RESUMO
MMP13 gene expression increases up to 2000-fold in mineralizing dental pulp cells (DPCs), with research previously demonstrating that global MMP13 deletion resulted in critical alterations in the dentine phenotype, affecting dentine-tubule regularity, the odontoblast palisade, and significantly reducing the dentine volume. Global MMP13-KO and wild-type mice of a range of ages had their molar teeth injured to stimulate reactionary tertiary dentinogenesis. The response was measured qualitatively and quantitatively using histology, immunohistochemistry, micro-CT, and qRT-PCR in order to assess changes in the nature and volume of dentine deposited as well as mechanistic links. MMP13 loss affected the reactionary tertiary dentine quality and volume after cuspal injury and reduced Nestin expression in a non-exposure injury model, as well as mechanistic links between MMP13 and the Wnt-responsive gene Axin2. Acute pulpal injury and pulp exposure to oral fluids in mice teeth showed upregulation of the MMP13 in vivo, with an increase in the gene expression of Mmp8, Mmp9, and Mmp13 evident. These results indicate that MMP13 is involved in tertiary reactionary dentine formation after tooth injury in vivo, potentially acting as a key molecule in the dental pulp during dentine-pulp repair processes.
Assuntos
Dentinogênese , Metaloproteinase 13 da Matriz , Traumatismos Dentários , Animais , Camundongos , Dentinogênese/genética , Metaloproteinase 13 da Matriz/genética , Dente Molar , OdontoblastosRESUMO
The development and repair of dentin are strictly regulated by hundreds of genes. Abnormal dentin development is directly caused by gene mutations and dysregulation. Understanding and mastering this signal network is of great significance to the study of tooth development, tissue regeneration, aging, and repair and the treatment of dental diseases. It is necessary to understand the formation and repair mechanism of dentin in order to better treat the dentin lesions caused by various abnormal properties, whether it is to explore the reasons for the formation of dentin defects or to develop clinical drugs to strengthen the method of repairing dentin. Molecular biology of genes related to dentin development and repair are the most important basis for future research.
Assuntos
Dentinogênese , Odontoblastos , Dentina , Dentinogênese/genética , Odontogênese/genéticaRESUMO
Direct pulp capping is an effective treatment for preserving dental pulp against carious or traumatic pulp exposure via the formation of protective reparative dentin by odontoblast-like cells. Reparative dentin formation can be stimulated by several signaling molecules; therefore, we investigated the effects of secreted frizzled-related protein (SFRP) 1 that was reported to be strongly expressed in odontoblasts of newborn molar tooth germs on odontoblastic differentiation and reparative dentin formation. In developing rat incisors, cells in the dental pulp, cervical loop, and inner enamel epithelium, as well as ameloblasts and preodontoblasts, weakly expressed Sfrp1; however, Sfrp1 was strongly expressed in mature odontoblasts. Human dental pulp cells (hDPCs) showed stronger expression of SFRP1 compared with periodontal ligament cells and gingival cells. SFRP1 knockdown in hDPCs abolished calcium chloride-induced mineralized nodule formation and odontoblast-related gene expression and decreased BMP-2 gene expression. Conversely, SFRP1 stimulation enhanced nodule formation and expression of BMP-2. Direct pulp capping treatment with SFRP1 induced the formation of a considerable amount of reparative dentin that has a structure similar to primary dentin. Our results indicate that SFRP1 is crucial for dentinogenesis and is important in promoting reparative dentin formation in response to injury.
