Acellular dermal matrix and cross-linking modification method thereof
Through cross-linking reactions catalyzed by ultrasound and TG enzyme, the decellularized dermal matrix is modified, which solves the problems of poor mechanical properties and excessive degradation speed, and achieves high biocompatibility and significantly improved mechanical properties. It is suitable for Various clinical applications.
Patent Information
- Application Number
- CN202411580700.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-02-07
AI Technical Summary
The existing decellularized dermal matrix has problems such as poor mechanical properties and fast degradation speed in clinical applications, which limits its wide application in clinical practice.
Using a method that can form covalent bonds, the decellularized dermal matrix is modified through cross-linking reactions catalyzed by sonication and glutamine transaminase (TG enzyme), avoiding the problems caused by toxic chemical cross-linking and physical cross-linking. .
The crosslinked modified decellularized dermal matrix obtained has excellent moisture and heat stability, mechanical and mechanical properties, biocompatibility and other related properties, which significantly improves its performance in thermal denaturation temperature, thermal shrinkage temperature and tensile strength, ensuring Its durability and stability in different clinical applications.
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Figure CN119386275A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical materials, and in particular relates to an acellular dermal matrix and a cross-linking modification method thereof. Background Art
[0002] Acellular dermal matrix (ADM) is a biomaterial widely used in tissue engineering and regenerative medicine, and its application in skin substitutes and wound dressings has attracted widespread attention. ADM removes cellular components from dermal tissue and retains the structure and function of the extracellular matrix (ECM), thus providing an ideal three-dimensional scaffold for cell adhesion, proliferation and differentiation. However, ADM has problems such as poor mechanical properties and rapid degradation in practical applications, which limits its widespread clinical application.
[0003] Compared with other dressings, ADM has significant advantages in biocompatibility and bioactivity. Although traditional synthetic dressings have excellent mechanical properties, their biocompatibility and bioactivity are often insufficient, which may lead to slow wound healing and poor results. ADM can more effectively promote tissue repair and improve treatment effects. In addition, compared with collagen dressings from other sources, ADM has been decellularized and has lower immunogenicity, reducing the risk of allergic reactions and immune rejection.
[0004] However, there are some challenges in the practical application of natural collagen ADM as a wound dressing. First, the sources of natural collagen materials are diverse, and strict control is required during the purification process to ensure its purity and safety. Second, the degradation rate of natural collagen is relatively fast, and it may lose its supporting function prematurely during the wound healing process. In addition, although natural collagen has good biocompatibility, allergy testing is still required to prevent possible allergic reactions in individual patients.
[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention
[0006] In order to solve the problems existing in the prior art, the present invention provides a method for cross-linking and modifying acellular dermal matrix and the like. The present invention is characterized in that a method capable of forming a covalent bond is adopted to avoid the cytotoxicity and the problem of reduced mechanical properties of materials caused by toxic chemical cross-linking (such as glutaraldehyde, formaldehyde, ethylenediamine) and physical cross-linking, and the reaction conditions are mild. The product obtained by the method of the present invention has excellent related properties such as wet heat stability, mechanical properties, and biocompatibility.
[0007] The solution of the present invention is to provide a method for cross-linking and modifying acellular dermal matrix, the method comprising the following steps:
[0008] (1) subjecting the acellular dermal matrix to ultrasonic treatment in a solution to obtain a pretreated acellular dermal matrix;
[0009] (2) adding a pH regulator and a metal ion solution to a glutamine transaminase (TG enzyme) solution, and obtaining a mixed solution after the solution is uniformly dissolved;
[0010] (3) Immersing the pretreated acellular dermal matrix in the mixed solution for reaction, and dehydrating the mixture after reaction, thereby obtaining a cross-linked and modified acellular dermal matrix.
[0011] Preferably, in step (1), the acellular dermal matrix comprises acellular porcine dermal matrix, acellular bovine dermal matrix (including acellular fetal bovine dermal matrix), acellular tilapia dermal matrix, acellular deep-sea fish dermal matrix or acellular sheep dermal matrix.
[0012] Preferably, in step (1), the solution is ultrapure water, water for injection, low-concentration ethanol solution (10-30 vol%), PBS solution or low-concentration glutamine transaminase solution (20-40 mmol / L).
[0013] Preferably, in step (1), the ultrasonic treatment is performed at a power of 60 to 100% for 15 to 40 minutes.
[0014] Preferably, in step (2), the concentration of the glutamine transaminase solution is 40 to 240 mmol / L.
