Material containing acellular matrix and preparation process thereof

Abstract

The invention relates to a material containing an acellular matrix and a preparation process thereof, and belongs to the technical field of biological materials. The decellularized matrix adopts ammonium hydroxide to crack lipid, Triton X-100 membrane removal protein and nuclease to degrade nucleic acid to thoroughly eliminate immunogenicity and synchronously reserve bioactive molecules such as type I collagen, growth factors and the like, a porous structure is constructed by combining supercritical CO2 degreasing and freeze-drying technologies, and the porous structure can be used for preparing the biomedical material. And the degradation rate is regulated and controlled by matching the natural restriction enzyme cutting sites with the hydrolysis characteristics of the silk fibroin. The hydrogel system takes an oxidized hyaluronic acid-thiolated chitosan dynamic covalent bond network as a core, the tensile strength is enhanced after silk fibroin beta-folding reinforcement, the hydrogel system has the lubricity of hyaluronic acid and the broad-spectrum antibacterial property of chitosan / N-sodium lauroyl sarcosinate, and amino-RGD-laminin triple cell adhesion sites promote ECM deposition; furthermore, nano calcium phosphate particles are compounded to form a nano reinforced interface, so that the mechanical property and the antibacterial property of the material are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials and relates to a material containing acellular matrix and a preparation process thereof. Background Art

[0002] Decellularized extracellular matrix (dECM) is a natural biomaterial that removes cellular and antigenic components from tissues through physical, chemical, or biological methods, while retaining the three-dimensional structure of the extracellular matrix (ECM) and its active components, such as adhesion proteins and proteoglycans. The ECM is the microenvironment for cell survival and is composed of various components, including collagen, proteoglycans, and elastin. It provides structural support and signal transmission for tissues and organs. Due to their excellent biocompatibility, biodegradability, and ability to induce cell differentiation, proliferation, and tissue formation, dECM materials show great potential in areas such as tissue repair, organ transplantation, and regenerative medicine.

[0003] Despite the many advantages of acellular matrix materials, existing preparation methods still have the following defects: Traditional physical and chemical methods, while removing cellular components, will damage the ultrastructure of the extracellular matrix to varying degrees, affecting the bioactivity and mechanical properties of the material. Existing decellularization methods easily lead to the loss of components such as soluble proteoglycans and glycoproteins, reducing the bioactivity of the material. Some acellular matrix materials have insufficient mechanical properties and are difficult to meet the requirements for material stability and support in clinical applications. Heterogeneous acellular matrix materials may retain immunogenic components, such as α-galactosidase, which can trigger immune reactions and tissue rejection. Summary of the Invention

[0004] The purpose of the present invention is to provide a material containing acellular matrix and a preparation process thereof.

[0005] The purpose of the present invention can be achieved through the following technical solutions: A preparation process of a material containing acellular matrix, wherein the specific steps of the preparation process are as follows: S1: 4-6 parts by weight of oxidized hyaluronic acid and 2-4 parts by weight of thiolated chitosan were mixed and added to 90-100 parts by weight of PBS buffer containing ethanol, followed by addition of 1-3 parts by weight of acellular matrix, and irradiated with UV for 30-50 s to obtain solution A; S2: 1-3 parts by weight of silk fibroin and 0.5-1 parts by weight of nano-calcium phosphate were added to solution A, sonicated for 30-60 minutes, and then freeze-dried at -45°C for 6-8 hours. Powder B was obtained by ball milling for 2-4 hours. S3: Add 10-20 parts by weight of powder B to 100-120 parts by weight of deionized water, add 4-6 parts by weight of sodium N-lauroyl sarcosinate, and let stand at 40-45° C. for 10-30 minutes to obtain the acellular matrix-containing material.

[0006] Furthermore, the preparation method of the decellularized matrix is ​​as follows: S2-1: Dissolve 0.8-1 parts by weight of ammonium hydroxide and 1-1.2 parts by weight of Triton X-100 in 100-120 parts by weight of phosphate buffer (pH 7.4), then add 10-15 parts by weight of animal dermal fragments, and incubate at 25°C with constant temperature and shaking for 4-6 hours to obtain a mixed solution A; S2-2: Add 3-5 parts by weight of deoxyribonuclease I and 1-2 parts by weight of ribonuclease A to mixed solution A at 4°C, then transfer to a 37°C constant temperature incubator and let it stand for 12-16 hours, then wash with deionized water and dry; S2-3: The dried fragments were added to a 5% by volume ethanol solution, extracted with supercritical CO2 fluid for 2 to 4 hours, and then freeze-dried at -30°C for 10 to 14 hours to obtain the acellular matrix.

