An autologous biofilm that promotes wound repair after breast augmentation surgery and its application

Abstract

This invention relates to an autologous active biomembrane for promoting the repair of breast augmentation surgery wounds and its application, belonging to the field of medical materials technology, and suitable for skin wound repair scenarios. This invention provides an active biomembrane loaded with an ICA-TGF-DEF fusion protein, wherein the ICA-TGF-DEF fusion protein is selected from any of the amino acid sequences shown in SEQ ID NO:4-6. The biomembrane uses collagen as the membrane matrix, and then the ICA-TGF-DEF fusion protein shown in any of SEQ ID NO:4-6 is cross-linked with the collagen membrane matrix to obtain an autologous active biomembrane for promoting the repair of breast augmentation surgery wounds. In terms of physical properties, this biomembrane has a high expansion rate, porosity, and high protein loading. In terms of biological activity, this biomembrane can significantly enhance cell adhesion and proliferation, and can also create an antibacterial wound microenvironment to promote tissue repair, making it particularly suitable for wound repair after breast augmentation surgery, accelerating tissue healing and reducing the risk of infection and fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to an autologous active biomembrane that promotes the repair of breast augmentation surgery wounds and its application. Background Technology

[0002] In recent years, with the development of plastic surgery techniques, breast augmentation, as a common procedure to improve appearance, has seen a year-on-year increase in demand. However, breast augmentation wounds often suffer from slow healing, susceptibility to infection, and scar formation, which not only prolongs the patient's recovery time but also increases the risk of postoperative complications. Traditional biomembranes used in postoperative wound repair suffer from poor biocompatibility and limited functionality, making them unsuitable for repairing complex wounds. Therefore, improving the surface properties of biomembranes and loading them with active proteins to further enhance their performance in wound repair has become a current research hotspot.

[0003] The crucial roles of bioactive molecules such as intercellular adhesion molecules (ICAM), transforming growth factor-β (TGF-β), and defensins in wound repair have been extensively studied. ICAM enhances cell adhesion and migration, promoting cell aggregation and tissue remodeling in the wound area; TGF-β plays a significant role in regulating cell proliferation, differentiation, and fibrosis; and defensins exert their antibacterial effect by directly disrupting bacterial cell membranes. However, when used alone, these bioactive molecules face numerous limitations in terms of stability, activity persistence, and ease of application, making it difficult to meet the repair needs of complex wounds. Therefore, integrating ICAM, TGF-β, and defensins using fusion protein technology to form a multifunctional fusion protein with adhesion-promoting, tissue repair-regulating, and antibacterial functions provides a new technological pathway for wound repair.

[0004] Biomembranes based on multifunctional fusion proteins can overcome the shortcomings of traditional biomembranes in terms of single functionality, sustained bioactivity, and structural stability. By immobilizing the intercellular adhesion molecule TGF-β-defensin (ICA-TGF-DEF) fusion protein onto autologous biomembranes (such as collagen or fibrin membranes containing autologous components), not only can cell adhesion and proliferation be significantly enhanced, but the wound microenvironment can also be regulated to promote tissue repair, while providing broad-spectrum antibacterial protection. This multifunctional biomembrane is particularly suitable for wound repair after breast augmentation surgery, accelerating tissue healing while reducing the risk of infection and fibrosis, providing an efficient and safe innovative solution for the field of plastic surgery. Summary of the Invention

[0005] To address the shortcomings of existing biomembranes used for breast augmentation wound repair in terms of cell adhesion, tissue repair, and antibacterial properties, this invention first provides an autologous biomembrane that promotes breast augmentation wound repair. This biomembrane is loaded with an intercellular adhesion molecule-transforming growth factor-β-defensin (ICA-TGF-DEF) fusion protein;

[0006] In some embodiments, the fusion protein is selected from any of the amino acid sequences shown in SEQ ID NO:4-6.

