阅读说明：本技术 一种脱细胞真皮基质组织工程支架及其制备方法 (Acellular dermal matrix tissue engineering scaffold and preparation method thereof ) 是由 陈维明 白玉龙 陈金发 周小垚 胡凯 姚杰 侯立存 李淼 骆井万 汪晶晶 于 2020-05-12 设计创作，主要内容包括：本发明涉及组织工程支架技术领域,尤其涉及一种脱细胞真皮基质组织工程支架及其制备方法。本发明将异种或异体皮浸入盐溶液中去除表皮组织,得到真皮组织；将真皮组织依次进行灭活处理和脱细胞处理,得到真皮基质；将所得真皮基质顺次进行干燥和细化处理,得到真皮基质纤维；将真皮基质纤维与分散液混合后依次进行冷冻干燥和交联处理,即得脱细胞真皮基质组织工程支架。本发明提供的脱细胞真皮基质组织工程支架,制备时无需酶处理和辅助材料的添加,保持了脱细胞真皮基质优异的生物相容性；且孔隙率高,形状可塑性强,有利于细胞的长入和营养物质的渗透,满足复杂形状缺损的修复。(The invention relates to the technical field of tissue engineering scaffolds, in particular to an acellular dermal matrix tissue engineering scaffold and a preparation method thereof. The invention immerses the xenogenous or xenogenous skin into saline solution to remove the epidermal tissue, and then the dermal tissue is obtained; sequentially inactivating and decellularizing the dermal tissue to obtain a dermal matrix; sequentially drying and thinning the obtained dermal matrix to obtain dermal matrix fibers; mixing the dermal matrix fiber and the dispersion liquid, and then sequentially carrying out freeze drying and crosslinking treatment to obtain the acellular dermal matrix tissue engineering scaffold. The acellular dermal matrix tissue engineering scaffold provided by the invention does not need enzyme treatment and addition of auxiliary materials during preparation, and maintains excellent biocompatibility of the acellular dermal matrix; and the porosity is high, the shape plasticity is strong, the cell growth and the nutrient substance permeation are facilitated, and the repair of complex shape defects is met.)

1. A preparation method of an acellular dermal matrix tissue engineering scaffold is characterized by comprising the following steps:

(1) immersing the xenogenic or xenogenic skin into a saline solution to remove epidermal tissue to obtain dermal tissue;

(2) sequentially inactivating and decellularizing the dermal tissue to obtain a dermal matrix;

(3) sequentially drying and thinning the obtained dermal matrix to obtain dermal matrix fibers;

(4) mixing the dermal matrix fiber and the dispersion liquid, and then sequentially carrying out freeze drying and crosslinking treatment to obtain the acellular dermal matrix tissue engineering scaffold.

2. The preparation method according to claim 1, wherein the mass-to-volume ratio of the xenogenic or xenogenic skin to the salt solution in the step (1) is 1g: 4-6 mL, the salt solution is sodium chloride solution, and the concentration of the sodium chloride solution is 1-2 mol/L.

3. The preparation method according to claim 1 or 2, wherein the inactivation treatment in the step (2) is inactivation with alcohol, the mass-to-volume ratio of the dermal tissue to the alcohol is 1g: 4-6 mL, the volume concentration of the alcohol is 70-80%, and the time of the inactivation treatment is 1-4 h.

4. The preparation method according to claim 1 or 2, wherein the acellular treatment in the step (2) is to soak the dermal tissue in an alkaline solution and a polyethylene glycol octylphenyl ether aqueous solution, the mass-to-volume ratio of the dermal tissue to the alkaline solution is 1g: 4-6 mL, the soaking time is 6-24 h, and the concentration of the alkaline solution is 0.5-2 mol/L; the mass-volume ratio of the dermal tissue to the polyethylene glycol octyl phenyl ether aqueous solution is 1g: 5-15 mL, the soaking time is 12-24 h, and the concentration of the polyethylene glycol octyl phenyl ether aqueous solution is 0.05-0.15%.

5. The preparation method according to claim 1, wherein the dispersion liquid in the step (4) is one or more of water, physiological saline and tertiary butanol, and the mass-to-volume ratio of the dermal matrix fibers to the dispersion liquid is 1g: 40-60 mL.

