S100a6 knockdown promotes the differentiation of dental epithelial cells toward the epidermal lineage instead of the odontogenic lineage.

Tooth development is a complex process involving various signaling pathways and genes. Recent findings suggest that ion channels and transporters, including the S100 family of calcium-binding proteins, may be involved in tooth formation. However, our knowledge in this regard is limited. Therefore, this study aimed to investigate the expression of S100 family members and their functions during tooth formation. Tooth germs were extracted from the embryonic and post-natal mice and the expression of S100a6 was examined. Additionally, the effects of S100a6 knockdown and calcium treatment on S100a6 expression and the proliferation of SF2 cells were examined. Microarrays and single-cell RNA-sequencing indicated that S100a6 was highly expressed in ameloblasts. Immunostaining of mouse tooth germs showed that S100a6 was expressed in ameloblasts but not in the undifferentiated dental epithelium. Additionally, S100a6 was localized to the calcification-forming side in enamel-forming ameloblasts. Moreover, siRNA-mediated S100a6 knockdown in ameloblasts reduced intracellular calcium concentration and the expression of ameloblast marker genes, indicating that S100a6 is associated with ameloblast differentiation. Furthermore, S100a6 knockdown inhibited the ERK/PI3K signaling pathway, suppressed ameloblast proliferation, and promoted the differentiation of the dental epithelium toward epidermal lineage. Conclusively, S100a6 knockdown in the dental epithelium suppresses cell proliferation via calcium and intracellular signaling and promotes differentiation of the dental epithelium toward the epidermal lineage.

Mesoscale structural gradients in human tooth enamel.

The outstanding mechanical and chemical properties of dental enamel emerge from its complex hierarchical architecture. An accurate, detailed multiscale model of the structure and composition of enamel is important for understanding lesion formation in tooth decay (dental caries), enamel development (amelogenesis) and associated pathologies (e.g., amelogenesis imperfecta or molar hypomineralization), and minimally invasive dentistry. Although features at length scales smaller than 100 nm (individual crystallites) and greater than 50 µm (multiple rods) are well understood, competing field of view and sampling considerations have hindered exploration of mesoscale features, i.e., at the level of single enamel rods and the interrod enamel (1 to 10 µm). Here, we combine synchrotron X-ray diffraction at submicrometer resolution, analysis of crystallite orientation distribution, and unsupervised machine learning to show that crystallographic parameters differ between rod head and rod tail/interrod enamel. This variation strongly suggests that crystallites in different microarchitectural domains also differ in their composition. Thus, we use a dilute linear model to predict the concentrations of minority ions in hydroxylapatite (Mg2+ and CO32-/Na+) that plausibly explain the observed lattice parameter variations. While differences within samples are highly significant and of similar magnitude, absolute values and the sign of the effect for some crystallographic parameters show interindividual variation that warrants further investigation. By revealing additional complexity at the rod/interrod level of human enamel and leaving open the possibility of modulation across larger length scales, these results inform future investigations into mechanisms governing amelogenesis and introduce another feature to consider when modeling the mechanical and chemical performance of enamel.

Automated High-Throughput Biological Sex Identification from Archeological Human Dental Enamel Using Targeted Proteomics.

Biological sex is key information for archeological and forensic studies, which can be determined by proteomics. However, the lack of a standardized approach for fast and accurate sex identification currently limits the reach of proteomics applications. Here, we introduce a streamlined mass spectrometry (MS)-based workflow for the determination of biological sex using human dental enamel. Our approach builds on a minimally invasive sampling strategy by acid etching, a rapid online liquid chromatography (LC) gradient coupled to a high-resolution parallel reaction monitoring (PRM) assay allowing for a throughput of 200 samples per day (SPD) with high quantitative performance enabling confident identification of both males and females. Additionally, we developed a streamlined data analysis pipeline and integrated it into a Shiny interface for ease of use. The method was first developed and optimized using modern teeth and then validated in an independent set of deciduous teeth of known sex. Finally, the assay was successfully applied to archeological material, enabling the analysis of over 300 individuals. We demonstrate unprecedented performance and scalability, speeding up MS analysis by 10-fold compared to conventional proteomics-based sex identification methods. This work paves the way for large-scale archeological or forensic studies enabling the investigation of entire populations rather than focusing on individual high-profile specimens. Data are available via ProteomeXchange with the identifier PXD049326.

