Figure 7.

Periosteal cells expanded with AMA form more bone in vivo

(A) H&E staining of ectopic implants, seeded with mPDCs treated with vehicle (veh) or 10 μM AMA for 3 weeks and implanted for 8 weeks (right panel is magnification of boxed area). b, bone; f, fibrous tissue; g, scaffold granule (n = 8).

(B) Quantification of the bone volume formed (n = 8).

(C and D) Osterix and Hoechst (DNA) staining of ectopic implants seeded with β-actin-GFP⁺ periosteal cells and implanted for 8 weeks (C, n = 4) with quantification of the percentage of β-actin-GFP⁺ cells within the Osterix⁺ population (D, n = 4). Magnifications of the boxed area show Osterix staining or merged pictures; white arrows point at β-actin-GFP⁺/Osterix⁺ cells.

(E) Viability of mPDCs, labeled prior to implantation with CellTracker Green CMFDA. Viability of CMFDA⁺ cells was determined after implantation for 3 days by annexin V-propidium iodide flow cytometry (n = 4).

(F–H) CD31 staining of ectopic implants, seeded with mPDCs and implanted for 8 weeks (F; right panel is magnification of boxed area; scale bars, 200 μm; n = 8), with quantification of the number of blood vessels per mm² (G) and blood vessel surface area (H) (n = 8).

(I and J) Visualization (I, methylene blue staining) and quantification of colonies formed by CMFDA⁺/PI⁻ cells isolated 3 days after implantation and cultured for 1 week (J, n = 6).

Data are means ± SD. ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 versus veh (unpaired two-tailed Student’s t test). Scale bars, 200 μm (A, C) and 1 cm (I).