# Shape-Morphing Photo-Crosslinked Alginate Hydrogels via Digital Light Processing 4D Printing

Michel Habib, Ariella Liberati, Naomi Nieswic, Jade Berthelot, Hichem Seriket, Frédéric Demoly, Audrey Tourrette, Tahmer Sharkawi, Sébastien Blanquer

► To cite this version:

Michel Habib, Ariella Liberati, Naomi Nieswic, Jade Berthelot, Hichem Seriket, et al. Shape-Morphing Photo-Crosslinked Alginate Hydrogels via Digital Light Processing 4D Printing. Applied Materials Today, 2026, 48 (103064), 10.1016/j.apmt.2025.103064. hal-05444082

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# Shape-Morphing Photo-Crosslinked Alginate Hydrogels
## via Digital Light Processing 4D Printing

Michel Habib<sup>a,b</sup>, Ariella Liberati<sup>a</sup>, Naomi Nieswic<sup>a</sup>, Jade Berthelot<sup>a</sup>, Hichem Seriket<sup>c</sup>, Frédéric Demoly<sup>c,d</sup>, Audrey Tourrette<sup>b</sup>, Tahmer Sharkawi<sup>a*</sup>, Sebastien Blanquer<sup>a*</sup>

*Corresponding authors: tahmer.sharkawi@umontpellier.fr; sebastien.blanquer@umontpellier.fr

### Keywords

4D printing, actuator, stereolithography, dual-crosslinking, smart devices, anisotropic swelling, biomaterials

### Abstract

The development of smart biomaterials capable of dynamic shape transformation is a key challenge in advancing 4D printing for biomedical applications. In particular, hydrogel systems must combine biocompatibility with precise control over volumetric changes to enable functionalities such as tissue scaffolds, responsive implants, or drug delivery devices. In this study, methacrylated alginate was prepared for 3D photo-crosslinked hydrogels using digital light processing (DLP). Hydrogels were fabricated with either uniform isotropic or heterogeneous anisotropic photo-crosslinking patterns. A secondary level of physical crosslinking was introduced through coordination of alginate guluronate blocks with Ca²⁺ ions, resulting in controlled hydrogel contraction. Volumetric changes, water uptake, and compression properties were first investigated on isotropic hydrogels with varying degrees of photo-crosslinking. Results showed that increasing the degree of photo-crosslinking restricted swelling in phosphate buffered saline (PBS) and contraction in calcium solution, due to reduced

<footnote><sup>a</sup> ICGM, University of Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote><sup>b</sup> CIRIMAT, Université Toulouse, Toulouse INP, CNRS, Université de Toulouse, Toulouse, France</footnote>

<footnote><sup>c</sup> ICB UMR 6303 CNRS, Université Marie et Louis Pasteur, UTBM, 90010 Belfort Cedex, France</footnote>

<footnote><sup>d</sup> Institut universitaire de France (IUF)</footnote>
mesh sizes within the hydrogel network. Furthermore, anisotropic photo-crosslinking enabled spatially programmed volumetric deformations, allowing tailored shape morphing upon swelling or contraction. This effect was demonstrated in 3D-printed films with gradient crosslinking, which exhibited bidirectional bending when alternately exposed to PBS and CaCl₂ solution. More complex deformations were achieved in tubular structures and porous cubes by designing specific crosslinking gradients. Finally, cytocompatibility assays performed on fibroblast NIH 3T3 cells confirmed the non-cytotoxicity of the 4D-printed devices, highlighting their potential for biomedical applications.

# 1. Introduction

4D printing of soft materials such as hydrogels represents an advanced fabrication approach that integrates the programmable shape deformation of 3D-printed structures under an external stimulus with the intrinsic swelling behavior of the hydrogel matrix.[1,2] Depending on the selected matrix, the external stimulus can vary from temperature, pH, ionic strength, light, or magnetic and electric fields. When exposed to such stimuli, 3D-printed hydrogels undergo time-dependent changes in shape or size, thereby introducing the ‘fourth-dimension’ in 4D printing technology.[3,4] This fabrication technique holds great promise for smart biomedical applications, including patient-specific implants, smart device or controlled drug delivery systems.[5–10] For such biomedical applications, the selection of a biocompatible material is crucial. Among the various candidates, alginate-based hydrogels have emerged as one of the most widely used biopolymers, particularly as cell substrate models, in tissue engineering, and other types of biomedical devices.[11]

Sodium alginate is a naturally occurring polysaccharide composed of statistic mannuronate (M) and guluronate (G) units. It has a remarkable ability to form a physically-crosslinked network between the G units in the presence of multivalent cations such as $Fe^{3+}$, $Cu^{2+}$, $Ca^{2+}$, and $Mn^{2+}$.[12] The physically crosslinked network can dissociate in physiological media due to sodium-calcium ion exchange. Moreover, the hydroxyl and carboxyl groups present on alginate backbone can be easily functionalized enabling the formation of a chemically crosslinked network.[13] Chemically crosslinked alginate is most commonly processed by photo-polymerization following alginate methacrylation, resulting in stable hydrogels suitable for biomedical applications.[14] Moreover, this chemical crosslinking process can be conveniently achieved during 3D printing using photo-polymerization techniques, allowing for the creation of more complex hydrogel architectures.
Photo-printing of hydrogels has already led to successful development of smart actuators capable of controlled and programmed shape deformations.[15,16] This manufacturing technique highlights the potential of smart hydrogels, paving the way for innovative applications of 4D printing in advanced biomedical devices.[14–16] For alginate 4D photo-printing, the most common reported technique in the literature is direct ink writing (DIW), where the resin is extruded through a needle and cured under UV layer by layer to obtain the 3D structure. Cao *et al.* developed alginate-based actuators with a gradient crosslinking using DIW.[20] The system consists of a bilayer film where each layer required a different formulation in order to obtain an anisotropic profile that leads to a shape change. Before the printing procedure, a complete rheological study was performed on the formulations of the bio-ink in order to adjust the printer parameters for high resolution printing. Similarly, Constante *et al.* fabricated circular bilayer films composed of a photo-crosslinked alginate as the first layer and a poly(caprolactone) as the second layer.[21] The two-step process produces self-folding thin films, with the diameter of the resulting tubes being controllable by adjusting calcium concentrations. In addition to bilayer systems, actuating mechanism can also be generated by an anisotropic swelling behavior across the surface of a single material. Lai *et al.* generated an anisotropic swelling degree in alginate films by modifying the number of layers printed followed by a drying step.[22] They controlled the shape change by modifying the pattern orientation of the different areas of the layer, while the films remained 2D structures. Another example of a self-folding 2D film based on alginate was reported by Kirillova *et al.*[23] Their understanding was that their crosslinking gradient resulted from the different light penetration between the top and the bottom of the film. Thus, the actuating mechanism originates only from light diffusion within the formulation.

