Injectable microparticulated acellular dermal matrix, process for its preparation and use

Abstract

The application provides an injectable microparticulate acellular dermal matrix and a preparation method and application thereof, and belongs to the technical field of preparation of acellular dermal matrixes.The preparation method of the microparticulate acellular dermal matrix comprises the following steps: (1) taking a dermal layer, performing homogenization and defatting treatment, and then performing decellularization treatment; (2) freezing under the condition of liquid nitrogen at-196 DEG C for 1-2 minutes; (3) placing in a cold trap circulating medium, controlling the temperature to be below-50 DEG C, and performing ball milling under the temperature condition by using milling balls with different sizes; (4) after the ball milling is completed, screening the obtained product in step (3), and directly collecting the product in a sterile collection container by using a turbine airflow to form a low-pressure area and storing the product, so that the microparticulate acellular dermal matrix is obtained.The microparticulate acellular dermal matrix obtained by the method has good uniformity, high product quality and high stability, and has good injectability, and can be used for minimally invasive surgical treatment of primary premature ejaculation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of preparation of acellular matrix microparticles, and particularly relates to an injectable microparticulated acellular dermal matrix, a preparation method thereof and application thereof. BACKGROUND

[0002] In recent years, acellular matrix materials have been widely used in the biomedical industry, and the product forms have become increasingly diversified. For example, from the initial sheet form, microparticles, gels (obtained by pepsin digestion of acellular matrix microparticles), and other new forms have been gradually developed. These newly developed forms can better meet the needs of clinical minimally invasive surgery.

[0003] Due to its natural reticular collagen scaffold structure, acellular matrix materials generally have a tough texture. In the process of microparticulating the acellular matrix, the existing conventional microparticulating equipment and process often fail to meet the needs of industrialized mass production, mainly in that: 1) the microparticulating efficiency is low; 2) the particle size of the microparticles is not uniform; 3) heating causes protein denaturation or inactivation of active factors; and 4) metal tanks can easily cause heavy metal pollution. Therefore, improving the production efficiency of acellular matrix microparticles, ensuring the uniformity of the particle size and activity of the products, and realizing industrialized mass production have become a major problem in the production and processing of acellular matrix microparticles.

[0004] At present, acellular dermal matrix is increasingly used in urology and male reproductive system diseases, such as penile enlargement, hypospadias, ureteral stricture, etc. Acellular dermal matrix is prepared into a three-dimensional scaffold structure through decellularization, and can induce the migration of surrounding cells to crawl on it after being implanted into the human body. It has good biocompatibility, and its degradation products contain various active ingredients such as collagen, elastin, fibronectin and various growth active factors, which can promote tissue regeneration.

[0005] For example, patent document CN108114319A discloses an acellular allogeneic dermal matrix and its application in penile dorsal nerve isolation. The patent method is a multi-step collaborative operation of enzyme treatment, surfactant ultrasonic treatment, DNA degradation treatment, etc., to prepare an acellular allogeneic dermal matrix material, which is made into an internal biological sleeve for the treatment of premature ejaculation. However, this acellular allogeneic dermal matrix has the following shortcomings: 1) mostly from cadaver skin, with limited sources, high ethical risk, and not conducive to large-scale production; 2) mostly aged and unhealthy dermal tissue; 3) risk of virus transmission; 4) large surgical wound, prone to sexual dysfunction; 5) not acellular dermal matrix microparticles, not injectable and minimally invasive, and its application efficacy cannot be comparable to that of acellular dermal matrix microparticles.

