CN118022055A - Preparation method of collagen composite membrane with high strength, high stability and high biosafety - Google Patents
Preparation method of collagen composite membrane with high strength, high stability and high biosafety Download PDFInfo
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 68
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- 239000002131 composite material Substances 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 68
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 68
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Abstract
The invention belongs to the field of medical tissue engineering materials, and provides a preparation method of a collagen composite membrane with high strength, high stability and high biosafety, which comprises the following steps: mixing tilapia skin collagen with hydrochloric acid solution to form a protein acid solution; dissolving spider silk protein in water, and mixing with the protein acid solution to obtain a mixed solution; regulating the pH value of the mixed solution to be neutral, standing and drying to obtain a composite membrane; and hydrating the composite membrane, immersing the composite membrane in a solution containing natural polyphenol compounds, and drying to obtain the collagen composite membrane. The suture-resistant collagen composite membrane prepared by the invention can be applied to the repair of the dura mater/spinal membrane and the promotion of the repair of membrane tissue injury, and has excellent application prospect.
Description
Technical Field
The invention belongs to the field of medical tissue engineering materials, and particularly relates to a collagen composite membrane material with high strength, high stability and high biosafety and a preparation method thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Collagen is a major structural protein in biological connective tissue, and has excellent biocompatibility, bioactivity and biodegradability. Therefore, the protein has good application prospect in the preparation of tissue engineering materials. However, the pure collagen material obtained by the conventional method is often low in strength and poor in stability, and is difficult to apply to practice.
The introduction of the artificial high molecular material or the use of the chemical cross-linking agent is a common collagen material strengthening means in the past, and can effectively improve the defects in the aspect of the physical and chemical properties of the collagen material, but also has various adverse effects. The artificial high molecular materials such as polylactic acid, PEG and the like have poor biodegradability, are difficult to be absorbed and degraded by tissues after being implanted into the body, and can cause chronic inflammation of the tissues and pain translation of patients after being implanted for a long time, and can also obviously inhibit complete regeneration and functional recovery of the tissues although rejection phenomenon is not caused in a short time. The introduction of chemical cross-linking agents such as glutaraldehyde can form covalent chemical bonds among collagen molecules, so that the degradation resistance of the collagen material is effectively improved, and the defect of mechanical properties of the material is improved to a certain extent. However, chemical cross-linking agents within the material are difficult to completely remove, and their significant biotoxicity can lead to secondary damage and calcification of surrounding tissues after implantation of the material into the body.
Spider silk proteins have received considerable attention in the past because of their excellent mechanical properties. Because of the strong consciousness and aggressiveness of spiders, spider silk proteins are difficult to obtain through large-scale cultivation like fibroin, so that the obtaining of recombinant spider silk proteins through heterologous expression of bioengineering bacteria is a main current acquisition way. However, high molecular weight spider silk proteins are difficult to obtain in large quantities because of their high sequence reproducibility and rich hydrophobic structures, and their recombinant protein products are also poorly water-soluble. The inventor provides a high-efficiency expression method and a water-solubility method of high-molecular-weight recombinant spider silk protein in a patent (202111661047.9) filed before, which provides a certain help for the research and development of related materials.
The natural plant polyphenol compounds such as tea polyphenol, anthocyanin and the like have good biological safety, and have clear in-vivo metabolic pathway and no toxic or side effect. Meanwhile, the natural polyphenol compounds are proved to have good anti-inflammatory and antibacterial beneficial functions in a plurality of researches. However, the improvement effect of the compounds on the physicochemical properties of protein materials is still freshly reported.
Disclosure of Invention
In order to solve the problems, the invention provides a simple and easy preparation method of the collagen composite membrane with high strength, high biocompatibility and high stability. The suture-resistant collagen composite membrane can be applied to the repair of the dura mater/the spinal membrane and the promotion of the repair of the membrane tissue injury, and has excellent application prospect.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect of the present invention, there is provided a method for preparing a collagen composite membrane with high strength, high stability and high biosafety, comprising:
Mixing tilapia skin collagen with hydrochloric acid solution to form a protein acid solution;
dissolving spider silk protein in water, and mixing with the protein acid solution to obtain a mixed solution;
regulating the pH value of the mixed solution to be neutral, standing and drying to obtain a composite membrane;
and hydrating the composite membrane, immersing the composite membrane in a solution containing natural polyphenol compounds, and drying to obtain the collagen composite membrane.
In some embodiments, the natural polyphenol compound is selected from at least one of tea polyphenol, anthocyanin, procyanidine, EGCG, catechin.
In some embodiments, the natural polyphenol compound is present in the natural polyphenol compound-containing solution at a mass concentration of 0.75% to 1%.
In some embodiments, the natural polyphenol compounds are EGCG and catechin in a concentration ratio of 2:1-1.2.
In some embodiments, the specific step of hydrating comprises: immersing the composite membrane in water until the membrane is completely soaked and whitened, absorbing excessive liquid, repeating for multiple times, and drying to obtain the final product.
In some embodiments, the concentration of the mixture is 2-4mg/ml.
In some embodiments, the mass ratio of fish skin collagen to spider silk protein is from 5 to 6:1.
In some embodiments, the spider silk protein is 24-fold, 48-fold, 72-fold, or 96-fold.
It should be noted that the spider silk protein of the present invention may be prepared by the method of example 1 in patent CN114292322 a.
More specifically, it includes:
1) Weighing 30mg of tilapia skin collagen, fully mixing the tilapia skin collagen with 2.9ml of 0.1-0.2M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein acid solution with the concentration of 10 mg/ml; weighing 6mg of 24-fold spider silk protein, dissolving in 100ul of water, and mixing with the protein acid solution to obtain a final concentration of 2mg/ml; slowly adding 2M NaOH to enable the final pH value to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film. The hydrated protein film is placed in a 1% (w/v) tea polyphenol solution, and is kept standing overnight at 4 ℃, taken out and dried.
2) Weighing 30mg of tilapia skin collagen, fully mixing the tilapia skin collagen with 2.9ml of 0.1-0.2M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein acid solution with the concentration of 10 mg/ml; weighing 6mg of 48-fold spider silk protein, dissolving in 100ul of water, and mixing with the protein acid solution to obtain a final concentration of 2mg/ml; slowly adding 2M NaOH to regulate the pH of the solution to enable the final pH to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film. The hydrated protein film is placed in 1% (w/v) anthocyanin solution, and the protein film is left standing overnight at 4 ℃, taken out and dried.
3) Weighing 30mg of fish skin collagen, fully mixing the fish skin collagen with 2.9ml of 0.1-0.2M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein acid solution with the concentration of 10 mg/ml; weighing 72 times spider silk protein 6mg, dissolving in 100ul water, mixing with the above protease solution, and final concentration of 2mg/ml; slowly adding 2M NaOH to regulate the pH of the solution to enable the final pH to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film. The hydrated protein film is placed in 1% (w/v) procyanidine solution, and left standing overnight at 4 ℃, taken out and dried.
4) Weighing 30mg of fish skin collagen, fully mixing the fish skin collagen with 2.9ml of 0.1-0.2M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein acid solution with the concentration of 10 mg/ml; weighing 96 times of spider silk protein 6mg, dissolving in 100ul of water, and mixing with the above protease solution to obtain final concentration of 2mg/ml; slowly adding 2M NaOH to regulate the pH of the solution to enable the final pH to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film. The hydrated protein film is placed in 0.5% (w/v) EGCG+0.25% (w/v) catechin solution, and the solution is kept standing overnight at 4 ℃, taken out and dried.
In a second aspect of the present invention, there is provided a collagen composite membrane prepared by the above method.
In a third aspect, the invention provides the use of a natural polyphenol compound for increasing the degradation resistance of a collagen composite membrane.
The beneficial effects of the invention are that
(1) On the premise of not introducing an artificial high polymer material, the invention effectively improves the mechanical property of the collagen membrane by combining the hydration method of the water-dissolved high ploidy spider silk protein and the membrane. The obtained collagen composite film can bear the stretching of the suture after the suture is penetrated, and the lateral stretching resistance can reach more than 100 MPa. The composite membrane material can be applied to various tissue injury filling/repairing scenes including the dura mater/the dura mater, and the dilemma that the traditional collagen membrane material is difficult to bear suturing is eliminated.
(2) By means of interaction of tea polyphenol, anthocyanin, procyanidine, polyphenol natural compounds such as EGCG, catechin and the like and protein side chains, the collagen composite membrane effectively improves the degradation resistance of the collagen composite membrane on the premise of not using EDC/NHS and glutaraldehyde chemical cross-linking agents, and the degradation resistance time can reach more than 20 days. The method effectively avoids biotoxicity caused by cross-linking of materials based on a conventional chemical method. Meanwhile, the anti-inflammatory, antibacterial, antioxidant and tissue injury repair promoting capabilities of the material can be effectively improved.
(3) The preparation method is simple, has strong practicability and is easy to popularize.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Fig. 1: collagen membrane (Collagen), collagen-spidroin membrane (Collagen & SP), and Collagen spidroin membrane (collegen & SP (Aquation)) after hydration, changes in the structural regularity of the proteins within the membrane;
fig. 2: collagen membrane (Collagen), collagen-spidroin membrane (Collagen & SP) and typical tensile strength results of Collagen spidroin membrane after hydration (Collagen & SP (Aquation));
Fig. 3: collagen-spidroin membrane (C) was compared to the difference in stability of collagen-spidroin membrane soaked with anthocyanin (c+a), tea polyphenol (c+tp), catechin (c+c), EGCG (c+egcg), catechin and EGCG (c+egcg+cg).
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Interpretation of the terms
In the present invention, EGCG means: epigallocatechin gallate.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1
Weighing 30mg of tilapia skin collagen, fully mixing the tilapia skin collagen with 2.9ml of 0.1M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein solution with the concentration of 10 mg/ml; weighing 72 times spider silk protein 6mg, dissolving in 100ul water, mixing with the above protease solution, and final concentration of 2mg/ml; slowly adding 2M NaOH to enable the final pH value to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film (collagen-spider silk protein film) after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film, thus obtaining the hydrated collagen spider silk protein film. Placing the hydrated protein membrane into 1% (w/v) tea polyphenol solution, standing overnight at 4deg.C, taking out, and drying to obtain collagen-spider silk protein membrane soaked with tea polyphenol (C+TP).
The test results of the existing collagen membrane, the collagen-spider silk protein membrane prepared by the method and the hydrated collagen spider silk protein membrane are shown in fig. 1 and 2, and therefore, the invention can effectively improve the mechanical properties of the collagen membrane by combining the hydration method of the water-dissolved high ploidy spider silk protein and the membrane.
Example 2
Weighing 30mg of tilapia skin collagen, fully mixing the tilapia skin collagen with 2.9ml of 0.1M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein solution with the concentration of 10 mg/ml; weighing 72 times spider silk protein 6mg, dissolving in 100ul water, mixing with the above protease solution, and final concentration of 2mg/ml; slowly adding 2M NaOH to regulate the pH of the solution to enable the final pH to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film. Placing the hydrated protein membrane into 1% (w/v) anthocyanin solution, standing overnight at 4deg.C, taking out, and drying to obtain collagen-spider silk protein membrane soaked with anthocyanin (C+A).
Example 3
Weighing 30mg of fish skin collagen, fully mixing the fish skin collagen with 2.9ml of 0.1M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein acid solution with the concentration of 10 mg/ml; weighing 72 times spider silk protein 6mg, dissolving in 100ul water, mixing with the above protease solution, and final concentration of 2mg/ml; slowly adding 2M NaOH to regulate the pH of the solution to enable the final pH to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film. Placing the hydrated protein membrane into 1% (w/v) procyanidine solution, standing at 4deg.C overnight, taking out, and drying to obtain collagen-spider silk protein membrane soaked with procyanidine.
