阅读说明：本技术 一种新型人工皮肤及其制备方法 (Novel artificial skin and preparation method thereof ) 是由 高秀岩 曲梁静 姜红 于 2020-03-20 设计创作，主要内容包括：本发明提供一种新型人工皮肤及其制备方法。该新型人工皮肤制备方法具体如下：取新鲜动物离体皮肤,通过氢氧化钠、DNA酶、胰蛋白酶、十二烷基硫酸钠、过氧乙酸、PBS等一系列处理后获得脱细胞真皮基质,超声混合将氧化石墨烯与脱细胞真皮基质溶液混匀,再通过富集处理后得到一种新型人工皮肤。本发明能够生产成分均一、稳定的脱细胞真皮基质,添加氧化石墨烯增加了基质物理强度,富集处理为表皮干细胞的黏附、爬行及生长提供了优良环境,对创面愈合具有显著效果。该新型人工皮肤的应用,不仅能阻止细菌对受损创面的入侵、破坏,防止创面感染,而且能有选择性的诱导干细胞向皮肤细胞分化,促进创面皮肤再生,提高移植成功率。(The invention provides a novel artificial skin and a preparation method thereof. The preparation method of the novel artificial skin comprises the following steps: taking fresh animal in-vitro skin, carrying out a series of treatments such as sodium hydroxide, DNA enzyme, trypsin, sodium dodecyl sulfate, peracetic acid, PBS and the like to obtain an acellular dermal matrix, carrying out ultrasonic mixing to uniformly mix graphene oxide and an acellular dermal matrix solution, and carrying out enrichment treatment to obtain the novel artificial skin. The invention can produce the acellular dermal matrix with uniform and stable components, the addition of the graphene oxide increases the physical strength of the matrix, the enrichment treatment provides excellent environment for the adhesion, crawling and growth of epidermal stem cells, and the invention has obvious effect on wound healing. The application of the novel artificial skin can not only prevent bacteria from invading and damaging the damaged wound surface and prevent wound surface infection, but also selectively induce stem cells to differentiate towards skin cells, promote the regeneration of the wound surface skin and improve the success rate of transplantation.)

1. A novel artificial skin and a preparation method thereof, the specific steps are as follows:

(1) soaking fresh in-vitro animal skin in a sodium hydroxide solution, washing with deionized water, and scraping redundant body hair and subcutaneous attached adipose tissues;

(2) mechanically crushing the obtained epidermal tissue into small pieces, oscillating and washing the small pieces for 3-5 times by using deionized water, and draining the water;

(3) sequentially oscillating the tissue obtained in the step (2) by using DNA enzyme, trypsin-EDTA, sodium dodecyl sulfate and peracetic acid, and rinsing the tissue by using deionized water;

(4) treating epidermal tissue with PBS solution by oscillation, rinsing with deionized water, draining, and freeze drying;

(5) dissolving the obtained acellular dermal matrix in a pepsin solution, and performing oscillation treatment at 37 ℃ to obtain an acellular dermal matrix solution; dropwise adding sodium hydroxide with proper amount while oscillating to adjust the pH of the solution to be neutral;

(6) ultrasonically and uniformly mixing graphene oxide powder and the obtained acellular dermal matrix solution by using an ultrasonic cell disruptor under the ice bath condition to obtain a uniform graphene oxide-acellular dermal matrix solution;

(7) pouring the treated graphene oxide-acellular dermal matrix solution into a smooth flat-bottom mold, and freeze-drying to obtain a loose and porous acellular dermal matrix;

(8) enriching the above acellular dermal matrix by soaking waste foreskin tissue or skin sheet specimen in calcium-magnesium-free phosphate buffer solution (PBS, pH 7.2) for 3 times, removing subcutaneous fat, and cutting into 0.5 × 1cm²Washing with PBS, adding into 0.25% trypsin solution, digesting at 4 deg.C for 18-22 hr, taking out, removing epidermis from dermis, placing in a small bottle containing 5m L DMEM culture solution, gently blowing the culture solution with a dropper to disperse epidermal cells into the culture solution, filtering with a filter screen, removing residual epidermis to obtain culture solution of epidermal cells, repeatedly flowing the culture solution containing epidermal cells through the prepared acellular dermal matrix by a negative pressure suction device to efficiently enrich cell growth factors in the matrix material, and drying at low temperature to obtain the final productA novel artificial skin is provided.

2. The method according to claim 1, wherein the excised skin of the animal in step (1) is derived from epidermis of mammal such as human, pig, rabbit, cow, sheep, rat, etc., and the concentration of the sodium hydroxide solution is 0.05-0.5 mol/L, and the soaking time is 5-10 h.

3. The method according to claim 1, wherein the DNase in the step (3) has a concentration of 40-60U/m L, the trypsin solution is a mixed solution containing 0.05-0.5% w/v of trypsin and 0.05-0.25% w/v of EDTA, and the shaking treatment time is 1-6 hours.

4. The preparation method according to claim 1, wherein the concentration of the sodium dodecyl sulfate in the step (3) is 0.02-0.5% w/v, and the shaking treatment time is 1-6 h; the volume fraction of the peroxyacetic acid is 0.2-1.0%, and the oscillation treatment time is 0.5-2 h.

5. The method of claim 1, wherein the PBS solution in step (4) is treated with shaking for 1-2h, and the treatment is repeated 3-5 times.

6. The preparation method according to claim 1, wherein the concentration of the pepsin solution in the step (5) is 0.2-0.8 mg/m L, the shaking treatment time is 22-30 h, the neutral pH is 6.8-7.5, and sodium hydroxide is added while shaking.

7. The preparation method according to claim 1, wherein the mass fraction of the graphene oxide in the step (6) is 0.25-0.5%; the ultrasonic cell disruption instrument is operated under the conditions of 25% strength, 2min ultrasonic time, 4s interval time and ice bath.

8. The method according to claim 1, wherein the temperature of the low-temperature drying in the step (8) is 28 to 32 ℃.

9. A novel artificial skin, characterized in that it is prepared by the method for preparing a novel artificial skin according to any one of claims 1 to 8.

10. The novel artificial skin is infiltrated by a small amount of fresh blood or body fluid of a patient before use, so that immunological rejection caused in the use process can be reduced, and the use effect of the novel artificial skin is better than that of deionized water or normal saline.

Technical Field

The invention belongs to the field of tissue engineering biomaterials, and particularly relates to novel artificial skin and a preparation method thereof.

Background

Skin is an important tissue organ for covering and protecting body surface, and skin defects and abnormalities caused by inflammation, ulcer, burn, congenital malformation and the like are common phenomena in clinic. At present, the method of autologous skin transplantation is mostly adopted for the tissue defect, but the source of autologous skin is very limited, and secondary damage can be caused to a patient when the material is taken, so that the treatment process is influenced. In view of this, the tissue engineering skin is the most potential treatment method, and the acellular dermal matrix as a natural extracellular matrix material has good mechanical properties, histocompatibility and lower immunogenicity, is a good matrix for tissue repair and reconstruction, is also a natural scaffold material commonly used in tissue engineering research, and has been widely used as skin and soft tissue wound repair.

The graphene oxide is a novel carbon material with excellent performance, the structure of the graphene oxide spans the typical scale of half of chemistry and material science, the graphene oxide can be regarded as a non-traditional soft material, and the graphene oxide has the characteristics of a polymer, a colloid, a thin film and an amphoteric molecule, the graphene oxide has very strong surface activity and wettability due to the introduction of an oxygen-containing active group ①, a stable dispersion solution can be formed in a common solvent, ② has good hydrophilicity, dispersibility and compatibility and can be used as a reinforcing filling material to avoid the use of a chemical cross-linking reagent, the excellent performance determines the high use value of the graphene oxide, a large number of ③ research results show that GO has excellent antibacterial activity on various common bacteria, the antibacterial capability of GO can be well utilized due to the appropriate action time and concentration and the combination of the GO and a nano compound, ④ GO has very strong surface adsorption capability and high biological safety at the same time, adsorbs serum protein to form a protein inclusion body, shows very low cytotoxicity on cells, the surface of GO is modified by polyethylene glycol, chitosan, hyaluronic acid and the like, almost has no toxicity of functional graphene oxide matrix, the high adhesion strength, the adhesion and the growth of the cell is accelerated, the wound surface of the cell is promoted by the adhesion, the adhesion of the graphene oxide, the wound surface of the skin is not only is promoted, but the cell adhesion and the cell is not only the cell adhesion of the cell is not enough.

The cell growth factor is a multifunctional strong cell factor and plays an important role in promoting the metabolism of fibroblasts and the formation of collagen. The cell growth factor is combined with a cell surface specific receptor to regulate and control division, reproduction and growth differentiation of skin epithelial cells, endothelial cells and matrix cells, can promote rapid growth and reproduction of cells related to skin injury, regulate synthesis, secretion and decomposition of intercellular matrix, promote regeneration of stratum corneum cells, accelerate repair of stratum corneum and matrix layers of skin and promote growth of skin cells of a human body. The enrichment technology selectively retains cell growth factors and other components beneficial to skin regeneration during material adhesion through a proper mesh structure and good surface adhesion performance of a matrix material, promotes the growth of periwound cells and the generation of blood vessels, induces stem cells to differentiate to skin cells, and has a promotion effect on wound healing.

The patent (CN 201910132697.0) discloses a preparation method of a collagen-graphene oxide-fat acellular matrix microcarrier, wherein α -amylase aqueous solution and acetic acid are homogenized to obtain a fat acellular matrix solution, the fat acellular matrix solution and the collagen-graphene oxide solution are mixed to serve as a dispersion phase and are introduced into a microfluidic chip, n-decanol containing span-80 is introduced into the microfluidic chip as a continuous phase to obtain acellular matrix-collagen-graphene oxide droplets wrapped with n-decanol, and the microcarrier is obtained by cross-linking through a cross-linking liquid.

Disclosure of Invention

The invention aims to provide a novel artificial skin and a preparation method thereof, and the novel artificial skin containing abundant structural proteins, polysaccharides, adhesive proteins and growth factors for promoting skin regeneration is obtained by carrying out a series of treatments on the skin of the same or different animals. The application of the artificial skin can not only prevent bacteria from invading and damaging the damaged wound surface and prevent the wound surface from being infected, but also the enriched cell growth factors can selectively induce stem cells to differentiate to skin cells, promote the regeneration of the wound surface skin and improve the success rate of transplantation.

The technical scheme provided by the invention is as follows: the preparation method of the novel artificial skin comprises the following specific steps:

1. the preparation of the acellular dermal matrix comprises the following steps:

a. soaking fresh in-vitro animal skin in a sodium hydroxide solution, washing with deionized water, and scraping redundant body hair and subcutaneous attached adipose tissues;

b. mechanically crushing the obtained epidermal tissue into small pieces, oscillating and washing the small pieces for 3-5 times by using deionized water, and draining the water;

c. c, oscillating the tissue obtained in the step b by using DNA enzyme, trypsin-EDTA, sodium dodecyl sulfate and peracetic acid in sequence, and rinsing the tissue by using deionized water;

d. treating epidermal tissue with PBS solution by oscillation, rinsing with deionized water, draining, and freeze drying;

2. preparation of acellular dermal matrix solution: dissolving the acellular dermal matrix obtained in the step 1 in a pepsin solution, and performing oscillation treatment at 37 ℃ to obtain an acellular dermal matrix solution; dropwise adding sodium hydroxide with proper amount while oscillating to adjust the pH of the solution to be neutral;

3. preparing a graphene oxide-acellular dermal matrix solution: ultrasonically and uniformly mixing graphene oxide powder and the acellular dermal matrix solution obtained in the step 2 by using an ultrasonic cell disruptor under an ice bath condition to obtain a uniform graphene oxide-acellular dermal matrix solution;

4. preparing novel artificial skin:

a. pouring the treated graphene oxide-acellular dermal matrix solution into a smooth flat-bottom mold, and freeze-drying to obtain a loose and porous acellular dermal matrix;

b. the above-mentioned fine powder is removedEnriching the dermis matrix, collecting waste foreskin tissue or skin sheet specimen, soaking in calcium-magnesium-free phosphate buffer solution (PBS, pH 7.2) for 3 times, removing subcutaneous fat, and cutting into 0.5 × 1cm²The cells are separated from the dermis after being sliced, washed by PBS, then put into 0.25 percent trypsin solution, digested for 18-22 h at 4 ℃, taken out, the epidermis is taken off from the dermis, the cells are placed in a small bottle containing 5m L domestic DMEM culture solution, the culture solution is blown by a dropper gently to disperse the epidermal cells into the culture solution, the filter screen is used for filtering, the residual epidermis is discarded, the culture solution of the epidermal cells is obtained, the culture solution containing the epidermal cells repeatedly flows through the prepared acellular dermal matrix through a negative pressure suction device, cell growth factors are efficiently enriched in the matrix material, and the novel artificial skin is obtained after low-temperature drying.

As a preferred technical scheme of the invention, the animal in-vitro skin in the step 1a is derived from the epidermis of mammals such as human, pig, rabbit, cow, sheep, rat, etc., the concentration of the sodium hydroxide solution is 0.05-0.5 mol/L, and the soaking time is 5-10 h.

As a preferable technical scheme of the invention, the concentration of the DNase used in the step 1c is 40-60U/m L, the trypsin solution is a mixed solution containing 0.05-0.5% w/v of trypsin and 0.05-0.25% w/v of EDTA, and the oscillation treatment time is 1-6 h.

As a preferred technical scheme of the invention, the concentration of the sodium dodecyl sulfate used in the step 1c is 0.02-0.5% w/v, and the oscillation treatment time is 1-6 h; the volume fraction of the peroxyacetic acid is 0.2-1.0%, and the oscillation treatment time is 0.5-2 h.

As a preferred technical scheme of the invention, the PBS solution used in the step 1d is treated for 1 to 2 hours by shaking and is repeated for 3 to 5 times.

As a preferred technical scheme of the invention, the concentration of the pepsin solution used in the step 2 is 0.2-0.8 mg/m L, the oscillation treatment time is 22-30 h, the neutral pH value is 6.8-7.5, and sodium hydroxide is added while oscillation is carried out.

As a preferred technical scheme, the mass fraction of the graphene oxide used in the step 3 is 0.25-0.5%; the ultrasonic cell disruption instrument is operated under the conditions of 25% strength, 2min ultrasonic time, 4s interval time and ice bath.

As a preferred technical scheme of the invention, the temperature of the low-temperature drying in the step 4b is 28-32 ℃.

As a preferred technical scheme of the invention, the novel artificial skin is infiltrated by a small amount of fresh blood or body fluid of a patient before use, so that immunological rejection reaction caused in the use process can be reduced, and the use effect of the novel artificial skin is better than that of deionized water or normal saline.

As a preferable technical scheme of the invention, the novel artificial skin is prepared by adopting the preparation method of the novel artificial skin.

Compared with the prior art, the invention has the following advantages:

(1) according to the invention, the immunogenic substances in the skin tissue are removed by crushing and then removing the cells, so that the components of the skin tissue extracellular matrix can be completely reserved, the excellent cell removing effect can be achieved, and the reagents with mild properties are used in the cell removing process, so that the active components of the skin extracellular matrix are reserved to the greatest extent;

(2) after natural materials are subjected to cell removal, the natural materials are treated by a certain cross-linking agent to have good mechanical properties, good three-dimensional structures, good histocompatibility and low immunogenicity, and the method is a common method for tissue engineering research. However, the existing methods have certain problems, the use of a chemical cross-linking agent causes certain pollution to the environment, the subsequent treatment increases the cost, and the reagent residue also causes serious cytotoxicity, thereby greatly influencing the clinical use. The acellular dermal matrix is a degradable material commonly used in tissue engineering, but has insufficient mechanical strength and rapid degradation; the graphene oxide has good mechanical properties, but lacks the functions of adhesion and growth of nearby cells; the effective combination of the two can produce the acellular dermal matrix with uniform components and excellent performance, which not only increases the material strength, but also is more favorable for promoting the adhesion, crawling and growth of stem cells and accelerating the repair of wound injury. Graphene oxide is a novel carbon material with excellent performance, the structure of the graphene oxide spans a half of the typical scale of chemistry and material science, and the graphene oxide can be regarded as a non-traditional soft material and has the characteristics of polymers, colloids, thin films and amphoteric molecules. Due to the introduction of the oxygen-containing active group, the graphene oxide has strong surface activity and wettability, can form a stable dispersion solution in a common solvent, has good hydrophilicity, dispersibility and compatibility, and can be used as a reinforcing filling material, and the excellent performance of the series of the graphene oxide determines that the graphene oxide has high use value;

(3) the enrichment treatment technology utilizes the loose and porous structure and good surface adhesion performance of the matrix material, selectively retains the components such as cell growth factors beneficial to skin regeneration during material adhesion, promotes the growth of cells around wounds and the generation of blood vessels, induces the differentiation of stem cells to skin cells, promotes the rapid growth and the propagation of cells related to skin injury, regulates the synthesis, the secretion and the decomposition of intercellular matrix, accelerates the repair of the stratum corneum and the matrix layer of the skin, promotes the growth of skin cells of a human body, and has specific promotion effect on wound healing.

Detailed Description