A decellularized matrix aerogel repair material and a preparation method and application thereof

Abstract

The present application belongs to the field of biomedical materials and tissue engineering, and particularly relates to a kind of acellular matrix aerogel repair material and its preparation method and application.Further, the present application relates to the comprehensive preparation method of the material, especially the extremely low immunogenicity acellular process combined with physical, chemical and supercritical fluid technologies, and the unique aerogel forming technology based on supercritical drying.Further, the present application relates to the application of the material, mainly related to soft tissue repair, medical cosmetology, bone / cartilage injury repair field.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering, specifically relating to a decellularized matrix aerogel repair material and its preparation method, as well as its application in soft tissue repair, medical aesthetics, bone / cartilage injury repair and other fields. Background Technology

[0002] The demand for surgical implant materials is growing in the field of tissue damage repair and regenerative medicine, especially in aesthetic shaping (such as rhinoplasty) and bone and cartilage repair. Ideal implant materials should possess good biocompatibility, biodegradability, and appropriate mechanical properties to provide support.

[0003] Currently, commonly used implant materials in clinical practice mainly include autologous tissue, allogeneic / xenogeneic tissue, and synthetic materials. While autologous tissue (such as autologous cartilage and bone) is the "gold standard," its availability is limited, and it can cause secondary damage to the donor site. Allogeneic or xenogeneic tissue carries risks of disease transmission and immune rejection. Although synthetic materials (such as polylactic acid and polycaprolactone) can have their mechanical properties customized, their bioinertness and potential inflammatory reactions caused by degradation products limit their application.

[0004] Decellularized extracellular matrix (dECM) materials offer hope for solving the aforementioned problems. By removing cellular components from tissues, dECM retains the natural components and three-dimensional structure of the extracellular matrix, exhibiting good biocompatibility and regeneration-inducing capabilities. However, existing dECM materials still face two major challenges:

[0005] First, immunogenicity control is incomplete. Traditional decellularization methods (such as simply using SDS or Triton X-100) may not completely remove immunogenic substances such as cell debris, nucleic acids, and lipids, potentially triggering a host immune response, affecting repair efficacy, and posing safety risks.

[0006] Second, limitations in material form and performance. Conventional scaffold materials such as dECM patches have uneven pore structures, loose structures, and insufficient mechanical properties. They often require the use of chemical cross-linking agents to enhance stability, which may introduce toxicity and reduce biocompatibility. dECM hydrogels have problems such as poor mechanical strength, rapid in vivo degradation, and difficulty in pre-shaping. dECM powder particles are not easy to shape and are prone to migration after injection.

[0007] Therefore, there is an urgent need in this field to develop a new type of dECM-based material that must meet the following requirements: (1) having extremely low immunogenicity to ensure implantation safety at high concentrations and intensities; (2) possessing superior three-dimensional porous structure and mechanical properties to support cell ingrowth and tissue regeneration; (3) being able to be precisely sculpted and shaped to meet the needs of surgical procedures; and (4) having controllable degradation properties that can match the rate of tissue repair. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned defects of the prior art and provide a decellularized matrix aerogel repair material with extremely low immunogenicity, high biocompatibility, excellent three-dimensional porous structure and surgical sculptability, wherein the aerogel is prepared from a decellularized matrix with extremely low immunogenicity.

[0009] The present invention also aims to provide a rigorous and reproducible method for preparing decellularized matrix aerogel repair materials. This method, through a series of innovative process combinations, ensures the complete removal of cellular components and immunogenic substances while maximally preserving the natural structure and bioactivity of the extracellular matrix, ultimately forming a structurally stable aerogel using supercritical CO2 technology.

[0010] Furthermore, the preparation method of the decellularized matrix aerogel repair material includes the following steps:

[0011] (1) Preparation of decellularized matrix with extremely low immunogenicity;

[0012] (2) The extremely low immunogenic decellularized matrix described in step (1) is pulverized to prepare decellularized matrix microparticles;

[0013] (3) Prepare the decellularized matrix microparticles described in step (2) into a decellularized matrix pregel;

[0014] (4) Prepare decellularized matrix aerogel from the decellularized matrix pregel described in step (3).

[0015] Preferably, the method for preparing the extremely low immunogenic decellularized matrix in step (1) includes a comprehensive processing procedure: starting from raw material pretreatment, it undergoes repeated freeze-thaw cycles, low / high osmotic treatment, treatment with a series of organic solvents (ethanol, methanol / ether, acetone), peracetic acid sterilization, supercritical CO2 fluid permeation pretreatment, treatment with a series of enzymes (trypsin) and detergents (Triton X-100, SDS, CHAPS, sodium deoxycholate), nuclease treatment, and finally, thorough cleaning. This combined strategy can systematically remove various immunogenic substances and prepare an extremely low immunogenic decellularized matrix.

[0016] More preferably, the method for preparing the extremely low immunogenic decellularized matrix in step (1) includes the following steps:

[0017] 1) Clean and cut the raw materials for pretreatment;

[0018] 2) Repeatedly freeze-thaw the pretreated material;

[0019] 3) Treat with hypotonic and / or hypertonic saline solutions after repeated freeze-thaw cycles;

[0020] 4) After step 3), treat sequentially with ethanol, methanol / ether mixture, acetone, and peracetic acid;

[0021] 5) After step 4), treat with supercritical CO2 fluid percolation;

[0022] 6) After step 5), the product is treated sequentially with trypsin, Triton X-100, sodium dodecyl sulfate (SDS), 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS), sodium deoxycholate (SD), and nuclease;

[0023] 7) After step 6), treat with physiological saline and / or phosphate buffer, followed by purified water and / or sterile water for injection.

[0024] More preferably, each of the treatment methods in steps 3)-4) or 6)-7) can be one or more of immersion, vibration, perfusion, and ultrasound.

[0025] More preferably, the repeated freeze-thaw cycles described in step 2) are performed in two temperature ranges: below -5°C and above 2°C, for a minimum of one cycle.

[0026] More preferably, the treatment with hypotonic and / or hypertonic saline solution in step 3) shall last for at least 10 minutes;

[0027] More preferably, in step 4), the treatment with ethanol is for at least 10 minutes; and / or the treatment with methanol / ether mixture is for at least 20 minutes; and / or the treatment with acetone is for at least 20 minutes; and / or the treatment with peracetic acid is for at least 10 minutes.

[0028] More preferably, the supercritical CO2 fluid permeation in step 5) is permeation with 7.38 MPa supercritical CO2 fluid at 31.1°C for no less than 30 minutes;

[0029] More preferably, in step 6), the treatment with trypsin is for at least 10 minutes; and / or the treatment with Triton X-100 is for at least 30 minutes; and / or the treatment with sodium dodecyl sulfate (SDS) is for at least 30 minutes; and / or the treatment with CHAPS is for at least 30 minutes; and / or the treatment with SD is for at least 30 minutes; and / or the treatment with nuclease is for at least 1 hour.

[0030] More preferably, in step 7), the treatment with physiological saline and / or phosphate buffer for at least 30 minutes; and / or the treatment with purified water and / or sterile water for injection for at least 30 minutes.

[0031] More preferably, the extremely low immunogenic decellularized matrix material in step (1) is derived from one or more organs, tissues or cells of humans or animals.

[0032] According to another aspect of the present invention, the preparation of the decellularized matrix pregel in step (3) adopts a multi-enzyme stepwise enzymatic digestion method: collagenase, neutral protease and pepsin are used sequentially for digestion, and a uniform and stable pregel solution is formed by precisely controlling the pH (first alkalizing to ≥8.5 to permanently inactivate pepsin, and then adjusting back to physiological pH) and ion concentration.

