Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH) expression is positively correlated with phosphorylated focal adhesion kinase (FAK) Tyr397. However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr397 were expressed in bone marrow cells and that pFAK Tyr397 was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr397 were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr397 time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK)-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b+ bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.

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Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling.

a b s t r a c tPurpose of the research: Tooth germ development involves multiple events, including cell proliferation and cell differentiation. Connective tissue growth factor (CTGF/CCN2) is a signaling protein involved in tooth germ development, and we investigated how it is expressed and what roles it may have in primary cultures of mesenchymal cells derived from the developing tooth germ. We also examined the expression of CCN2 in a human odontogenic myxofibroma, a benign tumor of odontogenic mesenchymal origin, and considered the possible roles of CCN2 in the development of myxofibromas. Materials and methods: Mesenchymal cells of early bell-stage tooth germs were isolated from Day-90 bovine embryos and placed in primary culture. A resected specimen from a patient with odontogenic myxofibroma was prepared for immunohistochemical studies. Principal results: The CCN2 expression level in proliferating odontogenic mesenchymal cells freshly isolated from the early bell stage of developing bovine tooth germs and placed in primary culture was 3 times higher than that in the confluent non-proliferating cells. Recombinant CCN2 significantly increased the proliferation and type I collagen expression in odontogenic mesenchymal cells in primary culture. Immunohistochemical analysis on myxofibroma case revealed that CCN2 was detectable in MIB-1, a cellular marker of proliferation-positive odontogenic mesenchymal cells adjacent to capillary blood vessels and in the endothelial cells of the vessels in the tumor. Major conclusion: CCN2 signaling would influence the proliferation of and extracellular matrix production by dental mesenchymal cells. Our results suggest that the same mechanisms of CCN2 action toward dental mesenchymal cells would also be operative in the odontogenic myxofibroma microenvironment.