CN110772669A - A bioink for 3D printing artificial skin - Google Patents

Abstract

The invention relates to a bio-ink for 3D printing artificial skin, belonging to the technical field of tissue engineering, comprising a component A for constructing an epidermis layer, a component B for constructing a dermis layer, and a component B for constructing an acellular matrix scaffold. Component C, wherein, component A includes a first seed cell, a first carrier hydrogel, a first growth factor sustained-release gel containing traditional Chinese medicine ingredients, the first carrier hydrogel and a first growth factor sustained-release gel The mass ratio of the gel is (60:1)-(60:8); the B component includes the second seed cell, the second carrier hydrogel and the second growth factor slow-release gel. It is added with Chinese medicinal ingredients such as dried blood and white leeks, and growth factor slow-release gel, and seed cells are extracted from the patient's own skin. The invention has good biocompatibility, can effectively promote cell proliferation and regeneration and wound healing, has good mechanical and fluid properties, and can be used in 3D printing skin and other related fields.

Description

A bioink for 3D printing artificial skin

technical field

The present invention relates to a bioink for 3D printing artificial skin.

Background technique

In China, 26 million people suffer from different degrees of burns every year. Among the burn patients, 49% are disabled and 8% are permanently disabled. For the general treatment methods for first- and second-degree burns, scalding ointment and antibiotics are mainly used. However, for patients with third-degree burns and deep second-degree burns whose wounds cannot heal by themselves, skin grafting is required. According to statistics, there are more than 3.2 million patients who need skin transplantation every year, and about 200,000 patients need to use skin replacement materials in a large area. According to international statistics, the average amount is 5000 cm ² per person, and the total demand is nearly 109 cm ² . However, due to limited medical technology, large skin grafting area, and high price, autologous transplantation cannot meet the needs of patients, and the treatment cycle is long, which seriously affects the daily life of patients. In recent years, the development of 3D bioprinting has provided new ideas for skin transplantation, bringing dawn to patients with skin diseases and skin-related industries.

3D printing technology is a new type of digital forming technology based on computer three-dimensional digital imaging technology and multi-level continuous printing. 3D bioprinting technology is based on 3D printing, a technology that uses living cells as raw materials to print living tissues, which can achieve precise control, layer-by-layer printing using living cells as raw materials, and build skin models in vitro for skin disease research. , therapeutic or topical drug development. 3D bioprinting provides an efficient and automated method for printing skin with layered structures, which can control the number of layers, cell density and precise placement of cells and materials in the printed skin, establish intercellular connections, and promote intercellular parasites in the printed tissue. Secretory effects and cell-to-cell interactions, and printing itself does not affect the biological behavior of cells. By 3D printing artificial skin, not only the time and cost of skin culture can be reduced, but also through 3D scanning, the artificial skin has a higher fit with the wound than traditional autologous transplantation, which can improve the speed of wound healing and shorten the treatment period. And if it is printed with the patient's own cells, it will greatly reduce the risk of immune rejection in the body.

The most important thing for 3D printing skin is the raw material used for 3D bioprinting, namely bioink. Bio-inks mostly use hydrogels produced from collagen as raw materials. Although with the continuous development of 3D printing skin technology, the materials for 3D printing skin are becoming more and more extensive, and there are also a large number of papers and patents related to 3D printing artificial skin bio-inks in China, but the existing bio-inks are not yet fully satisfied. need. For example: (1) The skin stem cell extraction technology and 3D scanning modeling technology mentioned in the invention patent "A Bioprinted Fully Customized Skin and Its Preparation Method" (Publication No. CNIO8392676A). Although through 3D scanning, fully customized skin that conforms to specific parts of the human body and specific 3D features can be printed, which improves the fit and success rate of artificial skin and wounds, but the extraction of stem cells does not take into account immune rejection and exogenous stem cells. and cannot be completely differentiated. (2) The invention patent "A method of constructing skin tissue based on biological 3D printing" (Publication No. CN106860918A) uses collagen sponge to structure artificial skin, although it can shorten the wound healing period and enhance the skin elasticity after wound healing, but in There is a case of bacterial contamination during the printing process.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a bio-ink for 3D printing artificial skin, so as to improve the printing efficiency and quality of the artificial skin.

In order to solve the above-mentioned technical problems, the technical scheme of the present invention is as follows:

A bioink for 3D printing artificial skin, including component A for building epidermis, component B for building dermis, and component C for building acellular matrix scaffolds, wherein component A Including the first carrier hydrogel containing the first seed cell, the first growth factor slow-release gel containing the Chinese medicinal composition, the mass ratio of the first carrier hydrogel and the first growth factor slow-release gel is (60 : 1)-(60:8); The B component includes a second growth factor slow-release gel, a second carrier hydrogel containing a second seed cell, the second carrier hydrogel and the second growth factor slow-release gel The mass ratio of the released gel was (30:1)-(30:4).

Further, the first seed cells are stem cells obtained from autologous skin tissue, and selecting autologous epidermal stem cells can greatly reduce immune rejection during transplantation.

Further, the second seed cells include epidermal mesenchymal stem cells, adipose stem cells and hair follicle stem cells obtained from autologous skin tissue, and selecting autologous stem cells can greatly reduce immune rejection during transplantation.

