Root Dentin Formation Research Articles

Hemicentin-1 is an essential extracellular matrix component during tooth root formation by promoting mesenchymal cells differentiation.

Introduction: Root dentin formation is an important process in tooth development. We tried to identify potential genes that regulate root dentin formation which could be potentially used for the regeneration and repair of defective or damaged dental roots. Methods: Tissues harvested from the labial and lingual sides of mouse incisors were used for microarray analysis. Gene ontology (GO) analysis of differentially expressed genes indicated the critical role of extracellular matrix in the discrepancy of dentin formation between root and crown, for which hemicentin-1 (Hmcn1) was selected as the target gene. Single-cell RNA sequencing analysis the expression pattern of Hmcn1 at different developmental stages in mouse molars. The spatiotemporal expression of HMCN1 in mouse incisors and molars was detected by immunohistochemical staining. The functions of HMCN1 in human dental pulp cells, including proliferation, differentiation and migration, were examined in vitro by CCK8 assay, BrdU assay, wound-healing assay, ALP staining and alizarin red staining, respectively. Results: It was showed that HMCN1 expression was more pronounced in papilla-pulp on the root than crown side in mouse incisors and molars. In vitro experiments presented inhibited dentinogenesis and migration after HMCN1-knockdown in human dental pulp cells, while there was no significant difference in proliferation between the HMCN1-knockdown group and control group. Discussion: These results indicated that HMCN1 plays an important role in dentinogenesis and migration of pulp cells, contributing to root dentin formation.

Semaphorin-RhoA signaling regulates HERS maintenance by acting against TGF-β-induced EMT.

Hertwig's epithelial root sheath (HERS) plays a role in root dentin formation. It produces the epithelial rests of Malassez (ERM) for the induction of periodontal tissue development during root formation. Although ERM is thought to be caused by epithelial-mesenchymal transition (EMT), the mechanism by which HERS is maintained as epithelium is unknown. Here, we aimed to elucidate the molecular mechanisms regulating the relationship between HERS maintenance and ERM development. To understand the relationship between HERS and ERM development during root formation, we observed the developing molar root using cytokeratin14 (CK14) Cre/tdTomato mice via stereomicroscopy. The relationship between semaphorin and transforming growth factor (TGF) signaling in the maintenance of HERS and ERM development was examined using CK14cre/R26-tdTomato mice and a HERS cell line. tdTomato-positive cells were observed on HERS and the migrating cells from HERS. The migrating cells showed reduced E-cadherin expression. In contrast, HERS cells expressed semaphorin receptors and active RhoA. Semaphorin signaling was associated with RhoA activation and cell-cell adhesion, while TGF-β induced decreased E-cadherin and active RhoA expression, and consequently enhanced cell migration. HERS induces root formation by controlling epithelial maintenance and EMT through the opposing effects of semaphorin and TGF-β signaling.

Apexogenesis & Apexification

Apexogenesis and apexification constitute two alternatives possibilities to endodontic therapies. Apexogenesis is a phenomenon implicating a vital pulp. The thin dentin walls of the large canals place the tooth at greater risk for root fracture. Root lengthening is associated with tooth eruption. The treatment objective is to maximize the opportunity for apical development and closure. This is known as apexification that is occurring when the pulp is non-vital, infected or not. Apexification enhanced the continued root dentin formation inside the lumen, linked with apical closure and possibly with radicular dental pulp regeneration. A new treatment option of revascularization has recently been introduced after triple antibiotic therapy. It involves disinfecting the root canal system. After laceration of the periapex with a file until bleeding occurs, it provides a matrix of blood clot into which cells could grow. In parallel, it involves the sealing of the coronal access.

BMP-Smad Signaling Regulates Postnatal Crown Dentinogenesis in Mouse Molar.