[0015] Preferably, in step (2), the pH regulator is an acid regulator or a base regulator;
[0016] And / or, the acid regulator is acetic acid or dilute hydrochloric acid;
[0017] And / or, the alkaline regulator is a potassium hydroxide solution or a sodium hydroxide solution;
[0018] And / or, after adding a pH adjuster, the pH is 5.0 to 7.0.
[0019] Preferably, in step (2), the metal ion in the metal ion solution is K + , Ca 2+ or Mg 2+ One of;
[0020] And / or, the concentration of the metal ions is 10 mmol / L.
[0021] It should be emphasized that the metal ion solution should be added in time so that the enzyme and the metal ion solution can jointly catalyze the reaction, and ultimately achieve the effect of significantly improving the thermal denaturation temperature, thermal shrinkage temperature and tensile strength of the modified acellular dermal matrix.
[0022] Preferably, in step (3), the pretreated acellular dermal matrix is immersed in 10 to 20 times the mass of the mixed solution, completely immersed in an environment of 2 to 8°C for 2 to 12 hours, placed in an environment of 25 to 55°C, reacted for 2 to 16 hours, and then dehydrated in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
[0023] It should be emphasized that irradiation or ethylene oxide is generally used for sterilization in post-processing.
[0024] Based on the same technical concept, another embodiment of the present invention is to provide a cross-linked and modified acellular dermal matrix obtained by the above method.
[0025] The beneficial effects of the present invention are:
[0026] The cross-linked modified acellular dermal matrix of the present invention has high biocompatibility, is well compatible with human tissues, and reduces immune rejection reactions. Through optimized cross-linking reactions, the dressing can effectively promote cell proliferation and tissue regeneration, and reduce healing time. It has significant improvements in tensile strength, thermal denaturation temperature, and thermal shrinkage temperature, ensuring its durability and stability in different clinical applications. In addition, no toxic by-products are produced during the cross-linking reaction, ensuring safe use. The modified acellular dermal matrix obtained by the present invention can be used for the preparation of high-end dressings, hemostatic membrane materials, biological tissue patches, soft tissue repair materials, tissue engineering scaffold materials, etc., and has broad clinical application prospects. More specifically:
[0027] (1) The method of the present invention cross-links and modifies the acellular dermal matrix by transglutaminase, thereby giving it excellent chemical cross-linking activity.
[0028] (2) Acellular dermal matrix is the main component protein of the extracellular matrix. Its application to the wound surface can promote the release of a variety of active growth factors from the wound tissue, induce the activity of fibroblasts, promote the regeneration and arrangement of collagen in the body, enhance the absorption of exudates and oxygen exchange, induce the production and arrangement of fibroblasts and collagen fibers, promote the production of granulation tissue and the formation of blood vessels and new scar tissue, and achieve wound repair and healing. The cross-linking modification of transglutaminase and acellular dermal matrix-based materials can play a synergistic role in promoting healing between the two.
[0029] (3) Since TGase is a natural enzyme, the product generated after cross-linking has low immunogenicity, reducing the immune response that may be caused after implantation. Compared with some existing chemical cross-linking agents (such as glutaraldehyde), ADM cross-linked by TGase causes less inflammatory response in the body and is more conducive to tissue repair and regeneration.
[0030] (4) TG enzyme can work at physiological temperature and neutral pH conditions, avoiding the damage to the material caused by high temperature or extreme pH conditions. The mildness of the TG enzyme cross-linking reaction makes it easy to operate and the reaction conditions are easy to control, reducing the complexity and risk of the experiment. In addition, the TG enzyme reaction does not generate toxic byproducts, avoiding the safety hazards that may be caused by chemical cross-linking agents.
[0031] (5) TG enzyme is derived from organisms, which ensures the excellent biocompatibility of the acellular dermal matrix, mild reaction, and no harmful byproducts, which largely compensates for the negative impact of cross-linking on cell proliferation rate.
[0032] (6) As a natural enzyme, TGase has a lower raw material cost than chemical crosslinkers (such as glutaraldehyde and EDC / NHS). TGase is usually extracted from renewable biological sources and has a lower procurement cost than synthetic chemical agents. The crosslinking process is relatively simple and does not require high temperature or extreme reaction conditions, thus saving energy consumption and equipment investment costs. In addition, the crosslinking process produces few by-products and low waste disposal costs, which meets modern environmental protection requirements. The main advantages of TGase in the crosslinking modification of acellular dermal matrix are its excellent biocompatibility, high specificity, mild reaction conditions, low immunogenicity and safety, which make it significantly superior in medical applications and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0034] Figure 1 This is a comparison chart of heat shrinkage temperatures.
[0035] Figure 2 It is a comparison chart of tensile strength.
