In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwig's epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-β regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-β was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-β1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-β1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-β signaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.

Our results suggest that both fibrillin-1 and fibrillin-2 expression is required to form thick oxytalan fibers in PDL. Based on the expression patterns for fibrillin-1 and fibrillin-2, they have different functions during tooth root and PDL development. Early expression of fibrillin-2 may regulate dental epithelial cell behavior during root and PDL development.

It is important to understand the different mechanisms involved in anterior hard and posterior soft palate development to prevent and treat patients with cleft palate. Genetic analyses of humans and genemutated mice with cleft palate have shown that TGF-β signaling has a critical role in palatogenesis. However, the intracellular signaling pathway of TGF-β during palatogenesis in the anterior-posterior axis has not yet been fully understood. In the present study, the expression patterns of intracellular molecules Smad2/3 and phosphop38 (Pp38) were examined at embryonic days 13.5, 14.0, and 14.5 (E13.5, E14.0, and E14.5) in mice. It was found that Smad3 was activated in anterior palatal mesenchyme and in the medial edge epithelium (MEE) region, with TGF-β3 expressed at E13.5. On the other hand, Pp38 was more expressed in posterior palatal mesenchyme and strongly expressed in the entire palatal epithelium at E13.5. These opposing expression patterns between Smad3 and Pp38 in palatal mesenchyme were also observed at E14.0. Interestingly, Pp38 expression was inhibited in MEE from E14.0. Generally, from E14.5, the tissue specificities of hard and soft palate started showing their characteristics following the activation of cell differentiation in palatal mesenchyme, and the medial edge seam (MES) of the palatal epithelium started to disappear for fusion to occur. At this stage, Smad3 was also more expressed in anterior palatal mesenchyme, while expression of Pp38 was activated in posterior palatal mesenchyme. Pp38 expression was inhibited, but Smad3 was activated in the MES. These results suggest that TGF-β signaling plays various roles, such as in cell proliferation and differentiation of palatal mesenchyme and in the disappearance of the MES, through different intracellular signaling pathways in anteriorposterior palatal mesenchyme and epithelium.

The periodontal ligament (PDL) is fibrous tissue that maintains the connective space between tooth root and alveolar bone while avoiding the root resorption and the ankylosis. Fibrillin is one of the components of the elastic system fibers which exist in PDL as oxytalan fibers, and it plays an important role in organizing PDL with collagen fibers. The aim of the present study was to evaluate the effect of applying fibrillin protein for PDL regeneration using a mouse tooth replantation model. The ratio of root resorption and ankylosis was not improved in a replanted tooth soaked in fibrillin/PBS solution as compared to being soaked in PBS only. Interestingly, replantation with fibrillin and propylene glycol alginate (fibrillin/PGA) induced less root resorption and ankylosis than were observed with teeth replanted with PGA only. The PDL space between tooth root and alveolar bone in the fibrillin/PGA was clearly wider than that with PGA only. This regenerated PDL with Azan staining showed the meshwork structures including rich fibers in the horizontal direction. Furthermore, immunohistochemical expression of periostin was strongly detected in the entire regenerated PDL in the fibrillin/ PGA. These results suggest that fibrillin promotes the formation of fibrous connective tissue in a PDL replantation and might be a good candidate as regenerative material for the PDL.