An injectable acellular matrix and its preparation method and application

Abstract

The present application relates to a kind of injectable acellular matrix and its preparation method and application, it includes acellular matrix particle and mixed medium, the mixed medium is hydrogel, the hydrogel is linked to the acellular matrix particle, the hydrogel has imine bond, borate ester bond and guluronic acid structure.In joint cavity can quickly repair the gel breakage due to joint activity, long-term maintenance three-dimensional network structure, avoids the problem that traditional hydrogel is easily broken in joint movement, failure.Effectively solve the core pain points that existing hydrogel is easily lost under the flushing of synovial fluid, difficult to long-term residence injury site.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to an injectable decellularized matrix, its preparation method, and its application. Background Technology

[0002] Articular cartilage is a complex and specialized structure that covers the ends of bones in a joint, providing a low-friction contact surface for joint movement within a certain range. Articular cartilage has poor self-repair capabilities, especially when significant damage occurs, leading to joint pain and loss of joint function, often progressing to osteoarthritis. The knee joint is the most complex joint in the human body and bears the greatest load, making it most susceptible to osteoarthritis.

[0003] Current clinical treatments for knee osteoarthritis typically involve oral nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, and / or intra-articular injections of hyaluronic acid, sodium hyaluronate, etc., whose effects are limited to pain suppression or physical lubrication. Furthermore, existing medical hydrogels are usually prepared using horseradish peroxidase as a cross-linking agent, exhibiting good biocompatibility; these hydrogels contain components with a brush-like structure, providing good lubrication and facilitating the treatment of osteoarthritis; however, they have poor adhesion, lack cartilage repair properties, and cannot fundamentally address the problem of cartilage damage. Summary of the Invention

[0004] The purpose of this invention is to provide an injectable decellularized matrix to solve the problem that existing joint injection preparations cannot adhere well to the surface of damaged cartilage, only have a lubricating effect but no repair function, and therefore require multiple injections.

[0005] The first objective of this invention is to provide an injectable decellularized matrix comprising decellularized matrix particles and a mixing medium, wherein the mixing medium is a hydrogel. The hydrogel is bonded to the decellularized matrix particles, and the hydrogel has an imine bond, a borate ester bond, and a guluronic acid structure.

[0006] Specifically, the process of attaching the hydrogel to the decellularized matrix particles includes: (1) Mix the decellularized matrix particles with sodium oxidized alginate to complete the pre-crosslinking and obtain a sol suspension; (2) The sol-state suspension is reacted with 3-aminophenylboronic acid to obtain a first-layer dynamic network self-healing gel; (3) The first dynamic network self-healing gel is reacted with dopamine hydrochloride grafted with hyaluronic acid to obtain a second dynamic network self-healing gel.

[0007] Preferably, in step (1), the mass ratio of sodium oxidized alginate to decellularized matrix particles is (2-3):(0.8-1.2); the particle size of the decellularized matrix particles is 100-500 μm.

[0008] Preferably, in step (2), the mass ratio of 3-aminophenylboronic acid to sodium alginate is (2-2.5); in step (3), the mass ratio of dopamine hydrochloride grafted hyaluronic acid to 3-aminophenylboronic acid is (5-10):1.

[0009] Preferably, the decellularized matrix particles are obtained by sequentially subjecting the decellularized matrix raw material to decellularization treatment, biological enzyme treatment, virus inactivation and deep cleaning, drying and grinding; the decellularized matrix raw material is a mixture of one or more of animal skin, human skin, animal visceral tissue, human visceral tissue, animal perinatal tissue and human perinatal tissue.

[0010] Furthermore, the decellularization process involves cutting the decellularized matrix raw material, adding it to a decellularization solution for treatment, and then washing it. The bio-enzyme treatment involves adding the decellularized matrix raw material, which has undergone decellularization treatment, to a bio-enzyme solution for treatment and washing. The virus inactivation and deep cleaning involve adding a virus inactivating agent solution to the decellularized matrix raw material that has been treated with biological enzymes and then cleaning it. The drying process involves freeze-drying decellularized matrix raw materials that have undergone virus inactivation and deep cleaning. The grinding process involves placing the dried, decellularized matrix material into a grinding machine for grinding.

[0011] More preferably, the decellularization solution is a mixture of one or more of the following: ionic surfactant solutions and nonionic surfactant solutions; the biological enzyme solution is a lipase, deoxyribonuclease, trypsin, or pepsin solution; and the virus inactivating agent is a mixture of one or more of the following: peracetic acid, hydrogen peroxide, peroctanoic acid, and ethanol.

[0012] A second objective of the present invention is to provide a method for preparing the above-mentioned injectable decellularized matrix, wherein the hydrogel is chemically bonded to the decellularized matrix particles.

[0013] A third objective of this invention is to provide an application of the above-mentioned injectable decellularized matrix for the preparation of medical materials for treating knee arthritis.

