An acellular matrix microparticle freeze-dried implant and its preparation method and application

Abstract

The present invention provides a decellularized matrix microparticle freeze-dried implant, a preparation method and an application thereof, belonging to the technical field of soft tissue filling materials. The decellularized matrix microparticle freeze-dried implant is composed of a decellularized matrix, a suspending agent and a freeze-drying protectant, wherein the mass ratio of the decellularized matrix, the suspending agent and the freeze-drying protectant is 0.5-10:0.5-10:0.5-6; the particle size of the cell matrix freeze-dried implant is 10 μm-1000 μm. The decellularized matrix microparticle freeze-dried implant of the present invention retains the natural three-dimensional structure and natural active ingredients, reduces the immune rejection reaction caused by the raw material source after the injection product is clinically used, and is suitable for injection into irregular defects for filling and repair, injection into the deep subcutaneous dermis to the superficial subcutaneous layer to repair moderate to severe wrinkles or folds, and has a good repair effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of soft tissue filling materials, and particularly relates to a decellularized matrix microparticle freeze-dried implant and a preparation method and application thereof. Background Art

[0002] Collagen is a reticular structure formed by the interweaving of connective tissues with elastin and proteoglycans, which generates a certain mechanical strength and is the material basis for supporting the human body curve and presenting a straight posture. Collagen can maintain the elasticity of blood vessel walls, prevent blood vessel rupture and embolism, improve the lubrication of joints, cartilage and ligaments, and relieve symptoms such as joint stiffness, soreness and hydrops. About 70% of the dermis in human skin is composed of collagen. As people age, the content of human collagen will gradually decrease, and the skin will lose elasticity, become thinner and age, and at the same time, conditions such as wrinkles, aging, sagging and skin spots will occur. Currently, through collagen injection, a small amount of collagen is implanted subcutaneously by a non-surgical method, which can stimulate the regeneration of human own fibroblasts and collagen. In a short time, it can accurately eliminate dense fine wrinkles on the face, 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 an anti-aging effect. The supplementary effects of regenerative materials during the injection process mainly include two types: collagen (human-like, porcine-derived) fillers and materials that stimulate collagen regeneration.

[0003] Decellularized matrix is a natural extracellular matrix biomaterial with multiple collagen components, mainly type I collagen, and also contains a certain amount of bioactive components and factors, such as polysaccharide proteins such as FN and LN, and 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 decellularized matrix microparticles obtained by processing the decellularized matrix have high cell affinity and excellent biological properties, can induce tissue regeneration, and are used for the repair of soft tissue defects in various parts. In clinical research, the soft tissue filling and repair material of decellularized matrix microparticles is mixed with an injection agent and applied to the repair of soft tissue defects, such as the repair of skin tissue damage and the filling and repair of soft tissues in the oral and maxillofacial region. It not only plays a supporting role in structural remodeling and volume filling, but also can induce the construction of the structure of the recipient itself. The gradually generated new structure will coordinate with the surrounding normal tissues, thus playing an orthopedic role and restoring the normal appearance.

[0004] The acellular matrix microparticles prepared by the existing methods may not meet the injection-grade requirements in terms of particle size when simply ground into powder, and problems such as aggregation and sedimentation will occur during the reconstitution and dispersion processes, resulting in uneven distribution and difficult injection; the acellular matrix microparticle products prepared by adding a suspending agent have irregular appearance after freeze-drying, poor morphology after sub-packaging, are difficult to be commercialized, and have unstable reconstitution and dispersion properties; for the acellular matrix microspheres prepared by the water-oil emulsification method, the oil-phase reaction system mostly uses organic reagents, and most of them are components with toxic and side effects. Harmful components will be introduced into the interior during the microsphere formation process, and it is difficult to remove the residual solvents, which affects the safety of the final product.

