A composite biomaterial and a preparation method and application thereof
By mixing porcine peritoneum and porcine fascia lata in a specific ratio and preparing composite biomaterials using a two-stage enzymatic hydrolysis and irradiation sterilization method, the structural damage and biocompatibility problems caused by preservatives in existing ECM materials during sterilization have been solved, achieving highly efficient skin repair and whitening effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUBEI HENGAN PHARMA
- Filing Date
- 2026-01-09
- Publication Date
- 2026-05-12
AI Technical Summary
Existing ECM materials suffer from triple helix structure destruction due to high-energy irradiation during sterilization, degrading into random small molecule peptides, resulting in reduced bioactivity. Furthermore, the addition of preservatives may cause biocompatibility issues.
A composite biomaterial is prepared by mixing porcine peritoneum and porcine fascia lata in a specific ratio and then performing a two-stage enzymatic hydrolysis and irradiation sterilization process. This process retains a high proportion of the natural triple helix structure and avoids the addition of chemical preservatives.
After effective sterilization, it retains a high content of natural triple helix structure, which improves the safety and bioactivity of biomaterials, promotes fibroblast migration and proliferation, achieves efficient skin repair, and may play a whitening role.
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Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical materials technology, specifically to a composite biomaterial and its preparation method and application. Background Technology
[0002] The extracellular matrix (ECM) is a crucial component of the cellular microenvironment, primarily composed of collagen, elastin, and glycosaminoglycans. The triple helix structure of collagen is key to its biological functions, such as promoting cell adhesion, proliferation, and differentiation. ECM materials derived from mammals like pigs, due to their structural similarity to humans, have been widely used in skin repair and wound dressings. However, existing commercially available ECM products have significant drawbacks. First, preservatives are typically added to preserve the product and prevent microbial growth; residues of these chemicals may cause biocompatibility issues or allergic reactions. Second, gamma-ray or electron beam irradiation is commonly used to ensure product sterility. While irradiation sterilization is reliable, its high energy disrupts the triple helix structure of collagen, causing it to degrade into randomly coiled small peptides, resulting in a very low content (typically less than 30%) of bioactive, intact ECM components in the product. This severely weakens the inherent biological functions of ECM materials, affecting their effectiveness in skin repair.
[0003] Therefore, developing an ECM biomaterial that can retain a high proportion of the natural triple helix structure after effective sterilization and does not require the addition of chemical preservatives has become an urgent technical problem to be solved in this field. Summary of the Invention
[0004] To address the aforementioned deficiencies in this field, this application aims to provide a composite biomaterial, its preparation method, and its application.
[0005] According to one aspect of this application, a method for preparing a composite biomaterial is provided, comprising:
[0006] Porcine peritoneum and porcine fascia lata were pretreated to obtain dried porcine peritoneum and dried porcine fascia lata.
[0007] The dried porcine peritoneum and dried porcine fascia lata were mixed, and a buffer solution was added for homogenization to obtain a composite homogenate.
[0008] Adjust the pH of the composite homogenate to alkaline, and add trypsin for the first stage of enzymatic hydrolysis;
[0009] Adjust the pH of the composite homogenate to acidic, and add pepsin for a second-stage enzymatic hydrolysis;
[0010] The composite homogenate after the second-stage enzymatic hydrolysis was subjected to salting out and centrifugation. The precipitate was collected and sterilized to obtain the composite biomaterial.
[0011] According to some embodiments of this application, the mass ratio of dried porcine peritoneum to dried porcine fascia lata is 1:(1-5).
[0012] According to some embodiments of this application, the mass ratio of dried porcine peritoneum to dried porcine fascia lata is 1:3.
[0013] According to some embodiments of this application, sterilization includes: redissolving the precipitate in an acidic solution, adding phosphate buffer solution and mixing, and then performing irradiation sterilization at an irradiation dose of 15-25 kGy.
[0014] According to some embodiments of this application, the alkaline conditions are pH 8.0-9.5.
[0015] The acidic conditions are pH 1.5-2.5.
