CN110772669A - Biological ink for 3D printing of artificial skin - Google Patents
Biological ink for 3D printing of artificial skin Download PDFInfo
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Abstract
The invention relates to biological ink for 3D printing of artificial skin, which belongs to the technical field of tissue engineering and comprises 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 first seed cells, first carrier hydrogel and 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 seed cell, a second carrier hydrogel and a second growth factor slow-release gel. The gel is added with Chinese medicinal components such as dragon's blood, ampelopsis japonica and the like and growth factor sustained-release gel, and seed cells are extracted from the skin of a patient. The invention has good biocompatibility, can effectively promote cell proliferation and regeneration and wound healing, has good mechanical and hydrodynamic properties, and can be used in the related fields of 3D printed skin and the like.
Description
Technical Field
The invention relates to biological ink for 3D printing of artificial skin.
Background
In China, 2600 million people burn and scald at different degrees each year, and 49% of the burned patients have disability and 8% have lifelong disability. The general method for treating I and II degree burn mainly comprises smearing scald ointment and taking antibiotic medicines. However, for patients with III degree burn and deep II degree burn whose wound surface can not be self-healed, skin grafting operation is required. According to statistics, more than 320 ten thousand patients needing skin transplantation every year need to use the skin substitute materials in a large area, about 20 ten thousand patients need to use the skin substitute materials in a large area, and the average of the skin substitute materials is 5000cm per person according to international statistics
2The total demand is calculated to be about 109cm
2. However, due to the limitations of limited medical technology, large skin grafting area, high price and the like, the autograft can not meet the requirements of patients, and the treatment period is long, thus seriously affecting the daily life of the patients. In recent years, the development of 3D bioprinting provides a new idea for skin transplantation and brings eosin for skin disease patients and skin related industries.
The 3D printing technology is a novel digital forming technology based on a computer three-dimensional digital imaging technology and multi-level continuous printing. The 3D biological printing technology is a technology for printing living tissues by taking living cells as raw materials on the basis of 3D printing, can realize accurate control, can print layer by taking the living cells as the raw materials, and can be used for researching and treating skin diseases or researching and developing skin external drugs by constructing a skin model in vitro. Biological 3D printing provides an efficient and automatic method for printing skin with a layered structure, the number of layers and the cell density of the printed skin can be controlled, cells and materials can be accurately placed, intercellular connection is established, the paracrine effect among the cells in the printed tissue and the interaction among the cells are promoted, and the biological behavior of the cells is not influenced by the printing. Through 3D printing artificial skin, not only can reduce skin culture time and cost, through three-dimensional scanning, artificial skin is higher than traditional autograft and the degree of agreeing with of wound moreover, can improve wound healing's speed, shortens treatment cycle. And if the patient autologous cells are used for printing, the risk of immunological rejection of the body is greatly reduced.
The most important for 3D printing skin is the raw material for 3D bio-printing, i.e. bio-ink. The biological ink mostly uses hydrogel produced by using collagen as a raw material. Although 3D printing skin technology is continuously developed, 3D printing skin materials are more and more extensive, and a large number of papers and patents related to 3D printing artificial skin biological ink exist in China, the existing biological ink cannot completely meet the use requirement. Such as: (1) the invention patent relates to a skin stem cell extraction technology and a three-dimensional scanning modeling technology mentioned in 'a biological printing full-custom skin and a preparation method thereof' (publication number CNIO 8392676A). Although the fully-customized skin which meets the specific part and specific three-dimensional characteristics of a human body can be printed through three-dimensional scanning, the attaching property and success rate of the artificial skin and a wound are improved, the problems of immunological rejection, incomplete differentiation of exogenous stem cells and the like are not considered during the extraction of the stem cells. (2) The invention patent "a method for constructing skin tissue based on biological 3D printing" (publication No. CN106860918A) discloses that although the artificial skin with collagen sponge structure can shorten the wound healing period and enhance the skin elasticity after wound healing, bacterial contamination exists in the printing process.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides biological ink for 3D printing of artificial skin, so as to improve the printing efficiency and quality of the artificial skin.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a bio-ink for 3D printing of artificial skin comprises an A component for constructing an epidermal layer, a B component for constructing a dermal layer and a C component for constructing an acellular matrix scaffold, wherein the A component comprises a first carrier hydrogel containing first seed cells, 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 slow-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 slow-release gel is (30: 1) - (30: 4).
Furthermore, the first seed cells are stem cells taken from autologous skin tissues, and the selection of the autologous epidermal stem cells can greatly reduce immune rejection reaction in the transplanting process.
Furthermore, the second seed cells comprise epidermal mesenchymal stem cells, adipose-derived stem cells and hair follicle stem cells which are taken from autologous skin tissues, and the immune rejection reaction in the transplanting process can be greatly reduced by selecting the autologous stem cells.
Generally, the carrier hydrogel includes, but is not limited to, fibrin, collagen, silk fibroin, hyaluronic acid, chitosan, extracellular matrix, polycaprolactone, polyethylene glycol, polylactic acid-glycolic acid copolymer, etc., and in contrast, fibrin has good biocompatibility and cell adhesion capability, but has low structural strength and lacks long-term stability; collagen has low immunogenicity and good biocompatibility, can be degraded by tissues in vivo, can be combined with integrin to enhance cell adhesion and proliferation capacity, but has poor mechanical properties and needs to be chemically modified or crosslinked with other materials; the silk fibroin has good biocompatibility and mechanical property, but has slow degradation rate in organisms, thereby influencing the regeneration and recovery of skin; the hyaluronic acid and other materials can be used for improving the material performance by crosslinking, but the mechanical property of the hyaluronic acid is poor; polycaprolactone has good tensile hardness, biocompatibility and stability, but has poor elasticity and weak rheological property; the polylactic acid-glycolic acid copolymer material and the degradation product thereof have good biocompatibility, but the cell adhesion is poor, thereby influencing the proliferation and regeneration of stem cells. In summary, from the consideration of degradability and degradation rate, biocompatibility, mechanical property and rheological property, collagen and acellular matrix material are preferred by combining the cross-linking mode, cross-linking site and density of the scaffold material, and the influence factors such as porosity, pore size and swelling ratio of hydrogel formed by the material and tissue cells.
Collagen is the most important material of human extracellular matrix, and types I, II and IV collagen are common in human skin, wherein the amino acid in the collagen mainly consists of α -amino acid, the glycine content is 34 wt%, the hydroxyproline content is about 10 wt%, and the proline content is about 12 wt%.
Furthermore, the carrier hydrogel mainly comprises a type I collagen solution, a sodium alginate solution and an acellular skin matrix solution according to the weight ratio of (18-22): (18-22): 1 by volume ratio.
Furthermore, 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%.
Furthermore, the growth factor sustained-release gel comprises a sustained-release gel carrier and a growth factor. Further, the sustained-release gel carrier includes, but is not limited to, one or more of polyglycolic acid PGA, polylactic acid PLA, copolymers of polylactic acid and polyglycolic acid PLGA, polylactone PCL, polylactide, collagen, gelatin, chitosan, hyaluronic acid, and the like, wherein the polyglycolic acid PGA, the polylactic acid PLA, the copolymers of polylactic acid and polyglycolic acid PLGA are synthetic degradable biopolymer materials approved by FDA for the earliest certification and applied to human body, and can be used as carrier materials of growth factors, and have good spheronization properties. The PLGA copolymer is prepared by mixing 50% of PGA and PLA respectively, combines the advantages of the PGA and PLA, can be completely degraded, but has high strength and prolonged degradation time; the degradation time can be precisely adjusted, but there is a disadvantage of poor biocompatibility. The collagen and other natural materials have good biocompatibility and no antigenicity, can promote cell adhesion and proliferation, and can be made into different shapes according to requirements. The collagen has the advantages of rich source, low immunogenicity, good biocompatibility, good degradability, participation in tissue repair and reconstruction and the like.
The microsphere sustained-release system has better performance than the traditional sustained-release carrier material, and the microsphere structure can protect the drug from being damaged by the external environment and has stable performance, so the sustained-release action time can be prolonged. In summary, the combination of PLGA microspheres and collagen is preferably used as the sustained release gel carrier.
Furthermore, the growth factor sustained-release gel comprises PLGA microspheres, collagen and growth factors, wherein the growth factors are 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 chemotactic factor to generate chemotactic signal, so that cells and protein matrix can gather on wound surface, and can repair wound, and promote mitosis and proliferation of related cells. EGF is also mitogen of fibroblast and vascular endothelial cell, and can promote synthesis of matrix components such as collagen and fibrin, and accelerate wound healing. KGF-2 can specifically accelerate gene transcription, gene replication and protein synthesis of wound tissue cells, thereby promoting mitosis, proliferation, differentiation, migration and the like of epithelial cells.
Furthermore, for the second growth factor sustained release gel, the growth factors comprise vascular endothelial growth factor VEGF, platelet derived endothelial cell growth factor PDGF, transforming growth factor β (TGF- β) and basic fibroblast growth factor bFGF, the VEGF has the functions of promoting vascular endothelial proliferation and stem cell differentiation, and can promote vascular permeability to be increased, the PDGF has multiple biological functions, can stimulate the division and proliferation of various cells and the production of extracellular matrix, and is mitogen and chemotactic factor of various cells, the TGF- β can promote the growth of fibroblasts, osteoblasts and Schwann cells, can promote the expression and the inhibition degradation of extracellular matrix such as collagen and fibronectin, can accelerate wound healing, and is beneficial to the angiogenesis.
Further, the Chinese medicinal components include, but are not limited to, dragon's blood, ampelopsis japonica, codonopsis pilosula, poria cocos, white peony root, sanguisorba officinalis, calamine, phellodendron amurense, cnidium monnieri, piper longum, calomel and the like, and the dragon's blood and the ampelopsis japonica are preferable in terms of efficacy, cost and the like.
The external application of xuejie can stop bleeding, promote granulation and heal wound, and is mainly used for treating abdominal mass, traumatic injury, blood stasis and swelling pain, traumatic hemorrhage, unhealed ulcer and other symptoms. Studies show that the dragon's blood also has the function of promoting the differentiation of hair follicle stem cells. Studies of Zhangdong Pink duckweed, Cao Jian Chun, Ju, and the like find that the dragon's blood tissue regeneration promoting paste can promote the formation of new blood vessels and the growth of granulation tissues and improve the local nutritional state by increasing the expression of VEGF (vascular endothelial growth factor) of the ulcer wound of a diabetic rat, so as to promote the healing of the ulcer wound. And also has obvious promotion effect on the proliferation of wound tissue cells, can accelerate the re-epithelization of the wound and promote the healing of the wound. Radix Ampelopsis has effects of clearing heat and detoxicating, resolving carbuncle and resolving hard mass, healing sore and promoting granulation, and is mainly used for treating carbuncle, cellulitis, back, furuncle, burn and scald, etc. Studies prove that the ampelopsis japonica also has an inhibiting effect on candida albicans, staphylococcus aureus and the like.
Further, the traditional Chinese medicine components comprise dragon's blood and Japanese ampelopsis, and the mass ratio of the dragon's blood to the Japanese ampelopsis is 3: 5.
further, 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.
Further, the preparation method of the first growth factor sustained-release gel comprises the following steps:
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.
Further, 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.
The component C comprises sigma collagen, sodium alginate and gelatin in terms of mechanical strength, porosity, cell proliferation promoting capacity, performance change of the material at lower concentration and the like. Selecting collagen, sodium alginate and gelatin to carry out cross-linking treatment under certain conditions. The prepared gel has the following characteristics after the hydrodynamics test and the cytotoxicity test: good biocompatibility and biodegradability. Can combine with various growth factors, thereby promoting the proliferation and differentiation process of peripheral cells; the adhesion degree with cells is high; the mechanical strength is moderate, and the anti-compression capacity is certain; the elasticity is better; collapse is not easy to occur in the process of forming the acellular matrix scaffold in the printing process.
Further, the preparation method of the component C 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.
According to the invention, the biological ink is added with the traditional Chinese medicine components such as dragon's blood, ampelopsis japonica and the like to improve the wound healing and repairing capability; the growth factor sustained-release gel with a microsphere structure is adopted to promote the fusion, growth and repair of skin and prolong the sustained-release action time; seed cells are extracted from the skin of a patient, so that the immune rejection of the patient in the transplanting process is greatly reduced, and the wound recovery and the regeneration of related tissues at the transplanted skin are facilitated; gel components such as sodium alginate, collagen, gelatin and the like for improving the mechanical property are added, so that collapse is not easy to occur in the printing process. The biological ink can greatly improve the printing efficiency and quality of artificial skin, reduce the period of transplanting the skin of a patient, and has important significance in the research and development of products in skin-related industries such as the research and treatment of skin diseases, beauty treatment and the like.
The biological ink for 3D printing artificial skin has moderate mechanical property and biocompatibility, can reduce immune rejection reaction in the transplanting process, can improve wound healing and repairing capacity and promote skin fusion growth, has good mechanical and fluidic properties, and has popularization significance in relevant fields such as 3D printing skin and the like.
Drawings
FIG. 1 is a graph showing the change in the compressive modulus of the acellular carrier gels prepared in examples 1 to 3, with different mass ratios.
FIG. 2 is a graph showing the degradation rate at day 15 of the decellularized carrier gels prepared in examples 1 to 3, in which the mass ratios are different.
FIG. 3 is a gel of the decellularized carrier prepared in examples 1, 4 and 5, with different concentrations of CaCl
2Crosslinking of the solutionIn the case of (2), a graph showing a change in the relationship between the breaking strength.
Detailed Description
The following description describes alternative embodiments of the invention to teach one of ordinary skill in the art how to make and use the invention. Some conventional aspects have been simplified or omitted for the purpose of teaching the present invention. Unless otherwise specified, percentages are generally to be understood as mass percentages.
A preparation method of bio-ink for 3D printing of artificial skin comprises the following steps:
s1: preparing an enzymolysis solution: preparing 2g/L neutral protease solution, 0.25% trypsin and 0.02% EDTA by using D-Hanks solution, filtering, sterilizing and storing at 4 ℃ for later use.
S2: preparing a type IV collagen culture bottle: dissolving human IV type collagen in 0.1% acetic acid solution, filtering, sterilizing, and storing at 4 deg.C. Spreading 1-2ml of prepared IV type collagen in a culture bottle, standing overnight, discarding the supernatant, rinsing with 0.01mol/L PBS solution for 3-4 times, drying in a drying oven at 40 ℃, and sterilizing for 2h by ultraviolet irradiation for later use.
S3: extraction of epidermal stem cells: taking autologous normal skin pieces, washing with PBS solution containing double antibiotics (penicillin and streptomycin) for 5-6 times, and removing subcutaneous tissue under aseptic condition. Cutting the skin into pieces of 1.0cm × 1.0cm, placing in low-sugar DMEM/F12 culture medium containing neutral proteolytic enzyme, digesting at 4 deg.C for 14-16h, removing supernatant, and separating epidermis and dermis layers. Cutting epidermis, digesting with 0.25% trypsin at 37 deg.C for 15min, adding DMEM containing 10% fetal calf serum or human autologous serum to stop digestion, blowing, grinding with 200 mesh screen, and filtering. The filtrate was centrifuged (1000r/min) for 5min and cells were collected. Discarding supernatant, adding epidermal stem cell culture medium, gently blowing to obtain single cell suspension, inoculating into culture bottle pre-paved with IV type collagen, incubating at 37 deg.C for 15min, sucking out culture solution and non-adherent cells, discarding non-adherent cell components, washing with D-Hanks solution for 2 times, adding appropriate amount of epidermal stem cell culture medium, and culturing in a carbon dioxide incubator at 37 deg.C and 5% volume fraction. Sucking out the culture solution after 24h, washing with PBS for 1 time, adding the epidermal stem cell culture medium, and placing in a carbon dioxide incubator with the volume fraction of 5% at 37 ℃ for continuous culture. Then, the solution is changed 1 time every 48h, and the cell growth condition is observed under an optical lens.
S4, identifying epidermal stem cells, namely, taking partial cells, washing the partial cells for 3 times by PBS, fixing the partial cells for 30min by 4% paraformaldehyde at room temperature, carrying out immunocytochemical staining on cells K19 and β 1 integrin respectively by a two-step immunohistochemical detection method, simultaneously replacing primary antibody by PBS to serve as blank control, and avoiding light as much as possible in the steps.
S5: preparation of the first carrier hydrogel: taking a collagen I solution with the concentration of 0.4 percent and a sodium alginate solution with the concentration of 2 percent at room temperature, and mixing the two solutions in a volume ratio of 1: 1, uniformly mixing, controlling the pH of the solution to be 6-7.45, and preparing the collagen-seaweed biogel. Adding 30% of acellular skin matrix solution and seed cells obtained from S3 into collagen-seaweed biogel, wherein the collagen-seaweed biogel and the acellular skin matrix solution are 20: 1 volume ratio, stirring uniformly to make the seed cell concentration be 1X 10
5-1×10
7And (4) preparing the epidermal layer carrier hydrogel.
S6, grinding dragon' S blood and ampelopsis japonica into powder, and mixing the following raw materials: ampelopsis japonica ═ 3: 5 (mass ratio) to obtain the traditional Chinese medicine components;
s7 preparation of epidermal growth factor sustained-release gel (first growth factor sustained-release gel):
① 0.001, 0.001% -0.002% of EGF solution, 0.00001% -0.001% of KGF-2 solution as an inner water phase and 20% -70% of PLGA dichloromethane solution as an oil phase, obtaining the EGF-KGF-2-PLGA solution by a multiple emulsion method, removing an organic solvent, centrifuging (1000r/min) for 5min, precipitating, and then freeze-drying to obtain the EGF-KGF-2-PLGA microspheres.
② adding type I collagen into 0.1% -0.3% acetic acid solution, stirring well, and preparing into collagen solution.
③ adding the microspheres prepared in the step ① and the traditional Chinese medicine components obtained in the step S6 into a collagen solution, uniformly stirring, pre-freezing for 8-30 h at-70 ℃, then carrying out freeze drying, solidifying for 20-40 min by using 60-90% ethanol, and carrying out freeze drying again to obtain the collagen carrier embedded with the EGF-KGF-2-PLGA microspheres.
S8 extraction of dermal mesenchymal stem cells: taking autologous normal skin pieces, washing with PBS solution containing double antibiotics (penicillin and streptomycin) for 5-6 times, and removing subcutaneous tissue under aseptic condition. Cutting the skin into pieces of 1.0cm × 1.0cm, placing in low-sugar DMEM/F12 culture medium containing neutral proteolytic enzyme, digesting at 4 deg.C for 14-16h, removing supernatant, and separating epidermis and dermis layers. Rinsing the dermis layer for 2 times by PBS, cutting, culturing in a low-sugar DMEM/F12 culture medium containing 1% fetal calf serum or human autologous serum and 0.1% type I collagenase at 37 ℃ in a carbon dioxide culture box with the volume fraction of 5%, adding PBS containing 2% fetal calf serum or human autologous serum after 10h to stop digestion, blowing, and grinding and filtering by 200-mesh and 350-mesh sieves respectively. The filtrate was centrifuged (1500r/min) for 5min and cells were collected. Discarding the supernatant, adding DMEM/F12 medium containing 10% fetal calf serum or human autologous serum, gently blowing and beating to prepare single cell suspension, inoculating into a culture bottle at a proper density for culture, changing the solution 1 time every 48h, digesting with enzymolysis solution when the cells reach 80-90% fusion, inoculating the separated cells into a new culture bottle for continuous culture, and then changing the solution 1 time every 60 h.
S9: identification of dermal mesenchymal stem cells: the 3 rd generation of well-grown cells of dermal origin cultured and amplified in monolayer culture were fixed with 4% paraformaldehyde for 10 min. Thereafter, 0.2% Triton X-100 was added dropwise, incubated at 25 ℃ for 10min, and washed with PBS. After blocking with 10% fetal calf serum or human autologous serum for 1h, CD90, CD105 antibody and vimentin were added dropwise and left overnight at 4 ℃. After 24h, goat anti-mouse IgG FITC or IgG-Texas Red is added dropwise, the cells are incubated for 1h at room temperature, then the cell nuclei are stained with DAPI, and the cells are observed under a fluorescence microscope.
S10: extraction of hair follicle stem cells: hair follicle stem cells are extracted from the epidermis of the autologous hindbrain in a sterile room. The hair follicle stem cells are inoculated into DMEM/F12 culture medium and are placed in a carbon dioxide incubator with the temperature of 37 ℃ and the volume fraction of 5 percent for culture.
S11: extraction of adipose-derived stem cells: aseptically collecting adipose tissue, washing with PBS containing double antibiotics (penicillin and streptomycin) at 4 deg.C for 5-6 times, and aseptically removing blood vessel and connective tissue. Cutting adipose tissue into small pieces of 0.2cm × 0.2cm, digesting with 0.2% type I collagenase at 37 deg.C for 15min, adding isovolume of low sugar DMEM/F12 medium containing 10% fetal calf serum or human autologous serum to stop digestion, blowing, grinding with 200 mesh screen, and filtering. The filtrate was centrifuged (1000r/min) for 5min and cells were collected. Discarding the supernatant, adding 3mL of erythrocyte lysate, blowing for 5min, centrifuging (1000r/min) for 5min, discarding the supernatant, repeatedly washing with PBS, resuspending the cells in DMEM culture solution containing 10% fetal calf serum, inoculating to a culture dish, and culturing in a carbon dioxide incubator with the volume fraction of 5% at 37 ℃. Changing the solution for 1 time every 48h, digesting with enzymolysis solution when the cells grow to 75% -90% fusion, inoculating the separated cells into a new culture bottle for continuous culture, and then changing the solution for 1 time every 72 h.
S12: identification of adipose-derived stem cells: the 3 rd generation cell with good growth is taken and inoculated on a 24-well plate with proper density, after 24, the 4-well cell with good growth is taken, 2 wells are taken as an experimental group, and 2 wells are taken as a control group. Discarding the culture medium, washing with PBS for 2-3 times, fixing with 4% paraformaldehyde for 20min, washing with PBS for 3 times, adding fluorescein isothiocyanate FITC-labeled CD34 and CD44 antibodies into the experimental components, incubating at 4 deg.C in dark for 2h, washing with PBS, and observing under a fluorescence microscope.
S13: preparation of carrier hydrogel: taking a collagen I solution with the concentration of 0.5 percent and a sodium alginate solution with the concentration of 3 percent at room temperature, and mixing the two solutions in a volume ratio of 1: 1, uniformly mixing, controlling the pH of the solution to be 6-7.45, and preparing the collagen-seaweed biogel. Adding 30% of acellular skin matrix solution and the relevant seed cells obtained in S8-12 into collagen-seaweed biogel, wherein the collagen-seaweed biogel and the acellular skin matrix solution are 20: 1 volume ratio, stirring uniformly to make the seed cell concentration be 1X 10
5-1×10
7And (c) one/ml, to prepare a carrier hydrogel for the dermal layer (second carrier hydrogel).
S14: preparation of dermal layer growth factor sustained-release gel (second growth factor sustained-release gel):
① 0.001.001% -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 oil phase, obtaining VEGF-PDGF-TGF- β -bFGF-PLGA solution by a multiple emulsion method, removing organic solvent, centrifuging (1000r/min) for 5min, precipitating, and freeze-drying to obtain the VEGF-PDGF-TGF- β -bFGF-PLGA microspheres.
② adding type I collagen into 0.1% -0.3% acetic acid solution, stirring well, and preparing into collagen solution.
③ adding the microspheres prepared in step ① into collagen solution, stirring uniformly, pre-freezing for 8-30 h at-70 ℃, then freeze-drying, solidifying for 20-40 min by using 60-90% ethanol, and freeze-drying again to obtain the collagen carrier for embedding VEGF-PDGF-TGF- β -bFGF-PLGA microspheres.
The component A is added with a first carrier hydrogel and a first growth factor slow-release gel according to the proportion; the component B is added with a second growth factor slow-release gel and a second carrier hydrogel according to the proportion. When the skin is printed by the ink-jet type biological printing technology, the component A is added into the epidermal layer ink box, and the component B is added into the dermal layer ink box.
S15: preparation of acellular matrix gel:
① gelatin was dissolved in PBS buffer to form a 10% solution, sodium alginate was dissolved in PBS buffer to form a 2% solution, residual acetic acid was neutralized with NaOH, respectively, pH was adjusted to 7.2, and batch sterilization was performed in an oven at 60 ℃.
② A certain mass of Sigma collagen type I powder was weighed, acetic acid was added to a final concentration of 30mg/mL, and the mixture was stirred and placed in a refrigerator at 4 ℃ overnight.
③ mixing the materials evenly according to the mass ratio of the gelatin solution to the collagen solution to the sodium alginate solution of 1: 1: 4, and sterilizing at high temperature.
④, the three-dimensional model is reconstructed by three-dimensional modeling software to generate STL file, and the STL file is imported into a 3D biological printer for layering and setting of printing parameters, and then printing is started.
⑤ after the printing of the model is finished, a proper amount of 5% CaCl is dripped on the surface of the bracket
2Solution, crosslinking for 30 s. After the mold is completely formed, sucking away the excessive CaCl
2And repeatedly washing the stent for three times by using PBS buffer solution, adding a proper amount of PBS solution, and putting the stent into a carbon dioxide incubator.
Example 1
The preparation method of the C component and the acellular matrix scaffold of the bio-ink for 3D printing of the artificial skin comprises the following steps:
① dissolving gelatin in PBS to obtain 10 wt% solution, dissolving sodium alginate in PBS to obtain 2 wt% solution, neutralizing residual acetic acid with NaOH, adjusting pH to 7.2, and sterilizing at 60 deg.C in oven.
② A certain mass of Sigma collagen type I powder was weighed, acetic acid was added to a final concentration of 30mg/mL, and the mixture was stirred and placed in a refrigerator at 4 ℃ overnight.
③ mixing the gelatin solution, the collagen solution and the sodium alginate solution uniformly according to the mass ratio of 1: 1: 2, and sterilizing at high temperature.
④, the three-dimensional model is reconstructed by three-dimensional modeling software to generate STL file, and the STL file is imported into a 3D biological printer for layering and setting of printing parameters, and then printing is started.
⑤ after the printing of the model is finished, a proper amount of 5% CaCl is dripped on the surface of the bracket
2Solution, crosslinking for 30 s. After the mold is completely formed, sucking away the excessive CaCl
2And repeatedly washing the stent for three times by using PBS buffer solution, adding a proper amount of PBS solution, and putting the stent into a carbon dioxide incubator.
Example 2
The preparation method of the C component and the acellular matrix scaffold of the bio-ink for 3D printing of the artificial skin comprises the following steps:
① gelatin was dissolved in PBS buffer to form a 10% solution, sodium alginate was dissolved in PBS buffer to form a 2% solution, residual acetic acid was neutralized with NaOH, respectively, pH was adjusted to 7.2, and batch sterilization was performed in an oven at 60 ℃.
② A certain mass of Sigma collagen type I powder was weighed, acetic acid was added to a final concentration of 30mg/mL, and the mixture was stirred and placed in a refrigerator at 4 ℃ overnight.
③ mixing the materials evenly according to the mass ratio of the gelatin solution to the collagen solution to the sodium alginate solution of 1: 1: 4, and sterilizing at high temperature.
④, the three-dimensional model is reconstructed by three-dimensional modeling software to generate STL file, and the STL file is imported into a 3D biological printer for layering and setting of printing parameters, and then printing is started.
⑤ after the printing of the model is finished, a proper amount of 5% CaCl is dripped on the surface of the bracket
2Solution, crosslinking for 30 s. After the mold is completely formed, sucking away the excessive CaCl
2And repeatedly washing the stent for three times by using PBS buffer solution, adding a proper amount of PBS solution, and putting the stent into a carbon dioxide incubator.
Example 3
The preparation method of the C component and the acellular matrix scaffold of the bio-ink for 3D printing of the artificial skin comprises the following steps:
① gelatin was dissolved in PBS buffer to form a 10% solution, sodium alginate was dissolved in PBS buffer to form a 2% solution, residual acetic acid was neutralized with NaOH, respectively, pH was adjusted to 7.2, and batch sterilization was performed in an oven at 60 ℃.
② A certain mass of Sigma collagen type I powder was weighed, acetic acid was added to a final concentration of 30mg/mL, and the mixture was stirred and placed in a refrigerator at 4 ℃ overnight.
③ mixing the materials evenly according to the mass ratio of the gelatin solution to the collagen solution to the sodium alginate solution of 1: 1: 8, and sterilizing at high temperature.
④, the three-dimensional model is reconstructed by three-dimensional modeling software to generate STL file, and the STL file is imported into a 3D biological printer for layering and setting of printing parameters, and then printing is started.
⑤ after the printing of the model is finished, a proper amount of 5% CaCl is dripped on the surface of the bracket
2Solution, crosslinking for 30 s. After the mold is completely formed, sucking away the excessive CaCl
2And repeatedly washing the stent for three times by using PBS buffer solution, adding a proper amount of PBS solution, and putting the stent into a carbon dioxide incubator.
Example 4
The preparation method of the C component and the acellular matrix scaffold of the bio-ink for 3D printing of the artificial skin comprises the following steps:
① gelatin was dissolved in PBS buffer to form a 10% solution, sodium alginate was dissolved in PBS buffer to form a 2% solution, residual acetic acid was neutralized with NaOH, respectively, pH was adjusted to 7.2, and batch sterilization was performed in an oven at 60 ℃.
② A certain mass of Sigma collagen type I powder was weighed, acetic acid was added to a final concentration of 30mg/mL, and the mixture was stirred and placed in a refrigerator at 4 ℃ overnight.
③ mixing the materials evenly according to the mass ratio of the gelatin solution to the collagen solution to the sodium alginate solution of 1: 1: 4, and sterilizing at high temperature.
④, the three-dimensional model is reconstructed by three-dimensional modeling software to generate STL file, and the STL file is imported into a 3D biological printer for layering and setting of printing parameters, and then printing is started.
⑤ after the printing of the model is finished, a proper amount of 2% CaCl is dripped on the surface of the bracket
2Solution, crosslinking for 30 s. After the mold is completely formed, sucking away the excessive CaCl
2And repeatedly washing the stent for three times by using PBS buffer solution, adding a proper amount of PBS solution, and putting the stent into a carbon dioxide incubator.
Example 5
The preparation method of the C component and the acellular matrix scaffold of the bio-ink for 3D printing of the artificial skin comprises the following steps:
① gelatin was dissolved in PBS buffer to form a 10% solution, sodium alginate was dissolved in PBS buffer to form a 2% solution, residual acetic acid was neutralized with NaOH, respectively, pH was adjusted to 7.2, and batch sterilization was performed in an oven at 60 ℃.
② A certain mass of Sigma collagen type I powder was weighed, acetic acid was added to a final concentration of 30mg/mL, and the mixture was stirred and placed in a refrigerator at 4 ℃ overnight.
③ mixing the materials evenly according to the mass ratio of the gelatin solution to the collagen solution to the sodium alginate solution of 1: 1: 4, and sterilizing at high temperature.
④, the three-dimensional model is reconstructed by three-dimensional modeling software to generate STL file, and the STL file is imported into a 3D biological printer for layering and setting of printing parameters, and then printing is started.
⑤ after the printing of the model is finished, a proper amount of 8% CaCl is dripped on the surface of the bracket
2Solution, crosslinking for 30 s. After the mold is completely formed, sucking away the excessive CaCl
2And repeatedly washing the stent for three times by using PBS buffer solution, adding a proper amount of PBS solution, and putting the stent into a carbon dioxide incubator.
The acellular matrix gels prepared in examples 1-3 were tested for compressive modulus at 37 ℃ and their degradation rates are shown in FIG. 1 and FIG. 2.
The acellular matrix gels prepared in examples 1, 4 and 5 were tested for breaking strength in an environment of 37 deg.C, and the results are shown in FIG. 3.
The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.
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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