Fig. 1

In vitro and in vivo analysis of rMSCs-CM pro-osteogenic potential. (A) Top panel. Relative expression levels of osteogenic markers (Runx2 and Alpl) in primary rat MSCs were assessed using semi-quantitative PCR after 48-h of pre-treatment with either rCM-Smurf1 or rCM-Ctrl. Bottom panel. Left graph illustrates the alkaline phosphatase (AP) activity in rMSCs cells preconditioned with rCM-Smurf1 or rCM-Ctrl. Quantification of in vitro mineralization is shown in the bottom panel, right part, of the figure. (B) Alizarin Red staining measuring mineralization was performed every four days during osteogenic differentiation. The images display results from representative samples. For all graphs, results are presented as means ± SEM. (n = 3) with each sample analyzed in technical triplicates. (C) Masson’s trichrome staining of ectodermically implanted scaffolds 8 weeks after implantation. Images show histological analysis of sections obtained from decalcified implants. Collagen of the extracellular bone matrix stained in dark blue. White arrowheads indicate osteocytes‐like cells surrounded by lacunae and immersed in the mineralized matrix (Magnification × 4). A higher magnification shows osteocyte cells surrounded by osteocytic lacunae in CM-Smurf1 (Magnification × 9). Histological sections of the ectodermic scaffolds stained by immunohistochemistry with specific antibody for ALPL and OCN (Magnification × 4). Graphs show the quantification of new bone formation and ALPL and OCN levels observed in the histological sections. Results are presented as means ± SEM. *:p-value < 0.05; **: p-value < 0.01; ***:p-value < 0.001. (n = 3)