[0007] In some embodiments, the biomembrane is prepared by using collagen as a matrix and immobilizing the fusion protein on the membrane surface or inside through chemical cross-linking. This autologous active biomembrane not only possesses excellent 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, making it particularly suitable for wound repair after breast augmentation surgery.

[0008] In some 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), which can synergistically promote the regeneration and healing of wound tissue.

[0009] In some embodiments, the fusion protein is prepared using genetic engineering techniques and then purified before being loaded onto a collagen matrix. The collagen matrix uses natural, autologous collagen raw materials, which not only possess good biocompatibility but also significantly improve the loading efficiency of the fusion protein and its stability in the wound environment through cross-linking technology.

[0010] This invention also provides a method for preparing an autologous active biomembrane to promote wound repair after breast augmentation surgery. The method includes the following steps: first, preparing a collagen gel matrix, and optimizing the membrane's expansion rate and porosity by adjusting the collagen solution concentration and cross-linking agent ratio; then, preparing a fusion protein ICA-TGF-DEF using recombinant DNA technology, purifying it while maintaining its activity; finally, immobilizing the fusion protein onto the collagen matrix through chemical cross-linking or physical adsorption to obtain an autologous active biomembrane loaded with the fusion protein. The prepared biomembrane can continuously release the fusion protein in the wound environment, thereby providing long-term stable tissue repair and antibacterial effects.

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

[0012] In some embodiments, the autologous biofilm of the present invention provides a safe and efficient solution for postoperative wound repair after breast augmentation by significantly improving 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] This invention is the first to introduce the intercellular adhesion molecule-transforming growth factor-β-defensin (ICA-TGF-DEF) fusion protein into a biomembrane, thereby achieving a comprehensive enhancement of cell adhesion, proliferation and antibacterial properties in wound repair through synergistic effects;

[0015] This 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 biomembrane has a high protein loading capacity and stability, and can continuously release fusion proteins, providing long-term bioactivity support after surgery.

[0017] The autologous biofilm of the present invention is particularly suitable for the repair of wounds after breast augmentation surgery, significantly improving healing efficiency and reducing the incidence of infection and fibrosis. Attached Figure Description

[0018] Figure 1 Detection of swelling rate of active biomembranes loaded with different fusion proteins.

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

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

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

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

[0023] Figure 6 Cell adhesion rate detection of active biomembranes loaded with different fusion proteins.

[0024] Figure 7 Biofilm antibacterial 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 Implementation

[0026] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0027] When designing fusion proteins, the functional requirements of biological membranes and protein properties are considered comprehensively, and three functional modules are selected: cell adhesion function, tissue regeneration function, and antibacterial function.

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

[0029] Transforming growth factor-β (TGF-β) is a multifunctional cytokine that plays a crucial role in various biological processes, including cell proliferation, differentiation, migration, apoptosis, immune regulation, and extracellular matrix production. TGF-β also plays a key role in embryonic development, tissue repair, immune responses, and the 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 disrupting pathogen membranes and regulating the host immune system.

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

[0032] Based on the sequence information of intercellular adhesion molecules (ICAMs, Homo sapiens) recorded in NCBI, specifically the 2-precursor functional domain of ICAMs (NCBI Reference Sequence: NP_001093258.1), the following was selected:

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

[0034] Based on the sequence information of transforming growth factor-β (TGF-β) recorded in NCBI, transforming growth factor-beta [Homo sapiens], GenBank: AAA36738.1, its functional domain was selected:

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

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

[0037] Taking into account the functional requirements of biomembranes and the properties of proteins, a biologically active fusion protein (ICA-TGF-DEF) was constructed by selecting intercellular adhesion molecules for cell adhesion function, transforming growth factor-β for tissue regeneration function, and defensins for antibacterial function.