[0005] Therefore, it is urgent to research acellular matrix microparticles with stable product particle size, good freeze-drying formability, easy reconstitution and dispersion, safety and reliability, and meeting the injection requirements. Summary of the Invention

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

[0007] The present invention provides a freeze-dried implant of acellular matrix microparticles, which is characterized in that the freeze-dried implant of acellular matrix microparticles is composed of an acellular matrix, a suspending agent and a lyoprotectant, wherein the mass ratio of the acellular matrix, the suspending agent and the lyoprotectant is 0.5-10:0.5-10:0.5-6; the particle size of the freeze-dried implant of acellular matrix microparticles is 10 μm-1000 μm.

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

[0009] The particle size of the freeze-dried implant of acellular matrix microparticles is 30 μm-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, glyceryl trioleate, sodium alginate;

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

[0013] The acellular matrix is prepared from an acellular matrix membrane, and the acellular matrix membrane is an acellular matrix fresh membrane or an acellular matrix freeze-dried membrane.

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

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

[0016] Furthermore, the acellular basement membrane is any one or more of an acellular small intestinal submucosa fresh membrane or its lyophilized membrane, an acellular dermis fresh membrane or its lyophilized membrane, an acellular adipose tissue fresh membrane or its lyophilized membrane, an acellular bladder fresh membrane or its lyophilized membrane, and an acellular amniotic membrane fresh membrane or its lyophilized membrane that has been disinfected, defatted, digested with trypsin, and acellularized.

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

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

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

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

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

[0022] (4) The water content ≤ 10%;

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

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

[0025] The present invention also provides a method for preparing the above-mentioned acellular matrix microparticle lyophilized implant, and the method includes the following steps: grinding the acellular matrix, mixing it with a suspending agent, a lyoprotectant, and water, and performing high hydrostatic pressure treatment to obtain an acellular matrix slurry; sub-packaging, lyophilizing, and sealing to obtain the product.

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

[0027] Further, the conditions for the high hydrostatic pressure treatment are as follows: treatment is carried out successively at a pressure of 0 bar to 600 bar until the material completes one full cycle at the highest pressure; preferably, the conditions for the high hydrostatic pressure treatment are as follows: treatment is carried out successively at a pressure of 100 bar to 400 bar until the material completes one full cycle at the highest pressure.

[0028] Further, the conditions for the high hydrostatic pressure treatment are as follows: treatment is carried out successively at pressures of 100 bar, 200 bar, 300 bar, and 400 bar until the material completes one full cycle at the highest pressure; alternatively, the conditions for the high hydrostatic pressure treatment are as follows: one cycle is carried out at a pressure of 100 bar.

[0029] Further, the conditions for the freeze-drying are as follows: drying is carried out with temperature rising at -40 to 30 °C until the water content ≤ 15%, more preferably, until the water content ≤ 10%; preferably, the conditions for the freeze-drying are as follows: after pre-freezing at -40 °C for 3 - 5 h, then maintaining at a temperature rising condition of -40 to 0 °C for 3 - 7 h, and then continuing to dry at 25 - 30 °C for 3 - 5 h; more preferably, the conditions for the freeze-drying are as follows: after pre-freezing at -40 °C for 4 h, then maintaining at a temperature rising condition of -40 to 0 °C for 6 h, and then continuing to dry at 25 °C for 4 h.

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

[0031] The present invention also provides the use of the above acellular matrix particle freeze-dried 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 acellular matrix particle freeze-dried implant in the preparation of medical aesthetic products such as the treatment of facial fine lines, correction of facial depressions, filling of tear troughs, facial sculpting, dermal or subcutaneous filling, wound healing, and anti-scarring.

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

[0034] The present invention for the first time uses hydrostatic pressure treatment in combination with a suspending agent and a lyoprotectant to prepare an acellular matrix particle freeze-dried product with regular appearance, easy for long-term storage, and easy to be reconstituted into an injectable homogenate before use.

[0035] The present invention micronizes the acellular matrix material, retaining the looseness and three-dimensional space of the material. Its unique three-dimensional network 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 has developed a micronized acellular matrix material suitable for tissue filling and repair. It is a kind of acellular matrix particle product with a suitable particle size for injection filling, easy to store, high reconstitution and dispersion efficiency before use, and can be evenly mixed with other injectables when necessary. It is suitable for filling and repairing the superficial dermis, deep dermis or subcutaneous tissue, as well as irregularly defective tissues, and can better meet the requirements of tissue repair and regeneration.

