Dental Mesenchyme Research Articles

The Role of Discoidin Domain Receptor 2 in Tooth Development

Collagen signaling is critical for proper bone and tooth formation. Discoidin domain receptor 2 (DDR2) is a collagen-activated tyrosine kinase receptor shown to be essential for skeletal development. Patients with loss of function mutations in DDR2 develop spondylo-meta-epiphyseal dysplasia (SMED), a rare, autosomal recessive disorder characterized by short stature, short limbs, and craniofacial anomalies. A similar phenotype was observed in Ddr2-deficient mice, which exhibit dwarfism and defective bone formation in the axial, appendicular, and cranial skeletons. However, it is not known if Ddr2 has a role in tooth formation. We first defined the expression pattern of Ddr2 during tooth formation using Ddr2-LacZ knock-in mice. Ddr2 expression was detected in the dental follicle/sac and dental papilla mesenchyme of developing teeth and in odontoblasts and the periodontal ligament (PDL) of adults. No LacZ staining was detected in wild-type littermates. This Ddr2 expression pattern suggests a potential role in the tooth and surrounding periodontium. To uncover the function of Ddr2, we used Ddr2 slie/slie mice, which contain a spontaneous 150-kb deletion in the Ddr2 locus to produce an effective null. In comparison with wild-type littermates, Ddr2 slie/slie mice displayed disproportional tooth size (decreased root/crown ratio), delayed tooth root development, widened PDL space, and interradicular alveolar bone defects. Ddr2 slie/slie mice also had abnormal collagen content associated with upregulation of periostin levels within the PDL. The delayed root formation and periodontal abnormalities may be related to defects in RUNX2-dependent differentiation of odontoblasts and osteoblasts; RUNX2-S319-P was reduced in PDLs from Ddr2 slie/slie mice, and deletion of Ddr2 in primary cell cultures from dental pulp and PDL inhibited differentiation of cells to odontoblasts or osteoblasts, respectively. Together, our studies demonstrate odontoblast- and PDL-specific expression of Ddr2 in mature and immature teeth, as well as indicate that DDR2 signaling is important for normal tooth formation and maintenance of the surrounding periodontium.

FAM20A is essential for amelogenesis, but is dispensable for dentinogenesis.

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.

Mesenchymal Sufu Regulates Development of Mandibular Molars via Shh Signaling

Sonic hedgehog (Shh) in dental epithelium regulates tooth morphogenesis by epithelial-mesenchymal signaling transduction. However, the action of Shh signaling regulation in this process is not well understood. Here we find that mesenchymal Suppressor of Fused (Sufu), a major negative regulator of Shh signaling, plays an important role in modulating the tooth germ morphogenesis during the bud-to-cap stage transition. Deletion of Sufu in dental mesenchyme by Dermo1-Cre mice leads to delayed development of mandibular molar into cap stage with defect of primary enamel knot (EK) formation. We show the disruption of cell proliferation and programmed cell death in dental epithelium and mesenchyme in Sufu mutants. Epithelial-specific adhesion molecule E-cadherin is evidently reduced in the bilateral basal cells of tooth germ at E14.5. The cells in the presumptive EK, predominantly expressing P-cadherin, appear stratified but fail to condense. Moreover, the transcripts of primary EK marker genes, including Shh, Fgf4, and p21, are significantly decreased compared to controls. In contrast, we find that deficiency of Sufu results in elevation of Shh signaling in mesenchyme, indicated by the significant upregulation of Gli1 and Ptch1. Meanwhile, the expression of Bmp4 and Fgf3, the critical factors of mesenchymal-epithelial induction, is significantly inhibited in dental mesenchyme. Furthermore, the expression of Runx2 experiences a transient decrease at the bud stage. Taken together, these data suggest that mesenchymal Sufu is necessary for tuning the Shh signaling, which may act as an upstream modulator of Bmp4 and Fgf3 to coordinate the interplay between the dental mesenchyme and epithelium of tooth germ.