6. The preparation method according to claim 1 or 5, wherein the crosslinking treatment in the step (4) is chemical crosslinking or physical crosslinking, and the time of the crosslinking treatment is 4-8 h.

7. The method according to claim 6, wherein the cross-linking agent for chemical cross-linking is one or more of glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and genipin.

8. The method of claim 6, wherein the physical crosslinking comprises one or more of thermal crosslinking, ultraviolet crosslinking, and radiation crosslinking.

9. An acellular dermal matrix tissue engineering scaffold prepared by the preparation method of any one of claims 1 to 8.

10. The acellular dermal matrix tissue engineering scaffold according to claim 9, characterized in that it is a three-dimensional porous structure.

Technical Field

The invention relates to the technical field of tissue engineering scaffolds, in particular to an acellular dermal matrix tissue engineering scaffold and a preparation method thereof.

Background

The acellular dermal matrix is an extracellular matrix obtained by carrying out acellular treatment on xenogenic or xenogenic skin, and is widely applied to the fields of burns, plastic surgery, tissue engineering scaffolds and the like due to good biocompatibility. Chinese patent CN109675112A discloses a method for preparing human acellular dermal matrix, the acellular allogeneic dermal matrix prepared by the method is of a sheet structure, the gap is compact, and the requirements of rapid cell growth and the use of the acellular allogeneic dermal matrix as a three-dimensional structure tissue engineering scaffold and a filler with a complex shape are difficult to meet. Therefore, the acellular dermal matrix with the sheet structure is constructed into a three-dimensional structure and high-porosity scaffold, so that the defects of the traditional sheet structure dermal matrix in the application aspect of three-dimensional tissue engineering scaffolds are overcome, cells can grow rapidly, and the tissue repair process is accelerated.

In order to increase the space size of the acellular dermal matrix, chinese patent CN102258807B discloses a method for adjusting the pore size of the porcine acellular dermal matrix for tissue engineering, but the method requires the addition of anionic biomacromolecules or cationic biomacromolecules into the dermal matrix, thereby changing the original composition of the dermal matrix. In addition, in order to realize the three-dimension of the dermal matrix and obtain high porosity, chinese patent CN109821071A discloses an acellular dermal matrix-based hydrogel and a preparation method thereof, but the method needs to introduce pepsin, pancreatin and other protease digestion treatments, the enzyme treatment will digest proteins in the dermal matrix and reduce the content of proteoglycan and other components in the dermal matrix, and the enzyme treatment time is long and the process is complicated. Therefore, the development of the acellular dermal matrix which is simple, does not change the original components of the dermal matrix and has a three-dimensional high-porosity structure has important clinical medical value.

Disclosure of Invention

The invention aims to provide an acellular dermal matrix tissue engineering scaffold and a preparation method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of an acellular dermal matrix tissue engineering scaffold, which comprises the following steps:

(1) immersing the xenogenic or xenogenic skin into a saline solution to remove epidermal tissue to obtain dermal tissue;

(2) sequentially inactivating and decellularizing the dermal tissue to obtain a dermal matrix;

(3) sequentially drying and thinning the obtained dermal matrix to obtain dermal matrix fibers;

(4) mixing the dermal matrix fiber and the dispersion liquid, and then sequentially carrying out freeze drying and crosslinking treatment to obtain the acellular dermal matrix tissue engineering scaffold.

Preferably, the mass-volume ratio of the xenogenic or xenogenic skin to the salt solution in the step (1) is 1g: 4-6 mL, the salt solution is a sodium chloride solution, and the concentration of the sodium chloride solution is 1-2 mol/L.

Preferably, the inactivation treatment in the step (2) is performed by using alcohol, the mass-volume ratio of the dermal tissue to the alcohol is 1g: 4-6 mL, the volume concentration of the alcohol is 70-80%, and the time of the inactivation treatment is 1-4 h.

Preferably, the acellular treatment in the step (2) is to soak the dermal tissue in an alkaline solution and a polyethylene glycol octyl phenyl ether aqueous solution, the mass-volume ratio of the dermal tissue to the alkaline solution is 1g: 4-6 mL, the soaking time is 6-24 h, and the concentration of the alkaline solution is 0.5-2 mol/L; the mass-volume ratio of the dermal tissue to the polyethylene glycol octyl phenyl ether aqueous solution is 1g: 5-15 mL, the soaking time is 12-24 h, and the concentration of the polyethylene glycol octyl phenyl ether aqueous solution is 0.05-0.15%.

Preferably, in the step (4), the dispersion liquid is one or more of water, physiological saline and tertiary butanol, and the mass-to-volume ratio of the dermal matrix fibers to the dispersion liquid is 1g: 40-60 mL.

Preferably, the crosslinking treatment in the step (4) is chemical crosslinking or physical crosslinking, and the time of the crosslinking treatment is 4-8 h.

Preferably, the crosslinking agent for chemical crosslinking is one or more of glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and genipin.

Preferably, the physical crosslinking comprises one or more of thermal crosslinking, ultraviolet crosslinking, and radiation crosslinking.

The invention also provides the acellular dermal matrix tissue engineering scaffold prepared by the preparation method.

Preferably, the acellular dermal matrix tissue engineering scaffold is a three-dimensional porous structure.

The invention has the beneficial effects that:

the preparation method is simple and easy to implement and high in production efficiency. In the preparation process, protease (pepsin, pancreatin and the like) is not needed for digestion treatment, auxiliary materials are not needed to be added, the original component composition of the dermal matrix is not damaged, and the excellent biocompatibility of the acellular dermal matrix is kept.

The acellular dermal matrix tissue engineering scaffold is of a three-dimensional porous structure and high in porosity. The invention overcomes the defects and shortcomings of the traditional dermal matrix tissue engineering scaffold, the porosity is as high as 95%, and the high porosity of the scaffold is beneficial to the growth of cells and the penetration of nutrient substances, thereby promoting the tissue repair; and the acellular dermal matrix tissue engineering scaffold has strong shape plasticity, and can prepare the dermal matrix into a scaffold with a complex shape to meet the repair of complex-shaped defects.

Drawings

FIG. 1 is a sheet of acellular dermal matrix;

FIG. 2 is a SEM image of a sheet-like acellular dermal matrix;

FIG. 3 is a photograph showing the appearance of cotton-like acellular dermal matrix fibers;

FIG. 4 is a three-dimensional porous acellular dermal matrix scaffold;

fig. 5 is a SEM picture of a three-dimensional porous acellular dermal matrix scaffold.

Detailed Description

The invention provides a preparation method of an acellular dermal matrix tissue engineering scaffold, which comprises the following steps:

(1) immersing the xenogenic or xenogenic skin into a saline solution to remove epidermal tissue to obtain dermal tissue;

(2) sequentially inactivating and decellularizing the dermal tissue to obtain a dermal matrix;

(3) sequentially drying and thinning the obtained dermal matrix to obtain dermal matrix fibers;

(4) mixing the dermal matrix fiber and the dispersion liquid, and then sequentially carrying out freeze drying and crosslinking treatment to obtain the acellular dermal matrix tissue engineering scaffold.

In the invention, the acellular dermal matrix tissue engineering scaffold is preferably prepared by scraping off the hair of the heterogeneous or xenogenic skin with a blade, and taking the skin with a drum-type skin taking machine, wherein the thickness is preferably 0.5-2 mm, and more preferably 1 mm.

In the invention, the mass-to-volume ratio of the xenogenic or xenogenic skin to the salt solution in the step (1) is preferably 1g: 4-6 mL, and more preferably 1g:5mL, the salt solution is preferably a sodium chloride solution, and the concentration of the sodium chloride solution is preferably 1-2 mol/L, and more preferably 1-1.5 mol/L.

In the present invention, the xenogenic or xenogenic skins are preferably shaken on a shaker for 12 hours after being immersed in the saline solution, and the epidermis layer is gently removed with tweezers; after removing the epidermal tissue, the skin is preferably washed with purified water for 20-40 min, and more preferably for 30 min.

In the present invention, the inactivation treatment in step (2) is preferably inactivation with alcohol, the mass-to-volume ratio of the dermal tissue to alcohol is preferably 1g: 4-6 mL, and more preferably 1g:5mL, the volume concentration of the alcohol is preferably 70-80%, and more preferably 75%, and the time of the inactivation treatment is preferably 1-4 h, and more preferably 2-3 h.

In the invention, after the dermal tissue is subjected to alcohol inactivation, the dermal tissue is preferably washed with deionized water for 20-40 min, and more preferably for 30 min.

