Fig. 4. Examination of differentiation markers in Mmp13^–/– endochondral cartilage. (A) BrdU incorporation in 1-week-old tibia of wild-type and Mmp13^–/– mice. Animals were injected with 1 µg BrdU/g mouse 1 hour prior to sacrifice. A similar number of BrdU-positive cells (brown staining) can be observed in the proliferating chondrocyte (PC) zone, and in cells within the trabecular bone (TB) for wild-type and Mmp13^–/– bones, indicating that proliferation is not impaired in Mmp13^–/– mice. (B) In situ hybridization on 1-week-old metatarsals with probes to chondrocyte differentiation markers show normal expression domain of col2, but expanded expression of collagen type X and VEGF, indicating an increased late hypertrophic chondrocyte population in the Mmp13^–/– growth plate. In wild-type mice, expression of osteopontin (Osp) is restricted to only one row of hypertrophic chondrocytes in the growth plate of wild-type mice. The growth plate of Mmp13 ^–/– mice contains several rows of chondrocytes expressing Osp (broken line demarcates front of ossification). Safranin-O staining shows general morphology (HC, hypertrophic cartilage). Scale bars: 100 µm.