[0014] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: (1) In this invention, decellularized matrix (ECM) particles are mixed with hydrogel to react, so that sodium alginate (OSA) and 3-aminophenylboronic acid (APBA) form imine bonds, and APBA and dopamine hydrochloride grafted with hyaluronic acid (HA-DA) form borate ester bonds. These are all dynamic and reversible bonds, which gives the hydrogel shear-thinning properties and makes it injectable. At the same time, the reversibility of the dynamic bonds gives the product good self-healing ability, which can quickly repair gel damage caused by joint movement in the joint cavity and maintain the three-dimensional network structure for a long time, avoiding the problem of traditional hydrogels being prone to breakage and failure during joint movement.

[0015] (2) The present invention uses a mild decellularization technique to prepare decellularized matrix particles. Combined with precise control of particle size, the prepared decellularized matrix particles are stably dispersed in the mixed medium, which further ensures the repair effect of injectable decellularized matrix in the knee joint.

[0016] (3) The injectable decellularized matrix can be injected into the body and can also achieve in vivo Ca 2+ Response-based in-situ curing constructs a triple synergistic network, significantly improving gel retention and erosion resistance: OSA retains the guluronic acid (G block) structure of sodium alginate (SA); after injection into the joint cavity, naturally occurring Ca in the synovial fluid... 2+ It can form egg-box-type ion crosslinks with G blocks to achieve in-situ curing of hydrogels, making the gel structure more stable. This curing process does not require the addition of additional initiators or crosslinking agents, and is fully compatible with the physiological environment in the body. It effectively solves the core pain points of existing hydrogels that are easily lost under the flushing of synovial fluid and are difficult to stay in the injured site for a long time.

[0017] (4) The catechol structure in HA-DA can form a strong non-covalent interaction with the hydroxyl and amino groups on the cartilage surface in the wet environment of synovial fluid, so that the hydrogel adheres tightly to the cartilage and synovial surface and avoids gel displacement caused by joint movement. At the same time, the adhesion can reduce the scouring of the gel by synovial fluid, further prolong the gel residence time, and ensure that the repair effect continues to play a role, overcoming the shortcomings of traditional hydrogels with poor wet surface adhesion and easy displacement.

[0018] (5) The triple network of the hydrogel of the present invention (imine bond, borate bond, Ca) 2+ The synergistic effect of the -G block ionic bonds allows the gel degradation rate to be gently controlled according to the needs of joint repair; moreover, all components are biocompatible materials, free of toxic and harmful substances, and do not require harsh preparation conditions such as high temperature or photoinitiation. The preparation process is gentle and suitable for multiple in vivo injections, further improving the safety and applicability of the product in clinical applications.

[0019] (6) 3-Aminophenylboronic acid (APBA) acts as a linker, reacting with OSA and HA-DA, consuming its own different chemical bonds, proceeding independently and stepwise. The reaction between APBA and OSA involves an aldehyde-amine condensation reaction (forming an imine bond) between the amino group (-NH2) in the APBA molecule and the aldehyde group (-CHO) in the OSA molecule. This process consumes only the amino group of APBA, while the phenylboronic acid group (-B(OH)2) in its molecule is completely retained, used to react with the catechol structure in HA-DA to form a stable borate ester bond. This process only consumes the phenylboronic acid group of APBA and does not involve the amino group. This ensures that APBA simultaneously plays the dual role of "linking OSA" and "crosslinking HA-DA".

[0020] (7) Furthermore, by scientifically designing the order of addition of each component (OSA→ECM→excess APBA→excess HA-DA) and rationally controlling the excess ratio of APBA and HA-DA, the present invention further endows the hydrogel with significant additional beneficial effects, as follows: The method follows the sequence of "preparing the OSA-ECM substrate system first, then adding APBA, and finally adding HA-DA". First, the aldehyde groups of OSA react fully with the amino groups of ECM to ensure the anchoring of ECM particles and prevent particle sedimentation. Then, APBA is added to allow the remaining aldehyde groups of OSA to react fully with the amino groups of APBA to form imine bonds, ensuring that APBA is uniformly grafted onto the OSA molecular chain. Finally, HA-DA is added to fully interact with the APBA reserved in the system, which can avoid problems such as gel clumping and inability to inject caused by premature cross-linking.

[0021] The excess APBA design further ensures that the aldehyde groups of OSA are fully grafted with APBA, while reserving sufficient free phenylboronic acid groups to provide ample reaction sites for the subsequent formation of borate ester bonds with the catechol structure of HA-DA, avoiding insufficient secondary crosslinking due to insufficient APBA. The excess HA-DA design ensures that all free and grafted APBA in the system can react fully, while the remaining catechol groups of the excess HA-DA can ensure the adhesion of the hydrogel to the tissue in a humid environment. Attached Figure Description

[0022] The following sections will describe some specific embodiments of the invention in a detailed manner by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art will understand that these drawings are not necessarily drawn to scale.

