Understanding the role of orthopaedic implant dissolution ions in bone remodelling

The ions released from materials used in orthopaedic repair can affect bone remodelling. These ions can be released as unintentional by-products of implant wear or can be intentionally released as part of implant design (e.g. bioactive glasses, cements and hydrogel). Furthering the understandings of how these ions (with a focus on Cobalt (Co) and Silicate (Si)) interact in vitro in this study, these ions affect bone remodelling and will allow the development of new materials with improved outcomes following implantation. The effects of these ions on bone remodelling cells, specifically on monocytes, osteoblasts and osteoclasts were performed and potential mechanistic cellular pathways (the hypoxia inducible factor (HIF) pathway and Fenton reaction) were investigated. This included the development of a new in vitro model of bone resorption using a sub-clone of mouse macrophage cell-line (RAW264.7), creating a novel non- primary cell line that demonstrates bone resorbing activity. Co ions, at concentration range below 200μM reported in the bone implant interface of failed implants (25-100μM), were found to increase the sub-unit of Tartrate resistant acid phosphatase 5b (TRAP-5b) expression, this enzyme is osteoclast specific in the osteoclast sub-clone cell lines, hence correlated to increased osteolysis (p<0.001). Co ions also increased osteoblast expression of the osteoclastic differentiation factor RANKL, reactive oxygen species (ROS) generation and macrophage phagocytic activity (p<0.001). These results suggest that Co ions may contribute inflammatory osteolysis and cause aseptic implant failure. Furthermore, inhibition of the HIF pathway using echinomycin (an inhibitor of HIF-1a transcriptional factor binding), reduced these Co ion dependent osteoclastogenic and inflammatory effects, supplying evidence of Co ion stabilization of the HIF-1a pathway. Bioactive glasses release Si which hypothesise to encourage bone formation. There is, however, little understanding of how Si interacts with osteoclasts. A possible cellular mechanism for this inhibition was investigated, whereby Si was found (concentration dependently) to inhibit ROS generation in osteoclasts via the Fenton reaction. The addition of iron (II) ions (10 μM FeCl2) restored osteoclastogenesis (p<0.001), suggesting, for the first time, that Si chelation of iron, may be responsible for enhanced bone formation. These results furthered our understanding on how Co and Si released from materials influence bone remodelling and may enable the development of materials that control bone resorption and bone formation depending on the disease and patient

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Integrated Genomic Analysis of the Ubiquitin Pathway across Cancer Types

Protein ubiquitination is a dynamic and reversibleprocess of adding single ubiquitin molecules orvarious ubiquitin chains to target proteins. Here,using multidimensional omic data of 9,125 tumorsamples across 33 cancer types from The CancerGenome Atlas, we perform comprehensive molecu-lar characterization of 929 ubiquitin-related genesand 95 deubiquitinase genes. Among them, we sys-tematically identify top somatic driver candidates,including mutatedFBXW7with cancer-type-specificpatterns and amplifiedMDM2showing a mutuallyexclusive pattern withBRAFmutations. Ubiquitinpathway genes tend to be upregulated in cancermediated by diverse mechanisms. By integratingpan-cancer multiomic data, we identify a group oftumor samples that exhibit worse prognosis. Thesesamples are consistently associated with the upre-gulation of cell-cycle and DNA repair pathways, char-acterized by mutatedTP53,MYC/TERTamplifica-tion, andAPC/PTENdeletion. Our analysishighlights the importance of the ubiquitin pathwayin cancer development and lays a foundation fordeveloping relevant therapeutic strategies