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

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 Equal Channel Angular Pressing-Conform (ECAP-C), 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. Sterile Ti discs, 13.5 mm in diameter and polished to a 2 nm average surface roughness 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 and were stored at -80°C in RNAlater. All subsequent steps were identical for all samples. Samples on CG were used as the control for UG-CG comparisons.