Tooth Organ Research Articles

Biology and Cultural Importance of the Narwhal.

Though narwhal have survived multiple ice ages, including 2.5 Ma and the last interglacial period with warming temperatures, Arctic climate change during the Anthropocene introduces new challenges. Despite their evolutionary connection to Arctic Pleistocene fossils, narwhal archeocete ancestors from the Pliocene (Bohaskaiamonodontoides) and Miocene (Denebola and Odobenocetopsidae) inhabited warm waters. Narwhal Arctic adaptation holds valuable insights into unique traits, including thin skin; extreme diving capacity; and a unique straight, spiraled, and sensory tooth organ system. Inaccessible weather, ice conditions, and darkness limit scientific studies, though Inuit knowledge adds valuable observations of narwhal ecology, biology, and behavior. Existing and future studies in myriad fields of physical, chemical, biological, and genetic science, combined and integrated with remote sensing and imaging technologies, will help elucidate narwhal evolution, biology, and adaptation. When integrated with Qaujimajatuqangit, "the Inuit way of knowing," these studies help describe interesting biologic expressions of the narwhal.

Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts.

Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here, we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in the direction predicted by a hair regeneration model: Wnt10a and Grem2a increased tooth replacement rate, whereas Bmp6 and Dkk2 strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA sequencing of stickleback dental tissue showed that Bmp6 overexpression resulted in an upregulation of Wnt inhibitors. Together, these data support a model in which different epithelial organs, such as teeth and hair, share genetic circuitry driving organ regeneration.

Development of a novel ex vivo organ culture system to improve preservation methods of regenerative tissues

Recent advances in regenerative technology have made the regeneration of various organs using pluripotent stem cells possible. However, a simpler screening method for evaluating regenerated organs is required to apply this technology to clinical regenerative medicine in the future. We have developed a simple evaluation method using a mouse tooth germ culture model of organs formed by epithelial–mesenchymal interactions. In this study, we successfully established a simple method that controls tissue development in a temperature-dependent manner using a mouse tooth germ ex vivo culture model. We observed that the development of the cultured tooth germ could be delayed by low-temperature culture and resumed by the subsequent culture at 37 °C. Furthermore, the optimal temperature for the long-term preservation of tooth germ was 25 °C, a subnormothermic temperature that maintains the expression of stem cell markers. We also found that subnormothermic temperature induces the expression of cold shock proteins, such as cold-inducible RNA-binding protein, RNA-binding motif protein 3, and serine and arginine rich splicing factor 5. This study provides a simple screening method to help establish the development of regenerative tissue technology using a tooth organ culture model. Our findings may be potentially useful for making advances in the field of regenerative medicine.

Prevalence of premature loss of deciduous teeth and its relationship with gender among children from Acapulco, Guerrero: a cross-sectional study.

Premature loss of deciduous teeth is the exfoliation or extraction before physiological replacement with < 50% or < 75% of the root of the substitute tooth formed or if there is > 1 mm of alveolar bone covering the permanent successor tooth organ. This study aimed to estimate the prevalence of premature tooth loss in children and identify associated factors in a health center in Acapulco, Guerrero. We conducted a cross-sectional study in which we collected information from 109 clinical records of children examined from January 2019 to August 2021. Sociodemographic data of the children and parents were collected: socioeconomic level, non-pathological personal history, and the history of premature deciduous tooth loss. Multivariate analysis to identify factors associated with premature deciduous tooth loss was performed with CIETmap statistical software. Odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the strength of the association. The prevalence of premature loss of primary teeth was 40% (43/109). The leading cause was caries (84%, 36/43). The tooth organ with the highest loss occurrence was the lower right second molar (33%, 14/43). Gender was identified as an associated factor, with males having a higher risk of loss (ORa = 2.97; CI95% = 1.33-6.65). Our results were similar to those reported in other studies. Strategies aimed at health promotion directed at parents and children should be reinforced.

