Full-thickness skin model and construction method and application thereof

Abstract

The invention discloses a full-thickness skin model and a construction method and application thereof, and relates to the technical field of biomedical materials. The invention provides biological matrigel. The biological matrigel is prepared from halloysite nanotubes, sodium alginate, hyaluronic acid, I-type collagen, calcium chloride and DMEM (Dulbecco Modified Eagle Medium) powder. The biological matrigel disclosed by the invention can obviously promote fibroblasts in a corium layer to secrete abundant extracellular matrixes, increases sustainable mechanical strength and thickness of the corium layer, is used for simulating a corium structure in normal and inflammatory states, and is beneficial to adhesion of upper keratinocytes and construction of a full-thickness skin model. A full-thickness skin model constructed by adopting the biological matrigel disclosed by the invention is low in degradation efficiency and stable in structure, has bionic performance and can simulate a human skin structure; and the construction method of the full-thickness skin model is simple to operate, short in time consumption and low in cost, and the constructed skin 3D model is stable and can be used for treatment and mechanism research of chronic and inflammatory diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to a full-thickness skin model and a construction method and application thereof. Background Art

[0002] At present, most drug and cosmetic tests need to rely on two-dimensional cell level and animal experiments, but the two-dimensional cell culture level cannot correctly reflect the interaction between cells and the extracellular matrix, so the currently used 3D skin model mainly refers to the simulated full-thickness skin structure prepared in vitro using engineering materials and corresponding cell biology principles and methods. A good full-thickness skin model needs to be highly similar to the normal skin structure. First, it needs to have a complete epidermis and dermis structure. Secondly, it needs to have good mechanical performance and stability in biological properties. It can be used to replace the 3D culture system of human primary cells in vitro to detect the pathophysiological characteristics under different inflammatory conditions; it can also be used to replace human skin tissue to evaluate the safety and efficacy of drugs and chemical molecules.

[0003] In the study of bionic 3D skin tissue, it is necessary to find suitable dermal matrix components to make them have similar biocompatibility, suitable mechanical properties, and intrinsic material pore size that can support cell proliferation, migration, and adhesion. Therefore, the in vitro construction scheme of the dermis is an important research link. The commonly used dermal matrix is ​​mainly prepared with biomatrix glue made of special materials. However, the in vitro skin 3D model based on biomatrix glue has the following disadvantages: fast degradation efficiency, unstable retractability and mechanical properties, which leads to the non-repeatability of the in vitro model, thus affecting the construction of long-term inflammatory stimulation and inhibition models. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a full-thickness skin model and its construction method and application. The dermis layer of the full-thickness skin model is mixed with fibroblasts through biomatrix glue, which can be used to simulate the dermis structure under normal and inflammatory conditions, and is conducive to the adhesion of upper keratinocytes, so that the prepared full-thickness skin model has low degradation efficiency, stable structure, and more bionic performance, and can be used for the treatment and mechanism research of chronic and inflammatory diseases.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is: a biomatrix glue, comprising the following components in concentrations: 0.5-10 μg / mL halloysite nanotubes, 1.5%-5% sodium alginate, 8-12 mg / mL hyaluronic acid, 3-8 mg / mL type I collagen, 5-15 μmol / L calcium chloride and 10×DMEM powder.

[0006] The inventor of the present application has found through a large number of experiments that by adding halloysite nanotubes, sodium alginate, hyaluronic acid, type I collagen and calcium chloride to DMEM powder for compounding, a biomatrix glue of the present application is formed, which can promote fibroblasts to secrete rich extracellular matrix, promote fibroblast proliferation and migration, increase the mechanical strength and thickness of the dermis; and is conducive to the adhesion of epidermal keratinocytes, so that the inoculated epidermal cells proliferate and differentiate normally, forming a multi-layered epithelial-like structure, which is conducive to the rapid construction of a full-thickness skin model. In addition, it has been found through experiments that the biomatrix glue of the present invention has the biological activity of promoting wound healing and shortening epithelialization, and can be used for the regeneration and repair of various tissues.

[0007] Among them, halloysite nanotubes are a naturally occurring aluminosilicate nanotubes with a unique multilayer tubular structure, with an outer diameter usually between 50 and 70 nanometers, a length ranging from 200 nanometers to several micrometers, a large surface area, and a hollow lumen. These structural features give halloysite nanotubes a high surface area and enhanced mechanical strength. The present application adds halloysite nanotubes to the biomatrix glue, so that the halloysite nanotubes perform well in the interaction with cell components and growth factors, which helps the attachment and proliferation of cells. The rough surface of the halloysite nanotubes provides more contact area, promoting the anchoring and expansion of cells. The surface hydroxyl groups can form hydrogen bonds with cell surface proteins, enhancing cell adhesion and signal transduction. In addition, the improved mechanical properties of the halloysite nanotubes affect the intracellular mechanical transduction pathways, stimulate cell proliferation and migration, thereby providing a microenvironment similar to the natural extracellular matrix, supporting cell activity and tissue regeneration.

[0008] Adding calcium chloride to the biomatrix glue plays a role in promoting cross-linking and promoting the formation of the biomatrix glue structure. Sodium alginate is also added to the biomatrix glue of the present application, and the prepared biomatrix glue reaches a stable state; adding hyaluronic acid and type I collagen makes the biomatrix glue have the advantages of better promoting wound healing and shortening epithelialization, and is used for the regeneration and repair of various tissues. Adding DMEM powder and mixing it into the cell culture environment together can adjust the pH of the biomatrix glue.

[0009] Preferably, the DMEM powder also contains saturated sodium bicarbonate to adjust the pH value of the biomatrix glue.

[0010] The inventors of the present application have found that the biomatrix gel composed of the components in the above concentration range is more conducive to the proliferation and migration of fibroblasts, and is more conducive to the adhesion of epidermal keratinocytes, so that the inoculated epidermal cells can proliferate and differentiate normally to form a multi-layered epithelial-like structure, which is conducive to the rapid construction of a full-thickness skin model.

[0011] As a preferred embodiment of the biomatrix glue of the present invention, the concentration of halloysite nanotubes in the biomatrix glue is 0.8-5 μg / mL, and the concentration of hyaluronic acid is 9-11 mg / mL.

[0012] The present application adopts the above-mentioned concentration range of halloysite nanotubes and hyaluronic acid, and adds them to the biomatrix glue, which has better mechanical properties and is more conducive to the attachment, proliferation and migration of cells, so that the biomatrix glue can better promote wound healing, shorten the biological activity of epithelialization, and is conducive to the regeneration and repair of various tissues.

[0013] As a preferred embodiment of the biomatrix glue of the present invention, the concentration of the halloysite nanotubes in the biomatrix glue is 1 μg / mL, and the concentration of the hyaluronic acid is 10 mg / mL.

[0014] The use of the above-mentioned most preferred concentrations of halloysite nanotubes and hyaluronic acid can be more conducive to the proliferation and migration of fibroblasts, and more conducive to the adhesion of epidermal keratinocytes, so that the inoculated epidermal cells can proliferate and differentiate normally to form a multi-layered epithelial-like structure, which is conducive to the rapid construction of a full-thickness skin model.

[0015] The present invention also provides the use of the biomatrix glue in constructing a skin dermis layer or full-thickness skin model.

[0016] The biomatrix glue designed with the formula of the present application can promote fibroblasts to secrete rich extracellular matrix, which is beneficial to the formation of the skin dermis; at the same time, the biomatrix glue of the present application is beneficial to the adhesion of epidermal keratinocytes, so that the inoculated epidermal cells proliferate and differentiate normally to form a multi-layered epithelial-like structure, which is beneficial to the rapid construction of a full-thickness skin model.

[0017] The present invention also provides the use of the biomatrix glue in preparing a preparation for promoting tissue regeneration and repair.

[0018] The present invention also provides a method for constructing the dermis of the skin, comprising digesting and centrifuging cultured primary fibroblasts to obtain a fibroblast suspension, adding the biomatrix glue to the fibroblast suspension, mixing and incubating the mixture, and then adding a complete culture medium for culturing.

[0019] As a preferred embodiment of the method for constructing the dermis of the skin of the present invention, the density of the primary fibroblasts is 5-10×10 5 Pieces / mL.

[0020] As a preferred embodiment of the method for constructing the skin dermis layer of the present invention, the volume ratio of the fibroblast suspension to the biomatrix glue is 10:9.

[0021] As a preferred embodiment of the method for constructing the skin dermis layer of the present invention, the incubation is specifically incubated in a 37° C., 5% CO 2 incubator for 15 minutes.

[0022] The present invention also provides a skin dermis model, which is constructed by using the skin dermis construction method.

[0023] The present invention also provides a method for constructing a full-thickness skin model, comprising the following steps:

[0024] S1. Preparation of fibroblasts;

[0025] S2. Add the biomatrix glue to the fibroblasts and mix well to obtain a cell-glue premix; pipette the cell-glue premix into a culture dish, incubate it to form, and then add a complete culture medium and culture it statically to obtain a dermis;

[0026] S3. The cultured epidermal cells are digested and inoculated into the dermis of S2, and complete culture medium is added for culture to obtain the full-thickness skin model.

[0027] As a preferred embodiment of the method for constructing a full-thickness skin model of the present invention, the seeding density of the epidermal cells in step S3 is 1×10 6 Pieces / cm 2 .

[0028] As a preferred embodiment of the method for constructing a full-thickness skin model of the present invention, the culture in step S3 specifically includes replacing the culture medium every 2 days, and inoculating epidermal cells on the surface again, and culturing continuously for 5 days.

[0029] As a preferred embodiment of the method for constructing a full-thickness skin model of the present invention, the complete culture medium in steps S2 and S3 uses DMEM powder as the base liquid, and is supplemented with 10% FBS and 1% double antibody.

[0030] As a preferred embodiment of the method for constructing a full-thickness skin model of the present invention, the culture conditions in steps S2 and S3 are 37° C. and 5% CO 2 .

[0031] The present invention also provides a full-thickness skin model constructed by the construction method.

[0032] The present invention also provides application of the full-thickness skin model in the safety or efficacy evaluation of cosmetics or medicines.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The present invention provides a full-thickness skin model, which includes a dermis and an epidermis, has low degradation efficiency, a stable structure, and bionic properties, and can simulate the structure of human skin; and the construction method of the full-thickness skin model is simple to operate, short in time, and low in cost. The constructed 3D skin model is stable and can be used for the treatment of chronic and inflammatory diseases and the study of their mechanisms;

[0035] 2. The dermis in the full-thickness skin model of the present invention is obtained by incubating fibroblasts with biomatrix glue, wherein the biomatrix glue includes halloysite nanotubes, sodium alginate, hyaluronic acid, type I collagen, calcium chloride and DMEM powder, which can significantly promote the secretion of rich extracellular matrix by fibroblasts in the dermis, increase the sustainable mechanical strength and thickness of the dermis, and is used to simulate the dermis structure under normal and inflammatory conditions, and is conducive to the adhesion of upper keratinocytes, which is conducive to the construction of a full-thickness skin model;

[0036] 3. Animal experiments have shown that the biomatrix glue in the full-thickness skin model of the present invention has the function of promoting wound healing and shortening epithelialization time, and can be used for tissue regeneration and repair, especially wound healing, reducing scar formation, skin regeneration, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a graph showing the effect of different biological matrix gels on promoting the proliferation of fibroblasts;

[0038] Figure 2 This is the expansion rate test result diagram of Test Example 2;

[0039] Figure 3 The graphs are the histological section thickness results of the full-thickness skin models prepared in Example 2 and Comparative Example 2;

[0040] Figure 4 This is a diagram showing the results of evaluating the in vivo healing effects of different biomatrix glues on full-thickness skin wounds in mice;

[0041] Figure 5 Figure 2 shows the mechanical properties test results of different biomatrix glues. DETAILED DESCRIPTION

[0042] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. The experimental methods in the embodiments of the present invention that do not specify specific conditions are usually carried out according to conventional conditions in the art or according to conditions recommended by the manufacturer; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

[0043] like Figure 5 As shown, the present invention uses halloysite nanotubes, sodium alginate, hyaluronic acid, type I collagen, calcium chloride and DMEM powder to prepare a biomatrix glue ( Figure 5 The biomatrix glue can significantly promote the secretion of rich extracellular matrix by dermal fibroblasts, increase the sustainable mechanical strength and thickness of the dermis, and is used to simulate the dermis structure under normal and inflammatory conditions, and is conducive to the adhesion of upper layer keratinocytes, which is conducive to the construction of a full-thickness skin model ( Figure 5 (middle B).

[0044] The human primary fibroblasts in the embodiments of the present invention can be isolated and operated by existing techniques, and the detailed steps can be seen in the embodiments. The epidermal cells refer to human immortalized keratinocytes, purchased from iCell (Cat. No.: iCell-h066).

[0045] In the following examples and comparative examples, 10×DMEM powder (containing saturated sodium bicarbonate) is Catalog # D2902, sourced from Sigma Aldrich, USA.

[0046] The model number of halloysite nanotubes is #1332-58-7, which comes from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd.

[0047] Example 1

[0048] This embodiment provides a method for constructing a full-thickness skin model, comprising the following steps:

[0049] S1. Isolation and culture of human primary fibroblasts: Under sterile conditions, place the discarded foreskin specimen (about 4×1.5 cm) removed from surgery in a 50 mL centrifuge tube containing PBS with double antibodies (100 U / mL penicillin + 0.1 mg / mL streptomycin) and place on ice; place the foreskin in a clean bench and soak it in 75% ethanol for 3 minutes; place the foreskin tissue in a sterile tray and wash it with PBS solution containing double antibodies for 3 minutes × 3 times to remove blood clots and other impurities; keep the foreskin moist, and use ophthalmic scissors and curved scissors to remove subcutaneous blood clots and fat tissue; wash it with PBS solution containing double antibodies for 3 minutes × 3 times; cut the skin tissue into about 1x0.5 cm 2 The skin was cut into pieces of different sizes and placed in a 15 mL centrifuge tube. Three times the volume of Dispase II (2.5 u / mL) was added and placed in a 4°C refrigerator for overnight digestion. The skin was removed on the second day, the liquid containing Dispase II was discarded, and the skin was washed with PBS solution containing double antibodies for 3 minutes × 3 times. The epidermis was peeled off with ophthalmic forceps, and the dermis was cut into pieces as small as possible, about 0.1 × 0.1 cm 2; Use a dental probe to evenly place the tissue blocks on the wall of the culture dish at appropriate intervals, add 3 mL of complete culture medium (DMEM powder high glucose medium + 10% FBS + 1% double antibody), and culture in a constant temperature incubator at 37°C and 5% CO2; add 2 mL of complete culture medium after 4 hours and continue culturing; change the medium every 2-3 days, and after about 2-3 days, a small number of short spindle cells can be observed crawling out around the tissue blocks under an inverted microscope, which are human primary fibroblasts.

[0050] S2. Construction of the dermis: The fifth generation of human primary fibroblasts were obtained and digested and centrifuged to obtain about 1×10 6 / mL fibroblast suspension; slowly aspirate biomatrix gel (sodium alginate, hyaluronic acid, type I collagen (with 10×DMEM ratio of 10:1), CaCl2, halloysite nanotubes) and cell suspension into a test tube (the volume ratio of cell suspension to matrix gel is 1:9), vortex for 5s to fully mix to obtain cell-gel premix; aspirate 200μL of cell-gel premix into the central concave of the culture dish, put it into a 37°C, 5% CO2 incubator and incubate for 15 minutes to form the gel; take out the culture dish, aspirate 200μL of complete culture medium (DMEM powder + 10% FBS + 1% double antibody) onto the surface of the gel, and slowly drip it; put it back into the incubator for static culture, observe and change the medium, change it every 1-2 days, and the dermis layer is obtained.

[0051] The biomatrix glue includes the following components: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 5 mg / mL sodium alginate, 10 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / L CaCl2, and 1 μg / mL halloysite nanotubes (HNT).

[0052] S3. Construction of epidermis: Immortalized human keratinocytes were collected at a rate of 1×10 6 Pieces / cm 2 Inoculate on the surface of the dermis, add culture medium (DMEM powder + 10% FBS + 1% double antibody), change the medium every 2 days, and repeat at 1×10 6 Pieces / cm 2 Epidermal cells were spread on the surface of the dermis, and after 5 days of culture, a 3D skin tissue of stratified epithelium was obtained, namely the full-thickness skin model.

[0053] Example 2

[0054] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0055] The biomatrix glue of Example 2 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 5 mg / mL sodium alginate, 10 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / L CaCl2, and 0.5 μg / mL halloysite nanotubes (HNT).

[0056] Example 3

[0057] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0058] The biomatrix glue of Example 3 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 5 mg / mL sodium alginate, 10 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / L CaCl2, and 10 μg / mL halloysite nanotubes (HNT).

[0059] Example 4

[0060] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0061] The biomatrix glue of Example 4 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 1.5 mg / mL sodium alginate, 8 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 5 μmol / L CaCl2, and 0.5 μg / mL halloysite nanotubes (HNT).

[0062] Example 5

[0063] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0064] The biomatrix glue of Example 5 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 5 mg / mL sodium alginate, 12 mg / mL hyaluronic acid, 8 mg / mL type I collagen, 15 μmol / L CaCl2, and 10 μg / mL halloysite nanotubes (HNT).

[0065] Example 6

[0066] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0067] The biomatrix glue of Example 6 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 5 mg / mL sodium alginate, 9 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / L CaCl2, and 0.8 μg / mL halloysite nanotubes (HNT).

[0068] Example 7

[0069] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0070] The biomatrix glue of Example 6 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 5 mg / mL sodium alginate, 11 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / L CaCl2, and 5 μg / mL halloysite nanotubes (HNT).

[0071] Comparative Example 1

[0072] This comparative example provides a method for constructing a full-thickness skin model, comprising the following steps:

[0073] S1. Isolation and culture of primary human fibroblasts: the same method as that of isolation and culture of primary human fibroblasts in Example 1.