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

[0039] Preparation of the collagen gel matrix: Dissolve type I collagen powder in 0.1M acetic acid solution in an ice bath, stirring slowly for 4-6 hours until completely dissolved, to prepare a collagen solution with a concentration of 3-5 mg / mL. Place the collagen solution in an ice bath and slowly add 0.1M NaOH solution dropwise, while simultaneously monitoring the pH value of the solution with a pH meter and adjusting it to approximately 7.4. During the adjustment process, ensure the solution is kept at a low temperature (4℃) to avoid collagen denaturation at high temperatures. Transfer the neutralized collagen solution to a pre-designed mold or petri dish, ensuring uniform distribution. Place the mold or petri dish in a 37℃ incubator and let it stand for 1-2 hours. The collagen solution will gradually gel, forming a transparent and soft three-dimensional gel matrix.

[0040] Preparation of the fusion protein ICA-TGF-DEF: A custom expression plasmid for the ICA-TGF-DEF fusion protein (as shown in SEQ ID NO:4) was synthesized by Genscript Biotech Co., Ltd. The pET-28a-ICA-TGF-DEF expression vector with a HIS tag was selected, and NcoI and XhoI restriction enzyme sites were inserted to ensure directional gene insertion. The BL21(DE3) strain, capable of expressing the fusion protein, was transformed into the pET-28a-ICA-TGF-DEF expression plasmid using heat shock. Positive clones were screened on LB agar plates containing antibiotics (ampicillin). Positive clones were inoculated into 5-10 mL of LB medium (containing antibiotics) and cultured until OD600 = 0.6-0.8. IPTG (final concentration 0.1-1 mM) was added, and protein expression was induced at low temperature (16-25℃). Collect the induced bacterial cells and centrifuge them at 4°C and 5000-10000×g for 10 minutes to collect the cell pellet. Then, resuspend the bacterial cells in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM PMSF, pH 7.4). Place the bacterial suspension in an ice bath and sonicate it (30-50% power, 3-second pulses, 5-second intervals) for a total of 10 minutes. Centrifuge to remove insoluble matter (4°C, 12000×g, 30 minutes) and retain the soluble protein fraction. Soluble protein samples were added to pre-equilibrated Ni-NTA samples (equilibration buffer: 50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, pH 8.0). After binding, the samples were washed several times (buffer containing 20-40 mM Imidazole to remove non-specifically bound proteins). The target protein was eluted with elution buffer (containing 200-500 mM Imidazole), and then purified using an ion exchange column (Q-Sepharose). The anion / cation exchange medium was selected based on the protein's isoelectric point. After column equilibration, the sample was loaded, and a gradient elution method (0-500 mM NaCl) was used to collect the protein peak, yielding high-purity ICA-TGF-DEF fusion protein.

[0041] Collagen-loaded membranes containing the ICA-TGF-DEF fusion protein: The dried collagen gel matrix membrane was immersed in PBS buffer (pH 7.4) and gently agitated to remove any impurities or residues on the membrane surface. The membrane was equilibrated for at least 1 hour to ensure full hydration and restore biocompatibility. After drying the membrane at room temperature for 12 hours, the collagen matrix membrane was treated with an EDC / NHS crosslinking reaction for 30 minutes. The ICA-TGF-DEF fusion protein solution was then added dropwise to the membrane surface, kept moist, and incubated at 37°C for 2 hours to firmly load the protein onto the collagen membrane. The membrane was washed three times with sterile PBS to remove unbound protein, and then lyophilized at -20°C to obtain an active biomembrane, labeled as ICA-TGF-DEF-biomembrane.

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

[0043] Constructing a biologically active fusion protein (DEF-ICA-TGF):

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

[0045] Preparation of the collagen gel matrix: Dissolve type I collagen powder in 0.1M acetic acid solution in an ice bath, stirring slowly for 4-6 hours until completely dissolved, to prepare a collagen solution with a concentration of 3-5 mg / mL. Place the collagen solution in an ice bath and slowly add 0.1M NaOH solution dropwise, while simultaneously monitoring the pH value of the solution with a pH meter and adjusting it to approximately 7.4. During the adjustment process, ensure the solution is kept at a low temperature (4℃) to avoid collagen denaturation at high temperatures. Transfer the neutralized collagen solution to a pre-designed mold or petri dish, ensuring uniform distribution. Place the mold or petri dish in a 37℃ incubator and let it stand for 1-2 hours. The collagen solution will gradually gel, forming a transparent and soft three-dimensional gel matrix.

