundant protein in the body and is widely present in many tissues of the body. As a natural polymer material with a wide range of sources, collagen, especially type I collagen, is widely used in the repair of damaged tissues and the construction of artificial organs in tissue engineering due to its low immunogenicity, good biocompatibility, and biodegradability. It is worth noting that collagen exhibits the characteristics of high fiber orientation distribution in various tissues of the body. The arrangement direction of collagen fibers in bone tissue determines the location of hydroxyapatite deposition, and the orientation of longitudinal collagen fibers is related to the elastic modulus and strength of bone. Articular cartilage consists of three regions: (1) surface area, where collagen fibers are basically parallel to the surface; (2) middle area, where fibers are randomly distributed; and (3) deep area, where fibers are basically perpendicular to the joint surface. Collagen fibers in other tissues of the body, such as tendons and cornea, also exhibit different orientation characteristics. The orientation of collagen fibers determines the structural characteristics of tissues and is also the basis for the physiological functions of tissues and organs. The destruction of collagen fiber orientation in tissues will cause damage to tissue structure and affect the function of tissues and organs. Collagen fibers are molecular aggregates with special micro-nano structures formed by step-by-step self-assembly of procollagen molecules; and collagen fiber orientation refers to the characteristics of collagen fibers with highly consistent distribution and arrangement. Due to its anisotropic structural characteristics, oriented collagen materials have more bionic mechanical properties and biological activities, which can be more conducive to the regeneration and repair of diseased and defective tissues. For example, studies have found that oriented collagen fiber scaffolds can effectively repair tendons and other tissues. Therefore, the in vitro construction of bionic oriented structured collagen fiber biomaterials is an important research direction in regenerative medicine, biomaterials and tissue engineering.
[0004] The oriented manufacturing of collagen materials has always been an important direction of basic research on their application. Since the method of inclined flow injection was first reported in 1972, the manufacture of materials with oriented collagen fibers has received great attention, and many methods for manufacturing ordered oriented collagen materials have been developed, including strong magnetic field, electrospinning technology, electrochemical orientation electrophoresis technology, microfluidics technology, and freeze-thaw methods, which can manufacture oriented collagen materials to a certain extent. However, the above methods still have the problem of small scale of manufacturing materials, the manufacturing process will change or fail to form the characteristic micro-nano structure of collagen fibers, and electrospinning and freeze-thaw technology will cause collagen to denature to a certain extent, affecting the biological activity of collagen. How to manufacture large-scale, biologically active oriented collagen-based biomaterials is still facing many challenges. The present invention provides an oriented collagen-based biomaterial and its preparation method and application. Summary of the invention
[0005] The purpose of the present invention is to provide an oriented collagen-based biomaterial and its preparation method and application. Specifically, through the preparation processes of collagen fiber self-assembly, concentration, precooling, prefreezing, airflow-assisted orientation and mechanical stretching, combined with freeze drying, chemical crosslinking and other means, a collagen-based sponge or collagen membrane material with fiber oriented arrangement structural characteristics is constructed. The constructed oriented collagen-based material has good biocompatibility and biological activity, and can be used for the regeneration and repair of diseased and defective tissues and the construction of tissue-engineered artificial organs.
[0006] The present invention provides an oriented collagen-based biomaterial, the main feature of which is that the collagen-based material is composed of collagen fibers with oriented arrangement characteristics. The collagen fibers are collagen fibers with characteristic striated structures formed by collagen through self-assembly. The oriented arrangement characteristics of the collagen fibers are characterized by the distribution and arrangement of the collagen fibers with high consistency and anisotropic structural characteristics. The oriented collagen-based biomaterial has good biocompatibility and bioactivity, and can be used for the regeneration, repair, functional reconstruction and enhancement of diseased and defective tissues, and can also be used for the in vitro and in vivo construction of tissue-engineered artificial organs.
[0007] The present invention provides an oriented collagen-based biomaterial, wherein the collagen-based material can be a sponge-like or film-like structural material. The sponge-like collagen-based material has a multi-layer, porous sponge-like structure; the thickness of the sponge-like collagen-based material is 0.5-5.0 mm. The film-like collagen-based material has a thin film-like structural characteristic, and the thickness is 0.01-0.5 mm.
