Acellular matrix particle freeze-dried implant as well as preparation method and application thereof

Abstract

The invention provides an acellular matrix particle freeze-dried implant as well as a preparation method and application thereof, and belongs to the technical field of soft tissue filling materials. The acellular matrix particle freeze-drying implant consists of an acellular matrix, a suspending aid and a freeze-drying protective additive, wherein the mass ratio of the acellular matrix to the suspending aid to the freeze-drying protective additive is (0.5-10): (0.5-10): (0.5-6); and the particle size of the cell matrix freeze-drying implant is 10-1000 [mu] m. The acellular matrix particle freeze-dried implant retains a natural three-dimensional structure and natural active components, and reduces immunological rejection caused by raw material sources after clinical use of an injection product; the composition is suitable for being injected to irregular defect positions for filling and repairing, is injected to a subcutaneous corium layer deep layer to a subcutaneous superficial layer to repair moderate to severe wrinkles or wrinkles, and has a good repairing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of soft tissue filling materials, and in particular relates to a freeze-dried acellular matrix particle implant and a preparation method and application thereof. Background Art

[0002] Collagen is a mesh structure formed by the interweaving of connective tissue, elastin and polysaccharide proteins. It has a certain mechanical strength and is the material basis for supporting the curves of the human body and reflecting an upright posture. Collagen can maintain the elasticity of the blood vessel wall, prevent blood vessel rupture and embolism, improve the lubrication of joints, cartilage and ligaments, and relieve symptoms such as joint stiffness, soreness, and water accumulation. About 70% of the dermis in human skin is collagen. As we age, the collagen content in the human body will gradually decrease, and the skin will lose elasticity and become thinner and aged. At the same time, it will cause wrinkles, aging, sagging, and spots. At present, through collagen injection, a small amount of collagen is implanted subcutaneously by non-surgical means to stimulate the regeneration of the body's own fibroblasts and collagen. In a short period of time, dense and fine facial wrinkles can be accurately eliminated, such as wrinkles between the eyebrows, forehead, outer canthus of the eyes, corners of the mouth, cheeks, neck, etc., making the skin shiny and elastic to achieve anti-aging effects. The supplementary role of regenerative materials during the injection process mainly includes collagen (human-like and porcine) fillers and stimulated collagen regeneration materials.

[0003] Acellular matrix is ​​a natural extracellular matrix biomaterial with multiple collagen components, mainly type I collagen, and a certain amount of bioactive ingredients and factors, such as polysaccharide proteins such as FN and LN, as well as factors such as VEGF, TGF-β1, FGF, and EGF. It plays an important role in regulating cell adhesion, migration, proliferation, and differentiation, and can also promote tissue repair and regeneration. The acellular matrix microparticles obtained by acellular matrix treatment have high affinity for cells, excellent biological properties, can induce tissue regeneration, and are used for repairing soft tissue defects in various parts. In clinical studies, the soft tissue filling and repair materials of acellular matrix microparticles are mixed with injections and applied to the repair of soft tissue defects, such as skin tissue damage repair, oral and maxillofacial soft tissue filling and repair, etc., which not only play a supporting role in structural remodeling and volume filling, but also can induce the construction of the patient's own structure. The gradually generated new structure will coordinate with the surrounding normal tissues, thereby playing a role in correction and restoring normal appearance.

[0004] The acellular matrix microparticles prepared by existing methods may not meet the injection grade requirements by simply grinding them into powders. Agglomeration and sedimentation may occur during the re-dissolution and dispersion process, resulting in uneven distribution and difficulty in injection. The acellular matrix microparticle products prepared by adding suspending agents have irregular appearance after freeze-drying, poor morphology after packaging, are difficult to commercialize, and have unstable re-dissolution and dispersion properties. The acellular matrix microspheres prepared by water-oil emulsification have an oil phase reaction system that is mostly organic reagents, and most of them are toxic and side-effect components. Harmful components will be introduced into the microspheres during the microsphere formation process, and the residual solvents are difficult to remove, affecting the safety of the final product.

[0005] Therefore, it is urgent to develop decellularized matrix microparticles that have stable particle size, good freeze-drying formability, easy resolubility and dispersion, safety and reliability, and can meet injection requirements. Summary of the invention

[0006] The purpose of the present invention is to provide a freeze-dried acellular matrix microparticle implant and a preparation method and application thereof.

[0007] The present invention provides a decellular matrix microparticle freeze-dried implant, characterized in that the decellular matrix microparticle freeze-dried implant consists of a decellular matrix, a suspending agent and a lyophilization protectant, wherein the mass ratio of the decellular matrix, the suspending agent and the lyophilization protectant is 0.5-10:0.5-10:0.5-6; and the particle size of the decellular matrix microparticle freeze-dried implant is 10μm-1000μm.

