阅读说明：本技术 一种脱细胞真皮基质及其制备方法 (Acellular dermal matrix and preparation method thereof ) 是由 李东升 谢则平 邵月华 张淑敏 李诚博 宫世周 曲承蕾 申胜标 宋国梁 韩梦禹 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种脱细胞真皮基质及其制备方法,该脱细胞真皮基质以罗非鱼皮为原材料,经过去除鳞衣、脱细胞、溶液保存、辐照灭菌等工艺制备而成。制备方法包括：1)刮除鱼鳞、残肉及脂肪等杂质；2)化学溶胀后采用片皮机刮除鳞衣层；3)采用表面活性剂和酶溶液洗涤；4)漂白与消毒；5)采用含有抗氧化剂的高盐溶液浸泡,进行保存液置换；6)低温下低剂量辐照灭菌。本发明产品无需冷冻干燥处理,生产成本低,易于规模化生产,并且去除鳞衣工艺可以高效率、较彻底地除去黑色杂质,大大改善产品外观、提高产品的患者接受度。本发明同时公开了该保存液配方,该保存液可以降低辐照对产品性能的负面影响,确保产品在常温运输与保存下质量稳定。(The invention discloses an acellular dermal matrix and a preparation method thereof, wherein the acellular dermal matrix is prepared by taking tilapia skin as a raw material through the processes of removing scales, acellular preservation, solution preservation, irradiation sterilization and the like. The preparation method comprises the following steps: 1) removing fish scales, residual meat, fat and other impurities; 2) scraping the scale coating layer by using a peeling machine after chemical swelling; 3) washing with surfactant and enzyme solution; 4) bleaching and sterilizing; 5) soaking in high-salt solution containing antioxidant for replacement of the preservation solution; 6) sterilizing by low-dose irradiation at low temperature. The product of the invention does not need freeze drying treatment, has low production cost and easy large-scale production, and the process for removing the scale coat can remove black impurities more thoroughly with high efficiency, greatly improve the appearance of the product and improve the patient acceptance of the product. The invention also discloses a formula of the preservative fluid, which can reduce the negative influence of irradiation on the performance of products and ensure the stable quality of the products during normal-temperature transportation and storage.)

1. A method for preparing a tilapia skin acellular dermal matrix specifically comprises the following steps:

1) pretreatment: scraping off fish scales, residual meat, fat and other impurities on the surface of tilapia skin;

2) scraping scale: scraping the scale coating layer by using a peeling machine after chemical swelling, and then soaking the scale coating layer in a buffer solution;

3) and (3) cell removal: adopting a surfactant and a nuclease solution to wash by oscillation;

4) bleaching treatment: bleaching and disinfecting the fish skin by adopting a chemical bleaching agent;

5) replacement of the preservation solution: soaking the raw materials in a preservation solution containing an antioxidant and high-concentration salt;

6) and (3) post-treatment: the sterilization treatment is carried out at a lower temperature ranging from-50 ℃ to 8 ℃ with a lower irradiation dose ranging from 10kGy to 20 kGy.

2. The method of claim 1, wherein: in the step 2), the chemical swelling treatment is to soak the fish skin in a glycerol solution and treat the fish skin for 2 to 24 hours at a temperature of between 4 and 20 ℃; then spraying the fish skin with an acidic solution or an alkaline solution on the outer side of the fish skin, fully contacting for 5-30 minutes, and scraping the black scale coating layer on the surface of the fish skin by using a skin stripping machine.

3. The method of claim 2, wherein: the acid solution is 0.1-3% acetic acid solution, citric acid solution or hydrochloric acid solution, and the alkaline solution is 0.05-0.2M NaOH, KOH or ammonia water solution.

4. The method of claim 1, wherein: in the step 2), after the scale coat is scraped, the fish skin is placed in a buffer solution with the pH value of 6.0-8.0 to restore the fish skin to a compact state before swelling; or in the step 3), the nuclease solution is 20-200U/mL nuclease solution, the nuclease treatment solution ratio is 1:10-1:30(w/v), the oscillation speed is 100-, the dose of the low dose radiation ranges from 10kGy to 20 kGy.

