Establishing protein expression profiles involved in tooth development using a proteomic approach.

Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.

Role of osteopontin in the process of pulpal healing following tooth replantation in mice.

Introduction: The role of osteopontin (OPN) following severe injury remains to be elucidated, especially its relationship with type I collagen (encoded by the Col1a1 gene) secretion by newly-differentiated odontoblast-like cells (OBLCs). In this study, we examined the role of OPN in the process of reparative dentin formation with a focus on reinnervation and revascularization after tooth replantation in Opn knockout (KO) and wild-type (WT) mice. Methods: Maxillary first molars of 2- and 3-week-old-Opn KO and WT mice (Opn KO 2W, Opn KO 3W, WT 2W, and WT 3W groups) were replanted, followed by fixation 3-56 days after operation. Following micro-computed tomography analysis, the decalcified samples were processed for immunohistochemistry for Ki67, Nestin, PGP 9.5, and CD31 and in situ hybridization for Col1a1. Results: An intense inflammatory reaction occurred to disrupt pulpal healing in the replanted teeth of the Opn KO 3W group, whereas dental pulp achieved healing in the Opn KO 2W and WT groups. The tertiary dentin in the Opn KO 3W group was significantly decreased in area compared with the Opn KO 2W and WT groups, with a significantly low percentage of Nestin-positive, newly-differentiated OBLCs during postoperative days 7-14. In the Opn KO 3W group, the blood vessels were significantly decreased in area and pulp healing was disturbed with a failure of pulpal revascularization and reinnervation. Conclusions: OPN is necessary for proper reinnervation and revascularization to deposit reparative dentin following severe injury within the dental pulp of erupted teeth with advanced root development.

Role of chondroitin sulfate in the developmental and healing process of the dental pulp in mice.

Chondroitin sulfate proteoglycan (CSPG), one of the major extracellular matrices, plays an important part in organogenesis. Its core protein and chondroitin sulfate (CS) chain have a specific biological function. To elucidate the role of CS in the developmental and healing process of the dental pulp, we performed an experimental tooth replantation in CS N-acethylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. We also performed cell proliferation assay and qRT-PCR analysis for the WT and T1KO primary dental pulp cells using T1-siRNA technique and external CS. During tooth development, CS was diffusely expressed in the dental papilla, and with dental pulp maturation, CS disappeared from the differentiated areas, including the odontoblasts. In fully developed molars, CS was restricted to the root apex region colocalizing with Gli1-positive cells. In the healing process after tooth replantation, CD31-positive cells accumulated in the CS-positive stroma in WT molars. In T1KO molars, the appearance of Ki67- and Gli1-positive cells in the dental pulp was significantly fewer than in WT molars in the early healing stage, and collagen I-positive reparative dentin formation was not obvious in T1KO mice. In primary culture experiments, siRNA knockdown of T1 gene significantly suppressed cell proliferation in WT dental pulp cells, and the mRNA expression of cyclin D1 and CD31 was significantly upregulated by external CS in T1KO dental pulp cells. These results suggest that CS is involved in the cell proliferation and functional differentiation of dental pulp constituent cells, including vascular cells, in the healing process of dental pulp tissue after tooth injury.

Exploration of the role of the subodontoblastic layer in odontoblast-like cell differentiation after tooth drilling using Nestin-enhanced green fluorescent protein transgenic mice.

OBJECTIVES: Original odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice. METHODS: A groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1-14 days postoperative. RESULTS: Odontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP-positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1-2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp-dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3-5 to form reparative dentin. Nestin-GFP-positive cells at the pulp-dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(-)/Nestin(+) cells were intermingled in the newly differentiated OBLCs. CONCLUSIONS: The commitment of Nestin-GFP-positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp-dentin border and their differentiation into OBLCs.

Hypoxia-Responsive Oxygen Nanobubbles for Tissues-Targeted Delivery in Developing Tooth Germs.

Hypoxia is a state of inadequate supply of oxygen. Increasing evidence indicates that a hypoxic environment is strongly associated with abnormal organ development. Oxygen nanobubbles (ONBs) are newly developed nanomaterials that can deliver oxygen to developing tissues, including hypoxic cells. However, the mechanisms through which nanobubbles recover hypoxic tissues, such as developing tooth germs remain to be identified. In this study, tooth germs were cultured in various conditions: CO2 chamber, hypoxic chamber, and with 20% ONBs for 3 h. The target stages were at the cap stage (all soft tissue) and bell stage (hard tissue starts to form). Hypoxic tooth germs were recovered with 20% ONBs in the media, similar to the tooth germs incubated in a CO2 chamber (normoxic condition). The tooth germs under hypoxic conditions underwent apoptosis both at the cap and bell stages, and ONBs rescued the damaged tooth germs in both the cap and bell stages. Using kidney transplantation for hard tissue formation in vivo, amelogenesis and dentinogenesis imperfecta in hypoxic conditions at the bell stage were rescued with ONBs. Furthermore, glucose uptake by tooth germs was highly upregulated under hypoxic conditions, and was restored with ONBs to normoxia levels. Our findings indicate that the strategies to make use of ONBs for efficient oxygen targeted delivery can restore cellular processes, such as cell proliferation and apoptosis, glucose uptake, and hypomineralization in hypoxic environments.

Oxygen regulates epithelial stem cell proliferation via RhoA-actomyosin-YAP/TAZ signal in mouse incisor.

Stem cells are maintained in specific niches that strictly regulate their proliferation and differentiation for proper tissue regeneration and renewal. Molecular oxygen (O2) is an important component of the niche microenvironment, but little is known about how O2 governs epithelial stem cell (ESC) behavior. Here, we demonstrate that O2 plays a crucial role in regulating the proliferation of ESCs using the continuously growing mouse incisors. We have revealed that slow-cycling cells in the niche are maintained under relatively hypoxic conditions compared with actively proliferating cells, based on the blood vessel distribution and metabolic status. Mechanistically, we have demonstrated that, during hypoxia, HIF1α upregulation activates the RhoA signal, thereby promoting cortical actomyosin and stabilizing the adherens junction complex, including merlin. This leads to the cytoplasmic retention of YAP/TAZ to attenuate cell proliferation. These results shed light on the biological significance of blood-vessel geometry and the signaling mechanism through microenvironmental O2 to orchestrate ESC behavior, providing a novel molecular basis for the microenvironmental O2-mediated stem cell regulation during tissue development and renewal.

Responses of oral-microflora-exposed dental pulp to capping with a triple antibiotic paste or calcium hydroxide cement in mouse molars.

INTRODUCTION: Responses of oral-microflora-exposed dental pulp to a triple antibiotic paste (TAP), a mixture of ciprofloxacin, metronidazole, and minocycline in ointment with macrogol and propylene glycol, remain to be fully clarified at the cellular level. This study aimed to elucidate responses of oral-microflora-exposed dental pulp to capping with TAP in mouse molars. METHODS: A cavity was prepared on the first molars of 6-week-old mice to expose the dental pulp for 24 h. The exposed pulp was capped with TAP (TAP group) or calcium hydroxide cement (CH group), in addition to the combination of macrogol (M) and propylene glycol (P) (MP, control group), followed by a glass ionomer cement filling. The samples were collected at intervals of 1, 2, and 3 weeks, and immunohistochemistry for nestin and Ki-67 and deoxyuride-5'-triphosphate biotin nick end labeling (TUNEL) assay were performed in addition to quantitative real-time polymerase chain reaction (qRT-PCR) analyses. RESULTS: The highest occurrence rate of pulp necrosis was found in the control group followed by the CH group at Weeks 2 and 3, whereas the highest occurrence rate of healed areas in the dental pulp was observed in the TAP group at each time point. Tertiary dentin formation was first observed in the dental pulp of the TAP group at Week 2. In contrast, bone-like and/or fibrous tissues were frequently observed in the CH group. qRT-PCR analyses clarified that TAP activated the stem and dendritic cells at Weeks 1 and 2, respectively. CONCLUSIONS: The use of TAP as a pulp-capping agent improved the healing process of oral-microflora-exposed dental pulp in mouse molars.

