Artificial skin as well as preparation method and application thereof

Abstract

The invention discloses artificial skin as well as a preparation method and application thereof, and relates to the field of biomedical materials and tissue engineering. The artificial skin is obtained by co-culturing human-derived dermal cells and a degradable high polymer material fiber web. According to the invention, allogeneic human cells are utilized to prepare regenerated artificial skin taking human collagen as a main material, then immunogenicity such as heterologous DNA is removed through a decellularization process, residual components are very close to human autologous skin, no other heterologous polymer residues exist, and the biocompatibility is excellent. A fiber net adopted for co-culture is formed by weaving high polymer materials which are good in biocompatibility and can be completely degraded, the fiber net can be automatically degraded in the later period of culture, no high polymer material is left, and rejection reaction generated when artificial cells are implanted into a human body in the later period is avoided. The artificial skin can be produced in a standardized manner, and the uniformity among batches is good; the mask is rich in collagen, so that the skin is tough; the production cycle is controllable, the clinical immediate availability is strong, and the price is low.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials and tissue engineering, and particularly to an artificial skin and its preparation method and application. Background Art

[0002] As the largest organ of the human body, the skin is the first barrier for direct contact between the human body and the external environment, mainly playing a role in protecting the human body. It can not only effectively shield the harm of external harmful substances to the human body, but also ensure the balance of water and electrolytes in the human body and maintain body temperature. The human skin is composed of the epidermis, dermis and subcutaneous tissue, and the most important structure among them is the skin cells in the dermis and the collagen secreted by them, which play the most important elastic and barrier repair roles in the skin tissue. For large-area skin injuries caused by accidental burns or diseases, the barrier protection function of the skin may be damaged, seriously threatening human health. Autologous skin transplantation is an effective means for clinical treatment of large-area skin injuries, but it will cause new skin injuries to the patient and also face the problem of insufficient donors; while the transplantation of using other people's skin is also greatly limited due to factors such as insufficient donors, immune rejection and medical ethics issues.

[0003] In related technologies, to solve the above problems, some researchers have obtained artificial patches by tissue engineering methods, removing most of the immunogenicity from the dermal matrices of animal sources such as pigs and cows through decellularization treatment; or, after co-culturing with autologous living cells by electrospinning technology, an artificial skin with an electrospun membrane as the main structure is obtained for allogeneic transplantation. However, the above solutions still have problems: the decellularized dermal matrices of pigs, cows, etc. are from animal sources, so this method has high requirements for the screening of animal sources and it is difficult to achieve the uniformity of the whole process; moreover, there are certain differences in the amino acid sequences of structural proteins such as collagen in human skin and animal collagen. When using animal-derived products clinically, different degrees of heterologous rejection reactions and late calcification will occur, which may lead to secondary infections clinically and greatly increase the risk of microbial invasion. The artificial skin made of an electrospun membrane as the main structure supplemented with autologous living cells, although overcoming some biocompatibility problems, still has predictable rejection reactions after being implanted into the human body because its main structural framework is still an electrospun membrane material made of polymer materials. And its production process is complex, and the risk of bacterial contamination during the production process is very high; and autologous adipose stem cells need to be used, and the time period from obtaining adipose tissue by liposuction surgery to extracting stem cells and then making artificial skin is more than one month, often resulting in the problem that clinical patients need to wait for a long time, and it will also greatly increase the risk of clinical infection, so its usability is very poor.

[0004] Therefore, it is of great significance to solve the problems of heterologous protein rejection and calcification in the animal-derived acellular matrix technology in the related art, as well as a series of problems existing in the electrospinning combined with autologous cell technology, such as the implantation and residual rejection of polymer materials in the body, the long waiting period for patients, the need for secondary liposuction surgery, high cost, and the possibility of repeated production in case of one failure, and to provide an artificial skin without any other xenogeneic polymer residues, which can be mass-produced in a standardized manner and has strong clinical availability in a timely manner. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides an artificial skin, a preparation method and an application thereof, aiming to solve the problems of heterologous protein rejection and calcification in the animal-derived acellular matrix technology in the related art of artificial skin at present, as well as a series of problems existing in the electrospinning combined with autologous cell technology, such as the implantation and residual rejection of polymer materials in the body, the long waiting period for patients, the need for secondary liposuction surgery, high cost, and the possibility of repeated production in case of one failure.