[0008] Furthermore, the mass ratio of the decellularized matrix, the suspending agent and the lyophilization protective agent is 2-8:1-4:2-6;

[0009] The particle size of the acellular matrix microparticle freeze-dried implant is 30 μm to 800 μm;

[0010] The suspending agent is selected from any one or more of hyaluronic acid, carboxymethyl cellulose, hydroxypropyl methyl cellulose, collagen, gelatin, mannitol, alpha cyclodextrin, hydroxyethyl methyl cellulose, glycerol, triolein, and sodium alginate;

[0011] The lyoprotectant is selected from any one or more of sucrose, trehalose, citric acid, lactose, alginic acid, and polyelectrolyte solution;

[0012] The polyelectrolyte in the polyelectrolyte solution is one or more of oxidized cellulose, carboxymethyl chitin, carboxymethyl chitosan, chondroitin sulfate, trehalose, hyaluronic acid, sodium carboxymethyl cellulose, and polyglutamic acid;

[0013] The decellularized matrix is ​​prepared from a decellularized matrix membrane, and the decellularized matrix membrane is a fresh decellularized matrix membrane or a freeze-dried decellularized matrix membrane.

[0014] Furthermore, the particle size of the acellular matrix microparticle freeze-dried implant is 30 μm to 300 μm.

[0015] Furthermore, the mass ratio of the decellularized matrix, the suspending agent and the lyophilization protective agent is 2-6:1-3:1-4, preferably 2-5:2-3:3-4.

[0016] Furthermore, the decellularized basement membrane is any one or more of the following: decellularized small intestinal submucosal fresh film or its freeze-dried film, decellularized dermis fresh film or its freeze-dried film, decellularized adipose tissue fresh film or its freeze-dried film, decellularized bladder fresh film or its freeze-dried film, and decellularized amniotic membrane fresh film or its freeze-dried film, which have been sterilized, defatted, trypsinized, and decellularized.

[0017] Furthermore, the lyoprotectant is sucrose, and the mass ratio of the decellularized matrix, suspending agent and sucrose is 2:2:4; or, the lyoprotectant is sucrose and trehalose, and the mass ratio of the decellularized matrix, suspending agent, sucrose and trehalose is 5:3:2:1.

[0018] Furthermore, the acellular matrix microparticle freeze-dried implant satisfies at least one of the following conditions:

[0019] (1) Heavy metal content is not more than 5 μg / g;

[0020] (2) The residual amount of organic solvents shall not exceed 50 μg / g;

[0021] (3) After reconstitution, the osmotic pressure is 270-350 mOsm / kg;

[0022] (4) Water content ≤ 10%;

[0023] (5) Bacterial endotoxin content is not more than 5EU / g;

[0024] (6) pH value 6.8-7.6.

[0025] The present invention also provides a method for preparing the above-mentioned decellularized matrix microparticle freeze-dried implant, which comprises the following steps: grinding the decellularized matrix, mixing it with a suspending agent, a freeze-drying protective agent and water, and subjecting it to high fluid static pressure treatment to obtain a decellularized matrix slurry; packaging, freeze-drying, and sealing.

[0026] Furthermore, the grinding temperature is -80°C to 15°C, preferably -20°C to 30°C, and the grinding process includes but is not limited to crushing the decellularized matrix by grinding, impacting, shearing, etc. The particle size of the decellularized matrix after grinding is less than or equal to 2 mm, preferably less than or equal to 1 mm.

[0027] Furthermore, the conditions for the high fluid static pressure treatment are: treating at a pressure of 0 bar to 600 bar in sequence until the material has completed one complete cycle at the highest pressure; preferably, the conditions for the high fluid static pressure treatment are: treating at a pressure of 100 bar to 400 bar in sequence until the material has completed one complete cycle at the highest pressure.

[0028] Furthermore, the conditions for the high fluid static pressure treatment are: treating at pressures of 100 bar, 200 bar, 300 bar, and 400 bar in sequence until the material has completed one complete cycle at the highest pressure; or, the conditions for the high fluid static pressure treatment are: circulating once at a pressure of 100 bar.

[0029] Further, the freeze-drying conditions are: drying at a temperature of -40 to 30°C to a moisture content of ≤15%, more preferably, to a moisture content of ≤10%; preferably, the freeze-drying conditions are: pre-freezing at -40°C for 3-5 hours, maintaining at a temperature of -40 to 0°C for 3-7 hours, and then continuing to dry at 25 to 30°C for 3 to 5 hours; more preferably, the freeze-drying conditions are: pre-freezing at -40°C for 4 hours, maintaining at a temperature of -40 to 0°C for 6 hours, and then continuing to dry at 25°C for 4 hours.

