
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00301-8
10.1016/j.ultsonch.2024.107053
107053
Original Research Article
Spatial regulation of hydrogel polymerization reaction using ultrasound-driven streaming vortex
Kang Byungjun a
Shin Jisoo b
Kang Donyoung a
Chang Sooho a
Rhyou Chanryeol a
Cho Seung-Woo bcd
Lee Hyungsuk hyungsuk@yonsei.ac.kr
a⁎
a School of Mechanical Engineering, Yonsei University, Seoul 03722, Republic of Korea
b Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea
c Graduate Program of Nano Biomedical Engineering (NanoBME), Advanced Science Institute, Yonsei University, Seoul 03722, Republic of Korea
d Center for Nanomedicine, Institute for Basic Science (IBS), Seoul 03722, Republic of Korea
⁎ Corresponding author. hyungsuk@yonsei.ac.kr
04 9 2024
11 2024
04 9 2024
110 1070534 6 2024
15 8 2024
28 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Ultrasound is gaining attention as an alternative tool to regulate chemical processes due to its advantages such as high cost-effectiveness, rapid response, and contact-free operation. Previous studies have demonstrated that acoustic bubble cavitation can generate energy to synthesize functional materials. In this study, we introduce a method to control the spatial distribution of physical and chemical properties of hydrogels by using an ultrasound-mediated particle manipulation technique. We developed a surface acoustic wave device that can localize micro-hydrogel particles, which are formed during gelation, in a hydrogel solution. The hydrogel fabricated with the application of surface acoustic waves exhibited gradients in mechanical, mass transport, and structural properties. We demonstrated that the gel having the property gradients could be utilized as a cell-culture substrate dictating cellular shapes, which is beneficial for interfacial tissue engineering. The acoustic method and fabricated hydrogels with property gradients can be applied to design flexible polymeric materials for soft robotics, biomedical sensors, or bioelectronics applications.

Keywords

Surface Acoustic Wave
Streaming Vortex
Micro-hydrogel
Property Gradient
Cell Morphology
==== Body
pmc1 Introduction

Ultrasound, which is a high-frequency acoustic wave, has been widely utilized in the chemical engineering fields [1], [2], [3]. Acoustic cavitation techniques have been widely utilized to increase chemical reaction rates [4], [5], [6] and to reduce energy consumption [7], [8] by regulating chemical synthesis in the processing of materials such as oil, food, and wastewater [2], [9], [10], [11]. Researchers proposed methods for engineering functional materials by harnessing the movement of particles and flow facilitated by ultrasound [12], [13], [14], [15], [16], [17]. The ultrasound-mediated streaming flow in a solution could lead to a rapid mixing of chemicals for the synthesis of functional nanoparticles [12], [13], [14] or homogenize the chemical concentration of an electrolyte to prevent precipitation [15]. Not only streaming flows but acoustic radiation force (ARF) formed in a solution upon the application of an acoustic wave can be used to control spatial locations of particles [16], [17]. Particles in a solution subjected to ultrasound undergo ARFs and they are moved to be located at a local force minimum [16], [17]. Dynamic properties of a soft actuator, such as magnitude and direction, depended on the geometry of particles patterned by the ultrasound [16]. These suggest that ultrasound is beneficial in developing functional materials by engineering particles in a spatiotemporal manner.

Recently, a ultrasound technique is emerging as a tool to fabricate a tissue-like material for biomedical engineering applications. It was reported that a spatial patterning of cells using surface acoustic waves could help to construct in vitro tissues exhibiting a therapeutic efficacy in regenerating tissue at the defect site [17]. Replicating not only the cellular arrangement but physical properties of the tissue is prerequisite in tissue engineering. Soft living tissues such as heart, blood vessels, and muscles exhibit a relatively low stiffness and spatial gradient in physical properties [18]. The mechanical properties of tissue regulate cellular behaviors such as cell proliferation, cell migration, stem cell differentiation and thus tissue maturation [19], [20] via the mechanotransduction pathways [21], [22], [23]. Therefore, researchers have suggested methods for regulating the stiffness gradient by controlling hydrogel concentration using a microfluidic mixer or crosslinking intensity using a photocrosslinkable hydrogel [24]. It has been also demonstrated that the three-dimensional printing with temperature-responsive hydrogel produced tissue mimics with gradients in mechanical properties and cell density [25].

In this study, we developed an ultrasonic technique to engineer the physical properties of the polyacrylamide hydrogel (PAAM) by spatially manipulating micro-hydrogels formed in the middle of hydrogel polymerization. Hydrogel is a three-dimensional polymeric network containing water and has been widely utilized for energy devices [26], [27], bioelectronics [28], soft robotics [29], and regenerative medicine [17], [21]. We applied surface acoustic waves (SAWs) to generate local fluidic vortices that are able to capture microparticles of hydrogel in a solution. When the PAAM was formed in the presence of SAWs, the denser polymeric network of the PAAM was formed in the location of the vortices, and the coarser network was formed in other regions. Spatial variations of the mechanical properties and mass transport properties were verified through indentation tests using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments, respectively. The hydrogel matrix having two different stiffnesses prepared by the ultrasonic technique was used to culture types of cells for interface tissue engineering. In the experiments for human adipose-derived stems cells (ADSCs), we showed that their morphology, indicative of stem cell differentiation, was altered depending on the local mechanical properties of hydrogel as in the interfacial tissues composed of soft tissues like tendon and hard tissues such as bone. Our results suggest that the ultrasonic technique can be applied to fabricate functional soft materials for wide applications such as tissue engineering, drug delivery, or soft robotics.

2 Materials and methods

2.1 Preparation of the surface acoustic wave device

The SAW device, composed of 128° Y-cut X-propagating lithium niobite piezoelectric substrate and interdigital transducers deposited on the substrate, was fabricated via a conventional photolithography technique [30], [31]. The interdigital transducers of the SAW device were designed to generate a SAW with a wavelength of 280 μm.

A polydimethylsiloxane (PDMS; Sylgard 184, Dow Corning, Midland, MI, USA) solution was prepared by mixing the base solution and curing agent at a ratio of 10:1. A PDMS film was fabricated by pouring the mixed PDMS solution onto a silicon wafer followed by curing at 60 °C for 24 h. The PDMS film was cut using a custom-made die-cutter to prepare a PDMS mold. The mold was bonded to the SAW device using oxygen plasma treatment (CUTE-MP, Femto Science, Seoul, Republic of Korea) [32]. The thickness of the PDMS film was approximately 500 μm.

To prevent both solution evaporation and inhibition of the free radical polymerization of PAAM by the oxygen from the air [56], the PDMS mold was then enclosed using a coverglass after injecting a pre-gel solution. The acoustic fields in the solution were generated by applying electrical signals to the interdigital transducers using an arbitrary function generator (33622A, Keysight, Santa Rosa, CA, USA) with an RF amplifier (LZY-22+, Mini-Circuits, Brooklyn, NY, USA). The frequency of signals was 14 MHz.

2.2 Numerical analysis for ultrasound-driven streaming flow

Streaming flows produced by the SAW was estimated using a two-dimensional finite element method (FEM) model containing a rectangular domain of water [33]. The dimension of the water domain inside the PDMS mold was the same as that in the experiment. The application of the SAW to the center region of the mold was simulated by an oscillation boundary condition that vibrates perpendicular to the edge of the mold (Figure S1). The other sides of PDMS wall were assumed to have the acoustic impedance of PDMS, which is 984,300 Pa·s/m. No-slip condition was used at the wall of PDMS mold for streaming simulation.

The acoustic fields, including the pressure and streaming fields, were described by the thermoacoustic, continuity, and Navier-Stokes equations. The perturbation theory was used to obtain first-order and second-order terms in those equations, which describe the pressure field and the time-averaged streaming field, respectively [33]. ‘Thermoacoustics’ and ‘Laminar flow’ modules in COMSOL Multiphysics 5.3a (COMSOL, Stockholm, Sweden) were used to solve the equations.

2.3 Characterization of microparticle motion

To trace particle motion in a solution, fluorescent polystyrene microparticles with a diameter of 2.0 μm (Molecular Probes, Eugene, OR, USA) were used. Images of microparticles under a streaming field formed by the application of the SAW were obtained using a fluorescence upright microscope (Ni-U, Nikon, Tokyo, Japan) and a digital single-lens reflex camera (D5300, Nikon). The motion of microparticles was quantified by particle image velocimetry (PIV) analysis using an open-source software PIVlab V1.41 [34] built on Matlab R2017b (Mathworks, Natick, MA, USA). The flow speed at the center of the mold was analyzed by manually tracking the positions of microparticles using the software Fiji [35]. The concentration of the polystyrene microparticle was 0.05 % w/v (weight per volume) and 0.2 % w/v for the manual tracking analysis and the PIV analysis, respectively.

2.4 Fabrication of polyacrylamide hydrogel with a stiffness gradient

Acrylamide (AAM; Duksan, Ansan, Republic of Korea) and ammonium persulfate (APS; Sigma-Aldrich) powder were dissolved in DI water to prepare 40 % w/v AAM solution and 10 % w/v APS solution. The pregel solution was prepared by mixing AAM solution, 2 % w/v N,N'-Methylenebisacrylamide (BAAM; Sigma-Aldrich) solution, and DI water. Hydrogel polymerization, called gelation, was initiated by adding APS and tetramethylethylenediamine (TEMED, Sigma-Aldrich) solution to the pregel solution. The pregel mixture with a volume of 20 µL was injected into the PDMS mold in the SAW device. After covering the PDMS mold with a coverglass, electrical signals were applied to the interdigital transducers of the device. The concentration of AAM was chosen to be within the range utilized in previous studies [36], [37] that investigated the effect of substrate stiffness on behavior of cells in soft tissues. Since the elastic modulus of polyacrylamide hydrogel increases by both the concentration of AAM and concentration ratio between AAM and BAAM [38], we regulated the gel stiffness by controlling the AAM concentration while maintaining the ratio of AAM to BAAM concentration to be 30:1. When the hydrogel concentration was lower than 5 %, the polyacrylamide gel was not solidified as reported in previous studies [39], [40]. When the hydrogel concentration was higher than 12 %, we observed detachment of the coverglass from the hydrogel, which might be attributed to the increase in the osmotic pressure of the hydrogel [41]. It could lead to the exposure of the remaining hydrogel solution to oxygen preventing the free radical polymerization of the gel [56]. The final concentration of APS and TEMED were 0.05 % w/v and 0.1 % w/v, respectively. The voltage applied to the SAW device was 8 V.

2.5 Characterization of the mechanical and mass transport properties of hydrogel

The mechanical properties of PAAM was investigated by an indentation test using an AFM (SPA-400, Seiko Instruments, Tokyo, Japan) with a chamber filled with DI water to prevent gel dehydration. The elasticity of hydrogel was calculated by fitting the force-distance curve from the indentation test to the ‘Sneddon Model’ [42]F=2Eδ2tanαπ(1-υ2)

where F, E, δ, α, and υ are the force measured by AFM, elastic modulus of the hydrogel, indentation depth, angle defined by the geometry of tip (18°), and Poisson’s ratio of the hydrogel of 0.45 [43], respectively.

The diffusivity of solutes in the hydrogel was characterized by FRAP experiments [44], [45]. Fluorescein isothiocyanate–dextran (FITC-dextran; Sigma–Aldrich, St. Louis, MO, USA) with 19 kDa [46], which was smaller than the apparent pore size of polyacrylamide hydrogels of 20 nm [37], [47], was chosen as a solute. Hydrogel samples were immersed in the 1.0 mg/mL FITC-dextran solution for 24 h. Fluorescent images of FITC-dextran were obtained using a confocal fluorescence microscope (LSM 510 META, Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) with a 20x magnification objective (Plan-Apochromat, Carl Zeiss). Images were taken 30 μm apart from the surface of the hydrogel. The photobleaching of fluorescent molecules was conducted by irradiating a high-intensity laser to a circle-shaped bleach region.

The intensity of FITC-dextran was normalized using the following equation [48]Inormt=Iref_preIreft×Ibleach(t)Ibleach_pre

where theIbleach(t) and Ireft indicates the average fluorescence intensity of FITC-dextran in the bleach region and a reference region at time t after the photobleaching. The values Ibleach,pre and Iref,pre represents the average fluorescence intensity of FITC-dextran in the bleach region and a reference region before the photobleaching. The reference region was defined outside of the bleach region.

The characteristic diffusion time τD was obtained by fitting the normalized intensity to the following equation [46]Inormalized(t)=∑n=0n=∞-bnn!11+n[1+2tτD]

where b is the curve-fitting parameter. The diffusivity of FITC-dextran D was calculated as followsD=r24τD

where r is the radius of the bleach region of 47.15 μm.

2.6 Cell culturing on a stiffness gradient hydrogel

NIH-3T3 fibroblasts (ATCC, Rockville, MD, USA) were cultured in DMEM (Welgene, Daegu, Republic of Korea) supplemented with 10 % fetal bovine serum (Gibco, Grand Island, NY, USA) and 1 % penicillin/streptomycin (Gibco). Human ADSCs (Invitrogen, Carlsbad, CA, USA) were cultured in MesenPRO RS medium (Invitrogen) with MesenPro RS growth supplement (Invitrogen), 1 % GlutaMAX (Invitrogen), and 1 % penicillin/streptomycin (Invitrogen).

To facilitate the attachment of cells on polyacrylamide hydrogels, the gel surface was treated by sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-SANPAH) [49] and coated with fibronectin protein at a concentration of 1.0 mg/mL. Fibroblast cells and ADSCs were seeded on the hydrogels at a density of 500 cells/sample and incubated in cell culture medium.

2.7 Immunocytochemistry and image-based analysis

Cells were fixed with 4 % paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and permeabilized with 0.05 % Triton X-100 (Sigma-Aldrich) on culture day 2. The nuclei and cytoskeletal actin networks were stained using Hoechst 33342 (Sigma-Aldrich) and Alexa-488 Phalloidin (Invitrogen). The fluorescent images of stained cells were obtained using a fluorescence upright microscope (Ni-U, Nikon) and a CCD camera (DS-Q1Mc, Nikon).

To quantify cell morphologies, fluorescent images of the nucleus and actin were binarized using Fiji software (National Institutes of Health) [35]. The cells and nuclei were segmented by applying the ‘Analyze Particles’ function of Fiji software. The area Acell and perimeter Pcell of cell were calculated using the ‘Analyze Particles’ function of Fiji software on the binarized images of actin. The circularity of cell C was calculated as follows:C=4πAcellPcell2

Lengths of the major axis Lnuc,major and minor axis Lnuc,minor of the nucleus were obtained from the ellipsoidal fit of the nucleus using the ‘Analyze Particle’ function of Fiji software. The aspect ratio of nucleus AR was defined as a ratio between tow axes as follows:AR=Lnuc,majorLnuc,minor

2.8 Statistical analysis

The statistical significance was determined using an unpaired Student’s t-test.

3 Results

3.1 Design and characterization of the ultrasound device

PAAM hydrogel is widely utilized in various fields including biomedical engineering [39], [50], [51]. PAAM is formed by the co-polymerization of AAM and BAAM, which function as monomer and crosslinker, respectively [39], [50], [51]. In the early stage of polymerization, the AAM and BAAM molecules tend to form complex-shaped polymeric aggregates called micro-hydrogel particles [39], [50], [51]. Further polymerization increases the size of micro-hydrogels interconnecting them into a polymeric network [39], [50], [51]. We hypothesized that the spatial distribution of micro-hydrogels can be regulated by the acoustic field during the polymerization process and ultimately produce a local gradient in the hydrogel properties.

Previous studies have shown that the application of ultrasound can locally generate a fluidic vortex at which particles in the solution can be trapped [52], [53], [54]. In this study, we designed an acoustofluidic device that enabled the application of SAWs to a part of the hydrogel solution using a PDMS block (Fig. 1a and 1b). When the PDMS is placed on the SAW device, the propagating SAW, generated by the interdigital transducers on the piezoelectric substrate, is attenuated as transferred to the PDMS [55]. The SAW attenuation increases exponentially with the length of the interface between the device and PDMS block [56]. Consequently, the SAWs were transmitted into the hydrogel solution through the part of the PDMS block where the interface length was relatively short (Fig. 1a).Fig. 1 Acoustofluidic device for the local application of the surface acoustic wave. (a, b) Schematic (a) and photograph (b) of the device for the application of surface acoustic wave (SAW) in a local region of the hydrogel solution. The device consists of a piezoelectric substrate, interdigital transducers, and a polydimethylsiloxane (PDMS) mold. (c, d) Flow field in the solution characterized by the numerical simulation (c) and experiment (d). Colormap and arrow represent the speed and direction of acoustically-driven flow, respectively. Scale bars represent 500 μm. (e) Flow speed of particles in the center region at various voltages of 2, 4, 6, and 8 V (V) characterized by particle tracking method (n = 4–10 particles). Inset represents schematic of the region at which the speed was measured. Error bars represent one standard deviation.

In order to investigate the effect of the transmitted SAWs on the hydrogel solution in the mold, we conducted a computational simulation using FEM software COMSOL. Our simulation result found that when SAWs were transmitted only to the center region of the solution (Figure S1), unidirectional fluid jet was produced along the direction of the SAW propagation (Fig. 1c). At the same time, counterflow was produced to the direction opposite to the SAW transmission in the peripheral region due to the fluid continuity. The interaction between two opposite-directional flows produced streaming vortices at the interface of those flows. The flow pattern obtained in the computational results was similar compared with that observed in the experiments (Fig. 1d). The additional numerical analysis using the three-dimensional (3D) model showed that the magnitude of the acoustic fields is significantly varied by the Z-location, which might be attributed to the reflection of acoustic waves at the solution-glass interface (Figure S2a). However, the flow direction was not significantly changed by the Z-location (Figure S2b). The fluidic field at XY plane obtained from the analysis for the 3D model was similar to that from the 2D model. The speed of the unidirectional flow at the center region could be regulated by the input voltage applied to the SAW device (Fig. 1e). It was measured to be increased linearly proportional to the applied voltage ranging from 2 V to 8 V. We monitored displacements of fluorescent polystyrene microparticles caused by the streaming vortices to estimate motion of micro-hydrogels during the hydrogel polymerization. We could see, as the streaming vortices trapped microparticles, the fluorescence intensity at the center region decreased while that at the side region increased (Fig. 2a). These results indicate that the micro-hydrogel formed during hydrogel polymerization could be localized at the region of acoustically-driven streaming vortices (Fig. 2b).Fig. 2 Motion of microparticles by the long-term application of surface acoustic wave. (a) Time-lapse fluorescent images of the polystyrene microparticles in the fluid and magnified images for the side and center regions. Scale bars represent 500 μm. (b) Prediction of micro-hydrogel aggregation at the streaming vortices.

3.2 Fabrication of hydrogel with the application of surface acoustic wave

Viscosity of hydrogel solution increases with its polymerization [57], [58]. Therefore, we speculated that the displacement of microparticles in hydrogel can be diminished with the increased solution viscosity (Fig. 3a). When the polymer network is formed completely, the microparticles are trapped in the network and they can rarely be moved. According to the experimental measurements, the speed of the microparticles in the center region decreased during the gelation. It was significantly reduced to near zero at approximately 9 min after the polymerization began (Fig. 3b). In contrast, in the side region, the motion of the microparticles decreased remarkably at 4 min. This result indicates that the polymeric network was formed faster in the side region due to the aggregation and subsequent interconnection of the micro-hydrogels.Fig. 3 Fabrication of hydrogel with the application of surface acoustic wave. (a) Schematic of particle displacement in response to a constant force depending on the status of gelation. (b) Normalized speed of microparticles during the gelation in the center and side region (n = 10). The speed of microparticles was normalized by the speed at 0 min. Error bars represent one standard deviation. (c) Fluorescent image of FITC-dextran in the polyacrylamide hydrogel fabricated with the surface acoustic wave (PAAM w/ SAW) (left) and its fluorescence intensity profiles along the dotted line in the image (right). (d) Scanning electron microscopy image of the freeze-dried PAAM w/ SAW. (e) Illustration of microstructure and gel density of the PAAM w/ SAW at the corresponding region. (f) Schematic of the gelation of the PAAM hydrogel with the application of the SAW. (f-1) Formation of polymer chains and micro-hydrogels. (f-2) Translation of the polymer chains and micro-hydrogels into the vortex. (f-3) Formation of high-density gel network near the vortex. (f-4) Formation of low-density gel network between the vortices. Scale bars in (c) and (d) represent 100 μm. The monomer concentration of the gel was 5 % w/v.

We investigated the effect of micro-hydrogel localization by the streaming vortices on the microstructure of the hydrogel. The structure of the polyacrylamide hydrogel fabricated with the SAW (PAAM w/ SAW) was visualized using both fluorescence imaging for the FITC-dextran dye inside gel and scanning electron microscopy (SEM) imaging for pore structures on the surface of the gel. The fluorescence intensity of FITC-dextran, which is inversely proportional to the gel density [59], [60], [61], was measured to be lower in the side region compared to the center region (Fig. 3c). The surface SEM image of the PAAM w/ SAW revealed that the pore size was larger in the center region compared to the side region (Fig. 3d). These results indicate that the PAAM fabricated with SAW exhibits Janus structure properties with two different pore densities (Fig. 3e). In summary, when the flow is generated by the SAW, the polymer chains and micro-hydrogel particles are translated to the center of the vortices (Fig. 3f). The hydrogel is solidified in the vortex region as the aggregated particles are interconnected during the polymerization reaction. The remaining polymer chains and micro-hydrogel particles were then transported and gelated in the region between the vortices, forming a relatively coarse gel network.

3.3 Characterization of mechanical and chemical properties of the hydrogel

We conducted indentation and FRAP experiments to characterize the mechanical and mass transport properties of hydrogels, respectively [62], [63]. In indentation tests, local elastic moduli of the hydrogel at the center and side regions were estimated by analyzing force-indentation depth curves obtained using the AFM device (Fig. 4a) [21], [43], [64]. For the PAAM fabricated without SAW (PAAM w/o SAW), there was no significant difference in the elastic moduli measured in the center region compared to the side one at 5 %, 8 %, and 12 % concentration (Fig. 4b). In contrast, the PAAM w/ SAW exhibited two different densities depending on the measurement location. The elastic modulus at the center region was much lower compared to that at the side region. The difference between two elastic moduli was more significant at high PAAM concentration. The mass transport properties in the center and side areas of the PAAM w/ SAW were characterized using FRAP experiments [44]. Fluorescent dye molecules dissolved at a given position in the hydrogel were bleached by high-intensity lasers (Fig. 5a). We monitored the recovery of fluorescent intensity as unbleached fluorescent dye molecules diffused into the bleach region (Fig. 5a and 5b). The diffusivity of the dye molecules was estimated by analyzing the characteristic time of the intensity recovery [65]. The diffusivity in the PAAM w/ SAW was higher in the center region than in the side region, while that of FITC-dextran in the PAAM w/o SAW did not show a spatial difference (Fig. 5c). Increasing the monomer concentration resulted in a greater spatial difference in the mass transport properties of the gel fabricated with SAW. The rigidity gradient of the fabricated hydrogel might be attributed to the degree of concentration and aggregation of polymeric particles during the gelation process. We found that the rate of particle aggregation into the side region increased with the applied voltage (Figure S3), indicating that the rigidity gradient can be regulated by the applied voltage.Fig. 4 Mechanical properties of the gel fabricated with the surface acoustic wave. (a) Representative force–displacement curve in the indentation test for the polyacrylamide (PAAM) hydrogel fabricated without (w/o) and with (w/) the surface acoustic wave (SAW). Inset shows the schematic of the indentation test using an atomic force microscope (AFM). The monomer concentration of the PAAM was 5 % w/v (weight per volume). (b) Elastic modulus of the PAAM w/o SAW and w/ SAW at the monomer concentrations of 5 %, 8 %, and 12 % w/v (n = 24; ****p < 0.0001 versus w/ SAW-center group, via unpaired t-test). Error bars represent one standard deviation.

Fig. 5 Mass transport properties of the gel fabricated with the surface acoustic wave. (a) Schematic of the fluorescence recovery after photobleaching (FRAP) experiment in the polyacrylamide (PAAM) hydrogel and the corresponding fluorescence intensity and fluorescent images of 19 kDa FITC-dextran. Scale bar represents 100 μm. (b) Representative normalized fluorescence intensity of 19 kDa FITC-dextran in DI water, in the center and side region of the PAAM fabricated without the surface acoustic wave (PAAM w/o SAW), and in the center and side region of the PAAM fabricated with the surface acoustic wave (PAAM w/ SAW) during the fluorescence recovery of FITC-dextran due to the diffusion. The monomer concentration of the PAAM was 5 % w/v (weight per volume). (c) Diffusivity of 19 kDa FITC-dextran in the center and side region of the PAAM w/o SAW and w/ SAW at the monomer concentrations of 5 %, 8 %, and 12 % w/v (n = 12; ***p < 0.001, and ****p < 0.0001 versus w/ SAW-center group, via unpaired t-test). Error bars represent one standard deviation.

3.4 Application of the fabricated hydrogel with a density gradient for interface tissue engineering

Interfacial tissues, where low stiffness tissues such as brain, heart and muscle are connected with high stiffness tissues such as bone, tendon, and cartilage, are critical in biological systems. However, the construction of in vitro interfacial tissues remains challenging. We utilized the PAAM w/ SAW as a versatile platform for regulating cell phenotype spatially on the same substrate. The feasibility was tested by seeding NIH-3T3 fibroblast cells and hADSCs, which are both commonly found within interfacial tissues, onto the fibronectin-coated hydrogel (Fig. 6a). Since cells respond differently depending on the substrate stiffness [21], [66], [67], [68], [69], [70], we analyzed morphological characteristics of these cells as a function of local stiffness.Fig. 6 Application of the hydrogel fabricated with surface acoustic wave as a cell culture substrate. (a) Schematic of the cell seeding on the fabricated polyacrylamide hydrogel having a stiffness gradient. (b) Fluorescent images of the nucleus (blue) and actin (green) of 3T3 fibroblast cells (FBs) and human adipose-derived stem cells (hADSCs) at day 2. Scale bars represent 30 μm. (c) Schematic and image-based quantification of cell area, cell circularity, nucleus area, and nucleus aspect ratio for FBs and hADSCs in center and side region (n = 9–31; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus center group, via unpaired t-test).

When fibroblasts and hADSCs were seeded on the PAAM w/o SAW, both types of cells exhibited more elongated shapes on a stiffer hydrogel substrate (Figures S4 and S5), as reported in previous studies [71], [72], [73], [74]. When seeded on the PAAM w/ SAW, fibroblasts and hADSCs showed a location-dependent cell shape: a circular shape in the center region and an elongated shape in the side one (Fig. 6b). Cell morphology was quantitatively analyzed in terms of cell area, cell circularity, nucleus area, and nucleus aspect ratio (Fig. 6c). The cell areas of fibroblasts and hADSCs were larger in the side region than in the center region. The circularity of the cell shape was lower in the side region compared to the center one. The nucleus area of fibroblasts was measured to be larger in the side region relative to the center region. In contrast, hADSCs exhibited a similar size of nucleus independent of location. While there was no significant difference in the aspect ratios of the nuclei of fibroblasts at the center and side regions, those of hADSCs were higher in the side region than in the center region. We also observed that the cell density observed on day 7 after seeding was higher in the side region of the gel fabricated with the SAW in contrast to the one without the SAW (Figure S6). This result might be attributed to durotaxis in which cell migration is guided by rigidity gradients [75] and stiffness-dependent cellular functions, such as cell proliferation and cell–cell communication [76], [77]. These results show the potential application of a hydrogel fabricated using surface acoustic waves as a cell culture platform for multipotency by providing multiple mechanical environments to cells on the same substrate or for the study of intercellular communications in interfacial tissues. It will be particularly beneficial in constructing interfacial tissues consisting of hard and soft tissues such as cartilage-to-bone and muscle-to-neuron.

The ultrasonic technique for fabrication of hydrogel with gradients in properties can have advantages in some aspects compared to other methods. The microfluidic mixing technique is feasible for the fabrication of hydrogel with gradual stiffness gradient. However, it requires a continuous flow and a relatively large volume of sample consequentially [78]. The light-based method is advantageous in terms of spatial resolution but it is only applicable to photo-sensitive hydrogel. Our ultrasonic technique is able to fabricate a hydrogel with a steep gradient in physical properties for a small volume of hydrogel without requiring labelling on a hydrogel. In addition, we expect that our acoustic fields-based method for fabricating hydrogel with gradient stiffness could potentially be combined with the methods to regulate a spatial arrangement of cells in a hydrogel. The simultaneous replication of the gradient in physical properties and cell arrangement of in vivo tissue would be beneficial for creating functional tissues for drug screening and regenerative medicine [79], [80], [81].

Our acoustofluidic technique also has the potential to control the shape of the cells encapsulated in the 3D hydrogel network. In the solution under acoustic fields, cells can be rapidly deformed by physical acoustic forces including the acoustic radiation force and fluidic hydrodynamic force [82], [83], [84], [85], [86]. When the cells were immersed in the hydrogel solution during the gelation with the SAW, the acoustic fields applied to the solution increased the temperature of the solution [87], [88] up to around 30 ° (Figure S7a). The cell viability was not decreased by the application of SAW (Figure S7b), as in our previous study [89]. The cells in the 3D hydrogel could be encapsulated with the elongated shape having a higher aspect ratio and then spatially confined by the solidified gel (Figure S8). We expect that the elongation of cells in the hydrogel by the SAW may help to study how the morphological change of cells, which can occur in response to shear stress [90], regulates the cellular functions in tissues having anisotropic structures [90], [91], [92].

4 Conclusion

In this paper, we demonstrated the acoustofluidic method for controlling the local structural, physical, and chemical properties of hydrogels. Micro-hydrogel particles could be trapped at streaming vortices formed by the SAWs altering the local polymerization processes. Through fluorescent and electron microscopy imaging, we analyzed that the pore density of hydrogel was higher near the vortices. The denser microstructure of the gel at the vortices was further confirmed by the increased elastic modulus and reduced diffusivity in the gel, which were measured by the indentation and FRAP test, respectively. We also demonstrated that the hydrogel fabricated with SAWs could control the morphological characteristics of stem cells, offering benefits for interfacial tissue engineering. We believe that the acoustofluidic method for regulating the hydrogel polymerization can be utilized in engineering chemical reactions in flow-based reactors.

CRediT authorship contribution statement

Byungjun Kang: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Conceptualization. Jisoo Shin: Resources, Methodology, Investigation. Donyoung Kang: Methodology, Investigation, Formal analysis. Sooho Chang: Methodology, Investigation, Formal analysis. Chanryeol Rhyou: Methodology, Investigation. Seung-Woo Cho: Resources, Methodology, Investigation. Hyungsuk Lee: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Funding acquisition, Conceptualization.

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.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Data 1

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant (2021R1A2C2009070) funded by the Ministry of Science and ICT and the Institute of Convergence Science (ICONS) at Yonsei University.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2024.107053.
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References

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