KDF1 promotes ameloblast differentiation by inhibiting the IKK/IκB/NF-κB axis.

Enamel protects teeth from external irritation and its formation involves sequential differentiation of ameloblasts, a dental epithelial cell. Keratinocyte differentiation factor 1 (KDF1) is important in the development of epithelial tissues and organs. However, the specific role of KDF1 in enamel formation and corresponding regulatory mechanisms are unclear. This study demonstrated that KDF1 was persistently expressed in all stages of ameloblast differentiation, through RNAscope in situ hybridization. KDF1 expression in the mouse ameloblast cell line LS8 was demonstrated via immunofluorescence assay. KDF1 was knocked out in LS8 cells using the CRISPR/Cas-9 system or overexpressed in LS8 cells through lentiviral infection. In vitro ameloblast differentiation induction, quantitative reverse transcription PCR, western blot analysis, and alkaline phosphatase (ALP) assay indicated that knockout or overexpression of KDF1 in LS8 cells decreased or increased the mRNA and protein levels of several key amelogenesis markers, as well as ALP activity. Furthermore, liquid chromatography-mass spectrometry and co-immunoprecipitation analyses revealed that KDF1 can interact with the IKK complex, thereby inhibiting the NF-κB pathway. Suppressing NF-κB activity partially recovered the decreased ameloblast differentiation in LS8 cells induced by KDF1-knockout. This study demonstrated that KDF1 can promote ameloblast differentiation of LS8 cells by inhibiting the IKK/IκB/NF-κB axis, and is a potential target for functional enamel regeneration.

Recombinant human enamelin produced in Escherichia coli promotes mineralization in vitro.

BACKGROUND: Enamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent. RESULTS: In the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml. CONCLUSIONS: These findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.

Seriation of Enzyme-Functionalized Multilayers for the Design of Scalable Biomimetic Mineralized Structures.

Biomimetic hydroxyapatites are widely explored for their potential applications in the repair of mineralized tissues, particularly dental enamel, which is acellular and, thus, not naturally reformed after damage. Enamel is formed with a highly-controlled hierarchical structure, which is difficult to replicate up to the macroscale. A biomimetic approach is thus warranted, based on the same principles that drive biomineralization in vivo. Herein, a strategy for the design of enamel-like architectures is described, utilizing enzymes embedded in polyelectrolyte multilayers to generate inorganic phosphate locally, and provide a favorable chemical environment for the nucleation and growth of minerals. Moreover, a method is proposed to build up seriated mineral layers with scalable thicknesses, continuous mineral growth, and tunable morphology. Results show the outstanding growth of cohesive mineral layers, yielding macroscopic standalone fluoride and/or carbonate-substituted hydroxyapatite materials with comparable crystal structure and composition to native human mineralized tissues. This strategy presents a promising path forward for the biomimetic design of biomineral materials, particularly relevant for restorative applications, with an exquisite level of synthetic control over multiple orders of magnitude.

PMCA Ca2+ clearance in dental enamel cells depends on the magnitude of cytosolic Ca2.

Enamel formation (amelogenesis) is a two-step process whereby crystals partially grow during the secretory stage followed by a significant growth expansion during the maturation stage concurrent with an increase in vectorial Ca2+ transport. This requires tight regulation of cytosolic Ca2+ (c Ca2+ ) concentration in the enamel forming ameloblasts by controlling Ca2+ influx (entry) and Ca2+ extrusion (clearance). Gene and protein expression studies suggest that the plasma membrane Ca2+ -ATPases (PMCA1-4) are likely involved in c Ca2+ extrusion in ameloblasts, yet no functional analysis of these pumps has been reported nor whether their activity changes across amelogenesis. PMCAs have high Ca2+ affinity and low Ca2+ clearance which may be a limiting factor in their contribution to enamel formation as maturation stage ameloblasts handle high Ca2+ loads. We analyzed PMCA function in rat secretory and maturation ameloblasts by blocking or potentiating these pumps. Low/moderate elevations in c Ca2+ measured using the Ca2+ probe Fura-2-AM show that secretory ameloblasts clear Ca2+ faster than maturation stage cells through PMCAs. This process was completely inhibited by an external alkaline (pH 9.0) solution or was significantly delayed by the PMCA blockers vanadate and caloxin 1b1. Eliciting higher c Ca2+ transients via the activation of the ORAI1 Ca2+ channel showed that the PMCAs of maturation ameloblasts were more efficient. Inhibiting PMCAs decreased the rate of Ca2+ influx via ORAI1 but potentiation with forskolin had no effect. Our findings suggest that PMCAs are functional Ca2+ pumps during amelogenesis regulating c Ca2+ upon low and/or moderate Ca2+ stimulus in secretory stage, thus participating in amelogenesis.

