阅读说明：本技术 一种组织工程皮肤的制备方法 (Preparation method of tissue engineering skin ) 是由 陶冶 康晓菲 于 2019-11-27 设计创作，主要内容包括：一种组织工程皮肤的制备方法,属于组织工程技术领域。为了解决目前在制作组织工程皮肤支架材料以及细胞与支架材料的相互作用方式还存在缺陷的问题,本发明提供了一种组织工程皮肤的制备方法。本发明所述的组织工程皮肤包括如下步骤(1)纳米静电纺丝材料的制备(2)人体皮肤细胞的培养(3)细胞培养至纳米纤维材料上。利用本发明所述的组织工程皮肤的制备方法制备组织工程皮肤构建方法简单,操作步骤少,且获得良好的动物在体实验效果。(A preparation method of tissue engineering skin belongs to the technical field of tissue engineering. The invention provides a preparation method of tissue engineering skin, aiming at solving the problems that the existing preparation method of a tissue engineering skin scaffold material and the interaction mode of cells and the scaffold material have defects. The tissue engineering skin comprises the following steps of (1) preparing a nano electrostatic spinning material, (2) culturing human skin cells, and (3) culturing the cells on a nanofiber material. The method for preparing the tissue engineering skin is simple in construction method, few in operation steps and good in animal in-vivo experiment effect.)

1. A preparation method of tissue engineering skin is characterized in that skin cells are used as seed cells for culture and amplification, nano electrostatic spinning materials are used as a support, and the cultured and amplified cells are printed on the support through a 3D printing technology to obtain the tissue engineering skin, and the preparation method comprises the following specific steps:

(1) preparation of nano electrostatic spinning material

Preparing PVP spinning solution with the mass fraction of 10-16% and N, N-dimethylformamide DMF as a solvent, swelling the PVP spinning solution in a spinning bottle at 29-31 ℃ for 22-26 hours, and processing the spinning solution into a nano electrostatic spinning material with the thickness of 0.45-0.55 mm by using an electrostatic spinning machine;

(2) culture of human skin cells

Repeatedly washing skin tissue with PBS buffer solution containing antibiotics, and cutting to 2-4cm²Soaking in protease solution at 37 deg.C for 12-16h, separating epidermisAnd dermis, placing the epidermis in phosphate buffer solution with 1% of pancreatin by mass fraction, digesting for 30 minutes at 37 ℃ to prepare epidermal cell suspension;

digesting the dermal homogenate in collagenase type I at 37 ℃ for 30 minutes to prepare a dermal cell suspension;

respectively neutralizing enzyme liquid in epidermal cell suspension and dermal cell suspension by using a DMEM culture medium containing 1.5 mass percent of bovine serum, respectively filtering the neutralized epidermal cell suspension and dermal cell suspension, centrifuging the obtained filtrate to obtain epidermal primary cells and dermal primary cells, respectively inoculating the epidermal primary cells and the dermal primary cells in the DMEM culture medium containing fetal bovine serum for culture and then subculturing, and respectively collecting the epidermal cells and the dermal cells;

(3) culturing the cells on the nanofiber material to obtain the tissue engineering skin

And (3) printing the epidermal cell culture solution and the dermal cell culture solution obtained in the step (2) on the nano electrostatic spinning material obtained in the step (1) through a 3D printer to obtain the tissue engineering skin.

2. The method for preparing the tissue engineering skin according to claim 1, wherein the conditions for processing the spinning solution by the electrostatic spinning machine are as follows: 15-18kV voltage, the distance between the capillary tube and the collecting screen is 25-30cm, and the processing time is 2 minutes.

3. The method for preparing tissue engineering skin according to claim 1, wherein the 3D printing parameters are obtained by programming a 3D bioprinter by using AutoCAD software, designing the printing parameters according to the size of the nanofiber material, wherein the printing nozzle is 0.8-1.0mm, the printing line speed is 8-10 cm/min, the printing thickness is 1-2 mm, the number of printing layers is two, the printing temperature is 37 ℃, and the printing humidity is 60-70%.

