Ingenious Architecture and Coloration Generation in Enamel of Rodent Teeth.

Teeth exemplify architectures comprising an interplay of inorganic and organic constituents, resulting in sophisticated natural composites. Rodents (Rodentia) showcase extraordinary adaptations, with their continuously growing incisors surpassing human teeth in functional and structural optimizations. In this study, employing state-of-the-art direct atomic-scale imaging and nanoscale spectroscopies, we present compelling evidence that the release of material from ameloblasts and the subsequent formation of iron-rich enamel and surface layers in the constantly growing incisors of rodents are complex orchestrated processes, intricately regulated and independent of environmental factors. The synergistic fusion of three-dimensional tomography and imaging techniques of etched rodent́s enamel unveils a direct correlation between the presence of pockets infused with ferrihydrite-like material and the acid resistant properties exhibited by the iron-rich enamel, fortifying it as an efficient protective shield. Moreover, observations using optical microscopy shed light on the role of iron-rich enamel as a microstructural element that acts as a path for color transmission, although the native color remains indistinguishable from that of regular enamel, challenging the prevailing paradigms. The redefinition of "pigmented enamel" to encompass ferrihydrite-like infusion in rodent incisors reshapes our perception of incisor microstructure and color generation. The functional significance of acid-resistant iron-rich enamel and the understanding of the underlying coloration mechanism in rodent incisors have far-reaching implications for human health, development of potentially groundbreaking dental materials, and restorative dentistry. These findings enable the creation of an entirely different class of dental biomaterials with enhanced properties, inspired by the ingenious designs found in nature.

Single-cell transcriptome profiling reveals distinct expression patterns among genes in the mouse incisor dental pulp.

SOX transcription factors play key roles in cell differentiation and cell fate determination during development. Using single-cell RNA-sequencing data, we examined the expression profiles of Sox genes in the mouse incisor dental pulp. Our analysis showed that Sox4, Sox5, Sox9, Sox11, and Sox12 are mainly expressed in mesenchymal stem/stromal cells (MSCs) representing osteogenic cells at different stages of differentiation. We found that in several MSCs, Sox genes co-expressed with regulatory genes such as Sp7, Satb2, Msx1, Snai2, Dlx1, Twist2, and Tfap2a. In addition, Sox family genes colocalized with Runx2 and Lef1, which are highly enriched in MSCs undergoing osteoblast differentiation. A protein interaction network analysis uncovered that CREBBP, CEBPB, TLE1, TWIST1, and members of the HDAC and SMAD families are interacting partners of RUNX2 and LEF1 during skeletal development. Collectively, the distinct expression patterns of the SOX transcription factors suggest that they play essential regulatory roles in directing lineage-specific gene expression during differentiation of MSCs.

Molecular profiling of the vestibular lamina highlights a key role for Hedgehog signalling.

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

A novel junctional epithelial cell line, mHAT-JE01, derived from incisor epithelial cells.

OBJECTIVES: Junctional epithelium (JE) connects the tooth surface and gingival epithelium and adheres directly to the tooth enamel. JE plays an important role as a barrier preventing the invasion of exogenous bacteria and substances. However, the cellular characteristics of this epithelium have not been adequately described, because no useful in vitro experimental model exists for JE. METHODS: We generated a novel JE cell line, mHAT-JE01, using naturally immortalized dental epithelium derived from incisor labial cervical cells and by selecting cells that adhered to apatite. mHAT-JE01 was characterized by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction and compared with the gingival epithelial cell line, mOE-PE01. RESULTS: The mHAT-JE01 cells had a higher capacity for producing JE-specific markers than oral mucous epithelial cells. In addition, the presence of lipopolysaccharides from Porphyromonas gingivalis downregulated the expression of JE protein markers in mHAT-JE01 cells. CONCLUSIONS: This cell line is stable and presents the opportunity to characterize JE efficiently, which is essential for the prevention and treatment of periodontal disease.

Klf4 haploinsufficiency in Sp7+ lineage leads to underdeveloped mandibles and insufficient elongation of mandibular incisor.

The mandible is an important component of the craniofacial bones, whose development is regulated by complex molecular networks and involves the well-coordinated development of the bone, cartilage, and teeth. Previously, we demonstrated that Krüppel-like factor 4 (KLF4) promoted dentinogenesis and osteogenesis, but it was enigmatic whether Klf4 participated in the development of the mandible. In this study, the Sp7-Cre; Klf4f/+ mice exhibited underdeveloped mandibles and insufficient elongation of the mandibular incisor when compared with Klf4f/+ and Sp7-Cre mice. Moreover, morphological and molecular analysis showed that the alveolar bone mass was significantly decreased in KLF4 deficient mice, accompanied by reduced expression of osteoblast-related genes. Meanwhile, the KLF4 deficient mice had decreased expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) and no significant change of osteoprotegerin (OPG) in the alveolar bone near the mandibular incisor. Simultaneously, the osteoclastogenesis in the alveolar bone of KLF4 deficient mice was attenuated, which was demonstrated by a diminished number of tartrate-resistant acid phosphatase positive (TRAP+), matrix metallopeptidase 9 positive (MMP9+), and cathepsin K positive (CTSK+) multinucleated osteoclasts, respectively. Collectively, our study suggested that Klf4 participated in mandibular development, and Klf4 in Sp7+ lineage affected osteogenesis directly and osteoclastogenesis indirectly.

The chromatin accessibility landscape in the dental pulp of mouse molars and incisors.

The dental pulp is a promising source of progenitor cells for regenerative medicine. The natural function of dental pulp is to produce odontoblasts to generate reparative dentin. Stem cells within the pulp tissue originate from the migrating neural crest cells and possess mesenchymal stem cell properties with the ability to differentiate into multiple lineages. To elucidate the transcriptional control mechanisms underlying cell fate determination, we compared the transcriptome and chromatin accessibility in primary dental pulp tissue derived from 5-6-day-old mice. Using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq), we correlated gene expression with chromatin accessibility. We found that the majority of ATAC-seq peaks were concentrated at genes associated with development and cell differentiation. Most of these genes were highly expressed in the mouse dental pulp. Surprisingly, we uncovered a group of genes encoding master transcription factors that were not expressed in the dental pulp but retained open chromatin states. Within this group, we identified key developmental genes important for specification of the neural crest, adipocyte, neural, myoblast, osteoblast and hepatocyte lineages. Collectively, our results uncover a complex relationship between gene expression and the chromatin accessibility landscape in the mouse dental pulp.

Sox2 controls asymmetric patterning of ameloblast lineage commitment by regulation of FGF signaling in the mouse incisor.

Mouse incisors are covered by enamel only on the labial side and the lingual side is covered by dentin without enamel. This asymmetric distribution of enamel makes it possible to be abrased on the lingual side, generating the sharp cutting edge of incisor on the labial side. The abrasion of mouse incisors is compensated by the continuous growth throughout life. Epithelium stem cells responsible for its continuous growth are reported to localize within the labial cervical loop. The transcription factor Sox2 plays important roles in the maintenance of stem cell pluripotency and organ formation. We previously found that Sox2 mainly expressed in the dental epithelium. Besides, Sox2 has been reported to be a dental epithelium stem cell marker in the incisor. However, the exact mechanism of Sox2 controlling amelogenesis is still not quite clearly elucidated. Here we report that conditional deletion of Sox2 in the dental epithelium using Shhcre caused impaired ameloblast differentiation in the labial side and induced ectopic ameloblast-like cell differentiation on the lingual side. Abnormal FGF gene expression was detected by RNAscope in situ hybridization in the mutant incisor. Collectively, we speculate that asymmetric ameloblast lineage commitment of mouse incisor might be regulated by Sox2 through FGF signaling.

DSPP dosage affects tooth development and dentin mineralization.

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

Distinct Expression Patterns of Cxcl12 in Mesenchymal Stem Cell Niches of Intact and Injured Rodent Teeth.

Specific stem cell populations within dental mesenchymal tissues guarantee tooth homeostasis and regeneration throughout life. The decision between renewal and differentiation of stem cells is greatly influenced by interactions with stromal cells and extracellular matrix molecules that form the tissue specific stem cell niches. The Cxcl12 chemokine is a general marker of stromal cells and plays fundamental roles in the maintenance, mobilization and migration of stem cells. The aim of this study was to exploit Cxcl12-GFP transgenic mice to study the expression patterns of Cxcl12 in putative dental niches of intact and injured teeth. We showed that endothelial and stromal cells expressed Cxcl12 in the dental pulp tissue of both intact molars and incisors. Isolated non-endothelial Cxcl12+ dental pulp cells cultured in different conditions in vitro exhibited expression of both adipogenic and osteogenic markers, thus suggesting that these cells possess multipotent fates. Taken together, our results show that Cxcl12 is widely expressed in intact and injured teeth and highlight its importance as a key component of the various dental mesenchymal stem cell niches.

