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. 2020 Dec 18;8:611757. doi: 10.3389/fcell.2020.611757

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Copyright © 2020 Ehirchiou, Bernabei, Chobaz, Castelblanco, Hügle, So, Zhang, Busso and Nasi.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CD11b agonist reduces murine and human chondrocytes calcification in vitro. (A) Alizarin Red staining of WT primary murine chondrocytes stimulated with secondary CPP ± LA1 (20 μM) for 24 h in DMEM + 10% FBS. Pictures show one representative culture well of one experiment out of three independent experiments. The graph shows the percentage of Alizarin Red positive surface over the total surface, normalized on Vehicle treated cells. Values represent means ± SD of triplicates samples. (B) IL-6 and MCP-1 concentration in cell supernatants of WT primary chondrocytes stimulated with secondary CPP and LA1 at different concentration (0 μM - Vehicle-, 1 μM and 20 μM). Values represent means ± SD of triplicates samples; *p < 0.05, ***p < 0.001. (C) qRT-PCR of the indicated genes in murine chondrocytes stimulated with LA1 (20 μM) or its vehicle for 4 h. Values represent fold increase of LA1 treated chondrocytes over their Vehicle-treated cells, shown in means ± SD of triplicates samples; ****p < 0.0001. (D) qRT-PCR of the indicated genes in WT primary murine chondrocytes stimulated with secondary CPP ± LA1 (20 μM) for 4 h. Values represent fold increase in LA1 treated chondrocytes over Vehicle-treated cells, shown as means ± SD of triplicates samples; *p < 0.05, ***p < 0.001, ****p < 0.0001. (E) Quantification of Alkaline phosphatase activity in WT chondrocytes treated with secondary CPP ± LA1 for 6 h. Values represent fold increase of Alp activity in LA1 treated chondrocytes over Vehicle-treated cells treated, shown as means ± SD of triplicates samples; **p < 0.01. (F) Alizarin Red staining of human primary chondrocytes stimulated with secondary CPP and treated with LA1 or its Vehicle. Pictures show one representative culture well of one OA patient (P9) out of eight patients. The graph shows the corresponding Alizarin Red positive surface over the total surface, calculated in Adobe^® Photoshop^®, and then normalized. Lines connect the two conditions for each patient, values are calculated over CPP-Vehicle and represent means of triplicates samples; *p < 0.05. (G) IL-6 concentration in supernatants of human primary chondrocytes at point (F). Values represent means of triplicates samples; *p < 0.05.