Tubular Nanoclay-Enhanced Calcium Phosphate Mineralization and Assembly to Impart High Stiffness and Antimicrobial Properties.

Achieving superior mechanical properties of composite materials in artificially engineered materials is a great challenge due to technical bottlenecks in the size and morphological modulation of inorganic nanominerals. Hence, a "bioprocess-inspired fabrication" is proposed to create multilayered organic-inorganic columnar structures. The sequential assembly of halloysite nanotubes (HNTs), polyelectrolytes (PAAs), and calcium phosphates (CaPs) results in organic-inorganic structures. PAA plays a crucial role in controlling the formation of CaP, guiding it into amorphous particles with smaller nanosizes. The introduction of HNT induces the assembly and maturation of CaP-PAA, leading to the formation of a highly crystalline hydroxyapatite. Poly(vinyl alcohol) was then woven into HNT-encapsulated hydroxyapatite nanorods, resulting in composite materials with basic hierarchical structures across multiple scales. The fabricated composite exhibits exceptional hardness (4.27 ± 0.33 GPa) and flexural strength (101.25 ± 1.72 MPa), surpassing those of most previously developed biological hard tissue materials. Additionally, the composite demonstrates effective antibacterial properties and corrosion resistance, attributed to the dense crystalline phase of CaP. This innovative approach showcases the potential of clay minerals, particularly HNT, in the advancement of biomaterial design. The outstanding mechanical and antimicrobial properties of clay-based composites make them a promising candidate for applications in hard tissue repair, offering versatility in biomedicine and engineering.

The composition of the dental pellicle: an updated literature review.

Background: The dental pellicle is a thin layer of up to several hundred nm in thickness, covering the tooth surface. It is known to protect the teeth from acid attacks through its selective permeability and it is involved in the remineralization process of the teeth. It functions also as binding site and source of nutrients for bacteria and conditioning biofilm (foundation) for dental plaque formation. Methods: For this updated literature review, the PubMed database was searched for the dental pellicle and its composition. Results: The dental pellicle has been analyzed in the past years with various state-of-the art analytic techniques such as high-resolution microscopic techniques (e.g., scanning electron microscopy, atomic force microscopy), spectrophotometry, mass spectrometry, affinity chromatography, enzyme-linked immunosorbent assays (ELISA), and blotting-techniques (e.g., western blot). It consists of several different amino acids, proteins, and proteolytic protein fragments. Some studies also investigated other compounds of the pellicle, mainly fatty acids, and carbohydrates. Conclusions: The dental pellicle is composed mainly of different proteins, but also fatty acids, and carbohydrates. Analysis with state-of-the-art analytical techniques have uncovered mainly acidic proline-rich proteins, amylase, cystatin, immunoglobulins, lysozyme, and mucins as main proteins of the dental pellicle. The pellicle has protective properties for the teeth. Further research is necessary to gain more knowledge about the role of the pellicle in the tooth remineralization process.

In vitro dentin permeability and tubule occlusion of experimental in-office desensitizing materials.

OBJECTIVES: This study investigates the dentin permeability (by hydraulic conductance) and tubule occlusion (by confocal and scanning electron microscopies) of in-office desensitizing materials. MATERIALS AND METHODS: Bovine dentin blocks were immersed in EDTA to open dentinal tubules. Placebo varnish (PLA), fluoride varnish (FLU), NaF 5% + 5% nanoparticulate sodium trimetaphosphate varnish (TMP), universal adhesive system (SBU), S-PRG filler varnish (SPRG), Biosilicate (BIOS), and amelotin (AMTN) solution were the materials tested. After application, the specimens underwent an erosive-abrasive challenge. Dentin permeability was evaluated at T0 (initial), T1 (after treatment), and T2 (after challenge). Confocal and scanning electron microscopy (SEM) were used to evaluate, respectively, length and number of dentinal tubule occlusions and opened dentinal tubules, after challenge. Permeability and SEM data were analyzed by two-way repeated measures ANOVA and Tukey's tests. Confocal data were analyzed by one-way ANOVA, Tukey's test, and Kruskal-Wallis and Dunn's tests. Spearman and Pearson's correlation tests were also used. Significance level was set at 5%. RESULTS: At T1, the AMTN group showed the lowest permeability value, following the increasing order at T2: AMTN = SBU < BIOS = SPRG < TMP < FLU < PLA. The SBU group had the highest value of occluded dentinal tubule length. The AMTN group presented more occluded dentinal tubules compared to PLA and FLU. AMTN and SBU had the lowest values of opened dentin tubules. Results showed a negative correlation between the analyses. CONCLUSION: The SBU and AMTN solution were more effective in reducing dentin permeability by occluding dentin tubules. CLINICAL RELEVANCE: All materials reduced permeability after challenge, except fluoride varnish.

