CN112618799B

CN112618799B - Fish skin acellular dermal matrix and preparation method and application thereof

Info

Publication number: CN112618799B
Application number: CN202011580520.6A
Authority: CN
Inventors: 马通; 孙文全
Original assignee: University of Shanghai for Science and Technology
Current assignee: Beijing Ruijian Gaoke Biotechnology Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2022-05-27
Anticipated expiration: 2040-12-28
Also published as: CN112618799A

Abstract

The invention provides a fish skin acellular dermal matrix and a preparation method and application thereof, the preparation method comprises the steps of pretreating fresh fish skin, cleaning, disinfecting, acellular treatment, virus inactivation, heavy metal removal cleaning, protective solution treatment, terminal sterilization and the like, and the preparation method of the fish skin acellular dermal matrix material does not use a chemical cross-linking agent, does not need to introduce an organic solvent, can effectively control pollution risks of pyrogen, bacteria and the like, and has strong controllability of the preparation process; in addition, the acellular fish skin matrix material of the invention well retains the natural three-dimensional structure of the tissue, and can provide a good bionic microenvironment for the regeneration of cells or tissues; meanwhile, the good mechanical property and flexibility of the acellular matrix material can be kept, and the clinical requirements can be better met.

Description

Fish skin acellular dermal matrix and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a fish skin acellular dermal matrix and a preparation method and application thereof.

Background

Extracellular matrix materials (ECM) of homogeneous and heterogeneous tissues containing collagen as a main component have been used for repairing soft tissue defects and wounds in clinical medicine, and are widely used for the construction of tissue organs and organoids in the fields of tissue engineering and regenerative medicine. The application range of the material is wider and wider, and the demand is increased continuously. At present, various ECM products mainly use tissues and organs derived from terrestrial mammals such as pigs, cows and sheep and donated from human remains as raw materials. Allogenic ECM materials have been the gold standard for such materials, and have the ability to direct in situ tissue regeneration, help repair tissue defects and wounds after implantation into the human body. But the source of the variant is very limited, which can not meet the requirement and the price is expensive; at the same time, allogeneic tissues have their disadvantages (e.g., certain pathogens may be present), and therefore, strict screening of the donor source of remains is required to eliminate the possibility of the presence of infectious viruses (e.g., HIV, hepatitis C, etc.). The xenogeneic mammal ECM material has the advantages of easily obtained materials and controllable breeding and cultivation, but is accompanied with the problems of a plurality of potential risks of pathogen transmission such as Bovine Spongiform Encephalopathy (BSE), Transmissible Spongiform Encephalitis (TSE) and foot-and-mouth disease (FMD) and the limitation of various factors. In addition to ECM materials derived from allogeneic and xenogeneic tissue organs, ECM derived from collagen-rich fish-derived tissues (e.g., fish dermis and bleb) is becoming a research hotspot in this field as an alternative source for mammals.

The aquaculture industry also occupies an important position in the protein food industry, and a large amount of waste leftovers such as fish skin, fish bones, fish scales and the like generated in the aquaculture industry processing process cause resource waste and also cause the problems of environmental pollution and the like if the waste leftovers are not utilized. The fish skin contains abundant collagen, so that the fish skin is converted into a biomedical material with high added value, the economic value of the fish is improved, and the environmental pollution is reduced. Therefore, the development of collagen which can be used for biomedical materials from aquatic animals has wide application prospect.

At present, many research reports on methods for extracting collagen from fish and animals exist, the methods mainly comprise hot water extraction, acid extraction, alkaline extraction, salt extraction, enzymatic extraction and the like, and the extracted collagen is widely used as health food and some medical collagens. The research on the in vitro physical and chemical characterization of the extracted medical fish collagen shows that the fish collagen can be used as the physical and chemical basis of biomedical materials; the results of culture of in vitro osteoblasts and fibroblasts, rat subcutaneous animal experiments, biological evaluation of ISO-10993 series and the like also prove the biological safety, biocompatibility and biodegradation performance of the fish collagen. (references 1 to 5). However, collagen produced using these methods lacks the native tissue three-dimensional structure of the ECM; furthermore, since fish sources live in water, the thermal stability of the fish tissue ECM material itself is weak. Compared with the terrestrial mammals, the collagen extracted from the fish-derived tissue raw material has worse thermal stability due to the loss of tissue structure, and the temperature of the thermal denaturation starting point is even lower than the body temperature of the human body. When the material is implanted into a human body in clinical application, the material can be spontaneously denatured and rapidly absorbed and degraded by the body, so that the application of fish-derived collagen and fish-derived ECM in clinical application is limited. In order to improve the stability of the material, the extracted fish collagen is crosslinked by adopting a chemical crosslinking method, and the risk of toxic crosslinking agent residue is introduced.