[0016] The first-stage enzymatic hydrolysis takes 11-13 hours;
[0017] The second-stage enzymatic hydrolysis takes 11-13 hours;
[0018] The degree of hydrolysis for the first and second stages of enzymatic hydrolysis is 75%-85%.
[0019] According to some embodiments of this application, the degree of hydrolysis of the first-stage enzymatic hydrolysis and the second-stage enzymatic hydrolysis is 80%.
[0020] According to some embodiments of this application, the pretreatment includes: defatting and decellularizing porcine peritoneum and porcine fascia lata, followed by freeze-drying.
[0021] According to some embodiments of this application, the decellularization process includes:
[0022] The porcine peritoneum or porcine fascia lata was subjected to ultrasonic treatment in the first mixed solution, the second mixed solution, the third mixed solution, the fourth mixed solution, the fifth mixed solution, the sixth mixed solution, the first ethanol solution, and pure water, respectively, to obtain decellularized products;
[0023] The first mixed solution is prepared by mixing the following raw materials in parts by weight: Triton X-100: 0.8-1.2%; acetone: 5-10%; anhydrous ethanol: 15-25%; water balance;
[0024] The second mixed solution is prepared by mixing the following raw materials in parts by weight: Triton X-100: 1.5-2.0%; sodium carbonate: 2.0-3.0%; anhydrous ethanol: 70-76.5%; water balance;
[0025] The third mixed solution is prepared by mixing the following raw materials in parts by weight: sodium dodecyl sulfate: 2.0-3.0%; citric acid: 2.0-3.0%; anhydrous ethanol: 20-30%; water balance;
[0026] The fourth mixed solution is prepared by mixing the following raw materials in parts by weight: sodium dodecyl sulfate: 1.0-2.0%; acetic acid: 2.0-3.0%; ethanol: 20-30%; water balance;
[0027] The fifth mixed solution is a PBS solution with a pH of 7.0-7.5; the sixth mixed solution is an aqueous solution of sodium bicarbonate.
[0028] According to one aspect of this application, a composite biomaterial prepared by the above-described preparation method is provided, wherein the triple helix structure content of the sterilized composite biomaterial is greater than 70%.
[0029] According to another aspect of this application, the composite biomaterial prepared by the above preparation method is used in skin repair or whitening products.
[0030] Compared with the prior art, this application has at least the following beneficial effects:
[0031] The method for preparing composite biomaterials in this application involves mixing porcine peritoneal ECM and porcine fascia lata ECM of different sources and physical properties in a specific ratio, followed by enzymatic hydrolysis with two enzymes at different hydrolysis times. By controlling the hydrolysis time, a matrix system that can effectively resist radiation can be constructed.
[0032] The method for preparing the composite biomaterials in this application involves strict raw material processing, aseptic operation, and terminal irradiation sterilization. No chemical preservatives are added during the product manufacturing process, achieving a sterile state, which enhances biosafety and avoids potential tissue irritation.
[0033] The composite biomaterial of this application not only retains a high content of natural triple helix collagen (>70%), but also contains some bioactive small molecule peptides and amino acids produced by enzymatic hydrolysis. These components work together to better promote fibroblast migration and proliferation, accelerate collagen regeneration, thereby achieving efficient skin repair, and may exert a whitening effect by inhibiting tyrosinase and other pathways. Detailed Implementation
[0034] The technical solution of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0035] It should be particularly noted that similar substitutions and modifications made to this application are obvious to those skilled in the art, and they are all considered to be included in this application. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.
[0036] Unless otherwise specified, this application is conducted under standard conditions or conditions recommended by the manufacturer. The raw materials or excipients used, as well as the reagents or instruments used, whose manufacturers are not specified, are all conventional products that can be obtained commercially.
[0037] The following is a detailed description of this application.
[0038] In order to provide an ECM biomaterial that retains a high proportion of the natural triple helix structure after effective sterilization and does not require the addition of chemical preservatives, this application provides a composite biomaterial, its preparation method and application.
[0039] In some examples, the preparation method of the composite biomaterial of this application includes:
[0040] Porcine peritoneum and porcine fascia lata were pretreated to obtain dried porcine peritoneum and dried porcine fascia lata.
[0041] The dried porcine peritoneum and dried porcine fascia lata were mixed, and a buffer solution (such as PBS solution) was added for homogenization to obtain a composite homogenate.
[0042] Adjust the pH of the composite homogenate to alkaline, and add trypsin for the first stage of enzymatic hydrolysis;
[0043] Adjust the pH of the composite homogenate to acidic, and add pepsin for a second-stage enzymatic hydrolysis;
[0044] The composite homogenate after the second-stage enzymatic hydrolysis was subjected to salting out and centrifugation. The precipitate was collected and sterilized to obtain the composite biomaterial.
[0045] According to some embodiments of this application, the mass ratio of dried porcine peritoneum to dried porcine fascia lata is 1:(1-5).
[0046] According to some embodiments of this application, the mass ratio of dried porcine peritoneum to dried porcine fascia lata is 1:3.
[0047] According to some embodiments of this application, sterilization includes: redissolving the precipitate in an acidic solution (such as hydrochloric acid, acetic acid, citric acid), adding phosphate buffer solution and mixing, and then performing irradiation sterilization with an irradiation dose of 15-25 kGy.
[0048] The alkaline conditions are pH 8.0~8.5.
[0049] The acidic conditions are pH 1.5 to 2.5.
[0050] The first-stage enzymatic hydrolysis takes 11-13 hours;
[0051] The second-stage enzymatic hydrolysis takes 11-13 hours;
[0052] The degree of hydrolysis for the first and second stages of enzymatic hydrolysis is 75%-85%.
[0053] Furthermore, the degree of hydrolysis for both the first and second stage enzymatic hydrolysis is 80%.
[0054] Furthermore, the pretreatment includes: defatting and decellularizing porcine peritoneum and porcine fascia lata, followed by freeze-drying.
[0055] In some examples, the above decellularization process includes:
[0056] The porcine peritoneum or porcine fascia lata was subjected to a first ultrasonic treatment in a first mixed solution to obtain a first intermediate product.
[0057] The first intermediate product was subjected to a second ultrasonic treatment in the second mixed solution to obtain the second intermediate product.
[0058] The second intermediate product was subjected to a third ultrasonic treatment in the third mixed solution to obtain the third intermediate product.
[0059] The third intermediate product was subjected to a fourth ultrasonic treatment in the fourth mixed solution to obtain the fourth intermediate product.
[0060] The fourth intermediate product was subjected to a fifth ultrasonic treatment in the fifth mixed solution to obtain the fifth intermediate product.
[0061] The fifth intermediate product was subjected to a sixth ultrasonic treatment in the sixth mixed solution to obtain the sixth intermediate product.
[0062] The sixth intermediate product was sequentially immersed in the first ethanol solution and pure water for a seventh and eighth ultrasonic treatment to obtain a decellularized product.
[0063] The first mixed solution is prepared by mixing the following raw materials in parts by weight: Triton X-100: 0.8-1.2%; acetone: 5-10%; anhydrous ethanol: 15-25%; water balance;
[0064] The second mixed solution is prepared by mixing the following raw materials in parts by weight: Triton X-100: 1.5-2.0%; sodium carbonate: 2.0-3.0%; anhydrous ethanol: 70-76.5%; water balance;
[0065] The third mixed solution is prepared by mixing the following raw materials in parts by weight: sodium dodecyl sulfate: 2.0-3.0%; citric acid: 2.0-3.0%; anhydrous ethanol: 20-30%; water balance;
[0066] The fourth mixed solution is prepared by mixing the following raw materials in parts by weight: sodium dodecyl sulfate: 1.0-2.0%; acetic acid: 2.0-3.0%; ethanol: 20-30%; water balance;
[0067] The fifth mixed solution is a PBS solution with a pH of 7.0-7.5; the sixth mixed solution is an aqueous solution of sodium bicarbonate.
