Disclosure of Invention
In order to solve the technical problems, the invention provides the acellular matrix particle filling agent which has a natural biological structure, contains various proteins such as collagen, fibronectin, elastin and proteoglycan, can provide good microenvironment for cells, regulate cell behaviors, has good filling effect and long filling maintenance time, has proper particle size, and is convenient for clinical use and wide in application range.
The invention discloses a acellular matrix particle filler, which comprises the following components in percentage by mass:
acellular matrix microparticles: 2.50% -60.00%;
mixing gel phases: 15.00% -75.00%;
solvent: 7.50% -75.00%;
the mixed gel phase comprises a crosslinked gel phase and a non-crosslinked gel phase, and the mass ratio of the crosslinked gel phase to the non-crosslinked gel phase is 0.1:1-1:0.1.
preferably, the decellularized matrix particles have a particle size of 30-200 μm.
Preferably, the cross-linked gel phase is obtained by cross-linking the gel phase with a cross-linking agent solution according to mass fraction, wherein the gel phase is at least one selected from 0.2-5% of sodium hyaluronate, 0.2-5% of sodium polyglutamate, 0.1-10% of sodium carboxymethyl cellulose, 0.1-10% of hydroxypropyl methylcellulose sodium, 0.1-10% of sodium alginate and 0.1-10% of agarose, and the concentration of the solution of the gel phase is 5-17%;
the cross-linking agent solution is selected from one or two mixed solutions of glutaraldehyde 0.2-8%, EDC 0.1-1.0%, BDDE 0.1-1.3%, PEGDE 0.1-3% and genipin 0.1-0.8%, and the mass ratio of the mixed solutions is 1:0.1-3:1, a step of;
the mass ratio of the gel phase to the cross-linking agent solution is 1:1-1:12.
preferably, the non-crosslinked gel phase is selected from at least one of 0.2-5% of sodium hyaluronate, 0.2-5% of sodium polyglutamate, 0.1-10% of sodium carboxymethyl cellulose, 0.1-10% of hydroxypropyl methylcellulose sodium, 0.1-10% of sodium alginate and 0.1-10% of agarose, and the solution concentration of the non-crosslinked gel phase is 0.3-13%.
Preferably, the solvent is one selected from glycerol, physiological saline and water for injection.
The invention also discloses a preparation method of the acellular matrix particle filler, which sequentially comprises the following steps:
(1) Taking soft tissues, removing hair and subcutaneous tissues, mixing the soft tissues with feed liquid, performing vibration degreasing, and cleaning after vibration degreasing to obtain degreased soft tissues;
further, the mass ratio of the soft tissue to the feed liquid is 1:1-1:20, oscillating degreasing conditions are as follows: the oscillation frequency is 45-160r/min, the oscillation time is 2-16h, and the oscillation times are 1-3 times;
(2) Freezing the defatted soft tissue at-15 to-80deg.C for 6-36 hr, taking out, sealing, and treating with 10-40kGy radiation to obtain irradiated soft tissue;
(3) Alternately putting the irradiated soft tissue into a hypertonic solution and a hypotonic solution for oscillation, and cleaning after oscillation to obtain decellularized soft tissue;
further, the mass ratio of the soft tissue to the hypertonic solution is 1:2-1:20, the mass ratio of the soft tissue to the hypotonic solution is 1:2-1:20, a step of; alternating times are 1-5 times, the oscillation frequency is 45-160r/min, and the oscillation time is 3-20min;
(4) Decellularized soft tissue was mixed with a crosslinker solution at 1:1-1:12, oscillating and crosslinking with the mass ratio, the oscillating frequency being 45-100r/min and the oscillating time being 0.5-24h, so as to obtain crosslinked soft tissues;
(5) Deep low-temperature freezing and crushing are carried out on the crosslinked soft tissue, and screening is carried out, so that acellular matrix particles are obtained;
(6) Crosslinking the gel phase and the crosslinking agent solution according to the mass ratio for 2-24h, and cleaning to remove the redundant crosslinking agent solution to obtain a crosslinked gel phase; the crosslinked gel phase and the non-crosslinked gel phase were combined at 0.1:1-1: mixing at a mass ratio of 0.1 to obtain a mixed gel phase;
(7) Adding a solvent into the acellular matrix particles to obtain acellular matrix particle suspension fluid with the mass percentage of 10-70%, and mixing the acellular matrix particle suspension fluid with mixed gel according to the mass ratio of 1:0.2-3, and homogenizing to obtain the acellular matrix particle filler.
Preferably, in step (1), the soft tissue is selected from one of mammalian skin, small intestine, diaphragm, meninges, pericardium and fascia, and fish skin.
Preferably, in the step (1), the feed liquid is at least one selected from alcohol, isopropanol, acetone, n-propanol, petroleum ether and chloroform.
Preferably, in the step (3), the hypertonic solution comprises 0.29-18.5% of sodium chloride solution by mass percent, and the hypotonic solution is selected from one of water and buffer solution with pH value of 5.5-7.2.
Preferably, in the step (5), the process conditions of cryogenic freeze-crushing are as follows: freezing at-20deg.C to-78deg.C for 4-10 hr, transferring into liquid nitrogen, pre-cooling for 1-4 hr, and pulverizing; during crushing, liquid nitrogen is added to control the temperature, the rotating speed is 3000-30000r/min, each time is 5-10s, and the crushing time is 10-55 s.
The beneficial effects are that:
(1) The acellular matrix particle filler provided by the invention comprises acellular matrix particles, a crosslinked gel phase and a non-crosslinked gel phase, and provides excellent lubricity and injectability of the filler through a multiphase composite system, so that the acellular matrix particle filler can be uniformly and continuously injected, is convenient for operation, and is suitable for injection needles with different specifications. The multiphase system endows the filler with obvious instant effect and medium-long-term filling effect; the crosslinked gel phase and the uncrosslinked gel phase serve as carriers of the microparticles, so that on one hand, the function of instant volume support is achieved, and on the other hand, the crosslinked gel phase has a tissue filling effect in the middle of injection; the acellular matrix particles can cause moderate immune response of organisms and promote collagen regeneration, and on the other hand, the acellular matrix particles retain natural compact microstructures and crude fiber forms of biological materials, so that the acellular matrix particles are more excellent in degradability and filling effect.
(2) According to the invention, no strong alkali solution is used in the preparation process of the acellular matrix particles, and the mixed solution is used for degreasing, mild acellular and virus inactivation treatment, so that the damage of the preparation process to the natural tissue structure is effectively avoided, the acellular matrix particles have a natural compact microporous structure and a crude fiber form of biological materials, contain various proteins such as collagen, fibronectin, elastin and proteoglycan, can provide a good microenvironment for cells, regulate cell behaviors, and have excellent degradation resistance.
(3) Before the cell removing process, the invention firstly carries out freezing irradiation, protects the natural structure of biological materials, can kill viruses and achieves the aim of virus inactivation; meanwhile, the cell wall and the nucleic acid structure in the tissue can be destroyed, the treatment intensity of the subsequent decellularization process is reduced, and the efficiency is improved.
(4) The preparation method of the acellular matrix particle filler has the advantages of simple preparation process operation, suitability for large-scale production, terminal sterilization product, no microbial load and better safety.
Detailed Description
In order to more fully understand the technical content of the present invention, the technical solution of the present invention will be further described and illustrated with reference to specific embodiments.
All percentages, fractions and ratios are calculated on the total mass of the composition of the invention, unless otherwise indicated. All of the mass of the ingredients listed, unless otherwise indicated, are given to the active substance content and therefore they do not include solvents or by-products that may be included in commercially available materials. The term "mass percent" herein may be represented by the symbol "%".
All formulations and tests herein take place in an environment of 25 ℃, unless otherwise indicated.
The terms "comprising," "including," "containing," "having," or other variations thereof herein are intended to cover a non-closed inclusion, without distinguishing between them. The term "comprising" means that other steps and ingredients may be added that do not affect the end result. The term "comprising" also includes the terms "consisting of …" and "consisting essentially of …". The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as additional or optional ingredients, components, steps, or limitations of any of the embodiments described herein. The terms "efficacy," "performance," "effect," "efficacy" are not differentiated herein.
The preparation method of the acellular matrix particle filler comprises the following steps:
1. pretreatment: taking fresh skin, pericardium and other soft tissues of animals, and removing hair, subcutaneous tissue and the like. The soft tissue is selected from skin, small intestine, diaphragm, meninges, pericardium and fascia of mammal such as pig, cattle, sheep, donkey, horse, ostrich, etc., and skin of fish.
2. Degreasing: mixing one or more solutions of alcohol, isopropanol, acetone, n-propanol, petroleum ether and chloroform, wherein the mixing ratio is 1:0-3:5, according to the mass ratio of the soft tissue to the feed liquid (the mass ratio is as follows) of 1:1-1: and (3) performing oscillation degreasing for 1-3 times at a speed of 45-160r/min for 2-16h each time.
3. Cleaning: the mass ratio of the soft tissue to the water is 1:5-1:10 cleaning the residual degreasing mixed solution for 2-5 times, wherein each time lasts for 5-20min, and the oscillation frequency is 45-160r/min.
4. Freezing and irradiating: placing the cleaned soft tissue at-15deg.C to-80deg.C, freezing for 6-36 hr, transferring into a heat preservation box, adding ice box or dry ice or polymer cold storage agent, sealing, and performing 10-40kGy irradiation treatment.
Before the cell removing process, freezing irradiation is performed first, so that the natural structure of the biological material is protected, viruses can be killed, the purpose of virus inactivation is achieved, cell walls and nucleic acid structures in tissues can be destroyed, the treatment intensity of the subsequent cell removing process is reduced, and the efficiency is improved.
5. Decellularization: the hypertonic solution is 0.29-18.5% sodium chloride solution, the hypotonic solution is water or buffer solution with pH of 5.5-7.2, wherein 0.01-0.5% sodium hydroxide or 0.1-2% triton 100 or 0.01-1.0% EDTA can be added into the hypertonic solution. According to the mass ratio of the soft tissue to the solution of 1:2-1:20, carrying out alternating oscillation circulation between the hypertonic solution and the hypotonic solution for 1-5 times, and 3-20 min/time, wherein the frequency is 45-160r/min.
The concentration of the high-low permeability solution in the decellularization process is low, no strong alkali solution is used, and the damage of the preparation process to the natural tissue structure is effectively avoided.
6. Cleaning: the mass ratio of the soft tissue to the water is 1:5-1:20 is washed for three times, each time for 10-30min, and the frequency is 45-160r/min.
7. Crosslinking: the mass ratio of the soft tissue to the cross-linking agent solution is 1:1-1:12 adding a cross-linking agent solution, and oscillating and cross-linking for 0.5-24h at a speed of 45-100 r/min. Wherein the cross-linking agent is one or two mixed solutions of glutaraldehyde 0.2-8%, EDC 0.1-1.0%, BDDE 0.1-1.3%, PEGDE 0.1-3% and genipin 0.1-0.8%, and the mixing ratio is 1:0.1-1:3.
after the soft tissue is crosslinked, the natural porous structure integrity of the matrix material is maintained, the mechanical property is good, the filling effect is enhanced, meanwhile, the degradation of the soft tissue can be delayed, and the filling effect can be maintained for a long time.
8. Cleaning: the mass ratio of the soft tissue to the water is 1:10-1:20 cleaning the cross-linking agent, 45-160r/min, 3-8 times, 5-10 min/time, or standing for 4-24h, changing the liquid every 2-4h, and removing the residue of the cross-linking agent.
9. Freezing and crushing: the cross-linked soft tissue is frozen and crushed at a low temperature, and the process conditions of the cryogenic crushing treatment are as follows: freezing at-20deg.C to-78deg.C for 4-10 hr, transferring into liquid nitrogen, pre-cooling for 1-4 hr, and pulverizing; during crushing, proper amount of liquid nitrogen is added to control the temperature, the rotating speed is 3000-30000r/min, the crushing time is 10-55 s, each time is 5-10s, the crushing is stopped and started, particles with the particle size of 30-200 mu m are collected by sieving, and the particles with the particle size of more than 200 mu m are repeatedly subjected to the step 9.
The crosslinked soft tissue is frozen and crushed at a deep low temperature, rather than adopting a normal-temperature crushing means, so that the natural tissue structure in the acellular matrix particles can be better protected; the decellularized matrix particles have natural compact microporous structure and crude fiber morphology of biological materials, contain various proteins such as type I collagen, type III collagen and fibronectin, can provide good microenvironment for host cell ingrowth, stimulate tissues to form new collagen, and have excellent degradation resistance.
10. Preparation of non-crosslinked gel phase: weighing at least one of 0.2-5% of sodium hyaluronate, 0.2-5% of sodium polyglutamate, 0.1-10% of sodium carboxymethyl cellulose, 0.1-10% of hydroxypropyl methylcellulose sodium, 0.1-10% of sodium alginate and 0.1-10% of agarose, and preparing into gel with 0.3-13%.
11. Preparing a crosslinked gel phase: weighing at least one of 0.2-5% of sodium hyaluronate, 0.2-5% of sodium polyglutamate, 0.1-10% of sodium carboxymethyl cellulose, 0.1-10% of hydroxypropyl methylcellulose sodium, 0.1-10% of sodium alginate and 0.1-10% of agarose to prepare 5-17% of solution, adopting the same cross-linking agent used in the step 7, and according to the mass ratio of gel phase to cross-linking agent of 1:1-1:12, crosslinking for 2-24h according to the mass ratio of crosslinked gel phase to water of 1:10-1:30 standing, soaking the crosslinked gel phase, changing water every 1-2h, changing for 1-10 times, and removing the crosslinking agent.
12. Mixing the decellularized matrix microparticles with a gel: preparing 10-70% of acellular matrix particles into a particle suspension by using glycerol or normal saline or water for injection, and mixing the acellular matrix particle suspension with mixed gel according to the mass ratio of 1:0.2-1:3, mixing; wherein the mixed gel phase consists of a crosslinked gel phase and a non-crosslinked gel phase, and the proportion is 0.1:1-1:0.1.
the crosslinked gel phase and the uncrosslinked gel phase serve as carriers of the microparticles, so that on one hand, the function of instant volume support is achieved, and on the other hand, the crosslinked gel phase has a tissue filling effect in the middle of injection; the acellular matrix particles can cause moderate immune response of organisms and promote collagen regeneration, and on the other hand, the acellular matrix particles retain natural compact microstructures and crude fiber forms of biological materials, so that the acellular matrix particles are more excellent in degradability and filling effect.
13. Homogenizing: vacuumizing the mixed acellular matrix particle suspension fluid to 20-95KPa,2000-11000r/min, homogenizing for 10-150s to obtain 30-200 μm mixed particle fluid.
14. Filling and sterilizing: filling the mixed particulate fluid into a prefilled syringe and performing wet heat sterilization under the conditions of 121 ℃ and 8-15min to obtain the sterile acellular matrix particulate filler.
The particle size of the particles in the acellular matrix particle filler is 30-200 mu m, so that a good filling effect can be achieved; if the particle size is too large, filling is difficult, the appearance and texture of filling are poor, and the foreign body sensation is strong; if the particle size is too small, the filler is easily degraded and easily displaced, and thus the filler is difficult to achieve a long-acting filling effect.
The finally prepared acellular matrix particle filler comprises the following components in percentage by mass:
acellular matrix microparticles: 2.50% -60.00%;
mixing gel phases: 15.00% -75.00%;
solvent: 7.50% -75.00%;
the mixed gel phase comprises a crosslinked gel phase and a non-crosslinked gel phase, and the mass ratio of the crosslinked gel phase to the non-crosslinked gel phase is 0.1:1-1:0.1.
example 1
An acellular matrix particulate filler comprising, in mass fraction:
acellular matrix microparticles: 16%;
mixing gel phases: 60 percent;
solvent (water for injection): 24%
The mixed gel phase comprises a crosslinked gel phase and a non-crosslinked gel phase, and the mass ratio of the crosslinked gel phase to the non-crosslinked gel phase is 0.7:0.3.
the embodiment also provides a preparation method of the acellular matrix particle filler, which comprises the following preparation steps:
1. pretreatment: taking fresh pericardium of cattle, removing subcutaneous tissues and the like;
2. degreasing: mixing isopropanol and petroleum ether solution in a mixing ratio of 1:0.5, according to the mass ratio of the soft tissue to the feed liquid of 1:5, performing oscillation degreasing for 2 times at a speed of 60r/min for 7h each time;
3. cleaning: the mass ratio of the soft tissue to the water is 1:10, cleaning the residual degreasing mixed solution for 2 times, wherein the frequency is 60r/min each time for 20min;
4. freezing and irradiating: placing the cleaned soft tissue in-20deg.C, freezing for 24 hr, transferring into a heat preservation box, adding ice box, sealing, and performing 15kGy irradiation treatment;
5. decellularization: the hypertonic solution is 5.88% sodium chloride solution, the hypotonic solution is PBS solution with pH5.5, wherein 0.1% triton 100 is added to the hypertonic solution. According to the mass ratio of the soft tissue to the solution of 1:8, high-low permeability oscillation circulates for 3 times, 20 min/time, and the oscillation frequency is 65r/min;
6. cleaning: the mass ratio of the soft tissue to the water is 1:10, cleaning for three times, wherein each time is 10min, and the frequency is 120r/min;
7. crosslinking: the mass ratio of the soft tissue to the cross-linking agent is 1: and 4, adding a cross-linking agent solution, and oscillating and cross-linking for 5 hours at 50 r/min. Wherein the cross-linking agent is a mixed solution of glutaraldehyde of 1 percent and EDC of 0.5 percent, and the mixing proportion is as follows: 1:0.1
8. Cleaning: the mass ratio of the soft tissue to the water is 1:10 Washing for 5 times at 80r/min for 3 min/min or standing for 16h, changing liquid every 2h, and removing the residue of the crosslinking agent;
9. freezing and crushing: freezing soft tissue at-20deg.C for 10 hr, transferring to liquid nitrogen, pre-cooling for 1 hr, pulverizing, adding appropriate amount of liquid nitrogen, controlling temperature, pulverizing at 10000r/min for 20s, stopping for 10s each time, pulverizing, sieving, and repeating step 9;
10. preparation of non-crosslinked gel phase: weighing sodium hyaluronate and sodium carboxymethylcellulose, and preparing into 8% gel. Wherein, the sodium hyaluronate is 0.5 percent, and the sodium carboxymethylcellulose is 7.5 percent;
11. preparing a crosslinked gel phase: weighing sodium hyaluronate and sodium carboxymethylcellulose to prepare a 10% solution; wherein, the sodium hyaluronate is 7.5 percent, and the sodium carboxymethylcellulose is 2.5 percent; and (3) adopting the same cross-linking agent used in the step (7), wherein the mass ratio of the gel phase to the cross-linking agent is 1:3, crosslinking for 16h according to the mass ratio of crosslinked gel phase to water of 1:20, standing and soaking, changing water every 2 hours, changing for 5 times, and removing the cross-linking agent;
12. mixing the decellularized matrix microparticles with a mixing gel: preparing 40% of cell-free matrix particles into a particle suspension fluid by using water for injection, and mixing the cell-free matrix particle suspension fluid with mixed gel according to the mass ratio of 1:1.5, wherein the mixed gel phase consists of a crosslinked gel phase and a non-crosslinked gel phase, and the proportion is 0.7:0.3;
13. homogenizing: vacuumizing the mixed acellular matrix particle suspension fluid to 40KPa, performing homogenization for 85s to obtain 30-200 μm mixed particle fluid;
14. and (3) filling: filling the mixed particulate fluid into a pre-filled syringe;
15. and (3) sterilization: placing the prefilled syringe filled with the mixed particulate fluid into a damp heat sterilizer for sterilization, and performing sterilization at 121 ℃ for 8min to obtain the sterile acellular matrix particulate filler.
Example 2:
an acellular matrix particulate filler comprising, in mass fraction:
acellular matrix microparticles: 16.66%;
mixing gel phases: 16.67%;
solvent (glycerol): 66.67%
The mixed gel phase comprises a crosslinked gel phase and a non-crosslinked gel phase, and the mass ratio of the crosslinked gel phase to the non-crosslinked gel phase is 0.2:0.8.
the embodiment also provides a preparation method of the acellular matrix particle filler, which comprises the following preparation steps:
1. pretreatment: taking fresh skin of pigs, and removing hair, subcutaneous tissues and the like;
2. degreasing: taking an acetone solution, wherein the mass ratio of the soft tissue to the feed liquid (the mass ratio is as follows) is 1:5, carrying out oscillation degreasing for 2 times at a speed of 75r/min for 4 hours each time;
3. cleaning: the mass ratio of the soft tissue to the water is 1:10 cleaning the residual degreasing mixed solution for 3 times, wherein the vibration frequency is 75r/min for 10min each time;
4. freezing and irradiating: placing the cleaned soft tissue in-40 ℃, freezing for 8 hours, transferring to a heat preservation box, adding a high molecular cool storage agent, sealing, and adopting 10kGy irradiation treatment;
5. decellularization: the hypertonic solution was 1.5% sodium chloride solution and the hypotonic solution was 6.8 PBS solution. According to the mass ratio of the soft tissue to the solution of 1:8 high-low osmosis cycles are carried out for 5 times, 15 min/time, and the frequency is 120r/min;
6. cleaning: the mass ratio of the soft tissue to the water is 1:10 cleaning for three times, each time for 20min, with the frequency of 120r/min;
7. crosslinking: the mass ratio of the soft tissue to the cross-linking agent solution is 1:2 adding a cross-linking agent solution, and oscillating and cross-linking for 4 hours at 75 r/min. Wherein, the cross-linking agent is a mixed solution of 0.2% EDC and 0.2% genipin, and the mixing ratio is 1:0.2;
8. cleaning: the mass ratio of the soft tissue to the water is 1:10 cleaning the cross-linking agent, standing for 24 hours, changing the liquid every 2 hours, and removing the residue of the cross-linking agent;
9. freezing and crushing: freezing soft tissue at-78deg.C for 4 hr, transferring to liquid nitrogen, pre-cooling for 2 hr, pulverizing, adding appropriate amount of liquid nitrogen, controlling temperature, pulverizing at 17000r/min for 15s, stopping, pulverizing at 5s, sieving, and repeating step 9;
10. preparation of non-crosslinked gel phase: weighing sodium alginate and sodium polyglutamate, and preparing into gel with 2.5%. Wherein, the sodium polyglutamate is 0.5 percent and the sodium alginate is 2.0 percent;
11. preparing a crosslinked gel phase: weighing sodium alginate and sodium polyglutamate to prepare a 7% solution; wherein, sodium alginate is 4% and sodium polyglutamate is 3%; and (3) adopting the same cross-linking agent used in the step (7), wherein the mass ratio of the gel phase to the cross-linking agent is 1:5, crosslinking for 12h according to the mass ratio of crosslinked gel phase to water of 1:20 standing, soaking the crosslinked gel phase, changing water every 2 hours, changing for 4 times, and removing the crosslinking agent;
12. mixing the decellularized matrix microparticles with a mixing gel: preparing 20% of cell-free matrix particles into a particle suspension body by using glycerol, and mixing the cell-free matrix particle suspension body with mixed gel according to the mass ratio of 1:0.2, wherein the mixed gel phase consists of a crosslinked gel phase and a non-crosslinked gel phase, and the proportion is 0.2:0.8
13. Homogenizing: vacuumizing the mixed acellular matrix particle suspension fluid to 25KPa,2000r/min, homogenizing for 35s to obtain 30-200 μm mixed particle fluid;
14. and (3) filling: filling the mixed particulate fluid into a pre-filled syringe;
15. and (3) sterilization: placing the prefilled syringe filled with the mixed particulate fluid into a damp heat sterilizer for sterilization, and performing sterilization at 121 ℃ for 9min to obtain the sterile acellular matrix particulate filler.
Example 3
An acellular matrix particulate filler comprising, in mass fraction:
acellular matrix microparticles: 21.00%;
mixing gel phases: 75.00%;
solvent (water for injection): 9.00%
The mixed gel phase comprises a crosslinked gel phase and a non-crosslinked gel phase, and the mass ratio of the crosslinked gel phase to the non-crosslinked gel phase is 0.8:0.2.
the embodiment also provides a preparation method of the acellular matrix particle filler, which comprises the following preparation steps:
1. pretreatment: taking a bovine diaphragm, removing subcutaneous tissues and the like;
2. degreasing: mixing alcohol and isopropanol solution with a mixing ratio of 0.4:0.6, according to the mass ratio of soft tissue to feed liquid (the mass ratio is as follows) of 1: performing vibration degreasing for 2 times at 140r/min for 4 hours each time;
3. cleaning: the mass ratio of the soft tissue to the water is 1:10 cleaning the residual degreasing mixed solution for 2 times, wherein each time is 20min, and the oscillation frequency is 140r/min;
4. freezing and irradiating: placing the cleaned soft tissue in-80 ℃, freezing for 6 hours, transferring into a heat preservation box, adding dry ice, sealing, and adopting 25kGy irradiation treatment;
5. decellularization: the hypertonic solution is 3% sodium chloride solution and the hypotonic solution is water. According to the mass ratio of the soft tissue to the solution of 1: the cycle of 20 high-low osmosis is 5 times, 15 min/time, and the frequency is 140r/min;
6. cleaning: the mass ratio of the soft tissue to the water is 1:20 is cleaned for three times, 10min each time, and the frequency is 140r/min;
7. crosslinking: the mass ratio of the soft tissue to the cross-linking agent solution is 1:12 adding a cross-linking agent solution, and oscillating and cross-linking for 2h at 75 r/min. Wherein the cross-linking agent is 0.2% glutaraldehyde and 0.8% BDDE mixed solution, and the mixing proportion is 1:3, a step of;
8. cleaning: the mass ratio of the soft tissue to the water is 1:20 cleaning the cross-linking agent, 140r/min, 8 times, 5 min/time, and removing the residue of the cross-linking agent;
9. freezing and crushing: freezing soft tissue at-40deg.C for 7 hr, transferring to liquid nitrogen, pre-cooling for 3 hr, pulverizing, adding appropriate amount of liquid nitrogen, controlling temperature, pulverizing at 25000r/min for 55s each time, stopping, pulverizing at start-up, sieving, and repeating step 9;
10. preparation of non-crosslinked gel phase: sodium carboxymethyl cellulose and sodium polyglutamate are weighed to prepare gel with 13 percent. Wherein, the sodium polyglutamate is 1 percent and the sodium carboxymethylcellulose is 12 percent;
11. preparing a crosslinked gel phase: weighing sodium carboxymethyl cellulose and sodium polyglutamate to prepare 17% solution; wherein, sodium carboxymethyl cellulose is 12 percent and sodium polyglutamate is 5 percent; and (3) adopting the same cross-linking agent used in the step (7), wherein the mass ratio of the gel phase to the cross-linking agent is 1:12, crosslinking for 21h according to the mass ratio of crosslinked gel phase to water of 1:30, standing water, soaking a crosslinked gel phase, replacing water for 8 times every 1h, and removing a crosslinking agent;
12. mixing the decellularized matrix microparticles with a gel: preparing the decellularized matrix particles into 70% particle suspension by using normal saline, and mixing the decellularized matrix particle suspension with mixed gel according to a mass ratio of 1:3, mixing, wherein the mixed gel phase consists of a crosslinked gel phase and a non-crosslinked gel phase, and the proportion is 0.8:0.2;
13. homogenizing: vacuumizing the mixed acellular matrix particle suspension fluid to 95KPa,11000r/min, homogenizing for 150s to obtain 30-200 μm mixed particle fluid;
14. and (3) filling: filling the mixed particulate fluid into a pre-filled syringe;
15. and (3) sterilization: placing the prefilled syringe filled with the mixed particulate fluid into a damp heat sterilizer for sterilization, and performing sterilization at 121 ℃ for 15min to obtain the sterile acellular matrix particulate filler.
Comparative example 1: the difference from example 1 is that the mixed gel phase does not include a non-crosslinked gel phase, i.e., the decellularized matrix particulate filler has a composition of: 16% of decellularized matrix particles, 60% of crosslinked gel phase and 24% of solvent (water for injection).
Comparative example 2: the difference from example 1 is that the decellularized soft tissue has not been crosslinked with a crosslinking agent, i.e. step 7 is absent.
Comparative example 3: an injectable soft tissue bio-filler material was prepared according to patent CN 200510044005.5.
Comparative example 4: the difference from example 1 is that the mixed gel phase does not include a crosslinked gel phase.
Comparative example 5: the difference from example 1 is that the ratio of crosslinked gel phase to non-crosslinked gel phase in the mixed gel phase is 1.5:0.1.
comparative example 6: the difference from example 1 is that the ratio of crosslinked gel phase to non-crosslinked gel phase in the mixed gel phase is 0.1:2.
comparative example 7: the difference from example 1 is that the mass ratio of decellularized matrix microparticle suspension fluid to mixed gel phase is 1:0.1.
comparative example 8: the difference from example 1 is that the mass ratio of decellularized matrix microparticle suspension fluid to mixed gel phase is 1:5.
microstructure of acellular matrix particles
The cross section of the decellularized matrix particles was observed using a field emission scanning electron microscope. Firstly, freezing a sample in liquid nitrogen, drying in a freeze dryer, plating gold on the surface of the sample, and observing by using a scanning electron microscope; the test results are shown in fig. 1 and 2.
FIG. 1 is a scanning electron microscope image of decellularized matrix particles prepared in example 1; FIG. 2 is a scanning electron microscope image of the injectable soft tissue bio-filler prepared in comparative example 3.
The results in FIG. 1 show that the acellular matrix particles of example 1 have intact and dense collagen structures, no significant degradation is seen, and the acellular matrix remains a natural three-dimensional mechanism and is not destroyed. In FIG. 2, the injectable soft tissue bio-filler prepared in comparative example 3 has a loose tissue due to the serious destruction of the natural structure of the acellular matrix by the treatment with the strong alkali solution. By adopting the technical scheme provided by the invention, the immunogenicity is removed, the natural tissue structure can be protected from being damaged to the greatest extent, and the decellularized matrix particles have a natural compact micropore structure and a crude fiber form of the biological material.
Performance testing
The swelling degree, young modulus and pushing force of the filler and the comparative example prepared by the invention are tested, and the swelling degree is calculated by adopting a gravimetric method; young's modulus measurement is carried out by using a rheometer, the measurement parameter is 25 ℃, the shearing strain is 0.1%, and the frequency is scanned to be 10-0.1Hz; the push force was measured according to YY/T0962-2021, and the results are shown in Table 1 below.
Table 1 performance test data for each example and comparative example
The results of the performance test show that, compared with comparative example 1, example 1 has proper pushing force and lubricity due to the addition of the non-crosslinked gel phase, the lubricity and injectability of comparative example 1 are greatly reduced, particularly, the injection is very laborious, the pushing force can reach more than 25N, and a large amount of filling agent is instantaneously pushed out, so that the clinical operation is more risky and difficult. The decellularized soft tissue of comparative example 2 was not crosslinked, had more water binding groups, and had a relatively loose internal structure, so that the swelling degree was too high, resulting in significant local swelling after implantation. The comparative example 5 has less non-crosslinked gel phase than the applicable ratio, and the pushing force is doubled, thus increasing the injection risk and being inconvenient for clinical use. The cross-linked gel phase in comparative example 6 was less than the applicable proportion, and its swelling degree was significantly increased, decreasing filling comfort and increasing use risk. In comparative example 7, the mixed gel phase was reduced, the swelling degree and young's modulus were not significantly changed, but the pushing force was increased, increasing the operational risk.
In vitro degradation experiments
The products obtained in the examples and the comparative examples are degraded by 2500UI collagenase, supernatant is centrifugally taken, the collagen content is measured by referring to the method in Chinese pharmacopoeia, and the in vitro degradation rate at different times is calculated.
Table 2 in vitro degradation rates of examples and comparative examples
In vitro degradation data show that the acellular matrix particle filler prepared by the technical scheme provided by the invention is not obviously degraded in a short period, and the comparative example is equivalent to the in vitro degradation degree of the example; degradation of comparative examples 2, 3, 4 was significantly accelerated over time, since the acellular matrix of comparative example 2 was not crosslinked, resulting in very poor degradation resistance of the acellular matrix particles; in contrast, in comparative example 3, the natural structure of the tissue was severely damaged during the preparation of the decellularized matrix particles, and all of the tissue was completely degraded in 72 hours; comparative example 4 was not mixed with the crosslinked gel and the overall degradation resistance was slightly inferior. The mixed gel phase of comparative example 8 exceeded the applicable proportion and the overall degradation resistance was reduced. While example 1 degraded slowly, it shows that the acellular matrix particles, crosslinked gel phase and non-crosslinked gel phase of the invention can have moderate pushing force, facilitate clinical use, effectively delay degradation and enhance filling effect.
Filling effect test
Animal experiment scheme
10 SD rats were taken, each half of the male and female rats were shaved off the back hair, and the interval was 2cm, and the points were marked as A-F, 0.5mL of the gel prepared in examples 1-3 and comparative examples 1-3 were subcutaneously injected, and the filling effect was observed at 1 week and 3 months, as shown in FIGS. 3 and 4, respectively, wherein A: examples 1, B: examples 2, C: examples 3, D: comparative examples 1, E: comparative examples 2, F: comparative example 3.
The filling effect is compared with the following table 3.
Table 3 comparative filling effect
As can be seen from fig. 3, fig. 4 and table 3, the filled cell-free matrix particle filler of examples 1 to 3 of the present invention has full appearance, soft texture, and no fibrous capsule formed in the observation period, which indicates that the cell-free matrix particle filler provided by the present invention has no foreign matter reaction, is safe in product, has good filling effect, and can maintain good filling volume and filling effect when observed for 3 months and 12 months. The sample of comparative example 1 was filled, and the filling site was hard and different from the natural tissue, which resulted in a stiff and unnatural filling effect in practical application, and showed significant migration when observed for a long period of time (12 months), because comparative example 1 was a hard decellularized matrix microparticle and a crosslinked gel phase, contained no relatively soft, non-crosslinked gel phase, and had poor cohesion of the filler, and showed significant migration after in vivo implantation. The sample of comparative example 2 has poor degradation resistance due to the non-crosslinked acellular matrix particles, and the filling volume is significantly reduced when observed for a long period of time. The filler of comparative example 3 has a hard appearance and unnatural filling effect, and causes foreign body reaction and fibrous capsule due to its large particle size, and the filler of comparative example 3 has a certain degree of wandering in short-term and long-term observation, which easily causes unsightly bumps, bumps and other deformities in other parts in actual clinical use, and even serious infection.
The foregoing description is only an example to further illustrate the technical content of the present invention, so that the reader can easily understand the technical content, but the embodiments of the present invention are not limited thereto, and any technical extension or recreating according to the present invention is protected by the present invention.