Role of TNF in fracture healing in vivo Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing, indicated by increased % callus.

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Role of TNF in fracture healing in vivo Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing, indicated by increased % callus mineralization, at day 28 after operation in a dose‐dependent manner. Role of TNF in fracture healing in vivo Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing, indicated by increased % callus mineralization, at day 28 after operation in a dose‐dependent manner. Data are presented as mean ± SEM. Treatment with 1 ng TNF versus PBS control, ****P < 0.0001 by one‐way ANOVA with Dunnett's multiple comparisons test. Addition of rhTNF at the fracture site must be given within the first 24 h to augment healing, indicated by % callus mineralization. Data are presented as mean ± SEM. 1 ng TNF versus PBS control treatment on days 0 and 1, ***P = 0.0009 by one‐way ANOVA with Dunnett's multiple comparisons test. Treatment with systemic anti‐TNF or local rmIL‐10 led to impaired fracture repair at day 28, indicated by % callus mineralization. Data are presented as mean ± SEM. Treatment with PBS control versus anti‐TNF, *P = 0.037, PBS control versus rmIL‐10, *P = 0.037, by unpaired two‐sided t‐tests. Representative micro‐CT images at the fracture site showing (from left to right): lateral view of tibia, cross‐section, and cross and longitudinal sections with color overlay and 3D reconstruction. In the color overlays, the shade of blue corresponds to percentage mineralization: light blue denotes soft immature callus, whereas dark blue denotes hard mineralized callus. Scale bar, 2 mm. Representative ISH image (light field) showing TNF expression at the murine fracture site at 24 h after fracture. Scale bar, 250 μm. Red box indicates region of interest. High‐power micrographs of region of interest from (E): at 24 h after fracture, mTNF expression (white signal on dark field, above) co‐localized with polymorphonuclear cells found on the adjacent H&E section (below). Scale bar, 25 μm. Neutrophils were identified by their polymorphonuclear morphology as well as positive dark brown staining with anti‐neutrophil elastase. High‐power micrographs: at 7 days, TNF expression (white signal on dark field, above) co‐localized with F4/80‐positive cells (stained dark brown) extravasating from a blood vessel on the adjacent H&E section (below). Scale bar, 25 μm. James K Chan et al. EMBO Mol Med. 2015;7:547-561 © as stated in the article, figure or figure legend