Smad7 Regulates Dental Epithelial Proliferation during Tooth Development

Tooth morphogenesis involves dynamic changes in shape and size as it proceeds through the bud, cap, and bell stages. This process requires exact regulation of cell proliferation and differentiation. Smad7, a general antagonist against transforming growth factor–β (TGF-β) signaling, is necessary for maintaining homeostasis and proper functionality in many organs. While TGF-β signaling is widely involved in tooth morphogenesis, the precise role of Smad7 in tooth development remains unknown. In this study, we showed that Smad7 is expressed in the developing mouse molars with a high level in the dental epithelium but a moderate to weak level in the dental mesenchyme. Smad7 deficiency led to a profound decrease in tooth size primarily due to a severely compromised cell proliferation capability in the dental epithelium. Consistent with the tooth shrinkage phenotype, RNA sequencing (RNA-seq) analysis revealed that Smad7 ablation downregulated genes referred to epithelial cell proliferation and cell cycle G1/S phase transition, whereas the upregulated genes were involved in responding to TGF-β signaling and cell cycle arrest. Among these genes, the expression of Cdkn1a (encoding p21), a negative cell proliferation regulator, was remarkably elevated in parallel with the diminution of Ccnd1 encoding the crucial cell cycle regulator cyclin D1 in the dental epithelium. Meanwhile, the expression level of p-Smad2/3 was ectopically elevated in the developing tooth germ of Smad7 null mice, indicating the hyperactivation of the canonical TGF-β signaling. These effects were reversed by addition of TGF-β signaling inhibitor in cell cultures of Smad7−/− molar tooth germs, with rescued expression of cyclin D1 and cell proliferation rate. In sum, our studies demonstrate that Smad7 functions primarily as a positive regulator of cell proliferation via inhibition of the canonical TGF-β signaling during dental epithelium development and highlight a crucial role for Smad7 in regulating tooth size.

Sequential stimulation with different concentrations of BMP4 promotes the differentiation of human embryonic stem cells into dental epithelium with potential for tooth formation

BackgroundTooth loss caused by caries or injuries has a negative effect on human health; thus, it is important to develop a reliable method of tooth regeneration. Research on tooth regeneration has mainly focused on mouse pluripotent stem cells, mouse embryonic stem cells, and adult stem cells from various sources in mice, whereas little has examined the differentiation of human embryonic stem (hES) cells into dental epithelium (DE) and odontogenic potential in vivo.MethodsIn this study, we induced hES cells to differentiate into dental epithelium using different concentrations of bone morphogenetic protein 4 (BMP4). With 1 pM BMP4, the hES cells differentiated into oral ectoderm (OE). These cells were then stimulated with 30 pM BMP4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence showed the differentiation of OE and DE. The DE generated was mixed with embryonic day 14.5 mouse dental mesenchyme (DM) and transplanted into the renal capsules of nude mice. Thirty days later, the resulting tooth-like structures were examined by micro-computed tomography and hematoxylin and eosin staining.ResultsAfter 4 days of 1 pM BMP4 stimulation, Pitx1-positive OE formed. qRT-PCR and immunofluorescence revealed that induction with 30 pM BMP4 for 2 days caused the OE to differentiate into Pitx2/Dlx2/AMBN-positive DE-like cells. These cells also expressed β-catenin and p-Smad1/5/8, which are key proteins in the Wnt/β-catenin and Bmp signaling pathways, respectively. Thirty days after in vivo transplantation, six teeth with enamel and dentin had formed on the kidney.ConclusionsThese results show that hES cells differentiated into DE after sequential stimulation with different concentrations of BMP4, and the DE thus generated showed odontogenic potential.

Role of osteoprotegerin in the regulation of dental epithelial‑mesenchymal signaling during tooth development

Dental epithelial-mesenchymal signaling is crucial for tooth development, but the detailed mechanism is not fully understood. Using microarray analysis, it was revealed that the expression of osteoprotegerin, an important factor regulating bone remodeling, significantly increased after removal of the dental epithelium. Immunohistochemical staining revealed that osteoprotegerin expression within the dental mesenchyme was quite low during the prenatal period, but significantly increased after birth. To investigate the influence of osteoprotegerin upon tooth development, first-molar tooth germs from embryonic day 14.5 (E14.5) Chinese Kunming mice were treated with different concentrations of osteoprotegerin. It was revealed that osteoprotegerin could inhibit the expression of odontogenic markers while promoting the expression of osteogenic markers, thereby disrupting tooth morphogenesis. These findings were further supported by in vitro and in vivo cultures. Finally, quantitative reverse transcription-polymerase chain reaction and immunofluorescence studies revealed that, after osteoprotegerin treatment, the activity of the wingless/integrated (Wnt)/β-catenin pathway increased, indicating that increased osteoprotegerin expression in prenatal tooth development could lead to uncontrolled upregulation of the Wnt/β-catenin pathway.

Low temperature culture enhances ameloblastic differentiation of human keratinocyte stem cells.

Previous studies have demonstrated that several types of human stem cells of non-dental origin can be induced to differentiate into enamel-secreting ameloblasts after recombined with mouse embryonic dental mesenchyme. However, the successful rate of ameloblastic differentiation is about rather low, which presents a major obstacle for future stem cell-based whole tooth bioengineering. Previous studies have shown that cultures at reduced temperature could improve the differentiation capability of stem cells in tissue engineering. In this study, we systematically investigated the effects of low temperature on the viability, proliferation and stemness of human keratinocytes stem cells (hKSCs) in cell culture and further examined ameloblastic differentiation of the hKSCs in human-mouse recombinant chimeric tooth germs. Our results demonstrated that low temperature indeed reduces growth rate and maintains healthy undifferentiated morphology of hKSCs without any effects on cell viability. Moreover, examination of stemness makers revealed improved stemness of hKSCs cultured at low temperature with increased expression of stemness markers K15, CD29 and p63 and decreased expression differentiation marker K10, as compared to those cultured at 37°C. These low temperature treated hKSCs, when recombined with mouse embryonic dental mesenchyme, exhibited significantly increased rate (40%) of ameloblastic differentiation, as compared to that (17%) in tissue recombinants with those hKSCs treated at standard temperature. Our studies demonstrate that low temperature cell culture improves the stemness and plasticity of hKSCs, which in turn enhances ameloblastic differentiation capability of the stem cells in bioengineered teeth.

Antagonistic interaction between Ezh2 and Arid1a coordinates root patterning and development via Cdkn2a in mouse molars.

Patterning is a critical step during organogenesis and is closely associated with the physiological function of organs. Tooth root shapes are finely tuned to provide precise occlusal support to facilitate the function of each tooth type. However, the mechanism regulating tooth root patterning and development is largely unknown. In this study, we provide the first in vivo evidence demonstrating that Ezh2 in the dental mesenchyme determines patterning and furcation formation during dental root development in mouse molars. Mechanistically, an antagonistic interaction between epigenetic regulators Ezh2 and Arid1a controls Cdkn2a expression in the dental mesenchyme to regulate dental root patterning and development. These findings indicate the importance of balanced epigenetic regulation in determining the tooth root pattern and the integration of roots with the jaw bones to achieve physiological function. Collectively, our study provides important clues about the regulation of organogenesis and has general implications for tooth regeneration in the future.

ALK5 is essential for tooth germ differentiation during tooth development

ABSTRACTThe TGFβ superfamily of proteins participates in tooth development. TGFβ1 and TGFβ3 regulate odontoblast differentiation and dentin extracellular matrix synthesis. Although the expression of TGFβ family member ligands is well-characterized during mammalian tooth development, less is known about the TGFβ receptor, which is a heteromeric complex consisting of a type I and type II receptors. The molecular mechanism of ALK5 (TGFβR1) in the dental mesenchyme is not clear. We investigated the role of ALK5 in tooth germ mesenchymal cells (TGMCs) from the lower first molar tooth germs of day 15.5 embryonic mice. Human recombinant TGFβ3 protein or an ALK5 inhibitor (SD208) was added to the cells. Cell proliferation was inhibited by SD208 and promoted by TGFβ3. We found that SD208 inhibited TGMCs osteogenesis and dentinogenesis. Both canonical and noncanonical TGFβ signaling pathways participated in the process. TAK1, P-TAK1, p38 and P-p38 showed greater expression and SMAD4 showed less expression when ALK5 was inhibited. Our findings contribute to understanding the role of TGFβ signaling for the differentiation of mesenchymal stem cells derived from dental germ and suggest possible targets for optimizing the use of stem cells of dental origin for tissue regeneration.

