A tissue-engineered skin with multilayered epidermal cells, its preparation method and application

Abstract

The present invention discloses a tissue-engineered skin with multiple layers of epidermal cells, including a dermis layer and an epidermis layer, wherein the epidermis layer is formed by differentiation of epidermal seed cells laid flat on the dermis layer; wherein the epidermis layer includes multiple layers of epidermal cells. Due to the inclusion of multiple layers of epidermal cells, the skin has better mechanical properties and wear resistance, can effectively protect defective skin tissue, and avoid secondary injury and exposure infection of the wound surface. At the same time, the dermis layer of the tissue-engineered skin is obtained by 3D printing technology and cell culture, and has the required three-dimensional structure, and the polyethylene glycol guanidine double-modified chitosan used in the bio-ink during the printing process not only provides nutrients for the differentiation and proliferation of living cells, but also can induce epidermal cells to form a multilayer structure, and has certain antibacterial properties to avoid microbial contamination. In addition, the preparation process of the tissue-engineered skin is simple, time-saving, and can be prepared in a personalized and batch manner, with good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of regenerative medicine engineering technology, and more specifically, to a tissue engineering skin having multiple layers of epidermal cells, and a preparation method and application thereof. Background Art

[0002] The development and application of tissue-engineered skin is an important progress in the field of wound repair in recent years. Tissue-engineered skin provides more options for traditional autologous skin transplantation models. Existing technologies have developed a variety of tissue-engineered skin substitutes, and some products have been put on the market. Some skin substitutes can be used as temporary wound coverings with biological activity, which significantly promote epithelial hyperplasia and crawling on the wound surface, shortening the wound healing time; another part of the skin substitute can permanently replace the epidermis or induce the formation of new dermis, which is expected to provide sufficient skin sources and improve the quality of wound repair. Although tissue-engineered skin has made great progress, there are still many problems in the clinical application process. For example, the source of decellularized matrix is ​​limited, the preparation method is complicated, and the construction of tissue-engineered skin takes a long time. In addition, the fragility and thinness of the epidermis, poor mechanical strength, and difficulty in effectively protecting the wound surface are also one of the main reasons for the limitation of tissue-engineered skin in clinical application.

[0003] Bio-3D printing technology refers to a method that can locate and assemble biomaterials or cell units according to the principle of additive manufacturing under the drive of a digital three-dimensional model to manufacture products such as medical devices, tissue engineering scaffolds, and tissue organs. At present, bio-3D printing technology is often used to construct tissue engineering skin in batches. For example, patent ZL201410259459.3 discloses a 3D printed tissue engineering skin that is quickly constructed and a preparation method thereof, wherein seed cells are arranged and printed in a flat manner in the corresponding positions on the front of the decellularized allogeneic dermal matrix by a 3D printer according to the anatomical structure of normal skin. The tissue engineering skin can be completed quickly in a short time, but the allogeneic dermal matrix needs to remove the same epidermis through bioengineering and can only be obtained after decellularization. Its source of acquisition is limited and expensive. And the dermal matrix does not have the ability to further promote the proliferation of epidermal cells.

[0004] How to obtain tissue-engineered skin with an epidermal layer of a certain thickness more simply and economically is a problem that needs to be solved urgently in the existing technology. Summary of the invention

[0005] One object of the present invention is to provide a tissue-engineered skin having multiple layers of epidermal cells, including a dermis layer and an epidermis layer. The epidermis layer includes multiple layers of epidermal cells, and the skin has better mechanical properties and wear resistance, and can effectively protect defective skin tissue and avoid secondary injury and exposure infection of the wound surface.

[0006] Another object of the present invention is to provide a method for preparing tissue-engineered skin with multiple layers of epidermal cells, wherein the dermis layer is obtained by 3D printing technology, and the epidermis seed cells are laid flat on the dermis layer to obtain the epidermis layer. The polyethylene glycol-guanidine double-modified chitosan contained in the bio-ink during the 3D printing process has antibacterial and biocompatibility, which can promote the growth of living cells and has a certain antibacterial effect to avoid microbial contamination.

[0007] Another object of the present invention is to provide a tissue-engineered skin having multiple layers of epidermal cells for use in preparing skin repair biomaterials.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A tissue engineering skin with multiple layers of epidermal cells comprises a dermis layer and an epidermis layer, wherein the epidermis layer is formed by differentiation of epidermal seed cells laid flat on the dermis layer; wherein the epidermis layer comprises multiple layers of epidermal cells.

[0010] Preferably, the epidermis layer comprises 2-8 layers of epidermal cells;

[0011] Preferably, the epidermis layer comprises 3-6 layers of epidermal cells;

[0012] In traditional tissue engineering skin, epidermal cells are further cultured on the defiberized allogeneic skin tissue or the constructed tissue engineering dermal scaffold, but the obtained epidermal cells are monolayer, with insufficient mechanical strength and unsatisfactory wear resistance. The present invention provides a tissue engineering skin having an epidermal layer including 2-8 layers of epidermal cells, which has better mechanical properties and wear resistance, can effectively protect defective skin tissue, and avoid secondary injury and exposure infection of the wound surface.

[0013] In some specific examples, the epidermal cells may be mouse epidermal cells, human keratinocytes or epidermal stem cells, and the dermal cells may be mouse fibroblasts or human skin fibroblasts. It will be appreciated by those skilled in the art that other types of suitable epidermal cells and dermal cells may be selected according to the actual application requirements of tissue engineered skin, and the present invention is not limited thereto.

[0014] Different from the prior art, which uses a limited and expensive allogeneic dermal matrix, the dermal layer in the present invention is obtained by 3D printing. In a preferred example, the dermal layer is obtained by dispersing dermal seed cells into biological ink, obtaining a dermal base by 3D printing technology, placing the dermal base in a dermal cell culture medium containing an anionic crosslinker for crosslinking, and then removing the anionic crosslinker and continuing to culture in the dermal cell culture medium to obtain a dermal structure; further preferably, the biological ink includes collagen, gelatin, polyethylene glycol-guanidine double modified chitosan and a solvent. Among them, the solvent is selected from a buffer solution, physiological saline, cell culture medium or sterile water.

[0015] The dermal seed cells are dispersed in the bio-ink, and after the 3D printing process, a dermal substrate of a specific three-dimensional shape containing the dermal seed cells can be obtained. After cross-linking and curing in the dermal cell culture medium containing anionic cross-linking agents, the dermal seed cells differentiate to form the dermal layer.

[0016] The bio-ink used in the present invention contains polyethylene glycol-guanidine double-modified chitosan, which is an active ingredient that can promote the differentiation and growth of dermal seed cells and epidermal active cells. Therefore, when the epidermal cell seeds are spread on the dermis, the dermis can induce the epidermal cells to differentiate, increase the number of cell layers, and thicken the epidermis, thereby improving the problem that the tissue engineering skin epidermis is fragile and thin, and it is difficult to effectively cover the wound surface. At the same time, polyethylene glycol-guanidine double-modified chitosan also has a certain antibacterial effect, avoiding microbial contamination during the proliferation of skin cells.

[0017] Preferably, the mass fraction of collagen in the bio-ink is 0.2-0.5%, the mass fraction of gelatin is 2-6%, and the mass fraction of polyethylene glycol-guanidine double-modified chitosan is 2-6%.

[0018] For example, the mass fraction of collagen includes but is limited to 0.25%, 0.30%, 0.35%, 0.40% or 0.45% etc.; the mass fraction of gelatin includes but is limited to 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or 5.5% etc.; the mass fraction of polyethylene glycol-guanidine double-modified chitosan includes but is limited to 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or 5.5% etc.

[0019] More preferably, the sum of the mass fractions of the gelatin and the polyethylene glycol-guanidine double-modified chitosan is 8%.

