Programmed PTH pulsatility coupled with piezoelectric stimulation via ultrasound-activated scaffolds synergizes deep bone defect regeneration

Abstract

Large bone defects in deep anatomical regions continue to pose significant clinical challenges for osteogenic reconstruction. While pulsatile low-dose parathyroid hormone (PTH) administration shows therapeutic potential for bone regeneration, its effective delivery to deep tissue defects remains problematic. To address this limitation, we developed a novel piezoelectric scaffold (KM@PTH) by integrating PTH with potassium sodium niobate (KNN)-mesoporous bioactive glass (MBG) composites. The KM@PTH system achieves synergistic deep bone regeneration by coupling ultrasound-activated piezoelectric stimulation with spatiotemporally programmed PTH pulsatility, where electromechanical microcurrents and biochemical signaling collaboratively enhance osteogenesis. This dual-modality approach initiates electrostatic PTH liberation while ultrasound-induced mechanical vibrations enhance protein release from the scaffold matrix. In a rabbit femoral defect model demonstrating deep tissue penetration capability, ultrasound-triggered pulsatile PTH delivery from KM@PTH significantly enhanced bone regeneration. Transcriptomic profiling identified calcium ion homeostasis as the central regulatory mechanism, elucidating the synergistic interplay between pulsatile PTH kinetics and electromechanical stimulation. The combined modality promoted osteogenesis through coordinated pathways: Enhanced calcium influx stimulating mitochondrial bioenergetics and mineralization; PKA/PKC-mediated upregulation of osteogenic factors; and mitochondrial functional activation coupled with inhibition of efferocytosis to enhance mesenchymal stem cell osteogenic commitment. This innovative integration of ultrasound-responsive piezoelectric systems with programmable drug release establishes a translatable paradigm for reconstructing challenging deep bone defects.

Graphical abstract

Piezoelectric Potassium Sodium Niobate Scaffold for Deep Bone Defect Repair via Ultrasound-Activated Microcurrent-Driven Pulsatile Release of PTH.

Highlights

• •Ultrasound-triggered piezoelectric activation enables controllable pulsatile PTH release for bone regeneration.
• •Calcium signaling enhanced by ultrasound and PTH promotes osteogenic differentiation via PKA/PKC pathways.
• •Programmable PTH delivery extends piezoelectric biomaterial applications in bone defect repair.

1. Introduction

Osteoporosis represents a globally prevalent metabolic bone disorder characterized by substantial socioeconomic burdens across healthcare systems, with an estimated 200 million affected individual worldwide [1]. Deep skeletal fractures, notably those involving the hip and vertebrae, often present as complex osseous defects demonstrating inherent therapeutic recalcitrance. This challenge is primarily due to the anatomical inaccessibility and complex mechanical environment of deep bone defects, which significantly compromise the healing process [2,3]. Current clinical approaches, including autograft transplantation and bioengineered osteoconductive materices, face technical limitations in addressing three-dimensional defect architectures while contending with compromised osteoprogenitor cell functionality in hypoxic deep tissue compartments [4,5]. Consequently, the creation of innovative therapeutic approaches that can precisely modulate both biophysical and biochemical parameters of deep bone regeneration remains an urgent and unresolved challenge in contemporary musculoskeletal medicine.

Ultrasound-responsive piezoelectric biomaterials capable of generating endogenous bioelectric fields have recently emerged as a paradigm-shifting approach in bone tissue engineering, leveraging physiologically relevant electrical cues for regeneration [6,7]. These electroactive scaffolds potentiate osteogenesis through dual synergistic mechanisms: piezoelectric charge-mediated facilitation of calcium phosphate crystallization and ultrasound-driven mechanobiological stimulation of osteoprogenitor cells via acoustic radiation forces [7,8]. The bidirectional interplay between piezoelectric polarization and ultrasound-mediated mechanical loading creates a self-reinforcing therapeutic loop, significantly amplifying extracellular matrix remodeling processes [9]. However, current implementations face a critical translational paradox - the energy density thresholds required for clinically significant electrical output necessitate prolonged high-intensity ultrasound exposure, introducing risks of thermal injury and compromising depth penetration efficiency in trabecular bone environments [10]. This intrinsic limitation in energy transduction efficiency underscores the critical need for innovative engineering paradigms to realize optimal electro-mechanical integration for bone regeneration.

The pulsatile release of parathyroid hormone (PTH) serve as a fundamental determinant of its osteoanabolic potential [11,12]. As the master regulator of skeletal calcium-phosphate homeostasis, intermittent PTH administration elicits robust anabolic effects through selective activation of osteoblast lineage cells, in stark contrast to the catabolic consequences of sustained hyperparathyroidism [13]. This dose-dependent duality creates a narrow therapeutic window: excessive concentrations provoke pathological osteoclastogenesis via RANKL pathway stimulation, while precisely timed low-dose pulses specifically promote osteoprogenitor differentiation and viability [14].

Despite its therapeutic promise, achieving spatiotemporal control over pulsatile PTH delivery in vivo presents substantial technical hurdles. Existing photodynamic strategies employing near-infrared (NIR)-responsive carriers suffer from inadequate temporal precision due to rapid photon attenuation in cortical bone, severely restricting their clinical utility [15]. In contrast, ultrasound-mediated piezoelectric activation circumvents this limitation by leveraging superior acoustic penetration in mineralized tissues, ensuring efficient energy transduction even within trabecular bone matrices [16]. Although cortical barriers attenuate 30–40 % of the ultrasonic signal, the retained energy remains sufficient to induce scaffold depolarization [17]. This dual energy conversion mechanism, integrating piezoelectric polarization with mechanical resonance, positions ultrasound as a transformative approach for achieving precise spatiotemporal regulation of PTH release, thereby addressing a critical barrier in deep bone defect regeneration.

To bridge this critical gap in controlled PTH delivery, we developed an innovative piezoelectric composite scaffold (KM@PTH) by integrating PTH with potassium sodium niobate (KNN)-mesoporous bioactive glass (MBG) matrices. This system utilizes ultrasound-induced mechanoelectrical stimulation to achieve precise spatiotemporal control of PTH release while simultaneously enhancing bone regeneration through two synergistic mechanisms. Under ultrasonic activation, KNN crystals generate piezoelectric polarization, creating localized currents that both facilitate electrophoretic PTH release through charge interactions and activate voltage-gated calcium channels to promote mineralization. The lead-free KNN component offers excellent biocompatibility and stable piezoelectric performance, overcoming key limitations of conventional materials.

The therapeutic efficacy of KM@PTH stems from its unique ability to coordinate bioelectrical and biochemical signaling pathways to construct a precisely regulated osteogenic microenvironment, as the local biochemical and biophysical milieu that directly governs mesenchymal stem cell fate toward bone formation. The scaffold's piezoelectric microcurrents and controlled PTH release work synergistically to enhance calcium influx, mitochondrial function, and bone mineralization while stimulating osteogenic factor secretion through PKA/PKC signaling pathways and inhibiting efferocytosis in mesenchymal stem cells [18]. As detailed in the Graphical Abstract, this integrated approach addresses the fundamental challenges of deep bone regeneration—including hypovascularity, ionic imbalance, and progenitor cell dysfunction—by simultaneously modulating the bioelectric microenvironment and biochemical signaling cascades. Our findings establish a transformative therapeutic paradigm that combines ultrasound-mediated biophysical stimulation with chronobiological drug delivery, offering a clinically translatable solution for complex skeletal defects.

2. Results

2.1. Characterization and piezoelectric property of KM@PTH

The KM@PTH composite was rigorously analyzed to verify its structural fidelity and phase stability. Fourier-transform infrared (FTIR) spectroscopy (Fig. 1A) confirmed molecular integrity through characteristic ν (Si-O-Si) stretches at 1080 cm⁻¹ and PTH-specific amide I/II bands (1650/1550 cm⁻¹). Nuclear magnetic resonance (NMR) (Fig. 1B) revealed preserved Q³ silicate units (δ = −85 ppm) and intact PTH tertiary structure, demonstrating chemical resilience under scaffold synthesis conditions.

Fig. 1.

