CN113827774A

CN113827774A - A biological tissue engineering regeneration scaffold technology and application method prepared from scaleless freshwater fish skin

Info

Publication number: CN113827774A
Application number: CN202111218808.3A
Authority: CN
Inventors: 常鹏; 唐明强; 王鸿涛
Original assignee: Shenyang Elite Blue Medical Technology Co ltd
Current assignee: Shengjing Hospital of China Medical University
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2021-12-24

Abstract

The invention belongs to the field of biological materials and technical methods in the field of regenerative medicine, in particular to a biological tissue engineering regeneration scaffold technology and application method prepared based on scaleless freshwater fish skin. Including the use of the excellent histocompatibility of scaleless freshwater fish (mirror carp, etc.), and the preparation of bioscaffold materials widely used in the field of tissue regeneration after screening and breeding and other safe processing technologies. The treated fish skin material retains the complete structure and activity of the fish skin collagen without losing the unsaturated fatty acids in the fish skin that promote the healing and regeneration of human tissue. Especially after specific design (thickness and porosity, etc.), it can be used to load micro-tissue regeneration structural units, and it can be applied to regeneration fields such as skin, liver, bone and fat, which has very important clinical value.

Description

Biological tissue engineering regeneration scaffold technology prepared from scale-free freshwater fish skin and application mode

Technical Field

The invention relates to the research fields of medical auxiliary instruments, medical biomaterials, biological tissue engineering and regenerative medicine, in particular to a biological tissue engineering regenerative scaffold technology prepared from scale-free freshwater fish skin and an application mode.

Background

10% -20% of full-thickness skin defects can be defined as very large wounds or severe wounds that can cause rapid loss of body fluids and severe infections, endangering the life of the patient. Under the condition of limited autologous skin resources, the main challenge of treating the oversized wound is to complete wound covering and construct skin with the functions of hair follicles, sweat glands and the like at one time. At present, clinically, the skin (epidermis and partial dermis) in a self-supply area is taken, the skin is manually or mechanically crushed to prepare the microskin, the wound with the surface area more than 10 times of that of the microskin can be covered by amplification, skin attachments such as hair follicles, sweat glands and the like do not exist, the healed scar has serious poor appearance, the wound area cannot perspire, and the scar is often broken and has pain and pruritus, so that great pain is brought to a patient.

Researchers have tried to treat large-area skin defects with full-thickness micro-particle skin or columnar skin containing hair follicles, but without the support of a tissue engineering scaffold, full-thickness micro-particle skin cannot grow three-dimensionally, and the repair effect is not ideal. We have found that the skin is arranged in the form of structural units arranged in equally spaced, staggered isosceles triangles, this natural arrangement providing the skin with the best flexibility and extensibility.

Therefore, we propose the concept of Minimum Functional Unit (MFUS): by collecting the miniature full-layer columnar autologous skin tissue, hair follicles, sweat glands and other skin accessories are contained in the minimum diameter of the miniature full-layer columnar autologous skin tissue, so that the miniature full-layer columnar autologous skin tissue can be used for constructing new skin with complete skin functions, and the new skin has good appearance and has the skin functions of perspiration and the like. Meanwhile, the method can be applied to organs with similar structural unit characteristics such as fat, liver, bones and the like. MFUS can be planted in tissue engineering scaffolds as a 'microtissue seed', repair tissue defects, and perform organ reconstruction.

It has been confirmed that fish skins can be used for skin wound repair, show good histocompatibility and wound healing promotion effects, but they are generally used as wound dressings and hemostatic materials, and wounds heal in the form of scars. In the related patents at present, fish skin is mostly extracted from collagen and used for preparing a chemically cross-linked collagen scaffold material, and a fish skin acellular dermal matrix material is also used as a wound dressing, but the designs cannot meet the requirements of the current scaffold for regenerating tissues and organs in the aspects of antibacterial property, supporting strength, porosity and collagen activity.

The invention mainly utilizes natural collagen components in fish skin to prepare a tissue engineering scaffold for assisting MFUS in organ regeneration. A fish skin treatment technology suitable for micro tissue regeneration is developed, the fish skin treatment technology is suitable for complete and functional repair of large-area full-layer skin defects, and a scaffold material with complete collagen structure and controllable porosity and supporting strength can be provided for regeneration of other tissues and organs.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a biological tissue engineering regeneration scaffold technology prepared from scale-free freshwater fish skin and an application mode, and solves the problems that after the large-area skin defect is treated at present, scars are serious, skin attachments such as hair follicles and sweat glands do not exist, perspiration cannot occur, scars of wound surfaces are frequently broken, pain and pruritus are caused, and the appearance is poor. And the scaffold material with complete collagen structure and controllable porosity and supporting strength can also be provided for the regeneration of other tissues and organs.

