CN107320781B

CN107320781B - Tissue engineering skin containing living cells and preparation method thereof

Info

Publication number: CN107320781B
Application number: CN201710562163.2A
Authority: CN
Inventors: 刘少辉; 车七石
Original assignee: Guangzhou Rainhome Pharm and Tech Co Ltd
Current assignee: Guangzhou Rainhome Pharm and Tech Co Ltd
Priority date: 2017-07-11
Filing date: 2017-07-11
Publication date: 2020-03-10
Anticipated expiration: 2037-07-11
Also published as: CN107320781A

Abstract

The invention belongs to the technical field of tissue engineering, and particularly relates to a tissue engineering skin containing living cells and a preparation method thereof, wherein the tissue engineering skin comprises an epidermal layer, a simulated cortex layer and a dermal layer, the simulated cortex layer comprises keratin cells, fibroblasts and a biological scaffold material, and the density ratio of the keratin cells to the fibroblasts is 0.1-0.2: 1; the biological scaffold material consists of gelatin, chitosan, glutamine peptide and hyaluronic acid according to the mass ratio of 6-7:1.5-2:0.5-1:1, and the prepared tissue skin has good biocompatibility, good plasticity and mechanical strength and can better repair wound surfaces.

Description

Tissue engineering skin containing living cells and preparation method thereof

Technical Field

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

Background

Tissue engineering (tissue engineering) is an emerging interdisciplinary discipline for the construction of tissue or organ substitutes in vitro or in vivo, using a combination of cell biology, engineering and materials, for the repair, maintenance or improvement of body morphology and function. The tissue engineering skin is an artificial skin substitute constructed by utilizing the principle of tissue engineering, is used for skin transplantation, and can effectively solve the problems of skin defect repair and insufficient skin supply in clinic.

Skin tissue engineering is the earliest and most mature field in tissue engineering, and the first tissue engineering product approved by FDA for marketing is tissue engineered skin. Clinical skin ulcers include diabetic ulcers, venous refractory ulcers, pyoderma gangrenosum ulcers and ulcers caused by other diseases, and the like, and the conventional treatment method cannot achieve satisfactory effects, but the effect is obvious after the artificial skin treatment.

At present, various skin products are published and used for clinical treatment to obtain a certain curative effect, but some defects such as poor compatibility, rejection reaction, long culture period, easy blister formation after transplantation, easy scar contracture, water loss in skin and the like exist, and in order to enable the tissue engineering skin to be closer to the human skin, more and more researches are carried out to innovatively improve the tissue engineering skin.

Chinese patent No. CN102462864B discloses a method for constructing tissue engineering skin, which uses human epidermal stem cells and dermal fibroblasts as seed cells, and uses the same acellular dermal matrix modified by human placenta type IV collagen as a scaffold, and compositely constructs an active double-layer tissue engineering skin by using a gas-liquid interface separation culture method. The tissue engineering skin constructed by the method is closer to the structure of natural skin, wherein the structure of a dermal collagen scaffold is complete, and a plurality of layers of cells with different differentiation degrees are arranged in an epidermal layer, so that the morphological requirements of the tissue engineering skin are met, but the culture medium is added with fetal calf serum, so that rejection reaction is easy to occur.

Chinese patent application publication No. CN104740689A discloses a composite tissue engineering skin containing living cells and a preparation method thereof. The composite tissue engineering skin not only contains living cells, but also contains a degradable polymeric material protective layer, so that the isolation capability of the composite tissue engineering skin is enhanced, the cost is reduced, and the quality guarantee period is prolonged. Overcomes the defects of high cost, short shelf life and poor isolation capability of tissue engineering skin containing living cells in the prior art. However, the degradable polymeric material protective layer used in the invention has long degradation time in human body, and is easy to cause poor effect of wound dressing.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention discloses tissue engineering skin containing living cells and a preparation method thereof, and the prepared tissue skin has good biocompatibility, plasticity and mechanical strength and can better repair wound surfaces.

The specific technical scheme of the invention is as follows:

a tissue engineering skin containing living cells comprises an epidermal layer, a simulated cortex and a dermal layer, wherein the simulated cortex comprises keratin cells, fibroblasts and a biological scaffold material, and the density ratio of the keratin cells to the fibroblasts is 0.1-0.2: 1; the biological scaffold material consists of gelatin, chitosan, glutamine peptide and hyaluronic acid according to the mass ratio of 6-7:1.5-2:0.5-1: 1.

The simulated cortex is added between the epidermal layer and the dermis layer, so that the contact between the epidermal layer and the simulated cortex is tighter, the defect that the epidermal layer and the dermis layer are easy to peel off due to direct contact is avoided, and experiments prove that keratin cells and fibroblast cells contained in the simulated cortex grow disorderly, so that the toughness of the tissue engineering skin can be effectively improved, and the wound healing effect is improved; the biological scaffold material used by the invention is added with the glutamine peptide, so that the decomposition of the biological scaffold material is facilitated, and experiments show that the added glutamine peptide can improve the toughness and elasticity of skin, is favorable for improving the mechanical strength of the biological scaffold material when being applied to the biological scaffold material, has better affinity with human body, is favorable for having good affinity with wound surfaces when being applied to tissue engineering skin, and can provide nutrients for subsequent cell growth.

Further, the preparation method of the simulated cortex comprises the following steps: mixing biological scaffold material and DMEM culture medium at 4 deg.C at volume ratio of 1:1, adding epidermal growth factor and basic fibroblast growth factor, inoculating mixed solution of fibroblast and keratinocyte with total inoculation density of 2 × 10⁶Per ml, wherein the density ratio of keratinocyte to fibroblast is 0.1-0.2:1, 37 deg.C, 5% CO₂Culturing in an incubator for 4-5 days, and then placing in a culture dish for solidification to form a simulated cortex;

the dosage ratio of the epidermal growth factor, the basic fibroblast growth factor and the DMEM medium is 2-3ng:12-15ng:1 ml.

Further, the biological scaffold material is composed of gelatin, chitosan, glutamine peptide and hyaluronic acid according to the mass ratio of 6.5:1.75:0.75: 1.

Further, the preparation method of the biological scaffold material comprises the following steps: preparing gelatin into 10% gelatin aqueous solution at 50 ℃, dissolving chitosan into 2% acetic acid solution to prepare 2% chitosan acetic acid solution, mixing the two solutions, adding glutamine peptide and hyaluronic acid, stirring uniformly, standing for 24-36 hours, casting the obtained mixed solution on a horizontal polyvinyl chloride plate with the surface temperature of 20 ℃ to form a film, finally soaking for 12 hours by using sodium hydroxide solution with the pH value of 7.2-7.4, and airing to obtain the chitosan/chitosan composite material.

Furthermore, the invention also provides a preparation method of the tissue engineering skin containing the living cells, which comprises the following steps:

(1) obtaining keratinocyte and fibroblast

(2) Preparing a simulated skin layer

Mixing biological scaffold material and DMEM culture medium at 4 deg.C at volume ratio of 1:1, adding epidermal growth factor and basic fibroblast growth factor, inoculating mixed solution of fibroblast and keratinocyte with total inoculation density of 2 × 10⁶Per ml, wherein the density ratio of keratinocyte to fibroblast is 0.1-0.2:1, 37 deg.C, 5% CO₂Culturing in an incubator for 4-5 days, and then placing in a culture dish for solidification to form a simulated cortex; the dosage ratio of the epidermal growth factor, the basic fibroblast growth factor and the DMEM culture medium is 2-3ng:12-15ng:1 ml;

(3) preparation of the dermis layer

Inoculating fibroblasts to the surface of the simulated cortex obtained in the step (2), adding a fibroblast culture medium, and inoculating at a density of 1 × 10⁶/ml，37℃，5％CO₂Culturing in incubator for 1-3 days to obtain dermis-artificial cortex;

(4) preparation of the skin layer

Inoculating keratinocyte onto the surface of the simulated cortex of the dermis-simulated cortex obtained in the step (3), adding epidermal cell culture medium, and inoculating at a density of 1 × 10⁶/ml，37℃，5％CO₂Culturing in incubator for 6-8 days to obtain dermis-simulated cortex-epidermis layer;

(5) construction of tissue engineered skin

The dermis-simulated cortex-epidermal layer obtained in the step (4) is laid on the culture mediumAdding 1 × 10 of water into the bottom of the culture dish^- ⁴In DMEM medium containing mol/L calcium chloride, at 37 deg.C and 5% CO₂Culturing in incubator for 6-8 days.

Further, the invention also provides a method for obtaining the keratin cells and the fibroblasts in the step (1), which comprises the following steps:

the transcription factors OCT4, SOX2, KLF4 and C-MYC were introduced into human urine cells at a cell density of 1X 10⁶Inoculating in pluripotent stem cell induction culture medium, at 37 deg.C and 5% CO₂Culturing in an incubator, changing the culture solution every day, and continuously culturing for 25-30 days to obtain induced pluripotent stem cells; inducing pluripotent stem cells at a cell density of 1X 10⁶Inoculating in mesenchymal stem cell induction culture medium, at 37 deg.C and 5% CO₂Culturing in an incubator, changing the culture solution every 2 days, and continuously culturing for 14-16 days to obtain mesenchymal stem cells;

acquisition and amplification of keratin cells: mesenchymal stem cells were cultured at a density of 1X 10⁶Inoculating to keratinocyte induction culture medium, differentiating to obtain keratinocyte, removing keratinocyte induction culture medium when the growth of keratinocyte reaches 80%, cleaning with DMEM/F12 culture medium, adding DPBS containing 0.5mM EDTA for digestion for 5min, centrifuging at 400r/min for 5min to obtain single keratinocyte, inoculating the single keratinocyte to epidermal cell culture medium at an inoculation density of 1 × 10⁶/ml，37℃，5％CO₂Culturing in an incubator;

obtaining and expanding fibroblasts: mesenchymal stem cells were cultured at a density of 1X 10⁶Inoculating in fibroblast induction culture medium, differentiating to obtain fibroblast, removing fibroblast induction culture medium when fibroblast grows to 80%, cleaning with DMEM/F12 culture solution, adding DPBS containing 0.5mM EDTA for digestion for 5min, centrifuging at 400r/min for 5min to obtain fibroblast single cell, inoculating into fibroblast culture medium at an inoculation density of 1 × 10⁶/ml，37℃，5％CO₂Culturing in incubator.

Further, the epidermal cell culture medium is: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, hydrocortisone 5ng/mL, cholera toxin 1×10^-10mol/L, insulin 0.05ng/mL, adenine 1.8X 10^-4mol/L, penicillin 100IU/mL, transferrin 10 mug/mL, glutamic acid 5 mug/mL, epidermal growth factor 10 mug/mL, ethanolamine 1X 10^-5mol/L；

The fibroblast culture medium comprises: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, insulin 5ng/mL, hydrocortisone 300 μ g/mL, adenine 15 μ g/mL, vitamin C120 μ g/mL, basic fibroblast growth factor 8 μ g/mL, transferrin 10 μ g/mL;

the keratin cell induction culture medium comprises: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, penicillin 100IU/mL, streptomycin 100 μ g/mL, epidermal growth factor 20ng/mL, insulin 10 μ g/mL, tretinoin 8 μ g/mL, calcium chloride 6 μ g/mL;

the fibroblast induction culture medium is prepared by taking DMEM/F12 as a basic culture medium, and the additives and the dosage of the additives are 50 mu g/mL of bovine pituitary extract, 100IU/mL of penicillin, 100 mu g/mL of streptomycin, β L of 15ng/mL of transforming growth factor, 20ng/mL of basic fibroblast growth factor, 10 mu g/mL of insulin iron selenium transfer protein and 0.1 mu mol/L of dexamethasone;

the induction culture medium of the pluripotent stem cells comprises: the fibroblast culture medium is taken as a basic culture medium, and the additives and the dosage are as follows: 2 mmol/L-glutamine, 0.02mmol/mL non-essential amino acid;

the mesenchymal stem cell induction culture medium comprises: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50. mu.g/mL, L-glutamine 2 mmol/L.

The weight ratio of DMEM/F12 is 2: 1.

the epidermal culture medium and the dermal culture medium used in the invention adopt a commercial DMEM and F12 mixed improved culture medium, the F12 culture medium contains a plurality of trace elements, the DMEM culture medium has high nutrient content, and the DMEM culture medium is mixed with the bovine pituitary extract to replace the traditional serum culture medium, thereby reducing the content of antigen and reducing the infection of virus.

The tissue engineering skin structure containing the living cells prepared by the invention is a dermis-simulated cortex-epidermis layer, wherein the simulated cortex contains a disordered growth layer of fibroblasts and keratin cells, the epidermis layer and the dermis layer are respectively cultured on two sides of the simulated cortex, so that the tissue engineering skin obtained by culture has a layered structure and is not easy to delaminate, the mechanical strength of the tissue engineering skin is improved, the fibroblasts in the dermis layer after transplantation and bovine pituitary extract existing in a culture medium can promote wound healing, various secreted growth factors can accelerate the interactive growth of the wound cells and the fibroblasts, the tissue engineering skin structure has good affinity and short wound healing time, and in addition, the biological scaffold material adopted by the invention can not only protect the wound from external pollution, but also has good biodegradability.

Compared with the prior art: the invention overcomes the defects that the dermis layer of the single-layer living cell tissue engineering skin is easily infected by external bacteria and has poor water retention capacity in the prior art, also overcomes the defects that the double-layer living cell tissue engineering skin has high cost, poor compatibility and difficult absorption of biological scaffold materials, and belongs to a three-layer skin structure to ensure that the mechanical property of the skin is stronger.

Detailed Description

The present invention will be further illustrated by the following examples. The components adopted by the invention all belong to conventional products, wherein the weight ratio of chitosan: CAS.148411-57-8, available from great warrior pharmaceutical chemical (group) Inc. of Wuhan, Dahua; DMEM, F12: shanghaineidae Biotech Co., Ltd; bovine pituitary extract: BPE brand: ScienCell, cat # 0713; insulin: CAS: 11061-68-0, Hubei Xinkang pharmaceutical chemical Co., Ltd.

EXAMPLE 1 Medium types and compositions

An epidermal cell culture medium is: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, hydrocortisone 5ng/mL, cholera toxin 1 × 10^-10mol/L, insulin 0.05ng/mL, adenine 1.8X 10^-4mol/L, penicillin 100IU/mL, transferrin 10 mug/mL, glutamic acid 5 mug/mL, epidermal growth factor 10mu.g/mL ethanolamine, 1X 10^-5mol/L；

A fibroblast culture medium is: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, insulin 5ng/mL, hydrocortisone 300 μ g/mL, adenine 15 μ g/mL, vitamin C120 μ g/mL, basic fibroblast growth factor 8 μ g/mL, transferrin 10 μ g/mL;

a keratin cell induction medium is as follows: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, penicillin 100IU/mL, streptomycin 100 μ g/mL, epidermal growth factor 20ng/mL, insulin 10 μ g/mL, tretinoin 8 μ g/mL, calcium chloride 6 μ g/mL;

a fibroblast induction culture medium is prepared from DMEM/F12 as basic culture medium, additives including ox pituitary extract 50 μ g/mL, penicillin 100IU/mL, streptomycin 100 μ g/mL, transforming growth factor β L15ng/mL, basic fibroblast growth factor 20ng/mL, insulin iron selenium transfer protein 10 μ g/mL, and dexamethasone 0.1 μmol/L;

a pluripotent stem cell induction culture medium comprises: the fibroblast culture medium is taken as a basic culture medium, and the additives and the dosage are as follows: 2 mmol/L-glutamine, 0.02mmol/mL non-essential amino acid;

a mesenchymal stem cell induction culture medium is: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, L-glutamine 2 mmol/L;

the weight ratio of DMEM/F12 is 2: 1.

example 2 tissue engineering skin containing Living cells

The tissue engineering skin containing the living cells comprises an epidermal layer, a simulated cortex and a dermal layer, wherein the simulated cortex comprises keratin cells, fibroblasts and a biological scaffold material, and the density ratio of the keratin cells to the fibroblasts is 0.15: 1; the biological scaffold material consists of gelatin, chitosan, glutamine peptide and hyaluronic acid according to the mass ratio of 6.5:1.75:0.75:1, and the preparation method comprises the following steps: preparing gelatin into a gelatin aqueous solution with the mass fraction of 10% at 50 ℃, dissolving chitosan into an acetic acid solution with the mass fraction of 2% to prepare an acetic acid solution with the mass fraction of 2% chitosan, mixing the two solutions, adding glutamine peptide and hyaluronic acid, uniformly stirring, standing for 30 hours, then casting the obtained mixed solution on a horizontal polyvinyl chloride plate with the surface temperature of 20 ℃ to form a film, finally soaking for 12 hours by using a sodium hydroxide solution with the pH value of 7.3, and airing to obtain the chitosan;

the preparation method of the tissue engineering skin containing the living cells,