[0020] In the bio-ink used for bio-3D printing of tissue engineering dermis, collagen and double-modified chitosan are used to simulate and provide cell matrix components suitable for fibroblast growth; at the same time, double-modified chitosan and the positively charged parts such as amino and guanidine groups in gelatin can also form a stable scaffold structure through electrostatic interactions with anionic crosslinkers. In addition, the strength of the material after gelation and its effect on cell activity need to be considered during bio-3D printing and epidermal cell induced growth. Considering various factors, the mass fractions of the three components need to be limited to a specific range to ensure that tissue-engineered skin with a multi-layered epidermal cell structure is obtained.

[0021] In some preferred examples, the collagen is rat tail type I collagen; and the gelatin is type A gelatin with a gel strength of 240 to 300 Bloom g.

[0022] Gelatin is a product of partial hydrolysis of natural collagen and a mixture of polymer chains of different lengths. Therefore, it will not form a solution when dispersed in a solvent, but will form a colloid and transform into a gel by cooling. The Bloom parameter, i.e., the gel strength, is an important method for analyzing the gelation ability of gelatin. The Bloom parameter of commercial gelatin is usually in the range of 50-300. Usually, gelatin with a high Bloom parameter has a higher melting temperature and gel point, resulting in a shorter gel time. In biological 3D printing, especially extrusion printing, on the one hand, it is required that the bio-ink can be fully dissolved in order to mix with living cells. At the same time, it is necessary to form a pre-gel solution with a certain viscosity in the low-temperature nozzle so that it can be printed and further cross-linked and cured. The gel strength of gelatin is precisely the use of the reversible sol-gel transition of the gelatin component in the bio-ink, which can be used to disperse cells and form quickly in a short time.

[0023] Polyethylene glycol-guanidine double-modified chitosan has an important effect on the differentiation and proliferation of dermal and epidermal cells, so choosing the right polyethylene glycol-guanidine double-modified chitosan is the key.

[0024] The ideal biomaterials that can be used for biological 3D printing should not only have basic printability, but also good solubility, biocompatibility and biodegradability. When chitosan-based biomaterials are used as bio-inks, they are not only required to be fully soluble so that they can be fully mixed with the cell-containing culture medium, but also to be cross-linked and cured after printing. On the one hand, polyethylene glycol-guanidine double-modified chitosan needs to rely on the introduction of polyethylene glycol segments to improve the solubility of natural chitosan, and on the other hand, it is also required to retain the rich positive charge in the guanidine functional group so as to further cure and form with a negatively charged cross-linking agent. The molecular weight of chitosan is limited to ensure that there are sufficient functional groups in its skeleton to participate in the grafting reaction; and the range of the grafting rate of polyethylene glycol segments and guanidine functional groups is to balance the ratio of positive charge to hydrophilic structure, so that the obtained double-modified chitosan has printability, formability and biocompatibility at the same time.

[0025] The molecular weight of the chitosan main chain in the polyethylene glycol-guanidine double-modified chitosan of the present invention is 500,000-1,000,000 Da, and the molecular weight of the polyethylene glycol segment is 550-5,000 Da; further preferably, the grafting rate of the polyethylene glycol segment on the polyethylene glycol-guanidine double-modified chitosan is 50-75%, and the grafting rate of the guanidine functional group is 10-20%.

[0026] For example, the molecular weight of the chitosan main chain includes, but is not limited to, 100,000-900,000 Da, 200,000-800,000 Da, 300,000-700,000 Da, 400,000-600,000 Da or 500,000 Da;

[0027] The molecular weight of the polyethylene glycol segment includes, but is not limited to, 750-4500Da, 1000-4000Da, 1500-3500Da, 2000-3000Da or 2500Da;

[0028] The grafting rate of the polyethylene glycol chain segment is 55%, 60%, 65%, 70% or 73%, etc.;

[0029] The grafting rate of the guanidine functional group is 11%, 13%, 15%, 17%, 18% or 19%, etc.

[0030] The polyethylene glycol-guanidine double-modified chitosan selected in the present invention includes but is not limited to polyethylene glycol-monoguanidine double-modified chitosan, polyethylene glycol-biguanidine double-modified chitosan or polyethylene glycol-arginine double-modified chitosan, etc. Of course, those skilled in the art can also select other types of polyethylene glycol-guanidine double-modified chitosan, and the present invention is not limited thereto.

