CN116813934A

CN116813934A - 4D printable calcium ion response self-healing hydrogel and preparation method and application thereof

Info

Publication number: CN116813934A
Application number: CN202310753524.7A
Authority: CN
Inventors: 朱卉; 金钊锐; 秦娜娜; 朱胜涛; 孙逸文; 贺健康; 李涤尘
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2023-06-25
Filing date: 2023-06-25
Publication date: 2023-09-29

Abstract

The invention discloses a 4D printable calcium ion response self-healing hydrogel and a preparation method and application thereof, wherein the method comprises the following steps: uniformly mixing carboxymethyl chitosan solution and oxidized sodium alginate solution, and performing a crosslinking reaction to obtain 4D printable calcium ion response self-healing hydrogel; the oxidized sodium alginate solution is prepared by dissolving oxidized sodium alginate in phosphate buffer solution; the carboxymethyl chitosan solution is prepared by dissolving carboxymethyl chitosan in phosphate buffer solution; when the carboxymethyl chitosan solution is mixed with the oxidized sodium alginate solution, the solute mass ratio is (3-4): 1. the hydrogel disclosed by the invention has relatively simple components and better self-healing capability, can respond to shrinkage by using the stimulus of divalent calcium ions to realize 4D printing, does not influence human bodies, has the characteristic of quick response to external stimulus and excellent biocompatibility, and can be widely applied to printing of fine structures such as tissue implants in the future.

Description

4D printable calcium ion response self-healing hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical material production and preparation, and particularly relates to 4D printable calcium ion response self-healing hydrogel and a preparation method and application thereof.

Background

Biological 3D printing technology has been used for constructing complex tissue organs in recent years, and has shown great potential in the field of implantable medical devices, however, the traditional 3D printing mode only considers the initial state of the printed biological structure, does not pay attention to the dynamic change of the biological structure, and cannot simulate the internal mechanism of a human body. Compared with the 3D printing mode, the 4D printing increases the time conversion dimension. The printed biological structure may change its size, shape or function over time when subjected to external physical, chemical or biological stimuli.

Hydrogels, which are important 4D bioprinting materials, exhibit excellent biocompatibility, can mimic extracellular matrix (ECM), and provide a similar biological environment for cells. For bio-4D printing technology, the choice of hydrogel ink is critical, and hydrogels useful as 4D printing bio-ink for biomedical applications should meet several basic requirements at the same time: (1) the hydrogel has good biocompatibility; (2) Can be quickly glued, and has the characteristics of shear thinning and self-healing; (3) adjustable mechanical properties; (4) has stimulus responsiveness.

Shuhong et al (CN 115403888A) developed a novel 4D printing ink, which was 4D printed using magnetizable single domain NdFeB particles and metal dynamic coordination bonds and intermolecular chain-to-chain hydrogen bonding. A temperature-responsive 4D printing intelligent hydrogel material is developed by the general name of gaku jiao et al (CN 111331836 a), and a precise layered structure is prepared through ultraviolet light curing. Both the 4D printing hydrogels developed above have better stimulus response, but the components adopted by the method are complex and the steps are complex. Meanwhile, the magnetic hydrogel is metal-based material with magnetism, and needs to deform under the action of an external magnetic field, so that the magnetic hydrogel can be used as a human implant to influence a human body, and the metal-based material has poor performance in terms of biocompatibility. The temperature responsive hydrogel material requires a certain temperature change stimulus, which affects the growth of the cells. Meanwhile, most hydrogels currently existing have no self-healing capacity or poor self-healing capacity.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide the 4D-printable calcium ion response self-healing hydrogel, the preparation method and the application thereof, the hydrogel composition is relatively simple, the self-healing capability is good, the hydrogel composition can shrink under the stimulus response of divalent calcium ions, 4D printing is realized, the influence on a human body is avoided, and meanwhile, the hydrogel composition has the characteristics of rapid response by external stimulus and excellent biocompatibility, and can be widely applied to printing of fine structures such as tissue implants in the future.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a preparation method of 4D printable calcium ion response self-healing hydrogel comprises the following steps:

uniformly mixing carboxymethyl chitosan solution and oxidized sodium alginate solution, and performing a crosslinking reaction to obtain the 4D printable calcium ion response self-healing hydrogel;

the oxidized sodium alginate solution is prepared by dissolving oxidized sodium alginate in phosphate buffer solution;

the carboxymethyl chitosan solution is prepared by dissolving carboxymethyl chitosan in phosphate buffer solution;

when the carboxymethyl chitosan solution is mixed with the oxidized sodium alginate solution, the solute mass ratio is (3-4): 1.

