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Tissue-engineered skin containing blood vessels and hair follicle structures based on 3D printing and preparation method thereof

A tissue-engineered skin, 3D printing technology, applied in the field of regenerative medicine engineering, can solve problems such as construction, and achieve the effects of strong regeneration ability, small rejection reaction, good three-dimensional network structure and elasticity

Active Publication Date: 2018-09-14
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some 3D printed skin materials have been prepared, it is still not possible to construct complete blood vessels and hair follicles and other appendages in the skin materials.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Isolation and culture of human epidermal stem cells.

[0039] The digested skin was carefully separated from the epidermis and dermis with ophthalmic tweezers, and the epidermis was immersed in 0.25% trypsin and digested at 37°C for 30 minutes to make it a cell suspension. Add DMEM containing 10% FBS to terminate the digestion, centrifuge at 1000r / min for 5min at a radius of 15cm, and discard the supernatant.

[0040] Take the centrifuged cell suspension and place it in a 60mm culture dish, add 2-5mL culture medium (containing DMEM, 10ng / mL EGF, 5μg / mL insulin, 100μg / mL gentamycin), and put it in 37℃, 5% CO 2 Culture in an incubator with saturated humidity, change the medium after 24 hours, and change the medium every 2 days thereafter. On the 19th day of culture, the epidermal stem cells were connected into sheets, and when they reached 80% confluence, they were subcultured.

[0041] Use a straw to suck up the remaining culture medium in the culture dish, ...

Embodiment 2

[0042] Example 2: Isolation and culture of dermal papilla cells.

[0043] Add glutamine, 2 mmol / L Hepes (hydroxyethylpiperazineethanesulfonic acid), 10 μg / L hydrocortisone, 10 mg / L transferrin, 10μg / L sodium selenite, 10 5 U / L penicillin and 100mg / L streptomycin were prepared as hair follicle culture medium, and bovine insulin 10mg / L was added before use.

[0044] The scalp was rinsed three times with PBS, scrubbed and disinfected with 75% ethanol gauze, moved to another sterile petri dish, and rinsed with PBS again. The scalpel is used to cut and separate the scalp to individual follicular units one by one.

[0045] The hair follicles with full dermal papillae, smooth and round shape, and darker color were selected with the naked eye, and the complete hair follicle units with complete dermal papillae and no separation of inner and outer root sheaths were selected again under the microscope, and placed in a 24-well plate (1 root / well). Add the prepared WilliamsE medium, 0.5...

Embodiment 3

[0046] Example 3: Isolation and culture of bone marrow mesenchymal stem cells.

[0047] Bone marrow was collected by bone marrow puncture under aseptic conditions. The bone marrow was gently blown and suspended with PBS, centrifuged at 1500r / min for 10 minutes, the supernatant and fat layer were discarded, and PBS was added to make a single cell suspension by blowing and blowing. Slowly add Percoll separation solution with a relative density of 1.073 along the tube wall, and centrifuge at 1500r / min for 20min to obtain a milky white cloudy mononuclear cell layer.

[0048] The monocytes were washed 3 times with PBS, collected by centrifugation, resuspended in DMEM / F12 medium containing 100 mL / L fetal bovine serum for culture, the medium was changed every other day, and unattached cells were discarded.

[0049] After the adherent cells reached 80-90% confluence, they were purified and amplified. Pour out the old culture medium, add trypsin, place in the incubator for 2-3 minutes...

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Abstract

The invention relates to tissue-engineered skin containing blood vessels and hair follicle structures based on 3D printing and a preparation method thereof. The tissue-engineered skin is composed of an epidermal layer, an acellular dermal scaffold and a dermis layer, wherein in the epidermal layer, epidermal stem cells are used seed cells, the seed cells are printed on the upper surface of the acellular dermal scaffold through a 3D printer after being compounded with a hydrogel carrier to differentiate and form a normal epidermal structure, in the dermis layer, mesenchymal stem cells, vascularendothelial cells, dermal papilla cells and adipose-derived stem cells are used as seed cells, the seed cells and a hydrogel carrier are compounded, through the 3D printer, gelatin slow-release micropheres compounded with cytokines are printed on the lower surface of the acellular dermal scaffold, and meanwhile, the hydrogel compound of the seed cells is printed in the gelatin slow-release micropheres to form a dermis structure with a three-dimensional spatial structure.

Description

technical field [0001] The invention belongs to the technical field of regenerative medicine engineering, and relates to human skin organs constructed by tissue engineering methods, in particular to tissue engineered skin containing blood vessels and hair follicle structures constructed by 3D printing. Background technique [0002] Skin is an important organ that covers the surface of the human body to protect the human body. However, various diseases such as extensive burns, surgery, trauma, skin ulcers, etc. can cause skin damage. For some large and severe skin defects, autologous transplantation is generally used for treatment now. But this approach not only creates new damage to the donor site, but is often limited by the limited source of autologous skin. At the same time, there are often differences in color, texture, and even function between the graft and the graft site. [0003] Tissue engineering is the combination of engineering and life sciences to research an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/60A61L27/40A61L27/36A61L27/38A61L27/56A61L27/54A61L27/50B33Y80/00B33Y70/00B33Y10/00
CPCA61L27/362A61L27/3804A61L27/3808A61L27/3834A61L27/50A61L27/54A61L27/56A61L27/60A61L2300/414A61L2300/602A61L2430/40B33Y10/00B33Y70/00B33Y80/00
Inventor 于保锋曾思衡傅松涛解军李静静袁洋洋姚志坚张丽娜王萱马培元田九博穆秀丽孟涛涛
Owner SHANXI MEDICAL UNIV
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