However, all these examples of the literature focus on 2D alginate films capable of deformation produced through various manufacturing techniques, and reports of complex 3D hydrogel actuators are notably less common. Yet, the use of additive manufacturing techniques becomes particularly significant when leveraging their ability to create complex 3D structures.[24] Specifically, additive manufacturing by photo-polymerization methods, such as stereolithography (SLA) and DLP techniques, enable the convenient fabrication of complex 3D architectures with specific deformation capabilities.[25,26] Indeed, one of the advantages of DLP printing is the ability to spatially control the chemical photo-crosslinking density not only on simple film-like structures but especially on more complex 3D architectures.[27,28] By controlling the grayscale profile of each layer, the crosslinking profile and density can be tuned within different areas of the printed object, which translates into different shape deformations
upon swelling or contraction. Kuang *et al* introduced grayscale DLP (g-DLP) using grayscale light pattern and a two-stage curing ink to build functionally graded lattice structures with controlled buckling and deformation sequence.[29] Built on this strategy, Montgomery *et al* developed a methodology to enhance accuracy of g-DLP using pixel-level grayscale to control round features from sharp pixels, enabling the fabrication of functional and structural parts with smaller features and smoother faces.[30] Jiang *et al*, obtained various deformation profiles by designing a well-tuned crosslinking profile that induces site-selective deformation on polyethylene glycol diacrylate (PEG) based resin.[31] Similarly, Ji *et al* controlled the degree of bending of 2-hydroxyethyl methacrylate based hydrogels by modifying the photo-crosslinking time on a tubular structure.[32] Finally, adjusting the degree of photo-crosslinking through UV exposure is another strategy to induce actuator deformation. For example, Ren *et al* created a crosslinking gradient by varying the curing time in composites made from commercial resin and fumed silica,[33] while Odent *et al* explored anisotropy-encoded hydrogel actuators by manipulating the photo-crosslinking degree in PNiPAAm-based resin, among other factors.[34] However, this gradient approach remains underexplored and is not yet widely adopted for hydrogels. Therefore, this research aims to explore the remarkable potential of the DLP technique to create crosslinking gradients combined with the well-known ability of alginate hydrogels to form reversible interactions with multivalent cations, with the goal of enabling shape transformation through controlled swelling effects as illustrated in **Figure 1**. While our previous work was initially demonstrated on simple 2D films, this innovative dual-crosslinking approach lays the groundwork for the development of bio-sourced and biocompatible 4D-printed hydrogels. In the present work, various 3D chemically photo-crosslinked alginate hydrogels were produced using DLP printing. The mechanical properties as well as the volumetric changes of the hydrogels were evaluated for various degrees of crosslinking. 4D-printed structures exhibiting controlled shape deformation is achieved by managing the crosslinking gradient during the DLP curing process. Cytotoxicity assays on the dual crosslinked hydrogels were done on NIH-3T3 fibroblast cells as preliminary biocompatibility for potential use as 4D-printed smart biomedical devices.
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Schematic illustration of DLP 4D printing to obtain various crosslinking degrees and the resulting network before and after ionic stimulation.</figcaption></figure>

## 2. Materials and Methods

### 2.1. Materials

Low viscosity sodium alginate (LVA), methacrylic anhydride (MA), calcium chloride (CaCl₂), ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA 4Na⁺, 2H₂O), Orange-G, and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (France). Triethylamine (TEA) was purchased from Fisher Chemicals (France). Milli-Q water (conductivity = 18.2 mΩ.cm at 23 °C) was used during this study. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was synthesized as described by Majima *et al.*[35] All chemicals were used without further purification unless mentioned otherwise. Culture media and additives were provided by Thermofisher (Gibco, France), Live/Dead cytotoxicity kit L3224 was purchased from Thermo Fisher (Invitrogen™, France), and NIH-3T3 CRL-1658TM cells were provided by ATCC (France).

### 2.2. Methods

#### Resin Formulation and 3D Printing

All structures were printed by a DLP printer (Max X27, Asiga Australia). The resins were prepared by dissolving the appropriate amounts of methacrylated alginate (Alg-MA) to obtain 6, 8, and 10% w/w aqueous solutions. The alginate methacrylation is described in our previous work and reported in supplementary information (see ¹H NMR in **Figure S1**).[13] LAP was added at 3.75% w/w with respect to the alginate and Orange G at 0.2% w/v as photo-absorber.
Orange G was added in order to control the UV light penetration for high resolution printing. UV intensity for DLP was fixed at 10 mW/cm² (after optimization **Figure S2**) and the printing layer thickness was 100 µm for all samples.

### Uniform Printing Conditions

The swelling behavior, volumetric change and the mechanical properties were tested on 3D-printed cylinders (8 mm diameter and 4 mm thickness). To obtain different degrees of photo-crosslinking, the printing time of the individual 100 µm layer was chosen through the working curve (**Figure S2**) in order to optimize the UV penetration depths. The crosslinking time per 100 µm layer of each formulation is summarized in **Table 1**. Upon printing, the hydrogels were washed in PBS.

### Gradient Printing Conditions

To obtain an anisotropic swelling behavior, a crosslinking gradient is implemented by varying the crosslinking time over the thickness of the designs generated by the Rhinoceros 3D software. To maintain a proper resolution, the gradient starts with the over-crosslinked section and decreases over the thickness when applicable. The details of each gradient section are provided in the discussion for the films, porous cubes, and cylinder designs.

### Gel Content Measurements

Gel content is calculated as the ratio of the dry mass of the hydrogel after washing and the total amount of polymers present in the hydrogel upon curing (resin concentration times volume of design). As seen, by increasing the degree of photo-crosslinking, the gel content increases for all formulations due to a higher efficiency in the crosslinking.

### Cyclic Swelling Studies

The equilibrium cyclic swelling degrees were measured starting from the washed hydrogels. The cycles comprised of the following sequences: (1) 5% w/v aq. CaCl₂ solution, and (2) PBS to mimic physiological conditions. For each measurement, the hydrogel was left to swell in the specific media for 24 h at 25 °C. When submerged in PBS, the solution was often changed to rinse out the Ca²⁺ ions properly. The swelling degree was calculated using Equation 1. The values are represented as the mean of triplicate sets.

$$Q = \frac{m_{swollen at time t}}{m_{dry}} \qquad (1)$$
where $m_{swollen at time t}$ denotes the mass of the sample at each measured time point and $m_{dry}$ the mass of the sample after it has been completely dried.

