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Objective Fluoride (F)-releasing restoratives typically are either weak mechanically or release only low levels of F ions. The objectives of this study were to: (1) develop a novel photo-cured nanocomposite with strong mechanical properties and high levels of sustained F ion release via a two-step “co-precipitation + spray-drying” technique to synthesize CaF2 nanoparticles (nCaF2); and (2) investigate the effect of spray-drying treatment after co-precipitation of nCaF2 on mechanical properties and F ion release of composite. Methods Two types of CaF2 particles were synthesized: A co-precipitation method yielded CaF2cp; “co-precipitation + spray-drying” yielded nCaF2cpsd. Composites were fabricated with fillers of: (1) 0% CaF2 + 70% glass; (2) 10% CaF2cp + 60% glass; (3) 15% CaF2cp + 55% glass; (4) 20% CaF2cp + 50% glass; (5) 10% nCaF2cpsd + 60% glass; (6) 15% nCaF2cpsd + 55% glass; and (7) 20% nCaF2cpsd + 50% glass. A commercial F-releasing nanocomposite served as control. Results The nCaF2cpsd had much smaller particle size (median = 32 nm) and narrower distribution (22–57 nm) than CaF2cp (median = 5.25 μm, 162 nm–67 μm). The composite containing nCaF2cpsd had greater flowability, flexural strength, elastic modulus and hardness than CaF2cp composite and commercial control composite. At 84-day immersion in water, the nanocomposites containing 20% nCaF2cpsd had 65 times higher cumulative F release, and 77 times greater long-term F-release rate, than commercial control. Conclusions A novel two-step “co-precipitation + spray-drying” technique of synthesizing nCaF2 was developed. The photo-cured nanocomposite containing 20% nCaF2cpsd possessed strong mechanical properties and excellent long-term F-release ability, and hence is promising for dental restoration applications to inhibit secondary caries.

Objectives. This study is aimed at investigating the root and root canal morphology by cone beam computed tomography (CBCT) and palatal furcation groove of the buccal root by microcomputed tomography (micro-CT) of maxillary first premolars in a Chinese subpopulation. Methods. This study assessed CBCT images of 440 patients aged 14-80 years. Based on Vertucci’s classification, the number of roots and the canal configuration were determined. Forty-eight maxillary first premolars with furcation grooves were analyzed by micro-CT in patients aged 18-25 years. Results. Based on the CBCT assay, 70.22% and 29.32% of maxillary first premolars were 1 root and 2 roots, respectively. The configuration indicated statistical difference ( P < 0.05 ) between male and female patients. The most common canal type was type IV and was found in 44.32% of cases, followed by type I in 27.84%, and then type II in 20.57%. Root bifurcations had 40.13% prevalence which was distributed more in the middle third than in the cervical and the apical third. For the micro-CT study, 95.83% of the furcation groove configuration was found in the bifurcated maxillary first premolars. The length varied from 1.02 to 7.63 mm. The mean depth of this groove was 0.57 mm in the root coronal, 0.47 mm in the root middle, and 0.22 mm in the root apical level. Palatal dentin width was smaller than 1 mm. Conclusion. The anatomy of the root and root canal system and the irregular wall width of maxillary first premolars with furcation grooves may help dentists to understand the anatomical morphology and improve the outcomes of endodontic treatment.

Objective Pit and fissure sealants with antibacterial and remineralization properties have broad application prospects in caries prevention. The objectives of this study were to: (1) develop a novel pit and fissure sealant containing CaF2 nanoparticles (nCaF2) and dimethylaminohexadecyl methacrylate (DMAHDM); and (2) investigate the effects of nCaF2 and DMAHDM on biofilm response and fluoride (F) ion release for the first time. Methods Helioseal F was used as a control. Bioactive sealants were formulated with DMAHDM and nCaF2. Flow properties, enamel shear bond strength, hardness and F ion releases were measured. Streptococcus mutans (S. mutans) biofilms were grown on sealants. Biofilm metabolic activity, lactic acid production, colony-forming units (CFU), and pH of biofilm culture medium were measured. Results Adding 5% DMAHDM and 20% nCaF2 did not reduce the paste flow and enamel bond strength, compared to control (p Significance The new bioactive pit and fissure sealant with nCaF2 and DMAHDM achieved high fluoride release and strong antibacterial performance. This novel fluoride-releasing and antibacterial sealant is promising to inhibit caries and promote the remineralizaton of enamel and dentin.

