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. 2018 Jun 18;115(27):E6135–E6144. doi: 10.1073/pnas.1805159115

Fig. 5.

Fig. 5.

Bone-like mineral phase inhibits EO in vivo. Safranin-O (S.O) (A) and H&E staining (B) of control and BBHAp constructs after 2, 4, and 8 wk following ectopic implantation in nude mice. Control samples showed GAG-rich cartilaginous matrix, which remodels into bone through EO after 8 wk. While the cartilaginous matric was remarkably absent in the BBHAp constructs at all time points, the bony ossicles were formed after 8 wk via IMO. The magnified area is denoted with a dashed black rectangle in the images. (C) In situ hybridization for visualization of human nuclei using an Alu probe. The region within the section that was probed using Alu is denoted by the dashed black rectangles in B. The presence of Alu+ nuclei (arrows) confirmed that the formation of bone in both conditions involves the participation of transplanted human MSCs. Explanted BBHAp constructs after 4 wk lack expression of characteristics of bone formation via EO, such as collagen type II (Col II) (D), MMP-9 (E), and TRAP (F). Coll II and MMP-9 are visualized as brown-colored regions positive for horseradish peroxidase activity. TRAP-positive areas in the control section are indicated by an arrow. (G) Three-dimensional reconstructed μCT images after 8 wk of in vivo implantation confirms the presence of mineralized tissue in both conditions. (H) Degree of maturation in mineralized tissue is visualized by an X-ray attenuation heat map. (I) Bone mineral density in both conditions calculated based on X-ray attenuation. ***P < 0.001. (J) SEM confirming the presence of BBHAp coating even after 8 wk of implantation. Magnified image of the region within the rectangular box on the Top is shown in the Bottom. (K) Fluorescent microscopy images of the area represented by the dashed circle in B (8-wk time point), which corresponds to bone deposits after staining for mouse CD45+ cells, showing the presence of immune cells in close proximity to ectopic bone. Magnified image of the region within the rectangular box on the Top is shown in the Bottom. (L) Visual representation of a proposed mechanism showing the counteractive effects of the PTH1R and IGF1 signaling pathways on hyperstimulation of CaSR between days 2 and 21 in vitro preceding the formation of bone via IMO in BBHAp constructs upon implantation.