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Method for constructing tissue engineering skin with physiological function

A tissue-engineered skin and construction method technology, applied in the field of tissue-engineered skin construction, can solve the problems of unavoidable immune rejection and disease transmission, easy shedding and necrosis of epidermal epithelium, and low flexibility.

Active Publication Date: 2019-02-12
LUOYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Toughness and mechanical properties of tissue-engineered skin are far from those of natural skin, with poor elasticity and low flexibility, and the epidermis is easy to fall off
(3) Another important factor that limits the application of artificial composite skin in the treatment of large area burns is that the blood supply of the artificial skin is insufficient, and the epidermis is prone to exfoliation and necrosis (Auger FA, Berthod F, Moulin V. et al. Tissue-engineered skin substitutes from in vitro constructs to in vivo applications.Biotechnol.Apppl.Biochem.2004 39,263-275)
(4) At present, the tissue-engineered composite skin that is available through commercial channels, the seed cells of Apligraft are derived from the foreskin of newborns, which are allogeneic cells, and the occurrence of immune rejection and disease transmission after transplantation cannot be avoided, especially in AIDS
However, there is no report on the construction of tissue-engineered skin using hair follicle stem cells and hair follicle dermal fibroblasts as seed cells

Method used

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  • Method for constructing tissue engineering skin with physiological function
  • Method for constructing tissue engineering skin with physiological function
  • Method for constructing tissue engineering skin with physiological function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Embodiment 1, the separation and culture of hair follicle dermal fibroblasts and hair follicle stem cells are:

[0037] ①Take goat ear skin samples 0.5×0.5cm 2 , use a lancet to scrape off the dirt on the skin surface in a purification workbench, disinfect with 75% (v / v) alcohol twice, and then rinse with D-Hank's solution 3 to 5 times, each time for 5 minutes.

[0038] ②Cut the skin sample in step ① into small strips with ophthalmic scissors, add 2.4U / mL Dispase, digest at 4°C for 15 hours, pull out the hair follicles with ophthalmic tweezers, cut out the prominence of the hair follicles under a stereo microscope, and place Digest in 0.05% trypsin at 37°C for 1.5h, stop the reaction, centrifuge at 1200r / min, collect the cells and inoculate them in 100μg / mL type IV collagen-coated culture dishes, collect the upper unattached cutin after 25min The forming cells and hair follicle dermal fibroblasts were cultured in another culture dish, and the conditioned medium was adde...

Embodiment 2

[0039] Embodiment 2, the construction of tissue engineered skin

[0040] ①Mix 7 volumes of 4.5mg / mL bovine type I collagen solution and 2 volumes of 5×DMEM medium, adjust the pH value to 7.2 with 1M NaOH, and then add 1 volume of hair follicle dermal fibroblasts for conditioned culture solution, mixed evenly, and made into cell gel, in which the density of hair follicle dermal fibroblasts in the conditioned medium was 5×10 6 cells / mL, and then 3.6 mL of cell gel was added dropwise on a human amniotic membrane scaffold with a diameter of 3 cm.

[0041] ②After the cell gel is solidified, add 2mL conditioned medium and place in CO 2 In an incubator, 37°C, 5% CO 2 1. Cultivate for 4 days under saturated humidity conditions, and change the medium every 2 days.

[0042] ③After 4 days, hair follicle stem cells were inoculated on the surface of the cell gel, and the cell inoculation density was 5×10 6 piece / cm 2 , cultured with serum-free conditioned medium containing 50 μg / mL vi...

Embodiment 3

[0044] Embodiment 3, the construction of tissue engineered skin

[0045] The construction method of Example 3 is basically the same as that of Example 2, except that the cell inoculation density of hair follicle stem cells in step ③ is 1×10 6 piece / cm 2 , the concentration of vitamin C in the serum-free conditioned medium was 40 μg / mL.

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Abstract

The invention discloses a method for constructing tissue engineering skin with a physiological function. Hair follicle stem cells and hair follicle dermal fibroblasts are used as seed cells of the tissue engineering skin, and a cell gel mixed liquid is dropwise added on a human amniotic membrane stent. The human amniotic membrane stent can effectively prevent the contraction of collagen gel, and the human amniotic membrane and organism tissues have very good compatibility during transplantation. At the beginning of transplantation, the human amniotic membrane stent can absorb some nutrients ofthe organism to provide the nutrition for the hair follicle stem cells and the hair follicle dermal fibroblasts and promote the hair production. In the invention, an acute full-thickness skin defectmodel is made by using Guanzhong dairy goat as an experimental animal, and the constructed tissue engineering skin is auto-transplanted. After 2 months of transplantation, the wound recovers well andthe hair grows. After 5 months of transplantation, the wound recovers well and the hair growth situation is good. After 9 months of transplantation, the wound recovers basically.

Description

technical field [0001] The invention relates to a method for constructing tissue-engineered skin with physiological functions, and belongs to the technical field of constructing artificial organs by tissue-engineering methods for biomedical engineering. Background technique [0002] Over the past few decades, the development of skin tissue engineering has made great progress. Tissue engineered skin has made important contributions to saving the lives of patients with deep and large area burns, and improving the healing rate of diabetic patients and elderly patients with varicose veins. . However, due to the shortcomings of tissue-engineered skin itself, this tissue-engineered skin can only be a temporary skin substitute. With the progress of human society, the wounded and sick, especially those with large area and deep burns, are no longer satisfied with the therapeutic effect provided by the commonly used clinical treatment methods. On the surface, it should be similar to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61L27/38A61L27/60A61L27/50
CPCA61L27/3804A61L27/3886A61L27/3895A61L27/50A61L27/60C12N5/0698C12N2500/38
Inventor 杨学义张沛史明艳郭燕杰马西亚
Owner LUOYANG NORMAL UNIV
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