[0030] Furthermore, the freeze-drying is carried out under vacuum conditions.

[0031] The present invention also provides the use of the above-mentioned freeze-dried acellular matrix microparticle implant in the preparation of soft tissue filling materials, tissue repair materials and scar repair materials.

[0032] The present invention also provides the use of the above-mentioned freeze-dried acellular matrix microparticle implant in the preparation of medical cosmetic products such as the treatment of facial fine lines, correction of facial depressions, tear trough filling, facial sculpting, dermal or subcutaneous filling, wound healing, and anti-scar products.

[0033] The present invention also provides a soft tissue filling material and / or tissue repair material, which may include the above-mentioned decellularized matrix freeze-dried implant and a reconstitution solvent.

[0034] The present invention adopts hydrostatic pressure treatment in combination with a suspending agent and a freeze-drying protective agent for the first time to prepare a freeze-dried product of acellular matrix microparticles which has a regular appearance, is easy to store for a long time, and is easy to re-dissolve into an injectable homogenate before use.

[0035] The present invention micronizes the decellularized matrix material, retains the bulkiness and three-dimensional space of the material, and its unique three-dimensional reticular structure can attract and recruit human tissue cells to proliferate and regenerate based on it, induce tissue growth, and achieve the clinical effect of repairing human soft tissue defects. The present invention develops a micronized decellularized matrix material suitable for tissue filling and repair, which has a suitable particle size for injection filling, is easy to store, has high efficiency of redissolution and dispersion before use, and can be uniformly mixed with other injections when necessary. It is suitable for filling and repairing superficial dermis tissue, deep dermis tissue or subdermis tissue, as well as irregular defective tissue, and can better meet the requirements of tissue repair and regeneration.

[0036] The beneficial effects of the freeze-dried acellular matrix microparticle implant of the present invention are as follows:

[0037] 1. In the freeze-dried implant of acellular matrix microparticles of the present invention, the injectable acellular matrix microparticles are cylindrical sponge solids with regular shapes.

[0038] 2. When in use, inject the liquid injection into the product to disperse the acellular matrix. The operation process is simple and can be dispersed into a homogenate within 1 minute with high dispersion efficiency.

[0039] 3. The injectable acellular matrix microparticle freeze-dried implant of the present invention contains biologically active extracellular matrix components, the main components of which are collagen types I, II, IV and VI in the acellular matrix, and also includes extracellular matrix components such as hyaluronic acid and aminopolysaccharides, while retaining the natural three-dimensional structure of the acellular matrix, which can be used as a scaffold to support tissue and cell growth. The active ingredients contained in it can induce and regulate cell mucosa, proliferation, migration and differentiation, and promote tissue repair and regeneration. The injectable acellular matrix microparticles can be used as a wound repair material on the one hand, and can be used as a substitute or filler for subsequent reconstruction of tissues such as medical beauty on the other hand.

[0040] 4. In the freeze-dried acellular matrix microparticle implant of the present invention, the particle size and ductility of the micronized acellular matrix ensure that the filler can be easily injected subcutaneously through a 27G fine needle to achieve injection filling. The injection belongs to soft tissue materials and can achieve the most natural effect after injection without nodules.

[0041] 5. The micronized decellularized matrix in the freeze-dried decellularized matrix microparticle implant of the present invention is injected into the treatment site, and the 3D microstructure acts as a cell scaffold, effectively promoting the proliferation of fibroblasts, thereby accelerating the synthesis of collagen by fibroblasts, helping to supplement collagen in the filling site, and also achieving the purpose of maintaining a persistent morphology.

[0042] 6. The collagen, glycoprotein, polysaccharide and active factors contained in the freeze-dried acellular matrix microparticle implant of the present invention serve as biological signal molecules and can effectively promote tissue regeneration and repair; and the ingredients are all required by the human body, can be completely absorbed, have no metabolic burden, and are both safe and effective.

[0043] 7. The freeze-dried implant of the acellular matrix microparticles of the present invention is easy to store, and the pre-filled freeze-dried sponge-like acellular matrix in the injection bottle can be stored in a natural environment not higher than 35° C., rather than being limited to low-temperature storage.

[0044] 8. The product of the present invention has stable particle size, good freeze-drying formability, is easy to re-dissolve and disperse, and meets the requirements of injectability.

