Decreased bone formation in type XII collagen–null mice. (A) Decreased mineralized nodule formation was observed in bone marrow cells derived from P30 Col12a1^−/− mice (n = 6) compared with wild-type controls (n = 5). Cells were cultured in osteogenic medium for 14, 21, and 28 d. Mineralized nodules represented by alizarin red staining are detected in wild-type cells after 14 d in culture, whereas nodules are virtually absent from Col12a1^−/− cells under the same conditions. At day 21, although nodules are formed in the Col12a1^−/− bone marrow cells, the nodule area is smaller than that in wild-type cultures. Similar nodule areas are detected at 28 d in culture. The mineralized nodule area is significantly decreased in Col12a1^−/− bone marrow cells at both day 14 and 21 in culture compared with wild-type controls. At day 28, the areas are comparable, indicating a delay in the absence of type XII collagen. (B–E) Bone morphometry in the periosteum of cross sections of tibia from P30 Col12a1^−/− (n = 5) and wild-type control (n = 4) mice was conducted using calcein double labeling. (B) Calcein label is detected as double lines (arrows). The boxed areas on the left are shown at higher magnification on the right. The distance between labels is decreased in Col12a1^−/− bone compared with wild-type control. (C) Mineral apposition rate is reduced ∼30% in Col12a1^−/− mice compared with wild-type controls. (D and E) The type XII collagen–null bones have ∼40% of the percentage of mineralized bone surface/total bone surface (D) and ∼50% of the bone formation rate (E) in Col12a1^−/− mice compared with wild-type controls. All of the differences are significant. *, P < 0.05; **, P < 0.01. Error bars are mean ± SD. Bars, 100 µm.