Figure 6.

The Combined In Vitro Priming by BMP-2 Treatment and Aggregation Led to Endochondral Bone Formation In Vivo

Histology on tissue constructs 3 weeks after in vivo subcutaneous implantation demonstrated that the simultaneous stimulation by aggregation and BMP-2 affected in vivo tissue formation.

(A) H&E staining displayed the presence hypertrophic chondrocytes (black arrows) next to denser ECM areas (white arrows).

(B) Denser AB stain lines the zones of hypertrophic chondrocytes (white arrows) surrounded by GAG-rich areas (white arrows).

(C) The MT stain confirmed the formation of denser areas (black arrows).

(D and E) TRAP-positive areas (black arrows) surrounding the hypertrophic chondrocytes suggest remodeling (D) and quantification (E).

(F) IHC for DIPEN showed breakdown of GAG-rich areas.

(G and H) IHC for S100 displayed more mature cartilage tissue in BMP-2 stimulated aggregates (G), confirmed by quantification (H).

(I and J) IHC for Ihh showed the presence of hypertrophic chondrocytes (I), which upon quantification was a 4-fold higher compared with 2D-stimulated cells (J).

(K) At 6 weeks, the endochondral tissue intermediate had developed into a mineralized ossicle.

(L) H&E staining confirmed de novo formed bone (black arrows) and bone marrow (red arrows), while MT staining displayed zones of mature bone (green arrows). TRAP staining suggested ongoing remodeling of immature bone (blue arrows) and IHC for hOCN confirmed the contribution of the implanted cells. Unlabeled scale bars, 20 μm. n = 4, ^∗p < 0.05, ^∗∗p < 0.01.