Screening of functionalized collagen membranes with a porcine periodontal regeneration model.

OBJECTIVES: Current methods for periodontal regeneration do not promote collagen fiber insertions into new bone and cementum. We used a pig wound model to screen different functionalized collagen membranes in promoting periodontal reattachment to root surfaces. METHODS: Treatment groups included (1) control with no membranes, (2) collagen-coated membranes, (3) membranes with insulin-like growth factor-1 (IGF-1), (4) membranes with amelotin, or (5) membranes attached with calcium phosphate cement (CPC), or with CPC combined with IGF-1. Flap procedures were performed on mandibular and maxillary premolars of each pig. RESULTS: Histomorphometric, micro-CT, and clinical measurements obtained at 4 and 12 weeks after surgery showed cementum formation on denuded roots and reformation of alveolar bone, indicating that the pig model can model healing responses in periodontal regeneration. Calcium phosphate cement simplified procedures by eliminating the need for sutures and improved regeneration of alveolar bone (p < 0.05) compared with other treatments. There was a reduction (p < 0.05) of PD only for the IGF group. Large observed variances between treatment groups indicated that a priori power analyses should be conducted to optimize statistical analysis. CONCLUSIONS: Pigs can model discrete elements of periodontal healing using collagen-based, functionalized membranes. Screening indicates that membrane anchorage with calcium phosphate cements improve regeneration of alveolar bone.

An SCPPPQ1/LAM332 protein complex enhances the adhesion and migration of oral epithelial cells: Implications for dentogingival regeneration.

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e-8 [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm2 to 63 dyn/cm2). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 ± 0.24 µm/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. STATEMENT OF SIGNIFICANCE: This work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration.

Amelotin Promotes Mineralization and Adhesion in Collagen-Based Systems.

Introduction: Periodontitis is characterized by the destruction of tooth-supporting tissues including the alveolar bone. Barrier membranes are used in dentistry for tissue regenerative therapy. Nevertheless, conventional membranes have issues related to membrane stability and direct induction of bone mineralization. Amelotin (AMTN), an enamel matrix protein, regulates hydroxyapatite crystal nucleation and growth. To apply an AMTN membrane in clinical practice, we investigated the mineralizing and adhesive effects of recombinant human (rh) AMTN in vitro using a collagen-based system. Methods: Collagen hydrogel incorporated with rhAMTN (AMTN gel) and rhAMTN-coated dentin slices were prepared. AMTN gel was then applied on a commercial membrane (AMTN membrane). Samples were incubated for up to 24 h in mineralization buffer, and the structures were observed. The peak adhesive tensile strength between the dentin and AMTN membrane was measured. Using an enzyme-linked immunosorbent assay, the release kinetics of rhAMTN from the membrane were investigated. Results: The AMTN gel resulted in the formation of hydroxyapatite deposits both onto and within the collagen matrix. Furthermore, coating the dentin surface with rhAMTN promoted the precipitation of mineral deposits on the surface. Interestingly, site-specific mineralization was observed in the AMTN membrane. Only 1% of rhAMTN was released from the membrane. Hence, the AMTN membrane adhered to the dentin surface with more than twofold greater tensile strength than that detected for a rhAMTN-free barrier membrane. Conclusions: RhAMTN can accelerate mineralization and adhesion in collagen-based systems. Furthermore, the AMTN membrane could inform the optimal design of calcified tissue regenerative materials. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00722-2.

