Janus 3D printed dynamic scaffolds for nanovibration-driven bone regeneration

The study addresses how to create dynamically responsive 3D printed scaffolds that can be remotely activated in biologically relevant environments to modulate cell behavior and promote bone regeneration. Traditional 3D scaffolds are largely static, while native tissues experience dynamic physical cues. Ultrasound is clinically used and can be applied externally, but existing approaches often deliver high-frequency LIPUS directly to cells or static constructs, limiting efficient mechanical transmission and in vivo translation. 4D printing has demonstrated shape changes but typically relies on irreversible, non-physiological triggers or non-biocompatible components. The authors hypothesize that phase-segregated, additively manufactured Janus scaffolds composed of PLA (active, deflecting) and PCL (damping) can function as ultrasound-transducer-like materials, producing controlled nanovibrations (amplitude and pulse) that enhance hBMSC proliferation and osteogenic differentiation via mechanotransductive pathways, suitable for remote, on-command activation.

The work builds on: (1) tissue engineering principles of active scaffolds and the importance of dynamic cues for cell fate; (2) prior demonstrations that mechanical, sonic, magnetic and electrical stimuli modulate cell responses; (3) clinical use of ultrasound in imaging and therapy, and studies of LIPUS for bone healing; (4) limitations of existing LIPUS approaches that apply MHz stimuli to static constructs and limited low-frequency implementations; (5) 4D printing advances showing morphological transformations often irreversible or using non-physiological triggers/materials. Prior low-frequency ultrasound-induced osteogenesis and nanoscale mechanotransduction studies suggest potential, but functional 3D dynamic scaffolds with realistic, externally applied ultrasound and translatable configurations remain underexplored. This work addresses these gaps by designing phase-segregated, biocompatible polymer scaffolds as transducer-like systems to tailor pulse characteristics relevant to cellular responses.

- Scaffold fabrication: Biodegradable PLA and PCL (Mw ~80 kDa) were twin-screw extruded at 150 °C, 100 rpm into blend filaments at ratios: 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 (PLA:PCL). Filaments were chopped (~5 mm) and fed to a fused deposition modeling (FDM) Bioscaffolder SYSENG (feed rate 500 mm/min, dispensing 30 rpm) using a 0.4 mm ID needle. Single-layer scaffolds: length 30 mm, strand distance 0.6 mm, layer thickness 0.25 mm; ends glued to petri dishes with UV-cured Norland Optical Adhesive 68 for fixation during culture and ultrasound. - Phase segregation characterization: SEM (gold-sputtered; FEI/Philips XL-30, 10 keV), polarized light microscopy, TEM (Epon-embedded, cryo-ultramicrotomy, FEI/Tecnai G2 Spirit BioTWIN, 80 keV), and light scanning microscopy. Rhodamine B and FITC were covalently attached to PCL and PLA, respectively, for phase visualization. - Discovery of Janus structures: At 50:50 PLA:PCL, homogeneous Janus fibers formed across the 3D structure with PLA area fraction 48.2% ± 8.2%. Lower/higher ratios produced dispersed phases via nucleation/spinodal decomposition with aspect ratios and particle sizes quantified. - Mechanical properties: Flexural modulus via 3-point bending on 2 mm extruded filaments (support span 1 cm; TA ElectroForce, 45 N load cell; strain rate 0.01 mm/s): Janus 759 ± 62 MPa; PCL 303 ± 70 MPa; PLA 2326 ± 36 MPa. Dynamic mechanical analysis (TA Q800) on 400 µm 3D printed fibers at 0.1 Hz and 25 °C: storage/loss modulus and damping (tan δ): PCL E′ 42.8 ± 5.9 MPa, E″ 4.9 ± 0.5 MPa, tan δ 11.3×10^-2 ± 0.4×10^-2; PLA E′ 81.6 ± 13.7 MPa, E″ 5.4 ± 1.1 MPa, tan δ 6.3×10^-2 ± 0.9×10^-2; Janus E′ 89.5 ± 12.2 MPa, E″ 6.8 ± 0.3 MPa, tan δ 7.6×10^-2 ± 0.4×10^-2. - Computational modeling: COMSOL Multiphysics simulated deflection of single-layered, end-fixed scaffolds in water using measured densities and flexural moduli. Circular acoustic source with intensity 60 Pa at 10, 20, or 40 kHz computed pressure variation and resulting flexural deflection. - Ultrasound stimulation setup: Kemo M048N generator (2–40 kHz) driving four Kemo P5123 mini piezo tweeters (2.5–45 kHz, 118 dB). Petri dishes placed atop speakers. Wave transmission characterized using a 0.2 mm needle hydrophone (NH0200, Precision Acoustics) with preamp/DC coupler to oscilloscope (Rigol DS1102); FFT separated carrier (~40 kHz) and material-dependent pulse. Media refreshed after each daily stimulation. - Nanoindentation under ultrasound: Piuma nanoindenter (MN2-TNI-ST, 0.5 N/m) measured scaffold deflection in aqueous media. After 500 nm indentation and 10 s hold, 40 kHz ultrasound was applied; deflection amplitude, pulse width, and PRF recorded. - Cell culture: hBMSCs (22-year-old male donor; passage 5) expanded in alpha-MEM + Glutamax + 10% FBS. Scaffolds sterilized (70% ethanol), PBS rinsed, vitronectin-coated (1 µg/cm^2). Seeding at 15,000 cells/cm^2 from 1.5×10^6 cells/mL concentrates. Proliferation studies in basal media ± ultrasound 30 min/day for 7 days at 0, 10, 20, 40 kHz. Matrix deposition in basal media + 0.2 mM L-ascorbic acid with 40 kHz, 30 min/day for 14 days. Osteogenic differentiation in alpha-MEM + Glutamax + 10% FBS + pen/strep + 0.2 mM L-ascorbic acid + 100 nM dexamethasone + 10 mM β-glycerophosphate with 40 kHz, 30 min/day for 21 days. For VGCC blockade, nifedipine 1 µM added. - Imaging and assays: Immunofluorescence for F-actin, DNA, fibronectin, collagen I; DHPR (L-VGCC) and RyR staining with LSM to assess coupling; quantification via CellProfiler and Fiji. Gene expression (RT-qPCR) for COL1A1, COL10A1, RUNX2, OCN, CACNA1C normalized to GAPDH using 2^-ΔCt. Calcium deposition: Alizarin red; morphology by SEM. Secreted factors: ATP (ATPlite), ALP (ELISA), osteocalcin (ELISA). Statistics via two-way ANOVA with appropriate post hoc tests; n ≥ 3 biological replicates.

- Phase segregation and Janus formation: PLA:PCL blends immiscible despite similar Hildebrand parameters; at 50:50, homogeneous Janus structures formed throughout printed fibers with PLA occupying 48.2% ± 8.2% of cross-sectional area. - Modeled ultrasound-induced deflection (end-fixed scaffolds in liquid): at 10/20/40 kHz, maximum deflections (nm) were PLA: 711 / 112 / 20.7; Janus: 207 / 79.3 / 19.8; PCL: 142 / 47.1 / 14.2. Higher frequency reduced deflection amplitude; softer PCL attenuated waves more. - Proliferation response (7 days, 30 min/day): 10 kHz decreased cell numbers across materials vs static. At 20 kHz, proliferation increased slightly for PCL and Janus and markedly for PLA. At 40 kHz, highest proliferation in PLA and Janus; PCL showed no significant change vs static. Quantitatively at 40 kHz: PLA 4492 ± 341 cells/cm^2 (519% ± 111% normalized), Janus 5219 ± 555 cells/cm^2 (234% ± 70%), PCL 1044 ± 97 cells/cm^2 (89% ± 18%). - ECM deposition (14 days, 40 kHz): Stimulated Janus scaffolds exhibited robust fibronectin-rich ECM; PLA showed mainly intracellular fibronectin despite increased proliferation; PCL deposited fibronectin with or without stimulation, with lower cell numbers. - Transducer-like behavior and pulse metrics (nanoindentation, 40 kHz): PLA (active) deflected with amplitude 53 ± 2 nm, pulse width 2 cycles (~0.49 s), PRF 1.1 Hz. PCL (damping) showed minimal, largely sinusoidal response (4 ± 1 nm). Janus combined behavior reduced amplitude to 36 ± 4 nm, halved pulse width to 1 cycle (~0.1 s), and increased PRF to 2.17 Hz. - Osteogenesis (21 days, 40 kHz): Stimulated Janus scaffolds formed dense collagen I networks; gene upregulation (COL1A1, RUNX2, OCN) on stimulated Janus and PCL vs static, with stronger effects on Janus. Calcium staining maximal on PCL and Janus; SEM indicated Ca-salt-like smooth crystals on PCL vs rounded porous, amorphous hydroxyapatite-like deposits on Janus, consistent with bone-like mineral maturation potential. - Secreted factors: Osteocalcin release increased with ultrasound for Janus and PLA but not PCL (lowest in PCL). ALP activity similar ± ultrasound but highest on Janus. ATP release was ~4-fold higher on stimulated Janus vs any other condition, consistent with mechanotransduction. - Mechanism via VGCC: CACNA1C (Ca_v1.2) expression increased ~3-fold only on stimulated Janus. DHPR staining present on PCL and Janus, but DHPR–RyR coupling observed only on Janus under stimulation. Nifedipine (1 µM) blockade reduced cell numbers across conditions and abolished ultrasound-induced increases in proliferation and osteogenic gene expression (COL1A1, COL10A1, RUNX2, OCN), eliminating differences between dynamic and static cultures, implicating L-type VGCC activation as necessary for the observed effects.

Designing PLA/PCL Janus scaffolds that mimic ultrasound transducers enabled precise control over nanovibration amplitude and, critically, pulse width and repetition. The Janus configuration, with PLA as the active deflecting phase and PCL as the damping phase, shortened pulse width and increased PRF relative to PLA alone, which correlated with enhanced hBMSC proliferation, ECM deposition, and osteogenic differentiation under remote ultrasound. While PLA alone promoted proliferation without osteogenic maturation and PCL showed minimal proliferation changes and chemical calcium salt deposition, Janus scaffolds uniquely supported both increased proliferation and bone-like matrix formation. Elevated ATP release and specific upregulation/activation of L-type VGCCs (CACNA1C expression, DHPR–RyR coupling), and the loss of effects under nifedipine blockade, demonstrate that ultrasound-induced mechanical pulses from Janus scaffolds activate VGCC-mediated mechanotransduction driving osteogenic programs. These findings address the central hypothesis by showing that externally applied, low-frequency ultrasound can remotely activate 3D printed scaffolds to deliver biologically effective, tunable mechanical cues, advancing 4D-like, on-command regenerative implants with potential translational relevance.

The study introduces an in situ phase-segregation strategy during FDM to create PLA/PCL Janus scaffolds with spatially controlled chemistries and dynamic, reversible deformation under externally applied ultrasound. By engineering the scaffold as a transducer-like composite (active PLA plus damping PCL), the authors tuned pulse characteristics of nanovibration, achieving enhanced hBMSC proliferation, ECM deposition, and osteogenic differentiation through L-type voltage-gated Ca2+ channel activation. Compared to single-component PLA or PCL, Janus scaffolds uniquely combined increased proliferation with bone-like mineral and matrix formation under stimulation. These results propose remotely activatable Janus scaffolds as promising dynamic implants for bone regeneration. Future work should evaluate in vivo performance, optimize ultrasound dosing (frequency, PRF, pulse width), assess long-term safety, and determine compatibility of standard sterilization methods with scaffold structure and function.

- In vivo applicability remains untested; translation to physiological environments and tissue integration requires animal studies. - Effects of common sterilization methods (ethylene oxide, gamma irradiation) on polymer structure and Janus morphology are unknown and need evaluation. - Differences between modeled and measured deflection amplitudes suggest unmodeled factors (incident/echo waves) affecting absolute responses. - Single donor hBMSC source and modest sample sizes (n=3 biological replicates) may limit generalizability; donor variability not assessed. - Ultrasound parameters were limited to specific frequencies (10, 20, 40 kHz) and exposure regimens (30 min/day); broader parameter space could further optimize outcomes.