Loss of Smad3 gives rise to poor soft callus formation and accelerates early fracture healing.

Smad3 is an intracellular signaling molecule in the transforming growth factor β (TGF-β) pathway that serves as a regulator of chondrogenesis and osteogenesis. To investigate the role of the TGF-β/Smad3 signaling in the process of fracture healing, an open fracture was introduced in mouse tibiae, and the histology of the healing process was compared between wild-type (WT) and Smad3-null (KO) mice. In KO mice, the bone union formed more rapidly with less formation of cartilage in the callus and eventually the fracture was repaired more rapidly than in WT mice. Alkaline phosphatase staining showed that osteoblastic differentiation in the fracture callus was promoted in KO mice. Additionally, TRAP staining and the TUNEL assay revealed that the induction of osteoclasts and apoptotic cells was significantly promoted in the healing callus of KO mice. Sox9 expression clearly decreased at both mRNA and protein levels in the early stage of fracture in KO mice. In contrast, the expression of genes for osteogenesis and osteoclast formation increased from day 5 until day 14 post-fracture in KO mice compared to WT mice. From these results, we concluded that the loss of TGF-β/Smad3 signaling promoted callus formation by promoting osteogenesis and suppressing chondrogenesis, which resulted in faster fracture healing.