[0008] The invention provides a method for preparing an oriented collagen-based biomaterial, which mainly comprises the following steps: self-assembly of collagen fibers, concentration of collagen fiber suspension, precooling of collagen fiber suspension, prefreezing of collagen fiber suspension, airflow-assisted orientation of collagen fibers and / or orientation manufacturing by mechanical stretching.
[0009] The present invention provides a method for preparing an oriented collagen-based biomaterial, which mainly comprises the following preparation process: S1, dissolving collagen in 3% acetic acid solution or 0.3% malonic acid solution, swelling at 4°C, stirring until fully swollen, obtaining a collagen swelling solution with a concentration of 1 mg-10 mg / mL, and storing at 4-8°C for later use; S2, take an appropriate amount of collagen swelling solution, transfer it to a centrifuge tube of appropriate size, add 10 x (0.1M) phosphate buffer (10 x PBS), the volume ratio of collagen swelling solution to 10 x PBS is 9:1, shake the mixture well and incubate it in a water bath at room temperature or 20-30℃ for 4-12 hours to allow collagen to self-assemble into collagen fibers; S3, after the collagen self-assembly is completed, low temperature (4°C) centrifugation is performed to deposit the collagen fibers, which are then suspended and washed with deionized water, and finally fully suspended with deionized water and concentrated to obtain a collagen fiber suspension with a concentration of 5-25 mg / mL, which is then precooled at 4-8°C for 4-12 hours; S4, transferring the concentrated pre-cooled collagen fiber suspension into a suitable square or rectangular container, preferably a stainless steel container, and pre-freezing it at -20-40°C for 4-12 hours; S5, carefully transferring the concentrated pre-frozen collagen fiber suspension block to a flat, pre-cooled substrate material, preferably stainless steel, glass, thick silicone plate or polymethacrylamide (PMMA) substrate material, stably placing it and fixing it by further freezing; S6, transferring the frozen collagen fibers and the base material to an airflow-assisted manufacturing device with a fixed wind direction and a stable and adjustable wind speed, adjusting the device temperature to 5-25°C, the wind speed to 0.5-2.0 m / s, and the wind direction to be perpendicular to one side of the square or rectangular collagen fibers and the base material, and continuing until the collagen-based material is completely dried to form a film-like material; S7, air drying may also be performed in the manner of S6 until the collagen-based material is dried into a hydrogel-like material, and then further pre-frozen and freeze-dried into a sponge-like material; S8, it is also possible to air-dry the collagen-based material in the manner of S7 until the collagen-based material dries into a hydrogel-like material, and then pre-freeze the sample and divide it into sections and transfer it to a silicone stretching box, and then stretch it on a stretching device, with the stretching direction perpendicular to the above-mentioned wind direction, until the sample is dry, or after completing a stretching procedure, transfer it to an airflow-assisted device for air-drying again, or obtain a sponge-like material by freeze-drying; S9, the preparation method can also be, after the concentrated pre-cooled collagen fiber suspension is transferred to a suitable square or rectangular container, it is divided during or after pre-freezing so that it can be adapted to the silicone stretching box container, after the pre-freezing is completed, it is transferred to a silicone box container, and then stretched on a stretching device until the sample is completely dry, or after completing a stretching procedure, it is transferred to an airflow-assisted device again for air drying, and a sponge-like material can also be obtained by freeze drying; S10, the collagen-based material can be a membrane-like or sponge-like material directly obtained by the above method, or can be a collagen-based material obtained by further chemical cross-linking.
[0010] The present invention provides an oriented collagen-based biomaterial, wherein the collagen may be type I collagen extracted from pig skin, cow skin, bovine tendon and rat tail tendon by acid swelling, enzyme degradation or decellularization, or type II collagen derived from pig or bovine articular cartilage; In a method for preparing an oriented collagen-based biomaterial provided by the present invention, the airflow-assisted orientation is performed by an airflow-assisted device, which consists of a fan with adjustable wind speed, a guide valve, a filter and a sample area; the airflow-assisted device is placed in an air bath box with adjustable temperature.
[0011] In the preparation method of an oriented collagen-based biomaterial provided by the present invention, the mechanical stretching procedure is that its stretching deformation ratio is 100-150%, the stretching frequency is 10-60 times / minute, and the stretching number is 5000-20000 times.
