Evaluation of osteoblast and osteoclast differentiation. Cell culture assay was performed using bone marrow cells flushed from the bilateral femurs and tibias of C57BL/6J (WT), transient receptor vanilloid 1 (TRPV1) knockout (V1KO), TRPV4 knockout (V4KO), and TRPV1 and TRPV4 double knockout (DKO) mice. (A) The images of wells with alkaline phosphatase (ALP)-stained or tartrate-resistant acid phosphatase (TRACP)-stained cultured cells from WT (a, e), V1KO (b, f), V4KO (c, g), and DKO (d, h) mice are shown. (B) The microscopic images of TRACP-stained cultured cells for WT (a) and DKO (b) mice. White arrows indicate multinucleated cells (scale bar = 500 μm). (C) The numbers of ALP-positive CFU-f colonies (a) and total CFU-f colonies (b) per well for each mouse strain and the percentage of ALP-positive CFU-f colonies/total CFU-f colonies (c) for each mouse strain are shown. TRACP-positive multinucleated cells (d), and TRACP-positive mononucleated cells (e) per well for each mouse strain are shown (n = 4 in each group). The data are presented as mean ± SEM. One-way ANOVA, *p < 0.05 and **p < 0.01 compared to WT; †p < 0.05 and ††p < 0.01 compared to V1KO; ‡‡p < 0.01 compared to V4KO.