[0036] The beneficial effects of the acellular matrix particle freeze-dried implant of the present invention:

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

[0038] 2. When in use, injecting a liquid injectable into the product can disperse the acellular matrix. The operation process is simple, and it can be dispersed into a homogeneous slurry within 1 minute, with high dispersion efficiency.

[0039] 3. The injectable acellular matrix particle freeze-dried implant of the present invention contains bioactive extracellular matrix components. The main components are type I, II, IV, and VI collagens in the acellular matrix, which also include extracellular matrix components such as hyaluronic acid and glycosaminoglycans. At the same time, it retains the natural three-dimensional structure of the acellular matrix and can be used as a scaffold for tissue and cell growth. The active components it contains can induce and regulate cell adhesion, proliferation, migration, and differentiation, and promote tissue repair and regeneration. On the one hand, this injectable acellular matrix particle can be used as a wound repair material, and on the other hand, it can be used as a substitute or filler for subsequent reconstructive tissues such as medical aesthetics.

[0040] 4. In the acellular matrix particle freeze-dried 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 injectable belongs to a soft tissue material and can achieve the most natural effect after injection without induration.

[0041] 5. Injecting the micronized acellular matrix in the acellular matrix particle freeze-dried implant of the present invention into the treatment site, the 3D microstructure plays the role of a cell scaffold, effectively promoting the proliferation of fibroblasts, and then accelerating the synthesis of collagen by fibroblasts, helping to supplement collagen in the filling site and also achieving the purpose of maintaining the form persistently.

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

[0043] 7. The acellular matrix particle freeze-dried implant of the present invention is convenient to store. The pre-filled and sealed 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 a stable particle size, good freeze-drying formability, is easy to redissolve and disperse, and meets the requirements of injectability.

[0045] 9. The heavy metal content of the acellular matrix particle freeze-dried 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 redissolution, the osmotic pressure is 270 - 350 mOsm / kg; the water content ≤ 10%; the bacterial endotoxin content is not more than 5 EU / g; the pH value is 6.8 - 7.6.

[0046] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can also be made.

[0047] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Freeze-drying effect of Comparative Sample 10.

[0049] Figure 2 : Freeze-drying effect of Comparative Sample 11.

[0050] Figure 3 : Freeze-drying effect of Comparative Sample 12.

[0051] Figure 4 : Freeze-drying effect of the sample in Example 2.

[0052] Figure 5 : Freeze-drying effect of the sample in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0053] The raw materials and equipment used in the present invention are all known products and 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 Biopharm Co., Ltd.

[0056] The acellular matrix freeze-dried membrane used in the embodiments of the present invention was obtained by disinfecting, defatting, digesting with pancreatin, acellularizing, and freeze-drying the small intestinal submucosa. The specific preparation method was as follows:

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

[0058] (2) Machine scraping: Use a sausage scraping machine to remove the main muscle layer. Place it in physiological saline and wash it 3 times, 5 minutes each time.

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

[0060] (4) Defatting: Completely immerse the tissue in a defatting solution composed of chloroform and methanol mixed in a volume ratio of 1:1, place it in a fume hood for 12 hours, and change the defatting solution once every 6 hours. After defatting is completed, discard the defatting solution, and then rinse it with deionized water to obtain defatted tissue.

[0061] (5) Pancreatin digestion: Immerse the defatted tissue in a 0.25% trypsin solution, and after treating it at 4°C for 12 hours, repeatedly wash it with deionized water to remove trypsin.

[0062] (6) Further acellularization and detergency with sodium dodecyl sulfate (SDS): 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 it with deionized water to remove SDS to obtain an acellular matrix (SIS) membrane.

[0063] Unfold and flatten the acellular matrix membrane, pre-freeze it at -20°C for 3 hours, and then freeze-dry it in a vacuum freeze dryer to obtain a sponge-like solid acellular matrix membrane freeze-dried membrane with regular shape and smooth surface.

