CN108144114A - For the 3D printing material of organizational project and the preparation method of Biodegradable scaffold material - Google Patents
For the 3D printing material of organizational project and the preparation method of Biodegradable scaffold material Download PDFInfo
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- CN108144114A CN108144114A CN201810009933.5A CN201810009933A CN108144114A CN 108144114 A CN108144114 A CN 108144114A CN 201810009933 A CN201810009933 A CN 201810009933A CN 108144114 A CN108144114 A CN 108144114A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0023—Heat
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
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- A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
- A61L2202/20—Targets to be treated
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Abstract
The present invention provides a kind of preparation method of 3D printing material for organizational project, includes the following steps：1) gelatin, sodium alginate are dissolved in water, mixing resulting mixture；2) mixture is transferred to sterilization container interior sealing, sterilization treatment is carried out using 60 80 DEG C of medium temperature interval sterilizations；3) seed cell is added in step 2) mixing in the mixture of sterilized processing, obtains 3D printing material.The present invention also provides a kind of preparation methods of the Biodegradable scaffold material for organizational project, include the following steps：By 3D printing material using 3D printer print carriage, calcium chloride solution is added in after the completion of printing, carries out stent curing.3D printing material prepared by this method can make printing after-poppet that excellent in shape be kept not collapse, and in germ-free condition, can be directly used for organizational project.
Description
Technical field
The present invention relates to field of tissue engineering technology, and in particular to a kind of to be dropped for the 3D printing material and biology of organizational project
Solve the preparation method of timbering material.
Background technology
Tissue engineering bracket material refers to that bio-tissue defect can be simultaneously implanted into tissue biopsy cell combination to be risen
To the material of substitution effect, according to the characteristic of biomaterial, two major class are can be divided into：Biodegradable and nonbiodegradable
Type.
Ideal organizational project Biodegradable scaffold material must have following characteristics：(1) good biocompatibility
And surface-active, conducive to the tactophily of cell, teratogenesis, unsafe to organism；(2) there is good conductibility and induction
Property, the degradation speed of material can be preferably controlled, it is made to repair speed with organism and is matched, and can induce the seed on stent
Cell differentiation is proliferated；(3) have suitable aperture and porosity, and be interconnected, conducive to nutriment in intrapore perfusion and
The discharge of metabolin makes it possible that cell grows into hole；(4) there is good mechanical strength and plasticity, material shapeable
Any shape, and graft can be supported to remain unchanged shape in transplantation site, until cambium grows into and substitutes transplanting completely
Object.
Sodium alginate (Sodium Alginate, SA) is a kind of polysaccharide extracted from natural brown alga, has good water
Dissolubility and biocompatibility can improve the function and survival rate of seed cell.Sodium alginate, which meets calcium ion, can combine rapidly shape
Into stable gel, which has comparatively ideal 3 D stereo porous structure, this cross-linking process is milder, to the damage of cell
Evil is smaller, and very rapid, can realize instantaneous crosslink in situ, becomes the ideal for directly carrying cell 3D printing
Timbering material.Alginate has facilitation for the maintenance of seed cell phenotype and the synthesis of proteoglycan.Gel network knot
Structure can provide sufficient attachment surface for cell, and cell growth is made to be conducive to cell in a kind of environment of more similar physiological status
It keeps activity and secretes a large amount of matrix.
But collapse phenomenon only can be generated during 3D printing by the use of sodium alginate as host material, it cannot keep
Ideal shape and structure, should enable material pass through printing syringe needle, and material is not caused to collapse after printing, keep one
Fixed shape, the preparation of material hydrogel state is very crucial.
Further, since material is cultivated in vitro after directly being printed jointly with cell, so must complete to disappear before printing
The process of poison sterilizing can make sodium alginate and gelatin materials be carbonized, can not dissolve using 170 DEG C of hot air sterilization in the prior art
Yu Shuizhong forms hydrogel, and damp and hot high pressure sterilization can make material directly be denaturalized into solid, be not used to 3D printing.
Therefore being badly in need of a kind of material preparation method makes the biodegradable printed material of preparation keep suitable water-setting gluey
State, and material is made to keep being in germ-free condition on the basis of original performance.
Invention content
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of 3D printing material for organizational project
Preparation method, 3D printing material prepared by this method can make printing after-poppet that excellent in shape be kept not collapse, and in sterile shape
State can be directly used for organizational project.
Technical solution provided by the present invention is：
For the preparation method of the 3D printing material of organizational project, include the following steps：
1) gelatin, sodium alginate are dissolved in physiological saline, mixing resulting mixture；
2) mixture is transferred to sterilization container interior sealing, sterilization treatment is carried out using 60-80 DEG C of medium temperature interval sterilization；
3) seed cell is added in step 2) mixing in the mixture of sterilized processing, obtains 3D printing material.
In above-mentioned technical proposal, sterilization treatment is carried out using medium temperature interval sterilization, temperature range is at 60-80 DEG C, at this
Under temperature condition, with regard to that can reach elimination Most bacterial, and the temperature of this range is relatively low, will not cause sea within 15 minutes or more
Mosanom is carbonized with gelatin materials as under the conditions of 170 DEG C of hot-air sterilizations, will not so that material is such as damp and hot at 121 DEG C
Generally directly denaturation becomes solid to sterilization.75% ethyl alcohol is immersed using the composite material after having printed in the prior art to disappear
Poison, even if used calcium chloride crosslinking curing mistake still can cause material dissolving, cave in it is shapeless.Ultraviolet irradiation sterilization is only
The disinfection of material surface can be carried out, the sterilizing of material deep layer cannot be carried out.The medium temperature interval sterilization that the present invention uses can be tieed up
The hydrogel state of biodegradable printed material is held, while sterilization effect can be reached again.
Preferably, the step 1) gelatin a concentration of 0.02-0.1g/ml in the mixture.Further preferably 0.04-
0.08g/ml。
Preferably, the step 1) sodium alginate a concentration of 0.05-0.15g/ml in the mixture.Further preferably
0.07-0.09g/ml。
Preferably, being additionally added hydroxyapatite in the step 1) mixture, the hydroxyapatite is in the mixture
A concentration of 0.02-0.05g/ml.
Preferably, when the step 1) gelatin and sodium alginate mixing resulting mixture, temperature is 40-70 DEG C.
Preferably, it is characterized in that, step 2) the medium temperature interval sterilization refers to：One was carried out every 12-24 hours
Secondary disinfection, 15-30 minutes each, coprocessing 2-4 times.Further preferably, it is primary every for 24 hours, each 20-30min.
Preferably, the step 3) seed cell is added to a concentration of 1 × 10 in mixture6-10×106cells/
ml。
Preferably, the step 3) seed cell includes mesenchymal stem cell (mesenchymal stem
Cells, MSCs), the cartilage cell that is induced differentiation by mesenchymal stem cell or osteoblast.
The present invention also provides a kind of preparation method of the Biodegradable scaffold material for organizational project, including walking as follows
Suddenly：
By 3D printing material using 3D printer print carriage, calcium chloride solution is added in after the completion of printing, stent is carried out and consolidates
Change；The 3D printing material is prepared by above-mentioned preparation method.
Preferably, a concentration of 0.02-0.05g/ml of the calcium chloride solution.
Compared with the existing technology, beneficial effects of the present invention are embodied in：
(1) the 3D printing material obtained by preparation method provided by the present invention has excellent moulding effect, energy after printing
Original shape is enough kept not collapse.
(2) preparation method provided by the present invention carries out sterilization treatment using medium temperature interval sterilization, does not destroy 3D printing
Material primary characteristic, while reach sterilization effect again, with being used for 3D printing after mixing with cells, and can directly be trained after printing
It supports.
Description of the drawings
Fig. 1 is that mixing adds in the photograph of cell/scaffold complex printed after bone marrow mesenchymal stem cells in embodiment 7
Piece；
Fig. 2 be embodiment 8 in mixing add in lure Cartilage culture after 4 weeks bone marrow mesenchymal stem cells printing lure cartilage
The photo of cell/scaffold complex；
Fig. 3 is luring into for the bone marrow mesenchymal stem cells printing that mixing is added in after luring skeletonization culture 4 weeks in embodiment 9
The photo of bone/scaffold complex；
Fig. 4 is printed for sodium alginate/glutin compound rest and cell compatibility lab diagram.
Specific embodiment
With reference to specific embodiment, the invention will be further described.
Experiment material：Gelatin (gelatin from porcine skin), purchased from sigma, G2500-500G, product goods
Number：9000-70-8, product code：1001221796；Sodium alginate (alginic acid sodium salt from brown
Algae), purchased from sigma, low viscosity, product article No.：9005-38-3, product code：1069301112.
Embodiment 1：The preparation of 3D printing material
It weighs 0.5g gelatin and adds in 10ml physiological saline, after 70 DEG C dissolve by heating, add 0.8g sodium alginates, magnetic force stirs
It mixes dissolving and is configured to the mixed sols of 10ml sodium alginates containing 0.08g/ml and 0.05g/ml gelatin as printed material.By material
Beaker is respectively charged into, rim of a cup tightens, the sterilizing of medium temperature interval, 70 DEG C, and 30min/ times, primary every for 24 hours, totally 3 times, short-term room temperature is put
It puts for use, as 3D printing material.
Embodiment 2：The preparation of 3D printing material
It weighs 0.5g gelatin and adds in 10ml physiological saline, after 70 DEG C dissolve by heating, add 1.0g sodium alginates, magnetic force stirs
It mixes dissolving and is configured to the mixed sols of 10ml sodium alginates containing 0.1g/ml and 0.05g/ml gelatin as printed material.By material
Beaker is respectively charged into, rim of a cup tightens, the sterilizing of medium temperature interval, 70 DEG C, and 30min/ times, primary every for 24 hours, totally 3 times, short-term room temperature is put
It puts for use, as 3D printing material.
Embodiment 3：The preparation of 3D printing material
It weighs 0.8g gelatin and adds in 10ml physiological saline, after 60 DEG C dissolve by heating, add in 0.5g sodium alginates, add
0.4g hydroxyapatites, magnetic agitation dissolving are configured to 10ml sodium alginates containing 0.05g/ml, 0.08g/ml gelatin and 0.04g/
The mixed sols of ml hydroxyapatites is as printed material.Material is respectively charged into beaker, rim of a cup tightens, the sterilizing of medium temperature interval,
75 DEG C, 20min/ times, primary every for 24 hours, totally 2 times, short-term room temperature is for use, as 3D printing material.
Embodiment 4：The preparation of 3D printing material
It weighs 0.2g gelatin and adds in 10ml physiological saline, after 70 DEG C dissolve by heating, add 1.0g sodium alginates, magnetic force stirs
It mixes dissolving and is configured to the mixed sols of 10ml sodium alginates containing 0.10g/ml and 0.02g/ml gelatin as printed material.By material
Beaker is respectively charged into, rim of a cup tightens, and the sterilizing of medium temperature interval, 65 DEG C, 30min/ times, primary every 20h, totally 4 times, short-term room temperature is put
It puts for use, as 3D printing material.
Embodiment 5：The preparation of 3D printing material
It weighs 0.5g gelatin and adds in 10ml physiological saline, after 70 DEG C dissolve by heating, add 0.8g sodium alginates, magnetic force stirs
It mixes dissolving and is configured to the mixed sols of 10ml sodium alginates containing 0.08g/ml and 0.05g/ml gelatin as printed material.By material
Beaker is respectively charged into, rim of a cup tightens, and the sterilizing of medium temperature interval, 70 DEG C, 30min/ times, primary every 12h, totally 4 times, short-term room temperature is put
It puts for use, as 3D printing material.
Embodiment 6：The preparation of 3D printing material
It weighs 0.5g gelatin and adds in 10ml physiological saline, after 70 DEG C dissolve by heating, add 0.8g sodium alginates, magnetic force stirs
It mixes dissolving and is configured to the mixed sols of 10ml sodium alginates containing 0.08g/ml and 0.05g/ml gelatin as printed material.By material
Beaker is respectively charged into, rim of a cup tightens, and the sterilizing of medium temperature interval, 70 DEG C, 30min/ times, primary every 18h, totally 3 times, short-term room temperature is put
It puts for use, as 3D printing material.
Embodiment 7：Mesenchymal stem cell/scaffold complex printing
Bone marrow mesenchymal stem cells are pressed 5 × 106Cells/ml is added in the 3D printing material of the preparation of embodiment 1 and mixes
It is even, it is fitted into printing barrel, centrifugation, 200g, 2 minutes, removes the bubble in material, upper 3D printer printing, it is suitable to select
Print syringe needle.It is 8 layers to print the number of plies, as shown in Figure 1a, whole to keep shape intact, does not occur collapsing situation, be transparent.It beats
0.025g/ml calcium chloride solutions are added in after having printed, ionomer is carried out 3 minutes with timbering material, completes stent solidification process, production
As shown in Figure 1 b, complex is creamy white to get Biodegradable scaffold material object.
Laboratory apparatus：3D biomaterials printing instrument (BioScaffolder 2.1).
Bone marrow mesenchymal stem cells：Extracted from 1 monthly age new zealand white rabbit marrow, by density-gradient centrifugation method and
Stationary culture separation obtains, and expands to the 4th generation, for testing.
Embodiment 8：Lure cartilage cell/scaffold complex printing
Cartilage cell will be lured by 1 × 106Cells/ml is added to mixing in the 3D printing material of the preparation of embodiment 2, is packed into
It prints in barrel, centrifugation, 200g, 2 minutes, removes the bubble in material, suitable print needle is selected in upper 3D printer printing
Head.It is 20 layers to print the number of plies, as shown in Figure 2 a, whole to keep shape intact, does not occur collapsing situation, be transparent.It has printed
0.025g/ml calcium chloride solutions are added in afterwards, are carried out ionomer 3 minutes with timbering material, are completed stent solidification process, product is such as
Shown in Fig. 2 b, complex is creamy white to get Biodegradable scaffold material.
Lure cartilage cell：The 3rd generation bone marrow mesenchymal stem cells being separately cultured are being lured into Cartilage culture base culture 4 weeks
Afterwards, cartilage cell has been formed through Toluidine blue staining identification, has digested to test.
Embodiment 9：Lure osteoblast/scaffold complex printing
Osteoblast will be lured by 1 × 106Cells/ml is added to mixing in the 3D printing material of the preparation of embodiment 3, is packed into
It prints in barrel, centrifugation, 200g, 2 minutes, removes the bubble in material, suitable print needle is selected in upper 3D printer printing
Head.It is 16 layers to print the number of plies, and as shown in Figure 3a, more not plus the material shape of hydroxyapatite keeps more preferable, does not occur collapsing feelings
Condition.0.03g/ml calcium chloride solutions are added in after having printed, ionomer is carried out 5 minutes with timbering material, stent is completed and cured
Journey, as shown in Figure 3b, compound arrangement of apertures is uniform for product, even if being not added with calcium chloride crosslinking can remain intact to get biology
Biodegradable bracket material.
Lure osteoblast：The 3rd generation bone marrow mesenchymal stem cells being separately cultured are being lured into osteogenic culture 4 weeks
Afterwards, the Mineral nodules formed after osteogenic induction are dyed with von kossa decoration methods to identify whether induced synthesis
Skeletonization digests to test.
Comparative example 1：The preparation of the Biodegradable scaffold material of non-sterilization treatment
It weighs 0.5g gelatin and adds in 10ml physiological saline, after 70 DEG C dissolve by heating, add 0.8g sodium alginates, magnetic force stirs
It mixes dissolving and is configured to the mixed sols of 10ml sodium alginates containing 0.08g/ml and 0.05g/ml gelatin as printed material.It is short-term normal
Temperature is placed for use, as 3D printing material.
Bone marrow mesenchymal stem cells are pressed 10 × 106Cells/ml is added in the 3D printing material of above-mentioned preparation and mixes
It is even, it is fitted into printing barrel, centrifugation, 200g, 2 minutes, removes the bubble in material, upper 3D printer printing, it is suitable to select
Print syringe needle.0.025g/ml calcium chloride solutions are added in after having printed, ionomer is carried out 3 minutes with timbering material, completes stent
Solidification process is to get cell/scaffold complex.The not sterilized compound is added in into low sugar DMEM culture mediums, 37 DEG C, 5%
CO2Incubator is cultivated, and muddiness occurs in culture medium within second day, and the situation of bacterial growth is presented, can only boil discarding, it is impossible to
It is cultivated for, to prevent laboratory pollution.
Performance test 1
For the 3D printing material prepared by embodiment 1-6, all material can keep hydrogel state, in gelatine content
During for 0.02g/ml, mixing material naked eyes sight is relatively dilute, and when gelatine content is 0.08g/ml, mixing material is relatively thick.
Short-term place there are no bacterium colony growth on material, and not carry out having fungus growth in the mixing material of medium temperature interval sterilizing.
Performance test 2：Cell upgrowth situation on stent detects
Mesenchymal stem cell/scaffold complex prepared by selection example 7, is rinsed well with phosphate buffer
Afterwards, a part of stent is put into 6 well culture plates, adds in low sugar DMEM culture mediums, 37 DEG C, 5%CO2Incubator is cultivated, such as
Shown in Fig. 4 a, cell growth is good, and after cultivating 2 days, culture medium color becomes crocus, liquid clarification from red, and prompting does not have
Pollution, and due to cell metabolism, make Medium's PH Value in acidity.
A remaining part adds in PBS, puts 37 DEG C, 5%CO2Incubator is cultivated, and observes the stent whether there is solution subsidence
And pollution condition, such as Fig. 4 b, the stent after being cured in calcium chloride solution is immersed in PBS, and 37 DEG C are placed 1 week, stent knot
Structure is complete, does not occur solution subsidence phenomenon, the situation for also not occurring long bacterium.
In vitro culture is organized to be seen after 3 days using calcein-AM (Calcein-AM)/propidium iodide (PI) fluorescent staining
Examine cell growing state in the bracket.
It is as follows：
(1) it inhales and abandons supernatant in culture plate, with PBS (phosphate buffer) fully cleaning tape cytoskeletons 2 times；
(2) 5mL Calcein-AM (5 μM)/PI (3 μM) mixed liquor is added in 6 orifice plates, 37 DEG C are cultivated 15-30 minutes.
(3) it is washed twice with PBS (phosphate buffer) and removes extra dyestuff.
(4) the optical filter observation cell of fluorescence microscope 488nm, 543nm excitation wavelength.As a result see attached drawing 4c, cell
The dead dyeing (calcein-AM/PI) of cell work is carried out after being grown 7 days on stent：Living cells (green) quantity is significantly relatively dead thin
Born of the same parents' (red) are more, and what black arrow was directed toward is living cells, what white arrow was directed toward is dead cell, indistinct on picture to also have
Green is three-dimensional because of material, prompt cell and the timbering material of printing on growth conditions it is good.
Claims (9)
1. the preparation method of the 3D printing material for organizational project, which is characterized in that include the following steps：
1) gelatin, sodium alginate are dissolved in physiological saline, mixing resulting mixture；
2) mixture is transferred to sterilization container interior sealing, sterilization treatment is carried out using 60-80 DEG C of medium temperature interval sterilization；
3) seed cell is added in step 2) mixing in the mixture of sterilized processing, obtains 3D printing material.
2. the preparation method of the 3D printing material according to claim 1 for organizational project, which is characterized in that the step
Rapid 1) gelatin a concentration of 0.02-0.1g/ml in the mixture.
3. the preparation method of the 3D printing material according to claim 1 for organizational project, which is characterized in that the step
Rapid 1) sodium alginate a concentration of 0.05-0.15g/ml in the mixture.
4. the preparation method of the 3D printing material according to claim 1 for organizational project, which is characterized in that the step
It is additionally added hydroxyapatite in rapid 1) mixture, the hydroxyapatite a concentration of 0.02-0.05g/ml in the mixture.
5. the preparation method of the 3D printing material according to claim 1 for organizational project, which is characterized in that the step
Rapid 2) medium temperature interval sterilization refers to：Once sterilizing, 15-30 minutes each, coprocessing 2-4 times were carried out every 12-24 hours.
6. the preparation method of the 3D printing material according to claim 1 for organizational project, which is characterized in that the step
Rapid 3) seed cell is added to a concentration of 1 × 10 in mixture6-10×106cells/ml。
7. the preparation method of the 3D printing material according to claim 1 for organizational project, which is characterized in that the step
Rapid 3) seed cell includes mesenchymal stem cell, the cartilage cell induced differentiation by mesenchymal stem cell or skeletonization
Cell.
8. the preparation method of the Biodegradable scaffold material for organizational project, which is characterized in that include the following steps：
By 3D printing material using 3D printer print carriage, calcium chloride solution is added in after the completion of printing, carries out stent curing；Institute
3D printing material is stated to be prepared by any preparation method of claim 1~7.
9. the preparation method of the Biodegradable scaffold material according to claim 8 for organizational project, feature exist
In a concentration of 0.02-0.05g/ml of the calcium chloride solution.
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