1). Although the expression of pSmad 1/5/8 was decreased in cases of non-unions compared to fracture callus, it was still present in osteoblasts and hypertrophic chondrocytes of non-unions (Table 2 and Fig. 1, Fig. 2 and Fig. 3), confirming our previous report showing active BMP signaling in non-unions [8]. The expression of noggin and gremlin was present in all cell types of all specimens. On the other hand, BMP3 (generally referred to as a BMP-inhibitor)

and chordin were not expressed in chondrocytes (hypertrophic and non-hypertrophic) of non-unions. Results of the expression of Smad-6 and Smad-7 were mixed. Although both Smad-6 and Smad-7 are inhibitors, their expression did not follow the same pattern. When comparing sections http://www.selleckchem.com/products/dabrafenib-gsk2118436.html of fracture callus with those of non-unions, our results showed increased expression of Smad-6 in osteoblasts, hypertrophic and non-hypertrophic chondrocytes of non-unions, Smad-7 showed equal expression in osteoblasts of both fracture callus and non-unions, while decreased expression in hypertrophic and non-hypertrophic chondrocytes of non-unions. Representative staining images are shown in Fig. 2 and Fig. 3. In general, results of double and triple immunofluorescence staining showed co-localization of BMP ligands with inhibitors, in all sections of both fracture callus and non-unions. There was also decreased staining of BMP2

in the non-unions (representative images are shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7 and Fig. 8). A summary of the expression data is shown in Table 2. The results of this study AG-14699 support our hypothesis that the balance between expression of endogenous BMP ligands and BMP-inhibitors in non-unions is different than in normal fracture healing. Specifically, our results show that in chondrocytes, expression of BMP2 was markedly decreased in non-unions and that of BMP7 was almost completely absent. On the other hand, expression of BMP-inhibitors (noggin, gremlin, Smad-6 and Smad-7) was almost the same in osteoblasts, chondrocytes and fibroblasts Oxalosuccinic acid of both fracture callus

and non-unions. Although these data are consistent with our hypothesis, we had expected that this “imbalance” was due to an increased expression of BMP-inhibitors in non-unions. The current data suggest, however, that it is due to decreased expression of BMPs. In our previous study on delayed and non-unions, we demonstrated that BMP2, BMP4 and BMP7, BMPRs and pSmad 1/5/8 were present in most non-unions in osteoblasts and fibroblasts [8]. However, in that study, we did not specifically analyze the expression of these BMP-related proteins in cartilage cells and we did not compare our findings with those of normal fracture healing. The concept of imbalance between BMP ligands and their antagonists, being a potential cause of the development of non-unions, was first suggested by Niikura et al.