[0068] In some examples, the duration of the first ultrasound treatment was 1-3 hours; the duration of the second ultrasound treatment was 1-3 hours; the duration of the third ultrasound treatment was 1-3 hours; the duration of the fourth ultrasound treatment was 1-3 hours; the duration of the fifth ultrasound treatment was 1-2 hours; the duration of the third ultrasound treatment was 1-2 hours; and the duration of the fourth ultrasound treatment was 1-2 hours.
[0069] In some examples, the amount of lipid-soluble solution used was 5 times the tissue mass; the amount of 10% ethanol used was 5 times the tissue mass; and the amount of pure water used was 5 times the tissue mass. The amount of the first mixed solution used was 7 times the mass of the pretreated porcine fascia tissue; the amount of the second mixed solution used was 7 times the mass of the first intermediate product; the amount of the third mixed solution used was 7 times the mass of the first intermediate product; and the amount of the fourth mixed solution used was 7 times the mass of the first intermediate product.
[0070] In some examples, the lipid-soluble solvent is one or more of acetone, n-hexane, and ethyl acetate.
[0071] In some examples, after the above decellularization treatment, the residual extracellular matrix cells were ≤2 cells / field of view (slide staining method), DNA residue was ≤5μg / g, Triton X-100 residue was ≤10μg / g, and sodium dodecyl sulfate residue was ≤3mg / g.
[0072] This application creatively discovers that mixing porcine peritoneal ECM and porcine fascia lata ECM of different origins and physical properties in a specific ratio, followed by enzymatic hydrolysis with two different enzymes at different times, significantly shortens the traditional enzymatic hydrolysis time and significantly enhances the radiation tolerance of the composite system. Single materials hydrolyzed with a single enzyme have relatively singular sites of action. By utilizing different enzymes and their different sites of action in the hydrolysis reaction, the resulting composite hydrolysate contains different amino acids, peptides, and other biomolecules, significantly improving the reduction in triple helix content after high-dose irradiation of single materials with a single enzyme. A synergistic protective effect exists between the two ECM components after enzymatic hydrolysis with different enzymes; the mechanism may be that different amino acid types, collagen, matrix, and other components cross-link or form a more stable microstructure.
[0073] In the preparation method of this application, strict raw material processing, aseptic operation, and terminal irradiation sterilization are employed. No chemical preservatives are added during product production, achieving a sterile state and enhancing biosafety, thus avoiding potential tissue irritation. Furthermore, by precisely controlling the mixing ratio of the two raw materials and the enzymatic hydrolysis process (especially the degree of hydrolysis), high-quality products meeting expected performance can be stably produced. This addresses the problem of collagen triple helix content decreasing to below 30% after high-dose irradiation. The present invention, through composite membrane, dual-enzyme hydrolysis, and freeze-drying to retain some moisture, ensures that the final product maintains a stable triple helix content of over 70% even under high-dose irradiation conditions.
[0074] The composite biomaterial of this application not only retains a high content of natural triple helix collagen, but also contains some bioactive small molecule peptides and amino acids produced by enzymatic hydrolysis. These components work together to better promote fibroblast migration and proliferation, accelerate collagen regeneration, thereby achieving efficient skin repair, and may exert a whitening effect by inhibiting tyrosinase and other pathways.
[0075] The technical solution of this application will be further described below with reference to specific embodiments.
[0076] Example 1
[0077] After defatting and decellularizing, the porcine peritoneum and porcine fascia lata were freeze-dried to obtain dried porcine peritoneum and dried porcine fascia lata. The dried porcine peritoneum and dried porcine fascia lata were mixed in a 1:1 ratio and homogenized in a buffer solution to obtain a composite slurry.
[0078] Digest with trypsin in alkaline solution for 12 hours; digest with pepsin in acidic solution for 12 hours. Salt out the enzymatically digested products and centrifuge to collect the precipitate.
[0079] The precipitate was redissolved in an acidic solution, mixed with one volume of phosphate buffer, and then sterilized by irradiation at a dose of 15-25 kGy to obtain the composite biomaterial.
