Intelligent hydrogel with active shrinkage function as well as preparation method and application of intelligent hydrogel

Abstract

The invention belongs to the technical field of 3D printing materials, and particularly discloses intelligent hydrogel with an active shrinkage function and a preparation method and application thereof.The preparation method comprises the following steps that S1, 3-acrylamidophenylboronic acid is added into deionized water, and a 3-acrylamidophenylboronic acid solution is obtained; s2, tannic acid is added into the 3-acrylamido phenylboronic acid solution, and a tannic acid 3-acrylamido phenylboronic acid solution is obtained; s3, phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate and guar gum are sequentially added into the tannic acid 3-acrylamide phenylboronic acid solution, and the intelligent hydrogel with the active shrinkage function is obtained. According to the intelligent hydrogel with the active contraction function as well as the preparation method and the application, the problems of insufficient forming precision and structural resolution in an existing extrusion type 3D printing technology are solved, and a new technical path is provided for in-vitro bionic construction of fine tissues such as capillary networks and nerve microcatheters.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of 3D printing materials, in particular to an intelligent hydrogel with active shrinkage function and a preparation method and application thereof. BACKGROUND

[0002] Tissue engineering, as the core field of regenerative medicine, aims to repair or reconstruct damaged tissues and organs by constructing three-dimensional functional substitutes with biological activity, and provides a new treatment strategy for major tissue defects that traditional medical methods cannot cope with. In this process, biological 3D printing has become a key platform technology to achieve this goal, which provides unprecedented possibilities for constructing structure-biomimetic and function-personalized tissue engineering constructs by precisely integrating cells, biomaterials and bioactive factors.

[0003] Micro-extrusion printing, due to its wide compatibility with biological materials and simple and reliable process operation, has become one of the most popular and practical technical paths for constructing tissue engineering scaffolds. One of the key prerequisites for the success of this technology is that the biological ink must have excellent direct writing ability, that is, it can quickly maintain its shape after extrusion, resist collapse and flow, so as to accurately maintain the designed fiber morphology and three-dimensional structure. However, this basic requirement has become a major challenge in the development of current biological inks: most natural hydrogels with good biocompatibility are difficult to meet the stringent rheological requirements of stable direct writing due to their inherent low viscosity, slow gelation kinetics and other characteristics.

[0004] Even if the ink with direct writing ability is used, the direct writing printing technology itself also has significant inherent limitations: the final molding resolution is largely limited by the physical minimum extrusion line width of the printer nozzle. When complex microphysiological environments within the biomimetic constructs need to be constructed, such as reproducing the intricate luminal structure of capillary networks or simulating the precise guidance path of nerve endings, these fine features at the micron or even sub-micron level, traditional direct writing printing is not up to the task. This "precision gap" has become a key bottleneck restricting the development of high-biomimetic and high-functional tissue manufacturing.

[0005] In the prior art, the mainstream technical path to improve the printing resolution mainly focuses on the extreme optimization of the printing device itself, and the most typical strategy is to use a micro nozzle with a smaller caliber. However, this scheme has derived a series of new technical problems in practice: the finer nozzle will cause the extrusion resistance to increase exponentially, and the shear stress applied to the bio-ink (especially the ink with high cell density) will increase sharply, thereby seriously damaging the cell activity and function; at the same time, the micron-level nozzle is easily blocked by clusters or cell aggregates in the material, resulting in frequent interruption of the printing process and reduced reliability. These side effects brought by the "device-centered" idea highlight the physical limit faced by simply relying on hardware improvement, and also pose a more stringent challenge to the direct writing ability of the bio-ink - the ink must be smoothly extruded in the extremely fine nozzle, and then instantaneously solidified.

[0006] Therefore, beyond the traditional "device-first" optimization idea, it is necessary to seek breakthrough from the material system itself, and to develop a new type of bio-ink that has excellent direct writing ability and high biological activity, does not depend on ultra-precise printing equipment, but can essentially improve the forming precision. This has become a crucial and urgently needed research direction for the development of biological 3D printing technology. Among them, how to design and prepare an ideal hydrogel material that meets all the above stringent requirements is undoubtedly the core challenge faced by this research direction. SUMMARY

[0007] The purpose of the present application is to provide a smart hydrogel with active shrinkage function and a preparation method and application, to solve the problem of insufficient forming precision and structural resolution in the existing extrusion type 3D printing technology, and to provide a new technical path for the in vitro biomimetic construction of fine tissues such as capillary networks and nerve microcatheters.

