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Study Design. A study examining the clinical protocol of scoliosis wound irrigation, demonstrating Povidone-Iodine's (PVI) effect on human osteoblast cells. Primary and immortal cell line osteoblasts were treated with 0.35% PVI for 3 minutes, and analyzed for proliferation rate, oxidative capacity and mineralisation.Objective. To model spinal wound irrigation with dilute PVI in vitro, in order to investigate the effect of PVI on osteoblast proliferation, metabolism and bone mineralisation.Summary of Background Data. Previously PVI irrigation has been proposed as a safe and effective practice to avoid bacterial growth following spinal surgery. However, recent evidence in multiple cell types suggests that PVI has a deleterious effect on cellular viability and cellular function.Methods. Primary and immortalhuman osteoblast cells were exposed toeither PBS control or with 0.35% PVI for 3 min. Cellular proliferation was measured over the duration of 7 days by MTS assay. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and proton production rate (PPR) were analysed using a Seahorse XFe24 Bioanalyzer. Protein expression of the electron transport chain subunits CII-SDHB, CIII-UQRCR2 and CV-ATP5A were measured via Western blotting. Mineralised bone nodules were stained with alizarin red.Results. Expressed as a percentage of normal osteoblast proliferation, osteoblasts exposed to 0.35% PVI exhibited a significant 24% decrease in proliferation after 24 h. This was a sustained response, resulting in a 72% decline in cellular proliferation at 1 week. There was a significant reduction in OCR, ECAR, and PPR (p < 0.05), in osteoblasts that had been exposed to 0.35% PVI for 3 min, coupled with a marked reduction in the protein expression of CII-SDHB. Osteoblasts exposed to 0.35% PVI exhibited reduced bone nodule mineralisation compared to control PBS exposed osteoblasts (p < 0.01).Conclusion. PVI has a rapid and detrimental effect on human osteoblast cellular proliferation, metabolic function, and bone nodule mineralisation.Level of Evidence: N/ACopyright (C) 2016 Wolters Kluwer Health, Inc. All rights reserved.