[0031] The second aspect of the present invention provides a method for preparing the tissue-engineered skin having multiple layers of epidermal cells, such as Figure 1 As shown, the following steps are included:

[0032] S1. dispersing dermal seed cells in bio-ink, and then using 3D printing technology to obtain a dermal base, placing the dermal base in a dermal cell culture medium containing an anionic crosslinker for crosslinking, and then removing the anionic crosslinker and continuing to culture in the dermal cell culture medium to obtain a dermal layer;

[0033] S2. The dermis layer is placed in an epidermal cell culture medium, and the epidermal seed cell suspension is spread on the dermis surface to culture a tissue-engineered skin containing both a stable epidermis layer and a dermis layer.

[0034] like Figure 1 As shown, the preparation process mainly includes two steps. Step 1 is to use 3D printing technology to obtain tissue-engineered dermis, replacing the allogeneic dermal matrix in the existing technology, which is limited in source and expensive; Step 2 is to lay epidermal seed cells on the surface of the dermis layer, induce epidermal cell growth, and construct an epidermis layer with multiple cells. This preparation method has a simple process flow, short time consumption, high effect, and can meet personalized needs.

[0035] Preferably, the density of dermal seed cells in the bio-ink is 1×10 5 ~1.5×10 7 Pieces / mL;

[0036] Preferably, the density of epidermal seed cells in the epidermal seed cell suspension is 1×10 5 ~1.5×10 7 Pieces / mL.

[0037] Preferably, the anionic crosslinking agent in the dermal cell culture medium is one or more of sodium trimetaphosphate, sodium hexametaphosphate or sodium β-glycerophosphate, and the concentration of the anionic crosslinking agent is 100 to 300 mg / mL;

[0038] Preferably, the anionic crosslinking agent is sodium β-glycerophosphate, and its concentration is 200-300 mg / mL; preferably, the crosslinking temperature in S1 is 10-30° C., and the crosslinking time is 30-60 min.

[0039] Preferably, during the 3D printing process, after the bio-ink containing dermal seed cells is loaded into the printing cylinder, it needs to be placed in a printing water tank at a temperature of 4-25° C. for 20-45 minutes before the printing process is carried out.

[0040] The third aspect of the present invention provides the use of the tissue-engineered skin with multiple layers of epidermal cells in the preparation of skin repair biomaterials. The epidermal layer of the tissue-engineered skin in the present invention has multiple layers of cells, the skin has high mechanical strength, good wear resistance, and good protection for wound surface, and has better clinical application effect.

[0041] The beneficial effects of the present invention are as follows:

[0042] The present invention provides a tissue-engineered skin with multiple layers of epidermal cells, wherein the epidermal layer includes multiple layers of epidermal cells, and the skin has better mechanical properties and wear resistance, and can effectively protect defective skin tissue, and avoid secondary injury and exposure infection of the wound surface. At the same time, the dermis layer of the tissue-engineered skin is obtained by 3D printing technology and cell culture, and has the required three-dimensional structure, and the polyethylene glycol guanidine double-modified chitosan used in the bio-ink during the printing process not only provides nutrients for the differentiation and proliferation of living cells, but also can induce epidermal cells to form a multilayer structure, and has certain antibacterial properties, avoiding microbial contamination. In addition, the preparation process of the tissue-engineered skin is simple, time-saving, and can be prepared in a personalized and batch manner, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The specific implementation modes of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0044] Figure 1 The preparation process of tissue engineering skin in the present invention is shown.

[0045] Figure 2 A photograph of the tissue-engineered dermis layer prepared in Example 1 is shown.

[0046] Figure 3 The figure shows the survival status of the tissue-engineered dermal cells prepared in Example 1 at 3 days and 7 days (scale bar: 200 μm).

[0047] Figure 4 The tissue engineered skin prepared in Example 1 and Comparative Example 1 and their corresponding actual thickness are shown.

[0048] Figure 5 a) shows the HE staining results of the tissue-engineered skin sections prepared in Example 1 and Comparative Example 1 at the same magnification, b) shows the thickness of the tissue-engineered skin, and c) shows the number of surface cell layers of the tissue-engineered skin. DETAILED DESCRIPTION

[0049] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0050] Example 1

[0051] Dilute rat tail collagen to 2 mg / mL with sterile water, referred to as solution A;

[0052] Dissolve 0.3 g of type A gelatin and 0.5 g of polyethylene glycol monoguanidine double-modified chitosan in 10 mL of solution A to obtain a bio-ink that can be used for biological 3D printing, wherein the chitosan main chain molecular weight in the polyethylene glycol monoguanidine double-modified chitosan is 1 million Da, the polyethylene glycol molecular weight is 5000 Da, the monoguanidine grafting rate is 10%, and the polyethylene glycol segment grafting rate is 70%;

[0053] Human skin fibroblasts (HSF) were trypsinized, centrifuged and collected, and suspended in bio-ink to a cell concentration of 10 6 / mL, recorded as mixed solution B containing dermal cells;

[0054] The mixed solution B is loaded into the printing cylinder, the temperature of the printer temperature-controlled water tank is set to 4°C, and the printing cylinder is placed in the printer temperature-controlled water tank to cool for 30 minutes; the printing cylinder is taken out, moved to the printing arm and connected and fixed to the air pressure device, appropriate printing parameters are set, and printing is started; immediately after printing is completed, 2 mL of a cell-conditioned medium solution containing 300 mg / mL of sodium β-glycerophosphate is cross-linked at 20°C for 30 minutes, and then the culture medium is removed, and fresh culture medium without sodium β-glycerophosphate is added, and the culture is cultured in an incubator at 37°C with a CO2 content of 5% for one week to obtain tissue-engineered dermis, such as Figure 2 shown.

[0055] The tissue engineered dermis in Example 1 was fluorescently stained using a cell death and viability kit and observed under a confocal microscope. Figure 3 As shown, the cell morphology was good and the cell survival rate was above 80% at 3 days and 7 days.

[0056] The culture medium soaking the tissue-engineered dermis was removed, and human keratinocytes (HaCat) collected by centrifugation and suspended in epidermal culture medium were plated on the surface of the tissue-engineered dermis at a cell concentration of 10 6 / mL, and continue to culture in an incubator at 37℃ with a CO2 content of 5% for one week, and finally obtain tissue-engineered skin containing both dermis and epidermis. After sectioning and HE staining, it can be observed that the tissue-engineered skin contains 5 layers of epidermal cells, such as Figure 4 , 5 shown.

[0057] Example 2

[0058] Dilute rat tail collagen to 3 mg / mL with sterile water, referred to as solution A;

[0059] Dissolve 0.3 g of type A gelatin and 0.5 g of polyethylene glycol monoguanidine double-modified chitosan in 10 mL of solution A to obtain a bio-ink that can be used for biological 3D printing, wherein the chitosan main chain molecular weight in the polyethylene glycol monoguanidine double-modified chitosan is 1 million Da, the polyethylene glycol molecular weight is 2000 Da, the monoguanidine grafting rate is 10%, and the polyethylene glycol segment grafting rate is 69%;

[0060] Human skin fibroblasts (HSF) were trypsinized, centrifuged and collected, and suspended in bio-ink to a cell concentration of 10 6 / mL, recorded as mixed solution B containing dermal cells;

[0061] The mixed solution B is loaded into the printing cylinder, the temperature of the printer's temperature-controlled water tank is set to 4°C, and the printing cylinder is placed in the printer's temperature-controlled water tank to cool for 30 minutes; the printing cylinder is taken out, moved to the printing arm and connected and fixed to the air pressure device, appropriate printing parameters are set, and printing is started; immediately after printing is completed, 2 mL of a cell-conditioned medium solution containing 300 mg / mL of sodium β-glycerophosphate is used for cross-linking at 20°C for 30 minutes, and then the culture medium is removed, and fresh culture medium without sodium β-glycerophosphate is added, and the culture is cultured in an incubator at 37°C with a CO2 content of 5% for one week to obtain tissue-engineered dermis.

