[0076] Therefore, we used the 10-mm-thick ink tank to quantify the curing sizes (or printing resolutions) (Fig. 2E). First, increasing the scanning speed can improve the printing resolution. We observed overcuring in the longitudinal dimension (larger than design) at a slow scanning speed with all three ultrasound frequencies (2.05, 3.41, and 6.86 MHz), owing to excess heat accumulation. In comparison, undercuring (lower than design) was observed at high scanning speeds because of inadequate temperature increase at the two ends. The inplane curing size was determined by the joint effect of the FUS scanning speed and thermal diffusion. For instance, the in-plane curing size at 3.41 MHz was improved from 7.6 mm to 1.6 mm by increasing the scanning speed from 0.4 mm s^-1 to 0.8 mm s^-1. Similarly, the axial curing size was mostly determined by the FUS depth of focus and thermal diffusion. As an example, the 6.86-MHz FUS had a smaller depth of focus (1.69 mm) than the 2.05-MHz FUS (5.58 mm), but the sono-ink had a 10-fold acoustic absorption at 6.86 MHz and thus much higher heating efficiency. Eventually, the axial curing size ranged from 5.0 to 9.9 mm for the investigated printing parameters. The depth of focus here is defined as the size of the focal zone along the acoustic axis, in which the acoustic pressure drops to half of the peak value.

[0077] The printing resolution of DA VP can be flexibly adjusted by optimizing the FUS scanning speed, frequency, and power. Generally, the printing anisotropy (ratio of the axial and in-plane curing size) increased with the printing speed, mostly as a result of the decreased in-plane curing size (Fig. 2F). For example, when printing at 0.8 mm s^-1 by the 6.86-MHz FUS, the cross section of printed filaments exhibited an oval shape with an axial size of 8.8 mm, which was 100% larger than the inplane curing size (Fig. 2, G and H). The sonothermal curing mechanism also led to different microstructures within the FUS heating zone. The cured hydrogels displayed sub-micrometer pores in the PEGDA/PNIPAm matrix due to polymerization-induced phase separation, as shown by scanning electron microscopy (SEM) (Fig. 21). SEM images suggested melting of agar microparticles owing to a high temperature of >85°C at the center of the heating zone (Fig. 21, panel ii). However, the agar microparticles maintained a granular shape outside the center of the focal zone at various printing speeds and frequencies, similar to that achieved by bulk heating at 60° to 70°C. These results further excluded the presence of FUS-induced cavitation and confirmed the heat accumulation-based curing mechanism. Meanwhile, the enhanced surface quality of the printed filaments was confirmed by the uniform and smooth surface.

[0078] When performing multipath printing, the axial printing size (or printing thickness) increased, as a result of repeated FUS exposure along the acoustic axis at the scanned regions; however, the printing lengths were less sensitive to the number of printing paths. Additionally, the printing performance of DAVP was independent of the line-scanning direction of the 3D FUS printer. There were no significant differences in printing fidelity and mechanical properties (e.g., Young’s moduli: 1.4 ± 0.1, 1.2 ± 0.3, and 1.3 ± 0.1 MPa) using different scanning directions (0°, 45°, and 90°, respectively). Moreover, the printed sample at the 0° infill angle showed significantly higher stiffness than that by bulk heating (0.9 ± 0.1 MPa), likely because of the high temperature-induced fast matrix curing and agar welding.

DAVP of volumetric constructs and material generality

[0079] Deep penetration is the primary advantage of DAVP over conventional light-based 3D printing (Fig. 3A). For example, in a black-dyed sono-ink (0.5 w/w%), the penetration depths for the 2.05-, 3.41-, and 6.86-MHz ultrasound waves were 295.2, 86.8, and 28.2 mm at room temperature, respectively, which were, respectively, 600, 180, and 60 times as large as those for the 405-nm light (Dp = 0.48 mm) (Fig. 3B). As such, a large curing depth of 24 mm in the dyed sono-ink was achieved in 26 s at an axial scanning speed of 1mm s^-1 and 3.41-MHz FUS, whereas only a thin solid (2.4 mm) in the dye stained photoink was obtained after photocuring for 165 s. The large penetration depth of DAVP, regardless of the optical properties of the ink, allowed us to volumetrically print nontransparent 2D and 3D hydrogel constructs with different sizes and geometrical complexities, including letters and lattices with sharp comers and spirals and vessels with smooth surfaces and transitions. For example, a 10-layer honeycomb and a vascular network were printed at 3.41MHz (Fig. 3, C and D). A complex 3D hand model (67 mm by 53 mm by 10 mm) and a spider model (52 mm by 43 mm by 10 mm) were also printed at 6.86 MHz (Fig. 3, E and F).

[0080] Using dye-stained PEGDA-based sono-inks, we printed opaque-colored hydrogel composites for prototypes, including a set of three gears of different sizes and anassembled wheel set of three colored parts (Fig. 3G). Altering the phase-transition polymers with higher Tt, such as poly(N-isopropylmethacrylamide) (PNIPMAm), could facilitate printing at body temperature (37°C). Additionally, fluorescence- stained sono-ink (0.1 wt % rhodamine B) was used to print a high-fidelity branched vascular network shape (81 mm by 70 mm by 3 mm) (Fig. 3H). Further, a nanocomposite sono-ink consisting of 10 wt % nanoclay was exploited to print a four-layer tree shape (2 mm in thickness) (Fig. 31). DAVP was also used to print protein-based biomaterials, as suggested by the tough-hydrogel heart model using the GelMA-based sono-ink (Fig. 3J). Both PEGDA- and GelMA-based sono- inks and their key components exhibited zero to low cytotoxicity using NIH/3T3 fibroblasts as a model cell line. High cell viability (>99%) similar to the control [using Dulbecco’s phosphate-buffered saline (DPBS)] was observed after direct and indirect exposure to the sono-ink for up to 30 min (Fig. 3, K and L). Moreover, similar to the control cultured in a petri dish, the GelMA-based hydrogel enabled high viability (>99%) and healthy attachment and proliferation of post seeded mammalian cells of different types (Fig. 3M), indicating its favorable bioactivity.

DAVP for through-tissue printing and constructive minimally invasive medicine

[0081] As a proof of concept, we applied DAVP for high-speed and high-resolution through- tissue manufacturing and minimally invasive medicine (Fig. 4A). First, we illustrated the ex vivo through-tissue printing using soft tissues of different types and dimensions (table 2). For through-tissue printing, thick tissues (up to 17 mm thick) were placed on top of the sono-ink chamber in the FUS near field. We printed a bone-shaped construct at 2.05 MHz through an ex vivo porcine tissue phantom consisting of a skin layer (3 mm), a fat layer (5 mm), and a muscle layer (7 mm) (Fig. 4B). High-fidelity honeycombs were printed through a 15-mm-thick porcine tissue consisting of skin and muscle or a 17-mm-thick porcine liver tissue (Fig. 4C). Similarly, a hollow heart-shaped model was printed through a 17-mm-thick porcine kidney tissue (Fig. 4D).

Table 2: Tissue types, model structures, and printing parameters of deep-tissue DAVP. Different types of PEGDA-based sono-inks supplemented with 1.0 w/w% APS were used for all printing.

[0082] Nonvalvular atrial fibrillation is a prevalent cardiovascular disease related to the left atrial appendage (LAA) (H. Ueno, et al., J. Cardiol. 81, 420-428 (2023)). Open-chest surgery or transcatheter procedures can seal off the LAA to reduce the risk of thromboembolism. However, surgical LAA closure is severely invasive, and the treatment is often incomplete (D. R. Holmes Jr., et al., Mayo Clin. Proc. 97, 1525-1533 (2022)). We demonstrated proof-of-concept DAVP assisted LAA closure (Fig. 4E). We delivered the sono-ink through a catheter to the LAA of an ex vivo goat heart placed in the printing chamber. The sono-ink was then solidified using the 3D FUS printer at 3.41 MHz through a 12-mm thick heart wall. Precise FUS focus scanning enabled selective curing of the sono- ink within the entire LAA volume while sparing surrounding heart tissues. After treatment, the cured hydrogels completely occluded the LAA and bonded well with the tissue wall, which could tolerate reasonable distortions that mimicked the heart beating (Fig. 4F).

[0083] We further explored the potential of the DAVP technique for tissue reconstruction and regeneration, such as treating large bone defects (A. Stahl, Y. P. Yang, Tissue Eng. Part B Rev. 27, 539-547 (2021)). As an illustration, the PEGDA/agar/PNIPMAm nanocomposite sono-ink consisting of 5 w/w% hydroxyapatite (HAp) nanoparticles was formulated to print a bone scaffold for a hypothetical bone loss treatment (Fig. 4G). We used a chicken leg to create a fibula bone defect model (1 cm long). After injecting the nanocomposite sono-ink, we printed a beam-shaped HAp-laden composite material through the skin and muscle tissues (10 mm thick) to reshape the defective volume. The nanocomposite material could reconstruct the bone with seamless bonding to the native parts without influencing the surrounding tissues, as confirmed by ultrasound imaging (Fig. 4H).

[0084] We also demonstrated DAVP for therapeutic drug delivery by printing drug-eluting filler hydrogels on liver lesions (Fig. 41). Doxorubicin, a clinical chemotherapy drug for treating a wide range of cancers, such as breast cancer and hepatocellular carcinoma, was used as a model drug (L. Barraud et al., J. Hepatol. 42, 736-743 (2005)). PEGDA/agar/PNIPMAm sono-ink consisting of 1 mg mL^-1 of doxorubicin was formulated for printing chemotherapy drug-eluting hydrogels at the liver lesion sites at 37°C using FUS at various frequencies. DAVP enabled through-tissue printing and selective curing, as shown by the intimate hydrogel-tissue interfacial bonding (front side) and negligible burning of the intervening tissue (back side) (Fig. 4J). The drug in the hydrogel gradually released and diffused into the liver tissue (effective diffusivity: 8.7 x 10⁸ cm² s^-1), forming an ~3-mm thick effective therapeutic layer [5.8 mg mL^-1 concentration (L. Barraud et al., J. Hepatol. 42, 736-743 (2005))] within 7 days by modeling (Fig. 4K). We anticipated that this method could be used as post-ablative chemotherapy to improve cancer treatment.

[0085] We note that relatively high FUS energies were needed for through-tissue printing, mainly because of the acoustic attenuation by the intervening tissues. The acoustic energy loss might possibly result in overheating of the intervening tissues. To mitigate the risk of tissue overheating and to improve future in vivo printing efficiency, we developed a confocal-DAVP system in which the 3D FUS printer used two FUS transducers aligned in a crossbeam pattern, with their acoustic axes 90° apart and their foci overlapping (Figs. 5A- 5C). Taking advantage of the energy superposition of two acoustic foci, confocal-DAVP can achieve the curing temperature threshold in the combined foci, using roughly half of the output energy from each FUS transducer. Such a confocal configuration has two advantages: (i) the acoustic energy deposition in the local intervening position is reduced by -50%, and thus the overheating risk is minimized; (ii) the printing resolution and speed can be improved, owing to better sonothermal confinement within the overlapped foci. Indeed, the axial printing resolution was improved to 0.7 mm, and the printing speed reached 8 mm s^-1 (Fig. 5). Accordingly, complex models, including a leaf-shaped structure (55 mm by 35mm) and a vessel-like network (75 mm by 25 mm), were successfully printed using the confocal-DAVP, taking only 20 and 83 s, respectively.