[0080] Example 2
[0081] After defatting and decellularizing, the porcine peritoneum and porcine fascia lata were freeze-dried to obtain dried porcine peritoneum and dried porcine fascia lata. The dried porcine peritoneum and dried porcine fascia lata were mixed in a ratio of 1:2 and homogenized in a buffer solution to obtain a composite slurry.
[0082] Digest with trypsin in alkaline solution for 12 hours; digest with pepsin in acidic solution for 12 hours. Salt out the enzymatically digested products and centrifuge to collect the precipitate.
[0083] The precipitate was redissolved in an acidic solution, mixed with one volume of phosphate buffer, and then sterilized by irradiation at a dose of 15-25 kGy to obtain the composite biomaterial.
[0084] Example 3
[0085] After defatting and decellularizing, the porcine peritoneum and porcine fascia lata were freeze-dried to obtain dried porcine peritoneum and dried porcine fascia lata. The dried porcine peritoneum and dried porcine fascia lata were mixed in a ratio of 1:3 and homogenized in a buffer solution to obtain a composite slurry.
[0086] Digest with trypsin in alkaline solution for 12 hours; digest with pepsin in acidic solution for 12 hours. Salt out the enzymatically digested products and centrifuge to collect the precipitate.
[0087] The precipitate was redissolved in an acidic solution, mixed with one volume of phosphate buffer, and then sterilized by irradiation at a dose of 15-25 kGy to obtain the composite biomaterial.
[0088] Example 4
[0089] After defatting and decellularizing, the porcine peritoneum and porcine fascia lata were freeze-dried to obtain dried porcine peritoneum and dried porcine fascia lata. The dried porcine peritoneum and dried porcine fascia lata were mixed in a ratio of 1:4 and homogenized in a buffer solution to obtain a composite slurry.
[0090] Digest with trypsin in alkaline solution for 12 hours; digest with pepsin in acidic solution for 12 hours. Salt out the enzymatically digested products and centrifuge to collect the precipitate.
[0091] The precipitate was redissolved in an acidic solution, mixed with one volume of phosphate buffer, and then sterilized by irradiation at a dose of 15-25 kGy to obtain the composite biomaterial.
[0092] Example 5
[0093] After defatting and decellularizing, the porcine peritoneum and porcine fascia lata were freeze-dried to obtain dried porcine peritoneum and dried porcine fascia lata. The dried porcine peritoneum and dried porcine fascia lata were mixed in a ratio of 1:5 and homogenized in a buffer solution to obtain a composite slurry.
[0094] Digest with trypsin in alkaline solution for 12 hours; digest with pepsin in acidic solution for 12 hours. Salt out the enzymatically digested products and centrifuge to collect the precipitate.
[0095] The precipitate was redissolved in an acidic solution, mixed with one volume of phosphate buffer, and then sterilized by irradiation at a dose of 15-25 kGy to obtain the composite biomaterial.
[0096] Comparative Example 1
[0097] The preparation steps are the same as in Example 1, except that the raw material is only porcine peritoneum.
[0098] Comparative Example 2
[0099] The preparation steps are the same as in Example 1, except that the raw material is only porcine fascia lata.
[0100] Comparative Example 3
[0101] The preparation steps are the same as in Example 1, except that the raw material is only porcine bladder membrane.
[0102] Comparative Example 4
[0103] The preparation steps are the same as in Example 1, except that the raw material is only porcine small intestinal mucosa.
[0104] Comparative Example 5
[0105] The preparation steps are the same as in Example 1, except that the raw material is only porcine kidney membrane.
[0106] Comparative Example 6
[0107] The preparation steps are the same as in Example 1, except that the raw materials are porcine peritoneum, porcine fascia lata, porcine bladder membrane, small intestinal mucosa and porcine renal membrane.
[0108] Comparative Example 7
[0109] The preparation steps are the same as in Example 1, except that the raw materials are porcine peritoneum and porcine bladder membrane.