(1) obtaining keratinocyte and fibroblast

The method comprises the following specific steps: the transcription factors OCT4, SOX2, KLF4 and C-MYC were introduced into human urine cells at a cell density of 1X 10⁶Inoculating in pluripotent stem cell induction culture medium, at 37 deg.C and 5% CO₂Culturing in an incubator, changing the culture solution every day, and continuously culturing for 27 days to obtain induced pluripotent stem cells; inducing pluripotent stem cells at a cell density of 1X 10⁶Inoculating in mesenchymal stem cell induction culture medium, at 37 deg.C and 5% CO₂Culturing in an incubator, changing the culture solution every 2 days, and continuously culturing for 15 days to obtain mesenchymal stem cells;

(2) Preparing a simulated skin layer

Mixing biological scaffold material and DMEM culture medium at 4 deg.C at volume ratio of 1:1, adding epidermal growth factor and basic fibroblast growth factor, inoculating mixed solution of fibroblast and keratinocyte with total inoculation density of 2 × 10⁶Per ml, with a density ratio of keratinocyte to fibroblast of 0.15:1, 37 deg.C, 5% CO₂Culturing in an incubator for 4.5 days, and then placing in a culture dish for solidification to form a simulated cortex; the dosage ratio of the epidermal growth factor, the basic fibroblast growth factor and the DMEM medium is 2.5ng:13.5ng:1 ml;

(3) preparation of the dermis layer

Inoculating fibroblasts to the surface of the simulated cortex obtained in the step (2), adding a fibroblast culture medium, and inoculating at a density of 1 × 10⁶/ml，37℃，5％CO₂Culturing in incubator for 2 days to obtain dermis-simulated cortex; (4) preparation of the skin layer

Inoculating keratinocyte onto the surface of the simulated cortex of the dermis-simulated cortex obtained in the step (3), adding epidermal cell culture medium, and inoculating at a density of 1 × 10⁶/ml，37℃，5％CO₂Culturing in incubator for 7 days to obtain dermis-simulated cortex-epidermis layer;

(5) construction of tissue engineered skin

Spreading the dermis-simulated cortex-epidermal layer obtained in the step (4) at the bottom of a culture dish, and adding the mixture containing 1 × 10^- ⁴In DMEM medium containing mol/L calcium chloride, at 37 deg.C and 5% CO₂Culturing in incubator for 7 days.

Example 2 relates to a medium using the formulation provided in example 1.

Example 3A tissue engineered skin containing viable cells

The difference with the embodiment 2 is that the biological scaffold material consists of gelatin, chitosan, glutamine peptide and hyaluronic acid according to the mass ratio of 6:2:1:1, and the rest is the same as the embodiment 2.

Example 4A tissue engineered skin containing viable cells

The difference with the embodiment 2 is that the biological scaffold material consists of gelatin, chitosan, glutamine peptide and hyaluronic acid according to the mass ratio of 7:1.5:0.5:1, and the rest is the same as the embodiment 2.

Comparative example 1 tissue engineering skin containing living cells

The difference from the example 2 is that fibrin is used to replace glutamine peptide, and the biological scaffold material consists of gelatin, chitosan, fibrin and hyaluronic acid according to the mass ratio of 6.5:1.75:0.75:1, and the rest is the same as the example 2.

Comparative example 2 tissue engineered skin containing viable cells

The difference from example 2 is that there is no step of preparing a simulated cortex in the preparation method, and the biological scaffold material is used to replace the simulated cortex, and the obtained tissue engineering skin is a dermis-biological scaffold material-epidermis layer, and the other steps are the same as example 2.

Test example 1 measurement of content of viable cells

The living cell content in the tissue engineering skin prepared in the examples 2-4 and the comparative examples 1-2 of the present invention was measured by MTT method, and the living cell content was measured after the prepared tissue engineering skin was stored for 15 days and 40 days, respectively.

The test method comprises the following steps: will be 1mm²Cutting area of skin into small pieces, adding into culture solution, adding MTT (concentration of 5mg/ml, 10 times)⁶200ul cells) are incubated for 4 hours at 37 ℃, culture solution is discarded, the incubated skin is cut into pieces, DMSO is added to shake for 20 minutes at the normal temperature at 100 rpm, and the shaken supernatant is extracted and placed under an enzyme-labeling instrument to measure the OD value;

measuring the OD value of the living cells contained in the same area by the same method;

the content of living cells was defined as skin OD value/living cell OD value × 100%, and is specifically shown in table 1.

TABLE 1 live cell Activity results

	Example 2	Example 3	Example 4	Comparative example 1	Comparative example 2
						After 20 days	85％	83％	82％	72％	68
After 40 days	61％	58％	56％	45％	43％

As can be seen from Table 1, the content of viable cells of the tissue engineering skin prepared in examples 2-4 is maintained above 82% after being preserved for 20 days, and the content of viable cells of the tissue engineering skin prepared in comparative examples 1-2 is maintained above 56% after being preserved for 40 days, while the content of viable cells of the tissue engineering skin prepared in comparative examples 1-2 is greatly reduced after being preserved for the same time, which indicates that the preservation period of the tissue engineering skin provided by the invention is longer, and further indicates that the glutamine peptide in the preparation of the dermis-imitated layer and the biological scaffold material affects the cell activity of the tissue engineering skin.

Test example 2 animal transplantation test

Test materials: male Balb/c-nu mice (nude mice, available from Shanghai Sphere-Bikay laboratory animals Co., Ltd.) weighed 20. + -.1 g, 30 mice.

The test method comprises the following steps: pretreatment: after anesthesia of a nude mouse by virtue of intraperitoneal injection of chloral hydrate, skin hairs on the back are shaved off, a full layer of circular skin with the diameter of 6mm is removed on the median line of the back of the mouse by virtue of surgical scissors, and the injury thickness is uniform;

(1) transplanting: the tissue engineering skin prepared in the embodiments 2-4 and the comparative examples 1-2 of the invention is transplanted on the wound surface, and a positive control group is set: naturally healing, fixing the tissue engineering skin by adopting an intradermal suture technology after transplantation, and covering by aseptic application;

(2) and (3) postoperative treatment: all mice were fed in a single cage under the same conditions and the wound surface was observed after transplantation. The specific results are shown in table 2:

TABLE 2 healing time

Example 2

Example 3

Example 4

Comparative example 1

Comparative example 2

Control group

Area of healing (1 week)

69％

65％

67％

49％

46％

32％

Area of healing (2 weeks)

100％

99％

91％

90％

80％

Complete healing time/day

14

14.5

15

18

19

23

As can be seen from table 2, examples 1 to 3 healed faster than comparative examples 1 to 2 and the control group, and it was observed through the transplantation test that: control group: the wound has thin crust skin, dark red color, slow healing speed and easy damage, and after complete healing, the crust skin is sunken downwards, the skin is tight and tense, and the regenerated skin is not elastic when being lightly touched.

Comparative examples 1 to 2: the wound surface and the tissue engineering skin have good application effect, no water bubbles are generated, the crust skin is slightly elastic, and the new skin is slightly tighter than the normal skin by slight touch.

Examples 2 to 4: the wound surface and the tissue engineering skin have good application effect, no bubble is generated, the wound surface skin becomes elastic, and the new skin is lightly touched to be tight and has the same touch feeling as the normal skin.

After 4 weeks of transplantation, section observation of wound skin shows that the epidermis and dermis structures of the tissue engineering skin prepared by the embodiment 2-4 of the invention are well developed after transplantation and have the shape close to that of normal skin, while the healing speed of the tissue engineering skin prepared by the comparative example 1 is slow, the whole layer structure of the skin is thinner than that of the normal skin, and the whole layer structure of the skin of the tissue engineering skin prepared by the comparative example 2 is thicker than that of the normal skin after transplantation, which indicates that the lack of a simulated cortex can cause the proliferation of the tissue engineering skin cells.

Test example 3 elasticity and toughness test

Test subjects: wound skin obtained after 4 weeks of transplantation by the sample method described in test example 2;

sample method: taking 5 multiplied by 5mm square wound surface skin, applying force to two sides of the skin, and calculating the elasticity and toughness of the skin: force applied when skin breaks; elasticity: the size of the deformation (length change) when the skin is broken; specific results are shown in table 3.

Table 3 skin elasticity and toughness data results

As can be seen from Table 3, the tissue engineering skin prepared in examples 2-4 has greater elasticity and toughness, while the tissue engineering skin prepared in comparative examples 1-2 has slightly poor toughness and elasticity, the control group heals naturally, the growth rate of other internal tissues or blood vessels is slow, and the wound surface shrinks directly due to long healing time, so that the skin performance of the wound surface is poor.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. The tissue engineering skin containing the living cells is characterized by comprising an epidermal layer, a simulated cortex and a dermal layer, wherein the simulated cortex comprises keratin cells, fibroblasts and a biological scaffold material, and the density ratio of the keratin cells to the fibroblasts is 0.1-0.2: 1; the biological scaffold material consists of gelatin, chitosan, glutamine peptide and hyaluronic acid according to the mass ratio of 6-7:1.5-2:0.5-1: 1.

2. The tissue engineering skin containing living cells according to claim 1, wherein the preparation method of the simulated cortex comprises: mixing biological scaffold material and DMEM culture medium at 4 deg.C at volume ratio of 1:1, adding epidermal growth factor and basic fibroblast growth factor, inoculating mixed solution of fibroblast and keratinocyte with total inoculation density of 2 × 10⁶Per ml, wherein the density ratio of keratinocyte to fibroblast is 0.1-0.2:1, 37 deg.C, 5% CO₂Culturing in an incubator for 4-5 days, and then placing in a culture dish for solidification to form a simulated cortex; the dosage ratio of the epidermal growth factor, the basic fibroblast growth factor and the DMEM medium is 2-3ng:12-15ng:1 ml.

3. The tissue engineering skin containing living cells according to claim 1, wherein the biological scaffold material is composed of gelatin, chitosan, glutamine peptide and hyaluronic acid in a mass ratio of 6.5:1.75:0.75: 1.

4. The tissue engineering skin containing living cells according to claim 1 or 3, wherein the preparation method of the biological scaffold material comprises: preparing gelatin into 10% gelatin aqueous solution at 50 ℃, dissolving chitosan into 2% acetic acid solution to prepare 2% chitosan acetic acid solution, mixing the two solutions, adding glutamine peptide and hyaluronic acid, stirring uniformly, standing for 24-36 hours, casting the obtained mixed solution on a horizontal polyvinyl chloride plate with the surface temperature of 20 ℃ to form a film, finally soaking for 12 hours by using sodium hydroxide solution with the pH value of 7.2-7.4, and airing to obtain the chitosan/chitosan composite material.

5. The method for preparing tissue engineering skin containing living cells according to any one of claims 1 to 4, comprising the steps of:

(1) obtaining keratinocyte and fibroblast

(2) Preparing a simulated skin layer

(3) preparation of the dermis layer

(4) preparation of the skin layer

(5) construction of tissue engineered skin

Spreading the dermis-simulated cortex-epidermal layer obtained in the step (4) at the bottom of a culture dish, and adding the mixture containing 1 × 10^-4In DMEM medium containing mol/L calcium chloride, at 37 deg.C and 5% CO₂Culturing in incubator for 6-8 days.

6. The method for preparing tissue engineering skin containing living cells according to claim 5, wherein the method for obtaining keratin cells and fibroblasts in step (1) comprises:

acquisition and amplification of keratin cells: inoculating mesenchymal stem cells into keratinocyte induction culture medium at density of 1 × 106, differentiating to obtain keratinocyte, removing keratinocyte induction culture medium when the keratinocyte grows to 80%, washing with DMEM/F12 culture solution, adding DPBS containing 0.5mM EDTA for digestion for 5min, centrifuging at 400r/min for 5min to obtain single keratinocyte, inoculating the single keratinocyte into epidermal cell culture medium at density of 1 × 10⁶/ml，37℃，5％CO₂Culturing in an incubator;

7. The method for preparing tissue engineering skin containing living cells according to claim 6,

the epidermal cell culture medium comprises: DMEM/F12 is used as a basic culture medium,the additive and the dosage are as follows: bovine pituitary extract 50 μ g/mL, hydrocortisone 5ng/mL, cholera toxin 1 × 10^-10mol/L, insulin 0.05ng/mL, adenine 1.8X 10^-4mol/L, penicillin 100IU/mL, transferrin 10 mug/mL, glutamic acid 5 mug/mL, epidermal growth factor 10 mug/mL, ethanolamine 1X 10^-5mol/L；

the mesenchymal stem cell induction culture medium comprises: DMEM/F12 is used as a basic culture medium, and the additives and the dosage are as follows: bovine pituitary extract 50 μ g/mL, L-glutamine 2 mmol/L;

the weight ratio of DMEM/F12 is 2: 1.