[0023] Figure 1 This is an electron micrograph of the decellularized matrix particles in Example 2 of the present invention. Detailed Implementation

[0024] The present invention relates to an injectable decellularized matrix comprising decellularized matrix particles and a mixed medium, wherein the mixed medium is a hydrogel; when the mixed medium is a hydrogel, the hydrogel is bonded to the decellularized matrix particles, and the hydrogel has an imine bond, a borate ester bond, and a guluronic acid structure.

[0025] The process of attaching the hydrogel to the decellularized matrix particles includes: (1) mixing the decellularized matrix particles with sodium alginate oxide to complete pre-crosslinking and obtain a sol suspension; (2) reacting the sol suspension with 3-aminophenylboronic acid to obtain a primary dynamic network self-healing gel; and (3) reacting the primary dynamic network self-healing gel with dopamine hydrochloride grafted with hyaluronic acid to obtain a secondary dynamic network self-healing gel.

[0026] In step (1), the mass ratio of sodium alginate oxidized to decellularized matrix particles is (2-3):(0.8-1.2), and the particle size of the decellularized matrix particles is 100-500 μm. In step (2), the mass ratio of 3-aminophenylboronic acid to sodium alginate oxidized is (2-2.5):10; in step (3), the mass ratio of dopamine hydrochloride grafted hyaluronic acid to 3-aminophenylboronic acid is (5-10):1.

[0027] The decellularized matrix particles are obtained by sequentially processing the decellularized matrix raw material through decellularization, bio-enzyme treatment, virus inactivation, deep cleaning, drying, and grinding; the decellularized matrix raw material is a mixture of one or more of the following: animal skin, human skin, animal visceral tissue, human visceral tissue, animal perinatal tissue, and human perinatal tissue.

[0028] The decellularization process involves cutting the decellularized matrix material and adding it to a decellularization solution (concentration 0.05~0.2% (w / v), mg / ml, the same below) for treatment and washing. The decellularization solution is a mixture of one or more of ionic and nonionic surfactant solutions, preferably a nonionic surfactant solution, such as Triton X-100 solution (concentration 0.05~0.2% (w / v)). The ratio of the weight (kg) of the decellularized matrix material to the volume (L) of the decellularization solution is 1:(3-5).

[0029] The bio-enzyme treatment involves adding the decellularized matrix material, which has undergone decellularization treatment, to a bio-enzyme solution for treatment and washing. The bio-enzyme solution is a solution of lipase, deoxyribonuclease, trypsin, or pepsin (with physiological saline as the solvent); the concentration of the bio-enzyme is 200-500 U / mL (preferably lipase); the mass-to-volume ratio (w / v) of the decellularized matrix material to the bio-enzyme solution is 1:(3-5) (mg / ml).

[0030] The virus inactivation and deep washing involve adding a virus inactivating agent solution to the decellularized matrix raw material treated with biological enzymes for treatment and washing. The virus inactivating agent is a mixture of one or more of peracetic acid, hydrogen peroxide, peroctanoic acid, and ethanol, preferably peracetic acid. After adding physiological saline, the virus inactivating agent is prepared into a solution with a weight / volume concentration (w / v) of 0.1-0.3% (mg / ml, the same below), and the weight / volume ratio (w / v) of the decellularized matrix raw material treated with biological enzyme solution to the prepared virus inactivating agent is 1:(3-5).

[0031] The drying process involves freeze-drying decellularized matrix raw materials that have undergone virus inactivation and deep cleaning. The grinding process involves placing the dried, decellularized matrix material into a grinding machine for grinding.

[0032] The above-mentioned method for preparing injectable decellularized matrix includes the following steps: chemically bonding hydrogel to the decellularized matrix particles.

[0033] The above-mentioned injectable decellularized matrix is ​​used in the preparation of medical materials for the treatment of knee arthritis.

