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.

Influence of violet LED associated or not with peroxide gel on inflammation, mineralization, and collagen fiber maturation in dentin and pulp tissue.

OBJECTIVES: To evaluate the influence of violet LED, associated or not with a 17.5% hydrogen peroxide (HP) bleaching gel, on inflammation, mineralization in pulp tissue, and collagen fiber maturation in dentin and pulp tissue. MATERIALS AND METHODS: The maxillary molars of eighty Wistar rats were distributed into four groups (n = 10): CONT - without treatment; HP - 30 min application of 17.5% HP; LED - 20 min application of violet LED; and HP+LED - application of PH and violet LED. Rats were euthanized and jaws were processed for histologic and immunohistochemical evaluation (IL-17, IL-23, and osteocalcin) and picrosirius red immediately after (T0), and at 7 (T1), 15 (T2), and 30 days (T3) post-treatment, with Wilcoxon, Mann-Whitney, paired T-test, and T-test (α = 0.05). RESULTS: HP and HP+LED presented necrosis and severe inflammatory infiltrate. When compared to CONT group, LED presented severe osteocalcin (OCN) immunostaining in T2 and less immature fibers in T2 and T3. CONCLUSION: The violet LED caused no severe damage to the pulp tissue, increased IL-17 and IL-23 expression in T0 when associated with HP, and had no influence on pulp tissue mineralization, besides accelerating the maturation of collagen fibers of dentin. CLINICAL RELEVANCE: Violet LED therapy induced no inflammation in the pulp tissue of rats and played no role in pulp tissue fibrosis, besides accelerating the maturation of dentin collagen fibers.

Hydroxypropylmethylcellulose as a film and hydrogel carrier for ACP nanoprecursors to deliver biomimetic mineralization.

Demineralization of hard tooth tissues leads to dental caries, which cause health problems and economic burdens throughout the world. A biomimetic mineralization strategy is expected to reverse early dental caries. Commercially available anti-carious mineralizing products lead to inconclusive clinical results because they cannot continuously replenish the required calcium and phosphate resources. Herein, we prepared a mineralizing film consisting of hydroxypropylmethylcellulose (HPMC) and polyaspartic acid-stabilized amorphous calcium phosphate (PAsp-ACP) nanoparticles. HPMC which contains multiple hydroxyl groups is a film-forming material that can be desiccated to form a dry film. In a moist environment, this film gradually changes into a gel. HPMC was used as the carrier of PAsp-ACP nanoparticles to deliver biomimetic mineralization. Our results indicated that the hydroxyl and methoxyl groups of HPMC could assist the stability of PAsp-ACP nanoparticles and maintain their biomimetic mineralization activity. The results further demonstrated that the bioinspired mineralizing film induced the early mineralization of demineralized dentin after 24 h with increasing mineralization of the whole demineralized dentin (3-4 µm) after 72-96 h. Furthermore, these results were achieved without any cytotoxicity or mucosa irritation. Therefore, this mineralizing film shows promise for use in preventive dentistry due to its efficient mineralization capability.

Beyond dissolution: Xerostomia rinses affect composition and structure of biomimetic dental mineral in vitro.

Xerostomia, known as dry mouth, is caused by decreased salivary flow. Treatment with lubricating oral rinses provides temporary relief of dry mouth discomfort; however, it remains unclear how their composition affects mineralized dental tissues. Therefore, the objective of this study was to analyze the effects of common components in xerostomia oral rinses on biomimetic apatite with varying carbonate contents. Carbonated apatite was synthesized and exposed to one of the following solutions for 72 hours at varying pHs: water-based, phosphorus-containing (PBS), mucin-like containing (MLC), or fluoride-containing (FC) solutions. Post-exposure results indicated that apatite mass decreased irrespective of pH and solution composition, while solution buffering was pH dependent. Raman and X-ray diffraction analysis showed that the addition of phosphorus, mucin-like molecules, and fluoride in solution decreases mineral carbonate levels and changed the lattice spacing and crystallinity of bioapatite, indicative of dissolution/recrystallization processes. The mineral recrystallized into a less-carbonated apatite in the PBS and MLC solutions, and into fluorapatite in FC. Tap water did not affect the apatite lattice structure suggesting formation of a labile carbonate surface layer on apatite. These results reveal that solution composition can have varied and complex effects on dental mineral beyond dissolution, which can have long term consequences on mineral solubility and mechanics. Therefore, clinicians should consider these factors when advising treatments for xerostomia patients.

