Special AT-rich sequence-binding protein 2 (Satb2) synergizes with Bmp9 and is essential for osteo/odontogenic differentiation of mouse incisor mesenchymal stem cells.

OBJECTIVES: Mouse incisor mesenchymal stem cells (MSCs) have self-renewal ability and osteo/odontogenic differentiation potential. However, the mechanism controlling the continuous self-renewal and osteo/odontogenic differentiation of mouse incisor MSCs remains unclear. Special AT-rich sequence-binding protein 2 (SATB2) positively regulates craniofacial patterning, bone development and regeneration, whereas SATB2 deletion or mutation leads to craniomaxillofacial dysplasia and delayed tooth and root development, similar to bone morphogenetic protein (BMP) loss-of-function phenotypes. However, the detailed mechanism underlying the SATB2 role in odontogenic MSCs is poorly understood. The aim of this study was to investigate whether SATB2 can regulate self-renewal and osteo/odontogenic differentiation of odontogenic MSCs. MATERIALS AND METHODS: Satb2 expression was detected in the rapidly renewing mouse incisor mesenchyme by immunofluorescence staining, quantitative RT-PCR and Western blot analysis. Ad-Satb2 and Ad-siSatb2 were constructed to evaluate the effect of Satb2 on odontogenic MSCs self-renewal and osteo/odontogenic differentiation properties and the potential role of Satb2 with the osteogenic factor bone morphogenetic protein 9 (Bmp9) in vitro and in vivo. RESULTS: Satb2 was found to be expressed in mesenchymal cells and pre-odontoblasts/odontoblasts. We further discovered that Satb2 effectively enhances mouse incisor MSCs self-renewal. Satb2 acted synergistically with the potent osteogenic factor Bmp9 in inducing osteo/odontogenic differentiation of mouse incisor MSCs in vitro and in vivo. CONCLUSIONS: Satb2 promotes self-renewal and osteo/odontogenic differentiation of mouse incisor MSCs. Thus, Satb2 can cooperate with Bmp9 as a new efficacious bio-factor for osteogenic regeneration and tooth engineering.

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth.

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.

Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling.

Stem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche.

A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage.

The classical model of tissue renewal posits that small numbers of quiescent stem cells (SCs) give rise to proliferating transit-amplifying cells before terminal differentiation. However, many organs house pools of SCs with proliferative and differentiation potentials that diverge from this template. Resolving SC identity and organization is therefore central to understanding tissue renewal. Here, using a combination of single-cell RNA sequencing (scRNA-seq), mouse genetics and tissue injury approaches, we uncover cellular hierarchies and mechanisms that underlie the maintenance and repair of the continuously growing mouse incisor. Our results reveal that, during homeostasis, a group of actively cycling epithelial progenitors generates enamel-producing ameloblasts and adjacent layers of non-ameloblast cells. After injury, tissue repair was achieved through transient increases in progenitor-cell proliferation and through direct conversion of Notch1-expressing cells to ameloblasts. We elucidate epithelial SC identity, position and function, providing a mechanistic basis for the homeostasis and repair of a fast-turnover ectodermal appendage.

Transit amplifying cells coordinate mouse incisor mesenchymal stem cell activation.

Stem cells (SCs) receive inductive cues from the surrounding microenvironment and cells. Limited molecular evidence has connected tissue-specific mesenchymal stem cells (MSCs) with mesenchymal transit amplifying cells (MTACs). Using mouse incisor as the model, we discover a population of MSCs neibouring to the MTACs and epithelial SCs. With Notch signaling as the key regulator, we disclose molecular proof and lineage tracing evidence showing the distinct MSCs contribute to incisor MTACs and the other mesenchymal cell lineages. MTACs can feedback and regulate the homeostasis and activation of CL-MSCs through Delta-like 1 homolog (Dlk1), which balances MSCs-MTACs number and the lineage differentiation. Dlk1's function on SCs priming and self-renewal depends on its biological forms and its gene expression is under dynamic epigenetic control. Our findings can be validated in clinical samples and applied to accelerate tooth wound healing, providing an intriguing insight of how to direct SCs towards tissue regeneration.

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248.

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/ß-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.

Microdissection and Isolation of Mouse Dental Epithelial Cells of Continuously Growing Mouse Incisors.