[0006] Therefore, how to provide a preparation method of the acellular dermal matrix microparticles with good injectability, good application effect in minimally invasive surgery, good microparticulation effect and suitability for industrial production, so as to be better applied to the treatment of primary premature ejaculation, has become a technical problem to be solved. SUMMARY

[0007] The present application is to solve the above technical problems, and provides an injectable microparticulated acellular dermal matrix and a preparation method and application thereof. The technical purpose of the present application is to provide a preparation method of an injectable microparticulated acellular dermal matrix for treating primary premature ejaculation, which solves the defects that the existing method cannot prepare the injectable microparticulated acellular dermal matrix, and the particle size is not uniform, the biological activity is poor, and the product quality is poor in the microparticulation preparation process.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] A preparation method of an injectable microparticulated acellular dermal matrix for treating primary premature ejaculation, comprising the following steps:

[0010] (1) taking the dermal matrix raw material to be removed from cells to obtain the dermal layer, then homogenizing and defatting, and then decellularizing;

[0011] (2) freezing the product obtained in step (1) at-196℃ under liquid nitrogen for 1-2 minutes;

[0012] (3) taking out the product obtained in step (2) and placing it in a cold trap circulating medium, controlling the temperature to be below-50℃, and using different sizes of grinding balls to ball mill under this temperature condition, controlling the ratio of material to ball to be 1:40-80; controlling the mass ratio of different sizes of grinding balls to be 10mm:5mm:1mm=2-5:2-5:10-15;

[0013] (4) after ball milling is completed, sieving the product obtained in step (3), and then using a turbine airflow to form a low pressure area to directly collect the product in a sterile collection container for storage, thereby obtaining the injectable microparticulated acellular dermal matrix.

[0014] At present, minimally invasive surgery has gradually replaced traditional surgical operation. In the past, the acellular dermal matrix was implanted into the penis by surgical operation, which resulted in large surgical wound, slow recovery of the patient and obvious pain, and some patients had erectile dysfunction. The microparticulated acellular dermal matrix prepared by the above method can be mixed with physiological saline and injected into the penis subcutaneously through a 25G needle to achieve the effect of isolating the dorsal nerve, thereby realizing the minimally invasive surgical treatment of primary premature ejaculation.

[0015] Secondly, the traditional decellularization technology generally uses biological enzymes, which has the risk of introducing viruses and causing immune reactions in the human body, and the biological enzymes are expensive and have high production costs. The traditional decellularization technology also uses strong acid or strong base, which is not conducive to safe production. The preparation method provided by the application only uses conventional chemical reagents, the quality is controllable, the cost is low, and the production is safe.

[0016] In addition, the traditional laboratory freeze mill can only mill 2g at a time, and the microparticulate decellularized dermal matrix in the application can mill 10g at a time, which greatly improves the production efficiency and product uniformity.

[0017] Finally, the tank material of the traditional freeze mill is stainless steel or titanium alloy, which will fatigue after long-term work, leading to ion exchange or surface peeling, thereby introducing heavy metals. The tank material of the freeze mill in the application is nylon, which is safer.

[0018] In the above preparation method provided by the application, freezing the decellularized matrix material at-196 DEG C under liquid nitrogen for 1-2 minutes is more critical, which is to ensure that the decellularized matrix material is converted into a glass state before microparticulation. In addition, the temperature is controlled below-50 DEG C by using a cold trap circulating medium for ball milling, which is to avoid significant temperature rise during microparticulation, thereby causing protein deformation and inactivation of active factors.

[0019] By the method of the application, a microparticulate decellularized dermal matrix with a particle size of less than 0.5mm can be obtained, which has small product size, uniform particle size and high biological activity, and can well meet the clinical use requirements.

[0020] Therefore, in summary, the method for preparing the injectable microparticulate decellularized dermal matrix provided by the application has the following advantages:

[0021] 1. The preparation process of the microparticulate decellularized dermal matrix provided by the application has the advantages of large yield, low cost, and no introduction of heavy metal elements;

[0022] 2. The microparticulate decellularized dermal matrix prepared by the application can realize minimally invasive surgical treatment of primary premature ejaculation and reduce postoperative complications;

[0023] 3. Since the microparticulate decellularized dermal matrix has a three-dimensional porous structure, local anesthetics can be added after mixing, and injected subcutaneously to the penis to play a local controlled release role, further enhancing the therapeutic effect.