Fish collagen has good promotion and induction effects on soft tissue repair in animal experiments, and people like Li Q^[6]The mixture of chitosan and perch collagen is used for preventing brain tissue adhesion in a rabbit dura mater defect model, reducing the chance of inflammation, promoting the growth of fibroblasts and promoting tissue regeneration and healing. Stone R et al^[7]And Kjartansson H^[8]Similar therapeutic effects were achieved in the full-thickness skin burn wounds of pigs and in the variant skin of pigs using atlantic cod skin as a temporary covering.

To date, the U.S. FDA approved the first decellularized fish skin product, Kerecis^TMThe product is derived from decellularized Atlantic cod skin, contains abundant unsaturated fatty acids such as omega-3, shows excellent anti-inflammatory performance, and can effectively promote healing of chronic wound surface, Kerecis^TMHas good economical efficiency and clinical performance, reduces the risk of disease transmission, and has no cultural limitation in use. Kerecis^TMRelated patent US8613957 discloses a method FOR preparing a SCAFFOLD material (SCAFFOLD material FOR WOUND care and/OR OTHER TISSUE healing applications) comprising the steps of: removing fish scales, cleaning with antioxidant/protease inhibitor/protease/antibiotic buffer solution, removing cells, cleaning with solution, bleaching, treating with enzyme, and storing; the process has simple preparation method and short preparation period. The cold water marine fish ECM material has low heat stability and hidden trouble of poor clinical use effect on conventional wound surface and other soft tissue repair.

The invention patent CN 108355172A discloses a decellularized tilapia skin bionic matrix for soft tissue repair and a preparation method and application thereof, and the patent method comprises the steps of cleaning fresh tilapia skin, and removing redundant tissues and impurities; also because of the problem of low ECM stability of the tilapia skin, other remaining processing steps are carried out at 4 ℃, and the processing steps comprise rough finishing to remove part of redundancy, phosphate buffer solution processing to carry out microstructure protection, NaCl solution cleaning, repeated freeze thawing and primary decellularization, repeated microstructure protection, secondary decellularization processing of a washing reagent, NaCl solution cleaning, protective solution pretreatment and processing and forming; and finally sterilizing the completely packaged dry or wet acellular tilapia skin bionic matrix to obtain a finished product. The acellular tilapia skin bionic matrix for repairing soft tissues prepared by the method has a complete natural network structure and good flexibility. But the treatment process is complex and needs to be carried out in a low-temperature environment of 4 ℃ all the time. The invention patent CN 108478869A discloses a decellularized black carp skin bionic matrix for regeneration and repair, a preparation method and application thereof, which can be used as a bionic matrix material for regeneration and repair of various soft tissues, the method of the invention patent CN 108478869A is basically the same as that of CN 108355172A, and the black carp skin is used as a raw material. The invention patent CN 108187140A discloses a fish skin source acellular dermal matrix and a preparation method thereof, wherein the preparation method comprises the following steps: scraping fish skin and meat residue, cleaning with sterile phosphate buffer solution, cleaning with electrolytic water plasma for sterilization, degreasing with alkali solution, decolorizing with potassium permanganate and sodium bisulfite solution, treating with high and low permeability, treating with strong alkali for corrosion, repeatedly freezing and thawing, and lyophilizing and shaping. The preparation process of the patent has a short period, the cells are alternatively treated by osmotic pressure, and meanwhile, the cell components are removed by matching with a strong alkali ablation and repeated freeze thawing method, so that a relatively ideal effect of removing the cells is achieved.

The invention patent CN 102580141A discloses a method for preparing acellular dermal matrix dressing, which takes pigskin, cow leather or sheep skin as raw materials and comprises the following processing steps: preparing a faulted skin sheet, performing a virus inactivation process (consisting of peracetic acid with the mass concentration of 0.1-2.0% and sodium chloride with the mass concentration of 1.0-5.0%), degreasing, decellularizing, soaking in povidone iodine solution, freeze-drying, packaging and performing irradiation disinfection. The method has the advantages of short process time, high efficiency, and light damage to natural structure of dermal extracellular matrix. However, the raw material adopted by the process preparation is a terrestrial mammal dermis layer with higher thermal stability than the fish-derived material, and the stability of the dermis matrix is less influenced in the treatment process.

Reference to the literature

[1]Yamamoto K,Igawa K,Sugimoto K,et al.Biological safety of fish(tilapia)collagen[J].BioMed Research International,2014,2014:1-9.

[2]Yamada S,Yamamoto K,Ikeda T,et al.Potency of fish collagen as a scaffold for regenerative medicine[J].Journal of Biomedicine and Biotechnology,2014,2014(4976):302932.

[3]Li J,Wang M,Qiao Y,et al.Extraction and characterization of type I collagen from skin of tilapia(Oreochromisniloticus)and its potential application in biomedical scaffold material for tissue engineering[J].Process Biochemistry,2018,74:156-163.

[4]Sun L,Hou H,Li B,et al.Characterization of acid-and pepsin-soluble collagen extracted from the skin of Nile tilapia(Oreochromisniloticus)[J].International Journal of Biological Macromolecules,2017,99:8-14.

