<p>Purpose: Our group has been evaluating the function and biocompatibility of nanomaterials for medical applications in the field of orthopedics. We earlier presented on the safety of carbon nanohorns (CNHs) and nano-ferrite particles (NFPs) on osteoblasts. Although the cytotoxicity of CNHs was higher, the osteoblast status differed between the nanomaterials. This study evaluated the cytotoxicity and uptake status of NFPs on osteoblasts under the same conditions as the prior CNH exposure. Methods: The MC3T3-E1 mouse preosteoblastic cell line (MC cells) with (Cal) and without (Non) calcification stimulation was used for NFP cytotoxicity assessment, morphological evaluation of NFP intracellular uptake, and relative quantitative comparisons by flow cytometry (FCM). Results: The cytotoxicity of NFPs was comparable in the Cal and Non groups at 24 h post-exposure, but at 48 h, the Cal group exhibited significantly more cytotoxicity. NFP uptake was evident on optical microscopy in the Cal group only. FCM comparisons showed a slight increase in cellular uptake in the Non group and a significant increase in the Cal group versus each control. Discussion: The cellular response of MC cells to NFPs was considerably different to CNHs. It appears that the intracellular uptake mechanism of CNHs in the non-calcification state of MC cells does not function for NFPs, whereas calcification stimulation induces MC cells to uptake NFPs. These results suggest that the safety of nanomaterials on osteoblasts should be evaluated before and after functional changes caused by calcification stimulation.</p>