Hedgehog (Hh) signaling plays important roles in various development processes. This signaling is necessary for osteoblast formation during endochondral ossification. In contrast to the established roles of Hh signaling in embryonic bone formation, evidence of its roles in adult bone homeostasis is not complete. Here we report the involvement of Gli1, a transcriptional activator induced by Hh signaling activation, in postnatal bone homeostasis under physiological and pathological conditions. Skeletal analyses of Gli1 +/− adult mice revealed that Gli1 haploinsufficiency caused decreased bone mass with reduced bone formation and accelerated bone resorption, suggesting an uncoupling of bone metabolism. Hh-mediated osteoblast differentiation was largely impaired in cultures of Gli1 +/− precursors, and the impairment was rescued by Gli1 expression via adenoviral transduction. In addition, Gli1 +/− precursors showed premature differentiation into osteocytes and increased ability to support osteoclastogenesis. When we compared fracture healing between wild-type and Gli1 +/− adult mice, we found that the Gli1 +/− mice exhibited impaired fracture healing with insufficient soft callus formation. These data suggest that Gli1, acting downstream of Hh signaling, contributes to adult bone metabolism, in which this molecule not only promotes osteoblast differentiation but also represses osteoblast maturation toward osteocytes to maintain normal bone homeostasis.

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Bone defects affect patients functionally and psychologically and can decrease quality of life. To resolve these problems, a simple and efficient method of bone regeneration is required. Human dental pulp stem cells (DPSCs) have high proliferative ability and multilineage differentiation potential. In our previous study, we reported a highly efficient method to induce osteogenic differentiation using DPSC sheets treated with a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH)) in a mouse calvarial defect model. However, the localization of the DPSCs after transplantation remains unknown. Therefore, in this study, we investigated the localization of transplanted DPSCs in a mouse fracture model. DPSCs were collected from six healthy patients aged 18–29 years, cultured in normal medium (NM), osteogenic medium (OM), or OM with TH, and fabricated them into cell sheets. To evaluate the efficacy of fracture healing using DPSCs treated with OM+TH, and to clarify the localization of the transplanted DPSC sheets in vivo, we transplanted OM+TH-treated DPSC sheets labeled with PKH26 into mouse tibiae fractures. We demonstrated that transplanted OM+TH-treated DPSCs sheets were localized to the fracture site and facilitated bone formation. These results indicated that transplanted OM+TH-treated DPSCs were localized at fracture sites and directly promoted fracture healing.

Objective: The extraordinarily high bone densities identified in the hypoparathyroidism patients originating from PTH R25C mutation suggested the possibility that this modified protein has unique biologic effects and contributes to the gain of bone volume. Interestingly, western blot of cell lysates stably transfected with PTH R25C construct revealed that Cys25PTH(1–84) formed a dimer, presumably due to disulfide bonding of the cysteine residues. This study aims to study the characteristics of Cys25PTH(1–34) dimeric peptide (Dimer) both in vitro and in vivo for potential therapeutic application of Dimer as a novel anabolic agent. Methods: Identity of the chemically synthesized Dimer was confirmed by its molecular weight and purity using MS and HPLC, respectively. Basic characteristics, for example, ability to bind to PTHR and cAMP production were investigated using a variety of cells. In addition, the ligand-receptor internalization was investigated using TMR tagged Dimer. In vivo characteristics, such as calcemic and phosphatemic responses, pharmacokinetics and pharmacodynamics, were assessed in CD1 mice. The osteoanabolic effects of Dimer were assessed using a fracture-healing model, a calvarial-injection model and an OVX mouse model. Results:In vivo study showed that Dimer has similar calcemic and phosphatemic responses to PTH(1–34; WT). In cell assays, Dimer showed a similar cAMP production but slightly lower binding affinity compared to WT. Dimer-receptor complex was internalized into the cells. Surprisingly, Dimer showed a potent anabolic effect in the fracture-healing model in mice measured as the callus volume fraction by microCT. We also observed a comparable anabolic effect of Dimer in calvarial-injection model and OVX model. Conclusions: Dimeric Cys25PTH(1–84) peptide might play a substantial role in the high bone mass in hypoparathyroidism patients, originating from the PTH R25C mutation. This may be translated into the development of potential therapeutic modality for the treatment of osteoporosis and fracture healing using Dimer.