Example 4
4) Weighing 30mg of fish skin collagen, fully mixing the fish skin collagen with 2.9ml of 0.1M HCl, and stirring overnight at 4 ℃ in a refrigerator to prepare a protein acid solution with the concentration of 10 mg/ml; weighing 72 times spider silk protein 6mg, dissolving in 100ul water, mixing with the above protease solution, and final concentration of 2mg/ml; slowly adding 2M NaOH to regulate the pH of the solution to enable the final pH to be 6-8; finally, pouring the solution into a 6-hole plate, standing at room temperature for 15min to form gel, and drying to form a film after 2-3 days. After film formation, adding gel equal volume distilled water, standing until the film is completely soaked and whitened, absorbing excessive liquid, and repeating for two times. And drying again to finish the hydration process of the protein film. Placing the hydrated protein membrane into 0.5% (w/v) EGCG+0.25% (w/v) catechin solution, standing overnight at 4deg.C, taking out, and drying to obtain collagen-spider silk protein membrane soaked with catechin and EGCG (C+EGCG+CG).
Comparative example 1
The difference from example 4 is that the concentration of catechin was 0.75% (w/v), and EGCG was not added, to obtain a collagen-spider silk protein film impregnated with catechin (C+C).
Comparative example 2
The difference from example 4 is that EGCG concentration was 0.75% (w/v), and no catechin was added, to obtain a collagen-spider silk protein film impregnated with EGCG (C+EGCG).
The degradation resistance of the collagen-spidroin membrane after soaking the polyphenol compounds prepared in examples 1, 2,4 and comparative examples 1 and 2 was tested, as shown in fig. 3, and it was found that the degradation resistance of the collagen-spidroin membrane was better improved by using a combination of catechin and EGCG, which has a synergistic effect in enhancing the degradation resistance, as compared with other phenolic compounds.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the collagen composite membrane with high strength, high stability and high biosafety is characterized by comprising the following steps:
Mixing tilapia skin collagen with hydrochloric acid solution to form a protein acid solution;
dissolving spider silk protein in water, and mixing with the protein acid solution to obtain a mixed solution;
regulating the pH value of the mixed solution to be neutral, standing and drying to obtain a composite membrane;
and hydrating the composite membrane, immersing the composite membrane in a solution containing natural polyphenol compounds, and drying to obtain the collagen composite membrane.
2. The method for preparing a collagen composite membrane with high strength, high stability and high biosafety according to claim 1, wherein the natural polyphenol compound is at least one selected from the group consisting of tea polyphenol, anthocyanin, procyanidine, EGCG and catechin.
3. The method for producing a high-strength, high-stability and high-biosafety collagen composite membrane according to claim 1, wherein the mass concentration of the natural polyphenol compound in the solution containing the natural polyphenol compound is 0.75% to 1%.
4. The method for preparing a collagen composite membrane with high strength, high stability and high biosafety according to claim 1, wherein the natural polyphenol compound is EGCG and catechin, and the concentration ratio of the EGCG to the catechin is 2:1-1.2.
5. The method for preparing a collagen composite membrane with high strength, high stability and high biosafety according to claim 1, wherein the specific steps of hydration include: immersing the composite membrane in water until the membrane is completely soaked and whitened, absorbing excessive liquid, repeating for multiple times, and drying to obtain the final product.
6. The method for preparing a collagen composite membrane with high strength, high stability and high biosafety according to claim 1, wherein the concentration of the mixed solution is 2-4mg/ml.
7. The method for preparing the collagen composite membrane with high strength, high stability and high biosafety according to claim 1, wherein the mass ratio of the tilapia skin collagen to the spider silk protein is 5-6:1.
8. The method for preparing a high-strength, high-stability and high-biosafety collagen composite membrane according to claim 1, wherein the spider silk protein is 24-fold, 48-fold, 72-fold or 96-fold.
9. A collagen composite membrane prepared by the method of any one of claims 1-8.
10. The application of natural polyphenol compounds in improving the degradation resistance of collagen composite membranes.
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