A technology for preparing the regenerated scaffold of biologic tissue engineering from the skin of fresh water without scale includes special culturing and screening of fresh water fish without scale, preparing the skin of fish, application in biologic tissue engineering and auxiliary tissue organ regeneration.

Preferably, the scale-free freshwater fish screening comprises mirror carp (scientific name: Cyprinus carpio var. specularis), is a variety of European carp, has glossy cuticle, is smooth as if the surface of a mirror and is obvious in gloss, and is called mirror carp. The back fin is thicker in body shape, flatter in side, raised in head and back, smaller in head, larger in eyes, larger in body surface scales, arranged along the edge, 1 row of complete scales from the front end to the head of the back fin, 1 row of symmetrical continuous complete scales on two sides of the back fin, scales on the base of each fin, and a few scales on the side line of an individual. The lateral lines are mostly straight and not branched, and the ends of the lateral lines of individual individuals are provided with shorter branches. The body color varies depending on the habitats, and is generally tan on the back and yellowish on the body and abdomen. It is inhabited in the middle and lower layers of the water area, and mainly comprises the sediment and sand still water area of the eutrophic water area, so that the water area has the group swimming habit. The fish is omnivorous, and mainly comprises small invertebrates and benthonic animals. Is a good aquaculture breed suitable for popularization in China. Under the conditions of reasonable stocking density and excellent feeding condition, the growth speed of the mirror carps is very fast. The corium layer of the mirror carp skin contains I type collagen components similar to human tissues, and the epidermis layer is subjected to scale degeneration and variation and evolves into a thicker scaleless corium layer rich in grease, so that the mirror carp skin is suitable for covering the wound surface of human skin.

Preferably, female carps of the sexual maturity age of the mirror carps are 3-4 years old, and male carps are 2-3 years old. The temperature of propagation water is usually 17-25 ℃, and the optimal temperature is 19-22 ℃. In the screening step, the cultivation is carried out in a clean room, and in the feeding process, the water temperature of the feeding is gradually increased, and the collagen heat stable temperature of the dermis layer is increased.

Preferably, fish type I collagen has a remarkable characteristic of lower heat stability than that of terrestrial animals such as pigs and cows, and exhibits specificity to fish species. The low thermal stability means that the collagen can be denatured under the condition of human body temperature, loses partial biological activity and is not beneficial to wound repair. We find that the thermal denaturation temperature of the collagen of the cyprinus carpio is higher than that of other fish species and can reach about 32-38 ℃, and further the thermal denaturation temperature of the collagen of the cyprinus carpio is improved through high-temperature culture, so that guarantee is provided for the treatment of the wound surface of a human body.

Preferably, in the skin structure of the mirror carp, the dermis layer is suitable for preparing a biological tissue engineering scaffold for medical treatment, and the epidermis layer plays the roles of moisturizing and protecting the wound surface and is beneficial to wound healing. The skin fat content of the mirror carp is about 22 percent (2 to 10 percent of common fishes), the fat component contains unsaturated fatty acid, especially omega-3 unsaturated fatty acid, the natural bacteriostatic action is realized, and the wound surface can be protected. The mirror carp skin has low metabolic rate and is easy to deposit heavy metal, and in the later stage of a special feeding mode, an ionic silver component (at present, silver ions are mostly used as an antibacterial main component in high-grade wound dressing) is added into feed, and the deposited silver ionic component can further play a natural antibacterial role to protect skin wounds.

Preferably, in the process of obtaining and preparing the fish skin material, a sterile workshop is used for obtaining complete fish skin tissues, other sundries such as fish meat and the like are removed by a physical method, and the whole process is operated at the temperature of 4 ℃, so that the natural structure of the type I collagen is protected.

Preferably, during the preparation process of the fish skin material, high-concentration NaCl: 8 to 26.5 percent (the saturation concentration is 26.5 percent), and further dissolves ineffective components such as mucus, pigment, foreign protein and the like. (Fish skin collagen does not dissolve in NaCl).

Preferably, in the preparation process of the fish skin material, low-concentration acetic acid (0.01% -0.1%) is used to further swell and loosen the dermal layer of the fish skin, so that the thickness of the fish skin is increased, the porosity is improved, the wound surface is easy to ventilate, capillary vessels grow, and the wound surface healing is promoted.

