**1. Introduction**

The development of modern robotics/sensors [1], micro/nanomechanics [2], tissue engineering [3], and drug delivery [4] introduces an urgent demand for smart micro- or nanostructured machines or robots [5]. However, conventional 3D-printed constructs have fallen short of expectations, mainly due to their bulky volume and inability to mimic the dynamic human tissues. Thereby, the shape-reconfigurable hydrogels emerge as a new scientific frontier for cancer treatment [6], wound healing [7], or biomimetic applications [8–15] acting similar to artificial muscles [11], grippers [12], actuators, active origami or machines [13], swellable scaffolds [14], organic electronics [15], microneedles [16,17], etc.

These smart hydrogel devices would ideally possess the embeddable volume and the reconfigurable morphology to be applied [9,10]. The composition of smart hydrogels re-defines the environment-to-hydrogel interactions, the controllability of responsive behavior [13], and the dynamic mechanical properties to a large extent. In the context of biomimetic applications, hydrogel-based materials have showcased certain advantages

**Citation:** Tao, Y.; Lu, C.; Deng, C.; Long, J.; Ren, Y.; Dai, Z.; Tong, Z.; Wang, X.; Meng, S.; Zhang, W.; et al. Four-Dimensional Stimuli-Responsive Hydrogels Micro-Structured via Femtosecond Laser Additive Manufacturing. *Micromachines* **2022**, *13*, 32. https://doi.org/10.3390/ mi13010032

Academic Editors: Jie Yin, Yang Liu and Ping Zhao

Received: 15 November 2021 Accepted: 22 December 2021 Published: 27 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

such as softness [14], biocompatibility and degradability [18], and massive potential for cell adhesion and proliferation.

Many additive manufacturing methods have been demonstrated with the four-dimensional (4D) time-dependent shape reconfiguration to obtain controllable stimuli-responsive behavior [19]. Four-dimensional printing has revolutionized traditional three-dimensional printing manufacturing products worldwide. In literature reviews, the state-of-the-artwork of 4D printing generally integrates the time-dependent behavior of stimulus-responsive materials [20], the associated materials interacting with various stimuli (physical, chemical, and even biological signals) for pre-designed motion or actuation [21,22]. The already-known 4D examples can be found from two-photon stereolithography [23] to 3D printing [24,25], extrusion fabrication, or ink-writing methods [26,27].

Among the existing macroscopic 4D products and fabrications with a relative millimeter or sub-millimeter resolution, the TPP using the femtosecond laser as a light source captures roaring attention. As is known, the primary-stage products of TPP are mostly stationary without controllable stimuli-inspired properties or artificial shape morphing. We recently reported a carbon nanotube-doped hydrogel with swelling-to-shrinkage behavior using two-photon polymerization (TPP) [15], where the swellable hydrogel scaffold absorbs functional materials for semiconductor applications. Its confinement of nonlinear two-photon absorption (TPA) within the submicron focal volume provided an ultra-fine spatial resolution [28]. Flexible laser parameters and a computer-aided technique [29] promised a tunable formation quality in fabricating quasi-arbitrary three-dimensional (3D) devices. In a nutshell, TPP will be a predominant tool [14,23,28] for fast fabricating core devices in interdisciplinary research due to its structural diversity and selective resolution.

Micro/nanostructures fabricated via TPP are formed through high-density covalent bonding networks. The solidified structures, generally, cannot undergo large transformations such as soft/elastomer materials. Recently, the smart properties of hydrogels have been improved. For example, the traditional actuation uses a residual stress-driven method for thermal shape deformation [30,31]. Some researchers use a unique laser writing process for pre-designed shape morphing [32], or a doping carbon nanotube to enhance the light-responsive behavior [33,34], or use a bio-environment to tune the optical properties [35,36]. Although having achieved tremendous progress [29–36], the stimuliresponsive behavior still deserves further investigation for complex high-freedom shape reconfiguration. The material limitation and monotonous laser scanning methods are now restraining this micro/nanoscale additive manufacturing from innovative development and broadband applications.

Herein, to obtain direct fabricating and temporally controlling micro/nanoscale quasi-arbitrary 3D geometries without filling other functional particles, we tentatively perform the modified TPP (Figure 1) on the bio-compatible hyaluronic acid methacryloyl (HAMA, Figure 1a) [7,17,23] with polyethylene glycol diacrylate [37] for responsiveness (Figure 1a). The shifted laser-scanning space creates a hetero-distribution of polymeric degrees through the formed hierarchical micro/nanostructures (Figure 1c), leading to a controllable surface tension mismatch. Following the programmable shape morphing, we display and identify several reconfigurable micro-scale structures by applying external stimuli for the first time. Shown by the cell loading experiment (Figures 1c, S1 and S2), the hydrogels allow fibril cells to crawl on the surface freely after one week, and the summarized cell viability exceeds 98%, implying the as-prepared hydrogels to be ready for cell culture [6,38] or tissue engineering.

**Figure 1.** The schematic procedure of TPP incorporating stimuli-responsive hydrogels: (**a**) the main components used in the photoresist containing the methacryloyl-modified hyaluronic acid, PEG-da, and acrylamide as structural or functional materials rendering the hydrogel with stimuli-responsive ability; (**b**) illustration of the laser beam focused on the interface between material and substrate, the laser beam assembles the discrete functional polymers into a polymerized matrix, and the twophoton absorption process; (**c**) description of the used hetero-scanning TPP strategy, where the laser-scanned path forms highly crosslinked nanowires, while the interconnecting spacing is a lowdegree crosslinked area, and the 3D view illustrates an interlocking morphology; (**d**) phase image, fluorescence image, and the merged images of typical grating structure created by our proposed method; (**e**) the fluorescence and images of loading fibril cells on the prepared square hydrogels for 2 weeks, where the active cells crawled on the surface of the structure after one week, indicating a desirable adhesion and bio-compatibility for cell culture. Interestingly, the optical double-frequency technique is adopted here for generating **Figure 1.** The schematic procedure of TPP incorporating stimuli-responsive hydrogels: (**a**) the main components used in the photoresist containing the methacryloyl-modified hyaluronic acid, PEG-da, and acrylamide as structural or functional materials rendering the hydrogel with stimuli-responsive ability; (**b**) illustration of the laser beam focused on the interface between material and substrate, the laser beam assembles the discrete functional polymers into a polymerized matrix, and the twophoton absorption process; (**c**) description of the used hetero-scanning TPP strategy, where the laser-scanned path forms highly crosslinked nanowires, while the interconnecting spacing is a lowdegree crosslinked area, and the 3D view illustrates an interlocking morphology; (**d**) phase image, fluorescence image, and the merged images of typical grating structure created by our proposed method; (**e**) the fluorescence and images of loading fibril cells on the prepared square hydrogels for 2 weeks, where the active cells crawled on the surface of the structure after one week, indicating a desirable adhesion and bio-compatibility for cell culture.

the 532 nm green femtosecond laser beam during TPP fabrication for the first time. The double-frequency crystal is inserted in the path to decrease the optical power and wavelength simultaneously. We also use a micro-mechanics platform to characterize the mechanical behaviors of the TPP-fabricated hydrogels. A surface tension analysis expounds the mechanics' theoretical rationales during the controllable shape morphing. **2. Materials and Methods** Interestingly, the optical double-frequency technique is adopted here for generating the 532 nm green femtosecond laser beam during TPP fabrication for the first time. The double-frequency crystal is inserted in the path to decrease the optical power and wavelength simultaneously. We also use a micro-mechanics platform to characterize the mechanical behaviors of the TPP-fabricated hydrogels. A surface tension analysis expounds the mechanics' theoretical rationales during the controllable shape morphing.

#### *2.1. Material Preparation* **2. Materials and Methods**

#### Hyaluronic acid methacryloyl (HAMA, 30 wt%), 2-hydroxy-2-methylpropiophenone *2.1. Material Preparation*

(2 wt%, molecular structure seen in Supplementary Materials, Figure S3) [39,40], acrylamide (10%, Figure 1c), and poly(2-ethyl-2-oxazoline) diacrylates (PEG-da 475, 55 wt%) were mixed in phosphate-buffered saline (PBS, 4.8 wt%) for the responsive photoresist. Then, the mixture was pre-processed under 30 min ultra-sonication for dispersion and then was magnetically stirred at 800 rpm for 8 h. We purchased HAMA from Aladdin (Shanghai, P.R.C.), and PBS from HyClone (Logan, UT, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were not purified before usage. The whole fabrication procedure (including development and applying external stimuli) Hyaluronic acid methacryloyl (HAMA, 30 wt%), 2-hydroxy-2-methylpropiophenone (2 wt%, molecular structure seen in Supplementary Materials, Figure S3) [39,40], acrylamide (10%, Figure 1c), and poly(2-ethyl-2-oxazoline) diacrylates (PEG-da 475, 55 wt%) were mixed in phosphate-buffered saline (PBS, 4.8 wt%) for the responsive photoresist. Then, the mixture was pre-processed under 30 min ultra-sonication for dispersion and then was magnetically stirred at 800 rpm for 8 h. We purchased HAMA from Aladdin (Shanghai, China), and PBS from HyClone (Logan, UT, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were not purified before usage. The

whole fabrication procedure (including development and applying external stimuli) was carried out without light illumination. In the following experimental section, a family of the photoresists in different weight ratios of two monomers were prepared in the same procedure. was carried out without light illumination. In the following experimental section, a family of the photoresists in different weight ratios of two monomers were prepared in the same procedure.

#### *2.2. Laser System and TPP Additive Manufacturing 2.2. Laser System and TPP Additive Manufacturing*

*Micromachines* **2022**, *13*, x FOR PEER REVIEW 4 of 13

A barium metaborate crystal (Ba(BO2)2) transformed the 1064 nm wavelength femtosecond laser beam from a Ti:Sapphire femtosecond laser (Chameleon-Discovery, Coherent, CA, USA) into a green 532 nm beam (Figure 2) based on the double-frequency effect. Optical power deceased to mW level in the optical path. An expander and an acoustic-optics modulator (AOM), a half-wave plate, a Glan mirror, and an aperture slot were placed in the optical path following our previous femtosecond laser direct writing system [41] (Figure 2a). The terminal biological microscope (IX83, Olympus, Tokyo, Japan) contained a two-dimensional nanometer-step moving platform, in situ charge-coupled device (CCD), and dichroic mirror, where three 20×, 40×, and 100× oil-immersed objectives were optional for different focusing lengths. The normal data slicing technique processed digitalized models such as 3D additive manufacturing [42,43]. The linearly polarized, sequential, near-infrared laser pulse propagated along the bottom-to-top path into the photon-sensitive photoresist without masks for 3D fabrication [44]. A barium metaborate crystal (Ba(BO2)2) transformed the 1064 nm wavelength femtosecond laser beam from a Ti:Sapphire femtosecond laser (Chameleon-Discovery, Coherent, CA, USA) into a green 532 nm beam (Figure 2) based on the double-frequency effect. Optical power deceased to mW level in the optical path. An expander and an acousticoptics modulator (AOM), a half-wave plate, a Glan mirror, and an aperture slot were placed in the optical path following our previous femtosecond laser direct writing system [41] (Figure 2a). The terminal biological microscope (IX83, Olympus, Tokyo, Japan) contained a two-dimensional nanometer-step moving platform, in situ charge-coupled device (CCD), and dichroic mirror, where three 20×, 40×, and 100× oil-immersed objectives were optional for different focusing lengths. The normal data slicing technique processed digitalized models such as 3D additive manufacturing [42,43]. The linearly polarized, sequential, near-infrared laser pulse propagated along the bottom-to-top path into the photonsensitive photoresist without masks for 3D fabrication [44].

**Figure 2.** (**a**) The optical configuration of the double-frequency technique in ultrafast laser system for green laser beam; (**b**) the scanned straightforward nanowires and a pattern of curvy lines using the 532 nm femtosecond laser. **Figure 2.** (**a**) The optical configuration of the double-frequency technique in ultrafast laser system for green laser beam; (**b**) the scanned straightforward nanowires and a pattern of curvy lines using the 532 nm femtosecond laser.

During the TPP process [29,44], we carefully adjusted laser power for resonant twophoton absorption ranging from 1.4 to 5 mW to avoid carbonization or incomplete photopolymerization. The repetition rate of the ultrafast pulsed laser beam was 78 MHz with an approximate 100 fs pulse width. Subsequently, we three-dimensionally moved laser focus inside photoresist tightly on the substrate to form solid gelation. The available moving speeds of x- and y-axis were both 50 µm/s following the pre-designed straight or curvilinear path (Seen in the Video S1). After TPP, the unsolidified photoresist was washed away by rinsing in alcohol (purity > 99%) for over 5 min. During the TPP process [29,44], we carefully adjusted laser power for resonant twophoton absorption ranging from 1.4 to 5 mW to avoid carbonization or incomplete photopolymerization. The repetition rate of the ultrafast pulsed laser beam was 78 MHz with an approximate 100 fs pulse width. Subsequently, we three-dimensionally moved laser focus inside photoresist tightly on the substrate to form solid gelation. The available moving speeds of x- and y-axis were both 50 µm/s following the pre-designed straight or curvilinear path (Seen in the Video S1). After TPP, the unsolidified photoresist was washed away by rinsing in alcohol (purity > 99%) for over 5 min.

#### *2.3. Measurement 2.3. Measurement*

Substrates to be observed by Nova Nano SEM field emission electron microscope (SEM) with acceleration voltages of 5 KV were pre-coated with indium tin oxide semiconductor (ITO) film for electric conduction. The SEM software analyzed the size and spatial Substrates to be observed by Nova Nano SEM field emission electron microscope (SEM) with acceleration voltages of 5 KV were pre-coated with indium tin oxide semiconductor (ITO) film for electric conduction. The SEM software analyzed the size and

resolution. The imaging spectrometer (island-320, Teledyne Princeton Instruments,

spatial resolution. The imaging spectrometer (island-320, Teledyne Princeton Instruments, Princeton, NJ, USA) reflected the fluorescence images of hydrogel structures with a highresolution scientific complementary metal oxide semiconductor (sCMOS, KURO, Teledyne Princeton Instruments, Princeton, NJ, USA). The software of island-320 set the color of fluorescence images. A CCD camera installed on the digitalized inverted microscope (ix83, Olympus, Tokyo, Japan) recorded the dynamic responsiveness. The minimum time interval of each video was a single sec. The external water stimuli were realized by dropping deionized water onto the sample or blowing moisture from a humidifier to investigate the responsive behavior.

The pH variation was realized by adding the diluted hydrochloric acid into in-solution hydrogels (Figure S4). A light stimulus was applied using the same optical system to the fabrication. The heating condition was realized by placing the sample on the thermoelectric cooler with a digital control step size of 0.2 ◦C at a range from 20 to 40 ◦C. The solidified samples' heat/water micro-forces that occurred were measured by FT-MTA2 of FemtoTools (Switzerland) with FT-S10000-TP tungsten probes of 50 <sup>×</sup> <sup>50</sup> <sup>µ</sup>m<sup>2</sup> tip radius and 5 nN resolution. Young's Modulus of the solidified samples was measured using FT-S100-TP of <sup>2</sup> <sup>×</sup> <sup>2</sup> <sup>µ</sup>m<sup>2</sup> tip radius and at the same resolution.