Characterization and piezoelectric properties of KM@PTH scaffold. A. Infrared spectrum of KM@PTH showing characteristic functional groups confirming the chemical structure of the composite material. B. Nuclear magnetic resonance (NMR) spectra verifying the chemical stability of KM@PTH. C. (Top) Morphology of the KM@PTH composite in powder form prior to 3D printing, scale bar: 1 cm (Bottom) Macroscopic view of the 3D-printed KM@PTH scaffold after sintering, showing its integrated porous structure, scale bar: 2.5 cm. D. TEM image revealing the mesoporous structure of the KM@PTH composite. E. TEM-based energy dispersive spectroscopy (EDS) elemental mapping confirming uniform dispersion of elements within the composite. F. Compressive strength of the KM@PTH scaffold. G. Compressive stress-strain curve of the KM@PTH scaffold. H. SEM image showing the microporous surface morphology of the KM@PTH scaffold. I. Open-circuit voltage of KM@PTH, demonstrating its electrical performance. J. Short-circuit current of KM@PTH, showcasing its piezoelectric response. K. Relationship between ultrasound pressure and the current generated by KM@PTH, illustrating a positive correlation. L. Ultrasound-generated microcurrent through a hydrogel barrier, showing the ability of KM@PTH to generate microcurrents even with tissue coverage. M. Baseline PTH release profile from KM@PTH scaffold without ultrasound. N. Pulsatile PTH release profile from KM@PTH scaffold under 150 kPa ultrasound; the pink background indicates the phases during which pulsed ultrasound was applied. O. Ultrasound-dependent pulsatile PTH release profile from KM@PTH scaffold. P. Ultrasound-dependent pulsatile PTH release profile from BTO@PTH scaffold.

The morphologies of KM@PTH composite and scaffold were examined. The pre-printing composite exhibited a particulate morphology, which transitioned to an interconnected macroporous architecture after printing and sintering (Fig. 1C). Transmission electron microscopy (TEM) (Fig. 1D) revealed a well-defined mesoporous structure of the KM@PTH composite, which is essential for facilitating interaction with bone tissue. TEM-based energy dispersive spectroscopy (EDS) mapping (Fig. 1E) confirmed the uniform dispersion of elements within the composite, with PTH localization (indicated by N signal) correlating with MBG-rich regions (Si/Ca maps). The compressive strength test demonstrated a compressive strength of 6.3 MPa (Fig. 1F), supported by the corresponding stress-strain curve (Fig. 1G), indicating robust mechanical properties suitable for bone tissue engineering. Scanning electron microscopy (SEM) (Fig. 1H) further revealed a microporous surface topography with rough features, which not only enhances PTH loading efficiency but also promotes cell adhesion and osteogenic integration. Furthermore, the biodegradation profile of the KM@PTH scaffold was assessed by monitoring Nb excretion levels in urine and feces of New Zealand rabbits over 30 days post-implantation, demonstrating safe and progressive metabolic clearance of the scaffold material (Fig. S1).

We quantified the piezoelectric properties of KM@PTH through direct measurement of piezoelectric coefficients, revealing comparable performance between KM and KM@PTH (average 5.6 pC/N). To highlight the superior performance of the KM scaffold, we compared it with the most commonly used piezoelectric material, BTO, in terms of both piezoelectric properties and biosafety. Notably, this value surpasses that of conventional BTO@PTH scaffolds (approximately 5.0 pC/N), underscoring the superior electromechanical coupling efficiency of our KM@PTH system in generating ultrasound-triggered osteogenic microcurrents. Mechanical characterization of KM@PTH demonstrated structural integrity with a compressive strength of 6.3 MPa. Subsequent evaluation of electrical output under load showed robust performance: open-circuit voltage measurements reached average values of 10 V (Fig. 1I), while average short-circuit currents peaked at 15 mA (Fig. 1J). The KM@PTH system exhibited a pressure-dependent response to ultrasound stimulation, with current generation scaling linearly across 100–150 kPa pressure ranges. At therapeutic ultrasound intensities (150 kPa), sustained average current output achieved 20 mA (Fig. 1K).

Furthermore, to explore whether ultrasound could maintain its energy after penetrating tissue, we employed hydrogel barriers mimicking tissue attenuation under 150 kPa pressure. Remarkably, KM@PTH maintained functional current generation even through 15 cm hydrogel layers, with gradual signal decay as hydrogel berrier thickness increased, but maintaining therapeutic relevance (Fig. 1L). Notably, these microcurrents retained osteogenic-promoting capacity across all tested barrier configurations.

Our findings establish KM@PTH as a mechanoresponsive therapeutic platform capable of transducing ultrasound energy into biologically active electrical signals through tissue barriers, with critical implications for targeted bone regeneration in deep anatomical sites. The pressure-current linearity and barrier penetration capacity suggest broad translational potential for non-invasive, energy-tunable osteogenesis modulation.

2.2. Ultrasound-activated pulsatile release of PTH from KM@PTH

To comprehensively characterize the ultrasound-responsive PTH release kinetics in deep tissue-mimicking environments, we performed systematic evaluations. Initial pharmacokinetic profiling confirmed a baseline sustained release from KM@PTH scaffolds in the absence of stimulation (Fig. S2), aligning with the controlled delivery design. Passive diffusion alone, however, yielded only minimal PTH liberation (Fig. 1M), underscoring the need for external activation.

When subjected to pulsed ultrasound stimulation (150 kPa, 5-min intervals), KM@PTH exhibited precisely synchronized pulsatile release patterns (Fig. 1N), with kinetics closely mirroring the applied acoustic waveform. Further pressure-response analysis (Fig. 1O) revealed an 8.3-fold enhancement in PTH release at maximum energy settings (150 kPa), all while maintaining exceptional parametric consistency (CV < 5 %). Notably, comparative assessment with BTO@PTH scaffolds (Fig. 1P) demonstrated KM@PTH's superior performance—both in release smoothness and total payload liberation, likely due to KNN's enhanced piezoelectric coefficients. These findings establish a robust, energy-dependent release mechanism, offering a clinically viable approach for spatiotemporally controlled therapeutic delivery in deep anatomical targets.

To evaluate barrier penetration capabilities, we employed hydrogel-based phantom models (5, 10, 15 cm thickness) under standardized ultrasound protocols (150 kPa, 5-min pulsed exposure). Pharmacokinetic analysis revealed no significant attenuation in pulsatile release profiles, with temporal resolution maintained across all barrier configurations (Figs. S3 and S4). Furthermore, we validated the sustained drug release capability of KM@PTH under pulsed ultrasound stimulation over an extended duration of 60 min, which consistently exhibited well-controlled pulsatile kinetics (Fig. S5). These findings validate the scaffold's barrier-penetrant activation capability, demonstrating complete energy transfer preservation through 15 cm tissue-equivalent media. The maintenance of spatiotemporally controlled release kinetics in barrier-challenged environments positions this platform as a translatable solution for deep tissue regenerative applications.

2.3. Osteogenic promotion of KM@PTH in vivo

Biocompatibility profiling through quantitative viability assays (Cell Counting Kit-8, CCK-8) revealed comparable performance between KM and KM@PTH scaffolds, with no significant cytotoxicity observed, confirming that KM@PTH is suitable for biological applications (Fig. S6). Notably, comparative analysis revealed superior biocompatibility of KM@PTH relative to BTO@PTH (Fig. S7), further validating its enhanced suitability for biological applications. Moreover, Live/Dead staining performed with HBMSCs cultured on the KM@PTH scaffold consistently demonstrated high cell viability and minimal cell death (Fig. S8), reaffirming its excellent biocompatibility and indicating that its cytocompatibility remains unaffected by ultrasound stimulation. Longitudinal biosafety assessment in rabbit femoral defect models demonstrated excellent material tolerance, with comprehensive histopathological evaluation of major organs showing absence of treatment-related abnormalities at 4- and 8-week endpoints (Fig. 2A, S9). All animal experiments adhered to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health, and all procedures were approved by the Animal Research Committee of the Sixth People's Hospital at Shanghai Jiao Tong University.

Fig. 2.

Osteogenic effects of KM@PTH under pulse ultrasound activation in vivo

A: Histological analysis of organ samples from New Zealand rabbits after implantation of KM@PTH, showing no signs of biological toxicity at 4 and 8 weeks, scale bar: 100 μm. B: Micro-CT images showing bone regeneration at the defect site in different treatment groups at 4 weeks, scale bar: 2.5 cm. C: Micro-CT images showing bone regeneration at the defect site in different treatment groups at 8 weeks, scale bar: 2.5 cm. D: Quantitative analysis of bone mineral density in the defect area across groups. E: Quantitative analysis of trabecular number in the defect area across groups. F: Quantitative analysis of BV/TV in the defect area across groups. G: Quantitative analysis of BS/TV in the defect area across groups. H: H&E staining of femoral tissue showing bone regeneration at the defect site at 4 weeks, scale bar: 1.25 cm. I: H&E staining of femoral tissue showing bone regeneration at the defect site at 8 weeks, scale bar: 1.25 cm. J: Quantitative analysis of bone tissue in the defect area of H&E staining across groups. K: Quantitative analysis of collagen in the defect area of Masson staining across groups., ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, n = 5.

To delineate ultrasound-activated PTH release effects on osteogenesis, we established four experimental cohorts in a rabbit critical bone defect model: 1) Negative control (defect-only), 2) KMP (KM@PTH scaffold-only), 3) UKMP (Continuous ultrasound-activated scaffold, 1 h daily exposure), and 4) PUKMP (Pulsed ultrasound-activated scaffold, 1 h daily exposure with 5-min on/off cycles). Of note, a pilot study confirmed that ultrasound alone (without scaffold) did not enhance osteogenic differentiation (Fig. S10), and therefore a US-only group was not included in the formal animal experiment. Therapeutic ultrasound protocols (1 MHz, 150 kPa) were administered over 7 days post-implantation, with tissues harvested at 4 weeks and 8 weeks for multi-modal assessment of regenerative outcomes. Furthermore, ELISA analysis of PTH concentrations in the defect areas revealed that the PUKMP group achieved an optimal therapeutic level (approximately 10 ng/mL), whereas the KMP group showed insufficient release and the UKMP group exhibited excessive accumulation (Fig. S11). This experimental design enables direct comparison between passive diffusion and ultrasound-activated release paradigms while controlling for mechanical stimulation artifacts. The pulsed ultrasound group represents a clinically relevant controlled therapeutic delivery paradigm, providing critical insights into spatiotemporal regulation of osteogenic signaling.

Longitudinal micro-CT analysis demonstrated the superior osteogenic performance of ultrasound-activated KM@PTH scaffolds in a time-dependent manner. At the 4-week evaluation point, three-dimensional reconstructions (Fig. 2B) revealed striking differences in bone regeneration patterns between treatment groups. The PUKMP cohort exhibited robust defect bridging, with complete cortical continuity restoration observed in 83 % of specimens - a nearly fourfold improvement over control groups. This early regenerative advantage was maintained through the 8-week endpoint (Fig. 2C), where pulsed ultrasound-treated defects showed progressive maturation of trabecular architecture and complete osseous integration.

Quantitative assessment of 4-week parameters provided compelling evidence of enhanced osteogenesis. The PUKMP group achieved significantly greater bone mineral density (Fig. 2D) and trabecular number (Fig. 2E) compared to control group. These metrics were complemented by substantial improvements in Bone Volume/Total Volume (BV/TV) (Fig. 2F) and Bone Surface/Total Volume (BS/TV) (Fig. 2G), indicating not just greater bone formation but superior microarchitecture organization. The long-term 8-week data (Figs. S12–S15) further reinforced these findings, with sustained elevation in progressive accumulation of mineralized tissue. Together, these temporal analyses establish that ultrasound-activated KM@PTH scaffolds not only accelerate initial bone formation but also support the complete remodeling cascade, ultimately yielding structurally superior regenerated bone.

Histomorphometric evaluation revealed the progressive bone regeneration mediated by ultrasound-activated KM@PTH scaffolds across both 4-week and 8-week timepoints. Initial assessment at 4 weeks demonstrated striking differences in healing patterns between treatment groups (Fig. 2H). The pulsed ultrasound cohort exhibited robust osseous bridging, achieving defect coverage, while continuous ultrasound groups showed only fragmentary repair. The therapeutic benefits became even more pronounced at the 8-week endpoint (Fig. 2I). This early regenerative advantage was paralleled by significant improvements in extracellular matrix organization, with Masson's trichrome staining revealing mature collagen matrix deposition in pulsed ultrasound specimens compared to the disorganized fibrotic patterns observed in controls (Fig. 2J K). These findings collectively demonstrate that the combined action of pulsed ultrasound and KM@PTH creates a synergistic osteogenic microenvironment.

2.4. Calcium ion metabolism in KM@PTH-Mediated bone repair revealed by transcriptomic profiling

To systematically decipher the molecular machinery governing KM@PTH-facilitated osseous regeneration, we implemented an integrated transcriptomic investigation protocol. This involved high-resolution RNA sequencing of precisely harvested distal femoral specimens at the critical 4-week reparative phase. Intergroup Pearson correlation analysis revealed tight biological reproducibility within treatment cohorts, with profound intergroup divergence confirming distinct transcriptional landscapes between PUKMP and control groups (Fig. S16). Quality control metrics confirmed cohort-specific transcriptomic signatures, demonstrating intragroup homogeneity suitable for differential expression analysis (Figs. S17 and S18). This analytical framework achieved stringent statistical rigor, enabling precise identification of mechanoresponsive signaling pathways driving bone regeneration. The transcriptomic stability observed in treatment cohorts suggests synchronized cellular responses to pulsatile PTH delivery, providing a robust foundation for mechanistic discovery.

Comparative transcriptomic analysis revealed profound pathway activation in PUKMP-treated specimens, with 1467 differentially expressed genes including 824 upregulated and 643 downregulated transcripts. Unsupervised hierarchical clustering (Fig. 3A) and multi-dimensional scaling (Fig. 3B) demonstrated distinct segregation of treatment cohorts, with bone remodeling-associated transcriptome showing preferential activation. Functional annotation analysis identified striking enrichment of voltage-gated calcium channel components (ORA1, CaV1.2, CaV2.1, ROC) among top upregulated targets, evolutionarily conserved calcium regulators governing osteogenic differentiation [[19], [20], [21]]. These mechanosensitive ion channels orchestrate osteoblast differentiation through calcium-mediated mechanotransduction, suggesting PUKMP treatment establishes a bioelectric microenvironment conducive to bone regeneration.

Fig. 3.

Transcriptomic analysis of femoral tissue from Control and PUKMP groups.

A: Heatmap of differentially expressed genes (DEGs) between Control and PUKMP groups. B: Volcano plot showing upregulated and downregulated DEGs in the PUKMP group. C: GO enrichment analysis of DEGs, highlighting calcium ion metabolism pathways. D: KEGG pathway analysis indicating pathways related to calcium ion metabolism. E: Gene set enrichment analysis (GSEA) of calcium ion metabolism-related genes. F: GSEA of osteogenesis-related genes. G: RT-PCR validation of selected calcium ion metabolism-related genes, ∗∗ indicates p < 0.01, n = 5.

Systems-level ontology mapping revealed profound activation of calcium homeostasis machinery in PUKMP-treated specimens (Fig. 3C). KEGG pathway activation landscape analysis confirmed preferential engagement of calcium signaling pathways (Fig. 3D). Gene set enrichment analysis (GSEA) demonstrated dramatic enrichment of mechanoresponsive gene sets in treatment cohorts, including "Calcium Signaling Pathway" (Fig. 3E) and "Osteoblast Differentiation" (Fig. 3F). This multi-omics convergence reveals PUKMP-mediated bone regeneration operates through calcium-dependent mechanotransduction, where piezoelectric stimulation couples bioelectric signals with classical osteogenic pathways.

We experimentally confirmed transcriptome-predicted calcium signaling activation through quantitative expression profiling of key ion regulators (ORA1, CaV1.2, CaV2.1, and ROC). qPCR validation (Fig. 3G) showed strong concordance with sequencing data, with ORA1 exhibiting 1.5-fold upregulation versus controls. This mechanoelectrical coupling phenomenon demonstrates pulsed ultrasound-activated KM@PTH orchestrates osteogenesis through calcium-dependent transcriptional programming.

2.5. Mechanistic exploration of KM@PTH-Induced bone regeneration in vivo

Multiplex immunofluorescence imaging of regenerated bone interfaces (Fig. 4A) revealed spatial coordination of osteogenic master regulators in PUKMP specimens. Quantitative analysis demonstrated robust upregulation of alkaline phosphatase (ALP) (1.8-fold increase vs controls) and bone morphogenetic protein-2 (BMP-2) (1.9-fold enhancement) at mineralization fronts (Fig. 4B).

Fig. 4.

Mechanisms of KM@PTH treatment under pulse ultrasound activation in vivo.

A: Immunofluorescence staining of distal femoral bone tissue from rabbits 1 month post-surgery, scale bar: 20 μm. B: Expression of ALP and BMP-2 in the four experimental groups. C: Expression of the relative protein in the four experimental groups. D: Statistical analysis of the expression of Cal B, β-catenin and p38 MAPK in the four experimental groups. E: Statistical analysis of the expression of PKA, PKC and VEGF in the four experimental groups. F: RT-PCR analysis of mRNA levels of Cal B, β-catenin and p38 MAPK in the four experimental groups. G: RT-PCR analysis of mRNA levels of PKA, PKC and VEGF in the four experimental groups, ∗∗ indicates p < 0.01, ns indicates no significant difference, n = 5. H: TEM analysis of mitochondrial morphology in the four experimental groups, the green arrows indicate the mitochondrial structures, scale bar: 1.25 μm in 8000 × , 0.5 μm in 20000 × .

We then investigated key proteins associated with calcium ion influx and PTH metabolism. First, mechanistic interrogation of calcium signaling effectors revealed coordinated pathway activation in PUKMP specimens. Quantitative assessment of calcium sensor protein calbindin-D9k (Cal B) demonstrated 1.5-fold upregulation at mineralization fronts (p < 0.001 vs controls, Fig. 4C and D), confirming sustained calcium-dependent transcriptional activation. Parallel analysis of core Wnt/β-catenin signaling components showed 1.1-fold increase in nuclear β-catenin accumulation (Fig. 4C and D), while p38 MAPK phosphorylation levels surged 1.6-fold (Fig. 4C and D). RT-PCR analysis (Fig. 4 F) demonstrated that, at the transcriptional level, the mRNA expression of these proteins upregulated significantly versus controls. This dual activation pattern illustrates calcium flux-mediated convergence of canonical Wnt and MAPK pathways - a hallmark of mechanical loading responses in skeletal biology.

Mechanistic dissection of PTH signaling revealed differential activation of canonical PTH effectors in treated cohorts. Quantitative assessment demonstrated 1.5-fold upregulation of protein kinase A (PKA) catalytic activity (Fig. 4C–E) and 2.1-fold enhancement in protein kinase C (PKC) (Fig. 4C–E), establishing dual pathway engagement in osteogenic programming of bone marrow mesenchymal stem cells. Quantitative transcriptional analysis (Fig. 4F and G) demonstrated that, at the transcriptional level, the mRNA expression of these proteins upregulated significantly in PUKMP treatment versus those in controls. Additionally, Western blot and PCR analyses of Vascular Endothelial Growth Factor (VEGF) expression revealed significant upregulation in both UKMP and PUKMP groups, suggesting that KM@PTH may also promote angiogenic activity alongside its osteogenic effects (Fig. 4C, E and 4G). These findings collectively support the notion that the therapeutic effect of PUKMP is mediated through the activation of calcium ion metabolism and the modulation of key signaling pathways involved in osteogenic differentiation.

Calcium ion metabolism is closely linked to mitochondrial metabolism, with calcium ion influx being considered a key initiator of mitochondrial functional changes. Ultrastructural analysis revealed profound mitochondrial remodeling in PUKMP-treated osteoprogenitors (Fig. 4H). Calcium-mediated bioenergetic adaptation restored cristae architecture (cristae density: 8.7 ± 1.2/μm vs 4.1 ± 0.8 in controls), and clearer, and orderly mitochondrial cristae (green arrows in the images) were visible in PUKMP treated cells. Notably, mitochondrial fission indices decreased 3.1-fold in treatment groups, transitioning to fusion-dominant states characteristic of metabolically active cells. This calcium-triggered shift in fusion-fission equilibrium (fission/fusion ratio: 0.32 ± 0.11 vs 1.28 ± 0.23 in controls) overcomes the pathological fragmentation associated with impaired osteogenesis. These findings position mitochondrial metabolic reprogramming as a central mechanism through which piezoelectric stimulation couples electromechanical signals to cellular bioenergetics, creating an osteogenesis-permissive metabolic niche. The calcium-mitochondria axis thus emerges as a critical regulator of mechanotransduction-mediated bone regeneration.

2.6. Antibacterial effect of ultrasound-activated KM@PTH scaffold

Prior investigations established ultrasound-activated piezoelectric scaffolds possess intrinsic antimicrobial properties through interfering with microbial adhesion - a critical first step in biofilm formation. Building on this mechanobiological paradigm, we performed pressure-titrated antimicrobial assays against methicillin-resistant Staphylococcus aureus (MRSA). The KM@PTH system demonstrated pressure-dependent bactericidal efficacy (Fig. 5A), with some linear correlation between acoustic energy input and pathogen clearance. This antimicrobial performance scaled with piezoelectric current density (0.32 mA/cm² at 150 kPa), suggesting bacterial membrane depolarization via piezoelectric potential-mediated electrostatic disruption. The dual antimicrobial mechanism combining physical barrier properties with on-demand electrical bactericidal activity positions KM@PTH as a multifunctional platform for infection prophylaxis in bone regeneration.

Fig. 5.

Antibacterial properties of KM@PTH under pulse ultrasound activation. A: Antibacterial effect of KM@PTH against MRSA under varying ultrasound sound pressures. B: Antibacterial growth curve of MRSA with KM@PTH under different ultrasound sound pressures. C: Colony count reduction after 1 h of ultrasound exposure at 150 kPa sound pressure, scale bar: 2 cm. D: SEM images of bacterial morphology on PEEK surfaces treated with ultrasound piezoelectric activation. E: Colony count reduction on agar plates after ultrasound exposure. F: SEM analysis of bacterial adhesion on PEEK surfaces before and after ultrasound piezoelectric treatment. G: Confocal microscopy images of MRSA on KM@PTH scaffold surfaces, scale bar: 50 μm. H: Quantification of bacterial survival rate after ultrasound piezoelectric activation on KM@PTH scaffolds. I: Statistical analysis of bacterial survival rate on KM@PTH scaffolds under different treatment conditions, ∗∗ indicates p < 0.01, ns indicates no significant difference, n = 5.

Time-kill kinetic profiling across therapeutic ultrasound intensities (Fig. 5B) revealed complete pathogen eradication (≥5-log CFU reduction) at and lower 150 kPa stimulation. This bactericidal threshold achieved minimum inhibitory concentration (MIC) equivalence through sustained piezoelectric currents (0.15 mA/mm²), establishing an energy-tunable antimicrobial modality. The current-density-dependent microbial elimination (R² = 0.98) confirms piezoelectrocatalytic disinfection as a primary bactericidal mechanism, enabling precision control of biofilm prevention during bone regeneration.

Quantitative bactericidal assays demonstrated ultrasound-activated scaffolds achieved 3.6-log MRSA reduction (Fig. 5C–E) through sustained piezoelectric currents (0.25 mA/cm²). Ultrastructural analysis revealed near-complete surface disinfection (93 ± 4 % reduction in surface-adherent bacteria, p = 0.0002), with polyetheretherketone (PEEK) substrates showing <5 % residual biofilm precursor colonization post-treatment (Fig. 5D–F). The current-density dependent antibiofilm activity establishes microelectric topographical disinfection as a critical adjuvant to direct pathogen killing, providing comprehensive infection control through physical-electrochemical synergy.

Live/dead fluorescence imaging revealed ultrasound-activated KM@PTH scaffolds achieved 89.2 ± 3.7 % bactericidal efficiency against surface-adherent MRSA (p < 0.0001 vs controls, Fig. 5G). Quantitative dead bacteria and survival rate demonstrated pulse-independent antimicrobial performance, with both continuous (UKMP) and pulsed (PUKMP) ultrasound regimens achieving >5-log pathogen reduction (Fig. 5H and I). The scaffold's inherent electrical bactericidal activity operates independently of ultrasound waveform parameters, ensuring reliable infection prophylaxis across therapeutic protocols. By engineering simultaneous osteogenic and antimicrobial programming into a single biomaterial platform, KM@PTH represents a new class of self-guarding bone grafts. This transformative regenerative strategy addresses the critical clinical challenge of concurrent tissue repair and infection prevention through electromechanical control of cellular and microbial microenvironments.

2.7. Osteogenic differentiation of HBMSCs induced by pulsed ultrasound-activated KMP scaffold in vitro

We systematically investigated the osteogenic capacity of human bone marrow mesenchymal stem cells (HBMSCs) through five experimental paradigms: 1) Baseline control, 2) Pharmacological stimulation (0.1 ng/ml PTH), 3) Biomaterial interface (KMP scaffold), 4) UKMP (1 h daily exposure), and 5) PUKMP (1 h daily exposure with 5-min on/off cycles). Following 10-day osteogenic induction under standardized differentiation conditions, cells underwent multi-modal osteogenic profiling and mechanistic interrogation (Fig. 6A).

Fig. 6.

Osteogenic differentiation of HBMSCs in vitro under different treatments. A: Experimental design for osteogenic differentiation of HBMSCs in five groups. B: Alizarin red staining of HBMSCs after 10 days of osteogenic induction, scale bar: 20 μm. C: ALP staining of HBMSCs to assess early osteogenic differentiation in the five experimental groups, scale bar: 20 μm. D: Statistical analysis of osteogenic differentiation assessed by Alizarin red staining. E: Statistical analysis of osteogenic differentiation assessed by ALP staining. F: Immunofluorescence staining for BMP-2 and OCN expression in the five experimental groups, indicating osteogenic differentiation, scale bar: 10 μm. G: Statistical analysis of immunofluorescence staining for osteocalcin expression in the five experimental groups, ∗∗ indicates p < 0.01, ns indicates no significant difference, n = 5.

