CN108114324A - A kind of vitro cytotoxicity test method for the method and the stent that skin tissue engineering scaffold is prepared based on 3D biometric print technologies - Google Patents
A kind of vitro cytotoxicity test method for the method and the stent that skin tissue engineering scaffold is prepared based on 3D biometric print technologies Download PDFInfo
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Abstract
The invention discloses a kind of vitro cytotoxicity test method for the method and the stent that skin tissue engineering scaffold is prepared based on 3D biometric print technologies, the preparation of high strength fibre element nanofiber/gelatin-compounded hydrogel including being used for print carriage, the technique of 3D tissue engineering brackets printing, the crosslinking process of stent.The present invention solves tissue engineering bracket high porosity, high-precision requirement using 3D biometric print technologies.Filler materials of the CNF as GEL plays an important role of raising GEL mechanical strengths, and the stent after printing is crosslinked using immersion in genipin solution.Skin tissue engineering scaffold prepared by this method has good mechanical property, and it has no toxic side effect, without immune rejection, tissue engineering bracket is prepared with convenient and efficient, easily controllable advantage using 3D printing technique simultaneously, it and can be according to the depth of patient's wound, size, shape carry out personalized customization.
Description
Technical field
The present invention relates to a kind of methods and the stent that skin tissue engineering scaffold is prepared based on 3D biometric print technologies
Vitro cytotoxicity test method, and in particular to one kind has biocompatibility, biodegradability, bioactivity and meets group
The preparation of the composite material of weaver's engineering support requirement and 3D printing technique adaptable therewith.
Background technology
Repair of tissue defect and organ transplant are one of greatest breakthroughs in 20th century medical history.Biological 3D printing technique
Appearance the patient of organ transplant and tissue repair is necessarily needed to bring new dawn to be several.
3D printing is also referred to as rapid shaping technique, is born in late 1980s.This is a kind of based on material stacking
The manufacturing technology of method, it is considered to be a manufacturing significant achievement in the past 20 years.It is one and collects mechanical engineering, computer aided manufacturing
Design is helped, engineering technology, Layered Manufacturing Technology, Numeric Control Technology, material science, the science of laser technology can be direct,
Quickly, the prototype of a certain function or manufactured parts is directly changed into product exactly, high efficiency is realized for production
Means are provided with low cost.
The research and selection of timbering material are the important research contents of tissue engineering bracket, and natural macromolecular material gelatin has
There are good biocompatibility, biodegradability and bioactivity, can preferably induce the self-recovery of compromised skin, but
Mechanical property is poor, and degradation speed is too fast under the action of various enzymes in vivo, and cambium does not grow good stent also and caved in,
Therefore, it is important to find a kind of method that can improve its mechanical property.
The gelatin composite material with excellent mechanical properties is printed as with three-dimensional porous knot using rapid shaping technique
The skin tissue engineering scaffold of structure can provide new approach for skin injury reparation.
The content of the invention
The purpose of the present invention is in view of the deficienciess of the prior art, providing a kind of based on the preparation of 3D biometric prints technology
The vitro cytotoxicity test method of the method for skin tissue engineering scaffold and the stent.
The object of the invention is achieved through the following technical solutions：One kind prepares skin tissue engineering based on 3D biometric print technologies
The method of stent, it is characterized in that, comprise the following steps：
（1）Gelatin（Gelatin, GEL）The preparation of solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until bright
Glue all dissolves, and obtains gelatin solution；
（2）It is cellulose nano-fibrous（Cellulose nanofibers, CNF）/ gelatin（Gelatin, GEL）Composite hydrogel
It prepares：Cellulose nano-fibrous solution is added in gelatin solution, using solution blended process；
（3）The 3D printing of tissue engineering bracket：By cellulose nano-fibrous/gelatin（CNF/GEL）Compound hydrogel material is poured into
Into 3D printer barrel, then barrel temperature, workbench temperature, needle diameter, extrusion pressure, nozzle walking speed are set
Degree, aperture parameters, complete the 3D printing of tissue engineering bracket；
（4）The crosslinking of tissue engineering bracket：Printed stent is inserted in genipin solution and is impregnated, being rinsed repeatedly after taking-up can
Directly use or it is cold deposit it is spare.
Further, step（1）In, the mass concentration of the gelatin solution is 2~15%（Mass percent）.
Further, step（2）The preparation of cellulose nano-fibrous/gelatin-compounded hydrogel, the fiber
Plain nanofiber solution, mass concentration 1%-8%, cellulose nano-fibrous loading are 3~20%, i.e. nano-cellulose
（Dry weight）In blend（Dry weight refers to cellulose nano-fibrous and gelatin）In mass percent be 3~20%.
Further, step（2）The preparation of cellulose nano-fibrous/gelatin-compounded hydrogel, blending temperature are
25~45 DEG C, when the blending time is 1~4 small.
Further, step（3）The 3D printing of the tissue engineering bracket, in 3 d structure model, upper and lower layer
Angle is 1~90（Angle between the silk of adjacent two layers）, the thickness of layering is 120~400 μm.
Further, step（3）The 3D printing of the tissue engineering bracket, barrel temperature are 3~25 DEG C, platform temperature
Spend for 1~25 DEG C, needle diameter is 150~400 μm, and extrusion pressure is 0.01~0.6Mpa, the nozzle speed of travel for 3~
50mm/s, aperture are 100~500 μm.
Further, step（4）The concentration of the crosslinking of the tissue engineering bracket, wherein genipin solution for 0.1~
10mM。
Further, step（4）The crosslinking of the tissue engineering bracket, stent are immersed in genipin solution, crosslinking the
The crosslinking temperature in one stage is 1~10 DEG C, and when crosslinking time is 2~10 small, crosslinking second stage temperature is 25~50 DEG C, crosslinking
When time is 5~48 small.
Further, step（4）The crosslinking of the tissue engineering bracket, cold condition of storage of depositing are：Ethylene oxide is close
It is honored as a queen, in -80~-15 DEG C of sterile storages.
The above-mentioned vitro cytotoxicity test method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies, it is special
Sign is, before repopulating cell, stent is positioned under r rays and irradiates 2h progress sterilization treatments；Then stent soaks under the conditions of 37 DEG C
Not in the medium overnight, then per 3T3 cell suspending liquids are added in hole, be put into 37 DEG C, gas concentration lwevel 5%, humidity 95%
Incubator in cultivated；Proliferative conditions of the 3T3 in stent finally are evaluated using CCK-8, using microplate reader in 450nm wavelength
OD values are detected at place.The performance of stent is judged by comparing the difference of experimental group and blank group absorbance, if experimental group OD values are remote
Less than blank group OD values, illustrate that 3T3 upgrowth situations on stent are poor；If experimental group OD values illustrate 3T3 close to blank group OD values
Upgrowth situation is good on stent；If experimental group OD values are connect much larger than blank group OD values, illustrate that stent cell growth has very
Good proliferation.
Compared with prior art, the present invention has the following advantages and beneficial effects：
（1）CNF used in the present invention can pass through from many natural plants, agricultural waste material or from from microcrystalline cellulose
Conventional chemical processes obtain, and improve the surcharge of plant fiber；
（2）CNF used herein is inserted in GEL matrix, the mechanical strength of hydrogel greatly improved as reinforcing material,
And nontoxic no effect, there is good biocompatibility；
（3）The present invention prepares skin tissue engineering scaffold by 3D printing technique, and not only technique is convenient, and can be according to patient
The size of wound, shape is different, completes personalized customization；
（4）Tissue engineering bracket prepared by the present invention has complicated porous internal structure, and porosity is not less than 80%, hole connection
Rate is not less than 80%, has good gas permeability and absorbability, may advantageously facilitate the growth of cell, blood vessel and tissue.
The present invention solves tissue engineering bracket high porosity, high-precision requirement using 3D biometric print technologies.CNF makees
For the filler material of GEL, play an important role of to improve GEL mechanical strengths, the stent after printing is carried out using immersion in genipin solution
Crosslinking.Skin tissue engineering scaffold prepared by this method has good mechanical property, and has no toxic side effect, anti-without immune rejection
Should, while tissue engineering bracket is prepared with convenient and efficient, easily controllable advantage using 3D printing technique, and can basis
The depth of patient's wound, size, shape carry out personalized customization.
Description of the drawings
Fig. 1 is the skin tissue engineering scaffold that embodiment 2 prints；
Fig. 2 is the skin tissue engineering scaffold that embodiment 3 prints；
Fig. 3 is the tissue engineering bracket after 4 genipin cross-linked of embodiment；
Fig. 4 is the vitro cytotoxicity experimental result for the stent that embodiment 5 is completed after printing.
Specific embodiment
Below by embodiment, the present invention is further illustrated, but therefore the scope of the present invention is not confined to down
Row embodiment, but limited by the claims and specification of the present invention.
1. embodiment 1
（1）Gelatin（Gelatin, GEL）The preparation of solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until bright
Glue all dissolves；
（2）It is cellulose nano-fibrous（cellulose nanofibers）/ gelatin（Gelatin, GEL）The system of composite hydrogel
It is standby：In GEL solution, a certain amount of CNF solution is added in so that GEL concentration is that 3%, CNF solid-fillings amount is 3%, 30 DEG C of conditions
When lower blending and stirring 1 is small；
（3）The 3D printing of tissue engineering bracket：The CNF/GEL compound hydrogel materials of above-mentioned preparation are entered to 3D printer barrel
In, it is 3 DEG C to set barrel temperature, platform temperature is 1 DEG C, needle diameter is 150 μm, extrusion pressure 0.08MPa, nozzle walking speed
10mm/S, 100 μm of aperture are spent, completes the 3D printing of tissue engineering bracket；
（4）The crosslinking of tissue engineering bracket：Printed stent is inserted in the genipin solution that concentration is 0.2mM, 4 DEG C of conditions
It is lower impregnate 4 it is small when, it is rear 25 DEG C impregnate 24 it is small when, taking-up is rinsed well rear spare repeatedly with ultra-pure water.
2. embodiment 2
（1）Gelatin（Gelatin, GEL）The preparation of solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until bright
Glue all dissolves；
（2）It is cellulose nano-fibrous（cellulose nanofibers）/ gelatin（Gelatin, GEL）The system of composite hydrogel
It is standby：In GEL solution, a certain amount of CNF solution is added in so that GEL concentration is that 5%, CNF solid-fillings amount is 5%, 30 DEG C of conditions
When lower blending and stirring 2 is small；
（3）The 3D printing of tissue engineering bracket：The CNF/GEL compound hydrogel materials of above-mentioned preparation are entered to 3D printer barrel
In, it is 5 DEG C to set barrel temperature, platform temperature is 3 DEG C, needle diameter is 150 μm, extrusion pressure 0.1MPa, nozzle walking speed
10mm/S, 150 μm of aperture are spent, completes the 3D printing of tissue engineering bracket；
（4）The crosslinking of tissue engineering bracket：Printed stent is inserted in the genipin solution that concentration is 0.2mM, 4 DEG C of conditions
It is lower impregnate 4 it is small when, it is rear 40 DEG C impregnate 18 it is small when, taking-up is rinsed well rear spare repeatedly with ultra-pure water.
3. embodiment 3
（1）Gelatin（Gelatin, GEL）The preparation of solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until bright
Glue all dissolves；
（2）It is cellulose nano-fibrous（cellulose nanofibers）/ gelatin（Gelatin, GEL）The system of composite hydrogel
It is standby：In GEL solution, a certain amount of CNF solution is added in so that GEL concentration is that 6%, CNF solid-fillings amount is 8%, 35 DEG C of conditions
When lower blending and stirring 3 is small；
（3）The 3D printing of tissue engineering bracket：The CNF/GEL compound hydrogel materials of above-mentioned preparation are entered to 3D printer barrel
In, it is 8 DEG C to set barrel temperature, platform temperature is 5 DEG C, needle diameter is 220 μm, extrusion pressure 0.1MPa, nozzle walking speed
15mm/S, 200 μm of aperture are spent, completes the 3D printing of tissue engineering bracket；
（4）The crosslinking of tissue engineering bracket：Printed stent is inserted in the genipin solution that concentration is 0.3mM, 4 DEG C of conditions
It is lower impregnate 4 it is small when, it is rear 40 DEG C impregnate 12 it is small when, taking-up is rinsed well rear spare repeatedly with ultra-pure water.
4. embodiment 4
（1）Gelatin（Gelatin, GEL）The preparation of solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until bright
Glue all dissolves；
（2）It is cellulose nano-fibrous（cellulose nanofibers）/ gelatin（Gelatin, GEL）The system of composite hydrogel
It is standby：In GEL solution, a certain amount of CNF solution is added in so that GEL concentration is that 7%, CNF solid-fillings amount is 10%, 40 DEG C of items
When blending and stirring 4 is small under part；
（3）The 3D printing of tissue engineering bracket：The CNF/GEL compound hydrogel materials of above-mentioned preparation are entered to 3D printer barrel
In, it is 10 DEG C to set barrel temperature, platform temperature is 5 DEG C, needle diameter is 220 μm, extrusion pressure 0.2MPa, nozzle walking speed
20mm/S, 300 μm of aperture are spent, completes the 3D printing of tissue engineering bracket；
（4）The crosslinking of tissue engineering bracket：Printed stent is inserted in the genipin solution that concentration is 0.4mM, 4 DEG C of conditions
It is lower impregnate 3 it is small when, it is rear 40 DEG C impregnate 24 it is small when, taking-up is rinsed well rear spare repeatedly with ultra-pure water.
5. embodiment 5
（1）Gelatin（Gelatin, GEL）The preparation of solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until bright
Glue all dissolves；
（2）It is cellulose nano-fibrous（cellulose nanofibers）/ gelatin（Gelatin, GEL）The system of composite hydrogel
It is standby：In GEL solution, a certain amount of CNF solution is added in so that GEL concentration is that 6%, CNF solid-fillings amount is 10%, 40 DEG C of items
When blending and stirring 4 is small under part；
（3）The 3D printing of tissue engineering bracket：The CNF/GEL compound hydrogel materials of above-mentioned preparation are entered to 3D printer barrel
In, it is 15 DEG C to set barrel temperature, platform temperature is 8 DEG C, needle diameter is 220 μm, extrusion pressure 0.2MPa, nozzle walking speed
30mm/S, 350 μm of aperture are spent, completes the 3D printing of tissue engineering bracket；
（4）The crosslinking of tissue engineering bracket：Printed stent is inserted in the genipin solution that concentration is 0.5mM, 4 DEG C of conditions
It is lower impregnate 2 it is small when, it is rear 45 DEG C impregnate 12 it is small when, taking-up is rinsed well rear spare repeatedly with ultra-pure water.
6. mechanical strength test：
The assay method of hydrogel intensity is as follows：Hydrogel is prepared into a diameter of 15mm, is highly the cylindric of 10mm, then
Hydrogel on electronic universal test machine for mechanism obtained by testing example 1- embodiments 5 crushes intensity, as shown in table 1：
The mechanical property for the hydrogel that table 1 is prepared for specific embodiment.
7. vitro cytotoxicity is tested：
Blank group：3T3 l cells.
Experimental group：For the tissue engineering bracket hydrogel of 3D printing, 3T3 l cells.
It, will before repopulating cell（Made of embodiment 5）Stent, which is positioned under r rays, irradiates 2h progress sterilization treatments.Then
Stent submerges in the medium overnight under the conditions of 37 DEG C, then per 3T3 cell suspending liquids are added in hole, be put into 37 DEG C, 5% 2
It aoxidizes, cultivated in the incubator of 95% humidity.Then 3T3 is evaluated in blank group and the multiplication feelings of experimental group using CCK-8
Condition using microplate reader at 450nm wavelength, detects OD values, and the test result is as shown in Figure 4.By comparing experimental group and blank
The difference of absorbance is organized to judge the performance of stent, if experimental group OD values are much smaller than blank group OD values, illustrates that 3T3 is raw on stent
Long situation is poor；If experimental group OD values illustrate that 3T3 upgrowth situations on stent are good close to blank group OD values；If experimental group OD
Value is connect much larger than blank group OD values, illustrates that stent cell growth has extraordinary proliferation.
Claims (10)
1. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies, it is characterized in that, comprise the following steps：
（1）The preparation of gelatin solution：Gelatin is added in phosphate buffer, is stirred continuously until gelatin all dissolvings, obtains bright
Sol solution；
（2）The preparation of cellulose nano-fibrous/gelatin-compounded hydrogel：It is added in gelatin solution cellulose nano-fibrous molten
Liquid, using solution blended process；
（3）The 3D printing of tissue engineering bracket：Cellulose nano-fibrous/gelatin-compounded hydrogel material is poured into 3D printer
In barrel, then barrel temperature, workbench temperature, needle diameter, extrusion pressure, the nozzle speed of travel, aperture parameters are set,
Complete the 3D printing of tissue engineering bracket；
（4）The crosslinking of tissue engineering bracket：Printed stent is inserted in genipin solution and is impregnated, being rinsed repeatedly after taking-up can
Directly use or it is cold deposit it is spare.
2. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1,
It is characterized in, step（1）In, the mass concentration of the gelatin solution is 2~15%.
3. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1,
It is characterized in, step（2）The preparation of cellulose nano-fibrous/gelatin-compounded hydrogel, described is cellulose nano-fibrous
Solution, mass concentration 1%-8%, cellulose nano-fibrous loading are 3~20%.
4. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1,
It is characterized in, step（2）The preparation of cellulose nano-fibrous/gelatin-compounded hydrogel, blending temperature are 25~45 DEG C, altogether
Do time for 1~4 it is small when.
5. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1,
It is characterized in, step（3）The 3D printing of the tissue engineering bracket, in 3 d structure model, angle between the silk of adjacent two layers
For 1~90, the thickness of layering is 120~400 μm.
6. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1,
It is characterized in, step（3）The 3D printing of the tissue engineering bracket, barrel temperature are 3~25 DEG C, and platform temperature is 1~25
DEG C, needle diameter is 150~400 μm, and extrusion pressure is 0.01~0.6Mpa, and the nozzle speed of travel is 3~50mm/s, and aperture is
100~500 μm.
7. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1,
It is characterized in, step（4）The concentration of the crosslinking of the tissue engineering bracket, wherein genipin solution is 0.1~10mM.
8. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1 or 7,
It is characterized in that step（4）The crosslinking of the tissue engineering bracket, stent are immersed in genipin solution, are crosslinked the first stage
Crosslinking temperature is 1~10 DEG C, and when crosslinking time is 2~10 small, crosslinking second stage temperature is 25~50 DEG C, crosslinking time 5
~48 it is small when.
9. a kind of method that skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to claim 1,
It is characterized in, step（4）The crosslinking of the tissue engineering bracket, cold condition of storage of depositing are：After ethylene oxide sealing, in -80
~-15 DEG C of sterile storages.
10. skin tissue engineering scaffold is prepared based on 3D biometric print technologies according to any one of claim 1-9
Vitro cytotoxicity test method, it is characterized in that, before repopulating cell, stent is positioned over irradiation 2h under r rays and is carried out at sterilizing
Reason；Then stent submerges in the medium overnight under the conditions of 37 DEG C, then adds in 3T3 cell suspending liquids in every hole, is put into 37
DEG C, cultivated in the incubator of gas concentration lwevel 5%, humidity 95%；Finally multiplication of the 3T3 in stent is evaluated using CCK-8
Situation using microplate reader at 450nm wavelength, detects OD values.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810088401.5A CN108114324A (en) | 2018-01-30 | 2018-01-30 | A kind of vitro cytotoxicity test method for the method and the stent that skin tissue engineering scaffold is prepared based on 3D biometric print technologies |
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|---|---|---|---|
| CN201810088401.5A CN108114324A (en) | 2018-01-30 | 2018-01-30 | A kind of vitro cytotoxicity test method for the method and the stent that skin tissue engineering scaffold is prepared based on 3D biometric print technologies |
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| CN108114324A true CN108114324A (en) | 2018-06-05 |
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| CN201810088401.5A Pending CN108114324A (en) | 2018-01-30 | 2018-01-30 | A kind of vitro cytotoxicity test method for the method and the stent that skin tissue engineering scaffold is prepared based on 3D biometric print technologies |
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| CN111544657A (en) * | 2020-05-11 | 2020-08-18 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method of cell 3D printing biological ink with good printability |
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| CN110171127A (en) * | 2018-06-29 | 2019-08-27 | 浙江大学 | A kind of 3D printing system |
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| CN111544657A (en) * | 2020-05-11 | 2020-08-18 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method of cell 3D printing biological ink with good printability |
| CN111544657B (en) * | 2020-05-11 | 2022-01-11 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method of cell 3D printing biological ink with good printability |
| CN112194801A (en) * | 2020-09-02 | 2021-01-08 | 中国农业大学 | Preparation and application of biocompatible polysaccharide-based 3D printing gel |
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