[0046] Preparation of the DEF-ICA-TGF fusion protein: A custom expression plasmid for the DEF-ICA-TGF fusion protein (SEQ ID NO:4) was synthesized by Genscript Biotech Co., Ltd. The pET-28a-DEF-ICA-TGF expression vector with a HIS tag was selected, and NcoI and XhoI restriction enzyme sites were inserted to ensure directional insertion of the gene fragment. The BL21(DE3) strain capable of expressing the fusion protein was transformed into the BL21(DE3) strain using heat shock. Positive clones were screened on LB agar plates containing antibiotics (ampicillin). Positive clones were inoculated into 5-10 mL of LB medium (containing antibiotics) and cultured until OD600 = 0.6-0.8. IPTG (final concentration 0.1-1 mM) was added, and protein expression was induced at low temperature (16-25℃). Collect the induced bacterial cells and centrifuge them at 4°C and 5000-10000×g for 10 minutes to collect the cell pellet. Then, resuspend the bacterial cells in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM PMSF, pH 7.4). Place the bacterial suspension in an ice bath and sonicate it (30-50% power, 3-second pulses, 5-second intervals) for a total of 10 minutes. Centrifuge to remove insoluble matter (4°C, 12000×g, 30 minutes) and retain the soluble protein fraction. Soluble protein samples were added to pre-equilibrated Ni-NTA samples (equilibration buffer: 50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, pH 8.0). After binding, the samples were washed several times (the buffer contained 20-40 mM Imidazole to remove non-specifically bound proteins). The target protein was eluted with elution buffer (containing 200-500 mM Imidazole), and then purified using an ion exchange column (Q-Sepharose). The anion / cation exchange medium was selected based on the protein's isoelectric point. After column equilibration, the sample was loaded, and a gradient elution method (0-500 mM NaCl) was used to collect the protein peak, yielding high-purity DEF-ICA-TGF fusion protein.

[0047] Deposition of DEF-ICA-TGF fusion protein onto collagen-loaded membranes: The dried collagen gel matrix membrane was immersed in PBS buffer (pH 7.4), gently agitated to remove any impurities or residues on the membrane surface, and equilibrated for at least 1 hour to ensure full hydration and restore biocompatibility. After drying the membrane at room temperature for 12 hours, the collagen matrix membrane was treated with an EDC / NHS crosslinking reaction for 30 minutes. A DEF-ICA-TGF fusion protein solution was added dropwise to the membrane surface, kept moist, and incubated at 37°C for 2 hours to firmly load the protein onto the collagen membrane. The membrane was washed three times with sterile PBS to remove unbound protein, and then lyophilized at -20°C to obtain an active biomembrane, labeled as DEF-ICA-TGF-biomembrane.

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

[0049] Constructing a biologically active fusion protein (TGF-DEF-ICA):

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

[0051] Preparation of the collagen gel matrix: Dissolve type I collagen powder in 0.1M acetic acid solution in an ice bath, stirring slowly for 4-6 hours until completely dissolved, to prepare a collagen solution with a concentration of 3-5 mg / mL. Place the collagen solution in an ice bath and slowly add 0.1M NaOH solution dropwise, while simultaneously monitoring the pH value of the solution with a pH meter and adjusting it to approximately 7.4. During the adjustment process, ensure the solution is kept at a low temperature (4℃) to avoid collagen denaturation at high temperatures. Transfer the neutralized collagen solution to a pre-designed mold or petri dish, ensuring uniform distribution. Place the mold or petri dish in a 37℃ incubator and let it stand for 1-2 hours. The collagen solution will gradually gel, forming a transparent and soft three-dimensional gel matrix.