Preferably, in the preparation process of the fish skin material, pepsin 0.1-1.0 w/v is further used for dissolving the exposed collagen terminal side amino acid antigen (main antigen causing human immune reaction) after swelling and loosening, and the intact collagen is not damaged.

Preferably, during the preparation process of the fish skin material, high-concentration NaCl: 8-26.5%, dissolves residual impurity protein such as parvalbumin, and the hypertonic condition also has natural bactericidal effect, and the bacteria can denature and die. During the preparation process of the fish skin material, the fish skin material is soaked in a sterile solution containing the carbonate sugar of the sprite, the residual acetic acid in the steps can be neutralized by the carbonate, and the sugar is beneficial to the growth of the wound surface in the later period.

Preferably, in the preparation process of the fish skin material, 0.8% of normal saline is used for soaking for 1 hour (the volume ratio is 1: 100), then the fish skin material is transferred into a 10 x 2cm sterile resin vessel disc and is prepared into a frozen tissue engineering scaffold at the temperature of-20 ℃, and when the fish skin material is applied, the fish skin material is directly used for wound treatment after ice blocks are dissolved, so that the transportation and the storage are facilitated.

Preferably, in the preparation process of the fish skin material, the frozen fish skin is further placed into a low-temperature negative-pressure freeze dryer to be prepared into the freeze-dried material. When in use, the ointment is soaked in 0.8 percent normal saline for 5 minutes, and the ointment is used for treating wound surfaces after the elasticity and moisture are recovered. Further increase the shelf life after lyophilization.

Preferably, in the preparation process of the fish skin material, the fish skin tissue engineering material is disinfected by ultraviolet rays; sterilizing the fish skin tissue engineering material by using III type iodine solution, chlorhexidine and the like; sterilizing and moisturizing the fish skin tissue engineering material by using Meibao scald ointment, metronidazole gel and the like; the glycerol component product is used for sterilizing and moisturizing the fish skin tissue engineering material.

Preferably, in the application of the biological tissue engineering regeneration field, the fish skin material can be applied to wound surfaces formed by chronic diseases such as burns, traumas, diabetes and the like, is beneficial to the tissue repair property of natural collagen, and is a natural wound surface biological dressing.

Preferably, the application in the field of biological tissue engineering regeneration is to punch a lattice on a support, punch a lattice in an isosceles triangle arrangement, implant a columnar skin micro-tissue (skin regeneration minimum function unit, MFUS) in the hole, make the MFUS grow three-dimensionally under the support assistance, and construct a skin with functionality after the wound surface is healed.

Preferably, the application mode of the auxiliary tissue organ regeneration is that the stent loads a liver micro tissue structure unit, a bone micro tissue structure unit and a fat micro tissue structure unit which are similar structure unit forms, and the regeneration of the tissue organ is assisted by adopting modes of multilayer superposition, microsphere blending and the like.

Drawings

FIG. 1 is a view showing that a mirror carp skin tissue engineering scaffold of the present invention is loaded with a rat skin minimal function unit to treat a large area skin defect wound surface; 1 rat back 23% full-layer skin defect wound surface, 2 rat normal skin (wound edge), 3 mirror carp skin tissue engineering scaffold, and 4 rat skin minimum function unit MFUS.

FIG. 2 is a schematic view of an electron microscope showing the thickness change of a mirror carp skin scaffold after a swelling step according to the present invention; dissolving 5 acetic acid with 10% concentration for six hours; dissolving 6 acetic acid with 3% concentration for six hours; after dissolving for six hours at the concentration of 7 acetic acid 1%, after dissolving for six hours at the concentration of 8 acetic acid 0.1%, after dissolving for six hours at the concentration of 9 acetic acid 0.01%, and after dissolving for six hours at the concentration of 10 distilled water.

FIG. 3 illustrates the detection of material properties according to the present invention; 11, carrying out statistics on the porosity of different experimental groups after swelling; 12 after swelling, the Fourier infrared spectrum analysis of different experimental groups analyzes the bond.

