Multi-omics analysis of oral bacterial biofilm on titanium oxide nanostructure modified implant surface: In vivo sequencing-based pilot study in beagle dogs

Peri-implantitis, the major cause of implant failure, is an inflammatory destructive disease due to the dysbiotic polymicrobial communities at the peri-implant sites. Therefore, it is highly warranted to develop the implant materials with antimicrobial properties and investigate their effects on oral microbiota. However, most of the relevant studies were performed in vitro, and insufficient to provide the comprehensive assessment of the antimicrobial capacity of the implant materials in vivo. Herein, we introduce an innovative approach to evaluate the in vivo antibacterial properties of the most commonly used implant materials, titanium with different nanostructured surfaces, and investigate their antibacterial mechanism via the next-generation sequencing (NGS) technology. We firstly prepared the titanium implants with three different surfaces, i) mechanical polishing (MP), ii) TiO(2) nanotubes (NT) and iii) nanophase calcium phosphate embedded to TiO(2) nanotubes (NTN), and then characterized them using scanning electron microscopy (SEM), energy-dispersive X-ray spectrometer (EDS), X-ray photoelectron spectroscopy (XPS), confocal laser scanning microscopy (CLSM) and surface hydrophilicity analysis. Afterwards, the implants were placed in the beagle dogs’ mouths to replace the pre-extracted premolar and molar teeth for eight weeks through implant surgery. The supra- and sub-mucosal plaques were collected and subjected to 16S rRNA gene/RNA sequencing and data analysis. It was found that the nanostructured surfaces in NT and NTN groups showed significantly increased roughness and decreased water contact angles compared to the MP group, while the XPS data further confirmed the successful modifications of TiO(2) nanotubes and the subsequent deposition of nanophase calcium phosphate. Notably, the nanostructured surfaces in NT and NTN groups had limited impact on the diversity and community structure of oral microbiota according to the 16S rRNA sequencing results, and the nanostructures in NTN group could down-regulate the genes associated with localization and locomotion based on Gene Ontology (GO) terms enrichment analysis. Moreover, the differentially expressed genes (DEGs) were associated with microbial metabolism, protein synthesis and bacterial invasion of epithelial cells. Taken together, this study provides a new strategy to evaluate the antibacterial properties of the biomedical materials in vivo via the high-throughput sequencing and bioinformatic approaches, revealing the differences of the composition and functional gene expressions in the supra- and sub-mucosal microbiome.