Three-Dimensional Printed Multiresponsive Structures of Smart Hydrogel

Abstract

Hydrogels, as soft smart materials, have been widely used in the manufacturing of responsive biomedical structures. The manufacturing process of smart hydrogels is critical for their application; however, the production efficiency and precision of the extrusion-based 3D printing method for the fabrication of responsive structures of gelatin-based hydrogels have not been extensively studied. In this study, a gelatin-based shape memory hydrogel was designed and prepared with the addition of sodium alginate, tannic acid, and sodium iron ethylene diamine tetra acetic acid to improve its mechanical strength and photothermal properties. The printability of the hydrogel based on extrusion-based 3D printing was studied under different mixture ratios of hydrogel components. The appropriate printing parameters and formulas of the hydrogel were investigated to determine the structural strength of printing structures. Furthermore, the shape memory properties of printed structures under different printing conditions were studied. The biomedical applications of the shape memory behavior of printed structures were explored. The proposed hydrogels and manufacturing process allow the realization of 3D printed shape memory smart hydrogel structures and provide details and solutions for the design and integration of multifunctional hydrogels.

Introduction

In recent years, responsive hydrogel structures with a 3D structure have been widely used in the fields of tissue engineering, in vitro organs, soft robots, material transport, and artificial muscles.^1–5 The regional 3D structural characteristics of the smart hydrogel in space, by accepting external environmental stimuli and achieving specific dynamic changes, enable the hydrogel to adapt to dynamic environmental changes for applications such as flexible grippers and responsive manipulation.^6,7 Compared to traditional processing methods such as hydrogel masks, fixture fixation, and extrusion,^8,9 the hydrogel 3D printing method can balance manufacturing efficiency with structural accuracy and the ability to create complex structures, which will enable responsive hydrogel structures to be used in a wider range of applications. It is reported that 3D printed PNIPAAm hydrogels allow temperature-controlled shape changes.^10–12

However, the 3D printing of gelatin-based smart multiresponsive hydrogel structures with improved mechanical properties remains challenging. To complete the 3D printed structure of natural gelatin-based hydrogels, the excellent rheology and cross-linking properties of hydrogels and the continuous optimization of the printing parameters are required.^13–15

In addition, the above requirements also affect the printability of the hydrogel,¹⁶ printing efficiency, shape memory performance, and mechanical strength after cross-linking. Therefore, printable smart responsive hydrogels are required to overcome many problems, including poor rheological properties, low shape memory efficiency, and deficiency in mechanical strength. In addition, the printability, shape memory, and mechanical properties of conventional hydrogels after cross-linking do not fully satisfy the design of multiresponsive structures and the properties of conventional photothermal hydrogels do not support 3D printing, and are usually processed by coating and injection.^17–19

To achieve responsive functionality and biocompatibility of the hydrogel, gelatin was used as a precursor material. Gelatin is a biocompatible, biodegradable, and fully absorbable natural biopolymer.²⁰ Benefitting from a triple helix structure in the gelatin network that can dissociate at temperatures up to 35–37°C, this controls the transformation of the permanent and temporary shape of the hydrogel.²¹ In addition, gelatin-based hydrogels show mechanical properties and swelling capacity similar to soft tissues and provide excellent biocompatibility.²² The use of sodium alginate into gelatin can improve the mechanical strength of gelatin hydrogels, and regulate the rheological properties of the pre-printing solution.^10,23–25

In addition, the Ca²⁺ ion was used to create coordination interactions with sodium alginate molecular chains to form temporary cross-links.²⁶ The curing of the hydrogel is mainly due to ionic cross-linking by the combination of sodium alginate and divalent cations in the component. The biopolymer chain is composed of β-d-mannuronate (M) and α-l-guluronate (G) linked through 1-4-glycosidic bonds,²⁷ as shown in Figure 1a. In a solution of alginate, blocks of M monomers form weak junctions with divalent cations. However, the interactions between G monomer blocks and divalent cations form tightly held junctions.

FIG. 1.

Principle of hydrogel curing. (a) Chemical structures of G and M alginate subunits and their arrangement in the biopolymer chain; (b) egg-box model.

The mechanism consists of the coordination of divalent ions with four carboxyl groups to form an egg-box arrangement,^28,29 as shown in Figure 1b. However, conventional hydrogels, which are usually composed of a single network of a hydrophilic polymer, are soft, weak, and brittle.³⁰ To improve the structural strength of the hydrogel and the shape memory effect of the hydrogel, tannic acid (TA) was added to the hydrogel system as a phenolic cross-linking agent.³¹ This benefits from the high molecular weight of TA with a variety of phenolic functional groups, where the o-diphenyl hydroxyl group can cross-link with the hydroxyl and amino groups in the gelatin functional group to form strong hydrogen bonds and enhance the strength of the hydrogel structure.³²

However, too strong of an interaction will flocculate gelatin and TA into granularity, which is not conducive to hydrogel printing, while the electrostatic interaction force between sodium alginate and gelatin can optimize the flocculation condition between gelatin and TA.^9,33 To enable the hydrogel system to achieve a photothermal effect, traditional photothermal schemes usually consider the use of graphene oxide, carbonnanotubes, or carbon nanoparticles, but this does not allow hydrogels to consider biocompatibility. Therefore, Fe³⁺ ions were introduced into it for complexation with TA in this study.³⁴

The complexes produced by the complexation reaction act as good photothermal agents, enabling remote control of the composite hydrogel and the use of sodium iron ethylene diamine tetra acetic acid (sodium iron ethylene diamine tetra acetic acid [EDTA]) as an Fe³⁺ ion carrier to ensure the biocompatibility of the overall hydrogel system. However, although the composite hydrogel can theoretically solve the problems of improving the printability and mechanical properties of traditional hydrogel materials and poor shape memory efficiency, the key parameters of the composite hydrogel, such as the ratio concentration, printing parameters, mechanical strength, and shape memory effect, have not been systematically studied.

In this study, based on the basic principles of composite hydrogels, TA and sodium iron EDTA were introduced into the gelatin-sodium alginate network to investigate the rheological properties of different components of composite hydrogels to determine the appropriate component concentrations and to investigate the printability and mechanical strength of extrusion-based 3D printing with different printing parameters. The efficiency of shape memory recovery of hydrogel structures was also investigated for different printing parameters. This provides a new reference for the realization of shape memory hydrogel structures.

Materials and Methods

Ethical statement

This study was conducted with approval from the Biomedical Ethics Committee of Anhui Medical University (20210317), and was performed in accordance with established guidelines.

Hydrogel ink preparation

Then, 15% gelatin (99% Biotech grade; Shanghai Macklin Biochemical Co., Ltd.) was used for the experiments, heated in a water bath by stirring at 80°C and allowed to completely dissolve the gelatin. Subsequently, 3% sodium alginate (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) was added to the gelatin solution, heated and stirred until fully incorporated, and the hydrogel became sticky. 8% of TA (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) was added to the gelatin-sodium alginate. With the addition of TA, the hydrogel exhibits flocculent coalescence and appears white.

Continuous stirring was continued until the flocculent coalescence disappeared, and the hydrogel became a white soluble gel. Then, 3% sodium iron EDTA (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) was added to the soluble gels and stirred thoroughly. The white gels show a purple color with the addition of sodium iron EDTA. After complete mixing and blending, it forms a homogeneous soluble state. Then, the hydrogel ink was loaded into the syringe, and the temperature was allowed to drop and print.

Shear viscosity experiment

The rheological properties of the biomaterial ink were measured by a rheometer (DISCOVERY HR-2) equipped with a plate with a diameter of 60 mm and a truncation gap distance of 50 μm. To measure the viscosity, these inks were loaded with steady-rate sweeps within a shear rate range of 100–1000 s⁻¹.

Mechanical testing of the hydrogels

In this work, the tensile testing of the hydrogel was performed using a Universal Tensile Strength Tester (REGER GEM-10; Shenzhen REGER Instrument Co.). The sample was printed with a 10 × 30 mm structure under the same conditions and cross-linked by curing, and then the width, length, and thickness were measured with a micrometer. The load cell is 10 N, and the strain rate is 5 mm/min. Stress and strain were calculated according to the following equations:

$$\sigma \left(MPa\right)=\frac{P\left(N\right)}{w\left(mm\right)\times d\left(mm\right)}$$

$$\epsilon =\frac{l}{l_0}\times 100\%$$

where σ, ɛ, P, w, d, l, and l₀ represent the stress, strain, load, mat width, mat length, extension length, and gauge length, respectively. Here, the values of w and d are the initial dimensions, so the stress values reported are engineering stress. Young's modulus can be calculated from stress–strain curves.

Biocompatibility test

Human renal epithelial cells (293T) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The cells were seeded in polystyrene tissue culture dishes and incubated at 37°C in a 5% CO₂ cell incubator. The medium was changed daily, and cells were dissociated and passaged 1:3 every 4 days using trypsin-EDTA.

Before co-culture, print scaffolds were first immersed in 75% ethanol and completely sterilized by irradiation for 1 h under ultraviolet light, washed three times with phosphate-buffered saline, and incubated overnight in 24-well plates containing DMEM. The cell suspension was then seeded on the scaffolds at a volume of 2 × 10⁵ cells/scaffold in 1 mL DMEM (1 scaffold was placed in each well of the 24-well plate). Scaffolds for cell inoculation were incubated at 37°C, 5% CO₂, and the medium was changed every other day.