[0052] Preparation of the fusion protein TGF-DEF-ICA: A custom expression plasmid for the TGF-DEF-ICA fusion protein, as shown in SEQ ID NO:4, was synthesized by Genscript Biotech Co., Ltd. The pET-28a-TGF-DEF-ICA expression vector with a HIS tag was selected, and NcoI and XhoI restriction enzyme sites were inserted to ensure directional insertion of the gene fragment. The BL21(DE3) strain, capable of expressing the fusion protein, was transformed into the pET-28a-TGF-DEF-ICA expression plasmid using heat shock. Positive clones were screened on LB agar plates containing antibiotics (ampicillin). Positive clones were inoculated into 5-10 mL of LB medium (containing antibiotics) and cultured until OD600 = 0.6-0.8. IPTG (final concentration 0.1-1 mM) was added, and protein expression was induced at low temperature (16-25℃). Collect the induced bacterial cells and centrifuge them at 4°C and 5000-10000×g for 10 minutes to collect the cell pellet. Then, resuspend the bacterial cells in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM PMSF, pH 7.4). Place the bacterial suspension in an ice bath and sonicate it (30-50% power, 3-second pulses, 5-second intervals) for a total of 10 minutes. Centrifuge to remove insoluble matter (4°C, 12000×g, 30 minutes) and retain the soluble protein fraction. Soluble protein samples were added to pre-equilibrated Ni-NTA samples (equilibration buffer: 50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, pH 8.0). After binding, the samples were washed several times (the buffer contained 20-40 mM Imidazole to remove non-specifically bound proteins). The target protein was eluted with elution buffer (containing 200-500 mM Imidazole), and then purified using an ion exchange column (Q-Sepharose). The anion / cation exchange medium was selected based on the protein's isoelectric point. After column equilibration, the sample was loaded, and a gradient elution method (0-500 mM NaCl) was used to collect the protein peak, yielding high-purity TGF-DEF-ICA fusion protein.

[0053] TGF-DEF-ICA fusion protein loaded onto collagen membranes: The dried collagen gel matrix membrane was immersed in PBS buffer (pH 7.4), gently agitated to remove any impurities or residues on the membrane surface, and equilibrated for at least 1 hour to ensure full hydration and restore biocompatibility. After drying the membrane at room temperature for 12 hours, the collagen matrix membrane was treated with an EDC / NHS crosslinking reaction for 30 minutes. A TGF-DEF-ICA fusion protein solution was added dropwise to the membrane surface, kept moist, and incubated at 37°C for 2 hours to firmly load the protein onto the collagen membrane. The membrane was washed three times with sterile PBS to remove unbound protein, and then lyophilized at -20°C to obtain an active biomembrane, labeled as TGF-DEF-ICA-biomembrane.

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

[0055] Expansion rate test experiment:

[0056] The biomembranes loaded with fusion proteins prepared in Examples 1-3 were cut into standard samples of 20mm × 20mm size, and the initial mass (W0) of the samples in the dry state was recorded. The samples were then immersed in phosphate-buffered saline (PBS) at 37°C, ensuring complete submersion, for 24 hours. After immersion, the samples were removed, and the surface was gently wiped with filter paper to remove any adhering liquid, avoiding any residual liquid affecting the measurement results. The wet mass (Wt) of the samples was measured using a high-precision balance, see [link to relevant documentation]. Figure 1 .