[0034] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] Example 1

[0037] This embodiment provides an injectable decellularized matrix, its preparation method, and its application, as detailed below: I. Preparation of decellularized matrix particles: 1) Decellularization treatment First, the fresh fetal membranes were peeled off, cleaned, cut into small pieces, and drained. Then, Triton X-100 solution (Triton X-100 dissolved in saline solution to a concentration of 0.2% (w / v), mg / ml, hereinafter the same) was added, and the mixture was placed on a shaker at 70 rpm for 48 hours. After treatment, the membranes were washed with purified water. The ratio of fetal membrane material weight (kg) to Triton X-100 solution volume (L) was 1:3. 2) Bioenzyme treatment After cleaning, the fetal membranes were drained, and lipase (enzyme concentration of 200 U / mL, solvent of physiological saline) was added. The membranes were placed in a shaker at 70 rpm and treated for 48 hours. Then, they were washed with purified water. The ratio of fetal membrane material weight (kg) to enzyme solution volume (L) was 1:3. 3) Virus inactivation and deep cleaning After cleaning, the fetal membranes were drained and treated with peracetic acid virus inactivation agent for 4 hours (the concentration of virus inactivation agent (peracetic acid) was 0.1% (w / v), and the solvent was physiological saline). The treated fetal membranes were then rinsed with ultrapure water. The ratio of fetal membrane material weight (kg) to virus inactivation agent volume (L) was 1:3. 4) Drying The fetal membranes processed in the above steps are placed in a vacuum dryer for freeze drying to obtain a highly bioactive decellularized fetal membrane matrix. 5) Preparation of decellularized matrix particles The decellularized fetal membrane matrix was placed in a grinder and ground. The grinding rate was set to 10 CPS, the cycle time was 2 min, the cooling time was 1 min, and the number of cycles was 3. The matrix was then sieved to obtain decellularized matrix particles (particle size 100-500 μm).

[0038] II. Preparation of injectable decellularized matrix: 1) Oxidation reaction of sodium alginate Weigh 1g of sodium alginate, dissolve it in water to a concentration of 1wt%, stir at room temperature for 4 hours, and adjust the pH of the solution to 3 with HCl. Weigh 0.5g of sodium periodate, dissolve it in water to a concentration of 5wt%, and slowly add the sodium periodate aqueous solution dropwise to the sodium alginate aqueous solution. Stir in the dark for 6 hours, and add ethylene glycol to terminate the reaction. Pour 300mL of anhydrous ethanol into the above reaction solution, stir until a white precipitate appears, discard the supernatant, and wash the precipitate twice with anhydrous ethanol. Dissolve the precipitate in water, transfer it to a dialysis bag, dialyze for 3 days, and freeze-dry to obtain OSA powder. 2) Anchoring of decellularized matrix particles Weigh 0.5g of OSA powder and dissolve it in PBS (phosphate buffered saline) to a concentration of 1wt%. Stir to dissolve and adjust the pH to 7-7.4 (to obtain an OSA powder PBS solution). Weigh fetal membrane decellularized matrix powder (ECM) and add it to the OSA powder PBS solution, making the mass ratio of OSA powder to ECM 2:0.8. Stir the mixture for 30 minutes to complete the pre-crosslinking and obtain a sol suspension (i.e., OSA-ECM suspension). 3) Preparation of a single-layer dynamic network gel Weigh 0.1g of 3-aminophenylboronic acid (APBA) and slowly add it to the aforementioned OSA-ECM suspension. Stir the mixture at room temperature for 60 minutes to obtain a single-layer dynamic network self-healing gel (OSA-ECM-APBA). 4) Grafting reaction of HA-DA Weigh 3g of hyaluronic acid, dissolve it in deionized water, adjust the pH to 5, add 0.25g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.25g of N-hydroxysuccinimide (NHS) to the solution, stir for 1h, then add 2g of dopamine hydrochloride, vacuum and nitrogen-filled, stir at room temperature in an anaerobic environment for 24h, transfer the reactants to a dialysis bag for analysis and purification, freeze-dry after purification to obtain the product HA-DA; 5) Preparation of dual dynamic network gel Weigh 0.5g of HA-DA powder, dissolve it in PBS to a final concentration of 0.5wt%, and add it dropwise to the aforementioned OSA-ECM-APBA. Stir well to obtain a dual dynamic network self-healing gel, which is then ready for injection as a decellularized matrix.

[0039] The above-mentioned application of injectable decellularized matrix: The injectable decellularized matrix is ​​injected into the joint cavity, where it reacts with the Ca in the synovial fluid. 2+ Ionic crosslinking is formed to obtain a triple crosslinked gel.

[0040] Example 2

[0041] This embodiment provides an injectable decellularized matrix, its preparation method, and its application, as detailed below: I. Preparation of decellularized matrix particles: 1) Decellularization treatment First, the fresh fetal membranes were peeled off, cleaned, cut into small pieces, and drained. Then, Triton X-100 solution (Triton X-100 dissolved in saline solution to prepare a 0.1% (w / v) solution, mg / ml, the same below) was added, and the mixture was placed on a shaker at 85 rpm for 30 hours. Afterward, it was washed with purified water. The ratio of fetal membrane material weight (kg) to Triton X-100 solution volume (L) was 1:4. 2) Bioenzyme treatment After cleaning, the fetal membranes were drained and treated with lipase (enzyme concentration of 300 U / mL, solvent: physiological saline). The membranes were placed on a shaker at 85 rpm and treated for 30 hours. The membranes were then rinsed with purified water. The ratio of fetal membrane material weight (kg) to enzyme solution volume (L) was 1:4. 3) Virus inactivation and deep cleaning After cleaning, the fetal membranes were drained and treated with peracetic acid virus inactivation agent for 5 hours (the concentration of virus inactivation agent (peracetic acid) was 0.2% (w / v), and the solvent was physiological saline). The treated fetal membranes were then rinsed with ultrapure water. The ratio of fetal membrane material weight (kg) to virus inactivation agent volume (L) was 1:4. 4) Drying The fetal membranes processed in the above steps are placed in a vacuum dryer for freeze drying to obtain a highly bioactive decellularized fetal membrane matrix. 5) Preparation of decellularized matrix particles The decellularized fetal membrane matrix was ground in a grinder at a grinding rate of 20 CPS for 3 minutes, followed by a 2-minute cooling period. The mixture was repeated twice, then sieved to obtain decellularized matrix particles with a particle size of 100-500 μm (see attached image). Figure 1 (As shown).

[0042] II. Preparation of injectable decellularized matrix: 1) Oxidation reaction of sodium alginate Weigh 1.5 g of sodium alginate, dissolve it in water to a concentration of 2 wt%, stir at room temperature for 5 h, and adjust the pH of the solution to 4 with HCl; weigh 0.7 g of sodium periodate, dissolve it in water to a concentration of 7 wt%, slowly add the sodium periodate aqueous solution to the sodium alginate aqueous solution, stir in the dark for 7 h, and add ethylene glycol to terminate the reaction. Pour 300 mL of anhydrous ethanol into the above reaction solution, stir until a white precipitate appears, discard the supernatant, wash the precipitate 3 times with anhydrous ethanol; dissolve the precipitate in water, transfer it to a dialysis bag, dialyze for 4 days, and freeze-dry to obtain OSA powder; 2) Anchoring of decellularized matrix particles Weigh 0.8g of OSA powder, add PBS to dissolve to a concentration of 2 wt%, stir to dissolve, and adjust the pH to 7-7.4 (to obtain an OSA powder PBS solution). Weigh fetal membrane decellularized matrix powder (ECM), where the OSA powder:ECM mass ratio is 2.5:1. Stir the mixture for 30 min to complete pre-crosslinking and obtain a sol suspension. 3) Preparation of a single-layer dynamic network gel Weigh 0.2 g of 3-aminophenylboronic acid (APBA) and slowly add it to the aforementioned sol-state suspension. Stir the mixture at room temperature for 60 min to obtain a single-layer dynamic network self-healing gel (OSA-ECM-APBA). 4) Grafting reaction of HA-DA Weigh 4 g of hyaluronic acid, dissolve it in deionized water, adjust the pH to 5.5, add 0.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.35 g of N-hydroxysuccinimide (NHS) to the solution, stir for 1 h, then add 2 g of dopamine hydrochloride, vacuum and nitrogen-filled, stir at room temperature in an anaerobic environment for 24 h, transfer the reactants to a dialysis bag for analysis and purification, freeze-dry after purification to obtain the product HA-DA; 5) Preparation of dual dynamic network gel Weigh 1.5 g of HA-DA powder, dissolve it in PBS to a final concentration of 0.7 wt%, and add it dropwise to OSA-ECM-APBA. Stir well to obtain a dual dynamic network self-healing gel, which is then ready for injection as a decellularized matrix.

[0043] The aforementioned injectable decellularized matrix is ​​used to prepare medical materials for treating knee osteoarthritis. Specifically, the injectable decellularized matrix is ​​injected into the joint cavity to react with the calcium in the synovial fluid. 2+ Ionic crosslinking is formed to obtain a triple crosslinked gel, thus enabling its application.

[0044] Example 3

[0045] This embodiment provides an injectable decellularized matrix, its preparation method, and its application, as detailed below: I. Preparation of decellularized matrix particles: 1) Decellularization treatment First, the fresh fetal membranes were peeled off, cleaned, cut into small pieces, and drained. Then, Triton X-100 solution was added (Triton X-100 was mixed with saline solution to prepare a 0.2% (w / v) solution, mg / ml, the same below), and placed on a shaker at 100 rpm for 48 hours. After treatment, the membranes were washed with purified water. The ratio of fetal membrane material weight (kg) to solution volume (L) was 1:5. 2) Bioenzyme treatment After cleaning, the fetal membranes were drained and treated with lipase (enzyme concentration of 500 U / mL, solvent of physiological saline). The membranes were placed on a shaker at 100 rpm and treated for 48 hours. The membranes were then rinsed with purified water. The ratio of fetal membrane material weight (kg) to enzyme solution volume (L) was 1:5. 3) Virus inactivation and deep cleaning After cleaning, the fetal membranes were drained and treated with peracetic acid virus inactivation agent for 6 hours (the concentration of virus inactivation agent (peracetic acid) was 0.3% (w / v), and the solvent was physiological saline). The treated fetal membranes were then rinsed with ultrapure water. The ratio of fetal membrane material weight (kg) to virus inactivation agent volume (L) was 1:5. 4) Drying The fetal membranes processed in the above steps are placed in a vacuum dryer for freeze drying to obtain a highly bioactive decellularized fetal membrane matrix. 5) Preparation of decellularized matrix particles The decellularized fetal membrane matrix was placed in a grinder and ground. The grinding rate was set to 30 CPS, the cycle time was 4 min, the cooling time was 3 min, and the cycle number was 3 times. The matrix was then sieved to obtain decellularized matrix particles with a particle size of 100-500 μm.

[0046] II. Preparation of injectable decellularized matrix: 1) Oxidation reaction of sodium alginate Weigh 2 g of sodium alginate, dissolve it in water to a concentration of 3 wt%, stir at room temperature for 6 h, and adjust the pH of the solution to 5 with HCl. Weigh 1 g of sodium periodate, dissolve it in water to a concentration of 10 wt%, and slowly add the sodium periodate aqueous solution dropwise to the sodium alginate aqueous solution. Stir in the dark for 8 h, and add ethylene glycol to terminate the reaction. Pour 300 mL of anhydrous ethanol into the above reaction solution, stir until a white precipitate appears, discard the supernatant, and wash the precipitate 3 times with anhydrous ethanol. Dissolve the precipitate in water, transfer it to a dialysis bag, dialyze for 5 days, and freeze-dry to obtain OSA powder.

[0047] 2) Anchoring of decellularized matrix particles Weigh 1.5g of OSA powder and dissolve it in PBS to a concentration of 3 wt%. Stir to dissolve and adjust the pH to 7-7.4 (to obtain an OSA powder PBS solution). Weigh out decellularized fetal membrane matrix powder (ECM) with an OSA powder:ECM mass ratio of 3:1.2. Stir the mixture for 30 min to complete pre-crosslinking and obtain a sol suspension. 3) Preparation of a single-layer dynamic network gel Weigh 0.3 g of 3-aminophenylboronic acid (APBA) and slowly add it to the aforementioned OSA-ECM suspension. Stir the mixture at room temperature for 60 min to obtain a single-layer dynamic network self-healing gel (OSA-ECM-APBA). 4) Grafting reaction of HA-DA Weigh 5g of hyaluronic acid, dissolve it in deionized water, adjust the pH to 6, add 0.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.5g of N-hydroxysuccinimide (NHS) to the solution, stir for 1h, then add 4g of dopamine hydrochloride, vacuum and nitrogen-filled, stir at room temperature in an anaerobic environment for 24h, transfer the reactants to a dialysis bag for analysis and purification, freeze-dry after purification to obtain the product HA-DA; 5) Preparation of dual dynamic network gel Weigh 3g of HA-DA powder, dissolve it in PBS to a final concentration of 1 wt%, and add it dropwise to OSA-ECM-APBA. Stir well to obtain a dual dynamic network self-healing gel, which is then ready for injection as a decellularized matrix.

[0048] The above-mentioned application of injectable decellularized matrix: The injectable decellularized matrix is ​​injected into the joint cavity, where it reacts with the Ca in the synovial fluid. 2+ Ionic crosslinking is formed to obtain a triple crosslinked gel, thus enabling its application.

[0049] Comparative Example 1 This comparative example provides an injectable decellularized matrix, its preparation method, and its application, which are basically the same as those in Example 2, except that in step 3), 0.3 g of PBA (phenylboronic acid) is used as a linker in the preparation of the first-order dynamic network gel.

[0050] Comparative Example 2 This comparative example provides an injectable decellularized matrix, its preparation method, and its application, which are basically the same as those in Example 2, except that the preparation order of the injectable decellularized matrix in step two is adjusted. Steps 2)-5) are adjusted as follows: 2) Preparation of a single-layer dynamic network gel Weigh 0.8g of OSA powder and dissolve it in PBS to a concentration of 2 wt%. Stir to dissolve and adjust the pH to 7-7.4. Weigh 0.2g of 3-aminophenylboronic acid (APBA) and slowly add it to the sol suspension. Stir at room temperature for 60 min to obtain a single-layer dynamic network self-healing gel (OSA-APBA). 3) Grafting reaction of HA-DA Weigh 4 g of hyaluronic acid, dissolve it in deionized water, adjust the pH to 5.5, add 0.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.35 g of N-hydroxysuccinimide (NHS) to the solution, stir for 1 h, then add 3 g of dopamine hydrochloride, vacuum and nitrogen-filled, stir at room temperature in an anaerobic environment for 24 h, transfer the reactants to a dialysis bag for analysis and purification, freeze-dry after purification to obtain the product HA-DA; 4) Preparation of dual dynamic network gel Weigh 1.5g of HA-DA powder, dissolve it in PBS to a final concentration of 0.7 wt%, add it dropwise to OSA-APBA, stir well to obtain a dual dynamic network self-healing gel. Weigh 0.32 g of decellularized fetal membrane matrix particles (OSA powder: ECM mass ratio = 2.5:1), add them to the dual dynamic network self-healing gel, and stir thoroughly to mix.