ABSTRACTDentinogenesis, a formation of dentin by odontoblasts, is an essential process during tooth development. Bone morphogenetic proteins (BMPs) are one of the most crucial growth factors that contribute to dentin formation. However, it is still unclear how BMP signaling pathways regulate postnatal crown and root dentinogenesis. BMPs transduce signals through canonical Smad and non‐Smad signaling pathways including p38 and ERK signaling pathways. To investigate the roles of Smad and non‐Smad signaling pathways in dentinogenesis, we conditionally deleted Bmpr1a, which encodes the type 1A receptor for BMPs, to remove both Smad and non‐Smad pathways in Osterix‐expressing cells. We also expressed a constitutively activated form of Bmpr1a (caBmpr1a) to increase Smad1/5/9 signaling activity without altered non‐Smad activity in odontoblasts. To understand the function of BMP signaling during postnatal dentin formation, Cre activity was induced at the day of birth. Our results showed that loss of BmpR1A in odontoblasts resulted in impaired dentin formation and short molar roots at postnatal day 21. Bmpr1a cKO mice displayed a reduction of dentin matrix production compared to controls associated with increased cell proliferation and reduced Osx and Dspp expression. In contrast, caBmpr1a mutant mice that show increased Smad1/5/9 signaling activity resulted in no overt tooth phenotype. To further dissect the functions of each signaling activity, we generated Bmpr1a cKO mice also expressing caBmpr1a to restore only Smad1/5/9 signaling activity. Restoring Smad activity in the compound mutant mice rescued impaired crown dentin formation in the Bmpr1a cKO mice; however, impaired root dentin formation and short roots were not changed. These results suggest that BMP‐Smad signaling in odontoblasts is responsible for crown dentin formation, while non‐Smad signaling may play a major role in root dentin formation and elongation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Expression of CPNE7 during mouse dentinogenesis.

Interactions between the ectodermal and mesenchymal tissues are the basis of the central mechanism regulating tooth development. Based on this epithelial-mesenchymal interaction (EMI), we demonstrated that copine-7 (CPNE7) is secreted by preameloblasts and regulates the differentiation of mesenchymal cells of dental or non-dental origin into odontoblasts. However, the precise expression patterns of CPNE7 in the stages of tooth development have not yet been elucidated. The aim of the present study was to establish the spatiotemporal expression pattern of CPNE7 during mouse tooth development. To examine the spatiotemporal expression patterns of CPNE7 during mouse tooth development, we investigate the distribution of CPNE7 in the embryonic and postnatal developing mouse tooth. Immunohistochemistry, in situ hybridization, real-time PCR, and western blot analysis are performed to investigate the CPNE7 expression pattern during tooth development of the mandibular mouse first molar. During the initiation stage (bud stage), CPNE7 protein expression is observed in the dental epithelium but not yet in the dental mesenchyme. At E18 (bell stage), expression of CPNE7 protein and mRNA is primarily observed in ectomesenchymal cells of dental papilla. At P7 (crown formation stage), CPNE7 is localized in differentiating odontoblasts but weak expression is detected in mature ameloblasts. These findings suggest that CPNE7 secreted by dental epithelium induces the differentiation of ectomesenchymal cells into preodontoblast in concert with EMI. CPNE7 is clearly expressed in differentiating odontoblasts and the odontoblast process during dentinogenesis, but is no longer expressed in fully differentiated odontoblasts. Furthermore, CPNE7 is expressed in the Hertwig's epithelial root sheath (HERS) and in the facing preodontoblasts during root dentin formation. Taken together, these results illustrate the dynamic expression of CPNE7 during tooth development and suggest its important function in entire stages of tooth development.

Essential Roles of Bone Morphogenetic Protein-1 and Mammalian Tolloid-like 1 in Postnatal Root Dentin Formation

IntroductionMutations in the proteinase bone morphogenetic protein-1 (BMP1) were recently identified in patients with osteogenesis imperfecta, which can be associated with type 1 dentinogenesis imperfecta. BMP1 is co-expressed in various tissues and has overlapping activities with the closely related proteinase mammalian tolloid-like 1 (TLL1). In this study we investigated whether removing the overlapping activities of BMP1 and TLL1 affects the mineralization of tooth root dentin. MethodsFloxed alleles of the BMP1 and TLL1 genes were excised via ubiquitously expressed Cre induced by tamoxifen treatment beginning at 3 days of age (harvested at 3 weeks of age) or beginning at 4 weeks of age (harvested at 8 weeks of age). Multiple techniques, including x-ray analysis, double-labeling with calcein and alizarin red stains for measurement of dentin formation rate, and histologic and immunostaining assays, were used to analyze the dentin phenotype. ResultsBMP1/TLL1 double knockout mice displayed short and thin root dentin, defects in dentin mineralization, and delayed tooth eruption. Molecular mechanism studies revealed accumulation of collagens in dentin and a sharp reduction in non-collagenous proteins such as dentin matrix protein 1 and dentin sialophosphoprotein. Furthermore, we found a strong reduction in tartrate-resistant acid phosphatase, which is likely caused by defects in bone cells. ConclusionsBMP1/TLL1 appear to play crucial roles in maintaining extracellular matrix homeostasis essential to root formation and dentin mineralization.