[0033] Preferably, the method for preparing the decellularized matrix pregel in step (3) includes the following steps:

[0034] 1) Use collagenase solution to enzymatically degrade the cell matrix at 20~42℃ for no less than 20 minutes until no particles are visible to the naked eye;

[0035] 2) Use a neutral protease solution to detach the cell matrix by shaking at 20~42℃ for no less than 20 minutes until a colorless or off-white mucus is obtained;

[0036] 3) Use a pepsin solution containing hydrochloric acid and / or acetic acid at pH 1.5-2.5 to lyse the cell matrix by shaking at 20-42°C for no less than 20 minutes, thereby lysing exogenous enzymes and / or other immunogenic sequences;

[0037] 4) Adjust the acidic enzymatic hydrolysate to pH ≥ 8.5 with sodium hydroxide solution to inactivate pepsin;

[0038] 5) Adjust the pH of the alkaline enzymatic hydrolysate to 6.0-8.0 using hydrochloric acid and / or acetic acid;

[0039] 6) Adjust the enzyme hydrolysate to 270-360 mOsmol / kg with physiological saline and / or phosphate buffer;

[0040] 7) Mix well and remove air bubbles. Let stand at 20~37℃ for no less than 15 minutes to obtain decellularized matrix pregel.

[0041] According to another aspect of the present invention, the formation of the decellularized matrix aerogel in step (4) employs supercritical CO2 technology: including mold shaping, water washing and desalting, ethanol gradient dehydration, and finally drying using supercritical CO2 fluid. This method avoids the ice crystal damage and structural collapse that may occur with conventional freeze drying, and can achieve physical strengthening without chemical crosslinking agents, thereby obtaining an aerogel with intact porous structure, enhanced mechanical properties, and better retention of biological activity.

[0042] Preferably, the method for preparing the decellularized matrix aerogel in step (4) includes the following steps:

[0043] 1) Place the decellularized matrix pregel in a mold and let it stand at 20~37℃ for no less than 15 minutes to shape it, thus obtaining a decellularized matrix gel block;

[0044] 2) Soak / dialyze the pregel of decellularized matrix with sterile water for injection for no less than 2 hours, changing the water at least once during the period, in order to remove small molecules such as salts from the gel block;

[0045] 3) Soak the decellularized matrix pregel with a gradient of 50%, 75%, 90%, and 100% ethanol for at least 20 minutes at each concentration to remove water from the gel;

[0046] 4) Permeate the gel with supercritical CO2 fluid at 10-14 MPa and 40-50℃ for no less than 2 hours to remove ethanol. This step also has a sterilization effect.

[0047] 5) Maintain in 5-10 MPa CO2 at 40-120℃ for no less than 2 hours to obtain decellularized matrix aerogel.

[0048] This aerogel has the characteristics of porous network structure, extremely light weight, high strength, and high biocompatibility.

[0049] According to another aspect of the present invention, the method for preparing the decellularized matrix pregel may further use a crosslinking agent to enhance the material properties; and / or may further composite with calcium hydroxyphosphate and / or synthesize polymeric materials to enhance the material properties.

[0050] According to another aspect of the present invention, the present invention also provides a decellularized matrix aerogel repair material, which is prepared by the aforementioned method for preparing decellularized matrix aerogel repair materials.

[0051] According to another aspect of the present invention, the present invention also provides an extremely low immunogenic decellularized matrix, which is prepared by the aforementioned method for preparing an extremely low immunogenic decellularized matrix.

[0052] According to another aspect of the present invention, an aerogel repair material is provided, along with a method for preparing the decellularized matrix aerogel repair material and / or the clinical application of the aforementioned extremely low immunogenic decellularized matrix, particularly in the fields of soft tissue repair, medical aesthetics, and bone / cartilage injury repair where high biocompatibility and mechanical property adjustability are required. Preferably, it is used in the preparation of materials for soft tissue repair, medical aesthetics, and bone / cartilage injury repair. More preferably, the material is an implantable material. Even more preferably, the material is used by injection.