[0008] To achieve the above-mentioned purpose, the present application provides a preparation method of a smart hydrogel with active shrinkage function, comprising the following steps: S1, 3-acrylamidophenylboronic acid is added to deionized water and stirred until completely dissolved to obtain a 3-acrylamidophenylboronic acid solution; S2, tannic acid is added to the 3-acrylamidophenylboronic acid solution obtained in S1 and stirred until completely dissolved to obtain a tannic acid 3-acrylamidophenylboronic acid solution; S3, lithium phenyl-2,4,6-trimethylbenzoyl phosphinate and guar gum are sequentially added to the tannic acid 3-acrylamidophenylboronic acid solution obtained in S2, and stirred until uniformly mixed to obtain a smart hydrogel with active shrinkage function.

[0009] Preferably, in S1, the concentration of 3-acrylamidophenylboronic acid in the 3-acrylamidophenylboronic acid solution is 0.005-0.015 g / mL.

[0010] Preferably, in S1, the stirring is specifically as follows: at 50-55 and vortex stirring for 30-40 minutes.

[0011] Preferably, in S2, the concentration of tannic acid in the tannic acid 3-acrylamidophenylboronic acid solution is 0.01-0.03 g / mL.

[0012] Preferably, in S2, the stirring is specifically as follows: at 50-55 and vortex stirring for 10-15 minutes.

[0013] Preferably, S3 is specifically as follows: Lithium phenyl-2,4,6-trimethylbenzoylphosphinate of 3-4 mg / mL is added to the tannic acid 3-acrylamidophenylboronic acid solution, vortex stirring at room temperature for 5-10 minutes, then guar gum of 0.02-0.04 g / mL is added to the tannic acid 3-acrylamidophenylboronic acid solution, vortex stirring at room temperature for 20-25 minutes, and then centrifugation is performed at a speed of 1200-1500 rpm to obtain the intelligent hydrogel with active contraction function.

[0014] The guar gum and 3-acrylamidophenylboronic acid in the hydrogel form borate ester bonds, and when in contact with a neutral or acidic water environment, the dynamic covalent borate ester bonds are selectively hydrolyzed and broken, resulting in a decrease in the crosslinking degree of the polymer network; this process simultaneously triggers the entropic elastic recoil of the guar gum molecular chain and the strong hydrophobic interaction generated by the protonation of the phenylboronic acid group, and the synergistic effect of the two effects together generates a driving force directed to the interior of the network, thereby efficiently expelling the free water wrapped in the grid, and finally exhibiting rapid volume contraction on the macroscopic level.

[0015] The application further provides an intelligent hydrogel with active contraction function.

[0016] The application further provides an application of the intelligent hydrogel with active contraction function in printing of a tissue engineering scaffold.

[0017] Preferably, the application method is as follows: The intelligent hydrogel is loaded into a syringe, an extrusion type 3D printer is used to print according to preset printing parameters, after the printing is completed, light crosslinking solidification is performed by using ultraviolet light, and a printed scaffold is obtained; The printed scaffold is placed in a water environment, and a contracted printed scaffold with uniform size change and complete structure is obtained.

[0018] Preferably, the printing parameters are specifically as follows: Extrusion rate 2-4 mL / min, layer thickness 0.4-0.6 mm, printing speed 10-15 mm / s, skip speed 15-20 mm / s, filling density 8-15%, and filling mode is linear.

[0019] Preferably, the wavelength of the ultraviolet light is 405 nm, and the intensity is 5-20 mW / cm 2 .

[0020] Therefore, the application adopts the above-mentioned intelligent hydrogel with active shrinkage function, preparation method and application, and has the following beneficial effects: (1) In view of the core bottleneck that the existing extrusion printing causes insufficient printing structure resolution due to the size of the nozzle and the extrusion line width limitation, the application utilizes the unique feature of the hydrogel material that it spontaneously shrinks after forming, has excellent 3D printing performance, and the unique active shrinkage feature can improve the forming resolution of the extrusion printing structure, realize micro-scaffold processing far beyond the inherent precision of the nozzle, form a post-printing finishing effect, thereby significantly improving the fineness of the formed structure without replacing the precision nozzle, using the existing conventional diameter printing nozzle, conveniently manufacturing complex three-dimensional microstructures far beyond the inherent resolution limit, providing key technical support for constructing highly biomimetic capillary blood vessel networks, nerve conduits and other complex microstructures, thereby significantly enhancing the guidance and regulation ability of the tissue engineering scaffold on cell behavior.

[0021] (2) The active shrinkage behavior of the application is an internal, material-driven physical process, which does not rely on complex external energy stimulation (such as light, heat) or chemical treatment, nor does it rely on complex and precise peripheral equipment, and the preparation process is simple, low in cost and highly practical, greatly reducing the equipment cost and technical threshold of high-precision 3D printing, and greatly promoting the popularization and application of the technology in the field of tissue engineering research and regenerative medicine.

[0022] The technical solutions of the application will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the scanning electron microscope image of the intelligent hydrogel of embodiment one of the application, a kind of intelligent hydrogel with active shrinkage function and preparation method and application; Figure 2 is the shear rate result graph of the intelligent hydrogel of embodiment one of the application, a kind of intelligent hydrogel with active shrinkage function and preparation method and application; Figure 3 is the strain result graph of the intelligent hydrogel of embodiment one of the application, a kind of intelligent hydrogel with active shrinkage function and preparation method and application; Figure 4is a CCK-8 result chart of L-929 cell culture by the intelligent hydrogel extraction liquid of the intelligent hydrogel with active contraction function and the preparation method and application embodiment one of the present application; Figure 5 is a direct writing printing performance verification chart of the intelligent hydrogel with active contraction function and the preparation method and application embodiment one of the present application, wherein (a) is linear printing, (b) is surface printing, and (c) is bionic printing; Figure 6 is a comparison chart of the lattice structure of the printed scaffold before and after contraction of the intelligent hydrogel with active contraction function and the preparation method and application embodiment one of the present application, wherein (a) is the printed scaffold before soaking, and (b) is the printed scaffold after soaking for 4 days; Figure 7 is a contraction rate chart of the overall size and wire diameter of the lattice structure of the printed scaffold of the intelligent hydrogel with active contraction function and the preparation method and application embodiment one of the present application. DETAILED DESCRIPTION

[0024] The technical solutions of the present application are further described below through the drawings and embodiments.

[0025] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0026] Embodiment one An intelligent hydrogel (MTG) with active contraction function, and the preparation method is as follows: (1) Take a clean and dry centrifuge tube, and dissolve 0.1 g of 3-acrylamidophenylboronic acid (MPBA) in 10 ml of deionized water at a concentration of 1% (w / v) under vortex stirring at 50 for 30 minutes to obtain a 3-acrylamidophenylboronic acid solution.

[0027] (2) Add 0.2 g of tannic acid (TA) to the 3-acrylamidophenylboronic acid solution at a concentration of 2% (w / v) under vortex stirring at 50 for 10 minutes to obtain a tannic acid 3-acrylamidophenylboronic acid solution (MT).

[0028] (3) Add 0.04 g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 0.3 g of guar gum (GG) to the tannic acid 3-acrylamidophenylboronic acid solution (MT) in sequence under vortex stirring at room temperature, and stir for 5 minutes and 20 minutes, respectively, and then centrifuge at 1200 rpm to obtain the intelligent hydrogel (MTG) with active contraction function.

[0029] The application of the intelligent hydrogel (MTG) in the printing of tissue engineering scaffolds is as follows: The MTG is loaded into a 5ml syringe, and a printing needle with a diameter of 250 is selected.

[0030] A 30mm x 30mm cube is printed in a culture dish using an extrusion type 3D printer, and the printing parameters are as follows: extrusion rate: 4mL / min, layer thickness: 0.5mm, printing speed: 13mm / s, skip speed: 17mm / s, filling density: 10%, and filling mode: linear.

[0031] After printing, the printed scaffold is cured by light crosslinking using ultraviolet light (wavelength 405nm, intensity 5-20mW / cm 2 ), and the curing time is 2 minutes. The printed scaffold is shown in (a) of FIG. 1. Figure 5

[0032] The printed scaffold is immersed in deionized water to make it in a suspended state, and after 4 days, it is taken out, as shown in (b) of FIG. 1. Figure 5

[0033] Example Two An intelligent hydrogel (MTG) with active contraction function is prepared as follows: (1) A clean and dry centrifuge tube is taken, and 0.05g of 3-acrylamidophenylboronic acid (MPBA) is dissolved in 10ml of deionized water at a concentration of 0.5% (w / v) and stirred at 50 for 30 minutes to obtain a 3-acrylamidophenylboronic acid solution.

[0034] (2) 0.1g of tannic acid (TA) is added to the 3-acrylamidophenylboronic acid solution at a concentration of 1% (w / v) and stirred at 50 for 10 minutes to obtain a tannic acid 3-acrylamidophenylboronic acid solution (MT).

[0035] (3) 0.03g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 0.2g of guar gum (GG) are sequentially added to the tannic acid 3-acrylamidophenylboronic acid solution (MT), and stirred at room temperature for 5 minutes and 20 minutes, respectively, and then centrifuged at 1200rmp to obtain an intelligent hydrogel (MTG) with active contraction function.

[0036] Example Three An intelligent hydrogel (MTG) with active contraction function is prepared as follows: ​​(1) Take a clean and dry centrifuge tube, dissolve 0.15 g 3-acrylamidophenylboronic acid (MPBA) in 10 ml deionized water at a concentration of 1.5% (w / v) at 50 rpm vortex for 30 minutes to obtain a 3-acrylamidophenylboronic acid solution.

[0037] (2) Add 0.3 g tannic acid (TA) to the 3-acrylamidophenylboronic acid solution at a concentration of 3% (w / v) at 50 rpm vortex for 10 minutes to obtain a tannic acid 3-acrylamidophenylboronic acid solution (MT).

[0038] (3) Add 0.04 g lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 0.4 g guar gum (GG) to the tannic acid 3-acrylamidophenylboronic acid solution (MT) in sequence, vortex at room temperature, stir for 5 minutes and 20 minutes respectively, and then centrifuge at 1200 rpm to obtain an intelligent hydrogel with active contraction function (MTG).

[0039] Test test 1. Intelligent hydrogel structure analysis The intelligent hydrogel of Example 1 was scanned by electron microscope, and the scanning photograph is shown in Figure 1 . The hydrogel has a typical three-dimensional porous network structure, and the pores are interconnected to form open channels. This porous structure is the key basis for the excellent biological activity and functionality of the hydrogel of the present application. This unique microstructure has the following effects: (1) provides sufficient space for cell growth, proliferation and migration; (2) facilitates efficient penetration of nutrients and oxygen; (3) is suitable for use as a controllable drug release carrier.

[0040] The intelligent hydrogel of Example 1 was tested by rheological scanning, and the shear rate scanning results are shown in Figure 2 , and the strain scanning results are shown in Figure 3 . As can be seen from Figure 2 , MGT shows a significant shear thinning behavior, and its apparent viscosity decreases sharply with increasing shear rate, ensuring excellent extrudability. As can be seen from Figure 3 , the MGT ink shows a solid-like behavior with G'>G'' at rest (low strain region), thereby maintaining the structure to prevent collapse; while under printing shear (high strain region), its G' decreases and G'' rises and reverses, realizing the transition to a fluid-like state to ensure smooth extrusion.

[0041] 2. Biocompatibility test L-929 cells (mouse fibroblasts) were cultured in the extract of the smart hydrogel of Example One, and cell viability was recorded, with results as shown in Figure 4 Cell viability was maintained at a high level throughout the entire culture period, and no decrease in viability due to toxicity was observed. Specifically, from the first day to the third day, cell viability remained stable, and from the third day to the fifth day, cell viability showed a significant growth trend, indicating that the cells had well adapted to the environment; this strongly proves that the conditions not only ensure the basic survival of the cells, but also actively promote the healthy proliferation of the cells. This excellent biocompatibility is a key basis for the safe application of the material of the present application in the fields of tissue engineering, drug delivery, or biological manufacturing, etc.

[0042] 3. Evaluation of water gel direct writing printing The smart hydrogel (MGT) prepared in Example One was subjected to linear, planar, and biomimetic direct writing printing, and the printing method was consistent with the application method of Example One. The printing results are shown in Figure 5

[0043] As can be seen from (a) in Figure 5 , the lines printed linearly are continuous, uniform, and smooth in surface, without breakage or beading phenomenon, indicating that the ink has excellent extrusion stability and linear retention ability. As can be seen from (b) in Figure 5 , the planar printing can be seen to have accurate hydrogel filament tracks, regular stacking, good interlayer fusion, and no collapse, proving that the ink has precise forming accuracy and shape fidelity in the two-dimensional plane. As can be seen from (c) in Figure 5 , a complex three-dimensional biomimetic structure resembling a nose was successfully printed, with clear outline and distinct features, fully demonstrating the ability of the hydrogel ink to support complex overhanging structures in three-dimensional space, and excellent printability. In summary, the hydrogel ink exhibits excellent direct writing printing performance from one-dimensional lines to two-dimensional planes, and even complex three-dimensional structures.

