Active collagen load guided tissue regeneration membrane and preparation method thereof

Abstract

The invention relates to an active collagen loaded guided tissue regeneration membrane and a preparation method thereof, belongs to the technical field of medical materials, and is suitable for tissue repair scenes. Collagen is used as a matrix, a reinforcement is a silk protein-collagen-elastin fusion protein cross-linked composite sponge with an amino acid sequence as shown in SEQ ID NO: 4, after the matrix and the reinforcement are compounded, chemical cross-linking treatment can be performed once, and the obtained product is the active collagen loaded guided tissue regeneration membrane. The membrane has excellent tensile strength and elastic modulus mechanical properties, is small in swelling after absorbing water, can avoid tissue swelling, is low in degradation rate, and is suitable for tissue repair needing long-time healing, and meanwhile, the EGF-loaded guided tissue regeneration membrane has the effects of remarkably promoting human skin fibroblast proliferation and total collagen expression.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to an active collagen-loaded guided tissue regeneration membrane and a preparation method thereof, which is suitable for promoting tissue regeneration and repair. Background Art

[0002] Guided Tissue Regeneration Membrane (GTR membrane) is a biomaterial used to promote tissue repair and regeneration, and is widely used in dentistry, orthopedics and other fields. This membrane usually creates a suitable environment to promote the growth of specific cells and tissue repair by covering or isolating the damaged area. Its main function is to prevent inappropriate cells from entering the repair area through physical barrier action, while providing support for the required cells, thereby accelerating the tissue regeneration process. The materials of guided tissue regeneration membranes usually have good biocompatibility and degradability, and can be degraded within a certain period of time and gradually replaced by new tissue.

[0003] Guided tissue regeneration membranes, as important materials in tissue engineering, are widely used to repair various tissue defects. However, existing guided tissue regeneration membranes have multiple problems in practical applications. First, their expansion rate is too high, resulting in insufficient stability and support of the membrane, affecting the repair effect of the tissue. Secondly, the low tensile strength makes the membrane prone to deformation or breakage under load, limiting its reliability in long-term use. In addition, the low elastic modulus also makes it impossible for the membrane to provide sufficient mechanical support during the tissue repair process, thereby failing to effectively promote the growth and repair of new tissues. In addition, current guided tissue regeneration membranes generally have a low degradation rate, which causes the material to degrade too slowly in the body, which may cause a long-term biovolume burden.

[0004] In addition to the deficiencies in mechanical properties, the biocompatibility of guided tissue regeneration membranes is also an important factor affecting their effectiveness. At present, guided tissue regeneration membranes with poor cell adhesion ability are difficult to support uniform growth of cells on the membrane surface, resulting in low efficiency of cell migration and proliferation during tissue repair. Cell adhesion and growth are crucial for tissue regeneration, especially for complex tissue defects that require a long time to repair. Therefore, improving the cell adhesion of the membrane is the key to enhancing its tissue repair function. However, current guided tissue regeneration membranes generally fail to fully consider the improvement of cell adhesion, thereby restricting the activity of the membrane in tissue regeneration.

[0005] In order to solve the above problems, in recent years, researchers have begun to learn from the active characteristics of silk protein, collagen and elastin to improve the performance of guided tissue regeneration membranes. Silk protein, collagen and elastin are natural polymer materials with good biocompatibility, degradability and biological activity. By introducing the active properties of these natural proteins, researchers hope to improve the swelling, tensile strength, elastic modulus and degradation characteristics of guided tissue regeneration membranes, while improving the adhesion ability of cells. These proteins have excellent cell adhesion and proliferation support effects, which can promote the effective combination of membranes with surrounding tissues, thereby promoting tissue regeneration. Therefore, through the guided modification and combination of these natural materials, the overall performance of guided tissue regeneration membranes can be significantly improved, thereby meeting higher clinical needs. Summary of the invention

[0006] In order to solve the above problems, the present invention first provides a method for preparing an active collagen-loaded guided tissue regeneration membrane, which can improve the mechanical properties, cell adhesion ability and stability of the membrane and is suitable for various occasions requiring tissue repair.

[0007] The method comprises the following steps: step S1: preparation of a collagen gel matrix; step S2: preparation of a composite sponge cross-linked with silk protein, collagen and elastin fusion protein; and step S3: preparation of a guided tissue regeneration membrane by chemically cross-linking the collagen gel and the composite sponge.

[0008] In certain embodiments, the silk protein-collagen-elastin fusion protein is synthesized by genetic engineering methods, and the fusion protein can provide abundant cell adhesion sites and good biocompatibility to promote cell growth on the membrane and tissue regeneration.

[0009] The present invention also provides a preparation method, which comprises the following steps: in step S1, type I collagen is dissolved and the concentration is adjusted by using a glacial acetic acid solution to ensure that the collagen can be evenly dissolved; in step S2, a silk protein-collagen-elastin fusion protein is expressed in a BL21 (DE3) strain by an expression plasmid, and the fusion protein is purified by a Ni-NTA agarose gel column; in step S3, a composite sponge with a good structure is prepared by freeze drying and cross-linking treatment, and the composite sponge is cross-linked with a collagen gel to obtain the guided tissue regeneration membrane.

[0010] Finally, the present invention provides an application, which is the application of the guided tissue regeneration membrane in products for bone tissue regeneration or soft tissue support, which can provide support for tissues, promote cell growth and tissue repair, and is particularly suitable for complex tissue defects that require a longer time to heal.

[0011] In certain embodiments, the guided tissue regeneration membrane has excellent tensile strength, elastic modulus, low swelling, high stability and low degradation rate, which can avoid tissue swelling and support long-term healing process, while providing a surface suitable for cell adhesion and promoting tissue regeneration.

[0012] In certain embodiments, the guided tissue regeneration membrane loaded with EGF has the efficacy of promoting the proliferation of human skin fibroblasts and the expression of total collagen.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects: it can provide a guided tissue regeneration membrane with good mechanical properties and biocompatibility, and the preparation process is simple and suitable for large-scale production. In addition, the present invention combines silk protein, collagen and elastin for the first time to develop a new type of high-efficiency guided membrane with better mechanical strength and cell support, which is particularly suitable for clinical tissue repair; at the same time, the above-mentioned guided tissue regeneration membrane loaded with EGF has the effect of significantly promoting the proliferation of human skin fibroblasts and the expression of total collagen. DETAILED DESCRIPTION

[0014] In order to make the technical problems to be solved, technical solutions and advantages of the present invention more clear, they are described in detail below in conjunction with specific embodiments.

[0015] The β-folded structure of the silk protein crystal region is composed of highly ordered, hydrogen-bonded polypeptide chains. Its tight molecular stacking gives silk protein excellent mechanical properties, such as high strength and thermal stability, which is the key to the excellent material properties of silk. The typical repeating sequence unit is: GAGAGS (SEQ ID NO: 1);

[0016] The core domain of collagen is composed of triple helical structure (stability and strength), non-helical terminal peptide (cross-linking effect), repeat sequence (molecular assembly) and modified region (enhanced function). These domains together determine the multifunctionality of collagen in tissue support, signal transduction and biomechanical properties. The typical repeat sequence unit is: GSLGIQGPQGPPGKEGQRGRRGKTGPPGKPGPPGPPGPPG IQGIHQTLGGYYNKDNKGNDEHEAGGLKGDKGETGLPGFPGSVGPKGQKGEPGEPFTKGEKGDRGEPGVIGSQGVKGEPGDPGPPGLIGSPGLKGQQGSAG SMGPRGPP (SEQ ID NO: 2);

[0017] The hydrophobic region of elastin provides the core mechanism for the reversible stretching of elastin; during the stretching process, the molecular chain is forced to stretch, overcoming the entropy drive of the random coil state. During relaxation, the hydrophobic amino acids reassemble through hydrophobic drive to restore the original state of the molecule; the hydrophobic region of elastin is mainly composed of the following types of repeating sequences: GVGAP (SEQ ID NO: 3);

[0018] Example 1 Preparation of Silk Protein-Collagen-Elastin Loaded Guided Tissue Regeneration Membrane

[0019] Preparation of collagen gel matrix: Weigh type I collagen and dissolve it in 0.5M glacial acetic acid solution to a concentration of 1-5 mg / mL; slowly stir at 4°C for 12-24 hours until it is completely dissolved to obtain a uniform collagen solution. Use NaOH to adjust the pH of the collagen solution to 7.2-7.4, pour the solution into the mold, balance the pH at room temperature, transfer to 37°C for incubation for 30-60 minutes, and observe the completion of gelation.

[0020] Preparation of Si-Col-El fusion protein: According to the structural characteristics of silk protein, collagen and elastin, silk protein-collagen-elastin fusion protein (Si-Co-El) was constructed:

[0021] (SEQ ID NO:4).

[0022] The expression plasmid for expressing the Si-Col-E1 fusion protein as shown in SEQ ID NO: 4 was synthesized and customized by GenScript Biotechnology Co., Ltd., wherein the pET-28a-Si-Col-E1 expression vector with HIS tag was selected, and the NcoI and XhoI restriction enzyme sites were inserted at the same time to ensure the directional insertion of the gene fragment; the BL21 (DE3) strain capable of expressing the fusion protein was used to transform the pET-28a-Si-Col-E1 expression plasmid into the BL21 (DE3) strain by heat shock; the positive clones were screened on the LB plate containing antibiotics (ampicillin); the transformed bacteria were cultured in LB medium and shaken at 37°C; IPTG (final concentration 1mM) was added and induced to express at 25°C for 4h; after induction, the bacteria were collected by centrifugation (4000x g, 10 minutes) and stored at -20°C. Lysis buffer (50mMTris-HCl, 300mM NaCl, 10mM Imidazole, pH 8.0) was used. Ultrasonic treatment or high-pressure homogenizer was used to lyse the bacteria; the lysate was obtained by centrifugation (12000 x g, 20 minutes). Ni-NTA agarose gel column was used to elute the eluate containing Imidazole (250 mM Imidazole, 50 mM Tris-HCl), and the eluate was subjected to SDS-PAGE electrophoresis to detect the molecular weight of Si-Col-El fusion protein to ensure successful expression.

[0023] Preparation of Si-Col-El fusion protein sponge: Si-Col-El fusion protein solution is injected into a specific square mold, and the thickness is controlled at 1-5mm; frozen at -80℃ for 4 hours, transferred to a vacuum freeze dryer and freeze-dried for 12-24 hours to form a porous structure sponge. Use 0.1% (v / v) glutaraldehyde crosslinker and react at room temperature for 12 hours for chemical crosslinking; after crosslinking, thoroughly wash the residual crosslinker with deionized water. Use ultrasonic treatment or fine-tune the freeze-drying program to adjust the pore size in the range of 50-300μm.

[0024] Composite cross-linking of collagen gel matrix and sponge reinforcement: The prepared Si-Col-El fusion protein sponge is immersed in collagen solution (1-2 mg / mL) and adsorbed for 30 minutes to ensure that collagen fully penetrates into the sponge pores. The collagen gel matrix and Si-Col-El fusion protein are alternately stacked into a multilayer membrane to avoid stratification and shedding; a mild cross-linking agent Genipin 0.5% (w / v) is used at 37°C for 6-12 hours for cross-linking treatment. 10% glycerol is added as a protective agent during the cross-linking process to reduce embrittlement during the cross-linking process, and the cross-linked product is repeatedly washed with deionized water to remove the unreacted cross-linking agent to obtain a regenerated membrane.

[0025] Freeze drying and molding of the membrane: the regenerated membrane is placed in a mold and frozen at -80°C for 4-6 hours; vacuum freeze drying is performed for 12-24 hours to obtain a uniform regenerated membrane, which is marked as Si-Col-El regenerated membrane.

[0026] Example 2 Preparation of Silk Protein-Collagen Loaded Guided Tissue Regeneration Membrane

[0027] According to the structural characteristics of silk protein and collagen, silk protein-collagen fusion protein (Si-Co) is constructed:

[0028] GAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGA GSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGGGGSGSLGIQGPQGPPGKEGQRGRRGKTGPPGKPGPPGPPGPPGIQGIHQTLGGYYNKDNKGNDEHEAGGLKGDKGETGL PGFPGSVGPKGQKGEPGEPFTKGEKGDRGEPGVIGSQGVKGEPGDPGPPGLIGSPGLKGQQGSAGSMGPRGPPGSLGIQGPQGPPGKEGQRGRRGKTGPPGKPGPPGPPGPPG IQGIHQTLGGYYNKDNKGNDEHEAGGLKGDKGETGLPGFPGSVGPKGQKGEPGEPFTKGEKGDRGEPGVIGSQGVKGEPGDPGPPGLIGSPGLKGQQGSAGSMGPRGPP(SEQ ID NO:5).

[0029] The preparation of the silk protein-collagen loaded guided tissue regeneration membrane is the same as the preparation method of Example 1, except that the fusion protein shown in SEQ ID NO: 5 is selected to finally obtain a Si-Co regeneration membrane.

[0030] Example 3 Preparation of collagen-elastin loaded guided tissue regeneration membrane

[0031] According to the structural characteristics of elastin and collagen, collagen-elastin fusion protein (Co-El) is constructed:

[0032] GSLGIQGPQGPPGKEGQRGRRGKTGPPGKPGPPGPPGPPGIQGIHQTLGGYYNKDNKGNDEHEAGGLKGDKGETGLPGFPGSVGPKGQKGEPGEPFTKGEKGDRGEPGV IGSQGVKGEPGDPGPPGLIGSPGLKGQQGSAGSMGPRGPPGSLGIQGPQGPPGKEGQRGRRGKTGPPGKPGPPGPPGPPGIQGIHQTLGGYYNKDNKGNDEHEAGGLKG DKGETGLPGFPGSVGPKGQKGEPGEPFTKGEKGDRGEPGVIGSQGVKGEPGDPGPPGLIGSPGLKGQQGSAGSMGPRGPPGGGGSGVGAPGVGAPGVGAPGVGAPGVGA PGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAPGVGAP(SEQ ID NO:6).

[0033] The preparation of the collagen-elastin loaded guided tissue regeneration membrane is the same as the preparation method of Example 1, except that the fusion protein shown in SEQ ID NO: 6 is selected to finally obtain the Co-El regeneration membrane.

[0034] Comparative Example 1 Preparation of guided tissue regeneration membrane loaded with natural silk protein

[0035] Weigh type I collagen powder, dissolve it in 0.1M glacial acetic acid, adjust the concentration to 1-5mg / mL, stir at 4°C for 12 hours until completely dissolved, and use NaOH to adjust the pH to 7.4 to avoid solution degradation. Pour the solution into the mold, balance the pH at room temperature, transfer to 37°C for incubation for 30-60 minutes, and observe the completion of gelation.

[0036] Take silk fibers from Bombyx mori and add 0.02M Na 2 CO 3Boil in a water bath for 30 minutes to remove sericin, wash 3 times and dry. Dissolve clean silk fibers in 9.3M LiBr solution, adjust the solution concentration to 10% (w / v), and stir at 60°C for 6 hours. Use a dialysis bag (molecular weight cutoff 3.5kDa) to dialyze in deionized water for 48 hours to obtain a silk protein solution with a concentration of 4-6%; inject the silk protein solution into a specific square mold, and control the thickness to 1-5mm; freeze at -80°C for 4 hours, transfer to a vacuum freeze dryer and freeze-dry for 12-24 hours to form a porous structure silk sponge. Use 0.1% (v / v) glutaraldehyde crosslinker and react at room temperature for 12 hours for chemical crosslinking; after crosslinking, thoroughly wash the residual crosslinker with deionized water. Use ultrasonic treatment or fine-tune the freeze-drying program to adjust the pore size in the range of 50-300μm.

[0037] The prepared silk sponge was immersed in a collagen solution (1-2 mg / mL) and adsorbed for 30 minutes to ensure that the collagen fully penetrated into the sponge pores. The collagen gel matrix and the silk sponge were alternately stacked into a multilayer film to avoid stratification and shedding; a mild crosslinking agent Genipin 0.5% (w / v) was used at 37°C for 6-12 hours for crosslinking treatment. 10% glycerol was added as a protective agent during the crosslinking process to reduce embrittlement during the crosslinking process, and the crosslinked product was repeatedly washed with deionized water to remove the unreacted crosslinking agent to obtain a regenerated membrane. The regenerated membrane was placed in a mold and frozen at -80°C for 4-6 hours; vacuum freeze-dried for 12-24 hours to obtain a uniform regenerated membrane, which was marked as a silk regenerated membrane.

[0038] Comparative Example 2 Preparation of guided tissue regeneration membrane loaded with natural elastin

[0039] Weigh type I collagen powder, dissolve it in 0.1M glacial acetic acid, adjust the concentration to 1-5mg / mL, stir at 4°C for 12 hours until completely dissolved, and use NaOH to adjust the pH to 7.4 to avoid solution degradation. Pour the solution into the mold, balance the pH at room temperature, transfer to 37°C for incubation for 30-60 minutes, and observe the completion of gelation.

[0040] The middle layer tissue of the pig aorta was taken, cut into small pieces, treated with 0.1M NaOH solution at 37°C for 2 hours to remove cells and impurities, washed to neutrality, and then added with 0.1M phosphate buffer (pH 8.0) and elastase (1mg / mL), and enzymatic hydrolysis was carried out at 37°C for 12-16 hours. The precipitate was removed by centrifugation, the supernatant was collected, the concentration was adjusted to 3mg / mL and filtered for sterilization. Dialysis was performed in deionized water for 48 hours using a dialysis bag (molecular weight cutoff 3.5kDa) to obtain a natural elastin solution with a concentration of 4-6%; the natural elastin solution was injected into a specific square mold with a thickness controlled at 1-5mm; frozen at -80°C for 4 hours, transferred to a vacuum freeze dryer for freeze drying for 12-24 hours to form a porous structure elastin sponge. Glutaraldehyde crosslinking agent 0.1% (v / v) was used to react at room temperature for 12 hours for chemical crosslinking; after crosslinking, the residual crosslinking agent was thoroughly washed with deionized water. Use sonication or fine-tune the lyophilization procedure to adjust the pore size to a range of 50-300 μm.

[0041] The prepared elastin sponge was immersed in a collagen solution (1-2 mg / mL) and adsorbed for 30 minutes to ensure that the collagen fully penetrated into the sponge pores. The collagen gel matrix and the silk sponge were alternately stacked into a multilayer membrane to avoid stratification and shedding; a mild crosslinking agent Genipin 0.5% (w / v) was used at 37°C for 6-12 hours for crosslinking treatment. 10% glycerol was added as a protective agent during the crosslinking process to reduce embrittlement during the crosslinking process, and the crosslinked product was repeatedly washed with deionized water to remove the unreacted crosslinker to obtain a regenerated membrane. The regenerated membrane was placed in a mold and frozen at -80°C for 4-6 hours; vacuum freeze-dried for 12-24 hours to obtain a uniform regenerated membrane, which was marked as the Elastin regenerated membrane.

[0042] Example 4 Performance testing of guided tissue regeneration membranes based on different protein loadings

[0043] The guided tissue regeneration membranes prepared in the above Examples 1-3 and Comparative Examples 1-2 were tested.

[0044] The expansion rate of the membrane guided by different proteins was tested: the thickness of the dry membrane was measured after being soaked in pure water or saline for 10 minutes and divided by the thickness of the dry membrane. The obtained ratio was the expansion rate.

[0045] Tensile strength test of tissue regeneration membranes based on different proteins: The membrane was cut into standard dumbbell-shaped specimens (ISO527-2 standard, thickness 0.5mm, narrow section width 5mm, length 25mm), and placed on an electronic universal material testing machine. Under environmental conditions of 25°C and 50% humidity, the specimen was loaded at a tensile rate of 1mm / min, and the stress-strain curve was recorded during the tensile process until the specimen broke. The tensile strength (σmax=Fmax / A) was obtained by calculating the maximum tensile force (Fmax) divided by the initial cross-sectional area (A).

[0046] Elastic modulus test of tissue regeneration membranes guided by different proteins: The membrane samples were cut into standardized rectangular strips (10mm×30mm, thickness 0.5mm) and tested with an electronic universal material testing machine. Under an environment of 25°C and 50% humidity, the membrane samples were clamped between the clamps of the testing machine to ensure that the sample was evenly stressed. Force was applied at a constant tensile rate of 1mm / min, and the stress-strain curve was recorded. The stress (σ=F / A) and strain (ε=ΔL / L 0 ) linear relationship, and the slope of the elastic interval was used to calculate the elastic modulus (E = σ / ε).

[0047] Table 1 Performance data of Examples 1-3 and Comparative Examples 1-2

[0048]

[0049] Note: Compared with comparative example 1, a p<0.05, b p<0.01; compared with control 2, c p<0.05, d p<0.01

[0050] The results in Table 1 show that in terms of swelling rate, the silk-collagen-elastin loaded membrane has the lowest swelling rate, enabling it to provide higher structural stability and biocompatibility in tissue repair while avoiding potential adverse interactions between materials and tissues, making it an ideal choice for tissue regeneration scenarios that require high-precision control and long-term stable support.

[0051] In terms of tensile strength, the silk-collagen-elastin loaded membrane has the highest tensile strength and is suitable for tissue repair with high mechanical requirements; while the natural elastin loaded membrane has the lowest tensile strength but is suitable for flexible tissue repair applications.

[0052] In terms of elastic modulus, the silk protein-collagen-elastin loaded membrane has a higher elastic modulus, combining both rigidity and elasticity; the natural elastin loaded membrane has the lowest elastic modulus, with outstanding flexibility but insufficient rigidity.

[0053] Taking into account the above-mentioned performances of expansion rate, tensile strength and elastic modulus, the guided tissue regeneration membrane loaded with silk protein-collagen-elastin has the best performance and is widely used in tissue repair scenarios that require high strength, elasticity and high stability.

[0054] Detection of enzyme degradation rate of different protein-guided tissue regeneration membranes: Each membrane was cut into 10 mm diameter discs and the dry weight (W 0 ) and record its initial weight. Use phosphate buffered saline (PBS) containing 0.1% collagenase as the environment for enzyme degradation. Keep the medium pH at 7.4 and the temperature at 37°C to simulate the environment in the human body. Each membrane was placed in 20 mL of degradation medium and placed on a shaker (50 rpm) to ensure uniform degradation. Samples were taken after 1 day, 3 days, 7 days, 14 days, 21 days and 28 days. Take out the membrane each time you take a sample, wash it 3 times with distilled water to remove residual enzymes, and weigh the degraded dry weight (W) after air drying. t ).

[0055] Calculate the degradation rate using the formula:

[0056]

[0057] Table 2 Degradation rate under enzyme degradation environment (PBS + collagenase):

[0058]

[0059] The results in Table 2 show that compared with other loaded membranes, the silk protein-collagen-elastin loaded membrane has the slowest degradation rate, which enables the membrane to maintain its structure and function in the body for a long time and provide continuous mechanical support. It is suitable for tissue repair that requires a long time to heal (such as bone tissue regeneration or soft tissue support) to avoid excessive degradation of the material, resulting in loss of support when tissue repair is not completed.

[0060] Cell adhesion experiment based on different protein-guided tissue regeneration membranes: Each membrane was prepared with a diameter of 10 mm and sterilized with ethanol, and then placed in a 24-well plate. The membrane was washed three times with phosphate buffered saline (PBS) to remove residual disinfectant. Human mesenchymal stem cells (hMSCs) were used as the test cell line. The cells were incubated at 2×10 4 Inoculate the membrane surface at a density of 100 μL / well and add 500 μL complete culture medium (containing 10% fetal bovine serum and 1% antibiotics) to ensure that the membrane is completely immersed. Incubate at 37°C and 5% CO 2 The cells were cultured in an incubator for 4 hours, 12 hours, and 24 hours to test the cell adhesion at different time points. CCK-8 assay: 10 μL of CCK-8 reagent was added at each time point, incubated at 37°C for 2 hours, and the absorbance (OD value) at 450 nm was measured to quantify the number of adhered cells.

[0061] Table 3 OD value of cell adhesion detected by CCK-8 (450nm)

[0062]

[0063] The results in Table 3 show that the Si-Col-El regenerated membrane has the highest cell adhesion rate, which is due to the synergistic effect of silk protein, collagen and elastin, providing abundant adhesion sites and flexibility; the performance of Si-Co regenerated membrane and Col-El regenerated membrane is slightly worse, but can still support good cell adhesion and expansion; the adhesion performance of Silk regenerated membrane and Elastin regenerated membrane is the lowest, which may be due to the lack of synergistic effect of composite proteins, resulting in insufficient surface adhesion sites or excessive rigidity.

[0064] Example 5 Experimental study on the biological activity of cross-linked epidermal growth factor with different protein-loaded guided tissue regeneration membranes

[0065] High-purity EGF is prepared using recombinant technology, and EGF is encapsulated using slow-release carrier chitosan nanoparticles. EGF and the carrier are mixed in an appropriate proportion, and EGF nanoparticles are prepared by emulsification-solvent evaporation method or ion crosslinking method, and the particle size is controlled at 100-200nm to ensure its slow-release performance. EGF nanoparticles are uniformly coated on the surface of the guided tissue regeneration membrane of Examples 1-3 and Comparative Examples 1-2, and EGF is fixed by electrostatic adsorption or physical binding. The membrane is slightly cross-linked with low-concentration EDC / NHS to further fix the EGF nanoparticles to avoid its too fast release in the body, ensure that the cross-linking conditions are mild, and maintain the biological activity of EGF, and finally prepare epidermal growth factor-loaded guided tissue regeneration membranes, which are respectively labeled as Si-Col-El-EGF regeneration membrane, Si-Co-EGF regeneration membrane, Col-El-EGF regeneration membrane, Silk-EGF regeneration membrane and Elastin-EGF regeneration membrane.

[0066] Human skin fibroblasts (HDFs) were selected as experimental cells and divided into 5 experimental groups and 5 blank control groups (not loaded with epidermal growth factor). Each group of cells was inoculated on the surface of the membrane material with a culture density of 5000 cells / cm2. A low serum culture medium containing 1% FBS was used to simulate the cell stress environment to highlight the proliferation-promoting effect of EGF. CCK-8 reagent was used to detect cell proliferation, and the OD value of each group of cells was measured on the 7th day to evaluate the proliferation ability; at the same time, ELISA was used to quantitatively analyze the total collagen content secreted in the cell culture medium.

[0067] Proliferation rate = (OD of experimental group - OD of control group) / OD of control group × 100%

[0068] Table 4 Proliferation rate results of human dermal fibroblasts (HDFs)

[0069]

[0070] The results in Table 4 show that compared with other EGF-loaded regeneration membranes, the EGF-loaded Si-Col-El regeneration membrane significantly promoted the proliferation of human skin fibroblasts.

[0071] Table 5 Total collagen expression levels in human dermal fibroblasts (HDFs)

[0072]

[0073] The results in Table 5 show that compared with other EGF-loaded regeneration membranes, the EGF-loaded Si-Col-El regeneration membrane significantly promoted the total secretion of collagen, verifying the key role of the EGF-loaded Si-Col-El regeneration membrane in stimulating collagen regeneration.

[0074] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an active collagen-loaded guided tissue regeneration membrane, characterized in that: The following steps are involved: Step S1 is the preparation of collagen gel matrix; Step S2 is the preparation of composite sponge cross-linked with silk protein-collagen-elastin fusion protein; Step S3 is the preparation of guided tissue regeneration membrane by chemical cross-linking of collagen gel and composite sponge.

2. The preparation method according to claim 1, characterized in that: The silk protein-collagen-elastin fusion protein is selected from the amino acid sequence shown in SEQ ID NO:

4.

3. The preparation method according to claim 1 or 2, characterized in that: Step S1: Preparation of collagen gel matrix includes: dissolving type I collagen in 0.5M glacial acetic acid solution, adjusting the concentration to 1-5 mg / mL, slowly stirring at 4°C for 12-24 hours until completely dissolved, adjusting the pH to 7.2-7.4 with NaOH, transferring to a mold, and incubating at 37°C for 30-60 minutes for gelation.

4. The preparation method according to any one of claims 1 to 3, characterized in that The silk protein-collagen-elastin fusion protein in step S2 is expressed in the BL21 (DE3) strain via an expression plasmid, induced by IPTG, and purified via a Ni-NTA agarose gel column.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The composite sponge in step S3 is prepared by injecting a silk protein-collagen-elastin fusion protein solution into a mold, freezing and vacuum freeze-drying to form a porous structure sponge, and further cross-linking treatment with glutaraldehyde cross-linking agent to obtain the composite sponge, which is then alternately stacked and cross-linked with collagen gel to obtain a guided tissue regeneration membrane.

6. Use of the guided tissue regeneration membrane prepared according to the method according to any one of claims 1 to 5 in preparing products for bone tissue regeneration or soft tissue support.

7. A guided tissue regeneration membrane loaded with EGF, characterized in that: The guided tissue regeneration membrane is prepared by the method according to any one of claims 1-5.

8. Use of the guided tissue regeneration membrane according to claim 7 in the preparation of medical beauty products for promoting the proliferation of human skin fibroblasts and the expression of total collagen.

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