CN107998451A - A kind of 3D printing preparation method of skin tissue engineering scaffold and the vitro cytotoxicity test method of the stent - Google Patents
A kind of 3D printing preparation method of skin tissue engineering scaffold and the vitro cytotoxicity test method of the stent Download PDFInfo
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Abstract
The present invention provides a kind of 3D printing preparation method of skin tissue engineering scaffold and the vitro cytotoxicity test method of the stent, including for timbering material the preparation of dialdehyde based nano-fiber element, the preparation of gelatin solution, dialdehyde based nano-fiber element/gelatin-compounded hydrogel preparation, the printing of 3D tissue engineering brackets the step of.The present invention solves tissue engineering bracket high porosity, high-precision requirement using 3D biometric print technologies.DAC is crosslinked by schiff base reaction as crosslinking agent, with GEL to be formed network structure and makes 3D printing tissue engineering bracket that there is excellent mechanical property to be not easily broken, while also improves the added value of plant fiber.DAC/GEL hydrogels have good biocompatibility, have no toxic side effect, and without immune rejection, and outside degradable characteristic, also have bioactivity, realizing for the growth and differentiation and cell function to cell is highly beneficial.
Description
Technical field
The present invention relates to a kind of preparation of the high-strength material for skin tissue engineering scaffold printing, and it is adapted therewith
3D biometric print techniques.
Background technology
Ulcer caused by skin is wound and chronic disease is group the main reason for causing defect of skin and function to lose
Its treatment that develops into for knitting engineering technology provides new approaches, becomes research in recent years hot spot.Organizational project prepared by conventional method
Stent procedures are cumbersome, and each Process Precision is not easy to hold, and the stent performance difference of making is very big, can not arbitrarily change walking for stent
To and hole.The appearance of 3D printing technique offer the possibility to solve the above problem, and has rapid shaping, sample standard, rule
The advantages that modulus is produced, and the stent of different scales can be printed by varying various parameters.
The 3D biometric print materials of skin tissue engineering are divided into manually and into two major class of material and natural macromolecular material.People
Geosynthetics sentence has good mechanical property and plasticity, easy printing shaping, but its catabolite to cause inflammation around more,
Biocompatibility is poor.Natural polymer has good biocompatibility, biodegradability and bioactivity, can be more preferable
Induction compromised skin self-recovery, but day mechanical property is poor, and degradation speed is too fast under the action of various enzymes in vivo,
Stent has caved in when cambium is also not long good.
Therefore, a kind of material with good biocompatibility, biodegradability, bioactivity and mechanical property is found
Material, and it is particularly important that 3D printing preparation process adaptable therewith.Once solving the problems, such as this, which will quickly apply to
In the treatment of skin injury.
The content of the invention
The purpose of the present invention is in view of the deficienciess of the prior art, provide it is a kind of for dermal scaffold 3D printing,
Biological hydrogel with biocompatibility, and the vitro cytotoxicity of biological 3D printing technique adaptable therewith and the stent
Test method.
The object of the invention is achieved through the following technical solutions：A kind of 3D printing preparation method of skin tissue engineering scaffold,
Its feature is comprising the following steps：
（1）Dialdehyde based nano-fiber element（DAC）Preparation：In nano-cellulose（Cellulose nanocrystals, CNC）It is molten
Sodium metaperiodate is added in liquid, lucifuge reaction, by centrifugation, dialyse to the free ion in solution removes completely, obtains dialdehyde base
Nano-cellulose solution；
（2）The preparation of gelatin solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously and is obtained after being completely dissolved
Gelatin solution；
（3）Dialdehyde based nano-fiber element（DAC）/ gelatin（GEL）The preparation of composite hydrogel：By dialdehyde based nano-fiber element solution
It is blended with gelatin solution, after stirring evenly rapidly, stands hatching crosslinking；
（4）The printing of 3D tissue engineering brackets：Calculating is imported in 3D printer after designing scaffold three-dimensional structural model；In barrel
Middle loading dialdehyde based nano-fiber element/gelatin-compounded hydrogel material, then sets barrel temperature, workbench temperature, syringe needle
Diameter, extrusion pressure, the nozzle speed of travel, aperture parameters, after the 3D printing for completing tissue engineering bracket, directly use or refrigerate
It is spare.
Further, step（1）In, the dialdehyde based nano-fiber element can be that bar-shaped short whisker or linear nano are fine
Dimension, the mass concentration of the dialdehyde based nano-fiber element solution obtained is 2%~10%.
Further, step（1）In, the dialdehyde based nano-fiber element is by nano-cellulose through sodium periodate oxidation system
, the solid masses ratio of nano-cellulose and sodium metaperiodate is 1:1~3, reaction temperature is 20~80 DEG C, the reaction time for 3~
20 it is small when, after reaction through centrifugation, dialyse repeatedly into dialdehyde based nano-fiber element solution free ion completely remove untill.
Further, step（2）In, the mass concentration of the gelatin solution is 2%~10%.
Further, the preparation of dialdehyde based nano-fiber element/gelatin-compounded hydrogel, dialdehyde based nano-fiber element
Solution and the volume ratio of gelatin solution blending are 1:9、2:8、 3:7、4:6、5:5、6:4、7:3、8:2、9:Any one group in 1
Ratio.
Further, step（3）The preparation of the dialdehyde based nano-fiber element/gelatin-compounded hydrogel, blending temperature
For 20~40 DEG C, brooding time is 1~5h, and incubation temperature is 15~40 DEG C, 200~2000rpm of mixing speed, after obtain dialdehyde
Based nano-fiber element/gelatin-compounded hydrogel, in hatching process, dialdehyde based nano-fiber element molecule passes through Schiff with gelatin molecule
Alkali reaction crosslinks.
Further, step（4）The printing of the 3D tissue engineering brackets, in 3 d structure model, adjacent two layers
Angle between silk is 1~90, and the thickness of layering is 120~400 μm.
Further, step（4）The printing of the 3D tissue engineering brackets, barrel temperature are 5~35 DEG C, workbench
Temperature is 1~30 DEG C, and needle diameter is 150~400 μm, and extrusion pressure be 0.1~0.4Mpa, the nozzle speed of travel is 10~
40mm/s, aperture are 100~500 μm.
Further, suddenly（4）The 3D tissue engineering brackets porosity is not less than 75%, and gap connected ratio is not less than
75%；The 3D tissue engineering bracket stored frozen conditions are：- 80~-15 DEG C of ethylene oxide pack, sterile storage.
The vitro cytotoxicity test method of stent in the 3D printing preparation method of above-mentioned skin tissue engineering scaffold, 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；Then proliferative conditions of the 3T3 in stent are evaluated using CCK-8, using microplate reader in 450nm wavelength
Place, detects OD values.The performance of stent is judged by the difference of comparative experiments 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.
The present invention can be rapidly completed skin branch according to depth, the shape of different patient's wounds using 3D biometric print technologies
The personalized customization of frame, its porosity are not less than 75%, and porosity communication rate is not less than 75%.Compared with prior art, present invention tool
Have the advantage that and beneficial effect：
（1）Crosslinking agents of the DAC used in the present invention as GEL, can be from agricultural waste material, or leads to from from microcrystalline cellulose
Cross conventional chemical processes and obtain CNC, by being prepared after sodium periodate oxidation, improve the surcharge of plant fiber.
（2）DAC and GEL obtains DAC/GEL hydrogels after hatching is crosslinked in the present invention, has good mechanical performance,
Biodegradability, and biocompatibility.
（3）The present invention prepares skin tissue engineering scaffold by 3D printing technique, convenient and efficient with technique, easy to operate
Advantage, and can be according to the size of patient's wound, shape is different, completes personalized customization.
（4）Tissue engineering bracket prepared by the present invention, has good appearance, and has complicated porous internal structure,
Porosity is not less than 75%, and hole connected ratio is not less than 75%, has good gas permeability and absorbability, may advantageously facilitate cell, blood
The growth of pipe and tissue.
The present invention provides a kind of method that strong mechanical performance tissue engineering bracket is prepared based on 3D biometric prints, including with
In the dialdehyde based nano-fiber element of timbering material（Dialdehyde cellulose nanocrystals, DAC）Preparation, bright
Sol solution（Gelatin, GEL）Preparation, dialdehyde based nano-fiber element（DAC）/ gelatin（GEL）The preparation of composite hydrogel, 3D
The step of tissue engineering bracket prints.The present invention solves tissue engineering bracket high porosity, height using 3D biometric print technologies
The requirement of precision.DAC is crosslinked by schiff base reaction as crosslinking agent, with GEL to be formed network structure and makes 3D printing tissue
Engineering rack has excellent mechanical property and is not easily broken, while also improves the added value of plant fiber.DAC/GEL water-settings
Glue has good biocompatibility, has no toxic side effect, and without immune rejection, and outside degradable characteristic, also has bioactivity,
Realizing for growth and differentiation and cell function to cell is highly beneficial.
Brief 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 skin tissue engineering scaffold that embodiment 4 prints；
Fig. 4 is the vitro cytotoxicity experimental result for the stent that embodiment 5 is completed after printing.
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）Dialdehyde based nano-fiber element（DAC）Preparation：The mass ratio of CNC and sodium metaperiodate is 1:0 DEG C of lucifuge reaction 6 of Isosorbide-5-Nitrae is small
When, by centrifugation, dialyse to the free ion in solution removes completely；
（2）The preparation of gelatin solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until gelatin all dissolves；
（3）Dialdehyde based nano-fiber element（DAC）/ gelatin（GEL）The preparation of composite hydrogel：By concentration be 4% DAC solution with
The GEL solution that concentration is 4% is with volume ratio 1:1 blending, after stirring evenly rapidly, when 37 DEG C of hatchings 2 are small；
（4）The printing of 3D tissue engineering brackets：Load DAC/GEL hydrogels in barrel, barrel temperature is 10 DEG C, platform temperature
It is 200 μm for 5 DEG C, needle diameter, extrusion pressure 0.1Mpa, nozzle speed of travel 15mm/S, completes the 3D of tissue engineering bracket
Printing.
2. embodiment 2
（1）Dialdehyde based nano-fiber element（DAC）Preparation：The mass ratio of CNC and sodium metaperiodate is 1:2,40 DEG C of lucifuge reactions 4 are small
When, by centrifugation, dialyse to the free ion in solution removes completely；
（2）The preparation of gelatin solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until gelatin all dissolves；
（3）Dialdehyde based nano-fiber element（DAC）/ gelatin（GEL）The preparation of composite hydrogel：By concentration be 5% DAC solution with
The GEL solution that concentration is 5% is with volume ratio 4:6 blendings, after stirring evenly rapidly, when 37 DEG C of hatchings 3 are small；
（4）The printing of 3D tissue engineering brackets：Load DAC/GEL hydrogels in barrel, barrel temperature is 8 DEG C, platform temperature
It is 220 μm for 8 DEG C, needle diameter, extrusion pressure 0.15Mpa, nozzle speed of travel 20mm/S, completes the 3D of tissue engineering bracket
Printing.
3. embodiment 3
（1）Dialdehyde based nano-fiber element（DAC）Preparation：The mass ratio of CNC and sodium metaperiodate is 1:2,40 DEG C of lucifuge reactions 5 are small
When, by centrifugation, dialyse to the free ion in solution removes completely；
（2）The preparation of gelatin solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until gelatin all dissolves；
（3）Dialdehyde based nano-fiber element（DAC）/ gelatin（GEL）The preparation of composite hydrogel：By concentration be 5% DAC solution with
The GEL solution that concentration is 5% is with volume ratio 6:4 blendings, after stirring evenly rapidly, when 30 DEG C of hatchings 4 are small；
（4）The printing of 3D tissue engineering brackets：Load DAC/GEL hydrogels in barrel, barrel temperature is 15 DEG C, platform temperature
To spend for 5 DEG C, needle diameter be 220 μm, extrusion pressure 0.25Mpa, nozzle speed of travel 30mm/S, completes tissue engineering bracket
3D printing.
4. embodiment 4
（1）Dialdehyde based nano-fiber element（DAC）Preparation：The mass ratio of CNC and sodium metaperiodate is 1:2,50 DEG C of lucifuge reactions 8 are small
When, by centrifugation, dialyse to the free ion in solution removes completely；
（2）The preparation of gelatin solution：In phosphate buffer（PBS）Middle addition gelatin, is stirred continuously until gelatin all dissolves；
（3）Dialdehyde based nano-fiber element（DAC）/ gelatin（GEL）The preparation of composite hydrogel：By concentration be 6% DAC solution with
The GEL solution that concentration is 6% is with volume ratio 4:6 blendings, after stirring evenly rapidly, when 37 DEG C of hatchings 2 are small；
（4）The printing of 3D tissue engineering brackets：Load DAC/GEL hydrogels in barrel, barrel temperature is 15 DEG C, platform temperature
It is 300 μm for 5 DEG C, needle diameter, extrusion pressure 0.2Mpa, nozzle speed of travel 30mm/S, completes the 3D of tissue engineering bracket
Printing.
5. embodiment 5
（1）Dialdehyde based nano-fiber element（DAC）Preparation：The mass ratio of CNC and sodium metaperiodate is 1:2,50 DEG C of lucifuge reactions 5 are small
When, by centrifugation, dialyse to the free ion in solution removes completely；
（2）The preparation of gelatin solution：In phosphate buffer（PBS）It is middle to add a certain amount of gelatin, it is stirred continuously until gelatin is whole
Dissolving；
（3）Dialdehyde based nano-fiber element（DAC）/ gelatin（GEL）The preparation of composite hydrogel：It is 4% by concentration
The GEL solution that DAC solution and concentration are 4% is with volume ratio 3:7 blendings, after stirring evenly rapidly, when 37 DEG C of hatchings 2 are small；
（4）The printing of 3D tissue engineering brackets：Load DAC/GEL hydrogels in barrel, barrel temperature is 18 DEG C, platform temperature
It is 220 μm for 10 DEG C, needle diameter, extrusion pressure 0.3Mpa, nozzle speed of travel 40mm/S, completes the 3D of tissue engineering bracket
Printing；
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 made from testing example 1-5 crushes intensity on electronic universal test machine for mechanism, as shown in table 1：
7. vitro cytotoxicity is tested：
Blank group：3T3 l cells.
Experimental group：For the tissue engineering bracket hydrogel of 3D printing, 3T3 l cells.
Before repopulating cell, stent is positioned under r rays and irradiates 2h progress sterilization treatments.Then stent is under the conditions of 37 DEG C
Submergence in the medium overnight, then per 3T3 cell suspending liquids are added in hole, be put into 37 DEG C, 5% carbon dioxide, 95% humidity
Cultivated in incubator.Then existed using CCK-8 evaluations 3T3 using microplate reader in the proliferative conditions of blank group and experimental group
At 450nm wavelength, OD values are detected, as shown in Figure 4.Stent is judged by the difference of comparative experiments group and blank group absorbance
Performance, if experimental group OD values are much smaller than blank group OD values, illustrates that 3T3 upgrowth situations on stent are poor；If experimental group OD values connect
Nearly blank group OD values, illustrate that 3T3 upgrowth situations on stent are good；If experimental group OD values are connect much larger than blank group OD values, explanation
Stent cell growth has extraordinary proliferation.
Claims (10)
1. a kind of 3D printing preparation method of skin tissue engineering scaffold, its feature are comprising the following steps：
（1）The preparation of dialdehyde based nano-fiber element：Add sodium metaperiodate in nano-cellulose solution, lucifuge reaction, by from
The heart, dialysis remove completely to the free ion in solution, obtain dialdehyde based nano-fiber element solution；
（2）The preparation of gelatin solution：Gelatin is added in phosphate buffer, is stirred continuously after being completely dissolved that to obtain gelatin molten
Liquid；
（3）The preparation of dialdehyde based nano-fiber element/gelatin-compounded hydrogel：By dialdehyde based nano-fiber element solution and gelatin solution
Blending, after stirring evenly rapidly, stands hatching crosslinking；
（4）The printing of 3D tissue engineering brackets：Calculating is imported in 3D printer after designing scaffold three-dimensional structural model；In barrel
Middle loading dialdehyde based nano-fiber element/gelatin-compounded hydrogel material, then sets barrel temperature, workbench temperature, syringe needle
Diameter, extrusion pressure, the nozzle speed of travel, aperture parameters, after the 3D printing for completing tissue engineering bracket, directly use or refrigerate
It is spare.
2. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（1）In, the dialdehyde based nano-fiber element can be bar-shaped short whisker or linear nano fiber, the dialdehyde base Nanowire obtained
The mass concentration of the plain solution of dimension is 2%~10%.
3. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（1）In, the dialdehyde based nano-fiber element is made by nano-cellulose through sodium periodate oxidation, nano-cellulose and periodic acid
The solid masses ratio of sodium is 1:1~3, reaction temperature be 20~80 DEG C, the reaction time for 3~20 it is small when, after reaction pass through from
Untill the heart, free ion of dialysing repeatedly into dialdehyde based nano-fiber element solution remove completely.
4. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（2）In, the mass concentration of the gelatin solution is 2%~10%.
5. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（3）In, the preparation of dialdehyde based nano-fiber element/gelatin-compounded hydrogel, dialdehyde based nano-fiber element solution and gelatin
The volume ratio of solution blending is 1:9、2:8、 3:7、4:6、5:5、6:4、7:3、8:2、9:Any one group of ratio in 1.
6. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（3）The preparation of the dialdehyde based nano-fiber element/gelatin-compounded hydrogel, blending temperature are 20~40 DEG C, brooding time 1
~5h, incubation temperature be 15~40 DEG C, 200~2000rpm of mixing speed, after obtain dialdehyde based nano-fiber element/it is gelatin-compounded
Hydrogel, in hatching process, dialdehyde based nano-fiber element molecule is crosslinked with gelatin molecule by schiff base reaction.
7. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（4）The printing of the 3D tissue engineering brackets, in 3 d structure model, the angle between the silk of adjacent two layers is 1~90,
The thickness of layering is 120~400 μm.
8. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（4）The printing of the 3D tissue engineering brackets, barrel temperature are 5~35 DEG C, and workbench temperature is 1~30 DEG C, and syringe needle is straight
Footpath is 150~400 μm, and extrusion pressure is 0.1~0.4Mpa, and the nozzle speed of travel is 10~40mm/s, and aperture is 100~500 μ
m。
9. a kind of 3D printing preparation method of skin tissue engineering scaffold according to claim 1, it is characterized in that, step
（4）The 3D tissue engineering brackets porosity is not less than 75%, and gap connected ratio is not less than 75%；The 3D organizational project branch
Frame stored frozen condition is：- 80~-15 DEG C of ethylene oxide pack, sterile storage.
10. stent in the 3D printing preparation method of the skin tissue engineering scaffold 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 3T3 cell suspending liquids in every hole, is put into 37
DEG C, cultivated in the incubator of gas concentration lwevel 5%, humidity 95%；Then propagation of the 3T3 in stent is evaluated using CCK-8
Situation, using microplate reader at 450nm wavelength, detects OD values.
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