[0110] Comparative Example 8
[0111] The preparation steps are the same as in Example 1, except that the raw materials are porcine peritoneum and small intestinal mucosa.
[0112] Comparative Example 9
[0113] The preparation steps are the same as in Example 1, except that the raw materials are porcine peritoneum and porcine renal membrane.
[0114] Comparative Example 10
[0115] The preparation steps are the same as in Example 1, except that the raw materials are porcine fascia lata and porcine bladder membrane.
[0116] Comparative Example 11
[0117] The preparation steps are the same as in Example 1, except that the raw materials are porcine fascia lata and porcine small intestinal mucosa.
[0118] Comparative Example 12
[0119] The preparation steps are the same as in Example 1, except that the raw materials are porcine fascia lata and porcine renal membrane.
[0120] Comparative Example 13
[0121] The preparation steps are the same as in Example 1, except that the raw materials are porcine bladder membrane and porcine small intestinal mucosa.
[0122] Comparative Example 14
[0123] The preparation steps are the same as in Example 1, except that the raw materials are porcine bladder membrane and porcine kidney membrane.
[0124] Comparative Example 15
[0125] The preparation steps are the same as in Example 1, except that the raw materials are porcine small intestinal mucosa and porcine kidney membrane.
[0126] Test case
[0127] 1. The integrity rate of the triple helix structure and the molecular weight distribution (molecular weight > 90KD) after irradiation sterilization in the test examples and comparative examples are shown in Table 1.
[0128] Table 1. Triple helix structure integrity and molecular weight distribution
[0129]
[0130] According to the test data in the table above, the synergistic effect of the two ECM sources (porcine peritoneum and porcine fascia lata) allows the final product to maintain more than 70% of the triple helix structure content after being irradiated with an effective sterilization dose (15~25kGy), which is much higher than the less than 30% after treatment with a single material.
[0131] 2. Perform biological evaluation on the product obtained in Example 3.
[0132] 2.1 One hundred subjects were included and compared with the recombinant collagen group (CAS NO.1375784-79-4). The test items included skin stratum corneum water content, erythema area, transepidermal water loss (TEWL) value, skin redness score, dryness score, elasticity coefficient R value, firmness F4 value, F3 / F4 value, dermal density, total dermal density, and crow's feet wrinkle length. The test instruments and test indicators are shown in Table 2, and the test results are shown in Tables 3 and 4.
[0133] Table 2. Testing Instruments and Test Indicators
[0134]
[0135] Table 3. Test results compared to before use
[0136]
[0137] Note: Improvement rate = (Test values at each test time point (T2, T3, T4) - Initial value (T0)) / Initial value (T0) * 100%.
[0138] Table 4. Test results compared to the control group
[0139]
[0140] 2.2 The levels of Integrin β1, a cell proliferation and adhesion-related factor, and α-SMA, a cell differentiation-related factor, in fibroblasts were detected. The results are shown in Tables 5, 6, and 7.
[0141] Table 5. Summary of EDU staining analysis results
[0142]
[0143] Table 6. Summary of Integrin β1 Immunofluorescence Analysis Results
[0144]
[0145] Table 7. Summary of α-SMA Immunofluorescence Analysis Results
[0146]
[0147] As can be seen from the test data in Tables 3, 4, 5, 6, and 7, the composite biomaterial of this application can better promote fibroblast migration and proliferation, accelerate collagen regeneration, and thus achieve a highly efficient skin repair effect.
[0148] 3. The decellularized product from Example 1 after decellularization treatment was subjected to residue detection. The detection method is as follows:
[0149] Cell residue detection: section staining method (H&E staining)
[0150] DNA residue: fluorescence method
[0151] Triton X-100 Residue: High Performance Liquid Chromatography
[0152] Sodium dodecyl sulfate (SDS) residue: Ultraviolet spectrophotometry
[0153] The test results are shown in Table 8.
[0154] Table 8. Residual Test Results
[0155]
[0156] As can be seen from the data in Table 8, the decellularization method with a specific ratio provided in this application thoroughly removes cells and DNA. At the same time, it can greatly reduce the residue of chemical reagents, improve the tolerance rate in human trials, and reduce the various degrees of allergic or irritant reactions (such as itching, erythema, etc.) that occur with traditional methods.
