Autologous active biological membrane for promoting wound repair in breast augmentation and application of autologous active biological membrane

Abstract

The invention relates to an autologous active biological membrane for promoting wound repair in breast augmentation and application of the autologous active biological membrane, belongs to the technical field of medical materials, and is suitable for skin wound repair scenes. The invention provides an active biological membrane loaded with ICA-TGF-DEF fusion protein, the ICA-TGF-DEF fusion protein is selected from an amino acid sequence as shown in any one of SEQ ID NO: 4-6, the biological membrane takes collagen as a membrane matrix, and then the ICA-TGF-DEF fusion protein as shown in any one of SEQ ID NO: 4-6 and the collagen membrane matrix are subjected to crosslinking treatment to obtain the active biological membrane loaded with the ICA-TGF-DEF fusion protein. The self-active biological membrane for promoting wound repair in breast augmentation is obtained through a biological membrane preparation method, in the aspect of physical performance, the biological membrane has high expansion rate, porosity and high protein loading capacity, in the aspect of biological activity, the biological membrane can remarkably enhance cell attachment and proliferation, and an antibacterial wound microenvironment can be created to promote tissue repair; the dressing is especially suitable for wound repair after breast augmentation, and can accelerate tissue healing and reduce infection and fibrosis risks.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to an autologous active biofilm for promoting breast augmentation wound repair and application thereof. Background Art

[0002] In recent years, with the development of plastic surgery technology, breast augmentation surgery, as a common surgery to improve appearance, has seen an increasing demand year by year. However, breast augmentation wounds usually have problems such as slow healing, easy infection and scar formation, which not only prolongs the patient's recovery time, but also increases the risk of postoperative complications. The application of traditional biofilms in postoperative wound repair is difficult to meet the needs of complex wound repair due to their poor biocompatibility and single function. Improving the surface properties of biofilms and loading active proteins to further improve their performance in wound repair has become a hot topic in current research.

[0003] The key roles of bioactive molecules such as intercellular adhesion molecules (ICAM), transforming growth factor-β (TGF-β) and defensins in wound repair have been widely studied. ICAM can enhance cell adhesion and migration, promote cell aggregation and tissue remodeling in the wound area; TGF-β has a significant role in regulating cell proliferation, differentiation and fibrosis; defensins have antibacterial effects by directly destroying bacterial cell membranes. However, when these bioactive molecules are used alone, there are many limitations in terms of stability, activity persistence and ease of application, which makes it difficult to meet the repair needs of complex wounds. Therefore, ICAM, TGF-β and defensins are integrated through fusion protein technology to form a multifunctional fusion protein with adhesion promotion, tissue repair regulation and antibacterial functions, which provides a new technical path for wound repair.

[0004] Biofilms based on multifunctional fusion proteins can make up for the shortcomings of traditional biofilms in terms of single functionality, sustained biological activity and structural stability. By fixing the intercellular adhesion molecule-TGF-β-defensin (ICA-TGF-DEF) fusion protein on autologous biofilms (such as collagen membranes or fibrin membranes containing autologous components), it can not only significantly enhance cell attachment and proliferation, but also regulate the wound microenvironment to promote tissue repair, while providing broad-spectrum antibacterial protection. This multifunctional active biofilm is particularly suitable for wound repair after breast augmentation surgery. It can not only accelerate tissue healing, but also reduce the risk of infection and fibrosis, providing an efficient and safe innovative solution for the field of plastic surgery. Summary of the invention

[0005] In order to solve the deficiencies of the biofilm used for breast augmentation wound repair in the prior art in terms of cell adhesion, tissue repair and antibacterial performance, the present invention first provides an autologous active biofilm that promotes breast augmentation wound repair. The biofilm is loaded with intercellular adhesion molecule-transforming growth factor-β-defensin (ICA-TGF-DEF) fusion protein;

[0006] In certain embodiments, the fusion protein is selected from the amino acid sequence shown in any one of SEQ ID NOs: 4-6.

[0007] In certain embodiments, the preparation method of the biofilm is: collagen is used as a matrix, and the fusion protein is fixed on the surface or inside of the membrane by chemical cross-linking. The autologous active biofilm not only has good physical properties (such as high expansion rate, porosity and high protein loading), but also exhibits significant biological activity, including the ability to promote cell attachment and proliferation, inhibit bacterial infection and accelerate tissue repair, and is therefore particularly suitable for wound repair after breast augmentation surgery.

[0008] In certain embodiments, the fusion protein ICA-TGF-DEF combines the function of intercellular adhesion molecule (ICA) in promoting cell adhesion and migration, the role of transforming growth factor-β (TGF-β) in regulating cell proliferation and differentiation, and the broad-spectrum antibacterial activity of defensin (DEF), and can synergistically promote the regeneration and healing of wound tissue.

[0009] In certain embodiments, the fusion protein is prepared by genetic engineering technology and loaded into a collagen matrix after purification. The collagen matrix uses natural autologous collagen material, which has good biocompatibility and can significantly improve the loading efficiency of the fusion protein and its stability in the wound environment through cross-linking technology.

[0010] The present invention also provides a method for preparing an autologous active biofilm that promotes breast augmentation wound repair, the method comprising the following steps: first preparing a collagen gel matrix, optimizing the expansion rate and porosity of the membrane by adjusting the concentration of the collagen solution and the ratio of the crosslinking agent; then preparing the fusion protein ICA-TGF-DEF by recombinant DNA technology, maintaining its activity after purification; finally, fixing the fusion protein to the collagen matrix by chemical crosslinking or physical adsorption to obtain an autologous active biofilm loaded with the fusion protein. The prepared biofilm can continuously release the fusion protein in the wound environment, thereby providing long-term stable tissue repair and antibacterial effects.

[0011] Finally, the present invention provides the application of the above-mentioned autologous active biological membrane in the field of medical beauty, including but not limited to the preparation of medical beauty products that promote cell proliferation and adhesion, functional dressings for breast augmentation wound repair, or other medical biomaterials.

[0012] In certain embodiments, the autologous active biofilm of the present invention provides a safe and efficient solution for wound repair after breast augmentation surgery by significantly improving the wound healing speed and reducing the risk of postoperative infection and fibrosis.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects:

[0014] The present invention introduces the intercellular adhesion molecule-transforming growth factor-β-defensin (ICA-TGF-DEF) fusion protein into the biofilm for the first time, and achieves the comprehensive improvement of cell adhesion, proliferation and antibacterial performance in wound repair through synergistic effects;

[0015] The present invention uses autologous collagen as the membrane matrix material, which has good biocompatibility and can effectively reduce the risk of foreign body rejection;

[0016] The biofilm has a high protein loading capacity and stability, can continuously release fusion protein, and provide long-term biological activity support after surgery;

[0017] The autologous active biofilm of the present invention is particularly suitable for repairing wounds after breast augmentation surgery, significantly improving healing efficiency and reducing the incidence of infection and fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Swelling rate assay of active biofilms loaded with different fusion proteins.

[0019] Figure 2 Porosity detection of active biofilms loaded with different fusion proteins.

[0020] Figure 3 Tensile strength test of active biofilms loaded with different fusion proteins.

[0021] Figure 4 Protein loading rate detection of active biofilms loaded with different fusion proteins.

[0022] Figure 5 Cell proliferation assay of active biofilms loaded with different fusion proteins.

[0023] Figure 6 Cell attachment rate assay of active biofilms loaded with different fusion proteins.

[0024] Figure 7 Biofilm inhibition assay of active biofilms loaded with different fusion proteins.

[0025] Figure 8 Detection of wound healing rate of active biofilms loaded with different fusion proteins. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0027] When designing fusion proteins, the functional requirements of biofilms and protein properties were comprehensively considered, and three functional modules of cell adhesion, tissue regeneration and antibacterial functions were selected;

[0028] Intercellular Adhesion Molecules (ICAMs) are a class of glycoproteins expressed on the cell surface, which are mainly involved in cell-to-cell adhesion and immune response. ICAMs regulate cell-to-cell interactions and cell migration by binding to receptors such as integrins, and are widely involved in biological processes such as inflammatory response, immune response, tissue development, and repair.

[0029] Transforming Growth Factor-β (TGF-β) is a multifunctional cytokine that plays an important role in a variety of biological processes, including cell proliferation, differentiation, migration, apoptosis, immune regulation, and extracellular matrix production. TGF-β plays a key role in embryonic development, tissue repair, immune response, and tumor microenvironment.

[0030] Defensins are a class of natural antimicrobial peptides with broad-spectrum antibacterial, antiviral and immunomodulatory activities. They play an important role in resisting infection, maintaining barrier function and promoting wound healing by physically destroying pathogen membranes and regulating the host immune system.