[0024] Further, the acellular dermal matrix raw material in step (1) includes skin, pericardium, small intestinal submucosa, bladder basement membrane, amnion or cornea, and the raw material is derived from human, pig, horse, dog, sheep, cow or fish; preferably, the homogenization step is to add triton, anhydrous sodium carbonate and deionized water to the acellular dermal matrix, and the triton, anhydrous sodium carbonate and deionized water are in a mass ratio of 1:5-20:250-1000; preferably, the homogenization treatment is to homogenize the mixture in a homogenizer at 5000 rpm for 10 minutes; preferably, the defatting treatment is to add a defatting solution for defatting treatment, and the defatting solution includes at least one of isopropyl alcohol, acetone, ethanol and dimethylbenzene, and the concentration of the defatting solution is 90-100 wt%, and the defatting treatment step is to shake at 180 rpm for 2 hours on a shaking table.

[0025] Further, the acellular treatment step is to add an acellular solution for acellular treatment, and the acellular solution is selected from at least one of Triton X-100, hydroxyethyl piperazine ethanesulfonic acid, polyethylene glycol octylphenyl ether, sodium deoxycholate, sodium dodecylaminopropionate or fatty alcohol polyoxyethylene ether; preferably, the concentration of the acellular solution is 0.1-1 wt%, and the acellular treatment step is to shake at 180 rpm for 4-24 hours on a shaking table; preferably, the DNA content after the acellular treatment is less than 50 ng / mg.

[0026] Further, the cold trap circulating medium in step (3) includes anhydrous ethanol or liquid nitrogen.

[0027] Further, the ball-to-material ratio in step (2) is 1:40, and the mass ratio of the different sizes of grinding balls is controlled to be 10 mm:5 mm:1 mm=2:2:10.

[0028] Further, the vibration frequency of the ball mill is controlled to be 800-1000 times / minute.

[0029] Further, the ball milling time in step (2) is 8-15 minutes.

[0030] Further, the sieving in step (3) is to pass through a 60-100 mesh sieve.

[0031] Further, the air flow in step (3) is formed by an air blower.

[0032] The second object of the present application is to provide an injectable micronized acellular dermal matrix prepared by the method as described above.

[0033] The third object of the present application is to provide the use of the injectable micronized acellular dermal matrix as described above in the treatment of primary premature ejaculation, which is prepared into a penile dorsal nerve isolation surgical material.

[0034] Compared with the prior art, the present application has the following advantages:

[0035] (1) The present application provides a preparation method of injectable microparticulate acellular dermal matrix, which is simple, controllable and suitable for industrial production, and solves the problem of poor microparticulation of the existing acellular dermal matrix and the inability to achieve injectability.

[0036] (2) The microparticulate acellular dermal matrix prepared by the present application has the advantages of uniform particle size, high biological activity, high product quality and small differences between batches, and can well meet the clinical application, and has excellent application prospect in the treatment of primary premature ejaculation. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a preparation process route map of the microparticulate acellular dermal matrix of the present application.

[0038] Figure 2 It is an immunogenic substance detection of the microparticulate acellular dermal matrix (mADM);

[0039] Figure 3 It is a scanning electron microscope image of mADM;

[0040] Figure 4 It is the growth factor detection result of mADM;

[0041] Figure 5 It is the degradation result of mADM after intradermal injection for 1 week;

[0042] Figure 6 It is the degradation result of mADM after intradermal injection for 2 weeks;

[0043] Figure 7 It is the degradation result of mADM after intradermal injection for 4 weeks;

[0044] Figure 8 It is the degradation result of mADM after intradermal injection for 8 weeks;

[0045] Figure 9 It is the result of treating primary premature ejaculation by isolating the dorsal nerve of the penis with mADM. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is described in detail below with examples, it is necessary to point out that the following examples are only used to explain and describe the present application, and do not limit the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above disclosure still fall within the scope of the present application.

[0047] Example 1

[0048] As Figure 1 shown in the production process, the present application provides a preparation method of microparticulate acellular dermal matrix, comprising the following steps:

[0049] (1) taking pig dermal matrix raw material to be removed cells, obtaining dermal layer, then homogenizing and defatting, and then decellularizing;

[0050] (2) freezing the product obtained in step (1) at -196℃ under liquid nitrogen for 1 minute;

[0051] (3) taking the product obtained in step (2) and placing it in a cold trap circulating medium (absolute ethanol), controlling the temperature at -50℃, and using different sizes of grinding balls to ball mill under this temperature condition, the vibration frequency of ball milling is 800 times / minute, the material ball ratio is controlled at 1:40; the mass ratio of different sizes of grinding balls is controlled at 10mm:5mm:1mm=2:2:10; the ball milling time is 8 minutes;

[0052] (4) after ball milling is completed, the product obtained in step (4) is passed through a 60-mesh sieve, and then the microparticulate acellular matrix is attracted to a low pressure area by a turbine airflow and directly enters a sterile collection container for storage, thereby obtaining the injectable microparticulate acellular dermal matrix (denoted as mADM).

[0053] Example 2

[0054] A preparation method of microparticulate acellular dermal matrix, comprising the following steps:

[0055] (1) taking pig dermal matrix raw material to be removed cells, obtaining dermal layer, then homogenizing and defatting, and then decellularizing;

[0056] (2) freezing the product obtained in step (1) at -196℃ under liquid nitrogen for 2 minutes;

[0057] (3) taking the product obtained in step (2) and placing it in a cold trap circulating medium (absolute ethanol), controlling the temperature at -52℃ or below, and using different sizes of grinding balls to ball mill under this temperature condition, the vibration frequency of ball milling is 1000 times / minute, the material ball ratio is controlled at 1:80; the mass ratio of different sizes of grinding balls is controlled at 10mm:5mm:1mm=5:5:15; the ball milling time is 15 minutes;

[0058] (4) after ball milling is completed, the product obtained in step (3) is passed through a 100-mesh sieve, and then the microparticulate acellular matrix is attracted to a low pressure area by a turbine airflow and directly enters a sterile collection container for storage.

[0059] Example 3

[0060] A method for preparing a micro-particleized acellular dermal matrix, comprising the following steps:

[0061] (1) taking pig dermal matrix raw material to be removed cells, obtaining dermal layer, then homogenizing and defatting, and then decellularizing;

[0062] (2) freezing the product of step (1) at -196℃ under liquid nitrogen for 2 minutes

[0063] (3) taking the product of step (2) and placing it in a cold trap circulating medium (liquid nitrogen), controlling the temperature at -60℃, and using different sizes of grinding balls to perform ball milling at this temperature, the vibration frequency of ball milling being 900 times / minute, the material-ball ratio being controlled at 1:60, and the mass ratio of different sizes of grinding balls being controlled at 10mm:5mm:1mm=3:4:12, the ball milling time being 10 minutes;

[0064] (4) after ball milling is completed, passing the product of step (3) through an 80-mesh sieve, and then using a turbine airflow to attract the micro-particleized acellular dermal matrix into a low-pressure area and directly into a sterile collection container for storage.

[0065] Example 4

[0066] A method for preparing a micro-particleized acellular dermal matrix, comprising the following steps:

[0067] (1) taking pig dermal matrix raw material to be removed cells, obtaining dermal layer, then homogenizing and defatting, and then decellularizing;

[0068] (2) freezing the product of step (1) at -196℃ under liquid nitrogen for 1-2 minutes;

[0069] (3) taking the product of step (1) and placing it in a cold trap circulating medium (liquid nitrogen), controlling the temperature at -55℃, and using different sizes of grinding balls to perform ball milling at this temperature, the vibration frequency of ball milling being 850 times / minute, the material-ball ratio being controlled at 1:70, and the mass ratio of different sizes of grinding balls being controlled at 10mm:5mm:1mm=4:5:13, the ball milling time being 12 minutes;

[0070] (4) after ball milling is completed, passing the product of step (3) through an 80-mesh sieve, and then using a turbine airflow to attract the micro-particleized acellular dermal matrix into a low-pressure area and directly into a sterile collection container for storage.

[0071] Experimental exploration example 1

[0072] The micro-particleized acellular dermal matrix was prepared according to the method of Example 1, and the particle size distribution under different freezing conditions was compared, and the results are shown in Table 1.

[0073] Table 1

[0074]

[0075] As shown in Table 1, the particle size of the micro-particulate acellular dermal matrix obtained after liquid nitrogen freezing is far below the required 0.5 mm, can smoothly pass through the clinical 25G needle, and the particle size is more uniform, and the product quality is more stable.