[0045] 9. The heavy metal content of the freeze-dried acellular matrix microparticle implant of the present invention is not more than 5 μg / g; the residual amount of each organic solvent does not exceed 50 μg / g; after reconstitution, the osmotic pressure is 270-350 mOsm / kg; the water content is ≤10%; the bacterial endotoxin content is not more than 5 EU / g; and the pH value is 6.8-7.6.

[0046] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0047] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Freeze-drying effect of comparison sample 10.

[0049] Figure 2 : Freeze-drying effect of comparison sample 11.

[0050] Figure 3 : Freeze-drying effect of comparison sample 12.

[0051] Figure 4 : Example 2 sample freeze-drying effect.

[0052] Figure 5 : Example 1 sample freeze-drying effect. DETAILED DESCRIPTION

[0053] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0054] Unless otherwise specified, the operations of the present invention are carried out at room temperature (25±10° C.).

[0055] Hyaluronic acid was purchased from Shandong Freda Pharmaceutical Group Co., Ltd.

[0056] The acellular matrix freeze-dried film used in the embodiment of the present invention is obtained by disinfecting, defatting, digesting with trypsin, decellularizing and freeze-drying the submucosal layer of the small intestine. The specific preparation method is as follows:

[0057] (1) Pretreatment: Take a pair of fresh pig small intestines, wash them with saline, and disinfect them with peracetic acid.

[0058] (2) Machine scraping: Use a scraper to remove the main muscle layer. Wash it in saline for 3 times, 5 minutes each time.

[0059] (3) Manual scraping: Use a scraper to scrape off as much of the remaining muscle layer as possible.

[0060] (4) Degreasing: The tissue was completely immersed in a degreasing solution made by mixing chloroform and methanol in a ratio of 1:1 by volume and placed in a fume hood for 12 hours. The degreasing solution was replaced every 6 hours. After degreasing, the degreasing solution was discarded and the tissue was rinsed with deionized water to obtain the defatted tissue.

[0061] (5) Trypsin digestion: Soak the defatted tissue in a 0.25% trypsin solution at 4°C for 12 h, and then wash repeatedly with deionized water to remove the trypsin.

[0062] (6) Sodium dodecyl sulfate (SDS) for further decellularization and descaling: Prepare a 0.3% SDS solution, immerse the material in the SDS solution, let it stand at room temperature for 4 hours, and then repeatedly wash with deionized water to remove SDS to obtain a decellularized matrix (SIS) membrane.

[0063] The decellularized matrix membrane was spread out and flattened, pre-frozen at -20°C for 3 hours, and freeze-dried in a vacuum freeze dryer to obtain a sponge-like solid decellularized matrix membrane freeze-dried membrane with a regular shape and a smooth surface.

[0064] In addition to the above-mentioned method, the decellularized matrix freeze-dried membrane can also be prepared by other conventional operations known to those skilled in the art.

[0065] The above-mentioned freeze-dried acellular matrix membrane can be used as the raw material, or fresh membrane that has not been freeze-dried can be directly used as the raw material.

[0066] Example 1: Preparation of freeze-dried acellular matrix microparticle implants of the present invention

[0067] The above-mentioned freeze-dried acellular matrix membrane is ground, hyaluronic acid is used as a suspending agent, one or more of sucrose and trehalose are used as freeze-drying protective agents, and water for injection is used as a solvent. The mixture is mixed according to the ratio shown in Table 1 (the mass ratio of freeze-dried acellular matrix membrane, hyaluronic acid, sucrose, and trehalose is 5:3:2:1), and high hydrostatic pressure treatment is performed to obtain a cell-free matrix slurry; the cell-free matrix slurry is filled into a 3 ml specification vial by an automatic filling machine, 1 ml per vial, and half-stoppered; freeze-dried, and then the freeze dryer is run half-stoppered, the sample is collected to a capping machine, and sealed to obtain a cell-free matrix microparticle freeze-dried implant.

[0068] The above-mentioned high fluid static pressure condition is: one cycle at a pressure of 100 bar.

[0069] The freeze-drying conditions are as follows: pre-freeze at -40°C for 4 hours, maintain at a temperature of -40 to 0°C for 6 hours, and then continue drying at 25°C for 4 hours.

[0070] Example 2: Preparation of the freeze-dried acellular matrix microparticle implant of the present invention

[0071] The above-mentioned freeze-dried acellular matrix membrane is ground, hyaluronic acid is used as a suspending agent, one or more of sucrose and trehalose are used as freeze-drying protective agents, and water for injection is used as a solvent. The mixture is mixed according to the ratio shown in Table 1 (the mass ratio of freeze-dried acellular matrix membrane, hyaluronic acid, and sucrose is 2:2:4), and high hydrostatic pressure treatment is performed to obtain a cell-free matrix slurry; the cell-free matrix slurry is filled into a 3 ml specification vial by an automatic filling machine, 1 ml per vial, and half-stoppered; freeze-dried, and then the freeze dryer is run half-stoppered, the sample is collected to a capping machine, and sealed to obtain a cell-free matrix microparticle freeze-dried implant.