[0012] In the preparation method of an oriented collagen-based biomaterial provided by the present invention, the chemical crosslinking is to use a crosslinking agent such as glutaraldehyde, genipin, carbodiimide, etc. to crosslink collagen fibers; the air-dried or freeze-dried membrane-like or sponge-like collagen-based material can be immersed in a crosslinking agent solution for crosslinking; or the crosslinking agent solution can be added during the stretching process in the above-mentioned S8 or S9 for stretching and crosslinking.
[0013] The present invention provides an oriented collagen-based biomaterial, wherein the collagen fibers have an oriented arrangement characteristic in which the collagen fibers are distributed and arranged with high consistency, have anisotropic structural characteristics, and also have anisotropic mechanical properties.
[0014] The invention provides an oriented collagen-based biomaterial and a preparation method thereof. Since the preparation method is performed under mild conditions, growth factors, active polypeptides and other small molecule bioactive substances can be introduced during the preparation process to improve the biological activity of the collagen-based biomaterial. The present invention provides an oriented collagen-based biomaterial, which has good biocompatibility and bioactivity and can be used for the regeneration, repair, functional reconstruction and enhancement of diseased and defective tissues. Preferably, the air-dried collagen membrane-like material can be used as a dura mater repair material, a hernia repair patch, a posterior scleral reinforcement material, a tendon repair material, a guided tissue regeneration material, etc.; the collagen sponge-like material can be used to construct tissue engineering artificial organs in vitro through cell culture or the like, or to study the effects and mechanisms of oriented collagen materials on cell proliferation and differentiation. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The schematic diagram of the airflow-assisted manufacturing device system of the present invention is shown in FIG. The airflow-assisted manufacturing device system is mainly composed of a temperature-controllable air bath box, a fan, a filter, a guide valve, an airflow channel and a sample processing area.
[0016] Figure 2 The scanning electron microscopy result of the collagen material prepared in Example 1 shows that the prepared sample is composed of collagen fibers, which are arranged in an orderly manner and have obvious oriented arrangement characteristics.
[0017] Figure 3 This is the scanning electron microscopy result of the collagen material prepared in Example 2. It can be seen that the prepared sample is composed of collagen fibers, and the collagen fibers are arranged in an orderly manner with obvious oriented arrangement characteristics.
[0018] Figure 4 The scanning electron microscopy result of the collagen material prepared in Example 3 shows that the prepared sample is composed of collagen fibers, which are arranged in an orderly manner and have obvious oriented arrangement characteristics.
[0019] Figure 5 The tensile test results of the collagen material prepared in Example 4 are shown. It can be seen that due to the oriented arrangement of the material, its mechanical properties show anisotropic characteristics. The tensile strength and elastic modulus of the collagen-based material in the direction perpendicular to the airflow are significantly higher than those in the airflow direction. This shows that the airflow-assisted orientation is not promoted by the shear force of the airflow. DETAILED DESCRIPTION
[0020] The present invention is now further described in detail in conjunction with embodiments so that those skilled in the art can better understand and implement the present invention.
[0021] Example 1: (1) 36 mL of 0.6% collagen was added to a centrifuge tube and then 4 mL of 10 x PBS was added. The mixture was incubated at room temperature overnight. The collagen incubation solution was placed in a 30°C water bath and incubated for 3 hours. (2) The collagen incubation solution was centrifuged at 6000 rpm for 15 minutes. After the supernatant was removed, the collagen fibers were fully suspended with 10 mL of deionized water and then transferred to a stainless steel plate (4 x 4 cm2 ) and freeze at -20℃. (3) Take out the frozen collagen and transfer it to the PMMA bracket. After further freezing and fixing at -20℃, transfer it to the airflow-assisted device, adjust the system temperature to 5℃ and the wind speed to 0.5m / s, and take it out after the collagen fiber material is completely air-dried. Carefully peel off the collagen membrane from the PMMA sheet, take out the sample with scissors and observe it under a scanning electron microscope.
[0022] Example 2: (1) 36 mL of 0.6% collagen was added to a centrifuge tube and then 4 mL of 10 x PBS was added. The mixture was incubated at room temperature for 1 hour and then placed in a 30°C water bath for another 5 hours. (2) The collagen incubation solution was centrifuged at 6000 rpm for 15 minutes. The supernatant was removed and washed with deionized water. The collagen fibers were suspended with 20 mL of deionized water and transferred to a stainless steel plate (4 x 4 cm 2 ) and placed in a -20℃ freezer. (3) Take out the frozen collagen and transfer it to the PMMA bracket. After further freezing and fixing at -20℃, transfer it to the airflow-assisted device, adjust the system temperature to 5℃ and the wind speed to 1.5m / s, and take it out after the collagen fiber material is completely air-dried. Carefully peel off the collagen membrane from the PMMA sheet and take the sample for scanning electron microscopy observation.
[0023] Example 3: (1) 36 mL of 0.6% collagen was added to a centrifuge tube and then 4 mL of 10 x PBS was added. The mixture was incubated at room temperature for 1 hour and then placed in a 30°C water bath for another 5 hours. (2) The collagen incubation solution was centrifuged at 6000 rpm for 15 minutes. The supernatant was removed and washed with deionized water. The collagen fibers were suspended with 20 mL of deionized water and transferred to a stainless steel plate (4 x 4 cm 2 ) and placed in -20℃ freezer. (3) Take out the frozen collagen and transfer it to the PMMA bracket. After further freezing and fixing at -20℃, transfer it to the airflow-assisted device, adjust the system temperature to 15℃, adjust the wind speed to 1.5m / s, until the collagen fiber material becomes a hydrogel, and place it at -20℃ to freeze again. Use a surgical blade to cut the sample and transfer it to a silicone stretching box. Place it on a mechanical stretching device and stretch it 10,000 times. The stretching deformation ratio is 130% and the stretching frequency is 10 times / minute. After the stretching is completed, the sample is frozen again and transferred to the PMMA plate and air-dried again until it is completely dry. Take the sample for scanning electron microscopy observation.
[0024] Example 4: (1) 36 mL of 0.6% collagen was added to a centrifuge tube and then 4 mL of 10 x PBS was added. The mixture was incubated at room temperature for 1 hour and then placed in a 30°C water bath for another 5 hours. (2) The collagen incubation solution was centrifuged at 6000 rpm for 15 minutes. The supernatant was removed and washed with deionized water. The collagen fibers were suspended with 20 mL of deionized water and transferred to a stainless steel plate (4 x 4 cm 2) and placed in a -20℃ refrigerator for freezing. (3) Take out the frozen collagen and transfer it to the PMMA bracket. After further freezing and fixing at -20℃, transfer it to the airflow-assisted device, adjust the system temperature to 5℃, adjust the wind speed to 1.5m / s, and take it out after the collagen fiber material is completely air-dried. (4) Use genipin cross-linking solution to cross-link the collagen membrane material. Genipin cross-linking solution concentration: 0.45% genipin-75% ethanol solution. Take 40mL of genipin cross-linking solution and place it in a centrifuge tube, then immerse the air-dried collagen membrane material, seal the centrifuge tube and place it in a 37℃ water bath for cross-linking for 5 days, and the cross-linking is completed. After taking out the material, rinse it thoroughly with deionized water and freeze-dry it. The material was subjected to tensile testing using an INSTRON 3345 universal material testing machine at a tensile speed of 1 mm / min.
[0025] With the above embodiments of the present invention as inspiration, through the above description, relevant personnel can make various changes and modifications within the scope of the technical idea of the present invention. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.
Claims
1. An oriented collagen-based biomaterial and its preparation method and application, characterized in that It is constructed through the processes of collagen fiber self-assembly, concentration, pre-cooling, pre-freezing, airflow-assisted orientation and mechanical stretching, combined with freeze-drying, chemical cross-linking and other means to obtain a collagen-based sponge or collagen membrane material with fiber-oriented structural characteristics.
2. The oriented collagen-based biomaterial according to claim 1, characterized in that It is mainly composed of collagen fibers with a characteristic striated structure formed by self-assembly.
3. The oriented collagen-based biomaterial according to claim 1, characterized in that It is mainly composed of collagen fibers that are distributed and arranged in an oriented manner with high consistency and has an anisotropic structural characteristic.
4. The oriented collagen-based biomaterial according to claim 1, characterized in that The material is mainly composed of oriented collagen fibers and has anisotropic structure and anisotropic mechanical properties.
5. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The method comprises the following steps: (1) dissolving collagen in an acetic acid solution with a mass concentration of 3% or a malonic acid solution with a mass concentration of 0.3%, swelling at 4°C, stirring until fully swollen, obtaining a collagen swelling solution with a concentration of 1 mg-10 mg / mL, and storing at 4-8°C for later use; (2) taking an appropriate amount of the collagen swelling solution, transferring it to a centrifuge tube of a suitable size, adding 10 x (0.1M) phosphate buffer (10 x PBS), mixing the collagen swelling solution with 10 x The volume ratio of PBS is 1:
9. After the mixture is shaken, it is incubated in a water bath at 20-30°C for 4-12 hours to allow the collagen to self-assemble into collagen fibers; (3) After the collagen self-assembles into collagen fibers, it is centrifuged and washed at low temperature, suspended in deionized water, and concentrated into a 5-25 mg / mL collagen fiber suspension, which is pre-cooled at 4-8°C for 4-12 hours; (4) The concentrated pre-cooled collagen fiber suspension is transferred to a suitable square or rectangular container and pre-frozen at -20-40°C for 4-12 hours; (5) The pre-frozen collagen fibers are transferred to a flat pre-cooled base material, stably placed, and frozen and fixed; (6) The frozen collagen fibers are transferred to an airflow-assisted manufacturing device, the device temperature is adjusted to 5-25°C, the wind speed is 0.5-2.0 m / s, and the air drying is continued until the collagen-based material is completely dried to form a film-like material.
6. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The method may also include the following steps: after the collagen fiber self-assembly, concentration, pre-cooling, pre-freezing, and airflow-assisted manufacturing are performed according to the above method, air-drying is continued until the collagen-based material becomes a hydrogel-like material, and further freeze-drying is performed to form a sponge-like material.
7. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The method may also include the following steps: after the collagen fibers are self-assembled, concentrated, pre-cooled, pre-frozen, and airflow-assisted manufactured according to the above method, air-dried continuously until the collagen-based material becomes a hydrogel-like material, and then the sample is pre-frozen and transferred to a silicone stretching box, and then mechanically stretched on a stretching device, with the stretching direction perpendicular to the above wind direction, until the sample is dry, or after completing a stretching procedure, the sample is transferred to an airflow-assisted device for air drying again. Sponge-like materials can also be obtained by freeze-drying.
8. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The method may also include the following steps: self-assembling, concentrating and pre-cooling collagen fibers according to the above method, transferring the concentrated and pre-cooled collagen fiber suspension to a suitable square or rectangular container, transferring it to a silica gel box container after pre-freezing, and then stretching it on a stretching device until the sample is completely dry, or transferring it to an airflow-assisted device for air drying after completing a stretching procedure. Sponge-like materials can also be obtained by freeze-drying.
9. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The collagen-based material can be a membrane-like or sponge-like material directly obtained by the above method, or can be a collagen-based material obtained by further chemical cross-linking.
10. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The airflow-assisted orientation is carried out in an airflow-assisted device, which is composed of a fan with adjustable wind speed, a guide valve, a filter and a sample area; the airflow-assisted device is placed in an air bath box with adjustable temperature.
11. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The mechanical stretching procedure has a stretching deformation ratio of 100-150%, a stretching frequency of 10-60 times / minute, and a stretching number of 5000-20000 times.
12. The oriented collagen-based biomaterial and the preparation method thereof according to claim 4, characterized in that The collagen-based material can be obtained by chemical cross-linking, wherein the chemical cross-linking is to immerse the air-dried or freeze-dried membrane-like or sponge-like collagen-based material in a cross-linking agent solution such as glutaraldehyde, genipin, carbodiimide, etc. for cross-linking; It can also be obtained by adding a crosslinking agent solution to the above-mentioned collagen-based material during the mechanical stretching process, and performing stretching and crosslinking.
13. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The collagen-based material can be type I collagen from pig skin, cow skin, cow tendon and rat tail tendon extracted by acid swelling, enzyme degradation or decellularization, or type II collagen from pig or cow articular cartilage.
14. The oriented collagen-based biomaterial and the preparation method thereof according to claim 1, characterized in that The collagen-based material can introduce growth factors, active polypeptides and other small molecule bioactive substances during the preparation process to improve the bioactivity of the collagen-based biomaterial.
15. The use of an oriented collagen-based biomaterial according to claim 1, characterized in that The oriented collagen-based biomaterial can be used for the regeneration, repair, functional reconstruction and enhancement of diseased and defective tissues and the construction of artificial organs in tissue engineering.