Fig 2: OMD inhibits osteoclastogenesis through its direct interaction with RANKL and balances bone remodeling. a Solid phase binding assay on the capture of RANKL by OMD. RANKL was coated on a plate followed by OMD addition. On the left: Binding assay with different concentrations of OMD (1 000 to 15.65 ng·mL−1 by serial 2X dilution), with 0.2 μg·mL−1 of coated RANKL (red curve) and negative control without RANKL (blue curve). On the right: Binding assay with different concentrations of coated RANKL (800 to 6.25 ng·mL−1) and 0.5 μg·mL−1 of given OMD (pink curve); negative control without OMD (purple curve). Wilcoxon test was performed with differences being considered significant at P values < 0.05 (*P < 0.05, **P ≤ 0.01). b Assay of the effect of OMD on primary murine osteoclast culture. The peripheral blood mononuclear cells were collected from murine bone marrow and differentiated into osteoclasts with M-CSF and RANKL. Osteoclasts were counted after 4 days of differentiation following a TRAP staining. Each point represents a mouse, n = 7. The osteoclast count was represented in percentage of cells with the corresponding control set as 100%. Blue arrows point at osteoclasts. Scale bar = 50 µm. The data were plotted as a box plot showing all points. One-way ANOVA was performed with differences being considered significant at P values <0.05 (*P < 0.05). c Level of P1NP and TRAcP 5b measured in the serum of KO, WT and UP mice. The data were plotted as a box plot showing all points with n = 12 for the KO and WT, and n = 10 for the UP. One-way ANOVA was performed when the distribution was Gaussian and Kruskal–Wallis was performed when the distribution was not Gaussian with differences being considered significant at P values < 0.05 (*P < 0.05, **P ≤ 0.01, ****P ≤ 0.000 1). d Schematic representation of the mechanism of OMD on osteoclastogenesis. Osteoblasts secrete RANKL which binds to the RANK receptor on the membrane of pre-osteoclasts to induce their differentiation into osteoclasts. In parallel, osteoblasts also secrete OMD which displays the ability to capture RANKL and would prevent its binding to RANK

Fig 3: Localization of OMD in the murine knee joint. a Immunostaining of OMD (in brown) in the knee joint (medial tibial plateau) of KO, WT, and UP male mice at 4, 8, and 16 months. Scale bar = 100 µm. Zoom on specific areas from WT of 16 months. Scale bar = 25 µm for (b) and 50 µm for (c, d). Representative pictures with n = 3 for each group. b Uncalcified articular cartilage (ac) and calcified cartilage (cc), separated by the tidemark (td—dotted line); chondrocytes (arrowheads). c Subchondral bone (sb), growth plate (gp) and lining cells (lc). d Metaphysis of the tibia showing the cortical bone (cb), the outer medial tibial side and the inner tibial side facing the bone marrow are indicated with (*)

Fig 4: Effect of Omd on the spontaneous development of subchondral bone sclerosis. a Histomorphometry of the subchondral bone on Safranin-O Fast Green of the knee joint of male mice at 8 and 16 months was performed with QuPath on the lateral and medial plateaus of the tibia separately. At 8 months: n = 8 for each genotype; at 16 months: n = 7 for the KO, n = 7 for the WT, and n = 9 for the UP. The data were plotted as a box plot showing all points. Two-way ANOVA was performed with differences being considered significant at P values <0.05 (*P < 0.05, **P ≤ 0.01). b Representative picture of the Safranin-O Fast Green of the knee joint of male mice showing the subchondral bone area for the lateral and medial plateaus of the tibia separately. Scale bar = 100 µm. c µCT of the subchondral bone of the tibia of the mice at 16 months. The pink asterisk indicates the lateral plateau and the blue asterisk indicates the medial plateau

Fig 5: Long bone structural phenotype of Omd-deficient mice. A) Representative μCT reconstructions of the distal metaphyseal trabecular bone compartment of the right femora from 12-week-old, female Omd+/+ and Omd-/- mice and a full bone representation with the analyzed region of interest indicated in red. Scale bar = 0.5 or 1 mm as indicated. B) μCT-based quantification of the bone volume per tissue volume of the distal metaphyseal trabecular compartment of the right femora from mice with the indicated age, gender, and genotype. C) Representative μCT reconstructions of the mid-diaphyseal cortical bone compartment of the right femora from 12-week-old, female Omd+/+ and Omd-/- mice. Black line with long dashes: Outer cortical limit of the Omd+/+ sample, green line with short dashes: Inner cortical limit of the Omd+/+ sample. Scale bar = 0.5 mm. The red overlay on the full femora depicted on the left indicates the analyzed region of interest. Scale bar = 1 mm. D) μCT-based quantification of the cortical thickness of the mid-diaphyseal cortical compartment of the right femora from mice with the indicated age, gender, and genotype. E) μCT-based quantification of the inner and outer cortical diameter of the mid-diaphyseal cortical compartment of the right femora from mice with the indicated age, gender, and genotype. F) Representative μCT reconstructions of the mid-diaphyseal cortical bone compartment of the left tibiae and humeri from 12-week-old, female Omd+/+ and Omd-/- mice, as well as full bone representations with the analyzed regions of interest indicated in red. Scale bar = 0.5 or 1 mm as indicated. G) μCT-based quantification of the cortical thickness of the mid-diaphyseal cortical compartment of the left tibiae and humeri of 12-week-old female Omd+/+ and Omd-/- mice. H) μCT-based quantification of the inner and outer cortical diameter of the mid-diaphyseal cortical compartment of the left tibiae and humeri of 12-week-old female Omd+/+ and Omd-/- mice. Data are presented as box plots with dots for individual datapoints, n = 5-7. Data were analyzed by Student t-test. * P < .05, ** P < .01, *** P < .001.