[0072] The conditions of the high fluid static pressure treatment are: circulating the material at pressures of 0 bar, 100 bar, 200 bar, 300 bar and 400 bar until the material is fully circulated once at the highest pressure.

[0073] The freeze-drying conditions are as follows: pre-freeze at -40°C for 4 hours, maintain at a temperature of -40 to 0°C for 6 hours, and then continue drying at 25°C for 4 hours.

[0074] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0075] Experimental Example 1: Screening of the composition and content of the freeze-dried acellular matrix microparticle implant of the present invention

[0076] 1. Experimental samples

[0077] The freeze-dried acellular matrix microparticle implants prepared in Example 1 and Example 2;

[0078] According to the method of Example 1, the only difference is that the composition and ratio are adjusted according to Table 1 to obtain the decellularized matrix microparticle freeze-dried implant comparative samples 1-3.

[0079] The method of Example 2 was followed, except that the composition and ratio were adjusted according to Table 1 to obtain a cell-free matrix microparticle freeze-dried implant comparative sample 4.

[0080] 2. Experimental Methods

[0081] The appearance of the above-mentioned acellular matrix microparticle freeze-dried implant was observed, and it was dispersed with 1 mL of injection water and shaken to compare the effects of the content of the suspending agent and the lyoprotectant in the acellular matrix microparticle freeze-dried implant on the product formability and the re-dissolution and dispersion time.

[0082] 3. Experimental results

[0083] The results are shown in Table 1.

[0084] It can be seen from the test results of comparative samples 1-3 that when the SIS content is relatively high (5%), only sucrose is used as a freeze-drying protective agent, and the amount used is higher than 6%. Regardless of whether high-pressure treatment is performed or not, the product cannot be formed. It can be seen from the test results of comparative sample 4 that when the SIS content is reduced to 1%, the product is an irregular cylindrical shape and is re-dissolved in 3 minutes, which is inefficient.

[0085] In Example 2 of the present invention, hyaluronic acid is selected as a suspending agent, sucrose is a lyophilization protective agent, and the SIS content is low. When the mass ratio of SIS, hyaluronic acid and sucrose is 2:2:4, the prepared freeze-dried sponge has good formability, the product is a regular cylindrical shape, and the re-dissolution and dispersion time can be as fast as 30s, forming an injectable demicronized cell matrix suspension.

[0086] In Example 1 of the present invention, trehalose is added as a freeze-drying protective agent, so that the amount of the decellularized matrix added to the product can be increased to 5%, the freeze-dried implant has a regular appearance, and the re-dissolution and dispersion time is acceptable.

[0087] The above experimental results show that the freeze-dried acellular matrix microparticle implants obtained under the specific composition and proportion of Examples 1-2 of the present invention are easy to shape, can be re-dissolved and dispersed, and can meet the requirements of being injectable.

[0088] Table 1 Results of composition, ratio screening and re-dissolution and dispersion time

[0089]

[0090]

[0091] Note: The % in the table indicates the mass percentage of the component relative to the solvent.

[0092] Experimental Example 2: Screening of high hydrostatic pressure treatment conditions

[0093] 1. Experimental samples

[0094] The freeze-dried acellular matrix microparticle implants prepared in Example 1 and Example 2;

[0095] The method of Example 2 was followed, except that the cycle pressure of the high hydrostatic pressure treatment was adjusted accordingly according to Table 2, to obtain comparative samples 5-9 of the lyophilized implant of decellularized matrix microparticles.

[0096] 2. Experimental Methods

[0097] The particle size of the freeze-dried acellular matrix microparticle implants obtained in each group was tested by a laser particle size analyzer.

[0098] The appearance of the above-mentioned acellular matrix microparticle freeze-dried implant was observed, and it was dispersed and shaken with 1 mL of water for injection to compare the effect of high hydrostatic pressure treatment conditions on the re-dissolution and dispersion effect of the product.

[0099] 3. Experimental results

[0100] The results are shown in Table 2.

[0101] Table 2 Effect of different high pressure conditions on particle size and product re-dissolution and dispersion effect

[0102]

[0103]

[0104] (1) Comparing the test results of Example 2 and Comparative Samples 5-9, it can be seen that when the SIS content is 2%:

[0105] When the pressure of the high hydrostatic pressure treatment is 0 bar, 100 bar and 200 bar, the product is prone to form lumps after reconstitution after freeze-drying, and a vortexer is required for dispersion, which is inefficient;

[0106] When the pressure increases to 300 bar, then to 0 bar, 100 bar, 200 bar, and 300 bar, the freeze-dried product will disperse, and the dispersion time is 2 min.

[0107] When the pressure was 0bar, 100bar, 200bar, 300bar and 400bar, freeze-dried products with excellent molding characteristics were prepared. The appearance was neat and flat, the dispersion efficiency of the product after reconstitution was significantly improved, the reconstitution time was only 30 seconds, and the obtained suspension had good uniformity and no agglomeration.

[0108] As the pressure continued to increase to 0 bar, 100 bar, 200 bar, 300 bar, 400 bar and 600 bar, the product could be dispersed within 2 minutes.

[0109] When the pressure was 100 bar, 200 bar, 300 bar, 400 bar, 600 bar and 800 bar, or 0 bar, 100 bar, 200 bar, 300 bar and 400 bar, and maintained at 600 bar for 2 cycles, the particles of the prepared micronized decellularized matrix agglomerated during the re-dispersion and dispersion process after freeze-drying.

[0110] (2) From the test results of the samples in Example 1, it can be seen that when the SIS content is high, 5%:

[0111] In combination with the various freeze-drying protective agents of the present invention, freeze-drying and forming can be achieved after one cycle at a pressure of 100 bar, with regular shape, high re-dissolution and dispersion efficiency, and the particle size of the retained particles is 50 to 800 μm.

[0112] The above experimental results show that when the SIS content is 2%, the optimal conditions for high hydrostatic pressure treatment are: circulating the material at 0 bar, 100 bar, 200 bar, 300 bar and 400 bar pressures. The product obtained under this condition has high re-dissolution and dispersion efficiency, the time is 30 seconds, and the suspension is uniform. Continue to increase to 600 bar, the re-dissolution and dispersion time is 2 minutes.

[0113] When the SIS content is high, 5%, and multiple freeze-drying protective agents of the present invention are used, the high hydrostatic pressure treatment condition is: one cycle at 100 bar pressure. The product obtained under this condition has a regular shape and high re-dispersion and dispersion efficiency.

[0114] Experimental Example 3: Screening of freeze-drying conditions

[0115] 1. Experimental samples

[0116] The freeze-dried acellular matrix microparticle implant prepared in Example 2;

[0117] The method of Example 2 was followed, except that the freeze-drying conditions were adjusted accordingly according to Table 3, to obtain freeze-dried acellular matrix microparticle implant comparative samples 10-12.

[0118] 2. Experimental Methods

[0119] The appearance of the above-mentioned freeze-dried implant of acellular matrix microparticles was observed, and the water content was tested, and the effects of freeze-drying conditions on the freeze-drying effect and water content of the product were compared.

[0120] 3. Experimental results

[0121] The results are shown in Table 3.

[0122] Table 3 Freeze-drying process related parameters and freeze-drying effect and water content

[0123]

[0124] The experimental results show that the acellular matrix microparticle freeze-dried implant product prepared under the freeze-drying conditions described in Example 2 of the present invention (i.e., pre-freezing at -40°C for 4 hours, maintaining at a temperature of -40 to 0°C for 6 hours, and then continuing to dry at 25°C for 4 hours) has good formability, a regular and smooth appearance, and a low water content of 10%.

[0125] Experimental Example 4: Subchronic toxicity evaluation

[0126] To confirm the safety of the product, a subchronic systemic toxicity evaluation is conducted according to the method recommended in GB / T16886.11-2011 "Biological Evaluation of Medical Devices Part 11: Systemic Toxicity Test".

[0127] 40 SD rats were selected for the experiment and randomly divided into two groups: an experimental group and a control group. The animals in the experimental group were subcutaneously injected with the test sample (i.e., the sample obtained by reconstituted the freeze-dried implant of the acellular matrix microparticles prepared in Example 2 with 1 mL of injection water). Four implantation sites were set for each animal, and 0.5 ml of the sample was subcutaneously injected at each implantation site; the control group was injected with 0.9% sodium chloride injection. The experimental period was 90 days in total. The general state and toxicity manifestations of the animals in each group were observed and recorded. Including changes in the skin, fur, eyes, mucous membranes, and the time, severity and duration of changes in respiration, circulation, autonomic and central nervous systems, body movements and behavioral patterns, etc., and blood was collected from the abdominal aorta at the end of the observation for the detection of hematological and clinical biochemical indicators, and the main organs were taken for histopathological examination.

[0128] According to clinical observation and pathological results, it was determined that under the experimental conditions, no obvious clinical toxicity symptoms were observed in the test group animals, and the weight of the animals was not significantly different from that of the control group animals during the same period. At the end of the experiment, there were no significant differences in the hematology, clinical biochemistry and organ indexes of the test group animals compared with those of the control group animals. Both the gross pathological observation results and the histopathological test results showed that no abnormal changes caused by the test samples were observed. Under the experimental conditions, no subchronic systemic toxicity was observed in the test samples.

[0129] The above experimental results show that the freeze-dried acellular matrix microparticle implant of the present invention does not cause subchronic systemic toxicity and has high safety.

[0130] Experimental Example 5: Performance Test

[0131] In order to better illustrate that the micronized decellularized matrix of the present invention has components and structural characteristics that are compatible with tissue and cell growth, can support cell migration and growth, support the transportation of signal molecules, nutrients and metabolites, and induce tissue regeneration, the sample obtained by re-dissolving the decellularized matrix microparticle freeze-dried implant prepared in Example 1 with 1 mL of injection water was used as the test sample, and an intradermal implantation experiment was carried out.

[0132] Take a New Zealand rabbit, male, four weeks old, prepare the back skin and disinfect it, then inject 0.05 ml of the test sample homogenate into the back skin of the New Zealand rabbit. After 4 weeks and 13 weeks of implantation, remove the skin papule and the surrounding 0.5 cm skin tissue along the mark. Avoid compression of the skin papule when taking the sample, and immediately put the removed skin tissue into a 50 ml centrifuge tube filled with 10% neutral formalin. After the tissue is fixed, dehydrate, make it transparent, slice it, and stain it with hematoxylin and eosin. After sealing, image it under a tissue section pathology scanner, and the obtained tissue staining results are shown in the attached figure.

[0133] The results showed that the decellularized matrix particles still existed 13 weeks after implantation, the compatibility between cells, tissues and particles was good, the fibrous capsule wall at the material / tissue interface disappeared, the cells were distributed more evenly, slight capillary neoplasia was occasionally observed, and no inflammatory cells were observed, proving that SIS can support the migration and growth of tissues and cells, and support the transport of signal molecules, nutrients and metabolites.

[0134] Experimental Example 6: Performance comparison between the freeze-dried acellular matrix microparticle implant of the present invention and existing products

[0135] a.Maintaining time improvement

[0136] Table 4 Maintenance time

[0137]

[0138] The present invention adopts micronized SIS, which not only retains the microstructure and components of SIS itself, but also improves the stability after injection.

[0139] b. Redissolution and dispersibility advantages:

[0140] Table 5 Redispersion and dispersibility

[0141]

[0142] c. Activity, regeneration and metabolic advantages:

[0143] Table 6 Activity, regeneration and metabolism

[0144]

[0145]

[0146] The above results show that compared with existing products, the freeze-dried acellular matrix microparticle implant of the present invention has significant advantages.

[0147] In summary, the present invention provides a decellular matrix microparticle freeze-dried implant and a preparation method thereof. The decellular matrix microparticle freeze-dried implant of the present invention has natural collagen and various factors unique to the decellular matrix material. After being measured by a factor content detection kit, it contains 500 mg / g of type III collagen, 1266 pg / g of bFGF, 513 pg / g of EGF, 1481 pg / g of VEGF, and 28 pg / g of KGF; the decellular matrix microparticle freeze-dried implant is in the form of a freeze-dried sponge, and after being re-dissolved and dispersed, it is a suspension of micronized decellular matrix; after being detected by a laser particle size analyzer, the particle size D of the re-dissolved microparticles is 30-100 μm, and the concentration is 5-100 mg / mL.

[0148] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited by the embodiments. Any other changes, combinations, substitutions and simplifications made without departing from the spirit and principles of the present invention shall be considered as equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A freeze-dried acellular matrix microparticle implant, characterized in that: The decellular matrix microparticle freeze-dried implant consists of a decellular matrix, a suspending agent and a freeze-dried protective agent, wherein the mass ratio of the decellular matrix, the suspending agent and the freeze-dried protective agent is 0.5-10:0.5-10:0.5-6; the particle size of the decellular matrix microparticle freeze-dried implant is 10μm-1000μm.

2. The freeze-dried acellular matrix microparticle implant according to claim 1, characterized in that: The mass ratio of the decellularized matrix, the suspending agent and the lyophilization protective agent is 2-8:1-4:2-6; The particle size of the freeze-dried acellular matrix microparticle implant is 30 μm to 800 μm; The suspending agent is selected from any one or more of hyaluronic acid, carboxymethyl cellulose, hydroxypropyl methyl cellulose, collagen, gelatin, mannitol, alpha cyclodextrin, hydroxyethyl methyl cellulose, glycerol, triolein, and sodium alginate; The lyoprotectant is selected from any one or more of sucrose, trehalose, citric acid, lactose, alginic acid, and polyelectrolyte solution; The polyelectrolyte in the polyelectrolyte solution is one or more of oxidized cellulose, carboxymethyl chitin, carboxymethyl chitosan, chondroitin sulfate, trehalose, hyaluronic acid, sodium carboxymethyl cellulose, and polyglutamic acid; The decellularized matrix is ​​prepared from a decellularized matrix membrane, and the decellularized matrix membrane is a fresh decellularized matrix membrane or a freeze-dried decellularized matrix membrane.

3. The freeze-dried acellular matrix microparticle implant according to claim 2, characterized in that: The mass ratio of the decellularized matrix, the suspending agent and the lyophilization protective agent is 2-6:1-3:1-4, preferably 2-5:2-3:3-4; The decellularized basement membrane is any one or more of the following: decellularized small intestinal submucosal fresh membrane or its freeze-dried membrane, decellularized dermis fresh membrane or its freeze-dried membrane, decellularized adipose tissue fresh membrane or its freeze-dried membrane, decellularized bladder fresh membrane or its freeze-dried membrane, and decellularized amnion fresh membrane or its freeze-dried membrane, which have been subjected to disinfection, defatting, pancreatic enzyme digestion, and decellularization treatments.

4. The freeze-dried acellular matrix microparticle implant according to claim 2, characterized in that: The lyoprotectant is sucrose, and the mass ratio of the decellularized matrix, suspending agent and sucrose is 2:2:4; or, the lyoprotectant is sucrose and trehalose, and the mass ratio of the decellularized matrix, suspending agent, sucrose and trehalose is 5:3:2:

1.

5. The freeze-dried acellular matrix microparticle implant according to any one of claims 1 to 4, characterized in that: The acellular matrix microparticle freeze-dried implant meets at least one of the following conditions: (1) Heavy metal content is not more than 5 μg / g; (2) The residual amount of organic solvents shall not exceed 50 μg / g; (3) After reconstitution, the osmotic pressure is 270-350 mOsm / kg; (4) Water content ≤ 10%; (5) Bacterial endotoxin content is not more than 5EU / g; (6) pH value 6.8-7.

6.

6. A method for preparing the freeze-dried acellular matrix microparticle implant according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: grinding the decellularized matrix, mixing it with a suspending agent, a freeze-drying protective agent and water, and subjecting it to high fluid static pressure treatment to obtain a decellularized matrix slurry; and packaging, freeze-drying and sealing.

7. The method according to claim 6, characterized in that The conditions for the high fluid static pressure treatment are: treating at pressures of 0 bar to 600 bar in sequence until the material has completed one complete cycle at the highest pressure; preferably, the conditions for the high fluid static pressure treatment are: treating at pressures of 100 bar to 400 bar in sequence until the material has completed one complete cycle at the highest pressure; more preferably, the conditions for the high fluid static pressure treatment are: treating at pressures of 100 bar, 200 bar, 300 bar, and 400 bar in sequence until the material has completed one complete cycle at the highest pressure; or, the conditions for the high fluid static pressure treatment are: circulating once at a pressure of 100 bar.

8. The method according to claim 6, characterized in that The freeze-drying conditions are: drying at a temperature of -40 to 30°C to a water content of ≤15%, more preferably, to a water content of ≤10%; preferably, the freeze-drying conditions are: pre-freezing at -40°C for 3-5 hours, maintaining at a temperature of -40 to 0°C for 3-7 hours, and then continuing to dry at 25 to 30°C for 3 to 5 hours; more preferably, the freeze-drying conditions are: pre-freezing at -40°C for 4 hours, maintaining at a temperature of -40 to 0°C for 6 hours, and then continuing to dry at 25°C for 4 hours.

9. Use of the freeze-dried acellular matrix microparticle implant according to any one of claims 1 to 5 in the preparation of soft tissue filling materials, tissue repair materials, and scar repair materials.

10. Use of the freeze-dried acellular matrix microparticle implant according to any one of claims 1 to 5 in the preparation of medical cosmetic products such as treatment of facial fine lines, correction of facial depressions, tear trough filling, facial sculpting, dermal or subcutaneous filling, wound healing, and anti-scar products.

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