Tissue regeneration aims to create active scaffolds mimicking native tissue functions. Early approaches focused on mechanical and structural properties and bioactive factors. However, cells respond to dynamic, not static, stimuli. While dynamic physical stimuli (mechanical, sonic, magnetic, electrical) show promise in vitro, in vivo application requiring external, non-invasive stimulation is limited. Ultrasound stimulation, widely used clinically, shows potential for bone fracture healing but requires improved systems for efficient wave transmission. Low-intensity pulsed ultrasound (LIPUS) at MHz frequencies is applied directly to cells or constructs which remain static at these frequencies. Low-frequency ultrasound stimulation of cell substrates has shown promise for osteogenic differentiation, but this hasn't been fully exploited in functional 3D scaffolds or in vivo. Additive manufacturing (3D printing) offers potential for creating patient-customized scaffolds, and "4D printing" incorporates changes in morphology in response to external triggers. However, existing 4D printing methods often involve irreversible transformations unsuitable for physiological environments. This research demonstrates a concept using ultrasound to create dynamic 3D-printed scaffolds, addressing limitations of current approaches.

The literature review highlights the limitations of existing tissue engineering strategies. Early efforts primarily focused on static scaffolds with mechanical and structural properties and bioactive factors. However, the dynamic nature of cellular responses to mechanical cues was largely ignored. Researchers have investigated the use of various dynamic stimuli, including mechanical, sonic, magnetic, and electrical, in vitro. Yet, translating these successes into in vivo applications remains challenging, mainly due to the need for non-invasive external stimulus delivery. Ultrasound stimulation has shown promise in this regard, particularly in bone regeneration applications. However, current techniques often lack the precision and efficiency needed to optimally transmit the mechanical vibrations to the cells. This study aims to address these limitations by developing a novel 3D-printed scaffold that can be activated remotely using ultrasound.

The researchers developed a novel additive manufacturing strategy to create scaffolds with spatially controlled chemistries. They used a blend of polycaprolactone (PCL) and poly(D,L)-lactide (PLA), two biodegradable and biocompatible polymers. By exploiting phase segregation during the fused deposition modeling (FDM) 3D printing process, they generated scaffolds with distinct PCL and PLA phases, including Janus structures at a 50:50 PLA:PCL ratio. The phase segregation process was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), polarized optical microscopy (POM), and light scanning microscopy (LSM). The mechanical properties of the resulting scaffolds, including flexural modulus, storage modulus, loss modulus, and tangent delta, were measured using a mechanical tester and dynamic mechanical thermal analyzer. Computational modeling using COMSOL Multiphysics software simulated the deflection of the scaffolds under ultrasound stimulation at different frequencies. The experimental setup involved a Kemo M048N ultrasound generator and piezoelectric tweeters, with the scaffolds submerged in liquid media during stimulation. A hydrophone was used to characterize the transmitted ultrasound waves. Human bone marrow-derived stromal cells (hBMSCs) were seeded onto the scaffolds, and cell proliferation, matrix deposition, and osteogenic differentiation were assessed under different ultrasound stimulation conditions (frequencies and durations). Immunofluorescence staining and gene expression analysis were performed to investigate the mechanisms underlying the observed effects. Finally, the role of voltage-gated calcium ion channels (VGCCs) in the osteogenic differentiation was explored using pharmacological inhibition.

Phase segregation during 3D printing produced scaffolds with various structures, including Janus structures at a 50:50 PLA:PCL ratio. Computational modeling and experimental measurements showed that PLA-rich scaffolds exhibited greater deflection under ultrasound stimulation compared to PCL-rich scaffolds. Ultrasound stimulation at 40 kHz resulted in the most significant increase in cell proliferation, especially on PLA and Janus scaffolds. The Janus scaffolds, with their combination of PLA (active material) and PCL (damping material), exhibited a shorter pulse width and higher pulse repetition frequency compared to PLA-only scaffolds. Culture of hBMSCs on Janus scaffolds for 14 days under ultrasound stimulation (40 kHz) led to enhanced fibronectin-rich extracellular matrix (ECM) deposition compared to static controls. Cultures on Janus scaffolds for 21 days under ultrasound stimulation showed increased collagen I network formation, elevated expression of osteogenic markers (Collagen I, RunX2, osteocalcin), higher calcium deposition displaying the morphology of amorphous hydroxyapatite, and increased ATP release, indicating enhanced osteogenic differentiation. Blocking L-type voltage-gated calcium ion channels (VGCCs) with nifedipine inhibited the osteogenic differentiation response, confirming the role of these channels in the ultrasound-mediated stimulation. Immunofluorescence staining showed that the DHPR subunit of L-VGCC was present in cells on Janus scaffolds and that these channels were coupled to RyR receptors, proving a direct effect of mechanical stimulation on osteogenic differentiation.

The findings demonstrate that the use of ultrasound to activate 3D-printed Janus scaffolds effectively enhances bone regeneration. The unique Janus structure, acting as a transducer, optimizes the transmission of mechanical nanovibrations, leading to increased cell proliferation and osteogenic differentiation. The observation that blocking L-type VGCCs abolishes the beneficial effects of ultrasound stimulation highlights the crucial role of mechanosensitive ion channels in the cellular response. This study advances 4D bioprinting by demonstrating a method to generate on-demand dynamic scaffolds remotely activated by non-invasive stimuli. The results support the potential of this approach for clinical translation.

This study introduces a novel approach to 4D bioprinting by generating Janus scaffolds that exhibit on-demand dynamic behavior upon ultrasound stimulation. The unique structural design and material properties of the Janus scaffolds optimize the delivery of nanovibrations, enhancing cell proliferation and osteogenic differentiation. The crucial role of L-type voltage-gated calcium ion channels in this process has been established. Further research should focus on in vivo studies to assess the clinical potential and explore long-term biocompatibility.

The study primarily focuses on in vitro experiments. Further in vivo studies are needed to validate the findings and evaluate the long-term effects. The effect of common sterilization methods on the structural integrity of the scaffolds requires further investigation. The study uses one specific type of hBMSC; investigating the response of other cell types would broaden the applicability of the findings.