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

[0065] The above acellular matrix membrane freeze-dried membrane can be used as a raw material, or a fresh membrane without freeze-drying can be directly used as a raw material.

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

[0067] Grind the above acellular matrix membrane freeze-dried membrane, use hyaluronic acid as a suspending agent, use one or more of sucrose and trehalose as a freeze-drying protectant, use water for injection as a solvent, and mix according to the ratio shown in Table 1 (the mass ratio of the acellular matrix membrane freeze-dried membrane, hyaluronic acid, sucrose, and trehalose is 5:3:2:1), and perform high hydrostatic pressure treatment to obtain an acellular matrix slurry; fill the acellular matrix slurry into 3 ml vials with an automatic filling machine, 1 ml per vial, semi-cork; freeze-dry, then run the freeze-dryer to semi-cork, collect the samples to the capping machine, and seal to obtain the acellular matrix microparticle freeze-dried implant.

[0068] The conditions of the above high hydrostatic pressure are: circulate once under a pressure of 100 bar.

[0069] The conditions of the above freeze-drying are: pre-freeze at -40 °C for 4 h, then maintain for 6 h under the temperature-rising condition of -40 to 0 °C, and then continue to dry at 25 °C for 4 h.

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

[0071] Grind the above acellular matrix membrane freeze-dried membrane, use hyaluronic acid as a suspending agent, use one or more of sucrose and trehalose as a freeze-drying protectant, use water for injection as a solvent, and mix according to the ratio shown in Table 1 (the mass ratio of the acellular matrix membrane freeze-dried membrane, hyaluronic acid, and sucrose is 2:2:4), and perform high hydrostatic pressure treatment to obtain an acellular matrix slurry; fill the acellular matrix slurry into 3 ml vials with an automatic filling machine, 1 ml per vial, semi-cork; freeze-dry, then run the freeze-dryer to semi-cork, collect the samples to the capping machine, and seal to obtain the acellular matrix microparticle freeze-dried implant.

[0072] The conditions of the above high hydrostatic pressure treatment are: circulate the material under pressures of 0 bar, 100 bar, 200 bar, 300 bar, and 400 bar until the material completes one full cycle under the highest pressure.

[0073] The conditions of the above freeze-drying are: pre-freeze at -40 °C for 4 h, then maintain for 6 h under the temperature-rising condition of -40 to 0 °C, and then continue to dry at 25 °C for 4 h.

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

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

[0076] 1. Experimental samples

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

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

[0079] According to the method of Example 2, the difference is only that the composition and ratio are adjusted correspondingly according to Table 1 to obtain comparative sample 4 of the acellular matrix particle freeze-dried implant.

[0080] 2. Experimental method

[0081] Observe the appearance of the above-mentioned acellular matrix particle freeze-dried implant, disperse it with 1 mL of injection water, and shake it to compare the effects of the suspending agent and the lyoprotectant content in the acellular matrix particle freeze-dried implant on the product formability and the reconstitution and dispersion time.

[0082] 3. Experimental results

[0083] The results are shown in Table 1.

[0084] From the test results of comparative samples 1-3, it can be seen that when the SIS content is relatively high (5%), only sucrose is used as the lyoprotectant and the dosage is higher than 6%, the product cannot be formed regardless of whether high-pressure treatment is carried out; from the test results of comparative sample 4, it can be seen that when the SIS content is reduced to 1%, the product is in an irregular cylindrical shape and the reconstitution time is 3 minutes, with low efficiency.

[0085] In Example 2 of the present invention, hyaluronic acid is selected as the suspending agent and sucrose is selected as the lyoprotectant, and when the SIS content is low and the mass ratio of SIS, hyaluronic acid to sucrose is 2:2:4, the prepared freeze-dried sponge has good formability, the product is in a regular cylindrical shape, the reconstitution and dispersion time can be as fast as 30 s, and an injectable de-micronized cell matrix suspension is formed.

[0086] In Example 1 of the present invention, trehalose is added as the lyoprotectant, which can increase the addition amount of the acellular matrix in the product to 5%, the appearance of the freeze-dried implant is regular, and the reconstitution and dispersion time is acceptable.