[5]Song W K,Liu D,Sun L L,et al.Physicochemical an Biocompatibility Properties of Type I Collagen from the Skin of Nile tilapia(Oreochromisniloticus)for Biomedical Applications[J].Marine Drugs,2019,17(3):137.https://doi.org/10.3390/md17030137

[6]Li Q,Mu L,Zhang F,et al.A Novel Fish Collagen Scaffold as Dural Substitute[J].Materials Science&Engineering C,2017,80(nov.):346-351.

[7]Stone R,Larson D,Wall J,et al.522Omega-3Rich Fish Skin Grafts Reduce Donor Skin Requirements for Full Thickness Burns[J].Journal of Burn care&Research:Official Publication of the American Burn Association,2018,39(suppl_1):S234-S235.

[8]Kjartansson H,Jeffery S,Baldursson B T,et al.118Omega-3rich Fish Skin Grafts in the Treatment of Full Thickness Burns:A Comparative Trial of Fish Skin and Cadaver Skin in a Porcine Model[J].Journal of Burn Care&Research:Official Publication of the American Burn Association(suppl_1):S65-S65.

Disclosure of Invention

Aiming at the problems that the prepared fish-derived collagen and fish-derived acellular matrix material in the prior art have poor structural property and low thermal stability; often if chemical cross-linking treatment is not used, it is not stable enough in the temperature range of human body and may denature spontaneously, leading to too fast absorption and degradation by the body. Therefore, the invention aims at providing a preparation method for improving the thermal stability of the fish skin acellular dermal matrix; the method combines the washing agent, the neutral salt solution and the physical extrusion imbibition treatment in the acellular process to avoid the damage of the acellular dermal matrix structure, and the stability of the acellular fish dermal matrix is further improved by the combined treatment of two times of hypertonic salt solution and peroxyacetic acid virus inactivation before and after the acellular treatment.

Another object of the present invention is to provide the above-mentioned acellular dermal matrix for fish skin.

It is still another object of the present invention to provide the use of the above-mentioned skin acellular dermal matrix.

In order to achieve the above primary object, the solution of the present invention is:

a preparation method of a fish skin acellular dermal matrix comprises the following steps:

(1) and (3) fish skin pretreatment: cleaning fresh fish skin, scraping fish scales and residual meat, removing redundant tissues, impurities and cortical tissues with pigments on the surface, and cleaning with purified water;

(2) and cleaning: soaking the fish skin raw material for 0.5-8h by using 0.5-5 wt% of sodium chloride solution or PBS buffer solution containing 0.5-5 wt% of sodium chloride in a material-liquid ratio of 1:3-1:20, removing waste liquid, and soaking for one or more times;

(3) primary virus inactivation and stability improvement treatment: cutting the cleaned fish skin material into required size, soaking in a first mixed solution composed of 0.05-1 wt% of peroxyacetic acid and 0.5-10 wt% of salt solution with a material-liquid ratio of 1:3-1:20, and removing waste liquid after soaking for 0.5-1 h;

(4) and (3) cell removal treatment: treating the fish skin material with a cell removing solution with a material-liquid ratio of 1:3-1:20 for 8-40h, and extruding and sucking the fish skin material in the cell removing solution; the first cell removing time is 8-40h, the fresh cell removing solution is replaced for the second time, and the waste liquid is removed after the second time of treatment for 2-8 h;

(5) and cleaning: soaking in neutral salt solution at a material-to-liquid ratio of 1:3-1:20 for 0.5-2h, removing waste liquid, and soaking for one or more times;

(6) and performing secondary virus inactivation and stability improvement treatment: treating for 2-6h by using a first mixed solution consisting of 0.05-1 wt% of peroxyacetic acid and 0.5-10 wt% of salt solution in a feed-liquid ratio of 1:3-1:20, and removing waste liquid;

(7) and removing heavy metal and cleaning: washing in a second mixed solution composed of 5-50mmol/L Ethylene Diamine Tetraacetic Acid (EDTA) solution and 0.1-2 wt% neutral salt solution in a material-to-liquid ratio of 1:3-1:20, wherein the first washing time is 0.5-2h, the second time is carried out by replacing fresh solution and then treating for 8-24h, and removing waste liquid, namely removing heavy metals through chelation;

(8) and treating the protective solution: soaking the acellular matrix in a protective solution in a material-liquid ratio of 1:3-1:20 for 0.5-6h, and removing waste liquid;

(9) and sterilizing a wet sample sterile packaging terminal: adding a small amount of protective solution into the sample obtained in the step (8), packaging, and performing gamma radiation sterilization to obtain a wet finished product;

(10) and (3) sterilizing a dry sample at an aseptic packaging terminal: and (3) washing the sample in the step (7) with sterile water for 1-3 times according to the material-liquid ratio of 1:3-1:20, removing waste liquid, packaging in a breathable Tyvek packaging bag, freeze-drying, and sterilizing with Ethylene Oxide (EO) to obtain a dry finished product.

Further, in the step (1), the raw material of the fish skin is selected from at least one of salmon, tilapia, cod, flatfish, basha, eel skin and catfish.

Further, in the steps (3) and (6), the salt solution has a pH of 5.5 to 9.0, and the salt in the salt solution is selected from one or more of sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate, sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, potassium chloride, and magnesium chloride.

In the steps (3) and (6), the structure of the extracellular matrix material is often greatly influenced in the raw material disinfection and sterilization process in the prior art, so that the stability of the extracellular matrix material is reduced; the method utilizes the saline solution and the peroxyacetic acid to simultaneously treat and improve the stability of the fish-derived tissue extracellular matrix, thereby not only avoiding the influence on the structure of the extracellular matrix material, but also improving the stability of the extracellular matrix material; further simplifying the preparation, better keeping the integrity of the natural structure of the tissue and keeping good mechanical property and flexibility. The short-time disinfection and sterilization process in the step (3) can effectively kill viruses and bacteria on the surface of the fish skin raw material so as to ensure that microorganisms cannot breed in the subsequent material treatment process. And (6) further removing viruses and bacteria by long-time disinfection and sterilization, and effectively improving the stability of extracellular matrix materials.

Further, in the step (4), the cell-removing solution is composed of a detergent and a neutral salt solution. Wherein the detergent is selected from more than one of 0.1-3 wt% sodium deoxycholate solution, 0.1-1 wt% polyethylene glycol p-isooctyl phenyl ether (triton), 0.1-5 wt% sodium dodecyl sulfate solution and 0.1-1 wt% 3- [3- (cholestyrylpropyl) dimethylamino ] propanesulfonic acid inner salt; the neutral salt solution is selected from more than one of sodium chloride solution, phosphate buffer solution, hydroxyethyl piperazine ethyl sulfate acid solution, tris (hydroxymethyl) aminomethane hydrochloride solution and ethylene diamine tetraacetic acid disodium solution.

Further, in the steps (5) and (7), the neutral salt solution is selected from one or more of sodium chloride solution, phosphate buffer solution, hydroxyethyl piperazine acetic acid solution and Tris-HCl solution.

Further, in step (7), disodium Ethylenediaminetetraacetate (EDTA), a chelating agent with strong chelating ability, may be used for heavy metal ions (including those that are difficult to move) (heavy metal ions such as copper (Cu)²⁺) Cadmium (Cd)²⁺) Mercury (Hg) and mercury (Hg)²⁺) Lead (Pb)²⁺) Manganese (Mn)²⁺) Nickel (Ni)²⁺) Zinc (Zn), zinc (Zn)²⁺)、Chromium (Cr)³⁺) Etc.) to carry out chelation or coordination reaction to form a stable, dissolved and movable complex which is removed by washing and diluting; in addition, some metal ions such as calcium (Ca)²⁺) Or magnesium (Mg)²⁺) Divalent metal ions are also often activators of some enzymes, and metal chelating agents such as EDTA can effectively remove the ions existing in extracellular matrix by chelating the divalent metal ions with the metal chelating agents, and inhibit the activity of the enzymes and the catalytic action of the ions on the autoxidation of materials.

Further, in the step (8), the protective solution is selected from one or more of a sodium chloride solution and a 2-15 wt% glycerol solution. Wherein, the sodium chloride solution can be replaced by PBS buffer solution. The glycerol solution is prepared from Hank's solution, D-Hank's solution or small molecule hyaluronic acid solution and glycerol.

In order to achieve the other purpose, the solution of the invention is as follows:

the fish skin acellular dermal matrix is obtained by the preparation method.

To achieve the above further object, the solution of the present invention is:

an application of the fish skin acellular dermal matrix in the aspects of tissue engineering and regenerative medicine.

Further, the fish skin acellular dermal matrix can guide endogenous tissue regeneration in soft tissue repair by cell infiltration growth and revascularization.

Further, soft tissue repair includes burn and scald wounds, ulcer wounds, hernia repair, dural repair, urethral/bladder/pelvic floor reconstruction, periodontal tissue repair, skin defect repair, and cosmetic reshaping.

Due to the adoption of the scheme, the invention has the beneficial effects that:

firstly, the preparation method of the fish skin acellular dermal matrix material does not use a chemical cross-linking agent, does not need to introduce an organic solvent, can effectively control pollution risks of pyrogen, bacteria and the like, has strong controllability of the preparation process, has better thermal stability than fish source raw materials, and is more suitable for repairing and regenerating human soft tissues.

Secondly, the preparation method can be used together with the treatment of decellularization, virus inactivation, sterilization and the like, the prepared decellularized fish skin matrix material has smooth surface, soft texture and consistent luster, the natural three-dimensional structure of the tissue is well kept, and a good bionic microenvironment can be provided for the regeneration of the cell or the tissue; meanwhile, the good mechanical property and flexibility of the acellular matrix material can be kept, and the clinical requirements can be better met; impurities are removed cleanly, cells are removed cleanly, the content of heterologous DNA is lower, and the safety of clinical use is improved. In addition, in animal verification experiments, the acellular fish skin matrix material with improved thermal stability has good biocompatibility and no adverse inflammatory reaction, so that in-situ tissue regeneration can be guided and wound healing can be accelerated.

Drawings

FIG. 1 is a DSC comparison of the tilapia skin acellular dermal matrix, NaOH-treated tilapia skin and tilapia skin raw material of example 1 of the present invention.

Fig. 2 is a comparative staining pattern of the tilapia skin acellular dermal matrix of example 1 and tilapia skin raw material HE (both images a and C are tilapia skin raw material, and both images B and D are tilapia skin acellular dermal matrix).

Fig. 3 is a comparison of the tilapia skin acellular dermal matrix of example 1 of the present invention and a scanning electron microscope fiber photograph of tilapia skin raw material.

FIG. 4 is a graph of HE staining of the tilapia skin acellular dermal matrix of example 1 of the present invention inducing cell ingrowth (small dots) and neovascularization (indicated by arrows) after implantation for 14 days in rat full-thickness skin defect repair experiment.

FIG. 5 is a comparison graph of the Salmon skin acellular dermal matrix of example 2 of the present invention and Salmon skin raw material DSC.

FIG. 6 is a DSC comparison of the salmon skin acellular dermal matrix of comparative example 1 of the present invention with salmon skin raw material.

FIG. 7 is a DSC comparison of the tilapia skin acellular dermal matrix of comparative example 2 of the present invention and tilapia skin raw material.

Detailed Description

The invention provides a fish skin acellular dermal matrix and a preparation method and application thereof.

The present invention will be further described with reference to the following examples.

Example 1:

the preparation method of the tilapia skin acellular dermal matrix comprises the following steps:

(1) and (3) fish skin pretreatment: cleaning fresh tilapia skin, scraping fish scales and residual meat, removing redundant tissues, impurities and cortical tissues with pigment on the surface, and cleaning with purified water.

(2) And cleaning: and (3) treating the waste liquor for 1 hour by using a 0.9 wt% sodium chloride solution with the feed-liquor ratio of 1:10, and repeating the steps for 2 times.

(3) Primary virus inactivation and stability improvement treatment: the cleaned fish skin material was cut into 3cm × 3cm, and then soaked in a first mixed solution (pH 7.5) of 0.1 wt% peracetic acid and 2 wt% sodium phosphate solution at a material-to-solution ratio of 1:10 for 0.5h, and the waste liquid was removed.

(4) And (3) cell removal treatment: carrying out decellularization treatment for 20 hours by using a decellularization solution (pH 7.4) which is composed of 0.2 wt% of triton and 10mmol/L of disodium ethylene diamine tetraacetate in a material-to-liquid ratio of 1:10, extruding and sucking the fish skin material in the decellularization solution, and removing waste liquid; and replacing the fresh cell-free solution, treating for 4 hours again, removing the waste liquid, and repeating the steps.

(5) And cleaning: and (3) treating the mixture for 0.5h by using 0.9 wt% sodium chloride solution with the material-to-liquid ratio of 1:10, removing waste liquid, and repeating the steps for 2 times.

(6) And performing secondary virus inactivation and stability improvement treatment: a first mixed solution consisting of 0.2 wt% of peroxyacetic acid and 2 wt% of sodium phosphate solution (pH 7.5) in a feed-to-liquid ratio of 1:10 is treated for 4 hours, and then waste liquid is removed.

(7) And removing heavy metal and cleaning: washing with a second mixed solution of 10mmol/L disodium Ethylene Diamine Tetraacetate (EDTA) solution and PBS buffer solution (0.01mol/L, pH 7.4) at a ratio of 1:10, wherein the first washing time is 0.5h, replacing fresh solution for the second time, and treating for 20h to remove waste liquid.

(8) And treating the protective solution: soaking the prepared acellular matrix in a protective solution consisting of 10 wt% of glycerol solution and 0.9 wt% of sodium chloride solution in a material-to-liquid ratio of 1:10 for 2h, and removing waste liquid.

(9) And aseptic packaging and sterilizing of the wet sample: and (4) adding a small amount of protective solution into the sample obtained in the step (8), packaging, and sterilizing by gamma radiation to obtain a wet finished product.

The residual amount of DNA in the acellular matrix material is an indirect index for measuring the acellular degree, and the low residual amount of DNA indicates that the acellular degree is high. The tilapia skin raw material which is cleaned by fresh fish skin, fish scales and residual meat, redundant tissues, impurities and cortical tissues with pigments on the surface is removed, and the content of DNA of the tilapia skin raw material is lower and is 6.25 +/-2.06 ng/mg. The residual amount of DNA of the tilapia skin acellular matrix prepared by the method of the embodiment is only 1.32 +/-0.38 ng/mg (Table 1).

The mechanical strength of the material is one of important indexes for measuring the structural damage degree of the acellular matrix in the preparation process, and the damage of the acellular matrix structure reduces the mechanical strength of the material. In the experiment, the tensile strength of the tilapia skin acellular dermal matrix and the tilapia skin raw material is compared by using an Instron electronic universal tester (3340 series). The measurement was carried out at a drawing speed of 150mm/min and a gauge length of 40 mm. The sample is cut into 1cm multiplied by 7cm, the two ends of the sample are placed on a clamp of a testing machine to be clamped after being washed by 0.9% sodium chloride solution, and the fracture part of the tested material is required to be positioned within the range of the gauge length. The acellular matrix of tilapia mossambica prepared by the method of this example retained the mechanical strength of the raw material of fish skin (table 1).

TABLE 1 comparison table of DNA content and mechanical properties of tilapia skin acellular dermal matrix and tilapia skin raw material

The initial denaturation temperature is one of the measures for the stability of acellular matrix, and the damage of the acellular matrix structure during the preparation process can reduce the stability of the acellular matrix. Fig. 1 is a Differential Scanning Calorimetry (DSC) comparison graph of the tilapia skin acellular dermal matrix prepared in example 1 and tilapia skin raw material. The tilapia skin acellular dermal matrix material prepared by the method of the embodiment improves the thermal stability of tilapia skin raw materials. The sodium hydroxide solution is a known common method for decellularization and virus inactivation in the preparation process of the animal-derived tissue matrix, and the heat stability of the tilapia skin matrix is greatly reduced by sodium hydroxide treatment, namely the heat stability of the tilapia skin treated by the sodium hydroxide is far lower than that of a tilapia skin raw material.

Histopathological sectioning methods conventional methods for assessing tissue structure. Fig. 2 is a comparison of the tissue structure of cross-section HE staining of tilapia skin acellular dermal matrix prepared in example 1 and tilapia skin raw material. Wherein, the picture A and the picture C are the raw materials of tilapia skin, and the picture B and the picture D are the acellular dermal matrix of tilapia skin. The HE staining pattern is grey collagen fibers and small circles indicate nuclei. The tilapia skin acellular dermal matrix prepared by the embodiment has no obvious cell nucleus, and the cell components are removed cleanly; the acellular dermal matrix retains the natural three-dimensional tissue infrastructure intact. After the decellularization, virus inactivation and sterilization treatment, the distance between the collagen fiber bundles of the tilapia skin decellularized tissue matrix is increased, the structure of the collagen fiber bundles is still clear and compact, the phenomenon of scattering and blurring is avoided, and the tilapia skin decellularized tissue matrix is more suitable for the immersion growth and revascularization of cells.

FIG. 3 is an SEM comparison of the tilapia skin acellular dermal matrix and the tilapia skin raw material. The tilapia skin acellular dermal matrix prepared in the example 1 well retains the natural tissue ultrastructure, and impurities are removed completely.

To verify the tissue regeneration performance of the tilapia skin acellular dermal matrix prepared in example 1, we performed an SD rat full-thickness skin defect model experiment. 2 round wound surfaces with phi of 1.8cm and full-layer skin defect are cut on the back of an SD rat, a tilapia skin acellular matrix is cut into square blocks with the length of 2.0-2.5cm, one wound surface is covered with the tilapia skin acellular matrix after being washed and wetted by normal saline, and the other wound surface is covered with sterile gauze dressing as a control; then, a 3M polyurethane dressing is used for covering (moisture preservation and bacterium isolation), and binding and fixing are carried out. The experimental animals were sacrificed at three time points (7d, 14d, 21d) in sequence, wound recovery was recorded, wound healing rate was determined and pathological observations were made. FIG. 4 is a graph of HE staining of the tilapia skin acellular dermal matrix inducing cell ingrowth (small dots) and neovascularization (indicated by arrows) after 14 days of implantation in rat full-thickness skin defect repair experiments. The experimental results show that: the tilapia skin acellular matrix can be used for repairing the wound surface without adverse inflammatory reaction, can guide cell growth and new blood vessel generation, promote wound healing and guide tissue regeneration, namely the tilapia skin acellular dermal matrix induces cell ingrowth and new blood vessel generation in the wound healing process; the wound healing rate was higher at each time point than the sterile gauze dressing control group.

Example 2:

the preparation method of the salmon skin acellular dermal matrix of the embodiment comprises the following steps:

(1) and fish skin pretreatment: cleaning fresh salmon skin, scraping scales and residual meat, removing redundant tissues, impurities and cortical tissues with pigment on the surface, and cleaning with purified water.

(2) And cleaning: and (3) treating the mixture for 1 hour by using a 0.9 wt% sodium chloride solution with the ratio of material to liquid being 1:6, removing waste liquid, and repeating the steps for 2 times.

(3) Virus inactivation, sterilization and stability improvement treatment: the cleaned fish skin material was cut into 5cm × 5cm, and then soaked in a first mixed solution (pH 7.6) of 0.1 wt% peracetic acid and 4 wt% potassium phosphate solution at a feed-to-solution ratio of 1:8 for 0.5h, and the waste liquid was removed.

(4) And (3) cell removal treatment: carrying out decellularization treatment on a decellularized solution consisting of 0.2 wt% of triton, 0.01mol/L PBS buffer solution and 10mmol/L disodium ethylene diamine tetraacetate solution in a material-to-liquid ratio of 1:6 for 24h, extruding and sucking the material in the decellularized solution, and removing waste liquid.

(5) And cleaning: and (3) treating the mixture for 1 hour by using a 0.9 wt% sodium chloride solution with the feed-liquid ratio of 1:8, removing waste liquid, and repeating the steps for 2 times.

Fig. 5 is a Differential Scanning Calorimetry (DSC) comparison graph of the salmon skin acellular dermal matrix prepared in example 2 and salmon skin raw materials. The salmon skin acellular dermal matrix prepared by the embodiment improves the thermal stability of the salmon skin raw material.

Comparative example 1:

the preparation method of the salmon skin acellular dermal matrix of the comparative example comprises the following steps:

(1) and fish skin pretreatment: cleaning fresh salmon skin, scraping fish scales and residual meat, removing redundant tissues, impurities and cortical tissues with pigments on the surface, and cleaning with purified water.

(2) And cleaning: after 1h of treatment with a 1:8 stock-to-liquor ratio PBS solution (0.01mol/L, pH 7.4, containing 0.8 wt% sodium chloride), the waste was removed and the above procedure was repeated 1 time.

(3) And (3) cell removal treatment: cutting the cleaned fish skin material into 1cm multiplied by 1cm, then carrying out decellularization treatment on the fish skin material for 18h by using a decellularization solution consisting of 0.2 wt% of triton, 1mol/L sodium chloride solution and 0.01mol/L PBS buffer solution with the material-to-liquid ratio of 1:8, extruding and sucking the fish skin material in the decellularization solution, and removing waste liquid; and replacing the fresh cell-free solution, treating for 3 hours again, removing the waste liquid, and repeating the steps.

(4) Virus inactivation, sterilization and stability improvement treatment: after a first mixed solution (pH 7.0) consisting of 0.1 wt% of peroxyacetic acid and 4 wt% of sodium chloride solution in a feed-to-liquid ratio of 1:8 is treated for 6h, waste liquid is removed.

(5) And cleaning: treating the mixture for 1 hour by PBS buffer solution (0.01mol/L, pH 7.4 and containing 0.8 wt% of sodium chloride) with the ratio of the material to the liquid being 1:8 to remove waste liquid; the above steps were repeated 2 times.

Fig. 6 is a Differential Scanning Calorimetry (DSC) comparison of the salmon skin acellular dermal matrix prepared in comparative example 1 with salmon skin raw material. The method of the comparative example is used for improving the thermal stability of the salmon acellular dermal matrix material.

Comparative example 2:

(1) and fish skin pretreatment: cleaning fresh tilapia skin, scraping fish scales and residual meat, removing redundant tissues, impurities and cortical tissues with pigment on the surface, and cleaning with purified water.

(4) Virus inactivation, sterilization and stability improvement treatment: the waste liquid is removed after the first mixed solution (pH 7.0) consisting of 0.1 wt% of peroxyacetic acid and 4 wt% of sodium chloride solution with the feed-liquid ratio of 1:8 is treated for 1 hour.

(5) And cleaning: treating the mixture for 1 hour by PBS buffer solution (0.01mol/L, pH 7.4 and containing 0.8 wt% of sodium chloride) with the ratio of the material to the liquid being 1:8 to remove waste liquid; the above steps are repeated for 2 times

Fig. 7 is a Differential Scanning Calorimetry (DSC) comparison graph of the tilapia skin acellular dermal matrix prepared in comparative example 2 and a raw material of tilapia skin. The tilapia skin acellular dermal matrix prepared by the comparative example improves the thermal stability of the tilapia skin raw material.

As can be seen from the above, in examples 1 and 2, the method of the fish skin acellular dermal matrix is to perform a preliminary virus inactivation treatment and then to perform an acellular treatment, while in comparative examples 1 and 2, the method of the fish skin acellular dermal matrix is to perform an acellular treatment and then to perform a virus inactivation treatment after the raw material is pretreated and washed. The adjustment of the above process in the comparative example was to verify that the key step for improving the stability of dermal matrix was the virus inactivation treatment process, and the virus inactivation before and after the decellularization did not affect the improvement of the stability of the fish skin raw material. In addition, although bacteria do not readily grow in the decellularized solution, the decellularized solution does not have the effect of sterilization. Therefore, the fish skin acellular dermal matrix material prepared by the method has better thermal stability than the fish source raw material, and the key step of improving the thermal stability of the material is further verified to be that the first mixed solution consisting of 0.05-1 wt% of peroxyacetic acid and 0.5-10 wt% of salt solution is used for disinfection and sterilization treatment.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.

Claims

1. A preparation method of a fish skin acellular dermal matrix is characterized by comprising the following steps: which comprises the following steps:

(1) and fish skin pretreatment: scraping scales and residual meat from fresh fish skin, and cleaning;

(2) and cleaning: soaking the fish skin raw material for 0.5-8h by using 0.5-5 wt% of sodium chloride solution or phosphate buffer solution containing 0.5-5 wt% of sodium chloride in a material-to-liquid ratio of 1:3-1:20, removing waste liquid, and soaking at least once;

(3) and primary virus inactivation treatment: cutting the cleaned fish skin material, soaking the cut fish skin material in a first mixed solution which is composed of 0.05-1 wt% of peroxyacetic acid and 0.5-10 wt% of salt solution in a material-liquid ratio of 1:3-1:20, and removing waste liquid after soaking treatment for 0.5-1 h;

(5) and cleaning: soaking with neutral salt solution at a material-to-liquid ratio of 1:3-1:20 for 0.5-2h, removing waste liquid, and soaking at least once;

(6) and performing virus inactivation treatment again: treating for 2-6h in a first mixed solution consisting of 0.05-1 wt% of peroxyacetic acid and 0.5-10 wt% of salt solution in a feed-liquid ratio of 1:3-1:20, and removing waste liquid;

(7) and removing heavy metal and cleaning: washing in a second mixed solution composed of 5-50mmol/L disodium ethylene diamine tetraacetate solution and 0.1-2 wt% neutral salt solution in a material-to-liquid ratio of 1:3-1:20, wherein the first washing time is 0.5-2h, the second time is carried out by replacing fresh solution and then treating for 8-24h, and removing waste liquid;

(8) and treating the protection solution: soaking the acellular matrix in a protective solution in a material-liquid ratio of 1:3-1:20 for 0.5-6h, and removing waste liquid;

(9) and sterilizing the wet sample at an aseptic packaging terminal: packaging and irradiating the sample in the step (8) for sterilization to obtain a wet finished product;

(10) and sterilizing a dry sample at an aseptic packaging terminal: washing the sample in the step (7) with sterile water for 1-3 times according to the material-liquid ratio of 1:3-1:20, removing waste liquid, packaging, freeze-drying, and sterilizing to obtain a dry finished product;

in the steps (3) and (6), the pH value of the salt solution is 5.5-9.0, and the salt in the salt solution is selected from more than one of sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate, sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, potassium chloride and magnesium chloride;

in the step (4), the cell-removing solution consists of a detergent and a neutral salt solution; the detergent is selected from more than one of 0.1-3 wt% of sodium deoxycholate solution, 0.1-1 wt% of polyethylene glycol p-isooctyl phenyl ether, 0.1-5 wt% of sodium dodecyl sulfate solution and 0.1-1 wt% of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt; the neutral salt solution is selected from more than one of sodium chloride solution, phosphate buffer solution, hydroxyethyl piperazine ethyl sulfate acid solution, tris (hydroxymethyl) aminomethane hydrochloride solution and ethylene diamine tetraacetic acid disodium solution.

2. The method of claim 1, wherein: in the step (1), the raw material of the fish skin is selected from more than one of salmon, tilapia, cod, flatfish, basha, eel and catfish.

3. The production method according to claim 1, characterized in that: in the steps (5) and (7), the neutral salt solution is selected from more than one of a sodium chloride solution, a phosphate buffer solution, a hydroxyethyl piperazine acetic acid sulfuric acid solution and a tris hydrochloride solution.

4. The method of claim 1, wherein: in the step (8), the protective solution is selected from more than one of sodium chloride solution and 2-15 wt% of glycerol solution;

the glycerol solution is prepared from Hank's solution, D-Hank's solution or hyaluronic acid solution and glycerol.

5. A fish skin acellular dermal matrix is characterized in that: which is obtained by the production method according to any one of claims 1 to 4.

6. Use of the fish skin acellular dermal matrix according to claim 5 for the preparation of products for tissue engineering and regenerative medicine.

7. Use according to claim 6, characterized in that: the fish skin acellular dermal matrix guides endogenous tissue regeneration in soft tissue repair through cell invasion growth and revascularization.

8. Use according to claim 7, characterized in that: the soft tissue repair comprises burn and scald wound surfaces, ulcer wound surfaces, hernia repair, dura mater repair, urethra/bladder/pelvic floor reconstruction, periodontal tissue repair, skin defect repair and cosmetic reshaping.