TVH-19, a synthetic peptide, induces mineralization of dental pulp cells in vitro and formation of tertiary dentin in vivo.

Functional peptides derived from the active domains of odontogenesis-related proteins have been reported to promote dental hard tissue regeneration. The purpose of this study was to evaluate the effects of an artificially synthesized peptide, TVH-19, on odontoblast differentiation and tertiary dentin formation in indirect pulp capping (IPC) using in vitro and in vivo experiments. TVH-19 did not exhibit any effect on the proliferation of human dental pulp cells (hDPCs) but significantly promoted cell migration, compared with the control (p < 0.05). TVH-19-treated hDPCs showed significantly higher alkaline phosphatase (ALP) activity and stronger alizarin red staining (ARS) reactivity than the control group (p < 0.05). TVH-19 also upregulated the mRNA and protein expression levels of odontogenic genes. After generating IPC in rats, the samples of teeth were studied using micro-computed tomography (Micro-CT), hematoxylin & eosin (HE) staining, and immunohistochemical staining to investigate the functions of TVH-19. The in vivo results showed that TVH-19 induced the formation of tertiary dentin, and reduced inflammation and apoptosis, as evident from the downregulated expression of interleukin 6 (IL-6) and cleaved-Caspase-3 (CL-CASP3). Overall, the results of our study suggest that TVH-19 induces differentiation of hDPCs, promotes tertiary dentin formation, relieves inflammation, and reduces apoptosis, indicating the potential applications of TVH-19 in IPC.

Novel metformin-containing resin promotes odontogenic differentiation and mineral synthesis of dental pulp stem cells.

This represents the first report on the development of metformin-containing dental resins. The objectives were to use the resin as a carrier to deliver metformin locally to stimulate dental cells for dental tissue regeneration and to investigate the effects on odontogenic differentiation of dental pulp stem cells (DPSCs) and mineral synthesis. Metformin was incorporated into a resin at 20% by mass as a model system. DPSC proliferation attaching on resins was evaluated. Dentin sialophosphoprotein (DSPP), dentin matrix phosphoprotein 1 (DMP-1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (Runx2) genes expressions were measured. ALP activity and alizarin red staining (ARS) of mineral synthesis by the DPSCs on resins were determined. DPSCs on metformin-containing resin proliferated well (mean ± SD; n = 6), and the number of cells increased by 4-fold from 1 to 14 days (p > 0.1). DSPP, ALP, and DMP-1 gene expressions of DPSCs on metformin resin were much higher than DPSCs on control resin without metformin (p < 0.05). ALP activity of metformin group was 70% higher than that without metformin at 14 days (p < 0.05). Mineral synthesis by DPSCs on metformin-containing resin at 21 days was 9-fold that without metformin (p < 0.05). A novel metformin-containing resin was developed, achieving substantial enhancement of odontoblastic differentiation of DPSCs and greater mineral synthesis. The metformin resin is promising for deep cavities and perforated cavities to stimulate DPSCs for tertiary dentin formation, for tooth root coatings with metformin release for periodontal regeneration, and for root canal fillings with apical lesions to stimulate bone regeneration.

Effect of Two Remineralizing Agents on Initial Caries-like Lesions in Young Permanent Teeth: An in Vitro Study.

AIM: To compare the effect of nano-hydroxyapatite (9000 ppm F) and casein phosphopeptide-amorphous calcium phosphate fluoride (900 ppm F) pastes on initial enamel carious lesions of young permanent teeth. MATERIALS AND METHODS: Sixty extracted young premolars with a standardized window on enamel were immersed in a demineralizing solution for 48 hours to produce subsurface enamel lesions. They were divided into three groups according to remineralizing agents (n = 20) group I: nano-hydroxyapatite paste; group II: casein phosphopeptide-amorphous calcium phosphate fluoride paste; and group III: control (without an agent). The enamel surface microhardness (SMH) was measured at baseline, after the incipient enamel lesion, and after treatment. Additional twenty young premolars were selected and prepared as mentioned above for surface morphology evaluation by scanning electron microscope (SEM). RESULTS: No significant difference was found in mean surface microhardness in teeth treated with nano-hydroxyapatite paste and those treated with casein phosphopeptide-amorphous calcium phosphate fluoride p = 0.26. SEM showed improvement in surface defects of demineralized enamel in the two test groups. CONCLUSION: Nano-hydroxyapatite and casein phosphopeptide-amorphous calcium phosphate fluoride pastes were effec -tive in rehardening the initial enamel caries lesions in young permanent teeth. CLINICAL SIGNIFICANCE: The best strategy for caries management is to focus on the methods of improving the reminer-alization process with the aid of the remineralizing agents. The current study compared the remineralizing effect of two remineralizing agents. Within the limitations of the study, both remineralizing agents were effective for remineralization of early caries-like lesions.

Regenerating a monoblock to obturate root canalsvia a mineralising strategy.

To develop a novel strategy for sealing and obturating dental root canals by tooth-like tissue regeneration, premolars with mature root apices were freshly collected, and root canals were prepared by following the clinical protocols in vitro. The teeth were immersed in supersaturated calcium and phosphate solution containing gallic acid and fluoride. At certain intervals, the dental roots were taken out, and their mineral precipitates were characterised by scanning electron microscopy, energy-dispersive spectroscopy mapping, X-ray diffraction and transmission electron microscopy. The cytocompatibility of the mineralizing products were evaluated with rabbit bone-marrow-derived mesenchymal stem cells in vitro. Results showed that the precipitates were mainly composed of fluoridated hydroxyapatite with ahexagonal prism morphology. Fluoridated hydroxyapatite initially nucleated and grew from the root canal dentine surface to the root canal centre. The fluoridated hydroxyapatite precipitate and root canal dentine intergraded together such that the interface became hardly distinguishable. The fluoridated hydroxyapatite precipitate grew into and obturated the dentinal tubules. In the root canal, the regenerated fluoridated hydroxyapatite densely packed and bundled together with a c-axis extension. After 7 days of mineralisation, the root canal was completely obturated, and the apical foramen was sealed. The mineralizing products had good biocompatibility with the cells, and the cells grew well on the mineralized surface. Biomimetic mineralisation strategy provides a novel means to regenerate tooth-like tissue to seal the root canal system permanently other than by passive synthetic material filling.

Effects of silicate-based composite material on the proliferation and mineralization behaviors of human dental pulp cells: An in vitro assessment.

The objective of this study was to investigate the effects of a silicate-based composite material on proliferation and mineralization of human dental pulp cells (hDPCs), which was compared with those of calcium hydroxide (Ca(OH)2, CH) and tricalcium silicate (Ca3SiO5, C3S). HDPCs were cultured with CH, C3S and tricalcium silicate/dicalcium silicate (Ca3SiO5/Ca2SiO4, C3S/C2S) composites extract. The CCK-8 assay showed that the composite material stimulated the proliferation of hDPCs. The odontogenic marker genes and DSPP protein expression were more significantly up-regulated by the C3S/C2S composite material compared with pure CH and C3S. HDPCs cultured with composite material extract exert stronger ALP activity and alizarin red S staining. C3S/C2S composite material was advantageous over pure C3S by showing enhanced ability to stimulate the proliferation and odontogenic differentiation of hDPCs, suggesting that the C3S/C2S composite materials possess desirable biocompatibility and bioactivity, and might be a new type of pulp-capping agent and dentin alternative materials.

Effect of antibiotics and NSAIDs on cyclooxygenase-2 in the enamel mineralization.

The objective of this study was to determine whether the use of the most commonly prescribed antibiotics and non-steroidal anti-inflammatory drugs in childhood could disturb enamel mineralization. Forty-two Swiss mice were divided into seven groups: controls; amoxicillin; amoxicillin/clavulanate; erythromycin; acetaminophen; ibuprofen and celecoxib, to inhibit cyclooxygenase 2 (COX2). SEM-EDX analysis was conducted on all cusps of the third molars. Calcium (Ca), phosphorus (P), aluminum, potassium, sodium, magnesium and chlorine were quantified. The stoichiometric Ca/P molar ratios were calculated. Immunohistochemical quantification of COX2 in incisors was carried out by image analysis using COX2-specific immunostaining. Groups treated with antibiotics showed no significant differences in the content of the chemical elements. Only acetaminophen and celecoxib showed a significant decrease in Ca and P compared with the control samples. Ca/P ratios showed no difference. Groups treated with amoxicillin, amoxicillin/clavulanate, erythromycin and acetaminophen showed significantly lower amounts of immunoreactive COX2 at the enamel organ maturation stage of the mouse incisors. Our results suggest that COX2 is involved in the maturation stage of the enamel organ and that its inhibition would appear to alter amelogenesis, producing hypomineralization.