[0053] The present invention has the following beneficial effects:

[0054] 1. Extremely low immunogenicity: A comprehensive decellularization process combining physical, chemical, enzymatic, and supercritical fluid technologies can completely remove key immunogenic substances such as cell debris, nucleic acids, and lipids, laying the foundation for safe implantation. The decellularized matrix can have a DNA content as low as 0.11 ng / mg dry weight, far lower than conventional preparation techniques.

[0055] 2. Superior microstructure and mechanical properties: Aerogels formed based on supercritical CO2 drying technology have high porosity, high specific surface area and good three-dimensional interconnected network structure. Their mechanical strength can be adjusted by pre-gel concentration, mold molding and pressure strength, etc., to meet the needs of different repair scenarios.

[0056] 3. Excellent biocompatibility and degradability: The material is derived from natural ECM and the preparation process is mild, preserving bioactivity to the greatest extent. In vitro and in vivo experiments have demonstrated its good cell compatibility, controllable degradation rate, and good compatibility with tissue repair processes.

[0057] 4. Excellent processability and clinical applicability: The material is in solid block form, which can be easily sculpted with a scalpel or laser to achieve personalized shaping and precise implantation through surgery. It is particularly suitable for fields that require complex shapes and stable support, such as rhinoplasty and bone defect filling. Attached Figure Description

[0058] Figure 1 : Very low immunogenic decellularized dermal matrix.

[0059] Figure 2 : Very low immunogenicity decellularized liver matrix.

[0060] Figure 3 : Routine decellularized liver matrix.

[0061] Figure 4 Decellularized matrix aerogel.

[0062] Figure 5 Micrograph of decellularized matrix aerogel (HE staining).

[0063] Figure 6 : In vitro degradation trend of decellularized matrix aerogel.

[0064] Figure 7 The expression of inflammatory factors induced by implanted materials in the body.

[0065] Figure 8 Before and after images of decellularized matrix aerogel used in nasal augmentation. Detailed Implementation

[0066] The present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0067] Example 1: Preparation of a cell-free matrix of porcine dermis and porcine liver with extremely low immunogenicity

[0068] 1. Preparation of decellularized porcine dermal matrix with extremely low immunogenicity:

[0069] Take fresh pig skin, remove subcutaneous fat and hair under sterile conditions, rinse repeatedly with PBS solution, and cut into 2cm × 3cm tissue blocks.

[0070] (1) Repeated freeze-thaw cycles: The tissue block was placed at -80℃ for 2 hours and then transferred to 4℃ for 2 hours to thaw. This is one cycle, and a total of 2 cycles are performed.

[0071] (2) Osmotic pressure treatment: First, shake with purified water (hypotonic) for 30 minutes, then shake with 1 M NaCl solution (hypertonic) for 30 minutes.

[0072] (3) Series of solvent treatments: sequentially treated with 75% ethanol for 30 minutes; treated with methanol: ether (1:1, v / v) mixed solution for 1 hour; and treated with acetone for 1 hour.

[0073] (4) Disinfection treatment: Shake with 1% peracetic acid solution for 30 minutes.

[0074] (5) Supercritical CO2 treatment: The tissue treated with peracetic acid was transferred to a supercritical extraction device and treated with supercritical CO2 fluid for 1 hour at 31.1℃ and 7.38 MPa.

[0075] (6) Enzyme and detergent series treatments: After supercritical CO2 treatment, the following solutions were sequentially used in a shaking incubator at 37°C: 0.25% trypsin solution for 30 minutes; 1% Triton X-100 solution for 2 hours; 0.5% SDS solution for 2 hours; 1% CHAPS solution for 2 hours; and 0.5% sodium deoxycholate (SD) solution for 2 hours. Each treatment was followed by thorough washing with PBS.

[0076] (7) Nucleic acid digestion: After series of treatments with enzyme and detergent, the enzyme was digested with PBS buffer containing 50 U / mL DNase and 1 U / mL RNase at 37°C for 4 hours.