[0057] Calculate the expansion rate using the following formula:

[0058]

[0059] Figure 1 The results showed that the expansion rate of the biomembrane loaded with ICA-TGF-DEF fusion protein was much higher than that of the other two biomembranes. This indicates that the biomembrane loaded with ICA-TGF-DEF fusion protein with a higher expansion rate can absorb more water or solution, making it suitable for applications requiring high water absorption, 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 testing experiment:

[0061] The biomembranes loaded with fusion proteins prepared in Examples 1-3 were cut into samples measuring 10 mm × 10 mm × 0.1 mm. The samples were placed in a sealed container, and anhydrous ethanol without air bubbles was added, ensuring complete immersion. The container was then placed under vacuum for 30 minutes to remove any residual air bubbles and ensure sufficient ethanol penetration into the biomembrane pores. After complete ethanol immersion, the samples were removed, and the surface liquid was gently wiped off with filter paper. The wet mass (W) was recorded. wet The samples were dried in a 50°C oven to constant weight, and the dry weight (W) was recorded. dry Record the volume (V) of the sample when it is completely dry. sample The thickness is obtained using a film thickness gauge, and the volume is calculated. (See...) Figure 2 .

[0062] Porosity is calculated using the following formula:

[0063]

[0064] Where ρ is the density of ethanol (0.789 g / cm³). 3 ).

[0065] Figure 2 The results showed that the porosity of the biomembrane loaded with ICA-TGF-DEF fusion protein was much higher than that of the other two biomembranes. This indicates that the biomembrane loaded with ICA-TGF-DEF fusion protein with high porosity has a larger pore space, which can better promote the exchange of gas and solute, and is suitable for tissue repair scenarios.

[0066] Tensile strength test experiment:

[0067] The biomembranes loaded with fusion proteins prepared in Examples 1-3 were used to prepare samples using solution casting, with a membrane thickness controlled at 1 mm. These samples were then cut into standard strips of 30 mm × 5 mm. An Instron 3345 electronic universal tensile testing machine was used, equipped with a 10 N load sensor. The tensile speed was set to 10 mm / min, the clamping distance to 20 mm, and the testing environment controlled at 25°C and 50% humidity. The samples were clamped between the two clamps of the tensile testing machine, ensuring the samples were centered and flat. The instrument was started to perform the tensile test, and the maximum tensile strength value (i.e., the stress value at fracture) of the sample was recorded. Each set of samples was tested 5 times, and the average value and standard deviation were recorded. Figure 3 .

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

[0069] Fusion protein loading rate detection experiment:

[0070] The biomembranes loaded with fusion proteins prepared in Examples 1-3 were cut into 10 mm × 10 mm pieces, and the dry membrane mass (W0) was accurately weighed. The pieces were immersed in 2 mL of phosphate-buffered saline (PBS, pH 7.4) containing 1% SDS and incubated at 37°C with shaking for 4 hours to ensure complete release of the fusion protein into the solution. The extract was then used to determine the protein concentration (C, unit: μg / mL) in the solution using a BCA protein quantification kit according to a standard curve. (See [link to BCA protein quantification kit]). Figure 4 .

[0071] The protein loading rate of each biomembrane was calculated using the formula:

[0072]

[0073] Where V is the extraction volume (2 mL). Each sample was tested three times, and the average value was taken.

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

[0075] Cell proliferation detection experiment:

[0076] The biomembranes loaded with fusion proteins prepared in Examples 1-3 were cut into 10mm × 10mm pieces, placed in 96-well plates, and washed three times with sterile PBS to remove residual impurities, followed by UV sterilization for 30 minutes. L929 fibroblasts were used, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / well. 4 Cells / well: Add 100 μL of cell suspension to each well, ensuring direct cell contact with the biomembrane. Incubate the 96-well plate at 37°C with 5% CO2 for 24 hours. Use untreated bare membranes as a negative control. After incubation, add 10 μL of CCK-8 reagent to each well and incubate for another 6 hours. Measure the absorbance (OD) of each well at intervals using a microplate reader at a wavelength of 450 nm. See [link to microplate reader]. Figure 5 .