[0087] The above experimental results show that the acellular matrix particle freeze-dried implants obtained under the specific composition and ratio of Examples 1-2 of the present invention are easy to form, can be reconstituted and dispersed, and meet the requirements of injectability.

[0088] Table 1 Results of composition, ratio screening and reconstitution and dispersion time

[0089]

[0090]

[0091] Note: % in the table represents the mass percentage of the composition relative to the solvent.

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

[0093] 1. Experimental samples

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

[0095] According to the method of Example 2, the only difference is that the circulating pressure of the high hydrostatic pressure treatment is adjusted correspondingly according to Table 2 to obtain the comparative samples 5-9 of the acellular matrix particle freeze-dried implants.

[0096] 2. Experimental methods

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

[0098] The appearance of the above-mentioned acellular matrix particle freeze-dried implants was observed, and they were dispersed with 1 mL of injection water and shaken to compare the influence of the high hydrostatic pressure treatment conditions on the reconstitution and dispersion effect of the products.

[0099] 3. Experimental results

[0100] The results are shown in Table 2.

[0101] Table 2 Influence of different high-pressure conditions on particle size and reconstitution and dispersion effect of particles

[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 pressures of the high hydrostatic pressure treatment are 0 bar, 100 bar, and 200 bar in sequence, the reconstituted product after freeze-drying is prone to form lumps and needs to be dispersed using a vortex mixer, with low efficiency;

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

[0107] When the pressures are 0 bar, 100 bar, 200 bar, 300 bar, and 400 bar, freeze-dried products with excellent forming characteristics are prepared, with neat and flat appearance. The dispersion efficiency of the reconstituted product is significantly improved, the reconstitution time is only 30 seconds, and the obtained suspension has good uniformity and no agglomeration phenomenon.

[0108] When the pressure continues to increase, and is 0 bar, 100 bar, 200 bar, 300 bar, 400 bar, and 600 bar, the product can be dispersed, and the time used is 2 min.

[0109] When the pressure is 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 micronized acellular matrix prepared has particle aggregation during the reconstitution and dispersion process after lyophilization.

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

[0111] In combination with various lyoprotectants of the present invention, it can be lyophilized and formed after 1 cycle of pressure cycling at 100 bar, with regular shape, high reconstitution and dispersion efficiency, and the retained particle size is 50 - 800 μm.

[0112] The above experimental results show that when the SIS content is 2%, the preferred conditions for high hydrostatic pressure treatment are: cycling the material at 0 bar, 100 bar, 200 bar, 300 bar and 400 bar pressures. The product obtained under this condition has high reconstitution and dispersion efficiency, with a time of 30 s and a uniform suspension. Continuing to increase to 600 bar, the reconstitution and dispersion time is 2 min.

[0113] When the SIS content is relatively high, at 5%, in combination with various lyoprotectants of the present invention, the conditions for high hydrostatic pressure treatment are: cycling at 100 bar pressure for 1 time. The product obtained under this condition has a regular shape and high reconstitution and dispersion efficiency.

[0114] Experimental Example 3. Screening of lyophilization conditions

[0115] 1. Experimental samples

[0116] The acellular matrix particle lyophilized implant prepared in Example 2;

[0117] According to the method of Example 2, the difference is only that the lyophilization conditions are adjusted correspondingly according to Table 3 to obtain the comparative samples 10 - 12 of the acellular matrix particle lyophilized implant.

[0118] 2. Experimental method

[0119] Observe the appearance of the above acellular matrix particle lyophilized implant and measure the water content. Compare the effects of lyophilization conditions on the lyophilization effect and water content of the product.

[0120] 3. Experimental results

[0121] The results are shown in Table 3.

[0122] Table 3. Parameters related to the lyophilization process, as well as the lyophilization effect and water content

[0123]

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

[0125] Experimental Example 4. Sub-chronic toxicity evaluation

[0126] To confirm the safety of the product, sub-chronic systemic toxicity evaluation was carried out according to the method recommended in GB / T 16886.11-2011 "Biological evaluation of medical devices - Part 11: Systemic toxicity test".