[0007] Furthermore, the animal dermis fragments in S2-1 are one or more of pig dermis fragments, bovine dermis fragments or sheep dermis fragments, and the diameter of the animal dermis fragments is less than 1 mm.

[0008] Furthermore, during the supercritical CO2 extraction process in S2-3, the CO2 flow rate is 3~5 L / min, the temperature is 30~36°C, and the pressure is 10~15 MPa.

[0009] Furthermore, the PBS buffer in the S1 contains 5% ethanol by volume.

[0010] Furthermore, the ultraviolet wavelength in S1 is 365 nm and the power density is 10 mW / cm².

[0011] Furthermore, the ball milling speed in S2 is 100-200 r / min.

[0012] Furthermore, the enzyme activity of deoxyribonuclease I in the S2-2 is 2000 U / mg, and the enzyme activity of ribonuclease A is 50 U / mg.

[0013] The invention discloses an application of a material containing acellular matrix, wherein the material containing acellular matrix can be applied to the fields of medical dressing and biological filling material.

[0014] The aldehyde groups provided by oxidized hyaluronic acid react with the thiol groups of thiolated chitosan under ultraviolet irradiation to form a Schiff base network, significantly improving the tensile strength and fracture strain of the hydrogel while retaining the inherent biocompatibility and lubricity of hyaluronic acid, providing a low-friction microenvironment for cell migration. Furthermore, the introduction of thiol groups enhances the cationic properties of chitosan, achieving a broad-spectrum antibacterial effect by disrupting bacterial cell membranes and chelating metal ions, significantly reducing the risk of wound infection.

[0015] The addition of silk fibroin further strengthens the cross-linked network through hydrogen bonding and β-sheet formation, enhancing the material's tensile strength. Incubation at 40-45°C promotes the transition of silk fibroin to a thermodynamically stable β-sheet conformation, stabilizing the three-dimensional structure while preventing high-temperature damage to bioactive components. The amino groups of chitosan and the RGD sequence of silk fibroin provide dual cell adhesion sites, which, combined with the laminin component of the dECM, form a three-dimensional cell-responsive interface, promoting extracellular matrix deposition and accelerating tissue regeneration.

[0016] The addition of nano-calcium phosphate, whose nano-particle size can accurately reproduce the inorganic structure of natural bone tissue, constructs a bionic microenvironment for osteoblasts, activates the osteogenic signaling pathway through the release of calcium and phosphorus ions, and systematically promotes the process of cell adhesion, proliferation and differentiation; in the silk fibroin-acellular matrix composite system, a nano-enhanced interface is formed through the synergistic effect of hydrogen bonds and ionic bonds, which improves the compressive strength of the material. At the same time, its microcrack barrier properties effectively delay stress failure and extend the service life of the material; in terms of degradation kinetics regulation, nano-calcium phosphate achieves controllable degradation through ion sustained release, and its metabolites participate in the body's calcium-phosphorus cycle, avoiding inflammatory reactions caused by acidic by-products; the high specific surface area characteristics of nano-calcium phosphate provide rich cell recognition sites, strengthen the material-cell interface interaction, and the released calcium ions further activate the endothelial cell calcium signaling pathway; in addition, the calcium ion-mediated antibacterial mechanism builds an infection defense barrier by destroying the bacterial membrane potential balance.

[0017] Through a gentle decellularization process, ammonium hydroxide provides an alkaline environment that effectively disrupts the cell membrane lipid bilayer, promoting cell lysis and dissolving intercellular acidic proteins. Triton X-100, a nonionic surfactant, disrupts hydrophobic interactions, dissolving membrane proteins and effectively removing cell debris. Phosphate buffer maintains physiological osmotic pressure, preventing excessive swelling of the ECM.

[0018] The addition of deoxyribonuclease and ribonuclease A can specifically degrade residual DNA / RNA and eliminate immunogenicity. Deionized water washing can simultaneously remove enzyme proteins and degradation products. The bioactive molecules of the natural ECM, such as type I collagen, fibronectin, and growth factors, are completely retained, simulating the natural tissue microenvironment and significantly promoting cell proliferation and directional differentiation. The natural enzyme cleavage sites retained in the decellularized matrix work synergistically with the slow hydrolysis properties of silk fibroin to achieve a precise match between the material degradation rate and the tissue regeneration rate, avoiding foreign body reactions.

[0019] Supercritical CO2 fluid combined with a 5% ethanol solution for degreasing effectively removes neutral lipids. Freeze-drying at -30°C induces directional ice crystal growth, forming a controlled porous structure that promotes vascular ingrowth and nutrient penetration. Sodium N-lauroylsarcosinate modulates the material's surface charge and wettability, enhancing cell adsorption efficiency. Its mild antimicrobial properties further reduce the risk of infection.