5. The method of claim 4, wherein: the antioxidant in the preservation solution is D-sodium erythorbate, or sodium ascorbate, or a mixture of the two in any proportion; the chloride is sodium chloride, potassium chloride, calcium chloride or a mixture of the three in any proportion.

6. A tilapia skin acellular dermal matrix is characterized in that: the tilapia skin is used as a raw material and is prepared by the method of claim 1.

7. A preservation solution for an acellular dermal matrix, characterized in that: it contains antioxidants and high concentrations of chloride salts.

8. The preservation solution according to claim 7, characterized in that: the antioxidant is D-sodium erythorbate, or sodium ascorbate, or a mixture of the two in any proportion; the chloride is sodium chloride, potassium chloride, calcium chloride or a mixture of the three in any proportion; the concentration of the chloride is 100mg/mL-350 mg/mL.

Technical Field

The invention belongs to the field of biomedical materials, and relates to a novel tilapia skin acellular fish skin matrix material, a preparation method thereof and a preservation solution used in the preparation process.

Background

Fish gelatin raw material has received increasing attention in recent years due to its good biocompatibility and no risk of zoonotic viral transmission. Patent WO 2011/042794 proposes a decellularized fish skin matrix which is prepared from cod skin as a raw material by decellularization with a chemical reagent, digestion with trypsin, freeze-drying and sterilization with ethylene oxide. The process has the advantages that large-scale artificial culture of the cod is not realized, the raw material source is limited, the requirement of a freeze drying process on equipment is high, and large-scale production is difficult. Patent CN108355172A describes a preparation method of a tilapia skin acellular matrix, but the production process is complicated, no black scale coat removing process step is provided, and the protective solution is physiological buffer solution, cell culture solution, tissue preservation solution, hyaluronic acid solution, chitosan solution, dextran solution, glycerol and other reagents, and it is not verified whether the protective solution can be used for normal temperature preservation of wet samples. Therefore, the development of a clinical instant acellular dermal matrix material which has a large amount of raw material supply, is easy for scale enlargement of the production process, can remove black scales, can be directly stored at normal temperature without freeze drying, and becomes a problem to be solved urgently in the industrialization of the medical collagen at present.

Disclosure of Invention

The invention aims to research a novel acellular fish skin matrix by using tilapia skin as a raw material. The acellular fish skin matrix has excellent thermal stability without cross-linking with chemical reagents, does not need to be dried in the production process, and can be transported and stored at normal temperature. The product can be used as wound dressing, surgical patch, soft tissue filling and repairing material, etc., and has wide clinical application prospect.

The invention discloses a method for preparing a tilapia skin acellular dermal matrix, which specifically comprises the following steps:

1) pretreatment: scraping off fish scales, residual meat, fat and other impurities on the surface of tilapia skin;

2) scraping scale: scraping the scale coating layer by using a peeling machine after chemical swelling, and then soaking the scale coating layer in a buffer solution;

3) and (3) cell removal: adopting a surfactant and a nuclease solution to wash by oscillation;

4) bleaching treatment: bleaching and disinfecting the fish skin by adopting a chemical bleaching agent;

5) replacement of the preservation solution: soaking the raw materials in a preservation solution containing an antioxidant and high-concentration salt;

6) and (3) post-treatment: and (4) carrying out sterilization treatment at low temperature by adopting lower irradiation dose.

In the method, in step 2), the chemical swelling treatment is to soak the fish skin in a glycerol solution and treat the fish skin at 4-20 ℃ for 2-24 hours; then spraying the fish skin with an acidic solution or an alkaline solution on the outer side of the fish skin, fully contacting for 5-30 minutes, and scraping the black scale coating layer on the surface of the fish skin by using a skin stripping machine. The acid solution is 0.1-3% acetic acid solution, citric acid solution or phosphoric acid solution, and the alkaline solution is 0.05-0.2M NaOH, KOH or ammonia water solution. After the scale coat is scraped, the fish skin is placed in a buffer solution with the pH value of between 6.0 and 8.0 to restore the fish skin to a compact state before swelling.

In the above method, specifically in step 3), the nuclease solution is a nuclease solution with a concentration of 20-200U/mL, the nuclease treatment feed-liquid ratio is 1:10-1:30(w/v), the oscillation speed is 100-250rpm, and the washing time is 2-24 hours.