Promoting mineralization at biological interfaces Ex vivo with novel amelotin-based bio-nano complexes.

Interfacial failure at the resin-dentin interface is a significant disadvantage of resin-based dental restoration. In this study, we created bio-inspired bio-nano complexes using the enamel protein amelotin (AMTN) or AMTN with an engineered collagen-binding site (AMTN-Col) to coat hydroxyapatite nanoparticles (HANP). The resulting nano-bio complexes, AMTN-HANP and AMTN-Col-HANP, were evaluated for their ability to promote collagen mineralization. Our study comprises three separate phases.In phase I, developing a method for functionalizing HANP with AMTN/AMTN-Col was explored. HANP were synthesized and characterized using TEM, SAED-TEM, XRD and ATR-FTIR. The nanoparticles were functionalized with AMTN or AMTN-Col. The successful coating of the nanoparticles with the proteins was confirmed using a TEM image of immunogold-labelled samples.In phase II of the study, the mineralization potential of the synthesized bio-nano complexes was studied using model systems consisting of simulated body fluid (SBF), polymerized collagen gels, and dentin disks prepared from human extracted molars. Mineral formation in SBF was recorded with a light scattering assay using a microplate reader on 8 replicates of each sample per study time point. Statistical analysis was performed using one-way ANOVA and the Tukey test. Significance was assigned at P â€‹< â€‹0.01. The extent of mineral formation on collagen gel and remineralization of demineralized dentin was studied with SEM. Accelerated mineral formation collagen mineralization of bio-nano complexes treated samples were observed in all model systems.In phase III of the study, the clinical utilization of AMTN/AMTN-Col coated HANP in bio-integration and enhancing the bond strength of a resin-based dental restoration and the dentin interface was investigated. The bio-nano complexes were applied as a pretreatment on dentin disks prepared from human extracted molars prior to the composite resin restoration. The micro-shear bond strength test was done on 8 samples per treatment group (a total of 32 samples). Statistical analysis on shear bond strength was performed using one-way ANOVA and the Tukey test. Significance was assigned at P â€‹< â€‹0.01. Shear bond strength values indicated that pretreatment of dentin with the bio-nano complexes before adhesive application significantly improved shear bond strength. Conclusion: We have shown that AMTN based bio-nano complexes promote mineral formation on collagenous interfaces. Our findings can be the basis of new bio-inspired, bio-nano materials that may improve dental restoration longevity by enhancing the stability and integrity of the dentin-composite resin interface.

The use of hydroxyapatite toothpaste to prevent dental caries.

Dissolution of hydroxyapatite from the tooth structure at low pH can lead to the irreversible destruction of enamel and dentin, which if left untreated can result in pain and tooth loss. Hydroxyapatite toothpastes contain hydroxyapatite particles in micro- or nanocrystalline form that have been shown to deposit and restore demineralized enamel surfaces. As such, they are currently being explored as a fluoride-free anti-caries agent. This narrative review article aims to summarize the recent findings of the research investigating the remineralization potential of HAP toothpaste in vitro, in situ and in vivo, as well as some other applications in dentistry.

Uniaxial Hydroxyapatite Growth on a Self-Assembled Protein Scaffold.

Biomineralization is a crucial process whereby organisms produce mineralized tissues such as teeth for mastication, bones for support, and shells for protection. Mineralized tissues are composed of hierarchically organized hydroxyapatite crystals, with a limited capacity to regenerate when demineralized or damaged past a critical size. Thus, the development of protein-based materials that act as artificial scaffolds to guide hydroxyapatite growth is an attractive goal both for the design of ordered nanomaterials and for tissue regeneration. In particular, amelogenin, which is the main protein that scaffolds the hierarchical organization of hydroxyapatite crystals in enamel, amelogenin recombinamers, and amelogenin-derived peptide scaffolds have all been investigated for in vitro mineral growth. Here, we describe uniaxial hydroxyapatite growth on a nanoengineered amelogenin scaffold in combination with amelotin, a mineral promoting protein present during enamel formation. This bio-inspired approach for hydroxyapatite growth may inform the molecular mechanism of hydroxyapatite formation in vitro as well as possible mechanisms at play during mineralized tissue formation.