Root dentin anomaly and a PLG mutation

We report a Thai girl affected with plasminogen deficiency, Type I. Ligneous conjunctivitis was first observed when she was one-month-old. The newly recognized findings include tapered incisor roots as a result of thin root dentin, generalized short tooth roots, and mandibular prognathism. Mutation analysis of PLG demonstrated homozygous c.1193G>A missense mutation. The parents were heterozygous for c.1193G>A mutation. The c.1193G>A mutation is novel and predicted to cause amino acid substitution p.Cys398Tyr. Thin root dentin in the patient who was affected with PLG mutation and immunolocalization of Plg during early root development in mice imply the role of plasminogen in root dentin formation.

Apical Papilla Stem Cells and its Significance in Regeneration of Dental Tissues

The use of allogeneic stem cells strongly extends the range of stem cell applications in dentistry1; stem cells have the capacity to divide and differentiate into various other cells of other tissues (multipotency) 2. The search for stem cells other than bone marrow has given rise to more interest in dental tissues. Human dental stem/progenitor cells (collectively termed dental stem cells [DSCs]) that have been isolated and characterized include dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla (SCAP), periodontal ligament stem cells, and dental follicle progenitor cells3. The SCAP stem cells appear to be the source of odontoblasts that are responsible for the formation of root dentin. Conservation of these stem cells when treating immature teeth may allow continuous formation of the root to completion. This article reviews current findings on the isolation and characterization of these stem cells. The potential role of these stem cells in the following respects will be discussed: (1) their contribution in continued root maturation in endodontically treated immature teeth with periradicular periodontitis or abscess and (2) their potential utilization for pulp/dentin regeneration and bioroot engineering4.

Tooth root dentin mineralization defects in a mouse model of hypophosphatasia.

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP(i) ), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP(i) , resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl(-/-) mice, a model for infantile HPP. In Alpl(-/-) mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl(-/-) mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl(-/-) molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl(-/-) dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl(-/-) mice, was present in the generally unmineralized Alpl(-/-) mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN-encoding Spp1 gene in Alpl(-/-) mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral-targeting human TNAP (ENB-0040) to Alpl(-/-) mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP(i) during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients.

Basic Fibroblast Growth Factor Enhances Stemness of Human Stem Cells from the Apical Papilla

IntroductionStem cells from the apical papilla (SCAP) are a type of mesenchymal stem cells found in the developing tissue, apical papilla, of immature permanent teeth. Studies have shown that SCAP are likely to be a source of primary odontoblasts that are responsible for the formation of root dentin. Basic fibroblast growth factor (bFGF) is a signaling molecule and pleiotropic growth factor involved in tooth root development, and it promotes proliferation of a variety of cell types. The effects of bFGF on SCAP, however, have not been examined. MethodsWe investigated the regulatory effects of bFGF on the proliferation and differentiation potential of human SCAP in vitro. Changes in the cell cycle and proliferation, colony-forming unit–fibroblastic formation, alkaline phosphatase (ALP) activity, osteogenic/dentinogenic differentiation, and stem cell gene makers of SCAP, cultured in the presence or absence of bFGF, were evaluated. ResultsTreatment with 5 ng/mL bFGF significantly increased SCAP proliferation and their colony-forming unit–fibroblastic formation efficiency. The growth factor also increased the expression of STRO-1 and the stem cell gene makers Nanog, Oct4, Sox2, and Rex1 in SCAP. In contrast, bFGF reduced the ALP activity, mineral nodule formation, and the expression of ALP, osteocalcin, bone sialoprotein, and dentin sialophosphoprotein. When SCAP cultures were expanded in the presence of bFGF for 1 week, subsequent stimulation of the osteogenic/dentinogenic condition resulted in enhanced differentiation. ConclusionsUnder certain conditions, bFGF enhances SCAP stemness by up-regulating stem cell gene expression, increasing proliferation ability, and potentiating differentiation potency.