Fluoride varnishes containing sodium trimetaphosphate reduce enamel demineralization in vitro.

OBJECTIVE: This study evaluated the effects of fluoride varnishes containing sodium trimetaphosphate (TMP) on bovine enamel demineralization in vitro. MATERIAL AND METHODS: Enamel bovine discs were randomly assigned into six groups (n = 20/group): placebo, 2.5% NaF, 2.5% NaF/5% TMP, 5% NaF, 5% NaF/5% TMP, and a commercial formulation (Duraphat, 5% NaF). Varnishes were applied on all enamel discs and kept for 6 h. Loosely and firmly bound fluoride formed on/in enamel after treatment were analyzed in 10 discs from each group. The other 10 discs were subjected to a pH-cycling regimen for 7 days, and analyzed for surface (SH) and cross-sectional hardness (ΔKHN), as well as for loosely and firmly bound fluoride in/on enamel. Data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls' test (p < .05). RESULTS: The lowest SH change and ΔKHN were observed for the 5%NaF/5%TMP varnish, which was significantly different from all the other groups. Both fluoridated varnishes containing TMP promoted significantly lower SH change and ΔKHN when compared with their counterparts without TMP. Loosely and firmly bound fluoride was significantly lower in groups treated with varnishes containing TMP. CONCLUSION: TMP and fluoride added to varnishes have a synergistic effect against enamel demineralization in vitro.

Dentine-pulp tissue engineering in miniature swine teeth by set calcium silicate containing bioactive molecules.

OBJECTIVE: The present study aims to investigate whether reparative dentinogenesis could be guided at central pulpal sites or at a distance from the amputated pulp of miniature pig teeth, by using set calcium silicate-based carriers containing human recombinant bioactive molecules. DESIGN: Pulp exposures were performed in 72 permanent teeth of 4 healthy miniature swine. The teeth were capped with pre-manufactured implants of set calcium silicate-based material containing BMP-7, TGFß1 or WnT-1, for 3 weeks. Conical-shaped intrapulpal implants were exposed in the central pulp core, while disc-shaped extrapulpal implants were placed at a distance from the amputated pulp. Implants without bioactive molecules were used as controls. Thickness and forms of new matrix mineralized deposition were assessed histologically at post-operative periods of 3 weeks by light microscopy. RESULTS: Intrapulpal applications: Calcified structures composed of osteodentine were found in contact with the BMP-7 implants. An inhomogeneous calcified tissue matrix was found around the WnT-1 carriers. A two-zone calcified structure composed of osteodentine and a thicker tubular matrix zone was seen at the TGFß1 carrier-pulp interface. Extrapulpal applications: The space between WnT-1 implants and pulp periphery had been invaded by soft tissue with traces of calcified foci. Thick calcified structures composed of osteodentine were found surrounding pulp exposure sites in response to application of BMP-7. Spindle-shaped cells associated with atubular calcified matrix or elongated polarized cells associated with tubular dentine-like matrix were found along the cut dentinal walls of the TGFß1 group. CONCLUSION: The present experiments indicated that set calcium silicate could be used as carrier for biologically active molecules. TGFß1 was shown to be an effective bioactive molecule in guiding tertiary dentine formation.

Assessing the Penetrating Abilities of Experimental Preparation with Dental Infiltrant Features Using Optical Microscope: Preliminary Study.

BACKGROUND: The aim of the infiltration technique is to penetrate demineralized enamel with a low viscosity resin. Icon® (DMG) is the first ever and so far the only dental infiltrant. Bacteriostaticity is one of the properties that should be inherent in dental infiltrants, but Icon lacks this feature. OBJECTIVES: The aim of the preliminary study was to properly choose a dye which would allow us to assess the penetrating abilities of our own, experimental preparation with features of a dental infiltrant with bacteriostatic properties and to compare using an optical microscope the depth of infiltration of the designed experimental preparation with the infiltrant available on the market. MATERIAL AND METHODS: The preparation is supposed to infiltrate decalcified human enamel and be assessed with an optical microscope. Eosin, neutral fuchsine and methylene blue were added to experimental preparation with dental infiltrant features and to Icon® (DMG) in order to assess the depth of penetration of the experimental solution into the decalcified layers of enamel. RESULTS: The experimental solution mixes well with eosin, neutral fuchsine, and methylene blue. CONCLUSIONS: During the preliminary study, the authors concluded that the experimental solution mixes well with methylene blue, neutral fuchsine, and eosin. An addition of eosin to a preparation which infiltrates inner, demineralized enamel layers, facilitates the assessment of such a preparation with an optical microscope. A designed experimental solution with the main ingredients, i.e., 2-hydroxyethyl methacrylate (HEMA) and tetraethylene glycol dimethacrylate (TEGDMA) with a ratio of 75% to 25% penetrates the demineralized (decalcified) inner parts of the enamel and polymerizes when exposed to light. In order to assess the infiltration of the experimental solution into the demineralized enamel layers, it is required to improve the measurement techniques that utilize optical microscopy.

[Effect of root canal sealers on biocompatibility of human periodontal ligament cells].

OBJECTIVE: To compare the effects of three root canal sealers with respect to time on biocompatibility of human periodontal ligament cells (hPDLCs).The sealers included zinc oxide and eugenol based sealers (ZOE), epoxy resin sealers (ERS) and silicone based sealers (SBS). METHODS: hPDLCs were primarily cultured,with the method combining of tissue explant and enzymatic digestion. The cells were then exposed to different extract fluids: (1) ZOE extracted for 24 h group ;(2) ZOE extracted for 1 week group;(3)ZOE extracted for 2 weeks group;(4) ERS extracted after 24 h group; (5) ERS extracted after 1 week group; (6) ERS extracted after 2 weeks group; (7) SBS extracted after 24 h group; (8) SBS extracted after 1 week group; (9) SBS extracted after 2 weeks group; (10) Dulbecco modified Eagle's medium/F12 (DMEM/F12) as negative control group. Cell morphology was observed under an inverted microscope.Cell proliferation was measured by methyl-thiazol-diphenyltetrazolium (MTT) assay. ALP assay kit was used for measuring alkaline phosphatase (ALP) activity. Sealers of 2 weeks' setting time were then immersed in an osteogenic medium for examination of mineral nodules and calcium deposits. RESULTS: Considering the relative growth rate(RGR),ZOE was severely to moderately cytotoxic(RGR:13.6%-39.9%), while ERS was slightly or not cytotoxic (RGR: 87.6%-95.3%).Only SBS did not show any cytotoxicity after setting (RGR: 91.8%-106.7%). The setting time influenced the cytotoxicity of ERS which decreased after 1 week. Considering the ALP activity,there was no difference between SBS group and control group (F=3.397,P=0.053). According to the results of calcium deposits, ZOE:D562 nm=0.180±0.050,ERS: D562 nm=2.968±0.201,SBS:D562 nm=3.623±0.039,Control:D562 nm=3.477±0.102,the ranking of ALP activity and calcium deposits was as follows: ZOE

Biocompatibility Evaluation of EndoSequence Root Repair Paste in the Connective Tissue of Rats.

INTRODUCTION: The aim of this study was to evaluate the subcutaneous connective tissue response to EndoSequence root repair paste (Brasseler, Savannah, GA) compared with mineral trioxide aggregate (MTA). METHODS: Thirty-six Wistar rats each received 3 sterile tubes, containing 1 of the tested materials and control. The animals were killed 1, 3 and 6 weeks after implantation. The specimens were evaluated histologically for type of inflammation, intensity and extent of inflammatory cells, foreign body reaction, fibrous capsule thickness, perivascular fragments, calcific deposits and vascular congestion. RESULTS: EndoSequence provoked severe inflammation after 1 week, which was significantly different from MTA and control (P ˂ .05), with fragmented particles and foreign body reaction. MTA showed tissue-tolerance features almost comparable to control. CONCLUSIONS: EndoSequence was significantly more irritating than MTA and control at 1 and 3 weeks in terms of severity and extent of inflammation. After 6 weeks it displayed more biocompatible characteristics.

Grading and quantification of dental fluorosis in zebrafish larva.

OBJECTIVE: The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment. MATERIALS AND METHODS: After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique. RESULTS: With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis. CONCLUSIONS: Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis.

Mineralization Effect of Hyaluronan on Dental Pulp Cells via CD44.

