Figure 6.

iOBs implanted in vivo form mineralized bone structure. Acellular Matrigel, fibroblast Matrigel mixtures and iOB Matrigel mixtures were subcutaneously implanted into immunodeficient mice (n = 4 per group). (A) Fibroblasts and iOBs that had been cultured in osteogenic medium in vitro for 20 days were mixed with equal volume of Matrigel and then subcutaneously implanted into the back of 7-week-old nude mice. Four weeks later, representative microCT x-ray and 3D images of explanted cell lumps show significant mineralized structure in iOB implant and limited radiodense region in fibroblast implant. (B) Serial cryosections were made in iOB and fibroblast of four- week implants. Mineralized structures were visualized using Alizerin Red S and Von Kossa staining. The nucleus of implanted fibroblasts and iOBs were stained with DAPI. Live GFP positive signal showed Col2.3GFP⁺ expression. Matrix protein osteocalcin was stained by immunofluorescence. (C) Representative microCT x-ray and 3D images of 12-week explanted ossicles from iOB implants. Serial sections were made from paraffin-embedded iOB implant samples. Cortical bony like structures were shown in H&E and Masson Aniline Blue staining.