Effect of Amelotin on Bone Growth in the Murine Calvarial Defect Model.

Amelotin (AMTN) is a protein that is expressed during the maturation of dental enamel and has important role in enamel hydroxyapatite mineralization. However, it is not well understood whether AMTN has a strong mineral-promoting ability in bone. In this study, the effect of AMTN on bone healing was investigated using mice calvarial defect model in vivo, and the expression of bone marker genes and cell proliferation were investigated to clarify the role of AMTN in bone mineralization using mouse osteogenic cells (MC3T3-E1) in vitro. Collagen membranes, with or without recombinant human (rh) AMTN, were applied to calvarial defects created on the parietal bones of C57BL/6N mice. Microcomputed tomography and histological observation revealed that the defect largely filled with mineralized tissue by the rhAMTN-containing membrane in eight weeks. Moreover, CD31 positive cells were observed in the newly formed mineralized tissue and around the rhAMTN-containing membrane. In the presence of rhAMTN, the expression of the Spp1 gene in MC3T3-E1 cells significantly increased within ten days in an osteoinductive medium. Moreover, rhAMTN significantly enhanced MC3T3-E1 cell proliferation. These findings indicate that AMTN positively influences bone repair by promoting hydroxyapatite mineralization.

Early bone formation in mini-lateral window sinus floor elevation with simultaneous implant placement: An in vivo experimental study.

OBJECTIVE: To investigate the early bone formation in beagles with mini-lateral window sinus floor elevation and simultaneous implant placement. MATERIAL AND METHODS: Six beagles were selected for the split-mouth design procedures. In each animal, one maxillary recess received a 5 mm-diameter mini-round lateral osteotomy (test group), and the contralateral maxillary recess received a large rectangular osteotomy (10 mm long and 8 mm wide), (control group). Simultaneous implant installation was executed on bilateral maxillary recesses. Tetracycline and calcein dyes were administered on the 14th, 13th days and the 4th, 3rd days prior to sacrifice, respectively. After 8 weeks of healing, the beagles were euthanized for fluorescent labeling and histomorphometric analyses. RESULTS: In both groups, new bone formation initiated from the circumferential native bone of the maxillary recesses and extended toward the central sub-recess cavities. The maxillary recesses with the mini-window procedures exhibited superior mineral apposition rate, bone formation rate, and the percentage of new bone area to those of the group exposed to large osteotomy procedure (p < .05). While there was no significant difference in the value of bone-to-implant contact, the mini-window group displayed a tendency for an increase in this aspect (p > .05). Bone formation rate and new bone amount were not statistically correlated with bone-to-implant contact (p > .05). CONCLUSION: The hypothesis that mini-lateral window sinus floor elevation with simultaneous implant placement would improve early new bone formation in augmented sinus compared with large lateral window procedure is accepted.

Follicular dendritic cell-secreted protein gene expression is upregulated and spread in nifedipine-induced gingival overgrowth.

Follicular dendritic cell-secreted protein (FDC-SP) is secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium (JE). Its expression could be controlled during inflammatory process of gingiva; however, responsible mechanism for gingival overgrowth and involvement of FDC-SP in clinical condition is still unclear. We hypothesized that JE-specific genes are associated with the initiation of drug-induced gingival enlargement (DIGE) called gingival overgrowth, and investigated the changes of JE-specific gene's expression and their localization in overgrown gingiva from the patients. Immunohistochemical analysis revealed that the FDC-SP localization was spread in overgrown gingival tissues. FDC-SP mRNA levels in GE1 and Ca9-22 cells were increased by time-dependent nifedipine treatments, similar to other JE-specific genes, such as Amelotin (Amtn) and Lamininß3 subunit (Lamß3), whereas type 4 collagen (Col4) mRNA levels were decreased. Immunocytochemical analysis showed that FDC-SP, AMTN, and Lamß3 protein levels were increased in GE1 and Ca9-22 cells. Transient transfection analyses were performed using luciferase constructs including various lengths of human FDC-SP gene promoter, nifedipine increased luciferase activities of -345 and -948FDC-SP constructs. These results raise the possibility that the nifedipine-induced FDC-SP may be related to the mechanism responsible for gingival overgrowth does not occur at edentulous jaw ridges.

