The non-canonical BMP and Wnt/ß-catenin signaling pathways orchestrate early tooth development.

BMP and Wnt signaling pathways play a crucial role in organogenesis, including tooth development. Despite extensive studies, the exact functions, as well as if and how these two pathways act coordinately in regulating early tooth development, remain elusive. In this study, we dissected regulatory functions of BMP and Wnt pathways in early tooth development using a transgenic noggin (Nog) overexpression model (K14Cre;pNog). It exhibits early arrested tooth development, accompanied by reduced cell proliferation and loss of odontogenic fate marker Pitx2 expression in the dental epithelium. We demonstrated that overexpression of Nog disrupted BMP non-canonical activity, which led to a dramatic reduction of cell proliferation rate but did not affect Pitx2 expression. We further identified a novel function of Nog by inhibiting Wnt/ß-catenin signaling, causing loss of Pitx2 expression. Co-immunoprecipitation and TOPflash assays revealed direct binding of Nog to Wnts to functionally prevent Wnt/ß-catenin signaling. In situ PLA and immunohistochemistry on Nog mutants confirmed in vivo interaction between endogenous Nog and Wnts and modulation of Wnt signaling by Nog in tooth germs. Genetic rescue experiments presented evidence that both BMP and Wnt signaling pathways contribute to cell proliferation regulation in the dental epithelium, with Wnt signaling also controlling the odontogenic fate. Reactivation of both BMP and Wnt signaling pathways, but not of only one of them, rescued tooth developmental defects in K14Cre;pNog mice, in which Wnt signaling can be substituted by transgenic activation of Pitx2. Our results reveal the orchestration of non-canonical BMP and Wnt/ß-catenin signaling pathways in the regulation of early tooth development.

Antibacterial and antibiofilm activities of eugenol from essential oil of Syzygium aromaticum (L.) Merr. & L. M. Perry (clove) leaf against periodontal pathogen Porphyromonas gingivalis.

The antibacterial effect and mechanism of eugenol from Syzygium aromaticum (L.) Merr. & L. M. Perry (clove) leaf essential oil (CLEO) against oral anaerobe Porphyromonas gingivalis were investigated. The results showed that eugenol, with content of 90.84% in CLEO, exhibited antibacterial activity against P. gingivalis at a concentration of 31.25 µM. Cell shrink and lysis caused by eugenol were observed with Scanning Electron Microscopy (SEM). The release of macromolecules and uptake of fluorescent dye indicated that the antibacterial activity was due to the ability of eugenol to permeabilize the cell membrane and destroy the integrity of plasmatic membrane irreversibly. In addition, eugenol inhibited biofilm formation and reduced preformed biofilm of P. gingivalis at different concentrations. The down-regulation of virulence factor genes related to biofilm (fimA, hagA, hagB, rgpA, rgpB, kgp) explained that eugenol suppressed biofilm formation at the initial stage. These findings suggest that eugenol and CLEO may be potential additives in food and personal healthcare products as a prophylactic approach to periodontitis.

Intra-epithelial requirement of canonical Wnt signaling for tooth morphogenesis.

Multiple Wnt ligands are expressed in the developing tooth and play important and redundant functions during odontogenesis. However, the source of Wnt ligands and their targeting cells and action mechanism in tooth organogenesis remain largely elusive. Here we show that epithelial inactivation of Gpr177, the mouse Wntless (Wls) whose product regulates Wnt sorting and secretion, leads to arrest of tooth development at the early cap stage and abrogates tooth-forming capability of the dental epithelium. Gpr177 in the epithelium is necessary for the activation of canonical Wnt signaling in the dental epithelium and formation of a functional enamel knot. Epithelial deletion of Gpr177 results in defective gene expression and cellular behavior in the dental epithelium but does not alter odontogenic program in the mesenchyme. Furthermore, deletion of Axin2, a negative intracellular regulator of canonical Wnt signaling, rescues the tooth defects in mice carrying Gpr177 mutation in the dental epithelium. Together with the fact that active Wnt canonical signaling is present predominantly in the dental epithelium during tooth development, our results demonstrate that Gpr177-mediated Wnt ligands in the dental epithelium act primarily in an intra-epithelial context to regulate enamel knot formation and subsequent tooth development.

Antisolvent fabrication of monodisperse liposomes using novel ultrasonic microreactors: Process optimization, performance comparison and intensification effect.

Liposomes as drug carriers for the delivery of therapeutic agents have triggered extensive research but it remains a grand challenge to develop a novel technology for enabling rapid and mass fabrication of monodisperse liposomes. In this work, we constructed a novel ultrasonic microfluidic technology, namely ultrasonic microreactor (USMR) with two different conjunction structure (co-flow and impinge flow, corresponding to USMR-CF and USMR-IF, respectively), to prepare uniform liposomes by antisolvent precipitation method. In this process, the monodisperse liposomes with tunable droplet sizes (DS) in 60-100 nm and a polydispersity index (PDI) less than 0.1 can easily be achieved by tuning the total flow rate, flow rate ratio, ultrasonic power, and lipid concentration within the two USMRs. Impressively, the USMR-IF is superior for reducing the PDI and tuning DS of the liposomes over the USMR-CF. More importantly, the ultrasonic can effectively reduce DS and PDI at the low TFR and support the IF-micromixer in reducing the PDI even at a high TFR. These remarkable performances are mainly due to the rapid active mixing, fouling-free property and high operation stability for USMR-IF. In addition, diverse lipid formulations can also be uniformly assembled into small liposomes with narrow distribution, such as the prepared HSPC-based liposome with DS of 59.6 nm and PDI of 0.08. The liposomes show a high stability and the yield can reach a high throughput with 108 g/h by using the USMR-IF at an initial lipid concentration of 60 mM. The results in the present work highlight a novel ultrasonic microfluidic technology in the preparation of liposomes and may pave an avenue for the rapid, fouling-free, and high throughput fabrication of different and monodisperse nanomedicines with controllable sizes and narrow distribution.

Follistatin controls the number of murine teeth by limiting TGF-ß signaling.

Supernumerary teeth are common developmental anomalies of dentition. However, the factors and mechanisms driving their formation remain largely unknown. Here, we report that conditional knockout of Fst, encoding an antagonist for the transforming growth factor ß (TGF-ß) signaling pathway, in both oral epithelium and mesenchyme of mice (Fst CKO ) led to supernumerary upper incisor teeth, arising from the lingual dental epithelium of the native teeth and preceded by an enlarged and split lingual cervical loop. Fst-deficiency greatly activated TGF-ß signaling in developing maxillary incisor teeth, associated with increased epithelium cell proliferation. Moreover, Fst CKO teeth exhibited increased expression of Tbx1, Sp6, and Sox2, which were identified as direct targets of TGF-ß/SMAD2 signaling. Finally, we show that upregulation of Tbx1 in response to Fst-deficiency was largely responsible for the formation of extra teeth in Fst CKO mice. Taken together, our investigation indicates a novel role for Fst in controlling murine tooth number by restricting TGF-ß signaling.

Periodic Nanoneedle and Buffer Zones Constructed on a Titanium Surface Promote Osteogenic Differentiation and Bone Calcification In Vivo.

Rapid and effective bone mineralization at the bone/implant interface is required for successful orthopedic and dental implants. In this study, two periodic microscale functionalized zones on titanium (MZT) are created, namely, nanoneedle zones and buffer zones. The aim of this design is to provide spatially regulated topographical cues on titanium to enhance the efficacy of bone regeneration. This goal is achieved using a versatile and effective technique in which nanoneedle structures are hydrothermally constructed on the surface of titanium sheets, after which selective laser irradiation is used to construct buffer zones. The zonal structures of the MZT overcome the suppressive effect of the nanoneedle film on osteoblasts. Additionally, the MZT exhibits zone-selective apatite deposition and protein adsorption. The accelerated in vitro osteoblast differentiation and nodule deposition on the MZT are confirmed. Elemental analysis of the bone nodules formed by the osteoblasts growing on the titanium and MZT demonstrates they have different compositions. Histological and scanning electron microscope analysis of the bone formation on in vivo implants shows that this process is also enhanced by the MZT implant. The concept of constructing functionalized zones on titanium implant could facilitate future research on improving the design of orthopedic and dental implant surfaces.