Mouse incisors are regenerative tissues, which grow continuously throughout life and are good model for the study of epithelial stem cells. The study of dental epithelial stem cells allows investigation of a variety of basic biological processes in the context of the stem cells. The ability to analyze dental epithelial stem cells in vitro has emerged as a powerful tool to understand how teeth are constructed and the signaling pathways that regulate ameloblast developmental processes. Here, we describe in detail our protocols for the culture of dental epithelial stem cells and the production of the cell lines. These techniques allow us to reproduce the differentiation process of ameloblasts and estimate the effect of specific genes ex vivo, as well as are a tool for studies on the mechanisms of normal and abnormal amelogenesis. They may also be applied to studies on other aspects of developmental biology and regenerative medicine using stem cells.

Isolation of Dental Stem Cell-Enriched Populations from Continuously Growing Mouse Incisors.

Continuous growth of the rodent incisor is enabled by epithelial and mesenchymal stem cells (ESCs and MSCs) which unceasingly replenish enamel and dentin, respectively, that wear by persistent animal gnawing. Lineage tracing studies have provided evidence that ESCs contribute to all epithelial lineages of the tooth in vivo. Meanwhile, in the mouse incisor, MSCs continuously contribute to odontoblast lineage and tooth growth. However, in vitro manipulation of ESCs has shown little progress, mainly due to lack of appropriate protocol to successfully isolate, culture, expand, and differentiate ESCs in vitro without using the co-culture system. In this chapter we describe the isolation of the Sox2-GFP+ cell population that is highly enriched in ESCs. Isolated cells can be used for various types of analyses, including in vitro culture, single cell-related analyses, etc. Furthermore, we describe ways to obtain populations enriched in the incisor MSCs using FACS sorting of antibody-labeled cells. Easily accessible FACS sorting enables easy and relatively fast isolation of the cells labeled by the fluorescent protein.

Runx1-Stat3 signaling regulates the epithelial stem cells in continuously growing incisors.

Rodent incisors grow permanently and the homeostasis of enamel production is maintained by a continuous supply of epithelial progenitors from putative stem cells in the cervical loop. We herein report that Runx1 regulates the Lgr5-expressing epithelial stem cells and their subsequent continuous differentiation into ameloblasts. Mice deficient in epithelial Runx1 demonstrate remarkable shortening of the incisors with underdevelopment of the cervical loop and enamel defects. In this mutant cervical loop, the proliferation of the dental epithelium was significantly disturbed and the expression of Lgr5 and enamel matrix proteins was remarkably downregulated. Interestingly, the expression of Socs3, an inhibitor of Stat3 signaling, was upregulated and Stat3 phosphorylation was suppressed specifically in the mutant cervical loop. The expression of Lgr5 and the enamel matrix protein in the wild-type incisor germs is disturbed by pharmaceutical Stat3 inhibition in vitro., of. Conversely, pharmaceutical activation of Stat3 rescues the defective phenotypes of the Runx1 mutant with upregulated Lgr5 and enamel matrix protein genes. The present results provide the first evidence of the role of Runx1 regulates the Lgr5-expressing epithelial stem cells and differentiation of ameloblast progenitors in the developing incisors. Our study also demonstrates that Stat3 modulates the Runx1-Lgr5 axis in the cervical loop.

Regulation of Mesenchymal Stem to Transit-Amplifying Cell Transition in the Continuously Growing Mouse Incisor.

In adult tissues and organs with high turnover rates, the generation of transit-amplifying cell (TAC) populations from self-renewing stem cells drives cell replacement. The role of stem cells is to provide a renewable source of cells that give rise to TACs to provide the cell numbers that are necessary for cell differentiation. Regulation of the formation of TACs is thus fundamental to controlling cell replacement. Here, we analyze the properties of a population of mesenchymal TACs in the continuously growing mouse incisor to identify key components of the molecular regulation that drives proliferation. We show that the polycomb repressive complex 1 acts as a global regulator of the TAC phenotype by its direct action on the expression of key cell-cycle regulatory genes and by regulating Wnt/ß-catenin-signaling activity. We also identify an essential requirement for TACs in maintaining mesenchymal stem cells, which is indicative of a positive feedback mechanism.

Role of the Inferior Alveolar Nerve in Rodent Lower Incisor Stem Cells.