[0020] Beneficial effects of the present invention: (1) A gentle three-step process involving ammonium hydroxide lysis of cell membrane lipids, Triton X-100 removal of membrane proteins, and nuclease degradation of DNA / RNA completely eliminates immunogenic components while fully retaining bioactive molecules such as type I collagen, fibronectin, and growth factors in the natural ECM, mimicking the natural tissue microenvironment. Combining supercritical CO2 degreasing with freeze-drying directional pore formation technology promotes vascular ingrowth and nutrient penetration. Furthermore, through the synergistic effect of natural enzyme cleavage sites and the hydrolysis properties of silk fibroin, a precise match between material degradation rate and tissue regeneration process is achieved, significantly reducing the risk of foreign body reaction.

[0021] (2) Oxidized hyaluronic acid and thiolated chitosan form a dynamic covalent bond network through a Schiff base reaction, which is combined with the β-folded structure of silk fibroin to enhance the tensile strength of the hydrogel while retaining the biocompatibility and lubricating properties of hyaluronic acid. Nano-calcium phosphate is combined with silk fibroin-acellular matrix to form an enhanced interface, improving mechanical properties and delaying failure. It can also avoid inflammatory reactions, provide cell recognition sites, activate endothelial cell signals, and have a synergistic antibacterial effect. The cationic properties of chitosan and sodium N-lauroyl sarcosine synergize to achieve broad-spectrum antibacterial effects and promote extracellular matrix deposition through the amino-RGD sequence-laminin triple adhesion site. Incubation at 40-45°C stabilizes the three-dimensional structure while avoiding thermal damage. DETAILED DESCRIPTION

[0022] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0023] In the Examples of the present invention and the Comparative Examples: Deoxyribonuclease I: purchased from Beijing Baier Ruisen Biotechnology Co., Ltd., with an enzyme activity of 2000 U / mg; Ribonuclease A: purchased from Beijing Baier Ruisen Biotechnology Co., Ltd., with an enzyme activity of 50 U / mg; Oxidized hyaluronic acid: purchased from Guangzhou Carbon Technology Co., Ltd., molecular weight HA: 5k; Thiolated chitosan: purchased from Guangzhou Carbon Technology Co., Ltd., purity 95%; Silk fibroin: purchased from Hubei Yiling Biotechnology Co., Ltd. Nano calcium phosphate: purchased from Philips Chemical (Zhejiang) Co., Ltd. Sodium N-lauroylsarcosinate: purchased from Hubei Xinghengkang Chemical Technology Co., Ltd. Ammonium hydroxide: purchased from Beijing Solebow Technology Co., Ltd. Triton X-100: purchased from Nantong Chenrun Chemical Co., Ltd.

[0024] Example 1 A preparation process of a material containing acellular matrix, wherein the specific steps of the preparation process are as follows: S1: 5 parts by weight of oxidized hyaluronic acid and 3 parts by weight of thiolated chitosan were mixed and added to 95 parts by weight of PBS buffer containing 5% ethanol. 2 parts by weight of acellular matrix were then added. The mixture was irradiated with UV light for 40 s at a wavelength of 365 nm and a power density of 10 mW / cm² to obtain solution A. S2: 2 parts by weight of silk fibroin and 0.7 parts by weight of nano-calcium phosphate were added to solution A, ultrasonicated for 45 min, and then freeze-dried at -45°C for 7 h. The powder was then ball-milled for 3 h at a speed of 150 rpm to obtain powder B. S3: 15 parts by weight of powder B were added to 110 parts by weight of deionized water, and 5 parts by weight of sodium N-lauroyl sarcosinate were added, and the mixture was allowed to stand at 43° C. for 20 minutes to obtain the acellular matrix-containing material.

[0025] The preparation method of the decellularized matrix is ​​as follows: S2-1: 0.9 parts by weight of ammonium hydroxide and 1.1 parts by weight of Triton X-100 were dissolved in 110 parts by weight of phosphate buffer having a pH of 7.4, followed by the addition of 12 parts by weight of pig dermal fragments having a diameter of less than 1 mm, and the mixture was kept at a constant temperature of 25°C and shaken for 5 hours to obtain a mixed solution A; S2-2: 4 parts by weight of deoxyribonuclease I and 1.5 parts by weight of ribonuclease A were added to the mixed solution A at 4°C, and then transferred to a 37°C constant temperature incubator for 14 hours, washed with deionized water, and dried; S2-3: The dried fragments were added to a 5% volume fraction ethanol solution, extracted with supercritical CO2 fluid for 3 hours at a CO2 flow rate of 4 L / min, a temperature of 33°C, and a pressure of 13 MPa, and then freeze-dried at -30°C for 12 hours to obtain the decellularized matrix.

[0026] Example 2 A preparation process of a material containing acellular matrix, wherein the specific steps of the preparation process are as follows: S1: 4 parts by weight of oxidized hyaluronic acid and 2 parts by weight of thiolated chitosan were mixed and added to 90 parts by weight of PBS buffer containing 5% ethanol by volume. 1 part by weight of acellular matrix was then added. The mixture was irradiated with UV light for 30 s at a wavelength of 365 nm and a power density of 10 mW / cm² to obtain solution A. S2: 1 part by weight of silk fibroin and 0.5 parts by weight of nano-calcium phosphate were added to solution A, and the mixture was ultrasonicated for 30 min. The mixture was then freeze-dried at -45°C for 6 h and ball-milled for 2 h at a speed of 100 rpm to obtain powder B. S3: 10 parts by weight of powder B were added to 100 parts by weight of deionized water, and 4 parts by weight of sodium N-lauroyl sarcosinate were added, and the mixture was allowed to stand at 40° C. for 10 minutes to obtain the acellular matrix-containing material.

[0027] The preparation method of the decellularized matrix is ​​as follows: S2-1: 0.8 parts by weight of ammonium hydroxide and 1 part by weight of Triton X-100 were dissolved in 100 parts by weight of phosphate buffer with a pH of 7.4, followed by the addition of 10 parts by weight of pig dermal fragments with a diameter of less than 1 mm, and the mixture was kept at a constant temperature of 25°C and shaken for 4 hours to obtain a mixed solution A; S2-2: 3 parts by weight of deoxyribonuclease I and 1 part by weight of ribonuclease A were added to the mixed solution A at 4°C, and then transferred to a 37°C constant temperature incubator for 12 hours, washed with deionized water, and dried; S2-3: The dried fragments were added to a 5% volume fraction ethanol solution, extracted with supercritical CO2 fluid for 2 h at a CO2 flow rate of 3 L / min, a temperature of 30 °C, and a pressure of 10 MPa, and then freeze-dried at -30 °C for 10 h to obtain the decellularized matrix.

[0028] Example 3 A preparation process of a material containing acellular matrix, wherein the specific steps of the preparation process are as follows: S1: 6 parts by weight of oxidized hyaluronic acid and 4 parts by weight of thiolated chitosan were mixed and added to 100 parts by weight of PBS buffer containing 5% ethanol by volume. Subsequently, 3 parts by weight of acellular matrix were added and irradiated with UV light for 50 s at a wavelength of 365 nm and a power density of 10 mW / cm² to obtain solution A. S2: 3 parts by weight of silk fibroin and 1 part by weight of nano-calcium phosphate were added to solution A, and the mixture was sonicated for 60 min. The mixture was then freeze-dried at -45°C for 8 h and ball-milled for 4 h at a speed of 200 rpm to obtain powder B. S3: 20 parts by weight of powder B were added to 120 parts by weight of deionized water, and 6 parts by weight of sodium N-lauroyl sarcosinate were added, and the mixture was allowed to stand at 45° C. for 30 minutes to obtain the acellular matrix-containing material.

[0029] The preparation method of the decellularized matrix is ​​as follows: S2-1: Dissolve 1 part by weight of ammonium hydroxide and 1.2 parts by weight of Triton X-100 in 120 parts by weight of phosphate buffer (pH 7.4), then add 15 parts by weight of pig dermis fragments with a diameter of less than 1 mm, and place at a constant temperature of 25°C with shaking for 6 hours to obtain a mixed solution A; S2-2: 5 parts by weight of deoxyribonuclease I and 2 parts by weight of ribonuclease A were added to the mixed solution A at 4°C, and then transferred to a 37°C constant temperature incubator for 16 hours, washed with deionized water, and dried; S2-3: The dried fragments were added to a 5% volume fraction ethanol solution, extracted with supercritical CO2 fluid for 4 hours at a CO2 flow rate of 5 L / min, a temperature of 36°C, and a pressure of 15 MPa, and then freeze-dried at -30°C for 14 hours to obtain the decellularized matrix.

[0030] Comparative Example 1 Silk fibroin was not added in the preparation process of the material containing the acellular matrix, and the remaining steps were the same as those in Example 1.

