Fig 1.
Workflow used to generate network analysis. Kits, software and instrumentation used to generate data for each step are shown in parentheses.
Table 1.
qPCR primers.
Fig 2.
Dark grey markers are those with adequate coverage to use in comparative analyses, genes shown in light grey were disregarded. Red font indicates genes used as indicators of cell identity. S1 Fig is the same curve annotated with differentially expressed and those of interest in mechanotransduction and osteogenesis.
Fig 3.
Expression levels of the five genes were normalized using Actb levels, which was not differentially expressed in the dataset. The 2-ΔΔCt method was used to calculate the differences and included three replicates of each sample. Those with asterisks are included in Fig 4.
Table 2.
Differentially expresses genes (p(adj) ≤ 0.05).
Table 3.
Differentially expressed (p≤0.05) non-coding RNAs.
Fig 4.
Biological process gene ontology terms enrichment and network analysis.
Of the 432 differentially expressed genes with p≤0.05, 117 are retained in this network. Diamond-shaped nodes are GO biological process terms, larger diamonds indicate higher levels of significance, p(adj) value for each GO node is shown. Round nodes are genes, blue nodes are downregulated and red are upregulated. Bolded gene abbreviations in the figure are discussed in the text and full gene names are given in Table 4. S3 Table in S1 File provides this information for all 117 genes.
Table 4.
Network genes discussed in text.
Fig 5.
Mechanosensation and transduction model for bio-activating materials.
5A. This model focuses on a single physical characteristic of Ti, the RGBL/cell. 5B. First contact involves the extracellular matrix (ECM) domain of integrin α (Itgα) with the substrate, which includes a 5 nm layer of TiO2. Contact causes the association with an integrin ß chain (Itg ß), setting off a complex cascade of protein activation in the cytoplasm. 5C. Mechanosensation transmits the force through focal adhesions (FA) and other cytoplasmic protein complexes. Eventually, the signal reaches the linker of nucleoskeleton and cytoskeleton (LINC) complex embedded in the nuclear envelope (NE). 5D. Chromatin remodeling response involves the differential condensation and opening of chromatin. 5E. RNA transcripts are processed depending upon their function and, if required, transported out of the nucleus through nuclear pores (NP). 5F. Inside-out signal transduction includes regulation by the RNA interference (RNAi), transport to a ribosome, passage through the ER (endoplasmic reticulum) and Golgi, any post-translational modifications, and transport to the appropriate compartment. 5G. Integration of the Ti into bone requires the modification of collagen molecules in the ECM in concert with the maturation of FAs. 5H. The response of the substrate and subsequent cell are topics requiring additional research. Not shown in this model is the role of primary cilia.