In the present invention, in the acellular treatment in step (2), the dermal tissue is preferably soaked in an alkaline solution and a polyethylene glycol octylphenyl ether aqueous solution, the mass-to-volume ratio of the dermal tissue to the alkaline solution is preferably 1g: 4-6 mL, more preferably 1g:5mL, the soaking time is preferably 6-24 h, more preferably 12-18 h, and the concentration of the alkaline solution is preferably 0.5-2 mol/L, more preferably 1-2 mol/L.

In the present invention, the alkaline solution may preferably be an alkaline solution such as sodium hydroxide or potassium hydroxide.

In the present invention, the mass-to-volume ratio of the dermal tissue to the aqueous solution of polyethylene glycol octylphenyl ether is preferably 1g:5 to 15mL, more preferably 1g:8 to 12mL, the soaking time is preferably 12 to 24 hours, more preferably 18 to 20 hours, and the concentration of the aqueous solution of polyethylene glycol octylphenyl ether is preferably 0.05 to 0.15%, more preferably 0.1 to 0.15%.

In the present invention, the dermal tissue may be soaked in the alkaline solution and then soaked in the aqueous solution of octyl phenyl ether of polyethylene glycol, or vice versa.

In the invention, the cell removal treatment is preferably performed by shaking on a shaking table, the shaking temperature is preferably 36-38 ℃, the shaking speed is preferably 37 ℃, and the shaking speed is preferably 170-190 r/min, and the shaking speed is preferably 180 r/min.

In the invention, after the acellular treatment of the dermal tissue, the dermal tissue is preferably washed by deionized water to remove the acellular fluid, and the washing time is preferably 20-40 min, and more preferably 30 min.

In the invention, the drying in the step (3) is preferably freeze drying, and the thinning treatment is preferably to put the dermal matrix into liquid nitrogen and then put the dermal matrix into an ultralow temperature pulverizer to pulverize, wherein the pulverizing time is preferably 5-15 min, and more preferably 10 min.

In the present invention, the dispersion liquid in step (4) is preferably one or more of water, physiological saline and tert-butanol, and the mass-to-volume ratio of the dermal matrix fibers to the dispersion liquid is preferably 1g:40 to 60mL, and more preferably 1g:50 to 55 mL.

In the present invention, the freeze-drying in step (4) is preferably performed in a vacuum freeze-dryer, and the drying time is preferably 6 to 18 hours, and more preferably 12 hours.

In the invention, the crosslinking treatment in the step (4) is chemical crosslinking or physical crosslinking, and the time of the crosslinking treatment is preferably 4 to 8 hours, more preferably 5 to 7 hours, and even more preferably 6 hours.

In the present invention, the crosslinking agent for chemical crosslinking is preferably one or more of glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, genipin.

In the present invention, the ratio of the dermal matrix fibers to the crosslinking agent is preferably 1g:15 to 25ml, more preferably 1g:18 to 22ml, and still more preferably 1g:20 ml.

In the invention, the glutaraldehyde is preferably an aqueous solution with the concentration of 3-5%, and is further preferably an aqueous solution with the concentration of 4%; the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is preferably an alcohol solution with a concentration of 0.1-1% (the alcohol concentration is 90%), and more preferably an alcohol solution with a concentration of 0.5-1% (the alcohol concentration is 90%); the genipin is preferably an alcohol solution with a concentration of 0.5 to 2% (alcohol concentration of 90%), and more preferably an alcohol solution with a concentration of 1.5 to 2% (alcohol concentration of 90%).

In the invention, the physical crosslinking preferably comprises one or more of thermal crosslinking, ultraviolet crosslinking and irradiation crosslinking, and the thermal crosslinking is preferably carried out at a high temperature of 100-200 ℃.

In the invention, after the crosslinking treatment is finished, deionized water is preferably used for washing for 20-40 min, the crosslinking agent is removed, and the vacuum freeze-drying machine is used for drying for 6-18 h, further preferably deionized water is used for washing for 30min, and the vacuum freeze-drying machine is used for drying for 12 h.

In the present invention, the temperature of all freeze-drying is preferably-60 to-20 ℃, more preferably-50 to-30 ℃, and still more preferably-40 ℃.

The invention also provides the acellular dermal matrix tissue engineering scaffold prepared by the preparation method.

In the invention, the acellular dermal matrix tissue engineering scaffold is a three-dimensional porous structure.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.