Total RNA-sequencing Detects Transcriptomic Differences in Mouse Preosteoblast Response to Bulk Nanostructured Titanium.

Rational: Titanium is the most widely used alloy family in dental and orthopedic implants due to its natural ability to integrate into bone, but cytotoxic alloying elements are required in titanium for its mechanical properties to match the functionality of natural bone in high-load bearing applications. Recent advances in nanostructuring, such as Continuous Equal Channel Angular Pressing (C-ECAP), reduce the grain size and increase the strength of pure grades of titanium, thereby eliminating the need for cytotoxic elements and increasing cytocompatibilty as measured by traditional cell biology techniques. Transcriptomic profiling of cells grown on conventional coarse grain (CG) versus nanostructured ultrafine grain (UG) surfaces can simultaneously enhance our understanding of genomics and biomaterials, facilitating the development of a new generation of implantable materials. Objective: We initiated this pilot project to develop a workflow for transcriptional profiling of both protein coding and non-coding RNAs (ncRNAs) in cells grown on Ti discs that are characterized for grain size, 12.1 μm for CG Ti and 0.28 μm for UG Ti. Methods: Two Ti substrates were designed for cell growth in 24-well culture dishes, CG and UG. Mouse pre-osteoblasts were cultured on Ti disc for 72 hours. Cells were harvested and total-RNA extracted. Ribosomal RNA subtraction was performed prior to Illumina sequencing library construction. Single-end 50 base pairs reads were subjected to Illumina quality control and the resulting reads were mapped to the mouse genome and annotations with Genomic Short-read Nucleotide Alignment Program (GSNAP) and HTseq-count. Read count files were normalized and statistically analyzed with empirical analysis of digital gene expression data in R (EdgeR) and filtered for low expression levels using Zipf’s Power Law. Transcripts with adequate coverage and significant expression differences were subjected to GO Biological Process term enrichment and network analyses with the Cytoscape App ClueGO. Results: Gene expression differences with an FDR≤0.05 are limited to six genes, five of which are likely ncRNAs. Of the 432 transcripts with significant differences at p≤0.05 117 form an interconnected network of genes and 12 GO biological processes (P(adj) ≤ 0.05). Biological processes that connect to predominately upregulated genes include cell division, mRNA metabolic processes, chromatin modification and protein localization to the cytoskeleton, while downregulated processes include regulation of apoptosis, of p38MAK cascade, and response to molecules of bacterial origin. Conclusions: After 72 hours, the transcriptional profile of cells on UG Ti confirms more rapid cell division than on CG. These results demonstrate that informative transcriptional profiling of cells growing on substrates that differ only in their crystallographic structure can be accomplished by a workflow that includes standard cell biology techniques, transcriptomic profiling, data mining, and network analyses. Sterile Ti discs, 13.5 mm in diameter, polished to a 2 nm average surface roughness (Ra), were arrayed in a non-tissue culture treated 24-well cell culture plate using a block design, with 12 CG and 12 UG disks. Wells were re-fed after 24 hours and after 72 hours Ti discs were transferred to new plates for standard cell harvesting protocols. The cells from four wells were consolidated into one sample resulting in three biological replicates for each sample. Consolidated samples were stored at -80°C in RNAlater and all subsequent steps were identical for all samples. Samples on CG were used as the control for UG-CG comparisons. This project was partially supported by grants from the National Center for Research Resources (5P20RR016480-12) and the National Institute of General Medical Sciences (8 P20 GM103451-12) from the National Institutes of Health.