[0031] Example 1 Preparation of active biofilm loaded with ICA-TGF-DEF fusion protein

[0032] According to the sequence information of intercellular adhesion molecules (ICAMs, Intercellular Adhesion Molecules) recorded by NCBI, intercellular adhesion molecule 2 precursor [Homo sapiens], NCBI Reference Sequence: NP_001093258.1, select its functional domain:

[0033] SDEKVFEVHVRPKKLAVEPKGSLEVNCSTTCNQPEVGGLETSLDKILLDEQAQWKHYLVSNISHDTVLQCHFTCSGKQESMNSNVSVYQPPRQVILTLQPTLVAVGKSFTIECRVPTVEPLDSLTLFLLF RGNETLHYETFGKAAPAPQEATATFNSTADREDGHRNFSCLAVLDLMSRGGNIFHKHSAPKMLEIYEPVSDSQMVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQRMGTYGVRAAWRRLPQAFRP(SEQ ID NO:1);

[0034] According to the sequence information of transforming growth factor-β (TGF-β, Transforming Growth Factor-β) recorded by NCBI, GenBank: AAA36738.1, select its functional domain:

[0035] FKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH(SEQ ID NO:2);

[0036] According to the NCBI sequence information of defensins, beta-defensin 104 precursor [Homosapiens], NCBI Reference Sequence: NP_525128.2: MQRLVLLLAIS LLLYQDLPVRSEFELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRKW DESLLNRTKP (SEQ ID NO: 3).

[0037] Taking into account the functional requirements of biofilm and the properties of proteins, intercellular adhesion molecules were selected as cell adhesion function, transforming growth factor-β as tissue regeneration function and defensin as antibacterial function to construct a fusion protein with biological activity (ICA-TGF-DEF):

[0038] SDEKVFEVHVRPKKLAVEPKGSLEVNCSTTCNQPEVGGLETSLDKILLDEQAQWKHYLVSNISHDTVLQCHFTCSGKQESMNSNVSVYQPPRQVILTLQPTLVAVGKSFTIECRVPTVEPLDSLT LFLFRGNETLHYETFGKAAPAPQEATATFNSTADREDGHRNFSCLAVLDLMSRGGNIFHKHSAPKMLEIYEPVSDSQMVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQRMGTYGVRAAWRRLPQ AFRPGGGGSGGGGSFKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETV PKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCHGGGGSGGGGSMQRLVLLLAISLLLYQDLPVRSEFELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRKWDESLLNRTKP(SEQ ID NO:4).

[0039] Preparation of collagen gel matrix: Dissolve type I collagen powder in 0.1M acetic acid solution in an ice bath and stir slowly for 4-6 hours until completely dissolved to prepare a collagen solution with a concentration of 3-5mg / mL. Place the collagen solution in an ice bath and slowly add 0.1M NaOH solution drop by drop, while using a pH meter to monitor the pH value of the solution and adjust it to close to 7.4. During the adjustment process, ensure that the solution is always at a low temperature (4°C) to avoid denaturation of collagen at high temperatures. Transfer the neutralized collagen solution to a preset mold or culture dish to ensure that the solution is evenly distributed. Place the mold or culture dish in a 37°C incubator and let it stand for 1-2 hours. The collagen solution will gradually gel to form a transparent and soft three-dimensional gel matrix.

[0040] Preparation of fusion protein ICA-TGF-DEF: GeneScript Biotech Co., Ltd. synthesized and customized an expression plasmid expressing the ICA-TGF-DEF fusion protein as shown in SEQ ID NO: 4, wherein the pET-28a-ICA-TGF-DEF expression vector with a HIS tag was selected, and NcoI and XhoI restriction enzyme sites were inserted at the same time to ensure the directional insertion of the gene fragment; a BL21 (DE3) strain capable of expressing the fusion protein was used to heat-shock transform the pET-28a-ICA-TGF-DEF expression plasmid into the BL21 (DE3) strain; positive clones were screened on an LB plate containing antibiotics (ampicillin); positive clones were inoculated into 5-10 mL LB medium (containing antibiotics) and cultured to OD600 = 0.6-0.8. IPTG (final concentration 0.1-1 mM) was added, and protein expression was induced at low temperature (16-25°C). Collect the bacteria after induced expression, use a centrifuge at 4°C, 5000-10000×g for 10 minutes to collect the cell precipitate; then resuspend the bacteria in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM PMSF, pH7.4), place the bacterial suspension in an ice bath, use an ultrasonic disruptor (power 30-50%, each pulse 3 seconds, interval 5 seconds), a total time of 10 minutes, centrifuge to remove insoluble matter (4°C, 12000×g, 30 minutes), and retain the soluble protein portion. The soluble protein sample was added to the pre-equilibrated Ni-NTA sample (equilibration solution was 50mM Tris-HCl, 300mM NaCl, 20mMImidazole, pH 8.0), and washed several times after binding (the buffer contained 20-40mM Imidazole to remove non-specific binding proteins); the target protein was eluted with elution buffer (containing 200-500mM Imidazole), and then the protein was purified using an ion exchange column (Q-Sepharose). The anion / cation exchange medium was selected according to the isoelectric point of the protein, and after equilibration of the column, the sample was loaded, and the protein peak was collected by gradient elution (0-500mM NaCl) to obtain a high-purity ICA-TGF-DEF fusion protein.

[0041] ICA-TGF-DEF fusion protein loaded on collagen membrane: Soak the dried collagen gel matrix membrane in PBS buffer (pH 7.4), shake gently to remove impurities or residual substances that may exist on the membrane surface, and balance the membrane for at least 1 hour to fully hydrate and restore biocompatibility. After drying the membrane at room temperature for 12 hours, the collagen basement membrane was treated with EDC / NHS cross-linking reaction for 30 minutes. Add the fusion protein ICA-TGF-DEF solution to the membrane surface, keep it moist, and incubate at 37°C for 2 hours to firmly load the protein on the collagen membrane. Rinse the membrane three times with sterile PBS to remove unbound proteins, freeze-dry at -20°C to obtain an active biofilm, and mark it as ICA-TGF-DEF-biofilm.

[0042] Example 2 Preparation of active biofilm loaded with DEF-ICA-TGF fusion protein

[0043] Construction of biologically active fusion protein (DEF-ICA-TGF):

[0044] MQRLVLLLAISLLLYQDLPVRSEFELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRKWDESLLNRTKPGGGSGGGGSSDEKVFEVHVRPKKLAVEPKGSLEVNCSTTCNQPEVGGLETSLDKILLDEQAQWKHYLVSNISHDTVLQCHFT CSGKQESMNSNVSVYQPPRQVILTLQPTLVAVGKSFTIECRVPTVEPLDSLTLFLFRGNETLHYETFGKAAPAPQEATATFNSTADREDGHRNFSCLAVLDLMSRGGNIFHKHSAPKMLEIYEPVSDSQMVIIVTVVSVLLSLFVTSVLLCFIF GQHLRQQRMGTYGVRAAWRRLPQAFRPGGGGSGGGGSFKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 5).

[0045] Preparation of collagen gel matrix: Dissolve type I collagen powder in 0.1M acetic acid solution in an ice bath and stir slowly for 4-6 hours until completely dissolved to prepare a collagen solution with a concentration of 3-5mg / mL. Place the collagen solution in an ice bath and slowly add 0.1M NaOH solution drop by drop, while using a pH meter to monitor the pH value of the solution and adjust it to close to 7.4. During the adjustment process, ensure that the solution is always at a low temperature (4°C) to avoid denaturation of collagen at high temperatures. Transfer the neutralized collagen solution to a preset mold or culture dish to ensure that the solution is evenly distributed. Place the mold or culture dish in a 37°C incubator and let it stand for 1-2 hours. The collagen solution will gradually gel to form a transparent and soft three-dimensional gel matrix.

[0046] Preparation of fusion protein DEF-ICA-TGF: GeneScript Biotech Co., Ltd. synthesized and customized the expression plasmid for expressing the DEF-ICA-TGF fusion protein as shown in SEQ ID NO: 4, wherein the pET-28a-DEF-ICA-TGF 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 heat-shock transform the pET-28a-DEF-ICA-TGF expression plasmid into the BL21 (DE3) strain; positive clones were screened on LB plates containing antibiotics (ampicillin); positive clones were inoculated into 5-10 mL LB medium (containing antibiotics) and cultured to OD600 = 0.6-0.8. IPTG (final concentration 0.1-1 mM) was added, and protein expression was induced at low temperature (16-25°C). Collect the bacteria after induced expression, use a centrifuge at 4°C, 5000-10000×g for 10 minutes to collect the cell precipitate; then resuspend the bacteria in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM PMSF, pH7.4), place the bacterial suspension in an ice bath, use an ultrasonic disruptor (power 30-50%, each pulse 3 seconds, interval 5 seconds), a total time of 10 minutes, centrifuge to remove insoluble matter (4°C, 12000×g, 30 minutes), and retain the soluble protein portion. The soluble protein sample was added to the pre-equilibrated Ni-NTA sample (equilibration solution was 50mM Tris-HCl, 300mM NaCl, 20mMImidazole, pH 8.0), and washed several times after binding (the buffer contained 20-40mM Imidazole to remove non-specific binding proteins); the target protein was eluted with elution buffer (containing 200-500mM Imidazole), and then the protein was purified using an ion exchange column (Q-Sepharose). The anion / cation exchange medium was selected according to the isoelectric point of the protein, and after equilibration of the column, the sample was loaded, and the protein peak was collected by gradient elution (0-500mM NaCl) to obtain a high-purity DEF-ICA-TGF fusion protein.

[0047] DEF-ICA-TGF fusion protein loaded on collagen membrane: Soak the dried collagen gel matrix membrane in PBS buffer (pH 7.4), shake gently to remove impurities or residual substances that may exist on the membrane surface, and balance the membrane for at least 1 hour to fully hydrate and restore biocompatibility. After drying the membrane at room temperature for 12 hours, the collagen basement membrane was treated with EDC / NHS cross-linking reaction for 30 minutes. Add the fusion protein DEF-ICA-TGF solution on the membrane surface, keep it moist, and incubate at 37°C for 2 hours to firmly load the protein on the collagen membrane. Rinse the membrane three times with sterile PBS to remove unbound proteins, freeze-dry at -20°C to obtain an active biofilm, which is labeled as DEF-ICA-TGF-biofilm.

[0048] Example 3 Preparation of active biofilm loaded with TGF-DEF-ICA fusion protein

[0049] Construction of biologically active fusion protein (TGF-DEF-ICA):

[0050] FKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAIS VLYFDDSSNVILKKYRNMVVRACGCHGGGGSGGGGSMQRLVLLLAISLLLYQDLPVRSEFELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRKWDESLLNRTKPGGGGSGGGGSSDEKVFE VHVRPKKLAVEPKGSLEVNCSTTCNQPEVGGLETSLDKILLDEQAQWKHYLVSNISHDTVLQCHFTCSGKQESMNSNVSVYQPPRQVILTLQPTLVAVGKSFTIECRVPTVEPLDSLTLFLFRGN ETLHYETFGKAAPAPQEATATFNSTADREDGHRNFSCLAVLDLMSRGGNIFHKHSAPKMLEIYEPVSDSQMVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQRMGTYGVRAAWRRLPQAFRP(SEQ ID NO:6).

[0051] Preparation of collagen gel matrix: Dissolve type I collagen powder in 0.1M acetic acid solution in an ice bath and stir slowly for 4-6 hours until completely dissolved to prepare a collagen solution with a concentration of 3-5mg / mL. Place the collagen solution in an ice bath and slowly add 0.1M NaOH solution drop by drop, while using a pH meter to monitor the pH value of the solution and adjust it to close to 7.4. During the adjustment process, ensure that the solution is always at a low temperature (4°C) to avoid denaturation of collagen at high temperatures. Transfer the neutralized collagen solution to a preset mold or culture dish to ensure that the solution is evenly distributed. Place the mold or culture dish in a 37°C incubator and let it stand for 1-2 hours. The collagen solution will gradually gel to form a transparent and soft three-dimensional gel matrix.

[0052] Preparation of fusion protein TGF-DEF-ICA: GeneScript Biotech Co., Ltd. synthesized and customized the expression plasmid for expressing the TGF-DEF-ICA fusion protein as shown in SEQ ID NO: 4, wherein the pET-28a-TGF-DEF-ICA 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 heat-shock transform the pET-28a-TGF-DEF-ICA expression plasmid into the BL21 (DE3) strain; positive clones were screened on LB plates containing antibiotics (ampicillin); positive clones were inoculated into 5-10 mL LB medium (containing antibiotics) and cultured to OD600 = 0.6-0.8. IPTG (final concentration 0.1-1 mM) was added, and protein expression was induced at low temperature (16-25°C). Collect the bacteria after induced expression, use a centrifuge at 4°C, 5000-10000×g for 10 minutes to collect the cell precipitate; then resuspend the bacteria in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM PMSF, pH7.4), place the bacterial suspension in an ice bath, use an ultrasonic disruptor (power 30-50%, each pulse 3 seconds, interval 5 seconds), a total time of 10 minutes, centrifuge to remove insoluble matter (4°C, 12000×g, 30 minutes), and retain the soluble protein portion. The soluble protein sample was added to the pre-equilibrated Ni-NTA sample (equilibration solution was 50mM Tris-HCl, 300mM NaCl, 20mMImidazole, pH 8.0), and washed several times after binding (the buffer contained 20-40mM Imidazole to remove non-specific binding proteins); the target protein was eluted with elution buffer (containing 200-500mM Imidazole), and then the protein was purified using an ion exchange column (Q-Sepharose). The anion / cation exchange medium was selected according to the isoelectric point of the protein. After equilibration of the column, the sample was loaded, and the protein peak was collected by gradient elution (0-500mM NaCl) to obtain a high-purity TGF-DEF-ICA fusion protein.

[0053] TGF-DEF-ICA fusion protein loaded on collagen membrane: Soak the dried collagen gel matrix membrane in PBS buffer (pH 7.4), shake gently to remove impurities or residual substances that may exist on the membrane surface, and balance the membrane for at least 1 hour to fully hydrate and restore biocompatibility. After drying the membrane at room temperature for 12 hours, the collagen basement membrane was treated with EDC / NHS cross-linking reaction for 30 minutes. Add the fusion protein TGF-DEF-ICA solution to the membrane surface, keep it moist, and incubate at 37°C for 2 hours to firmly load the protein on the collagen membrane. Rinse the membrane three times with sterile PBS to remove unbound proteins, freeze-dry at -20°C to obtain an active biofilm, and mark it as TGF-DEF-ICA-biofilm.

[0054] Example 4 Detection of various indicators of biofilms loaded with different fusion proteins

[0055] Expansion rate detection experiment:

[0056] Cut the biofilm loaded with fusion protein prepared in Examples 1-3 into standard samples with a size of 20 mm × 20 mm, and record the initial mass (W0) of the samples in the dry state. Soak the samples in phosphate buffered saline (PBS) at 37°C, ensuring that the samples are completely immersed, and leave them for 24 hours. Take out the soaked samples, and gently wipe them with filter paper to remove the liquid attached to the surface to avoid the measurement results being affected by excess liquid residue. Use a high-precision balance to measure the wet mass (Wt) of the sample, see Figure 1 .

[0057] The expansion rate is calculated according to the following formula:

[0058]

[0059] Figure 1 The results showed that the swelling rate of the active biofilm loaded with ICA-TGF-DEF fusion protein was much higher than that of the other two active biofilms, indicating that the active biofilm loaded with ICA-TGF-DEF fusion protein with a higher swelling rate can absorb more water or solution, and is suitable for application scenarios that require high water absorption performance, such as wound dressings, moisturizing materials and soft tissue engineering scaffolds. At the same time, it can maintain a moist environment, which helps promote cell migration and tissue repair.

[0060] Porosity detection experiment:

[0061] The biofilm loaded with fusion protein prepared in Example 1-3 was cut into samples with a size of 10 mm × 10 mm × 0.1 mm. The sample was placed in a sealed container and bubble-free anhydrous ethanol was added to ensure that the sample was completely immersed. The container was placed in a vacuum environment for 30 minutes to remove the bubbles remaining inside the sample and ensure that ethanol fully entered the pores of the biofilm. The sample completely soaked with ethanol was taken out, and the surface liquid was gently wiped with filter paper, and the wet mass (W) was recorded. wet The sample was dried in an oven at 50°C until constant weight was obtained and the dry mass (W dry ). Record the volume of the sample when it is completely dry (V sample ), obtain the thickness through the film thickness measuring instrument and calculate the volume, see Figure 2 .

[0062] The porosity was calculated according to the following formula:

[0063]

[0064] Where ρ is the density of ethanol (0.789 g / cm 3 ).

[0065] Figure 2 The results showed that the porosity of the active biofilm loaded with ICA-TGF-DEF fusion protein was much higher than that of the other two active biofilms, indicating that the active biofilm loaded with ICA-TGF-DEF fusion protein with high porosity has a larger pore space, which can better promote the exchange of gases and solutes and is suitable for tissue repair scenarios.

[0066] Tensile strength test experiment:

[0067] The biofilm loaded with fusion protein prepared in Examples 1-3 was prepared by solution casting to prepare samples, the film thickness was controlled to be 1 mm, and it was cut into standard strip samples of 30 mm×5 mm. An electronic universal tensile tester (Instron 3345) was used, a 10N load sensor was installed, the stretching speed was set to 10 mm / min, the clamping distance was 20 mm, and the test environment was controlled at 25°C and 50% humidity. The sample was clamped between the two clamps of the stretching machine to ensure that the sample was centered and flat, and the instrument was started for tensile testing, and the maximum tensile strength value of the sample (i.e., the stress value when stretched to fracture) was recorded. Each group of samples was tested 5 times, and the average value and standard deviation were taken. Figure 3 .

[0068] Figure 3 The results showed that the active biofilm loaded with ICA-TGF-DEF fusion protein and the other two membranes had good tensile strength, meeting the basic requirements of biofilm.

[0069] Fusion protein loading rate detection experiment:

[0070] Cut the biofilm loaded with fusion protein prepared in Example 1-3 into small pieces of 10 mm × 10 mm, and accurately weigh the dry film mass (W0). Soak the sample in 2 mL of phosphate buffered saline (PBS, pH 7.4) containing 1% SDS, and incubate at 37°C for 4 hours with shaking to ensure that the fusion protein is completely released into the solution. Take the extract and use the BCA protein quantification kit to determine the protein concentration in the solution according to the standard curve (C, unit: μg / mL), see Figure 4 .

[0071] The protein loading rate of each biofilm was calculated according to the formula:

[0072]

[0073] Wherein, V is the volume of the extract (2 mL). Each group of samples was tested 3 times and the average value was taken.

[0074] Figure 4 The results showed that the protein loading rate of the active biofilm loaded with ICA-TGF-DEF fusion protein was much higher than that of the other two active biofilms, which means that the active biofilm loaded with ICA-TGF-DEF fusion protein with a high loading rate can provide more bioactive proteins, which may significantly improve the treatment efficiency in scenarios such as promoting tissue repair, anti-inflammation, antibacterial, or regulating immune response. At the same time, a higher loading rate may prolong the duration of protein release, thereby achieving longer-lasting biological signal stimulation.

[0075] Cell proliferation assay:

[0076] The biofilm loaded with fusion protein prepared in Example 1-3 was cut into small pieces of 10 mm × 10 mm, placed in a 96-well plate, washed three times with sterile PBS to remove residual impurities, and then sterilized with ultraviolet light for 30 minutes. L929 fibroblasts were used and the cell concentration was adjusted to 1 × 10 4 Cells / well, add 100 μL of cell suspension to each well to ensure that the cells are in direct contact with the biofilm. Place the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours. The untreated bare membrane is used as a negative control group. After the incubation, add 10 μL of CCK-8 reagent to each well and continue incubation for 6 hours. Use an enzyme reader to measure the absorbance value (OD value) of each well at intervals, and set the wavelength to 450nm. Figure 5 .

[0077] Figure 5 The results showed that the active biofilm loaded with ICA-TGF-DEF fusion protein promoted cell proliferation much more effectively than the other two active biofilms (P<0.01), indicating that the biofilm loaded with fusion protein still maintained significant biological activity in promoting proliferation.

[0078] Cell attachment rate detection experiment:

[0079] The biofilm loaded with fusion protein prepared in Example 1-3 was cut into 1 cm × 1 cm segments and placed in a 24-well plate. An equal number of fibroblasts (1 × 10 5 / well), incubated in DMEM culture medium containing 10% FBS at 37°C and 5% CO2 for 4 hours. After the incubation, use PBS buffer to gently wash to remove non-attached cells, of which the untreated bare membrane was used as a negative control; the biofilm coated with pure BSA was used as a positive control. The relative number of cells attached to each group of biofilms was determined by the CCK-8 method. CCK-8 working solution (10% CCK-8 dissolved in culture medium) was added to each well and incubated at 37°C for 1 hour. The supernatant was collected and the absorbance (OD value) was measured at 450nm using a microplate reader. Calculation of cell attachment rate: Taking the amount of attached cells in the bare membrane group as the benchmark, the relative attachment rates of each experimental group were compared, see Figure 6 .

[0080] Figure 6 The results showed that compared with the untreated control group (bare membrane), the biofilms loaded with different fusion proteins significantly improved the cell attachment rate, and the active biofilm loaded with ICA-TGF-DEF fusion protein performed best. This result indicates that the type of fusion protein has an important influence on the cell attachment performance of the biofilm.

[0081] Biofilm antibacterial test:

[0082] The biofilm loaded with fusion protein prepared in Examples 1-3 was cut into 1 cm × 1 cm pieces and placed in 24-well plates. The untreated bare biofilm was used as a negative control; the biofilm coated with pure BSA was used as a positive control. The target strain Escherichia coli was selected and amplified to the logarithmic growth phase, and the bacterial solution concentration was adjusted to 1 × 10 6 CFU / mL. Add 1 mL of bacterial solution to each well and incubate at 37°C for 24 hours. After incubation, transfer the liquid in each well to a sterile centrifuge tube, gradiently dilute the bacterial solution and spread it on an LB agar plate, culture at 37°C for 18 hours, and count the colony forming units (CFU). At the same time, wash the surface of the biofilm with PBS and scrape the attached bacteria, then spread and culture again to evaluate the amount of attached bacteria. The bare membrane without fusion protein loading was used as the negative control group. The antibacterial effect was evaluated by calculating the inhibition rate (inhibition rate = (control group CFU-experimental group CFU) / control group CFU×100%), see Figure 7 .

[0083] Figure 7The results showed that compared with the untreated control group (bare membrane), the biofilms loaded with different fusion proteins significantly reduced the survival rate of bacteria, among which the active biofilm loaded with ICA-TGF-DEF fusion protein showed the best antibacterial performance. This result shows that there are significant differences in the antibacterial performance of biofilms loaded with different fusion proteins.

[0084] Wound repair effect experiment:

[0085] A Sprague-Dawley rat model was used to simulate the full-thickness skin wound repair experiment of breast augmentation surgery. The rats were randomly divided into 4 groups (n=6 in each group): a bare membrane control group, an active biofilm group loaded with ICA-TGF-DEF fusion protein, an active biofilm group loaded with DEF-ICA-TGF fusion protein, and an active biofilm group loaded with TGF-DEF-ICA fusion protein. A full-thickness circular wound with a diameter of about 8 mm was established on the back of the rat, and the biofilm of the corresponding group was covered on the wound and fixed with a dressing. The biofilm was replaced once a day and observed for 14 days. The wound area was calculated by image analysis software to evaluate the repair process. Changes in wound area: Wound healing was recorded by taking photos every day, and the healing rate was calculated using ImageJ software (healing rate = initial wound area - remaining wound area / initial wound area × 100%), see Figure 8 .

[0086] Figure 8 The results showed that the wound healing rate of the active biofilm group loaded with ICA-TGF-DEF fusion protein was significantly higher than that of other groups. This indicates that ICA (cell adhesion signal), TGF (transforming growth factor) and DEF (defensin) in the active biofilm loaded with ICA-TGF-DEF fusion protein work synergistically to optimize the regenerative microenvironment of the wound by promoting cell migration, adhesion, proliferation and antibacterial activity, thereby supporting the functional regeneration of skin tissue.

[0087] 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 autologous active biofilm for promoting breast augmentation wound repair, characterized in that: The following steps are involved: Step S1: preparation of collagen gel matrix; Step S2: preparation of fusion protein; Step S3: preparation of autologous active biofilm by chemical cross-linking of collagen gel and fusion protein, wherein the fusion protein is composed of intercellular adhesion molecule, transforming growth factor-β and defensin.

2. The preparation method according to claim 1, characterized in that: The fusion protein is selected from the amino acid sequence shown in any one of SEQ ID NOs: 4-6.

3. An autologous active biofilm for promoting breast augmentation wound repair, characterized in that: The biofilm is a fusion protein loaded with an amino acid sequence as shown in any one of SEQ ID NOs: 4-6.

4. An autologous active biofilm for promoting breast augmentation wound repair, characterized in that: The biofilm is prepared by the method according to any one of claims 1-2.

5. Use of the biofilm according to claim 3 or 4 in the preparation of medical beauty products that promote cell proliferation and adhesion.

6. Use of the biofilm according to claim 3 or 4 in the preparation of medical beauty products for promoting breast augmentation wound repair.

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