Early tissue engineering skin for wound treatment and preparation method thereof
阅读说明：本技术 一种用于创面治疗的早期组织工程皮肤及其制备方法 (Early tissue engineering skin for wound treatment and preparation method thereof ) 是由 吴训伟 张群 白福响 温洁 冷雪 于 2020-08-05 设计创作，主要内容包括：本发明属于生物医学技术领域,具体涉及一种用于创面治疗的早期组织工程皮肤。研究发现：皮肤干细胞构建的短期培养的双层组织工程皮肤含有更多的活性细胞,凋亡通路激活较少,移植后的皮肤结构更好,还有皮脂腺和毛囊产生。研究表明,短期培养的双层组织工程皮肤在体内移植后能产生更好的皮肤结构,临床上可能有利于伤口更好的愈合。另外,短期培养的组织工程皮肤可大大缩短制备时间及成本,本研究为其临床应用提供了充分的理论及研究依据。(The invention belongs to the technical field of biomedicine, and particularly relates to early tissue engineering skin for wound treatment. The research finds that: the short-term cultured double-layer tissue engineering skin constructed by the skin stem cells contains more active cells, the activation of an apoptosis pathway is less, the structure of the transplanted skin is better, and sebaceous glands and hair follicles are generated. Research shows that the double-layer tissue engineering skin cultured in a short period can generate a better skin structure after being transplanted in vivo, and is possibly favorable for better healing of wounds clinically. In addition, the tissue engineering skin cultured in a short period can greatly shorten the preparation time and cost, and the research provides sufficient theory and research basis for clinical application of the tissue engineering skin.)
1. The application of the early tissue engineering skin in wound treatment is characterized in that the culture time of the early tissue engineering skin is 5-9 days.
2. The use of claim 1, wherein the early stage tissue engineered skin is a double layer tissue engineered skin.
3. The use of claim 2, wherein the bilayer of tissue engineered skin comprises: collagen layer and epidermal layer.
4. The use of claim 3, wherein the collagen layer comprises: a cell-free collagen layer and a dermal cell collagen layer.
5. The use of claim 1, wherein the early stage tissue engineered skin is prepared by the following method:
preparing a cell-free collagen layer;
constructing a collagen matrix containing dermal stem cells above the acellular collagen layer;
establishing an epidermal cell layer;
and culturing the tissue engineering skin for 5-9 days.
6. The use according to claim 5, wherein the specific steps for preparing the acellular collagen layer are: mixing MEM, bovine type I collagen, FBS, L-glutamine and DMEM, adjusting the pH value to 7.2-7.3, and uniformly mixing to form a mixture;
adding the mixture into a culture plate, and adding 5-5.5% of CO at 37-37.5 DEG C2The mixture was gelled by incubation in an incubator.
7. The use according to claim 5, wherein the specific steps for constructing the collagen matrix containing dermal stem cells are: mixing MEM, bovine type I collagen, FBS, L-glutamine and DMEM, adjusting pH to 7.2, adding fibroblast, and mixing; adding the mixture to a gelled acellular collagen matrix, and adding 5-5.5% CO at 37-37.5 deg.C2The culture box is used for culturing to ensure that the cell matrix is completely gelatinized, and the fibroblast is added for continuous culture to ensure that the collagen matrix contracts and tends to be stable.
8. The use of claim 5, wherein the particular step of establishing the epidermal cell layer is: resuspending the epidermal stem cells in DMEM, uniformly and slowly inoculating the cells on contracted collagen gel, and then carrying out 5-5.5% CO treatment at 37-37.5 DEG C2The culture box is incubated to ensure that the epidermal stem cells are completely adhered and are attached to the matrix to form a confluent cell monolayer.
9. The early tissue engineering skin is characterized in that the culture time of the early tissue engineering skin is 5-9 days.
10. A method for preparing early tissue engineering skin is characterized by comprising the following steps:
preparing a cell-free collagen layer;
constructing a collagen matrix containing dermal stem cells above the acellular collagen layer;
establishing an epidermal cell layer;
and culturing the tissue engineering skin for 5-9 days.
