A full-thickness skin model and its construction method and application

Abstract

The present invention discloses a full-thickness skin model and its construction method and application, and relates to the technical field of biomedical materials. The present invention provides a biomatrix glue, comprising halloysite nanotubes, sodium alginate, hyaluronic acid, type I collagen, calcium chloride and DMEM powder. The biomatrix glue of the present invention 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. The full-thickness skin model constructed using the biomatrix glue of the present invention has a slow degradation efficiency, a stable structure, and biomimetic 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 and mechanism research of chronic and inflammatory diseases.

Description

Technical Field

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

[0002] Currently, most drug and cosmetic testing relies on two-dimensional cell culture and animal experiments. However, two-dimensional cell culture cannot accurately reflect the interactions between cells and the extracellular matrix. Therefore, the currently used 3D skin models mainly refer to simulated full-thickness skin structures 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 normal skin structure. First, it needs to have a complete epidermal and dermal structure. Second, it needs to have good mechanical efficiency and stability in terms of biological properties. It can be used to replace the 3D culture system of human primary cells in vitro to detect 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 biomimetic 3D skin tissue, it is necessary to find a suitable dermal matrix component that has similar biocompatibility, suitable mechanical properties, and an intrinsic material pore size that can support cell proliferation, migration, and adhesion. Therefore, the in vitro construction of the dermis is a key research step. Currently, the commonly used dermal matrix is ​​mainly formulated with special material biomatrix glue. However, the current in vitro skin 3D models based on biomatrix glue have the following shortcomings: rapid degradation efficiency, unstable retractility and mechanical properties, resulting in the non-reproducibility of the in vitro model, which affects the construction of long-term inflammatory stimulation and inhibition models. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art by providing a full-thickness skin model, its construction method, and its application. The dermis of the full-thickness skin model is mixed with fibroblasts via biomatrix glue, which can be used to simulate the dermal structure under normal and inflammatory conditions and facilitate the adhesion of upper-layer keratinocytes. The resulting full-thickness skin model has low degradation efficiency, a stable structure, and biomimetic properties, making it suitable for the treatment of chronic and inflammatory diseases and for studying their mechanisms.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: a biomatrix glue comprising the following components at the following 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] After extensive experiments, the inventors of this application have discovered that by adding halloysite nanotubes, sodium alginate, hyaluronic acid, type I collagen, and calcium chloride to DMEM powder for compounding, a biomatrix glue is formed. This biomatrix glue can promote the secretion of rich extracellular matrix by fibroblasts, promote fibroblast proliferation and migration, increase the mechanical strength and thickness of the dermis; and facilitate the adhesion of epidermal keratinocytes, allowing the inoculated epidermal cells to proliferate and differentiate normally, forming a multilayered epithelial-like structure, which is conducive to the rapid construction of a full-thickness skin model. In addition, experiments have found 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 type of naturally occurring aluminosilicate nanotubes with a unique multilayer tubular structure. Their outer diameter is typically between 50 and 70 nanometers, and their length ranges from 200 nanometers to several micrometers. They have a large surface area and contain 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 interacting with cellular components and growth factors, which helps cell attachment and proliferation. The rough surface of the halloysite nanotubes provides more contact area, promoting cell anchoring and expansion. 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 mechanical transduction pathways within the cells, stimulating cell proliferation and migration, thereby providing a microenvironment similar to the natural extracellular matrix, supporting cell activity and tissue regeneration.

[0008] Calcium chloride is added to the biomatrix glue to promote cross-linking and promote the formation of the biomatrix glue structure. Sodium alginate is also added to the biomatrix glue of the present application to achieve a stable biomatrix glue. The addition of hyaluronic acid and type I collagen gives the biomatrix glue the advantages of better promoting wound healing and shortening epithelialization, making it suitable for the regeneration and repair of various tissues. DMEM powder is added and mixed into the cell culture environment to adjust the pH of the biomatrix glue.

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

[0010] The inventors of the present application have found that the biomatrix glue 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, forming 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 uses halloysite nanotubes and hyaluronic acid in the above-mentioned concentration range and adds them to the biomatrix glue, which has better mechanical properties and is more conducive to cell attachment, proliferation and migration, so that the biomatrix glue can better promote wound healing, shorten the biological activity of epithelialization, and facilitate the regeneration and repair of various tissues.