Reduced enamel epithelium-derived cell niche in the junctional epithelium is maintained for a long time in mice.

BACKGROUND: Although numerous reports have demonstrated that the junctional epithelium (JE) is derived from the reduced enamel epithelium (REE), the fate of the REE-derived JE remains controversial. The present study elucidated the fate of the REE-derived JE and the cell dynamics of stem/progenitor cells in the JE following tooth eruption. METHODS: Mandibular first molar germs (embryonic days 15 to postnatal 1-day-old) were transplanted into the socket of 2-week-old mice after extraction of the upper first molars of B6 wild-type (WT) and green fluorescent protein (GFP) transgenic mice. After analysis by µ-CT, paraffin sections were processed for immunohistochemistry for Nestin, Ki67 and GFP. We also performed chasing analysis using BrdU-administered TetOP-H2B-GFP mice. RESULTS: Amelogenesis progressed normally in the cervical areas, and the structure of the JE was like that in normal tooth development. The JE was GFP-negative in transplantations using GFP transgenic mice as the host, and the oral epithelium (OE) showed a positive reaction. By contrast, the JE remained GFP-positive throughout the experimental period in transplantations using GFP transgenic mice as the donor. Chasing analysis revealed that H2B-GFP- and 5-Bromo-2'-deoxyuridine (BrdU)-labeled cells in the basal side of the JE translocated to oral side of the JE as the chasing time increased. Some label-retaining cells remained at the supra-basal cell layer in the JE. CONCLUSION: These results suggest that REE-derived cell niche in the JE is maintained for a long time following tooth eruption. Therefore, the JE may be available as the source of the odontogenic epithelium.

Loss of autophagy in chondrocytes causes severe growth retardation.

Chondrogenesis is accompanied by not only cellular renovation, but also metabolic stress. Therefore, macroautophagy/autophagy is postulated to be involved in this process. Previous reports have shown that suppression of autophagy during chondrogenesis causes mild growth retardation. However, the role of autophagy in chondrocyte differentiation still largely remains unclear. Here, we show the important role of autophagy on chondrogenesis. The transition of mesenchymal cells to chondrocytes was severely impaired by ablation of Atg7, a gene essential for autophagy. Mice lacking Atg7 after the transition exhibited phenotypes severer than mutant mice in which Atg7 was removed before the transition. Atg7-deficient chondrocytes accumulated large numbers of glycogen granules, hardly proliferate and died specifically in the proliferative zone without any ER-stress signal. Our results suggest that the suppression of autophagy in prechondrogenic cells drives compensatory mechanism(s) that mitigate defective chondrogenesis, and that autophagy participates in glycogenolysis to supply glucose in avascular growth plates.Abbreviations: DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; SQSTM1/p62: sequestosome 1; STBD1: starch-binding domain-containing protein 1.

Three-dimensional configuration of apical epithelial compartments including stem cell niches in guinea pig cheek teeth.

