BACKGROUND: MSX1 sequence variants have been known to cause human tooth agenesis (TA) with or without orofacial clefts. However, their roles during the whole processes of tooth development are not fully understood. This study aimed to characterize a 4-membered family with TA carrying a novel MSX1 pathogenic variant and investigate the disease mechanism.
METHODS: The authors conducted whole exome analysis to define the disease-causing sequence variant. They performed microcomputed tomography, morphometric analyses, transcriptome profiling, and molecular characterization to study the affected teeth and the gene variant.
RESULTS: The authors identified an MSX1 pathogenic variant, p.Glu232∗, in affected family members with TA and concomitant orodental anomalies, namely, prominent maxillary labial frenum, central incisor diastema, median maxillary anterior alveolar cleft, tooth fusion, mandibular molar dysmorphology, thin dentin layer, and slender dental roots. MSX1-defective teeth were not apparently microdontic but had thin dentin layers. The mandibular molars showed a homeotic transformation to maxillary counterparts. Genes involved in extracellular matrix organization and dentinogenesis, such as DMP1 and MMP20, were downregulated in dental pulp tissues of MSX1-defective teeth. The p.Glu232∗-truncated MSX1 properly localized to the nucleus but partially lost its transactivation ability. Analyzing reported cases indicated that truncation sequence variants within the homeobox domain of MSX1 caused a more severe TA phenotype than those outside of the homeobox domain, probably due to dominant negativity compared with haploinsufficiency.
CONCLUSIONS: This study provides in vivo evidence that MSX1 contributes to developmental processes of various orodental tissues in humans.
CONCLUSIONS: Clinically, hypertrophic labial frenum, incisor diastema, and median maxillary anterior alveolar cleft might be considered diagnostic for MSX1-associated TA.

Previous studies have reported successful clinical outcomes after regenerative endodontic procedures (REPs) for immature permanent teeth with pulpal infection. However, it remains unclear whether the procedures promote true regeneration or repair. This case report describes the histologic and electron microscopic characteristics of a human immature permanent premolar with a chronic apical abscess that was treated with an REP. Tooth #20 of a 9-year-old girl underwent an REP. At the 6-year follow-up, the patient was asymptomatic, and closure of the apex and thickening of the dentinal walls were observed. However, 16 years after the procedure, apical periodontitis recurred, necessitating apical surgery. The resected root fragments were obtained during the surgery and analyzed using micro-computed tomography, light microscopy, and scanning electron microscopy. Distinct dentinal tubules and interglobular dentin were observed in the regenerated hard tissue. Cementum-like tissue and a root canal were also observed in the apical fragment. The regenerated root tissue in this case exhibited a structure similar to the native root structure. Therefore, we believe that cell-free REPs possess regenerative potential for teeth diagnosed with pulp necrosis and chronic apical abscess.

Cluster of differentiation 146 (CD146) is known to localize in stem cells and precursor cells of various tissues. In this study, to analyze the function of CD146 in odontoblast differentiation, immunohistochemical localization of CD146 was examined during rat molar tooth development and after cavity preparation. At the cap and bell stages, many CD146-positive cells were visible around the blood vessels in the dental papillae. On Postnatal day 2, osterix-positive odontoblasts were arranged in the dentin sialoprotein (DSP)-positive predentin, and many CD146-positive cells were observed near these odontoblasts with blood vessels. Some perivascular CD146-positive cells overlapped with Smad4-positive cells. However, the immunoreactivity for alpha-smooth muscle actin (α-SMA), one of the markers for undifferentiated cells, was negligible. Furthermore, the number of these cells decreased in the dental pulp on Postnatal day 28. On Day 4 after cavity preparation, Osterix-positive odontoblasts appeared lining the reparative dentin. Most of the blood vessels near the reparative dentin showed immunoreactivities for CD146. Reparative odontoblasts actively formed DSP-positive dentin matrix because these cells were positive for Smad4 and Osterix, but not for α-SMA. After 7 days, the number of CD146-positive cells near blood vessels decreased in the dental pulp beneath the cavity. These results suggest that the CD146 is expressed in the perivascular area of the dental pulp and induces vascularization in the vicinity of dentin formation, and some CD146-positive cells are activated by the bone morphogenetic protein signaling pathway and differentiate into odontoblasts in the early stages of dentin formation and repair.

Novel technologies and platforms have allowed significant breakthroughs in dental pulp tissue engineering. The development of injectable scaffolds that can be combined with stem cells, growth factors, or other bioactive compounds has enabled the regeneration of functional dental pulps able to secrete dentin in preclinical and clinical studies. Similarly, cell-homing technologies and scaffold-free strategies aim to modulate dental pulp self-regeneration mediated by resident stem cells and can evade some of the technical challenges related to cell-based tissue engineering strategies. This article will discuss emerging technologies and platforms for the clinical applications of dental pulp tissue engineering.

In recent years, there has been increasing interest in the treatment of bone defects using undifferentiated mesenchymal stem cells (MSCs) in vivo. Recently, dental pulp has been proposed as a promising source of pluripotent mesenchymal stem cells (MSCs), which can be used in various clinical applications. Dentin is the hard tissue that makes up teeth, and has the same composition and strength as bone. However, unlike bone, dentin is usually not remodeled under physiological conditions. Here, we generated odontoblast-like cells from mouse dental pulp stem cells and combined them with honeycomb tricalcium phosphate (TCP) with a 300 μm hole to create bone-like tissue under the skin of mice. The bone-like hard tissue produced in this study was different from bone tissue, i.e., was not resorbed by osteoclasts and was less easily absorbed than the bone tissue. It has been suggested that hard tissue-forming cells induced from dental pulp do not have the ability to induce osteoclast differentiation. Therefore, the newly created bone-like hard tissue has high potential for absorption-resistant hard tissue repair and regeneration procedures.

OBJECTIVE: To identify odontogenesis-promoting compounds and examine the molecular mechanism underlying enhanced odontoblast differentiation and tooth formation.
METHODS: Five different nymphaeols, nymphaeol B (NB), isonymphaeol B (INB), nymphaeol A (NA), 3\'-geranyl-naringenin (GN) and nymphaeol C (NC) were isolated from the fruit of Macaranga tanarius. The cytotoxic effect of nymphaeols on human DPSCs was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of nymphaeols on odontoblast differentiation was analysed with Alizarin Red S staining and odontoblast marker expression was assessed using real-time polymerase chain reaction and Western blot analysis. The molecular mechanism was investigated with Western blot analysis. In order to examine the effect of INB on dentine formation in the developing tooth germ, INB-soaked beads were placed under the tooth bud explants in the collagen gel; thereafter, the tooth bud explant-bead complexes were implanted into the sub-renal capsules for 3 weeks. Tooth root formation was analysed using micro-computed tomography and histological analysis. Data are presented as mean ± standard error (SEM) values of three independent experiments, and results are compared using a two-tailed Student\'s t-test. The data were considered to have statistical significance when the P-value was less than 0.05.
RESULTS: Three of the compounds, NB, INB, and GN, did not exert a cytotoxic effect on human DPSCs. However, INB was most effective in promoting the deposition of calcium minerals in vitro (P < 0.001) and induced the expression of odontogenic marker genes (P < 0.05). Moreover, this compound strongly induced the phosphorylation of mitogen-activated protein (MAP) kinases and protein kinase B (AKT) (P < 0.05). The inhibition of p38 MAP, c-Jun N-terminal kinase (JNK), and AKT substantially suppressed the INB-induced odontoblast differentiation (P < 0.001). In addition, isonymphaeol B significantly induced the formation of dentine and elongation of the tooth root in vivo (P < 0.05).
CONCLUSIONS: Prenylflavonoids, including INB, exerted stimulatory effects on odontoblast differentiation and tooth root and dentine formation via the MAP kinase and AKT signalling pathways. These results suggest that nymphaeols could stimulate the repair processes for dentine defects or injuries.

A previous study suggested that fibroblast growth factor (FGF) signaling plays an important role in dentin formation during tooth development. In this study, to examine dentin formation after tooth eruption involving secondary and tertiary dentin, we analyzed the expression patterns and expressing cells of Fgfr1, -2c, and -3c in mouse maxillary first molars (M1). Since it is difficult to recover the mRNAs from mineralized tissues, we tested methods for extraction after fixation and decalcification of teeth. We successfully obtained consistent results with quantitative real-time PCR (qPCR) using β-actin transcripts for validation. qPCR for Dentin sialo phosphoprotein (Dspp), Fgfr1, -2c, and -3c transcripts was performed on mice at ages of 2-20 weeks. The results showed that the highest expression levels of Dspp and Fgfr2c occurred at 2 weeks old followed by lower expression levels after 4 weeks old. However, the expression levels of Fgfr1 and Fgfr3c were constant throughout the experimental period. By in situ hybridization, Dspp, Fgfr1, and Fgfr3c transcripts were detected in odontoblasts at ages of 2 and 4 weeks. In addition, Dspp and Fgfr1 transcripts were detected in odontoblasts facing reactionary dentin at 8 weeks old. These results suggest that FGF-FGFR signaling might be involved in the regulation of odontoblasts even after tooth eruption, including secondary and tertiary dentin formation. Moreover, our modified method for extracting mRNA from mineralized tissues after fixation and decalcification successfully produced consistent results.

Recently, dental pulp has been attracting attention as a promising source of multipotent mesenchymal stem cells (MSCs) for various clinical applications of regeneration fields. To date, we have succeeded in establishing rat dental pulp-derived cells showing the characteristics of odontoblasts under in vitro conditions. We named them Tooth matrix-forming, GFP rat-derived Cells (TGC). However, though TGC form massive dentin-like hard tissues under in vivo conditions, this does not lead to the induction of polar odontoblasts. Focusing on the importance of the geometrical structure of an artificial biomaterial to induce cell differentiation and hard tissue formation, we previously have succeeded in developing a new biomaterial, honeycomb tricalcium phosphate (TCP) scaffold with through-holes of various diameters. In this study, to induce polar odontoblasts, TGC were induced to form odontoblasts using honeycomb TCP that had various hole diameters (75, 300, and 500 μm) as a scaffold. The results showed that honeycomb TCP with 300-μm hole diameters (300TCP) differentiated TGC into polar odontoblasts that were DSP positive. Therefore, our study indicates that 300TCP is an appropriate artificial biomaterial for dentin regeneration.

Bone morphogenetic protein 2 (Bmp2) is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta (DGI), showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. Here we describe the generation of an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko-dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2flox/flox-dp) cells.

BACKGROUND: Regeneration of the pulp-dentin complex is the penultimate goal of regenerative endodontic procedures (REPs). Histological outcomes have demonstrated reparative tissue formation in human teeth extracted post-REPs. However, lack of accurate characterization has precluded identification of the true nature of tissues formed post-REP.
METHODS: Here, we present 2 case reports of tooth #29 and #9 treated with REPs and demonstrate their clinical, radiographic, and histological outcomes.
RESULTS: Clinical outcomes revealed healing of apical periodontitis in both teeth and re-establishment of vitality responses in tooth #29. Moreover, radiographic assessments using 2D and 3D-volumetric analyses demonstrate considerable increase in root development for both teeth. Further, histological outcomes evaluated using Hematoxylin and Eosin and immunohistochemical staining demonstrates presence of vascular and lymphatic structures as well as immune cell markers indicative of regeneration of an immunocompetent pulp. Lastly, examination of hard tissue deposition shows dentin-like tissue in parts of tooth #29 demonstrating for the first time, regeneration of a pulp-dentin complex post-REP.
CONCLUSIONS: Collectively, this is the first study demonstrating recapitulation of several tissues commonly found as part of a pulp-dentin complex in teeth treated with REPs.