Conclusions

[0086] Leveraging the deep-penetration capability of FUS waves, low acoustic streaming, and rapid sono-polymerization of the viscoelastic self-enhancing sono-inks, we have developed a DAVP technique that can volumetrically build constructs with high printing fidelity and resolution in the absence of a build platform. The use of a thermally responsive adaptive acoustic absorber resolves the conflict between acoustic streaming and deep penetration upon FUS exposure. The self-enhancing sono-ink and nonlinear acoustic propagation collectively enhanced the sonothermal heating at the FUS focus for fast and selective material solidification as the building voxel. The heat accumulation-based curing mechanism resulted in anisotropic printing resolution at a millimeter scale, which may be further improved by optimizing printing parameters of FUS frequency and scanning speed and by using the confocal dual-transducer configuration. The deep penetration of FUS waves allows the volumetric fabrication of opaque (nano)composites and printing through centimeter-thick tissues that are not attainable through state-of-the-art light-based printing techniques. The self-enhancing sono-ink design can be generalized for different systems, greatly expanding the materials library for acoustic printing techniques.

Materials and Methods

Materials

[0087] Polyethylene glycol)-diacrylate (PEGDA, M_n = 700 Da, 455008), agar (MQ200 sourced from algae, A1296), gelatin from porcine skin (Type-A, 50-100 kDa, 300-bloom, G2500), methacrylic anhydride (MAA, 94%, 276685), A-isopropylacrylamide (NIPAm, 415324), A,A-diethylacrylamide (DEAm, 773212), A-isopropylmethacrylamide (NIPMAm, 423548), 2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959,410896), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 95%, 00889), nanoclay (hydrophilic bentonite, 682659), hydroxyapatite (HAp, 702153), rhodamine B (Rh-B, >95%, R6626), and N,N,N' ,N' -tetramethylethylenediamine (T9281) were obtained from Sigma-Aldrich. Ammonium persulfate (APS, 98%, 54106) was purchased from Alfa Aesar. Polydimethylsiloxane (PDMS) (Sylgard Silicone Elastomer 184) was ordered from Dow chemical. Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), trypsin-EDTA, and antibiotic- antimycotic solution (Anti- Anti, lOOx) were supplied from Life Technologies. 4’,6-diamidino-2- phenylindole (DAPI) (D1306), Live/Dead Viability/Cytotoxicity Kit (L3224), Alexa Fluor 488-phalloidin (A12379), and Alexa Fluor 594-phalloidin (A12381) were purchased from Thermo Fisher.

Low critical solution temperature (LCST)-type polymers synthesis [0088] Low critical solution temperature (LCST)-type polymers can dissolve in aqueous solutions at temperatures lower than the LCST, and the polymers become hydrophobic above the LCST due to the coil-to-globule phase-transition. We synthesized several LCST- type polymers with tunable LCST (20.9-38.6 °C) by free-radical polymerization.

[0089] I. Poly isopropylacrylamide) (PNIPAm). PNIPAm was synthesized by

photopolymerizing the NIP Am monomer in water. Briefly, 1.8 g (15.91 mmol) of NIP Am was dissolved in a glass vial with deionized (DI) water to obtain 9.0 wt% solution. Then, 3.0 mg (13.4 |imol) of Irgacure 2959 was introduced to the above solution. After vortex mixing, the obtained precursor solution was degassed by vacuum for 10 minutes. The precursor was polymerized in ice water with an ultraviolet (UV) lamp (-100 mW cm^-2, X = 360-480 nm, OmniCure S2000, Excelitas Technologies) for 1 hour (30 minutes on each side). Using the Mark- Houwink parameters (/ = IO^{-4 24} dL g’¹, and a - 0.78) of PNIPAm (M. Wang et al., Nat. Commun. 13, 3317 (2022)), the evaluated viscometric molecular weight (Af_v) was -25 kDa. This as -polymerized PNIPAm solution can be directly used to formulate sono-inks. Purified PNIPAm was obtained by dialysis and freeze-drying. To do this, the solution was first dialyzed in dialysis membrane tubing (M_w cut-off = 12-14 kDa, Spectrum Laboratories) against DI water at 30°C for 3 days. The DI water was replaced at least twice daily. The dialyzed solution was filtered through a vacuum filter (0.22 pm in pore size, Millipore) and freeze-dried at -40 °C. The obtained foams were kept at -20 °C before use.

[0090] II. Poly (A, A-diethylacrylamide) (PDEAm). PDEAm was synthesized in water following the same protocol above. Briefly, 1.8 g (14.15 mmol) of NIP Am was dissolved in a glass vial with DI water to obtain 9.0 wt% solution supplemented with 3.0 mg (13.4 pmol) of Irgacure 2959. The precursor was photopolymerized in ice water by an UV lamp for 1 hour (30 minutes on each side). Copolymers of poly(NIPAm-co-DEAm) (1/2, w/w) were synthesized by photo-polymerizing a 20-mL solution (9 wt%) containing 1.2 g of NIP Am and 0.6 g of DEAm supplemented with 3 mg (13.4 pmol) of Irgacure 2959. Similarly, 9 wt% of poly(NIPAm-co-DEAm) (2/1, w/w) was prepared by photo-polymerization. The obtained solutions were directly used to formulate sono-inks.

[0091] III. Polv(N-isopropylmethacrylamide) (PNIPMAm). PNIPMAm with a higher LCST was synthesized by thermally polymerizing the NIPMAm monomer in water. Briefly, 1.8 g (14.15 mmol) of NIPMAm was dissolved in a glass vial with DI water to obtain a 9.0 wt% solution supplemented with 6.0 mg (26.4 pmol) of APS. The precursor was photopolymerized by heating at 50 °C for 24 hours. Using the same method, copolymers of poly(NIPAm-co-NIPMAm) (1/2, w/w) and poly(NIPAm-co-NIPMAm) (2/1, w/w) were synthesized by polymerizing 20 mL of aqueous precursor solutions at 9 wt% in total containing NIP Am and NIPMAm supplemented with 6.0 mg (26.4 pmol) of APS. After synthesis, the opaque suspension was dissolved in a 4 °C fridge and mixed to form a homogenous solution. The solutions were directly used to formulate the sono-inks.

Sono-ink preparation (Fig. 6)

[0092] Sono-inks were prepared by mixing vinyl oligomers, LCST-type acoustic absorbers, and initiators, as well as rheological modifiers, as needed. Vinyl oligomers from synthetic or natural material sources, LCST-type acoustic absorbers of different phase-transition temperatures, and rheological modifiers of various types can be combined to formulate sono-inks for multiple applications. The typical preparation procedures for sono-inks are below:

[0093] L PEGDA-based sono-inks. PEGDA oligomer was used to prepare PEGDA-based sonoinks. In brief, 30 g of agar powder was suspended in 105 g of DI water. After heating the paste at 70 °C oven for 5 minutes, 100 g of 9.0 wt% PNIPAm solution and 60 g of PEGDA were added and manually mixed to get a homogenous suspension. Later, the predetermined amount of APS was dissolved in 5-mL DI water and mixed with the ink matrix. Consequently, thermally curable sono-inks with the composition of PEGDA/agar/PNIPAm (20/10/3, w/w/w) and various APS contents (0.5-2.0 w/w%) were obtained as PEGDA-based sono-inks. The obtained sono-inks were gently centrifuged to remove the air bubbles before use. Besides, PEGDA-based sono-inks, including PEGDA/agar/PNIPAm (40/10/3, w/w/w), PEGDA/agar/PNIPMAm (20/10/3, w/w/w), PEGDA/agar/poly(NIPMAm-co-NIPMAm, 1/2) (20/10/3, w/w/w), and

PEGDA/nanoclay/PNIPAm (20/10/3, w/w/w) supplemented with 1.0 w/w% APS were prepared using the same protocol. The sono-inks with initiators were stored in a 4 °C fridge and warmed to room temperature before use. The sono-ink matrix is generally referred to PEGDA/agar/PNIPAm (20/10/3, w/w/w%) unless otherwise noted. [0094] II. Gelatin methacryloyl (GelMA)-based sono-ink. First, GelMA was synthesized following previously published protocols (M. Wang et al. , Nat. Commun. 13, 3317 (2022)). Briefly, 10 g of type-A gelatin was dissolved in (DPBS lx) at 50 °C to form a 10 wt% solution, and 5 mL of MAA was added dropwise into the above solution using a syringe pump for 30 minutes (Fig. 6B). Then, the reaction was maintained at 50 °C for 2 additional hours. Subsequently, the reaction mixture was diluted with preheated 100 mL of DPBS and further stirred for 15 minutes at 50 °C. The obtained solution was dialyzed at 40 °C for 5 days. The DI water was replaced at least twice daily during the dialysis. Lastly, the dialyzed solution was filtered, freeze-dried, and stored at -20 °C before use.

[0095] Then, the lyophilized GelMA foam was dissolved in DI water at 50 °C for a 20wt% GelMA stock solution. To the GelMA solution, a predetermined amount of as-synthesized 9 wt% PNIPAm solution and additional DI water was added and manually mixed at 30 °C for 5 minutes to obtain the GelMA/PNIPAm (10/4, w/w) solution. Afterward, pre-dissolved APS (in a minimal amount of water) was introduced to the above mixture to obtain the GelMA-based sono-ink with 1.0 w/w% APS. The sono-ink was centrifuged for air-bubble removal and stored at 4 °C. Before use, the gelled GelMA-based sono-ink was preheated in a water bath (-30 °C) for 5 minutes to form a fluid.

[0096] III. Formulating sterilized sono-inks. Sterilized PEGDA-based sono-ink and GelMAbased sono-ink were also formulated to evaluate biocompatibility and bioactivity. For PEGDA-based sono-ink, 1.0 g of PEGDA and 0.15 g PNIPAm foams were dissolved in 3.35 g of DPBS followed by homogeneously mixing 0.5 g of agar powder. Using the lyophilized and foams, 20.0 wt% GelMA and 8.0 wt% PNIPAm stock solutions in DPBS were mixed in 1/1 (w/w) ratio followed by mixing at 30 °C for 5 minutes to prepare the GelMA-based sono-ink (10/4, w/w). The above solutions were sterilized by heating (70 °C) and cooling (4 °C) (10 minutes each) for 4 cycles. Meanwhile, a 5 wt% APS stock solution in DPBS was prepared and filtered (0.22 pm). Then, different volumes of APS solution were added to obtain the sterilized sono-ink with different APS concentrations, i.e., 0, 0.5, 1 w/w%.

Focused ultrasound (FUS) transducer and pressure measurement [0097] The FUS transducer is a bowl-shaped piezoelectric ceramic transducer. FUS transducer transmits and focuses acoustic energy into a small focal volume with very high acoustic intensity. FUS has increased clinical use as a treatment modality, enabling noninvasive tissue heating and ablation for numerous applications (G. ter Haar, et al., International journal of hyperthermia 23, 89-104 (2007)). The acoustic field and acousticenergy output of the FUS system is critical to better understanding the interaction with sono-inks and essential for numerical modeling.

[0098] I. FUS transducer. Two annular FUS transducers (H-148 and H-102, Sonic Concepts, Inc.) with the same geometries (active diameter: 64 mm, radius of curvature: 63.2 mm, diameter: 22.6 mm, and central hole diameter: 20 mm) were used in this work. H-148 (FUS- 1) and H-102 (FUS-2) run at the fundamental frequencies of 1.1 MHz and 2.05 MHz, and third harmonics frequencies of 3.41 MHz and 6.86 MHz, respectively. The conversion efficiency of acoustic power from electrical power was 85% for both FUS transducers. The FUS transducers were driven by function generators in combination with radio-frequency (RF) power-amplifiers (Electronics & Innovation, Ltd., 325LA). The peak output voltage from the function generator was 0.5-1 V, which was then amplified by 240 times to get the peak driving voltage (V_p) unless otherwise noted.

[0099] IL Acoustic pressure measurement. The acoustic pressure of the FUS transducer was measured by a fiber hydrophone (RP acoustics, FOPH 2000) using a similar approach in the literature (Y. Zhou, et al., The Journal of the Acoustical Society of America 120, 676-685 (2006)). The output of the FOPH was first recorded by a digital oscilloscope. The signal was converted to pressure based on the specification of the manufacturer. To do this, FUS transducers were mounted at the bottom of a water tank filled with degassed and deionized water at 25 °C. The FUS transducer axis and the position of the FUS focus were determined by searching for the location of maximum signal strength. The peak positive acoustic pressures were recorded with the confocal alignment of the probe hydrophone tip and FUS focus. Adjusting the V_p, we obtained the voltage-dependent peak acoustic pressure. Meanwhile, the acoustic field along the lateral direction of the FUS focal zone was measured by a line scan with a step size of A -0.25 mm.

Rheological characterizations (Figs. 7, 8, and 15) [00100] The dynamic viscosity and viscoelasticity of sono-inks and their components were measured on a DHR-3 rheometer (TA Instruments) with a 40-mm-diameter, 1 -degree core plate geometry (steel Peltier plate). The gap height was set to 200 pm for the particle- free low-viscosity samples (such as aqueous PEGDA and LCST-type polymer solutions). A larger gap of 1 mm was employed for high-viscosity sono-inks. The bubble-free samples were loaded on the lower plate, and any excess samples were trimmed after lowering the gap height. The apparent viscosities of aqueous PNIPAm solutions of various mass content were measured from 0.01 to 10 s’¹ using a steady-state shear rate-sweep at 25 °C. The M_v of PNIPAm macromolecules were calculated by the concentration-dependent zero-viscosity data using the Mark-Houwink-Sakaruda parameters. Steady-state temperature sweeps were conducted at a shear rate of 1 s’¹ over 25 to 40 °C. Oscillatory temperature sweeps were performed from 15 to 37 °C with a shear strain of 1% and a frequency of 6.28 rad s’¹. Oscillatory temperature sweeps were carried out from 15 to 40 °C at a frequency of 6.28 rad s’¹ and a strain of 1%. Oscillatory frequency sweeps were carried out from 0.05 to 100 Hz (or 0.341 to 628 rad s’¹) with a strain of 1% at 25 and 40 °C for different sono-inks. To determine the gelation time of sono-inks, oscillatory time-sweeps were performed at a frequency of 6.28 rad s’¹ and a shear strain of 1% using various temperatures ranging from 45 to 60 °C (5 °C interval). Gelation was assumed to occur when viscosity reached 1000 Pa.s (K. De La Caba, et al. , Eur. Polym. J. 33, 19-23 (1997).). Additionally, gelation time It_gei) was also determined at the time marked with the sharp reduction on tan 6 curves. The gelation activation energy (E_gei) values were evaluated from ln(t_ge/) versus reciprocal temperature curve by fitting with the Arrhenius equation (S. Mortimer, et al., Macromolecules 34, 2973-2980 (2001)). Normal rheological tests used APS-free samples (Figs. 7). Sono-inks supplemented with 1.0 w/w% APS were used for gelation measurements (Fig. 15).

Acoustic streaming measurement (Fig. 9)

[00101] The acoustic streaming of different inks near the FUS focus was visualized by tracking the mass flow of a dye-stained droplet. Streaming tests were performed for both unconfined and confined conditions. For the unconfined test, the ink was loaded in an acrylic chamber with a sample height of 5 mm. The sample was supported by a membrane (~10 pm in thickness, polyethylene-based plastic wrap) attached to the bottom while the top surface was exposed to the air. In the case of the confined test, the ink was completely sealed in the ink tank (50 x 50 x 5 mm³) by the membranes at the top and bottom sides. The FUS focus was set above the membrane and close to the center of the ink. Then, a drop of pink dye (Opaque Airbrush Paint, Createx Colors) was carefully injected into the testing medium near the focal region. After that, 3.41-MHz FUS driven by V_p =156 V was turned on for approximately 30 seconds. The acoustic streaming visualized by the dye-mixing process was captured using a digital camera from the top.

Acoustic attenuation coefficient characterizations (Fig. 10)

[00102] I. Setup and measurement procedures. Acoustic waves interact with the materials during propagation. In most fluids, acoustic wave shows acoustic attenuation by absorption (-70%) and scattering (-30%). The acoustic energy will be dissipated and converted into thermal energy, termed the sono-thermal effect. The ratio of acoustic absorption to scattering depends on the physical properties of materials and is generally hard to determine precisely. To roughly evaluate the capability of acoustic absorption, we measured the acoustic attenuation coefficient (a) using a customized setup. The setup employed two water-immersion longitudinal plane-wave transducers (V213, Olympus) with a frequency band of 30 MHz. The function generator was used to drive a transducer as the transmitter with sinusoidal waveforms with three frequencies of 2.05 MHz, 3.41 MHz, and 6.86 MHz. The transmitted signal was received by the other transducer (receiver), followed by an amplifier (ZFL-500+, Mini Circuits). An ink chamber (1-2 cm in thickness) was placed between the two transducers. The whole system was placed in a thermostatic water bath to facilitate measurement at different temperatures. The acoustic transmission signal would decrease with acoustic attenuation. The attenuation by water was recorded as a reference. As a result, relative acoustic attenuation could be obtained.

[00103] II. Measurement procedures and data analyses. The a values of different fluids, including the PEGDA-based sono-ink (without APS) and their individual components, were measured at two temperatures (25 °C and 37 °C) and three ultrasound frequencies (2.05 MHz, 3.41 MHz, and 6.86 MHz). The sono-ink was loaded into the chamber, with the two-end sealed by a 10-pm membrane. The transducers and the ink chamber were carefully aligned using a three-axis stage before the test, and the sample was equilibrated in the water bath at the designed temperature for 10 minutes. The signals were recorded by an oscilloscope (DS1202Z RIGOL). The acoustic transmission signal in water was recorded as a reference. The attenuation coefficients of the sample were analyzed by the Eqn. SI as below (H. Mori, et al., Ultrasonics 83, 171-178 (2018)):

(Eqn. SI)

, where is the ultrasound frequency, L is the sample path length, A is the signal amplitude, and the subscriptions ‘sam’ and ‘ref’ stand for the sample and reference (water), respectively.

Sono-thermal heating measurements (Figs. 11 and 12)

[00104] FUS energy absorption at the focal region can raise the temperature locally, leading to the formation of a heating-zone. To visualize the heating-zone, we used an infrared (IR) camera (Seek Thermal Compact PRO) to capture the in-plane temperature map on the membrane using a bottom-up FUS printer setup. The degassed water was loaded in a rectangular ink tank (5-mm-thick) and encapsulated by thin membranes. The ink tank was attached to a three-axis motion stage, and the distance between the FUS transducer was calibrated before the test. The FUS focus was set inside the ink tank close to the top membrane. The ink tank was mounted with the topside membrane exposed to the air. The IR camera captured the temperature maps during heating and cooling with the FUS turned on and off. The IR thermal image was also taken on a preheated silicon tube of known sizes to calibrate the dimensions of the heating-zone. The IR thermal images and photographs were continuously captured with the same approach for single-point FUS exposure and single-line FUS scanning.

FUS printer setup and control

[00105] Lab-built FUS printers were assembled by integrating computer-controlled motorized translation stages with FUS transducers driven by function generators and power amplifiers. The FUS transducer and an ink tank encapsulated with membranes (10-p.m thick) were immersed in a water tank filled with degassed DI water. The motorized translation stage could be modified from a computer-numerical-control (CNC) machine or remodeled from a commercial fused deposition modeling (FDM) printer. The CNC machine was made of a two-axis translation system with a printer-head traveling area of 410 x 400 mm². A commercial FDM printer (Creality Ender 3, printing size: 220 x 220 x 250 mm³) was also modified for the FUS printer by replacing the heating nozzle with the FUS transducer. The FUS printer can be implemented by using a single transducer or dual transducers (for confocal-DAVP).

[00106] I. Single-transducer FUS printer.

[00107] The single-transducer FUS printer was assembled using either bottom-up or top-down configurations. The top-down acoustic 3D printer used the three-axis translation system of the FDM printer, and the FUS transducer was mounted on the translation stage facing down to the water tank. This acoustic 3D printer can be used for printing 3D objects with a maximum scanning volume of 150 x 150 x 100 mm³. The bottom-up FUS printer used CNC to control the movement of the ink tank above the FUS transducer that was mounted on the bottom of the water tank. The FUS transducer was driven by a function generator with a 1-Hz repetition rate and 90% duty cycle. An RF amplifier was employed to amplify the output driving voltage (fig. 16).

[00108] II. Dual-transducer confocal FUS printer setup.

[00109] To reduce the energy deposition in the intervening materials and enhance total acoustic energy at the focus, we also developed a dual-transducer confocal FUS printer. To do so, two FUS transducers (H-148 as FUS-1 and H-102 as FUS-2) fixed on two mounts were aligned in a crossbeam pattern with their acoustic axes 90-degree apart and their foci overlapped. To achieve overlapped foci, one mount was fixed on the bottom of the water tank, and the other was attached to a three-axis stage that can adjust the transducer position. A needle transducer was used to facilitate the confocal alignment of the two FUS transducers. The two FUS transducers were driven independently. Two sets of function generators and RF amplifiers were used to drive the FUS transducers to synchronize the acoustic wave transmission. FUS-1 and FUS-2 were run at the third harmonic frequency of 3.41 MHz and fundamental frequency of 2.98 MHz, respectively. The superposition of acoustic energy from the two FUS transducers led to much higher acoustic energy inside the overlapped foci than the outside. Altogether, the unique confocal design can reduce the required energy output from each individual transducer and mitigate tissue overheating. Consequently, faster printing speed and improved axial resolution were achieved (fig. 17).

[00110] Examples 2 - 14 describe a variety of sono-inks prepared by the inventors, and applications for these sono-inks.

Example 2: Formulation of PEGDA-based Sono-Ink for hydrogel printing

[00111] A sono-ink is prepared using 20 wt% polyethylene glycol) diacrylate (PEGDA), 10 wt% agar, and 3 wt% PNIPAm, supplemented with 1.0 wt% APS. The ink was mixed thoroughly, and stored at 4°C until use. When exposed to focused ultrasound waves at 3.41 MHz, the ink undergoes rapid polymerization in a localized region, forming solid 3D structures.

Example 3: Formulation of PEGDA-based Sono-ink for stiff hydrogels

[00112] A sono-ink is prepared using 40 wt% polyethylene glycol) diacrylate (PEGDA), 10 wt% agar, and 3 wt% PNIPAm, supplemented with 1.0 wt% APS. The ink was mixed thoroughly, and stored at 4°C until use. When exposed to focused ultrasound waves at 3.41 MHz, the ink undergoes rapid polymerization in a localized region, forming solid 3D structures.

Example 4: GelMA-based Sono-ink for cell-seeding

[00113] GelMA (20 wt%) is combined with 8 wt% PNIPAm with mixing ratio of 1:1 (w:w) and 1 wt% APS to create a sono-ink suitable for biomedical applications. The ink is formulated followed by ultrasound exposure for printing biocompatible scaffolds with cellseeding potential.

Example 5: PEGDA/PNIPMAm Sono-ink for biomedical applications

[00114] Composition: 20 wt% polyethylene glycol) diacrylate (PEGDA); 10 wt% agar; 3 wt% poly(N-isopropylmethacrylamide) (PNIPMAm); 1.5 wt% APS.

[00115] Application: Designed for printing thermally responsive scaffolds at body temperature (37 °C). The PNIPMAm ensures enhanced acoustic absorption while providing higher phase transition temperatures (LCST around 38°C), ideal for printing through tissues or in bioactive environments.

Example 6: GelMA-based sono-Ink with nanoclay for tissue engineering

[00116] Composition: 15 wt% gelatin methacryloyl (GelMA); 10 wt% nanoclay; 5 wt% poly(N-isopropyl acrylamide) (PNIPAm); 1.0 wt% APS.

[00117] Application: Suitable for bioprinting complex 3D hydrogel constructs for tissue scaffolds. Nanoclay improves the mechanical properties and ensures proper acoustic attenuation, making it ideal for high-resolution, biologically compatible prints.

Example 7 ; PEGDA/PNIPAm-Co-DEAm copolymer sono-Ink for flexible electronics

[00118] Composition: 30 wt% polyethylene glycol) diacrylate (PEGDA); 5 wt% agar;

5 wt% poly(NIPAm-co-DEAm) copolymer (2:1 weight ratio); 1.0 wt% APS.

[00119] Application: This ink is designed for printing flexible electronics or devices that require thermally responsive materials. The copolymer system allows tuning of the LCST to control phase transition temperatures, providing versatility in thermal control during the printing process.

Example 8: Hydroxyapatite (HAp)-laden sono-Ink for bone tissue regeneration

[00120] Composition: 25 wt% PEGDA; 5 wt% hydroxyapatite (HAp) nanoparticles; 3 wt% PNIPAm; 1.0 wt% APS.

[00121] Application: This sono-ink is optimized for bone regeneration applications. The inclusion of hydroxyapatite promotes osteoconductivity, while PNIPAm aids in ultrasound-driven phase transitions, ensuring accurate polymerization in bone tissue engineering.

Example 9: GelMA/PNIPAm Nanocomposite sono-ink for soft tissue scaffolds

[00122] Composition: 10 wt% GelMA; 3 wt% poly(N-isopropyl acrylamide) (PNIPAm); 1.0 wt% APS. [00123] Application: This ink is intended for printing soft tissue scaffolds with lower stiffness and good bioactivity.

Example 10: PEGDA/GelMA hybrid sono-ink for multi-functional bioprinting

[00124] Composition: 20 wt% polyethylene glycol) diacrylate (PEGDA); 10 wt% gelatin methacryloyl (GelMA); 3 wt% PNIPAm; 1.5 wt% APS.

[00125] Application: This hybrid ink is tailored for printing bioactive hydrogels, combining the mechanical strength of PEGDA with the biological functionality of GelMA. It is ideal for bioprinting applications, including tissue scaffolds and regenerative medicine.

Example 11: Chemotherapy drug-loaded PEGDA sono-ink for drug delivery

[00126] Composition: 20 wt% PEGDA; 10 wt% agar; 3 wt% PNIPAm; 1 mg mL¹ doxorubicin; 1.0 wt% APS.

[00127] Application: This ink is designed for printing drug delivery systems. The high biocompatibility of PEGDA, combined with the thermal responsiveness of PNIPAm, makes this ink ideal for delivering anti-cancer drugs at specific targeted locations via focused ultrasound.

Example 12: PNIPAm-Co-NIPMAm-based sono-ink for body-temperature bioprinting

[00128] Composition: 20 wt% PEGDA; 3 wt% poly(N-isopropyl acrylamide-co-N- isopropyl methacrylamide) (PNIPAm-co-NIPMAm); 10 wt% agar; 1.5 wt% APS.

[00129] Application: Designed for printing at body temperature (37°C) for in vivo bioprinting applications. The copolymer PNIPAm-co-NIPMAm provides reversible temperature sensitivity, making it ideal for biomedical applications in controlled environments.

Example 13: PEGDA/nanoclay sono-ink for high-strength nanocomposites

[00130] Composition: 40 wt% PEGDA; 10 wt% hydrophilic nanoclay; 3 wt% PNIPAm; 2.0 wt% APS. [00131] Application: This sono-ink is engineered for producing high-strength nanocomposite structures. Nanoclay improves the mechanical properties of the printed material, making it suitable for structural applications, such as load-bearing implants or prosthetics.

Example 14: GelMA/PNIPAm for Cell-Laden Bioprinting

[00132] Composition: 15 wt% gelatin methacryloyl (GelMA); 5 wt% poly(N-isopropyl acrylamide) (PNIPAm); 1.0 wt% APS.

[00133] Application: Ideal for cell-laden bioprinting, where encapsulated cells are printed into complex scaffolds. GelMA provides a biologically active environment, while PNIPAm ensures precise control over acoustic absorption and polymerization.

[00134] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMS What is claimed is:

1. A sono-ink composition, comprising an acrylate oligomer, an acoustic absorber, and a thermal initiator.

2. The sono-ink composition of claim 1, wherein the acrylate oligomer comprises a natural polymer.

3. The sono-ink composition of claim 1, wherein the acrylate oligomer comprises gelatin methacryloyl (GelMA).

4. The sono-ink composition of claim 1, wherein the acrylate oligomer comprises a synthetic polymer.

5. The sono-ink composition of claim 1, wherein the acrylate oligomer comprises polyethylene glycol)-diacrylate (PEGDA).

6. The sono-ink composition of claim 1 , further comprising a rheology modifier.

7. The sono-ink composition of claim 6, wherein the rheology modifier comprises agar or hydrophilic nanoclay.

8. The sono-ink composition of claim 1, wherein the acoustic absorber comprises poly(N-isopropyl acrylamide) (PNIPAm) or poly(N-isopropyl methacrylamide) (PNIPMAm).

9. The sono-ink composition of claim 1, wherein the thermal initiator comprises ammonium persulfate.

10. The sono-ink composition of claim 1, wherein the acrylate oligomer is about 15 to 25 weight%, the rheology modifier is about 5 to 15 weight%, and the acoustic absorber is about 2 to 5 weight%.

11. The sono-ink composition of claim 1, wherein the composition further comprises a bone-repair additive.

12. The sono-ink composition of claim 1, wherein the composition further comprises a therapeutic additive.

13. A method of deep-penetrating volumetric printing, comprising: a) providing a volume of a sono-ink composition, comprising a acrylate oligomer, an acoustic absorber, and a thermal initiator; b) directing a focused acoustic projection of ultrasound waves into the volume of the sono-ink, wherein the focused acoustic projection of ultrasound waves has an intensity and frequency sufficient to activate the thermal initiator so that local polymerization is achieved at a desired region within the volume of sono-ink; and c) optionally repeating step b wherein the focused acoustic projection of ultrasound waves is directed to selected regions within the sono-ink, until a three-dimensional object is formed.

14. The method of claim 13, wherein the method comprises b) directing at least two focused acoustic projections of ultrasound waves into the volume of the sono-ink, the at least focused acoustic projections including a first focused acoustic projection of ultrasound waves and a second focused acoustic projection of ultrasound waves, wherein each of the first and second focused acoustic projections of ultrasound waves are directed into the sono-ink in a direction orthogonal to the other, and wherein each focused acoustic projection of ultrasound waves has an intensity and frequency sufficient to activate the thermal initiator so that local polymerization is achieved only at the intersection of the two focused acoustic projections of ultrasound waves that is sufficient to activate the thermal initiator combined; and c) optionally repeating step b wherein the first and second focused acoustic projections of ultrasound waves are directed to selected regions within the sono-ink, until a three- dimensional object is formed.

15. The method of claim 13, wherein the acrylate oligomer comprises gelatin methacryloyl (GelMA).

16. The method of claim 13, wherein the acrylate oligomer comprises polyethylene glycolj-diacrylate (PEGDA).

17. The method of claim 16, wherein the sono-ink composition further comprises a rheology modifier,

18. The method of claim 17, wherein the rheology modifier comprises agar or hydrophilic nanoclay.

19. The method of claim 13, wherein the acoustic absorber comprises poly(N-isopropyl acrylamide) (PNIPAm) or poly(N-isopropyl methacrylamide) (PNIPMAm).

20. The method of claim 13, wherein the thermal initiator comprises ammonium persulfate.

21. The method of claim 13, wherein the three-dimensional object is a tissue construct.

22. The method of claim 13, wherein the volume of sono-ink is provided in vivo.

23. The method of claim 13, wherein the frequency of the ultrasound is from about 0.5

MHz to about 10 MHz.

24. The method of claim 13, wherein a robotic arm and ultrasound imaging is used to guide focused acoustic projection of ultrasound waves into the volume of the sono-ink.