[0006] An embodiment of the first aspect of the present invention provides an artificial skin, which is obtained by co-culturing human-derived dermal cells with a fiber mesh of a biodegradable polymer material.

[0007] The artificial skin according to the embodiment of the first aspect of the present invention has at least the following beneficial effects: The artificial skin provided by the present invention is obtained by co-culturing human-derived dermal cells with a fiber mesh of a biodegradable polymer material. The present invention uses allogeneic human-derived cells to produce a regenerated artificial skin mainly made of human-derived collagen through tissue engineering, and then removes immunogenic substances such as heterologous DNA through an acellular process. The remaining components of the artificial cells are very close to those of human autologous skin (for example, including human-derived collagen, elastin, collagen fibers and other structural proteins), and there is no residue of any other xenogeneic polymer, and the biocompatibility is excellent. The fiber mesh used in the co-culture is woven from a polymer material with good biocompatibility and complete degradability, and can be automatically degraded in the later stage of culture, and no polymer material will remain in the harvested product, avoiding the rejection reaction when the artificial cells are implanted into the human body in the later stage. Moreover, the above artificial skin can be mass-produced in a standardized manner, with good uniformity between batches, and there are no problems of heterologous protein rejection and calcification; it is rich in collagen, the skin is tough, and the biocompatibility is good; the production cycle is controllable, the clinical availability in a timely manner is strong, and the price is low. The present invention completely changes the existing methods and paths for obtaining artificial cells, and the obtained products can simulate the composition of human autologous skin to the greatest extent, providing another idea for developing more paths for obtaining artificial skin, promoting the research on skin transplantation and skin injury processes, and having extremely high scientific research significance and clinical application value.

[0008] In some embodiments of the present invention, the thickness of the artificial skin is 0.5 to 1.5 mm.

[0009] In some embodiments of the present invention, the content of collagen in the artificial skin is greater than 50%, preferably greater than 70%. The composition of human skin consists of the epidermis, dermis, and subcutaneous tissue, and the most important structure among them is the skin cells in the dermis and the collagen secreted by them, which play the most important elastic and barrier repair roles in the skin tissue. The artificial skin provided by the present invention can simulate the composition of human autologous skin to the greatest extent, mainly composed of collagen, elastin, and collagen fibers, etc. The content of collagen is greater than 50%, even up to 70%, and the artificial skin is tough. Moreover, the above-mentioned collagen is human-derived collagen, which conforms to the structure composition of human autologous skin, has excellent biocompatibility, and will not cause heterologous rejection or late calcification when used in clinical products, avoiding the risk of secondary infection in clinical practice.

[0010] In some embodiments of the present invention, the dermal cells are isolated from the umbilical cord. Specifically, the dermal cells can be primary cells isolated from the umbilical cord, or passage cells obtained after the primary cells are cultured and stably proliferated. The present invention isolates dermal cells from the umbilical cord discarded after neonatal delivery through a compliant ethical method, and performs culture and stable proliferation to establish a library. The above approach can not only avoid ethical risks but also obtain a sufficient number of cells without the problem of insufficient donors.

[0011] In some embodiments of the present invention, the material of the degradable polymer material fiber mesh includes at least one of polycaprolactone (PCL), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polyhydroxyalkanoates (PHA), and collagen. The fiber mesh is woven from the above-mentioned completely degradable polymers, and has good biocompatibility; moreover, the natural degradation period of the above materials is 1.5 to 2 months, and it can be automatically degraded in the later stage of artificial skin culture, without leaving any polymer materials in the harvested products, avoiding rejection reactions when artificial cells are implanted into the human body later.

[0012] In some embodiments of the present invention, the thickness of the degradable polymer material fiber mesh is 0.5 to 1.6 mm.

[0013] In some embodiments of the present invention, the density of the degradable polymer material fiber mesh is 15 to 30 g / m 2 。

[0014] An embodiment of the second aspect of the present invention provides a method for preparing an artificial skin, including the steps:

[0015] S100. Weave a biodegradable polymer material into a biodegradable polymer fiber mesh, and fix the fiber mesh in a cell culture container after sterilization treatment;

[0016] S200. Inoculate human-derived dermal cells on the fiber mesh, and add a dermal cell culture medium to culture for a predetermined time period one;

[0017] S300. Replace the dermal cell culture medium with a secretory medium, and continue to culture for a predetermined time period two to obtain a sheet of skin tissue;

[0018] S400. Perform decellularization treatment on the sheet of skin tissue to obtain the artificial skin;

[0019] Wherein, the secretory medium includes a dermal cell culture medium and additives, and the additives include human serum albumin (HSA), human platelet lysate (HPL), and transforming growth factor β1 (TGF-β1).

[0020] According to the preparation method of the second aspect embodiment of the present invention, it has at least the following beneficial effects: Through tissue engineering and regenerative medicine methods, the present invention inoculates human-derived dermal cells isolated in vitro onto a loose fiber mesh woven from a biodegradable polymer material with good biocompatibility, and after culturing for a period of time, a tissue containing cells and matrix such as collagen is formed, and then decellularization treatment is performed to obtain tissue-engineered artificial skin. The present invention uses different cell culture media at different culture stages to achieve the purpose of promoting cell proliferation in the early stage and promoting cells to secrete structural proteins such as collagen in the later stage. The harvested artificial skin is rich in collagen, has toughness, and has good biocompatibility. The present invention uses the decellularization process to remove immunogenic substances such as heterologous DNA. The remaining components of the artificial skin are very close to those of human autologous skin (for example, containing structural proteins such as human-derived collagen, elastin, and collagen fibers), and there is no residue of any other foreign polymers, and the biocompatibility is excellent. The fiber mesh inoculated with dermal cells is woven from a biodegradable polymer material with good biocompatibility, and can be automatically degraded in the later stage of culture, without leaving any polymer material in the harvested product, avoiding rejection reactions when the artificial skin is implanted into the human body later. Moreover, the above preparation method can achieve standardized production, the production cycle is controllable, the clinical availability of the product is strong, and the price is low. The preparation method of the present invention changes the existing ways and paths of obtaining artificial skin, and the obtained product can simulate the composition of human autologous skin to the greatest extent, providing another idea for developing more ways to obtain artificial skin, and can promote the research of skin transplantation and skin injury processes, and has extremely high scientific research significance and clinical application value.

[0021] In some embodiments of the present invention, the thickness of the artificial skin is 0.5 to 1.5 mm.

[0022] In some embodiments of the present invention, the content of collagen in the artificial skin is greater than 50%, preferably greater than 70%. The composition of human skin consists of the epidermis, dermis and subcutaneous tissue, and the most important structure among them is the skin cells in the dermis and the collagen secreted by them, which play the most important elastic role and barrier repair role in the skin tissue. The artificial skin provided by the present invention can simulate the composition of human autologous skin to the greatest extent, and is mainly composed of collagen, elastin, collagen fibers, etc. The content of collagen is greater than 50%, even up to 70%, and the artificial skin is tough. Moreover, the above-mentioned collagen is human-derived collagen, which conforms to the structure composition of human autologous skin, has excellent biocompatibility, and will not cause heterologous rejection reaction or late calcification when used in clinical products, avoiding the risk of secondary infection in clinical practice.

[0023] In some embodiments of the present invention, the material of the degradable polymer material fiber mesh includes at least one of polycaprolactone (PCL), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polyhydroxyalkanoates (PHA), and collagen. The fiber mesh is woven from the above-mentioned completely degradable polymers and has good biocompatibility; moreover, the natural degradation period of the above materials is 1.5 to 2 months, and it can be automatically degraded in the later stage of artificial skin culture, without leaving any polymer materials in the harvested products, avoiding rejection reactions when artificial cells are implanted into the human body later.

[0024] In some embodiments of the present invention, the thickness of the degradable polymer material fiber mesh is 0.5 to 1.6 mm.

[0025] In some embodiments of the present invention, the density of the degradable polymer material fiber mesh is 15 to 30 g / m 2 。

[0026] In some embodiments of the present invention, in step S100, after irradiating and sterilizing the fiber mesh, it is aseptically unpacked in a Class A biosafety cabinet, and then fixed in a 150 mm cell culture dish with a high-viscosity collagen or gelatin solution, and air-dried overnight in the safety cabinet for standby.

[0027] In some embodiments of the present invention, the dermal cells are isolated from the umbilical cord. Specifically, the dermal cells can be primary cells isolated from the umbilical cord, or subcultured cells obtained after culturing and stable proliferation of the primary cells. In the present invention, dermal cells are isolated from the umbilical cord discarded after neonatal delivery through an ethical and compliant approach, and then cultured and stably proliferated to establish a cell bank. The above approach can not only avoid ethical risks but also obtain a sufficient number of cells, without the problem of insufficient donors.