[0077] (8) Final washing: After nuclease digestion, wash with PBS three times with shaking for 30 minutes each time; finally wash with sterile water for injection twice for 30 minutes each time.

[0078] The resulting decellularized dermal matrix is ​​soft and white. Figure 1 The DNA residue was found to be 0.16 ng / mg dry weight (Table 1).

[0079] 2. Preparation of decellularized matrix from porcine liver with extremely low immunogenicity: Fresh porcine liver was first soaked and washed with PBS. Subsequent processing procedures were basically the same as for porcine dermal fillers.

[0080] Similar to other methods, but because liver tissue is more fragile, cutting it into small pieces is not the optimal option. Due to its complete vascular network, transvascular perfusion can efficiently produce extremely low immunogenic porcine liver decellularized matrix. The decellularization fluid and processing time are consistent; the liver matrix is ​​processed via transvascular perfusion, while the dermis is cut into pieces and then shaken. The resulting liver decellularized matrix is ​​soft and white, and the local detail image shows that the morphology of the intrinsic liver lobules is basically intact. Figure 2 The DNA residue was found to be 0.11 ng / mg dry weight (Table 1).

[0081] Example 2: Preparation of conventional decellularized matrix

[0082] Taking decellularized pig liver matrix as an example. Fresh pig liver was first soaked and washed with PBS. Decellularization was performed using a transvascular perfusion method. Virus inactivation was achieved by perfusion with 1% peracetic acid for 30 minutes, followed by perfusion with 1% Triton X-100 solution for 12 hours, perfusion with 1% SDS solution for 24 hours, perfusion with PBS buffer containing 50 U / mL DNase and 1 U / mL RNase for 8 hours in a 37°C incubator, perfusion with PBS for 1.5 hours, and perfusion with injection water for 1 hour. The resulting decellularized liver matrix was soft and dark white; detailed images showed that the inherent lobular morphology had begun to collapse and be destroyed. Figure 3The DNA residue was found to be 19.78 ng / mg dry weight (Table 1), which meets the industry's basic requirements for decellularized matrix (<50 ng / mg dry weight). However, the DNA residue is 179.82 times that of the extremely low immunogenicity liver decellularized matrix of this invention, which increases the risk in the application of high-concentration implant materials.

[0083]

[0084] Example 3: Preparation of decellularized matrix microparticles

[0085] The freeze-dried porcine dermis and decellularized porcine liver matrix prepared in Example 1 were used separately...

[0086] The particles were pulverized using a cryogenic grinder pre-cooled with liquid nitrogen. The pulverized powder was then passed through a 60-mesh sieve to collect decellularized matrix microparticles with a particle size of less than 250 micrometers, which were then dispensed for use.

[0087] Example 4: Preparation of decellularized matrix pregel and aerogel (using porcine dermis as an example; the operation steps are the same for liver source).

[0088] 1. Pregel preparation:

[0089] (1) Collagen hydrolysis: 4.0g of decellularized dermal matrix microparticles were suspended in 100 mL of PBS containing 0.4% collagenase (type II) and digested at 37°C and 150 rpm for 1 hour until the solution was homogeneous and free of obvious particles.

[0090] (2) Neutral protease digestion: Add 0.2% neutral protease to the final concentration and continue to digest at 37°C with shaking for 1 hour until the solution becomes a homogeneous viscous liquid.

[0091] (3) Pepsin digestion: Adjust the pH of the solution to 2.5 with 1M HCl, add pepsin with a final concentration of 0.2%, and digest at 37°C with shaking for 1 hour.

[0092] (4) Pre-gelation: Under ice bath conditions, slowly adjust the pH of the solution to 8.5 with 1M NaOH and let it stand for 30 minutes. Then, adjust the pH back to 7.0 with 1M HCl. Add an appropriate amount of 10X PBS to adjust the solution to physiological osmotic pressure, and then adjust the matrix concentration to 35 mg / mL according to the overall solution addition in each step. Remove air bubbles from the solution and let it stand at 37°C for 20 minutes to form a homogeneous decellularized matrix pre-gel.