Linking African herbivore community enamel isotopes and environments: challenges, opportunities, and paleoecological implications.

Paleoenvironmental reconstructions of fossil sites based on isotopic analyses of enamel typically rely on data from multiple herbivore taxa, with the assumption that this dietary spectrum represents the community's isotopic range and provides insights into local or regional vegetation patterns. However, it remains unclear how representative the sampled taxa are of the broader herbivore community and how well these data correspond to specific ecosystems. Verifying these underlying assumptions is essential to refining the utility of enamel isotopic values for paleoenvironmental reconstructions. This study explores potential links between modern herbivore community carbon isotopic enamel spectra, biome types, and climate in sub-Saharan Africa. This region is one of the most comprehensively isotopically sampled areas globally and is of particular relevance to hominin evolution. Our extensive data compilation reveals that published enamel isotopic data from sub-Saharan Africa typically sample only a small percentage of the taxa documented at most localities and that some biome types (e.g., subtropical savannas) are dramatically overrepresented relative to others (e.g., forests) in these modern data sets. Multiple statistical analyses, including linear models and cluster analyses, revealed weak relationships of associated mammalian herbivore enamel isotopic values, biome type, and climate parameters. These results confound any simple assumptions about how community isotopic profiles map onto specific environments, highlighting the need for more precise strategic approaches in extending isotopic frameworks into the past for paleoecological reconstructions. Developing more refined modern analogs will ultimately allow us to more accurately characterize the isotopic spectra of paleo-communities and link isotopic dietary signatures to specific ecosystems.

Co-exposure to fluoride and sulfur dioxide induces abnormal enamel mineralization in rats via the FGF9-mediated MAPK signaling pathway.

Fluoride (F) and sulfur dioxide (SO2) contamination is recognized as a public health concern worldwide. Our previous research has shown that Co-exposure to F and SO2 can cause abnormal enamel mineralization. Ameloblastin (AMBN) plays a crucial role in the process of enamel mineralization. However, the process by which simultaneous exposure to F and SO2 influences enamel formation by regulating AMBN expression still needs to be understood. This study aimed to establish in vivo and in vitro models of F-SO2 Co-exposure and investigate the relationship between AMBN and abnormal enamel mineralization. By overexpressing/knocking out the Fibroblast Growth Factor 9 (FGF9) gene, we investigated the impact of FGF9-mediated Mitogen-Activated Protein Kinase (MAPK) signaling on AMBN synthesis to elucidate the mechanism underlying the induction of abnormal enamel mineralization by F-SO2 Co-exposure in rats. The results showed that F-SO2 exposure damaged the structure of rat enamel and ameloblasts. When exposed to F or SO2, gradual increases in the protein expression of FGF9 and phosphorylated p38 mitogen-activated protein kinase (p-P38) were observed. Conversely, the protein levels of AMBN, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) were decreased. AMBN expression was significantly correlated with FGF9, p-ERK, and p-JNK expression in ameloblasts. Interestingly, FGF9 overexpression reduced the levels of p-ERK and p-JNK, worsening the inhibitory effect of F-SO2 on AMBN. Conversely, FGF9 knockout increased the phosphorylation of ERK and JNK, partially reversing the F-SO2-induced downregulation of AMBN. Taken together, these findings strongly demonstrate that FGF9 plays a critical role in F-SO2-induced abnormal enamel mineralization by regulating AMBN synthesis through the JNK and ERK pathways.

The flapless approach with and without enamel matrix derivatives for the treatment of intrabony defects: A randomized controlled clinical trial.

AIM: To compare the clinical and radiographic outcomes of flapless procedure alone or in combination with enamel matrix derivatives (EMD) in the treatment of deep intrabony defects. MATERIALS AND METHODS: Forty-six patients re-evaluated after non-surgical therapy were randomly assigned to the test (flapless with EMD) or control group (flapless alone). Clinical measurements were recorded pre-surgery and at 6 and 12 months after surgery, and radiographic measurements were taken pre-surgery and after 12 months. RESULTS: Forty-six patients completed the study. Improvements were observed in both groups at 12 months for mean clinical attachment level (CAL) gain, with significant differences between test (3.9 ± 1.1 mm) and control groups (3.0 ± 1.2) (p = .017). Probing pocket depth (PPD) reduction (4.0 ± 0.7 vs. 3.3 ± 1.4 mm) was also near to statistical significance (p = .051). Also, more sites achieved successful composite outcome measure (final PPD ≤ 4 mm and CAL gain ≥3 mm) for the regenerative treatment in the flapless + EMD group (82.6% vs. 52.2%; p = .028). In terms of radiographic outcomes, EMD yielded a greater defect bone fill than flapless treatment alone (3.0 ± 1.0 mm vs. 1.8 ± 1.5 mm; p < .001). CONCLUSIONS: The additional application of EMD during the flapless procedure for intrabony defects slightly improved clinical and radiographic outcomes. CLINICALTRIALS: gov identification number: NCT05456555.

Dental caries in children and vitamin D deficiency: a narrative review.

Dental caries represents one of the most prevalent health problems in childhood. Numerous studies have assessed that vitamin D deficiency is highly related to dental caries in primary and permanent teeth in children. The aim of this study is to elaborate a narrative review about proposed mechanisms by which vitamin D deficiency interacts with dental caries process in children. Vitamin D deficiency during pregnancy may cause intrauterine enamel defects, and through childhood is accompanied by insufficient activity of antibacterial peptides, decreased saliva secretion, and a low level of calcium in saliva.  Conclusion: In conclusion, vitamin D deficiency would increase the risk of caries in the primary and/or permanent dentition. Relationship between vitamin D deficiency and dental caries is evident enough for vitamin D deficiency to be considered as a risk factor for dental caries in children. Optimal levels of vitamin D throughout pregnancy and childhood may be considered an additional preventive measure for dental caries in the primary and permanent dentition.

The Notch-mediated circuitry in the evolution and generation of new cell lineages: the tooth model.

The Notch pathway is an ancient, evolutionary conserved intercellular signaling mechanism that is involved in cell fate specification and proper embryonic development. The Jagged2 gene, which encodes a ligand for the Notch family of receptors, is expressed from the earliest stages of odontogenesis in epithelial cells that will later generate the enamel-producing ameloblasts. Homozygous Jagged2 mutant mice exhibit abnormal tooth morphology and impaired enamel deposition. Enamel composition and structure in mammals are tightly linked to the enamel organ that represents an evolutionary unit formed by distinct dental epithelial cell types. The physical cooperativity between Notch ligands and receptors suggests that Jagged2 deletion could alter the expression profile of Notch receptors, thus modifying the whole Notch signaling cascade in cells within the enamel organ. Indeed, both Notch1 and Notch2 expression are severely disturbed in the enamel organ of Jagged2 mutant teeth. It appears that the deregulation of the Notch signaling cascade reverts the evolutionary path generating dental structures more reminiscent of the enameloid of fishes rather than of mammalian enamel. Loss of interactions between Notch and Jagged proteins may initiate the suppression of complementary dental epithelial cell fates acquired during evolution. We propose that the increased number of Notch homologues in metazoa enabled incipient sister cell types to form and maintain distinctive cell fates within organs and tissues along evolution.

Effect of combining aminomethacrylate and fluoride against erosive and abrasive challenges on enamel and dentin.

This study evaluated the effect of solutions containing aminomethacrylate copolymer (AA) and sodium fluoride (F; 225 ppm F-) or fluoride plus stannous chloride (FSn; 225 ppm F-, 800 ppm Sn2+) against enamel and dentin erosion/abrasion. Solutions F, FSn, AA, F+AA, FSn+AA, and deionized water as negative control were tested. Bovine enamel and dentin specimens (n = 13/solution/substrate) underwent a set of erosion-abrasion cycles (0.3% citric acid [5 min, 4×/day], human saliva [1 h, 4×/day], brushing [15 s, 2×/day], and treatments [2 min, 2×/day]) for each of five days. Initial enamel erosion was evaluated using Knoop microhardness after the first and second acid challenge on day 1, and surface loss with profilometry after day 5. KOH-soluble fluoride was assessed. Data were analyzed with ANOVA/Tukey tests. The combination of fluoride and AA resulted in higher protection against enamel erosion, whereas this was not the case for the combination of AA and FSn. All treatments protected against enamel and dentin loss. The lowest surface loss values were observed with F+AA and FSn+AA. The polymer did not significantly influence the KOH-soluble fluoride formation on enamel/dentin specimens. The aminomethacrylate copolymer effectively enhanced the efficacy of sodium fluoride against initial erosion and improved the control of enamel and dentin wear of F and FSn solutions.

Deep dental phenotyping and a novel FAM20A variant in patients with amelogenesis imperfecta type IG.

OBJECTIVES: To identify etiologic variants and perform deep dental phenotyping in patients with amelogenesis imperfecta (AI). METHODS: Three patients of two unrelated families were evaluated. Genetic variants were investigated by exome and Sanger sequencing. An unerupted permanent third molar (AI1) from Patient1 and a deciduous first molar (AI2) from Patient2, along with three tooth-type matched controls for each were characterized. RESULTS: All three patients harbored biallelic pathogenic variants in FAM20A, indicating AI1G. Of the four identified variants, one, c.1231C > T p.(Arg411Trp), was novel. Patient1 possessed the largest deletion, 7531 bp, ever identified in FAM20A. In addition to hypoplastic enamel, multiple impacted teeth, intrapulpal calcification, pericoronal radiolucencies, malocclusion, and periodontal infections were found in all three patients, gingival hyperplasia in Patient1 and Patient2, and alveolar bone exostosis in Patient3. Surface roughness was increased in AI1 but decreased in AI2. Decreased enamel mineral density, hardness, and elastic modulus were observed in AI1 enamel and dentin and AI2 dentin, along with decreased phosphorus, increased carbon, and increased calcium/phosphorus and carbon/oxygen ratios. Severely collapsed enamel rods and disorganized dentin-enamel junction were observed. CONCLUSIONS: We report a novel FAM20A variant and, for the first time, the defective mineral composition and physical/mechanical properties of AI1G teeth.

Efficacy of OFD with EMD for treatment of periodontal defects: A systematic review and meta-analysis.

OBJECTIVES: In order to enhance clinical improvement of periodontal defects, the addition of enamel matrix derivatives (EMD) to open flap debridement (OFD) has been investigated. The aim of this systematic review is to figure out whether such a combination, in comparison to the treatment with OFD alone has some effects on the following outcomes: clinical attachment level gain, probing depth reduction, and gingival recessions increase. METHODS: Electronic databases (PubMed, Embase, Web of Science, and Cochrane) were searched for randomized controlled trials in humans addressing the use of a combination of OFD and EMD versus a control group with OFD alone for the treatment of periodontal defects, with a minimum of 6 months of follow-up; meta-analysis and trial sequential analysis were then performed. RESULTS: From a total of 204 records screened by title and abstract, 13 studies were read full-text and eight out of them included in the meta-analysis. Some significant differences have been demonstrated both for clinical attachment level gain and probing depth reduction between test and control groups. CONCLUSIONS: In the treatment of periodontal defects, the addition of EMD to OFD seems to be beneficial in terms of clinical attachment level gain, probing depth reduction, promoting periodontal regeneration. However, such results should be considered with caution because of the small number of studies included in the meta-analysis and their heterogeneity.

Optical penetration models for practical prediction of femtosecond laser ablation of dental hard tissue.

OBJECTIVES: To develop and practically test high-precision femtosecond laser ablation models for dental hard tissue that are useful for detailed planning of automated laser dental restorative treatment. METHODS: Analytical models are proposed, derived, and demonstrated for practical calculation of ablation rates, ablation efficiency and ablated morphology of human dental enamel and dentin using femtosecond lasers. The models assume an effective optical attenuation coefficient for the irradiated material. To achieve ablation, it is necessary for the local energy density of the attenuated pulse in the hard tissue to surpass a predefined threshold that signifies the minimum energy density required for material ionization. A 1029 nm, 40 W carbide 275 fs laser was used to ablate sliced adult human teeth and generate the data necessary for testing the models. The volume of material removed, and the shape of the ablated channel were measured using optical profilometry. RESULTS: The models fit with the measured ablation efficiency curve against laser fluence for both enamel and dentin, correctly capturing the fluence for optimum ablation and the volume of ablated material per pulse. The detailed shapes of a 400-micrometer wide channel and a single-pulse width channel are accurately predicted using the superposition of the analytical result for a single pulse. CONCLUSIONS: The findings have value for planning automated dental restorative treatment using femtosecond lasers. The measurements and analysis give estimates of the optical properties of enamel and dentin irradiated with an infrared femtosecond laser at above-threshold fluence and the proposed models give insight into the physics of femtosecond laser processing of dental hard tissue.

Developmental Defects of Enamel: A Bibliometric Analysis of the Top 100 Most-Cited Papers.

BACKGROUND: Developmental defects of enamel (DDE) are a result of disturbances during formation and maturation of the enamel. Evaluating the most-cited DDE papers can provide important tools that point to the gaps and strengths of this important topic in dentistry. SUMMARY: This bibliometric study analyzed the 100 most-cited papers on DDE. Using a combined keyword search strategy, the 100 most-cited papers were selected in the Web of Science Core Collection. Papers that addressed any type of DDE were included. The extracted data were title, number of citations, study theme, authorship, journal, type of DDE, type of dentition (primary or permanent), type of diagnosis, study design, year, and country of publication. The bibliometric networks were generated through VOSviewer software. The 100 papers had a range from 78 to 459 citations. The main themes of studies were etiopathogenesis (53%), prevalence and incidence (22%), and diagnosis (8%). The authors with the highest number of citations were Goodman AH and Rose JC (459 citations). Most articles were published in dental journals (47%). The most studied types of DDE were fluorosis and amelogenesis imperfecta in the permanent dentition (47%). Observational (24%) and non-systematic reviews (24%) were the most common study designs and ranged from 1977 to 2019. The country with the highest number of publications was the USA (41%). KEY MESSAGES: Most of the top 100 DDE papers were about fluorosis and amelogenesis imperfecta, with top papers from three continents with English as the native language. This topic is of great importance in dentistry, and the need for further studies is highlighted, especially regarding the diagnosis and treatment of some DDEs.

Hemoglobin Protects Enamel against Intrinsic Enamel Erosive Demineralization.

INTRODUCTION: This study investigated the changes in the acquired enamel pellicle (AEP) proteome when this integument is formed in vivo after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), and a statherin-derived peptide (StN15), or their combination and then exposed to an intrinsic acid challenge. The effectiveness of these treatments in preventing intrinsic erosion was also evaluated. METHODS: Ten volunteers, after prophylaxis, in 5 crossover phases, rinsed with the following solutions (10 mL, 1 min): control (deionized water-H2O) - group 1, 0.1 mg/mL CaneCPI-5 - group 2, 1.0 mg/mL HB - group 3, 1.88 × 10-5M StN15 - group 4, or a blend of these - group 5. Following this, AEP formation occurred (2 h) and an enamel biopsy (10 µL, 0.01 m HCl, pH 2.0, 10 s) was conducted on one incisor. The biopsy acid was then analyzed for calcium (Arsenazo method). The vestibular surfaces of the other teeth were treated with the same acid. Acid-resistant proteins in the residual AEP were then collected and analyzed quantitatively via proteomics. RESULTS: Compared to control, treatment with the proteins/peptide, mixed or isolated, markedly enhanced acid-resistant proteins in the AEP. Notable increases occurred in pyruvate kinase PKM (11-fold, CaneCPI-5), immunoglobulins and submaxillary gland androgen-regulated protein 3B (4-fold, StN15), Hb, and lysozyme C (2-fold, StN15). Additionally, a range of proteins not commonly identified in the AEP but known to bind calcium or other proteins were identified in groups treated with the tested proteins/peptide either in isolation or as a mixture. The mean (SD, mM) calcium concentrations released from enamel were 3.67 ± 1.48a, 3.11 ± 0.72a, 1.94 ± 0.57b, 2.37 ± 0.90a, and 2.38 ± 0.45a for groups 1-5, respectively (RM-ANOVA/Tukey, p < 0.05). CONCLUSIONS: Our findings demonstrate that all treatments, whether using a combination of proteins/peptides or in isolation, enhanced acid-resistant proteins in the AEP. However, only HB showed effectiveness in protecting against intrinsic erosive demineralization. These results pave the way for innovative preventive methods against intrinsic erosion, using "acquired pellicle engineering" techniques.

Characterization of a Novel TiF4 Inclusion Complex and in vitro Evaluation of Its Effect on Inhibiting Enamel Demineralization.

INTRODUCTION: Titanium tetrafluoride (TiF4) is an anticariogenic agent with high remineralizing potential. However, the acidic pH of TiF4 solution can limit its clinical application. The present study aimed to prepare and characterize a new TiF4-dendrimer inclusion complex and evaluate its ability to inhibit enamel demineralization under pH cycling conditions. METHODS: PEG-citrate dendrimer and TiF4-dendrimer inclusion complex were synthesized and their molecular structures were evaluated using Fourier-transform Infrared Spectroscopy (FTIR), Hydrogen Nuclear Magnetic Resonance (HNMR), and Liquid Chromatography-Mass Spectrometry (LC-MS) tests. Forty-eight enamel samples were prepared and randomly divided into four groups: distilled water (negative control), TiF4 solution (T), dendrimer solution (D), and TiF4-dendrimer solution (TD). The microhardness of the samples was measured initially. Next, the samples underwent pH cycling, were exposed to the solutions, the microhardness was measured again, and microhardness loss was calculated. EDX analysis was performed on the surface and cross-sectional segments of the samples. RESULTS: The microhardness loss was significantly higher in control (-65.1 ± 6.0) compared to other groups. No significant difference was observed between T (-47.9 ± 5.6) and D (-41.7 ± 12.0) and also D and TD (-40.5 ± 9.4) in this regard. Microhardness loss was significantly higher in T compared to TD group. The TD samples showed similar fluoride and titanium content in both surface and subsurface regions, while the T group had higher concentrations in the surface region. Moreover, the TD solution had a higher pH of 3.4 compared to the T solution's pH of 1.1. CONCLUSION: No significant difference was observed between the efficacy of TiF4-dendrimer and TiF4 solution in inhibiting demineralization while TiF4-dendrimer solution had the added advantage of having a higher pH.

Acquired Pellicle and Biofilm Engineering by Rinsing with Hemoglobin Solution.

INTRODUCTION: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the "acquired pellicle engineering" concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization. OBJECTIVE: Combining in vivo and in vitro proof-of-concept protocols, we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization. METHODS: In the in vivo studies, 10 volunteers, in 2 independent experiments, rinsed (10 mL,1 min) with deionized water-negative control or 1.0 mg/mL Hb. The AEP and biofilm formed along 2 or 3 h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA next-generation sequencing (NGS). In in vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with (1) phosphate-buffered solution (PBS), (2) 0.12% chlorhexidine, (3) 500 ppm NaF, (4) 1.0 mg/mL Hb, (5) 2.0 mg/mL Hb, and (6) 4.0 mg/mL Hb. The biofilm was cultivated for 5 days. Resazurin, colony forming units (CFU), and transversal microradiography were performed. RESULTS: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU, and enamel demineralization compared to PBS. CONCLUSION: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering.