CN113476667A

CN113476667A - Fish skin acellular dermal matrix scaffold and preparation method and application thereof

Info

Publication number: CN113476667A
Application number: CN202110842811.6A
Authority: CN
Inventors: 王园园; 李八方; 李青; 代元坤; 宋文山
Original assignee: Qingdao Lanyuan Bioengineering Co ltd; Qingdao Marine Biomedical Research Institute Co Ltd
Current assignee: Qingdao Marine Biomedical Research Institute Co Ltd
Priority date: 2021-07-26
Filing date: 2021-07-26
Publication date: 2021-10-08

Abstract

The invention relates to the field of biomedical materials, in particular to a fish skin acellular dermal matrix scaffold and a preparation method and application thereof. The method comprises the following steps: pretreating fish skin to obtain a dermis skin sheet; and (3) respectively carrying out virus inactivation, decoloration, degreasing, immunogenicity removal, crosslinking, freeze-drying and shaping, incision and sterilization on the dermis sheet. The method of the invention reduces the damage to the collagen structure of the fish skin dermal matrix scaffold to the maximum extent, and the collagen fiber is arranged neatly, has uniform and regular pores and strong hydrophilicity, can be quickly vascularized, and has good mechanical property, biocompatibility and tissue induction capability. The material is widely used for replacement and repair treatment of dermal defects.

Description

Fish skin acellular dermal matrix scaffold and preparation method and application thereof

Technical Field

The invention relates to the field of biomedical materials, in particular to a fish skin acellular dermal matrix scaffold and a preparation method and application thereof.

Background

The skin is the largest organ in the human body and has an extremely important barrier function: on one hand, the loss of water, electrolyte and other substances in the body is prevented; on the other hand, the invasion of the outside harmful substances is prevented. When the superficial layer of skin or small area of skin is damaged, the body can regenerate new skin. However, if a large area of deep skin is wounded, the skin cannot be repaired by itself, and then autologous skin transplantation is usually performed. Currently, a composite method of Acellular Dermal Matrix (ADM) and autologous epidermal graft, which is a Dermal substitute for a full-thickness skin defect wound, is a promising method for full-thickness skin defect repair requiring skin grafting.

ADM is obtained by removing antigen components such as epidermal layer and cells in animal skin from natural skin through a series of physical and chemical methods, and retaining dermal matrix components. ADM can be used for repairing burn and wound surfaces, repairing mucosa and filling soft tissue. The sources of autologous skin and allogenic skin are very limited, and acellular dermal matrixes of xenogenic skin (such as pig, cow leather and the like) become hot spots of current tissue repair research, but research and development of fish skin tissues as acellular dermal matrixes are not common. And a series of immune rejection reactions easily occur in the process of repairing the dermal tissue. Acellular dermal scaffolds can be prepared in a variety of ways in order to remove all unwanted cellular components from the dermis while preserving the integrity of the collagen network, etc., in the dermis to the maximum extent.

Various methods for preparing an acellular dermal matrix are disclosed in the prior art, and for example, patent application publication No. CN201350162Y discloses an acellular allogeneic dermal matrix tissue patch, but the methods for preparing the acellular dermal matrix are not described in detail; patent application publication No. CN1775189 uses repeated treatments of mammalian skin with sodium hydroxide solution and detergent to remove cytogenetic material DNA from the tissue, and then uses detergent to remove lipid material from the cell membrane, but is not effective in removing cell debris; patent application publication No. CN102781485A discloses a scaffold material for wound care and/or other tissue healing applications and a method for preparing the same, but the method does not provide a virus inactivation process, does not remove immunogenic substances, i.e., fats, easily causes chemical residues using various chemical agents, thereby causing immune response, and is complicated in preparation and operation process and long in time; patent application publication No. CN111084900A discloses a method for preparing a novel acellular fish skin matrix, but it is only used as a wound dressing and not as a dermal scaffold in complex therapy.

In the preparation process of ADM, a virus inactivation process and a decellularization process are main process and technical difficulties, and the virus, cell-free fragments and DNA components are required to be completely removed or inactivated from tissues, and meanwhile, the three-dimensional structure of a natural decellularized scaffold is completely reserved. Most of the existing ADM preparation methods cannot completely remove DNA components, and have the disadvantages of complicated preparation procedures, long time consumption, and poor hydrophilicity of most ADMs, which affects cell infiltration; too dense, resulting in slow vascularization; the patch is not strong in sticking property, so that the skin patch is not easy to survive and the skin transplantation fails; too fast degradation, later wound contraction, etc.

Although relevant researches on the fish skin acellular dermal matrix are rarely seen in China, the biocompatibility researches on the fish collagen show that the fish collagen has lower antigenicity and can not cause obvious anaphylactic reaction. And the risk of spreading viruses by humans and animals in the fish skin acellular dermal matrix is far lower than that of spreading viruses by pigs and cattle hide, and the clinical safety is higher. In addition, China is a fishery big country, and with the expansion of aquaculture scale and the development of related processing industry, medical supplies with high added value can be formed by reasonably developing wastes of a plurality of aquatic products, so that the environment is protected.

Therefore, the invention aims to provide a novel acellular dermal matrix scaffold material which has low immunogenicity, good hydrophilicity, rapid vascularization, improved survival rate of skin grafting and proper degradation period and dermal regeneration speed and a preparation method thereof aiming at the defects of the prior art.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a fish skin acellular dermal matrix scaffold and a preparation method and application thereof.