[0031] Comparative Example 2 Nano-calcium phosphate was not added in the preparation process of the material containing the acellular matrix, and the remaining steps were consistent with those in Example 1.

[0032] Comparative Example 3 Thiolated chitosan was not added in the preparation process of the material containing the acellular matrix, and the remaining steps were the same as those in Example 1.

[0033] Comparative Example 4 The preparation method of the decellularized matrix does not include steps S2-3, and the remaining steps are the same as those in Example 1.

[0034] Comparative Example 5 In the preparation process of the material containing the acellular matrix, sodium N-lauroyl sarcosinate was not added, and the remaining steps were the same as those in Example 1.

[0035] Performance Testing The tensile strength and elongation at break of the examples and comparative examples were tested according to GB / T 1040.3-2006 at a tensile rate of 50 mm / min. The results are shown in the following table:

[0036] The antibacterial rates of the examples and comparative examples were tested according to GB / T 21510-2008. The initial bacterial solution concentration was 10 7 cfu / mL, the culture time was 24 h, and the antibacterial results were as follows:

[0037] The data of the examples and comparative examples show that the material containing the acellular matrix prepared by the present invention has good mechanical properties and has good antibacterial effects on both Gram-negative bacteria and Gram-positive bacteria.

[0038] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A process for preparing a material containing an acellular matrix, characterized in that: The specific steps of the preparation process are as follows: S1: 4-6 parts by weight of oxidized hyaluronic acid and 2-4 parts by weight of thiolated chitosan were mixed and added to 90-100 parts by weight of PBS buffer containing ethanol, followed by addition of 1-3 parts by weight of acellular matrix, and irradiated with UV for 30-50 s to obtain solution A; S2: 1-3 parts by weight of silk fibroin and 0.5-1 parts by weight of nano-calcium phosphate were added to solution A, and the mixture was sonicated for 30-60 min. The mixture was then freeze-dried at -45°C for 6-8 h, and ball-milled for 2-4 h to obtain powder B. S3: Add 10-20 parts by weight of powder B to 100-120 parts by weight of deionized water, add 4-6 parts by weight of sodium N-lauroyl sarcosinate, and let stand at 40-45° C. for 10-30 minutes to obtain the acellular matrix-containing material.

2. The process for preparing a material containing acellular matrix according to claim 1, characterized in that: The preparation method of the decellularized matrix is ​​as follows: S2-1: Dissolve 0.8-1 parts by weight of ammonium hydroxide and 1-1.2 parts by weight of Triton X-100 in 100-120 parts by weight of phosphate buffer (pH 7.4), then add 10-15 parts by weight of animal dermal fragments, and place at 25°C with constant temperature and shaking for 4-6 hours to obtain a mixed solution A; S2-2: Add 3-5 parts by weight of deoxyribonuclease I and 1-2 parts by weight of ribonuclease A to mixed solution A at 4°C, then transfer to a 37°C constant temperature incubator and let it stand for 12-16 hours, then wash with deionized water and dry; S2-3: The dried fragments were added to a 5% by volume ethanol solution, extracted with supercritical CO2 fluid for 2 to 4 hours, and then freeze-dried at -30°C for 10 to 14 hours to obtain the acellular matrix.

3. The process for preparing a material containing acellular matrix according to claim 2, characterized in that: The animal dermis fragments in S2-1 are one or more of pig dermis fragments, bovine dermis fragments or sheep dermis fragments, and the diameter of the animal dermis fragments is less than 1 mm.

4. The process for preparing a material containing acellular matrix according to claim 2, characterized in that: During the supercritical CO2 extraction process in S2-3, the CO2 flow rate is 3~5 L / min, the temperature is 30~36°C, and the pressure is 10~15 MPa.

5. The process for preparing a material containing acellular matrix according to claim 1, characterized in that: The PBS buffer in the S1 contains 5% ethanol by volume.

6. The process for preparing a material containing acellular matrix according to claim 1, characterized in that: The ultraviolet wavelength in S1 is 365 nm and the power density is 10 mW / cm².

7. The process for preparing a material containing acellular matrix according to claim 1, characterized in that: The ball milling speed in S2 is 100-200 r / min.

8. The process for preparing a material containing acellular matrix according to claim 2, characterized in that: The enzyme activity of deoxyribonuclease I in the S2-2 is 2000 U / mg, and the enzyme activity of ribonuclease A is 50 U / mg.

9. A material containing an acellular matrix, characterized in that: The method is prepared by the preparation process according to any one of claims 1 to 8.

10. Use of the material containing acellular matrix according to claim 9, characterized in that: The material containing the acellular matrix can be applied to the fields of medical dressings and biological filling materials.

Images