preferably, the preparation process of the oxidized sodium alginate comprises the following steps:

dropwise adding a sodium periodate aqueous solution into the sodium alginate ethanol suspension, and stirring in the dark to react to be sticky; then adding excessive glycol to terminate the reaction; dialyzing the obtained solution in deionized water, and freeze-drying to obtain oxidized sodium alginate;

wherein the mass volume ratio of the sodium alginate to the ethanol is 200:1, and the concentration of the sodium periodate aqueous solution is 0.25mol/L; the molecular weight cut-off for dialysis was 6-8kDa.

Preferably, the concentration of the oxidized alginate solution is 60-80mg/mL, and the solvent is phosphate buffer with pH=7.0-7.3;

the concentration of the carboxymethyl chitosan solution is 60-80mg/mL, and the solvent is phosphate buffer solution with pH=7.0-7.3.

Preferably, the carboxymethyl chitosan is O-carboxymethyl chitosan, and the phosphate buffer solution is Du's phosphate solution.

Preferably, the temperature during the crosslinking reaction is 30-37℃and the time is 30-60 seconds.

Preferably, the preparation method further comprises a process of crosslinking and curing the 4D-printable calcium ion-responsive self-healing hydrogel at room temperature.

The invention provides 4D-printable calcium ion response self-healing hydrogel, which is prepared by the preparation method disclosed by the invention.

The 4D printable calcium ion response self-healing hydrogel disclosed by the invention can realize 4D printing through calcium ion response.

Preferably, the process of 4D printing includes:

3D printing is carried out on the 4D-printable calcium ion response self-healing hydrogel, and a 3D printing piece is obtained;

immersing the 3D printing piece into a divalent calcium ion solution to carry out secondary crosslinking and deformation, so as to realize 4D printing;

the divalent calcium ion concentration is 0.1-2M, and the crosslinking time is not more than 30min.

Preferably, the 4D printable calcium ion responsive self-healing hydrogel is used to print tissue implants including nerve conduits and vascular microchannels.

The invention has the following beneficial effects:

the invention utilizes the characteristic that oxidized sodium alginate aldehyde group can react with amino groups on carboxymethyl chitosan to generate Schiff base, and generates a hydrogel polymerization network based on the principle that Schiff base can quickly form dynamic imine bonds between aldehyde groups and amino groups under mild conditions. The hydrogel is a shear-thinning self-healing hydrogel, is suitable for being used as biological ink, does not need a rheology modifier or a cross-linking agent, and has good biocompatibility; the component content of the oxidized alginate-carboxymethyl chitosan hydrogel is regulated to enable the oxidized alginate-carboxymethyl chitosan hydrogel to have the characteristic of adjustable mechanical properties. The invention utilizes the characteristic of stimulus response shrinkage of carboxymethyl chitosan to divalent calcium ions to realize 4D printing. The invention takes carboxymethyl chitosan and sodium alginate as main raw materials, and takes arthropod and insect crusta and brown algae as raw materials respectively, and the two main raw materials are widely distributed in nature, easy to obtain, low in cost and excellent in biocompatibility.

Drawings

FIG. 1 (a) is a physical diagram of self-healing of 4D printable calcium ion responsive self-healing hydrogel prepared in example 1 of the present invention; FIG. 1 (b) is a graph showing the self-healing properties of a 4D printable calcium ion responsive self-healing hydrogel rheology test prepared in example 1 of the present invention;

FIG. 2 (a) is a porous structure diagram of the invention in example 3, experimental group 1 extrusion type 3D printing; FIG. 2 (b) is a porous structure diagram of the experimental group 2 extrusion type 3D printing of the invention in example 3; FIG. 2 (c) is a porous structure diagram of the experimental group 3 extrusion type 3D printing of the invention;

