Fig. 8.

Histological staining and fluorescent imaging of the calvarial defect to assess new bone formation. (A) Toluidine blue, (B) van Gieson, and (C) Masson staining of undecalcified femoral sections at 12 weeks. IB, immature bone; MB, mature bone; BV, blood vessel; FT, fibrous tissue. (D) Representative images showing calcein and alizarin red staining. (E) Immunofluorescence staining for CD31 (green) and CD206 (red) in decalcified bone tissues. Nuclei were labeled using DAPI (blue). (F) Quantitative evaluation of MAR at 4–6 weeks post-implantation. (G) Quantitative analysis of the newly formed blood vessels at 12 weeks post-implantation. (H) Quantitative analysis of the CD206 positively stained cells. **P < 0.01, *P < 0.05, n = 3.