## Expansion Coefficient measurements

The volumetric change of the hydrogels was assessed by evaluating the dimensional change in different medias (PBS and CaCl₂ solutions). The expansion coefficient is calculated as the ratio of the dimensions of the swollen hydrogels and the dimensions of the reference design (Equations 2 and 3).

$$ \alpha_{xy} = \frac{\text{diameter at swollen state}}{\text{diameter of STL design}} \qquad (2) $$

$$ \alpha_z = \frac{\text{thickness at swollen state}}{\text{thickness of STL design}} \qquad (3) $$

where $\alpha_{xy}$ and $\alpha_z$ represents the expansion coefficient in the xy-axes of the cylindrical sample and z-axis, respectively.

## Compression Testing

Compression tests on the 3D-printed cylindrical samples (8 x 4 mm) swollen hydrogels were performed on an Instron 3366L5885 mechanical tester equipped with a 100 N load cell. Hydrogels were taken at different points of the cycling swelling and the compression tests were done after measuring the dimensions. The compression speed was set at 1 mm.s⁻¹ with 200 ms sampling time. Each value is represented by the mean ± SD (n = 3). The Young's modulus was calculated from the slope of the initial 3% deformation in the linear domain. Mesh size of the hydrogels (ξ) at different degrees of photo-crosslinking was calculated following the rubber elasticity theory (RET) assuming incompressible elasticity according to the following equation:

$$ \xi = \left( \frac{E R T}{3 N_A} \right)^{1/3} \qquad (4) $$

where E is the compression modulus, $N_A$ is the Avogadro constant (6.022 ×10²³), R is the gas constant (8.314 J/K mol) and T is the temperature (298 K).
## Photo-Rheology Analyses

Photo-rheology was done on a ThermoFisher HAAKE MARS 60 rheometer using 20 mm parallel circular plate-plate geometry. The measurements were obtained during 500 s irradiation under 0.5 W/cm² UV mercury lamp equipped with a 320-480 nm filter. Before testing, the samples were prepared to obtain 8% w/w Alg-MA solutions containing 3.75% w/w LAP and 0.2% w/v Orange G. The gap was fixed at 100 µm corresponding to the printing layer thickness and the temperature fixed at 20 °C. Results are obtained under a constant load of 0.1 N, strain of 1% and 1 Hz frequency to ensure the linear viscoelastic domain.

Mesh size (ξ) was calculated according to Flory-Rehner rubber elasticity theory:[36,37]

$$ \xi = \left( \frac{RT}{G'N_A} \right)^{1/3} \qquad (5) $$

where G' is the storage modulus, N_A is the Avogadro constant (6.022 ×10²³), R is the gas constant (8.314 J/K mol) and T is the temperature (298 K).

## Radius of Curvature Prediction

The radius of curvature of bisectional systems was predicted using the following simplified Timoshenko's equation[38]:

$$ \frac{1}{\rho} = \frac{24 (\Delta\alpha)(1 + m)^2}{h(3(1 + m)^2 + (1 + mn)(m^2 + \frac{1}{mn}))} \qquad (6) $$

Where ρ is the radius of curvature, Δα is the difference of xy expansion between the section X4 and X1, m is the ration between the thicknesses of section X4 to section X1, h is the total thickness of the sample and n the ration of Young's modulus of section X1 to section X2.

## Finite Element Simulation

Finite element (FE) simulations were performed using the commercial software Abaqus version 2023 ("Dassault Systèmes"). The computational model employed a well-established thermal analogy approach to simulate the coupled diffusion-deformation behavior of the alginate hydrogels.[39–41] This numerical strategy leverages the mathematical equivalence between the governing equations of mass diffusion and heat transfer, enabling the use of commercially available coupled temperature-displacement solvers to model chemo-mechanical phenomena in swelling hydrogels.[42,43] In this framework, the calcium ion concentration field c<sub>Ca2+</sub> was
mapped to a pseudo-temperature field T, while the experimentally measured volumetric
expansion coefficients (α<sub>xy</sub>) were implemented as coefficients of thermal expansion β in the
material constitutive model. Specifically, the swelling behavior in PBS solution (where c<sub>Ca<sup>2+</sup></sub> =
0 mM) was modeled with positive expansion coefficients derived from experimental
measurements: β<sub>X1</sub> = 0.27, corresponding to α<sub>xy</sub> = 1.27, and β<sub>X4</sub> = 0.11, corresponding to α<sub>xy</sub> =
1.11. The shrinking behavior in CaCl<sub>2</sub> solution (c<sub>Ca<sup>2+</sup></sub> = 50 mM, 5% w/w) was represented by
negative coefficients: β<sub>X1</sub> = -0.30 (estimated from Ca<sup>2+</sup> complexation behavior) and β<sub>X4</sub> = -0.15,
consistent with the differential shrinking trends observed experimentally and the calcium
content measurements showing 2.7% w/w for X1 and 1.7% w/w for X4 in the dry state. The
temperature-dependent elastic moduli were calibrated to match the compression test results
obtained in PBS and Ca<sup>2+</sup> environments: E<sub>X1</sub> ranging from 10 kPa (PBS) to 50-60 kPa (Ca<sup>2+</sup>),
and E<sub>X4</sub> from 60 kPa (PBS) to 70 kPa (Ca<sup>2+</sup>), as reported in **Figure 3**. The bilayer film geometry
(33 × 22 × 1 mm for the rectangular design) was discretized using 8-node coupled temperature-
displacement brick elements (C3D8T) with a mesh size of 1 mm, resulting in approximately
1,452 elements. The diffusion coefficients for Ca<sup>2+</sup> transport were approximated through
thermal conductivity parameters, accounting for the mesh size differences between X1 (22,100
g/mol in PBS, decreasing to ~5,500 g/mol in Ca<sup>2+</sup>) and X4 (7,000 g/mol in PBS) as calculated
using Flory-Rehner theory. Self-contact with a friction coefficient of 0.1 was included to handle
the large deformations during rolling. The simulation employed two sequential steps: (i) PBS
swelling inducing initial rolling toward the X4 side, and (ii) CaCl<sub>2</sub> exposure triggering reverse
rolling toward the X1 side. This thermal analogy approach has been extensively validated for
hydrogel systems [44–46] and offers significant computational advantages over direct
implementation of poroelastic or triphasic theories, while maintaining thermodynamic
consistency.[42]

Titration Analyses

The calcium content in the hydrogels, submerged in 5% w/w CaCl₂ solution, was quantified by colorimetric titration with EDTA in presence of Eriochrome Black T. Briefly, hydrogels photo-crosslinked at X1, X2, X3, and X4 were left in CaCl₂ solution for 24 hours then placed in 10 mL PBS to release the calcium for 24 hours. The hydrogels were then removed (and dried) and the appropriate amount of PBS was added to compensate the quantity absorbed by the hydrogels. The pH of the solutions was adjusted to 11 with 1 M NaOH. 1 mL of the solution was titrated with 0.01 M EDTA solution. Equilibrium point is reached upon color change from dark blue to dark red.
## Cytotoxicity Assays

Cytotoxicity assays were performed on extracts from printed hydrogel actuators swollen in PBS and CaCl₂ solution according to the ISO 10993 standard. The hydrogel samples were sterilized in ethanol for 24 hours and then left to swell in filter sterilized PBS or CaCl₂. Extracts were prepared by incubating the hydrogel samples in a complete cell culture medium (Dulbecco's Modified Eagle's medium 4.5 g L⁻¹ D-glucose supplemented with 1 v/v% L-glutamine, 10 v/v% fetal bovine serum, and 1 v/v% penicillin / streptomycin) at a hydrogel concentration of 0.1 mg/mL for 24 hours at 37 °C. Hydrogel samples were studied once immersed in PBS or in 5% w/w CaCl₂.

NIH-3T3 fibroblast cells were maintained in complete cell culture media at 37 °C in a humidified atmosphere with 5 % CO₂. For the assay, cells were seeded into 12-well plates at a density of 10⁵ cells/well and allowed to adhere for 24 hours.

After 24 hours of seeding, the culture media was replaced with 2 mL of hydrogel extracts. Control group includes cells treated with fresh culture medium (negative control) and cells treated with 70 % ethanol (positive control). After 24 hours of incubation at 37 °C with the extracts, cells were washed with PBS, detached using trypsin-EDTA, resuspended in PBS and stained using Live/Dead kit for 20 min at room temperature. Live/Dead assays were analyzed using a flow cytometer (FACS AccurciC6+ with Ex 488 nm; emission filters: 530/30 nm for live cells, and 610/20 nm for dead cells). The results were presented as mean ± standard deviation of three independent experiments.

## Statistical Analysis

All data are represented as the mean ± standard deviation unless otherwise specified. Statistical significance was assessed using two-tailed t-test assuming unequal variances. The t-test was conducted using Microsoft Excel (2021) with a significance threshold at p < 0.5.

# 3. Results and Discussion

## 3.1. Study of the Photo-Crosslinked Alginate Hydrogel

Alginate hydrogels are commonly formed through physical crosslinking with multivalent cations such as Ca²⁺.[47] However, these physically crosslinked hydrogel systems exhibit low stability in the physiological environment, where Ca²⁺ decomplexation occurs through exchange with Na⁺ or other monovalent ions, thereby limiting their applicability. To overcome this limitation, chemical crosslinking of the alginate macromolecules produces more stable gels
that can further be ionically crosslinked to tune certain properties of the hydrogels. By grafting methacrylate moieties onto the hydroxyl groups, a photo-crosslinkable resin can be produced, while the carboxyl groups remain available for complexation with Ca²⁺ ions.

In our research, we used sodium alginate with a molecular weight of 55,000 g.mol⁻¹, as this choice balances mechanical properties and viscosity effectively. Higher molecular weight alginates, such as those with molecular weights of 172,000 g.mol⁻¹ and 320,000 g.mol⁻¹, exhibited weaker mechanical properties and were unsuitable for increased concentrations due to their high viscosity, which limits the 3D DLP printing.[13] The degree of methacrylation achieved was 140% relative to a sugar moiety of alginate as determined by ¹H NMR (Figure S1), ensuring both robust mechanical properties and high printing resolution. Based on these studies, the 8% w/w photo-crosslinked alginate hydrogels were the easiest to formulate, provided the best photo-printing conditions, and exhibited the most promising volumetric changes. We therefore focus the investigation on this hydrogel concentration. To obtain various degrees of photo-crosslinking (Xn), the curing time per 100 µm layer are summarized in Table 1 after optimizing the printing conditions (Figure S2).

<table><caption>**Table 1.** Irradiation time of the 100 µm printing layer to obtain different degrees of photo-crosslinking (Xn) for 8% w/w Alg-MA.</caption><thead><tr><th rowspan="2">Degree of photo-crosslinking (Xn)</th><th colspan="4">Curing time</th></tr><tr><th>X1</th><th>X2</th><th>X3</th><th>X4</th></tr></thead><tbody><tr><td>8% w/w</td><td>8 s</td><td>15 s</td><td>25 s</td><td>40 s</td></tr></tbody></table>

## 3.2. Physical Properties of the Photo-Crosslinked Hydrogel and Impact on the Dual Crosslinking.

The influence of the printing conditions was investigated through swelling measurements and the volumetric change with respect to the designed dimensions of the 3D structures (cylinders with 8 mm of radius and 4 mm of thickness). In addition, such measurements were also performed to investigate the effect of the secondary crosslinking in the presence of Ca²⁺ ions.

As seen in Figure 2a, increasing the degree of crosslinking Xn gradually from X1 to X4, reduces the swelling ratio in PBS from Q = 31 to Q = 15, which is due to a densification of the network, thereby reducing the PBS uptake. This is confirmed by the increase in gel content,
which gradually rises from ≈ 60% for hydrogels crosslinked at X1 to ≈ 97% for hydrogels crosslinked at X4, implying a corresponding difference in network density and mesh sizes (Figure 2b). Moreover, it should be noted that X4 was also deliberately chosen as the maximum crosslinking level.

Mesh sizes have been calculated following Flory-Rehner theory (Equation S1) and later on using the rubber elasticity theory (Equation 4). Mesh sizes represent a number-average molecular weight of the linear chains between two network nodes (Mc). The results show a significant decrease in the mesh size when varying the crosslinking density, as it decreases from 22,100 g/mol for X1 to 7,000 for X4 (Table S1). Once immersed in Ca²⁺ solution, secondary ionic crosslinking occurs between the guluronate units, which tends to further densify the network, reduces the mesh size, and results in expulsion of water. Interestingly, the swelling ratio for all Xn is similar, with values close to Q ≈ 7. In consequence, the mesh sizes measured using Flory-Rehner theory are similar in the network generated by dual crosslinking and reach around 5,500 g/mol. This suggests that a maximum level of dual crosslinking is achieved and the swelling properties of the network reaches a threshold. This effect should be observable through changes in Ca²⁺ complexation regarding the Xn. Consequently, we measured the Ca²⁺ content of each dual crosslinked hydrogel using colorimetric titration. As shown in Figure 2d, the Ca²⁺ content in the dry state of the hydrogel is 2.7% w/w when photo-crosslinked at X1. This value gradually decreases to 1.7% w/w as the Xn increases to X4. The reduction in Ca²⁺ complexation in the hydrogels when the Xn increases can be explained by the progressively limited chain mobility, which hinders the ability of the different guluronate units to further complex Ca²⁺ ions. Moreover, SEM images (Figure S3) reveal a distinguished aspect of the hydrogel's pores between the two swollen states. In PBS, the network demonstrates a thin film like structures whereas upon dual crosslinking, the walls become denser similarly to what reported by Li et al. in dual crosslinked Agar/PAAc hydrogels.[48] Furthermore, it should be noted that the EDS analyses show calcium distribution throughout the hydrogel not homogeneous, with a higher concentration at the surface than in the core (Figure S4). This effect arises from surface network densification during dual crosslinking, which hinders diffusion toward the interior of the material, resulting in a gradient with more calcium at the surface.

This secondary ionic crosslinking is reversible and can be removed in PBS due to the Ca²⁺-Na⁺ ion exchange, which increases the swelling ratio to almost its initial state. This confirms that the dual crosslinking reaches a maximum limit, and the network cannot be further crosslinked,
regardless of the level of the chemical photo-crosslinking. Shrinking and swelling kinetics were recorded for all hydrogels (Figure S5). As we previously demonstrated, altering the crosslinking time does not affect the time required to reach equilibrium, because the swelling mechanism of dual crosslinking alginate hydrogels follows a non-Fickian diffusion.[13]

Besides studying water uptake, it is important to distinguish the properties of the generated network and the shape change aspect of the hydrogels upon shrinking in Ca²⁺ solution. Therefore, we also demonstrated that the volumes of the printed hydrogels show a difference in the deformation regarding the Xn. To quantify such volume deformation, the expansion coefficients in xy axes (α<sub>xy</sub>) (Figure 2c) and z axe (α<sub>z</sub>) (Figure S6) were measured for each crosslinking condition. Since α<sub>xy</sub> is later used in the actuator system to calculate the theoretical bending motion based on Timoshenko’s principle (Equation 6), we primarily focused on α<sub>xy</sub> to illustrate the volume transformation.[38] As expected, higher Xn lead to less shrinkage in PBS, with expansion coefficient α<sub>xy</sub> values ranging from 1.27 to 1.11 following the trend α(X4) < α(X3) < α(X2) < α(X1). This aligns with the idea that denser hydrogels have less chain mobility and therefore less potential for volume deformation. However, since the initial swelling ratio varies significantly depending on the degree of photo-crosslinking, while a threshold is reached once immersed in Ca²⁺ solution (Figure 2a), the resulting volume deformation after dual crosslinking differs notably. Therefore, upon exposure to a Ca²⁺ rich solution generating the secondary ionic crosslinking, the hydrogels shrink at different rates, resulting in varying final diameters (Figure 2a and Figure S7). These values correlate with the volumetric change and expansion coefficient trend, where in Ca²⁺ solution, hydrogels with higher Xn exhibit a lower shrinking behavior α(X1) < α(X2) < α(X3) < α(X4). The expansion coefficient in the z-axis α<sub>z</sub> was found to follow the same trend than α<sub>xy</sub> (Figure S6).

From a unique design, several volumetric changes can be obtained in the equilibrium swelling state in PBS and Ca²⁺ solution by simply tuning the printing process and varying the photo-crosslinking time. The fact that the volumetric change varies with Xn allows the design of controlled shape deformation, which is a very attractive feature, notably for building hydrogel structures with an anisotropic crosslinking profile.

Finally, the swelling behavior of X1 and X4 hydrogels over five swelling/shrinking cycles was monitored to assess their stability when alternately immersed in PBS and Ca²⁺ solutions (Figure S8). Both formulations exhibited stable and reproducible swelling in PBS, systematically returning to the same dimensions at each cycle. However, the shrinking behavior in Ca²⁺ gradually became less efficient, particularly for sample X4. This trend can be attributed to
progressive changes in Ca²⁺ diffusion kinetics during repeated cycling: although a 24 h equilibration time was sufficient for the first cycle, polymer-chain rearrangements occurring upon cycling likely alter the effective diffusion pathways, causing later cycles to require longer times to reach true equilibrium. This effect is more pronounced in highly photopolymerized hydrogels, where reduced molecular mobility further slows equilibration. Consistent with this interpretation, similar behavior is also observed for X1, although only after a greater number of cycles. These results indicate that this fatigue-like effect must be considered depending on the intended application, particularly when several numbers of swelling/shrinking cycles is expected during use.

The study of the expansion coefficient of the hydrogels in different media provides crucial insight to engineer and design hydrogel 3D-actuators with programmed and predicted shape deformations by implementing a crosslinking gradient. In PBS, increasing the photo-crosslinking time will result in a lower expansion trend due to the higher photo-crosslinking density of the network. On the other hand, when submerged in Ca²⁺ solution, hydrogels at higher Xn show a lower shrinking behavior (Figure 2e). The denser the network becomes, the more hindered Ca²⁺ diffusion into the matrix will become which was verified by quantifying the Ca²⁺ by colorimetric titration.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2** (a) Swelling ratio respectively in PBS and Ca²⁺. (b) Gel content of the hydrogels obtained from the different alginate concentrations at various degrees of photo-crosslinking. (c) the expansion coefficient of Alg-MA hydrogels when swollen in PBS and Ca²⁺ solution. (d)</figcaption></figure>
Calcium weight percentage in the dry hydrogels as obtained from colorimetric titration for freeze dried hydrogels photo-crosslinked at X1, X2, X3, and X4 after being submerged in a 5% w/w Ca²⁺ solution and reswollen in PBS. (e) Schematic illustration of the evolution of the gel content, swelling behavior, and calcium content as the photo-crosslinking degree varies.

### 3.3. Mechanical Properties Studies Related to the Crosslink Density

The influence of varying Xn (from X1 to X4) as well as the impact of dual crosslinking on the mechanical properties of the 3D-printed structures were investigated. As seen in **Figure 3a**, gradually increasing the degree of photo-crosslinking from X1 to X4 results in higher stress at break in PBS, increasing from 20 to 75 kPa, while the deformation remains similar around 25% for all the Xn. The strength of the network is also depicted by the evolution of the Young's modulus, which increases significantly and gradually from 10 to 60 kPa as Xn increases. Therefore, the progression of mechanical properties of the printed hydrogels typically correlates with the Xn. As the network density increases, mechanical resistance and stiffness also increase. Consequently, mesh sizes have been calculated using the rubber elasticity theory (Equation 4). By increasing the Xn from X1 to X4, the mesh size decreases from 7.6 nm to 4.3 nm confirming the increased crosslinking density observed in measurements (**Table S1**).