In the above method, particularly in step 4), the chemical bleaching agent is an aqueous solution of 0.5-5% by mass of hydrogen peroxide or an ethanol solution of 0.5-5% by mass of peracetic acid, preferably, an aqueous solution of 3% by mass of hydrogen peroxide; wherein the ratio of the chemical bleaching agent to the material liquid is 1:10-1:30(w/v), and the treatment time is 1-6 hours.

In the above method, specifically in step 5), the solution used for the replacement of the preservation solution contains an antioxidant at a concentration of 0.2mg/mL-5mg/mL and a chloride at a concentration of 100mg/mL-350 mg/mL. The antioxidant is D-sodium erythorbate, or sodium ascorbate, or a mixture of the two in any proportion; the chloride is sodium chloride, potassium chloride, calcium chloride or a mixture of the three in any proportion.

In the above method, particularly in step 6), the temperature of the low-temperature irradiation ranges from-50 ℃ to 8 ℃, preferably, the temperature is-20 ℃; the dose of the low dose radiation is in the range of 10kGy to 20kGy, preferably the dose is 12 kGy.

The invention also discloses a tilapia skin acellular dermal matrix which is prepared by taking tilapia skin as a raw material through the processes of mechanical scale removal, nuclease acellular treatment, high-concentration salt solution preservation, low-temperature low-dose irradiation sterilization and the like.

In addition, a preservation solution of the acellular dermal matrix is also within the scope of the present invention. The preservation solution contains antioxidant with pH value close to neutral and high-concentration chloride salt. The antioxidant is D-sodium erythorbate, or sodium ascorbate, or a mixture of the two in any proportion; preferably, the antioxidant is sodium D-erythorbate; the chloride is sodium chloride, potassium chloride, calcium chloride or a mixture of the three in any proportion; preferably, the chloride is sodium chloride; further, the chloride is present at a concentration of 100mg/mL to 350mg/mL, preferably at a concentration of 200 mg/mL.

In a word, the preparation method of the acellular fishskin matrix of tilapia fishskin provided by the invention has the following technical advantages:

(1) compared with the traditional decellularization technology, the invention comprehensively adopts the technologies of enzyme method treatment, irradiation protective agent, low-temperature irradiation and the like, and the prepared wet tilapia skin decellularization matrix has higher thermal stability, does not need freeze drying treatment and is easy for large-scale production;

(2) the innovative black scale removing technology comprehensively applies swelling treatment and automatic equipment scraping technology, the fish skin becomes thick and the black scale becomes soft after swelling, the operation of a splitting machine is easy, and the fish skin can be restored to a compact state after being placed in a buffer solution after being split. The process can remove the black scale coat with high efficiency and more thoroughly on the premise of keeping the original mechanical properties of the fish skin, greatly improve the appearance of the product and improve the patient acceptance of the product;

(3) the preservation solution of the high-concentration salt composite antioxidant is developed, the formula is simple and easy to obtain, the high-concentration salt can ensure that bacteria are not easy to breed before the product is sterilized, the solution cannot be frozen in the low-temperature sterilization process, the damage to collagen caused by repeated freeze thawing can be avoided, and the better heat stability of the sample can be maintained after the sterilization. And the antioxidant is used as an irradiation protective agent to reduce the oxidative damage of free radicals to products. Therefore, the product prepared by the process does not need cold chain management and can be transported and stored at normal temperature;

(4) compared with the biological material of terrestrial animal source such as acellular pigskin or cow hide, the acellular tilapia skin has no risk of spreading viruses of zoonosis, has lower production cost, does not need hydration before use compared with a freeze-dried preparation formulation, is more convenient for clinical use, and is easy to popularize and apply in large clinical range.

Detailed Description

The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified. Unless otherwise specified, the concentration of a solution prepared with a liquid is expressed in terms of volume ratio (v/v), the concentration of a solution prepared with a solid is expressed in terms of weight to volume ratio (w/v), and the unit form of the mass to volume ratio described herein is g/mL.