Typically, carrier hydrogels include, but are not limited to, fibrin, collagen, silk fibroin, hyaluronic acid, chitosan, acellular extracellular matrix, polycaprolactone, polyethylene glycol, poly(lactic-co-glycolic acid) In contrast, although fibrin has good biocompatibility and cell adhesion ability, its structural strength is low and lacks long-term stability; collagen has low immunogenicity and good biocompatibility, and can be absorbed by in vivo tissues. Degradation, can combine with integrins to enhance cell adhesion and proliferation, but poor mechanical properties, must be chemically modified or cross-linked with other materials; silk fibroin has good biocompatibility and mechanical properties, but in vivo The degradation rate is slow, which affects the regeneration and recovery of the skin; the cross-linking of hyaluronic acid and other materials can improve the material properties, but its mechanical properties are poor; polycaprolactone has good tensile hardness, biocompatibility and stability , but the elasticity is poor and the rheological properties are not strong; the polylactic acid-glycolic acid copolymer material itself and its degradation products have good biocompatibility, but the cell adhesion is poor, which affects the proliferation and regeneration of stem cells. In summary, considering the degradability and its degradation rate, biocompatibility, mechanical properties and rheological properties, the cross-linking method, cross-linking site and density of the scaffold material, the water composition of the material and tissue cells are considered. The porosity, pore size, swelling ratio and other influencing factors of the gel are preferably collagen and acellular matrix materials.

Collagen is the most important material of human extracellular matrix. Type I, II and IV collagen are common in human skin. The amino acids in collagen are mainly composed of α-amino acids, the content of glycine is 34wt%, hydroxyproline is about 10wt%, and proline is about 12wt%. The above-mentioned required collagen must be in a sterile state, and considering that the collagen may be denatured after being sterilized by physical or chemical means, type I collagen is preferred. Further, considering the improvement of the mechanical properties of collagen, due to the good biocompatibility of sodium alginate, the wide range of sources, the displaceable divalent ions and the gel properties, the type I collagen mixed with sodium alginate solution and acellular matrix is preferred. combination.

Further, the carrier hydrogel is mainly formed by mixing type I collagen solution, sodium alginate solution and acellular skin matrix solution in a volume ratio of (18-22):(18-22):1.

Further, the concentration of the type I collagen solution is 0.4-0.5 wt %, the concentration of the sodium alginate solution is 2-3 wt %, and the concentration of the acellular skin matrix solution is 28-32 wt %.

Preferably, the concentration of the type I collagen solution is 0.4 wt %, the concentration of the sodium alginate solution is 2 wt %, and the concentration of the acellular skin matrix solution is 30 wt %.

Preferably, for the second carrier hydrogel, the concentration of the type I collagen solution is 0.5 wt %, the concentration of the sodium alginate solution is 3 wt %, and the concentration of the acellular skin matrix solution is 30 wt %.

Further, the growth factor slow-release gel includes two parts, the slow-release gel carrier and the growth factor. Further, sustained-release gel carriers include but are not limited to polyglycolic acid PGA, polylactic acid PLA, polylactic acid and polyglycolic acid copolymer PLGA, polyglycolactone PCL, polylactide, collagen, gelatin, chitosan and One or more of hyaluronic acid, etc., of which polyglycolic acid PGA, polylactic acid PLA, polylactic acid and polyglycolic acid copolymer PLGA are the earliest FDA-certified synthetic degradable biopolymer materials that agree to be applied to the human body , can be used as the carrier material for growth factors, and the spheroidization performance is good. PLGA copolymer is mixed by 50% of polyglycolic acid PGA and polylactic acid PLA. It combines the advantages of both and can be completely degraded, but the strength is high and the degradation time is prolonged; the degradation time can be precisely adjusted, but there is a biological phase. The disadvantage of poor tolerance. Natural materials such as collagen have good biocompatibility, no antigenicity, can promote cell adhesion and proliferation, and can be made into different shapes as needed. Among them, collagen is rich in sources, and has the advantages of low immunogenicity, good biocompatibility, degradability, and participation in tissue repair and reconstruction.

The microsphere sustained-release system has better performance than the traditional sustained-release carrier material. The microsphere structure can protect the drug from the damage of the external environment and has stable performance, so it can prolong the sustained-release time. To sum up, the combination of PLGA microspheres and collagen is preferred as the composition of the sustained-release gel carrier.

Further, the growth factor slow-release gel includes PLGA microspheres, collagen and growth factors, the growth factors being one or more of EGF, KGF-2, VEGF, PDGF, TGF-β, and bFGF.

Further, for the first growth factor sustained-release gel, the growth factors include epidermal growth factor EGF and keratinocyte growth factor KGF-2. EGF can be used as a chemokine to generate chemotactic signals, so that cells and protein matrices can aggregate on the wound surface, repair the wound, and can also promote the mitosis and proliferation of related cells. EGF is also a mitogen of fibroblasts and vascular endothelial cells, which can promote the synthesis of matrix components such as collagen and fibrin, and accelerate wound healing. KGF-2 can specifically accelerate the gene transcription, gene replication and protein synthesis of wound tissue cells, thereby promoting mitosis, proliferation, differentiation and migration of epithelial cells.

Further, for the second growth factor sustained-release gel, the growth factors include vascular endothelial growth factor VEGF, platelet-derived endothelial cell growth factor PDGF, transforming growth factor beta (TGF-beta) and basic fibroblast growth factor bFGF. VEGF has the effect of promoting vascular endothelial proliferation and stem cell differentiation, and can promote the increase of vascular permeability. PDGF has a variety of biological functions, can stimulate various types of cell division, proliferation and the production of extracellular matrix, is a mitogen and chemokine of a variety of cells. TGF-β can promote the growth of fibroblasts, osteoblasts and Schwann cells, and can also promote the expression and inhibit degradation of extracellular matrix such as collagen and fibronectin, which can accelerate wound healing and facilitate cell repair. bFGF has the effect of promoting angiogenesis, and may also promote the chemotaxis and aggregation of fibroblasts and other cells to the wound surface, thereby promoting the growth of granulation and blood vessel formation in the wound surface, accelerating the division and proliferation of cells, and shortening the healing time of the wound surface; it can also accelerate the wound tissue. The gene transcription, gene replication and protein synthesis of cells promote the mitosis, proliferation and differentiation of fibroblasts and epithelial cells; it can promote the migration of macrophages, mesenchymal cells, endothelial cells and fibroblasts to the wound site.

Further, the traditional Chinese medicine composition includes but is not limited to Dracula sinensis, white radish, Codonopsis pilosula, Poria, white peony, Burnet, calamine, Phellodendron, Cnidium, longum, light powder, etc., from aspects such as efficacy and cost. Considered, Draconis and white leeks are preferred.

External application of Dermatophaga can stop bleeding and regenerate muscles, astringe sores, and treat bruises, bruises, congestion, swelling and pain, traumatic bleeding, and ulcers that do not converge. Studies have found that dried blood can also promote the differentiation of hair follicle stem cells. Zhang Dongping, Cao Jianchun, Ju Shang, etc. found that Xuejieshengji ointment can promote the healing of ulcer wounds by increasing the expression of VEGF in the ulcer wounds of diabetic rats, promoting the formation of new blood vessels and the growth of granulation tissue, and improving the local nutritional status. . It also has a significant promoting effect on the proliferation of wound tissue cells, can accelerate the re-epithelialization of the wound, and promote wound healing. It has the functions of clearing away heat and detoxifying, dispelling carbuncle and dissipating knots, astringing sores and promoting muscle growth. Studies have shown that white leeks also have inhibitory effects on Candida albicans and Staphylococcus aureus.

Further, the components of the traditional Chinese medicine include dried blood and white leeks, and the mass ratio thereof is 3:5.

Further, the preparation method of carrier hydrogel comprises the following steps:

Take type I collagen solution with a concentration of 0.4-0.5wt% and a sodium alginate solution with a concentration of 2-3wt%, mix them evenly, and control the pH of the solution to be 6-7.45 to prepare a collagen-algae biogel;

The acellular skin matrix solution and seed cells with a concentration of 28-32wt% are added to the collagen-algae biogel, and the mixture is evenly mixed, so that the seed cell concentration is 1×10 ⁵ -1×10 ⁷ cells/ml, to prepare carrier hydrogel.

Further, the preparation method of the first growth factor slow-release gel comprises the following steps:

① Using 0.001-0.002wt% EGF solution and 0.00001-0.001wt% KGF-2 solution as the inner water phase, and 20-70wt% PLGA dichloromethane solution as the oil phase, EGF-KGF-KGF- 2-PLGA solution, remove the organic solvent, centrifuge, precipitate, and then freeze-dry to obtain EGF-KGF-2-PLGA microspheres;

Among them, in the centrifugation process, the rotating speed is 900-1100r/min, and the centrifugation time is 4-6min;

②Add type I collagen to the acetic acid solution with a concentration of 0.1-0.3wt%, stir evenly to obtain a collagen solution;

③Add the EGF-KGF-2-PLGA microspheres and traditional Chinese medicine ingredients prepared in step ① into the collagen solution, stir evenly, pre-freeze at -70°C for 8h-30h, and then freeze-dry at a concentration of 60 After solidifying the 90% ethanol solution for 20min-40min, freeze-drying is performed again to obtain the first growth factor slow-release gel embedded with EGF-KGF-2-PLGA microspheres.

Further, the preparation method of the second growth factor slow-release gel comprises the following steps:

①0.001-0.002wt% VEGF solution, 0.0001-0.001wt% PDGF solution, 0.00001-0.001wt% TGF-β solution, 0.0001-0.001wt% bFGF solution as inner water phase, 20-70wt% PLGA dichloromethane solution as In the oil phase, the VEGF-PDGF-TGF-β-bFGF-PLGA solution was obtained by double emulsification method, and the organic solvent was removed, followed by centrifugation, precipitation, and then freeze-dried to obtain VEGF-PDGF-TGF-β-bFGF-PLGA microparticles. ball;

②Add type I collagen to 0.1-0.3wt% acetic acid solution, stir evenly, and prepare a collagen solution;

③Add the VEGF-PDGF-TGF-β-bFGF-PLGA microspheres prepared in step ① into the collagen solution, stir well, pre-freeze at -70°C for 8h-30h, and then freeze-dry, using 60-90wt% After the ethanol is solidified for 20min-40min, freeze-drying is performed again to obtain the second growth factor sustained-release gel embedded with VEGF-PDGF-TGF-β-bFGF-PLGA microspheres.

The C components include sigma collagen, sodium alginate and gelatin in terms of the material's mechanical strength, porosity, ability to promote cell proliferation, and changes in material properties at lower concentrations. Collagen, sodium alginate and gelatin were selected for cross-linking treatment under certain conditions. The prepared gel should have the following characteristics after hydrodynamic test and cytotoxicity test: good biocompatibility and biodegradability. Can be combined with a variety of growth factors to promote the proliferation and differentiation of surrounding cells; high adhesion to cells; moderate mechanical strength, with a certain compressive ability; good elasticity; the printing process forms acellular matrix scaffolds During the process, it is not easy to collapse.

Further, the preparation method of C component comprises the steps:

Dissolve gelatin and sodium alginate in PBS buffer to obtain a gelatin solution with a concentration of 10wt%;

Dissolving sodium alginate in PBS buffer to obtain a sodium alginate solution with a concentration of 2wt%;

Adjust the pH value of described gelatin solution, sodium alginate solution respectively to 7.2, then carry out sterilization treatment, standby;

Weigh type I Sigma collagen powder and add it to acetic acid to obtain a collagen solution with a final concentration of 30 mg/mL.

The gelatin solution, the collagen solution and the sodium alginate solution are uniformly mixed in a mass ratio of 1:1:3-5, and after sterilization, the C component is obtained.

In the present invention, traditional Chinese medicine ingredients such as dried blood and white leeks are added to the bioink to improve the wound healing and repair ability; the growth factor slow-release gel with microsphere structure is used to promote the fusion, growth and repair of the skin, and prolong the slow-release action time; Seed cells are extracted from the patient's own skin, which greatly reduces the patient's own immune rejection during the transplantation process, which is conducive to the recovery of the wound at the transplanted skin and the regeneration of related tissues; adding gel components that improve mechanical properties such as sodium alginate and collagen And gelatin, etc., so that it is not easy to collapse during the printing process. The bio-ink of the present invention can greatly improve the printing efficiency and quality of artificial skin, reduce the period of skin transplantation for patients, and is of great significance in the research and treatment of skin diseases, and product development in skin-related industries such as beauty.

The bioink for 3D printing artificial skin of the present invention has moderate mechanical properties and biocompatibility, can reduce immune rejection during transplantation, can also improve wound healing and repair ability, promote skin fusion and growth, and has the advantages of Good mechanical and fluid properties are of great significance in 3D printing skin and other related fields.

Description of drawings

Figure 1 is a graph showing the relationship between the compressive modulus of the acellular carrier gels prepared in Examples 1-3 under different mass ratios.

Figure 2 is a graph showing the relationship between the degradation rates on the 15th day of the acellular carrier gels prepared in Examples 1-3 under different mass ratios.

Figure 3 is a graph showing the relationship between the breaking strengths of the acellular carrier gels prepared in Examples 1, 4 and 5 under the condition of cross-linking with different concentrations of CaCl ₂ solutions.

Detailed ways

The following description describes alternative embodiments of the invention to teach those of ordinary skill in the art how to implement and reproduce the invention. In order to teach the technical solutions of the present invention, some conventional aspects have been simplified or omitted. Unless otherwise specified, the percentage should generally be understood as the mass percentage.

A preparation method of bioink for 3D printing artificial skin, comprising the following steps:

S1: Enzymatic hydrolysis solution configuration: prepare 2g/L neutral protease solution, 0.25% trypsin and 0.02% EDTA with D-Hanks solution, filter and sterilize and store at 4°C for later use.

S2: Preparation of type IV collagen culture flask: Dissolve human type IV collagen in acetic acid solution with a volume fraction of 0.1%, filter and sterilize, and store at 4°C for later use. Spread 1-2ml of prepared type IV collagen in a culture flask overnight, discard the supernatant, rinse 3-4 times with PBS solution with a concentration of 0.01mol/L, dry in a drying oven at 40°C, and irradiate with ultraviolet light. 2h for disinfection.

S3: Extraction of epidermal stem cells: Autologous normal skin slices were taken, washed with PBS solution containing double antibiotics (penicillin, streptomycin) for 5-6 times, and the subcutaneous tissue was removed under sterile conditions. Cut the skin pieces into 1.0cm×1.0cm size, put them in low-sugar DMEM/F12 medium containing neutral proteolytic enzymes, digest at 4°C for 14-16h, discard the supernatant, and separate the epidermis and dermis. Cut the epidermis, digest with 0.25% trypsin at 37°C for 15 min, add DMEM containing 10% fetal bovine serum or human autologous serum to terminate the digestion, and filter with a 200-mesh sieve after pipetting. The filtrate was centrifuged (1000 r/min) for 5 min and the cells were collected. Discard the supernatant, add epidermal stem cell medium and gently pipette to make a single-cell suspension, inoculate it in a culture flask pre-coated with type IV collagen at an appropriate density, and incubate at 37 °C for 15 min, aspirate the culture medium and non-adherent cells. Discard the non-adherent cell components, wash twice with D-Hanks solution, add an appropriate amount of epidermal stem cell culture medium, and culture in a 37°C, 5% carbon dioxide incubator. After 24 hours, the culture medium was aspirated, rinsed once with PBS, and then added with epidermal stem cell medium, and placed in a 37° C., 5% carbon dioxide incubator to continue culturing. After that, the medium was changed every 48 h, and the cell growth was observed under a light microscope.

S4: Identification of epidermal stem cells: take part of the cells, wash 3 times with PBS, fix with 4% paraformaldehyde at room temperature for 30 minutes, and perform two-step immunohistochemical detection for K19 and β1 integrin immunocytochemical staining; Antibody was used as blank control. Avoid the above steps as much as possible.

S5: Preparation of the first carrier hydrogel: at room temperature, take type I collagen solution with a concentration of 0.4% and sodium alginate solution with a concentration of 2%, mix them evenly at a volume ratio of 1:1, and control the pH of the solution to be 6 -7.45, modulated into collagen-algae biogel. The acellular skin matrix solution with a concentration of 30% and the seed cells obtained from S3 were added to the collagen-algae biogel, wherein the collagen-algae biogel and the acellular skin matrix solution were mixed thoroughly at a volume ratio of 20:1 and stirred well , the seed cell concentration was 1×10 ⁵ -1×10 ⁷ cells/ml, and the epidermal carrier hydrogel was prepared.

S6: the dried blood and the white stalk are ground into powder, the dried sage: the ratio (mass ratio) of 3:5 to obtain the traditional Chinese medicine composition;

S7: Preparation of Epidermal Growth Factor Sustained-Release Gel (First Growth Factor Sustained-Release Gel):

①0.001%-0.002% EGF solution, 0.00001%-0.001% KGF-2 solution as inner water phase, 20%-70% PLGA dichloromethane solution as oil phase, EGF-KGF-2 obtained by double emulsion method -PLGA solution, remove the organic solvent, centrifuge (1000r/min) for 5min, precipitate, and then freeze-dry to obtain EGF-KGF-2-PLGA microspheres.

②Add type I collagen to 0.1%-0.3% acetic acid solution, stir evenly, and prepare a collagen solution.

③Add the microspheres prepared in step ① and the traditional Chinese medicine ingredients obtained in S6 into the collagen solution, stir evenly, pre-freeze at -70°C for 8h-30h, then freeze-dry, and use 60%-90% ethanol for solidification After 20min-40min, freeze-drying is performed again to obtain a collagen carrier embedded with EGF-KGF-2-PLGA microspheres.

S8: Extraction of Dermal Mesenchymal Stem Cells: Autologous normal skin slices were taken, washed with PBS solution containing double antibiotics (penicillin, streptomycin) for 5-6 times, and the subcutaneous tissue was removed under sterile conditions. Cut the skin pieces into 1.0cm×1.0cm size, put them in low-sugar DMEM/F12 medium containing neutral proteolytic enzymes, digest at 4°C for 14-16h, discard the supernatant, and separate the epidermis and dermis. The dermis was rinsed twice with PBS, minced, and then placed in a low-glucose DMEM/F12 medium containing 1% fetal bovine serum or human autologous serum and 0.1% type I collagenase, and placed in a 37°C incubator with a volume fraction of 5% carbon dioxide. After 10 hours, PBS containing 2% fetal bovine serum or human autologous serum was added to terminate the digestion. The filtrate was centrifuged (1500 r/min) for 5 min and the cells were collected. Discard the supernatant, add DMEM/F12 medium containing 10% fetal bovine serum or human autologous serum and gently pipet to make a single cell suspension, inoculate it in a culture flask at an appropriate density, and change the medium every 48h. When the cells reached 80-90% confluence, they were digested with enzymatic hydrolysis solution, and the separated cells were inoculated into new culture flasks to continue culturing, and then the medium was changed every 60 hours.

S9: Identification of Dermal Mesenchymal Stem Cells: Take well-grown third-generation monolayer culture-expanded dermal-derived cells, and fix with 4% paraformaldehyde for 10 minutes. After that, 0.2% Triton X-100 was added dropwise, incubated at 25°C for 10 min, and then rinsed with PBS. After blocking with 10% fetal bovine serum or human autologous serum for 1 h, CD90, CD105 antibody and vimentin were added dropwise, and the cells were kept at 4°C overnight. After 24 hours, goat anti-mouse IgG FITC or IgG-Texas Red was added dropwise, incubated at room temperature for 1 hour, and then the nuclei were stained with DAPI and observed under a fluorescence microscope.

S10: Extraction of hair follicle stem cells: In a sterile room, hair follicle stem cells were extracted from autologous hindbrain epidermis. The hair follicle stem cells were inoculated into DMEM/F12 medium and cultured in a 37°C incubator with a volume fraction of 5% carbon dioxide.

S11: Extraction of adipose stem cells: Take adipose tissue under aseptic conditions, wash with PBS solution containing double antibiotics (penicillin, streptomycin) at 4°C for 5-6 times, and remove blood vessels and connective tissue under aseptic conditions. Cut the adipose tissue into small pieces of 0.2cm×0.2cm size in a petri dish, digest it with 0.2% collagenase type I at 37°C for 15 min, and add an equal volume of low-glucose DMEM/F12 containing 10% fetal bovine serum or human autologous serum for culture The digestion was terminated, and after pipetting, it was filtered through a 200-mesh sieve. The filtrate was centrifuged (1000 r/min) for 5 min and the cells were collected. After discarding the supernatant, add 3 mL of erythrocyte lysate, pipetting for 5 min, centrifuge (1000 r/min) for 5 min, discard the supernatant, wash repeatedly with PBS, resuspend the cells in DMEM medium containing 10% fetal bovine serum, and inoculate them into a culture dish. Placed in a 37°C, volume fraction of 5% carbon dioxide incubator. The medium was changed every 48h, and when the cells grew to 75%-90% confluence, they were digested with enzymolysis solution, and the separated cells were inoculated into new culture flasks to continue culturing, and then the medium was changed every 72h.

S12: Identification of adipose stem cells: Take the well-grown third-generation cells and inoculate them in a 24-well plate at an appropriate density. After 24, take the well-grown cells in 4 wells, 2 wells are used as the experimental group, and 2 wells are used as the control group. The culture medium was discarded, washed with PBS for 2-3 times, fixed with 4% paraformaldehyde for 20 min, and then washed with PBS for 3 times. The experimental group was added with FITC-labeled CD34 and CD44 antibodies, and kept at 4°C. Incubate in light for 2 h, wash with PBS, and observe under a fluorescence microscope.

S13: Preparation of carrier hydrogel: Take type I collagen solution with a concentration of 0.5% and a sodium alginate solution with a concentration of 3% at room temperature, mix them evenly in a volume ratio of 1:1, and control the pH of the solution to be 6-7.45 , modulated into collagen-algae biogel. The acellular skin matrix solution at a concentration of 30% and the relevant seed cells obtained in S8-12 were added to the collagen-algae biogel, wherein the collagen-algae biogel and the acellular skin matrix solution were sufficient at a volume ratio of 20:1 Mix and stir evenly so that the seed cell concentration is 1×10 ⁵ -1×10 ⁷ cells/ml to prepare a dermal layer carrier hydrogel (second carrier hydrogel).

S14: Preparation of dermal growth factor sustained-release gel (second growth factor sustained-release gel):

①0.001%-0.002% VEGF solution, 0.0001%-0.001% PDGF solution, 0.00001%-0.001% TGF-β solution, 0.0001%-0.001% bFGF solution as inner water phase, 20%-70% PLGA dichloromethane solution As the oil phase, the two obtained VEGF-PDGF-TGF-β-bFGF-PLGA solution by double emulsion method, removed the organic solvent, centrifuged (1000r/min) for 5min, precipitated, and then lyophilized to obtain VEGF-PDGF-TGF - β-bFGF-PLGA microspheres.

②Add type I collagen to 0.1%-0.3% acetic acid solution, stir evenly, and prepare a collagen solution.

③Add the microspheres prepared in step ① into the collagen solution, stir evenly, pre-freeze at -70°C for 8h-30h, then freeze-dry, and use 60%-90% ethanol to solidify for 20min-40min, and then again Freeze-drying is performed to obtain a collagen carrier for embedding VEGF-PDGF-TGF-β-bFGF-PLGA microspheres.

The first carrier hydrogel and the first growth factor sustained-release gel are added in the A component according to the proportions; the B component is added with the second growth factor sustained-release gel and the second carrier hydrogel according to the proportions. When the skin is printed by inkjet bioprinting technology, the A component is added to the epidermis ink cartridge, and the B component is added to the dermal layer ink cartridge.

S15: Preparation of acellular matrix gel:

① Dissolve gelatin in PBS buffer to form a 10% solution, dissolve sodium alginate in PBS buffer to form a 2% solution, neutralize the residual acetic acid with NaOH, adjust the pH to 7.2, and place in an oven at 60°C Intermittent sterilization.

②Weigh a certain mass of type Ⅰ Sigma collagen powder, add acetic acid, the final concentration is 30mg/mL, stir, and after stirring, put it in a 4°C refrigerator overnight.

③ According to the mass ratio of gelatin solution:collagen solution:sodium alginate solution is 1:1:4, mix evenly, and carry out high temperature sterilization.

④ The constructed 3D model is reconstructed with 3D modeling software to generate an STL file, imported into a 3D bioprinter for layering and setting of printing parameters, and then printing is started.

⑤ After the model printing is completed, drop an appropriate amount of 5% CaCl ₂ solution on the surface of the scaffold, and cross-link for 30s. When the model is completely formed, the excess CaCl ₂ solution is sucked away, and the scaffold is repeatedly washed three times with PBS buffer, an appropriate amount of PBS solution is added, and it is placed in a carbon dioxide incubator.

Example 1

A bioink for 3D printing artificial skin, the preparation of its C component and acellular matrix scaffold includes the following steps:

① Dissolve gelatin in PBS buffer to form a 10wt% solution; dissolve sodium alginate in PBS buffer to form a 2wt% solution; neutralize the residual acetic acid with NaOH, adjust the pH to 7.2, and put it in a 60°C oven intermittently Sterilize.

②Weigh a certain mass of type Ⅰ Sigma collagen powder, add acetic acid, the final concentration is 30mg/mL, stir, and after stirring, put it in a 4°C refrigerator overnight.

③ According to the mass ratio of gelatin solution:collagen solution:sodium alginate solution is 1:1:2, mix well, and carry out high temperature sterilization.

④ The constructed 3D model is reconstructed with 3D modeling software to generate an STL file, imported into a 3D bioprinter for layering and setting of printing parameters, and then printing is started.

⑤ After the model printing is completed, drop an appropriate amount of 5% CaCl ₂ solution on the surface of the scaffold, and cross-link for 30s. When the model is completely formed, the excess CaCl ₂ solution is sucked away, and the scaffold is repeatedly washed three times with PBS buffer, an appropriate amount of PBS solution is added, and it is placed in a carbon dioxide incubator.

Example 2

A bioink for 3D printing artificial skin, the preparation of its C component and acellular matrix scaffold includes the following steps:

① Dissolve gelatin in PBS buffer to form a 10% solution, dissolve sodium alginate in PBS buffer to form a 2% solution, neutralize residual acetic acid with NaOH respectively, adjust the pH to 7.2, and intermittently in a 60°C oven Sterilize.

②Weigh a certain mass of type Ⅰ Sigma collagen powder, add acetic acid, the final concentration is 30mg/mL, stir, and after stirring, put it in a 4°C refrigerator overnight.

③ According to the mass ratio of gelatin solution:collagen solution:sodium alginate solution is 1:1:4, mix evenly, and carry out high temperature sterilization.

④ The constructed 3D model is reconstructed with 3D modeling software to generate an STL file, imported into a 3D bioprinter for layering and setting of printing parameters, and then printing is started.

⑤ After the model printing is completed, drop an appropriate amount of 5% CaCl ₂ solution on the surface of the scaffold, and cross-link for 30s. When the model is completely formed, the excess CaCl ₂ solution is sucked away, and the scaffold is repeatedly washed three times with PBS buffer, an appropriate amount of PBS solution is added, and it is placed in a carbon dioxide incubator.

Example 3

A bioink for 3D printing artificial skin, the preparation of its C component and acellular matrix scaffold includes the following steps:

① Dissolve gelatin in PBS buffer to form a 10% solution, dissolve sodium alginate in PBS buffer to form a 2% solution, neutralize residual acetic acid with NaOH respectively, adjust the pH to 7.2, and intermittently in a 60°C oven Sterilize.

②Weigh a certain mass of type Ⅰ Sigma collagen powder, add acetic acid, the final concentration is 30mg/mL, stir, and after stirring, put it in a 4°C refrigerator overnight.

③ According to the mass ratio of gelatin solution:collagen solution:sodium alginate solution is 1:1:8, mix well, and carry out high temperature sterilization.

④ The constructed 3D model is reconstructed with 3D modeling software to generate an STL file, imported into a 3D bioprinter for layering and setting of printing parameters, and then printing is started.

⑤ After the model printing is completed, drop an appropriate amount of 5% CaCl ₂ solution on the surface of the scaffold, and cross-link for 30s. When the model is completely formed, the excess CaCl ₂ solution is sucked away, and the scaffold is repeatedly washed three times with PBS buffer, an appropriate amount of PBS solution is added, and it is placed in a carbon dioxide incubator.

Example 4

A bioink for 3D printing artificial skin, the preparation of its C component and acellular matrix scaffold includes the following steps:

① Dissolve gelatin in PBS buffer to form a 10% solution, dissolve sodium alginate in PBS buffer to form a 2% solution, neutralize residual acetic acid with NaOH respectively, adjust the pH to 7.2, and intermittently in a 60°C oven Sterilize.

②Weigh a certain mass of type Ⅰ Sigma collagen powder, add acetic acid, the final concentration is 30mg/mL, stir, and after stirring, put it in a 4°C refrigerator overnight.

③ According to the mass ratio of gelatin solution:collagen solution:sodium alginate solution is 1:1:4, mix evenly, and carry out high temperature sterilization.

④ The constructed 3D model is reconstructed with 3D modeling software to generate an STL file, imported into a 3D bioprinter for layering and setting of printing parameters, and then printing is started.

⑤ After the model printing is completed, drop an appropriate amount of 2% CaCl ₂ solution on the surface of the scaffold, and cross-link for 30 s. When the model is completely formed, the excess CaCl ₂ solution is sucked away, and the scaffold is repeatedly washed three times with PBS buffer, an appropriate amount of PBS solution is added, and it is placed in a carbon dioxide incubator.

Example 5

A bioink for 3D printing artificial skin, the preparation of its C component and acellular matrix scaffold includes the following steps:

① Dissolve gelatin in PBS buffer to form a 10% solution, dissolve sodium alginate in PBS buffer to form a 2% solution, neutralize residual acetic acid with NaOH respectively, adjust the pH to 7.2, and intermittently in a 60°C oven Sterilize.

②Weigh a certain mass of type Ⅰ Sigma collagen powder, add acetic acid, the final concentration is 30mg/mL, stir, and after stirring, put it in a 4°C refrigerator overnight.

③ According to the mass ratio of gelatin solution:collagen solution:sodium alginate solution is 1:1:4, mix evenly, and carry out high temperature sterilization.

④ The constructed 3D model is reconstructed with 3D modeling software to generate an STL file, imported into a 3D bioprinter for layering and setting of printing parameters, and then printing is started.

⑤ After the model printing is completed, drop an appropriate amount of 8% CaCl ₂ solution on the surface of the scaffold, and cross-link for 30 s. When the model is completely formed, the excess CaCl ₂ solution is sucked away, and the scaffold is repeatedly washed three times with PBS buffer, an appropriate amount of PBS solution is added, and it is placed in a carbon dioxide incubator.

The compressive modulus of the acellular matrix gels prepared in Examples 1-3 was tested at 37° C., and the results were shown in FIG. 1 , and the degradation rates were tested, and the results were shown in FIG. 2 .

The breaking strength of the acellular matrix gels prepared in Examples 1, 4 and 5 was tested at 37°C, and the results are shown in Figure 3 .

It should be understood that these embodiments are only used to illustrate the present invention more clearly, but not to limit the scope of the present invention. After reading the present invention, those skilled in the art will recognize various equivalent forms of the present invention. The modifications fall within the scope defined by the appended claims of this application.

Claims (10)

1. The bio-ink for 3D printing of the artificial skin is characterized by comprising a component A for constructing an epidermal layer, a component B for constructing a dermal layer and a component C for constructing an acellular matrix scaffold, wherein the component A comprises a first carrier hydrogel containing first seed cells and a first growth factor slow-release gel containing traditional Chinese medicine components, and the mass ratio of the first carrier hydrogel to the first growth factor slow-release gel is (60: 1) - (60: 8); the component B comprises a second growth factor sustained-release gel and a second carrier hydrogel containing second seed cells, and the mass ratio of the second carrier hydrogel to the second growth factor sustained-release gel is (30: 1) - (30: 4).

2. The bio-ink according to claim 1, wherein the seed cells are stem cells taken from autologous skin tissue.

3. The bio-ink according to claim 1, wherein the carrier hydrogel is mainly composed of a type i collagen solution, a sodium alginate solution, and a acellular skin matrix solution in a ratio of (18-22): (18-22): 1 by volume ratio.

4. The bio-ink according to claim 3, wherein the concentration of the collagen type I solution is 0.4 to 0.5 wt%, the concentration of the sodium alginate solution is 2 to 3 wt%, and the concentration of the acellular skin matrix solution is 28 to 32 wt%.

5. The bio-ink according to claim 1, wherein the growth factor sustained release gel comprises PLGA microspheres, collagen and a growth factor, and the growth factor is one or more of EGF, KGF-2, VEGF, PDGF, TGF- β and bFGF.

6. The bio-ink according to claim 1, wherein the Chinese herbal medicine components comprise dragon's blood and Japanese ampelopsis, and the mass ratio of dragon's blood to Japanese ampelopsis is 3: 5.

7. the bio-ink according to claim 1, wherein the preparation method of the carrier hydrogel comprises the following steps:

mixing collagen I solution with concentration of 0.4-0.5 wt% and sodium alginate solution with concentration of 2-3 wt%, controlling pH of the solution at 6-7.45, and concocting to obtain collagen-seaweed biogel;

adding 28-32 wt% of acellular skin matrix solution and seed cells into the collagen-seaweed biogel, and mixing uniformly to make the concentration of the seed cells be 1 × 10 ⁵-1×10 ⁷And (4) preparing carrier hydrogel.

8. The bio-ink according to claim 1, wherein the first growth factor sustained-release gel is prepared by a method comprising the steps of:

①, taking 0.001-0.002 wt% EGF solution and 0.00001-0.001 wt% KGF-2 solution as internal water phase and 20-70 wt% PLGA dichloromethane solution as oil phase, preparing EGF-KGF-2-PLGA solution by double emulsion method, removing organic solvent, centrifuging, precipitating, and freeze drying to obtain EGF-KGF-2-PLGA microsphere;

wherein, in the centrifugation process, the rotating speed is 900-;

② adding type I collagen into 0.1-0.3 wt% acetic acid solution, and stirring to obtain collagen solution;

③, adding the EGF-KGF-2-PLGA microspheres prepared in the step ① and traditional Chinese medicine components into a collagen solution, uniformly stirring, pre-freezing for 8-30 h at-70 ℃, then freeze-drying, solidifying for 20-40 min by using an ethanol solution with the concentration of 60-90%, and freeze-drying again to obtain the first growth factor sustained-release gel embedded with the EGF-KGF-2-PLGA microspheres.

9. The bio-ink according to claim 1, wherein the preparation method of the second growth factor sustained-release gel comprises the following steps:

① 0.001, 0.001-0.002 wt% of VEGF solution, 0.0001-0.001 wt% of PDGF solution, 0.00001-0.001 wt% of TGF- β solution, 0.0001-0.001 wt% of bFGF solution as an inner water phase, and 20-70 wt% of PLGA dichloromethane solution as an oil phase, wherein the VEGF-PDGF-TGF- β -bFGF-PLGA solution is obtained by the two through a multiple emulsion method, the organic solvent is removed, centrifugation and precipitation are carried out, and then the VEGF-PDGF-TGF- β -bFGF-PLGA microspheres are obtained through freeze drying;

② adding type I collagen into 0.1-0.3 wt% acetic acid solution, stirring to obtain collagen solution;

③ adding the VEGF-PDGF-TGF- β -bFGF-PLGA microspheres prepared in the step ① into a collagen solution, uniformly stirring, pre-freezing for 8-30 h at-70 ℃, then freeze-drying, solidifying for 20-40 min by using 60-90 wt% of ethanol, and freeze-drying again to obtain the second growth factor sustained-release gel embedded with the VEGF-PDGF-TGF- β -bFGF-PLGA microspheres.

10. The bio-ink according to claim 1, wherein the preparation method of the C component comprises the following steps:

dissolving gelatin and sodium alginate in PBS buffer solution to obtain 10 wt% gelatin solution;

dissolving sodium alginate in PBS buffer solution to obtain sodium alginate solution with concentration of 2 wt%;

respectively adjusting the pH values of the gelatin solution and the sodium alginate solution to 7.2, and then performing sterilization treatment for later use;

weighing I type Sigma collagen powder, adding the powder into acetic acid to obtain a collagen solution with the final concentration of 30mg/mL, stirring, and putting the solution into a refrigerator at 4 ℃ for overnight standby;

mixing the gelatin solution, the collagen solution and the sodium alginate solution according to the ratio of 1: 1: 3-5, and sterilizing to obtain the component C.

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