CN109385393A - A kind of 3D printing skin model and its construction method - Google Patents
A kind of 3D printing skin model and its construction method Download PDFInfo
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- CN109385393A CN109385393A CN201811237581.5A CN201811237581A CN109385393A CN 109385393 A CN109385393 A CN 109385393A CN 201811237581 A CN201811237581 A CN 201811237581A CN 109385393 A CN109385393 A CN 109385393A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
- C12N5/0698—Skin equivalents
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- C12N2502/00—Coculture with; Conditioned medium produced by
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
- C12N2502/091—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells melanocytes
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
- C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
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- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1352—Mesenchymal stem cells
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- C12N2513/00—3D culture
Abstract
The present invention provides a kind of 3D printing skin model and its construction method, belongs to tissue engineering technique field.The subcutaneous layer of fat of 3D printing skin model is provided with the first through hole of multiple internal run-throughs, and first through hole runs through the two sides up and down of subcutaneous layer of fat, and skin corium is provided with the second through-hole of multiple internal run-throughs, and the internal diameter of the second through-hole is sequentially increased from top to bottom.The construction method of 3D printing skin model includes the following steps: the skin image for extracting patient, and carries out three-dimensional reconstruction, obtains the three-dimensional digital model of three-dimensional digital model or direct construction skin model.Support construction, subcutaneous layer of fat, skin corium and epidermis are printed successively with Z-type or unistage type printing path according to three-dimensional digital model and obtain 3D printing skin model.Use culture medium culture 3D printing skin model.The 3D printing skin model that this construction method obtains can be such that cell divides equally in the material, and obtained skin model is closer to human body real skin structure.
Description
Technical field
The present invention relates to tissue engineering technique fields, in particular to a kind of 3D printing skin model and its building side
Method.
Background technique
Skin is the maximum organ of human body, has important physics, chemistry and biology barrier function.By wound, burn
And full thickness dermal caused by disunion ulcer is clinically very common.Compared to traditional treatment method, skin substitutes
The appearance of object provides an ideal approach for the treatment of defect of skin, passes through the transplanting of Graftskin, Ke Yiti
The repairing effect and healing quality of the high surface of a wound.But such presently commercially available product, material property is poor, function is simple, and prepares
Complex process, production save and transportation cost is high, is difficult to realize efficient individuation customization and clinical expansion.
In recent years, with the continuous development of biotechnology and tissue engineering technique, various Graftskin technologies of preparing are not
It is disconnected to emerge in large numbers.Wherein, the biological 3D printing technique for being integrated with material and cell technology is most with prospects, can be according to impaired skin
Skin design feature prints the bionical organization engineering skin bracket of height, realizes the timely reparation of the surface of a wound, hence it is evident that shortens skin and repairs
The multiple time improves surgical effect, avoids the generation of secondary damage and immunological rejection.In addition, being passed in terms of toxicity test in vitro
The safety evaluatio of system mainly uses animal model, but there are evaluation cycle length, poor repeatability and species variations for zoopery
The disadvantages of.And 3D skin model is controllably easy to quantitatively with result due to altitude simulation human body real skin, and with experiment condition
The advantages that and be paid more and more attention.
The skin model that existing 3D printing obtains and real skin gap are larger.
Summary of the invention
The first object of the present invention is to provide a kind of 3D printing skin model, constructs, connect convenient for the distribution of blood vessel
The skin model of nearly real skin.
The second object of the present invention is to provide a kind of construction method of 3D printing skin model, is layered and carries out printing structure
It builds, obtained skin model constructs convenient for the distribution of blood vessel, obtains the skin model close to real skin.
Based on above-mentioned first purpose, the present invention adopts the following technical solutions is realized:
A kind of 3D printing skin model, including epidermis, skin corium, subcutaneous layer of fat and the branch set gradually from top to bottom
Support structure；
Subcutaneous layer of fat is provided with the first through hole of multiple internal run-throughs, first through hole through subcutaneous layer of fat up and down two
Side, skin corium are provided with the second through-hole of multiple internal run-throughs, and the internal diameter of the second through-hole is sequentially increased from top to bottom.
Further, in preferred embodiments of the present invention, above-mentioned support construction includes basal layer and increasing layer, increase layer around
Groove is formed set on the edge of basal layer, epidermis, skin corium and subcutaneous layer of fat are set in turn in groove from top to bottom.
Further, in preferred embodiments of the present invention, it is above-mentioned increase layer include multilayer set gradually from top to bottom plus
High unit, each increase unit have it is spaced it is multiple increase part, the two neighboring part of increasing for increasing unit is cross-linked.
Further, in preferred embodiments of the present invention, it is above-mentioned increase layer include it is multiple set gradually from top to bottom plus
High unit each increases unit includes Thermo-sensitive first and increases the second of part and non-Thermo-sensitive and increases part, first increase part and
Second increases the connection of part interval.
Further, in preferred embodiments of the present invention, above-mentioned subcutaneous layer of fat and skin corium are provided with multilayer, and second
The aperture of one end of the separate epidermis of through-hole is 10~200 μm, the aperture of one end of the separate subcutaneous layer of fat of the second through-hole
For 0.4 μm~40um.
Based on above-mentioned second purpose, the present invention adopts the following technical solutions is realized:
A kind of construction method of above-mentioned 3D printing skin model, includes the following steps:
(1), the skin image of human body is extracted, and carries out three-dimensional reconstruction, obtains three-dimensional digital model or direct construction skin
The three-dimensional digital model of model；
(2), support construction, subcutaneous fat are successively printed with Z-type or unistage type printing path according to three-dimensional digital model
Layer, skin corium and epidermis obtain 3D printing skin model；
(3), using culture medium culture 3D printing skin model.
Further, in preferred embodiments of the present invention, the material of above-mentioned printing support construction is selected from synthesis macromolecule material
Material, natural extracellular matrix material and one of synthesis high molecular material and the composite material of natural extracellular matrix material；
It is total selected from polycaprolactone, l-lactic acid, racemic poly lactose, poly lactic-co-glycolic acid to synthesize high molecular material
One of polymers；
Natural extracellular matrix material is selected from one of chitosan, collagen, gelatin.
Further, in preferred embodiments of the present invention, the material of above-mentioned printing subcutaneous layer of fat is fat deposit biology ink
Water；
Fat deposit bio-ink includes fat deposit host material, fat deposit seed cell and fat deposit active factors；
Fat deposit host material includes natural extracellular matrix and biological degradation polyalcohol；
Natural extracellular matrix is selected from collagen, gelatin, fibroin albumen, fibroin, hyaluronic acid, fibrinogen, sea
Alginic acid, cellulose, starch, at least two in chitin and chitosan；
Biological degradation polyalcohol is selected from the double acrylic acid of poly lactide-glycolide acid, polylactic acid, polyethylene glycol
The one of which of ester, polyglycolic acid and polyethylene terephthalate；
Fat deposit seed cell includes fat mesenchymal stem cell；
Fat deposit active factors include IBMX, biotin, insulin and Indomethacin；
The concentration of IBMX is 0.1~1.0mol/ml, and the concentration of biotin is 0.1~1.5mmol/ml, the concentration of insulin
For 5~15mol/ml, the concentration of Indomethacin is 100~200mmol/ml.
Further, in preferred embodiments of the present invention, the material of above-mentioned printing skin corium is skin corium bio-ink；
Skin corium bio-ink includes skin corium host material, skin corium seed cell and skin corium active factors；
Skin corium host material is native protein high molecular material or natural polysaccharide material or modified hydrogel material；
Native protein high molecular material is selected from one of collagen, gelatin, fibroin and fibrinogen hydrogel；
Natural polysaccharide material is selected from one of alginic acid, cellulose, starch, chitin and chitosan；
Skin corium seed cell includes fibroblast and vascular endothelial cell, the fibroblast and described intravascular
Chrotoplast ratio is 2:1~5:1；
Skin corium active factors are selected from one of VEGF, bFGF, aFGF, HGF, PDGF, PDGF or its derived protein；
The concentration of VEGF is 6~25ng/ml, and the concentration of bFGF is 0.1~1.0mg/ml, the concentration of aFGF is 0.1~
The concentration of 2.0mg/ml, HGF are 20~40ng/ml, and the concentration of PDGF is 0.1~1.0mg/ml.
Further, in preferred embodiments of the present invention, the material of above-mentioned printing epidermis is epidermis bio-ink；
Epidermis bio-ink includes epidermis seed cell and epidermis active factors；
Epidermis seed cell includes keratinocyte, melanocyte and hair follicle stem cells；
Epidermis active factors include be suitble to keratinocyte growth HKGS, insulin, bFGF, EGF, hydrogenation can
Pine, transferrins, glutamine, vitamin C and calcium chloride are suitble to the HGF and ET-1 and suitable hair follicle of melanocyte growth
The FGF-1 and FGF-2 of stem cell growth；
The concentration of HKGS is 10~100mg/ml, and the concentration of insulin is 5.0~55ng/ml, the concentration of EGF is 0.1~
The concentration of 15.0ng/ml, bFGF are 0.1~1.0mg/ml, and the concentration of hydrocortisone is 0.1~5.0 μ g/ml, transferrins
Concentration be 5.0~55.0ng/ml, the concentration of glutamine is 0.2~22.0g/l, and ascorbic concentration is 20~30ng/
Ml, the concentration of calcium chloride are 0.1Mm~20Mm；
The concentration of HGF is 20~40ng/ml, and the concentration of ET-1 is 1.0~20ng/ml；
The concentration of FGF-1 is 10~20pg/ml, and the concentration of FGF-2 is 20~40pg/ml.
Compared with prior art, the beneficial effect of the 3D printing skin model of presently preferred embodiments of the present invention offer includes:
Layering setting is carried out, subcutaneous layer of fat is provided with the first through hole of multiple internal run-throughs, and first through hole is through subcutaneous
The two sides up and down of fat deposit, are arranged the internal run-through of multiple first through hole and multiple first through hole, being capable of bionics skin fat deposit
Vascular distribution structure promote angiogenesis and regeneration, the conveying of nutriment and metabolic waste convenient for forming blood vessel access
Discharge.Skin corium is provided with the second through-hole of multiple internal run-throughs, and the internal diameter of the second through-hole is sequentially increased from top to bottom, is convenient for shape
At blood vessel access, meet the structure of human body real skin.
The beneficial effect of the construction method of 3D printing skin model provided by the invention includes: that can obtain above-mentioned 3D printing
Skin model meets the structure of human body real skin convenient for forming blood vessel access.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings and also belong to protection scope of the present invention.
Fig. 1 is the structural schematic diagram for the 3D printing skin model that the embodiment of the present invention 1 provides；
The first structure diagram of skin corium in the 3D printing skin model that Fig. 2 provides for the embodiment of the present invention 2；
Second structural schematic diagram of skin corium in the 3D printing skin model that Fig. 3 provides for the embodiment of the present invention 2；
The structural schematic diagram of basal layer in the 3D printing skin model that Fig. 4 provides for the embodiment of the present invention 1；
Increase the first structure diagram of layer in the 3D printing skin model that Fig. 5 provides for the embodiment of the present invention 1；
Increase the second structural schematic diagram of layer in the 3D printing skin model that Fig. 6 provides for the embodiment of the present invention 1；
The structure of the Z-type printing path of subcutaneous layer of fat in the 3D printing skin model that Fig. 7 provides for the embodiment of the present invention 2
Schematic diagram；
The single line section printing path of subcutaneous layer of fat in the 3D printing skin model that Fig. 8 provides for the embodiment of the present invention 2
Structural schematic diagram.
Icon: 110- epidermis；120- skin corium；130- subcutaneous layer of fat；140- support construction；131- first
Through-hole；The second through-hole of 121-；141- basal layer；142- increases layer；143- groove；1421- increases unit；1422- adds
High part；1423- first increases unit；1424- second increases unit；1425- third increases unit；1426- first increases
Part；1427- second increases part；1428- third increases part.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
3D printing skin model provided in an embodiment of the present invention and its construction method are specifically described below.
Embodiment 1
Referring to Fig. 1,3D printing skin model includes the epidermis 110 set gradually from top to bottom, skin corium 120, skin
Lower fat deposit 130 and support construction 140.Subcutaneous layer of fat 130 is provided with multiple first through hole 131 through upper and lower two sides, very
Cortex 120 is provided with the second through-hole 121 of multiple internal run-throughs, and the internal diameter of the second through-hole 121 is sequentially increased from top to bottom.
Layering setting is carried out, subcutaneous layer of fat 130 is provided with the first through hole 131 of multiple internal run-throughs, first through hole 131
Through the two sides up and down of subcutaneous layer of fat 130, the internal run-through of multiple first through hole 131 and multiple first through hole 131, energy are set
The vascular distribution structure of enough bionics skin fat deposits promotes angiogenesis and regeneration, nutriment convenient for forming blood vessel access
The discharge of conveying and metabolic waste.Fig. 2 and Fig. 3 are please referred to, skin corium 120 is provided with the second through-hole 121 of multiple internal run-throughs,
The internal diameter of second through-hole 121 is sequentially increased from top to bottom, convenient for forming blood vessel access, meets the structure of human body real skin.
Specifically, referring to Fig. 4, support construction 140 includes basal layer 141 and increases layer 142, increase layer 142 and be set around
The edge of basal layer 141 forms groove 143, and epidermis 110, skin corium 120 and subcutaneous layer of fat 130 are set gradually from top to bottom
In in groove 143.
It, can be by epidermis 110, skin corium 120 and subcutaneous layer of fat by basal layer 141 and the setting for increasing layer 142
130 claddings are got up, nutriment and the transport channel of discharged waste when supporting, and being capable of providing culture.
Basal layer 141 includes the multi-layer fiber layer being fixedly connected sequentially from the bottom to top together, and each fibrous layer includes more
The fiber filament of a parallel setting, the fiber filament of adjacent two layers fibrous layer are staggered, and basal layer 141 forms the three-dimensional space to interweave
Between reticular structure, so as to the setting of other layer of structure of skin model.
Adjacent two layers or multi-layer fiber layer constitute a fiber element, in multiple fiber elements, at least one fiber
Fiber filament in one layer of fibrous layer in unit is parallel with the fiber filament in other one or more fiber elements and non-coplanar sets
It sets.And since the fiber filament in multiple fiber elements parallel and non-coplanar (or not face) is arranged, thus, institute in basal layer 141
The multiple holes formed and not coaxial setting, thus cell is not easy to slide from the hole of basal layer 141 to 141 bottom of basal layer.Extremely
The cross sectional shape in the hole that multiple fiber filaments of few two fibre layers are staggered to form is polygon or round." polygon " herein can
Think triangle, quadrangle (including square, rectangle, diamond shape, parallelogram), hexagon etc..
The shape of the basal layer 141 is multiedge cylinder or cylindrical body." multiedge cylinder " herein can for triangular prism, cube
Body, hexagonal prisms etc..The shape of the basal layer 141 can be designed according to the shape of cell culture apparatus.
The outer surface layer of the basal layer 141 passes through hydrophilic treated.Specifically, such as: using plasma hydrophily processing, temperature
Quick property processing or bracket expoeridium collagen or extracellular matrix, to improve the bioactivity of rack surface, increase cell adhesion
Energy.
Optionally, increase unit 1421 including what multilayer was set gradually from top to bottom referring to Fig. 5, increasing layer 142, each
Increase unit 1421 have it is spaced it is multiple increase part 1422, the two neighboring part 1422 of increasing for increasing unit 1421 interlocks
Connection.The epidermis 110, skin corium 120 and subcutaneous layer of fat 130 being arranged on basal layer 141 can be supported.Together
When, the part 1422 of increasing being staggered is conducive to the conveying of nutriment and the discharge of metabolin.
Further, increasing layer 142 includes that first set gradually from top to bottom increases unit 1423, second increases unit
1424 and third increase unit 1425, first to increase unit 1423 include multiple first increasing part 1426, and second increases unit
1424 increase part 1427 including multiple second, and it includes that multiple thirds increase part 1428 that third, which increases unit 1425, and first increases part
1426, second increase part 1427 and third to increase part 1428 be cylindrical type, and it is horizontally disposed.First increases under the two of part 1426
The upper end for increasing part 1427 with two second is held to connect.Second increases two upper ends of part 1427 and two first are increased part 1426
Lower end connection, the second two lower ends for increasing part 1427 connect with the upper end that two thirds increase part 1428, if interlocked
Setting, is conducive to the conveying of nutriment and the discharge of metabolin.
In another embodiment, referring to Fig. 6, increase layer include it is multiple set gradually from top to bottom increase list
Member each increases unit includes Thermo-sensitive first and increases part and the second of non-Thermo-sensitive and increases part, and first increases part and second
Increase the connection of part interval.After printing is completed, heating to the property of can choose makes the first of Thermo-sensitive to increase part dissolution, can also not
It makes it dissolve, to be configured to layer is increased.
Optionally, subcutaneous layer of fat 130 and skin corium 120 are provided with multilayer, and the thickness of subcutaneous layer of fat 130 is not more than
1500 μm, meet the structure of human body real skin.Further, each layer of subcutaneous layer of fat 130 is respectively provided with multiple first through hole
131, run through inside each layer of multiple first through hole 131, the first through hole 131 of multilayer fat deposit is also internal to be run through, further
The vascular distribution structure of bionics skin fat deposit, convenient for forming blood vessel access, promote angiogenesis and regeneration, nutriment it is defeated
Send the discharge with metabolic waste.
Skin corium 120 with a thickness of 600~1500 μm, epidermis 110 with a thickness of 40~150 μm, the second through-hole 121
The aperture of one end far from epidermis 110 is 10~200 μm, the aperture of one end of the separate subcutaneous layer of fat of the second through-hole 121
It is 0.4~40 μm, meets the structure of human body real skin.
In the present embodiment, subcutaneous layer of fat 130 is the reticular structure being pre-designed, by the second through-hole on skin corium 120
121 be aperture gradually smaller structure from the bottom to top, is capable of forming compact texture, is uniformly distributed cell in the material, internal
Vascular access structure is conducive to the absorption of cytotrophy substance and the discharge of metabolic waste, promotes cell Proliferation, closer
Human body real skin structure.
Embodiment 2
The present embodiment provides a kind of construction methods for the 3D printing skin model that embodiment 1 provides, and include the following steps:
(1), the skin image of patient is extracted, and carries out three-dimensional reconstruction, obtains three-dimensional digital model.According to different patients
Situation, to image is extracted at the skin damage of patient, so that the actual conditions phase of the three-dimensional digital model finally obtained and patient
It is corresponding, the skin model being applicable in patient state of an illness that is obtaining.
(2), fat deposit bio-ink, skin corium bio-ink and epidermis bio-ink are configured.
Specifically, fat deposit bio-ink include fat deposit host material, fat deposit seed cell and fat deposit activity because
Son.
Optionally, fat deposit host material includes natural extracellular matrix and biological degradation polyalcohol.The outer base of n cell
Matter is selected from collagen, gelatin, fibroin albumen, fibroin, hyaluronic acid, fibrinogen, alginic acid, cellulose, starch, crust
At least two in element and chitosan；Biological degradation polyalcohol is selected from poly lactide-glycolide acid (PLGA), polylactic acid
(PLA), polyethylene glycol double methacrylate (PEDGA), polyglycolic acid (PGA) and polyethylene terephthalate (PET)
One of which.
Fat deposit seed cell includes fat mesenchymal stem cell, which is easy to draw materials, and yield is high and activity is not with year
Age increases and dies down.The inoculum density of fat deposit seed cell is 0.15 × 106~0.9 × 106A/cm2。
Fat deposit active factors are used to activate the different differentiation signal accesses of fat mesenchymal stem cell.Fat deposit mainly leads to
The growth factor that addition promotes adipose-derived stem cells to be differentiated to form fat deposit is crossed, basal medium is that DMEM adds 10% tire ox blood
Clearly, fat deposit active factors include IBMX, concentration be 0.1~1.0mol/ml (such as: 0.1mol/ml, 0.5mol/ml or
1.0mol/ml)；Biotin, concentration are 0.1~1.5mmol/ml (such as: 0.1mol/ml, 1mol/ml or 1.5mol/ml)；Pancreas
Island element, concentration are 5~15mol/ml (such as: 5mol/ml, 10mol/ml or 15mol/ml)；Indomethacin, concentration be 100~
200mmol/ml (such as: 100mmol/ml, 150mmol/ml or 200mmol/ml).Angiogenesis is by subcutaneous layer of fat
Inside is added while vascular endothelial cell is added promotees vascular fatty layer active factors, and induced lipolysis mescenchymal stem cell is to fat
Layer internal migration, proliferation form capillary network.
At the initial stage that fat deposit is formed, fat deposit active factors VEGF is added, makes itself and junket ammonia corresponding on endothelial cell
Acid kinase receptor combines, and causes cell differentiation, forms hyperplasia and form luminal structure.After late blood vessel generates, outside n cell
Matrix can maintain the luminal structure to be formed, and the later period, other active factors can be used for stablizing luminal structure.In fat deposit building process,
Adipose-derived stem cells, which can be secreted, promotees vasoactive factors (VEGF), promotes angiogenesis.
In the present embodiment, fat deposit bio-ink can by Cellular gels, cell encapsulation, cell vesicle, cell microsphere,
The multi phase states bio-ink system encapsulation technology such as cell suspension carries out preparing fat deposit bio-ink box, realizes fat deposit matrix material
The integration of material, fat deposit seed cell and fat deposit active factors material solves the problems, such as cell damage when printing, improves thin
The survival rate of born of the same parents, and maintain the directed differentiation ability of stem cell.
Specifically the preparation method comprises the following steps: being shredded subcutaneus adipose tissue with operating scissors, 0.1% I types of 37 DEG C of preheatings are then added
Clostridiopetidase A, and continue to digest 20min at this temperature, the culture medium containing serum is then added and terminates digestion；The screen to filtrate,
Supernatant is abandoned after 1000r/min centrifugation 8min, carries out viable count with trypan blue staining.The fetal calf serum containing 10% is added
(FBS) culture medium, which is resuspended, mixes, and inoculum density is adjusted to 1 × 104cells/cm2, it is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
Flow cytomery cell surface molecule marker, it is determined whether be mescenchymal stem cell.The primary culture of replacement every three days
Base, the adult adipose mesenchymal stem cell biological ink for taking P2 to be formed for cell as induction late blood vessel.
The fat mesenchymal stem cell in P2 generation and vascular endothelial cell are resuspended with active factors respectively, aggravate outstanding rear liquid
It is mixed with host material, forms fat deposit bio-ink, be gently mixed mixing, cell density is allocated as 1 × 106cells/
Ml so far obtains tissue adaptive type subcutaneous layer of fat bio-ink.
Specifically, skin corium bio-ink include skin corium host material, skin corium seed cell and skin corium activity because
Son.
Skin corium host material is the class cell epimatrix material of skin corium, and content is 500-1000 μ l, is provided to cell
Mild aqueous environments and suitable mechanical strength.Skin corium host material is native protein high molecular material or natural polysaccharide
Material or modified hydrogel material.Native protein high molecular material is in collagen, gelatin, fibroin and fibrinogen hydrogel
One kind；Natural polysaccharide material is selected from one of alginic acid, cellulose, starch, chitin and chitosan.
Skin corium seed cell includes fibroblast and vascular endothelial cell, fibroblast and vascular endothelial cell ratio
Example is 2:1~5:1 (such as: 2:1,3:1 or 5:1), and fibroblast is basic seed cell, for constructing corium confluent monolayer cells ink
Water, vascular endothelial cell is for constructing blood vessel bio-ink.The inoculum density of skin corium seed cell is 0.15 × 106~0.9
×106A/cm2(such as: 0.15 × 106A/cm2、0.5×106A/cm2Or 0.9 × 106A/cm2)。
It is 6~25ng/ml's (such as: 6ng/ml, 20ng/ml or 25ng/ml) that skin corium active factors, which are selected from concentration,
VEGF, concentration are the bFGF of 0.1~1.0mg/ml (such as: 0.1mg/ml, 0.5mg/ml or 1.0mg/ml), concentration be 0.1~
The aFGF of 2.0mg/ml (such as: 0.1mg/ml, 0.5mg/ml or 2.0mg/ml), concentration be 20~40ng/ml (such as: 20ng/
Ml, 30ng/ml or 40ng/ml) HGF, concentration be 0.1~1.0mg/ml (such as: 0.1mg/ml, 0.5mg/ml or 1.0mg/
Ml one of PDGF or PDGF derived protein).Optionally, 10% serum is added in DMEM culture medium, adds true
The cortical activity factor is cultivated.
Skin corium bio-ink can by skin corium host material, skin corium seed cell and skin corium active factors according to
Optimal proportion uniformly premixes configuration.Skin corium host material can use fibrinogen hydrogel, and degradation speed is fast, and
The penetrating power of oxygen and glucose is strong；Can also using other natural macromolecular materials by it is modified make its contain aldehyde radical or
Base of the hydrogel as the cortex bio-ink that comes true is formed by schiff base reaction after amino, aldehyde radical component and amino group mixing
Material, such as oxidized sodium alginate, oxidized dextran and oxidized hyaluronic acid contain aldehyde radical；Amination hyaluronic acid, amino
Change carboxylic chitosan etc. and has amino.
In the present embodiment, skin corium bio-ink can by Cellular gels, cell encapsulation, cell vesicle, cell microsphere,
The multi phase states bio-ink system encapsulation technology such as cell suspension prepares skin corium bio-ink box, it is ensured that cell is in bio-ink
Higher survival rate is maintained, and pays attention to the sterile and holding time of ink cartridge.
Specifically the preparation method comprises the following steps: isolated corium is cut into 0.5~1mm with operating scissors and surgical forceps3Tissue block, so
Portion of tissue block is placed directly in culture bottle bottom afterwards；1~2 DMEM culture medium of the drop containing 10%FBS is added to each piece of group
It knits, is placed in 37 DEG C, 5%CO24h is cultivated in incubator；The DMEM culture medium containing 10%FBS is added to continue to cultivate, changes within 3 days primary
Liquid.Tissue is removed after 5 days to continue to cultivate.Hereafter a subculture is replaced every three days, takes P2 for cell as fibroblast
Bio-ink.
Upper remaining dermal tissue block is put into containing in II solution of 1ml1.25mg/ml clostridiopetidase A, 37 DEG C, 5% CO2
40min is cultivated in incubator, and 2ml fetal calf serum is added and terminates reaction.Dermal tissue block is stirred with plastic forceps, is placed in 200 μm
Screen filtration, the speed of 1000rpm/min, 5min centrifugation remove supernatant, are resuspended with 5mlDMEM culture medium.It is inoculated in advance
It is coated in the culture dish of the 100mm of gelatin, in 37 DEG C, 5%CO2It is cultivated in incubator, takes P2 thin as blood vessel endothelium for cell
Born of the same parents' bio-ink.
Subcutaneus adipose tissue is shredded with operating scissors, 0.1% Type I collagen enzyme of 37 DEG C of preheatings is then added, and is warm herein
Continue to digest 20min under degree, the culture medium containing serum is then added and terminates digestion；The screen to filtrate, after 1000r/min is centrifuged 8min
Supernatant is abandoned, carries out viable count with trypan blue staining.The DMEM culture medium containing 10%FBS is added, mixing is resuspended, is inoculated with close
Degree is adjusted to 1 × 104cells/cm2, it is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Flow cytomery cell surface molecule
Marker, it is determined whether be fat mesenchymal stem cell.A subculture is replaced every three days, takes P2 for cell as between fat
Mesenchymal stem cells bio-ink.
The fibroblast in P2 generation and vascular endothelial cell are resuspended with respective active factors respectively, after resuspension
Liquid and host material mixing form skin corium bio-ink and at blood vessel bio-ink, two kinds of bio-inks are mixed.True
In the building of cortex, the ratio of fibroblast and vascular endothelial cell is 2:1, in mixing material cell density be 1 ×
105cells/ml so far obtains tissue adaptive type skin corium standard biological ink.
Specifically, epidermis bio-ink includes epidermis seed cell, epidermis active factors and deionized water.
Epidermis seed cell includes keratinocyte, melanocyte and hair follicle stem cells, and optionally, cutin is formed carefully
Born of the same parents are basic epidermis seed cell, and melanocyte and hair follicle stem cells according to demand, are selectively added.
Such as: seed cell inoculum density is 0.10 × 106~1.0 × 106A/cm2(such as: 0.10 × 106A/cm2、
0.5×106A/cm2Or 01.0 × 106A/cm2), keratinocyte and melanocyte additional proportion are 15:1~3:1 (example
Such as: 15:1,3:1 or 5:1), the ratio of hair follicle stem cells and keratinocyte be 1:5~1:30 (such as: 1:5,1:20 or 1:
30) epidermis of the epidermis bio-ink printing, finally obtained can be more nearly the real skin of human body.
Epidermis active factors are arranged in basal medium, meanwhile, the epidermis activity in basal medium is set
The factor includes the HKGS for being suitble to keratinocyte growth, insulin, bFGF, EGF, hydrocortisone, transferrins, glutamy
Amine, vitamin C and calcium chloride are suitble to the HGF and ET-1 of melanocyte growth and the FGF-1 of suitable hair follicle stem cells growth
And FGF-2.The selection of epidermis active factors is related with the selection of epidermis seed cell, has corresponding relationship.
Wherein, basal medium includes one or more of K-SFM, Epilife, DMEM, F12, MCDB53 mixing group
At wherein DMEM:F12 content range is 3:1~1:3 (such as: 1:1,3:1 or 1:3).It is suitble to keratinocyte growth
HKGS concentration is 10~100mg/ml (such as: 10mg/ml, 20mg/ml or 100mg/ml)；Insulin concentration is 5.0~55ng/
Ml (such as: 5ng/ml, 20ng/ml or 55ng/ml)；EGF concentration be 0.1~15.0ng/ml (such as: 0.1ng/ml, 10ng/
Ml or 15ng/ml)；BFGF concentration is 0.1~1.0mg/ml (such as: 0.1mg/ml, 0.5mg/ml or 1mg/ml)；Hydrogenation can
Loose concentration is 0.1~5.0 μ g/ml (such as: 0.1 μ g/ml, 3 μ g/ml or 5.0 μ g/ml)；Transferrin concentrations be 5.0~
55.0ng/ml (such as: 5ng/ml, 20ng/ml or 55ng/ml)；Glutamine concentration be 0.2~22.0g/l (such as: 0.2g/
L, 10g/l or 22.0g/l), vitamin C concentration is 20~30ng/ml (such as: 20ng/ml, 25ng/ml or 30ng/ml)；Chlorine
The concentration for changing calcium is 0.1Mm~20Mm (such as: 0.1Mm, 10Mm or 20Mm).The HGF concentration for being suitble to melanocyte growth is 20
~40ng/ml (such as: 20ng/ml, 30ng/ml or 40ng/ml)；ET-1 concentration be 1.0~20ng/ml (such as: 1ng/ml,
20ng/ml or 15ng/ml).Be suitble to hair follicle stem cells growth FGF-1 concentration be 10~20pg/ml (such as: 10pg/ml,
12pg/ml or 20pg/ml)；FGF-2 concentration is 20~40pg/ml (such as: 20pg/ml, 32pg/ml or 40pg/ml).
By keratinocyte, melanocyte, hair follicle stem cells, the active factors for being suitble to keratinocyte growth, fit
The active factors of the active factors and the growth of suitable hair follicle stem cells that close melanocyte growth are proportionally uniformly configured to one kind
Epidermis bio-ink.
Similar embodiment may also is that different epidermis seed cell and its corresponding epidermis active factors
Independent be blended configures series of tables cortex bio-ink, i.e., keratinocyte and the growth of suitable keratinocyte it is active because
Son, deionized water are blended to form keratinocyte bio-ink by optimal proportion, melanocyte and suitable melanocyte
The active factors of growth, deionized water are blended to form melanocyte bio-ink according to optimal proportion, hair follicle stem cells and suitable
Close the active factors of hair follicle stem cells growth, deionized water is blended to form hair follicle cell bio-ink according to optimal proportion.
In the present embodiment, epidermis bio-ink can by Cellular gels, cell encapsulation, cell vesicle, cell microsphere,
The multi phase states bio-ink system encapsulation technology such as cell suspension prepares epidermis bio-ink box, it is ensured that cell is in bio-ink
Higher survival rate is maintained, and pays attention to the sterile and holding time of ink cartridge.
Specifically the preparation method comprises the following steps: the skin for removing subcutaneus adipose tissue is cut into 2cm × 1cm strip, 3 are rinsed with PBS
It after~5 times, is put into 0.1% trypsin solution, is placed in cold digestion 15h in 4 DEG C of refrigerators；Gently will with scalpel and surgical forceps
Epidermis and corium are peeled away, and the corium isolated is placed in spare to prepare skin corium in sterile centrifugation tube.
Epidermis is put in 0.25% trypsase-EDTA digestion solution of 37 DEG C of preheatings, maintains 37 DEG C of digestion 2min,
Then the culture medium containing serum is added and terminates digestion, the screen to filtrate, 800r/min abandons supernatant after being centrifuged 8min, is separately added into K-
SFM (Gibco) and growth factor mixture are centrifuged again, are abandoned supernatant, are carried out viable count with trypan blue staining.Use K-
Cell, which is resuspended, for SFM (Gibco) basal medium and growth factor additive mixes, and inoculum density is adjusted to 1 × 104cells/
cm2, it is placed in 37 DEG C, 5%CO2It is cultivated in incubator.A subculture is replaced every three days, takes P2 for keratinocyte and black
Plain cell is spare.
The skin for removing subcutaneus adipose tissue is cut into 0.25 × 0.25cm2Small tissue blocks, be added with DMEM it is diluted
12.5mg/ml neutral proteinase II, 4 DEG C overnight, removes epidermis, hair follicle is extracted from corium, is rinsed 3 times using PBS.It will take
Under hair follicle 15min is digested at 37 DEG C with the EDTA of 0.25% pancreatin+0.02%, then with the DMEM containing 10%FBS
Digestion is terminated, is blown and beaten repeatedly, the screen to filtrate, supernatant is abandoned after 800r/min centrifugation 8min, is separately added into K-SFM (Gibco) and life
Long factor cocktails are centrifuged again, are abandoned supernatant, are carried out viable count with trypan blue staining.With the basis K-SFM (Gibco)
Cell, which is resuspended, for culture medium and growth factor additive mixes, and inoculum density is adjusted to 1 × 104cells/cm2, be placed in 37 DEG C,
5%CO2It is cultivated in incubator.A subculture is replaced every three days, takes P2 spare for hair follicle stem cells.
By the epidermal keratinocytes in P2 generation, melanocyte and hair follicle stem cells, cultivated with the basis K-SFM (Gibco)
Base and growth factor additive resuspension cell gross density are 2 × 106Cells/ml, keratinocyte and melanocyte quantity
Than mixing (can also be according to ethnic different, the selective adjustment of progress, such as: the melanocyte increase of black race) for 4:1, hair follicle is dry
Cell quantity needs hair and other accessory organ's attributes to be adjusted according to the later period, so far obtains tissue adaptive type epidermis mark
Quasi- bio-ink.
(3), support construction, subcutaneous fat are successively printed with Z-type or unistage type printing path according to three-dimensional digital model
Layer, skin corium and epidermis obtain 3D printing skin model.
Firstly, by the material of support construction, fat deposit bio-ink, skin corium bio-ink and epidermis bio-ink point
It is not packed into corresponding barrel.
Support construction is printed, the material for printing support construction is selected from synthesis high molecular material, natural extracellular matrix material
With one of the composite material of synthesis high molecular material and natural extracellular matrix material；Synthesis high molecular material, which is selected from, gathers oneself
One of lactone, l-lactic acid, racemic poly lactose, poly lactide-glycolide acid；Natural extracellular matrix material
Material is selected from one of chitosan, collagen, gelatin.
The sterilizable material of 10ml support construction is packed into sterile barrel, is 80~100 DEG C in nozzle temperature；Platform temperature
It is 10~20 DEG C；Pressure is 0.3~0.5MPa；Print speed is 5~7mm/s；Filling spacing is 0.75~0.85mm；Printable layer
Thickness is 0.18~0.35mm；The printing of structure is supported under the print conditions that printing needle diameter is 0.21~0.41 μm.
The whole height of support construction is 10~15mm, and support construction base layer thickness is 3~5mm, increases layer with a thickness of 5
~10mm.Support construction basal layer periphery is formed as round, and shape is cylinder, support construction entirety outer surface by collagen or
Other extracellular matrix hydrophilic treateds.
In the present embodiment, increasing layer can discontinuously be printed, and formed and spaced multiple increased part.Or increase layer by
Two or more printed material carries out alternately continuous printing, and one of material has Thermo-sensitive, 20~40
Become liquid under conditions of DEG C, such as: collagen, alginic acid or gelatin, optionally, gelatin is as pass structure forming material.According to
According to the condition of culture of skin, become liquid outflow at 37 DEG C, to form the pass structure for increasing layer.
Continue to print subcutaneous layer of fat on the support structure: fat deposit ink is packed into the barrel of biological 3D printing equipment
It is interior, it is 4~25 DEG C in nozzle temperature；Platform temperature is 0~37 DEG C；Pressure is 0.06~0.58MPa；Print speed be 2~
15mm/s；Filling spacing is 0.5~1.4mm；Printing thickness is 0.05~0.12mm；Printing needle diameter is 80~340 μm
The printing of subcutaneous layer of fat is carried out under print conditions.
Optionally, the printing path of fat deposit can be Z-type or single line section (such as Fig. 7 be Z-type printing path, and Fig. 8 is single line
Section printing path) or other printable paths, print the number of plies >=1 layer, wherein Z-type structure is after 1 layer of printing, in Z-type path
Hole in, be filled printing with the bio-ink that Thermo-sensitive material (such as gelatin) and cell form；Single line segment structure is being beaten
After printing the number of plies >=1 and≤3 layers, it is filled printing using the bio-ink that Thermo-sensitive material (such as gelatin) and cell form,
After above two printing path is planned, alternating expression printing is carried out, foundation structure is fat deposit bio-ink composition, filling knot
Structure is needed using Thermo-sensitive material (such as gelatin), which can promote cell Proliferation and the later period is degradable, is facilitated inside fat
Form blood vessel access.
Continue to print skin corium on subcutaneous layer of fat: skin corium ink be packed into the barrel of biological 3D printing equipment,
It is 4~25 DEG C in nozzle temperature；Platform temperature is 0~37 DEG C；Pressure is 0.06~0.58MPa；Print speed is 2~15mm/
s；Filling spacing is 0.5~1.4mm；Printing thickness is 0.05~0.12mm；Under conditions of printing needle diameter is 80~340 μm
Carry out the printing of skin corium.
Optionally, the printing path of skin corium can be Z-type or single line section or other printable paths, and the printing number of plies >=
1 layer, wherein in Z-type structural path or the hole in single line section path, be filled printing with Thermo-sensitive material (such as: gelatin).Make
Alternating expression printing is carried out with Z-type structural path or two kinds of single line section path printing path, foundation structure is skin corium bio-ink
Composition, interstitital texture are needed using Thermo-sensitive material (such as gelatin), which can promote cell Proliferation and the later period is degradable, have
Help form blood vessel access inside fat.
Continue to print epidermis on skin corium: epidermis ink being packed into the barrel of biological 3D printing equipment, sprayed
Head temperature is 0~25 DEG C；Platform temperature is 0~37 DEG C；Pressure is 0.06~0.58MPa；100~1500 μ s of duration of valve opening, spray
Penetrate the printing that epidermis is carried out under conditions of every drop volume is 25~35nl.
Support construction, subcutaneous layer of fat, skin corium and epidermis obtain 3D printing skin model after successively printing completion,
Using culture medium culture 3D printing skin model, skin model is obtained, reparation and the test of external toxicity for clinic skin.
The specific training method of skin model are as follows: by the multilayer vitro skin model after the completion of printing, with 0.2%~5%
(w/v) sterile calcium chloride solution is crosslinked 1~10min, is then rinsed 3 times with phosphate buffer (PBS), is added to Skins culture
Submergence culture in base, example of spatial compartmentalizationis liquid add the dedicated serum free medium of skin model, are put into 37 after culture 7 days
DEG C, 5%CO2Incubator in carry out the critical culture of solution-air 5~14 days, replace a liquid medium every three days, finally
To skin model.
Wherein, the support construction of skin model is by organic-biological high molecular polymer either inorganic bio institute group
At main function is support skin model.Skin model when in use, needs to remove support construction.Fat deposit is by between fat
Mesenchymal stem cells, vascular endothelial cell and natural extracellular matrix are formed, and have internal porous structure up and down.Corium
Layer is made of fibroblast, vascular endothelial cell and natural macromolecular material, wherein skin corium bottom part aperture diameter 10um, on
Layer aperture 0.4um.Epidermis is made of keratinocyte, melanocyte and hair follicle stem cells, has compact texture, and
Include corresponding accessory organ, such as hair.
Fat mesenchymal stem cell, fibroblast, keratinocyte and vascular endothelial cell in skin model, can
To be constructed accordingly for particular race crowd, and its cell origin can utilize the skin at any position of human skin
Tissue.Skin model, fat deposit with a thickness of 1500 μm, for skin corium with a thickness of 600 μm, skin layer thickness is 400 μm.
The 3D printing skin model that printing is completed is by incubation under liquid level, being added the rush blood vessel that induction of vascular generates
Growth factor after complete epidermis to be formed, skin corium, subcutaneous layer of fat and support construction, carries out solution-air culture, obtains
Skin model.
It joined fibroblast and vascular endothelial cell in the skin corium bio-ink of skin model, not only have and
The advantages that source enriches, easily obtains, growth rate is fast, also has multinomial differentiation capability, can secrete various kinds of cell during the cultivation process
The factor promotes fibroblast proliferation simultaneously to synthesize I type and III Collagen Type VI, the blood vessel access of building facilitate nutriment exchange and
The discharge of metabolic waste, the bionical true skin corium structure of human body, while being formed to epidermis layering and hair follicle, there is paracrine to make
With being conducive to late-stage differentiation at having functional multilayered structure and accessory organ.
Using more spray head biology 3D printing techniques, fat deposit is printed as to pre-designed reticular structure, by skin corium
It is printed as aperture gradually smaller structure from the bottom to top, while filling printing inside fat deposit and skin corium, it is internal convenient for being formed
Organ, such as blood vessel.Epidermis is printed as compact texture.This structure can be such that cell is uniformly distributed in the material；Internal blood
Tube passage structure is conducive to the absorption of cytotrophy substance and the discharge of metabolic waste, promotes cell Proliferation, closer to people
Body real skin structure；Fibroblast and the direct role and influence of fat mesenchymal stem cell can be made to upper layer epidermis, had
Conducive to the proliferation and differentiation of epidermal layer cells.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of 3D printing skin model, which is characterized in that including the epidermis, skin corium, subcutaneous set gradually from top to bottom
Fat deposit and support construction；
The subcutaneous layer of fat is provided with the first through hole of multiple internal run-throughs, and the first through hole runs through the subcutaneous layer of fat
Two sides up and down, the skin corium is provided with the second through-hole of multiple internal run-throughs, and the internal diameter of second through-hole is from top to bottom
It is sequentially increased.
2. 3D printing skin model according to claim 1, which is characterized in that the support construction includes basal layer and adds
High level, it is described increase layer and be set around the edge of the basal layer form groove, the epidermis, the skin corium and described subcutaneous
Fat deposit is set in turn in from top to bottom in the groove.
3. 3D printing skin model according to claim 2, which is characterized in that it is described increase layer include multilayer from top to bottom
What is set gradually increases unit, each increases unit and has and spaced multiple increases part, the two neighboring institute for increasing unit
It states and increases part and be cross-linked.
4. 3D printing skin model according to claim 2 or 3, which is characterized in that the layer of increasing includes multiple from upper
What is set gradually under increases unit, and the unit of increasing includes the first of Thermo-sensitive increasing the second of part and non-Thermo-sensitive
Increase part, described first, which increases part, increases part interval with described second and connect.
5. described in any item 3D printing skin models according to claim 1~3, which is characterized in that the subcutaneous layer of fat and
The skin corium is provided with multilayer, and the aperture of one end far from the epidermis of second through-hole is 10~200 μm, institute
The aperture for stating one end far from the subcutaneous layer of fat of the second through-hole is 0.4 μm~40um.
6. a kind of construction method of the described in any item 3D printing skin models of Claims 1 to 5, which is characterized in that including such as
Lower step:
(1), the skin image of human body is extracted, and carries out three-dimensional reconstruction, obtains three-dimensional digital model or direct construction skin model
Three-dimensional digital model；
(2), support construction, subcutaneous fat are successively printed with Z-type or unistage type printing path according to the three-dimensional digital model
Layer, skin corium and epidermis obtain the 3D printing skin model；
(3), using 3D printing skin model described in culture medium culture.
7. construction method according to claim 6, which is characterized in that the material for printing the support construction is selected from synthesis height
In the composite material of molecular material, natural extracellular matrix material and synthesis high molecular material and natural extracellular matrix material
It is a kind of；
It is total that the synthesis high molecular material is selected from polycaprolactone, l-lactic acid, racemic poly lactose, poly lactic-co-glycolic acid
One of polymers；
The natural extracellular matrix material is selected from one of chitosan, collagen, gelatin.
8. construction method according to claim 6, which is characterized in that the material for printing the subcutaneous layer of fat is fat deposit
Bio-ink；
The fat deposit bio-ink includes fat deposit host material, fat deposit seed cell and fat deposit active factors；
The fat deposit host material includes natural extracellular matrix and biological degradation polyalcohol；
The natural extracellular matrix is selected from collagen, gelatin, fibroin albumen, fibroin, hyaluronic acid, fibrinogen, sea
Alginic acid, cellulose, starch, at least two in chitin and chitosan；
The biological degradation polyalcohol is selected from the double acrylic acid of poly lactide-glycolide acid, polylactic acid, polyethylene glycol
The one of which of ester, polyglycolic acid and polyethylene terephthalate；
The fat deposit seed cell includes fat mesenchymal stem cell；
The fat deposit active factors include IBMX, biotin, insulin and Indomethacin；
The concentration of the IBMX is 0.1~1.0mol/ml, and the concentration of the biotin is 0.1~1.5mmol/ml, the pancreas islet
The concentration of element is 5~15mol/ml, and the concentration of the Indomethacin is 100~200mmol/ml.
9. construction method according to claim 6, which is characterized in that the material for printing the skin corium is skin corium biology
Ink；
The skin corium bio-ink includes skin corium host material, skin corium seed cell and skin corium active factors；
The skin corium host material is native protein high molecular material or natural polysaccharide material or modified hydrogel material；
The native protein high molecular material is selected from one of collagen, gelatin, fibroin and fibrinogen hydrogel；
The natural polysaccharide material is selected from one of alginic acid, cellulose, starch, chitin and chitosan；
The skin corium seed cell includes fibroblast and vascular endothelial cell, the fibroblast and described intravascular
Chrotoplast ratio is 2:1~5:1；
The skin corium active factors are selected from one of VEGF, bFGF, aFGF, HGF, PDGF, PDGF or its derived protein；
The concentration of the VEGF is 6~25ng/ml, and the concentration of the bFGF is 0.1~1.0mg/ml, and the concentration of the aFGF is
The concentration of 0.1~2.0mg/ml, the HGF are 20~40ng/ml, and the concentration of the PDGF is 0.1~1.0mg/ml.
10. construction method according to claim 6, which is characterized in that it is raw for epidermis to print the material of the epidermis
Object ink；
The epidermis bio-ink includes epidermis seed cell and epidermis active factors；
The epidermis seed cell includes keratinocyte, melanocyte and hair follicle stem cells；
The epidermis active factors include HKGS, the insulin, bFGF, EGF, hydrogenation for being suitble to the keratinocyte growth
Cortisone, transferrins, glutamine, vitamin C and calcium chloride, be suitble to the melanocyte growth HGF and ET-1 and
It is suitble to the FGF-1 and FGF-2 of the hair follicle stem cells growth；
The concentration of the HKGS is 10~100mg/ml, and the concentration of the insulin is 5.0~55ng/ml, the concentration of the EGF
For 0.1~15.0ng/ml, the concentration of the bFGF is 0.1~1.0mg/ml, and the concentration of the hydrocortisone is 0.1~5.0
μ g/ml, the concentration of the transferrins are 5.0~55.0ng/ml, and the concentration of the glutamine is 0.2~22.0g/l, institute
Stating ascorbic concentration is 20~30ng/ml, and the concentration of the calcium chloride is 0.1Mm~20Mm；
The concentration of the HGF is 20~40ng/ml, and the concentration of the ET-1 is 1.0~20ng/ml；
The concentration of the FGF-1 is 10~20pg/ml, and the concentration of the FGF-2 is 20~40pg/ml.
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| SE1950711A1 (en) * | 2019-06-13 | 2020-12-14 | Cellink Ab | 3d bioprinted skin tissue model |
| CN112263357A (en) * | 2020-10-25 | 2021-01-26 | 无锡聚火医疗器械有限公司 | Preparation method of medical artificial skin |
| CN113082286A (en) * | 2021-04-07 | 2021-07-09 | 江南大学 | Three-layer bionic skin stent based on 3D printing technology and preparation method thereof |
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| CN114540275A (en) * | 2022-02-23 | 2022-05-27 | 合肥学院 | Skin biological printing ink and preparation method and application thereof |
| CN115305231A (en) * | 2022-08-19 | 2022-11-08 | 上海大学 | Multi-process composite artificial skin preparation method |
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| CN116077737A (en) * | 2023-04-07 | 2023-05-09 | 云南云科特色植物提取实验室有限公司 | Artificial skin containing vascular structure and preparation method thereof |
| CN116099056A (en) * | 2023-04-07 | 2023-05-12 | 云南云科特色植物提取实验室有限公司 | Artificial skin containing bionic texture structure and preparation method thereof |
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| CN116077737A (en) * | 2023-04-07 | 2023-05-09 | 云南云科特色植物提取实验室有限公司 | Artificial skin containing vascular structure and preparation method thereof |
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