[0062] The culture medium soaking the tissue-engineered dermis was removed, and human keratinocytes (HaCat) collected by centrifugation and suspended in epidermal culture medium were plated on the surface of the tissue-engineered dermis at a cell concentration of 10 6 / mL, and continue to culture in an incubator at 37°C with a CO2 content of 5% for one week, and finally obtain tissue-engineered skin containing both dermis and epidermis. After sectioning and HE staining, it can be observed that the tissue-engineered skin contains three layers of epidermal cells.

[0063] Example 3

[0064] Dilute rat tail collagen to 5 mg / mL with sterile water, referred to as solution A;

[0065] Dissolve 0.3 g of type A gelatin and 0.5 g of polyethylene glycol monoguanidine double-modified chitosan in 10 mL of solution A to obtain a bio-ink that can be used for biological 3D printing, wherein the chitosan main chain molecular weight in the polyethylene glycol monoguanidine double-modified chitosan is 1 million Da, the polyethylene glycol molecular weight is 1000 Da, the monoguanidine grafting rate is 10%, and the polyethylene glycol segment grafting rate is 72%;

[0066] Human skin fibroblasts (HSF) were trypsinized, centrifuged and collected, and suspended in bio-ink to a cell concentration of 10 6 / mL, recorded as mixed solution B containing dermal cells;

[0067] The mixed solution B is loaded into the printing cylinder, the temperature of the printer's temperature-controlled water tank is set to 4°C, and the printing cylinder is placed in the printer's temperature-controlled water tank to cool for 30 minutes; the printing cylinder is taken out, moved to the printing arm and connected and fixed to the air pressure device, appropriate printing parameters are set, and printing is started; immediately after printing is completed, 2 mL of a cell-conditioned medium solution containing 300 mg / mL of sodium β-glycerophosphate is used for cross-linking at 20°C for 30 minutes, and then the culture medium is removed, and fresh culture medium without sodium β-glycerophosphate is added, and the culture is cultured in an incubator at 37°C with a CO2 content of 5% for one week to obtain tissue-engineered dermis.

[0068] The culture medium in which the tissue-engineered dermis was immersed was removed, and human keratinocytes (HaCat) collected by centrifugation and suspended in epidermal culture medium were spread on the surface of the tissue-engineered dermis at a cell concentration of 10^6 cells / mL. The cells were cultured in an incubator at 37°C with a CO2 content of 5% for one week, and finally a tissue-engineered skin containing both the dermis and the epidermis was obtained. After sectioning and HE staining, it was observed that the tissue-engineered skin contained three layers of epidermal cells.

[0069] Example 4

[0070] Dilute rat tail collagen to 2 mg / mL with sterile water, referred to as solution A;

[0071] 0.2 g of type A gelatin and 0.6 g of polyethylene glycol biguanide double-modified chitosan were dissolved in 10 mL of solution A to obtain a bio-ink that can be used for biological 3D printing, wherein the molecular weight of the chitosan main chain in the polyethylene glycol biguanide double-modified chitosan is 500,000 Da, the molecular weight of polyethylene glycol is 1000 Da, the grafting rate of biguanide groups is 12%, and the grafting rate of polyethylene glycol segments is 65%;

[0072] Mouse fibroblasts (L929) were trypsinized, centrifuged and collected, and the cells were suspended in bio-ink to a cell concentration of 1.5×10 6 / mL, recorded as mixed solution B containing dermal cells;

[0073] The mixed solution B is loaded into the printing cylinder, the temperature of the printer's temperature-controlled water tank is set to 8°C, and the printing cylinder is placed in the printer's temperature-controlled water tank to cool for 45 minutes; the printing cylinder is taken out, moved to the printing arm and connected and fixed to the air pressure device, appropriate printing parameters are set, and printing is started; immediately after printing is completed, 2 mL of a cell-conditioned medium solution containing 200 mg / mL of sodium β-glycerophosphate is used for cross-linking at 16°C for 45 minutes, and then the culture medium is removed, and fresh culture medium without sodium β-glycerophosphate is added, and the culture is cultured in an incubator at 37°C with a CO2 content of 5% for one week to obtain tissue-engineered dermis.

[0074] The culture medium soaking the tissue-engineered dermis was removed, and mouse epidermal cells collected by centrifugation and suspended in epidermal culture medium were spread on the surface of the tissue-engineered dermis at a cell concentration of 1.5×10 6 / mL, and continue to culture in an incubator at 37°C with a CO2 content of 5% for one week, and finally obtain tissue-engineered skin containing both dermis and epidermis. After sectioning and HE staining, it can be observed that the tissue-engineered skin contains three layers of epidermal cells.

[0075] Example 5

[0076] Dilute rat tail collagen to 5 mg / mL with sterile water, referred to as solution A;

[0077] 0.4 g of type A gelatin and 0.4 g of polyethylene glycol arginine double-modified chitosan were dissolved in 10 mL of solution A to obtain a bio-ink that can be used for biological 3D printing, wherein the molecular weight of the chitosan main chain in the polyethylene glycol arginine double-modified chitosan is 200,000 Da, the molecular weight of polyethylene glycol is 2000 Da, the arginine grafting rate is 20%, and the polyethylene glycol segment grafting rate is 60%;

[0078] Human skin fibroblasts (HSF) were trypsinized, centrifuged and collected, and the cells were suspended in bio-ink to a cell concentration of 1.5 × 10 7 / mL, recorded as mixed solution B containing dermal cells;

[0079] The mixed solution B is loaded into the printing cylinder, the temperature of the printer's temperature-controlled water tank is set to 6°C, and the printing cylinder is placed in the printer's temperature-controlled water tank to cool for 30 minutes; the printing cylinder is taken out, moved to the printing arm and connected and fixed to the air pressure device, appropriate printing parameters are set, and printing is started; immediately after printing is completed, 3 mL of a cell-conditioned medium solution containing 100 mg / mL of sodium β-glycerophosphate is cross-linked at 25°C for 60 minutes, and then the culture medium is removed, and fresh culture medium without sodium β-glycerophosphate is added, and the culture is cultured in an incubator at 37°C with a CO2 content of 5% for one week to obtain tissue-engineered dermis.

[0080] The culture medium soaking the tissue-engineered dermis was removed, and epidermal stem cells collected by centrifugation and suspended in epidermal culture medium were spread on the surface of the tissue-engineered dermis at a cell concentration of 1.5×10 7 / mL, and continue to culture in an incubator at 37°C with a CO2 content of 5% for one week, and finally obtain tissue-engineered skin containing both dermis and epidermis. After sectioning and HE staining, it can be observed that the tissue-engineered skin contains 4 layers of epidermal cells.

[0081] Comparative Example 1

[0082] Dilute rat tail collagen to 2 mg / mL with sterile water, referred to as solution A;

[0083] Dissolve 0.3 g of type A gelatin in 10 mL of solution A to obtain bio-ink that can be used for biological 3D printing;

[0084] Human skin fibroblasts (HSF) were trypsinized, centrifuged and collected, and suspended in bio-ink to a cell concentration of 10 6 / mL, recorded as mixed solution B containing dermal cells; the mixed solution B is loaded into the printing cylinder, the temperature of the printer's temperature-controlled water tank is set to 4°C, and the printing cylinder is placed in the printer's temperature-controlled water tank to cool for 30 minutes; the printing cylinder is taken out, the printing cylinder is moved to the printing arm and connected and fixed with the air pressure device, appropriate printing parameters are set, and printing is started; immediately after printing is completed, 2mL of a cell-conditioned medium solution containing 300mg / mL of sodium β-glycerophosphate is cross-linked at 20°C for 30min, and then the culture medium is removed, and a fresh culture medium without sodium β-glycerophosphate is added, and the culture is cultured in an incubator at 37°C with a CO2 content of 5% for one week to obtain tissue-engineered dermis.

[0085] The culture medium soaking the tissue-engineered dermis was removed, and human keratinocytes (HaCat) collected by centrifugation and suspended in epidermal culture medium were plated on the surface of the tissue-engineered dermis at a cell concentration of 10 6 / mL, and continue to culture in an incubator at 37℃ with a CO2 content of 5% for one week, and finally obtain tissue-engineered skin containing both dermis and epidermis. After sectioning and HE staining, it can be observed that the tissue-engineered skin contains only a single layer of epidermal cells, such as Figure 4 , 5 shown.

[0086] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A tissue-engineered skin having multiple layers of epidermal cells, characterized in that: It includes a dermis layer and an epidermis layer, wherein the epidermis layer is formed by differentiation of epidermal seed cells laid flat on the dermis layer; wherein the epidermis layer includes multiple layers of epidermal cells; The epidermis layer includes 2-8 layers of epidermal cells; The dermis layer is prepared by dispersing dermis seed cells into biological ink, obtaining a dermis base through 3D printing technology, placing the dermis base in a dermis cell culture medium containing an anionic crosslinker for crosslinking, then removing the anionic crosslinker, and continuing to culture in the dermis cell culture medium to obtain a dermis structure; wherein the biological ink includes collagen, gelatin, polyethylene glycol-guanidine double-modified chitosan and a solvent; the mass fraction of collagen in the biological ink is 0.2-0.5%, the mass fraction of the gelatin is 2-6%, and the mass fraction of the polyethylene glycol-guanidine double-modified chitosan is 2-6%; The gelatin is type A gelatin with a gel strength of 240 to 300 Bloom g; The molecular weight of the chitosan main chain in the polyethylene glycol-guanidine double-modified chitosan is 500,000-1,000,000 Da, and the molecular weight of the polyethylene glycol segment is 550-5,000 Da; the grafting rate of the polyethylene glycol segment on the polyethylene glycol-guanidine double-modified chitosan is 50-75%, and the grafting rate of the guanidine functional group is 10-20%; The anionic crosslinking agent is sodium β-glycerophosphate.

2. The tissue engineered skin according to claim 1, characterized in that: The epidermis layer includes 3-6 layers of epidermal cells.

3. The tissue engineered skin according to claim 1, characterized in that: The collagen is rat tail type I collagen.

4. The tissue engineered skin according to claim 1, characterized in that: The polyethylene glycol-guanidine double-modified chitosan is selected from one or more of polyethylene glycol-monoguanidine double-modified chitosan, polyethylene glycol-biguanidine double-modified chitosan or polyethylene glycol-arginine double-modified chitosan.

5. A method for preparing tissue-engineered skin having multiple layers of epidermal cells as claimed in any one of claims 1 to 4, characterized in that: The steps include: S1. dispersing dermal seed cells in bio-ink, and then using 3D printing technology to obtain a dermal base, placing the dermal base in a dermal cell culture medium containing an anionic crosslinker for crosslinking, and then removing the anionic crosslinker and continuing to culture in the dermal cell culture medium to obtain tissue-engineered dermis; S2. The tissue-engineered dermis is placed in an epidermal cell culture medium, and the epidermal seed cell suspension is spread on the surface of the dermis to culture a tissue-engineered skin containing both a stable epidermal layer and a dermal layer.

6. The preparation method according to claim 5, characterized in that: The density of dermal seed cells in the bio-ink was 1×10 5 ~1.5×10 7 Pieces / mL; The density of epidermal seed cells in the epidermal seed cell suspension is 1×10 5 ~1.5×10 7 Pieces / mL.

7. The preparation method according to claim 5, characterized in that: The anionic crosslinking agent in the dermal cell culture medium is sodium β-glycerophosphate, and its concentration is 200-300 mg / mL; The cross-linking temperature in S1 is 10-30°C and the cross-linking time is 30-60 minutes.

8. Use of the tissue-engineered skin having multiple layers of epidermal cells as claimed in any one of claims 1 to 4 in the preparation of skin repair biomaterials.

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