OBJECTIVES: Continuously growing rodent incisors have an apically located epithelial stem cell compartment, known as an "apical bud" (AB). Few studies have described the morphological features of ABs and stem cell niches in continuously growing premolars/molars. We attempted to clarify the relationship between the three-dimensional configuration of ABs and the stem cell niches in guinea pig cheek teeth. METHODS: We perfusion-fixed four-week-old guinea pigs, then decalcified their premolars/molars to produce serial paraffin sections, which we immunostained for Sox2. We reconstructed the serial sections using image processing and analysis software. We processed undecalcified samples for scanning electron microscopy by KOH digestion. RESULTS: Two types of epithelia with M and Δ shapes surrounded the S-shaped dental papilla in the apical region of the premolars/molars, and there were three Sox2-positive epithelial bulges above the M- and Δ-shaped epithelia. Sox2-positive epithelial stem cell niches were restricted to the apical side, and cell proliferation and differentiation immediately proceeded in the crown-analogue dentin. The Sox2-positive epithelial stem cell niches were sparsely distributed and extended to the occlusal side. We also detected continuously proliferating cells in the cervical loop and Hertwig's epithelial root sheath of the root-analogue dentin. CONCLUSIONS: Our findings suggest that guinea pig cheek teeth have three ABs, and the complex configuration of these types of teeth may be attributed to the prompt formation of crown-analogue dentin followed by the long-term formation of root-analogue dentin.

Extracellular enzymatically synthesized glycogen promotes osteogenesis by activating osteoblast differentiation via Akt/GSK-3ß signaling pathway.

Glycogen is the stored form of glucose and plays a major role in energy metabolism. Recently, it has become clear that enzymatically synthesized glycogen (ESG) has biological functions, such as the macrophage-stimulating activity. This study aimed to evaluate the effect of ESG on osteogenesis. MC3T3-E1 cells were cultured with ESG, and their cell proliferative activity and osteoblast differentiation were measured. An in vivo study was conducted in which ESG pellets with BMP-2 were grafted into mouse calvarial defects and histomorphometrically analyzed for the new bone formation. To confirm the effect of ESG on bone growth in vivo, ESG was orally administered to pregnant mice and the femurs of their pups were examined. We observed that ESG stimulated cell proliferation and enhanced messenger RNA expression of osteocalcin and osteopontin in MC3T3-E1 cells. ESG was taken up by the cells associated with GLUT-1 and activated the Akt/GSK-3ß pathway. In vivo, the new bone formation in the calvarial defect was significantly accelerated by ESG and the maternal administration of ESG promoted fetal bone growth. In conclusion, ESG stimulates cell proliferation and differentiation of preosteoblasts via the activation of Akt/GSK-3ß signaling and promotes new bone formation in vivo, suggesting that ESG could be a useful stimulant for osteogenesis.

Craniofacial abnormality with skeletal dysplasia in mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase-1.

Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.

Nestin expression is differently regulated between odontoblasts and the subodontoblastic layer in mice.

The Nestin gene encodes type VI intermediate filament and is known to be expressed in undifferentiated cells during neurogenesis and myogenesis. To regulate Nestin expression, the first or second intron enhancer is activated in a tissue-dependent manner, for example, the former in mesodermal cells and the latter in neural stem cells. Although Nestin has also been used as a differentiation marker for odontoblasts during tooth development, how Nestin expression is regulated in odontoblasts remains unclear. Therefore, this study aimed to compare the expression patterns of Nestin-GFP (green fluorescent protein) with that of endogenous Nestin in developing teeth of Nestin-EGFP (enhanced GFP) transgenic mice, in which the second intron enhancer is connected with the EGFP domain, at postnatal 7d, 3w, and 8w. Immunohistochemical and in situ hybridization analyses revealed that endogenous Nestin protein and Nestin mRNA were intensely expressed in differentiated odontoblasts, while GFP immunoreactivity, which reflects the activity of Nestin second intron enhancer-mediated transcription, was mainly observed in the subodontoblastic layer. These results indicate that the first intron enhancer may be activated in differentiated odontoblasts. Intriguingly, Nestin-GFP expression in the subodontoblastic layer was found to be restricted to the coronal pulp of molars, which is susceptible to tooth injuries. Because the subodontoblastic layer serves as a reservoir of newly differentiated odontoblast-like cells upon exogenous stimuli to dentin, our findings suggest that the original odontoblasts and regenerated odontoblast-like cells may differently regulate Nestin expression.