In order to achieve the above object, a first aspect of the present invention provides a method for preparing a fish skin acellular dermal matrix scaffold, comprising the steps of:

1) the process for preparing the leather skin sheet comprises the following steps: puffing fish skin in an acid solution, removing epidermis and subcutaneous tissues after puffing to obtain a dermis layer, and then puffing the dermis layer in a saline solution to obtain a dermis layer skin sheet;

2) virus inactivation and decoloration processes: soaking the dermis leather sheet in a virus inactivation and decoloration solution for virus inactivation and decoloration treatment;

3) degreasing process: soaking the corium skin pieces treated in the step 2) in an alkali solution and a first surfactant solution respectively for degreasing;

4) and (3) an immunogenicity removal process: soaking the dermis layer skin pieces treated in the step 3) in a hypotonic solution, a trypsin solution and a second surfactant solution respectively to remove immunogenic substances; wherein the osmotic pressure of the hypotonic solution is less than 7.6 atmospheres;

5) and (3) a crosslinking process: crosslinking the corium skin sheet treated in the step 4) under the heating condition; or soaking the corium skin piece treated in the step 4) in a cross-linking agent solution for cross-linking treatment;

6) a freeze-drying and shaping process: washing the corium skin sheet treated in the step 5), and then putting the corium skin sheet into a freeze-drying agent solution for freeze-drying;

7) a notching process: sequentially carrying out penetrating incision and shaping on the corium layer skin sheet treated in the step 6);

8) the sterilization process comprises the following steps: and (3) carrying out radiation sterilization on the dermis layer skin slice treated in the step 7) to obtain the fish skin acellular dermis matrix scaffold.

In a second aspect, the invention provides a fish skin acellular dermal matrix scaffold prepared by the method described above.

In a third aspect, the present invention provides the use of a fish skin acellular dermal matrix scaffold as described above for the preparation of an acellular dermal matrix.

The method of the invention has the following characteristics:

1) according to the preparation method of the fish skin acellular dermal matrix scaffold, the fish skin is used as a raw material, so that the risk of carrying and spreading the zoonosis virus caused by using land mammal-derived materials such as pigs or cows is avoided; and the raw materials have wide sources, traceability and low price.

2) The method of the invention adopts an acid method for swelling to increase the thickness of the skin, so that the skin of the real leather layer is easy to obtain, the basement membrane is reserved, the growth of the epithelial layer is convenient, and the biocompatibility of the product is improved.

3) According to the method, the epidermal layer, the foreign proteins, the lipids and other immunogenic substances are removed, and the degreased dermal layer skin is respectively soaked in the hypotonic solution, the trypsin solution and the surfactant solution, so that the immunogenicity of the fish skin dermal matrix scaffold is reduced to the maximum extent, and the safety of the product is improved; the method of the invention ensures that the original structure of the material does not collapse by freeze-drying in the freeze-drying agent solution, ensures a certain porosity of the stent and is beneficial to the growth of blood vessels and cells; the method can generate stronger capillary attraction through the penetrable incision, can fully guide the penetration of the plasma of the wound surface, and provides sufficient nutrition for the autologous skin of the skin grafting at the early stage, thereby further improving the survival rate of the skin grafting.

4) The method of the invention reduces the damage to the collagen structure of the fish skin dermal matrix scaffold to the maximum extent, and the collagen fibers are arranged in order, have uniform and regular pores, strong hydrophilicity, can be quickly vascularized and have good mechanical property, so that the scaffold effect in the process of repairing dermal tissue can not be lost due to too fast degradation (the degradation period is proper to the regeneration speed of the dermis), and the invention has good biocompatibility and tissue induction capability. The material is widely used for replacement and repair treatment of dermal defects.

5) Compared with other virus inactivation methods, the method can inactivate viruses and play a role in chemical bleaching, can completely inactivate the viruses, has a killing role on both enveloped and non-enveloped viruses, can inactivate sealed products, has low damage to materials, cannot pollute the environment or generate substances with toxic and side effects after irradiation, and greatly reduces the risk of animal-derived medical instruments.

6) The fish skin acellular dermal matrix scaffold prepared by the invention is easy to store and transport.

7) The fish skin acellular dermal matrix scaffold prepared by the invention has the advantages of high hydration speed, time saving and easy infiltration, vascularization and tissue regeneration of later-stage cells when in use.

8) From the test examples 2, 4 and 5, it can be seen that the fish skin acellular dermal matrix scaffold provided by the invention has low immunogenicity; test example 5 shows that the fish skin acellular dermal matrix scaffold provided by the invention has good hydrophilicity, can be quickly vascularized so as to improve the survival rate of skin grafting, has a proper degradation period and dermal regeneration speed, and can avoid the characteristic of later-stage wound surface shrinkage.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

FIG. 1 is a photograph showing a real object of the acellular dermal matrix scaffold of fish skin according to example 1;

FIG. 2 is a reverse photograph of the fish skin acellular dermal matrix scaffold of example 1;

FIG. 3 is a front view of the scanning electron microscope of the dermal matrix scaffold of decellularized fish skin of example 1;

FIG. 4 is a back view of the scanning electron microscope of the dermal matrix scaffold with skin decellularized in the fish skin of example 1;

FIG. 5 is a scanning electron microscope cross-sectional image of the dermal matrix scaffold of decellularized fish skin of example 1;

FIG. 6 is a photograph of HE staining of the acellular dermal matrix scaffold of fish skin of example 1 at a magnification of 100 times;

FIG. 7 is a general view of the rat skin acellular dermal matrix scaffold implanted in situ for 4 weeks for healing of acute wound;

fig. 8 is a picture of HE staining of a fish skin acellular dermal matrix scaffold in situ implanted for 4 weeks for acute wound healing in rats with 400-fold magnification.

Figure 9 is the change in fiber bulk with and without lyophilizate solution (left unused, right used);

figure 10 is a graph of the change in thickness of a solution of the material lyophilizate of the same thickness before and after use (left before and right after use);

FIG. 11 is a graph of vascularization in a 2 week animal model experiment of non-incised material;

figure 12 is a graph of vascularization in a 2 week animal model test of the incision material.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the present invention provides a method for preparing a fish skin acellular dermal matrix scaffold, comprising the steps of:

According to the invention, the fish skin can be derived from any fish species, and can be fish of a scaled species or fish of a non-scaled species. Wherein, the fish of the scaled variety may include, but is not limited to, grass carp, snakehead, tilapia, and cod; the non-scaly breeds of fish may include, but are not limited to, eel, catfish, and eel.

Preferably, the skin is derived from fish of a species having a scale, more preferably tilapia.

According to the invention, when the skin is derived from a scaled species of fish, it is preferred that the skin is mechanically descaled prior to acid expansion. When the fish skin is derived from a non-scale variety of fish, the fish skin can be directly subjected to acid puffing.

It is well known that fish skin, whether of the scaly or non-scaly variety, includes epidermal, dermal and subcutaneous tissue. Preferably, the thickness of the dermal layer obtained after removal of the epidermis and subcutaneous tissue after acid bulking is 0.4-1.6mm, for example, 0.4mm, 0.6mm, 0.8mm, 1.0mm, 1.2mm, 1.4mm, 1.6 mm.

Wherein the epidermis and subcutaneous tissue of the fish skin can be removed using an electric skinning machine.

According to the present invention, in step 1), the acid solution may be any conventional acidic solution, but in order to effectively enhance the acid swelling effect, preferably, the acid solution is selected from any one or more of a hydrochloric acid solution, a sulfuric acid solution, a phosphoric acid solution, a formic acid solution and an acetic acid solution, and more preferably, hydrochloric acid.

The concentration of the acid in the acid solution may be selected from a wide range, and is preferably 0.01 to 1mol/L, and may be, for example, 0.01mol/L, 0.05mol/L, 0.06mol/L, 0.07mol/L, 0.08mol/L, 0.09mol/L, 0.1mol/L, 0.11mol/L, 0.12mol/L, 0.13mol/L, 0.14mol/L, 0.15mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.7mol/L, 0.8mol/L, 0.9mol/L, 1mol/L, more preferably 0.05 to 0.5mol/L, and still more preferably 0.08 to 0.12 mol/L.

The acid swelling time can be adjusted within a wide range according to the swelling effect, and is preferably 10 to 60min, for example, 10min, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, and 60min, preferably 15 to 25min, and more preferably 18 to 22 min.

The invention adopts an acid method for swelling to increase the thickness of the skin, so that the skin of the real leather layer is easy to obtain, and the basement membrane is reserved, thereby facilitating the growth of the epithelial layer and improving the biocompatibility of the product.

According to the invention, the salt solution can be selected within a wide range, as long as it is effective in de-bulking. In order to effectively improve the de-bulking effect, the salt solution is preferably selected from one or more of sodium chloride solution, potassium chloride solution and calcium chloride solution, and more preferably sodium chloride solution.

The salt concentration in the salt solution may be selected from a wide range, and is preferably 0.5 to 7% by weight, and for example, may be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, more preferably 0.5 to 5%, and still more preferably 0.8 to 1.2%.

The time for the de-bulking can be adjusted within a wide range according to the effect of the de-bulking, and preferably the time for the de-bulking is 0.5 to 3 hours, for example, 0.5 hour, 0.6 hour, 0.7 hour, 0.8 hour, 0.9 hour, 1 hour, 1.1 hour, 1.2 hour, 1.3 hour, 1.4 hour, 1.5 hour, 2 hour, 2.5 hour, 3 hour, preferably 0.5 to 2 hours, and more preferably 50 to 80 minutes.

According to the present invention, in step 2), the virus inactivation and decolorization solution may be a conventional substance capable of virus inactivation and decolorization of a product, preferably, in order to more effectively ensure the use effect of the prepared fish skin acellular dermal matrix scaffold, the virus inactivation and decolorization solution contains peroxide and optionally alkali, and more preferably, the virus inactivation and decolorization solution is a peroxide solution.

The peroxide may vary within wide limits and may include, for example, but is not limited to, peracetic acid, hydrogen peroxide, preferably hydrogen peroxide.

The concentration of the peroxide may be varied within a wide range, and is preferably 0.5 to 5% by weight, for example, 0.5%, 1%, 1.1%, 1.5%, 2%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 4%, 5%, more preferably 0.5 to 4%, and still more preferably 2.5 to 3.5%.

Among them, the base may be various bases conventionally used, and for example, may include, but not limited to, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate, and more preferably sodium hydroxide.

When the virus inactivation and decolorization solution contains a base, the concentration of the base is preferably 0.1 to 3% by weight, and for example, may be 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 1% by weight, 1.1% by weight, 1.5% by weight, 2% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3% by weight, and more preferably 0.1 to 0.5% by weight.

According to the present invention, the time of the virus inactivation and decolorization treatment can be adjusted within a wide range according to the effect, preferably 2 to 12 hours, more preferably 2 to 8 hours, and still more preferably 3 to 5 hours.

According to the present invention, in the step 3), the alkali solution may be a substance that generally provides an alkaline environment, and in order to further enhance the degreasing effect, preferably, the solute of the alkali solution is selected from at least one of sodium carbonate, sodium bicarbonate and sodium hydroxide, and more preferably, sodium carbonate. Preferably, the concentration of the solute in the alkali solution is 0.5 to 2 wt%, 0.5 wt%, 0.8 wt%, 1 wt%, 1.3 wt%, 1.5 wt%, 1.8 wt%, 2 wt%, more preferably 0.8 to 1.2 wt%.

According to the present invention, preferably, the first surfactant is at least one selected from sodium dodecyl sulfate, alkyl glucoside, triton x-100, sodium deoxycholate, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid, 4- (1,1,3, 3-tetramethylbutyl) phenyl-polyethylene glycol, and tert-octylphenoxypolyethoxyethanol, and more preferably alkyl glucoside.

Preferably, the concentration of the surfactant in the first surfactant solution is 0.1 to 5% by weight, and for example, may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, more preferably 0.2 to 2%, and still more preferably 1.3 to 1.7%. Within the concentration range of the invention, the toxicity brought by the surfactant can be effectively reduced.

Preferably, the total time for degreasing is 1-40h, more preferably 2-35h, even more preferably 4-30 h.

According to the present invention, the order of soaking the alkali solution and the first surfactant solution is not particularly limited, and is not in order. Preferably, the ratio of the time of soaking in the alkaline solution to the time of soaking in the first surfactant solution is 1: 2-3.

According to the present invention, in step 4), preferably, the hypotonic solution is water, and for example, may be deionized water, purified water, ultrapure water, and the like, and more preferably, ultrapure water.

According to the present invention, it is preferable that the concentration of trypsin in the trypsin solution is 0.01 to 2% by weight, for example, may be 0.01%, 0.03%, 0.05%, 0.08%, 0.1%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2% by weight; more preferably 0.025 to 1.5% by weight, and still more preferably 0.3 to 0.8% by weight.

According to the present invention, preferably, the second surfactant is at least one selected from sodium dodecyl sulfate, alkyl glucoside, triton x-100, sodium deoxycholate, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid, 4- (1,1,3, 3-tetramethylbutyl) phenyl-polyethylene glycol, and tert-octylphenoxypolyethoxyethanol, and more preferably, sodium deoxycholate.

Preferably, the concentration of the surfactant in the second surfactant solution is 0.5 to 5% by weight, and for example, may be 0.5%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, more preferably 1 to 3%, and still more preferably 1.5 to 2.5% by weight. Within the concentration range of the invention, the toxicity brought by the surfactant can be effectively reduced.

The method of the invention reduces the immunogenicity of the fish skin dermal matrix scaffold to the maximum extent by respectively soaking the dermis skin sheet in the hypotonic solution, the protease solution and the surfactant solution, thereby improving the safety of the product

According to the invention, the total time for removal of the immunogenic substance is preferably 6-70h, preferably 15-65h, more preferably 30-58 h.

According to the present invention, the order of the soaking of the hypotonic solution, the trypsin solution and the second surfactant solution is not particularly limited, nor is it divided into two. Preferably, the ratio of the time of soaking in the hypotonic aqueous alkaline solution, the time of soaking in the trypsin solution and the time of soaking in the second surfactant solution is 1: (1-2): (1-2).

According to the present invention, in step 5), when the heating method is used for crosslinking, the heating temperature can be selected within a wide range by those skilled in the art, and in order to effectively improve the properties of the final product, the heating conditions include: the temperature is 100 ℃ to 160 ℃ (for example, 100 ℃, 110 ℃, 120 ℃, 130 ℃, 140 ℃, 150 ℃ and 160 ℃), and the time is 24-72h (for example, 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h and 72 h).

According to the invention, in step 5), when crosslinking is carried out in a crosslinker solution, the crosslinker solution is preferably a genipin solution, a carbodiimide solution or a glutaraldehyde solution, more preferably a carbodiimide solution.

The concentration of the crosslinking agent in the crosslinking agent solution may vary within a wide range, and is preferably 0.1 to 3 wt%, and for example, may be 0.1 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.3 wt%, 1.5 wt%, 1.8 wt%, 2 wt%, 2.3 wt%, 2.5 wt%, 2.8 wt%, 3 wt%, and more preferably 0.4 to 0.6 wt%.

The time of the crosslinking treatment may be selected from a wide range, preferably 2 to 15 hours, and for example, may be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, preferably 10 to 15 hours.

According to the invention, in step 6), the washing is preferably carried out in ultrapure water, which may be washed, for example, 3 to 5 times.

According to the present invention, the lyophilizate solution is preferably at least one selected from the group consisting of an aqueous solution of dimethyl sulfoxide, an aqueous solution of t-butanol and an aqueous solution of cyclohexane, more preferably an aqueous solution of t-butanol.

According to the invention, the concentration of the lyophilizate in the lyophilizate solution can be selected within a wide range, preferably from 2 to 15% by weight, and can be, for example, 2% by weight, 2.2% by weight, 2.4% by weight, 2.6% by weight, 2.8% by weight, 3% by weight, 3.2% by weight, 3.4% by weight, 3.6% by weight, 3.8% by weight, 4% by weight, 5% by weight, 6% by weight, 7% by weight, 8% by weight, 9% by weight, 10% by weight, 11% by weight, 12% by weight, 13% by weight, 14% by weight, 15% by weight, preferably from 2 to 4% by weight.

The freeze-drying time can be selected in a wide range, preferably 30-180min, for example, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 85min, 100min, 120min, 140min, 160min, 180min, more preferably 40-60 min.

The method of the invention ensures that the original structure of the material does not collapse by freeze-drying in the freeze-drying agent freeze solution, ensures a certain porosity of the stent and is beneficial to the growth of blood vessels and cells.

According to the invention, in step 7), preferably, in order to further increase the effectiveness of the product, the penetrating cuts are penetrating semicircular cuts.

The invention adopts a mode of a penetrable incision to generate stronger capillary attraction, can fully guide the penetration of the plasma of the wound surface, and provides sufficient nutrition for the autologous skin of the skin grafting at the early stage, thereby further improving the survival rate of the skin grafting.

Preferably, the diameter of the incision is 0.5-2 mm; the distance between the centers of the notches is 1.5-3 mm.

The method of the invention can fully guide the penetration of the plasma of the wound surface through the incision, and further improve the survival rate of the skin grafting.

Preferably, the shape is any shape such as a circle, a square, or a rectangle.

According to the invention, in step 8), the radiation dose for radiation sterilization is preferably 15 to 25kGy, and may be, for example, 15kGy, 16kGy, 17kGy, 18kGy, 19kGy, 20kGy, 21kGy, 22kGy, 23kGy, 24kGy, 25kGy, 26kGy, 27kGy, 28kGy, 29kGy, 30kGy, and preferably 18 to 22 kGy.

The invention preferably adopts a method of double inactivation of peroxide and irradiation sterilization (electron accelerator), compared with other virus inactivation methods, the method not only can inactivate viruses, but also plays a role in chemical bleaching, can inactivate the viruses thoroughly, has a killing effect on enveloped and non-enveloped viruses, can inactivate sealed products, has low damage effect on materials, cannot pollute the environment or generate substances with toxic and side effects after irradiation, and greatly reduces the risk of animal-derived medical instruments.

In a second aspect, the present invention provides a fish skin acellular dermal matrix scaffold prepared by the above method.

The fish skin acellular dermal matrix scaffold is an extracellular matrix with a three-dimensional space frame structure, and comprises a relatively compact surface formed by a biological basement membrane and a relatively loose surface of collagen formed by a dermal scaffold, and a penetrating incision capable of promoting plasma permeation is preferably arranged on the fish skin acellular dermal matrix scaffold.

The present invention will be described in detail below by way of examples. In the following examples of the present invention,

the thickness of the skin is measured by a thickness gauge;

the defatting rate was measured by the total fat content method;

the efficiency of removing the impurity protein is measured by a method for measuring the content of the collagen by Kjeldahl determination of nitrogen;

the efficiency of removing the glycoprotein is measured by a method for measuring the total sugar content of the material;

the cell removal efficiency is measured by a method for quantifying extracted residual DNA;

the crosslinking strength is measured by a method of measuring the degree of crosslinking.

Example 1

This example illustrates the present invention for the preparation of a dermal matrix scaffold for skin decellularization and the preparation method thereof

1) The process for preparing the leather skin sheet comprises the following steps: taking fresh snakehead skin, mechanically descaling the snakehead skin, puffing the snakehead skin for 15min by using a 0.05M acetic acid solution, removing 0.2mm of epidermis and subcutaneous tissues of the snakehead skin by using an electric snakehead skin taking machine, preparing into a dermis layer skin sheet with the thickness of 0.8mm, and soaking the skin sheet in a 0.5 weight percent potassium chloride solution for puffing for 30 min;

2) virus inactivation and decoloration processes: soaking the corium skin piece in 1 weight percent peroxyacetic acid solution for 2 hours for virus inactivation and decoloration treatment;

3) degreasing process: soaking the corium skin piece obtained in the step 2) in 0.5 weight percent sodium hydroxide solution for 1h and in 0.2 weight percent TritonX-100 solution for 3h for degreasing;

4) and (3) an immunogenicity removal process: soaking the corium skin piece obtained in the step 3) in ultrapure water for 6 hours, 0.025 weight percent of trypsin solution for 12 hours and 1 weight percent of sodium deoxycholate solution for 12 hours to remove immunogenic substances such as DNA, foreign proteins, glycoprotein and the like;

5) and (3) a crosslinking process: placing the real leather skin sheet obtained in the step 4) in a vacuum drying oven at the temperature of 120 ℃ for high-temperature crosslinking for 24 hours for crosslinking treatment;

6) a freeze-drying and shaping process: soaking the corium skin sheet obtained in the step 5) in sterile ultrapure water, washing for 3 times, then placing the corium skin sheet in 2.0 weight percent of dimethyl sulfoxide for 30min, and freeze-drying;

7) a notching process: cutting the dermis layer skin sheet by using a cutting device, wherein the cut is a penetrating semicircular cut, the diameter of the cut is 0.5mm, and the center distance of the cut is 1.5 mm;

8) the sterilization process comprises the following steps: and packaging the dermis layer skin sheet, and then irradiating and sterilizing the dermis layer skin sheet by using an electron accelerator, wherein the irradiation dose is 15kGy, so as to obtain the fish skin acellular dermis matrix bracket.

Example 2

1) The process for preparing the leather skin sheet comprises the following steps: taking fresh tilapia skin, mechanically descaling tilapia skin, puffing with 0.1M hydrochloric acid solution for 20min, removing 0.25mm epidermis and subcutaneous tissue from the tilapia skin with an electric skin taking machine, making into dermis layer skin sheet with the thickness of 1.0mm, soaking the skin sheet in 1 wt% sodium chloride solution, and puffing for 60 min;

2) virus inactivation and decoloration processes: soaking the corium skin piece in 3 wt% hydrogen peroxide solution for 4h for virus inactivation and decolorization;

3) degreasing process: soaking the corium skin pieces obtained in the step 2) in a sodium carbonate solution with the weight percent of 1% and an alkyl glucoside solution with the weight percent of 1.5% respectively for degreasing, wherein the soaking time is 3 hours and 6 hours respectively;

4) and (3) an immunogenicity removal process: soaking the dermis skin sheet obtained in the step 3) in ultrapure water for 12 hours, 0.5 weight percent of trypsin solution for 12 hours and 2 weight percent of sodium deoxycholate solution for 16 hours to remove immunogenic substances such as DNA, foreign proteins, glycoprotein and the like;

5) and (3) a crosslinking process: placing the corium skin sheet obtained in the step 4) in 0.1 weight percent carbodiimide solution for crosslinking for 12 hours for crosslinking treatment;

6) a freeze-drying and shaping process: soaking the corium skin sheet in sterile ultrapure water, washing for 3 times, then placing in 3.0 wt% of tert-butyl alcohol for 50min, and freeze-drying;

7) a notching process: cutting the dermis layer skin sheet by using a cutting device, wherein the cut is a penetrating semicircular cut, the diameter of the cut is 1.0mm, and the center distance of the cut is 2.0 mm;

8) the sterilization process comprises the following steps: and packaging the dermis layer skin sheet, and then irradiating and sterilizing the dermis layer skin sheet by using an electron accelerator, wherein the irradiation dose is 20kGy, so as to obtain the fish skin acellular dermis matrix bracket.

Example 3

1) The process for preparing the leather skin sheet comprises the following steps: taking fresh catfish skin, mechanically descaling the catfish skin, puffing with 0.1M formic acid solution for 60min, removing 0.5mm epidermis and subcutaneous tissue from the catfish skin with an electric skin taking machine to obtain a dermis layer skin sheet with the thickness of 1.2mm, soaking the skin sheet in 5 wt% calcium chloride solution, and puffing for 2 h;

2) virus inactivation and decoloration processes: soaking the corium skin piece in a mixed solution of 2 weight percent of hydrogen peroxide solution and 0.1 weight percent of sodium hydroxide for 4 hours to inactivate viruses and decolor;

3) degreasing process: soaking the corium skin piece obtained in the step 2) in a 2 wt% sodium hydroxide solution for 5 hours and a 2 wt% sodium dodecyl sulfate solution for 10 hours for degreasing;

4) and (3) an immunogenicity removal process: soaking the dermis skin sheet obtained in the step 3) in ultrapure water for 16h, 1.5 wt% of trypsin solution for 18h and 2 wt% of sodium deoxycholate solution for 24h to remove immunogenic substances such as DNA, hybrid protein, glycoprotein and the like;

5) and (3) a crosslinking process: placing the corium skin sheet obtained in the step 4) into a glutaraldehyde solution with the mass percentage concentration of 0.5 wt% for crosslinking for 12 hours for crosslinking treatment;

6) a freeze-drying and shaping process: soaking the corium skin sheet in sterile ultrapure water, washing for 3 times, then placing in cyclohexane with the weight percentage of 8% for 80min, and freeze-drying;

7) a notching process: cutting the dermis layer skin sheet by using a cutting device, wherein the cut is a penetrating semicircular cut, the diameter of the cut is 2.0mm, and the center distance of the cut is 3.0 mm;

8) the sterilization process comprises the following steps: and packaging the dermis layer skin sheet, and then irradiating and sterilizing the dermis layer skin sheet by using an electron accelerator, wherein the irradiation dose is 25kGy, so as to obtain the fish skin acellular dermis matrix bracket.

Comparative example 1

Comparative example to illustrate a reference dermal matrix scaffold for skin decellularization and method of preparing the same

The preparation of the fish skin acellular dermal matrix scaffold was performed according to the method of example 2, except that in step 2) and step 8), the sterilization was performed without using the method of double inactivation of peroxide and electron accelerator irradiation sterilization, and Co60 irradiation sterilization was used, and the natural material after sterilization was largely damaged, poor in mechanical properties, too fast in later degradation, and failed to function as a scaffold.

Comparative example 2

The preparation of the fish skin acellular dermal matrix scaffold is carried out according to the method of example 2, except that in the step 7), a penetrating incision is not adopted, but the complete structure of the matrix is kept, and the method can affect the exudation of blood and effusion, easily cause poor adhesion of the matrix and a wound surface, further affect the generation of new tissues and prolong the time of autologous skin transplantation.

Comparative example 3

The preparation of the fish skin acellular dermal matrix scaffold was performed according to the method of example 2, except that, in step 6), no lyophilizate was used.

Fig. 9 and 10 show the change of the fiber porosity and thickness with and without the lyophilized agent solution, wherein the left side of the figure shows the dermal matrix scaffold prepared in comparative example 3, and the right side of the figure shows the dermal matrix scaffold prepared in example 2, which shows that the lyophilized agent solution is used to ensure that the original structure of the material is not collapsed, thereby ensuring that the scaffold has a larger porosity.

Comparative example 4

The preparation of the fish skin acellular dermal matrix scaffold was performed according to the method of example 2, except that the crosslinking step, i.e., the removal step 5), was not employed.

Test example 1

Basic performance test of fish skin acellular dermal matrix scaffold

The fish skin acellular dermal matrix scaffold prepared by the invention is a white membranous substance with a penetrating semicircular incision, the front surface is relatively smooth and compact (the fish skin acellular dermal matrix scaffold prepared by the embodiment 2 is shown in a figure 1), the back surface is rough (the fish skin acellular dermal matrix scaffold prepared by the embodiment 2 is shown in a figure 2), and the fish skin acellular dermal matrix scaffold has certain strength and toughness. The material is soft, and is convenient for surgical suture. Can be cut into different shapes and sizes according to requirements.

After the dermis skin sheet preparation and the virus inactivation process treatment, the pigment of the sample is removed; when the product is observed on a scanning electron microscope of JSM-7500 model manufactured by JEOL company of Japan, the front surface of the product is relatively compact (the fish skin acellular dermal matrix scaffold prepared in example 2 is shown in figure 3), the back surface of the product is relatively loose (the fish skin acellular dermal matrix scaffold prepared in example 2 is shown in figure 4), and the section photo shows that the product has a multilayer fiber structure and a certain porosity (the fish skin acellular dermal matrix scaffold prepared in example 2 is shown in figure 5); after paraffin embedding, tissue sectioning and HE staining, the ordered arrangement of collagen fiber layers, no epidermal layer and subcutaneous tissue layer, and no nuclear residue were observed by an optical microscope of olympus CX31RTSF model (the fish skin acellular dermal matrix scaffold prepared in example 2 is shown in fig. 6).

Test example 2

Quantitative determination of fish skin acellular dermal matrix scaffold DNA residue

The method comprises the following steps of extracting DNA from the fish skin acellular dermal matrix scaffolds prepared in the examples and the comparative examples, and measuring the content of the DNA remained in the fish skin acellular dermal matrix scaffold after acellular treatment, and comprises the following specific steps: a certain amount of the fish skin acellular dermal matrix scaffold is weighed, a genome DNA extraction kit (marine animal tissue genome DNA extraction kit, purchased from Beijing Tiangen Biochemical technology Co., Ltd.) is used for extracting DNA, and the extracted DNA is quantified by an ultramicro ultraviolet spectrophotometer (NanoDropTM One/OnEC, purchased from Thermo Fisher, USA), and the result is shown in Table 1.

TABLE 1 determination of DNA content

Compared with a material without cells, the residual amount of DNA after the cells are removed is greatly reduced, and the residual amount of the DNA of the dry weight of a treated product is less than 50ng/mg, thereby meeting the evaluation standard of the effectiveness of the cell-removed tissue.

Test example 3

Mechanical property detection of fish skin acellular dermal matrix scaffold

Part 4 was determined according to the national standard GBT1040.4-2006 tensile Properties of plastics: the mechanical properties of the material were determined under the test conditions for isotropic and orthotropic fiber-reinforced composites. The specific method comprises the following steps: the acellular dermal matrix scaffolds of fish skin before and after crosslinking in examples and comparative examples were lyophilized and sterilized, and then prepared into a dumbbell shape of 5.0cm × 0.5cm according to the standard, and then stretched at a speed of 20mm/min using an electronic universal tester ((WDW-1, available from jonnan friendly testing machine ltd.) to obtain a stress-strain curve, and the maximum stress was calculated from the stress-strain curve, as shown in table 2.

TABLE 2 maximum stress of the product before and after crosslinking

	uncrosslinked/MPa	Cross-linking/MPa	The lifting ratio%
				Example 1	6.48	18.6	187
Example 2	6.75	33.2	392
				Example 3	7.02	19.4	176
Comparative example 3	5.98	18.9	216
				Comparative example 4	5.46	10.2	87

The above results illustrate that: the stress of the fish skin acellular dermal matrix scaffold is obviously improved by crosslinking, and the fish skin acellular dermal matrix scaffold prepared by the embodiment has good mechanical property.

Test example 4

Biocompatibility evaluation of fish skin acellular dermal matrix scaffold

The fish skin acellular dermal matrix scaffold prepared in example 2 is evaluated to have no more than grade 1 cytotoxic response to L929 fibroblasts according to the method specified in GB/T16886; the delayed hypersensitivity test of the guinea pigs proves that no delayed hypersensitivity is caused; hemolytic tests of the ear arterial blood of the New Zealand white rabbit prove that no hemolytic reaction exists; in the rabbit pyrogen test, the temperature rise values of the rabbits are all lower than 0.6 ℃, and the total temperature rise is lower than 1.3 ℃, so that the pyrogen examination of the product is proved to be in accordance with the regulation; the acute toxicity test of the whole body of the mouse and the repeated contact toxicity test of the whole body of the mouse both prove that the product is qualified; therefore, the prepared fish skin acellular dermal matrix scaffold has good biocompatibility and meets the regulation requirements of III-class medical instruments.

Test example 5

Rat acute wound healing test of fish skin acellular dermal matrix scaffold

The fish skin acellular dermal matrix scaffold prepared in the example 2 is trimmed, the size and the shape meet the requirements, and the fish skin acellular dermal matrix scaffold is placed into physiological saline containing gentamicin for soaking for 15min for standby after being fully hydrated.

The specific operation is as follows: injecting muscle into SD rat with weight of 200g for anesthesia, fixing in prone position, shaving hair on back, sterilizing conventionally, and preparing a 2 cm-2 cm full-thickness skin defect wound surface on one side of rat spine to reach deep fascia; fully stopping bleeding of the wound by using an electric coagulation pen; placing the product on the wound surface with the compact surface facing upwards and the loose surface facing downwards, and covering the product with a 2 cm-2 cm self-body blade-thick skin sheet; sewing the product and the autologous skin together by using a suture line according to 3 needles on each side, and reserving a packed line; compacting the gap between the product and the wound surface, and packaging and fixing the product by using a suture line; rats were housed in a single cage.

A certain number of rats were sacrificed at 2, 4 and 8 weeks after surgery, paraffin embedding, tissue sectioning, HE staining, and observation of vascularization and cell proliferation in the graft area under an optical microscope. Calculating the wound healing rate and the wound shrinkage; wherein, the wound healing rate (%) is (original wound area-unhealed wound area)/original wound area, and the wound shrinkage rate is (total skin area-wound area at observation)/total skin area 100%. Scars were evaluated descriptively by the Vancouver scar scale.

For comparison, the general observation picture of the prepared fish skin acellular dermal matrix scaffold prepared by the method of comparative example 2 without incision after the rat is implanted in situ for 2 weeks for acute wound healing is shown in fig. 11, the vascularization state is general at 2 weeks, and the skin grafting condition is not satisfied, while the general observation picture of the fish skin acellular dermal matrix scaffold prepared by the method of example 2 after the rat is implanted in situ for 2 weeks for acute wound healing is shown in fig. 12, the material is well attached to the wound, the bottom of the scaffold is tightly connected with muscle tissue, the exudation is less, the shrinkage is not generated, the growth of bright red granulation tissue is seen, the wound margin skin is regular, and the subcutaneous hematocele, hydrops and other conditions are not seen, and the skin grafting operation can be performed at the moment. The incision can generate stronger capillary attraction, can fully guide the penetration of the plasma of the wound surface, and provides sufficient nutrition for the autologous skin of the skin grafting at the early stage, thereby further improving the survival rate of the skin grafting.

The general observation picture of the fish skin acellular dermal matrix scaffold prepared in example 2 after the rat acute wound healing in situ implantation for 4 weeks is shown in fig. 7, the skin sheet survives, the wound margin is regular, the combination with the surrounding tissues is good, the skin sheet is normal in color and luster, smooth in surface, soft in texture and good in elasticity, and the wound is only slightly contracted, which indicates that the prepared fish skin acellular dermal matrix scaffold can be used as a dermal regeneration template to promote the healing of the wound, the survival rate after the implantation is high, and the appearance and the function are satisfactory. The HE staining picture of the fish skin acellular dermal matrix scaffold prepared in example 2 after being implanted in situ for 4 weeks for acute wound healing of rats is shown in fig. 8, a large amount of fibroblasts infiltrate the inside of the scaffold, the fibroblasts grow along the scaffold, and a vascular structure can also be seen in the scaffold, which indicates that the scaffold can support infiltration of host fibroblasts and neovascularization after being implanted, and induces dermal tissue to regenerate and gradually degrade, so that scars can be reduced, and the quality of wound healing can be improved.

The experimental result shows that the inflammatory reaction of the fish skin acellular dermal matrix scaffold is lighter, the cell infiltration and blood vessel growth speed is higher, the wound healing rate is 100%, the wound shrinkage rate is 3.68%, the scores of the color, the blood vessel distribution and the softness of a scar are all 0, the score of the thickness is 1, and the scar is proved to be very light. Acute wound healing tests of rats show that the fish skin acellular dermal matrix scaffold and the autologous sword-thick skin composite transplantation have no obvious immunological rejection, high survival rate, low shrinkage rate and obvious appearance and function effects, are ideal biological templates for repairing dermal defects and have wide clinical application prospects.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

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

2) virus inactivation and decoloration processes: soaking the dermis leather sheet in a virus inactivation and decoloration solution for virus inactivation and decoloration treatment, wherein the virus inactivation and decoloration solution contains peroxide;

8) the sterilization process comprises the following steps: and (3) carrying out electron accelerator irradiation sterilization on the dermis layer skin sheet treated in the step 7) to obtain the fish skin acellular dermis matrix support.

2. The method according to claim 1, wherein in step 1), the skin is derived from any one of grass carp, snakehead, tilapia, cod, eel, catfish and eel;

preferably, the thickness of the dermis layer is 0.4-1.6 mm.

3. The method according to claim 1 or 2, wherein in step 1), the acid solution is selected from any one or more of hydrochloric acid solution, sulfuric acid solution, phosphoric acid solution, formic acid solution and acetic acid solution, and the concentration of acid in the acid solution is preferably 0.01-1 mol/L; and/or

The puffing time is 10-60 min; and/or

The salt solution is selected from any one or more of a sodium chloride solution, a potassium chloride solution and a calcium chloride solution, and the concentration of the salt in the salt solution is preferably 0.5-7 wt%; and/or

The de-bulking time is 0.5-3 h.

4. The method according to any one of claims 1 to 3, wherein in step 2), the virus inactivation and decolorization treatment is carried out for 2 to 12 hours; and/or

The concentration of the peroxide is 0.5-5 wt%; and/or

The peroxide is selected from peracetic acid and/or hydrogen peroxide;

preferably, the virus inactivation and decolorization solution further contains a base; the alkali is preferably selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate.

5. The method according to any one of claims 1 to 4, wherein in step 3), the solute of the alkali solution is selected from at least one of sodium carbonate, sodium bicarbonate and sodium hydroxide, and the concentration of the solute in the alkali solution is preferably 0.5 to 2 wt%; and/or

The first surfactant is at least one selected from sodium dodecyl sulfate, alkyl glucoside, TritonX-100, sodium deoxycholate, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, 4- (1,1,3, 3-tetramethylbutyl) phenyl-polyethylene glycol and tert-octylphenoxy polyethoxyethanol, and the concentration of the surfactant in the first surfactant solution is preferably 0.1-5 wt%;

preferably, the total time for degreasing is 1-40 h.

6. The method according to any one of claims 1 to 5, wherein in step 4), the hypotonic solution is ultrapure water; and/or

The concentration of trypsin in the trypsin solution is 0.01-2 wt%; and/or

The second surfactant is at least one selected from sodium dodecyl sulfate, alkyl glucoside, TritonX-100, sodium deoxycholate, 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, 4- (1,1,3, 3-tetramethylbutyl) phenyl-polyethylene glycol and tert-octylphenoxy polyethoxyethanol, and the concentration of the surfactant in the second surfactant solution is preferably 0.5-5 wt%;

preferably, the total time for removal of the immunogenic material is 6-70 h.

7. The method according to any one of claims 1-6, wherein in step 5), the heating conditions comprise: the temperature is 100-160 ℃, and the time is 24-72 h; and/or

The cross-linking agent solution is a genipin solution, a carbodiimide solution or a glutaraldehyde solution; and/or

The concentration of the cross-linking agent in the cross-linking agent solution is 0.1-3 wt%; and/or

The time of the cross-linking treatment is 2-15 h.

8. The method according to any one of claims 1 to 7, wherein in step 6), the lyophilizate solution is selected from at least one of an aqueous solution of dimethyl sulfoxide, an aqueous solution of tert-butanol and an aqueous solution of cyclohexane; and/or

The concentration of the lyophilizate in the lyophilizate solution is 2-15 wt%; and/or

The freeze-drying time is 30-180 min.

9. The method of any one of claims 1 to 8 wherein in step 7) the penetrating cut is a penetrating semicircular cut;

preferably, the diameter of the incision is 0.5-2 mm; the distance between the center of the cut is 1.5-3 mm;

preferably, the shaping is round, square or rectangular.

10. The method according to any one of claims 1 to 9, wherein the radiation dose for radiation sterilization in step 8) is 15-25 kGy.

11. The fish skin acellular dermal matrix scaffold prepared by the method of any one of claims 1 to 10.

12. Use of the fish skin acellular dermal matrix scaffold of claim 11 for the preparation of an acellular dermal matrix.