Objective Secondary caries and micro-cracks are the main limiting factors for dentin bond durability. The objectives of this study were to develop a self-healing adhesive containing dimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP), and investigate the effects of water-aging for 12 months on self-healing, dentin bonding, and antibacterial properties for the first time. Methods Microcapsules were synthesized with poly (urea-formaldehyde) (PUF) shells containing triethylene glycol dimethacrylate (TEGDMA) and N,N -dihydroxyethyl- p -toluidine (DHEPT). The adhesive contained 7.5% microcapsules, 10% DMAHDM, and 20% NACP (all mass). Specimens were water-aged at 37 °C for 1 day to 12 months. Dentin bond strength was measured using extracted human teeth. A single-edge-V-notched-beam (SEVNB) method was used to measure fracture toughness K IC and self-healing efficiency. A dental plaque microcosm biofilm model was used with human saliva as inoculum. Results The microcapsules + DMAHDM + NACP group showed no decline in dentin bond strength after water-aging for 12 months, which was significantly higher than that of other groups without DMAHDM ( p IC was obtained even after 12 months of water immersion, indicating that the self-healing ability was not lost in water-aging ( p > 0.1). The bacteria-killing ability of this adhesive did not decline from 1 day to 12 months ( p > 0.1), with biofilm CFU reduction by 3–4 orders of magnitude after the resin was water-aged for 12 months, compared to control resin. Significance This novel adhesive with triple merits of self-healing, antibacterial and remineralization functions showed an excellent long-term durability in water-aging for 12 months. This multifunctional adhesive has the potential for dental applications to heal cracks, inhibit bacteria, provide ions for remineralization, and increase the restoration longevity.

Objective Calcium phosphate cements (CPCs) mimic nanostructured bone minerals and are promising for dental, craniofacial and orthopedic applications. Vascularization plays a critical role in bone regeneration. This article represents the first review on cutting-edge research on prevascularization of CPC scaffolds to enhance bone regeneration. Methods This article first presented the prevascularization of CPC scaffolds. Then the co-culture of two cell types in CPC scaffolds was discussed. Subsequently, to further enhance the prevascularization efficacy, tri-culture of three different cell types in CPC scaffolds was presented. Results (1) Arg–Gly–Asp (RGD) incorporation in CPC bone cement scaffold greatly enhanced cell affinity and bone prevascularization; (2) By introducing endothelial cells into the culture of osteogenic cells (co-culture of two different cell types, or bi-culture) in CPC scaffold, the bone defect area underwent much better angiogenic and osteogenic processes when compared to mono-culture; (3) Tri-culture with an additional cell type of perivascular cells (such as pericytes) resulted in a substantially enhanced prevascularization of CPC scaffolds in vitro and more new bone and blood vessels in vivo, compared to bi-culture. Furthermore, biological cell crosstalk and capillary-like structure formation made critical contributions to the bi-culture system. In addition, the pericytes in the tri-culture system substantially promoted stability and maturation of the primary vascular network. Significance The novel approach of CPC scaffolds with stem cell bi-culture and tri-culture is of great significance in the regeneration of dental, craniofacial and orthopedic defects in clinical practice.

The dentin bonding often fails over time, leading to secondary caries and restoration failure. The objectives of this study were to develop an adhesive with dimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP), and investigate the effects of storage in artificial saliva for six months on the bonding durability, antibacterial activity, ion release and biofilm pH properties for the first time.DMAHDM was added at 5% (by mass) to Scotchbond Primer and Adhesive (SBMP). NACP was added at 10%, 20%, and 30% to SBMP adhesive. Dentin bonding durability, antibacterial activity against Streptococcus mutans biofilms, and calcium (Ca) and phosphate (P) ion liberation properties were investigated after 1 day and 6months of storage in artificial saliva.Dentin bond strength (n = 50) had 25% loss after 6 months of aging for SBMP control. However, SBMP + DMAHDM+10NACP and SBMP + DMAHDM+20NACP showed no loss in bond strength after storage in artificial saliva for 6 months. The DMAHDM + NACP incorporation method dramatically reduced the biofilm metabolic activity and acid production, and decreased the biofilm CFU by four orders of magnitude, compared to SBMP control, even after 6 months of aging (p0.05). DMAHDM + NACP had long-lasting Ca and P ion releases, and raised the biofilm pH to 6.8, while the control group had a cariogenic biofilm pH of 4.5.Incorporating DMAHDM + NACP in bonding agent yielded potent and long-lasting antibacterial activity and ions liberation ability, and much higher long-term dentin bond strength after 6-month of aging. The new bonding agent is promising to inhibit caries at the restoration margins and increase the resin-dentin bonding longevity.The novel bioactive adhesive is promising to protect tooth structures from biofilm acids and secondary caries. NACP and DMAHDM have great potential for applications to a wide range of dental materials to reduce plaque and achieve therapeutic effects.