[0028] In some embodiments of the present invention, the first predetermined time is 5 to 7 days.

[0029] In some embodiments of the present invention, in step S200, the human-derived dermal cells isolated from the umbilical cord are inoculated onto the fiber mesh at a density of 1 million to 5 million cells / mL. Then, a dermal cell culture medium is added to cover the fiber mesh, and the liquid level of the medium is about 1 cm higher than the fiber mesh. Then, it is placed in an incubator with a 5% CO2 concentration and 37 °C for 5 to 7 days. During this period, the cells rapidly proliferate and arrange internally on the fiber mesh, and begin to form a very thin cell mass covering the fiber mesh.

[0030] In some embodiments of the present invention, the second predetermined time is 3 to 6 weeks, preferably 4 to 6 weeks.

[0031] In some embodiments of the present invention, when culturing in the secretory medium, the medium is changed every 3 to 7 days, preferably every 3 to 5 days, and more preferably every 3 days.

[0032] In some embodiments of the present invention, based on the final concentration in the medium, the secretory medium includes at least one of (a1) to (a3):

[0033] (a1) The final concentration of human serum albumin (HSA) is 1 to 5 mg / mL;

[0034] (a2) The final concentration of human platelet lysate (HPL) is 1 (v / v)% to 5 (v / v)%;

[0035] (a3) The final concentration of transforming growth factor β1 (TGF-β1) is 5 to 15 ng / mL.

[0036] In some embodiments of the present invention, the dermal cell culture medium includes DMEM, DMEM / F-12, or RPMI-1640, but is not limited thereto. It can also be a medium with similar components or a commercial dermal cell culture medium.

[0037] In some embodiments of the present invention, the dermal cell culture medium is supplemented with auxiliary components.

[0038] In some embodiments of the present invention, the auxiliary components include at least one of fetal bovine serum (FBS), non-essential amino acids (NEAA), trace elements, and double antibiotics (penicillin / streptomycin).

[0039] In the present invention, different cell culture media are added at different culture stages to achieve the purpose of promoting cell proliferation in the early stage and promoting the secretion of collagen and elastin by cells in the later stage. After culturing in the dermal cell culture medium for 5 to 7 days, the cells rapidly proliferate and arrange internally on the fiber mesh, and begin to form a very thin cell mass covering the fiber mesh. At this time, the culture medium system is changed to a secretory medium, and the composition of the secretory medium is: the dermal cell culture medium is added with nutritional factors required for collagen production, including human serum albumin (concentration of 1 to 5 mg / mL), human platelet lysate (concentration of 1 (v / v)% to 5 (v / v)%), and TGF-β1 (concentration of 5 to 15 ng / mL). Culturing in this medium for 3 to 6 weeks, the culture medium is changed every 3 to 7 days. It can be observed that the polymer material fiber mesh gradually degrades in the first to second week, and the degraded part becomes the extracellular matrix such as collagen secreted by the cells, forming a white sheet-like skin tissue composed of cell masses, collagen and other extracellular matrices, and a small amount of fiber mesh material (content below 10%). After decellularization treatment, tissue-engineered artificial skin can be obtained, and the thickness of this artificial skin is 0.5 to 1.5 mm.

[0040] An embodiment of the third aspect of the present invention provides the application of the above artificial skin or the preparation method of the above artificial skin in at least one of (b1) to (b5):

[0041] (b1) Preparing a therapeutic or prophylactic drug for skin diseases;

[0042] (b2) Preparing a product for skin transplantation;

[0043] (b3) Screening a therapeutic or prophylactic drug for skin diseases;

[0044] (b4) Constructing a skin injury healing model;

[0045] (b5) Studying the molecular mechanism in the process of skin damage caused by diabetes and its complications;

[0046] The application is for non-diagnostic or therapeutic purposes.

[0047] An embodiment of the fourth aspect of the present invention provides at least one product of the following (c1) to (c3):

[0048] (c1) Therapeutic or prophylactic drugs for skin diseases;

[0049] (c2) Products for skin transplantation;

[0050] (c3) Reagents or kits for skin diseases;

[0051] The product contains the artificial skin described above or the artificial skin obtained by the above preparation method.