4. The method for preparing tissue engineering skin according to claim 1, wherein a drug is further added to the spinning solution of step (1).

5. The method of preparing tissue engineered skin of claim 4, wherein the drug is a mixture of one or more of penicillin, amoxicillin, azithromycin, roxithromycin, oxytetracycline, and tetracycline.

6. The method for preparing tissue engineering skin according to claim 5, wherein the drug is less than 3% by mass based on DMF.

7. The method of claim 1, wherein the PVP spinning solution of step (1) is 16% by mass based on DMF.

8. The method for preparing tissue engineered skin according to claim 1, wherein the swelling temperature in step (1) is 30 ℃ and the swelling time is 24 hours.

9. The method for preparing tissue engineering skin according to claim 2, wherein the voltage in step (1) is 15kV, and the distance between the capillary and the collecting screen is 30 cm.

10. The method for preparing tissue engineered skin according to claim 1, wherein the filtration in step (2) is performed by using a 100 μm filter screen.

Technical Field

The invention belongs to the technical field of tissue engineering, and particularly relates to a preparation method of tissue engineering skin.

Background

The skin is the largest tissue and organ of a human body, is nonspecific of the human body, is an immune barrier, causes serious health problems due to large-area skin defect, and even threatens life, however, the large-area skin defect repair is a medical problem, the conventional treatment means causes great physical and psychological pain to patients, infection is easily caused, the recovery speed is slow, the recovery scar cannot be controlled, and effective drug treatment cannot be carried out on the wound surface. People are always looking for ideal skin substitutes.

The skin tissue engineering is to construct a tissue substitute for repairing, maintaining and improving the functions of damaged tissues by utilizing the principles of biology and engineering, and the core of the skin tissue engineering is to create a three-dimensional growth scaffold, carry out in-vitro composite culture on cells separated from an organism to form an artificially regenerated dermis equivalent or skin equivalent, and transplant the dermis equivalent or skin equivalent to the damaged and reconstructed skin. The seed cells, scaffold material and the way in which the cells interact with the scaffold material are 3 basic elements for the construction of tissue engineered skin. In order to make the tissue engineering skin more realistic and reduce immune rejection, the problems existing in the three aspects are still to be further perfected and solved.

Disclosure of Invention

The invention provides a preparation method of tissue engineering skin, aiming at solving the problems that the existing preparation method of a tissue engineering skin scaffold material and the interaction mode of cells and the scaffold material have defects. The preparation method of the tissue engineering skin provided by the invention comprises the following steps of culturing and amplifying skin cells serving as seed cells, printing the cultured and amplified cells on a bracket by using a nano electrostatic spinning material as the bracket through a 3D printing technology to obtain the tissue engineering skin, and specifically comprises the following steps:

(1) preparation of nano electrostatic spinning material

Preparing PVP spinning solution with the mass fraction of 10-16% and N, N-dimethylformamide DMF as a solvent, swelling the PVP spinning solution in a spinning bottle at 29-31 ℃ for 22-26 hours, and processing the spinning solution into a nano electrostatic spinning material with the thickness of 0.45-0.55 mm by using an electrostatic spinning machine;

(2) culture of human skin cells

Repeatedly washing skin tissue with PBS buffer solution containing antibiotics, and cutting to 2-4cm²Soaking in protease solution at 37 deg.C for 12-16h, separating epidermis and dermis, digesting the epidermis in phosphate buffer solution containing 1% pancreatin at 37 deg.C for 30 min to obtain epidermis cell suspension;

digesting the dermal homogenate in collagenase type I at 37 ℃ for 30 minutes to prepare a dermal cell suspension;

respectively neutralizing enzyme liquid in epidermal cell suspension and dermal cell suspension by using a DMEM culture medium containing 1.5 mass percent of bovine serum, respectively filtering the neutralized epidermal cell suspension and dermal cell suspension, centrifuging the obtained filtrate to obtain epidermal primary cells and dermal primary cells, respectively inoculating the epidermal primary cells and the dermal primary cells in the DMEM culture medium containing fetal bovine serum for culture and then subculturing, and respectively collecting the epidermal cells and the dermal cells;

(3) culturing the cells on the nanofiber material to obtain the tissue engineering skin

And (3) printing the epidermal cell culture solution and the dermal cell culture solution obtained in the step (2) on the nano electrostatic spinning material obtained in the step (1) through a 3D printer to obtain the tissue engineering skin.

Preferably, the conditions for processing the spinning solution by the electrostatic spinning machine are as follows: 15-18kV voltage, the distance between the capillary tube and the collecting screen is 25-30cm, and the processing time is 2 minutes.

Preferably, the 3D printing parameters are that the 3D bioprinter is programmed by using AutoCAD software, the printing parameters are designed according to the size of the nanofiber material, a printing nozzle is 0.8-1.0mm, the printing linear speed is 8-10 cm/min, the printing thickness is 1-2 mm, the number of printing layers is two, the printing temperature is 37 ℃, and the printing humidity is 60-70%.

Preferably, a drug may be further added to the spinning solution of step (1).

Preferably, the drug is a mixture of one or more of penicillin, amoxicillin, azithromycin, roxithromycin, oxytetracycline and tetracycline.

Preferably, the mass fraction of the drug is less than 3% based on DMF.

Preferably, the mass fraction of the PVP spinning solution in the step (1) is 16% based on the DMF.

Preferably, the swelling temperature in step (1) is 30 ℃ and the swelling time is 24 hours.

Preferably, the voltage in step (1) is 15kV, and the distance between the capillary and the collecting screen is 30 cm.

Preferably, the filtration in step (2) is performed by using a 100 μm filter screen.

Advantageous effects

The nano electrostatic spinning material prepared by the invention can utilize the combination of different spinning materials to reduce the human body rejection reaction to the maximum extent, has excellent air permeability and water absorption, is easy to be absorbed by the skin, is well attached to the skin, and can be directly manufactured on the surface of the skin. The material has very small diameter and good small size effect. The skin can absorb materials and simultaneously provide a bracket for the recovery growth of cells, so that the generation of scars is reduced;

drug-embedded electrospun material: the problem that the skin cannot use the medicine in a large area can be solved by embedding the antibiotics and the medicine for inhibiting the immune reaction in the electrostatic spinning material. Can well control the drug concentration and conveniently carry out multiple times of drug administration. And different drugs can be embedded according to actual conditions, and different concentrations can be embedded, so that large-area uniform administration can be realized.

The cell culture technique has a very slow cell growth rate in the body skin, and is likely to cause a series of problems such as infection of the wound surface. The number of epidermal cells can be increased in geometric multiple by using a cell culture technology, and the cultured cells can act on the wound surface of a patient by using the technology for multiple times to promote the healing of the wound surface;

3D printing technology: the cultured cell culture solution is used as biological ink to print on the surface of the drug-embedded electrostatic spinning material, so that the cell concentration and the cell arrangement can be well controlled, scars can be beautified, and the healing of wound surfaces is accelerated.

Drawings

FIG. 1 shows that for nano electrostatic spinning materials prepared under different PVP concentrations, the concentration of PVP is 6% for A, 8% for B, 16% for C and 18% for D;

FIG. 2 shows that the voltage A of the nano electrostatic spinning material prepared under different voltages is 12kv, the voltage B is 16kv, the voltage C is 20kv, and the voltage D is 22 kv;

FIG. 3 shows that the distance between the capillary and the collecting screen is different, A is 15cm, B is 20cm, C is 25cm, and D is 28 cm;

FIG. 4 shows the nano-electrospinning materials prepared with or without drugs, wherein A, B, and C are the nano-electrospinning materials prepared under the conditions of examples 1,2, and 3, respectively, and D, E, and F are the nano-electrospinning materials prepared under the conditions of examples 4,5, and 6, respectively.

Detailed Description

The high polymer PVP used in the following examples was produced by Roche Negro Tech technologies, Inc.