Assuntos
Polpa Dentária/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Odontoblastos/metabolismo , Adolescente , Animais , Diferenciação Celular/genética , Polpa Dentária/fisiologia , Dentina/metabolismo , Dentina/fisiologia , Dentina Secundária/fisiologia , Dentinogênese/genética , Dentinogênese/fisiologia , Feminino , Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Odontoblastos/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/genética , Adulto JovemRESUMO
This study aimed to evaluate the effects of low-energy blue LED irradiation on the osteogenic differentiation of stem cells from the apical papilla (SCAPs). SCAPs were derived from human tooth root tips and were irradiated with 0 (control group), 1 J/cm2, 2 J/cm2, 3 J/cm2, or 4 J/cm2 blue light in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP), alizarin red staining, and real-time polymerase chain reaction (RT-PCR). The results of the MTT assay indicated that SCAPs in the LED groups exhibited a lower proliferation rate than those in the control group, and there were statistically differences between the 2 J/cm2, 3 J/cm2, and 4 J/cm2 groups and the control group (P < 0.05). The results of the ALP and alizarin red analyses showed that blue LED promoted osteogenic differentiation of the SCAPs. And 4 J/cm2 blue light upregulates the expression levels of the osteogenic/dentinogenic genes ALP, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and osteocalcin (OCN) in SCAPs. Our results confirmed that low-energy blue LED at 1 J/cm2, 2 J/cm2, 3 J/cm2, and 4 J/cm2 could inhibit the proliferation of SCAPs and promotes osteogenic differentiation of SCAPs. Further in vitro studies are required to explore the mechanisms of the effects by low-energy blue LED.
Assuntos
Diferenciação Celular/efeitos da radiação , Papila Dentária/citologia , Osteogênese/efeitos da radiação , Células-Tronco/citologia , Células-Tronco/efeitos da radiação , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Células Cultivadas , Dentinogênese/genética , Dentinogênese/efeitos da radiação , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMO
Tooth development is a complex process that is regulated precisely by several signalling pathways and transcription factors. GATA-binding protein 4 (GATA4) is a DNA binding transcription factor, and our previous study showed that GATA4 is a novel regulator of root development. However, it remains unclear whether GATA4 is necessary for odontoblast differentiation and dentin formation. Here, we evaluated the phenotypic changes of Wnt1-Cre;GATA4fl/fl mice. The mutant mice showed defective dentin and short root deformity. The odontoblasts lost polarity instead of exhibiting a shorter height and flattened morphology. Moreover, the expression of several molecules, such as DSPP, COL-1, DCN, and PCNA, were downregulated during mutant tooth development. In vivo, we injected lentivirus to overexpress GATA4 in mice root. The dentin formation and the expression of odonto/osteogenic markers (DSPP, COL-1, DCN) were enhanced in the GATA4 overexpression group. During the in vitro study, the ability of proliferation, migration and odonto/osteogenic differentiation was declined by GATA4 knockdown approach in human dental pulp stem cells (DPSCs). The expression of odonto/osteogenic markers (DSPP, BMP4, RUNX2, OSX, OPN, OCN) was reduced in the shGATA4 group, while overexpressing GATA4 in DPSCs promoted mineralization. Furthermore, an immunoprecipitation-mass spectrometry procedure was used to confirm the interaction between GATA4 and Fructose-1, 6-bisphosphatase 1 (FBP1). We used gain and lose-of-function to delineated the role of GATA4 in regulating FBP1 expression. Knocking down GATA4 in DPSCs resulted in decreased glucose consumption and lactate production. We used small hairpin RNA targeting FBP1 to reduce the expression of FBP1 in DPSCs, which significantly increased glucose consumption and lactate production. Together, the results suggested that GATA4 is important for root formation and odontoblast polarity, as it promotes the growth and differentiation of dental mesenchymal cells around the root and affects the glucose metabolism of DPSCs via the negative regulation of FBP1.
Assuntos
Dentina/metabolismo , Frutose-Bifosfatase/metabolismo , Fator de Transcrição GATA4/metabolismo , Raiz Dentária/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Dentinogênese/genética , Dentinogênese/fisiologia , Frutose-Bifosfatase/genética , Fator de Transcrição GATA4/genética , Gluconeogênese/genética , Gluconeogênese/fisiologia , Camundongos Knockout , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Odontoblastos/citologia , Odontoblastos/metabolismoRESUMO
OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.