[0044] 4. Analysis of printed scaffold lattice structure As can be seen from Figure 6 , the MGT ink exhibits excellent wet-state structural stability. After immersing the three-dimensional square lattice structure formed by printing in deionized water for 4 days, the morphology undergoes controllable changes. As can be seen by comparing the printed scaffold before and after immersion, the structure after immersion undergoes uniform shrinkage, but the regular macroscopic lattice shape and clear structure outline are well maintained, without distortion, collapse, or dissolution. This result shows that the structure constructed from the MGT ink of the present application can maintain a high degree of structural integrity in a water environment, and the size change is predictable and uniform, which provides a key guarantee for its long-term application in the biomedical field (such as tissue engineering scaffolds).

[0045] ​The shrinkage of the overall size of the lattice structure and the wire diameter was tracked for 96 hours, and the tracking results are shown in Figure 7

[0046] As can be seen from Figure 7 , the macroscopic overall length and the microscopic wire diameter of the structure will shrink, but the shrinkage rate of the macroscopic overall length is significantly lower than that of the wire diameter. This result shows that the overall size and structure shape of the printed structure can be well maintained during the curing process, and the shrinkage stress is mainly released through the densification of the wire at the microscopic scale, thereby effectively avoiding the severe deformation of the macroscopic structure and ensuring the high consistency of the formed structure with the design model.

[0047] Therefore, the present application adopts the above-mentioned intelligent hydrogel with active shrinkage function and its preparation method and application, solves the problem of insufficient forming precision and structure resolution in the existing extrusion type 3D printing technology, and provides a new technical path for the in vitro bionic construction of capillary network, nerve microcatheter and other fine tissues.

[0048] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application but not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the present application can still be modified or replaced by the equivalent, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.​

Claims

1. A method for preparing a smart hydrogel with active shrinkage function, characterized in that, The method comprises the following steps: S1, 3-acrylamidophenyl boronic acid is added to deionized water and stirred until completely dissolved to obtain a 3-acrylamidophenyl boronic acid solution; S2, tannic acid is added to the 3-acrylamidophenyl boronic acid solution obtained in S1 and stirred until completely dissolved to obtain a tannic acid 3-acrylamidophenyl boronic acid solution; S3, lithium phenyl-2,4,6-trimethylbenzoyl phosphinate and guar gum are sequentially added to the tannic acid 3-acrylamidophenyl boronic acid solution obtained in S2, and stirred until mixed uniformly to obtain an intelligent hydrogel with active contraction function.

2. The method for preparing a smart hydrogel with active shrinkage function according to claim 1, characterized in that, In S1, the concentration of 3-acrylamidophenyl boronic acid in the 3-acrylamidophenyl boronic acid solution is 0.005-0.015 g / mL.

3. The method for preparing a smart hydrogel with active shrinkage function according to claim 1, characterized in that, In S1, the stirring is specifically: at 50-55 stirring for 30-40 minutes.

4. The method for preparing a smart hydrogel with active shrinkage function according to claim 1, characterized in that, In S2, the concentration of tannic acid in the tannic acid 3-acrylamidophenyl boronic acid solution is 0.01-0.03 g / mL.

5. The method for preparing a smart hydrogel with active shrinkage function according to claim 1, characterized in that, In S2, the stirring is specifically: at 50-55 stirring for 10-15 minutes.

6. The method for preparing a smart hydrogel with active shrinkage function according to claim 1, characterized in that, S3 is specifically: 3-4 mg / mL of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate is added to the tannic acid 3-acrylamidophenyl boronic acid solution, vortexed and stirred at room temperature for 5-10 minutes, then 0.02-0.04 g / mL of guar gum is added to the tannic acid 3-acrylamidophenyl boronic acid solution, vortexed and stirred at room temperature for 20-25 minutes, and then centrifuged at a speed of 1200-1500 rpm to obtain an intelligent hydrogel with active contraction function.

7. An intelligent hydrogel prepared by the method of any one of claims 1-6.

8. Use of the smart hydrogel according to claim 7 in the printing of tissue engineering scaffolds, characterized by, The application method is as follows: The intelligent hydrogel is loaded into a syringe, an extrusion type 3D printer is used to print according to preset printing parameters, after printing, ultraviolet light is used for photocrosslinking curing to obtain a printed scaffold; The printed scaffold is placed in a water environment to obtain a contracted printed scaffold with uniform size change and complete structure.

9. Use according to claim 8, characterized in that, The printing parameters are specifically: The extrusion rate is 2-4 mL / min, the layer thickness is 0.4-0.6 mm, the printing speed is 10-15 mm / s, the skip speed is 15-20 mm / s, the filling density is 8-15%, and the filling mode is linear.

10. Use according to claim 8, characterized in that: The wavelength of the ultraviolet light is 405 nm, and the intensity is 5-20 mW / cm 2 .

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