Zinc balance is critical for NFI‐C mediated regulation of odontoblast differentiation

Zinc is trace element essential for diverse metabolic and cellular signaling pathways for the growth, development, and maintenance. Zinc deficiency is involved in bone malformations and oral disease. Mice deficient in zinc transporter Zip13 show connective tissue and skeletal disorders, abnormal incisor teeth, and reduced root dentin formation in the molar teeth and share a morphologically similar phenotype to nuclear factor I-C (NFI-C)-deficient mice. However, the precise function of zinc in NFI-C signaling-mediated odontoblast differentiation and dentin formation remains unclear. Here, we show that zinc stimulated the expression of metal transcription factor-1, but decreased NFI-C expression in odontoblastic MDPC-23 cells. Zinc also enhanced the phosphorylation of Smad2/3 (p-Smad2/3) and increased the binding efficiency of NFI-C and p-Smad2/3 in the cytoplasm. In contrast, zinc deficiency resulted in the accumulation of NFI-C into nucleus. Consequently, NFI-C had the biologic properties of a transcription factor, including DNA binding affinity for metallothionein-1 and the dentin sialophosphoprotein (DSPP) promoter, and transcriptional activation of the DSPP gene. Furthermore, zinc deficiency condition promoted DSPP expression in odontoblasts and dentin mineralization, while zinc sufficiency condition decreased DSPP expression and slightly delayed dentin mineralization. These data suggest that zinc equilibrium is required for odontoblast differentiation and dentin formation during dentinogenesis through the nuclear accumulation and modulation of NFI-C.

치근단 병소를 가진 영구치의 보존적 치수 치료

최근 미성숙 영구치의 치근단 유두에서 존재하는 미분화 줄기세포가 새로 발견되었고 이 줄기세포들은 치근 상아질의 형성에 관여하는 법랑모세포의 근원으로 보여진다. 미성숙 영구치를 다룰때 이 줄기세포들의 보존은 치근의 지속적인 형성을 완성시키게 한다. 따라서 치근단 염증을 동반한 미성숙 영구치에서 치근단의 최소한의 침습으로 임상 및 방사선적 치유 양상을 관찰할 수 있었기에 보고하고자 한다. 본 증례는 조선대학교 치과대학병원 소아치과에 내원한 만 10세 남아(하악 우측 소구치), 만 8세 여아(상악 좌측 소구치)에서 임상 및 방사선 검사결과 치수괴사와 치근단 농양으로 진단된 영구치에서 치근단부위의 기구조작을 제한하고 MTA(Mineral Trioxide Aggregate)를 치수강내에 적용하였다. 정기적으로 관찰한 결과 치근단 농양의 치유와 치근 발육 및 성장의 양상이 관찰되었다. 치수괴사와 치근단 농양을 가진 영구치에서 치근단부위의 치수 및 치유두를 보호함으로써 관찰기간동안 양호한 예후를 보였으며, 이는 어린환자의 미성숙영구치에서 보존적 치수치료의 시도에 대한 보다 많은 임상적 검증 및 장기간의 고찰이 필요할 것이다. Recently, undifferentiated stem cells which exist in dental papillae of immature permanent teeth were newly discovered and these stem cells appear to be the origin of ameloblasts associated with the formation of root dentin. When treating immature permanent teeth, the preservation of these stem cells induce the continuous formation of the root. Therefore, it is reported that minimal invasion to periapical region in immature permanent teeth with periapical inflammation resulted in good-healing pattern in clinical and radiographic examination. In this case, a 10 year-old boy(mandibular right premolar) and a 8 year-old girl(maxillary left premolar) who visited the department of pediatric dentistry at Chosun University Dental Hospital were diagnosed with pulp necrosis and periapical abscess in clinical and radiographic examination. Endodontic instrumentation to the periapical region was limited and MTA(Mineral Trioxide Aggregate) was applied into the pulp canal. The periodic checks showed healing of periapical abscess and the development and growth pattern of roots. In permanent teeth with pulp necrosis and periapical abscess, preservation of pulp and dental papillae in the periapical region showed good prognosis during the periodic examinations. Therefore, a lot of clinical examination and long-term evaluation of conservative pulp treatment in immature permanent teeth are expected to be necessary.

MRNA expression and protein localization of dentin matrix protein 1 during dental root formation

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation.

Ultrastructural characterization of mesenchymal and epithelial cells co-cultured from human dental root apical explants.

Previous studies have shown the role of cell-cell and cell-matrix interactions in the differentiation of the specific secretory cells of the tooth. In order to elucidate the mechanisms implicated in root dentin formation, we developed a co-culture system of human pulpal mesenchymal and epithelial root sheath cells. Root tips of premolars were cultured in Eagle's basal Medium supplemented with fetal calf serum, ascorbic acid, antibiotics and, for some of them, with sodium beta-glycerophosphate. After 60 days of culture, cells were prepared for light and electron microscopy. Three main cell types were observed: (1) polygonal mesenchymal cells showing a functional polarity and producing a dense network of tactoid collagenous fibers. The latter had a specific circular organization that delimited small lacunae around the cells and mineralized in the presence of beta-glycerophosphate; (2) spindle-shaped mesenchymal cells mainly localized inside epithelial-mesenchymal knots and synthesizing an abundant collagenous matrix; and (3) epithelial cells lying on the plastic culture dish, on the dense collagenous matrix, or on spindle-shaped cells. Epithelial cells deposited a structured basement membrane when they were lying on the plastic culture dish or on spindle-shaped cells. On the contrary, no basement membrane was found when epithelial cells were overlying the dense collagenous network. Immunoelectron microscopic analysis of type IV collagen and laminin indicated that these two specific basement membrane components were produced by all cell types. These results show that the co-culture system should be valuable for (1) studying the in vitro formation of human dental root hard tissues, (2) characterizing cell-cell and cell-matrix interactions implicated in dental basement membrane production, and (3) isolating populations of cells implicated in dental root formation.

根未完成永久歯の歯髄および歯根膜に及ぼす放射線照射の影響に関する実験的研究

Left mandibular fourth premolars were irradiated for 3, 000 R using 200kVp X-rays, and effect on the dental pulp and periodontal membrane was investigated histopathologically. The results were as follows : 1. One week after irradiation, the dental pulp showed normal findings except for inflammatory cellular infiltration limited to a portion of the coronal pulp. Marked changes were hardly seen in the root dentin, epithelial sheath of Hertwig, or periodontal membrane. 2. Two weeks after irradiation, findings of inflammatory cellular infiltration and reticular atrophy limited to the coronal pulp were observed. In the epithelial sheath of Hertwig, calcification of cementum was active and cementoblasts were arranged densely. In the periodontal membrane, fiber bundles of the periodontal membrane were running disorderedly in both the lingual and buccal periodontal membrane. 3. From one to two months after irradiation, inflammatory changes continued in the coronal pulp. Deposition of hyaline materials was seen around blood vessels. The apical pulp underwent hyaline degeneration, and calcareous degeneration was also observed on the blood vessel walls. The pulp chamber was still spacious and root dentin showed good growth. Collaterals of the root apex were increased in number. In the periodontal membrane, fibroblastes decreased slightly and the finding of hyalinization was beginning to be seen. In the alveolar process, inflammatory cellular infiltration extended diffusedly and the finding of resorption was seen in the bundle bone. 4. From four to six months after irradiation, reticular atrophy and hyaline degeneration were observed markedly in the dental pulp from the crown to the apex. At this period, root dentin was formed thickly and the pulp chamber was small ; however, obliteration of the apex by cemetum was still incomplete. The periodontal membrane, although healthy in part, underwent hyaline degeneration on the whole and disappeared in many portions. Tlhe bundle bone also showed the resorption image. The periodontal membrane directly under the apex in particular was replaced by fatty marrow. 5. Eight months after irradiation, the dental pulp on the whole showed marked degenerative changes. However the pulp chamber became small along with formation of root dentin. Obliteration of the apex was incomplete yet although it was tentatively obliterated with addition of cementum. The periodontal membrane underwent severe hyaline degeneration. At this period, images of new bundle bone were observed sporadically. The periodontal membrane directly under the apex were replaced extensively by fatty marrow. 6. On the basis of the foregoing findings, it was proved that although irradiation brought about marked changes in the dental pulp and periodontal membrane, formation and growth of the root progressed rather satisfactorily, but obliteration of the apex was incomplete.