[0051] Comparative Example 3 This comparative example provides an injectable decellularized matrix, its preparation method, and its application, which are basically the same as those in Example 2, except that: in the third step of the preparation of the injectable decellularized matrix, 0.05 g of 3-aminophenylboronic acid (APBA) is added, and in the fifth step, 0.2 g of HA-DA powder is added.

[0052] Comparative Example 4 This comparative example provides an injectable decellularized matrix, its preparation method, and its application, which are basically the same as those in Example 2, except that in step one, the preparation of the decellularized matrix particles: 2) Decellularization treatment Immerse the pretreated samples in the prepared SDS decellularization solution and shake at 37°C and 100 rpm for 48 hours, depending on the sample type. 3) Cleaning Discard the decellularization solution, rinse the sample repeatedly with PBS buffer, then soak and stand for 48 hours, changing the solution regularly to completely remove residual reagents.

[0053] Experimental Example 1: Cell Proliferation Assay

[0054] Samples prepared in Examples 1-3 and Comparative Examples 1-4 were used to detect cell proliferation at 37°C. Equal amounts of injectable decellularized matrix were added sequentially to 24-well plates, followed by MC3T3-E1 cell suspension. The plates were incubated at 37°C, with the medium changed every other day for 7 days. Proliferation was then detected using the MTT assay.

[0055]

[0056] Wherein OD0 represents the average absorbance of each well in the blank group at 570 nm; OD1 represents the average absorbance of each well in the test group at 570 nm.

[0057] Table 1. Cell proliferation rate (%) Example 1 101.1 Example 2 109.4 Example 3 104.7 Comparative Example 1 100.9 Comparative Example 2 99.5 Comparative Example 3 99.3 Comparative Example 4 53.5 The experimental results show that Examples 1-3 and Comparative Examples 1-3 used a mild decellularization technique, which can retain the natural active ingredients such as collagen, glycosaminoglycans (GAG), fibronectin, and laminin in the matrix, as well as cell adhesion sites, to the greatest extent. This can effectively promote the adhesion, proliferation, and matrix secretion of articular chondrocytes, and there are no chemical reagent residues, resulting in a good cell proliferation rate. Comparative Example 4 used SDS for decellularization, which damages the ECM, breaks the bonds between collagen and elastin, and leads to matrix fiber breakage and loosening of the structure. It also destroys bioactive ingredients and cell adhesion sites, and the presence of solvent residues leads to a poor cell proliferation rate.

[0058] Experimental Example 2: In vitro degradation test The samples prepared in Examples 1-3 and Comparative Examples 1-3 were freeze-dried and weighed, and the weight was recorded as W0. Water was added to fully swell the samples. The remaining solid samples were taken 1, 2, and 3 months after swelling, and the samples were freeze-dried and weighed as W1. The in vitro degradation rate was calculated.

[0059]

[0060] Table 2. Degradation percentage (%) of each sample group Example 1 10.1 39.6 69.3 Example 2 13.2 40.3 71.9 Example 3 14.4 43.5 74.9 Comparative Example 1 63.3 85.9 95.9 Comparative Example 2 30.2 52.0 76.2 Comparative Example 3 58.0 79.4 87.8 The experimental results show that all samples began to degrade over time. Samples in Examples 1-3 showed slow overall degradation at the 1-month time point, with some residue remaining by the 3-month time point, ensuring long-term repair effects. Comparative Examples 1-3 did not form a stable system and began to degrade significantly at the 1-month time point. By the 3-month time point, there were virtually no residues in Comparative Examples 1-3. The decellularized matrix particles in Comparative Example 2 showed poor anchoring effect, but the gel network system remained stable.

[0061] Experimental Example 3: Knee Cartilage Repair Experiment Twenty-seven New Zealand white rabbits were randomly divided into four groups: a control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group, with three rabbits in each group. An incision was made in the right knee joint, the medial joint capsule was cut to expose the joint cavity, and the patella was hyperextended and laterally dislocated. A cartilage defect model was created by drilling a hole (6 mm in diameter and 0.5 mm in depth) in the center of the medial femoral condyle using Kirschner wires, and the wound was sutured. Two weeks later, rabbits in each group were injected for the first time with 0.5 mL of the corresponding injectable decellularized matrix, and a second injection was given four weeks later. Animals were sacrificed 20 weeks after the second injection, and tissue samples were collected. Histological scoring was performed using the Wakitani scoring system, which consisted of five indicators: cell morphology (0-4 points), matrix staining (0-4 points), surface smoothness (0-2 points), thickness of newly formed cartilage (0-2 points), and integration with surrounding tissue (0-2 points). The scoring range was 0-14 points. The higher the score, the closer the repaired new tissue was to normal tissue. In addition, the P value was a probability value, and P < 0.01 indicated that the difference was extremely significant.