Downregulation of FGF Signaling by Spry4 Overexpression Leads to Shape Impairment, Enamel Irregularities, and Delayed Signaling Center Formation in the Mouse Molar.

FGF signaling plays a critical role in tooth development, and mutations in modulators of this pathway produce a number of striking phenotypes. However, many aspects of the role of the FGF pathway in regulating the morphological features and the mineral quality of the dentition remain unknown. Here, we used transgenic mice overexpressing the FGF negative feedback regulator Sprouty4 under the epithelial keratin 14 promoter (K14-Spry4) to achieve downregulation of signaling in the epithelium. This led to highly penetrant defects affecting both cusp morphology and the enamel layer. We characterized the phenotype of erupted molars, identified a developmental delay in K14-Spry4 transgenic embryos, and linked this with changes in the tooth developmental sequence. These data further delineate the role of FGF signaling in the development of the dentition and implicate the pathway in the regulation of tooth mineralization. © 2019 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

C/EBPß and YY1 bind and interact with Smad3 to modulate lipopolysaccharide-induced amelotin gene transcription in mouse gingival epithelial cells.

Junctional epithelium (JE) develops from reduced enamel epithelium during tooth formation and is critical for the maintenance of healthy periodontal tissue through ensuring appropriate immune responses and the rapid turnover of gingival epithelial cells. We have previously shown a relationship between inflammatory cytokines and expression of JE-specific genes, such as amelotin (AMTN), in gingival epithelial cells. Here, we elucidated the effects of Porphyromonas gingivalis-derived lipopolysaccharide (Pg LPS) on Amtn gene transcription and the interaction of transcription factors. To determine the molecular basis of transcriptional regulation of the Amtn gene by Pg LPS, we performed real-time PCR and carried out luciferase assays using a mouse Amtn gene promoter linked to a luciferase reporter gene in mouse gingival epithelial GE1 cells. Gel mobility shift and chromatin immunoprecipitation assays were performed to identify response elements bound to LPS-induced transcription factors. Next, we analyzed protein levels of the LPS-induced transcription factors and the interaction of transcription factors by western blotting and immunoprecipitation. LPS increased Amtn mRNA levels and elevated luciferase activities of constructs containing regions between -116 and -238 of the mouse Amtn gene promoter. CCAAT/enhancer-binding protein (C/EBP) 1-, C/EBP2- and Ying Yang 1 (YY1)-nuclear protein complexes were increased by LPS treatment. Furthermore, we identified LPS-modulated interactions with C/EBPß, YY1 and Smad3. These results demonstrate that Pg LPS regulates Amtn gene transcription via binding of C/EBPß-Smad3 and YY1-Smad3 complexes to C/EBP1, C/EBP2 and YY1 response elements in the mouse Amtn gene promoter.

Negative feedback by SNAI2 regulates TGFß1-induced amelotin gene transcription in epithelial-mesenchymal transition.