Constitutive activation of ß-catenin in odontoblasts induces aberrant pulp calcification in mouse incisors.

During dentin formation, odontoblast polarization ensures that odontoblasts directionally secrete dentin matrix protein, leading to tubular dentin formation; however, little is known about the major features and regulatory mechanisms of odontoblast polarization. In a study of epithelial cell polarization, ß-catenin was shown to serve as a structural component of cadherin-based adherens junctions to initiate cell polarity. However, the role of ß-catenin in odontoblast polarization has not been well investigated. In this study, we explored whether ß-catenin participated in odontoblast polarization to regulate the secretion of mineralization proteins. We established Col1-CreErt2; ß-catenin exon3fl/fl (CA-ß-catenin) mice, which constitutively activate ß-catenin in odontoblasts. CA-ß-catenin mice exhibited disorganization and depolarization of incisor odontoblasts. Moreover, the incisor dentin was hypomineralized, and ectopic calcification was found in mouse incisor pulp. In addition, by constitutive activation of ß-catenin, the expression levels of the core polarity molecule Cdc42 and its downstream polarity protein complex Par3-Par6-aPKC were decreased in the incisors of CA-ß-catenin mice. These findings suggest that ß-catenin plays an essential role in dentin formation by regulating odontoblast polarization.

Anomalous incisor morphology indicates tissue-specific roles for Tfap2a and Tfap2b in tooth development.

Mice possess two types of teeth that differ in their cusp patterns; incisors have one cusp and molars have multiple cusps. The patterning of these two types of teeth relies on fine-tuning of the reciprocal molecular signaling between dental epithelial and mesenchymal tissues during embryonic development. The AP-2 transcription factors, particularly Tfap2a and Tfap2b, are essential components of such epithelial-mesenchymal signaling interactions that coordinate craniofacial development in mice and other vertebrates, but little is known about their roles in the regulation of tooth development and shape. Here we demonstrate that incisors and molars differ in their temporal and spatial expression of Tfap2a and Tfap2b. At the bud stage, Tfap2a is expressed in both the epithelium and mesenchyme of the incisors and molars, but Tfap2b expression is restricted to the molar mesenchyme, only later appearing in the incisor epithelium. Tissue-specific deletions show that loss of the epithelial domain of Tfap2a and Tfap2b affects the number and spatial arrangement of the incisors, notably resulting in duplicated lower incisors. In contrast, deletion of these two genes in the mesenchymal domain has little effect on tooth development. Collectively these results implicate epithelial expression of Tfap2a and Tfap2b in regulating the extent of the dental lamina associated with patterning the incisors and suggest that these genes contribute to morphological differences between anterior (incisor) and posterior (molar) teeth within the mammalian dentition.

Oxygen regulates epithelial stem cell proliferation via RhoA-actomyosin-YAP/TAZ signal in mouse incisor.

Stem cells are maintained in specific niches that strictly regulate their proliferation and differentiation for proper tissue regeneration and renewal. Molecular oxygen (O2) is an important component of the niche microenvironment, but little is known about how O2 governs epithelial stem cell (ESC) behavior. Here, we demonstrate that O2 plays a crucial role in regulating the proliferation of ESCs using the continuously growing mouse incisors. We have revealed that slow-cycling cells in the niche are maintained under relatively hypoxic conditions compared with actively proliferating cells, based on the blood vessel distribution and metabolic status. Mechanistically, we have demonstrated that, during hypoxia, HIF1α upregulation activates the RhoA signal, thereby promoting cortical actomyosin and stabilizing the adherens junction complex, including merlin. This leads to the cytoplasmic retention of YAP/TAZ to attenuate cell proliferation. These results shed light on the biological significance of blood-vessel geometry and the signaling mechanism through microenvironmental O2 to orchestrate ESC behavior, providing a novel molecular basis for the microenvironmental O2-mediated stem cell regulation during tissue development and renewal.

