(A) Sponges embedded with cells differentiated in osteogenic medium (upper 2 panels) or loaded with undifferentiated MSCs (lower panels) were transplanted into immunocompromised mice, and recipient mice were treated with Bzb at 0.3 mg/kg i.p. for 10 doses. Bzb treatment had no observable impact on osteoblast-loaded sponges (upper 2 panels; stained with H&E). In MSC-loaded sponges (lower panels), osteoid and bone were increased upon Bzb treatment (yellow arrows), shown in low power (original magnification, ×40) or in high power (HP; original magnification, ×100). Bzb treatment also increased matrix-depositing cells, seen as cells (white arrows) adjacent to xylenol orange (XO) fluorescence (red) with DAPI nuclear counterstain. Increased bone upon Bzb treatment was also observed with trichrome stain and with alizarin red stain on control- and Bzb-treated sponges. Graph shows quantification of alizarin red–stained fraction of tissue in control- and Bzb-treated sponges. *P = 0.007; n = 3. (B) Mice were implanted intrafemorally with GFP⁺ MSCs and then treated with control or Bzb i.p. Upper panels show femurs containing implanted GFP⁺ MSCs after treatment. Femurs were stained with alkaline phosphatase (purple). Increased ectopic bone is seen in Bzb-treated bones (arrows). Lower 4 panels show bright-field and epifluorescence images of the same section at higher magnification. Original magnification, ×20 (upper 2 panels); ×100 (lower 4 panels). In saline-treated animals, most GFP⁺ cells remained Alkaline phosphatase negative (long arrow). Occasional GFP⁺Alkaline phosphatase–positive cells were seen lining the endosteal surface (short arrows). In contrast, in Bzb-treated animals, increased ectopic bone was observed. Ectopic alkaline phosphatase–positive cells derived from GFP⁺ MSCs (green) were frequently observed (lower 2 right panels, white arrows) not only next to the bone, but also in the marrow space. (C) In saline-treated animals, GFP⁺ cells retained fibroblastoid appearance and CD105 positivity (GFP, green; CD105, red; double labeled, yellow; double-labeled cells are shown with white arrows in panel). In Bzb-treated animals, most cells lost their CD105 positivity (green). Images are from a single optical slice, using a confocal microscope. Original magnification, ×100.