3D-printed zinc oxide nanoparticles modified barium titanate/hydroxyapatite ultrasound-responsive piezoelectric ceramic composite scaffold for treating infected bone defects

Abstract

Clinically, infectious bone defects represent a significant threat, leading to osteonecrosis, severely compromising patient prognosis, and prolonging hospital stays. Thus, there is an urgent need to develop a bone graft substitute that combines broad-spectrum antibacterial efficacy and bone-inductive properties, providing an effective treatment option for infectious bone defects. In this study, the precision of digital light processing (DLP) 3D printing technology was utilized to construct a scaffold, incorporating zinc oxide nanoparticles (ZnO-NPs) modified barium titanate (BT) with hydroxyapatite (HA), resulting in a piezoelectric ceramic scaffold designed for the repair of infected bone defects. The results indicated that the addition of ZnO-NPs significantly improved the piezoelectric properties of BT, facilitating a higher HA content within the ceramic scaffold system, which is essential for bone regeneration. In vitro antibacterial assessments highlighted the scaffold's potent antibacterial capabilities. Moreover, combining the synergistic effects of low-intensity pulsed ultrasound (LIPUS) and piezoelectricity, results demonstrated that the scaffold promoted notable osteogenic and angiogenic potential, enhancing bone growth and repair. Furthermore, transcriptomics analysis results suggested that the early growth response-1 (EGR1) gene might be crucial in this process. This study introduces a novel method for constructing piezoelectric ceramic scaffolds exhibiting outstanding osteogenic, angiogenic, and antibacterial properties under the combined influence of LIPUS, offering a promising treatment strategy for infectious bone defects.

Keywords: Antibacterial therapy; Bone regeneration; Low-intensity pulsed ultrasound; Piezoelectric ceramics; Zinc oxide nanoparticles.

Graphical abstract

Fig. 1

Characterization of different groups of piezoelectric ceramic samples A: The preparation processes of the 3D printed piezoelectric ceramic simples. B: Panoramic images of the 3D printed ceramic scaffolds. C: The SEM images of the 3D printed ceramic simples. D: The EDS mapping of the 3D printed ceramic simples. E: The EDX mapping of the 3D printed ceramic simples. F: The XRD pattern of the 3D printed ceramic simples. G: The water contact angle of the 3D printed piezoelectric ceramic simples at a RT. H: The morphology, amplitude and phase of the 3D printed piezoelectric ceramic simples. I: The relative dielectric (εr) of the 3D printed piezoelectric ceramic simples at a frequency of 1 KHz. J: The d₃₃ of the 3D printed piezoelectric ceramic simples.

Fig. 2

In vitro antibacterial properties of different groups of piezoelectric ceramic samples A: The process of antibacterial experiment of ceramic samples. B: The results of plate counting of the 3D printed ceramic simples. C: The quantitative results of CFU of the 3D printed ceramic simples. D: The live/dead bacterial staining of the 3D printed ceramic simples, green represents living bacteria and red represents dead bacteria. E: The quantitative proportion of dead bacteria of the 3D printed ceramic simples. F: The bacterial morphology scanning of the 3D printed piezoelectric ceramic simples. G: The plot growth curves of S. aureus after co-culture with 3D printed ceramic simples. H: The plot growth curves of E. coli after co-culture with 3D printed ceramic simples.

Fig. 3

In vitro biosafety of different groups of piezoelectric ceramic samples A: The process of in vitro biosafety test of ceramic samples. B: The results of CCK-8 assay of the 3D printed ceramic simples. C: The live/dead cell staining of the 3D printed ceramic simples, green represents living cells and red represents dead cells. D: The quantitative proportion of dead cells of the 3D printed ceramic simples. E: F-actin and nucleus results of DPSCs after 3 days of co-culture with different groups of ceramic samples, green represents F-actin and blue represents dead nucleus. F: Quantitative results of C/N ratio.

Fig. 4

In vitro osteogenic properties of different groups of piezoelectric ceramic samples A: The process of in vitro osteogenic properties test of ceramic samples. B: ALP staining assays results of DPSCs after 7 days of co-culture with different groups of ceramic samples. C: Color comparison of eluents harvested from different groups of ceramic samples after ALP staining assays. D: The quantitative analysis of eluents harvested from different groups of ceramic samples after ALP staining assays. E: Immunofluorescence staining assays results of DPSCs after 7 days of co-culture with different groups of ceramic samples (red for OPN, green for OCN, blue for nucleus). F: The quantitative analysis results of immunofluorescence staining of different groups of ceramic samples.

Fig. 5

In vitro angiogenic properties of different groups of piezoelectric ceramic samples A: The process of in vitro angiogenic properties test of ceramic samples. B: The cell scratch assay results of DPSCs in different groups of ceramic samples. C: The quantitative analysis of wound healing rate results in different groups of ceramic samples at different time points. D: The quantitative analysis of eluents harvested from different groups of ceramic samples after ALP staining assays. D: Immunofluorescence staining assays results of DPSCs after 7 days of co-culture with different groups of ceramic samples (red for VEGF, green for HIF-1α, blue for nucleus). E: The quantitative analysis results of immunofluorescence staining of different groups of ceramic samples.

Fig. 6

The results of transcriptomics analysis of osteogenesis A: The process of transcriptomics analysis of osteogenesis. B: Differential gene volcano plot of LP-ZnO@BT/HA group and control group. C: Differential gene heat map of LP-ZnO@BT/HA group and control group. D: Heat maps of some osteogenic differential genes in LP-ZnO@BT/HA group and control group. E: The KEGG enrichment of all differential gene. F: The KEGG enrichment of upregulated and downregulated genes (blue indicating downregulation, red indicating upregulation). G: Signaling pathways associated with osteogenesis analyzed in part by GSEA enrichment.

Fig. 7

In vivo osteogenic properties of different groups of piezoelectric ceramic samples A: The process of in vivo osteogenic properties test of ceramic samples. B: Two-dimensional images of bone defects combined with ceramic scaffolds and 3D reconstruction of the new bone tissue with/without ceramic scaffolds. C: The quantitative results of BV/TV of the 3D printed ceramic simples. D: The quantitative results of Tb.Th of the 3D printed ceramic simples. E: The quantitative results of Tb.N of the 3D printed ceramic simples. F: The quantitative results of Tb. Sp of the 3D printed ceramic simples. G: The H&E staining results of the 3D printed ceramic simples. H: The Masson's trichrome staining results of the 3D printed ceramic simples.

Fig. 8

Results of immunofluorescence staining of osteogenesis and angiogenesis in vivo A: TGF-β and ANG-1 protein expression in different ceramic scaffold groups (red for TGF-β, green for ANG-1, blue for nucleus). B: The quantitative analysis of TGF-β and ANG-1 protein expression in different ceramic scaffold groups. C: VEGF and HIF-1α protein expression in different ceramic scaffold groups (red for VEGF, green for HIF-1α, blue for nucleus). D: The quantitative analysis of VEGF and HIF-1α protein expression in different ceramic scaffold groups. E: Runx2 and COL1 protein expression in different ceramic scaffold groups (red for Runx2, green for COL1, blue for nucleus). F: The quantitative analysis of Runx2 and COL1 protein expression in different ceramic scaffold groups. G: OPN and OCN protein expression in different ceramic scaffold groups (red for OPN, green for OCN, blue for nucleus). H: The quantitative analysis of OPN and OCN protein expression in different ceramic scaffold groups.