[0093] 2. Aerogel preparation:

[0094] (1) Shaping and washing: Place the pre-gelled material into a polytetrafluoroethylene mold of a predetermined shape, cover it with a cover plate, apply a pressure of 0.5 MPa, and let it stand at 37°C for 2 hours to form the shape.

[0095] (2) Desalting and dehydration: Carefully remove the formed gel block and soak it in sterile water for injection for 4 hours, changing the water twice during the period to fully remove the salt; put the gel block into 50%, 75%, 90%, and 100% ethanol solutions in sequence, soaking for 30 minutes at each concentration to perform gradient dehydration.

[0096] (3) Supercritical Drying and Stabilization: The dehydrated gel block was transferred to a supercritical drying apparatus. It was treated with supercritical CO2 fluid for 2.5 hours at 45℃ and 12MPa to completely remove ethanol. Subsequently, the system temperature was raised to 80℃ and the pressure to 7 MPa, maintained for 2.5 hours to further stabilize the material structure. Finally, CO2 was slowly released to atmospheric pressure to obtain a dermal-derived decellularized matrix aerogel of the predetermined shape. Figure 4 Microscopic observation revealed that it possesses an excellent three-dimensional porous network structure. Figure 5 ).

[0097] Example 5: Characterization of material physical properties

[0098] Quasi-static uniaxial compressive mechanical properties were tested on the dermal and liver-derived aerogels prepared in Example 4 (densities of 30 mg / cm³, 40 mg / cm³, 50 mg / cm³, 60 mg / cm³, and 70 mg / cm³, respectively).

[0099] (1) Sample preparation: The aerogel block was prepared into a cylindrical sample with a diameter of 8 mm and a height of about 5 mm using a biopsy perforator (n=5).

[0100] (2) Test method: A universal testing machine equipped with a 20 mm diameter flat indenter was used to compress the specimen at a constant rate of 0.5 mm / min until the strain reached 60%. Load-displacement data were recorded.

[0101] (3) Data analysis: The data is converted into stress-strain curves. The compressive modulus is obtained by calculating the slope of the linear segment of the stress-strain curve.

[0102] The results are shown in Table 2.

[0103]

[0104] Data shows that dermal-derived aerogels exhibit a higher compressive modulus than liver-derived aerogels at the same density, which is related to the differences in composition and microstructure of the ECM from different tissues. The results indicate that by selecting matrices from different sources and adjusting preparation parameters, materials with different mechanical properties required for tissue repair can be obtained.

[0105] Example 6: Evaluation of in vitro degradation and cell compatibility of materials

[0106] Taking liver matrix material as an example, aerogel material of standard size and weight (2 cm square) was immersed in sterile PBS (pH=7.4, enzyme-free), aseptically sealed, and subjected to in vitro degradation experiments in a shaker at 37℃. Mass loss was calculated by periodic lyophilization and weighing. The results showed that the aerogel experienced slow mass loss (<5%) within 10 weeks, indicating good in vitro stability. Figure 6 The viability of mouse fibroblasts (L929) cultured with dECM material at 37°C, 60 rpm, and 72 hours was assessed using the MTT assay. The extraction medium was 90% MEM + 10% dECM.

[0107] BSA, blank control was cultured in extraction medium, negative control was sterile high-density polyethylene resin.

[0108] The extract was cultured at 37℃, 60 rpm, and for 72 hours. The positive control was cultured in 10% DMSO.

[0109] The results are shown in Table 3.

[0110]

[0111] The relative survival rate was the ratio of the average OD value of the test group to the average OD value of the blank control group. The results indicate that the material is non-cytotoxic and has good biocompatibility.