[0052] Other features and advantages of the present invention will be described in the subsequent specification, and in part will be obvious from the specification, or will be understood by implementing the present invention. The objectives and other advantages of the present invention can be realized and obtained through the structures specifically pointed out in the specification, claims, and drawings. Brief Description of the Drawings

[0053] Figure 1 Schematic diagram of the co - culture of cells and fiber mesh provided in Example 1 of the present invention;

[0054] Figure 2 Schematic diagram of the artificial skin provided in Example 1 of the present invention;

[0055] Figure 3 Schematic diagram of the HE staining result of the artificial skin provided in Example 1 of the present invention;

[0056] Figure 4 Schematic diagram of the MASSON staining result of the artificial skin provided in Example 1 of the present invention;

[0057] Figure 5 Schematic diagram of the artificial skin provided in Example 2 of the present invention;

[0058] Figure 6 Schematic diagram of the artificial skin provided in Example 3 of the present invention;

[0059] Figure 7 Schematic diagram of the artificial skin provided in Example 4 of the present invention;

[0060] Figure 8 Schematic diagram of the artificial skin provided in Comparative Example 1 of the present invention;

[0061] Figure 9 Schematic diagram of the artificial skin provided in Comparative Example 2 of the present invention. Detailed Description of the Invention

[0062] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0063] In the description of the present invention, the descriptions referring to terms such as "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0064] In the description of the present invention, unless otherwise specified, the numerical range "a~b" represents the abbreviated representation of any real number combination between a and b, where both a and b are real numbers. Unless otherwise specified, each reaction or operation step can be carried out in sequence or not in sequence. Preferably, the reaction method in the present invention is carried out in sequence.

[0065] For the experimental methods without specific conditions indicated in the following embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in this embodiment are, unless otherwise specified, reagents and materials obtained from commercial channels.

[0066] Example 1

[0067] This example provides an artificial skin with a thickness of 0.6 mm.

[0068] The preparation method is as follows:

[0069] (1) Prepare a degradable polymer material fiber mesh

[0070] Prepare the material for the degradable polymer fiber mesh. The material is poly(lactic-co-glycolic acid) (PLGA). Process the material into a fiber mesh shape. The wall thickness of the fiber mesh is 0.5~0.7 mm, and the gram weight is 18 g / m 2 .

[0071] (2) Sterilize the fiber mesh by irradiation and unpack it aseptically in a biosafety cabinet. Then soak it in a 10 mg / mL collagen solution in a 150 mm cell culture dish, and then air-dry it overnight in the biosafety cabinet so that the fiber mesh adheres to the bottom of the culture dish.

[0072] (3) The dermal cells isolated from discarded umbilical cords of infants (the acquisition method complies with relevant ethical regulations) are inoculated onto the fiber mesh air-dried in step (2) at a density of 3 million / mL. Then, complete medium for dermal cells (purchased from Procell, product number CM-H103) is added to cover the fiber mesh, with the liquid level about 1 cm higher. The culture dish is placed in an incubator with a 5% CO2 concentration and at 37 °C for 5 days.

[0073] During this period, the cells rapidly proliferate and arrange internally on the fiber mesh, and begin to form a very thin cell mass covering the fiber mesh. The picture of the co-culture of cells and the fiber mesh is as Figure 1 .

[0074] (4) The culture medium system is changed to a secretory medium, and the composition of the secretory medium is: complete medium for dermal cells + 2.5 mg / mL human serum albumin + 3 (v / v)% human platelet lysate + 10 ng / mL TGF-β1. The cells are cultured in this medium for 6 weeks, and the medium is changed every 3 days.

[0075] It can be observed that the high-molecular fiber mesh material gradually degrades starting from the second week. The degraded part becomes extracellular matrix such as collagen secreted by the cells, forming a white sheet-like skin tissue composed of cell masses, collagen and other extracellular matrix, and a small amount of fiber mesh material (about 5%).

[0076] (5) The obtained sheet-like skin tissue is subjected to decellularization treatment to obtain tissue-engineered artificial skin, and the thickness of this artificial skin is 0.6 mm.

[0077] The picture of the artificial skin obtained in this example is as Figure 2 shown. Then, the harvested artificial skin was respectively subjected to HE staining (as Figure 3 ) and MASSON staining (as Figure 4 ) of transverse sections for characterization. It can be seen that the distribution of cells in the skin is very uniform, and the cells are densely surrounded by collagen fibers.

[0078] After that, the collagen content in the artificial skin was measured. The method was as follows: The skin tissue was minced and digested with collagenase to obtain a collagen solution; since hydroxyproline is a specific amino acid of collagen, by measuring the hydroxyproline content in the solution, the proportion of collagen in it can be obtained after conversion.

[0079] After measurement, the collagen content in the artificial skin of this example was 70.8%.

[0080] Example 2

[0081] This embodiment provides an artificial skin, which is only different from that of Embodiment 1 in that the wall thickness of the fiber mesh formed by the degradable polymer material is about 1.5 mm thick. The rest are the same as those of Embodiment 1 and will not be elaborated here.

[0082] The thickness of the artificial skin obtained in this embodiment is 1.5 mm, and the picture is as Figure 5 shown.

[0083] It was measured (by the same method as in Embodiment 1) that the content of collagen in the artificial skin of this embodiment is 71.2%.

[0084] Embodiment 3

[0085] This embodiment provides an artificial skin, which is only different from that of Embodiment 1 in that the artificial skin samples are harvested after being cultured in the secretory medium for 3 weeks. The rest are the same as those of Embodiment 1 and will not be elaborated here.

[0086] The picture of the artificial skin obtained in this embodiment is as Figure 6 shown. At the same time, it was observed during the harvest that a complete sheet of skin could also be formed in this embodiment, but its toughness was slightly poor, and there were a few undegraded fiber materials remaining in the skin.

[0087] It was measured (by the same method as in Embodiment 1) that the content of collagen in the artificial skin of this embodiment is 62.5%.

[0088] Embodiment 4

[0089] This embodiment provides an artificial skin, which is only different from that of Embodiment 1 in that when culturing in the secretory medium, the time for changing the medium is changed from 3 days to 7 days, and the number of times of changing the medium is reduced from 14 times to 6 times (the culture period is the same 6 weeks). The rest are the same as those of Embodiment 1 and will not be elaborated here.

[0090] The picture of the artificial skin obtained in this embodiment is as Figure 7 shown. At the same time, by observing the culture process, it was found that the edges of the artificial cells began to shrink at the 4th week, and a complete sheet of skin could also be formed at the harvest, but its toughness was poor. It may be that the cells received insufficient nutrients, resulting in a poor development state.

[0091] It was measured (by the same method as in Embodiment 1) that the content of collagen in the artificial skin of this embodiment is 58.7%.

[0092] Comparative Example 1

[0093] This comparative example provides an artificial skin, which is only different from that of Embodiment 1 in that the secretory medium is not used, and the complete medium for dermal cells is used throughout the culture process. The rest are the same as those of Embodiment 1 and will not be elaborated here.

[0094] The picture of the artificial skin obtained in this comparative example is as Figure 8 shown. Although a relatively complete skin tissue was also formed after the cultivation, the formed tissue had very poor toughness and was very easy to have holes.

[0095] It was measured (by the same method as in Example 1) that the content of collagen in the artificial skin of this comparative example was 36.4%, and the collagen content was much lower than that in Example 1.

[0096] Comparative Example 2

[0097] This comparative example provides an artificial skin, and the difference from Example 1 is only that: the artificial skin sample was harvested after culturing in the secretory medium for 2 weeks. The rest are the same as in Example 1 and will not be elaborated here.

[0098] The picture of the artificial skin obtained in this comparative example is as Figure 9 shown. When observing at the time of harvest, the degradable material was still clearly visible. After decellularization treatment, the sample was completely loose and partially aggregated into small clusters.

[0099] It was measured (by the same method as in Example 1) that the content of collagen in the artificial skin of this comparative example was 26.4%, and the collagen content was much lower than that in Example 1.

[0100] Comparative Example 3

[0101] This comparative example provides an artificial skin, and the difference from Example 1 is only that: the co-culture was not carried out using the degradable polymer fiber mesh material. The rest are the same as in Example 1 and will not be elaborated here.

[0102] At the final harvest, no complete tissue was formed, and it was basically fragmented tissue. After decellularization treatment, the sample showed a loose and small cluster morphology.

[0103] It was measured (by the same method as in Example 1) that the content of collagen in the artificial skin of this comparative example was 35.7%, and the collagen content was much lower than that in Example 1.

[0104] The above is a specific description of the preferred embodiments of the present application, but the present application is not limited to the above embodiments. Those skilled in the art can also make various equivalent deformations or substitutions without departing from the spirit of the present application, and these equivalent deformations or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. An artificial skin, characterized in that, The artificial skin is obtained by co - culturing human - derived dermal cells with a biodegradable polymer material fiber mesh.

2. The artificial skin according to claim 1, characterized in that, The thickness of the artificial skin is 0.5 - 1.5 mm; and / or, the collagen content in the artificial skin is greater than 50%.

3. The artificial skin according to claim 1, characterized in that, The dermal cells are isolated from the umbilical cord; and / or, the material of the biodegradable polymer material fiber mesh includes at least one of polycaprolactone, polyethylene glycol, poly (lactic - co - glycolic acid), polyhydroxyalkanoate, and collagen; and / or, the thickness of the biodegradable polymer material fiber mesh is 0.5 - 1.6 mm; And / or, the density of the degradable polymer material fiber web is 15-30 g / m 2 .

4. A method for preparing artificial skin, characterized in that, It includes the steps: Weaving a biodegradable polymer material to form a biodegradable polymer material fiber mesh, and fixing the fiber mesh in a cell culture container after sterilization treatment; Inoculating human - derived dermal cells on the fiber mesh, and adding a dermal cell culture medium to culture for a predetermined time one; Replacing the dermal cell culture medium with a secretory medium, and continuing to culture for a predetermined time two to obtain a sheet - like skin tissue; Performing decellularization treatment on the sheet - like skin tissue to obtain the artificial skin; Wherein, the secretory medium includes a dermal cell culture medium and additives, and the additives include human serum albumin, human platelet lysate, and transforming growth factor β1.

5. The preparation method of the artificial skin according to claim 4, characterized in that, The thickness of the artificial skin is 0.5 - 1.5 mm; and / or, the collagen content in the artificial skin is greater than 50%.

6. The preparation method of the artificial skin according to claim 4, characterized in that, The dermal cells are isolated from the umbilical cord; and / or, the biodegradable polymer material includes at least one of polycaprolactone, polyethylene glycol, poly (lactic - co - glycolic acid), polyhydroxyalkanoate, and collagen; and / or, the thickness of the biodegradable polymer material fiber mesh is 0.5 - 1.6 mm; And / or, the density of the degradable polymer material fiber web is 15 to 30 g / m 2 .

7. The preparation method of the artificial skin according to claim 4, characterized in that The predetermined time one is 5 - 7 days; and / or, the predetermined time two is 3 - 6 weeks; and / or, when culturing in the secretory medium, the medium is changed every 3 - 7 days.

8. The preparation method of the artificial skin according to claim 4, characterized in that, Based on the final concentration in the medium, the secretory medium includes at least one of (a1)-(a3): (a1) The final concentration of human serum albumin is 1 - 5 mg / mL; (a2) The final concentration of human platelet lysate is 1 (v / v)% - 5 (v / v)%; (a3) The final concentration of transforming growth factor β1 is 5 - 15 ng / mL; and / or, the dermal cell culture medium includes DMEM, DMEM / F - 12, or RPMI - 1640; and / or, the dermal cell culture medium is added with auxiliary components, and the auxiliary components include at least one of fetal bovine serum, non - essential amino acids, trace elements, and double antibodies.

9. The application of the artificial skin according to any one of claims 1 - 3 or the preparation method of the artificial skin according to any one of claims 4 - 8 in at least one of (b1)-(b5): (b1) Preparing a therapeutic or prophylactic drug for skin diseases; (b2) Preparing a product for skin transplantation; (b3) Screening a therapeutic or prophylactic drug for skin diseases; (b4) Constructing a skin injury healing model; (b5) Studying the molecular mechanism in the process of skin injury caused by diabetes and its complications; The application is for non - diagnostic or therapeutic purposes.

10. At least one of the following products (c1) to (c3): (c1) Therapeutic or prophylactic drugs for skin diseases; (c2) Products for skin transplantation; (c3) Reagents or reagent kits for skin diseases; The product contains the artificial skin described in any one of claims 1-3 or the artificial skin obtained by the preparation method described in any one of claims 4-8.

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