[0036] Figure 3 This is a comparison chart of thermal denaturation temperatures.
[0037] Figure 4 This is a cytotoxicity test detection chart.
[0038] Figure 5 This is a diagram of the degradation resistance test.
[0039] Figure 6 It is a comparison chart of biological activity. DETAILED DESCRIPTION
[0040] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.
[0041] Example 1
[0042] This embodiment provides a method for cross-linking and modifying an acellular dermal matrix, comprising the following steps:
[0043] (1) The decellularized porcine dermal matrix was placed in ultrapure water for ultrasonic treatment for 0.5 hours to obtain a pretreated decellularized dermal matrix.
[0044] (2) A TG enzyme solution with a concentration of 41.15 mmol / L was prepared, and the pH value of the solution was adjusted to 5.0 using a dilute hydrochloric acid solution. The solution was stirred at room temperature until it was completely dissolved, and a 10 mmol / L calcium chloride solution was added to obtain a mixed solution.
[0045] (3) Adding the pretreated acellular dermal matrix to the mixed solution, completely immersing it in a 4°C environment for 2 hours, then placing it in a 25°C environment, reacting it for 4 hours, and dehydrating it in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
[0046] Example 2
[0047] This embodiment provides a method for cross-linking and modifying an acellular dermal matrix, comprising the following steps:
[0048] (1) The decellularized porcine dermal matrix was placed in ultrapure water for ultrasonic treatment for 0.5 hours to obtain a pretreated decellularized dermal matrix.
[0049] (2) A TG enzyme solution with a concentration of 82.3 mmol / L was prepared, and the pH value of the solution was adjusted to 6.5 using a potassium hydroxide solution. The solution was stirred at room temperature until it was completely dissolved, and a 10 mmol / L magnesium chloride solution was added to obtain a mixed solution.
[0050] (3) Adding the pretreated acellular dermal matrix to the mixed solution, completely immersing it in a 4° C. environment for 4 hours, then placing it in a 45° C. environment, reacting it for 8 hours, and dehydrating it in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
[0051] Example 3
[0052] This embodiment provides a method for cross-linking and modifying an acellular dermal matrix, comprising the following steps:
[0053] (1) The decellularized porcine dermal matrix was placed in ultrapure water for ultrasonic treatment for 0.5 hours to obtain a pretreated decellularized dermal matrix.
[0054] (2) A TG enzyme solution with a concentration of 82.3 mmol / L was prepared, and the pH value of the solution was adjusted to 5.5 using a dilute hydrochloric acid solution. The solution was stirred at room temperature until it was completely dissolved, and a 10 mmol / L calcium chloride solution was added to obtain a mixed solution.
[0055] (3) Adding the pretreated acellular dermal matrix to the mixed solution, completely immersing it in a 4°C environment overnight, then placing it in a 35°C environment, reacting for 8 hours, and then dehydrating it in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
[0056] Example 4
[0057] This embodiment provides a method for cross-linking and modifying an acellular dermal matrix, comprising the following steps:
[0058] (1) The decellularized porcine dermal matrix was placed in low concentration ethanol and ultrasonically treated for 0.5 hours to obtain a pretreated decellularized dermal matrix.
[0059] (2) A TG enzyme solution with a concentration of 165 mmol / L was prepared, and the pH value of the solution was adjusted to 6 using a potassium hydroxide solution. The solution was stirred at room temperature until it was completely dissolved, and a 10 mmol / L calcium chloride solution was added to obtain a mixed solution.
[0060] (3) Adding the pretreated acellular dermal matrix to the mixed solution, completely immersing it in a 4°C environment for 4 hours, then placing it in a 45°C environment to react overnight, and dehydrating it in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
[0061] Example 5
[0062] This embodiment provides a method for cross-linking and modifying an acellular dermal matrix, comprising the following steps:
[0063] (1) The decellularized porcine dermal matrix was placed in a PBS solution and subjected to ultrasonic treatment for 0.5 hours to obtain a pretreated decellularized dermal matrix.
[0064] (2) A TG enzyme solution with a concentration of 240 mmol / L was prepared, and the pH value of the solution was adjusted to 6 using a sodium hydroxide solution. The solution was stirred at room temperature until it was completely dissolved, and a 10 mmol / L magnesium chloride solution was added to obtain a mixed solution.
[0065] (3) Adding the pretreated acellular dermal matrix into the mixed solution, completely immersing it in a 4°C environment for 8 hours, then placing it in a 55°C environment for reaction for 7 hours, and dehydrating it in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
[0066] Example 6
[0067] This embodiment provides a method for cross-linking and modifying an acellular dermal matrix, comprising the following steps:
[0068] (1) The decellularized porcine dermal matrix was placed in injection water and ultrasonically treated for 0.5 hours to obtain a pretreated decellularized dermal matrix.