The Effect of Matrix for Regeneration of Enamel, Dentin and Pulp: A Systematic Review

The tooth structure does not have ability to regenerate on it’s own until there is introduction of some inducing factors. Several inducing factors present in the form of stem cells, scaffolds and growth factors which thereby, plays role in the formation of enamel, dentin and pulp. In order to stimulate these regenerative factors, certain amount of temperature, pressure and atmospheric humidity should be in control. Specific knowledge of proteins which is present in each of the part of tooth structure (enamel, dentin) is important in order to achieve the final outcome. The present review elaborates about the achievement of tooth structure matrix and organs by usage of specific amount of proteins, growth factors, stem cells

In the absence of a basal lamina, ameloblasts absorb enamel in a serumless and chemically defined organ culture system

ObjectivesTooth organ development was examined in a serumless, chemically defined organ culture system to determine whether morphological and functional development was identical to that in in vivo and serum-supplemented organ cultures. MethodsMouse mandibular first molar tooth organs at 16 days of gestation were cultured for up to 28 days in a Tronwell culture system using a serum-supplemented or serumless, chemically defined medium. After culture, specimens were processed for assessing tooth development using ultrastructural, immunohistochemical, and mRNA expression analyses. ResultsIn serum-supplemented conditions, inner enamel epithelial cells differentiated into secretory-stage ameloblasts, which formed enamel and reached the maturation stage after 14 and 21 days of culture, respectively. Ameloblasts deposited a basal lamina on immature enamel. Conversely, in serumless conditions, ameloblasts formed enamel on mineralized dentin after 21 days. Moreover, maturation-stage ameloblasts did not form basal lamina and directly absorbed mineralized enamel after 28 days of culture. RT-PCR analysis indicated that tooth organs, cultured in serumless conditions for 28 days, had significantly reduced expression levels of ODAM, amelotin, and laminin-322. ConclusionsThese results indicate that several differences were detected compared to the development in serum-supplemented conditions, such as delayed enamel and dentin formation and the failure of maturation-stage ameloblasts to form basal laminae. Therefore, our results suggest that some factors might be required for the steady formation of mineralized dentin, enamel, and a basal lamina. Additionally, our results indicate that a basal lamina is necessary for enamel maturation.

Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption.

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

Functional Expression of Sodium-Dependent Glucose Transporter in Amelogenesis

Glucose is an essential source of energy for mammalian cells and is transported into the cells by glucose transporters. There are 2 types of glucose transporters: one is a passive glucose transporter, GLUT (SLC2A), and the other is a sodium-dependent active glucose transporter, SGLT (SLC5A). We previously reported that the expression of GLUTs during tooth development is precisely and spatiotemporally controlled and that the glucose uptake mediated by GLUT1 plays a crucial role in early tooth morphogenesis and tooth size determination. This study aimed to clarify the localization and roles of SGLT1 and SGLT2 in murine ameloblast differentiation by using immunohistochemistry, immunoelectron microscopy, an in vitro tooth organ culture experiment, and in vivo administration of an inhibitor of SGLT1/2, phloridzin. SGLT1, which has high affinity with glucose, was immunolocalized in the early secretory ameloblasts and the ruffle-ended ameloblasts in the maturation stage. However, SGLT2, which has high glucose transport capacity, was observed in the stratum intermedium, papillary layer, and ameloblasts at the maturation stage and colocalized with Na+-K+-ATPase. The inhibition of SGLT1/2 by phloridzin in the tooth germs induced the disturbance of ameloblast differentiation and enamel matrix formation both in vitro (organ culture) and in vivo (mouse model). The expression of SGLT1 and SGLT2 was significantly upregulated in hypoxic conditions in the ameloblast-lineage cells. These findings suggest that the active glucose uptake mediated by SGLT1 and SGLT2 is strictly regulated and dependent on the intra- and extracellular microenvironments during tooth morphogenesis and that the appropriate passive and active glucose transport is an essential event in amelogenesis.

MicroRNA-875-5p plays critical role for mesenchymal condensation in epithelial-mesenchymal interaction during tooth development

Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/β-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Expression of new genes in vertebrate tooth development and p63 signaling.

p63 is an evolutionarily ancient transcription factor essential to vertebrate tooth development. Our recent gene expression screen comparing wild-type and "toothless" p63-/- mouse embryos implicated in tooth development several new genes that we hypothesized act downstream of p63 in dental epithelium, where p63 is also expressed. Via in situ hybridization and immunohistochemistry, we probed mouse embryos (embryonic days 10.5-14.5) and spotted gar fish embryos (14 days postfertilization) for these newly linked genes, Cbln1, Cldn23, Fermt1, Krt15, Pltp and Prss8, which were expressed in mouse and gar dental epithelium. Loss of p63 altered expression levels but not domains. Expression was comparable between murine upper and lower tooth organs, implying conserved gene functions in maxillary and mandibular dentitions. Our meta-analysis of gene expression databases supported that these genes act within a p63-driven gene regulatory network important to tooth development in mammals and more evolutionary ancient vertebrates (fish, amphibians). Cbln1, Cldn23, Fermt1, Krt15, Pltp, and Prss8 were expressed in mouse and fish dental epithelium at placode, bud, and/or cap stages. We theorize that these genes participate in cell-cell adhesion, cell polarity, and extracellular matrix signaling to support dental epithelium integrity, folding, and epithelial-mesenchymal cross talk during tooth development.

Landmark‐based 3D Morphometrics of the Developing Human Dentition in the Middle Trimester

Human primary tooth formation begins around 6 weeks in utero and by the time a baby is born, all 20 teeth have mineralized and are embedded in the jaw bone. Our study is addressing the period during which tooth morphogenesis and jaw development are taking place, the middle trimester. The tooth‐bone relationship involves inductive interactions between the condensed mesenchyme of the dental follicle surrounding the tooth germs and the non‐dental mesenchyme of the jaws. As teeth pass through bud, cap and bell stages, intramembranous bone is being deposited by osteoblasts but the patterning of the bone in relation to the teeth is not well understood. From studies in mouse, the tooth follicle is thought to induce bone and in turn the bone is thought to restrict tooth organ growth. Mouse molars are completely encased in bone as soon as the crown is completed. However in humans the bone surrounding tooth organs is fenestrated. The buccal and occlusal surfaces of the first molar (m1) and canine (c) are not covered by bone whereas the incisors (lateral incisor – i2, central incisor – i1) are surrounded by bone on buccal, lingual, gingival, mesial and distal. Therefore we wondered whether crypt growth is isometric or allometric and whether there were differences between the tooth morphotypes. To answer this question, we scanned 25 intact fetal jaws with microCT from a collection of human specimens maintained at UBC. Scans were imported into Amira (Thermo‐Fisher) and used for segmentation or landmark placement. Landmarks were placed in a consistent manner on all surfaces of the bony tooth crypt (Fig. B,D) and then exported to MorphoJ for morphological analyses (Fig. A,C). In addition segmented tooth volumes and linear dimensions were measured in Amira.The principal component and canonical variate analysis revealed that there are significant shape differences in the crypts between 12–14 weeks and 17–19 weeks. The intermediate period 15–16 weeks was not significantly different than the early or later timeframes. These trends matched segmented volumetric data for the tooth germs and mandible. Shifts in specific landmarks particularly the buccal landmarks adjacent to m1 and c were observed between the early and late period which is consistent with the lack of constraint by bone on those surfaces (Fig. A). Allometric growth in the occlusal gingival direction was also observed with increased depth to the crypts for all teeth over the developmental period (Fig. C). Some of these changes are consistent with reconstruction data from others showing relatively greater bone resorption on the internal surfaces of the crypt. Our data suggests that the all the tooth germs are interacting with the bone that will later surround the roots. The prominent buccal bulges of m1, plus the formation of the mental foramen on the buccal side of the m1 crypt, may require spatially restricted tissue interactionsSupport or Funding InformationFunded by the Faculty of Dentistry, UBC; Human ethics approval number H08‐0257This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Silver-Albumin Tissue Staining Protocol to Visualize Odontogenesis in Whole Embryos.

Visualizing tooth organs from their earliest inception as they actually appear in three dimensions has, until recently, been difficult due to the technical obstacle of imaging these tiny, translucent, low-density embryonic craniodental tissues. Related to this obstacle, quantifying craniodental morphology has been confounded by the time consuming need to physically section and then digitally photograph and reconstruct these images of tissues into 3D volumes. Here we provide a simple solution in the form of an overnight silver albumin tissue stain for whole embryos. Because it is differentially absorbed by embryonic tissues, this stain generates the contrast needed to detect and visualize unmineralized dental tissues. Stained specimens can be scanned using either desktop or synchrotron micro-computed tomography systems, generating digital 3D datasets of whole embryos that can immediately be used to assess dental morphology and histology. Craniodental structures can then be measured with high precision and accuracy using 3D image analysis software.

Sex estimation using sexually dimorphic amelogenin protein fragments in human enamel

Amelogenin genes are located on both X and Y sex chromosomes in humans and are a major focus of DNA-based sex estimation methods. Amelogenin proteins, AMELX_HUMAN and AMELY_HUMAN, are expressed in the tooth organ and play a major role in mineralization of enamel, the most taphonomically resistant, archaeologically persistent human tissue. We describe shotgun liquid chromatography mass spectrometry analysis of 40 enamel samples representing 25 individuals, including modern third molars and archaeological teeth from open-air contexts including permanent adult (400 to 7300 BP) and deciduous teeth (100 to 1000 BP). Peptides specific to the X-chromosome isoform of amelogenin were detected in all samples. Peptides specific to the sexually dimorphic Y-chromosome isoform were also detected in 26 samples from 13 individuals, across all time periods, including previously unsexed deciduous teeth from archaeological contexts. While the signal of each gene product can vary by more than an order of magnitude, we show close agreement between osteological and amelogenin-based sex estimation and thus demonstrate that the protein-based signal can be obtained reliably from open-air archaeological contexts dating to at least 7300 years ago. While samples with AMELY_HUMAN peptides are unambiguously male, samples with no AMELY_HUMAN signal may either be low signal male false negative samples or female samples. In order to estimate sex in these samples we developed a probability curve of female sex as a function of the logarithm of AMELX_HUMAN signal (p < 0.0001) using logistic regression. This is also the first demonstration using proteomics to estimate sex in deciduous teeth and pushes back the application of the method to teeth that are at least 7300 years old.

Effective Differentiation of Induced Pluripotent Stem Cells Into Dental Cells.

A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129-139, 2019. © 2018 Wiley Periodicals, Inc.

Bone Marrow Stromal Cells Promote Innervation of Bioengineered Teeth

Transplantation of bone marrow mesenchymal stem cells (BMDCs) into a denervated side of the spinal cord was reported to be a useful option for axonal regeneration. The innervation of teeth is essential for their function and protection but does not occur spontaneously after injury. Cultured reassociations between dissociated embryonic dental mesenchymal and epithelial cells and implantation lead to a vascularized tooth organ regeneration. However, when reassociations were coimplanted with a trigeminal ganglion (TG), innervation did not occur. On the other hand, reassociations between mixed embryonic dental mesenchymal cells and bone marrow–derived cells isolated from green fluorescent protein (GFP) transgenic mice (BMDCs-GFP) (50/50) with an intact and competent dental epithelium (ED14) were innervated. In the present study, we verified the stemness of isolated BMDCs, confirmed their potential role in the innervation of bioengineered teeth, and analyzed the mechanisms by which this innervation can occur. For that purpose, reassociations between mixed embryonic dental mesenchymal cells and BMDCs-GFP with an intact and competent dental epithelium were cultured and coimplanted subcutaneously with a TG for 2 wk in ICR mice. Axons entered the dental pulp and reached the odontoblast layer. BMDCs-GFP were detected at the base of the tooth, with some being present in the pulp associated with the axons. Thus, while having a very limited contribution in tooth formation, they promoted the innervation of the bioengineered teeth. Using quantitative reverse transcription polymerase chain reaction and immunostainings, BMDCs were shown to promote innervation by 2 mechanisms: 1) via immunomodulation by reducing the number of T lymphocytes (CD3+, CD25+) in the implants and 2) by expressing neurotrophic factors such as NGF, BDNF, and NT3 for axonal growth. This strategy using autologous mesenchymal cells coming from bone marrow could be used to innervate bioengineered teeth without treatment with an immunosuppressor such as cyclosporine A (CsA), thus avoiding multiple side effects.

Expression of desmosomal proteins and their implications during enamel organ morphogenesis

Background/Objective: The present article is between the fewest that describe the expression of desmosomal proteins in human tooth organ and to implicate desmosome and its components in tooth morphogenesis. Although present in all epithelia, desmosomes are particularly down regulated in undifferentiated, dedifferentiated and malignant cells. Methodology: In the present article, using immunohistochemical staining, we report the expression patterns of 6 desmosomal components; Desmoplakin, plakoglobin, desmoglein, plakophilin-1, plakophilin-2, and plakophilin-3. The dental, oral and ectodermal epithelia were investigated in 7 and 9 weeks' human embryos. Results: The expression of these components appeared to associate with cell differentiation, stage of development and epithelial type. The fundamental desmosomal proteins, desmoplakin, plakoglobin and desmoglein, produced almost identical profiles, suggesting that desmosomes are absent or under developed in negative and weak reacting areas respectively. Whilst the accessory component plakophilin-1 was always absent, plakophilin-2 and -3 were strongly labeled in the embryonic epidermis and moderately expressed in the oral epithelium. In the developing enamel organ, gradients of fundamental protein expressions were produced being strong towards the oral epithelium and weak/negative around the inner enamel epithelium. These gradients were also produced using antibodies to plakophilin-2 and -3 although the majority of enamel organ cells were unlabeled. Conclusion: Mirror image symmetry was foundwas found for the distribution patterns of these markers. Furthermore, the left-right asymmetry was found both for the maxillary and mandibular tooth germs, thus bringing further evidence implicating desmosome and its components in the mechanisms regulating tooth morphogenesis.