Bio-activating ultrafine grain titanium: RNA sequencing reveals enhanced mechano-activation of osteoconduction on nanostructured substrates

Abstract

Titanium is essentially absent from biological systems yet reliably integrates into bone. To achieve osseointegration, titanium must activate biological processes without entering cells, defining it as a bio-activating material. Nanostructuring bulk titanium reduces grain size, increases strength, and improves other quantifiable physical properties, including cytocompatibility. The biological processes activated by increasing grain boundary availability were detected with total RNA-sequencing in mouse pre-osteoblasts grown for 72 hours on nanometrically smooth substrates of either coarse grain or nanostructured ultrafine grain titanium. The average grain boundary length under cells on the conventional coarse grain substrates is 273.0 μm, compared to 70,881.5 μm for cells adhered to the nanostructured ultrafine grain substrates; a 260-fold difference. Cells on both substrates exhibit similar expression profiles for genes whose products are critical for mechanosensation and transduction of cues that trigger osteoconduction. Biological process Gene Ontology term enrichment analysis of differentially expressed genes reveals that cell cycle, chromatin modification, telomere maintenance, and RNA metabolism processes are upregulated on ultrafine grain titanium. Processes related to immune response, including apoptosis, are downregulated. Tumor-suppressor genes are upregulated while tumor-promoting genes are downregulated. Upregulation of genes involved in chromatin remodeling and downregulation of genes under the control of the peripheral circadian clock implicate both processes in the transduction of mechanosensory information. Non-coding RNAs may also play a role in the response. Merging transcriptomics with well-established mechanobiology principles generates a unified model to explain the bio-activating properties of titanium. The modulation of processes is accomplished through chromatin remodeling in which the nucleus responds like a rheostat to grain boundary concentration.

Commercialization of bulk nanostructured metals and alloys

“…7 Researchers have extensively explored the interaction of nanostructured alloys with physiological systems, which have been summarized in recent reviews. 2,[8][9][10] The results of these investigations demonstrate benefits including increased cell adhesion, proliferation, and differentiation for a wide range of cell types. Most recently, Reiss et al sequenced the RNA of bone-forming cells on nanostructured titanium to discover the mechanism by which the metal surface grain boundaries enhance osteoconduction.…”

“…Most recently, Reiss et al sequenced the RNA of bone-forming cells on nanostructured titanium to discover the mechanism by which the metal surface grain boundaries enhance osteoconduction. 10 The combination of enhanced biological properties with the superior mechanical properties of ultrafine grain alloys makes the pursuit of commercialization for trauma, orthopedic, and dental highly attractive.…”

Short-term high fat diet alters genes associated with metabolic and vascular dysfunction during adolescence in rats: a pilot study

“…The resulting data files were downloaded and opened with JMP version 14 (SAS Institute, Cary, NC, USA) for data filtering and further analyses. Zipf’s power law is a graphic method employed to model complex phenomena ( Adamic, 2011 ) that has been used to identify low count theshold in environmental metagenomic shotgun sequence data ( Reiss, Guerra & Makhnin, 2016 ) and in a study of cell response to nanostructured titanium ( Reiss et al, 2020 ). Transcription profiles of tissue qualifies as a complex system and a low count cutoff was determined by a Ziph graph, in which the log abundance per gene, as measured counts per million (CPM) for all samples, is graphed against the log rank of each gene ( Fig.…”

“…Network analysis can help detect the relationships between the biological processes and changes in gene expression relevant to the experiment. For example, using a workflow with two conditions, each with two replicates that consisted of pooled cells exposed to titanium of a specific grain size, gene expression differences revealed changes in biological processes associated with mechanotransduction ( Reiss et al, 2020 ). Cytoscape and associated applications combine data-mining applications with network analysis that facilitate the visualization of data, resulting in an improved understanding of data ( Bindea et al, 2009 ; Cline et al, 2007 ).…”

Nanostructured Bulk Titanium with Enhanced Properties—Strategies and Prospects for Dental Applications

“…The RNA sequencing techniques revealed that the processes responsible for division are upregulated, whereas the undesirable reactions (immune response, apoptosis) are downregulated in the material with a large number of grain boundaries (UFG Ti). [48] In addition to accelerating the biological response, the biomaterial's surface has to be resistant to bacterial adhesion. Generally, it was observed that nanostructure enhance the proliferation of both osteoblast and bacteria cells, [47,49,50] but different results can be also found in the literature.…”