Fig. 8

Pinch loss upregulates Rankl in HZ chondrocytes and promotes osteoclast formation in vitro and in bone. a–f Tartrate-resistant acid phosphatase (TRAP) staining. Tibial sections from 6-week-old male control and dKO mice were used for TRAP staining (a, d). The osteoclast surface/bone surface (Oc.S/BS) (c, f) and osteoclast number/bone perimeter (Oc.N/BPm) (b, e) of the primary and secondary spongiosa bones from mice of the two genotypes were measured using Image-Pro Plus 7.0 (c–g). The arrow indicates osteoclasts located on the trabecular bone surface. Scale bar, 50 μm. *P < 0.05, ***P < 0.001. N = 5 mice per group. Student’s t test. The results are expressed as the mean ± s.d. g–j In vitro osteoclast formation. Primary BMMs from 6-week-old control and dKO male mice were first cultured in proliferation medium (α-MEM containing 10% FBS and 10 ng·mL⁻¹ human recombinant M-CSF) for 3 days followed by differentiation medium (proliferation medium plus 50 ng·mL⁻¹ human recombinant RANKL) for 4–9 days and then subjected to TRAP staining (g). TRAP⁺ MNCs (≥3, 10, or 30 nuclei) per well were scored (h–j). The data are expressed as the mean ± s.d. Student’s t test. **P < 0.01. Scale bar, 100 μm. k ELISA of serum levels of collagen type I cross-linked C-telopeptide (CTX1). Sera were collected from 6-week-old male control and dKO mice and subjected to ELISA for CTX1. N = 5 control mice; N = 6 dKO mice. Student’s t test. The results are expressed as the mean ± s.d. l IHC staining of tibial sections from 6-week-old male control and dKO mice with an anti-Rankl antibody. Scale bar, 50 μm