FIG. 4 is a photograph of protein electrophoresis of the material of the present invention; 13 protein electrophoresis molecular weight marking band; 14 fresh mirror carp skin protein component; 15 scaffold material sample-1 protein component; 16 scaffold material-like-2 protein component.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-4, the present invention provides a technical solution: a technology for preparing the regenerated scaffold of biologic tissue engineering from the skin of fresh water without scale includes special culturing and screening of fresh water fish without scale, preparing the skin of fish, application in biologic tissue engineering and auxiliary tissue organ regeneration. The invention applies the excellent histocompatibility of the scale-free freshwater fish (such as mirror carp and the like), prepares the biological scaffold material which can be widely applied to the field of tissue regeneration through safe processing technologies such as screening and breeding, and the processed fish skin material not only retains the complete structure and activity of fish skin collagen, but also retains unsaturated fatty acid and the like of the fish skin to promote healing and regeneration components, particularly the specially designed thickness and porosity, is used for loading a micro-tissue regeneration structural unit, is applied to the regeneration fields of skin regeneration, liver regeneration, bone regeneration, adipose tissue and the like in the future, and has very important clinical application value.

Example 1.

In this embodiment: 1 rat back 23% full-layer skin defect wound surface, 2 rat skin (wound edge), 3 mirror carp skin tissue engineering scaffold, 4 rat skin minimum function unit MFUS. After the mirror carp skin after being specially bred and screened, the tissue engineering scaffold after freeze-drying and the mirror carp skin scaffold are soaked in normal saline, and holes of 2mm are used for loading the minimum functional unit for skin tissue regeneration. Dissolving 5 acetic acid with 10% concentration for six hours; dissolving 6 acetic acid with 3% concentration for six hours; after dissolving for six hours at the concentration of 7 acetic acid 1%, after dissolving for six hours at the concentration of 8 acetic acid 0.1%, after dissolving for six hours at the concentration of 9 acetic acid 0.01%, and after dissolving for six hours at the concentration of 10 distilled water. And combining bonds among different experimental groups for porosity statistics after 11 mirror carp skin is swelled and different experimental groups for Fourier infrared spectrum analysis after 12 mirror carp skin is swelled. 13-16 mirror carp skin and bracket protein electrophoresis results.

In this example, mirror carp fries were raised in 20 ℃ water and the water temperature was gradually increased to 25 ℃ over 3 years. The third year of feeding, the ionic silver component was added to the feed and the water in the culture, and drinking water with silver ion concentration lower than 0.1ppm did not cause adverse effect on the organism according to the fourth edition of the drinking water quality directive Standard of the world health organization newly issued by WHO (ppm is an expression method of the solute mass fraction of the solution, 1 milligram of solute in 1 liter of water solution, g/m3 or mg/L). In the preparation process of the fish skin, a sterile workshop is used for obtaining complete fish skin tissues, other sundries such as fish meat and the like are removed by a physical method, and the whole process is operated at the temperature of 4 ℃, so that the natural structure of the type I collagen is protected.

In this example, a high concentration of NaCl: 8 percent, and further dissolves ineffective components such as mucus, pigment, foreign protein and the like. Low-concentration acetic acid (0.3-0.01%) is used to further swell and loosen the dermal layer of fish skin, and pepsin 0.1-1 w/v is used to dissolve the exposed amino acid antigen at the end of collagen (the main antigen causing immune reaction of human body) without damaging the intact collagen. In the preparation process of the fish skin material, high-concentration NaCl: 6-10 percent of the raw materials are dissolved, residual impurity proteins such as parvalbumin and the like are soaked in the solution of the components of the tobamon and the sugar, 0.9 percent of normal saline is used for soaking for 1 hour (the volume ratio is 1: 100), then the raw materials are transferred into a sterile resin vessel tray of 10 multiplied by 2cm to prepare a frozen tissue engineering bracket at the temperature of minus 20 ℃, and the frozen tissue engineering bracket is directly used for wound treatment after ice blocks are dissolved during application and is beneficial to transportation and storage. Or placing the frozen fish skin into a low-temperature negative pressure freeze dryer to prepare the freeze-dried material. When in use, the composition is soaked in 0.9 percent normal saline for 5 to 30 minutes, and then further applied after the elasticity and the moisture are recovered.

In this example, it was further found by scanning electron microscopy that the mirror carp skin scaffold material was able to form a desired void ratio, and the optimum concentration of the swollen acetic acid solution was determined to be 0.1% for six hours. The structure of the collagen after 0.1% swelling is kept intact by using Fourier infrared spectrum detection, and the concentration of acetic acid higher than the concentration has destructive effect on the collagen components. Furthermore, the tissue engineering scaffold obtained by the treatment process can preserve the complete collagen peptide chain structure by applying protein electrophoresis.

In the embodiment, the mirror carp skin tissue engineering scaffold is loaded with the rat skin minimum functional unit to treat a large-area skin defect wound; constructing 23% of full-layer skin defect wound surface 1 on the back of the SD rat; rat skin (wound margins) 2; designing the aperture of a radius dot matrix of a mirror carp skin tissue engineering scaffold 3; loading a rat skin minimal function unit MFUS 4; constructing novel tissue engineering skin for skin tissue regeneration.

Example 2.

The tissue engineering scaffold after the tilapia skin is freeze-dried after being specially fed and screened is prepared by the following steps: soaking tilapia skin scaffolds in normal saline; carrying out a tilapia skin bracket folding toughness test; testing the tensile toughness of the tilapia skin support; and (4) testing the torsion toughness of the tilapia skin bracket.

In this example, after the tilapia skin is descaled, a freeze-dried scaffold is obtained by applying the similar steps described above; soaking in 0.9% physiological saline for 5 min, and recovering elasticity and water for further application.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

The biological tissue engineering regeneration scaffold technology and the application mode prepared from the scale-free freshwater fish skin are different from other similar patents in the following steps:

patent number

Examples 1 to 2

CN105755078 B

CN103320485 B

CN104474589 B

CN108355171 B

CN108187140 B

CN1061104 00 A

Status of state

Patent granting

Patent publication

Name (R)

Scale-free fresh water fish skin Prepared biological tissue engineering Process regeneration stent technology and applications thereof

Medical grade Of fish skin collagen Preparation method and applications thereof

Medical graduate student Fish skin for material Preparation method of collagen Method of

Guide group Regenerated membrane and method for preparing the same And applications thereof

Acellular dermal matrix Guided tissue regeneration membrane Material and preparation method thereof Method and application

Fish skin source stripper Cell dermal matrix And method for preparing the same

Skin care product Recycled material And preparation thereof Method

Species of fish

Scale-free freshwater fish

Fish skin without special purpose

Artificial polymer Fiber

Fish skin without special purpose

Pigskin, luofi Fish skin

Feeding sieve Selecting

High temperature raising/ionic silver resisting Fungus breeding

Is free of

Dissolution test Agent for treating cancer

No toxicity and residue

Extracting the reagent to obtain Easily remains in parts

Extracting reagent components Is easy to remain

Is free of

The components of the extracting reagent are easy to extract Residue is remained

Extracting reagent components Is easy to remain

Extraction reagent Easily residual components Leave behind

Swelling method Formula (II)

Retention of macroscopic microstructure

Completely dissolve

Is free of

Acellular matrix, free of Activity of

Is complicated with Long term

Strong support Degree of rotation

Good taste

Difference (D)

Can be used for

Difference (D)

Support hole Adjustment of gap rate Node (C)

Is adjustable

Is free of

Limited adjustment

Is free of

Tissue engineering worker Program micro group Tissue transplantation Applications of

Is free of

Bone defect repair

Is free of

Wound healing Dressing material

Claims

1. A biological tissue engineering regeneration scaffold technology prepared from scale-free freshwater fish skin and an application mode are characterized in that: the scale-free freshwater fish variety with a specific breeding mode is selected, the characteristics of natural collagen components and scale-free structures of the fish variety are applied, the materials are free of harmful reagent residues through the technologies of skin taking, impurity protein and impurity components removing, swelling, freeze drying and the like, the materials are prepared into a biological tissue engineering regeneration bracket 3, and the biological tissue engineering regeneration bracket is matched with a micro-tissue functional unit 4 and applied to tissue and organ regeneration 1 and 2.

2. The biological tissue engineering regeneration scaffold technology and the application mode prepared from the fish skin of the scale-free fresh water fish according to claim 1 are characterized in that: the scale-free freshwater fish includes but is not limited to (1) non-scaly mirror carp (academic name: Cyprinus carpio var. specularis); (2) grass carp, snakehead, catfish, eel; (3) other non-scale freshwater fish species; (4) the descaled tilapia, salmon and other varieties; (5) through the selection of specific culture technology.

3. The scale-free freshwater fish of claim 2, wherein: the non-scaly mirror carp (scientific name: Cyprinus carpio var. specularis); the weight is more than 200 g; raising with clean water source, raising the temperature of raising water with dissolved oxygen over 5mg/L, no parasite, no impurity, no bacteria, no heavy metal pollution, etc. and with fish skin collagen thermal denaturation temperature higher than 36 deg.c; the other scale-free freshwater fish species all reach the feeding standard.

4. The scale-free freshwater fish of claim 2, wherein: the selective specific breeding technology comprises the following steps of breeding in a special mode in a breeding stage, aiming at being applied to the field of medical health in the later period, and comprising but not limited to: (1) raising the temperature of the feeding water; (2) adding antibacterial components such as ionic silver in feed and culture water; (3) other special treatment measures aimed at clinical applications.

5. The biological tissue engineering regeneration scaffold technology and the application mode prepared from the fish skin of the scale-free fresh water fish according to claim 1 are characterized in that: the fish skin includes but is not limited to (1) the skin of the side of the fish, the back of the fish and the abdomen; (2) swim bladder, fish intestine, bone tissue, etc.; (3) the thickness of the fish skin is more than 0.3 mm; (4) the fish skin is rich in fatty acid; (5) no leukoderma, ulcer and other diseases; (6) the available tissues of fin and tail.

6. The biological tissue engineering regeneration scaffold technology and the application mode prepared from the fish skin of the scale-free fresh water fish according to claim 1 are characterized in that: biological tissue engineering regeneration scaffolds include, but are not limited to, (1) wound biological dressings; (2) wound surface films and micro-needle hemostatic materials; (3) wound surface artificial skin; (4) an acellular dermal matrix scaffold; (5) a biological tissue engineering scaffold carrier; (6) skin regeneration minimum functional unit carriers; (7) liver, bone, adipose tissue regeneration minimal functional unit vector, and the like.

7. The tissue engineering regeneration scaffold of claim 6, wherein: applied to partial or full-thickness defects of human skin; wound surface caused by chronic diseases such as burn, trauma, operation, diabetes, etc.; the auxiliary regeneration bracket material of other human tissue organs such as the minimum functional unit of skin tissue regeneration, the minimum functional unit of liver tissue regeneration, the minimum functional unit of bone tissue regeneration and the like and the regeneration and reconstruction medicine is applied.

8. The tissue regeneration minimum functional unit of claim 6, wherein: human tissues such as skin, liver, bones and the like have the characteristics of structural unit form, and the minimum acquisition method, the minimum acquisition radius and the minimum acquisition volume can be determined through statistical calculation; acquisition above this standard can create unnecessary trauma to the tissue; less than this criterion, cannot contain fully functional regenerative tissue; the minimum unit micro-organizational structure characteristics include, but are not limited to: skin, liver, bone, fat, etc.; the stent material is needed to assist the regeneration thereof; the donor area heals depending on the self-regeneration capacity; the functional unit can realize volume and function regeneration under the assistance of tissue engineering scaffold materials by virtue of self regeneration potential after transplantation.

9. The acquisition method according to claim 8, characterized in that: full-automatic and semi-automatic devices are applied; the method is quick and efficient to obtain, and meanwhile, the damage to a supply area is minimized; including but not limited to electric trephines, electric pin headers, skin micro-tissue extraction devices, etc.

10. The biological tissue engineering regeneration scaffold technology and the application mode prepared from the fish skin of the scale-free fresh water fish according to claim 1 are characterized in that: techniques and applications include, but are not limited to: high-concentration sodium chloride solution, very-low-concentration acetic acid (acetic acid solution), carbon dioxide acid-base neutralization technology, freezing preservation technology, freeze-drying preservation technology, ultraviolet, solution and gel disinfection technology and the like are applied.

11. The techniques and applications of claim 10, wherein: cryopreservation techniques include, but are not limited to: directly or after desalting, decalcification and depigmentation, immersing the fish skin tissue in purified water or normal saline, freezing at-20 ℃, freezing, transporting, and thawing, and applying to the fields.

12. The techniques and applications of claim 10, wherein: lyophilization preservation techniques include, but are not limited to: the product is prepared into a freeze-drying bracket by applying a vacuum negative pressure drying device at the temperature of-20 ℃, is stored in an aseptic and sealed manner, and is applied to the field.

13. The techniques and applications of claim 10, wherein: uv, solution, gel disinfection techniques include, but are not limited to: sterilizing the fish skin tissue engineering material by using ultraviolet rays; sterilizing the fish skin tissue engineering material by using III type iodine solution, chlorhexidine and the like; sterilizing and moisturizing the fish skin tissue engineering material by using Meibao scald ointment, metronidazole gel and the like; the glycerol component product is used for sterilizing and moisturizing the fish skin tissue engineering material, and other treatment modes relate to sterility and moisturizing.