Junctional epithelium (JE) demonstrates biological responses with the rapid turnover of gingival epithelial cells. The state occurs in inflammation of gingiva and wound healing after periodontal therapy. To understand the underlying mechanisms and to maintain homeostasis of JE, it is important to investigate roles of JE-specific genes. Amelotin (AMTN) is localized at JE and regulated by inflammatory cytokines and apoptotic factors that represent a critical role of AMTN in stabilizing the dentogingival attachment, which is an entrance of oral bacteria. In this study, we demonstrated that the AMTN gene expression was regulated by SNAI2 and transforming growth factor ß1 (TGFß1)-induced epithelial-mesenchymal transition (EMT) that occurs in wound healing and fibrosis during chronic inflammation. SNAI2 downregulated AMTN gene expression via SNAI2 bindings to E-boxes (E2 and E4) in the mouse AMTN gene promoter in EMT of gingival epithelial cells. Meanwhile, TGFß1-induced AMTN gene expression was attenuated by SNAI2 and TGFß1-induced SNAI2, without inhibition of the TGFß1-Smad3 signaling pathway. Moreover, SNAI2 small interfering RNA (siRNA) rescued SNAI2-induced downregulation of AMTN gene expression, and TGFß1-induced AMTN gene expression was potentiated by SNAI2 siRNA. Taken together, these data demonstrated that AMTN gene expression in the promotion of EMT was downregulated by SNAI2. The inhibitory effect of AMTN gene expression was an independent feedback on the TGFß1-Smad3 signaling pathway, suggesting that the mechanism can be engaged in maintaining homeostasis of gingival epithelial cells at JE and the wound healing phase.

Resveratrol derivative-rich melinjo seed extract induces healing in a murine model of established periodontitis.

BACKGROUND: Products of internal defense systems, like pro-inflammatory cytokines, reactive oxygen species, and leukocytes, are released which attack periodontal bacteria in periodontitis, but at the same time, lead to tissue destruction as well. We hypothesize that resveratrol derivative-rich melinjo seed extract (MSE), an edible plant extract that has antioxidant properties, should promote healing of periodontal bone loss and modulating immune-inflammatory systems that leads periodontal tissue destruction. METHODS: We used an experimentally induced periodontitis (EP) model in mice. Ligatures were placed first for development of EP (15 days). MSE was intraperitoneally administrated (0.001% (w/w)) to reverse bone loss that had already occurred in established EP and mice were then sacrificed (day 17, 20 and 22). RESULTS: Morphometric outcomes revealed lower bone-loss in the MSE groups compared to control. Immunohistochemistry assays demonstrated lower oxidative stress in MSE groups. MSE also inhibited M-CSF/sRANKL mediated osteoclast formation and down-regulated osteoclast activity. CONCLUSIONS: Treatment with MSE in EP actually caused healing of bone, and these effects are probably related to decreases in local oxidative damage and osteoclast activity. Given MSE's positive effects on osteodifferentiation as well, these findings suggest that MSE could be a useful therapeutic agent for the management of periodontitis.

The enamel protein ODAM promotes mineralization in a collagen matrix.

Purpose/aim of the study: Odontogenic ameloblast-associated protein (ODAM) is predominantly expressed during the maturation stage of enamel formation and interacts strongly with amelotin (AMTN). AMTN is involved in enamel mineralization, but the effect of ODAM on mineralization has not been investigated. This study determined whether ODAM was able to induce hydroxyapatite (HA) mineralization in modified simulated body fluid (SBF) and in a collagen matrix in vitro. MATERIALS AND METHODS: To monitor the kinetics of calcium phosphate mineralization, recombinant human (rh) ODAM protein in SBF buffer was incubated at 37°C and a light-scattering assay was conducted at intervals. To investigate the nucleation of ODAM in collagen matrix, the ODAM-impregnated collagen hydrogel was incubated in SBF buffer for 24 hours. Bovine serum albumin (BSA) was used as negative control. Mineral deposits were visualized using electron microscopy. RESULTS: The presence of rh-ODAM protein in SBF resulted in higher light-scattering values after 18-24 hours. Calcium phosphate precipitates were observed on the surface of the ODAM-treated, but not BSA-treated collagen hydrogel after 24 hours in SBF. TEM and SAED analyses showed that these crystals consisted of needle-like HA. CONCLUSION: Similar to AMTN, ODAM is able to promote HA nucleation in a dose-dependent manner in SBF, and even outside of its biological context in vitro.

