Anodizing could be used for bio-functionalization of the surfaces of titanium alloys. In this study, we use anodizing for creating nanotubes on the surface of porous titanium alloy bone substitutes manufactured using selective laser melting. Different sets of anodizing parameters (voltage: 10 or 20V anodizing time: 30min to 3h) are used for anodizing porous titanium structures that were later heat treated at 500°C. The nanotopographical features are examined using electron microscopy while the bioactivity of anodized surfaces is measured using immersion tests in the simulated body fluid (SBF). Moreover, the effects of anodizing and heat treatment on the performance of one representative anodized porous titanium structures are evaluated using in vitro cell culture assays using human periosteum-derived cells (hPDCs). It has been shown that while anodizing with different anodizing parameters results in very different nanotopographical features, i.e. nanotubes in the range of 20 to 55nm, anodized surfaces have limited apatite-forming ability regardless of the applied anodizing parameters. The results of in vitro cell culture show that both anodizing, and thus generation of regular nanotopographical feature, and heat treatment improve the cell culture response of porous titanium. In particular, cell proliferation measured using metabolic activity and DNA content was improved for anodized and heat treated as well as for anodized but not heat-treated specimens. Heat treatment additionally improved the cell attachment of porous titanium surfaces and upregulated expression of osteogenic markers. Anodized but not heat-treated specimens showed some limited signs of upregulated expression of osteogenic markers. In conclusion, while varying the anodizing parameters creates different nanotube structure, it does not improve apatite-forming ability of porous titanium. However, both anodizing and heat treatment at 500°C improve the cell culture response of porous titanium.
The Fe(II) and 2-oxoglutarate dependent oxygenase Jmjd6 has been shown to hydroxylate lysine residues in the essential splice factor U2 auxiliary factor 65 kDa subunit (U2AF65) and to act as a modulator of alternative splicing. We describe further evidence for the role of Jmjd6 in the regulation of pre-mRNA processing including interactions of Jmjd6 with multiple arginine-serine-rich (RS)-domains of SR- and SR-related proteins including U2AF65, Luc7-like protein 3 (Luc7L3), SRSF11 and Acinus S', but not with the bona fide RS-domain of SRSF1. The identified Jmjd6 target proteins are involved in different mRNA processing steps and play roles in exon dependent alternative splicing and exon definition. Moreover, we show that Jmjd6 modifies splicing of a constitutive splice reporter, binds RNA derived from the reporter plasmid and punctually co-localises with nascent RNA. We propose that Jmjd6 exerts its splice modulatory function by interacting with specific SR-related proteins during splicing in a RNA dependent manner.