The canonical Wnt/β-catenin signaling pathway has been shown to play essential roles in tooth initiation and early tooth development. However, the role of Wnt/β-catenin signaling in cusp patterning and crown calcification in large mammals are largely unknown. In our previous study, miniature pigs were used as the animal model due to the similarity of tooth anatomy and replacement pattern between miniature pig and human. Dynamic gene expression of third deciduous molar (DM3) in miniature pig at early stages was profiled using microarray method and expression of Wnt genes was significantly correlate with odontogenesis. In the present study, dynamic expression patterns of Wnt/β-catenin signaling genes of DM3 at cap, early bell and late bell (secretory) stage were identified. We found that Lef1 and Axin2 were expressed in the enamel knot and underlying mesenchyme regions. Meanwhile, Dkk1 was expressed in the peripheral and lower parts of dental papilla, thus forming the potential Wnt signaling gradient. We also found that β-Catenin, Axin2 and Lef1 were expressed strongly in undifferentiated cells of the inner enamel epithelium (IEE), but weakly in differentiated ameloblasts. Furthermore, we found that both Wnt signaling read-out gene Lef1 and the inhibitor Dkk1 were co-expressed in the pre-odontoblasts. In conclusion, the spatiotemporal distribution and potential gradient of Wnt signaling may contribute to cusp patterning and crown calcification. These data may yield insight into future study of precise control of crown morphogenesis and regeneration in large mammals.

The tooth root transmits and balances occlusal forces through the periodontium to the alveolar bone. The periodontium, including the gingiva, the periodontal ligament, the cementum and the partial alveolar bone, derives from the dental follicle (DF), except for the gingiva. In the early developmental stages, the DF surrounds the tooth germ as a sphere and functions to promote tooth eruption. However, the morphological dynamics and factors regulating the differentiation of the DF during root elongation remain largely unknown. Miniature pigs are regarded as a useful experimental animal for modeling in craniofacial research because they are similar to humans with respect to dentition and mandible anatomy. In the present study, we used the third deciduous incisor of miniature pig as the model to investigate the factors influencing DF differentiation during root development. We found that the DF was shaped like a crescent and was located between the root apical and the alveolar bone. The expression levels of WNT5a, β-Catenin, and COL-I gradually increased from the center of the DF (beneath the apical foramen) to the lateral coronal corner, where the DF differentiates into the periodontium. To determine the potential regulatory role of the apical papilla on DF cell differentiation, we co-cultured dental follicle stem cells (DFSCs) with stem cells of the apical papilla (SCAPs). The osteogenesis and fibrogenesis abilities of DFSCs were inhibited when being co-cultured with SCAPs, suggesting that the fate of the DF can be regulated by signals from the apical papilla. The apical papilla may sustain the undifferentiated status of DFSCs before root development finishes. These data yield insight into the interaction between the root apex and surrounding DF tissues in root and periodontium development and shed light on the future study of root regeneration in large mammals.

Renewal of integumentary organs occurs cyclically throughout an organism's lifetime, but the mechanism that initiates each cycle remains largely unknown. In a miniature pig model of tooth development that resembles tooth development in humans, the permanent tooth did not begin transitioning from the resting to the initiation stage until the deciduous tooth began to erupt. This eruption released the accumulated mechanical stress inside the mandible. Mechanical stress prevented permanent tooth development by regulating expression and activity of the integrin β1-ERK1-RUNX2 axis in the surrounding mesenchyme. We observed similar molecular expression patterns in human tooth germs. Importantly, the release of biomechanical stress induced downregulation of RUNX2-wingless/integrated (Wnt) signaling in the mesenchyme between the deciduous and permanent tooth and upregulation of Wnt signaling in the epithelium of the permanent tooth, triggering initiation of its development. Consequently, our findings identified biomechanical stress-associated Wnt modulation as a critical initiator of organ renewal, possibly shedding light on the mechanisms of integumentary organ regeneration.

Retinoic acid (RA) signal is involved in tooth development and osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) are one of the useful MSCs in tissue regeneration. However, the function of RA in osteo/odontogenic differentiation of DPSCs remains unclear. Here, we investigated the expression pattern of RA in miniature pig tooth germ and intervened in the RA signal during osteo/odontogenic differentiation of human DPSCs. Deciduous canine (DC) germs of miniature pigs were observed morphologically, and the expression patterns of RA were studied by in situ hybridization (ISH). Human DPSCs were isolated and cultured in osteogenic induction medium with or without RA or BMS 493, an inverse agonist of the pan-retinoic acid receptors (pan-RARs). Alkaline phosphatase (ALP) activity assays, alizarin red staining, quantitative calcium analysis, CCK8 assay, osteogenesis-related gene expression, and in vivo transplantation were conducted to determine the osteo/odontogenic differentiation potential and proliferation potential of DPSCs. We found that the expression of RARβ and CRABP2 decreased during crown calcification of DCs of miniature pigs. Activation of RA signal in vitro inhibited ALP activities and mineralization of human DPSCs and decreased the mRNA expression of ALP, osteocalcin, osteopontin, and a transcription factor, osterix. With BMS 493 treatment, the results were opposite. Interference in RA signal decreased the proliferation of DPSCs. In vivo transplantation experiments suggested that osteo/odontogenic differentiation potential of DPSCs was enhanced by inversing RA signal. Our results demonstrated that downregulation of RA signal promoted osteo/odontogenic differentiation of DPSCs and indicated a potential target pathway to improve tissue regeneration.