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to early tissue engineering skin for wound treatment.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Skin damage due to accidents, diseases, chronic wounds, acute wounds, burns, etc. can damage the skin barrier, and can seriously lead to permanent disability or death of the injured person. Skin grafting is the most promising method for treating large area wounds, however, for patients with severe burns, autologous skin is very limited and autologous transplantation is not the optimal treatment option. For allograft transplantation, the foreign tissue also presents potential immune rejection problems. The use of tissue engineering skin substitutes has created new promise for patients in the treatment of acute and chronic skin wounds after the formal introduction of tissue engineering in the late 80 s of the 20 th century. A common method of preparing tissue engineered skin is to implant cells, including epidermal keratinocytes, dermal fibroblasts, and/or stem cells, on a biodegradable scaffold. In recent decades, human tissue engineered skin has evolved from a simple epidermal replacement to a complex full-thickness skin with a double-layered structure of epidermis and dermis. The double-layer tissue engineering skin constructed from autologous cell sources is very suitable for clinical use because it not only contains the full-layer structure of the skin, but also can form permanent fusion without immunological rejection after transplantation.
However, currently, there are still many limitations in the application and treatment of tissue engineering skin: on the one hand, regenerating skin with full function (protection, regulation and feel) remains entirely challenging. Tissue engineered skin has been able to achieve regeneration of skin appendages, including capillary networks, sensory innervation, adipose tissue and pigmentation, through condition control, but nevertheless, has failed to fully reconstitute intact skin function, particularly functional hair follicle and sweat gland regeneration. An important reason why cultured cells cannot regenerate hair follicles in vitro is that the cells lose trichogenicity during the culture process. For regenerative medicine research, it is important to develop an optimal system to maintain the regenerative capacity of cells in vitro. On the other hand, the construction of the full-thickness tissue engineering skin requires the culture of a large number of different types of cells, and the culture process is complex and time-consuming. The inventor finds that: when the double-layer tissue engineering skin is constructed by using the prior art, the epidermal and dermal cells usually need to be amplified and cultured for 2 to 4 weeks to obtain enough quantity, and the basic structure of the tissue engineering skin needs to be cultured for 3 weeks or more after being constructed. In clinical treatment, time consumption is a major limiting factor for tissue engineering skin applications.
Disclosure of Invention
In order to overcome the problems, the invention provides the tissue engineering skin cultured in a short term for wound treatment, and after the tissue engineering skin is transplanted, the tissue engineering skin cultured in a double-layer tissue engineering skin (hereinafter referred to as early-stage tissue engineering skin) cultured in a short term by adopting the technology has better transplanting effect than the tissue engineering skin (hereinafter referred to as late-stage tissue engineering skin) cultured in a longer term. The research finds that: the stratified epidermis with a normal structure is formed after the early and late tissue engineering skin transplantation, but the proliferation marker Ki-67 and the epidermal stem cell marker p63 in the epidermis formed by the early tissue engineering skin are higher in level. Transplantation of early tissue-engineered skin not only has a larger dermal thickness, a larger dermal cell density, and a larger number of neovessels, but also hair follicle formation was observed only in the skin formed after transplantation of early tissue-engineered skin. Compared with the later-stage tissue engineering skin, the early-stage tissue engineering skin expresses high-level p63, but the expression level of genes involved in activating an apoptosis pathway is lower.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides application of early tissue engineering skin in wound treatment, wherein the culture time of the early tissue engineering skin is 5-9 days.
The research of the invention finds that: the early double-layer tissue engineering skin constructed by the skin stem cells contains more active cells, the activation of an apoptosis pathway is less, the structure of the skin after transplantation is better, and sebaceous glands and hair follicles are generated. Research shows that the double-layer tissue engineering skin cultured in a short period can generate a better skin structure after being transplanted in vivo, and is possibly favorable for better healing of wounds clinically. In addition, the tissue engineering skin cultured in a short period can greatly shorten the preparation time and cost, and the research provides sufficient theory and research basis for clinical application of the tissue engineering skin.
According to a second aspect of the invention, the early tissue engineering skin is provided, and the culture time of the early tissue engineering skin is 5-9 days.
The invention shortens the culture time required by the construction of the double-layer tissue engineering skin, prepares the tissue engineering skin with complete functions in a short time, and has important significance for the clinical application of the tissue engineering skin.
In a third aspect of the present invention, there is provided a method for preparing early tissue engineering skin, comprising:
preparing a cell-free collagen layer;
constructing a collagen matrix containing dermal stem cells above the acellular collagen layer;
establishing an epidermal cell layer;
and culturing the tissue engineering skin for 5-9 days.