[0112] Example 7: Evaluation of in vivo degradation performance of materials

[0113] Materials were grouped into 6 groups: (A) liver-derived aerogel; (B) liver-derived micronized matrix; (C) liver-derived pregel; (D) dermal-derived aerogel; (E) dermal-derived micronized matrix; and (F) dermal-derived pregel. In this embodiment, the density of the aerogel and pregel was 35 mg / cm³. 3 The implanted volume is 0.2 cm. 3 The corresponding micronized matrix was prepared as a 35 mg / mL suspension using PBS, with an implantation volume of 200 μL, ensuring a dry weight of 7 mg for each implant. Animal surgery: SD rats were anesthetized and the material was implanted subcutaneously in their backs. The aerogel group underwent implantation of block-shaped material through a small incision; the pre-gel and microparticle groups were implanted via injection through the same incision. Results: Samples were collected periodically and observed using HE staining. Degradation time data are shown in Table 4.

[0114]

[0115] Data shows that the aerogel form can significantly slow down the degradation rate of dECM in vivo, making it more suitable for repair scenarios that require long-term support.

[0116] Example 8: Evaluation of in vivo inflammatory response of materials

[0117] Experimental animals were divided into 5 groups: (A) liver-derived aerogel; (B) liver-derived micronized matrix; (C) liver-derived pregel; (D) blank control group; and (E) PLLA-positive control group. The above materials were implanted into the experimental animals, and samples were collected at 1, 4, and 12 weeks after implantation. RNA was extracted, cDNA was reverse transcribed, and the gene expression levels of IL-1β and TNF-α in each group were detected by RT-qPCR. The results showed that the decellularized matrix material prepared in this invention did not induce high expression of inflammatory factors after subcutaneous injection, demonstrating its extremely low immunogenicity. Figure 7 ).

[0118] Example 9: Application of Materials in Surgical Procedures

[0119] Taking nasal tip reshaping as an example, rats were selected as experimental subjects, and their nasal structures were photographed and recorded before the operation.

[0120] photo( Figure 8 The implant shape was designed based on the rat's nasal condition. During the procedure, a pre-prepared decellularized matrix aerogel block (dermal source, density 50 mg / cm³), such as dECM, was removed from its sterile packaging. ® The decellularized matrix aerogel block was thoroughly infiltrated with sterile saline. On a sterile operating table, using a fine scalpel and sculpting tool, the material was sculpted into the desired graft shape. Through an incision at the tip of the nose, a suitable cavity was created subcutaneously at the tip of the nose. The sculpted aerogel implant was inserted and adjusted to the optimal position. The incision was sutured, disinfected, and an immediate postoperative photograph was taken. Figure 8 ).

[0121] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a decellularized matrix aerogel repair material, characterized by, Comprising the following steps: (1) Preparation of extremely low immunogenicity acellular matrix; (2) The extremely low immunogenicity acellular matrix of step (1) is crushed to prepare acellular matrix microparticles; (3) The acellular matrix microparticles of step (2) are prepared into acellular matrix pre-gel; (4) The acellular matrix pre-gel of step (3) is prepared into acellular matrix aerogel; The preparation method of the acellular matrix pre-gel in step (3) comprises the following steps: 1) Enzymatic de-cellularization of the acellular matrix at 20-42℃ for not less than 20 minutes with collagenase solution until no visible particles are observed; 2) Enzymatic de-cellularization of the acellular matrix at 20-42℃ for not less than 20 minutes with neutral protease solution until no color or white mucous is observed; 3) Enzymatic de-cellularization of the acellular matrix at 20-42℃ for not less than 20 minutes with pepsin solution containing hydrochloric acid and / or acetic acid at pH 1.5-2.5 to lyse exogenous enzymes and / or other immunogenic sequences; 4) Adjusting the pH of the acidic enzymatic solution to not less than 8.5 with sodium hydroxide solution to inactivate pepsin; 5) Adjusting the pH of the alkaline enzymatic solution to 6.0-8.0 with hydrochloric acid and / or acetic acid; 6) Adjusting the osmotic pressure of the enzymatic solution to 270-360 mOsmol / kg with physiological saline and / or phosphate buffer; 7) Mixing and removing air bubbles, and standing at 20-37℃ for not less than 15 minutes to obtain the acellular matrix pre-gel.