Epithelial loss of mitochondrial oxidative phosphorylation leads to disturbed enamel and impaired dentin matrix formation in postnatal developed mouse incisor.

The formation of dentin and enamel matrix depends on reciprocal interactions between epithelial-mesenchymal cells. To assess the role of mitochondrial function in amelogenesis and dentinogenesis, we studied postnatal incisor development in K320E-TwinkleEpi mice. In these mice, a loss of mitochondrial DNA (mtDNA), followed by a severe defect in the oxidative phosphorylation system is induced specifically in Keratin 14 (K14+) expressing epithelial cells. Histochemical staining showed severe reduction of cytochrome c oxidase activity only in K14+ epithelial cells. In mutant incisors, H&E staining showed severe defects in the ameloblasts, in the epithelial cells of the stratum intermedium and the papillary cell layer, but also a disturbed odontoblast layer. The lack of amelogenin in the enamel matrix of K320E-TwinkleEpi mice indicated that defective ameloblasts are not able to form extracellular enamel matrix proteins. In comparison to control incisors, von Kossa staining showed enamel biomineralization defects and dentin matrix impairment. In mutant incisor, TUNEL staining and ultrastructural analyses revealed differentiation defects, while in hair follicle cells apoptosis is prevalent. We concluded that mitochondrial oxidative phosphorylation in epithelial cells of the developed incisor is required for Ca2+ homeostasis to regulate the formation of enamel matrix and induce the differentiation of ectomesenchymal cells into odontoblasts.

EMILIN proteins are novel extracellular constituents of the dentin-pulp complex.

Odontoblasts and pulp stroma cells are embedded within supramolecular networks of extracellular matrix (ECM). Fibrillin microfibrils and associated proteins are crucial constituents of these networks, serving as contextual scaffolds to regulate tissue development and homeostasis by providing both structural and mechanical properties and sequestering growth factors of the TGF-ß superfamily. EMILIN-1, -2, and -3 are microfibril-associated glycoproteins known to modulate cell behaviour, growth factor activity, and ECM assembly. So far their expression in the various cells of the dentin-pulp complex during development, in the adult stage, and during inflammation has not been investigated. Confocal immunofluorescence microscopy and western blot analysis of developing and adult mouse molars and incisors revealed an abundant presence of EMILINs in the entire dental papilla, at early developmental stages. Later in development the signal intensity for EMILIN-3 decreases, while EMILIN-1 and -2 staining appears to increase in the pre-dentin and in the ECM surrounding odontoblasts. Our data also demonstrate new specific interactions of EMILINs with fibulins in the dentin enamel junction. Interestingly, in dentin caries lesions the signal for EMILIN-3 was significantly increased in inflamed odontoblasts. Overall our findings point for the first time to a role of EMILINs in dentinogenesis, pulp biology, and inflammation.

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.

Saliva proteomic patterns in patients with molar incisor hypomineralization.

Molar incisor hypomineralization (MIH) is an endemic pediatric disease with an unclear pathogenesis. Considering that saliva controls enamel remineralization and that MIH is associated with higher saliva flow rate, we hypothesized that the protein composition of saliva is linked to disease. To test this, we enrolled 5 children aged 6-14 years with MIH showing at least one hypersensitive molar and 5 caries-free children without hypomineralization. Saliva samples were subjected to proteomic analysis followed by protein classification in to biological pathways. Among 618 salivary proteins identified with high confidence, 88 proteins were identified exclusively in MIH patients and 16 proteins in healthy controls only. Biological pathway analysis classified these 88 patient-only proteins to neutrophil-mediated adaptive immunity, the activation of the classical pathway of complement activation, extracellular matrix degradation, heme scavenging as well as glutathione -and drug metabolism. The 16 controls-only proteins were associated with adaptive immunity related to platelet degranulation and the lysosome. This report suggests that the proteaneous composition of saliva is affected in MIH patients, reflecting a catabolic environment which is linked to inflammation.

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.

ATG7 is essential for secretion of iron from ameloblasts and normal growth of murine incisors during aging.

The incisors of rodents comprise an iron-rich enamel and grow throughout adult life, making them unique models of iron metabolism and tissue homeostasis during aging. Here, we deleted Atg7 (autophagy related 7) in murine ameloblasts, i.e. the epithelial cells that produce enamel. The absence of ATG7 blocked the transport of iron from ameloblasts into the maturing enamel, leading to a white instead of yellow surface of maxillary incisors. In aging mice, lack of ATG7 was associated with the growth of ectopic incisors inside severely deformed primordial incisors. These results suggest that 2 characteristic features of rodent incisors, i.e. deposition of iron on the enamel surface and stable growth during aging, depend on autophagic activity in ameloblasts. Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; CMV: cytomegalovirus; Cre: Cre recombinase; CT: computed tomography; FTH1: ferritin heavy polypeptide 1; GFP: green fluorescent protein; KRT5: keratin 5; KRT14: keratin 14; LGALS3: lectin, galactose binding, soluble 3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NCOA4: nuclear receptor coactivator 4; NRF2: nuclear factor, erythroid 2 like 2; SQSTM1: sequestosome 1.

Spontaneous Development of Dental Dysplasia in Aged Parp-1 Knockout Mice.

Poly(ADP-ribose) polymerase (Parp)-1 catalyzes polyADP-ribosylation using NAD+ and is involved in the DNA damage response, genome stability, and transcription. In this study, we demonstrated that aged Parp-1-/- mouse incisors showed more frequent dental dysplasia in both ICR/129Sv mixed background and C57BL/6 strain compared to aged Parp-1+/+ incisors, suggesting that Parp-1 deficiency could be involved in development of dental dysplasia at an advanced age. Computed tomography images confirmed that dental dysplasia was observed at significantly higher incidences in Parp-1-/- mice. The relative calcification levels of Parp-1-/- incisors were higher in both enamel and dentin (p < 0.05). Immunohistochemical analysis revealed (1) Parp-1 positivity in ameloblasts and odontoblasts in Parp-1+/+ incisor, (2) weaker dentin sialoprotein positivity in dentin of Parp-1-/- incisor, and (3) bone sialoprotein positivity in dentin of Parp-1-/- incisor, suggesting ectopic osteogenic formation in dentin of Parp-1-/- incisor. These results indicate that Parp-1 deficiency promotes odontogenic failure in incisors at an advanced age. Parp-1 deficiency did not affect dentinogenesis during the development of mice, suggesting that Parp-1 is not essential in dentinogenesis during development but is possibly involved in the regulation of continuous dentinogenesis in the incisors at an advanced age.

Effect of dental bleaching on pulp oxygen saturation in maxillary central incisors - a randomized clinical trial.

OBJECTIVE: To assess pulp oxygen saturation levels (SaO2) in maxillary central incisors after dental bleaching. MATERIALS AND METHODS: 80 participants (160 teeth) were randomly allocated to four groups: G1 In-office bleaching with two applications of 35% hydrogen peroxide (HP) (20 minutes), followed by at-home bleaching with 10% carbamide peroxide (CP) (2 hours/day for 16 days); G2 - Same protocol as G1, plus desensitizing toothpaste; G3 - In-office bleaching with 35% HP and one application of placebo gel (20 minutes), followed by at-home bleaching with 10% CP (2 hours/day for 16 days); and G4 - Same protocol as G3, plus desensitizing toothpaste. Pulp SaO2 levels were measured before (T0) and immediately after (T1) in-office bleaching; on the 5th (T2), 8th (T3), 12th (T4), and 16th days of at-home bleaching (T5); and on the 7th (T6) and 30th (T7) days. Mean (SD) pulp SaO2 levels were compared within groups by generalized estimating equations (GEE) and Student's t-test (P<0.05). RESULTS: Mean pulp SaO2 at T0 was 84.29% in G1, 84.38% in G2, 84.79% in G3, and 85.83% in G4. At T1, these values decreased to 81.96%, 82.06%, 82.19%, and 81.15% in G1, G2, G3, and G4 respectively, with significant difference in G4 (P<0.05). During home bleaching, pulp SaO2 levels varied in all groups, with 86.55%, 86.60%, 85.71%, and 87.15% means at T7 for G1, G2, G3, and G4, respectively; G2 presented significant difference (P<0.05). CONCLUSIONS: Pulp SaO2 level in maxillary central incisors was similar at baseline, reducing immediately after in-office bleaching, regardless of using desensitizing toothpaste and increasing at 30 days after dental bleaching.