In developing teeth, the sequential and reciprocal interactions between epithelial and mesenchymal tissues promote stem/progenitor cell differentiation. However, the origin of the stem/progenitor cells has been the subject of considerable debate. According to recent studies, mesenchymal stem cells originate from periarterial cells and are regulated by neurons in various organs. The present study examined the role of innervation in tooth development and rodent incisor stem/progenitor cell homeostasis. Rodent incisors continuously grow throughout their lives, and the lower incisors are innervated by the inferior alveolar nerve (IAN). In this study, we resected the IAN in adult rats, and the intact contralateral side served as a nonsurgical control. Sham control rats received the same treatment as the resected rats, except for the resection process. The extent of incisor eruption was measured, and both mesenchymal and epithelial stem/progenitor cells were visualized and compared between the IAN-resected and sham-operated groups. One week after surgery, the IAN-resected incisors exhibited a chalky consistency, and the eruption rate was decreased. Micro-computed tomography and histological analyses performed 4 wk after surgery revealed osteodentin formation, disorganized ameloblast layers, and reduced enamel thickness in the IAN-resected incisors. Immunohistochemical analysis revealed a reduction in the CD90- and LRIG1-positive mesenchymal cell ratio in the IAN-resected incisors. However, the p40-positive epithelial stem/progenitor cell ratio was comparable between the 2 groups. Thus, mesenchymal stem/progenitor cell homeostasis is more related to IAN innervation than to epithelial stem/progenitor cells. Furthermore, sensory nerve innervation influences subsequent incisor growth and formation.

A quiescent cell population replenishes mesenchymal stem cells to drive accelerated growth in mouse incisors.

The extent to which heterogeneity within mesenchymal stem cell (MSC) populations is related to function is not understood. Using the archetypal MSC in vitro surface marker, CD90/Thy1, here we show that 30% of the MSCs in the continuously growing mouse incisor express CD90/Thy1 and these cells give rise to 30% of the differentiated cell progeny during postnatal development. In adulthood, when growth rate homeostasis is established, the CD90/Thy1+ MSCs decrease dramatically in number. When adult incisors are cut, the growth rate increases to rapidly re-establish tooth length and homeostasis. This accelerated growth rate correlates with the re-appearance of CD90/Thy+ MSCs and re-establishment of their contribution to cell differentiation. A population of Celsr1+ quiescent cells becomes mitotic following clipping and replenishes the CD90/Thy1 population. A sub-population of MSCs thus exists in the mouse incisor, distinguished by expression of CD90/Thy1 that plays a specific role only during periods of increased growth rate.

Plasticity within the niche ensures the maintenance of a Sox2+ stem cell population in the mouse incisor.

In mice, the incisors grow throughout the animal's life, and this continuous renewal is driven by dental epithelial and mesenchymal stem cells. Sox2 is a principal marker of the epithelial stem cells that reside in the mouse incisor stem cell niche, called the labial cervical loop, but relatively little is known about the role of the Sox2+ stem cell population. In this study, we show that conditional deletion of Sox2 in the embryonic incisor epithelium leads to growth defects and impairment of ameloblast lineage commitment. Deletion of Sox2 specifically in Sox2+ cells during incisor renewal revealed cellular plasticity that leads to the relatively rapid restoration of a Sox2-expressing cell population. Furthermore, we show that Lgr5-expressing cells are a subpopulation of dental Sox2+ cells that also arise from Sox2+ cells during tooth formation. Finally, we show that the embryonic and adult Sox2+ populations are regulated by distinct signalling pathways, which is reflected in their distinct transcriptomic signatures. Together, our findings demonstrate that a Sox2+ stem cell population can be regenerated from Sox2- cells, reinforcing its importance for incisor homeostasis.

Transplantation of dental pulp stem cells improves long-term diabetic polyneuropathy together with improvement of nerve morphometrical evaluation.

BACKGROUND: Although previous reports have revealed the therapeutic potential of stem cell transplantation in diabetic polyneuropathy, the effects of cell transplantation on long-term diabetic polyneuropathy have not been investigated. In this study, we investigated whether the transplantation of dental pulp stem cells (DPSCs) ameliorated long-term diabetic polyneuropathy in streptozotocin (STZ)-induced diabetic rats. METHODS: Forty-eight weeks after STZ injection, we transplanted DPSCs into the unilateral hindlimb skeletal muscles. Four weeks after DPSC transplantation (i.e., 52 weeks after STZ injection) the effects of DPSC transplantation on diabetic polyneuropathy were assessed. RESULTS: STZ-induced diabetic rats showed significant reductions in the sciatic motor/sensory nerve conduction velocity, increases in the current perception threshold, and decreases in capillary density in skeletal muscles and intra-epidermal nerve fiber density compared with normal rats, all of which were ameliorated by DPSC transplantation. Furthermore, sural nerve morphometrical analysis revealed that the transplantation of DPSCs significantly increased the myelin thickness and area. DPSC-conditioned media promoted the neurite outgrowth of dorsal root ganglion neurons and increased the viability and myelin-related protein expression of Schwann cells. CONCLUSIONS: These results indicated that the transplantation of DPSCs contributed to the neurophysiological and neuropathological recovery from a long duration of diabetic polyneuropathy.

Involvement of IGF-2, IGF-1R, IGF-2R and PTEN in development of human tooth germ - an immunohistochemical study.

Insulin-Like Growth Factor 2 (IGF-2) is a peptide hormone essential for prenatal growth and development. IGF-2 exerts its mitogenic effects via Insulin-Like Growth Factor 1 Receptor (IGF-1R), and is eliminated by binding to Insulin-Like Growth Receptor 2 (IGF-2R). IGF-2 is also negatively regulated by Phosphatase and Tensin Homolog (PTEN), a phosphatase mutated in various tumors. Not much is known about the interplay between these factors during human odontogenesis. In this study, expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN were analyzed by double immunofluorescence in incisor human tooth germs during the foetal period of development between the 7th and 20th gestational week. Throughout the investigated period, IGF-2 was mostly expressed in enamel organ, whereas mild to moderate expression of PTEN could be seen in dental papilla and parts of enamel organ. Expression of IGF-1R was ubiquitous and displayed strong intensity throughout the entire enamel organ. In contrast, expression of IGF-2R had rather erratic pattern in enamel organ and dental papilla alike. Expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN in highly proliferative cervical loops, as well as in differentiating pre-ameloblasts and pre-odontoblasts of cusp tip region during the early and late bell stages when enamel organ acquires definitive shape, indicate importance of these factors in crown morphogenesis of human incisor. Taken together, our data suggest the involvement of IGF-2, IGF-1R, IGF-2R and PTEN in temporo-spatial patterning of basic cellular processes (proliferation, differentiation) during normal tooth development. They are also relevant for improving knowledge of molecular basis of human odontogenesis.

CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in the dental stem cell niche.

Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche.

The enamel knot-like structure is eternally maintained in the apical bud of postnatal mouse incisors.

OBJECTIVES: The boundary where inner and outer enamel epithelia meet is referred to as the cervical loop (CL) in molar tooth germs. In contrast, rodent incisors are continuously growing: the labial side of the teeth is covered with enamel (crown-analog), and the lingual side is covered with cementum (root-analog). These results in the appearance of CL in the frontal sections apart from the apical end. However, many researchers have used the term "labial CL" to indicate the apical end of incisors. DESIGN: This study investigated the gene expression patterns for the enamel knot signaling center in tooth morphogenesis to clarify the difference between "labial CL" and molar CL. We examined the three-dimensional expression patterns of markers for the enamel knot including fibroblast growth factor 4 (Fgf4), sonic hedgehog (Shh), Msx2, and P21 in frontal sections of murine mandibular incisors. RESULTS: Serial frontal sections from the apical end of the postnatal incisor clearly demonstrated the existence of enamel knot-like structures composed of supra-inner enamel epithelium and stellate reticulum in the "labial CL". This structure includes the expression of all examined markers for enamel knots such as Fgf4, Shh, Msx2, and P21. CONCLUSIONS: The molar tooth germ-like structure is maintained indefinitely in the "labial CL". Therefore, the "labial CL" is not equivalent to the molar CL. The existence of an EK-like structure in the apical end of incisors implies that the usage of "labial CL" may be inappropriate for indicating this region. The "apical bud" could be used as an alternative term.

Three-dimensional Imaging Reveals New Compartments and Structural Adaptations in Odontoblasts.

In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes.

Interaction between fibronectin and ß1 integrin is essential for tooth development.

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a ß1 integrin-neutralizing antibody, suggesting that fibronectin-ß1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and ß1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed ß1 integrin conditional knockout mice (Intß1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and ß1 integrin is important for ameloblast differentiation and enamel formation.