[0076] Experimental exploration example 2

[0077] The effects of different material-ball ratios on the crushing of the acellular dermal matrix (material amount: 5 g, dry method) were compared, and the results are shown in Table 2.

[0078] Table 2

[0079]

[0080] As shown in Table 2, with the increase of the material-ball ratio, the ball milling particle size shows a downward trend and is more uniform, but after increasing to 1:40, the particle size change is no longer obvious. However, the more the balls increase, the less the material is relatively, and the greater the overall weight, the greater the machine loss, which is not conducive to industrial production. Therefore, in order to ensure the yield and service life, the material-ball ratio is set to 1:40.

[0081] Experimental exploration example 3

[0082] The effects of different sizes of grinding balls on the crushing of the acellular dermal matrix (material-ball ratio 1:40, dry method) were compared, and the results are shown in Table 3.

[0083] Table 3

[0084]

[0085]

[0086] As shown in Table 3, when the mass ratio of different grinding ball sizes is 10 mm:5 mm:1 mm=2-5:2-5:10-15, the particle size of the crushed product is smaller and more uniform, and when the mass ratio is 2:2:10, the particle size range is the smallest, which is more conducive to the micro-particulate micro-injection of the acellular matrix in the clinic. Therefore, it is recommended that the mass ratio of the grinding ball size is 10 mm:5 mm:1 mm=2:2:10.

[0087] Experimental example 1

[0088] Taking Example 1 as an example, the properties of the micro-particulate acellular dermal matrix prepared in the examples of the application were examined. The results obtained in the other examples are similar to those of Example 1.

[0089] (I) The immunogenic substances of the obtained micro-particulate acellular dermal matrix were measured, and the obtained DNA and fat contents are shown in Table 4. Figure 2 Table 4

[0090] In order to evaluate the immunogenicity removal effect, it is known from the literature that the DNA content of the acellular matrix material should be <50 ng / mg tissue dry weight, and the fat content of the acellular SIS is less than 7% according to the United States Pharmacopoeia, and from Figure 2 It can be seen that the immunogenicity content of the microparticulate acellular dermal matrix prepared by the method of the application meets the requirements.

[0091] (II) Transmission electron microscopy

[0092] The microparticulate acellular dermal matrix prepared by the method of the application was observed by transmission electron microscopy, and the results are shown in Figure 3 It can be found that the material presents a fibrous honeycomb shape (drug-loaded basis), and the classic triple helix structure of collagen fibers can be seen. Figure 3

[0093] (III) Growth factor detection

[0094] 1. The microparticulate acellular dermal matrix prepared by the method of the application was subjected to growth factor detection, and the growth factor detection method was as follows:

[0095] Purchased porcine epidermal growth factor (EGF) ELISA kit, porcine fibroblast growth factor (FGF) ELISA kit and porcine vascular endothelial growth factor (VEGF) ELISA kit for quantitative detection of EGF, FGF and VEGF, respectively.

[0096] The specific method is as follows:

[0097] 1. Weigh 0.15 g of sample into a 1.5 mL centrifuge tube, add 1 mL of phosphate buffer to the vortex mixer and shake for 5 min;

[0098] 2. Centrifuge at 4000 rpm for 15 min, and take the supernatant for detection;

[0099] 3. Take the kit from the medical refrigerator and equilibrate at room temperature for 1 h;

[0100] 4. Use the kit that has been equilibrated to room temperature, take out the required board and install it on the microplate;

[0101] 5. Set the standard hole, blank hole and sample hole, add 50 μL of different concentrations of standard to the standard hole, add 50 μL of sample supernatant to the sample hole; add 50 μL of sample diluent to the blank hole;

[0102] 6. Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard hole and sample hole, seal the reaction hole with a sealing film, and incubate at 37°C in a gas bath thermostat oscillator for 60 min;

[0103] ​7. Discard the liquid, pat dry with absorbent paper, add full of washing liquid to each well, stand for 1 min, shake off the washing liquid, pat dry with absorbent paper, repeat the washing of the plate for 5 times;

[0104] 8. Add 50 μL of substrate to each well, incubate at 37℃ for 15 min in dark;

[0105] 9. Add 50 μL of termination liquid to each well, measure the OD value of each well at 450 nm within 15 min;

[0106] Take the standard concentration as the horizontal coordinate and the corresponding OD value as the vertical coordinate, draw a standard linear regression curve, and calculate the concentration value of each sample according to the curve equation.

[0107] The test results are shown in Figure 4 From Figure 4 it can be seen that the EGF content in the microparticulate acellular dermal matrix prepared in Example 1 of the present application is 1.03±0.13 ng / g; the FGF content is 1.65±0.12 ng / g; and the VEGF content is 0.43±0.05 ng / g, proving that the microparticulate acellular dermal matrix obtained by the method of the present application has abundant growth factors and can further promote the repair of postoperative wounds.

[0108] 2. With refrigerant circulation (cold trap temperature <-50℃), 10 batches of ball milling experiments were carried out, and the test results are shown in Table 4.

[0109] Table 4

[0110]

[0111] As can be seen from Table 4, the biological activity of the obtained acellular matrix microparticles is high and the stability of each batch is good by using the refrigerant circulation method for temperature control of the present application. The EGF content is 0.90±0.13 ng / g; the FGF content is 1.61±0.21 ng / g; and the VEGF content is 0.23±0.04 ng / g, and there is no significant decrease in each growth factor after continuous crushing, and the uniformity is good, which can meet the requirements of industrial production.

[0112] 3. Without refrigerant circulation, i.e. without temperature control step, directly ball milling, examining the influence of 10 batches of ball milling experiments on the activity of growth factors, and the test results are shown in Table 5.

[0113] Table 5

[0114]

[0115] As can be seen from Table 5, under the refrigerant-free cycle, with the increase of the crushing times, the growth factors all show a significant downward trend due to the increase of the tank temperature. When the crushing is performed for 10 times, the EGF, FGF and VEGF contents are decreased by 74.8%, 58.4% and 61.1% respectively, which leads to the decrease of the product performance, and the differences of the acellular matrix microparticles prepared in different batches are large.

[0116] Experimental Example 2

[0117] 1. The injectability of the acellular matrix microparticles mADM prepared in the embodiment of the present application (taking Example 1 as an example) was studied

[0118] After the physiological saline was fully mixed with the material liquid according to the specified ratio, the push force test was carried out by using different specifications of needle, and the results are shown in Table 6.

[0119] Table 6

[0120]

[0121]

[0122] As can be seen from Table 6, the mADM with the concentration of 100 mg / mL can be successfully injected by using the 25G needle, which proves the injectability of the product and the prospect of clinical application.

[0123] 2. Effect of the ball mill tank material on the heavy metals and trace elements of the product

[0124] The results of the full element analysis by ICP-MS are shown in Table 7.

[0125] Table 7

[0126]

[0127] It is found that no matter the stainless steel tank or the titanium alloy tank, the heavy metals or trace elements are occasionally high, and are mostly concentrated in the first sample of the first day. The nylon itself does not contain heavy metals, so the risk of exceeding the heavy metals or trace elements is greatly reduced.

[0128] 3. New Zealand rabbit intradermal degradation test

[0129] The microparticulate acellular dermal matrix is injected intradermally, the concentration is 100 mg / mL, and the injection volume is 0.2 mL. After the operation, the degradation of the product is determined by B-ultrasound, and the degradation after 1 week, 2 weeks, 4 weeks and 8 weeks is examined, and the results are shown in Table 8. Figures 5-8

[0130] Figures 5-8 ​​It can be seen that the product can still be observed after 8 weeks of intradermal injection of microparticulate acellular dermal matrix, indicating that the product has a long degradation period, ensuring the durability of the product's efficacy. In addition, no adverse reactions such as redness, edema, infection, necrosis, etc. were observed at the injection site during the test period, proving that the product has good biological safety.

[0131] 4. Using mADM for penile dorsal nerve isolation in the treatment of primary premature ejaculation

[0132] Two primary premature ejaculation rat models were screened out by observation method, the fur on the perineum was carefully removed, the surgical area was disinfected with new jieer disinfectant, and the penis was exposed after laying a single, a silk thread was used to fix the glans penis for traction to expose the surgical area, 100 mg / mL mADM was uniformly injected around the dorsal nerve using a 25G needle. Two weeks after the operation, the ejaculation latency was observed. The results are shown in Figure 9 and Table 8.

[0133] Table 8

[0134]

[0135] From Figure 9 The results of Table 8 show that the ejaculation latency of the two rats was significantly prolonged, indicating that the use of mADM for penile dorsal nerve isolation can effectively treat primary premature ejaculation.

Claims

1. A method for the preparation of injectable micronized acellular dermal matrix for the treatment of primary premature ejaculation, characterized by, The method comprises the following steps: (1) obtaining a dermal layer from a dermal matrix raw material from which cells are to be removed, then homogenizing and defatting the dermal layer, and then decellularizing the dermal layer; (2) freezing the product obtained in step (1) at -196°C for 1-2 minutes; (3) taking the product obtained in step (2) and placing it in a cold trap circulating medium comprising anhydrous ethanol or liquid nitrogen, controlling the temperature to be below -50°C, and then ball-milling the product at the temperature using balls of different sizes, with the ball-to-material ratio being controlled to be 1:40, the mass ratio of balls of different sizes being controlled to be 10 mm:5 mm:1 mm=2:2:10, the vibration frequency of the ball-milling being controlled to be 800-1000 times per minute, and the ball-milling time being controlled to be 8-15 minutes; (4) after the ball-milling is completed, sieving the product obtained in step (3), and then directly collecting the product in a sterile collection container using a turbine airflow to form a low-pressure zone, thereby obtaining the injectable microparticulate decellularized dermal matrix.

2. The production method according to claim 1, characterized by, The dermal matrix raw material from which cells are to be removed in step (1) comprises skin, pericardium, lower intestinal mucosa, bladder basement membrane, amniotic membrane, or cornea, and the raw material is derived from humans, pigs, horses, dogs, sheep, cattle, or fish.

3. The preparation method according to claim 1, characterized in that, The homogenization step is to add triton, anhydrous sodium carbonate, and deionized water to the decellularized dermal matrix, and then homogenize the mixture, with the mass ratio of triton, anhydrous sodium carbonate, and deionized water being 1:5-20:250-1000.

4. The production method according to claim 3, characterized by, The homogenization treatment is to homogenize the mixture in a homogenizer at 5000 rpm for 10 minutes.

5. The preparation method according to claim 1, characterized in that, The defatting treatment is to add a defatting solution to the mixture, with the defatting solution comprising at least one of isopropyl alcohol, acetone, ethanol, or dimethylbenzene, and the concentration of the defatting solution being 90-100 wt%, and the defatting treatment step being to shake the mixture on a shaker at 180 rpm for 2 hours.

6. The method of claim 1, wherein, The decellularization treatment step is to add a decellularization solution to the mixture, with the decellularization solution being selected from at least one of Triton X-100, hydroxyethylpiperazine ethanesulfonic acid, polyethylene glycol octylphenyl ether, sodium deoxycholate, sodium dodecylaminopropionate, or fatty alcohol polyoxyethylene ether.

7. The production method according to claim 6, wherein The concentration of the decellularization solution is 0.1-1 wt%, and the decellularization treatment step is to shake the mixture on a shaker at 180 rpm for 4-24 hours.

8. The method of claim 1, wherein, The DNA content of the decellularized product is less than 50 ng / mg.

9. The method of claim 1, wherein, The sieving in step (4) is to pass the product through a 60-100 mesh sieve.

10. The method of claim 1, wherein, The turbine airflow in step (4) is formed by an air blower.

11. An injectable microparticulate decellularized dermal matrix prepared by the method of any one of claims 1-10.

12. Use of injectable micromized acellular dermal matrix according to claim 11 for the preparation of a material for the treatment of primary premature ejaculation, characterized in that, The injectable microparticulate decellularized dermal matrix is prepared into a penile dorsal nerve isolation surgical material.

Images