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

[0014] The use of the above-mentioned optimal 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, forming 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 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, forming a multilayered 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 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 to obtain a cell-glue premix; pipette the cell-glue premix into a culture dish, incubate until formed, and then add complete culture medium and allow to stand for culturing to obtain a dermis;

[0026] S3. The cultured epidermal cells are digested and inoculated into the dermis layer 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 is specifically to replace the culture medium every 2 days, and to inoculate epidermal cells on the surface again, and to culture 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 using the construction method.

[0032] The present invention also provides the use 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 comprising the dermis and epidermis, which has low degradation efficiency, stable structure, and biomimetic properties, and can simulate the structure of human skin. The method for constructing the full-thickness skin model is simple to operate, short in time, and low in cost. The resulting 3D skin model is stable and can be used for the treatment of chronic and inflammatory diseases and research on their mechanisms.

[0035] 2. The dermis in the full-thickness skin model of the present invention is obtained by incubating fibroblasts with a biomatrix adhesive. The biomatrix adhesive includes halloysite nanotubes, sodium alginate, hyaluronic acid, type I collagen, calcium chloride, and DMEM powder. It 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. It also facilitates the adhesion of upper-layer keratinocytes and 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, and skin regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This figure shows the results of different biological matrix gels promoting the proliferation of fibroblasts;

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

[0039] Figure 3 Graph showing the thickness results of histological sections of the full-thickness skin models prepared in Example 2 and Comparative Example 2;

[0040] Figure 4 The figure shows the results of the in vivo evaluation of the 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 following examples further illustrate the above-mentioned content of the present invention through specific implementation methods. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. Experimental methods in the examples of the present invention, where specific conditions are not specified, generally follow conventional conditions in the art or conditions recommended by the manufacturer; materials, reagents, etc. used, unless otherwise specified, are commercially available. 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 biological matrix 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 dermal structure under normal and inflammatory conditions. It is also beneficial for the adhesion of upper layer keratinocytes and is conducive to the construction of a full-thickness skin model ( Figure 5 Middle B).

[0044] The human primary fibroblasts in the examples of the present invention can be isolated and operated using existing techniques, and the detailed steps can be found in the examples. 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 is 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 (approximately 4 × 1.5 cm) removed from surgery in a 50 mL centrifuge tube containing PBS containing double-antibody (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-antibody 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-antibody for 3 minutes × 3 times; cut the skin tissue into approximately 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 the skin was digested overnight in a 4°C refrigerator. The next day, the skin was removed, 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. 2Use a dental probe to evenly place tissue blocks on the wall of the culture dish at appropriate intervals. Add 3 mL of complete culture medium (DMEM powdered high-glucose medium + 10% FBS + 1% double-antibody) and culture in a constant-temperature incubator at 37°C and 5% CO2. After 4 hours, add 2 mL of complete culture medium and continue culturing. Change the medium every 2-3 days. After about 2-3 days, a small number of short spindle-shaped cells can be observed crawling out from the tissue blocks under an inverted microscope. These are primary human fibroblasts.

[0050] S2. Construction of the dermis: Primary human fibroblasts of passage 5 were obtained, digested and centrifuged to obtain approximately 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 5 seconds to fully mix to obtain cell-gel premix; aspirate 200 μL of cell-gel premix into the central concave of the culture dish, place in a 37°C, 5% CO2 incubator and incubate for 15 minutes to allow the gel to form; remove the culture dish, aspirate 200 μL of complete culture medium (DMEM powder + 10% FBS + 1% double antibody) onto the surface of the gel, and slowly add dropwise; return to 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 at the following concentrations: 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 (HNTs).

[0052] S3. Epidermal layer construction: 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 re-inoculate at 1×10 6 pieces / cm 2 Epidermal cells were plated on the surface of the dermis and cultured for 5 days to obtain 3D skin tissue of stratified epithelium, i.e., 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 other steps are the same as Example 1.

[0055] The biomatrix glue of Example 2 includes the following components at the following concentrations: 10× DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10× and diluted to a 1× concentration), 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 (HNTs).