The method can simply and quickly separate the human skin epidermal cells and the dermal stem cells, shortens the culture time required by the expansion of the skin stem cells, and still maintains the potential of the human skin cells for generating full-thickness skin with hair follicles after the human skin cells are expanded in a culture system.
The invention has the beneficial effects that:
(1) the research of the invention finds that: the early double-layer tissue engineering skin transplantation effect after transplantation is better. The stratified epidermis with normal structure is formed after the transplantation of the early and late tissue engineering skins, but the proliferation marker Ki-67 and the epidermal stem cell marker p63 in the epidermis formed by the early tissue engineering skins are higher in level. Transplantation of early tissue-engineered skin not only has a larger dermal thickness, a larger dermal cell density, and a larger number of neovessels, but also hair follicle formation was observed only in the skin after transplantation of early tissue-engineered skin. Compared with the later-stage tissue engineering skin, the early-stage tissue engineering skin expresses high-level p63, but the expression level of genes involved in activating an apoptosis pathway is lower.
(2) The method has the advantages of short culture time (5-9 days), simple method, strong practicability and easy popularization.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a flow chart of the preparation of early and late tissue engineered skin in example 1 of the present invention. a, establishing a layer of cell-free collagen in a transwell of a six-well plate. b, constructing a collagen matrix containing dermal stem cells on the upper part of the acellular collagen layer. c, the collagen matrix shrinks after being cultured in the incubator for 4 days. d, after the stroma is completely contracted and stabilized at day 4, adult epidermal stem cells are added to the stromal upper layer. e, adding an epidermization medium to each chamber. The medium was changed every 2 days until day 11. f, from day 12 onward, change to gas-liquid interface epidermization medium: the chamber was filled with medium and the interior of the chamber was kept dry. Tissue engineered skin was collected at four different time points during the culture, with 5-day and 9-day groups being early tissue engineered skin groups, and 14-day and 21-day groups being late tissue engineered skin groups.
FIG. 2 is a graph showing the overall effect of newly formed skin after early and late tissue engineering skin grafting in example 1 of the present invention. The area of successful transplantation showed marked pigmentation, the white dotted line indicating the border between the skin of the host mouse and the pigmented area of human transplanted skin, and early tissue engineered skin was able to produce a larger pigmented area after transplantation than later transplants. bar 5mm, p <0.05, p <0.01, n 3.
Fig. 3 is a comparison of the epidermis layer of newly formed skin after early and late tissue engineering skin grafting in example 1 of the present invention. All groups were able to form normal epidermal structures, but more Ki-67 and p 63-positively expressed cells were present in the epidermis formed after early tissue engineering skin transplantation compared to late tissue engineering skin. bar 50 μm, p <0.05, p <0.01, p <0.001, n 3.
FIG. 4 is a comparison of the dermis layer, blood vessels and appendages of newly formed skin after early and late tissue engineering skin grafts in example 1 of the present invention. The thickness of the dermis formed by the tissue engineering skin early transplantation group is larger than that of the dermis formed by the late group, the density of the dermal fibroblast is higher, and the number of the formed dermal capillaries is more than that of the dermis formed by the late group. The formation of mature hair follicles and sebaceous glands was observed only in the transplanted group of early tissue engineered skin. bar 50 μm, p <0.05, p <0.01, p <0.001, n 3.
FIG. 5 shows the differences in protein expression and molecular levels before early and late tissue engineering skin grafting in example 1 of the present invention. Compared with other groups, the tissue engineering skin group expresses the highest level of p63 in the 5-day group, the expression of p63 is gradually reduced along with the prolonging of the in vitro culture time, and the expression of activated Caspase-3 is increased along with the prolonging of the culture time. The analysis of qRT-PCR confirmed the analysis results of western blot: expression was decreased in the late group of p63, Bax was increased in the late group, and expression of the anti-apoptotic gene Bcl-2 was decreased in the late group. P <0.05, p <0.01, p <0.001, n-3.
FIG. 6 shows the basic structure of tissue-engineered skin cultured for a short period in example 1 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The invention provides application of early tissue engineering skin in wound treatment, wherein the culture time of the early tissue engineering skin is 5-9 days.
In some embodiments, the early stage tissue engineered skin is a double layer tissue engineered skin.
In some embodiments, the bilayer tissue engineered skin comprises: collagen layer and epidermal layer.
In some embodiments, the collagen layer comprises: a cell-free collagen layer and a dermal cell collagen layer.
In some embodiments, the early tissue engineered skin is prepared as follows:
preparing a cell-free collagen layer;
constructing a collagen matrix containing dermal stem cells above the acellular collagen layer;
establishing an epidermal cell layer;
and culturing the tissue engineering skin for 5-9 days.
In some embodiments, the specific steps for preparing the cell-free collagen layer are: mixing MEM, bovine type I collagen, FBS, L-glutamine and DMEM, adjusting the pH value to 7.2-7.3, and uniformly mixing to form a mixture;
adding the mixture into a culture plate, and adding 5-5.5% of CO at 37-37.5 DEG C2The mixture was gelled by incubation in an incubator.
In some embodiments, the specific steps for constructing the collagen matrix containing dermal stem cells are: mixing MEM, bovine type I collagen, FBS, L-glutamine and DMEM, adjusting pH to 7.2, adding fibroblast, and mixing; adding the mixture to a gelled acellular collagen matrix, and adding 5-5.5% CO at 37-37.5 deg.C2The culture box is used for culturing to ensure that the cell matrix is completely gelatinized, and the fibroblast is added for continuous culture to ensure that the collagen matrix contracts and tends to be stable.
In some embodiments, the specific steps for establishing the epidermal cell layer are: resuspending the epidermal stem cells in DMEM, uniformly and slowly inoculating the cells on contracted collagen gel, and then carrying out gel electrophoresis at 37-37.5 ℃ and 5-5 DEG C.5％CO2The culture box is incubated to ensure that the epidermal stem cells are completely adhered and are attached to the matrix to form a confluent cell monolayer.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Preparation of tissue engineering skin
In the previous research, the invention establishes a simple and rapid method to effectively separate the human skin epidermal cells and the dermal stem cells, shortens the culture time required for the expansion of the skin stem cells, and maintains the potential of the human skin cells for generating full-thickness skin with hair follicles after the human skin cells are expanded in a culture system. In this study, the present invention prepares skin stem cells for the construction of a bilayer tissue engineered skin. The double-layer tissue engineering skin comprises a collagen layer and an epidermal layer, wherein the collagen layer is divided into a cell-free collagen layer and a dermal cell collagen layer, and epidermal cells cover the top of the collagen layer. The preparation method is as follows (as shown in figure 1):
(1) in the transwell chamber of a six-well plate, a layer of cell-free collagen is established which has two main functions: on the one hand, the membrane can be used as an adhesion matrix for a cell layer, and on the other hand, the membrane can prevent cell collagen from excessively shrinking and separating from a basement membrane. The preparation steps are as follows: a mixture of 0.57ml volume of 10 XMEM, 3.4ml volume of 2.5mg/ml bovine type I collagen, 0.83ml volume of FBS, 55. mu.l volume of 200mM L-glutamine and 2.0ml volume of DMEM was prepared on ice, followed by saturated NaHCO3The pH was adjusted to 7.2. The mixture was mixed using a cooled straw to avoid the formation of bubbles during mixing. 1ml of the mixture was added to each well of the transwell in a 6-well plate, ensuring that it covered the entire bottom of the transwell. The plates were then incubated at 37 ℃ in 5% CO2The mixture will gel within 30 minutes after incubation in the incubator.
(2) A collagen matrix containing dermal stem cells is constructed over the acellular collagen layer. Dermal stem cells were adjusted to a final concentration of
(3) An epidermal cell layer was established. After the stroma was contracted and stabilized at day 4, adult epidermal stem cells were added to the stromal upper layer. 2 x 10 to6The epidermal stem cells of (4) were resuspended in 100. mu.l of DMEM, uniformly and slowly seeded onto a shrinking collagen gel, then incubated at 37 ℃ without medium, 5% CO2The incubator (2) was incubated for 60 minutes to completely adhere the epidermal stem cells. The epidermal stem cells attach to the matrix, forming a confluent cell monolayer.
(4) Tissue engineering skin culture at different stages. 12ml of the epithelialization medium (10 ml outside the chamber, 2ml inside the chamber) was added to each chamber. The medium was changed every 2 days until day 11. From day 12 onwards, the medium was changed to a gas-liquid interface epidermization medium: the chamber was filled with 7ml of medium, the interior of the chamber was kept dry, and the tissue was lifted to the gas-liquid interface to achieve complete stratification. The bottom of the chamber was maintained in contact with the medium every other day until the end of the 21 st day culture.
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