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

[0078] Cell adhesion rate detection experiment:

[0079] The biomembranes loaded with fusion proteins prepared in Examples 1-3 were cut into 1cm × 1cm fragments and placed in 24-well plates. An equal number of fibroblasts (1 × 10⁻⁶) were seeded onto the surface of each biomembrane. 5 Cells were incubated in DMEM medium containing 10% FBS at 37°C and 5% CO2 for 4 hours. After incubation, unattached cells were gently washed with PBS buffer to remove unattached cells. Untreated bare membranes served as negative controls, and biofilms coated with pure BSA served as positive controls. The relative number of cells attached to the biofilms in each group was determined by the CCK-8 assay. CCK-8 working solution (10% CCK-8 dissolved in the medium) was added to each well, and the biofilms were incubated at 37°C for 1 hour. The supernatant was collected, and the absorbance (OD value) was measured at 450 nm using a microplate reader. Cell attachment rate was calculated: the relative attachment rate of each experimental group was compared with the number of attached cells in the bare membrane group. Figure 6 .

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

[0081] Biofilm antibacterial assay:

[0082] The biofilms loaded with fusion proteins prepared in Examples 1-3 were cut into 1cm × 1cm pieces and placed in 24-well plates. Untreated bare membranes served as negative controls; biofilms coated with pure BSA served as positive controls. The target strain of *Escherichia coli* was amplified to the logarithmic growth phase, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 6 CFU / mL. Add 1 mL of bacterial suspension to each well and incubate at 37°C for 24 hours. After incubation, transfer the liquid from each well to a sterile centrifuge tube, serially dilute the bacterial suspension, and spread it onto LB agar plates. Incubate at 37°C for 18 hours, and count colony-forming units (CFU). Simultaneously, wash the biofilm surface with PBS and scrape off any attached bacteria, then spread and incubate again to assess the amount of attached bacteria. A bare membrane without fusion protein loading was used as a negative control. The antibacterial effect was assessed by calculating the inhibition rate (inhibition rate = (control group CFU - experimental group CFU) / control group CFU × 100%), see [link to relevant documentation]. Figure 7 .

[0083] Figure 7The results showed that, compared with the untreated control group (naked membrane), biofilms loaded with different fusion proteins significantly reduced bacterial survival, with the biofilm loaded with the ICA-TGF-DEF fusion protein exhibiting the best antibacterial performance. This indicates that biofilms loaded with different fusion proteins show significant differences in antibacterial properties.

[0084] Experiment on wound healing effect:

[0085] Using the Sprague-Dawley rat model, a full-thickness skin wound repair experiment simulating breast augmentation surgery was conducted. Rats were randomly divided into four groups (n=6 per 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 approximately 8 mm was created on the rat's back. The corresponding biofilm was covered on the wound and fixed with dressings. The biofilm was changed daily for 14 days. The wound area was calculated using image analysis software to evaluate the repair progress. Wound area changes: Wound healing was recorded daily by photographing the wound. The healing rate was calculated using ImageJ software (healing rate = initial wound area - remaining wound area / initial wound area × 100%). See [link to relevant documentation]. 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, thus supporting the functional regeneration of skin tissue.

[0087] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an autologous living biofilm for promoting the repair of breast augmentation wounds, characterized by, The method comprises the following steps: Step S1: preparation of a collagen gel matrix; Step S2: preparation of a fusion protein; Step S3: preparation of an autologous active biological membrane by chemical crosslinking of the collagen gel and the fusion protein; wherein the fusion protein is composed of intercellular adhesion molecules, transforming growth factor-β and defensins; and the fusion protein is selected from the amino acid sequences shown in any one of SEQ ID NO: 4-6.

2. The autologous living biomembrane for promoting wound repair after breast augmentation surgery obtained by the preparation method according to claim 1, characterized in that, The biological membrane is loaded with the fusion protein having the amino acid sequence shown in any one of SEQ ID NO: 4-6.

3. Use of the biological membrane according to claim 2 in the preparation of a medical and cosmetic product for promoting cell proliferation or adhesion.

4. Use of the biological membrane according to claim 2 in the preparation of a medical and cosmetic product for promoting the repair of a breast augmentation wound.

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