[0069] (2) A TG enzyme solution with a concentration of 165 mmol / L was prepared, and the pH value of the solution was adjusted to 5 using an acetic acid solution. The solution was stirred at room temperature until it was completely dissolved, and a 10 mmol / L magnesium chloride solution was added to obtain a mixed solution.
[0070] (3) Adding the pretreated acellular dermal matrix into the mixed solution, completely immersing it in a 4°C environment for 4 hours, then placing it in a 35°C environment for reaction for 12 hours, and dehydrating it in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
[0071] Test example
[0072] The uncrosslinked product (control group), genipin (competing product control), and the crosslinked acellular dermal matrix obtained in Example 2 (TG enzyme crosslinked sample) were tested. Figure 1 to Figure 3 It can be seen that the thermal denaturation temperature increased from 79.3°C to 89.6°C, the thermal shrinkage temperature increased from 63.3°C to 72.28°C, and the tensile strength increased from 16.4mPa to 22.3mPa, which indirectly indicates that the product is uniformly cross-linked, improves the mechanical properties and retains the original medical function of the acellular dermal matrix.
[0073] Depend on Figure 4 (The horizontal axis represents the dilutions of different concentrations) It can be seen that the in vitro cytotoxicity test was detected at level 0.
[0074] Depend on Figure 5 , Figure 6 It can be seen that the cross-linked modified acellular dermal matrix obtained in Example 2 of the present invention retains the triple helix structure unique to the undenatured dermal matrix, ensuring the biological activity of the acellular dermal matrix. The obtained acellular dermal matrix has excellent biodegradability and high safety, and can be widely used in the biomedical field.
[0075] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
Claims
1. A method for cross-linking and modifying an acellular dermal matrix, characterized in that: The method comprises the following steps: (1) subjecting the acellular dermal matrix to ultrasonic treatment in a solution to obtain a pretreated acellular dermal matrix; (2) adding a pH regulator and a metal ion solution to the glutamine aminotransferase solution, and obtaining a mixed solution after the solution is uniformly dissolved; (3) Immersing the pretreated acellular dermal matrix in the mixed solution for reaction, and dehydrating the mixture after reaction, thereby obtaining a cross-linked and modified acellular dermal matrix.
2. The method for cross-linking and modifying acellular dermal matrix according to claim 1, characterized in that: In step (1), the acellular dermal matrix includes acellular porcine dermal matrix, acellular bovine dermal matrix, acellular tilapia dermal matrix, acellular deep-sea fish dermal matrix or acellular sheep dermal matrix.
3. The method for cross-linking and modifying acellular dermal matrix according to claim 1, characterized in that: In step (1), the solution is ultrapure water, water for injection, low-concentration ethanol solution, PBS solution or low-concentration glutamine transaminase solution.
4. The method for cross-linking and modifying acellular dermal matrix according to claim 1, characterized in that: In step (1), the ultrasonic treatment is carried out at a power of 60 to 100% for 15 to 40 minutes.
5. The method for cross-linking and modifying acellular dermal matrix according to claim 1, characterized in that: In step (2), the concentration of the glutamine transaminase solution is 40 to 240 mmol / L.
6. The method for cross-linking and modifying acellular dermal matrix according to claim 1, characterized in that: In step (2), the pH regulator is an acid regulator or a base regulator; And / or, the acid regulator is acetic acid or dilute hydrochloric acid; And / or, the alkaline regulator is a potassium hydroxide solution or a sodium hydroxide solution; And / or, after adding a pH adjuster, the pH is 5.0 to 7.
0.
7. The method for cross-linking and modifying acellular dermal matrix according to claim 1, characterized in that: In step (2), the metal ion in the metal ion solution is K + , Ca 2+ or Mg 2+ One of; And / or, the concentration of the metal ions is 10 mmol / L.
8. The method for cross-linking and modifying acellular dermal matrix according to claim 1, characterized in that: In step (3), the pretreated acellular dermal matrix is immersed in 10 to 20 times the mass of the mixed solution, completely immersed in an environment of 2 to 8°C for 2 to 12 hours, placed in an environment of 25 to 55°C, reacted for 2 to 16 hours, and then dehydrated in a freeze dryer to obtain a cross-linked modified acellular dermal matrix.
9. The cross-linked modified acellular dermal matrix obtained by the method according to any one of claims 1 to 8.