[0157] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of this application. It should be noted that, for those skilled in the art, several improvements and modifications can be made to this application without departing from the principles of this application, and these improvements and modifications also fall within the protection scope of the claims of this application.
Claims
1. A method for preparing a composite biomaterial, characterized in that, include: Porcine peritoneum and porcine fascia lata were pretreated to obtain dried porcine peritoneum and dried porcine fascia lata. The dried porcine peritoneum and dried porcine fascia lata were mixed, and a buffer solution was added for homogenization to obtain a composite homogenate. Adjust the pH of the composite homogenate to alkaline, and add trypsin for the first stage of enzymatic hydrolysis; Adjust the pH of the composite homogenate to acidic, and add pepsin for a second-stage enzymatic hydrolysis; The composite homogenate after the second-stage enzymatic hydrolysis was subjected to salting out and centrifugation. The precipitate was collected and sterilized to obtain the composite biomaterial. The mass ratio of the dried porcine peritoneum to the dried porcine fascia lata is 1:(1-5). The first-stage enzymatic hydrolysis takes 11-13 hours; The second-stage enzymatic hydrolysis takes 11-13 hours; The degree of hydrolysis for the first and second enzymatic hydrolysis is 75%-85%.
2. The method for preparing the composite biomaterial according to claim 1, characterized in that, The mass ratio of the dried porcine peritoneum to the dried porcine fascia lata is 1:
3.
3. The method for preparing the composite biomaterial according to claim 1, characterized in that, The sterilization process includes: redissolving the precipitate in an acidic solution, mixing it with phosphate buffer, and then subjecting it to irradiation sterilization at a dose of 15-25 kGy.
4. The method for preparing the composite biomaterial according to claim 1, characterized in that, The alkaline conditions are pH 8.0-9.5; The acidic conditions are pH 1.5-2.
5.
5. The method for preparing the composite biomaterial according to claim 4, characterized in that, The degree of hydrolysis for the first and second enzymatic hydrolysis is 80%.
6. The method for preparing the composite biomaterial according to claim 1, characterized in that, The pretreatment includes: pig peritoneum and pig fascia lata, which are defatted, decellularized, and then freeze-dried.
7. The method for preparing the composite biomaterial according to claim 6, characterized in that, The decellularization process includes: The porcine peritoneum or porcine fascia lata was subjected to ultrasonic treatment in a first mixed solution, a second mixed solution, a third mixed solution, a fourth mixed solution, a fifth mixed solution, a sixth mixed solution, a first ethanol solution, and pure water, respectively, to obtain decellularized products; The first mixed solution is prepared by mixing the following raw materials in parts by weight: Triton X-100: 0.8-1.2%; acetone: 5-10%; anhydrous ethanol: 15-25%; water balance; The second mixed solution is prepared by mixing the following raw materials in parts by weight: Triton X-100: 1.5-2.0%; sodium carbonate: 2.0-3.0%; anhydrous ethanol: 70-76.5%; water balance; The third mixed solution is prepared by mixing the following raw materials in parts by weight: sodium dodecyl sulfate: 2.0-3.0%; citric acid: 2.0-3.0%; anhydrous ethanol: 20-30%; water balance; The fourth mixed solution is prepared by mixing the following raw materials in parts by weight: sodium dodecyl sulfate: 1.0-2.0%; acetic acid: 2.0-3.0%; anhydrous ethanol: 20-30%; water balance; The fifth mixed solution is a PBS solution with a pH of 7.0-7.5; the sixth mixed solution is an aqueous solution of sodium bicarbonate.
8. A composite biomaterial prepared by any one of the preparation methods described in claims 1-7, characterized in that, The composite biomaterial has a triple helix structure content of more than 70% after sterilization.
9. The application of a composite biomaterial prepared by any one of the preparation methods described in claims 1-7 in the preparation of skin repair or whitening products.