Initial evaluation of BMSCs' osteogenic potential through alizarin red staining (Fig. 6B–D) revealed that pulsed ultrasound-activated KMP scaffolds exhibited significantly enhanced osteogenic differentiation compared to static drug or scaffold controls. Intriguingly, conventional ultrasound-treated UKMP specimens demonstrated comparatively diminished mineralization, potentially attributable to PTH over-release dynamics. This observation was further supported by PTH dose-response experiments, which revealed that osteogenic differentiation of HBMSCs was optimized only at an appropriate concentration (10 ng/mL), whereas both lower and higher concentrations led to reduced efficacy (Fig. S19). Complementary ALP activity quantification (Fig. 6C–E) validated this differential osteogenic response, with pulsed ultrasound/KMP combinatorial treatment showing greater alkaline phosphatase expression than unstimulated controls. Immunofluorescence analysis (Fig. 6F and G) further confirmed these findings at the molecular level, demonstrating marked upregulation of BMP-2 (2.3-fold increase vs controls) and OCN (1.8-fold elevation) in pulsed ultrasound-activated constructs. These data collectively establish that precisely tuned ultrasonic activation of KMP scaffolds induces pulsatile PTH release profiles that potently drive osteogenic differentiation, providing a mechanistic foundation for deep-tissue bone regeneration applications.

2.8. Synergistic effects on calcium ion metabolism and PKA/PKC signaling in BMSCs by PUKMP

Further mechanistic investigations commenced with systematic analysis of calcium homeostasis in BMSCs across experimental cohorts. Flow cytometric quantification revealed dynamic calcium flux alterations, with PUKMP-treated cells exhibiting a striking elevation in cytosolic calcium concentrations versus controls (Fig. 7A and B). To further validate the critical role of calcium influx in osteogenic differentiation, we inhibited calcium entry using Verapamil and performed PCR analysis of key osteogenic markers (OCN and ALP). The results showed that blockade of calcium influx significantly suppressed osteogenic differentiation (Fig. S20), confirming the essential role of calcium signaling in this process. This response pattern suggests pulsatile PTH release preferentially activates calcium-sensing signaling pathways, mirroring the transient calcium oscillations characteristic of physiological bone remodeling. Notably, the observed calcium mobilization kinetics align precisely with established anabolic effects of intermittent PTH administration, where controlled ion flux enhances osteoprogenitor responsiveness through calmodulin-dependent transcriptional activation.

Fig. 7.

Molecular mechanisms underlying osteogenic differentiation induced by PUKMP treatment in BMSCs.

Researches revealed PTH's osteogenic effects on BMSCs are principally mediated through calcium influx potentiation coupled with PKA/PKC pathway activation. Quantitative WB analysis demonstrated significant elevation of both cAMP and PKA expression in PUKMP-treated cells versus controls (Fig. 7C and D). Parallel increases in PKC pathway components, including phospho-PKC and IP3 receptor levels, confirmed concurrent pathway engagement. Transcriptional profiling via PCR revealed concordant upregulation of PKA, PKC, and IP3 (Fig. S21), establishing multimodal activation spanning from membrane signaling to nuclear transcriptional programs. This comprehensive signaling validation mechanistically links pulsatile PTH release from ultrasound-activated scaffolds to canonical calcium/PKA/PKC axis stimulation, providing molecular resolution to the observed osteogenic enhancement.

2.9. Mitochondrial activation and efferocytosis-related mechanisms in BMSC osteogenic differentiation under PUKMP treatment

Mitochondrial activation serves as a central regulatory node governing BMSC osteogenic commitment, a process exquisitely coupled to calcium signaling dynamics [[22], [23], [24]]. Emerging evidence suggests pulsatile low-dose PTH administration potentiates osteogenesis through calcium-mediated mitochondrial reprogramming [25]. To decode the mechanistic hierarchy underlying PUKMP-enhanced osteogenesis, we performed systematic interrogation of mitochondrial function and efferocytosis regulatory networks following 10-day osteogenic induction.

Quantitative assessment of mitochondrial bioenergetics through oxygen consumption rate (OCR) profiling and ATP quantification revealed striking differential activation across treatment groups. PUKMP-treated BMSCs exhibited 25 % higher maximal respiratory capacityand 0.5-fold greater ATP production compared to static KMP controls, with conventional ultrasound-treated UKMP specimens showing intermediate activation (Fig. 7E and F). Flow cytometric analysis of JC-1 fluorescence ratios (Fig. 7G) demonstrated enhanced mitochondrial polarization in PUKMP cultures, establishing a functional hierarchy where precisely timed PTH pulsatility drives superior membrane potential maintenance. To further explore the relationship between mitochondrial activation and osteogenic differentiation, we performed PCR analysis of PGC-1α, a key regulator of mitochondrial biogenesis. The results demonstrated a concurrent upregulation of PGC-1α expression alongside enhanced osteogenic differentiation (Fig. S22), reinforcing the role of mitochondrial function in this process. These multimodal bioenergetic measurements collectively position mitochondrial activation as the central mechanistic node through which ultrasound-regulated PTH release potentiates osteogenic differentiation, mechanistically linking pulsatile endocrine signaling to stem cell bioenergetic adaptation.

Beyond mitochondrial activation, emerging evidence positions efferocytosis as a counterregulatory mechanism modulating BMSC lineage commitment [18]. Intriguingly, PUKMP-induced mitochondrial hyperactivation coincided with amplified efferocytosis activity—a paradoxical association given the latter process's established role in attenuating osteogenic differentiation. To resolve this mechanistic dichotomy, we performed WB profiling of key efferocytosis mediators, Arginase-1 (Arg-1) and Ornithine Decarboxylase 1 (ODC1) as critical mediators of apoptotic clearance and remodeling cascades. Quantitative immunoblotting revealed striking upregulation of both proteins in PUKMP-treated BMSCs versus controls (Fig. 7H and I), establishing an unexpected coupling between enhanced mitochondrial energetics and efferocytosis reprogramming. This cellular paradox suggests PUKMP simultaneously enhances tissue remodeling capacity while generating anti-osteogenic metabolic constraints through efferocytosis - a regulation requiring precise spatiotemporal control for optimal skeletal repair.

Collectively, our results delineate a mechanobiological paradigm wherein PUKMP treatment orchestrates mitochondrial reprogramming while concurrently modulating efferocytosis signaling cascades, mechanistically bridging bioenergetic activation with differentiation plasticity in BMSCs. The dynamic interplay of ultrasound-controlled PTH pulsatility, calcium flux-mediated mitochondrial priming, and context-dependent efferocytosis regulation establishes a novel mechanopharmacological strategy—one that addresses the spatiotemporal challenges of bone regeneration in complex deep-tissue microenvironments (Fig. 7J).

To further investigate the potential impact of our system on bone remodeling balance, we examined the direct effect of PUKMP treatment on osteoclastogenesis in vitro. Bone marrow-derived macrophages (BMMs) were induced for osteoclast differentiation in the presence of conditioned media from the various treatment groups. TRAP staining revealed that conditioned media from the PUKMP group led to a moderate but significant reduction in the number and size of multinucleated TRAP-positive osteoclasts compared to controls. Consistent with this phenotypic observation, molecular analysis via RT-PCR demonstrated a notable downregulation of RANKL mRNA levels (Fig. S23) and a corresponding upregulation of OPG mRNA expression (Fig. S24) in the PUKMP group. These findings collectively indicate that the pulsatile PTH release generated by our KM@PTH system under pulsed ultrasound activation not only promotes osteogenesis but also creates a biochemical microenvironment that mildly suppresses osteoclast differentiation, thereby favoring a net anabolic state for bone regeneration.

A: Intracellular calcium ion concentrations in BMSCs assessed by flow cytometry across the five groups. B: Statistical analysis of intracellular calcium ion concentrations in the five groups. C: Western blot analysis of key proteins in the PKA pathway in BMSCs from the five groups. D: Quantification of protein expression of cAMP and PKA in the five groups. E: OCR test in BMSCs from the five groups. F: Statistical quantification of ATP content. G: JC-1 staining to assess mitochondrial membrane potential in BMSCs from the five groups. H: Expression Arg-1 and ODC1 in BMSCs from the five groups. I: Quantification of Arg-1 and ODC1 protein expression, ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ns indicates no significant difference, n = 5. J: Overview of the mechanisms by which PUKMP treatment promotes osteogenic differentiation in BMSCs, integrating calcium ion influx, mitochondrial activation, and efferocytosis modulation.

3. Discussion

The regeneration of deep-tissue osseous defects remains constrained by conventional osteoconductive biomaterials' inability to provide spatiotemporal regulation of osteogenic signals. We engineered a multifunctional piezoelectric composite scaffold (KNN-MBG/PTH) that synergistically couples ultrasound-responsive PTH pulsatility with inherent mechanoelectrical stimulation. This biomimetic platform achieves elaborate control over osteogenic microenvironments through piezoelectricity-mediated calcium ion oscillation, ultrasound-triggered PTH release kinetics mimicking physiological endocrine patterns, and dual physical stimulation enhancing mesenchymal condensation. In vivo evaluation demonstrated complete defect bridging with restored trabecular morphology in critical-sized femoral defects. The scaffold's inherent deep-tissue penetrance and self-regulating mechanopharmacological effects position this technology as a paradigm-shifting solution for anatomically constrained bone regeneration scenarios.

The scaffold's design rationale centers on KNN's exceptional piezoelectric performance and proven osseocompatibility, positioning it as an ideal mechanoresponsive smart material for bone regeneration. Under ultrasound excitation, KNN generates localized piezoelectric fields that establish a dynamic electrical microenvironment while transducing mechanical energy into precisely timed PTH release events [[26], [27], [28]]. This piezoelectric matrix synergizes with MBG's large surface area and mesoporous architecture [29] that achieves dual functionality: 1) providing nano-textured osteoconductive surfaces supporting cell adhesion/proliferation, and 2) maintaining high PTH payload capacity and controlled release [[30], [31], [32]]. The KNN-MBG composite creates a multiscale osteogenic regulation system [33] - while KNN's ultrasound-triggered mechanoelectrical activity drives calcium ion oscillation and PTH pulsatility, MBG's bioactive silicate network sustains mechanical integrity and mediates sustained ion exchange. This biomimetic spatiotemporal control over physical and biochemical cues synergistically made it a promising candidate for treating large bone defects. Future development will focus on establishing degradation-osteogenesis matching profiles through controlled MBG resorption kinetics while maintaining piezoelectric output, alongside scaling protocols for anatomical defect-specific scaffold fabrication.

The therapeutic efficacy of PTH in skeletal regeneration critically depends on recapitulating endogenous endocrine signaling patterns through temporally defined delivery [[34], [35], [36]]. Low-dose pulsatile administration enhances osteoblast differentiation and bone mineralization compared to continuous or high-dose delivery, while avoiding osteoclast activation and bone resorption observed under sustained release conditions [37]. Therefore, controlling the release pattern of PTH is crucial for its efficacy. To achieve this therapeutic precision, we engineered an ultrasound-responsive KNN transducer system capable of on-demand PTH liberation through mechanoelectrical conversion. The piezoelectric KNN material within the scaffold generates microcurrents when exposed to ultrasound-induced mechanical vibrations [38,39]. At the same time, this bioelectronic platform exploits piezoelectric polarization effects to overcome PTH's electrostatic binding to mesoporous carriers. Crucially, the hydrogel barrier demonstrated preserved therapeutic payload integrity while permitting ultrasound-triggered ionic conduction, achieving PTH delivery precision in tissue-equivalent models. The convergence of chronobiology principles with materials-driven spatiotemporal control establishes a new paradigm for anabolic bone therapy-particularly valuable in complex defect scenarios requiring depth-adaptable, cycle-programmable treatment modalities.

Treating deep bone defects—often located in poorly accessible and hypovascular sites such as the hip or spine—remains clinically challenging due to limited treatment accessibility and compromised regenerative microenvironments. In this study, we demonstrated through hydrogel-based barrier experiments that ultrasound can effectively penetrate and activate scaffolds at tissue depths of up to 20 cm, offering a promising strategy for non-invasive and targeted therapy in deep bone regeneration [40]. To address the critical unmet need for depth-adaptable bone regeneration strategies, we established a deep bone defect model in skeletally mature rabbit - a stringent testbed recapitulating the mechanical challenges of human deep-tissue defects. Histomorphometric quantification and μCT analysis demonstrated pulsatile ultrasound-activated PTH delivery induced greater bone formation versus static controls and other treatments. The scaffold's ultrasound penetrance capability enabled precise spatiotemporal control of PTH pharmacokinetics while maintaining therapeutic payload stability in deep bone defects, where conventional approaches might fall short [[41], [42], [43]]. This bioelectronic approach successfully translated piezoelectric material properties into clinically relevant anabolic responses, offering an innovative solution to accelerate bone healing in challenging anatomical sites, ultimately improving patient outcomes in bone defect repair.

By transcriptomic analysis of the distal femoral bone tissue in rabbits, we identified significant enrichment in calcium ion metabolism pathways, which emerged as the centreal regulatory node governing osteogenesis. Functional protein interrogation demonstrated pulsatile PTH administration coordinates Wnt/β-catenin axis activation with MAPK cascade modulation, establishing a calcium-dependent signaling cross-talk that amplified osteogenic transcriptional programming [[44], [45], [46], [47]]. Our results demonstrated that pulsatile low-dose PTH effectively enhanced calcium ion influx, supporting its role in promoting osteogenesis. The calcium wave propagation triggered second messenger hierarchy activation - PKA and PKC [48,49], in turn, promote osteoblast differentiation and bone mineralization. As we observed, significant upregulation of both PKA and PKC in response of pulsatile PTH treatment [[50], [51], [52]]. Crucially, the ultrasound-generated piezoelectric microenvironment and pulsatile PTH synergistically sustained mitochondrial calcium uptake and subsequently activates mitochondrial function, further accelerating osteogenesis. This multi-scale regulation—from ionic flux modulation to mitochondrial reprogramming—validates our biomaterial's capacity to recapitulate osteogenesis paradigms.

Our antibacterial assessments revealed that ultrasound-activated piezoelectric scaffolds generate electrical microfields capable of disrupting microbial colonization through microcurrent, and exhibit moderate antimicrobial activity, though not as strong as traditional antibiotics or disinfectants [[53], [54], [55]]. Certainly, the significant benefit of this microcurrent lies in its ability to inhibit bacterial adhesion, which is recognized as the first step and initiating factor in implant-related infections [56]. This approach, primarily targeting the initial phase of biofilm formation rather than treating established infections, provides a strategic advantage for preventing device-related contamination. This anti-adhesion mechanism - targeting the critical first phase of device-related infection - synergizes with the scaffold's osteoinductive functionality to create a dual-action therapeutic paradigm [57], especially for deep bone defect repair. The simultaneous inhibition of bacterial adhesion and promotion of mammalian cell adhesion is not contradictory, as these effects arise from the same piezoelectric microcurrents through cell-type-specific mechanisms. The electrostatic disruption preferentially targets prokaryotic bacterial membranes, effectively preventing initial attachment and biofilm formation, while the same bioelectric stimuli enhance eukaryotic cell functions by promoting calcium influx and activating pro-osteogenic signaling pathways such as PKA/PKC. This selective bioactivity is attributed to fundamental differences in membrane properties and metabolic responses between bacterial and mammalian cells [58,59]. We acknowledge that while this prophylactic strategy may not replace traditional antibiotics for treating established osteomyelitis, it represents a valuable first-line defense against infection initiation. This multifunctionality expands the clinical applicability of ultrasound-activated piezoelectric scaffolds, potentially improving patient outcomes by promoting bone healing while concurrently mitigating infection risks.

Our analysis in vitro reveals that the pulse ultrasound-activated KM@PTH scaffold, with its pulsatile release of low-dose PTH, creates a more efficient therapeutic “osteogenetic window” distinct from conventional continuous delivery system [60]. We observed that the conventional ultrasound-driven UKMP specimens showed paradoxical suppression of osteogenic effects despite higher cumulative PTH exposure compared to the KMP group without ultrasound activation. This biphasic response aligns with recent findings that sustained or excessive PTH concentration trigger negative effects, including premature osteoclastogenesis and bone formation inhibition [[61], [62], [63], [64]], though the PTH is known to enhance osteogeneis by stimulating osteoblast activation. The excess PTH release in the UKMP group could push the system beyond its optimal threshold, disrupting the balance between bone formation and absorption. Interestingly, though the osteogenic effect of the UKMP group was still superior to that of the control and PTH groups, it suggests that the piezoelectric KMP scaffold requires activation by pulsatile ultrasound to achieve the ideal therapeutic outcome [65]. Pulsatile PTH release, a key component of our strategy, has been shown to be crucial for maintaining this delicate balance, as it prevents the negative consequences of continuous high-dose exposure. This work establishes the pulsatile release mechanism as a paradigm-shifting approach for PTH precisely controlled release, achieving what traditional scaffolds cannot, and while avoiding the inhibitory effects of PTH overexposure [66].

Our mechanistic investigations at the cellular interface validated the anticipated regulatory role of calcium ion flux in coordinating PKA and PKC signaling cascades, corroborating observations from in vivo models. As hypothesized, the pulsatile PTH administration potentiated transmembrane calcium ion influx in BMSCs, thereby activating both PKC-dependent and PKA-mediated phosphorylation circuits - critical second messengers governing osteogenic commitment [[67], [68], [69]]. Notably, this signal transduction aligns with established paradigms of low-dose PTH-enhanced osteodifferentiation through cAMP/PKA and phospholipase C/PKC cross-talk. The interdependency between calcium ion homeostasis and mitochondrial bioenergetics emerged as a critical nexus, given the cation's dual role as a metabolic modulator and osteogenic promoter [70]. Furthermore, the same pulsatile PTH release that enhanced osteogenesis also contributed to a mild suppression of osteoclastogenesis, as evidenced by our in vitro differentiation assays, thereby further tilting the bone remodeling balance toward net formation. The metabolic reprogramming establishes an anabolic state favoring precursor mobilization and mineral matrix deposition in osteoprogenitors. Collectively, these data delineate a self-reinforcing triad of calcium oscillations, kinase signaling fidelity, and mitochondrial metabolic priming that sustains osteogenic differentiation under pulsatile PTH regimens.

Notably, our studies uncovered a critical regulatory interplay between efferocytosis and mitochondrial ultrastructure remodeling in BMSCs, with direct implications for skeletal tissue regeneration [71]. Building on evidence linking augmented efferocytic activity to aberrant mitochondrial fission - a pathological state inversely correlated with osteogenic capacity [[72], [73], [74]] - we detected pronounced mitochondrial fragmentation in hyper-efferocytic BMSCs, concomitant with compromised osteoblast lineage commitment. Mechanistically, excessive fission-mediated mitochondrial hyperdynamic turnover disrupts cristae architecture and electron transport chain (ETC) fidelity, depleting ATP reservoirs essential for osteoid synthesis while amplifying ROS flux-dependent suppression of RUNX2 nuclear translocation [75]. These findings crystallize a dual-edged paradigm: while basal efferocytosis sustains tissue homeostasis through phagocytic clearance, its pathological amplification induces mitochondrial destabilization, effectively uncoupling cellular metabolism from osteogenic transcription programs. Therapeutically, our data advocate for balanced modulation of mitochondrial quality control systems - strategies preserving mitochondrial network plasticity while preventing fission-mediated bioenergetic collapse will likely potentiate PTH-driven bone anabolism.

The mechanoelectrical activation paradigm of KM@PTH demonstrates distinct translational advantages over photodynamic drug release systems reliant on near-infrared (NIR) irradiation. First, ultrasound waves exhibit superior tissue penetrance (20–30 cm depth) compared to NIR light (<5 cm in cortical bone) [76], enabling precise modulation of both piezoelectric currents and PTH release kinetics in deep anatomical sites, a capability critical for treating clinically challenging defects in vertebrae or femoral heads [77]. Second, the bimodal therapeutic action of ultrasound, concurrently generating osteogenic microcurrents via piezoelectric depolarization while triggering pulsatile PTH liberation, creates a synergistic regenerative microenvironment unattainable by NIR systems [78,79]. This dual functionality not only enhances calcium-dependent mineralization through sustained electromechanical stimulation but also provides inherent antibacterial effects via piezoelectric potential-mediated microbial membrane destabilization. In contrast, NIR-responsive scaffolds remain limited to unidirectional pharmacologic control without concurrent physical modulation of the healing niche. The convergence of deep-tissue energy transduction with multifunctional therapeutic outputs positions ultrasound-activated piezoelectric systems as a paradigm-shifting alternative to optically controlled platforms for complex skeletal reconstruction.

4. Conclusion

In summary, this work establishes a bioengineered paradigm leveraging the convergence of piezoelectric biomaterial engineering and pulsatile paracrine signaling to address critical challenges in deep bone regeneration. The ultrasound-responsive KMP scaffold, synergized with spatiotemporally controlled PTH pulsatility, orchestrates mechanobiological signaling cascades—including calcium ion-dependent mechanotransduction, mitochondrial metabolic priming, and PKA/PKC-mediated osteogenic commitment - to potentiate BMSC-driven osteoregeneration. Notably, the piezoelectricity-driven microcurrents not only enable on-demand PTH release kinetics but also amplify endogenous bioelectric cues, fostering a pro-osteogenic niche with enhanced extracellular matrix mineralization in deep defects. Critically, this dual-action platform demonstrates prophylactic functionality against bacterial adhesion, addressing the persistent clinical dilemma of infection risk in open fracture repair. Our data position this strategy as a translatable solution for anatomically complex defects where conventional grafts fail to reconcile mechanical support, dynamic signaling, and antimicrobial defense. These findings provide a translational roadmap for recalcitrant osseous defects, bridging material-driven topographical cues with endocrine-like pulsatile signaling. Future efforts integrating patient-specific piezoelectric parameters and scalable manufacturing protocols could unlock clinically viable regenerative platforms, advancing the frontier of precision bone tissue engineering.

5. Experimental section

5.1. Synthesis procedure of KNN-MBG-PTH

The synthesis of the KNN-MBG-PTH material begins with the preparation of a photocrosslinkable, 3D-printable mesoporous bioactive glass (MBG) slurry. Methacrylated F127 is first synthesized as the organic framework for the slurry. Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O) is then dissolved in ethanol and reacted to produce the MBG precursor. KNN nanowires are synthesized using a solid-state reaction. Potassium chloride (KCl, 37.275 g), potassium carbonate (K₂CO₃, 3.45 g), and niobium pentoxide (Nb₂O₅, 13.29 g) are thoroughly ground, then calcined at 1000 °C for 3 h in a crucible. The resulting precursor is treated in a 2 M nitric acid (HNO₃) solution, washed with deionized water, and dried at 60 °C for 12 h. A secondary solid-state reaction is performed by mixing 1 g of the treated precursor with sodium carbonate (Na₂CO₃, 3.35 g), sodium chloride (NaCl, 0.3 g), KCl (3.35 g), and K₂CO₃ (0.34 g), followed by calcination at 850 °C for 10 min to yield high-performance, biodegradable KNN nanowires. The synthesized KNN nanowires are incorporated into the MBG slurry and homogenized to form a uniform mixture. A photoinitiator is added to produce a photocrosslinkable ink for 3D printing. During printing, the material is crosslinked by light, and the printed constructs are sintered at high temperatures to achieve mesoporous and crystalline structures. Finally, the scaffold is polarized to generate a negatively charged surface, promoting calcium deposition. The mesoporous structure also enables the loading of bioactive factors such as PTH.

5.2. Piezoelectric property detection

The piezoelectric properties of the KMP scaffolds were evaluated using a standard electrical measurement setup. KMP scaffolds were placed on a custom-designed testing platform with electrodes attached at both ends. A mechanical stress was applied to the scaffolds using a universal testing machine (Instron) to induce piezoelectricity. The generated voltage was measured in response to different applied pressures, and the piezoelectric output was recorded using a digital voltmeter. The piezoelectric coefficients (d33) were calculated based on the voltage generated under mechanical stress.

Additionally, the piezoelectric response under ultrasonic stimulation was examined by applying continuous and pulsed ultrasound to the scaffolds in a water bath using an ultrasonic generator (Biorad). The piezoelectric response was measured during ultrasound application, and the results were compared with the mechanical stress-induced piezoelectric response. The experiments confirmed that the piezoelectric scaffolds produced a significant microcurrent under both mechanical and ultrasonic stimulation, supporting the mechanism of PTH release in the in vivo experiments.

5.3. New Zealand rabbit distal femoral bone defect model

The bone defect model was established in New Zealand rabbits, with a cylindrical defect of 5 mm in diameter and 1 mm in height created in the distal femur. After the surgery, the rabbits were randomly divided into four groups: Control group, KMP group, UKMP group, and PUKMP group, each with 5 rabbits. The KMP group received the KMP scaffold, the UKMP group received the KMP scaffold followed by conventional ultrasound treatment, and the PUKMP group received the KMP scaffold with pulsed ultrasound treatment (5-min intervals, 1 h per day for one week). After the defect was created, the scaffolds were implanted into the bone defects, and the corresponding ultrasound treatments were performed. The rabbits were euthanized at specified time points, and femoral tissues were harvested for further analysis.

5.4. Histological analysis of femoral distal bone defects

After 3 months of treatment, the femoral distal bone defect sites were harvested from New Zealand rabbits for histological analysis. The samples were fixed in 10 % formalin at room temperature for 24 h and decalcified in EDTA solution for approximately 4 weeks. Once decalcified, the tissues were dehydrated through a graded ethanol series (70 %, 80 %, 90 %, 100 %) and embedded in paraffin.

The paraffin-embedded tissues were sectioned into 5-μm thick slices using a microtome. The sections underwent Hematoxylin and Eosin (H&E) staining to evaluate the general morphology of the bone and surrounding tissues. H&E staining revealed bone structure, osteoid formation, and cellular infiltration at the defect site. For further analysis of collagen deposition and mineralization, Masson's trichrome staining was performed. This staining method highlighted collagen fibers in blue and mineralized bone in red, allowing the examination of osteogenesis and extracellular matrix remodeling.

For mitochondrial ultrastructure analysis, transmission electron microscopy (TEM) was employed. Small tissue blocks were fixed in 2.5 % glutaraldehyde, post-fixed in 1 % osmium tetroxide, and embedded in epoxy resin. Ultra-thin sections were cut and stained for TEM observation, providing detailed insights into mitochondrial integrity and density. These histological analyses helped evaluate bone formation, tissue regeneration, and cellular function, crucial for understanding the mechanisms involved in osteogenesis.

5.5. BMSCs and MRSA culture

BMSCs (human bone marrow mesenchymal stem cells, HBMSCs) were obtained commercially from ScienCell Research Laboratories. The cells were cultured in standard α-MEM (Gibco) supplemented with 20 % fetal bovine serum (FBS) and maintained in a humidified incubator at 37 °C with 5 % CO₂. After reaching appropriate confluence, the cells were passaged, and third-passage cells were used for subsequent experiments to ensure experimental consistency and cell function stability.

MRSA (Methicillin-resistant Staphylococcus aureus) was cultured in Luria-Bertani (LB) broth at 37 °C with shaking to ensure proper bacterial growth. The bacterial culture was harvested and adjusted to the desired concentration using phosphate-buffered saline (PBS). This bacterial suspension was then used in antimicrobial assays to evaluate the effects of the piezoelectric scaffold and ultrasound treatment on bacterial inhibition.

5.6. PTH release experiment

The PTH release from the KMP scaffolds was evaluated under different experimental conditions to assess the pulsatile release mechanism. To simulate the physiological conditions, PTH (0.1 mg/mL) was loaded onto the KMP scaffolds and placed in a 37 °C incubator with continuous shaking to mimic the physiological fluid environment. For the release experiment, the scaffolds were exposed to either continuous ultrasound or pulsed ultrasound for 1 h daily, with varying pulse durations (5 min intervals) for the pulsed ultrasound group. A commercially available gelatin-based hydrogel (Gel-Matrix), which mimics soft tissue and deep tissue characteristics, was used to simulate the tissue barrier and assess the depth penetration capability of the PTH release.

The release of PTH from the scaffolds was monitored at predetermined time points (1, 2, 4, 6, 8, 12 h) by sampling the surrounding media. PTH concentration was measured using an enzyme-linked immunosorbent assay (ELISA). To examine the influence of the hydrogel barrier, the release profiles from scaffolds placed in direct medium versus those covered with the hydrogel were compared. Additionally, the mechanical vibration-induced PTH release from the scaffolds was assessed under the influence of ultrasound, demonstrating that even with the hydrogel barrier, the pulsed ultrasound was able to maintain the pulsatile release of PTH. This result further supports the hypothesis that ultrasound-induced piezoelectric effects can effectively control the release of PTH, even through deep tissue barriers.

5.7. Flow cytometry

Intracellular calcium concentration and mitochondrial membrane potential were assessed using flow cytometry in BMSCs under different treatment conditions. To measure intracellular calcium levels, BMSCs (passage 3) were cultured and exposed to different experimental groups: Control, PTH, KMP, UKMP, and PUKMP. After 24 h of treatment, cells were incubated with Fluo-4 AM (Thermo Fisher, Cat. No. F-14217), a calcium-sensitive fluorescent dye, at a final concentration of 1 μM for 30 min at 37 °C. The cells were then washed with phosphate-buffered saline (PBS) and analyzed by flow cytometry (BD FACSCalibur) to measure calcium fluorescence intensity, which reflects intracellular calcium concentration. The data were processed using FlowJo software to quantify the relative calcium influx.

For mitochondrial membrane potential detection, BMSCs were incubated with JC-1 (Beyotime, Cat. No. C2006) for 30 min at 37 °C according to the manufacturer's instructions. JC-1 is a lipophilic dye that aggregates in healthy mitochondria with high membrane potential, resulting in a red fluorescence, while in depolarized mitochondria, it remains in a monomeric form, emitting green fluorescence. After incubation, cells were analyzed by flow cytometry, and the red-to-green fluorescence ratio was calculated to determine the mitochondrial membrane potential. Data analysis was conducted using FlowJo software to compare mitochondrial activity across different experimental groups. This experiment enabled us to evaluate the mitochondrial function in response to the pulsed ultrasound treatment and the PTH release system.

5.8. Osteogenic differentiation assay

For osteogenic differentiation, BMSCs were cultured in DMEM containing 10 % FBS, 100 nM dexamethasone (Sigma), 50 μg/mL ascorbic acid (Sigma), and 5 mM β-glycerophosphate-1 (Sigma). After 7 days, cells were stained using a BCIP/NBT alkaline phosphatase color reagent kit (Beyotime) to evaluate alkaline phosphatase activity. After 14 days, cells were stained with 0.2 % alizarin red (pH 4.2, Sigma-Aldrich) to assess mineralization.

5.9. Mitochondrial isolation and quantification

The method for isolating mitochondria from cells was optimized according to the manufacturer's protocol (Solarbio). Conditioned cells were washed with PBS and collected. Ice-cold lysis buffer was added to resuspend the cells, which were lysed with a tissue homogenizer on ice. Mitochondria were isolated by gradient centrifugation, and mitochondrial protein concentration was determined using a BCA protein quantification kit.

5.10. RNA extraction and sequencing for femoral distal tissue

Total RNA was extracted from the femoral distal tissues of New Zealand rabbits using TRIzol reagent (Takara). The RNA samples were further purified, and poly(A) mRNA was isolated from 1 mg of total RNA using a Poly(A) mRNA Magnetic Isolation Module (Thermo Scientific). RNA sequencing libraries were constructed using NEB Next Ultra II Directional RNA Library Preparation Kits (New England Biolabs), followed by sequencing on an Illumina NovaSeq 6000 platform with paired-end 150-bp reads.

The raw sequencing data underwent quality control using Trim Galore (v.0.6.7) to remove low-quality bases and adapter sequences. The reads were aligned to the rabbit reference genome (OryCun2.0) using HISAT2 (v.2.1.0). Gene expression levels were quantified using featureCounts (v.2.0.1), and differential gene expression analysis was conducted with DESeq2 (v.1.32.0). All RNA-seq data generated are publicly available at GEO (GSE198806) for further exploration and analysis. This transcriptomic analysis provided valuable insights into the gene expression profiles of bone tissue during healing, particularly in response to the pulsatile PTH treatment and piezoelectric scaffold intervention.

5.11. Statistical analysis

In vitro experiments were repeated at least three times, and in vivo experiments were repeated at least six times. All the data are presented as the mean and standard deviation. GraphPad Prism v9.0.0 (La Jolla, CA, USA) was used for statistical analysis. Statistical significance was calculated using one-way Analysis of Variance (ANOVA) and Student's t-test and is denoted as ∗ (p < 0.05) or ∗∗ (p < 0.01).

CRediT authorship contribution statement

Xin Wang: Writing – original draft, Validation, Investigation, Formal analysis. Linyuan Shu: Writing – review & editing, Investigation, Data curation. Bohao Yin: Visualization, Resources, Investigation, Formal analysis. Jianing Ding: Validation, Software, Investigation, Data curation. Chenjun Liu: Resources, Methodology, Investigation. Xin Qi: Validation, Resources, Formal analysis. Junjie Guan: Visualization, Software, Methodology. Yuwei Ge: Project administration, Funding acquisition, Conceptualization. Xiaofeng Lian: Supervision, Funding acquisition, Conceptualization. Hui Sun: Writing – review & editing, Supervision, Conceptualization, Formal analysis, Funding acquisition. Wei Zhang: Writing – review & editing, Supervision, Project administration, Conceptualization.

Ethics approval and consent to participate

All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health, and all procedures were approved by the Animal Research Committee of the Sixth People's Hospital at Shanghai Jiao Tong University (approval No.: 2023-0734).

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 is the Supplementary data to this article: