CN107137775A

CN107137775A - A kind of preparation method of the thermo-setting elastomer tissue engineering bracket with multistage pore structure

Info

Publication number: CN107137775A
Application number: CN201710348151.XA
Authority: CN
Inventors: 游正伟; 雷东
Original assignee: Donghua University
Current assignee: Donghua University; National Dong Hwa University
Priority date: 2017-05-17
Filing date: 2017-05-17
Publication date: 2017-09-08
Anticipated expiration: 2037-05-17
Also published as: CN107137775B

Abstract

The present invention relates to a kind of preparation method of the thermo-setting elastomer tissue engineering bracket with multistage pore structure, including：(1) thermosets is mixed with packing material, obtains mixing material；The model of cube network structure is built using CAD software, then mixing material is added in the heating chamber of 3D printer, initial support is obtained by 3D printing；(2) initial support in step (1) is subjected to heat cross-linking or photo-crosslinking, obtains thermo-setting elastomer tissue engineering bracket；Packing material is finally removed, is produced.The present invention solves thermoplasticity FDM and directly prints thermosets root problem, and prepared tissue engineering bracket has a multistage pore structure of controllable precise in structure, and method is simple, quick, suitable for a variety of biomaterials, has a good application prospect.

Description

A kind of preparation of the thermo-setting elastomer tissue engineering bracket with multistage pore structure Method

Technical field

The invention belongs to tissue engineering bracket field, more particularly to a kind of thermo-setting elastomer with multistage pore structure The preparation method of tissue engineering bracket.

Background technology

Tissue, the damage of organ are one of major diseases of serious threat human health, traditionally mainly pass through clinic The means such as organ transplant are treated.Pioneer Yuan-Cheng professors Fung of 1980s bioengineering are first Organizational project this term is started, the purpose is to the tissue or device with three-dimensional structure of inner or in vitro generation substitutability Official, the tissue for doing harm to or losing with reparation, regeneration of damaged, organ, so as to break through existing clinical medicine means to damaged tissues Or the limitation of organ treatment, including the limited amount of organ donation, the reaction of allosome rejection, the infection of potential virus, it is autologous " with Secondary injury of wound repair wound " etc..Tissue engineering technique is due to reach successfully repair of damaged tissues regeneration The purpose of sufferer is treated, more extensive concern had been obtained in past 20 years.

Tissue engineering bracket can play simulation natural extracellular matrix function there is provided suitable cell growth break up it is micro- See environment.Preferable tissue engineering bracket should possess following basic characteristics：(1) have suitable physical surface features and Biochemical property, to promote propagation and the differentiation of cell；(2) there is open, interconnected microcellular structure, it is thin to promote Born of the same parents' nutriment spreads the release with metabolite；(3) there is certain mechanical strength, to provide tissue growth supporting role； (4) there is good biocompatibility, it is ensured that to cytotoxic side effect and to human body non-immunogenicity；(5) have controllable Biological degradability, it is desirable to which degradation rate and the speed of regeneration match, and biological support is gradually degraded while regeneration Final be metabolized excretes.But the also very limited of clinic can be really applied at present, reason for that is a lot, one Important factor is exactly that the mechanical property between biomaterial and tissue is mismatched, and many tissues and organ of human body are such as Angiocarpy, lung, bladder etc. are all with favorable elasticity, while in the environment in lasting mechanical stimulation.Therefore with good Biocompatibility and degradability, the bioelastomer for the mechanical property that above-mentioned natural tissues can be simulated to a certain extent meet the tendency of and Raw, the mechanical stimulation of surrounding can be passed to cambium by these bioelastomers, can be extensive from the deformation of circulation repeatedly It is multiple, it is adaptable to the dynamic in vitro culture of cell and to be implanted in the dynamic mechanical environment of human body, will not be to the group of surrounding after implantation Knit generation mechanical damage.Just because of this various features, it is important that bioelastomer has rapidly become a class in organizational project Biomaterial, while also being applied in other related biomedical sectors.

One preferable bioelastomer will meet many requirements, except with excellent mechanical property, also requiring have Good biocompatibility and biological degradability, while bioactive molecule can preferably be combined, with good processing Property.The bioelastomer for meeting these conditions at present is also less, real to obtain the more limited of application, mainly or with PLA [poly (lactic acid), PLA], polyglycolic acid [poly (glycolic acid), PGA], and polycaprolactone Based on [polycaprolactone (PCL)] and its copolymer and derivative.In the bioelastomer that new development is got up, the poly- last of the ten Heavenly stems Diglyceride [Poly (glycerol sebacate), PGS] is a prominent representative.PGS bioelastomers were in more than 10 years Before, reported first by Wang etc., be to be introduced into one of bioelastomer of field of tissue engineering technology earliest, its appearance has driven it The development of its thermosetting bioelastomer.And PGS is in itself also due to its many excellent characteristic, such as have good internal degraded Performance, gradually corrodes from outside to inside, slowly equably degrades, and material is kept original geometric shape and power in a long time Performance is learned until being replaced by cambium, so as to can guarantee that the biomedical implants such as tissue engineering bracket prepared therefrom whole In individual degradation process, good integrality can be kept, due effect is persistently played.Therefore PGS, which is obtained, continues extensive research, And show good application prospect.With going deep into that PGS is studied, it has also been realised that also there are some defects, limitation in it It is further applied, and one of distinct issues are that it needs the violent cross linking conditions of high temperature high vacuum, can only typically lead to Die methods and etching method shaping are crossed, its processing method and application is greatly limited.