Example 1A method for preparing a acellular fish skin matrix

1. Pretreatment

Taking 100g of fresh tilapia skin, scraping off fish scales, residual meat and fat by using a knife, soaking for 6 hours at 10 ℃ by using 1% NaCl, wherein the material-liquid ratio is 1:10(w/v), repeating the steps once, and draining.

2. Descaling clothes

Firstly, soaking fish skin in enough 50 percent of glycerol solution, treating for 6 hours at 10 ℃ to fully swell the fish skin, and wiping off the glycerol solution on the surface; then spreading the fish skin on an operation table, spraying 0.1% acetic acid solution on the outer side of the fish skin by using an atomizer, fully contacting for 15 minutes, scraping a black scale coating layer on the surface of the fish skin by using a skin stripping machine, rinsing for 3 times by using pure water, and temporarily storing in phosphate buffer solution with pH7.4 to restore the compact state before swelling.

3. Surfactant washing

Draining the fish skin, adding 0.1% Triton X100 (w/w) solution at a ratio of 1:10(w/v), shaking at 10 deg.C and 100rpm for 24 hr, draining, rinsing with pure water for 3 times, and draining. This step can serve as a decellularization.

4. Enzymatic decellularization

Adding 50U/mL DNA enzyme solution into fish skin, at a feed-liquid ratio of 1:10(w/v), oscillating at 10 ℃ and 100rpm for 24 hours, and draining; rinsed 3 times with purified water and drained again. This step can be used for removing cells and antigenic substances such as nucleic acid.

5. Bleaching

A0.5% hydrogen peroxide solution was added to the skin of a fish in a feed-to-solution ratio of 1:10(w/v), and the mixture was shaken at 100rpm for 3 hours at 10 ℃ and then drained, followed by rinsing 3 times with a phosphate buffer solution having a pH of 7.4 and draining. Besides the bleaching function, the step also plays a certain role in sterilization and virus inactivation.

6. Replacement of preservative fluid

The preserving fluid is prepared by adopting water for injection, and the formula is as follows: 200mg/mL sodium chloride, 1mg/mL sodium ascorbate. Placing the fish skin treated in the step 5 in sufficient preservation solution, oscillating for 3 hours at 10 ℃ and 100rpm, and draining; repeating the above steps for 3 times to fully replace the interior of the sample with the preservation solution.

7. Post-treatment

Cutting fish skin into required size, sealing with medical packaging bag, placing in a medical incubator at-12 deg.C to-18 deg.C, and sterilizing by cobalt 60 irradiation with 15kGy dose.

Example 2A method for preparing a acellular fish skin matrix

1. Pretreatment

Taking 100g of frozen tilapia skin for thawing, soaking with 5% NaCl at 4 ℃ for 0.5 hour, the material-liquid ratio is 1:30(w/v), repeating the steps once, and draining.

2. Descaling clothes

Firstly, soaking fish skin in sufficient 10 percent of glycerol solution, treating for 24 hours at 4 ℃ to fully swell the fish skin, and wiping off the glycerol solution on the surface; then spreading the fish skin on an operation table, spraying 3% citric acid solution on the outer side of the fish skin by using an atomizer, fully contacting for 5 minutes, scraping the black scale coating layer on the surface of the fish skin by using a skin stripping machine, rinsing for 3 times by using pure water, and temporarily storing in MES buffer solution with pH6.0 to restore the compact state before swelling.

3. Surfactant washing

Adding 1% Tween 80(w/w) solution into fish skin at a ratio of 1:30(w/v) at 4 deg.C, shaking at 250rpm for 2 hr, draining, rinsing with pure water for 3 times, and draining.

4. Enzymatic decellularization

Adding 20U/mL nuclease solution into the fish skin, wherein the feed-liquid ratio is 1:30(w/v), oscillating at 4 ℃ and 250rpm for 2 hours, and draining; rinsed 3 times with purified water and drained again. This step can be used for removing cells and antigenic substances such as nucleic acid.

5. Bleaching

Adding 5% hydrogen peroxide solution into fish skin at a ratio of 1:30(w/v), standing at 4 deg.C for 6 hr, draining, rinsing with MES buffer solution (pH 6.0) for 3 times, and draining. Besides the bleaching function, the step also plays a certain role in sterilization and virus inactivation.