Assuntos
Compostos de Alumínio/administração & dosagem , Compostos de Cálcio/administração & dosagem , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Capeamento da Polpa Dentária/métodos , Polpa Dentária/fisiologia , Dentinogênese/genética , Transportador de Glucose Tipo 1/genética , Óxidos/administração & dosagem , Pulpotomia , Silicatos/administração & dosagem , Serina-Treonina Quinases TOR/genética , Animais , Combinação de Medicamentos , Expressão Gênica , Imunoquímica , Masculino , Dente Molar/cirurgia , Nestina/genética , Odontoblastos/fisiologia , Ratos , Ratos WistarRESUMO
Bone sialoprotein (BSP) is a member of the SIBLING family with essential roles in skeletogenesis. In the developing teeth, although the expression and function of BSP in the formation of acellular cementum and periodontal attachment are well documented, there are uncertainties regarding the expression and function of BSP by odontoblasts and dentin. Reporter mice are valuable animal models for biological research, providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. In the present study, we examined the expression of a BSP-GFPtpz reporter mouse line during odontoblast differentiation, reparative dentinogenesis, and bone. In the developing teeth, BSP-GFPtpz was expressed at high levels in cementoblasts but not in odontoblasts or dentin. In bones, the transgene was highly expressed in osteoblasts at an early stage of differentiation. Interestingly, despite its lack of expression in odontoblasts and dental pulp during tooth development, the BSP-GFPtpz transgene was detected during in vitro mineralization of primary pulp cultures and during reparative dentinogenesis following pulp exposures. Importantly, under these experimental contexts, the expression of BSP-GFPtpz was still exclusive to DSPP-Cerulean, an odontoblast-specific reporter gene. This suggests that the combinatorial use of BSP-GFPtpz and DSPP-Cerulean can be a valuable experimental tool to distinguish osteogenic from dentinogenic cells, thereby providing an avenue to investigate mechanisms that distinctly regulate the lineage progression of progenitors into odontoblasts versus osteoblasts.
Assuntos
Dentina Secundária , Dentinogênese , Osteogênese , Transgenes , Animais , Diferenciação Celular , Polpa Dentária , Dentinogênese/genética , Expressão Gênica , Sialoproteína de Ligação à Integrina/genética , Camundongos , Odontoblastos , Osteogênese/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Transgenes/genéticaRESUMO
Mutations in the gene encoding family with sequence similarity 20, member A (FAM20A) caused amelogenesis imperfecta (AI), in humans. However, the roles of FAM20A in amelogenesis and dentinogenesis are poorly understood. In this study, we generated a Fam20a knockout (Sox2-Cre;Fam20afl/fl) mouse model by crossing Fam20afl/fl mice with Sox2-Cre transgenic mice, in which Fam20a was ablated in both dental epithelium and dental mesenchyme. We found that these mice developed an enamel phenotype that resembles human AI associated with FAM20A mutations, but did not have apparent dentin defects. The secretory stage ameloblasts in the mandibular incisors from the Sox2-Cre;Fam20afl/fl mice were shorter and detached from the enamel matrix, and subsequently lost their polarity, became disorganized and formed numerous spherical extracellular matrices in place of normal enamel. At the molecular level, the Sox2-Cre;Fam20afl/fl mice displayed dramatically reduced expression levels of the genes encoding the enamel matrix proteins, but unaltered levels of the genes encoding the dentin matrix proteins. Moreover, Fam20a ablation resulted in a great decrease in FAM20C protein level, but it did not alter the intracellular localization of FAM20C protein in ameloblasts and odontoblasts. These results indicate that FAM20A is essential for amelogenesis, but is dispensable for dentinogenesis.