3D printing technique (also known as 3D rapid shaping techniques or RP), it refers under the control of the computer, impaired according to patient The data such as tissue or CAD (CAD) model of organ or computed tomography (CT), pass through the accurate of material 3D accumulates, and quickly manufactures the novel digital forming technique of arbitrarily complicated three-dimensional shape.Technology can not only realize material and patient The perfect matching of diseased region, and can in microstructure accuracy controlling material structure.FDM has as in 3D printing technique Representational one kind, its principle be using hot melt shower nozzle so that the material of molten condition by computer control path extrusion, Deposition, and coagulation forming, by layer by layer deposition, solidification, finally remove backing material, obtain required three-dimensional objects.The technology Feature is shaped article precision height, the good, non-environmental-pollution of surface quality etc., but it has the disadvantage that operation temperature is higher, also because of it Such principle and feature are the processing method as thermoplastic all the time.

Generally speaking, material and rack forming method are two key elements prepared by tissue engineering bracket, bioelastic The predicament solved to a certain extent on material of arising at the historic moment of body material, PGS also progressively turns into system due to its excellent performance One of ideal material of standby tissue engineering bracket, and FDM technology is used for for a long time as a kind of printing technique of thermoplastic Build the tissue engineering bracket of labyrinth.FDM printing techniques are all used as one kind all the time because of the principle of its melt molding Thermoplastic processing mode is used and studied, it is more difficult to applied to above-mentioned thermoset elastomer materials.By taking PGS as an example, wherein Under main difficulty：Prepolymer is that thermoplastic can bear plastic working i.e. with printability first, is then needed into one The high temperature of step, vacuum environment are crosslinked and solidify guarantor's type.But in second step, because prepolymer is in itself to thermo-responsive, Its mobility is greatly increased by heat energy, is deformed upon before crosslinking curing and destroys original structure, can not finally be beaten by 3D Print the elastomer that this mode processes this crosslinkings of PGS.Due to above-mentioned thermosets property and FDM thermoplasticity processings principle it Between incompatibility, cause at present have no using FDM print thermosetting biological support report, more have no FDM printing PGS elasticity Body support frame.

The content of the invention

The technical problems to be solved by the invention are to provide a kind of thermo-setting elastomer tissue with multistage pore structure The preparation method of engineering rack, this method solve thermoplasticity FDM and directly prints thermosets root problem, prepared group Weaver's engineering support has a multistage pore structure of controllable precise in structure, and method is simple, quick, suitable for a variety of biological materials Material, has a good application prospect.

A kind of preparation method of thermo-setting elastomer tissue engineering bracket with multistage pore structure of the present invention, bag Include：

(1) by thermosets and packing material in mass ratio 1:0.5-3 is mixed, and obtains mixing material；Utilize CAD software The model of cube network structure is built, then mixing material is added in the heating chamber of 3D printer, is obtained by 3D printing Initial support；

(2) initial support in step (1) is subjected to heat cross-linking or photo-crosslinking, obtains thermo-setting elastomer organizational project branch Frame；Packing material is finally removed, the thermo-setting elastomer tissue engineering bracket with multistage pore structure is produced；Wherein, it is multistage Pore structure includes two grades of macroporous structures of the gap generation between primary contour structure, fiber element diameter and fiber and filled out Fill the three-level microcellular structure produced after material is removed as template.

Thermosets in the step (1) is PGS, polyurethane or epoxy resin etc..

Packing material in the step (1) is salt particle, graphene, CNT (or other carbon materials), dioxy SiClx, hydroxyapatite (or other inorganic material), (or the higher polymerization of other other fusing points of nylon or makrolon Thing).

A diameter of 20~100 μm of the salt particle.

Hybrid mode in the step (1) is solvent mixing method or heating.

3D printing parameter in the step (1) is：40~100 DEG C of extrusion chamber temperature and nozzle temperature.

Heat cross-linking parameter in the step (2) is：The preliminary crosslinking curing 12-24h in 100 DEG C of vacuum drying oven, so The further solidification crosslinking in 120 DEG C -150 DEG C of vacuum drying oven afterwards.

The thermo-setting elastomer tissue engineering bracket with multistage pore structure obtained in the step (2) passes through freezing Drying is preserved.

Using PGS as thermosets, salt particle be used as the principle for illustrating the present invention exemplified by packing material：

The salt particle in the range of salt particle, screen cloth screening certain size is smashed with pulverizer, by salt particle and PGS prepolymers Mixed in different ratios, the printability in printing experiment is melted by actual 3D, including it is extrudability steady with initial configuration Qualitative, and the conformality in subsequent high temperature solidification process, integrated survey selects most suitable mixed proportion to meet PGS 3D The requirements of printing.Mixture is mounted in 3D printing in syringe, preferable print parameters are adjusted, it is desirable to can continuous uniform fiber Extrusion (good is extrudability), there is good initial configuration stability after the completion of printing.For PGS elastomeric tissue engineering branch Frame, devises multi-level pore structure.By the modelling to 3D printing, the personalized customizable excellent of 3D printing is utilized Gesture, can conveniently build the primary contour structure of support.By selecting needle sizes size and the design to printing path, to adjust Gap between the thickness and fiber of fiber element diameter, so as to two grades of macroporous structures of effective control support.It is another Aspect, the consumption and size of salt grain are mixed by adjusting, fiber element is distributed in adjust after salt grain is removed as template The porosity and void size of inner porosity, so as to the three-level microcellular structure of effectively control support.Then, PGS conducts A kind of representational thermosetting bioelastomer, it is necessary to could obtain stable three-dimensional configuration and mechanical property by hot setting crosslinking Energy.However, the crystallization temperature of PGS prepolymers itself is relatively low, viscosity, which can be reduced, at high temperature easily deforms upon.Therefore, salt grain Be mixed into during high-temperature cross-linking and serve the effect of very important physical support, and PGS prepolymers are served and are similar to The effect of adhesive.In order that the form and the PGS prepolymer supports of printing after hot conditions solidification are consistent, support first exists A certain degree of solidification is carried out at relatively low temperature, further solidification crosslinking is then carried out at relatively high temperatures.Finally, it will hand over Support after the completion of connection, is repeatedly immersed in ethanol distillation water mixed liquid and removes unpolymerized prepolymer, salt particle etc., so as to obtain There must be the 3D printing PGS elastomeric tissue engineering racks of multistage pore structure, then carry out freeze-drying and remove in support Moisture, in order to be preserved using with long-term.

Beneficial effect

The present invention solves thermoplasticity FDM and directly prints thermosets root problem, prepared tissue engineering bracket There is the multistage pore structure of controllable precise in structure, method is simple, quick, suitable for a variety of biomaterials, can be according to trouble The data such as the CT of person carry out the tissue engineering bracket needed for personalized customization, available for prepare people's auricle cartilage scaffold, myocardium sticking patch, And the multi-stage porous support needed for other organizational projects, have a good application prospect.

Brief description of the drawings

Fig. 1 is the process chart of embodiment；

Fig. 2 schemes for the SEM of different mixing proportion support；Wherein, A, D are respectively ratio 1：The section of 0.5 sample and table 50 times of electron microscopes in face；B, E are respectively ratio 1：The section of 1 sample and the 50 of surface times of electron microscopes；C, F are respectively ratio 1： The section of 2 sample and the 50 of surface times of electron microscopes；

Fig. 3 is PGS500 and PGS300 electron microscope；

Fig. 4 is crosslinked for the solidification of support；

Fig. 5 A-D are that support removes comparison diagram before and after salt particle；

Fig. 6 is support shear stress and the curve map of shear rate；

Fig. 7 is the batten high temperature conformality comparison diagram of different mixing proportion；

Fig. 8 contrasts for PGS300 and PGS500 pore size and diameter dimension；

Fig. 9 A-D are the mechanical property of PGS supports；

Figure 10 is the biological degradability of PGS supports；

Figure 11 a-l are the micro-phase action of PCLU elastomers and its support；

Mechanical properties and infrared spectrum analysis of Figure 12 a-d for PCLU supports.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Scope.

Embodiment 1

1. printed material prepares

The hybrid parameter of PGS prepolymers (Pre-PGS) and salt particle directly determines extrudability, initial when support is printed The pore structure of morphological stability, hot setting conformality and support, so that the performance of support is determined indirectly, including mechanical property Energy and biological degradability.The parameter of research needed for mixed process includes hybrid mode, diameter of mixed proportion and salt particle etc..

1.1 hybrid mode

Pre-PGS is thick at normal temperatures and viscosity is larger, with the increase of the salt ratio of mixing, the viscosity of mixture Also gradually increase, it is easy to produce the problem of mixing is uneven, it is therefore desirable to consider the viscosity of reduction mixture.Conventional reduction The method of viscosity has two kinds：Solvent method and heating.Therefore two methods mixing material is used, passes through pre-extruded experiment progress pair Than analysis, the method being more suitable for is selected to carry out subsequent experimental.

1.1.1 solvent mixing method

1st, by Pre-PGS and acetone with mass volume ratio 1:2 is uniform without heating stirring, and Pre-PGS total amounts are 8g, and acetone is The thick solution that cumulative volume is 20ml is obtained after 16ml, mixing；

2nd, NaCl be placed in pulverizer crush after, by the screen cloth of 200 mesh and 400 mesh sieve diameter 38~75 μm it Between salt grain；

3rd, salt grain is well mixed by a certain percentage with Pre-PGS, be fitted into injector syringe:

4th, stand and treat that acetone is placed in vacuum drying oven 30 DEG C after slightly volatilizing, 24h removes acetone；

5th, with the tentative extrusion of syringe.

1.1.2 heating

1st, the beaker that will be equipped with Pre-PGS is placed in heating in 60 DEG C of oil baths；

3rd, Pre-PGS is mixed with salt grain by different proportion, be fitted into injector syringe：

4th, with the tentative extrusion of syringe.

1.2 mixed proportion

2g Pre-PGS are weighed with assay balance to be placed in beaker, 60 DEG C of oil bath heatings.With pulverizer polishing salt grain, use The screen cloth of 200 mesh and 400 mesh screens a diameter of 38 μm~75 μm of salt particle, and 2g salt particles are weighed with assay balance, beaker is treated In Pre-PGS be changed into adding during transparent liquid, stirred with glass bar and prepare Pre-PGS：NaCl=1:1 it is mixed Compound, is fitted into 10ml one-shot injector, standby.In order to find suitable print scale, a series of mixing of ratios is designed Thing is printed, the ratio such as table 1 of mixture：

The Pre-PGS of table 1 and salt particle mixed proportion

	1:0.5	1:1	1:2	1:3
					Pre-PGS	2g	2g	2g	2g
Salt particle	1g	2g	4g	6g

2.3D prints Pre-PGS supports

2.1 3D printing models

Using the software building models of AutoCAD 2014, cube network structure is built, the length of side is 20mm, its micro grid Gap can be controlled by the path and parameter of printing.To embody the personalized customizable advantage of 3D printing, it can print each Complicated contour structure is planted, snowflake figure is have chosen wherein and is printed.

2.2 3D printing state modulators

The power supply of 3D printer is opened, before 3D printing is carried out, the nozzle and pressure ram of 3D printer is unloaded, cleaning is dry Only, nozzle and syringe needle are loaded onto, PGS and NaCl mixed material is put into barrel, pressure ram is then reinstalled.Open 1 number Heater, sets extrusion chamber temperature and nozzle temperature, double-clicks the 3D printer software on computer, makes computer and 3D printer phase Connection, the CAD model needed for selection 3D printing.Then parameter setting is carried out, extrusion chamber temperature and nozzle temperature difference are set all For 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, material extrudes the change of state at 65 DEG C.20G syringe needles are selected, floor height is 0.5, net Lattice filling width is 1.2mm, and x position is 90, and y location is 90, and Contour filling number of times is 0, and angle is 90 ° and 0 °.Click on XY axles Speed fills in corresponding XY axles movement velocity and T axle material extrusion speed, and wherein XY axles movement velocity is 2mm/s, and T axle material extrusion speed is 0.006mm/s, preservation clickstream data " it is determined that " and fill path.By x, y location is all transferred to 90, adjustment z-axis come adjust syringe needle from The height of receiver board, then by the xy shaft position back to zeros of printer, after temperature rises to designated value and stabilization, you can press Auto starts printing.

2.3 needle sizes

The model of extrusion syringe needle directly determines the diameter dimension of extrusion fiber element and the fineness of support, in order to improve The fineness of printing, has used the smaller syringe needle of internal diameter (22G), between the size and fiber element to reduce fiber element diameter Gap width.But salt particle size directly determines the extrusion performance of printed material with pinhead-sized matching, in salt grain On the premise of filler consumption is constant, in order that material is easier extrusion, the salt grain that particle diameter is smaller has been selected.

Salt grain from two kinds of different-diameters carries out contrast test, a diameter of 38~75 μm and 26~38 μm of salt particle and divides Do not printed with 20G and 22G syringe needles.Wherein printed using 20G syringe needles and a diameter of 38~75 μm of salt grains as filler Support is PGS 500；The support wherein printed as filler using 22G syringe needles and a diameter of 26~38 μm of salt grains is PGS 300.Two kinds of syringe needle parameter comparisons are shown in Table 2：

The 21G of table 2 and 22G syringe needle parameter comparisons

The solidification crosslinking and precipitation of 2.4 supports

Because support raw material are Pre-PGS and salt particle, the high-temperature cross-linking for carrying out second step is also needed to turn into PGS branch Frame.Although salt particle maintains the three-dimensional structure of support in print procedure as setting agent, with the rise of temperature, Pre-PGS viscositys progressively decline, and the structure of support is inevitably destroyed.Therefore one is selected suitably compared with low temperature Degree, at this temperature, support can keep its structure within a certain period of time, and crosslinking can also takes place in Pre-PGS.In Pre- PGS occurs after a certain degree of crosslinking, it is ensured that it still has after shape-retaining ability at high temperature, then high temperature rapid curing crosslinking.

It is polymerize according to the access and PGS of bibliography and solidifies the condition of crosslinking, is 100 by low temperature guarantor's type temperature setting DEG C, support enters traveling one in 100 DEG C of vacuum drying oven in preliminary crosslinking curing 12h, the vacuum drying oven for being then transferred into 150 DEG C The solidification crosslinking of step.

To remove salt particle and uncrosslinked polymer in support, support is immersed in absolute ethyl alcohol at ambient temperature 1 is pressed with distilled water:In the solution of 3 ratios mixing, mixed once liquid is changed within every 4 hours, is washed 3 times, after freezing 12 hours, be placed in- 20 DEG C, freeze-drying obtains the elastic body support frames of PGS in 50Pa freeze drier.

3. characterize and detection

The sign of 3.1 printabilities

Using the rheological property of the mixture of proof stress rheometer measurement different proportion, design module is divided into two portions Point：First is holding stage, and holding temperature is 45 DEG C, and rotating speed is 0, and soaking time is 300s；Second stage is that rotating speed increases rank Section, temperature is 45 DEG C, and rotating speed increases to 100rpm from 0, and the time is 600s.Each three groups of data of sample test are designed, are used Shear stress-shear rate curve the figure of four ratio samples of origin Software on Drawing.

The measure of 3.2 high temperature conformalitys

It is respectively 1 to prepare PGS and NaCl ratios:0.5,1:1,1:2,1:3 mixture, takes long 1cm, a diameter of 0.5cm Column model, mixture is packed into mould with scraper, is compacted, the material of each ratio prepares two battens, is placed in -20 DEG C refrigerator in freeze 30min, with ear of maize release obtain column batten.10min is incubated in the vacuum drying oven for being placed in 100 DEG C, is seen Its change in shape is examined, so as to help to select proper mixture ratio example.

The sign of 3.3 morphosis and test

The sign of supporting structure is detected by SEM, the form of support is observed, if deform, caves in and slightly It is thin whether uniform etc. so as to select proper mixture ratio example and solidification temperature, the distribution of pores of qualitative characterization's support and overall tie Structure.And the SEM figures of support are counted using ImageJ softwares, each 30~40 data of measurement is averaged and side Difference, with reference to the fiber element diameter and pore-size of Origin Software on Drawing block diagram quantitative analysis supports.

3.4 porosity

This experiment is using ethanol densimetry measurement porosity.Support is freeze-dried after constant weight 24h in drier, analysis Balance weighs its quality m0.Support is put into centrifuge tube, 12h in absolute ethyl alcohol is completely submerged in, measurement centrifuge tube, ethanol with The gross mass of support is m1.Carefully support is taken out from bottle with tweezers, the support of taking-up is filled ethanol liquid and do not dripped It is defined, support is placed on clean culture dish.The gross mass of the remaining ethanol that weighs with scale and centrifuge tube is m2.PGS density ps PGS, absolute ethyl alcohol density p ethanol at 20 DEG C.The porosity calculation formula of support is as follows：

3.5 Mechanics Performance Testings are with characterizing

Four layers of support are cut into 10*5mm rectangular specimens, its tensile break strength is tested and tension fracture elongation rate is right Afterwards under conditions of slightly less than tension fracture elongation rate, cyclic tension test, test loop number of times 10 times are carried out.With 16 layers of branch Frame carries out circulation compression verification, and deformation degree is 40%, and test loop number of times is 1 time, 10 times, 30 times, 50 times respectively.

3.6 biological degradability

Above-mentioned PGS supports are cut into 5mm*5mm sample, initial mass is weighed.Support is respectively put into equipped with 5ml esterases In the centrifuge tube of solution, it is placed in 37 DEG C of constant-temperature incubation casees.Respectively in 10min, 30min, 1h, 2h, 3h, 5h, by sample during 7h Take out, using being freeze-dried after distilled water rinse to constant weight, weigh the quality after sample breakdown.And observe sample using SEM Support form after degraded.

4. result

2.1.1 the hybrid mode of material

Pre-PGS is thick at normal temperatures and viscosity is larger, with the increase of the salt ratio of mixing, the viscosity of mixture Also gradually increase, it is easy to produce the problem of mixing is uneven, it is therefore desirable to consider the viscosity of reduction mixture.Conventional reduction The method of viscosity has two kinds：Solvent method and heating, are carried out comparative using both approaches.Experiment shows：Using solvent method Mixing, when taking out solution from vacuum drying oven, although acetone is substantially removed, without very big pungent smell, but passes through 12h, which is stood, finds that most of salt grain is deposited on bottom, mixes uneven with Pre-PGS, extrusion performance is poor.There are two in addition Defect is：First, it can not ensure that acetone is removed completely；2nd, acetone is constantly being volatilized during experimental implementation, and Pre-PGS acetone is mixed The volume for closing liquid is being continually changing, it is impossible to ensure the real ratio of Pre-PGS and NaCl particles.The material mixed using heating It is extrudability more stable than more uniform, while without other organic solvents, it is ensured that the biocompatibility of material.Therefore Subsequent experimental is carried out using heating.

2.1.2 the mixed proportion of material

Salt particle plays a part of setting agent and pore-foaming agent in the mixture, therefore salt grain is very few, it is impossible to plays sizing and makees With the structure of printing is easily caved in；Salt grain is excessive, and viscosity is too high, may result in printing difficulty, or even can not print. Accordingly, it would be desirable to which exploring a proper mixture ratio example can continue intactly to print, the structure of print carriage can be kept again, full On the basis of the two conditions of foot, with reference to the observation of SEM Electronic Speculum, the overall structure and microstructure of support are analyzed, Pre- is selected PGS and salt particle most suitable ratio.

From SEM figures, it can be seen that ratio is 1:0.5 sample is 35 DEG C in print temperature can be relatively easy to heated squeeze Go out, but because heating-up temperature and room temperature are approached so that mobility variations are small after cooling, yielding after shaping, the fiber in support Unit is distributed unclear in length and breadth, is sticked to each other between different layers to together.Ratio is 1:1 sample, temperature can be squeezed at 45~50 DEG C Go out, fiber overall distribution is clear, but the phenomenon that single fiber occurs necessarily collapsing downwards in unsupported region, by Action of Gravity Field Influence is larger.Ratio is 1:2 samples, can smoothly be extruded when temperature is 55 DEG C, and room temperature cooling aftershaping is stable, and fiber list Member distribution is clear, does not deform upon substantially under gravity, fibre section is into smooth circle, and shape-retaining ability is good.Ratio is 1:3 Sample, heating extrusion temperature rises to 90 DEG C, but there is the not smooth defect of extrusion, and fiber is extruded by intermittent type, and this is probably Salt content increase causes caused by Heated Flow difference.

2.1.3 3D printing parameter

2.1.3.1 print temperature

From the point of view of print procedure, with the rise of print temperature, the mobility of material is become better and better, and is at the same time printed The shape-retaining ability of support worse and worse, therefore will select a suitable temperature, i.e., at this temperature, and material can steady and continuous Uniform threadiness is extruded into, can be fixed up again on receiver board and keep shape during printing without deforming.From From the point of view of experimental result, at 40~50 DEG C, the mobile performance of material is very poor, and fiber extrusion is discontinuous, simultaneously because temperature is relatively low, pin Head point is not heated, and material is easily blocked in syringe needle.At 60~65 DEG C, the mobile performance of material is very good, so that printed Cheng Zhonghui causes fibre diameter to become big or fibrous fracture suddenly because of some slight changes of pressure or the rupture etc. of bubble, Temperature is not reduced in time after fiber reaches receiver board simultaneously, and mobile performance preferably, is affected by gravity and fiber is caved in, whole Individual supporting structure deforms.At 55 DEG C or so, mobile performance is more suitable, can continuously extrude, Simultaneous Stabilization reaches After receiver board, the reduction of temperature drop mobile performance can more stably maintain its fiber shape and supporting structure.Therefore 55 are used Temperature on the basis of DEG C, is ceaselessly finely tuned with the change of the process material state of printing, because in print procedure, material Material is extruded and heated for a long time in extrusion chamber, and a certain degree of change can all occur for state.Also needed in print procedure A main problem is easily to produce bubble in material, causes the unexpected fracture of fiber, thus before printing is started, it is necessary to Precharge is carried out, material stable a period of time in extrusion chamber is started printing again.

2.1.3.2 3D printing software parameters

The caramel fibre diameter of shower nozzle extrusion is except relevant with the size of syringe needle, and also relevant with two factors, one is raw material Rate of extrusion, two be shower nozzle rate travel.In the case of rate of extrusion is constant, shower nozzle rate travel is bigger, and fibre diameter is smaller；Spray In the case of head rate travel is constant, material extrusion speed is bigger, and fibre diameter is bigger.Therefore the basal rate of raw material is fixed, changed The rate travel of shower nozzle explores both proper ratios, from the point of view of experimental result, when T axle material extrusions speed is 0.006mm/s When, when shower nozzle rate travel is 2mm/, the diameter of fiber is more matched with needle sizes, this explanation T axle material extrusion speed and shower nozzle The ratio between rate travel ideal is about 0.003：1, the size of both can be changed on this basis and change print speed.But this two The value of person can not increase always, once because shower nozzle rate travel is excessive, fiber is difficult to be fitted well with receiver board, while fine Stuck up during dimension turnover because volume easily occurs for excessive velocities, supporting structure deformation.Floor height is related to needle sizes, 20G syringe needles it is interior Footpath is that can produce a certain degree of stretching in 0.61, but print procedure to fiber, and can be affected by gravity, therefore selection 0.5mm is used as floor height.In addition, general when printing starts set syringe needle more smaller than floor height from the height of receiver board, so as to material Material, which can be stablized, to be pasted on receiver board.

2.1.4 needle sizes

In this experiment, mechanical support phase when NaCl particles can be used as solidifying, is also used as pore-foaming agent, passes through Microcellular structure is formed after water-soluble removal.In order to further control the microstructure of fiber and the cast structure that becomes more meticulous, to salt grain chi Very little and extrusion nozzle size is adjusted with having matched.A, B are respectively PGS500 50 times of electron microscopes and 1000 times of electron microscopes；C, D is respectively that PGS300 50 times of electron microscopes and 1000 times of electron microscopes originally used 20G syringe needle, and corresponding uses particle diameter It is about 500 μm or so (PGS500) in fibre diameter made from 38 μm~75 μm of NaCl particles, fibrous inside and surface are uniform Distributed substantial amounts of microcellular structure, pore size matches with grain diameter.And when using 22G syringe needles printing extrusion, it is considered to Extrusion smoothness of the particle at shower nozzle, have selected particle diameter in 26~38 μm of NaCl particles (≤38 μm), obtains fiber list Also substantial amounts of micropore is evenly distributed with the support (PGS300) of a diameter of 300 μm or so of member, same fiber, multistage hole is formed Structure.But, in contrast PGS supports made from 22G syringe needles, fibre diameter is smaller so that fiber alignment is even closer, finely Du Genggao, while salt particle is smaller so that pore size is smaller, whole support specific surface area is bigger, is more beneficial for as tissue work Engineering support is used.

2.1.5 the solidification crosslinking of support

Fig. 4 left sides are uncured support, and the right is the support after solidification, it can be clearly seen that solidification front and back support Obvious change does not occur for global shape, and original defect still has after solidification, illustrates that support does not have again Generation is largely deformed.Illustrate that 100 DEG C of low temperature of design are tentatively crosslinked, the experimental program that 150 DEG C of high temperature is further crosslinked Efficiently solve the problem that thermoplasticity 3D printing technique prints heat cured PGS.

2.1.6 the precipitation of support

From the point of view of Fig. 5 A, C, support does not only have the macrovoid between grid, by the removing of salt particle on the surface of support Just inside all produces substantial amounts of hole, so as to constitute the multistage hole of support, is conducive to the breeding and growth of cell.And by B, D, can obtain the size of micropore with quantitative analysis and the size of salt particle matches, so that prove can be by being mixed into salt particle Diameter dimension controls the size of support micropore, and this is conducive to support in organizational project towards specific clinical application.

The sign of 2.2 supporting structures and detection

2.2.1 the sign of printability

Printability includes the stability of extrudability and initial configuration.Left figure is obtained according to proof stress rheometer test The data arrived draw the sample that each slope of a curve in shear stress and the curve map of shear rate, figure represents each ratio Viscosity under this shear rate, therefore from the point of view of whole piece curve and the shear rate specified, identical can be obtained Conclusion：With the increase of the ratio of salt particle in mixture, the viscosity of material is constantly increasing.And extruded in 3D fusions real In testing, with salt particle ratio increase, at same temperature, the extrudability of mixture worse and worse, and initial configuration Stability is become better and better, and this phenomenon can just be explained with above-mentioned viscosity.In general, the sample of first three ratio all passes through The regulation of print temperature obtains suitable printability, but Pre-PGS and NaCl ratios are 1：3 sample is at 90 DEG C Also it is difficult to good extrudability.Therefore from printability, 1:The mixture of 3 ratios is not suitable for 3D melting extrusions Printing prepares the elastic body support frames of PGS.

2.2.2 the measure of high temperature conformality

As can be seen from Figure 7 with the content increase of NaCl particles in mixture, the high temperature conformality of support gradually improves, Pure Pre-PGS, Pre-PGS and NaCl ratio are 1：0.5 and 1:Condition of the sample of 1 these three ratios in 100 DEG C of crosslinked at low temperature Under be still difficult to keep its shape, and Pre-PGS and NaCl ratios be 1:2 and 1:The sample of 3 the two ratios then can be certain Its shape is kept in degree.Therefore from high temperature conformality, 1:2 and 1:3 the two ratios are relatively adapted to, in conjunction with can beat Print property, 1:2 this ratio are optimal.

2.2.3 diameter and pore-size are counted

There is significant difference, fibrous inside and table in the diameter dimension that can quantitatively find out PGS300 and PGS500 from Fig. 8 The size of salt particle of the pore size in face with being mixed into matches.The pore size of fibre section is substantially than fiber surface Greatly, because fiber surface can only see a part for micropore and the salt grain on surface is generally covered by PGS, salt grain is caused The diameter of hole is smaller after precipitation or hole is not obvious.

2.2.4 the detection of brace aperture rate

The structure of three-dimensional rack has vital effect to its application in organizational project, if support is with higher Porosity and good connectivity, then be beneficial to the absorption of cell and the transport of propagation, nutriment and metabolic waste. The elastic body support frames of the PGS of this experimental design, primary contour structure, the fiber element produced by the modelling to 3D printing is straight The three-level microcellular structure that the two grades of macroporous structures and salt grain that gap between footpath and fiber is produced are produced after being removed as template The multistage hole of support is constituted together, the passage conveyed as cell growth needed nutrient matter and metabolic waste.Therefore PGS bullets The porosity of property body support frame is a vital parameter.

The porosity for the elastic body support frames of PGS that this test determines 3D printing using formula shown in experimental section.Such as the institute of table 3 Show, the porosity of three-dimensional hollow support prepared by this method is more than 66%, and mean porosities are 72.96%.

The porosity measurement of the support of table 3

M0	M1	M2	Porosity
				0.0579	1.8109	1.5632	0.7354
0.0259	1.9062	1.7806	0.7655
				0.0310	2.0333	1.8701	0.7834
0.0477	1.7894	1.6310	0.6630
				0.0611	1.8261	1.5497	0.7492
0.0593	1.5516	1.3431	0.6809

2.2.5 Mechanics Performance Testing and sign

Can be seen that from mechanical experimental results Fig. 9 (A), the tension fracture elongation rate of the supports of PGS 500 35% or so, Fracture strength is 80kPa or so, and the tension fracture elongation rate of the supports of PGS 300 is 40% or so, and fracture strength is 60kPa left It is right.Elongation is cyclic tension 10 times under conditions of 25%, shows typical elastic deformation curve and with good deformation Restorative (C, D), the compliance for possessing certain internal suture strength and internal dynamic mechanical environment.By circulating compression verification Can be seen that the elastic body support frame of this 3D printing has stronger fatigue durability, and the curve that multiple deformation recovers is essentially coincided, still The initial good elasticity (B) of holding, possesses and is stressed compression in vivo and can recover in time, can be kept well with tissue Matching.Circulation compression curve analysis to PGS 500 and PGS 300 understands that PGS300 compressive strength is than PGS 500 slightly Greatly, this is close relevant with PGS 300 fiber element arrangement more matter.What both supports were all kept within deformation degree 50% It is elastic deformation, i.e., compression curve is overlapped with release recovery curve, but after more than 50%, curve is not overlapped, and is recovered The force diagram of process is higher than compression process, shows the phenomenon (B) of " mechanics enhancing ".For this phenomenon, it may be possible to press When (being more than 50%) when contracting deformation degree is excessive, integrally become flattening, caused by lifting surface area increase.

2.2.6 biological degradability

Can be seen that the PGS supports of 3D printing have a good biological degradability from Figure 10 degradation curves, Preliminary degradation compared with It hurry up, subsequent degradation is in a basic balance, degradation rate is up to more than 90% after 5 hours.The electron microscope of sample can be seen that branch after degraded The degraded of frame is that internally progressively occur etchingization degraded from surface, and the microcellular structure size on surface progressively becomes big.

Embodiment 2

Prepare PCLU supports, method be the same as Example 1.The use of polycaprolactone glycol, HDI trimer is raw material, with salt particle Mixing printing solidify afterwards shaping, save prepolymer synthesis step, allow material unit in the structure that 3D is molded in a heated condition Reaction solidification.

As shown in figure 11, this method can also be satisfied with PCLU printing shaping, can prepare biological support and other are not advised Then shape, this heat cured PCLU is also a kind of elastomer, and (a-d) still can be quickly recovered by folding repeatedly.To branch Frame carries out Electronic Speculum test, and its fiber element is clear regularly arranged and stacks, and fibre section is rounded, has no that obvious fiber is collapsed Phenomenon, with good solidification shape-retaining ability, while substantial amounts of micropore is distributed with fiber surface and inside, support is integrally in multi-level Pore structure (e-l).

Mechanical test results to PCLU supports are as shown in figure 12：Its tensile break strength is 386kPa or so, and fracture is stretched Long rate is 80%, better than PGS supports.In 10 loop cycle extension tests and the circulation compression verification in 50 cycles loading and The curve matching degree of release is high, has shown excellent elastic and deformation restorative；Mechanical characteristics repeatedly circulate simultaneously after Curve does not change substantially, with good fatigue durability.For this PCLU elastomers, due to raw material use it is poly- oneself This degradable unit of lactone dihydric alcohol, it is contemplated that it possesses biological degradability.In addition, for the potential source biomolecule poison having in raw material Property NCO group, can determine whether after being analyzed by infrared spectrum measurement it is reacted be not present completely, it is medical poly- similar to other Urethane material and possess good biocompatibility.

Claims

1. a kind of preparation method of the thermo-setting elastomer tissue engineering bracket with multistage pore structure, including：

(1) by thermosets and packing material in mass ratio 1:0.5-3 is mixed, and obtains mixing material；Built using CAD software The model of cube network structure, then adds mixing material in the heating chamber of 3D printer, is obtained initially by 3D printing Support；

(2) initial support in step (1) is subjected to heat cross-linking or photo-crosslinking, obtains thermo-setting elastomer tissue engineering bracket； Packing material is finally removed, the thermo-setting elastomer tissue engineering bracket with multistage pore structure is produced；Wherein, multistage hole Structure includes the two grades of macroporous structures and filling material that the gap between primary contour structure, fiber element diameter and fiber is produced The three-level microcellular structure that material is produced after being removed as template.

2. a kind of preparation of thermo-setting elastomer tissue engineering bracket with multistage pore structure according to claim 1 Method, it is characterised in that：Thermosets in the step (1) is PGS, polyurethane or epoxy resin.

3. a kind of preparation of thermo-setting elastomer tissue engineering bracket with multistage pore structure according to claim 1 Method, it is characterised in that：Packing material in the step (1) is salt particle, graphene, CNT, silica, hydroxyl Apatite, nylon or makrolon.

4. a kind of preparation of thermo-setting elastomer tissue engineering bracket with multistage pore structure according to claim 3 Method, it is characterised in that：A diameter of 20~100 μm of the salt particle.

5. a kind of preparation of thermo-setting elastomer tissue engineering bracket with multistage pore structure according to claim 1 Method, it is characterised in that：Hybrid mode in the step (1) is solvent mixing method or heating.

6. a kind of preparation of thermo-setting elastomer tissue engineering bracket with multistage pore structure according to claim 1 Method, it is characterised in that：3D printing parameter in the step (1) is：40~100 DEG C of extrusion chamber temperature and nozzle temperature.

7. a kind of preparation of thermo-setting elastomer tissue engineering bracket with multistage pore structure according to claim 1 Method, it is characterised in that：Heat cross-linking parameter in the step (2) is：Tentatively it is crosslinked in 80 DEG C -100 DEG C of vacuum drying oven Solidify 12-24h, the then further solidification crosslinking in 120 DEG C -150 DEG C of vacuum drying oven.

8. a kind of preparation of thermo-setting elastomer tissue engineering bracket with multistage pore structure according to claim 1 Method, it is characterised in that：What is obtained in the step (2) has the thermo-setting elastomer tissue engineering bracket of multistage pore structure Preserved by being freeze-dried.