6. Replacement of preservative fluid

The preserving fluid is prepared by adopting water for injection, and the formula is as follows: 100mg/mL potassium chloride, 0.2mg/mL sodium ascorbate. Placing the fish skin treated in the step 5 in sufficient preservation solution, oscillating at the temperature of 4 ℃ and the rpm of 250 for 0.5 hour, and draining; repeating the above steps for 3 times to fully replace the interior of the sample with the preservation solution.

7. Post-treatment

Cutting fish skin into required size, sealing with medical packaging bag, placing in medical incubator at 2-8 deg.C, and sterilizing by cobalt 60 irradiation with dosage of 10 kGy.

Example 3A method for preparing a acellular fish skin matrix

1. Pretreatment

Taking 100g of fresh tilapia skin, removing scales, residual meat and fat, soaking with 3% NaCl at 20 ℃ for 12 hours at a feed-liquid ratio of 1:20(w/v), repeating the steps once, and draining.

2. Descaling clothes

Firstly, soaking fish skin in sufficient 80% glycerol solution, treating for 2 hours at 20 ℃ to fully swell the fish skin, and wiping off the glycerol solution on the surface; then spreading the fish skin on an operation table, spraying 0.4% sodium hydroxide solution on the outer side of the fish skin by using an atomizer, fully contacting for 30 minutes, scraping a black scale coating layer on the surface of the fish skin by using a skin stripping machine, rinsing for 3 times by using pure water, and temporarily storing in Tris-HCl buffer solution with pH of 8.0 to recover the compact state before swelling.

3. Surfactant washing

Adding 0.3% sodium deoxycholate solution into fish skin at a ratio of 1:20(w/v) at 20 deg.C, shaking at 175rpm for 48 hr, draining, rinsing with pure water for 3 times, and draining.

4. Enzymatic decellularization

Adding 200U/mL nuclease solution into the fish skin, wherein the feed-liquid ratio is 1:20(w/v), shaking at 175rpm at 20 ℃ for 6 hours, and draining; rinsed 3 times with purified water and drained again. This step can be used for removing cells and antigenic substances such as nucleic acid.

5. Bleaching

Adding ethanol solution containing 1% peroxyacetic acid into fish skin at a feed-liquid ratio of 1:20(w/v), treating at 20 deg.C for 1 hr, draining, rinsing with Tris-HCl buffer solution (pH 8.0) for 3 times, and draining. Besides the bleaching function, the step also plays a certain role in sterilization and virus inactivation.

6. Replacement of preservative fluid

The preserving fluid is prepared by adopting water for injection, and the formula is as follows: 350mg/mL calcium chloride and 5mg/mL sodium D-erythorbate. Placing the fish skin treated in the step 5 in sufficient preservation solution, oscillating at the temperature of 20 ℃ and the rpm of 175 for 1 hour, and draining; repeating the above steps for 3 times to fully replace the interior of the sample with the preservation solution.

7. Post-treatment

Cutting fish skin into required size, sealing with medical packaging bag, placing in a medical incubator at-44 deg.C to-50 deg.C, and sterilizing by electron beam irradiation with 20kGy dose.

Comparative example 1 preparation of tilapia skin acellular dermal matrix by using conventional acellular process

This comparative example was designed in particular for the purpose of examining the effect of the mechanical descaling, enzymatic decellularization and preservation solution employed in the present invention. By referring to the traditional production process adopted in patent 201811232737.0 and making slight modification, the method adopts manual scale scraping, alkaline washing for removing cells and direct radiation sterilization without adding protective liquid.

1. Pretreatment

Same as in step 1 of example 1.

2. Manual descaling

First, a piece of filter paper is taken to be fully soaked in 0.1% acetic acid solution, then fish skin is spread on the filter paper, the side containing the scaly clothing is fully contacted with the filter paper, the fish skin is taken out after 15 minutes, the black scaly clothing is scraped by a knife until the scaly clothing is basically scraped clean, and the fish skin is temporarily stored in phosphate buffer solution with pH7.4 after being rinsed for 3 times by pure water. With this manual skinning process, it took about 3 hours for 2 employees to process 100 fish skins, whereas with the mechanized process described in example 1, skinning with a skinning machine was completed in 1 hour.

3. Surfactant washing

Same as in step 3 of example 1.

4. Alkaline decellularization

Adding 0.05M NaOH solution into fish skin, at a feed-liquid ratio of 1:10(w/v), oscillating at 10 ℃ and 200rpm for 24 hours, and draining; rinsed 3 times with purified water and drained again. The step can play a role in decellularizing, removing antigen substances such as hybrid protein and the like, inactivating viruses and the like.

5. Bleaching

Same as step 5 of example 1.

6. Post-treatment

Packaging and irradiation sterilization are directly carried out without a preservative solution replacement link, and the specific process is the same as the step 7 of the example 1.

Comparative example 2 preparation of tilapia skin acellular dermal matrix by conventional sterilization process

This comparative example was designed in order to examine the effect of low-temperature, low-dose radiation sterilization. Namely, the sample is irradiated and sterilized at normal temperature, and the sterilization dosage adopts 25kGy commonly used in the medical industry. Specifically, steps 1 to 6 are the same as the corresponding processing steps of embodiment 1. And the step 7 post-processing method is adjusted as follows: cutting the fish skin into required size, sealing with a medical packaging bag, and placing in a medical incubator at normal temperature for 25kGy dosage of cobalt 60 irradiation sterilization.

Example 4 comparison of the Performance of the acellular dermal matrix of different Processes

The methods of the invention of example 1, example 2, example 3, comparative example 1 and comparative example 2 are respectively adopted to prepare the tilapia acellular dermal matrix, and the tilapia acellular dermal matrix is subjected to performance test:

1. and (3) detecting the residual quantity of the DNA: DNA residues were detected by the method defined in YY/T0606.25-2014.

2. And (3) appearance inspection: for each sample, 6 specimens (n ═ 6) were selected, the surface color was observed under normal lighting, and scored by: the total white, non-mottled spots are scored as 0, the few mottled spots are scored as 1, a certain number of mottled spots are scored as 2, and the many mottled spots are scored as 3. The lower the score, the better the removal of black scale.

3. Temperature of thermal denaturation: measuring with differential scanning calorimetry, shearing 5mg sample, wetting with physiological saline, placing in aluminum crucible, sealing, heating from 20 deg.C to 80 deg.C with empty crucible as reference, heating at temperature rate of 5 deg.C/mi n, and measuring with nitrogen flow of 20mL/mi n.

4. Determination of thermal stability: the three samples were placed in a 37 ℃ environment for stability testing and after 6 months the thermal denaturation temperature was determined again using the differential scanning calorimetry method described above.

5. Mechanical properties: the sample was cut into a dumbbell-shaped specimen, sufficiently wetted with physiological saline, and the tensile strength was measured on an electronic universal tester with the tensile speed set at 15mm/mi n.

TABLE 1 comparison of acellular skin matrix Performance of Tilapia and cod

The results show (table 1) that the properties of the samples of example 1, example 2 and example 3 of the present invention are close and have no significant difference, which indicates that the preparation method of the present invention can keep the quality of the product stable and controllable within a certain parameter range. In addition, the DNA residual quantity of the product has no significant difference with that of the product in the comparative example 1, and the DNA residual quantity meets the limit of 50ng/mg accepted by the industry, which shows that compared with the alkaline process, the enzymatic process can also achieve good effect of removing nucleic acid residues; the product prepared by the invention has an appearance inspection score which is obviously lower than that of the sample of comparative example 1 (p is less than 0.05), and the black scale removing effect is better; in addition, the thermal denaturation temperature and the tensile strength are obviously superior to those of the two comparative examples, which shows that the process of the invention can protect the thermodynamic and mechanical properties of the product to the maximum extent; the thermal stability experiment also verifies that after the sample is placed in a 37 ℃ environment for 6 months, the thermal denaturation temperature of the sample is not changed greatly, which shows that the thermodynamic property of the sample is quite stable, while the thermal denaturation temperatures of the other two comparative examples are obviously reduced, and the sample may have protein degradation behavior.

While particular embodiments of the present invention have been described, it will be understood by those skilled in the art that various changes and substitutions may be made thereto without departing from the spirit and scope of the invention. Such modifications and substitutions are intended to be included within the scope of the present invention as defined by the appended claims.