Cells were stained using the Calcein acetoxymethyl ester (AM)/propidium iodide (PI) kit according to the manufacturer's instructions and subjected to cell viability analysis. Calcein AM is provided by U.S. EVERBRIGHT (Suzhou, China). PI is provided by Dalian Meilun Biotechnology Co., Ltd. Calcein AM/PI staining was performed on the cells co-cultured with the scaffold on day 1, 3, and 5, and the cell status was observed by inverted fluorescence microscopy.

We examined the fluorescence images of the printed grid structure compared to three different regions of cells co-cultured for different days and calculated the number of live cells (green fluorescence) versus dead cells (red fluorescence) in each region using the Cell Counting Tool (ImageJ NIH). The survival rate of co-cultured cells was calculated as [live cells/(live cells + dead cells)*100%].

Print quality assessment

According to many experiments, for the purpose of quantitative assessment of the quality of printed filaments, we defined the dispersion rate of the filament as α. A term α was defined using the equation,

$$\alpha =\frac{W-d}{d}\times 100\%$$

where W is the filament width and d is the inner diameter of the nozzle. This parameter reflects the dispersion of the hydrogel during the printing process and the print quality.

Three-dimensional printer designer

Based on the proposed hydrogel composition and properties, a 3D printer was built in this study, which is based on a Cartesian coordinate system and driven by a linear stepper motor. The print syringe is fixed to the X-axis and connected at the piston to the I-DOT 2400 dispenser and the OTS-750 oil-free air compressor (Taizhou Otus Industrial & Trading Co., Ltd.) to provide stable extrusion air pressure to ensure that the print line can be kept stable, providing a maximum extrusion pressure of 30 psi. The print syringe is a 3 cc capacity dispensing syringe. A nozzle with an inner diameter of 0.26–0.51 mm can be used according to the resolution.

To ensure that the temperature and viscosity of the hydrogel ink rheology change with temperature, a heating sleeve and temperature control system are fixed outside the printing syringe, and a chiller and circulating water cooler are used to form a printing cold bed, which can ensure that the cold bed temperature is lower than 5°C and reduce the collapse and dispersion of hydrogel filaments during the printing process. The whole system is controlled by an Arduino Mega 2560 microcontroller and the improved open-source software Cura, as shown in Supplementary Figure S1.

Results and Discussion

The viscosity of the hydrogel affects the printability of the hydrogel. When the viscosity of the hydrogel is too high, it will not extrude smoothly at the nozzle and the nozzle will become clogged, resulting in problems with poor printing or intermittent printing.³⁵ When the viscosity of the hydrogel is low, the printed result will tend to distort and collapse. In this study, gradient concentrations of gelatin,^36–38 sodium alginate,^28,39,40 TA,^18,41 and sodium iron EDTA were set for the four components of the hydrogel by comparison with other articles. The specific component concentrations are shown in Table 1.

Table 1.

Hydrogel Ink Composition for Viscosity Test

Sample No.	Gelatin, %w/v	Sodium alginate, %w/v	TA, %w/v	Sodium iron EDTA, %w/v
1	10	3	8	3
2	15	3	8	3
3	20	3	8	3
4	15	3	5	3
5	15	3	11	3
6	15	5	8	3
7	15	8	8	3
8	15	3	8	5
9	15	3	8	8

EDTA, ethylene diamine tetra acetic acid; TA, tannic acid.

The experimental results show that the viscosity decreases significantly with the shear rate, indicating that the hydrogel has shear thinning properties, as shown in Supplementary Figure S2.

After many experiments, we found that 10% gelatin has poor shape fidelity and cannot be shaped during the printing process; 20% gelatin tends to clog the nozzle during the printing process. The concentration of sodium alginate at 3.5–10% can improve the resolution printing suitability and material structure stability, but will limit the cytocompatibility. To balance the biocompatibility and printing resolution, a 3% concentration of sodium alginate was chosen.²⁹ Considering the mechanical properties and cytotoxicity of the hydrogel, the addition of TA can enhance the mechanical properties of hydrogels, but an excessive TA concentration shows certain cytotoxicity.^18,41 In addition, too high a concentration of sodium iron EDTA will affect the self-healing properties of the hydrogel.³⁹

In particular, in this study, 15% gelatin, 3% sodium alginate, 8% TA, and 3% sodium iron EDTA composite hydrogels were used for printing. However, gelatin methacryloyl (GelMA), as a representative traditional hydrogel, usually has a low viscosity at room temperature or high temperature, which cannot meet the requirements of direct biological 3D printing.⁴²

The effect of printing parameters on the filament width and dispersion rate of hydrogels

The shear viscosity of the hydrogel exhibits shear thinning properties, which are more appropriate for hydrogel printing. If the applied stress is higher than its shear yield stress, it behaves like a liquid; otherwise, it behaves like a solid.⁴³

During material extrusion, the hydrogel material fluid is subjected to shear stress, resulting in the stretching of the hydrogel molecular chains in the nozzle, while at the exit of the nozzle, the disappearance of the shear stress and the return of the hydrogel molecular chains to their coiled state lead to the phenomenon of expansion,^43,44 and the hydrogel filament diameter was thicker than the inner diameter of the nozzle, as shown in Figure 2a and b. During the deposition process, the filament broadened upon deposition onto the platform due to gravity and the extrusion stress between the nozzle and platform, thereby creating filaments that were wider in width than in diameter, as shown in Figure 2c and d.

FIG. 2.

Extrusion expansion and deposition broadening of hydrogel. (a) Schematic of hydrogel filaments swelling during the printing process; (b) image of hydrogel filaments swelling during extrusion (scale bar: 1 mm); (c) schematic diagram of the broadening phenomenon during hydrogel filament deposition; (d) image of hydrogel filament broadening during deposition.

The width of the hydrogel filament affects the precision and resolution of the structure in the printing process. After extensive experiments, it was found that the degree of broadening during deposition was influenced by air pressure, printing speed, the distance from the nozzle to the substrate, printing temperature, and nozzle inner diameter.

The most direct factor affecting the filament width of printing is the air pressure, which exerts an extrusion force on the hydrogel.⁴⁵ When the air pressure exerts a force stronger than the static yield stress of the hydrogel, it pushes the hydrogel to extrude downward,⁴³ while affecting the shear stress and shear rate of the hydrogel inside the printing nozzle. To understand the changes in hydrogel mechanics during extrusion, we simulated the hydrogel shear stress and shear rate inside the nozzle for different air pressures.

Hydrogels are power-law fluids with shear thinning behavior. The hydrogel extrusion model under pressure is shown in the Figure 3a. The viscosity for the power-law fluid can be described by Refs. 13 and 46.

FIG. 3.

Simulation diagram of the effect of air pressure on hydrogel extrusion: (a) diagram of hydrogel extruded by air pressure, (b) curve fitting diagram of shear rate-viscosity of hydrogel, (c) simulation diagram of shear stress distribution of hydrogel in needle at air pressure of 5 psi, (d) simulation diagram of shear stress distribution of hydrogel in needle at air pressure of 15 psi, (e) simulation diagram of shear stress distribution of hydrogel in needle at air pressure of 25 psi, (f) comparison of experimental and simulated data on shear rate and shear stress of hydrogel in nozzle.

where n and m are the power-law index and power-law consistency coefficient, respectively $\dot{\gamma }$ is the shear rate, and $\eta$ is the viscosity for the power-law fluid. The m and n values of the applied hydrogel precursor solution were obtained by fitting the shear rate-viscosity curve of sample number 2, as shown in Figure 3b.

We performed finite element simulation of the process of hydrogel extrusion, and the inlet pressure was restricted to 5, 15, and 25 psi to obtain the flow rate and shear rate during the extrusion process. The results are shown in Supplementary Figure S3.

In addition, for a power-law fluid in a pipe, the shear stress (τ) is a function of the shear rate ($\dot{\gamma }$) as follows¹³:

The analysis is carried out by transferring Equation (2) into the finite element simulation as a limiting condition to obtain the distribution of the shear stress (τ), as shown in Figure 3c–e.

Furthermore, we extracted the shear rate and shear stress data from the simulation results for analytical plotting and compared them with the shear rate-shear stress curve obtained from rheological experiments. The comparison results are shown in Figure 3f. The results show that the simulation results are consistent with the experimental results. Since the viscosity of the hydrogel is negatively correlated with the shear stress and shear rate, if the air pressure for printing is higher, the viscosity of the hydrogel at the nozzle outlet will be lower, and the width of the extruded filament will be larger.

If the applied air pressure is too low, the shear stress and shear rate at the nozzle will be low, the concentration of the hydrogel at the nozzle outlet will be higher, and the width of the extruded hydrogel filament will be smaller. The simulation results fit the experimental data well (as shown in Fig. 3) and it support the conclusion that the pressure is critical for the printed filament, and only an appropriate air pressure can ensure that the filament is printed without interruption and excessive squeezing.

To obtain the most appropriate pressure, the influence of air pressure on the filament width and dispersion rate of the printing filament is further studied, and the result is shown in Figure 4. The nozzle used for printing has an inner diameter of 0.51 mm. When printed at an air pressure of 5 psi, the average width of the printed lines is 1.36 mm, the dispersion rate is 1.66, and it cannot print consistently at this stage. For air pressures up to 30 psi, the average filament width of the printed filament reaches 4.68 mm. This does not ensure good printing results and shape fidelity. Therefore, based on the experimental and simulation study, the appropriate printing air pressure is obtained and the range is 15–20 psi.

FIG. 4.

Influence of air pressure on the filament width and dispersion rate of printed filaments (d = 0.51 mm).

The printing speed of the hydrogel is the moving speed of the nozzle (v), which affects the extrusion distribution of the hydrogel per unit time, thus affecting the printed filament width diameter. Under constant air pressure, when the nozzle moves too slowly, the hydrogel ink accumulates. When the printing speed is too fast, the nozzle stretches the extruded filament, which makes the printed filament appear to have an uneven thickness. As the printing speed increases, the extruded filament is pulled further until it breaks. During the printing process, the filament width of the printed filament becomes thinner as the printing speed increases, as shown in Figure 5, and the dispersion rate of the printed filament decreases from 2.65 to 1.11. Considering the thickness of the printed filament and a reasonable range of filament widths, the appropriate printing speed in the experiment is 14–20 mm/s.

FIG. 5.

Influence of printing speed on the filament width and dispersion rate of printed filament (d = 0.51 mm and P = 15 psi).

The distance between the nozzle and substrate affects the filament width of the printing filament. Once a continuous filament is extruded, the print quality of filaments in terms of filament morphology, filament width, and print fidelity is influenced by the distance between the nozzle and substrate. When the distance between the nozzle and the substrate (D_h) was too short, below the height of the hydrogel filaments (H), the hydrogel filaments were overdeposited at the nozzle, which caused the filament width to be large and uncontrollable.

When the distance between the nozzle and the substrate reaches the appropriate distance, the hydrogel filaments change from extrusion to molding without delay, the morphology of the deposited filaments is well shaped, and the filament width is predictable and controllable. When the distance between the nozzle and the substrate is too high, the printing filament has a certain delay from extrusion to molding.⁴³ Under the effect of gravity and dragging, the filament is stretched, thinned, and even broken to form a droplet, as shown in Figure 6a. The relationship between the filament width of the printed filament and the distance from the nozzle to the substrate is shown in Figure 6b. When D_h was 0.1 mm, the hydrogel filaments were excessively deposited.

FIG. 6.

Relationship between the distance connecting the nozzle and the substrate and the print filament quality, (a) schematic of the distance between the nozzle and substrate affecting print quality; (b) the influence of the distance between the nozzle and substrate on the filament width and dispersion rate of the printed filament (d = 0.51, P = 15 psi, and v = 20 mm/s).

When D_h reaches 0.9 mm, discontinuous printing filaments appear for the first time. At D_h values higher than 1 mm, the printed hydrogel filaments become unstable, and printing discontinuities occur; when the D_h reaches 1.5 mm, the hydrogel filaments are completely unprintable and appear as dots of hydrogel accumulation. The width of the hydrogel filaments decreases with the distance from the nozzle to the substrate. When D_h <0.5 mm, right-angled hydrogel filaments were easier to print during printing. When D_h >0.9 mm, the corners of printed filaments were curved. The reason is that there was a lag time from extrusion of hydrogel filaments to adhesion on the substrate, the hydrogel filaments were relatively soft, and the sharp angle of printing was smooth. The appropriate distance between the nozzle and the substrate ranges from 0.3 to 0.5 mm.

Due to the gelatin contained in the hydrogel, the rheological properties of hydrogels vary with temperature.⁴⁵ Hydrogel rheology is related to the dispersion rate of printed filaments of hydrogels. As shown in Figure 7a, four temperatures were set for the experiment when printed. The filament width of the printed filament increases with temperature. The hydrogel at this time can no longer maintain the lines during printing. There is apparent collapse and spreading. Therefore, when hydrogels are used in applications of tissue engineering, due to 40°C as a nonfatal single-strike event for sublethal injuries, they can usually be repaired.⁴⁷ The printing temperature can be set in the range between 30°C and 40°C for tissue engineering printing, and in the realization of structural printing, considering the printing efficiency and printing effect, a better printing effect can be obtained when the printing temperature is 40°C.

FIG. 7.

The relationship between print temperature and nozzle on filament width. (a) Influence of printing temperature on filament width and dispersion rate (P = 15, psi v = 20 mm/s, d = 0.51 mm, and D_h = 0.3 mm). (b) Influence of inner diameter on filament width and dispersion rate (P = 15 psi, v = 20 mm/s, D_h = 0.3 mm, and T = 40°C).

The inner diameter of the nozzle directly affects the filament width of the printed filament, which in turn affects the accuracy of the printed structure. In this experiment, we used four inner diameters of nozzles (0.25, 0.34, 0.41, and 0.51 mm) and performed line printing under the same printing conditions. The experimental results show the influence of the inner diameter of the nozzle on the filament width and dispersion rate of printed filaments in Figure 7b. The width of the printed filament becomes thinner as the inner diameter of the nozzle becomes thinner. Different nozzles can be selected for printing according to the resolution requirements. This makes it possible to print smaller structures.

Printed structures and biocompatibility

Structural printing is the basis for transforming the model design into a printed solid. The printed filament fusion phenomena show that the filament width (D) becomes larger, and the pitch (D_L) gradually disappears, forming a flat structure, as shown in Figure 8a. A series of 30 × 30 mm structures was printed by four inner diameter nozzles (0.25, 0.34, 0.41, and 0.51 mm) at a distance of 1–3 mm, as shown in Supplementary Figure S4. The printing parameters were used as described above (P = 15 psi, v = 20 mm/s, D_h = 0.3 mm, and T = 40°C).

FIG. 8.

Hydrogel diffusion in printing. (a) Schematic of hydrogel filaments diffusing and merging due to the influence of gravity and surface tension; (b) corner diffusion at a 1 mm print distance with different inner diameters (0.25, 0.34, 0.41, and 0.51 mm) (scale bar: 10 mm); (c) corner diffusion length of the print structure with different nozzle inner diameters.

Through the printing process, it is found that under the action of gravity and surface tension, the hydrogel will accumulate in the corners, and the printing wire will spread and fuse. We printed a single-layer structure at a spacing of 1 mm with four nozzle inner diameters, as shown in Figure 8b. The corner accumulation effect of the hydrogel increases significantly with increasing nozzle inner diameter. The printed hydrogel filaments are completely fused when the nozzle inner diameter reaches 0.51 mm. Meanwhile, we compared the length of hydrogel diffusion at the corner, and the results are shown in Figure 8c. Hydrogel diffusion at the corners is minimal at an inner diameter of 0.25 mm. However, considering practical applications, an appropriate nozzle is used to guarantee the strength of printed scaffolds.

Based on the printed scaffold structure (10 mm width and 1 mm thickness), to evaluate the biocompatibility of printed hydrogel scaffolds, we co-cultured 293T cells with the printed scaffold and stained the cells with Calcein AM/PI, as shown in Supplementary Figure S5. The process of the biocompatibility test is demonstrated in the Supplementary Information. On the first, third, and fifth day of co-culture, green cells were viable cells and red cells were dead cells. With cell proliferation, the survival rate of cell was 72.5% on the first day, 85.27% on the fifth day, and 94.07% on the fifth day.

The results indicate that the scaffold printed based on this material has good biocompatibility. Since gelatin is a biocompatible, biodegradable, and fully absorbable natural biopolymer, the gelatin-based 3D printed smart structures could extend the biomedical applications of 3D printed multiresponsive smart hydrogel. While most of components have already been used in clinical, the proposed 3D printed hydrogel in this article bridges the gaps between the 3D printed smart hydrogels and clinical practice.

Hydrogel cross-linking strength

Hydrogel cross-link mainly relies on the exchange of Na⁺ ions in sodium alginate with divalent metal cations. The -COO group in sodium alginate is cross-linked by chelating reaction with metal cations, which causes the hydrogel precursor solution to transition from the sol state to the gel. CaCl₂ solution was used to provide Ca²⁺ as a cross-linking agent. The structural strength and sol-gel state of the hydrogel cross-linked with Ca²⁺ were studied. A 10 × 30 mm rectangular single-layer structure was printed, and the elastic modulus of the structure after cross-linking and curing was measured. We cross-linked the hydrogel structure with 2% CaCl₂ for 120, 180, and 240 s.

The result of measuring the elastic modulus is shown in Supplementary Figure S5a. The elastic modulus of the hydrogel and the cross-linking time are positively correlated. The main reason is that increasing the cross-linking time of Ca²⁺ promotes the production of calcium alginate, leading to a significant increase in the elastic modulus of the hydrogel.⁴⁸ The longer the cross-linking time is, the higher the cross-linking density of the hydrogel. The experiment revealed that the elastic modulus of the hydrogel could be regulated by changing the cross-linking time of Ca²⁺. In addition, we measured the elastic modulus of hydrogel structures cross-linked by 2%, 4%, and 6% Ca²⁺ concentrations for 120 s, and the measurement results are shown in Supplementary Figure S5b. The results showed that the elastic modulus of the hydrogel structure increased with increasing Ca²⁺ concentration.

In general, the elastic modulus of hydrogels can be considered to be dependent on the degree of cross-linking.⁴⁹ This indicates that the elastic modulus and mechanical properties of the hydrogel can be adjusted by cross-linking Ca²⁺ at different concentrations. Considering that the conventional GelMA hydrogel has a wide range of applications in experiments, we compared the elastic modulus of 10% GelMA hydrogel after curing with the elastic modulus of the hydrogel in this study. The results showed that the elastic modulus of the hydrogel in this study was three times that of the GelMA hydrogel, as shown in Supplementary Figure S6c and d.

Shape memory efficiency of the 3D printed hydrogel structure

Hydrogel uses gelatin as the precursor material, which is cured at low temperature and can maintain a solid state. At high temperature, the triple helix of gelatin depolymerizes and softens, keeping the temporary shape by applying an external force, and the triple helix of gelatin repolymerizes after the temperature decreases, thereby storing the applied stress.

When the temperature of the hydrogel increases again under the action of near-infrared (NIR) light, the triple helix structure of the gelatin is depolymerized again, the stored stress is released, and the previous shape is restored. The printing parameters affect the distribution of the hydrogel during the printing process, which in turn affects the shape memory efficiency of the printed structure after cross-linking and curing. The single-sided fan structure with a single layer was designed to explore the effect of 3D printing parameters on the shape memory efficiency of printed structures, as shown in Supplementary Figure S7.

The printing speed of the hydrogel affects the efficiency of the shape memory of the printed structure. During the hydrogel printing process, shear forces are applied to the extruded filaments at the nozzle as the nozzle moves. With other printing parameters consistent, the shear force exerted by the nozzle on the printed filament is related to the speed. The faster the nozzle moves, the greater the shear force it exerts. After curing, the stress is stored within the hydrogel filament. When the hydrogel is fixed to a temporary shape, under NIR irradiation, the hydrogel warms up due to the photothermal effect of the complex reaction of TA and Fe³⁺, the triple helix structure of the gelatin unspins, and the internal stresses of the hydrogel are released, exhibiting a hydrogel shape recovery effect.⁵⁰

Due to the fast movement of the nozzle, the corresponding internal stresses in the hydrogel structure are relatively high, corresponding to the higher efficiency of its shape memory recovery. Its shape recovery effect is shown in Figure 9a, and the experimental results show that the shape memory recovery efficiency of the hydrogel resulting from a printing speed of 20 mm/s is better compared with the hydrogel structure printed at a printing speed of 10 mm/s when the printing direction is 0°.

FIG. 9.

Hydrogel printing parameters affect shape memory efficiency. (a) Printing speed affects the shape memory efficiency; (b) distance between the hydrogel filaments affects the shape memory efficiency; (c) printing path affects the shape memory efficiency.

The distance between the hydrogel filaments affects the shape memory efficiency of the structure. In the experiment, we tested the angle change of shape recovery at the same time under the distance between the hydrogel filaments of 0.3 mm and 0.4 mm, as shown in Figure 9b. The results show that the recovery efficiency and speed of structure shape memory recovery at a distance of 0.4 mm are better than those at a distance of 0.3 mm. Compared to a 0.3 mm print distance, 0.4 mm requires fewer repeat prints and less total hydrogel content in the structure, for the same structure. Under the same NIR light irradiation conditions, it takes less time to increase the temperature to the glass transition temperature of the hydrogel.

To test the effect of the printing path on the shape memory efficiency of structures, two hydrogel printing model paths were designed, 0° and 90°, as shown in Figure 9c. In this study, 0° means that the print path of structures is in the 0° direction, and the same meaning applies for 90°. During the printing process, the other printing parameters were ensured consistently, and the shape memory efficiency of the 0° printing path and 90° printing path at the same time was tested.

The experimental results show that the angle recovery per unit time for structures in the 0° print path is better than 90°. This corresponds to the faster shape memory recovery efficiency with a 0° print path under the other printing conditions. The main reason for this is that the 0° print path is perpendicular to the folded traces. The direction of stress in the structure is the same as the direction of the printed filament. Due to the effect of internal stress, shape memory hydrogels require less external energy to achieve the same deformation.⁵¹ The phenomenon shows that the structure under the 0°path has a better shape memory efficiency than the structure under the 90° path.

Using composite hydrogels, we printed a double layer collapsible gripper that can grasp objects to demonstrate potential applications in the fabrication of responsive grippers. The hydrogel material used in this application is the same as the study material. As shown in Figure 10a and b, the main structure of the hydrogel is a four-point star structure, and the diameter of the four-corner structure plane is 3 cm. Using a water bath method, the hydrogel structure was heated to 50°C, and the gripper grabbed the object through the four-cornered planar structure.

FIG. 10.

Three-dimensional printed shape memory gripper that grabs and releases objects. (a) Three-dimensional modeling through CAD; (b) photos of 3D printed structures; (c) gripper grasps the object; (d) gripper relaxes under NIR light exposure; (e) 3D printed gripper to release objects (scale bar: 10 mm). NIR, near-infrared.