However, by immersing the printed hydrogel in a Ca²⁺ solution inducing an additional physical crosslinking, the mechanical properties are significantly improved, which logically results from a denser network (**Figure 3b**). As a result, dual crosslinked hydrogels exhibit higher deformations at break, reaching approximatively around 70% for all the Xn. The dual crosslinked hydrogels also demonstrate improved stresses at break reaching up to 900 kPa compared to a maximum of 80 kPa when swollen in PBS. Nevertheless, the progression of Young's modulus provides valuable insight on the properties of the network. Specifically, Young's modulus in Ca²⁺ solution increases for all Xn compared to hydrogels in PBS. However, the ratio between Young's modulus in PBS and in Ca²⁺ solution decreases significantly as Xn increases. This suggests that the main crosslinking at X1 is the physical, which gradually diminishes until X4, where photo-chemical crosslinking becomes dominant. However, as shown by the swelling properties of the network, which reach a threshold under dual crosslinking, Young's modulus also tends to be relatively close, ranging between 50 and 70 kPa with a large standard deviation.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 3** a) Stress-strain curves for 3D printed cylinders 8% w/w Alg-MA at Xn depth for 100 µm layer printing process. b) the Young's modulus of hydrogels at Xn in PBS and Ca²⁺ solution calculated as the slope of the stress-strain curves at 3% deformation and the ratio E₂/E₁ of the Young's moduli in calcium to PBS. The values are represented as the mean ± SD (n=3). c) Storage and loss modulus of 8% w/w Alg-MA resin during crosslinking under UV light and the evolution of the mesh size of the network. Circles refer to the mesh size of the hydrogels as calculated by RET from compression testing (Table S3).</figcaption></figure>

Photo-rheology on the resin indicated a significant time range between the crossover of the storage modulus (*G*') and loss modulus (*G''*) and the equilibrium as shown in **Figure 3c**. This demonstrates the ability to tune the hydrogels mesh sizes by varying the photo-crosslinking time. Consequently, the exposure times determined initially using the working curve, align perfectly with the evolution of the mesh size, confirming the network differences between X1 (8 s), X2 (15 s), X3 (25 s) and X4 (40 s).
These values of irradiation time correlate perfectly with the evolution of the mesh size under UV irradiation (photo-printing) allowing us to position the hydrogels' $X_n$ on the photo-rheogram (Figure 3c). The mechanical properties of these hydrogels are directly tied to the curing process, with longer photo-crosslinking times resulting in stronger and stiffer materials. The addition of $Ca^{2+}$ ions further strengthen the hydrogels by creating additional physical crosslinking points. Understanding these mechanical properties is key to designing hydrogel actuators, as it helps predict and control the bending deformation.[49]

### 3.4. 4D-Printed Actuators Based on Alginate Hydrogel

The volumetric changes induced by varying environmental conditions of such hydrogels can be harnessed to achieve shape deformation in 3D-printed designs. This capability enables the development of 4D-printed alginate-based hydrogels. We previously demonstrated that the key feature is the progressive decrease of $\alpha_{xy}$ when increasing the $X_n$ from X1 to X4 in PBS and the progressive shrinking behavior in $Ca^{2+}$ solution. Therefore, the variation in swelling behavior due to photo-crosslink density, further enhanced by the introduction of $Ca^{2+}$, can be exploited to generate gradients within a single structure printed by DLP. Various structures have been designed and fabricated using DLP, and the results are discussed in this section.

We initially start with simple film structures, similar to the studies on hydrogel actuators,[50] by incorporating a bisectional gradient on films and varying surface geometries: (i) square surface (22 mm x 22 mm x 1 mm), (ii) rectangular surface (33 mm x 22 mm x 1 mm), with one side featuring a regular topographical mesh, to easily identify the correct side visible in response to the stimulation medium (Figure 4). The introduction of topography into these films also enabled the investigation of shape deformation effects on film actuators. This is particularly relevant for potential applications as medical devices, such as in wound healing, where the topography could be adapted to enhance tissue adhesion.[51,52] By combining the volumetric expansion coefficient with the compression modulus that were established earlier, shape deformation in a bisectional actuator design can be predicted using Timoshenko's equation.

Therefore, through the modulation of $X_n$ in a bisectional film system, a curvature will be induced towards the section with the lowest expansion coefficient. Each section of film represents half of the total thickness, with one half-section photo-crosslinked at X1, X2, or X3 (section of the film containing the topography in the case of patterned films) and the other half-section photo-crosslinked at X4, respectively, to control the radius of curvature according to
Timoshenko's principle (Equation 6).[53] The radius of curvature is directly proportional to the $\Delta\alpha_{xy}$; the maximum curvature can be obtained for the highest $\Delta\alpha_{xy}$, which corresponds to films with section 1 photo-crosslinked at X4 and section 2 photo-crosslinked at X1. By modulating the $Xn$ of section 2 for X2 or X3, various bending curvatures can be obtained as seen in **Figure. 4a** leading to radii of curvatures similar to the predicted values obtained from Timoshenko's equation ranging between 3.1 to 8.1 mm.

Another parameter which can be adjusted to control the degree of bending is the total thickness of the printed hydrogel. A supplementary rectangular bisectional film (X4/X1) was printed with a thickness of 2 mm. The printed hydrogel films swell anisotropically due to the implemented gradient, with X1 expanding more than X4. This differential behavior between the two sections causes the hydrogel to roll up. Both hydrogels with thickness of 1 mm (**Figure 4**) and 2 mm (**Figure S9**) exhibit strong curvature toward the more crosslinked section which has a lower $\alpha_{xy}$ in PBS (corresponding to the non-patterned side, in case of films with topography). The radii of curvature obtained are 6.3 mm and 3.1 mm for thicknesses of 2 mm and 1 mm, respectively. The values obtained corresponds to the theoretical values from Timoshenko's model demonstrating the viability of the model for 4D-printed alginate hydrogels. It is important to note that all the films behave similarly with a rolling motion of the actuators, and such independently to the geometries or the presence of topography. As seen in **Figure 4b-d**, rectangular and square actuators were printed with and without the mesh design as well as a circular film where the same rolling motion is observed.
<figure><img src="image_11.png" /><figcaption>**Figure 4** (a) 4D-printed bisectional hydrogel actuators (33 mm x 22 mm x 1 mm) with different gradient profiles swollen in PBS at equilibrium and the radii of curvature obtained upon deformation which correlates to Timoshenko's mathematical model. (b) Design and dimensions of the square and rectangular films with and without mesh and a circular film. (c) face, and (d) side views of the rolled actuators in PBS.</figcaption></figure>

