Increased expression of osteogenic marker genes in ActRIIB-Fc-treated calluses at two weeks.

<p>Quantitative real-time PCR analyses of the two-week time point revealed higher expression of essential osteoblast markers osterix, runt-related transcription factor 2 and alkaline phosphatase. Expression of cathepsin K and tartrate resistant acid phosphatase decreased compared to PBS controls which could demonstrate impaired cartilage and bone resorption. Furthermore expression of Sox9 was also lower compared to controls. Expression levels of Sclerostin and Dkk-1, negative regulators of Wnt-signaling, were also lower in ActRIIB-Fc-treated mice. Expression of Smad1/5/8 target genes Id1 and Id3 were decreased as well. * = p<0.05, ** = p<0.01. n = 7 for PBS groups and 8 for ActRIIB-Fc groups.</p

Changes in body weight compared to the start of the experiment (%).

<p>Changes in body weight compared to the start of the experiment (%).</p

Immunohistochemical analyses of the calluses.

<p>Trends towards decreased number of cathepsin K+ cells in ActRIIB-Fc-treated mice were seen at both two and four weeks (A). The number of Runx2 positive cells was slightly increased at four weeks compared to PBS controls (p = 0.346) (B). There were no changes in the number of p-Smad1+ cells at either two or four weeks (C) but trends of increased number of p-Smad2+ cells were observed at two (p = 0.056) and four weeks (p = 0.209) due to ActRIIB-Fc-treatment (D). n = 8–9 for PBS 2 weeks, n = 10 for ActRIIB-Fc 2 weeks, n = 5–8 for PBS 4 weeks and n = 5–8 for ActRIIB-Fc 4 weeks.</p

ActRIIB-Fc robustly accelerated fracture healing and callus mineralization.

<p>(A-D) Representative radiographic and micro-computed cross-sectional images of the fracture callus of the PBS- and ActRIIB-Fc-treated mice at two and four weeks. There were no significant differences in bone structure between the groups at the two-week time point. (E-I) At four weeks, ActRIIB-Fc treatment resulted in greater increases bone volume/tissue volume, trabecular numbers and volumetric bone mineral density as well as decreased trabecular separation and structural model index-* = p<0.05, ** = p<0.01, *** = p<0.001. n = 8 for PBS 2 weeks, 7 for ActRIIB-Fc 2 weeks, 8 for PBS 4 weeks and 6 for ActRIIB-Fc 4 weeks.</p

Treatment with ActRIIB-Fc improves mechanical strength of calluses.

<p>ActRIIB-Fc increases callus strength in the four week groups compared to PBS controls in terms of (A) maximum load, (B) stiffness and (C) bending strength. No significant changes were noted between the two week groups. * = p<0.05 n = 6 for PBS 2 weeks, 8 for ActRIIB-Fc 2 weeks, 5 for PBS 4 weeks and 7 for ActRIIB-Fc 4 weeks.</p

Histological analysis of the calluses.

<p>Representative hematoxylin and eosin stained histological images of fracture calluses at four weeks. (A and C) Overview image of the callus (in black and white in order to distinguish the newly formed trabecular bone more easily) and (B and D) larger magnification of the trabecular bone within the callus at four weeks. Black arrows pinpoint newly formed trabeculae. At the two week time point, ActRIIB-Fc treatment caused increased (E) woven bone volume and (F) cartilage volume compared to PBS controls resulting in increased (G) mineralized tissue per tissue volume. At the four-week time point ActRIIB-Fc treatment greatly enhanced (H) bone volume/tissue volume and (J) trabecular numbers and decreased their (I) separation * = p<0.05 ** = p<0.01, *** = p<0.001. n = 6 for PBS 2 weeks, 6 for ActRIIB-Fc 2 weeks, 7 for PBS 4 weeks and 7 for ActRIIB-Fc 4 weeks.</p