When the temperature was decreased to 5°C, the hydrogel gripper kept the objects wrapped, as shown in Figure 10c. This requires a hydrogel with excellent retention force. When the object needs to be released and the gripper is irradiated with NIR light, the temperature of the hydrogel gripper increases and the hydrogel gripper loosens, as shown in Figure 10d and e. This application demonstrates the possibility of extrusion-based 3D printing of multiresponsive structures for bionic gripper applications.

It is possible to print structures to enable material transport through extrusion-based 3D printing techniques. This study demonstrates a new use of shape memory multiresponsive structures to enable carrier motion in narrow pathways. We designed complex pathways and printed quatrefoil-shaped structures, as shown in Figure 11a. The quatrefoil structures have a planar diameter of 15 mm, as shown in Figure 11b. After printing, the surface of the structure was sprayed with magnetic particles (iron oxide [II, III], 300–500 nm; Shanghai Aladdin Bio-Chem Technology Co., Ltd.) and then cross-linked and cured by the dropwise addition of a 10% CaCl₂ solution. Once curing was complete, the structure was heated to 50°C using a water bath, with the four sides of the structure wrapped in particulate matter to simulate encapsulated drugs, and then the shape was fixed after cooling.

FIG. 11.

Three-dimensional printing structures for deliveries. (a) Three-dimensional modeling through CAD; (b) photos of 3D printed four-corner structure; (c) structure moves under the action of magnetic field; (d) the structure unfolds under NIR light (scale bar: 10 mm).

When folded, the structure can be placed into narrow complex pathways. The structure is controlled by an external magnetic field and is able to move the hydrogel structure to any position within the pathway without contact, as shown in Figure 11c. When the structure is moved to a specified position, the structure is irradiated with NIR light. The structure heats up and unfolds within the channel in response to NIR light irradiation and releases the encapsulated particulate matter, as shown in Figure 11d. This application demonstrates a new use of extrusion-based 3D printed multiresponsive structures for substance transport in narrow pathways, with new applications in drug transport.

Conclusion

In this article, a gelatin-based multiresponsive hydrogel was proposed, and the photothermal response and shape memory were combined under the condition of biocompatibility. Meanwhile, a series of experiments were carried out to study the printability of the hydrogel. The results show that the air pressure, the moving speed of the nozzle, the distance between the nozzle and the substrate, the printing temperature, and the inner diameter of the nozzle are all important factors affecting the hydrogel extrusion.

A set of appropriate printing process parameters is proposed, and the influence of air pressure on hydrogel extrusion is analyzed by finite element method. Second, we printed a series of scaffolds to analyze the diffusion and fusion performance of hydrogels in the process of structure printing. Moreover, the printed scaffolds were co-cultured with 293T cells, and the cell proliferation was not affected during the culture process, indicating that this material has good biocompatibility. In addition, the mechanical properties of hydrogels under different cross-linking conditions were analyzed and compared with GelMA hydrogels. The results showed that the mechanical properties of hydrogels in this study were stronger than GelMA hydrogels.

By comparing the influence of printing parameters on the efficiency of structural shape memory under different printing conditions, the reasons of the influence of printing parameters on the recovery effect of structural shape memory are analyzed, and the influence of printing parameters on the recovery effect of structural shape memory is studied. The hydrogel and its fabrication process proposed in this study provide a new solution for 3D printing of shape memory smart hydrogel structures for biomedical applications, which provides a new reference for the design and integration of multifunctional hydrogels.

Authors' Contributions

L.C.: writing—original draft (lead), conceptualization (lead), and data curation (lead). Q.T.: formal analysis (lead). Y.Z.: visualization (lead). X.C.: resources (lead). A.M.: validation (equal). J.S.: investigation (equal). S.W.: investigation (equal). F.N.: resources (equal). L.Z.: funding acquisition (equal). Y.D.: funding acquisition (equal). Q.G.: funding acquisition (equal). G.L.: writing—review and editing (lead), funding acquisition (lead), and supervision (lead). R.Y.: writing—review and editing (lead), funding acquisition (lead), and supervision (lead).

Author Disclosure Statement

The authors declare that there is no conflict of interest.

Funding Information

This work was supported by the National Natural Science Foundation of China (Nos. 61973003, 61603002, 82172893), the Outstanding Youth Research Project in Universities of Anhui Province (2022AH030077), the Key Program in the Youth Elite Support Plan in Universities of Anhui Province (gxyqZD2020012), the Basic and Clinical Collaborative Research Improvement Project of Anhui Medical University (2019xkjT017), the Research Foundation of Anhui Institute of Translational Medicine (2021zhyx-C27), the Scientific Research Project of Education Department of Anhui Province (YJS20210284), the Key Program of Natural Science Project of Educational Commission of Anhui Province (KJ2021A0248), Grants for Scientific Research of BSKY (XJ201919), and the Key Research and Development Projects of Anhui Province (2022e07020047).