CN111084900A

CN111084900A - Preparation method and application of acellular fish skin matrix

Info

Publication number: CN111084900A
Application number: CN201811232737.0A
Authority: CN
Inventors: 李东升; 谢则平; 张淑敏; 李诚博; 宫世周; 曲承蕾; 申胜标; 宋国梁
Original assignee: Yantai Lanchuang Biotechnology Co ltd; Shandong International Biotechnology Park Development Co ltd
Current assignee: YANTAI LANCHUANG BIOTECHNOLOGY Co.,Ltd.
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2020-05-01

Abstract

The invention discloses a preparation method of a novel acellular fish skin matrix, which comprises the following steps: 1) washing tilapia skin to remove impurities and other pretreatment; 2) scraping off the scale coat after acid treatment; 3) washing with a surfactant to remove cells; 4) washing with alkali liquor; 5) chemical bleaching agent treatment; 6) drying and sterilizing. The acellular fish skin matrix prepared by the invention has the advantages of low production cost, high thermal stability, no risk of spreading viruses shared by human and livestock and the like, and creatively provides a scale coat removing technology, so that the clinical practicability of the product is greatly improved; and various technologies are comprehensively applied to carry out virus inactivation, thereby improving the biological safety of the product. The invention also discloses application of the acellular fish skin matrix as a wound dressing, which can promote wound healing and epithelization of new tissues.

Description

Preparation method and application of acellular fish skin matrix

Technical Field

The invention belongs to the field of biomedical materials, and relates to a preparation method of a novel acellular fish skin matrix material and application of the acellular fish skin matrix material as a wound dressing.

Background

The correct treatment of the surgical wound is one of the keys of success and failure of surgical operation treatment, and the good healing of the wound is the premise of the recovery of the body function after the wound, so that the research for accelerating the healing of the wound is very important, and the research on the wound dressing is one of the hot spots of the research. With the progress of wound healing research, it is recognized that the purpose of using a dressing is far beyond covering the wound surface, and that the dressing must also aid in wound healing. The development of functional dressings with wound healing promoting or hemostatic activity has therefore become a focus of current research. At present, the wound dressings clinically used in China have more brands, but according to clinical feedback, the nationwide recognition of better hemostasis effect is strong 'fast-growing yarn', but the wound dressings have no functional activity of promoting healing; the acellular dermal matrix or collagen sponge dressing with good healing promoting effect has the effects of hemostasis, tissue repair, healing promotion and the like, and is widely applied clinically, such as bovine-derived collagen sponge, porcine-derived acellular dermal matrix and the like.

A large number of studies and clinical practices show that collagen has good biocompatibility and wound healing promoting activity. However, mammalian collagen still carries the risk of transmitting animal diseases, such as bovine spongiform encephalopathy and foot and mouth disease. In addition, the use of mammalian collagen has been limited for religious reasons. In recent years, aquatic collagen has attracted attention because of its abundance and low cost.

The number of fish collagen medical products which are clinically used at present is small, and the acellular fish skin matrix Kerecis approved by FDA in 2013^TMTakes Atlantic cod as raw materialThe dressing is developed by adopting the patent technology of WO 2011/042794 and is mainly used as a dressing for the wound surface of chronic ulcer. In WO 2011/042794, the method comprises removing impurities from fish skin, cleaning, soaking in sterile PBS containing 50mM ascorbic acid and 500ppm streptomycin to stabilize the three-dimensional structure in matrix, removing cells with deoxycholic acid (or SDS and Triton X-100), digesting with Tris-HCl/trypsin mixed solution, treating with pre-freezing solution, freeze drying, and sterilizing with ethylene oxide.

However, large-scale artificial culture of cod is not realized at present, the traceability of raw materials of wild cod is poor, so that the raw materials for preparing medical products by using cod as a raw material are very limited, and the thermal stability of collagen of cod as a deep-sea fish is poor. In addition, the production process described in WO 2011/042794 is tedious and does not include virus inactivation process steps, so that development of a clean fish collagen product that has a large supply of raw materials and thermal stability and is close to mammals is a problem to be solved in the current fish collagen industrialization.

Disclosure of Invention

The invention aims to research a novel preparation method of acellular fish skin matrix by using tilapia skin as a raw material, and verify the performance of the acellular fish skin matrix as wound dressing.

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

1) washing tilapia skin to remove impurities and other pretreatment;

2) scraping off the scale coat after acid treatment;

3) washing with a surfactant to remove cells;

4) washing with alkali liquor;

5) chemical bleaching agent treatment;

6) drying and sterilizing.

In the above method, particularly in step 1), the pretreatment includes an operation unit for mechanical impurity removal and brine soaking. The saline water is NaCl or KCl water solution with the mass percentage of 1-5%, and the soaking time is 0.5-12 hours.

In the above method, specifically in step 2), before the peeling, an acid treatment is performed, that is, the filter paper is sufficiently soaked with an acid solution, and then the fish skin is spread on the filter paper, so that the side containing the peeling is sufficiently contacted with the filter paper for 1-30 minutes. The acid solution is 0.1-3% acetic acid solution, or 0.1-3% citric acid solution, or 0.1-3% phosphoric acid solution, or any combination of the three. Preferably, the treatment is carried out for 5 to 10 minutes with an acetic acid solution having a concentration of 0.1 to 3%. After the scaling, the fish skin is preserved in a buffer solution with pH 6.0-8.0, preferably a phosphate buffer solution with pH 7.4.

In the above method, specifically in step 3), the surfactant solution is an aqueous solution of 0.1% to 1% of Triton × 100, 0.1% to 1% of tween 80, or 0.1% to 1% of sodium deoxycholate by mass, preferably, 0.1% to 1% of Triton × 100; the material-liquid ratio of the washing is 1:10-1:30(w/v), the oscillation speed is 100-250rpm, and the washing time is 2-48 hours.

In the above method, particularly in step 4), the alkali solution is 0.05M to 0.2M NaOH, 0.05M to 0.2M KOH or 0.05M to 0.2M ammonia solution, preferably 0.05M to 0.2M NaOH; the material-liquid ratio of the alkali liquor washing is 1:10-1:30(w/v), the oscillation speed is 100-250rpm, and the washing time is 2-24 hours.

In the method, in step 5), the chemical bleaching agent is an aqueous solution containing 0.5-5% by mass of hydrogen peroxide or an ethanol solution containing 0.5-5% by mass of peroxyacetic acid, the ratio of the chemical bleaching agent to the treated solution is 1:10-1:30(w/v), the oscillation speed is 0-250rpm, and the treatment time is 1-5 hours. Preferably, the treatment is carried out with an aqueous solution of 0.5-5% hydrogen peroxide for 3 hours.

In the above method, specifically in step 6), the sterilization method is ethylene oxide sterilization, cobalt 60 irradiation sterilization or low-temperature plasma irradiation sterilization. Preferably, ethylene oxide sterilization is used in order to reduce damage to the product from irradiation.

In addition, the application of the acellular fish skin matrix produced by the invention and the application of the acellular fish skin matrix as a wound dressing is also within the protection scope of the invention. The wound dressing has the functional activity of promoting wound healing and epithelization of new tissues.

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 cold water fishes such as cod and the like, the tilapia skin has the advantages of wide source, traceable raw materials, higher thermal stability, better and excellent mechanical property and quality controllability;

(2) compared with the biological material from terrestrial animals, the tilapia skin has no risk of spreading viruses with zoonosis, has no limitation of religious contraindication, has lower production cost and can greatly reduce the medical cost of patients;

(3) the innovative scale coat removing technology integrates acid liquor soaking, mechanical scraping and chemical bleaching treatment, and on the premise of keeping the original mechanical performance of the fish skin, the black scale coat and the residual fish meat and fat are removed more thoroughly, so that the clinical practicability and safety of the product are improved;

(4) the surfactant, strong base and hydrogen peroxide are comprehensively adopted for virus inactivation treatment, so that the risk of residual viruses of the animal-derived medical material is reduced.

Drawings

FIG. 1 is a photograph of a real object of a decellularized fish skin matrix, which is a front photograph.

FIG. 2 is a scanning electron micrograph of the front surface of the acellular fish skin matrix.

FIG. 3 is a scanning electron micrograph of a cross-section of a decellularized fish skin matrix showing a multilayered fibrous structure.

FIG. 4 is a photograph of H & E staining of decellularized fish skin matrix at 40-fold magnification. Collagen fibers were arranged in bundles, and no nuclei were observed to remain therein.

FIG. 5 is a photograph of H & E staining at 40X magnification with some inflammatory cells and new capillaries infiltrated therein, of a decellularized fish skin matrix implanted subcutaneously in rats for 2 weeks.

FIG. 6 is a photograph of H & E staining at 40X magnification of a decellularized fish skin matrix implanted 4 weeks under the skin of rats with some fibroblasts growing into the interior of the sample.

Fig. 7 is a general observation of the experiment of acute wound on the back skin of rats with acellular fishskin matrix, showing wound healing of fishskin acellular matrix dressing, commercially available pigskin dressing and gauze dressing at 1 week, 2 weeks and 3 weeks after surgery.

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, removing scales, residual meat and fat, soaking with 1% NaCl at 15 ℃ for 6 hours at a material-liquid ratio of 1:10(w/v), repeating the steps once, and draining. The step is mainly to remove the stains on the surface of the fish skin by a mechanical method, thereby being beneficial to the subsequent operation.

2. Descaling clothes

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.

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 15 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. Washing with alkaline solution

Adding 0.05M NaOH solution into the fish skin, wherein the feed-liquid ratio is 1:10(w/v), oscillating at 15 ℃ 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

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 15 ℃ and then drained, followed by rinsing 3 times with pure water and draining. This step, in addition to the bleaching function, also serves a certain virus inactivation.

6. Post-treatment

Freeze-drying the fish skin in a vacuum freeze-drying machine for 48 hours, and finally sterilizing by ethylene oxide.

Example 2A method for preparing a acellular fish skin matrix

1. Pretreatment

Taking 100g of frozen tilapia skin, thawing, soaking with 5% NaCl at 10 ℃ for 0.5 hour, the material-liquid ratio is 1:30(w/v), repeating the steps once, and draining.

2. Descaling clothes

Firstly, taking a piece of filter paper to be fully soaked in 3% citric acid solution, then spreading fish skin on the filter paper to enable one side containing the scale to be fully contacted with the filter paper, taking out after 1 minute, scraping the black scale with a knife until the scale is basically scraped clean, rinsing with pure water for 3 times, and temporarily storing in MES buffer solution with pH 6.0.

3. Surfactant washing

Adding 1% Tween 80(w/w) solution into fish skin at a ratio of 1:30(w/v) at 10 deg.C, shaking at 250rpm for 2 hr, draining, rinsing with pure water for 3 times, and draining.

4. Washing with alkaline solution

Adding 0.2M KOH solution into the fish skin, wherein the material-liquid ratio is 1:30(w/v), oscillating at 10 ℃ and 250rpm for 2 hours, and draining; rinsed 3 times with purified water and drained again.

5. Bleaching

Adding 5% hydrogen peroxide solution into fish skin at a ratio of 1:30(w/v), standing at 10 deg.C for 1 hr, draining, rinsing with pure water for 3 times, and draining.

6. Post-treatment

Freeze-drying the fish skin in a vacuum freeze-drying machine for 36 hours, and finally performing irradiation sterilization by cobalt 60 to obtain the fish skin.

Example 3A method for preparing a acellular fish skin matrix

1. Pretreatment

Taking 100g of new tilapia skin, removing scales, residual meat and fat, soaking with 3% KCl 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, taking a piece of filter paper to be fully soaked in 0.6% acetic acid solution, then spreading fish skin on the filter paper, fully contacting one side containing the scaly clothing with the filter paper, taking out after 5 minutes, scraping the black scaly clothing by using a knife until the scaly clothing is basically scraped clean, rinsing with pure water for 3 times, and temporarily storing in Tris-HCl buffer solution with the pH value of 8.0.

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. Washing with alkaline solution

Adding 0.1M ammonia water solution into fish skin at a feed-liquid ratio of 1:20(w/v), oscillating at 175rpm for 12 hours at 20 ℃, and draining; then rinsed 3 times with pure water and drained.

5. Bleaching

Adding ethanol solution containing 1% peroxyacetic acid into fish skin at a ratio of 1:20(w/v), treating at 20 deg.C for 5 hr, draining, rinsing with pure water for 3 times, and draining.

6. Post-treatment

Freeze-drying the fish skin in a vacuum freeze-drying machine for 72 hours, and finally sterilizing by low-temperature plasma irradiation.

Example 4 basic Performance assays of acellular Fish skin matrices

The acellular fish skin matrix prepared by the invention is odorless, tasteless and white flake (figure 1), and shows that black scales and stains of a sample are thoroughly removed after the scales are scraped by acid treatment and chemically bleached; scanning electron micrographs of sample cross sections revealed multilayer fiber architecture (fig. 2); the samples were paraffin embedded, sectioned, H & E stained and their loose and ordered arrangement of collagen fibers in the samples was observed without residual nuclei (fig. 3); the DNA residue was determined according to the method specified in YY/T0606.25-2014, resulting in 1.39. + -. 0.69ng residual DNA per mg of dry sample.

Example 5 comparison of thermal stability and mechanical Properties of different acellular dermal matrices

The tilapia skin was replaced by cod skin, a decellularized cod skin matrix was prepared by the method described in example 1, and the performance advantages of the tilapia skin decellularized matrix prepared in example 1 were examined by using a commercially available pigskin dressing as a control:

1. and (3) denaturation temperature: measuring with differential scanning calorimetry, accurately weighing 5mg sample, thoroughly wetting with normal saline, placing in aluminum crucible, covering, sealing, heating from 20 deg.C to 80 deg.C with empty crucible as reference, and measuring with temperature rate of 2 deg.C/mi n and nitrogen flow of 20mL/mi n.

2. Tensile strength: 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.

The results show that (table 1) the denaturation temperature and the mechanical strength of the tilapia mossambica sample are obviously higher than those of the cod sample and are very close to those of the pigskin dressing, which shows that the tilapia mossambica skin has certain advantages as a raw material of an acellular dermal matrix in a plurality of aquatic products and is expected to replace a pigskin dressing product.

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

Example 6 evaluation of biocompatibility of acellular fishskin matrix

The acellular fish skin matrix was evaluated for cytotoxicity against L929 fibroblasts, hemolysis against rabbit ear arterial blood, and local reactions to subcutaneous implantation in the back of SD rats according to the method described in GB/T16886; the results showed that the cytotoxicity was grade 0, the hemolysis rate was 1.2%, the inflammatory response was mild at 2 weeks of subcutaneous implantation in rats, a large number of neovascularization occurred (FIG. 5), a large number of fibroblasts infiltrated the interior of the sample at 4 weeks of implantation (FIG. 6), and the cells were completely absorbed at 12 weeks of implantation. Therefore, the acellular fish skin matrix prepared by the invention has good biocompatibility and meets the regulatory requirements of dressing medical instruments.

Example 7 rat dorsal skin acute wound repair wound animal experiment with acellular fishskin matrix

In the experiment, the acellular fish skin matrix prepared in the embodiment 1 is soaked in sterile normal saline for 10min in advance, and is cut into 2cm multiplied by 2cm slices for use when being implanted. And a product on the market of the pigskin dressing is used as a positive control, and the sterile gauze dressing is used as a negative control.

The specific operation is as follows: 18 SD rats are male, the weight is 200-. After operation, 6 animals of the corresponding group were sacrificed at three time points (1 week, 2 weeks, 3 weeks), the wound area was measured to calculate the wound healing rate, and skin tissue of the wound area was taken and H & E staining was used to measure the neoepithelium length.

The experimental result shows that compared with the pigskin dressing and the gauze dressing, the acellular fish skin matrix dressing group has the advantages that the inflammatory response is slight, the arrangement of collagen fibers is orderly, the swelling degree of granulation tissues at the 1 st week is the lowest, the newly born epithelia at the 2 nd week is the most, the wound healing rate at the 3 rd week is 97.72%, the wound healing rate is obviously superior to 86.65% of the gauze dressing group and is equivalent to 97.27% of the pigskin dressing group, the wound healing promoting effect is very obvious in the final stage of wound (fig. 7), and the clinical development value is high.

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.

Claims

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

1) washing tilapia skin to remove impurities and other pretreatment;

2) scraping off the scale coat after acid treatment;

3) washing with a surfactant to remove cells;

4) washing with alkali liquor;

5) chemical bleaching agent treatment;

6) drying and sterilizing.

2. The method of claim 1, wherein: in the step 1), the pretreatment comprises an operation unit for mechanical impurity removal and brine soaking.

3. The method of claim 2, wherein: the saline water is NaCl or KCl water solution with the mass percentage of 1-5%, and the soaking time is 0.5-12 hours.

4. The method of claim 1, wherein: in the step 2), before the scale is scraped, acid treatment is carried out, namely, the water-absorbable material is fully soaked by acid solution, then the fish skin is spread on the water-absorbable material, and the side containing the scale is fully contacted with the filter paper for 1-30 minutes.

5. The method of claim 4, wherein: the material capable of absorbing water is filter paper; the acid solution is 0.1-3% acetic acid solution, citric acid solution or phosphoric acid solution, or any combination of the three.

6. The method of claim 1, wherein: in step 2), after scaling, the fish skin is preserved in a buffer solution with pH value of 6.0-8.0.

7. The method of claim 1, wherein: in the step 3), the surfactant solution is 0.1-1% aqueous solution of Triton X100, Tween 80 or sodium deoxycholate by mass percent, the washing material-liquid ratio is 1:10-1:30(w/v), the oscillation speed is 100-.

8. The method of claim 1, wherein: in the step 4), the alkali liquor is NaOH, KOH or ammonia water solution with the molar concentration of 0.05M-0.2M, the material-liquid ratio of the alkali liquor washing is 1:10-1:30(w/v), the oscillation speed is 100-250rpm, and the washing time is 2-24 hours.

9. The method of claim 1, wherein: in the step 5), the chemical bleaching agent is an aqueous solution of hydrogen peroxide with the mass percentage of 0.5-5% or an ethanol solution of peroxyacetic acid with the mass percentage of 0.5-5%, the ratio of the chemical bleaching agent to the treated material liquid is 1:10-1:30(w/v), the oscillation speed is 0-250rpm, and the treatment time is 1-5 hours.

10. The method of claim 1, wherein: in step 6), the sterilization method is ethylene oxide sterilization.

11. Use of the acellular fish skin matrix prepared according to the method of claim 1 in wound dressings.

12. Use according to claim 11, characterized in that: the wound dressing has the functions of promoting wound healing and new epithelization.