Mesenchymal Wnt/β-catenin signaling induces Wnt and BMP antagonists in dental epithelium

ABSTRACTPrevious studies indicated that the elevated mesenchymal Wnt/β-catenin signaling deprived dental mesenchyme of odontogenic fate. By utilizing ex vivo or pharmacological approaches, Wnt/β-catenin signaling in the developing dental mesenchyme was suggested to suppress the odontogenic fate by disrupting the balance between Axin2 and Runx2. In our study, the Osr2-creKI; Ctnnb1ex3f mouse was used to explore how mesenchymal Wnt/β-catenin signaling suppressed the odontogenic fate in vivo. We found that all of the incisor and half of the molar germs of Osr2-creKI; Ctnnb1ex3fmice started to regress at E14.5 and almost disappeared at birth. The expression of Fgf3 and Msx1 was dramatically down-regulated in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor and molar mesenchyme, while Runx2transcription was only diminished in incisor mesenchyme. Intriguingly, in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor epithelium, the expression of Noggin was activated, while Shh was abrogated. Similarly, the Wnt and BMP antagonists, Ectodin and Noggin were also ectopically activated in the E14.5 Osr2-creKI; Ctnnb1ex3f molar epithelium. Recombination of E13.5 Osr2-creKI; Ctnnb1ex3f molar mesenchyme with E10.5 and E13.5 WT dental epithelia failed to develop tooth. Taken together, the mesenchymal Wnt/β-catenin signaling resulted in the loss of odontogenic fate in vivo not only by directly suppressing odontogenic genes expression but also by inducing Wnt and BMP antagonists in dental epithelium.

Wnt Production in Dental Epithelium Is Crucial for Tooth Differentiation

The Wnt ligands display varied spatiotemporal expression in the epithelium and mesenchyme in the developing tooth. Thus far, the actions of these differentially expressed Wnt ligands on tooth development are not clear. Shh expression specifies the odontogenic epithelium during initiation and is consistently restricted to the dental epithelium during tooth development. In this study, we inactivate Wntless (Wls), the key regulator for Wnt trafficking, by Shh-Cre to investigate how the Wnt ligands produced in the dental epithelium lineage act on tooth development. We find that conditional knockout of Wls by Shh-Cre leads to defective ameloblast and odontoblast differentiation. WlsShh-Cre teeth display reduced canonical Wnt signaling activity in the inner enamel epithelium and the underlying mesenchyme at the early bell stage, as exhibited by target gene expression and BAT-gal staining. The expression of Wnt5a and Wnt10b is not changed in WlsShh-Cre teeth. By contrast, Wnt10a expression is significantly increased in response to epithelial Wls deficiency. In addition, the expression of Hedgehog signaling pathway components Shh, Gli1, and Patched1 was greatly decreased in WlsShh-Cre teeth. Epithelial Wls loss of function in Shh lineage also leads to aberrant cell proliferation in dental epithelium and mesenchyme at embryonic day 16.5; however, the cell apoptosis is unaffected. Moreover, we find that Decorin and Col1a1, the key markers for odontoblast differentiation that are downregulated in WlsShh-Cre teeth, act as direct downstream targets of the canonical Wnt signaling pathway by chromatin immunoprecipitation analysis. Additionally, Decorin and Col1a1 expression can be increased by lithium chloride (LiCl) treatment in the in vitro tooth explants. Taken together, our results suggest that the spatial expression of Wnt ligands within the dental epithelial lineage regulates the differentiation of tooth structures in later stages.

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.

Establishment of tooth blood supply and innervation is developmentally regulated and takes place through differential patterning processes.

Teeth are richly supported by blood vessels and peripheral nerves. The aim of this study was to describe in detail the developmental time-course and localization of blood vessels during early tooth formation and to compare that to innervation, as well as to address the putative role of vascular endothelial growth factor (VEGF), which is an essential regulator of vasculature development, in this process. The localization of blood vessels and neurites was compared using double immunofluorescence staining on sections at consecutive stages of the embryonic (E) and postnatal (PN) mandibular first molar tooth germ (E11-PN7). Cellular mRNA expression domains of VEGF and its signaling receptor VEGFR2 were studied using sectional radioactive insitu hybridization. Expression of VEGF mRNA and the encoded protein were studied by RT-PCR and western blot analysis, respectively, in the cap and early bell stage tooth germs, respectively. VEGFR2 was immunolocalized on tooth tissue sections. Smooth muscle cells were investigated by anti-alpha smooth muscle actin (αSMA) antibodies. VEGF showed developmentally regulated epithelial and mesenchymal mRNA expression domains including the enamel knot signaling centers that correlated with the growth and navigation of the blood vessels expressing Vegfr2 and VEGFR2 to the dental papilla and enamel organ. Developing blood vessels were present in the jaw mesenchyme including the presumptive dental mesenchyme before the appearance of the epithelial dental placode and dental neurites. Similarly, formation of a blood vessel plexus around the bud stage tooth germ and ingrowth of vessels into dental papilla at E14 preceded ingrowth of neurites. Subsequently, pioneer blood vessels in the dental papilla started to receive smooth muscle coverage at the early embryonic bell stage. Establishment and patterning of the blood vessels and nerves during tooth formation are developmentally regulated, stepwise processes that likely involve differential patterning mechanisms. Development of tooth vascular supply is proposed to be regulated by local, tooth-specific regulation by epithelial-mesenchymal tissue interactions and involving tooth target expressed VEGF signaling. Further investigations on tooth vascular development by local VEGF signaling, as well as how tooth innervation and development of blood vessels are integrated with advancing tooth organ formation by local signaling mechanisms, are warranted.

Trps1 transcription factor regulates mineralization of dental tissues and proliferation of tooth organ cells

Mutations of the TRPS1 gene cause trichorhinophalangeal syndrome (TRPS), a skeletal dysplasia with dental abnormalities. TRPS dental phenotypes suggest that TRPS1 regulates multiple aspects of odontogenesis, including the tooth number and size. Previous studies delineating Trps1 expression throughout embryonic tooth development in mice detected strong Trps1 expression in dental mesenchyme, preodontoblasts, and dental follicles, suggesting that TRPS dental phenotypes result from abnormalities in early developmental processes. In this study, Trps1+/− and Trps1−/− mice were analyzed to determine consequences of Trps1 deficiency on odontogenesis. We focused on the aspects of tooth formation that are disturbed in TRPS and on potential molecular abnormalities underlying TRPS dental phenotypes. Microcomputed tomography analyses of molars were used to determine tooth size, crown shape, and mineralization of dental tissues. These analyses uncovered that disruption of one Trps1 allele is sufficient to impair mineralization of dentin in both male and female mice. Enamel mineral density was decreased only in males, while mineralization of the root dental tissues was decreased only in females. In addition, significantly smaller teeth were detected in Trps1+/− females. Histomorphometric analyses of tooth organs showed reduced anterior-posterior diameter in Trps1−/− mice. BrdU-incorporation assay detected reduced proliferation of mesenchymal and epithelial cells in Trps1−/− tooth organs. Immunohistochemistry for Runx2 and Osx osteogenic transcription factors revealed changes in their spatial distribution in Trps1−/− tooth organs and uncovered cell-type specific requirements of Trps1 for Osx expression. In conclusion, this study has demonstrated that Trps1 is a positive regulator of cell proliferation in both dental mesenchyme and epithelium, suggesting that the microdontia in TRPS is likely due to decreased cell proliferation in developing tooth organs. Furthermore, the reduced mineralization observed in Trps1+/− mice may provide some explanation for the extensive dental caries reported in TRPS patients.

Distinctive role of ACVR1 in dentin formation: requirement for dentin thickness in molars and prevention of osteodentin formation in incisors of mice.

Dentin is a major component of teeth that protects dental pulp and maintains tooth health. Bone morphogenetic protein (BMP) signaling is required for the formation of dentin. Mice lacking a BMP type I receptor, activin A receptor type 1 (ACVR1), in the neural crest display a deformed mandible. Acvr1 is known to be expressed in the dental mesenchyme. However, little is known about how BMP signaling mediated by ACVR1 regulates dentinogenesis. To explore the role of ACVR1 in dentin formation in molars and incisors in mice, Acvr1 was conditionally disrupted in Osterix-expressing cells (designated as cKO). We found that loss of Acvr1 in the dental mesenchyme led to dentin dysplasia in molars and osteodentin formation in incisors. Specifically, the cKO mice exhibited remarkable tooth phenotypes characterized by thinner dentin and thicker predentin, as well as compromised differentiation of odontoblasts in molars. We also found osteodentin formation in the coronal part of the cKO mandibular incisors, which was associated with a reduction in the expression of odontogenic gene Dsp and an increase in the expression of osteogenic gene Bsp, leading to an alteration of cell fate from odontoblasts to osteoblasts. In addition, the expressions of WNT antagonists, Dkk1 and Sost, were downregulated and B-catenin was up-regulated in the cKO incisors, while the expression levels were not changed in the cKO molars, compared with the corresponding controls. Our results indicate the distinct and critical roles of ACVR1 between incisors and molars, which is associated with alterations in the WNT signaling related molecules. This study demonstrates for the first time the physiological roles of ACVR1 during dentinogenesis.

Expression of BMP2/4/7 during the odontogenesis of deciduous molars in miniature pig embryos.

Bone morphogenetic proteins (BMPs) play important roles in tooth development. However, their expression has not been studied in miniature pigs, which have many anatomical similarities in oral and maxillofacial region compared to human. This study investigated BMP2/4/7 expression patterns during deciduous molar development in miniature pigs on embryonic days (E) 40, 50, and 60. The mandibles were fixed, decalcified, and embedded before sectioning. H&E staining, immunohistochemistry, in situ hybridization using specific radionuclide-labeled cRNA probes, and real-time PCR were used to detect the BMP expression patterns during morphogenesis of the third deciduous molar. H&E staining showed that for the deciduous third molar, E40 represented the cap stage, E50 represented the early bell stage, and E60 represented the late bell stage or secretory stage. BMP2 was expressed in both the enamel organ and in the dental mesenchyme on E40 and E50 and was expressed mainly in pre-odontoblasts on E60. BMP7 expression was similar to BMP2 expression, but BMP7 was also expressed in the inner enamel epithelium on E60. On E40, BMP4 was expressed mainly in the epithelium, with some weak expression in the mesenchyme. On E50, BMP4 expression was stronger in the mesenchyme but weaker in the epithelium. On E60, BMP4 was expressed mainly in the mesenchyme. These data indicated that BMP2/4/7 showed differential spatial and temporal expression during the morphogenesis and odontogenesis of deciduous molars, suggesting that these molecules were associated with tooth morphogenesis and cell differentiation.

Efficient induction of functional ameloblasts from human keratinocyte stem cells

BackgroundAlthough adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study.MethodsCultured human keratinocyte stem cells (hKSCs) were treated with fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) for 18 h or 36 h prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH-soaked agarose beads into reconstructed chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and western blot analysis. Scanning electronic microscopy and a nanoindentation test were further employed to analyze the prism running pattern and mechanical properties of the regenerated enamel.ResultsTreatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human enamel. The nanoindentation test indicated that, although much softer than human child and adult mouse enamel, mechanical properties of the regenerated enamel improved as the culture time was extended.ConclusionsApplication of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human–mouse chimeric tooth germs.

Bone Morphogenetic Protein 2 Coordinates Early Tooth Mineralization

Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Odontoblast and ameloblast differentiation involves reciprocal and sequential epithelial-mesenchymal interactions. Similar to early tooth development, various Bmps are expressed during this process, although their functions have not been explored in detail. Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. In developing molars, Bmp2LacZ reporter mice revealed restricted expression of Bmp2 in early polarized and functional odontoblasts while it was not expressed in mature odontoblasts. Loss of Bmp2 in neural crest cells, which includes all dental mesenchyme, caused a delay in dentin and enamel deposition. Immunohistochemistry for nestin and dentin sialoprotein (Dsp) revealed polarization defects in odontoblasts, indicative of a role for Bmp2 in odontoblast organization. Surprisingly, pSmad1/5/8, an indicator of Bmp signaling, was predominantly reduced in ameloblasts, with reduced expression of amelogenin (Amlx), ameloblastin (Ambn), and matrix metalloproteinase (Mmp20). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry showed that loss of Bmp2 resulted in increased expression of the Wnt antagonists dickkopf 1 (Dkk1) in the epithelium and sclerostin (Sost) in mesenchyme and epithelium. Odontoblasts showed reduced Wnt signaling, which is important for odontoblast differentiation, and a strong reduction in dentin sialophosphoprotein (Dspp) but not collagen 1 a1 (Col1a1) expression. Mature Bmp2-deficient teeth, which were obtained by transplanting tooth germs from Bmp2-deficient embryos under a kidney capsule, showed a dentinogenesis imperfecta type II–like appearance. Micro–computed tomography and scanning electron microscopy revealed reduced dentin and enamel thickness, indistinguishable primary and secondary dentin, and deposition of ectopic osteodentin. This establishes that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

Mesenchymal Wnt/β-catenin signaling limits tooth number.

Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/β-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor AXIN2 lead to human tooth agenesis. We have investigated the functions of Wnt/β-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/β-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/β-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/β-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. Runx2, a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors Axin2 and Drapc1 in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that Axin2/Runx2 antagonistic interactions modulate the level of mesenchymal Wnt/β-catenin signaling, underlying the contrasting dental phenotypes caused by human AXIN2 and RUNX2 mutations.

Specificity Protein 7 Is Required for Proliferation and Differentiation of Ameloblasts and Odontoblasts.

The Sp7/Osterix transcription factor is essential for bone development. Mutations of the Sp7 gene in humans are associated with craniofacial anomalies and osteogenesis imperfecta. However, the role of Sp7 in embryonic tooth development remains unknown. Here we identified the functional requirement of Sp7 for dentin synthesis and tooth development. Sp7-null mice exhibit craniofacial dysmorphogenesis and are completely void of alveolar bone. Surprisingly, initial tooth morphogenesis progressed normally in Sp7-null mice. Thus the formation of alveolar bone is not a prerequisite for tooth morphogenesis. Sp7 is required for mineralization of palatal tissue but is not essential for palatal fusion. The reduced proliferative capacity of Sp7-deficient ectomesenchyme results in small and misshapen teeth with randomly arranged cuboidal preodontoblasts and preameloblasts. Sp7 promotes functional maturation and polarization of odontoblasts. Markers of mature odontoblast (Col1a, Oc, Dspp, Dmp1) and ameloblast (Enam, Amelx, Mmp20, Amtn, Klk4) are barely expressed in incisors and molar tissues of Sp7-null mice. Consequently, dentin and enamel matrix are absent in the Sp7-null littermates. Interestingly, the Sp7 expression is restricted to cells of the dental mesenchyme indicating the effect on oral epithelium-derived ameloblasts is cell-nonautonomous. Abundant expression of Fgf3 and Fgf8 ligand was noted in the developing tooth of wild-type mice. Both ligands were remarkably absent in the Sp7-null incisor and molar, suggesting cross-signaling between mesenchyme and epithelium is disrupted. Finally, promoter-reporter assays revealed that Sp7 directly controls the expression of Fgf-ligands. Together, our data demonstrate that Sp7 is obligatory for the differentiation of both ameloblasts and odontoblasts but not for the initial tooth morphogenesis. © 2018 American Society for Bone and Mineral Research.