OBJECTIVES: A nanoparticle-doped adhesive that can be controlled with magnetic forces was recently developed to deliver drugs to the pulp and improve adhesive penetration into dentin. However, it did not have bactericidal and remineralization abilities. The objectives of this study were to: (1) develop a magnetic nanoparticle-containing adhesive with dimethylaminohexadecyl methacrylate (DMAHDM), amorphous calcium phosphate nanoparticles (NACP) and magnetic nanoparticles (MNP); and (2) investigate the effects on dentin bond strength, calcium (Ca) and phosphate (P) ion release and anti-biofilm properties. METHODS: MNP, DMAHDM and NACP were mixed into Scotchbond SBMP at 2%, 5% and 20% by mass, respectively. Two types of magnetic nanoparticles were used: acrylate-functionalized iron nanoparticles (AINPs); and iron oxide nanoparticles (IONPs). Each type was added into the resin at 1% by mass. Dentin bonding was performed with a magnetic force application for 3 minutes, provided by a commercial cube-shaped magnet. Dentin shear bond strengths were measured. Streptococcus mutans biofilms were grown on resins, and metabolic activity, lactic acid and colony-forming units (CFU) were determined. Ca and P ion concentrations in, and pH of biofilm culture medium were measured. RESULTS: Magnetic nanoparticle-containing adhesive using magnetic force increased the dentin shear bond strength by 59% over SBMP Control (p < 0.05). Adding DMAHDM and NACP did not adversely affect the dentin bond strength (p > 0.05). The adhesive with MNP+DMAHDM+NACP reduced the Streptococcus mutans biofilm CFU by 4 logs. For the adhesive with NACP, the biofilm medium became a Ca and P ion reservoir. The biofilm culture medium of the magnetic nanoparticle-containing adhesive with NACP had a safe pH of 6.9, while the biofilm medium of commercial adhesive had a cariogenic pH of 4.5. CONCLUSIONS: Magnetic nanoparticle-containing adhesive with DMAHDM and NACP under a magnetic force yielded much greater dentin bond strength than commercial control. The novel adhesive reduced biofilm CFU by 4 logs and increased the biofilm pH from a cariogenic pH 4.5 to 6.9, and therefore is promising to enhance the resin-tooth bond, strengthen tooth structures, and suppress secondary caries at the restoration margins.

Decreased expression and increased phosphorylation of Forkhead box o1 (Foxo1) in ameloblasts were observed both in vivo and in vitro when treated by fluoride. The present study aims to investigate the possible relationship between Foxo1 and enamel matrix proteinases, matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), in NaF-treated ameloblasts. Ameloblast-like cells (LS8 cells) were exposed to NaF at selected concentration (0/2 mM) for 24 h. Gene overexpression and silencing experiments were used to up- and down-regulate Foxo1 expression. The expression levels of Foxo1, MMP20, and KLK4 were detected by quantitative real-time PCR and western blot. Dual luciferase reporter assay was performed to evaluate the regulation of Foxo1 on the transcriptional activity of KLK4 promoter. The results showed that KLK4 expression was decreased in LS8 cells treated by NaF, while MMP20 expression was not changed. Foxo1 activation led to significantly up-regulation of KLK4 in LS8 cells under NaF condition. Knockout of Foxo1 markedly decreased klk4 expression in mRNA level, and intensified inhibition occurred in LS8 cells when combined with NaF treatment. However, the variation trend of MMP20 was not clear. Dual luciferase reporter assay showed that Foxo1 activation enhanced the transcriptional activity of KLK4 promoter. These findings suggest that the decrease of Foxo1 expression induced by high fluoride was a cause for low KLK4 expression.

To investigate the prevalence and intra-oral distribution of dentine hypersensitivity (DH) and to evaluate the related risk factors.A total of 1320 subjects, aged between 20 and 69 years old, were selected from six communities in the urban areas of Xi'an, China. The data were collected by conducting individual interviews using a standard questionnaire; then, the clinical examination was performed for patients who reported about the discomforts they felt in their teeth when subjected to chemical, mechanical and thermal stimuli. Dentine hypersensitivity (DH) was diagnosed by a subject short, sharp pain in response to a blast of cold air from a triple syringe.While replying to the questionnaire, 445 subjects reported about signs of discomfort in the teeth. DH was diagnosed in 336 persons by clinic examination. Thus, the overall prevalence of DH was 33.7% in the questionnaire and 25.5% in the intraoral test. The prevalence of DH was higher in females (33.8%) than in males (22.2%). Furthermore, we found that the prevalence of DH was highest in the age group of 50-59 years (39.3%). The most common initiation factors were acid (37.7%) followed by cold stimuli (35.8%). In general, most subjects with sensitive teeth had a higher educational background.The prevalence of DH was 25.5% in the population of Xi'an City in China. More emphasis should give to middle-aged and old females while planning oral health intervention campaigns. In addition, premolars and cervical surfaces should be examined for the prevention of DH.