FIG. 3 shows that the hydrogel prepared in example 4 of the present invention has good mechanical properties after secondary crosslinking; repeatedly squeezing;

FIG. 4 shows the volume change trend of the hydrogel prepared in example 4 according to the present invention during soaking in solutions of different calcium chloride concentrations;

FIG. 5 (a) is a morphology diagram of a 3D printed porous structure of the hydrogel prepared in example 5 of the present invention, and FIG. 5 (b) is a partial enlarged view of the region of the white frame in FIG. 5 (a); FIG. 5 (c) is a morphology diagram of the 3D printing porous structure of the hydrogel prepared in example 5 of the present invention after being crosslinked by 0.1M calcium chloride solution, and FIG. 5 (D) is a partial enlarged view in the white frame region in FIG. 5 (c);

FIG. 6 (a) is a diagram showing the structure of a hydrogel prepared in Experimental group 1 of example 1, before shrinkage of a vascular model, which is used as a bio-ink for 3D printing by extrusion; FIG. 6 (b) is a diagram showing the structure of the hydrogel prepared in experimental group 1 of example 1 after shrinkage of a vascular model, which is an application of the hydrogel as a bio-ink by extrusion type 3D printing;

FIG. 7 (a) is a front view of the nerve conduit model, which is a schematic diagram showing the application of the hydrogel prepared in experiment set 2 of example 1 as a bio-ink in the 4D printing field; FIG. 7 (b) is a side view of the nerve conduit model, which is a schematic diagram showing the application of the hydrogel prepared in experimental group 2 of example 1 as a bio-ink in the 4D printing field.

Detailed Description

The invention is further described below with reference to the drawings and examples.

The preparation method of the 4D printable calcium ion response self-healing hydrogel comprises the following steps: the method for synthesizing oxidized sodium alginate and preparing oxidized sodium alginate-carboxymethyl chitosan hydrogel comprises the following specific implementation steps:

step (1), synthesizing oxidized sodium alginate:

dropwise adding a sodium periodate aqueous solution into the sodium alginate ethanol suspension, and stirring in the dark to react to be sticky; then adding excessive glycol to terminate the reaction; and dialyzing the obtained solution in deionized water for several days, and freeze-drying to obtain oxidized sodium alginate. And dissolving the obtained oxidized sodium alginate in a phosphate buffer solution to obtain an oxidized sodium alginate solution. Wherein the mass volume ratio of sodium alginate to ethanol is 200:1, and the concentration of the sodium periodate aqueous solution is 0.25mol/L; the molecular weight cut-off for dialysis was 6-8kDa. The concentration of the obtained oxidized alginate solution is 60-80mg/mL, and the solvent is phosphate buffer solution with pH=7.0-7.3; further preferably 70mg/mL.

Step (2), preparing oxidized sodium alginate-carboxymethyl chitosan hydrogel:

dissolving carboxymethyl chitosan in phosphate buffer solution to obtain carboxymethyl chitosan solution, uniformly mixing the carboxymethyl chitosan solution with the oxidized sodium alginate solution prepared in the step (1), and performing a crosslinking reaction to obtain oxidized sodium alginate-carboxymethyl chitosan hydrogel. Wherein, when the oxidized alginate solution and the carboxymethyl chitosan solution are mixed, the solute mass ratio of the oxidized alginate solution to the carboxymethyl chitosan solution is (3-4): 1. the concentration of the carboxymethyl chitosan solution is 60-80mg/mL, and the solvent is phosphate buffer solution with pH=7.0-7.3. Preferably, the carboxymethyl chitosan is O-carboxymethyl chitosan; further preferably 70mg/mL.

The hydrogel prepared in the step (2) can be directly used as 3D printing ink, and has self-repairing capability. The hydrogel prepared in the step (2) is used as printing ink and is printed, and the specific process is as follows (the printing support structure is taken as an example for illustration):

3D printing: and (3) transferring the hydrogel prepared in the step (2) into a needle tube, and extruding and printing the hydrogel by a 3D biological printer according to set printing parameters to obtain the hydrogel bracket structure.

And (3) structural transformation: structural transformation of divalent calcium ion stimulus response

Immersing the hydrogel bracket structure based on oxidized sodium alginate-carboxymethyl chitosan obtained by 3D printing into a divalent calcium ion solution with a certain concentration, so that the hydrogel bracket structure is subjected to secondary crosslinking and curing, and the hydrogel bracket structure can undergo volume shrinkage in a certain time in the crosslinking and curing process. Wherein the temperature of the crosslinking reaction is 30-37 ℃ and the time is 30-60 seconds. The divalent calcium ion concentration is 0.1-2M, and the specific time is not more than 30min. As the divalent calcium ion solution, a calcium chloride solution can be used. The calcium chloride solution is used as a post-crosslinking reagent to further improve the mechanical property of the hydrogel and delay the biodegradation time of the hydrogel; in addition, the introduced calcium ions also have the effects of hydrogel coagulation, wound healing promotion, angiogenesis and the like.

The hydrogel provided by the invention has controllable shrinkage under the stimulation of calcium ions, calcium ion crosslinking can enable oxidized alginate-carboxymethyl chitosan hydrogel scaffolds with different volumes to obtain 10% -20% of volume shrinkage, and the higher the concentration of the calcium ions, the larger the shrinkage degree is, so that ion stimulation response is realized, and the hydrogel with a fine structure is obtained. This phenomenon helps to obtain a complex biomimetic structure with finer volume in a simpler way through 4D bioprinting.

The hydrogel can be combined with other materials to form a composite structure after 3D extrusion printing, and meanwhile, the characteristic that the hydrogel generates volume shrinkage under the action of calcium ions is utilized to realize the 4D printing effect with controllable complex structure.

Example 1:

the preparation method of the 4D printable calcium ion response self-healing hydrogel comprises the following steps:

step one: preparation of oxidized alginate (ADA)

10g of sodium alginate was added to a brown laboratory glass bottle covered with tinfoil paper, and 50mL of ethanol solution was added to the brown laboratory glass bottle and stirred in a water bath at room temperature. 1.337g-4.011g sodium periodate is dissolved in 50mL deionized water in a dark place, slowly added into sodium alginate suspension in a dropwise manner, and stirred in the dark for reaction; after 5h-7h, adding 10mL of ethylene glycol to terminate the reaction; the obtained solution is poured into a dialysis bag, dialyzed in deionized water for 3 days and then freeze-dried, and the molecular weight cut-off of the dialysis bag is 6-8kDa, so that oxidized alginate (ADA) is obtained.

The content and reaction time of the sodium periodate adopted in the first step are shown in Table 1

TABLE 1

Step two: preparation of oxidized sodium alginate-carboxymethyl chitosan hydrogel

And (3) dissolving ADA-2 in the step I in the Du's phosphate solution, stirring overnight at a low speed to obtain an ADA solution, dissolving carboxymethyl chitosan (CMC) in the Du's phosphate solution, and fully stirring in a dark place to obtain the CMC solution. Slowly dripping CMC solution into ADA solution, mixing, crosslinking at room temperature for 30-60 s, and standing for solidification. The 4D calcium ion response hydrogel is prepared by adopting 7 groups of ADA and CMC with different volume concentrations and proportions, and the hydrogels of 7 groups of experimental groups can be uniformly gelled. The crosslinking time is related to the volume concentration and proportion, and the higher the carboxymethyl chitosan content in the hydrogel system, the higher the total polymer amount, so that the more crosslinking sites in the hydrogel are, and the shorter the crosslinking time is required.

The concentrations and ratios used in step two are shown in Table 2

TABLE 2

Test group	CMC w/v％	ADA w/v％	Crosslinking time
				1	1.50	4.50	50s
2	1.75	5.25	40s
				3	2.00	6.00	30s
4	1.20	4.80	60s
				5	1.40	5.60	45s
6	1.60	6.40	35s
				7	1.55	5.43	55s

Example 2: self-healing experiments were performed on the 4D calcium ion-responsive hydrogel prepared in example 1 above

The hydrogels prepared in the 7 experimental groups of example 1 all had self-healing properties. The hydrogel prepared in the experimental group 1 in example 1 was poured into a mold in a state of not being completely cured to prepare a cylindrical hydrogel sample, and after standing and curing, the cylindrical hydrogel sample was demolded and taken out, and gel fragments were obtained by cutting into two pieces with a knife. After the gel fragments were split and allowed to stand for ten minutes, a complete hydrogel was obtained as shown in FIG. 1 (a). The re-healed hydrogel is not easy to separate under pulling, has certain mechanical strength, and proves that the series of hydrogels have good self-healing performance.

In addition, the hydrogel prepared in the experimental group 2 of example 1 was subjected to a rheology recovery test to detect its mechanical properties and self-healing properties: a cylindrical hydrogel with a diameter of 25mm and a height of 2.5mm is placed on a rheometer, and the angular frequency is increased from 0.05rad/s to 500rad/s for frequency scanning, so as to obtain the relation between the angular frequency and the complex viscosity; the strain was continued at 0.2% for 300s, then the strain was raised to 20% for 20s, and the process was cycled twice, and alternate strain scans were performed to examine the self-healing properties of the hydrogels. As can be seen from fig. 1 (b), the viscosity of the hydrogel decreases with increasing angular frequency, being a shear-thinning fluid; FIG. 1 (b) demonstrates that the hydrogel can quickly return to its normal state after alternating strains, demonstrating good self-healing. Both figures demonstrate that the hydrogels can be used as extrusion printing materials.

Example 3: printing of carboxymethyl chitosan-oxidized sodium alginate hydrogel ink

Experiment group 1: setting the filling mode of a printing model to be 90 degrees in a cross vertical way, introducing the printing model into a 3D printer, printing by adopting the hydrogel prepared in the experimental group 1 in the embodiment 1, moving the hydrogel prepared in the experimental group 1 in the embodiment 1 down to a needle tube after crosslinking for 50s, removing bubbles of the hydrogel in the needle tube by centrifugation, printing 30 layers of brackets at room temperature, immersing the brackets in 0.1M calcium chloride solution for 30min after printing, and crosslinking; fig. 2 (a) shows a printed hydrogel scaffold, the 4D calcium ion-responsive hydrogel is uniform in structure and clear and complete in profile, demonstrating good printability of the hydrogel.

Experiment group 2: setting the filling mode of a printing model to be 90 degrees in a cross vertical way, introducing the hydrogel prepared by the experimental group 2 in the embodiment into a 3D printer for printing, transferring the hydrogel prepared by the experimental group 2 in the embodiment to a needle tube after crosslinking for 40s, removing bubbles of the hydrogel in the needle tube by centrifugation, printing 30 layers of brackets at room temperature, immersing the brackets in 0.1M calcium chloride solution for 30min after printing, and crosslinking; fig. 2 (b) shows a printed hydrogel scaffold, the 4D calcium ion-responsive hydrogel is uniform in structure, clear and complete in profile, demonstrating good printability of the hydrogel. As shown in fig. 3, the hydrogel has good mechanical properties after post-crosslinking; can be repeatedly extruded.

Experiment group 3: setting the filling mode of a printing model to be 90 degrees in a cross vertical way, introducing the printing model into a 3D printer, printing the hydrogel prepared by the experiment group 3 in the example, transferring the hydrogel prepared by the experiment group 3 in the example 1 to a needle tube after crosslinking for 30s, removing bubbles of the hydrogel in the needle tube by centrifugation, printing 30 layers of brackets at room temperature, immersing the brackets in 0.1M calcium chloride solution for 30min after printing, and crosslinking; fig. 2 (c) shows a printed hydrogel scaffold, the 4D calcium ion-responsive hydrogel is uniform in structure and clear and complete in profile, demonstrating good printability of the hydrogel.

Example 4: contractility verification of carboxymethyl chitosan-oxidized sodium alginate hydrogel

Experimental group 1: ADA-2 prepared in example 1 is dissolved in Du's phosphate solution, stirred at low speed overnight to obtain 70mg/mL of ADA solution, carboxymethyl chitosan (CMC) is dissolved in Du's phosphate solution, uniformly stirred in a dark place to obtain 70mg/mL of CMC, CMC solution is slowly dropped into ADA solution to be uniformly mixed, then transferred into a die with the diameter of 9mm and the height of 4mm, subjected to crosslinking reaction for 50 seconds at room temperature, and left to stand for solidification. The mass volume concentration of the carboxymethyl chitosan in the hydrogel is 1.75%, the mass volume concentration of the oxidized sodium alginate in the hydrogel is 5.25%, the mass volume concentration of the 3ADA-1CMC is 3:1. the samples were then immersed in 0.1M calcium chloride solution for 48 hours, and the volume of the samples was measured at intervals therebetween, the trend of which is shown in fig. 4.

Experiment group 2: ADA-2 prepared in example 1 is dissolved in Du's phosphate solution, stirred at low speed overnight to obtain 70mg/mL of ADA solution, carboxymethyl chitosan (CMC) is dissolved in Du's phosphate solution, uniformly stirred in a dark place to obtain 70mg/mL of CMC, CMC solution is slowly dropped into ADA solution to be uniformly mixed, then transferred into a die with the diameter of 9mm and the height of 4mm, subjected to crosslinking reaction for 50 seconds at room temperature, and left to stand for solidification. The mass volume concentration of the carboxymethyl chitosan in the hydrogel is 1.75%, the mass volume concentration of the oxidized sodium alginate in the hydrogel is 5.25%, the mass volume concentration of the 3ADA-1CMC is 3:1. the samples were then immersed in a 2M calcium chloride solution for 48 hours, during which time the volume of the samples was tested at intervals, the trend of which is shown in fig. 4.

Experiment group 3: ADA-2 prepared in example 1 is dissolved in Du's phosphate solution, stirred at low speed overnight to obtain 70mg/mL of ADA solution, carboxymethyl chitosan (CMC) is dissolved in Du's phosphate solution, uniformly stirred in a dark place to obtain 70mg/mL of CMC, CMC solution is slowly dropped into ADA solution to be uniformly mixed, then transferred into a die with the diameter of 9mm and the height of 4mm, subjected to crosslinking reaction for 50 seconds at room temperature, and left to stand for solidification. The mass volume concentration of the carboxymethyl chitosan in the hydrogel is 1.75%, the mass volume concentration of the oxidized sodium alginate in the hydrogel is 5.25%, the mass volume concentration of the 3ADA-1CMC is 3:1. the samples were then immersed in 5M calcium chloride solution for 48 hours, during which time the volume of the samples was tested at intervals, the trend of which is shown in fig. 4.

Example 5: 4D printing of carboxymethyl chitosan-oxidized sodium alginate hydrogel ink

ADA-2 prepared in example 1 is dissolved in Du's phosphate solution, stirred at low speed overnight to obtain 70mg/mL of ADA solution, carboxymethyl chitosan (CMC) is dissolved in Du's phosphate solution, uniformly stirred in a dark place to obtain 70mg/mL of CMC, the CMC solution is slowly dripped into the ADA solution to be uniformly mixed, and the mixture is subjected to crosslinking reaction at room temperature for 40 seconds to obtain 3ADA-1CMC hydrogel ink with the mass volume concentration of carboxymethyl chitosan in the hydrogel of 1.75% and the mass volume concentration of oxidized sodium alginate in the hydrogel of 5.25%. The filling mode of the solution setting printing model is 90 degrees crossed and vertical, the solution setting printing model is guided into a 3D printer, the prepared hydrogel is moved to a needle tube, 30 layers of supports are printed at room temperature, the inner diameter of a needle head is 0.33mm, the printing speed is 6mm/s, the printed hydrogel support is shown in fig. 5 (a), fig. 5 (b) is an enlarged view of the hydrogel support, the aperture of the hydrogel support is 145.46 mu m, and the distance between two holes is 86.03 mu m. The stent is then immersed in a 0.1M calcium chloride solution for 30min. The hydrogel after calcium ion response is shown in FIG. 5 (c), and the enlarged view of the hydrogel after calcium ion response is shown in FIG. 5 (d), wherein the pore diameter of the scaffold after shrinkage is 100.87 μm, and the distance between the two pores is 66.44. Mu.m. The divalent calcium ions interact with the carboxylic acid ions on the CMC chain. The repulsive forces between deprotonation in neutral or alkaline solutions keep the CMC chains in stretched form and part of the carboxyl groups of the hydrogel are occupied by calcium ions, the number of carboxyl groups being reduced so that hydrophilicity is reduced. The controllable contractility of the hydrogel ensures that the hydrogel has good application prospect in printing micro biological structures and organs such as micro blood vessels.

Example 6: calcium ion response self-healing hydrogel 4D printing blood vessel micro-channel model

The hydrogel prepared in experiment group 1 in example 1 was used as a supporting material, gelatin was used as a sacrificial material, the self-healing hydrogel and gelatin ink were respectively transferred to two needle tubes, and were mounted in a multi-head integrated 3D printing platform system, extrusion pressure was provided by an air pump, and the two inks were extruded through a 20G needle head, and a preset vascular model was compositely punched out, as shown in fig. 6 (a). After printing, the model was immersed in a calcium chloride solution at 37 ℃ or higher, gelatin was dissolved and flowed out as a sacrificial material at 37 ℃ or higher, and the ADA-CMC hydrogel was shrunk and deformed by the stimulation of divalent calcium ions, to obtain a vascular model having a fine structure as shown in fig. 6 (b).

Example 7: calcium ion response self-healing hydrogel 4D printing nerve conduit

Experiment group 1: the PCL melting ink and the ADA-CMC hydrogel ink are respectively placed in a temperature control syringe at 90 ℃ and a common extrusion injection syringe, extrusion pressure is provided by an air pump, and a 3D biological printing platform integrated by a plurality of spray heads is utilized for carrying out composite 4D printing of PCL fibers and the hydrogel. Firstly, a PCL diamond-shaped bracket printing model is constructed, the X-axis length is 15 cm, the Y-axis length is 15 cm, the line spacing is 0.4 cm, the applied voltage is 4500V, the air pressure is set to 300, the receiving distance is 2.5 cm, the number of layers is 5, and a stretchable PCL diamond-shaped micrometer fiber bracket is constructed by utilizing an electrostatic micro-nano printing technology. Secondly, a parallel line printing model is constructed, the X-axis length is 15 cm, the y-axis length is 15 cm, the line spacing is 0.4 cm, the layer height is 0.18 cm, the receiving distance is 0.15 cm, the number of layers is 2, data are imported into a 3D printer, 4D calcium ion response hydrogel prepared in the experiment group 2 in the example 1 is used for printing, and the data are accurately deposited on a PCL bracket. The diameter of the printing needle head 22G, the extrusion air pressure is 1600, the printing speed is 2.5mm/s, and the printing path is along the normal direction of the stretching direction of the PCL diamond-shaped bracket. After printing, the hydrogel/PCL composite scaffold was immersed in a 1M calcium chloride solution for crosslinking for 30 minutes. The hydrogel bracket contracts to drive the PCL bracket to self-curl to form the nerve conduit, as shown in fig. 7 (a) and 7 (b), the 4D printing nerve conduit has obvious conduit curling effect and clear and complete structure, and the 4D calcium ion responds to the hydrogel.

Experiment group 2: a parallel line printing model was constructed, the X-axis length was 15 cm, the y-axis length was 15 cm, the line spacing was 0.4 cm, the layer height was 0.18 cm, the accepted distance was 0.15 cm, the number of layers was 2, and the data was introduced into a 3D printer and printed using the 4D calcium ion responsive hydrogel prepared in experimental group 2 of example 1. The hydrogel is moved to a needle tube, bubbles of the hydrogel in the needle tube are removed through centrifugation, the hydrogel is installed in a 3D printing platform system, extrusion pressure is provided by an air pump, hydrogel ink is extruded through a 22G needle head, the movement speed and the extrusion pressure are continuously adjusted, the extrusion air pressure is 1600, and the printing speed is 2.5. So that the hydrogel ink can be written uniformly on the substrate. On this basis, the hydrogel is printed on a diamond-shaped PCL stent, and then a PCL parallel structure is printed on the formed hydrogel-PCL composite structure by a 3D printing technique, which can induce the growth of nerve cells. Finally, the scaffold was immersed in a 0.1M calcium chloride solution for crosslinking for 30 minutes. Hydrogel stents may self-curl to form a catheter.

Experiment group 3: a parallel line printing model was constructed, the X-axis length was 15 cm, the y-axis length was 15 cm, the line spacing was 0.4 cm, the layer height was 0.18 cm, the accepted distance was 0.15 cm, the number of layers was 2, and the data was introduced into a 3D printer and printed using the 4D calcium ion responsive hydrogel prepared in experimental group 2 of example 1. The hydrogel is moved to a needle tube, bubbles of the hydrogel in the needle tube are removed through centrifugation, the hydrogel is installed in a 3D printing platform system, extrusion pressure is provided by an air pump, hydrogel ink is extruded through a 22G needle head, the movement speed and the extrusion pressure are continuously adjusted, the extrusion air pressure is 1600, and the printing speed is 2.5. So that the hydrogel ink can be written uniformly on the substrate. On this basis, the hydrogel is printed on a diamond-shaped PCL stent, and then a PCL parallel structure is printed on the formed hydrogel-PCL composite structure by a 3D printing technique, which can induce the growth of nerve cells. Finally, the scaffold was immersed in a 2M calcium chloride solution for crosslinking for 30 minutes. Hydrogel stents may self-curl to form a catheter.

In conclusion, the oxidized sodium alginate-carboxymethyl chitosan hydrogel with excellent biocompatibility is prepared, and the hydrogel is combined with a 4D printing technology, so that the responsiveness of the carboxymethyl chitosan hydrogel to divalent cations is utilized, and only the concentration of mild divalent calcium ions is required to be regulated. And the influence of the concentration change of divalent calcium ions on the cell growth is relatively small, and the printing structure of the fine structure with the same-ratio reduced mechanical property improvement can be obtained while the good biocompatibility and degradability of the hydrogel are maintained. In addition, self-healing of hydrogels is achieved using Schiff base-based self-healing that can rapidly form dynamic imine bonds between aldehyde groups and amino groups under mild conditions. In addition, no photosensitizer is needed to be introduced in the preparation process, the system is simple, the raw materials are rich, the cost is low, and the industrialization is facilitated, so that the preparation method is an ideal human tissue implant preparation material.

Claims

1. The preparation method of the 4D printable calcium ion response self-healing hydrogel is characterized by comprising the following steps of:

2. the method for preparing the 4D printable calcium ion response self-healing hydrogel according to claim 1, wherein the preparation process of the oxidized sodium alginate comprises the following steps:

3. The method for preparing a 4D printable calcium ion responsive self-healing hydrogel according to claim 1, wherein the concentration of the oxidized alginate solution is 60-80mg/mL, and the solvent is phosphate buffer solution with ph=7.0-7.3;

4. The method for preparing 4D printable calcium ion responsive self-healing hydrogel according to claim 1, wherein the carboxymethyl chitosan is O-carboxymethyl chitosan, and the phosphate buffer is duchenne's phosphate solution.

5. The method for preparing a 4D printable calcium ion responsive self-healing hydrogel according to claim 1, wherein the temperature during the cross-linking reaction is between 30 and 37 ℃ for 30 to 60 seconds.

6. The method of preparing a 4D printable calcium ion responsive self-healing hydrogel according to claim 1, further comprising the step of cross-linking and curing the 4D printable calcium ion responsive self-healing hydrogel at room temperature.

7. A 4D printable calcium ion responsive self-healing hydrogel, characterized in that the 4D printable calcium ion responsive self-healing hydrogel is prepared by the preparation method of any one of claims 1-6.

8. Use of a 4D printable calcium ion responsive self-healing hydrogel according to claim 7, wherein the 4D printable calcium ion responsive self-healing hydrogel achieves 4D printing by calcium ion response.

9. The use of a 4D printable calcium ion responsive self healing hydrogel according to claim 8, wherein the process of 4D printing comprises:

immersing the 3D printing piece into a divalent calcium ion solution to carry out crosslinking reaction and deformation, so as to realize 4D printing;

the divalent calcium ion concentration is 0.1-2M, and the soaking time is not more than 30min.

10. The use of a 4D printable calcium ion responsive self-healing hydrogel according to claim 8, wherein the 4D printable calcium ion responsive self-healing hydrogel is used to print tissue implants comprising nerve conduits and vascular microchannels.