Sono-Activable Biocatalytic 3D-Printed Scaffolds for Sequential Osteosarcoma Therapy and Bone Regeneration

Osteosarcoma is a highly malignant bone tumor that primarily affects children and adolescents, predominantly in long bones. It is highly invasive, causing bone destruction, severe pain, disability, and even distant metastasis, necessitating combined systemic drug therapy and surgical intervention. Current mainstream limb-salvage therapies include complete tumor resection, bone defect reconstruction, and adjuvant chemo- or radiotherapy, which ideally remove the tumor while restoring limb function. However, the common chemoradiotherapy resistance of osteosarcoma often prevents microscopically margin-negative (R0) resections, leading to local recurrence and limb-salvage failure, frequently requiring secondary or multiple invasive surgeries, thereby severely compromising patient prognosis.

Biomimetic 3D-printed composite scaffolds offer a promising strategy for osteosarcoma limb-salvage therapy. Among them, calcium phosphate (CaP) ceramic scaffolds, such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), are prominent substitutes for bone grafts due to their close similarity to natural bone mineral composition, excellent biocompatibility, osteoconductivity, and osteoinductivity. However, osteosarcoma invasion, tumor metabolism-induced hyperactive osteolysis, and surgical resection create a bone-defect microenvironment with poor regenerative capacity, exceeding the intrinsic repair capability of these scaffolds. Existing scaffold modification strategies, including incorporation of bioactive factors or stem cells, partially address this issue but cannot simultaneously achieve tumor eradication and bone regeneration. They also lack intelligent microenvironmental adaptability, involve complex material design, and exhibit limited regenerative efficiency.

Recurrence of osteosarcoma or inflammatory trauma following limb-salvage surgery leads to markedly elevated H₂O₂ levels and hypoxic microenvironments in the defect site, disrupting redox homeostasis and causing sustained oxidative stress to endogenous stem cells, which severely impairs bone regeneration. Ideally, bone-defect repair materials should enable intelligent H₂O₂ catalysis: converting H₂O₂ into highly toxic reactive oxygen species (ROS) in the tumor microenvironment to induce tumor-cell apoptosis, while decomposing H₂O₂ into O₂ in the inflammatory bone-defect microenvironment to alleviate inflammation and restore osteogenesis–osteoclastogenesis balance. Current nanozyme-based biocatalytic materials, including metal oxides and noble-metal nanoparticles, are unable to simultaneously achieve intelligent H₂O₂-to-ROS and H₂O₂-to-O₂ transformation. Moreover, existing therapeutic strategies primarily focus on either antitumor or anti-inflammatory functions. No multifunctional biocatalytic materials have yet been developed specifically for osteosarcoma limb-salvage therapy. Therefore, designing versatile biocatalytic materials capable of intelligently eradicating tumors and restoring redox homeostasis represents a critical need for improving limb-salvage treatment.

In this study, we report, for the first time, the design and construction of sono-activable biocatalytic nanoparticle-modified 3D-printed hydroxyapatite scaffolds (HS-ICTO). By employing TiO₂ nanomaterials as semiconductor substrates, Ir clusters (ICTO) with Ti–O–Ir electronic coupling were engineered to achieve spatiotemporally controllable H₂O₂ catalysis. This enables efficient ROS generation for tumor eradication within the osteosarcoma microenvironment, while decomposing H₂O₂ into O₂ in the bone-defect microenvironment to promote bone regeneration. This platform realizes intelligent sequential therapy from tumor eradication to bone defect repair. The scaffold’s in vitro antitumor efficacy, oxidative stress modulation, osteoclastogenesis inhibition, as well as in vivo tumor suppression and bone defect reconstruction, were systematically evaluated, and its underlying catalytic mechanisms were elucidated.

HS-ICTO scaffolds with varying ICTO loadings (HS-ICTO-x, x = 0, 0.5, 1.0, 2.0 mg/mL) were prepared by wet evaporation-deposition of ICTO nanoparticles onto 3D-printed HA scaffolds (HS). Scaffold structures were comprehensively characterized using field-emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and X-ray absorption spectroscopy (XAS). Results showed that ICTO nanoparticles formed highly dispersed Ir clusters (~1.7 nm) on the TiO₂ surface, creating Ti–O–Ir chemical coupling and interfacial charge transfer. HS-ICTO maintained the interlocked, porous architecture of HS, with uniform distribution of Ti, Ir, Ca, P, and O elements. Importantly, ICTO deposition did not compromise the mechanical properties of HS (compressive strength: 2.02 ± 0.15 MPa), comparable to human trabecular bone. The ICTO nanoparticles exhibited excellent surface stability, with detachment occurring only under mechanical scraping or external energy disturbance.

Figure 2 | Morphology and structural characterization of HS-ICTO. a: Schematic diagram of the HS-ICTO scaffold from millimeter to atomic scale (gray: O; cyan: Ti; yellow: Ir); b: Representative SEM image of HS-ICTO; c, d: High-magnification SEM images of HS-ICTO; e: TEM image of ICTO; f: EDS element mapping of ICTO; g: High-resolution TEM image of ICTO; h: Ultra-high-magnification TEM image of ICTO; i: HAADF-STEM image of ICTO and corresponding FFT spectrum; j: XPS spectra of ICTO and Ir/C in the Ir 4f region; k: XPS spectra of ICTO and TO in the Ti 2p region; l: Normalized XANES spectrum of Ir L3 edge; m: k³-weighted Fourier transform spectrum of Ir L-edge EXAFS spectrum; n: Wavelet transform of k³-weighted EXAFS signal. R is the distance between the adsorbed atom and its neighboring atom, χ(k) is the EXAFS oscillation amplitude as a function of the photoelectron wavenumber k, and the color gradient is from orange (high signal intensity) to cyan (low signal intensity).

In this study, we successfully designed and fabricated ultrasound-activatable and biocatalytic 3D-printed hydroxyapatite scaffolds (HS-ICTO) using direct ink writing (DIW) bioprinting. Based on the concept of redox medicine and targeting H₂O₂ as a shared therapeutic node, HS-ICTO achieved intelligent sequential therapy for osteosarcoma eradication and bone defect regeneration. The main findings are summarized as follows:

1. In Vivo Antitumor Efficacy: The Ti–O–Ir interfacial chemical coupling and charge transfer of HS-ICTO are central to its multi-enzyme-like catalytic activity, enabling POD/OXD-like activity in the mildly acidic tumor microenvironment. Combined with ultrasound activation, HS-ICTO efficiently generates reactive oxygen species (ROS), depletes glutathione (GSH), and alleviates hypoxia, thereby disrupting tumor redox homeostasis and mitochondrial structure. This results in targeted, efficient osteosarcoma cell killing, with a significant in vivo tumor inhibition rate of 90.43% and excellent biosafety.

2. Redox Regulation and Stem Cell Protection: In the neutral bone defect microenvironment, HS-ICTO exhibits strong CAT-like activity, rapidly scavenging excessive H₂O₂ and continuously generating O₂. This effectively blocks H₂O₂-mediated oxidative stress, protecting bone marrow mesenchymal stem cell (BMSC) viability, migration, and osteogenic differentiation. Furthermore, by inhibiting H₂O₂/HIF-1α-dependent osteoclast differentiation signaling, HS-ICTO reduces osteoclast formation and bone resorption, restoring the balance between osteogenesis and osteoclastogenesis.

3. Bone Defect Regeneration: HS-ICTO preserves the porous structure and mechanical properties of 3D-printed hydroxyapatite scaffolds, matching human trabecular bone. The ICTO nanoparticles demonstrate excellent loading stability, enabling long-term catalytic activity in vitro and promoting new bone formation in rat cranial defects in vivo. HS-ICTO significantly enhances bone volume, bone mineral density, and trabecular number, improving the regenerative microenvironment of bone defects.

4. Integrated and Intelligent Therapy: HS-ICTO achieves a unified, intelligent strategy of “ultrasound-activated tumor eradication and biocatalytic bone regeneration,” eliminating the need for multiple invasive surgeries. This provides a novel approach for precise osteosarcoma therapy and tissue regeneration and offers important guidance for designing multifunctional bone implant materials.

Future work will focus on exploring the long-term biocompatibility of HS-ICTO and validating its efficacy in orthotopic osteosarcoma models and large animal bone defect models. Combined with clinical techniques such as high-intensity focused ultrasound (HIFU), this strategy could enable periodic local ultrasound activation to clear residual tumors postoperatively, reducing systemic toxicity and adverse effects.

• Journal: Nature Communications

• DOI: https://doi.org/10.1038/s41467-025-61377-x

• First Authors: Rong Xiao, Sutong Xiao (co-first authors)

• Corresponding Authors: Boqing Zhang, Li Qiu, Chong Cheng

• Affiliations: West China Hospital, Sichuan University; School of Polymer Science and Engineering, Sichuan University; School of Biomedical Engineering, Sichuan University, China

• Core Keywords: osteosarcoma, 3D-printed hydroxyapatite scaffold, ultrasound-activatable, biocatalytic, nanozyme, bone defect regeneration, sequential therapy

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