[0074] S2. Construction of the dermis: The fifth generation of human primary fibroblasts were obtained and digested and centrifuged to obtain about 1×10 6 / mL; slowly pipette biomatrix gel (sodium alginate, hyaluronic acid, type I collagen (with 10×DMEM powder in a ratio of 10:1), CaCl2) and cell suspension into a test tube, vortex for 5s to fully mix to obtain a cell-gel premix; pipette 200 μL of the cell-gel premix into the central concave of the culture dish, put it into a 37°C, 5% CO2 incubator and incubate for 15 minutes to allow the gel to form; take out the culture dish, pipette 200 μL of complete culture medium (DMEM powder + 10% FBS + 1% double antibody) onto the surface of the gel, and slowly drip it; put it back into the incubator for static culture, observe and change the medium, change it every 1-2 days, and the dermis layer is obtained.

[0075] The biomatrix glue includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate), 5 mg / mL sodium alginate, 10 mg / mL hyaluronic acid, 3 mg / mL type I collagen, and 10 μmol / L CaCl2.

[0076] S3. Construction of epidermal layer: Epidermal cells were collected at a rate of 1×10 6 Pieces / cm2 Inoculate on the surface of the dermis, add culture medium (DMEM powder + 10% FBS + 1% double antibody), change the medium every 2 days, and repeat at 1×10 6 Pieces / cm 2 Epidermal cells were spread on the surface of the dermis, and after 5 days of culture, a 3D skin tissue of stratified epithelium was obtained, namely the full-thickness skin model.

[0077] Comparative Example 2

[0078] This comparative example provides a method for constructing a full-thickness skin model, comprising the following steps:

[0079] S1. Isolation and culture of primary human fibroblasts: the same method as that of isolation and culture of primary human fibroblasts in Example 1.

[0080] S2. Construction of the dermis: The fifth generation of human primary fibroblasts were obtained and digested and centrifuged to obtain about 1×10 6 / mL; slowly pipette biomatrix glue (sodium alginate, hyaluronic acid, type I collagen (with 10×DMEM powder ratio of 10:1), CaCl2, carbon nanotubes (CNTs)) and cell suspension into a test tube, vortex for 5s to fully mix to obtain a cell-gel premix; slowly pipette sodium alginate, hyaluronic acid, type I collagen (with 10×DMEM powder ratio of 10:1), cell suspension CaCl2 and halloysite nanotubes into a test tube, vortex for 5s to fully mix to obtain a cell-gel premix; pipette 200 μL of the cell-gel premix into the central concave of the culture dish, put it into a 37°C, 5% CO2 incubator and incubate for 15 minutes to form the glue; take out the culture dish, pipette 200 μL of complete culture medium (DMEM powder + 10% FBS + 1% double antibody) onto the surface of the glue, and slowly drip it; put it back into the incubator for static culture, observe and change the medium, change it every 1-2 days, and obtain the dermis. The biomatrix glue includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate), 5 mg / mL sodium alginate, 10 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / LCaCl2, and 1 μg / mL carbon nanotubes (CNTs).

[0081] S3. Construction of epidermal layer: Epidermal cells were collected at a rate of 1×10 6 Pieces / cm 2 Inoculate on the surface of the dermis, add culture medium (DMEM powder + 10% FBS + 1% double antibody), change the medium every 2 days, and repeat at 1×10 6 Pieces / cm 2 Epidermal cells were spread on the surface of the dermis, and after 5 days of culture, a 3D skin tissue of stratified epithelium was obtained, namely the full-thickness skin model.

[0082] Comparative Example 3

[0083] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0084] The biomatrix glue of Comparative Example 3 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10×, and diluted to a concentration of 1×), 5 mg / mL sodium alginate, 10 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / L CaCl2, and 50 μg / mL halloysite nanotubes (HNT).

[0085] Comparative Example 4

[0086] Compared with Example 1, the difference is that the formula of the biomatrix glue is different (does not contain hyaluronic acid), and the remaining steps are the same as Example 1.

[0087] The biomatrix glue of Comparative Example 4 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10x and diluted to a concentration of 1X), 5 mg / mL sodium alginate, 3 mg / mL type I collagen, 10 μmol / LCaCl2, and 1 μg / mL halloysite nanotubes (HNT).

[0088] Comparative Example 5

[0089] Compared with Example 1, the difference is that the formula of the biomatrix glue is different, and the remaining steps are the same as Example 1.

[0090] The biomatrix glue of Comparative Example 5 includes the following components in concentration: 10×DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10x and diluted to a concentration of 1X), 5 mg / mL sodium alginate, 20 mg / mL hyaluronic acid, 3 mg / mL type I collagen, 10 μmol / L CaCl2, and 1 μg / mL halloysite nanotubes (HNT).

