Quantification of in vitro mineralisation using ion chromatography.

Analysis of in vitro mineralisation is an important tool in orthopedic research, allowing assessment of new therapeutic agents and devices; however, access to analytical equipment and accuracy of current methods can be a limiting factor. This current work investigated the use of calcium chelation with citric acid and subsequent analysis by ion chromatography as a method for accurately quantifying the extent of in vitro calcium deposition. Primary human osteoblasts were cultured on tissue culture plastic for 21 days under osteogenic conditions. At 3, 7, 14, and 21 days, alizarin red staining and citric acid calcium chelation of the cultures were performed. The use of alizarin red revealed increased calcium deposition over the culture period but was not sensitive enough to detect mineralisation at early time points after taking in to account background residual staining. The use of ion chromatography gave a limit of detection of 2 µg calcium, sensitive enough to detect mineralisation after 3 days, with no issues relating to background levels. We believe that the use of ion chromatography for quantifying in vitro mineralisation gives researchers an accurate, accessible, and cheap way of assessing novel technologies.

Identification of mesenchymal stromal cell survival responses to antimicrobial silver ion concentrations released from orthopaedic implants.

Antimicrobial silver (Ag+) coatings on orthopaedic implants may reduce infection rates, but should not be to the detriment of regenerative cell populations, primarily mesenchymal stem/stromal cells (MSCs). We determined intramedullary silver release profiles in vivo, which were used to test relevant Ag+ concentrations on MSC function in vitro. We measured a rapid elution of Ag+ from intramedullary pins in a rat femoral implantation model, delivering a maximum potential concentration of 7.8 µM, which was below toxic levels determined for MSCs in vitro (EC50, 33 µM). Additionally, we present in vitro data of the reduced colonisation of implants by Staphylococcus aureus. MSCs exposed to Ag+ prior to/during osteogenic differentiation were not statistically affected. Notably, at clonal density, the colony-forming capacity of MSCs was significantly reduced in the presence of 10 µM Ag+, suggesting that a subpopulation of clonal MSCs was sensitive to Ag+ exposure. At a molecular level, surviving colony-forming MSCs treated with Ag+ demonstrated a significant upregulation of components of the peroxiredoxin/thioredoxin pathway and processes involved in glutathione metabolism compared to untreated controls. Inhibition of glutathione synthesis using L-buthionine sulfoxamine eliminated MSC clonogenicity in the presence of Ag+, which was rescued by exogenous glutathione.

The variable toxicity of silver ions in cell culture media.

The elevated interest in silver ions (Ag+) as a broad spectrum antimicrobial for use on medical devices has increased the number and importance of in vitro biocompatibility testing, however little consideration is given to the culture environment in which the assessments are performed. The current investigation assessed the viability of mouse fibroblasts (L929) exposed to different concentrations of Ag+ in both Dulbecco's modified Eagle's medium (DMEM) and minimal essential medium Eagle, alpha modification (αMEM). We identified a significant increase in the EC50 of L929 cells exposed to Ag+ in αMEM compared to DMEM, which was matched by a corresponding decrease in Ag+ availability in αMEM at concentrations ≤400 µM, as detected by inductively coupled plasma mass spectrometry (ICP-MS). The reduced availability was not observed for Ag+ > 400 µM, the concentration above which caused in vitro cytotoxicity in L929 cells in αMEM; while linear quantification of Ag+ was observed in DMEM. Equilibration of the chloride and glucose components between media did not affect cytotoxicity on primary test cells; mesenchymal stromal cells (MSCs). Overall, our results present evidence of the importance of culture conditions on the in vitro evaluation of silver, with DMEM providing a reliable basal media in which to conduct assessments.