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Vascularized full-thickness tissue-engineered skin layer-by-layer assembled by hydrogel, nanofiber scaffolds and skin cells and preparation method thereof

A tissue engineering skin and nanofiber technology, applied in the field of polymer materials and biomedical materials, can solve the problems of unfavorable cell migration, proliferation, no significant improvement of cell distribution and wound healing promotion effect, and no wound healing effect, etc., to achieve Improve tissue distribution, promote wound healing, and reduce scar formation

Active Publication Date: 2019-03-29
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the nanofibers obtained by electrospinning provide a suitable surface morphology for cell adhesion and growth, which is conducive to the adhesion and growth of cells on the scaffold, the skin scaffold prepared by electrospinning is not conducive to the cell adhesion due to the small pore size. Migration and proliferation in the depth direction, and it is difficult to effectively regulate the distribution of cells on the fibrous scaffold, which limits the application of artificial skin scaffolds in the field of medical treatment of skin injuries
[0006] Although many researchers have developed a variety of artificial tissue-engineered skins, most of which are collagen gels or sponges, there is currently no nanofiber scaffold, hydrogel, and layer-by-layer self-assembly made of pure natural polymers. Relevant literature and patent reports on the application of technology to vascularized tissue engineering full-thickness skin. The patents with application numbers "201510631809.9" and "201610793440.6" respectively report a preparation method of high-strength and high-toughness hydrogel nanofibers and a seaweed Tissue-engineered skin constructed with sodium hydrogel scaffolds, but no related research on wound healing effect
The patents with the application numbers "201610499353.X" and "201611008057.1" respectively reported a micro-nano composite double-layer skin scaffold and its preparation method, and a flexible artificial skin and its preparation method, but did not significantly improve the cells in the tissue. Correlation research on distribution and wound healing promotion effect

Method used

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  • Vascularized full-thickness tissue-engineered skin layer-by-layer assembled by hydrogel, nanofiber scaffolds and skin cells and preparation method thereof
  • Vascularized full-thickness tissue-engineered skin layer-by-layer assembled by hydrogel, nanofiber scaffolds and skin cells and preparation method thereof
  • Vascularized full-thickness tissue-engineered skin layer-by-layer assembled by hydrogel, nanofiber scaffolds and skin cells and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, keratinocyte separation and purification

[0054] The foreskin was taken out and washed several times with sterile PBS to clarify it. After removing fascia and other tissues, cut into strips; add Dispase, and place in a refrigerator at 4°C for overnight digestion. The foreskin digested overnight was rewarmed at 37°C, and the epidermis and dermis were separated after removal, and the separated epidermis was washed in PBS. The isolated epidermis was digested with trypsin (containing EDTA) at 37°C. The digestive juice was filtered through a strainer, and the filtrate was collected and centrifuged. After washing with PBS, the cells were resuspended in K-SFM medium at 1×10 5 / cm 2 Cell density Cells were inoculated and cultured in a 37°C incubator. Change the medium for the first time after 2 days, and change the medium every 3 days thereafter.

Embodiment 2

[0055] Embodiment 2, circulating fibrocyte separation and purification

[0056] Add human lymphocyte separation medium to the L tube, centrifuge to the lower layer, dilute the blood with PBS, add the diluted blood to the L tube, and centrifuge to separate layers. Discard the upper layer of plasma, suck out the buffy coat layer and add it to a centrifuge tube, add PBS to dilute and centrifuge, repeat the above steps 3 times. Then resuspended in PBS, counted cells, at 1×10 6 / mL density inoculated in Petri dishes.

Embodiment 3

[0057] Embodiment 3, separation and purification of vascular endothelial cells

[0058] The HUVEC cell line purchased from Sciencell was resuscitated at 37°C, seeded in a 100mm culture dish, and after the cells were congested, trypsinized and passaged.

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Abstract

The invention discloses a vascularized full-thickness tissue-engineered skin layer-by-layer assembled by hydrogel, nanofiber scaffolds and skin cells and a preparation method thereof, and belongs to the technical fields of macromolecule materials and biomedical materials. Artificial tissue engineered skin consists of an epidermal layer and a corium layer, wherein the epidermal layer is formed by alternately stacking nanofiber scaffolds located above the corium layer with seed cells. The corium layer consists of lower-layer nanofiber scaffolds, upper-layer hydrogel scaffolds and three kinds ofseed cells, and the seed cells are distributed on the surface of the nanofiber scaffolds, inside the hydrogel and on the surface of the hydrogel. The vascularized full-thickness tissue-engineered skinis prepared by combination of an electrospinning technology and a macromolecular complexation technology with a fiber / cell layer-gel layer-fiber / cell layer-by-layer self-assembly technology. The artificial tissue-engineered skin with biological function can be used for regeneration and repair of various tissues, especially for wound healing, angiogenesis, scar formation reduction and the like.

Description

technical field [0001] The invention belongs to the technical field of polymer materials and biomedical materials, and in particular relates to a vascularized full-thickness tissue-engineered skin assembled layer by layer with hydrogel, nanofiber scaffolds and skin cells and a preparation method thereof. Background technique [0002] As the complex and largest organ of the human body, the skin has complex functions such as maintaining the physiological balance of internal organs, protecting the body from infection and damage, regulating moisture, body temperature, and sense of touch. Many types of injury, such as chemical and thermal burns, contusions and cuts, can injure the skin and compromise its integrity as a barrier. For superficial and small injuries, the skin can quickly repair itself. However, for deep and large-area open wounds (loss of part or all of the dermis or even subcutaneous tissue), it is very difficult to fill them only with granulation tissue produced b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/40A61L27/60A61F2/10A61L27/18A61L27/20A61L27/22A61L27/24A61L27/38A61L27/52
CPCA61F2/105A61L27/18A61L27/20A61L27/222A61L27/24A61L27/3804A61L27/3808A61L27/3813A61L27/3891A61L27/52A61L27/60A61L2300/30A61L2300/412A61L2400/12C08L67/04C08L5/16
Inventor 李学拥王红军黄容李跃军李金清李靖许利荣边永钎赵聪颖徐晓丽
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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