Leukemia Inhibitory Factor and Oncostatin M Influence the Mineral Phases Formed in a Murine Heterotopic Calcification Model: A Fourier Transform-Infrared Microspectroscopic Study

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Abstract
The study of bone mineralization processes is of considerable interest in understanding bone diseases and developing new therapies for skeletal disorders, particularly since bone homeostasis requires numerous cell types and a large cytokine network. Cell culture models of mineralization have often been used to study the cellular mechanisms of mineralization, but few data have been reported concerning the influence of extracellular matrix components and cytokines on the physicochemical properties of mineral. The purpose of this study was to analyze the effects of two cytokines, leukemia inhibitory factor (LIF) and oncostatin M (OSM), involved in bone metabolism on the physicochemical properties of bone mineral formed in a murine in vivo mineralization model. Murine bone marrow cells implanted under the kidney capsule in the presence or absence of cytokines led to heterotopic ossicle formation. A scanning electron microscopic microprobe revealed that heterotopic calcification had a lower (∼20%) Ca/P ratio after cytokine treatment as compared with the control without cytokine. Transmission electron microscopic analysis of cytokine‐treated ossicles showed numerous areas with low mineral density, whereas electron diffraction pattern revealed an apatitic phase. These areas were not observed in the absence of cytokine. Moreover, Fourier transform‐infrared microspectroscopy showed at the molecular level that the presence of either cytokine induced many microscopic areas in which short‐range order organization, such as incorporation of carbonate and crystallinity/maturity of ossicle mineral, were modified. LIF and OSM influenced mineral phase formation in the present model and may thus be key protagonists in bone mineral development and skeletal diseases.