[0056] Example 3

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

[0058] The biomatrix glue of Example 3 includes the following components at the following concentrations: 10× DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10× and diluted to a 1× concentration), 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 (HNTs).

[0059] Example 4

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

[0061] The biomatrix glue of Example 4 includes the following components at the following concentrations: 10× DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10× and diluted to a 1× concentration), 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 (HNTs).

[0062] Example 5

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

[0064] The biomatrix glue of Example 5 includes the following components at the following concentrations: 10× DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10× and diluted to a 1× concentration), 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 (HNTs).

[0065] Example 6

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

[0067] The biomatrix glue of Example 6 includes the following components at the following concentrations: 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 (HNTs).

[0068] Example 7

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

[0070] The biomatrix glue of Example 6 includes the following components at the following concentrations: 10× DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10× and diluted to a 1× concentration), 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 (HNTs).

[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: Primary human fibroblasts of passage 5 were obtained, digested and centrifuged to obtain approximately 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 5 seconds to mix thoroughly to obtain a cell-gel premix; pipette 200 μL of the cell-gel premix into the central concave of the culture dish, place it in a 37°C, 5% CO2 incubator and incubate for 15 minutes to allow the gel to form; remove 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 add dropwise; return it to 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 at the following concentrations: 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 CaCl 2 .

[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 re-inoculate at 1×10 6 pieces / cm 2 Epidermal cells were plated on the surface of the dermis and cultured for 5 days to obtain 3D skin tissue of stratified epithelium, i.e., 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: Primary human fibroblasts of passage 5 were obtained, digested and centrifuged to obtain approximately 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 5 seconds to fully mix to obtain 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 5 seconds to fully mix to obtain cell-gel premix; pipette 200 μL of cell-gel premix into the central concave of the culture dish, place in a 37°C, 5% CO2 incubator and incubate for 15 minutes to allow the gel to form; remove 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 add dropwise; return to the incubator for static culture, observe and change the medium, change it every 1-2 days to obtain the dermis. The biomatrix glue includes the following components at the following concentrations: 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 re-inoculate at 1×10 6 pieces / cm 2 Epidermal cells were plated on the surface of the dermis and cultured for 5 days to obtain 3D skin tissue of stratified epithelium, i.e., 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 other steps are the same as Example 1.

[0084] The biomatrix glue of Comparative Example 3 includes the following components at the following concentrations: 10× DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10× and diluted to a 1× concentration), 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 (HNTs).

[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 at the following concentrations: 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, 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 other steps are the same as Example 1.

[0090] The biomatrix glue of Comparative Example 5 includes the following components at the following concentrations: 10× DMEM powder (containing saturated sodium bicarbonate, DMEM powder is dissolved to a concentration of 10× and diluted to a 1× concentration), 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 (HNTs).

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

[0092] Specific experimental methods:

[0093] (1) CCK-8 Assay: 200 μL of the cell-gel premix of Examples 1-7 and Comparative Examples 1-5 were placed in a cell culture dish, and 200 μL of CCK-8 reagent was added. The well plate or dish was returned to the cell culture incubator and incubated for another 2 hours. The results were measured at 450 nm using a spectrophotometer and then analyzed. The cell viability or cytotoxicity of each treatment group was calculated based on the measured optical signal.

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

[0095] Specific experimental method: Obtain a 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, the sections are incubated with Ki67 primary antibody at 4°C overnight and then washed with PBS. After that, incubate the sections with fluorescently labeled secondary antibody 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 sections on a slide, add mounting medium and cover with a coverslip, and finally observe the sections under a fluorescence microscope to assess 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 glues 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 glues of Examples 6-7 are better than those of Examples 4-5 in improving the survival rate and cell proliferation of fibroblasts. In particular, 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 (HNTs), Comparative Example 4 does not contain hyaluronic acid, and the biomatrix glue of Comparative Example 2 uses carbon nanotubes (CNTs) instead of halloysite nanotubes (HNTs). 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, halloysite nanotubes (HNT) or hyaluronic acid concentration is excessive, and it is not within the scope of this application. The biomatrix glue prepared improves fibroblast survival rate or cell proliferation effect less than embodiment 1-7, illustrating that the halloysite nanotubes (HNT) or hyaluronic acid concentration of suitable concentration contribute to improving fibroblast survival rate or cell proliferation effect. Halloysite nanotubes (HNT) or hyaluronic acid can be able to create a kind of nanomatrix structure environment within the concentration range of this application, simulate extracellular matrix, thus promote the key processes such as attachment, migration and proliferation of cells. However, halloysite nanotubes (HNT) or hyaluronic acid are under 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 of the dry biomatrix glue sample (H dry ), then place the sample in distilled water or PBS and soak for a period of time (such as 24 hours) to ensure that it is fully expanded. Take out the expanded sample, remove excess water on 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 entire 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 the biomatrix gel in its hydrated state.

[0105] In addition, the swelling rate is the ratio of the volume or weight change of the biomatrix glue after absorbing water. It not only reflects the water absorption capacity of the biomatrix glue, but also affects its degradation performance. Biomatrix glues with higher swelling rates usually have a looser cross-linked network structure, making it easier for the degradation medium to penetrate and accelerate the degradation rate. In addition, biomatrix glues with high swelling rates 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 from the tests of 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 glues of Examples 1-7 of the present application have 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 without halloysite nanotubes (HNTs), Comparative Example 4 without hyaluronic acid, or Comparative Example 2 using carbon nanotubes (CNTs) instead of halloysite nanotubes (HNTs), the obtained biomatrix glue had 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 this application. The swelling rate of the prepared biomatrix glue is lower than 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 are combined with hyaluronic acid to significantly improve the expansion rate of biomatrix glue. The tubular structure of halloysite nanotubes (HNT) and the high viscosity of hyaluronic acid form a stable three-dimensional network together, which enhances 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 application rationally adjusts both concentrations to help achieve optimal performance. The hyaluronic acid within the concentration range of the application can promote the expansion rate of biomatrix glue, and simultaneously 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 sections. They were 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 was poor and a full-thickness skin model could not 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 methods: C57BL / 6 mice were randomly divided into three experimental and control groups, with three mice in each group. Each group fasted for 12 hours before surgery. The animals were anesthetized with an intramuscular injection of 2% sodium pentobarbital. The back skin was prepared and disinfected with iodine. A sterile drape was then placed on the back of the mice. A circular, full-thickness skin defect with a diameter of 0.8 cm was surgically incised on the back of the mice. 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. The dressing was changed every two days.

[0123] Wound images were captured with a digital camera on days 0, 2, 5, 7, and 14. Image analysis was performed using Java-based image processing and analysis software (ImageJ). Skin samples were collected on day 14 for histological evaluation. The number of pixels in the marked area was converted into the actual area to obtain wound area data for evaluating 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 the pictures of the wound healing process at different time points (day 0, day 2, day 5, day 7, day 14), and the groups are mainly the control group, the group without halloysite nanotubes (Comparative Example 1) and the group treated with halloysite nanotubes (Example 1). Since the CNTs in Comparative Example 2 easily formed colonies in vitro and could not be implanted in mice, they were not used.

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

[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 a Microtester G2 (CellScale). The biomatrix glue was compressed at a compression rate of 1 mm / min and a strain of 5%-10%. 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, and is conducive to the adhesion of upper layer keratinocytes, which is conducive to the construction of a full-thickness skin model.

[0131] Finally, it should be noted that the above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by persons skilled in the art without departing from the spirit and technical concepts disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A biomatrix glue, characterized in that It is composed of the following components 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, wherein 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 then incubated for shaping, and then a complete culture medium is added for culturing.

7. The method for constructing the dermis 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 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 dermis layer is constructed by the method for constructing the dermis layer of the skin according to any one of claims 6 to 8.

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 and mix them to obtain a cell-gel premix; pipette the cell-gel premix into a culture dish, incubate it until it forms, and then add complete culture medium and culture it statically to obtain a dermis layer; S3. The cultured epidermal cells are digested and inoculated into the dermis layer 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 10, wherein: The culture in step S3 specifically includes replacing the culture medium every 2 days, and re-inoculating epidermal cells on the surface, and culturing continuously for 5 days.

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

13. The method for constructing a full-thickness skin model according to claim 10, wherein: The culture conditions in steps S2 and S3 are 37° C. and 5% CO 2 .

14. A full-thickness skin model constructed using 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 pharmaceuticals.

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