[0062] Table 3. Cartilage repair score results for each group of samples. Example 1 12.63±0.87 ＜0.001 Example 2 11.88±0.96 ＜0.001 Example 3 12.74±0.81 ＜0.001 Comparative Example 1 3.91±0.87 ＜0.001 Comparative Example 2 3.87±0.79 ＜0.001 Comparative Example 3 4.01±0.95 ＜0.001 Blank group 13.82±0.69 ＜0.001 The experimental results show that, compared with the control group, Examples 1-3 significantly improved the repair effect of cartilage defects. This indicates that by providing bioactive factors through the fetal membrane matrix and through the special design of the preparation process and material structure, long-term tissue adhesion and repair functions can be achieved. In addition, the stability of the entire system ensures sufficient contact between the material and cells, and the synergistic effect of various structures and components achieves the purpose of cartilage repair. Among them, Examples 1-3 are stable systems with a triple gel network, ensuring adhesion and repair effects.

[0063] Comparative Examples 1-3 exhibited poor repair effects due to a lack of system stability and adhesion. Specifically, Comparative Example 1 used phenylboronic acid for gel network preparation, which could only form a gel network with HA-DA and could not connect with OSA-ECM, resulting in poor system stability. Comparative Example 2 adjusted the order of material addition, preventing sufficient aldehyde groups from cross-linking the ECM with the gel system, leading to poor anchoring of matrix particles. Comparative Example 3 did not add excessive amounts of APBA and HA-DA. On one hand, the amount of APBA as a linker was insufficient, resulting in weak dual-network connections; on the other hand, the amount of HA-DA was insufficient, resulting in a lack of excess catechol groups to adhere to the moist tissue, ultimately leading to poor overall system stability.

[0064] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An injectable decellularized matrix, characterized in that: It comprises decellularized matrix particles and a mixed medium, wherein the mixed medium is a hydrogel. The hydrogel is bonded to the decellularized matrix particles, and the hydrogel has an imine bond, a borate ester bond, and a guluronic acid structure. The process of attaching the hydrogel to the decellularized matrix particles includes: (1) Mix the decellularized matrix particles with sodium oxidized alginate to complete the pre-crosslinking and obtain a sol suspension; (2) The sol-state suspension is reacted with 3-aminophenylboronic acid to obtain a first-layer dynamic network self-healing gel; (3) React the first dynamic network self-healing gel with dopamine hydrochloride grafted with hyaluronic acid to obtain a second dynamic network self-healing gel. In step (1), the mass ratio of sodium oxidized alginate to decellularized matrix particles is (2-3):(0.8-1.2), and the particle size of the decellularized matrix particles is 100-500 μm; In step (2), the mass ratio of 3-aminophenylboronic acid to sodium alginate is (2-2.5):10; In step (3), the mass ratio of dopamine hydrochloride grafted with hyaluronic acid to 3-aminophenylboronic acid is (5-10):

1.

2. The injectable decellularized matrix according to claim 1, characterized in that: The decellularized matrix particles are obtained by sequentially processing the decellularized matrix raw material through decellularization, bio-enzyme treatment, virus inactivation, deep cleaning, drying, and grinding; the decellularized matrix raw material is a mixture of one or more of the following: animal skin, human skin, animal visceral tissue, human visceral tissue, animal perinatal tissue, and human perinatal tissue.

3. The injectable decellularized matrix according to claim 2, characterized in that: The decellularization process involves cutting the decellularization matrix raw material into pieces, adding it to a decellularization solution for treatment, and then washing it. The bio-enzyme treatment involves adding the decellularized matrix raw material, which has undergone decellularization treatment, to a bio-enzyme solution for treatment and washing. The virus inactivation and deep cleaning involve adding a virus inactivating agent solution to the decellularized matrix raw material that has been treated with biological enzymes and then cleaning it. The drying process involves freeze-drying decellularized matrix raw materials that have undergone virus inactivation and deep cleaning. The grinding process involves placing the dried, decellularized matrix material into a grinding machine for grinding.

4. The injectable decellularized matrix according to claim 3, characterized in that: The decellularization solution is a mixture of one or more of the following: ionic surfactant solutions and nonionic surfactant solutions; the biological enzyme solution is a lipase, deoxyribonuclease, trypsin, or pepsin solution; and the virus inactivating agent is a mixture of one or more of the following: peracetic acid, hydrogen peroxide, peroctanoic acid, and ethanol.

5. A method for preparing an injectable decellularized matrix as described in any one of claims 1 to 4, characterized in that: The hydrogel is chemically bonded to the decellularized matrix particles.

6. An application of the injectable decellularized matrix as described in any one of claims 1 to 4, characterized in that: Used to prepare medical materials for the treatment of knee osteoarthritis.

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