Assuntos
Amelogênese Imperfeita/genética , Amelogênese/genética , Proteínas do Esmalte Dentário/genética , Dentinogênese/genética , Ameloblastos/metabolismo , Amelogênese Imperfeita/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Esmalte Dentário/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Dentina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Odontoblastos/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
OBJECTIVES: To evaluate the effect of analogues of cationic peptides on the viability and the expression of phenotypic and genotypic markers of dentin mineralization in MDPC-23 odontoblast-like cells. MATERIALS AND METHODS: Cells were exposed to serial dilutions of analogues of cationic peptides hBD-3-1CV and KR-12-a5 compared to peptide LL-37 and their viability was assessed by methyltetrazolium assay. Next, peptides (0.78-62.5 µg/mL) were applied on the MDPC-23 cells for evaluating the total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition. Gene expression of mineralization markers (DSPP and DMP-1) was also determined by quantitative PCR. RESULTS: LL-37 and hBD-3-1CV treatment did not affect cellular viability at concentrations below 62.5 µg/mL. KR-12-a5 reduced cell viability above 31.25 µg/mL. TP production was similar for all groups compared with the control group, except by hBD-3-1CV (at 15.62 µg/mL). LL-37 (at 62.5 µg/mL) induced higher ALP activity than control and other experimental groups. LL-37 and hBD-3-1CV, at 62.5 µg/mL and KR-12-a5 at 31.25 µg/mL stimulated the highest deposition of mineralized nodule. Overall, no statistical differences were observed between the groups for DSPP-1 and DMP-1 expressions. CONCLUSIONS: LL-37 was the only peptide that induced both ALP activity and mineralized nodules deposition, without affecting cell viability. None of peptides tested induced the expression of DSPP or DMP-1, genes commonly involved in active dentin mineralization.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Dentinogênese , Proteínas da Matriz Extracelular , Odontoblastos , Fragmentos de Peptídeos , Fosfoproteínas , Sialoglicoproteínas , beta-Defensinas , Animais , Catelicidinas , Células Cultivadas , Dentina , Dentinogênese/genética , Proteínas da Matriz Extracelular/genética , Humanos , Camundongos , Peptídeos , Fosfoproteínas/genética , Sialoglicoproteínas/genéticaRESUMO
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.
Assuntos
Diferenciação Celular/genética , Dentinogênese/genética , Proteínas de Membrana/metabolismo , Dente Molar/crescimento & desenvolvimento , Dente/crescimento & desenvolvimento , Ameloblastos/citologia , Ameloblastos/metabolismo , Animais , Papila Dentária/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Camundongos , Dente Molar/metabolismo , Odontoblastos/citologia , Odontoblastos/metabolismo , Dente/metabolismoRESUMO
The mineralization-front theory is historically rooted in mineralization research fields for many decades. This theory is widely used to describe mineralization events in both osteogenesis and dentinogenesis. However, this model does not provide enough evidence to explain how minerals are propagated from the pulp-end dentin to dentin-enamel junction (DEJ). To address this issue, we modified the current research approaches by a) extending the mineral deposition windows of time from minutes to hours, instead of limiting the mineralization assay on days and weeks only; b) switching a regular fluorescent microscope to a more powerful confocal microscope; in which both mineral deposition rates and detail mineral labeling along with dentin tubules can be documented; and c) using reporter mice, including the Gli1-CreERT2 activated tomato and the 2.3 Col1-GFP to mark odontoblast processes combined with mineral dye injections. Our key findings are: 1) Odontoblast-processes, full of numerous mini-branches, evenly spread to entire dentin matrices with a high density of processes and a large diameter of the main process at the predentin-dentin junction; and 2) The minerals deposit along with entire odontoblast-processes and form many individual mineral collars surrounding odontoblast processes. As a result, these merged collars give rise to a single labeled line at the dentin-predentin junction, in which the dental tubules are wider in diameter and denser in odontoblast processes compared to other dentin areas. We therefore propose that it is the odontoblast-process that directly contributes to mineralization, which is not simply limited in the mineralization front at the edge of dentin and predentin, but occurs along with the entire odontoblast process. These new findings will shed new light on our understanding of dentin structure and function, as well as the mechanisms of mineralization.
Assuntos
Dentina/metabolismo , Dentinogênese/fisiologia , Animais , Calcificação Fisiológica/fisiologia , Dentinogênese/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Microscopia Confocal , Odontoblastos/citologia , Odontoblastos/metabolismoRESUMO
The capacity to fully replace teeth continuously makes zebrafish an attractive model to explore regeneration and tooth development. The requirement of attachment bone for the appearance of replacement teeth has been hypothesized but not yet investigated. The transcription factor sp7 (osterix) is known in mammals to play an important role during odontoblast differentiation and root formation. Here we study tooth replacement in the absence of attachment bone using sp7 zebrafish mutants. We analysed the pattern of tooth replacement at different stages of development and demonstrated that in zebrafish lacking sp7, attachment bone is never present, independent of the stage of tooth development or fish age, yet replacement is not interrupted. Without bone of attachment we observed abnormal orientation of teeth, and abnormal connection of pulp cavities of predecessor and replacement teeth. Mutants lacking sp7 show arrested dentinogenesis, with non-polarization of odontoblasts and only a thin layer of dentin deposited. Osteoclast activity was observed in sp7 mutants; due to the lack of bone of attachment, remodelling was diminished but nevertheless present along the pharyngeal bone. We conclude that tooth replacement is ongoing in the sp7 mutant despite poor differentiation and defective attachment. Without bone of attachment tooth orientation and pulp organization are compromised.
Assuntos
Dentinogênese/genética , Odontogênese/genética , Fator de Transcrição Sp7/fisiologia , Anormalidades Dentárias/genética , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/genética , Processo Alveolar/patologia , Animais , Animais Geneticamente Modificados , Polpa Dentária/patologia , Dentina/anormalidades , Dentinogênese/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Odontoblastos/patologia , Odontogênese/fisiologia , Osteoclastos/metabolismo , Regeneração , Fator de Transcrição Sp7/deficiência , Fator de Transcrição Sp7/genética , Raiz Dentária/patologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Dentinogenesis is a long and complex process not only in tooth development, but also throughout the lifespan. Reporter mice provided us a preferred model to study the dentin formation with characteristics of high sensitivity, visualization, and reliability, which makes the long-term and intricate period of dentinogenesis much clear. With the advent of different gene reporters, genetic engineering methods, and tissue specific promoters, various reporter mice can be created to solve different problems. OBJECTIVE: To understand the fundamental concepts and characteristics to use the reporter mice for dentinogenesis study. RESULTS: This review introduced the frequently used gene-based reporters, genetic engineering technologies, dentinogenesis-related promoters and the reporter mice commonly used in the dentin study, with the purpose of obtaining a better application of reporter mice and gaining more details about dentinogenesis. CONCLUSION: Reporter mice is a convenient and reliable model for studying dentinogenesis.
Assuntos
Dentinogênese/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes , Odontoblastos/metabolismo , Dente/crescimento & desenvolvimento , Animais , Técnicas de Inativação de Genes/métodos , Humanos , Camundongos TransgênicosRESUMO
Primary cilia, with highly regulated cellular sensory functions, play key roles in tissue development and function maintenance. Intraflagellar transport 140 (IFT140) is a subunit of IFT complex A, which is specialized for retrograde transportation in cilia. Mutations of Ift140 are usually associated with syndromic ciliopathy and may cause isolated diseases such as retinal dystrophy, short ribs, and polycystic kidney. However, the role of IFT140 in tooth development has not been well investigated. In this study, a close relationship between IFT140 and dentin formation is disclosed. During tooth development, IFT140 was highly expressed in odontoblasts. To further understand the role of IFT140 in dentinogenesis, Ift140flox/flox/Osx-Cre mouse was generated. The dentin thickness of Ift140flox/flox/Osx-Cre mouse is thinner and the dentin formation is slower than that in control. In vitro, deletion of IFT140 in odontoblasts led to poor odontogenic differentiation, abnormal primary cilia, and decreased Sonic hedgehog signaling molecules. More important, due to loss of primary cilia in odontoblasts by IFT140 deletion, reparative dentin formation was impaired in a tooth-drilling model. These results suggest that cilia gene IFT140 is essential in promoting dentin formation and reparation.
Assuntos
Proteínas de Transporte/genética , Dentinogênese/genética , Animais , Proteínas de Transporte/fisiologia , Células Cultivadas , Polpa Dentária/citologia , Dentinogênese/fisiologia , Imunofluorescência , Imuno-Histoquímica , Mandíbula/diagnóstico por imagem , Camundongos , Camundongos Mutantes , Odontoblastos/metabolismo , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Microtomografia por Raio-XRESUMO
In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost-/-). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost-/- and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost-/- mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost-/- pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost-/- pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost-/- dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.
Assuntos
Polpa Dentária/citologia , Dentinogênese/genética , Glicoproteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Compostos de Alumínio , Animais , Compostos de Cálcio , Resinas Compostas , Capeamento da Polpa Dentária/métodos , Combinação de Medicamentos , Glicoproteínas/deficiência , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dente Molar/cirurgia , Óxidos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Silicatos , Microtomografia por Raio-XRESUMO
In non-growing teeth, such as mouse and human molars, primary odontoblasts are long-lived post-mitotic cells that secrete dentine throughout the life of the tooth. New odontoblast-like cells are only produced in response to a damage or trauma. Little is known about the molecular events that initiate mesenchymal stem cells to proliferate and differentiate into odontoblast-like cells in response to dentine damage. The reparative and regenerative capacity of multiple mammalian tissues depends on the activation of Wnt/ß-catenin signaling pathway. In this study, we investigated the molecular role of Wnt/ß-catenin signaling pathway in reparative dentinogenesis using an in vivo mouse tooth damage model. We found that Axin2 is rapidly upregulated in response to tooth damage and that these Axin2-expressing cells differentiate into new odontoblast-like cells that secrete reparative dentine. In addition, the Axin2-expressing cells produce a source of Wnt that acts in an autocrine manner to modulate reparative dentinogenesis.
Assuntos
Proteína Axina/genética , Diferenciação Celular/genética , Dentinogênese/genética , Expressão Gênica , Odontoblastos/citologia , Odontoblastos/metabolismo , Via de Sinalização Wnt , Animais , Proliferação de Células , Doenças da Polpa Dentária/genética , Doenças da Polpa Dentária/metabolismo , Doenças da Polpa Dentária/patologia , Camundongos , Dente Molar/crescimento & desenvolvimento , Dente Molar/patologiaRESUMO
The muscle segment homeogenes Msx1 and Msx2 play a major role in tooth and bone formation. Periodontal osteoclast impairment also occurs in Msx2 null mutant mice, which is restored by overexpression of the receptor activator of NF-κB targeted in osteoclast lineage. Here, we investigated the role of Msx2 in dentinogenesis. Experiments were performed on Msx2(-/-) mice and the MDPC-23 odontoblastic cell line. After Msx2 gene silencing, real-time quantitative RT-PCR data showed significant overexpression of Runx2, Bglap, Dspp, and Alpl. Of three inhibitors of Wnt/ß-catenin signaling (Dkk1, SostDc1, and Sost/Sclerostin), only Sost was expressed in postnatal teeth and overexpressed in Msx2(-/-) tooth samples. Initial crown dentin formation-primary dentinogenesis-occurred fairly normally in Msx2(-/-) teeth, albeit with distorted cusp patterns. Later stages of tooth development were characterized by a deviation from secondary toward tertiary dentinogenesis with osteodentin formation and impaired dentin deposition leading to limited root elongation. In Msx2(-/-)/receptor activator of NF-κB-transgenic double mutants, the dentin phenotype, notably in the roots, was rescued and sclerostin levels were normalized. These data suggest that Msx2 may act indirectly on dentinogenesis by controlling osteoclast activity and the signaling network related to eruption, supporting and further extending the concept that Msx2 controls formation of mineralized tissues by inhibition of the Wnt/ß-catenin pathway; Sost in dentin and Dkk1 in bone, as previously demonstrated.
Assuntos
Dentinogênese/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/genética , Proteínas de Homeodomínio/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Dentina/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Glicoproteínas/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Odontoblastos/citologia , Osteoclastos/citologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Dente/crescimento & desenvolvimento , Erupção Dentária , Raiz Dentária/crescimento & desenvolvimento , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis.
Assuntos
Dentina/embriologia , Dentinogênese/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , Odontoblastos/citologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Cromatina/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Inativação Gênica , Células HEK293 , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Camundongos , MicroRNAs/biossíntese , Peptídeo Hidrolases/biossíntese , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/genética , Ratos , Sialoglicoproteínas/metabolismo , Fatores de Transcrição/genéticaRESUMO
Transforming growth factor ß (TGF-ß) signaling has been implicated in dentin formation and repair; however, the molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-ß signaling in dentin formation, we analyzed odontoblast-specific Tgfbr2 conditional knockout mice. The mutant mice had aberrant teeth with thin dysplastic dentin and pulpal obliteration, similar to teeth from human patients with dentinogenesis imperfecta type II and dentin dysplasia. In mutant, the odontoblasts lost their cellular polarity, and matrix secretion was disrupted after mantle dentin formation. As a consequence, the amount of predentin decreased significantly, and an ectopic fibrous matrix was formed below the odontoblast layer. This matrix gradually calcified and obliterated the pulp chamber with increasing age. Immunohistochemistry revealed decreased expression of alkaline phosphatase in mutant odontoblasts. In mutant dentin, Dsp expression was reduced, but Dmp1 expression increased significantly. Collagen type I, biglycan, and Dsp were expressed in the ectopic matrix. These results suggest that loss of responsiveness to TGF-ß in odontoblasts results in impaired matrix formation and pulpal obliteration. Our study indicates that TGF-ß signaling plays an important role in dentin formation and pulp protection. Furthermore, our findings may provide new insight into possible mechanisms underlying human hereditary dentin disorders and reparative dentin formation.
Assuntos
Calcificações da Polpa Dentária/genética , Odontoblastos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Fosfatase Alcalina/análise , Animais , Biglicano/análise , Polaridade Celular/genética , Colágeno Tipo I/análise , Displasia da Dentina/genética , Dentinogênese/genética , Dentinogênese Imperfeita/genética , Desmoplaquinas/análise , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/análise , Camundongos , Camundongos Knockout , Odontoblastos/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
FAM20C is an evolutionarily reserved molecule highly expressed in mineralized tissues. Previously we demonstrated that Sox2-Cre;Fam20C(fl/fl) mice, in which Fam20C was ubiquitously inactivated, had dentin and enamel defects as well as hypophosphatemic rickets. We also showed that K14-Cre;Fam20C(fl/fl) mice, in which Fam20C was specifically inactivated in the epithelium, had enamel defects but lacked hypophosphatemia and defects in the bone and dentin. These results indicated that the enamel defects in the Sox2-Cre;Fam20C(fl/fl) mice were independent of dentin defects and hypophosphatemia. To determine if the dentin defects in the Sox2-Cre;Fam20C(fl/fl) mice were associated with the enamel defects and hypophosphatemia, we crossed Fam20C(fl/fl) mice with Wnt1-Cre and Osr2-Cre transgenic mice to inactivate Fam20C in the craniofacial mesenchymal cells that form dentin and alveolar bone. The resulting Wnt1-Cre;Fam20C(fl/fl) and Osr2-Cre;Fam20C(fl/fl) mice showed remarkable dentin and alveolar bone defects, while their enamel did not show apparent defects. The serum FGF23 levels in these mice were higher than normal but lower than those in the Sox2-Cre;Fam20C(fl/fl) mice; they developed a mild type of hypophosphatemia that did not cause major defects in long bones. These results indicate that the dentin defects in the Sox2-Cre;Fam20C(fl/fl) mice were independent of the enamel defects.