Orthodontic force application upregulated pain-associated prostaglandin-I2/PGI2-receptor/TRPV1 pathway-related gene expression in rat molars.

This study aimed to analyze the mRNA expression and protein localization of prostaglandin I2 (PGI2) synthase (PGIS), the PGI2 receptor (IP receptor) and transient receptor potential cation channel, subfamily V, member 1 (TRPV1) in force-stimulated rat molars, toward the elucidation of the PGI2-IP receptor-TRPV1 pathway that is in operation in the pulp and possibly associated with orthodontic pain and inflammation. Experimental force was applied to the maxillary first and second molars by inserting an elastic band between them for 6-72 h. PGIS, PTGIR (the IP receptor gene), and TRPV1 mRNA levels in the coronal pulp were analyzed with real-time PCR. PGIS, IP receptor, and TRPV1 proteins were immunostained. The force stimulation induced significant upregulation of PGIS at 6-24 h, and PTGIR and TRPV1 at 6 and 12 h in the pulp. PGIS was immunolocalized in odontoblasts and some fibroblasts in the force-stimulated pulp. The IP receptor and TRPV1 immunoreactivities were detected on odontoblasts and some nerve fibers. It was concluded that PGIS, PTGIR, and TRPV1 in rat molar pulp were significantly upregulated shortly after the force application, and that the IP receptor was co-expressed on TRPV1-expressing nerves and odontoblasts. These findings suggest that the PGI2-IP receptor-TRPV1 pathway is associated with the acute phase of force-induced pulp changes involving odontoblasts and nerves.

Effects of pulpotomy using mineral trioxide aggregate on prostaglandin transporter and receptors in rat molars.

Mineral trioxide aggregate (MTA) is a commonly used dental pulp-capping material with known effects in promoting reparative dentinogenesis. However, the mechanism by which MTA induces dentine repair remains unclear. The aim of the present study was to investigate the role of prostaglandin E2 (PGE2) in dentine repair by examining the localisation and mRNA expression levels of its transporter (Pgt) and two of its receptors (Ep2 and Ep4) in a rat model of pulpotomy with MTA capping. Ep2 expression was detected in odontoblasts, endothelial cells, and nerve fibres in normal and pulpotomised tissues, whereas Pgt and Ep4 were immunolocalised only in the odontoblasts. Moreover, mRNA expression of Slco2a1 (encoding Pgt), Ptger2 (encoding Ep2), and Ptger4 (encoding Ep4) was significantly upregulated in pulpotomised dental pulp and trigeminal ganglia after MTA capping. Our results provide insights into the functions of PGE2 via Pgt and Ep receptors in the healing dentine/pulp complex and may be helpful in developing new therapeutic targets for dental disease.

Pannexin 3 regulates proliferation and differentiation of odontoblasts via its hemichannel activities.

Highly coordinated regulation of cell proliferation and differentiation contributes to the formation of functionally shaped and sized teeth; however, the mechanism underlying the switch from cell cycle exit to cell differentiation during odontogenesis is poorly understood. Recently, we identified pannexin 3 (Panx3) as a member of the pannexin gap junction protein family from tooth germs. The expression of Panx3 was predominately localized in preodontoblasts that arise from dental papilla cells and can differentiate into dentin-secreting odontoblasts. Panx3 also co-localized with p21, a cyclin-dependent kinase inhibitor protein, in preodontoblasts. Panx3 was expressed in primary dental mesenchymal cells and in the mDP dental mesenchymal cell line. Both Panx3 and p21 were induced during the differentiation of mDP cells. Overexpression of Panx3 in mDP cells reduced cell proliferation via up-regulation of p21, but not of p27, and promoted the Bone morphogenetic protein 2 (BMP2)-induced phosphorylation of Smad1/5/8 and the expression of dentin sialophosphoprotein (Dspp), a marker of differentiated odontoblasts. Furthermore, Panx3 released intracellular ATP into the extracellular space through its hemichannel and induced the phosphorylation of AMP-activated protein kinase (AMPK). 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an activator of AMPK, reduced mDP cell proliferation and induced p21 expression. Conversely, knockdown of endogenous Panx3 by siRNA inhibited AMPK phosphorylation, p21 expression, and the phosphorylation of Smad1/5/8 even in the presence of BMP2. Taken together, our results suggest that Panx3 modulates intracellular ATP levels, resulting in the inhibition of odontoblast proliferation through the AMPK/p21 signaling pathway and promotion of cell differentiation by the BMP/Smad signaling pathway.

Quiescent adult stem cells in murine teeth are regulated by Shh signaling.

The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed in the enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

Locally Produced BDNF Promotes Sclerotic Change in Alveolar Bone after Nerve Injury.

Brain-derived neurotrophic factor (BDNF), which is released due to nerve injury, is known to promote the natural healing of injured nerves. It is often observed that damage of mandibular canal induces local sclerotic changes in alveolar bone. We reported that peripheral nerve injury promotes the local production of BDNF; therefore, it was possible to hypothesize that peripheral nerve injury affects sclerotic changes in the alveolar bone. This study aimed to evaluate the effect of BDNF on osteogenesis using in vitro osteoblast-lineage cell culture and an in vivo rat osteotomy model. MC3T3-E1 cells were cultured with BDNF and were examined for cell proliferative activity, chemotaxis and mRNA expression levels of osteoblast differentiation markers. For in vivo study, inferior alveolar nerve (IAN) injury experiments and mandibular cortical osteotomy were performed using a rat model. In the osteotomy model, exogenous BDNF was applied to bone surfaces after corticotomy of the mandible, and we morphologically analyzed the new bone formation. As a result, mRNA expression of osteoblast differentiation marker, osteocalcin, was significantly increased by BDNF, although cell proliferation and migration were not affected. In the in vivo study, osteopontin-positive new bone formation was significantly accelerated in the BDNF-grafted groups, and active bone remodeling, involving trkB-positive osteoblasts and osteocytes, continued after 28 days. In conclusion, BDNF stimulated the differentiation of MC3T3-E1 cells and it promoted new bone formation and maturation. These results suggested that local BDNF produced by peripheral nerve injury contributes to accelerating sclerotic changes in the alveolar bone.

Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells.

Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4-12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.

The Semaphorin 4D-RhoA-Akt Signal Cascade Regulates Enamel Matrix Secretion in Coordination With Cell Polarization During Ameloblast Differentiation.

During tooth development, oral epithelial cells differentiate into ameloblasts in order to form the most mineralized tissue in the vertebrate body: enamel. During this process, ameloblasts directionally secrete enamel matrix proteins and morphologically change from low columnar cells to polarized tall columnar cells, both of which are essential for the proper formation of enamel. In this study, we elucidated the molecular mechanism that integrates ameloblast function and morphology. Immunohistochemistry revealed that the restricted expression of semaphorin 4D (Sema4D) and RhoA activation status are closely associated with ameloblast differentiation in mouse incisors. In addition, in vitro gain-of-function and loss-of-function experiments demonstrated that Sema4D acts upstream of RhoA to regulate cell polarity and amelogenin expression via the Plexin B1/Leukemia-associated RhoGEF (LARG) complex during ameloblast differentiation. Experiments in transgenic mice demonstrated that expression of a dominant-negative form of RhoA in dental epithelium hindered ameloblast differentiation and subsequent enamel formation, as well as perturbing the establishment of polarized cell morphology and vectorial amelogenin expression. Finally, we showed that spatially restricted Akt mediates between Sema4D-RhoA signaling and these downstream cellular events. Collectively, our results reveal a novel signaling network, the Sema4D-RhoA-Akt signal cascade, that coordinates cellular function and morphology and highlights the importance of specific spatiotemporally restricted components of a signaling pathway in the regulation of ameloblast differentiation. © 2016 American Society for Bone and Mineral Research.