Moreover, when the bisectional (33 mm x 22 mm, X1/X4) film is immersed in a Ca²⁺ rich solution, a reverse actuation is observed as predicted in **Figure 5a**. As shown and explained previously, hydrogels photo-crosslinked at X4 demonstrate a lower shrinking behavior than the one photo-crosslinked at X1, this forces the system to roll towards the X1 section (towards the patterned side for the structures with topographical mesh) as seen in **Figure 5b** and **Figure S9**, leading to curvatures of 4.9 mm and 12 mm for the hydrogel having 1 and 2 mm thickness respectively. Interestingly, an unexpected behavior was identified during the inverse rolling
transformation. The hydrogels not only reverse the rolling actuation from X1 to X4, which was
expected, but also exhibit an unexpected orthogonal curvature change in the xy plane of the film
(Figure 5c). The same orthogonal unfolding behavior was observed when switching the media
from Ca²⁺ solution to PBS as seen in **Figure 6** and **Figure S10**. Additionally, this behavior was
obtained independently from the geometries or patterned hydrogel films. Such behavior can be
clearly visualized in the movies (Supplementary Information SI-M1 and SI-M2). Initially
swollen in PBS, the hydrogel films roll towards the higher photo-crosslinked section (X4)
where the section photo-crosslinked at X1 is on the outside. This rolling motion generates a
stress profile with higher stresses in the curvature direction compared to the lateral direction.
Upon immersion in a Ca²⁺-rich solution, both sections of the hydrogel shrink simultaneously.
This shrinkage triggers an unfolding motion of the film, followed by an orthogonal refolding.
The actuator's motion begins at regions where the stress is minimal, allowing areas with high
stress to gradually relax. This sequence results in a dynamic bidirectional bending: the film first
unfolds in one direction due to the initial stress relief, and then refolds in a perpendicular
direction as the stresses redistribute. This complex motion showcases the actuator's ability to
respond adaptively to changes in its environment, optimizing its performance through the
controlled redistribution of stress.
<figure><img src="image_12.png" /><figcaption>**Figure 5.** (a) Comparison of FE simulation and physical shape morphing of the radius of curvature of 4D printed 33 mm x 22 mm x 1 mm bi-sectional actuators X1/X4 when swollen in PBS and in 5% w/w Ca²⁺ solution. (b) Timelapse unfolding of the bisection actuator when transferred from PBS to Ca²⁺ solution taken from Movie SI-M1, accompanied by FE simulations predicting the type of rolling. All scale bars: 5 mm.</figcaption></figure>

Additionally, to predict and validate the atypical transformation observed experimentally, we performed finite element (FE) simulations inspired by a well-established thermal-analogy computational model used to simulate the coupled diffusion–deformation behavior of alginate hydrogels. To verify the simulation accuracy, Timoshenko's analytical equation (Eq. 6) was implemented within the simulation code as a validation benchmark, calculating the theoretical radius of curvature based on the experimental material properties (Δα<sub>xy</sub>, elastic moduli E<sub>X1</sub> and
$E_{X4}$, layer thickness ratio *m*, and modulus ratio *n*). This built-in verification step confirmed excellent three-way agreement: $\rho_{Timoshenko} = 3.4$ mm (analytical prediction) $\approx \rho_{FE} \approx 3.4$ mm (FE simulation) $\approx \rho_{exp} = 3.1$ mm (experimental measurement) in PBS for the X1/X4 bisectional film, and similarly $\rho_{FE} \approx 5.5$ mm versus $\rho_{exp} = 4.9$ mm in CaCl₂ solution. These results validate the model's capability to capture the differential swelling-driven actuation mechanism and quantitatively predict the curvature radii for the bidirectional bending behavior. This built-in verification step confirmed excellent three-way agreement: $\rho_{Timoshenko} = 3.4$ mm (analytical prediction) $\approx \rho_{FE} \approx 3.4$ mm (FE simulation) $\approx \rho_{exp} = 3.1$ mm (experimental measurement) in PBS for the X1/X4 bisectional film, and similarly $\rho_{FE} \approx 5.5$ mm versus $\rho_{exp} = 4.9$ mm in CaCl₂ solution. These results validate the model's capability to capture the differential swelling-driven actuation mechanism and quantitatively predict the curvature radii for the bidirectional bending behavior.

However, it should be noted that the numerical model does not account for additional physical factors that are likely involved in the orthogonal axis inversion observed experimentally during the transition from PBS to Ca²⁺ solution, and vice versa (Figure 5c, Figure 6). Factors such as instability of intermediate equilibrium states during the unfolding-refolding transition, edge effects at the film boundaries creating asymmetric stress distributions, dynamic fluid-structure interactions in the aqueous medium, and friction between the hydrogel and the substrate during the complex rolling sequence may contribute to breaking the symmetry and driving the perpendicular axis reorientation observed experimentally. Therefore, future computational work incorporating these additional physics could provide a more comprehensive description of the complete axis inversion mechanism.
$CaCl_2 \rightarrow PBS$

<figure><img src="image_13.png" /><figcaption>**Figure 6.** Timelapse images of the rolling deformation of the 33 mm x 22 mm x 1 mm hydrogel actuator X1/X4 at 1 mm thickness when switching the media from $Ca^{2+}$ solution to PBS taken from Movie SI-M2, accompanied by FE simulations of the rolling effect over time in blue. All scale bars: 5 mm.</figcaption></figure>

As a second design, we investigated more volumetric 3D structures by designing a porous cube shown in **Figure S10a**. When subjected to homogeneous isotropic photo-crosslinking at X1, without any photo-crosslinking gradient, the cube exhibits a uniform swelling in PBS where its dimension increases by 27% from design and shrinks uniformly in $Ca^{2+}$ rich solution by 40% in size from its swollen state as observed in **Figure 7** according to the expansion coefficient previously determined.

In contrast, employing photo-crosslinking gradients results in a more sophisticated response. In this approach, the porous cube is photo-crosslinked with a gradient X4, X3, X2, and finally X1 across its compartments from bottom to top (**Figure 7**). This gradient introduces varying expansion coefficients ($\alpha_{xy}$) for each compartment. As a result, when immersed in PBS, the compartments of the porous cube will exhibit a gradient volumetric change according to the expansion coefficient $\alpha(X1) > \alpha(X2) > \alpha(X3) > \alpha(X4)$. This differential expansion transforms
the cubic structure into a trapezoidal shape, with the compartment photo-crosslinked at X1 (top) bulging outward more than the X4 compartment (bottom), as illustrated in **Figure 7**.

When this structure is subsequently submerged in a Ca²⁺ rich solution, secondary ionic crosslinking is introduced into the network. In this environment, the compartments shrink unequally according to the previously calculated expansion coefficients α(X1) < α(X2) < α(X3) < α(X4). This differential contraction reverses the initial observed trapezoidal deformation, demonstrating a reversible shape transformation. Consequently, the 4D-printed cube demonstrates a primary deformation in PBS and a reversible secondary deformation upon exposure to the Ca²⁺ rich solution. The varying Xn induce internal stresses that drive the structure to transition between the programmed shapes.

<figure><img src="image_14.png" /><figcaption>**Figure 7.** (a) Swelling behavior of a 3D-printed porous cube uniformly photo-crosslinked at X1 and with 4 compartments crosslinking profile (X1, X2, X3, and X4 from top to bottom), with the schematic illustration of the shape morphing of the hydrogels in blue. (b) Crosslinked network when swollen in PBS and 5% w/w Ca²⁺ solution. All scale bars: 1 cm.</figcaption></figure>
Another 3D structure, representing a tubular device, was designed and printed using the same 3D printing technique (Figure 8a, Figure S11b). When subjected to an isotropic crosslinking at X1, this uniform photo-crosslinking induces a homogeneous volume expansion in PBS and a subsequent contraction of approximately 40% upon immersion in a Ca²⁺ solution. The versatility of the material is further demonstrated through anisotropic crosslinking, wherein a gradual gradient from X1 to X4 generates a conical morphology upon swelling in PBS (Figure 8). This conical structure exhibits a reversible change in conicity when exposed to a Ca²⁺ rich solution, as observed in Figure 8b, showcasing the material's versatile controlled response to environmental stimulation.

Additionally, the potential for creating alternated deformations can be achieved through the strategic incorporation of photo-crosslinking compartments arranged in sequences such as X4-X1-X4 or X1-X4-X1. A triblock gradient X4-X1-X4 will result in a convex deformation due to the higher expansion of the middle part. Whereas the inverse deformation is obtained for X1-X4-X1 triblock gradient leading to a concave tubular structure. Furthermore, the deformation curvature switches direction upon submerging in Ca²⁺ solution. The X4-X1-X4 tubular structure tends to switch from convex to concave due to the lower expansion of X1 section than the X4 section while the X1-X4-X1 tubular structures tends to switch from concave to convex. This methodology enables the generation of complex printed architectures, highlighting the precision and versatility achievable through controlled crosslinking by DLP process. This approach enables a single design to achieve a range of controlled and dynamic shape deformations.

In hydrogel engineering, manipulating crosslinking profiles offers unprecedented control over shape deformations. This approach enables the realization of complex and dynamic transformations from a single 3D design. Such processing can only be obtained through additive manufacturing based on photo-polymerization.
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Figure. 8** (a) Photo-crosslinking gradient profiles implanted on a tubular structure and their deformations when swollen in (b) PBS and 5% w/w Ca²⁺ solution, accompanied by a 3D representation illustrating the anisotropic swelling in blue. All scale bars: 5 mm.</figcaption></figure>

**3.5. Cytotoxicity of the Dual Crosslinked Alginate Hydrogel**

Given the macroscopic size of the hydrogels and the specific Ca²⁺ concentration in the dual crosslinked network, it is important to evaluate the biocompatibility of the 4D-printed structures and determine whether the amount of released calcium could cause toxicity to the cells. Therefore, cytotoxicity was evaluated according to ISO-10993 on alginate actuators swollen in PBS and Ca²⁺ solutions. The dual crosslinked hydrogels are compared to printed hydrogels without Ca²⁺ using the bilayer actuator (photo-crosslinked at X1/X4). Live/Dead assays show that the cells remain viable in the medium containing the released Ca²⁺ that were complexed in the hydrogels, comparable to the positive control (Figure 9a). Quantification shows that NIH 3T3 fibroblast viability reaches 93% for the hydrogel swollen in PBS and decreases slightly, but not significantly, to 87% for the dual crosslinked actuator (Figure 9b). Moreover, as shown in the Figure 9c, the morphology of the cells does not change between the control medium and cells in contact of Ca²⁺, confirming the absence of toxicity.
Therefore, shape morphing alginate hydrogels hold great potential for environmental and biomedical applications [54,55], and more particularly, for applications where reversible, programmable deformation is needed. In biomedical contexts, the use of NIH 3T3 fibroblasts as a model cell system supports their prospective use as dynamic 3D scaffolds that could adapt geometry to guide tissue regeneration, modulate cell mechanical cues, or control exposure of cells to bioactive factors.

<figure><img src="image_17.png" /><figcaption>**Figure 9.** (a) NIH 3T3 cells as observed under an EVOS FL microscope after 24 hours incubation. (b) Extract cytotoxicity essays on hydrogel films swollen in PBS and Ca²⁺ solutions prior to extract (n=3) and (c) cells morphology after 24 hours incubation at 37 °C in the control</figcaption></figure>
group and with the extract. Red dotted line represents the 70 % threshold set by the European
standard. All scale bars: 400 µm.

**4. Conclusion**

Sodium alginate (Mw = 55,000 g.mol⁻¹) was functionalized with methacrylate units to obtain a photo-crosslinkable resin suitable for DLP 3D printing. Resin formulation and printing conditions were optimized to have a high resolution and a fast-printing time. By tuning the degree of photo-crosslinking of the 3D-printed structures, several dimensions at the equilibrium swollen state in PBS and Ca²⁺ solutions can be obtained from a single 3D design. It was observed that by increasing the degree of photo-crosslinking, the hydrogels swell in PBS and expand greater than the design where the hydrogels with the lowest degrees of photo-crosslinking have bigger dimensions. Moreover, it was observed that in Ca²⁺ solution the hydrogels shrink and by increasing the degree of photo-crosslinking, the shrinking behavior was reduced due to a lower calcium complexation quantified by colorimetric titration. By implementing a crosslinking gradient on a single architecture, for example on a hydrogel film, a bidirectional bending motion can be obtained between PBS and Ca²⁺ solutions. In addition, various precise and programmed transformations were obtained on porous cube and tubular structures by controlling the degree of photo-crosslinking profile. Lastly, the 4D-printed structures were observed to be non-cytotoxic for NIH 3T3 cells. This study confirms the potential of the alginate-based hydrogel to create 4D-printed structures suitable for biomedical applications.

**CRediT authorship contribution statement**

Michel Habib (Investigation, Methodology, Writing original draft), Ariella Liberati (Investigation, Methodology, Formal analysis), Naomi Nieswic (Methodology, Formal analysis), Jade Berthelot (Formal analysis), Frédéric Demoly (Validation, Writing-review editing), Audrey Tourrette (Project administration, Supervision, Validation, Writing-review editing), Tahmer Sharkawi (Conceptualization, Project administration, Supervision, Validation, Writing-review editing), Sebastien Blanquer (Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing-review editing)
**Declaration of competing interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Data availability**

Data will be made available on request.

**Acknowledgement**

This work has been supported by the Carnot Institute, "Institut Carnot Chimie Balard Cirimat ICCBC" and the National Research Agency (ANR) under the France 2030 program, "DIADEM Program" with grant number ANR-22-PEXD-0016 ("ARTEMIS").

**Supporting information**

The Supporting Information includes working curves for various formulations, gel fraction results, microscopic images of the hydrogels, swelling kinetics, diameter size evolution, mesh size calculations, and 4D printing actuation images. Additionally, it contains two videos showcasing the actuation behavior.

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