2. The method for preparing decellularized matrix aerogel repair material according to claim 1, characterized in that: The preparation method of the extremely low immunogenicity acellular matrix in step (1) comprises the following steps: 1) Washing and cutting the raw material for pretreatment; 2) Repeated freeze-thawing of the pretreated material; 3) Treatment with low-osmotic and / or high-osmotic salt solution after repeated freeze-thawing; 4) Sequential treatment with ethanol, methanol / ether mixture, acetone, and peroxiacetic acid after step 3); 5) Supercritical CO2 fluid infiltration treatment after step 4); 6) Sequential treatment with trypsin, triton X-100, sodium dodecyl sulfate, 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt, sodium deoxycholate, and nuclease after step 5); 7) Treatment with physiological saline and / or phosphate buffer followed by treatment with purified water and / or sterile water for injection after step 6).

3. The method of claim 2, wherein the decellularized matrix aerogel repair material is prepared by: The raw material of the extremely low immunogenicity acellular matrix in step (1) is derived from one or more of organs, tissues, or cells of humans or animals.

4. The method for preparing the decellularized matrix aerogel repair material according to claim 2, characterized in that: Further comprising one or more of the following steps: The repeated freeze-thawing in step 2) is performed at two temperature intervals below -5℃ and above 2℃ for not less than 1 cycle; The treatment with low-osmotic and / or high-osmotic salt solution in step 3) is for not less than 10 minutes; The ethanol treatment in step 4) is for not less than 10 minutes; and / or the methanol / ether mixture treatment is for not less than 20 minutes; and / or the acetone treatment is for not less than 20 minutes; and / or the peroxiacetic acid treatment is for not less than 10 minutes; The supercritical CO2 fluid infiltration in step 5) is with 7.38 MPa supercritical CO2 fluid at 31.1℃ for not less than 30 minutes; The trypsin treatment in step 6) is not less than 10 minutes; and / or the triton X-100 treatment is not less than 30 minutes; and / or the sodium dodecyl sulfate treatment is not less than 30 minutes; and / or the 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt treatment is not less than 30 minutes; and / or the sodium deoxycholate treatment is not less than 30 minutes; and / or the nuclease treatment is not less than 1 hour; The physiological saline and / or phosphate buffer treatment in step 7) is not less than 30 minutes; and / or the purified water and / or sterile water for injection treatment is not less than 30 minutes.

5. The method of claim 1-4, wherein: The preparation method of the acellular matrix aerogel in the step (4) comprises the following steps: 1) The acellular matrix pre-gel is placed in a mold and shaped at 20-37℃ for not less than 15 minutes to obtain an acellular matrix gel block; 2) The acellular matrix pre-gel is soaked / dialyzed with sterile water for not less than 2 hours, and the water is replaced for not less than 1 time to remove small molecules of salt in the gel block; 3) The acellular matrix pre-gel is gradiently soaked with 50%, 75%, 90%, and 100% ethanol for not less than 20 minutes for each concentration to remove water in the gel; 4) The gel is permeated with 10-14 MPa supercritical CO2 fluid at 40-50℃ for not less than 2 hours to remove ethanol in the gel, and this step also has a sterilization effect; 5) The gel is maintained in 5-10 MPa CO2 at 40-120℃ for not less than 2 hours to obtain an acellular matrix aerogel.

6. The method of claim 5, wherein the decellularized matrix aerogel repair material is prepared by: A crosslinking agent can be further used, and / or calcium hydroxyapatite and / or a synthetic polymer material can be further compounded to enhance the material performance.

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