[0127] Forty SD rats were selected for the test and randomly divided into two groups: the test group and the control group. The animals in the test group were subcutaneously injected with the test sample (i.e., the sample obtained by reconstituting the acellular matrix particle freeze-dried implant prepared in Example 2 with 1 mL of injection water), and 4 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 test period was 90 days in total. The general status and toxicity manifestations of the animals in each group were observed and recorded. Including the changes in skin, hair, eyes, mucous membranes, and the time, severity, and duration of the changes in respiration, circulation, autonomic and central nervous systems, body movement, and behavior patterns, etc. And blood was collected from the abdominal aorta at the observation end point for the detection of hematological and clinical biochemical indexes, and the main organs were taken for histopathological examination.

[0128] According to the clinical observation and pathological results, it was determined that under the test conditions, no obvious clinical toxicity symptoms were observed in the animals of the test group, and there was no significant difference in the body weight of the animals compared with the animals in the control group during the same period. At the end of the test, there was no significant difference in the hematology, clinical biochemistry, and organ indexes of the animals in the test group compared with the animals in the control group. The gross pathological observation results and histopathological detection results both showed that no abnormal changes caused by the test sample were observed. Under the test conditions, the test sample showed no sub-chronic systemic toxicity.

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

[0130] Experimental Example 5. Performance test

[0131] To better illustrate that the micronized decellularized matrix of the present invention has components and structural characteristics suitable for tissue and cell growth, can support cell migration and growth, support the transport of signaling molecules, nutrients and metabolites, and induce tissue regeneration. Using the sample obtained by reconstituting the freeze-dried implant of the decellularized matrix prepared in Example 1 with 1 mL of injection water as the test sample, an intradermal implantation experiment was carried out.

[0132] Take New Zealand rabbits, male, four weeks old. After shaving and disinfecting the back, 0.05 ml of the homogenate of the test sample of the present invention was injected intradermally into the back of the New Zealand rabbits. At 4 weeks and 13 weeks after implantation, the papules and the surrounding 0.5 cm of skin tissue were removed along the marks. When taking the materials, avoid compressing the papules. Immediately put the removed skin tissue into a 50 ml centrifuge tube containing 10% neutral formalin. After tissue fixation, dehydration, clearing, sectioning, and hematoxylin and eosin staining were carried out. After mounting the slides, imaging was performed under a tissue section pathological scanner, and the obtained tissue staining results are shown in the accompanying drawings.

[0133] The results showed that the decellularized matrix microparticles still existed 13 weeks after implantation, the compatibility between cells, tissues and microparticles was good, the fibrous capsule wall at the material / tissue interface disappeared, the distribution of cells was relatively uniform, slight capillary neovascularization was occasionally seen, and no inflammatory cells were found, proving that SIS can support the migration and growth of tissues and cells, and support the transport of signaling molecules, nutrients and metabolites.

[0134] Experimental Example 6. Performance comparison between the freeze-dried implant of the decellularized matrix microparticles of the present invention and existing products

[0135] a. Improvement in maintenance time

[0136] Table 4 Maintenance time

[0137]

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

[0139] b. Advantage in reconstitution dispersibility:

[0140] Table 5 Reconstitution dispersibility

[0141]

[0142] c. Advantages in activity, regeneration effect and metabolism:

[0143] Table 6 Activity, regeneration effect and metabolism

[0144]

[0145]

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

[0147] In summary, the present invention provides an acellular matrix particle freeze-dried implant and a preparation method thereof. The acellular matrix particle freeze-dried implant of the present invention has natural collagen and various factors unique to acellular matrix materials. 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 acellular matrix particle freeze-dried implant is in the form of a freeze-dried sponge and forms a suspension of micronized acellular matrix after reconstitution and dispersion; detected by a laser particle size analyzer, the reconstituted particle size D is 30-100 μm and the concentration is 5-100 mg / mL.

[0148] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the embodiments. Any other changes, combinations, substitutions, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent substitution methods and are all included in the protection scope of the present invention.

Claims

1. An acellular matrix microparticle freeze-dried implant, characterized in that, The acellular matrix particle freeze-dried implant is composed of an acellular matrix, a suspending agent, and a freeze-drying protectant. The suspending agent is hyaluronic acid, and the freeze-drying protectants are sucrose and trehalose. The mass ratio of the acellular matrix, the suspending agent, and the freeze-drying protectants is 2-5:2-3:3-4; the particle size of the acellular matrix particle freeze-dried implant is 10μm-1000μm; The preparation method of the acellular matrix particle freeze-dried implant includes the following steps: grinding the acellular matrix, mixing it with the suspending agent, the freeze-drying protectant, and water, and performing high hydrostatic pressure treatment to obtain an acellular matrix slurry; aliquoting, freeze-drying, and sealing to obtain the product.

2. The acellular matrix particle freeze-dried implant according to claim 1, wherein the particle size of the acellular matrix particle freeze-dried implant is 30μm-800μm; the acellular matrix is prepared from an acellular matrix membrane, and the acellular matrix membrane is an acellular matrix fresh membrane or an acellular matrix freeze-dried membrane.

3. The acellular matrix particle freeze-dried implant according to claim 2, wherein the acellular matrix membrane is any one or more of an acellular small intestinal submucosa fresh membrane or its freeze-dried membrane, an acellular dermis fresh membrane or its freeze-dried membrane, an acellular adipose tissue fresh membrane or its freeze-dried membrane, an acellular bladder fresh membrane or its freeze-dried membrane, and an acellular amniotic membrane fresh membrane or its freeze-dried membrane that have been disinfected, degreased, digested with trypsin, and acellularized.

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

1.

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

6.

6. A method for preparing the acellular matrix particle freeze-dried implant as described in any one of claims 1 to 5, characterized in that, The method includes the following steps: grinding the acellular matrix, mixing it with the suspending agent, the freeze-drying protectant, and water, and performing high hydrostatic pressure treatment to obtain an acellular matrix slurry; aliquoting, freeze-drying, and sealing to obtain the product.

7. The method according to claim 6 above, characterized in that, The conditions of the high hydrostatic pressure treatment are: sequentially treating at a pressure of 0bar-600bar until the material completes a full cycle at the highest pressure once.

8. The method according to claim 7, wherein The conditions of the high hydrostatic pressure treatment are: sequentially treating at a pressure of 100bar-400bar until the material completes a full cycle at the highest pressure once.

9. The method according to claim 8, wherein The conditions of the high hydrostatic pressure treatment are: sequentially treating at pressures of 100bar, 200bar, 300bar, and 400bar until the material completes a full cycle at the highest pressure once.

10. The method according to claim 6 above, characterized in that The conditions of the freeze-drying are: drying by heating at -40 to 30°C until the water content ≤ 15%.

11. The method according to claim 10 above, characterized in that, The conditions of the freeze-drying are: drying by heating at -40 to 30°C until the water content ≤ 10%.

12. The method according to any one of the preceding claims 10 or 11, characterized in that, The conditions of the freeze-drying are: pre-freezing at -40°C for 3-5h, then maintaining at a heating condition of -40 to 0°C for 3-7h, and then continuing to dry at 25-30°C for 3-5h.

13. The method according to claim 12 above, characterized in that, The lyophilization conditions are as follows: after pre-freezing at -40°C for 4 h, then maintaining for 6 h under the temperature-rising condition from -40°C to 0°C, and then continuing to dry at 25°C for 4 h.

14. Use of the acellular matrix particle lyophilized implant according to any one of claims 1 to 5 in the preparation of a tissue repair material.

15. The application according to claim 14, wherein The tissue repair material is a soft tissue filling material or a scar repair material.

16. Use of the acellular matrix particle lyophilized implant according to any one of claims 1 to 5 in the preparation of a medical aesthetic product.

17. The application according to claim 16, wherein The medical aesthetic product is a product for the treatment of facial fine lines, correction of facial depressions, facial sculpting, dermal or subcutaneous filling, wound healing or anti-scarring.

18. The application according to claim 17, characterized in that, The product for dermal or subcutaneous filling is a product for filling the tear trough.

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