[0091] Experimental Example 1: Proliferation-promoting effect of biomatrix gel on fibroblasts

[0092] Specific experimental methods:

[0093] (1) CCK-8 detection experiment: Take 200 μL of the cell-gel premix of Examples 1-7 and Comparative Examples 1-5 in a cell culture dish, and add 200 μL of CCK-8 reagent. Put the well plate or culture dish back into the cell culture incubator, continue incubation for 2 hours, and measure the results at 450nm using a spectrophotometer, and then perform data analysis; calculate the cell survival rate or cytotoxicity of each treatment group based on the measured optical signal.

[0094] (2) Detection of cell proliferation index Ki67

[0095] Specific experimental method: Obtain the biomatrix gel-tissue mixture, first fix it with 4% paraformaldehyde for 30 minutes and then wash it with PBS. Then incubate it in blocking solution for 30 minutes to block nonspecific binding sites. Subsequently, incubate the slices with Ki67 primary antibody at 4°C overnight and wash them with PBS. After that, incubate the slices with fluorescently labeled secondary antibodies for 30 minutes to 1 hour in the dark. After washing again, stain with DAPI for 5 minutes to mark the cell nucleus, and finally perform a quick PBS wash. Place the slices on a slide, add a sealing agent and cover the coverslip, and finally observe the slices under a fluorescence microscope to evaluate the cell proliferation status.

[0096] The results are shown in Table 1.

[0097] The results of the biomatrix prepared in Example 2 and Comparative Example 1 on the survival rate or proliferation of fibroblasts are as follows: Figure 1 shown.

[0098] Table 1

[0099] Group Cell viability Cell proliferation Example 1 115.5％ 114.19％ Example 2 103.44％ 101.20％ Example 3 104.88％ 101.88％ Example 4 108.58％ 110.22％ Example 5 107.89％ 109.87％ Example 6 110.21％ 111.34％ Example 7 113.46％ 112.09％ Comparative Example 1 79.26％ 76.00％ Comparative Example 2 76.26％ 75.20％ Comparative Example 3 82.33％ 80.07％ Comparative Example 4 81.57％ 78.22％ Comparative Example 5 88.51％ 87.89％

[0100] The results are shown in Table 1. The biomatrix glue of Examples 1-7 can improve the survival rate or proliferation of fibroblasts. When the concentration of halloysite nanotubes in the biomatrix glue is preferably 0.8-5 μg / mL and the concentration of hyaluronic acid is preferably 9-11 mg / mL, the biomatrix glue of Examples 6-7 is better than that of Examples 4-5 in improving the survival rate and cell proliferation of fibroblasts, especially the biomatrix glue of Example 1 has the best effect in improving the survival rate and proliferation of fibroblasts.

[0101] The biomatrix glue of Comparative Example 1 does not contain halloysite nanotubes (HNT), Comparative Example 4 does not contain hyaluronic acid, and the biomatrix glue in Comparative Example 2 uses carbon nanotubes (CNTs) instead of halloysite nanotubes (HNT). The fibroblast survival rate or cell proliferation effect is not as good as that of Example 1.

[0102] In comparative example 3 and comparative example 5, the concentration of halloysite nanotubes (HNT) or hyaluronic acid is excessive, which is not within the scope of the present application. The prepared biomatrix glue improves the survival rate of fibroblasts or the cell proliferation effect as poor as that of Examples 1-7, indicating that the concentration of halloysite nanotubes (HNT) or hyaluronic acid at a suitable concentration helps to improve the survival rate of fibroblasts or the cell proliferation effect. Halloysite nanotubes (HNT) or hyaluronic acid can create a nanomatrix structure environment within the concentration range of the present application, simulate the extracellular matrix, so as to promote the key processes such as the attachment, migration and proliferation of cells. However, halloysite nanotubes (HNT) or hyaluronic acid are at higher concentrations, and excessive HNT or hyaluronic acid may form a denser matrix, which may hinder the mobility of cells and limit proliferation.

[0103] Test Example 2: Expansion Rate Detection of Full-Thickness Skin Model

[0104] Specific experimental method: The method for calculating the expansion rate of biomatrix glue using relative height is as follows: First, measure the initial height (H) of the dry biomatrix glue sample. dry ), then place the sample in distilled water or PBS and soak for a period of time (e.g., 24 hours) to ensure that it is fully expanded. Take out the expanded sample, remove excess water from the surface, and immediately measure its expanded height (H swollen The expansion ratio is calculated by the following formula: Expansion ratio (%) = (H swollen -H dry ) / H dry x100. Throughout the process, the accuracy of all measurement steps and the consistency of sample conditions should be ensured to obtain reliable data. These steps can help evaluate the volume change and water absorption capacity of biomatrix gel in a hydrated state.

[0105] In addition, the swelling rate is the ratio of the volume or weight change of the biomatrix glue after absorbing water, which not only reflects the water absorption capacity of the biomatrix glue, but also affects its degradation performance. Biomatrix glue with a higher swelling rate usually has a looser cross-linked network structure, which makes the degradation medium easier to penetrate and accelerates the degradation rate. In addition, biomatrix glue with a high swelling rate can diffuse and discharge degradation products faster, further promoting the degradation process. In general, the swelling rate is positively correlated with the degradation rate. The higher the swelling rate, the faster the degradation rate.

[0106] The expansion rate results obtained by testing Examples 1-7 and Comparative Examples 1-5 are shown in Table 2.

[0107] The results of Example 2 and Comparative Example 2 are as follows Figure 2 shown.

[0108] Table 2

[0109]

[0110]

[0111] As can be seen from Table 2, the biomatrix glue of Examples 1-7 of the present application has a better swelling rate and a better degradation rate. When the concentration of halloysite nanotubes in the biomatrix glue is preferably 0.8-5 μg / mL and the concentration of hyaluronic acid is preferably 9-11 mg / mL, the swelling rate and degradation rate of the biomatrix glue of Examples 6-7 are better than those of Examples 4-5, especially Example 1 has the best effect.

[0112] However, in Comparative Example 1, which does not contain halloysite nanotubes (HNT), or Comparative Example 4, which does not contain hyaluronic acid, or in Comparative Example 2, the biomatrix glue uses carbon nanotubes (CNTs) instead of halloysite nanotubes (HNT), the obtained biomatrix glue has a lower expansion rate than Examples 1-7.

[0113] In Comparative Examples 3 and 5, the concentration of halloysite nanotubes (HNT) or hyaluronic acid is excessive and is not within the scope of the present application. The swelling rate of the prepared biomatrix glue is not as good as that of Examples 1-7, indicating that the appropriate concentration of halloysite nanotubes (HNT) or hyaluronic acid helps to improve the swelling rate of the biomatrix glue.

[0114] Halloysite nanotubes combined with hyaluronic acid can significantly improve the expansion rate of biomatrix glue, and the tubular structure of halloysite nanotubes (HNT) and the high viscosity of hyaluronic acid form a stable three-dimensional network together, which enhances the water retention capacity. At the same time, halloysite nanotubes (HNT) further improve the mechanical strength and expansion performance of the material as a physical crosslinking agent. The present application reasonably adjusts the concentration of both to help achieve optimal performance. The hyaluronic acid within the concentration range of the present application can improve the expansion rate of biomatrix glue, and at the same time coordinates with halloysite nanotubes (HNT) to form a more stable network structure. This synergistic effect improves the colloid in water absorption and mechanical strength, thereby achieving better performance.

[0115] Test Example 3: Thickness of histological sections of full-thickness skin model

[0116] Specific experimental method: The full-thickness skin models prepared in Example 1 and Comparative Example 2 were fixed with formalin, embedded in paraffin, and then cut into 5 μm slices, stained with H&E, dehydrated and washed, and finally sealed and photographed under a microscope to measure their thickness.

[0117] The results are as follows Figure 3 As shown, the upper epidermis formation effect of Comparative Example 2 is not good and a full-thickness skin model cannot be formed.

[0118] Experimental Example 4: Evaluation of the in vivo healing effect of biomatrix glue in full-thickness skin wounds in mice

[0119] Specific experimental method: C57BL / 6 mice were selected and randomly divided into an experimental group and a control group, with 3 mice in each group, for a total of 3 groups. Each group of animals was fasted for 12 hours before surgery. 2% sodium pentobarbital was injected intramuscularly for anesthesia, the back skin was prepared, the back skin was disinfected with iodine, and a sterile towel was laid. A circular full-thickness skin defect with a diameter of 0.8 cm was surgically cut on the back of the mouse. The following treatments were performed according to the grouping:

[0120] Control group: only gauze bandage was used;

[0121] Experimental group 1: The biomatrix glue of Example 1 was applied to the wound surface and bandaged with gauze, and the dressing was changed every two days;

[0122] Experimental Group 2: The biomatrix glue of Comparative Example 1 was applied to the wound surface and bandaged with gauze, and the dressing was changed every two days.

[0123] Wound images were taken with a digital camera on days 0, 2, 5, 7, and 14, and image analysis was performed using Java-based image processing and analysis software (ImageJ). Skin samples were collected on day 14 for histological evaluation, and the number of pixels in the marked area was converted into the actual area to obtain the wound area data to evaluate the healing effect.

[0124] Table 3

[0125] Group <![CDATA[Wound area day 0 5mm 2 > Example 1 76 Comparative Example 1 25

[0126] The results are shown in Table 3 and Figure 4 As shown, Figure A shows: pictures of the wound healing process, at different time points (day 0, day 2, day 5, day 7, day 14), the groups are mainly control groups, without halloysite nanotubes (Comparative Example 1) and halloysite nanotubes treatment groups (Example 1). Since the CNTs in Comparative Example 2 easily form colonies in vitro and cannot be implanted into mice, they are not used.

[0127] Figure B shows the changes in wound area during the healing process of different treatment groups. Each figure shows the average area of ​​each group of wounds at different time points (with color representing time). Figure C quantitatively shows the percentage of wound area of ​​each group at different time points (relative to the initial area on day 0). In summary, the wound healing rate of the group treated with halloysite nanotubes was the fastest, and the wound was almost completely healed on the 14th day.

[0128] Test Example 5: Mechanical properties of biomatrix glue

[0129] Specific experimental method: The compression modulus of the biomatrix glue (Example 1, Comparative Example 2) was measured using Microtester G2 (CellScale). The biomatrix glue was compressed at a compression speed of 1 mm / min and a strain of 5%-10%, and three parallel samples were set for each sample. The Young's modulus was evaluated by calculating the slope of the stress-strain curve.

[0130] The results are as follows Figure 5 The biomatrix glue of the present application increases the sustainable mechanical strength of the dermis, is used to simulate the dermis structure under normal and inflammatory conditions, is conducive to the adhesion of upper layer keratinocytes, and is conducive to the construction of a full-thickness skin model.

[0131] Finally, it should be noted that the above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A biomatrix glue, characterized in that: The following components were included at the following concentrations: 0.5-10 μg / mL halloysite nanotubes, 1.5-5 mg / mL sodium alginate, 8-12 mg / mL hyaluronic acid, 3-8 mg / mL type I collagen, 5-15 μmol / L calcium chloride, and 10× DMEM powder.

2. The biomatrix glue according to claim 1, characterized in that The concentration of the halloysite nanotubes in the biomatrix glue is 0.8-5 μg / mL, and the concentration of the hyaluronic acid is 9-11 mg / mL.

3. The biomatrix glue according to claim 2, characterized in that The concentration of the halloysite nanotubes in the biomatrix glue is 1 μg / mL, and the concentration of the hyaluronic acid is 10 mg / mL.

4. Use of the biomatrix glue according to any one of claims 1 to 3 in constructing a skin dermis or full-thickness skin model.

5. Use of the biomatrix glue according to any one of claims 1 to 3 in the preparation of a preparation for promoting tissue regeneration and repair.

6. A method for constructing the dermis of the skin, characterized in that: The cultured primary fibroblasts are digested and centrifuged to obtain a fibroblast suspension, the biomatrix glue according to any one of claims 1 to 3 is added to the fibroblast suspension, mixed and incubated for shaping, and then a complete culture medium is added for culturing.

7. The method for constructing the dermis layer of the skin according to claim 6, characterized in that: The concentration of the primary fibroblasts is 5-10×10 5 Pieces / mL.

8. The method for constructing the dermis layer of the skin according to claim 6, characterized in that: The volume ratio of the fibroblast suspension to the biomatrix glue is 10:

9.

9. A skin dermis model, characterized in that: The method for constructing the skin dermis layer according to any one of claims 6 to 8 is used to construct the skin dermis layer.

10. A method for constructing a full-thickness skin model, characterized in that: The following steps are involved: S1. Preparation of fibroblasts; S2. Add the biomatrix glue according to any one of claims 1 to 3 to the fibroblasts, mix well to obtain a cell-glue premix; pipette the cell-glue premix into a culture dish, incubate to form, then add a complete culture medium and culture statically to obtain a dermis layer; S3. The cultured epidermal cells are digested and inoculated into the dermis of S2, and complete culture medium is added for culture to obtain the full-thickness skin model.

11. The method for constructing a full-thickness skin model according to claim 8, characterized in that: The culturing in step S3 specifically includes replacing the culture medium every 2 days, and inoculating the epidermal cells on the surface again, and culturing continuously for 5 days.

12. The method for constructing a full-thickness skin model according to claim 8, characterized in that: The complete culture medium in steps S2 and S3 is based on DMEM powder and is supplemented with 10% FBS and 1% double antibody.

13. The method for constructing a full-thickness skin model according to claim 8, characterized in that: The culture conditions in steps S2 and S3 are 37° C. and 5% CO 2 .

14. A full-thickness skin model constructed by the construction method according to any one of claims 10 to 13.

15. Use of the full-thickness skin model according to claim 14 in the safety or efficacy evaluation of cosmetics or drugs.

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