Objective Human periodontal ligament stem cells (hPDLSCs) are promising for periodontal regeneration. However, to date, there has been no report of hPDLSC differentiation into the fibrogenic lineage. There has been no report demonstrating hPDLSC differentiation into all three (osteogenic, fibrogenic and cementogenic fibrogenic) lineages in the same report. The objectives of this study were to harvest hPDLSCs from the periodontal ligaments (PDL) of the extracted human teeth, and use the same vial of hPDLSCs to differentiate into all three (osteogenic, fibrogenic and cementogenic) lineages for the first time. Methods hPDLSCs were harvested from PDL tissues of the extracted premolars. The ability of hPDLSCs to form bone, cementum and collagen fibers was tested in culture mediums. Gene expressions were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Immunofluorescence, alizarin red (ARS), Xylenol orange, picro sirius red staining (PSRS), alcian blue staining (ABS) and alkaline phosphatase (ALP) staining were evaluated. Results In osteogenic medium, hPDLSCs had high expressions of osteogenic genes (RUNX2, ALP, OPN and COL1) at 14 and 21 days (15–20 folds of that of control), and produced mineral nodules and ALP activity (5 and 10 folds those of the control). hPDLSCs in fibrogenic medium expressed high levels of PDL fibrogenic genes (COL1, COL3, FSP-1, PLAP-1 and Elastin) at 28 days (20–70 folds of control). They were stained strongly with F-actin and fibronection, and secreted PDL collagen fibers (5 folds of control). hPDLSCs in cementogenic medium showed high expressions of cementum genes (CAP, CEMP1 and BSP) at 21 days (10–15 folds of control) and synthesized mineralized cementum (50 folds via ABS, and 40 folds via ALP staining, compared to those of control). Conclusions hPDLSCs differentiated into bone-, fiber- and cementum-forming cells, with potential for regeneration of periodontium to form the bone-PDL-cementum complex.

Objectives. To investigate the root morphology and root canal anatomy of maxillary first premolar using microscopic computed tomography (micro-CT). Methods. 324 maxillary first premolars were collected and scanned. The root and canal diameter, canal wall thickness, root taper, and cross-sectional shapes were determined in the single root with 1 canal (SR1C), single root with 2 canals (SR2C), and 2 roots with 2 canals (2R2C) by micro-CT. Results. The results showed that single-rooted maxillary premolars were more common than other types. The incidence of SR1C, SR2C, and 2R2C reached 25%, 26.39%, and 26.39%, respectively. Root and canal diameters and canal wall thickness were decreased from coronal third to apical foramen. The three parameters and canal taper showed increases from buccal and palatal (BP) to mesiodistal (MD) aspects. The root canal tapers were smallest of the middle third level. The findings showed the different variations in 2R2C teeth. The root canal cross-sectional morphology in maxillary first premolars is complicated, including round, oval, long oval, flat canal, and irregular canal shapes. The distribution varied in different aspects. Conclusion. Root canal morphology showed a wide variation and complicated structure. The single-rooted teeth were more common in the Chinese adolescent population, and the majority of maxillary first premolars have two canals.

Objectives The present study aimed to investigated the effect and mechanism of Ca2+ treatment on fluoride in ameloblast-lineage cells (ALCs). Materials and methods The effects of fluoride and different Ca2+ levels treatment on the proliferative activity, cell apoptosis, cell cycle, intracellular free Ca2+, were firstly determined. Kallikrein 4 (KLK4), glucose-responsive protein 78 (GRP78), Protein kinase R -like endoplasmic reticulum kinase (PERK), the α subunit of eukaryotic initiation factor 2 (eIF2α), activating transcription factor 4 (ATF4), CCAAT enhancer-binding protein homologous protein (CHOP), were investigated in ALCs. Results The proliferative activity was obviously inhibited under concentrations of single fluoride high than 1 mM, and indicated highest proliferation at single 2.5 mM Ca2+ concentration in ALC cells. In addition, we found that single fluoride markedly induced intracellular free Ca2+ increasing, G2/M phase arrest, apoptosis. GRP78 and endoplasmic reticulum stress pathway of PERK/eIF2α/ATF4/CHOP were significantly increased, while the proliferation and KLK4 were markedly reduced in ALCs. Ca2+ additional treatment can obviously reverse the effect of fluoride-induced apoptosis and inhibition of KLK4. The effect of GRP78 and endoplasmic reticulum stress pathway of PERK/eIF2α/ATF4/CHOP were also alleviated under Ca2+ additional treatment in ALCs. More important, the results of 2.5 mmol/L Ca2+ treatment on the proliferation, cell cycle and apoptosis suggest this concentration is relatively better to mediate the intracellular Ca2+ homeostasis in ALCs. Conclusions In sum, Ca2+-supplementation exerts antagonistic the toxic effects on fluoride and this inhibitory effect suggests the potential implications for Ca2+-supplementation on fluorosis.

Objectives To investigate the effect and mechanism of calcium on LS8 cell differentiation, especially on phosphatidylinositol 3 kinase (PI3K) /protein kinase B(AKT) pathway. Materials and methods Ameloblast-like LS8 cell line was used and additional 0–3.5 mmol/L calcium chloride was treated for 24 h, 48 h. Cell viability and morphological changes, cell cycle and associated regulatory proteins were analyzed. Results No significant effects on morphological changes were observed. Decreased cell viability and increased S phase cells were accompanied by the significant decrease of cyclin A and cyclin B proteins, and significant increase of cyclin D protein in LS8 cells. Additionally, kallikrein-4 and amelotin expressions were significantly increased. Finally, the levels of PI3K, AKT, p-AKT and forkhead box O3 (FOXO3) significantly downregulated after calcium treatment in LS8 cells. Conclusions Calcium inhibit proliferation and promotes differentiation in LS8 cells, this is closely related to the downregulation of PI3K/AKT signal in LS8 cells.

Secondary caries is a primary reason for dental restoration failures. The objective of this study was to investigate the remineralization of human dentin lesions in vitro via restorations using nanocomposites containing nanoparticles of amorphous calcium phosphate (NACP) or NACP and tetracalcium phosphate (TTCP) for the first time.NACP was synthesized by a spray-drying technique and incorporated into a resin consisting of ethoxylated bisphenol A dimethacrylate (EBPADMA) and pyromellitic glycerol dimethacrylate (PMGDM). After restoring the dentin lesions with nanocomposites as well as a non-releasing commercial composite control, the specimens were treated with cyclic demineralization (pH 4, 1h per day) and remineralization (pH 7, 23h per day) for 4 or 8 weeks. Calcium (Ca) and phosphate (P) ion releases from composites were measured. Dentin lesion remineralization was measured at 4 and 8 weeks by transverse microradiography (TMR).Lowering the pH increased ion release of NACP and NACP-TTCP composites. At 56 days, the released Ca concentration in mmol/L (mean±SD; n=3) was (13.39±0.72) at pH 4, much higher than (1.19±0.06) at pH 7 (p0.05). At 56 days, P ion concentration was (5.59±0.28) at pH 4, much higher than (0.26±0.01) at pH 7 (p0.05). Quantitative microradiography showed typical subsurface dentin lesions prior to the cyclic demineralization/remineralization treatment, and dentin remineralization via NACP and NACP-TTCP composites after 4 and 8 weeks of treatment. At 8 weeks, NACP nanocomposite achieved dentin lesion remineralization (mean±SD; n=15) of (48.2±11.0)%, much higher than (5.0±7.2)% for dentin in commercial composite group after the same cyclic demineralization/remineralization regimen (p0.05).Novel NACP-based nanocomposites were demonstrated to achieve dentin lesion remineralization for the first time. These results, coupled with acid-neutralization and good mechanical properties shown previously, indicate that the NACP-based nanocomposites are promising for restorations to inhibit caries and protect tooth structures.

Objective The aim of the present study was to select the optimal concentration of TiF 4 solution to facilitate the remineralization of early dentine caries lesions. Design Sixty human dentine specimens were cut and randomly divided into 6 groups (1%, 2%, 3%, 4% TiF 4 groups, 2.712% NaF group and distilled deionized water (DDW) control group). Artificial dentine caries-like lesions were created. After being subjected to fluoride treatment and immersed in remineralizing solution for 2 weeks, the specimens were observed by microCT, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Data were analysed using linear regression analysis ( P Results The lesion depths of the specimens treated by 2% TiF 4 solution were statistically less than those of the other groups. Further, the greyscale values of these lesion areas were greater. The 3% and 4% TiF 4 solutions caused further lesion demineralization. The 2.712% NaF solution seemed to be detrimental to remineralization during the experimental time, as the subsurface area remained hypomineralized with a thick precipitation layer on the surface. Conclusions The 2% TiF 4 solution demonstrated better remineralizing potency than did the other treatments.