INTRODUCTION: CD44 is a cell-surface glycoprotein involved in various cellular functions. Recent studies have suggested that CD44 is involved in early mineralization of odontoblasts. Hyaluronic acid (HA) is the principal ligand for receptor CD44. Whether and how HA regulated the mineralization process of dental pulp cells were investigated. METHODS: The effects of high-molecular-weight HA on differentiation and mineral deposition of dental pulp cells were tested by using alkaline phosphatase (ALP) activity assay and alizarin red S staining. Osteogenesis real-time polymerase chain reaction array, quantitative polymerase chain reaction, and Western blotting were performed to identify downstream molecules involved in the mineralization induction of HA. CD44 was knocked down and examined to confirm whether the mineralization effect of HA was mediated by receptor CD44. Immunohistochemistry was used to understand the localization patterns of CD44 and the identified downstream proteins in vivo. RESULTS: Pulse treatment of HA enhanced ALP activity and mineral deposition in dental pulp cells. Tissue-nonspecific ALP, bone morphogenetic protein 7 (BMP7), and type XV collagen (Col15A1) were upregulated via the HA-CD44 pathway in vitro. Immunohistochemistry of tooth sections showed that the staining pattern of BMP7 was very similar to that of CD44. CONCLUSIONS: Results of this study indicated that high-molecular-weight HA enhanced early mineralization of dental pulp cells mediated via CD44. The process involved important mineralization-associated molecules including tissue-nonspecific ALP, BMP7, and Col15A1. The findings may help develop new strategies in regenerative endodontics.

Apical Closure in Apexification: A Review and Case Report of Apexification Treatment of an Immature Permanent Tooth with Biodentine.

Materials such as calcium hydroxide paste and mineral trioxide aggregate are used in apexification treatment of immature permanent teeth, but the search for improved materials with higher characteristics of biocompatibility results in different materials. Biodentine is a tricalcium silicate cement that possesses adequate handling characteristics and acceptable mechanical and bioactivity properties. This report describes the case of a 9-year-old boy who was referred to the Department of Dental Clinic of Querétaro Autonomous University of Mexico. One month prior the patient had suffered a dental trauma of his upper left central incisor and had been treated by another dentist. The clinical diagnosis was previously initiated therapy and symptomatic apical periodontitis. The treatment was apexification with Biodentine. At follow-ups performed at 3, 6, and 18 months after treatment the tooth was asymptomatic. The cone-beam computed tomography scan at 18-month postoperative follow-up revealed continuity of periodontal ligament space, absence of periapical rarefactions, and a thin layer of calcified tissue formed apical to the Biodentine barrier. On the basis of sealing ability and biocompatibility, apexification treatment with Biodentine was applied in the present case report. The favorable clinical and radiographic outcome in this case demonstrated that Biodentine may be an efficient alternative to the conventional apexification materials.

Hypertension Undermines Mineralization-inducing Capacity of and Tissue Response to Mineral Trioxide Aggregate Endodontic Cement.

INTRODUCTION: This study evaluated the effect of hypertension on tissue response to and mineralization capacity of white and gray mineral trioxide aggregate (MTA) (Angelus Industry Ontological Products, Londrina, Brazil), an endodontic reparative cement. METHODS: Polyethylene tubes containing gray MTA, white MTA, or intermediate restorative material (positive control) or an empty tube (negative control) were implanted into the dorsal connective tissue of spontaneous hypertensive and Wistar rats (n = 12 each). Six rats in each group were sacrificed after 7 days, and the remainder after 30 days. Tubes with surrounding tissue were removed, and a histologic analysis was performed using hematoxylin-eosin and von Kossa staining and examination by polarized light microscopy. RESULTS: The inflammatory response to all materials was greater in hypertensive compared with normotensive rats (P < .05). Positive von Kossa staining and birefringent structures in polarized light were observed for both gray and white MTA (P > .05), but these were more pronounced in normotensive rats (P < .05). Necrotic areas with positive von Kossa staining were observed for intermediate restorative material. CONCLUSIONS: Hypertension undermines tissue repair and mineralization, which can negatively affect treatment outcome. Nonetheless, mineralization in response to MTA was observed even under hypertensive conditions.

Dentin matrix components extracted with phosphoric acid enhance cell proliferation and mineralization.

OBJECTIVE: Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations. METHODS: DMC were solubilized from powdered human dentin (7 days - 4°C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1×10(5)) and cultured for 24h in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0µg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10vol% Alamar blue solution, which was added to each well for 1h. Cell numbers were first measured by cell counting (Trypan blue; n=5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC (n=5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's (p≤0.05). RESULTS: There was a linear correlation between cell counts and Alamar blue fluorescence (R(2)>0.96 for both cell types) , verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0µg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10µg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts. SIGNIFICANCE: Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration.