Bozycki Lukasz, Komiazyk Magdalena, Mebarek Saida, Buchet Rene, Pikula Slawomir, Strzelecka-Kiliszek Agnieszka
Laboratory of Lipid Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences.
Université de Lyon; Université Lyon 1; L'insitut National des Sciences Appliquées (INSA) de Lyon; Ecole Supérieure de Chimie Physique Electronique (CPE) Lyon; Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR), L'institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS).
J Vis Exp. 2018 Jun 24(136):57423. doi: 10.3791/57423.
This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM). Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions. The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.
本视频展示了使用透射电子显微镜结合能量色散X射线微分析（TEM-EDX）来比较两种人类骨细胞系（hFOB 1.19和Saos-2）释放的囊泡中矿物质的状态。这些细胞系在用抗坏血酸（AA）和β-甘油磷酸酯（β-GP）处理后，经历从增殖到矿化的完全成骨转分化，并产生基质囊泡（MVs），这些囊泡可触发细胞外基质（ECM）中的磷灰石成核。基于茜素红S（AR-S）染色以及使用紫外线（UV）对细胞裂解物中的矿物质成分进行分析，或者使用TEM成像后进行EDX定量和离子映射对囊泡中的矿物质成分进行分析，我们可以推断骨肉瘤Saos-2细胞和成骨细胞hFOB 1.19细胞呈现出不同的矿化模式。Saos-2细胞比hFOB 1.19细胞矿化效率更高，产生更大的矿物质沉积物，这些沉积物在紫外线下不可见，但类似于羟基磷灰石（HA），因为它们有更多的钙和氟替代物。使用这些技术获得的结果使我们能够得出结论，矿化过程因细胞类型而异。我们提出，在细胞水平上，囊泡的起源和性质预先决定了矿物质的类型。