TGFß1-induced Amelotin gene expression is downregulated by Bax expression in mouse gingival epithelial cells.

Amelotin (AMTN) is induced upon initiation of apoptosis by transforming growth factor beta1 (TGFß1) and is mediated by Smad3 in gingival epithelial cells (GE1 cells). This upregulation of AMTN gene expression is temporary, and the mechanism responsible is still unclear. The present study investigated the transcriptional downregulation of TGFß1-induced AMTN gene expression in GE1 cells during the progression of apoptosis. To examine time-dependent changes in the levels of AMTN, Smad3 and Bax mRNA induced by TGFß1, real-time PCR analyses were performed. Immunocytochemistry was carried out to detect the expression of Smad3 and Bax. Transient transfection analyses were performed using mouse AMTN gene promoter constructs of various lengths including Smad response elements (SBEs), in the presence or absence of TGFß1. Changes in Smad3 binding to SBEs resulting from overexpression of Bax were examined using ChIP assays. Overexpression of Bax dramatically downregulated the levels of TGFß1-induced AMTN mRNA and transcription of the AMTN gene. Smad3 binding to SBEs in the mouse AMTN gene promoter was induced by overexpression of Smad3 or TGFß1, and this was inhibited by Bax overexpression. These results show that the levels of AMTN mRNA induced by TGFß1 and Smad3 are decreased by robust expression of Bax in gingival epithelial cells.

The Role of Epithelial Stat3 in Amelogenesis during Mouse Incisor Renewal.

The aim of this study was to evaluate the role of epithelial signal transducer and activator of transcription 3 (STAT3) in mouse incisor amelogenesis. Since Stat3 is expressed in the epithelial component of developing and adult mouse teeth, we generated and analyzed Krt14Cre/+;Stat3fl/fl mutant mice in which Stat3 was inactivated in epithelia including ameloblast progenitors and ameloblasts, the cells responsible for enamel formation. Histological analysis showed little enamel matrix in mutant incisors compared to controls. Delayed incisor enamel mineralization was demonstrated using micro-computed X-ray tomography analysis and was supported by an increase in the pre-expression distance of enamel-enriched proteins such as amelogenin, ameloblastin, and kallikrein-4. Lastly, scanning electron microscopy analysis showed little enamel mineralization in mutant incisors underneath the mesial root of the 1st molar; however, the micro-architecture of enamel mineralization was similar in the erupted portion of control and mutant incisors. Taken together, our findings demonstrate for the first time that the absence of epithelial Stat3 in mice leads to delayed incisor amelogenesis.

Isl1 Controls Patterning and Mineralization of Enamel in the Continuously Renewing Mouse Incisor.

Rodents are characterized by continuously renewing incisors whose growth is fueled by epithelial and mesenchymal stem cells housed in the proximal compartments of the tooth. The epithelial stem cells reside in structures known as the labial (toward the lip) and lingual (toward the tongue) cervical loops (laCL and liCL, respectively). An important feature of the rodent incisor is that enamel, the outer, highly mineralized layer, is asymmetrically distributed, because it is normally generated by the laCL but not the liCL. Here, we show that epithelial-specific deletion of the transcription factor Islet1 (Isl1) is sufficient to drive formation of ectopic enamel by the liCL stem cells, and also that it leads to production of altered enamel on the labial surface. Molecular analyses of developing and adult incisors revealed that epithelial deletion of Isl1 affected multiple, major pathways: Bmp (bone morphogenetic protein), Hh (hedgehog), Fgf (fibroblast growth factor), and Notch signaling were upregulated and associated with liCL-generated ectopic enamel; on the labial side, upregulation of Bmp and Fgf signaling, and downregulation of Shh were associated with premature enamel formation. Transcriptome profiling studies identified a suite of differentially regulated genes in developing Isl1 mutant incisors. Our studies demonstrate that ISL1 plays a central role in proper patterning of stem cell-derived enamel in the incisor and indicate that this factor is an important upstream regulator of signaling pathways during tooth development and renewal. © 2017 American Society for Bone and Mineral Research.