CN107137775B

CN107137775B - A kind of preparation method of thermosetting elastomer tissue engineering scaffold with multi-level pore structure

Info

Publication number: CN107137775B
Application number: CN201710348151.XA
Authority: CN
Inventors: 游正伟; 雷东
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2017-05-17
Filing date: 2017-05-17
Publication date: 2020-05-29
Anticipated expiration: 2037-05-17
Also published as: CN107137775A

Abstract

The invention relates to a preparation method of a thermosetting elastomer tissue engineering scaffold with a multi-level pore structure, comprising: (1) mixing a thermosetting material and a filling material to obtain a mixed material; using CAD software to build a model of a cubic network structure, and then Add the mixed material into the heating chamber of the 3D printer, and obtain the initial scaffold by 3D printing; (2) thermally crosslink or photocrosslink the initial scaffold in step (1) to obtain a thermosetting elastomer tissue engineering scaffold; finally remove the filling material, that is. The invention solves the fundamental problem of thermoplastic FDM directly printing thermosetting materials, the prepared tissue engineering scaffold has an accurate and controllable multi-level pore structure in structure, the method is simple and fast, suitable for various biological materials, and has good application prospects.

Description

Preparation method of thermosetting elastomer tissue engineering scaffold with multistage pore structure

Technical Field

The invention belongs to the field of tissue engineering scaffolds, and particularly relates to a preparation method of a thermosetting elastomer tissue engineering scaffold with a multistage pore structure.

Background

Tissue and organ damage is one of the major diseases seriously threatening human health, and is traditionally treated mainly by means of clinical organ transplantation and the like. The pioneer of bioengineering in the 20 th century and the 80 th era, the professor Yuan-Cheng Fu Lung, first initiated the term of tissue engineering, which aims to generate replaceable tissues or organs with three-dimensional structures in vivo or in vitro to repair and regenerate damaged or lost human tissues and organs, thereby breaking through the limitations of the existing clinical medical means on the treatment of damaged tissues or organs, including the limited number of donated organs, foreign body rejection, infection of potential viruses, autologous secondary injury of repairing wounds with wounds, and the like. Tissue engineering techniques have gained much attention over the last two decades due to their success in repairing and regenerating damaged tissues for the purpose of treating patients.

The tissue engineering scaffold can play a role in simulating a natural extracellular matrix and provide a micro environment suitable for cell growth and differentiation. An ideal tissue engineering scaffold should have several basic features: (1) has suitable physical surface morphology and biochemical properties to promote proliferation and differentiation of cells; (2) has an open, interconnected microporous structure to facilitate cellular nutrient diffusion and metabolite release; (3) has certain mechanical strength to provide tissue growth supporting function; (4) the biological compatibility is good, and no toxic or side effect on cells and immunogenicity on human bodies are ensured; (5) has controllable biodegradability, requires that the degradation rate is matched with the tissue regeneration rate, and the biological scaffold is gradually degraded while the tissue is regenerated and finally metabolized and discharged out of the body. However, the current clinical applications are very limited, and the reasons are many, one important factor is the mismatch of mechanical properties between the biomaterial and the human tissue, and many tissues and organs of the human body such as the cardiovascular system, the lung, the bladder, etc. have good elasticity and are in the environment of continuous mechanical stimulation. Therefore, the biological elastomers have good biocompatibility and degradability, can simulate the mechanical properties of the natural tissues to a certain extent and can be transported to the beginning, can transmit surrounding mechanical stimuli to the new tissues and recover from repeated deformation in a circulating way, are suitable for dynamic in-vitro culture of cells and implantation in the dynamic mechanical environment of a human body, and cannot generate mechanical damage to the surrounding tissues after implantation. Due to these characteristics, the bio-elastomer has rapidly become an important biomaterial in tissue engineering, and has also been applied in other related biomedical fields.

An ideal bio-elastomer is required to satisfy many requirements, and in addition to having excellent mechanical properties, it is also required to have good biocompatibility and biodegradability, and at the same time, it is required to be able to better bind with bioactive molecules and have good processability. At present, the bio-elastomers satisfying these conditions are still less, and the applications of the bio-elastomers are more limited, mainly polylactic acid (poly (lactic acid), PLA), polyglycolic acid (poly (glycolic acid), PGA), and polycaprolactone (pcl) and copolymers and derivatives thereof. Among the newly developed biologics, polyglycerol sebacate, PGS, is an outstanding representative. PGS bioelastomers, first reported by Wang et al over 10 years ago, were one of the first bioelastomers introduced into the field of tissue engineering, and their emergence has driven the development of other thermoset bioelastomers. The PGS is gradually eroded from outside to inside due to a plurality of excellent characteristics, such as good in-vivo degradation performance, and is slowly and uniformly degraded, so that the material can keep the original geometric form and mechanical property for a long time until the material is replaced by a new tissue, and the biomedical implant such as a tissue engineering scaffold prepared from the PGS can be ensured to keep good integrity and continuously play a due role in the whole degradation process. Therefore, PGS is continuously and widely researched and shows good application prospect. With the intensive research of PGS, people also recognize that the PGS has some defects and limits the further application of the PGS, and one of the outstanding problems is that the PGS needs severe crosslinking conditions with high temperature and high vacuum, and can be formed only by a mold method and an etching method, so that the processing method and the application of the PGS are greatly limited.

The 3D printing technique (also known as 3D rapid prototyping technique or RP) is a novel digital prototyping technique for rapidly manufacturing any complex three-dimensional shape by precise 3D stacking of materials under the control of a computer according to data such as a Computer Aided Design (CAD) model or Computed Tomography (CT) of a damaged tissue or organ of a patient. The technology not only can realize perfect matching of the material and the pathological change part of a patient, but also can accurately regulate and control the structure of the material on a microstructure. FDM is a representative one of 3D printing technologies, and its principle is to use a hot melt nozzle to extrude, deposit, solidify and form a material in a molten state according to a computer-controlled path, and then remove a support material after layer-by-layer deposition and solidification to obtain a desired three-dimensional product. The technical characteristics are high precision of formed products, good surface quality, no environmental pollution and the like, but the technical characteristics have the defect of higher operation temperature, and the principle and the characteristics are always used as a processing method of thermoplastic materials.

Generally, a material and a scaffold forming method are two key elements for preparing a tissue engineering scaffold, the difficulty in the material is solved to a certain extent by the transportation of a biological elastomer material, PGS is gradually one of ideal materials for preparing the tissue engineering scaffold due to the excellent performance of the PGS, and an FDM technology is used for constructing the tissue engineering scaffold with a complex structure for a long time as a printing technology of a thermoplastic material. The FDM printing technology has been used and studied as a thermoplastic material processing method because of its principle of melt molding, and is difficult to be applied to the above thermosetting elastomer material. Taking PGS as an example, the major difficulties are among others: firstly, the prepolymer is thermoplastic material which can bear plastic processing, namely, the prepolymer has printability, and then needs further high-temperature and vacuum environment for crosslinking to be solidified and shape-preserving. However, in the second step, since the prepolymer itself is sensitive to heat and the fluidity thereof is greatly increased by the heat energy, it is deformed before crosslinking and curing to destroy the original structure, and finally, it is impossible to process a crosslinked elastomer such as PGS by 3D printing. Due to the incompatibility between the properties of the thermosetting materials and the FDM thermoplastic processing principle, reports of printing thermosetting biological stents by using FDM are not seen at present, and PGS elastomer stents printed by using FDM are not seen at present.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a preparation method of a thermosetting elastomer tissue engineering scaffold with a multilevel pore structure, the method solves the fundamental problem of directly printing a thermosetting material by thermoplastic FDM, the prepared tissue engineering scaffold has an accurately controllable multilevel pore structure in structure, and the method is simple and rapid, is suitable for various biological materials, and has good application prospect.

The invention discloses a preparation method of a thermosetting elastomer tissue engineering scaffold with a multistage pore structure, which comprises the following steps:

(1) mixing a thermosetting material and a filling material according to a mass ratio of 1:0.5-3 to obtain a mixed material; utilizing CAD software to construct a model of a cubic reticular structure, then adding a mixed material into a heating cavity of a 3D printer, and obtaining an initial support through 3D printing;

(2) carrying out thermal crosslinking or photo crosslinking on the initial scaffold in the step (1) to obtain a thermosetting elastomer tissue engineering scaffold; finally, removing the filling material to obtain the thermosetting elastomer tissue engineering scaffold with the multistage pore structure; the multistage pore structure comprises a primary contour structure, a secondary macroporous structure generated by the diameters of fiber units and gaps among fibers, and a tertiary microporous structure generated after a filling material is removed as a template.

The thermosetting material in the step (1) is PGS, polyurethane or epoxy resin and the like.

The filling material in the step (1) is salt particles, graphene, carbon nanotubes (or other carbon materials), silica, hydroxyapatite (or other inorganic materials), nylon or polycarbonate (or other polymers with higher melting points).

The diameter of the salt particles is 20-100 μm.

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

The 3D printing parameters in the step (1) are as follows: the temperature of the extrusion cavity and the temperature of the nozzle are 40-100 ℃.

The thermal crosslinking parameters in the step (2) are as follows: primarily crosslinking and curing for 12-24h in a vacuum oven at 100 ℃, and further curing and crosslinking in a vacuum oven at 120-150 ℃.

And (3) preserving the thermosetting elastomer tissue engineering scaffold with the multistage pore structure obtained in the step (2) by freeze drying.

The principle of the invention is illustrated by taking PGS as the thermosetting material and salt particles as the filler material:

salt particles are smashed by a smashing machine, the salt particles in a certain size range are screened by a screen, the salt particles and the PGS prepolymer are mixed according to different proportions, and through the printability in an actual 3D fusion printing experiment, including the extrusion property, the stability of an initial form and the shape retention in a subsequent high-temperature curing process, the most appropriate mixing proportion is comprehensively considered and selected to meet various requirements of PGS 3D printing. The mixture is loaded in a needle cylinder for 3D printing, ideal printing parameters are adjusted, continuous and uniform fiber extrusion (good extrusion property) is required, and good initial form stability is achieved after printing is completed. For the PGS elastomer tissue engineering scaffold, a multi-level pore structure is designed. Through the model design to 3D printing, utilize 3D to print individualized customizable advantage, can conveniently construct the elementary profile structure of support. The size of the needle head and the design of the printing path are selected to adjust the diameter of the fiber unit and the gap between the fibers, so that the secondary macroporous structure of the bracket can be effectively controlled. On the other hand, the porosity and the gap size of the porous structure distributed in the fiber unit after the salt particles are removed as a template are adjusted by adjusting the using amount and the size of the mixed salt particles, so that the three-level microporous structure of the bracket can be effectively controlled. Next, PGS, a representative thermosetting bio-elastomer, must be cured and cross-linked at high temperature to obtain stable three-dimensional morphology and mechanical properties. However, PGS prepolymers themselves have a low crystallization temperature, and are easily deformed due to a decrease in viscosity at high temperatures. Thus, the incorporation of salt particles plays a very important physical supporting role during high temperature crosslinking, while the PGS prepolymer plays a role similar to that of a binder. In order to maintain the high temperature cured morphology consistent with the printed PGS prepolymer scaffold, the scaffold is first cured to some extent at a lower temperature and then further cured and crosslinked at a higher temperature. And finally, soaking the scaffold after crosslinking in ethanol and distilled water mixed liquor for multiple times to remove unpolymerized prepolymer, salt particles and the like, thereby obtaining the 3D printing PGS elastomer tissue engineering scaffold with the multistage pore structure, and then carrying out freeze drying to remove water in the scaffold so as to facilitate use and long-term storage.

Advantageous effects

The invention solves the fundamental problem of direct printing of thermosetting materials by thermoplastic FDM, the prepared tissue engineering scaffold has an accurate and controllable multi-level pore structure in structure, the method is simple and quick, the tissue engineering scaffold is suitable for various biological materials, the tissue engineering scaffold required by the patient can be personalized and customized according to the data of the patient such as CT and the like, the tissue engineering scaffold can be used for preparing human auricular cartilage scaffolds, myocardial patches and other multi-level pore scaffolds required by other tissue engineering, and the tissue engineering scaffold has good application prospect.

Drawings

FIG. 1 is a process flow diagram of an embodiment;

FIG. 2 is an SEM image of scaffolds with different mixing ratios; wherein A, D is the ratio 1: a 50-fold electron micrograph of the cross section and surface of the 0.5 sample; B. e is respectively the proportion of 1:1, cross-section and surface of the sample at 50 times electron micrograph; C. f is the ratio of 1:2 cross-section and surface of the sample at 50 times electron micrograph;

FIG. 3 is an electron micrograph of PGS500 and PGS 300;

FIG. 4 is a cured crosslink of a stent;

FIGS. 5A-D are comparative graphs of a stent before and after removal of salt particles;

FIG. 6 is a graph of stent shear stress versus shear rate;

FIG. 7 is a comparison graph of high temperature conformality of splines at different mixing ratios;

FIG. 8 is a comparison of pore size and diameter size of PGS300 and PGS 500;

FIGS. 9A-D show the mechanical properties of PGS scaffolds;

FIG. 10 is the biodegradability of PGS scaffolds;

FIGS. 11a-l are the micro-morphological structures of PCLU elastomers and their scaffolds;

FIGS. 12a-d are the mechanical properties and infrared spectroscopy analysis of PCLU scaffolds.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

1. Print material preparation

The mixing parameters of the PGS prepolymer (Pre-PGS) and the salt particles directly determine the extrudability, the initial form stability, the high-temperature curing shape retention property and the pore structure of the stent during printing, thereby indirectly determining the properties of the stent, including the mechanical property and the biodegradability. The parameters to be studied for the mixing process include the mixing mode, the mixing ratio, the diameter of the salt particles, and the like.

1.1 mixing mode

Pre-PGS is viscous and has a high viscosity at room temperature, and the viscosity of the mixture gradually increases with the increase of the salt ratio to be mixed, so that the problem of uneven mixing is likely to occur, and therefore, it is necessary to consider reducing the viscosity of the mixture. Two methods of reducing viscosity are commonly used: solvent methods and heating methods. Therefore, the materials are mixed by two methods, comparative analysis is carried out through a pre-extrusion experiment, and a more appropriate method is selected for subsequent experiments.

1.1.1 solvent mixing method

1. Uniformly stirring the Pre-PGS and acetone in a mass volume ratio of 1:2 without heating, wherein the total amount of the Pre-PGS is 8g, and the amount of the acetone is 16ml, and mixing to obtain a viscous solution with the total volume of 20 ml;

2. after NaCl is placed in a grinder to be ground, screening through 200-mesh and 400-mesh screens to obtain salt particles with the diameter of 38-75 mu m;

3. mixing the salt granules and Pre-PGS uniformly according to a certain proportion, and filling into a syringe:

4. standing until the acetone is slightly volatilized, placing the mixture in a vacuum oven at the temperature of 30 ℃ for 24 hours to remove the acetone;

5. trial extrusion was performed with a syringe.

1.1.2 heating method

1. Placing the beaker filled with the Pre-PGS in an oil bath at 60 ℃ for heating;

3. mixing Pre-PGS and salt granules according to different proportions, and filling into a syringe cylinder:

4. trial extrusion was performed with a syringe.

1.2 mixing ratio

2g of Pre-PGS was weighed on an analytical balance and placed in a beaker and heated in an oil bath at 60 ℃. Grinding salt particles by a grinder, screening the salt particles with the diameter of 38-75 mu m by using screens of 200 meshes and 400 meshes, weighing 2g of salt particles by using an analytical balance, adding the salt particles when Pre-PGS in a beaker becomes transparent liquid, and uniformly stirring the salt particles by using a glass rod to prepare the Pre-PGS: the NaCl 1:1 mixture was loaded into a 10ml disposable syringe and was ready for use. To find the appropriate print ratio, a series of ratios of the mixture were designed to print, for example, the ratio of the mixture in table 1:

TABLE 1 mixing ratio of Pre-PGS to salt particles

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

2.3D printing Pre-PGS Stent

2.13D printing model

And constructing a model by using AutoCAD 2014 software, constructing a cubic mesh structure, wherein the side length is 20mm, and the microscopic mesh gap can be controlled by a printed path and parameters. In order to embody the advantages of 3D printing, personalized and customizable printing, various complex outline structures can be printed, and snowflake graphics are selected for printing.

2.23D printing parameter control

The power of the 3D printer is turned on, the nozzle and the extrusion rod of the 3D printer are detached before 3D printing is carried out, the 3D printer is cleaned, the nozzle and the needle head are installed, the mixture of PGS and NaCl is loaded into the feeding cylinder, and then the extrusion rod is installed back. And opening the heating device of the No. 1 head, setting the temperature of the extrusion cavity and the temperature of the nozzle, double-clicking the 3D printer software on the computer, connecting the computer with the 3D printer, and selecting the CAD model required by 3D printing. And then setting parameters, namely setting the extrusion cavity temperature and the nozzle temperature to be 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ respectively to change the extrusion state of the material. A 20G needle was selected with a layer height of 0.5, a mesh fill width of 1.2mm, an x position of 90, a y position of 90, a contour fill count of 0, and angles of 90 ° and 0 °. And clicking the XY axis speed to fill the corresponding XY axis movement speed and T axis extrusion speed, wherein the XY axis movement speed is 2mm/s, the T axis extrusion speed is 0.006mm/s, and storing data points to click to determine and fill the path. The x and y positions are adjusted to 90, the height of the needle head away from the receiving plate is adjusted by adjusting the z axis, then the xy axis position of the printer is returned to zero, and after the temperature rises to a specified value and is stable, the Auto can be started to print.

2.3 needle size

The size of the extruding needle directly determines the diameter size of the extruded fiber unit and the fineness of the bracket, and in order to improve the fineness of printing, a needle (22G) with a smaller inner diameter is used for reducing the diameter size of the fiber unit and the gap width between the fiber units. However, the matching of the size of the salt particles and the size of the needle directly determines the extrusion performance of the printing material, and on the premise that the amount of the salt particle filler is not changed, the salt particles with smaller particle size are selected for easier extrusion of the material.

Two salt particles with different diameters are selected for comparison test, the diameter of the salt particles is 38-75 mu m and 26-38 mu m, and the salt particles are printed by 20G needles and 22G needles respectively. Wherein the PGS500 is a scaffold printed by using a 20G needle and salt particles with the diameter of 38-75 mu m as fillers; wherein the PGS300 is a stent printed by using a 22G needle and salt particles with the diameter of 26-38 mu m as fillers. A comparison of the two needle parameters is shown in table 2:

table 221G vs. 22G needle parameters

2.4 curing crosslinking and precipitation of scaffolds

Because the raw materials of the scaffold are Pre-PGS and salt particles, the PGS scaffold can be formed by high-temperature crosslinking in the second step. Although the salt particles maintain the three-dimensional structure of the scaffold as a setting agent during printing, the Pre-PGS viscosity gradually decreases with increasing temperature, and the structure of the scaffold is inevitably damaged. Thus, a suitably low temperature is selected at which the scaffold retains its structure for a certain period of time and the Pre-PGS begins to crosslink. After the Pre-PGS is crosslinked to a certain degree and is still shape-retaining at high temperature, the high-temperature rapid curing crosslinking is carried out.

According to the reference and the conditions of PGS polymerization and curing crosslinking, the low temperature shape keeping temperature is set as 100 ℃, the stent is subjected to primary crosslinking curing in a vacuum oven at 100 ℃ for 12h, and then transferred to a vacuum oven at 150 ℃ for further curing crosslinking.

In order to remove salt particles and non-crosslinked polymers in the scaffold, the scaffold is immersed in a solution of absolute ethyl alcohol and distilled water mixed according to a ratio of 1:3 at room temperature, the mixed solution is changed every 4 hours, the washing is carried out for 3 times, after the mixture is frozen for 12 hours, the mixture is placed in a freeze dryer with the temperature of minus 20 ℃ and the pressure of 50Pa for freeze drying, and the PGS elastomer scaffold is obtained.

3. Characterization and detection

3.1 characterization of printability

The rheological property of the mixture with different proportions is measured by adopting a controlled stress rheometer, and a design module is divided into two parts: the first is a heat preservation stage, the heat preservation temperature is 45 ℃, the rotating speed is 0, and the heat preservation time is 300 s; the second stage is a speed increasing stage, the temperature is 45 ℃, the speed is increased from 0 to 100rpm, and the time is 600 s. Three sets of data were designed and tested for each sample and origin software was used to plot shear stress versus shear rate curves for the four proportional samples.

3.2 measurement of high-temperature shape-retaining Property

Preparing a mixture with PGS and NaCl in the ratio of 1:0.5,1:1,1:2 and 1:3 respectively, taking a columnar model with the length of 1cm and the diameter of 0.5cm, filling the mixture into a mold by using a scraper, compacting, preparing two sample strips from the materials in each ratio, freezing in a refrigerator at the temperature of-20 ℃ for 30min, and pushing out by using a rod to obtain the columnar sample strips. The mixture was placed in a vacuum oven at 100 ℃ for 10min and observed for shape change to aid in the selection of the appropriate mixing ratio.

3.3 characterization and testing of morphological structures

The characterization of the scaffold structure is detected by SEM, the morphology of the scaffold is observed, whether deformation and collapse occur or not and whether the thickness is uniform or not are observed, so that the proper mixing ratio and curing temperature are selected, and the pore distribution and the overall structure of the scaffold are qualitatively characterized. And adopting ImageJ software to count the SEM images of the scaffold, measuring 30-40 data for each item to calculate the average value and the variance, and drawing a histogram by combining Origin software to quantitatively analyze the fiber unit diameter and the pore size of the scaffold.

3.4 porosity

The porosity was measured by ethanol density method in this experiment. After freeze-drying the scaffold, the scaffold was placed in a desiccator for a constant weight of 24h and weighed with an analytical balance to a mass m 0. Putting the bracket into a centrifuge tube, completely immersing the centrifuge tube in absolute ethyl alcohol for 12h, and measuring the total mass of the centrifuge tube, the ethyl alcohol and the bracket as m 1. Carefully taking out the bracket from the bottle by using tweezers, and placing the bracket on a clean culture dish until the bracket taken out is filled with ethanol liquid and does not drip. The remaining ethanol and the total mass of the centrifuge tube were weighed with balance m 2. PGS density ρ PGS, absolute ethanol density ρ ethanol at 20 ℃. The porosity calculation formula of the stent is as follows:

3.5 mechanical Property testing and characterization

The four-layer scaffold was cut into 10 x 5mm rectangular specimens, tested for tensile strength at break and tensile elongation at break and then subjected to a cyclic tensile test at slightly below tensile elongation at break for 10 cycles. The 16-layer scaffold was subjected to cyclic compression testing with a deformation degree of 40% for 1, 10, 30, and 50 test cycles, respectively.

3.6 biodegradability

The PGS scaffold was cut into 5mm by 5mm samples and the initial mass was weighed. The scaffolds were placed in centrifuge tubes containing 5ml of esterase solution, respectively, and placed in a 37 ℃ incubator. Taking out the sample at 10min, 30min, 1h, 2h, 3h, 5h and 7h respectively, rinsing with distilled water, freeze-drying to constant weight, and weighing the mass of the degraded sample. And observing the degraded stent shape by using SEM.

4. Results

2.1.1 mixing of materials

Pre-PGS is viscous and has a high viscosity at room temperature, and the viscosity of the mixture gradually increases with the increase of the salt ratio to be mixed, so that the problem of uneven mixing is likely to occur, and therefore, it is necessary to consider reducing the viscosity of the mixture. Two methods of reducing viscosity are commonly used: solvent method and heating method, using these two methods for comparison. Experiments show that: when the solution was taken out of the vacuum oven by solvent mixing, most of the salt particles were found to be deposited on the bottom by 12 hours of standing, and mixing with Pre-PGS was not uniform and extrusion performance was poor, although acetone was substantially removed without a strong pungent odor. There are also two other drawbacks: firstly, complete removal of acetone cannot be guaranteed; secondly, acetone is volatilized continuously in the experimental operation process, the volume of the Pre-PGS acetone mixed solution is changed continuously, and the real proportion of the Pre-PGS to the NaCl particles cannot be ensured. The materials mixed by the heating method are uniform, the extrusion is stable, and other organic solvents are not added, so that the biocompatibility of the materials is ensured. Therefore, subsequent experiments were performed using a heating method.

2.1.2 mixing ratio of materials

The salt particles play a role of a sizing agent and a pore-forming agent in the mixture, so that the salt particles are too few to play a sizing role, and a printed structure is easy to collapse; too much salt particles and too high viscosity may cause difficulty or even failure in printing. Therefore, an appropriate mixing ratio needs to be searched, the printing can be continuously and completely carried out, the structure of the printing support can be kept, the overall structure and the microstructure of the support are analyzed by combining the observation of an SEM (scanning electron microscope) on the basis of meeting the two conditions, and the optimal ratio of Pre-PGS to salt particles is selected.

From the SEM image, it can be seen that the sample with the ratio of 1:0.5 can be easily heated and extruded at the printing temperature of 35 ℃, but the heating temperature is close to the room temperature, so that the fluidity change is small after cooling, the deformation is easy after forming, the fiber units in the scaffold are not distributed in a clear manner, and different layers are adhered to each other. The proportion is 1:1, the fiber can be extruded at 45-50 ℃, the whole distribution of the fiber is clear, but a certain downward collapse phenomenon occurs in an unsupported area of a single fiber, and the influence of gravity is large. The sample with the ratio of 1:2 can be smoothly extruded at the temperature of 55 ℃, the molding is stable after the sample is cooled at room temperature, the fiber units are clearly distributed, the deformation basically does not occur under the action of gravity, the cross section of the fiber is in a smooth round shape, and the shape retention is good. The ratio of 1:3 samples, the temperature of the heated extrusion was raised to 90 ℃, but there was a defect of poor extrusion, and the fibers were extruded intermittently, which may be caused by poor heat fluidity due to increased salt content.

2.1.33D printing parameters

2.1.3.1 printing temperature

From the point of view of the printing process, the fluidity of the material is better and better as the printing temperature increases, and at the same time the shape retention of the printed support is poorer and worse, so that a suitable temperature is selected at which the material can be extruded stably and continuously into a uniform fibrous shape and can be fixed on a receiving plate and maintain the shape during printing without deformation. From experimental results, at 40-50 ℃, the material has poor flow property, discontinuous fiber extrusion, and the material is easy to block the needle head because the temperature is low and the needle head part is not heated. At 60-65 ℃, the flow property of the material is so good that the diameter of the fiber is suddenly increased or the fiber is broken due to slight changes of pressure or the breakage of bubbles in the printing process, meanwhile, the temperature of the fiber is not timely reduced after the fiber reaches the receiving plate, the flow property is good, the fiber is collapsed under the influence of gravity, and the whole support structure deforms. At about 55 ℃, the fluidity is proper, the continuous extrusion can be realized, the fluidity is reduced after the temperature is reduced after the continuous extrusion reaches the receiving plate, and the fiber shape and the bracket structure can be maintained stably. Therefore, 55 ℃ is adopted as the reference temperature, and the continuous fine adjustment is carried out along with the change of the material state in the printing process, because the material is extruded and heated in the extrusion cavity for a long time in the printing process, the state can be changed to a certain degree. A major problem in the printing process is that air bubbles are easily generated in the material, which causes sudden breakage of the fibers, so that the material needs to be pre-pressurized before printing is started, so that the material is stabilized in the extrusion chamber for a period of time before printing is started.

2.1.3.23D printing software parameters

The diameter of the caramel fiber extruded by the spray head is related to two factors besides the size of the needle head, wherein the two factors are the raw material extrusion speed and the spray head moving speed. Under the condition of constant extrusion speed, the larger the movement speed of the nozzle is, the smaller the fiber diameter is; under the condition that the moving speed of the spray head is constant, the larger the extrusion speed is, the larger the fiber diameter is. Therefore, to fix the basic velocity of the raw material, and to change the moving speed of the nozzle to find a suitable ratio between them, from the experimental results, when the T-axis extrusion speed is 0.006mm/s, and the moving speed of the nozzle is 2mm/s, the diameter of the fiber is matched with the size of the needle, which indicates that the ideal ratio of the T-axis extrusion speed to the moving speed of the nozzle is about 0.003: 1, the printing speed can be changed by changing the sizes of the two. However, the values of the two cannot be increased all the time, because once the moving speed of the nozzle is too high, the fiber is difficult to be well attached to the receiving plate, and meanwhile, the fiber is easy to warp due to too high speed when turning, and the support structure is deformed. The layer height is related to the needle size, the 20G needle has an inner diameter of 0.61, but some stretching of the fibers occurs during printing and is influenced by gravity, so 0.5mm is selected as the layer height. In addition, the printing is typically started with the needle slightly lower than the layer height from the receiving plate so that the material is stably attached to the receiving plate.

2.1.4 needle size

In the experiment, the NaCl particles can be used as a mechanical supporting phase during solidification and also can be used as a pore-forming agent, and a microporous structure is formed after the NaCl particles are removed by water dissolution. To further control the microstructure of the fibers and refine the cast structure, the salt grain size and extrusion nozzle size are adjusted and matched. A and B are respectively a 50-time electron microscope image and a 1000-time electron microscope image of PGS 500; c and D are respectively a 50-time electron microscope image and a 1000-time electron microscope image of the PGS300, a 20G needle is originally adopted, the diameter of the fiber prepared from NaCl particles with the particle size of 38-75 mu m is about 500 mu m (PGS500), a large number of micropore structures are uniformly distributed in the fiber and on the surface of the fiber, and the pore size is matched with the particle size of the particles. When a 22G needle head is adopted for printing and extruding, NaCl particles (less than or equal to 38 micrometers) with the particle size of 26-38 micrometers are selected in consideration of the extrusion smoothness of the particles at the spray head, a bracket (PGS300) with the fiber unit diameter of about 300 micrometers is obtained, and a large number of micropores are uniformly distributed in the fiber to form a multistage pore structure. However, compared with the PGS stent made of 22G needles, the fiber diameter is smaller, so that the fibers are arranged more closely and have higher fineness, and meanwhile, the smaller salt particles enable the micropore size to be smaller and the specific surface area of the whole stent to be larger, so that the PGS stent is more favorable for being used as a tissue engineering stent.

2.1.5 curing crosslinking of scaffolds

Fig. 4 shows the uncured stent on the left and the stent after curing on the right, and it is obvious that the overall shape of the stent before and after curing has not changed significantly, and the original defect still exists after curing, again indicating that the stent has not deformed to a large extent. The experimental scheme of preliminary crosslinking at low temperature of 100 ℃ and further crosslinking at high temperature of 150 ℃ is shown to effectively solve the problem of thermosetting PGS printed by the thermoplastic 3D printing technology.

2.1.6 precipitation of scaffolds

From fig. 5A and C, the scaffold not only has large pores between the grids, but also generates a large number of pores inside the surface of the scaffold by removing the salt particles, thereby forming multi-level pores of the scaffold, and facilitating the propagation and growth of cells. And through B and D, the size of the micropores can be quantitatively analyzed and matched with the size of the salt particles, so that the size of the micropores of the stent can be controlled by the diameter size of the mixed salt particles, and the method is favorable for the application of the stent to specific clinic in tissue engineering.

2.2 characterization and detection of scaffold Structure

2.2.1 characterization of printability

Printability includes extrudability and stability of the initial form. The left graph is a plot of shear stress versus shear rate plotted against the data obtained from the controlled stress rheometer test, where the slope of each curve represents the viscosity at that shear rate for each proportion of sample, and thus the same conclusion can be drawn from both the entire curve and a specified shear rate: as the proportion of salt particles in the mixture increases, the viscosity of the material increases continuously. In 3D melt extrusion experiments, as the proportion of salt particles increases, the extrudability of the mixture becomes worse and the stability of the initial form becomes better at the same temperature, which can be explained by the viscosity mentioned above. In summary, the first three ratios of samples were adjusted by printing temperature to obtain suitable printability, but the ratio of Pre-PGS to NaCl was 1: the sample of 3 had difficulty in having good extrudability even at 90 ℃. Thus, a 1:3 ratio mixture is not suitable for 3D melt extrusion printing to make PGS elastomer scaffolds from a printability perspective.

2.2.2 measurement of high-temperature shape-Retention

From FIG. 7, it can be seen that as the content of NaCl particles in the mixture increases, the high temperature conformality of the scaffold gradually becomes better, and the ratio of pure Pre-PGS, Pre-PGS to NaCl is 1: the samples with the three ratios of 0.5 and 1:1 still have difficulty in maintaining their shapes under the low temperature crosslinking condition at 100 ℃, while the samples with the two ratios of Pre-PGS to NaCl of 1:2 and 1:3 can maintain their shapes to some extent. Therefore, from the viewpoint of high temperature shape retention, the ratio of 1:2 to 1:3 is suitable, and the ratio of 1:2 is most desirable in combination with printability.

2.2.3 diameter and pore size statistics

It can be quantitatively seen from fig. 8 that there is a significant difference in the diameter size of PGS300 and PGS500, and that the micropore size of the fiber interior and surface matches the size of the salt particles incorporated. The pore size of the fiber cross section is significantly larger than that of the fiber surface because only a portion of the pores are visible on the fiber surface and the salt particles on the surface are typically covered with PGS, resulting in smaller pore diameters or insignificant pores after salt particles are extracted.

2.2.4 detection of scaffold porosity

The structure of the three-dimensional scaffold plays a crucial role in the application of the three-dimensional scaffold in tissue engineering, and if the scaffold has high porosity and good connectivity, the scaffold is beneficial to the adsorption and proliferation of cells and the transportation of nutrients and metabolic wastes. The PGS elastomer scaffold designed by the experiment forms a multistage pore of the scaffold together by a primary contour structure generated by the design of a 3D printed model, a secondary macroporous structure generated by the diameters of fiber units and gaps between fibers and a tertiary microporous structure generated after salt grains are removed as a template, and is used as a channel for conveying nutrient substances and metabolic waste required by cell growth. The porosity of the PGS elastomer scaffold is therefore a crucial parameter.

The porosity of the 3D printed PGS elastomer scaffold was determined using the formula shown in the experimental part. As shown in Table 3, the three-dimensional hollow scaffold prepared by the method has a porosity of above 66% and an average porosity of 72.96%.

TABLE 3 porosity determination of scaffolds

M0	M1	M2	Porosity of the material
				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 mechanical Property testing and characterization

From the results of the mechanical property tests shown in fig. 9(a), it can be seen that the tensile elongation at break of the PGS500 stent is about 35%, the breaking strength is about 80kPa, and the tensile elongation at break of the PGS300 stent is about 40%, and the breaking strength is about 60 kPa. The fabric is cyclically stretched for 10 times under the condition of 25 percent of elongation, shows a typical elastic deformation curve and has good deformation recovery (C, D), and has certain in-vivo suture strength and compliance of in-vivo dynamic mechanical environment. The cyclic compression test shows that the elastomer support printed by the 3D printing has stronger fatigue resistance, the curves of multiple deformation recovery are basically superposed, the initial good elasticity (B) is still kept, the elastomer support can be timely recovered after being compressed by stress in the body, and the elastomer support can keep good matching with tissues. Analysis of the cyclic compression curves of PGS500 and PGS300 revealed that the compression strength of PGS300 was slightly greater than that of PGS500, which is related to the more dense arrangement of the fiber units of PGS 300. Both stents maintained elastic deformation within 50% of the deformation degree, i.e. the compression curve coincided with the release recovery curve, but after exceeding 50%, the curves did not coincide, and the mechanical curve of the recovery process was higher than that of the compression process, thus showing the phenomenon of "mechanical reinforcement" (B). This phenomenon may be caused by an increase in the force-receiving area due to flattening of the entire structure when the compression set is too large (more than 50%).

2.2.6 biodegradability

As can be seen from the degradation curve of fig. 10, the PGS scaffold printed in 3D has good biodegradability, fast initial degradation, substantially balanced subsequent degradation, and a degradation rate of more than 90% after 5 hours. As can be seen from the electron microscope image of the degraded sample, the degradation of the bracket is that etching degradation gradually occurs from the surface to the inner part, and the size of the microporous structure on the surface is gradually increased.

Example 2

PCLU scaffolds were prepared in the same manner as in example 1. The method uses polycaprolactone diol and HDI trimer as raw materials, and the raw materials are mixed with salt particles for printing and then are cured and formed, so that the step of synthesizing prepolymer is omitted, and the raw material units are reacted and cured on a 3D formed structure under the heating condition.

As shown in FIG. 11, this method is also satisfactory for PCLU printing and molding, and can be used to prepare bioscaffolds and other irregular shapes, and the thermosetting PCLU is also an elastomer which can be rapidly recovered by repeated folding (a-d). The bracket is subjected to electron microscope test, fiber units are clearly and regularly arranged and stacked, the cross section of the fiber is circular, no obvious fiber collapse phenomenon is seen, the curing and shape-keeping performance is good, meanwhile, a large number of micropores are distributed on the surface and inside of the fiber, and the bracket is integrally in a multi-level pore structure (e-l).

The mechanical test results for PCLU scaffolds are shown in fig. 12: the tensile breaking strength is about 386kPa, and the breaking elongation is 80 percent, which is superior to that of a PGS bracket. The fitting degree of the loaded and released curves in the 10-period cyclic tensile test and the 50-period cyclic compression test is high, and excellent elasticity and deformation recovery are shown; meanwhile, the mechanical characteristic curve is basically unchanged after multiple cycles, and the fatigue resistance is good. For the PCLU elastomer, since the degradable unit polycaprolactone diol is used as the raw material, the PCLU elastomer is expected to have biodegradability. In addition, for NCO groups with potential biotoxicity in the raw materials, the NCO groups can be judged to be completely reacted and not exist after infrared spectrometry analysis, and the NCO groups are similar to other medical polyurethane materials and have good biocompatibility.

Claims

1. A preparation method of a thermosetting elastomer tissue engineering scaffold with a multistage pore structure comprises the following steps:

(1) mixing a thermosetting material and a filling material according to a mass ratio of 1:0.5-3 to obtain a mixed material; utilizing CAD software to construct a model of a cubic reticular structure, then adding a mixed material into a heating cavity of a 3D printer, and obtaining an initial support through 3D printing; wherein the thermosetting material is PGS, the filling material is salt particles,

(2) carrying out thermal crosslinking or photo crosslinking on the initial scaffold in the step (1) to obtain a thermosetting elastomer tissue engineering scaffold; finally, removing the filling material to obtain the thermosetting elastomer tissue engineering scaffold with the multistage pore structure; the multistage pore structure comprises a primary contour structure, a secondary macroporous structure generated by the diameters of fiber units and gaps among fibers, and a tertiary microporous structure generated after a filling material is removed as a template; the thermal crosslinking parameters are as follows: primarily crosslinking and curing for 12-24h in a vacuum oven at 80-100 ℃, and further curing and crosslinking in a vacuum oven at 120-150 ℃.

2. The method for preparing the thermosetting elastomer tissue engineering scaffold with the multilevel pore structure according to claim 1, wherein the method comprises the following steps: the diameter of the salt particles is 20-100 μm.

3. The method for preparing the thermosetting elastomer tissue engineering scaffold with the multilevel pore structure according to claim 1, wherein the method comprises the following steps: the mixing mode in the step (1) is a solvent mixing method or a heating method.

4. The method for preparing the thermosetting elastomer tissue engineering scaffold with the multilevel pore structure according to claim 1, wherein the method comprises the following steps: the 3D printing parameters in the step (1) are as follows: the temperature of the extrusion cavity and the temperature of the nozzle are 40-100 ℃.

5. The method for preparing the thermosetting elastomer tissue engineering scaffold with the multilevel pore structure according to claim 1, wherein the method comprises the following steps: and (3) preserving the thermosetting elastomer tissue engineering scaffold with the multistage pore structure obtained in the step (2) by freeze drying.