Angelo Putignano

A double‐blind randomized‐controlled trial comparing the desensitizing efficacy of a new dentifrice containing carbonate/hydroxyapatite nanocrystals and a sodium fluoride/potassium nitrate dentifrice

BACKGROUND AND AIM Several dentifrices have shown to be effective in reducing dentine hypersensitivity (DH), but more effective products are needed. The aim of the study was to evaluate the desensitizing efficacy of a new dentifrice based on zinc-carbonate hydroxyapatite (CHA) nanocrystals. METHODS AND MATERIALS Using a double-blind, randomized design, the new dentifrice was compared with potassium nitrate/fluoride dentifrice (active control). The participants DH was evaluated at baseline and after 4 and 8 weeks using airblast (primary outcome), tactile, cold water and subjective tests (secondary outcomes). RESULTS The final sample consisted of 70 subjects with baseline DH; 36 received the new dentifrice and 34 the control one. Both dentifrices were largely effective; the percentage of score reduction from baseline to 8 weeks was greater than 28% for all tests (and greater than 55% for the cold water test) in both groups. As compared with controls, experimental subjects had a significantly greater improvement in the airblast test score (mean percentage of reduction of 46.0%versus 29.4% in controls) and the subjective test score (47.5%versus 28.1%, respectively), with both differences already being significant after 4 weeks. In contrast, there was no significant difference between groups for either the tactile or cold water tests at any time point and with any outcome. CONCLUSIONS This study documented that the new dentifrice containing zinc-CHA nanocrystals significantly reduced dentinal hypersensitivity after 4 and 8 weeks, supporting its utility in clinical practice.

Immunohistochemical localization of dentin matrix protein 1 in human dentin

Dentin matrix protein 1 (DMP1) is a non-collagenous matrix protein with a recognized role in the formation of mineralized tissues such as dentin. The aim of this study was to analyze the distribution of DMP1 in human dentin by means of immunofluorescence and high-resolution immunogold labeling. Fully developed, sound human dentin specimens were submitted to fluorescence labeling and post-embedding immunolabeling techniques with a rabbit polyclonal antihuman DMP1 antibody followed by corresponding fluorochrome-conjugated or gold-conjugated secondary antibodies. Both immunofluorescence and immunogold labeling showed an intense labeling associated with the peritubular dentin. In addition, at the ultrastructural level, there was also a moderate and diffuse immunoreaction over intertubular dentin, and a weak labeling within predentin which increased in density towards the mineralization front. This study suggests that in adult human teeth, like in rodents, DMP1 is prevalently concentrated at the level of peritubular dentin and this feature is preserved also in fully developed-teeth. These data are consistent with what has been observed in rodents and suggest that DMP1 plays a role in maintenance of the dentin tubular space.

Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: a comparative in vivo study

Consumption of acidic foods and drinks and other factors that cause enamel wear are responsible for the daily enamel loss and degradation. Use of some toothpastes that have been showed to possess different properties of remineralisation and/or repair of the enamel surface may help to protect tooth enamel. The aim of this study was to evaluate whether the use of toothpaste containing Zn-carbonate hydroxyapatite (CHA) nanostructured microcrystals may exert remineralization/repair effects of the enamel surface. Two groups of patients, aged between 18 and 75 years, used a Zn-CHA nanocrystals-based toothpaste (experimental group) and a potassium nitrate/sodium fluoride toothpaste (active control group) for 8 weeks. At the end of this period, extractions were performed in five subjects per study group. Negative controls consisted of two subjects treated with non-specified fluoride toothpaste. Teeth were processed for morphological and chemical-physic superficial characterizations by means of Scanning Electronic Microscopy with Elementary analysis, X-Ray Diffraction analysis and Infrared analysis. In this study, the use of a Zn-CHA nanocrystals toothpaste led to a remineralization/repair of the enamel surface, by deposition of a hydroxyapatite-rich coating. On the other hand, the use of both a nitrate potassium/sodium fluoride and non-specified fluoride toothpastes did not appreciably change the enamel surface. In conclusion, this study demonstrates that the toothpaste containing Zn-CHA nanostructured microcrystals, differently from nitrate potassium/sodium fluoride and non-specified fluoride toothpastes, may promote enamel superficial repair by means of the formation of a protective biomimetic CHA coating.

Dicalcium phosphate (CaHPO4·2H2O) precipitation through ortho- or meta-phosphoric acid-etching: effects on the durability and nanoleakage/ultra-morphology of resin-dentine interfaces.

OBJECTIVES To compare the effects of two etching procedures using meta-phosphoric (MPA) or ortho-phosphoric acid (OPA) on dentine demineralisation, resin-dentine bonds durability and interface nanoleakage/ultra-morphology. METHODS Middle-dentine specimens were etched using 37% OPA (15s) or 40% MPA (60s) and submitted to infrared spectroscopy (FTIR) or ultra-morphology dye-assisted (calcium-staining) confocal microscopy (Ca-CLSM). A three-step etch-and-rinse adhesive was formulated, applied onto dentine and light-cured for 30s before composite build-up. After 24h, the dentine-bonded specimens were cut into 1mm(2) beams; half were immediately submitted to microtensile bond strength (μTBS) and half stored in DW for six months. The μTBS results were analysed with repeated-measures ANOVA and Tukeys test (p<0.05). Further teeth were bonded and prepared for interface nanoleakage/ultra-morphology confocal evaluation. RESULTS FTIR and Ca-CLSM analyses showed dicalcium phosphate dihydrate (Brushite) precipitation in MPA-etched dentine and on the bottom (front of demineralisation) of the OPA-etched dentine. Statistical analysis showed similar μTBS for both etching procedures after 24h. The μTBS of specimens in OPA-group dropped significantly (p<0.05) after six month; the specimens in the MPA group showed no statistically difference (p>0.05). CLSM depicted no evident sign of nanoleakage within the resin-dentine interface of the MPA-treated specimens, while the specimens in OPA-group presented intense nanoleakage and interface degradation. CONCLUSION The use of MPA (60s) as an alternative dentine conditioning agent in etch-and-rinse bonding procedures may be a suitable strategy to create more durable resin-dentine bonds.

Matrix Metalloproteinase-2 Expression Induced by Two Different Adhesive Systems on Human Pulp Fibroblasts

INTRODUCTION This study evaluated the expression of matrix metalloproteinase-2 (MMP-2) in primary cultures of human pulp fibroblasts (HPFs) when exposed to extracts from dentin-bonding systems. METHODS Polymerized resin disks of the bonding agent of a 2-step self-etch adhesive (TechBond, Isasan, Rovello Porro, Italy) or of the primer/bonding agent a 2-step etch-and-rinse adhesive (Optibond Solo; Sybron-Kerr, Orange, CA) were immersed in HPF culture medium for 24 or 96 hours. HPFs were incubated in the adhesive-conditioned or control (untreated) culture medium for 24 hours. Western blot and immunofluorescence analyses were performed to assay MMP-2 expression. RESULTS MMP-2 expression levels in HPFs cultured for 24 hours in culture medium were similar in both the control and experimental media groups showing a faint band at 67 kDa. Conversely, the HPFs incubated in the medium that contain polymerized resin disks for 96 hours showed increased MMP-2 expression compared with the untreated medium. The self-etch adhesive displayed the most pronounced induction of MMP-2 expression. These findings were confirmed by immunofluorescence analysis. CONCLUSIONS HPFs display increased MMP-2 expression after 96 hours of conditioning of the HPF culture medium with polymerized disks of dentin bonding systems. This MMP-2 expression/activation may represent a defence mechanism exhibited by HPFs towards monomers eluted from the dentin bonding systems.

Immunocytochemical detection of dentin matrix proteins in primary teeth from patients with dentinogenesis imperfecta associated with osteogenesis imperfecta

Dentinogenesis imperfecta determines structural alterations of the collagen structure still not completely elucidated. Immunohisto-chemical analysis was used to assay type I and VI collagen, various non-collagenous proteins distribution in human primary teeth from healthy patients or from patients affected by type I dentinogenesis imperfecta (DGI-I) associated with osteogenesis imperfecta (OI). In sound primary teeth, an organized well-known ordered pattern of the type I collagen fibrils was found, whereas atypical and disorganized fibrillar structures were observed in dentin of DGI-I affected patients. Expression of type I collagen was observed in both normal and affected primary teeth, although normal dentin stained more uniformly than DGI-I affected dentin. Reactivity of type VI collagen was significantly lower in normal teeth than in dentin from DGI-I affected patients (P<0.05). Expressions of dentin matrix protein-1 (DMP1) and osteopontin (OPN) were observed in both normal dentin and dentin from DGI-I affected patients, without significant differences, being DMP1 generally more abundantly expressed. Immuno labeling for chondroitin sulfate (CS) and biglycan (BGN) was weaker in dentin from DGI-I-affected patients compared to normal dentin, this decrease being significant only for CS. This study shows ultra-structural alterations in dentin obtained from patients affected by DGI-I, supported by immunocytochemical assays of different collagenous and non-collagenous proteins.

Antibiofilm Activity of Zinc-Carbonate Hydroxyapatite Nanocrystals Against Streptococcus mutans and Mitis Group Streptococci

Dental plaque is a complex multispecies biofilm consisting of a dense community of interacting bacteria (*10 cells/ mg) embedded in a self-produced polysaccharide matrix [1, 4]. Plaque control is critical for oral health, since plaque bacteria include pathogens involved in both dental caries and periodontal disease—the most prevalent human microbial diseases—in addition to a variety of opportunistic pathogens that cause endocarditis, bacteraemia and septicaemia [10]. Plaque formation follows a regimented pattern of colonisation steps that begin with specific adhesion of pioneer colonizers to the acquired pellicle on the tooth surface followed by adhesion of secondary colonizers through interbacterial coaggregation [6]. Streptococcus mitis, Streptococcus gordonii, Streptococcus oralis and Streptococcus sanguinis, all mitis group streptococci (MGS), are dominant pioneer species capable of irreversible attachment to the acquired pellicle by stereochemical interactions between bacterial adhesins and pellicle receptors [7]. MGS play a key role in early plaque formation and conversion to a community with the potential to cause caries and periodontal disease via interspecies coaggregation and selective recruitment of species [12]. Indeed, once MGS have adhered to the pellicle, they coaggregate in a specific manner with late colonizers—predominantly gram-negative anaerobic species involved in periodontitis such as Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella spp. and Treponema spp.—as well as with cariogenic streptococci of the mutans group such as S. mutans and S. sobrinus [5]. Mutualistic relationships are established between MGS and late colonizers. One of the best characterised coaggregation systems is the one where S. gordonii and S. oralis coaggregate with P. gingivalis, S. gordonii also providing metabolic support for P. gingivalis [9]. A thick biofilm hosting a community of interacting microorganisms eventually forms [2], whose composition becomes stable over time. Inhibition of early biofilm formation by MGS can thus help prevent dental plaque development. This study examines the anti-caries potential of nanocrystals of zinc-carbonate hydroxyapatite (Zn-CHA)—the active component of a recently investigated desensitizing toothpaste (BioRepair Plus; Coswell S.p.A., Funo, Bologna, Italy) [8], by assessing whether they affect biofilm formation by MGS and S. mutans strains in polystyrene microtiter plates at concentrations devoid of antibacterial activity. The streptococcal strains used in this study included: three strains from the American Type Culture Collection (ATCC) [S. oralis ATCC 10557 and S. sanguinis ATCC 10556 (both isolated from cases of endocarditis)], and [S. mutans ATCC 25175 (caries)] and two strains from the collection of the Department of Biomedical Sciences and Public Health of Polytechnic University of Marche [S. gordonii AN-10 and S. mitis AN-12 (dental plaque)]. Streptococci were routinely grown in blood agar base (Oxoid, Basingstoke, UK) supplemented with 5 % defibrinated sheep blood and Brain Heart (BH, Oxoid) broth at 37 C in presence of 5 % CO2. Minimum inhibitory concentrations (MICs) were determined by the agar assay, according to the guide lines of the C. Palmieri G. Magi B. Facinelli (&) Section of Microbiology, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Via Tronto 10/A, 60126 Ancona, Italy e-mail: b.facinelli@univpm.it

Spectroscopic and Mechanical Properties of a New Generation of Bulk Fill Composites

Objectives: The aims of this study were to in vitro evaluate the degree of conversion and the microhardness properties of five bulk fill resin composites; in addition, the performance of two curing lamps, used for composites polymerization, was also analyzed. Materials and Methods: The following five resin-based bulk fill composites were tested: SureFil SDR®, Fill Up!™, Filtek™, SonicFill™, and SonicFill2™. Samples of 4 mm in thickness were prepared using Teflon molds filled in one increment and light-polymerized using two LED power units. Ten samples for each composite were cured using Elipar S10 and 10 using Demi Ultra. Additional samples of SonicFill2, (3 and 5 mm-thick) were also tested. The degree of conversion (DC) was determined by Raman spectroscopy, while the Vickers microhardness (VMH) was evaluated using a microhardness tester. The experimental evaluation was carried out on top and bottom sides, immediately after curing (t0), and, on bottom, after 24 h (t24). Two-ways analysis of variance was applied to evaluate DC and VMH-values. In all analyses, the level of significance was set at p < 0.05. Results: All bulk fill resin composites recorded satisfactory DCs on top and bottom sides. At t0, the top of SDR and SonicFill2 showed the highest DCs-values (85.56 ± 9.52 and 85.47 ± 1.90, respectively), when cured using Elipar S10; using Demi Ultra, SonicFill2 showed the highest DCs-values (90.53 ± 2.18). At t0, the highest DCs-values of bottom sides were recorded by SDR (84.64 ± 11.68), when cured using Elipar S10, and Filtek (81.52 ± 4.14), using Demi Ultra. On top sides, Demi Ultra lamp showed significant higher DCs compared to the Elipar S10 (p < 0.05). SonicFill2 reached suitable DCs also on bottom of 5 mm-thick samples. At t0, VMH-values ranged between 24.4 and 69.18 for Elipar S10, and between 26.5 and 67.3 for Demi Ultra. Using both lamps, the lowest VMH-values were shown by SDR, while the highest values by SonicFill2. At t24, all DC and VMH values significantly increased. Conclusions: Differences in DC and VMH among materials are suggested to be material and curing lamp dependent. Even at t0, the three high viscosity bulk composites showed higher VMH than the flowable or dual curing composites.

Potential bone to implant contact area of short versus standard implants: An in vitro micro-computed tomography analysis

Aim:To compare the available potential bone-implant contact (PBIC) area of standard and short dental implants by micro–computed tomography (&mgr;CT) assessment. Methods:Three short implants with different diameters (4.5 × 6 mm, 4.1 × 7 mm, and 4.1 × 6 mm) and 2 standard implants (3.5 × 10 mm and 3.3 × 9 mm) with diverse design and surface features were scanned with &mgr;CT. Cross-sectional images were obtained. Image data were manually processed to find the plane that corresponds to the most coronal contact point between the crestal bone and implant. The available PBIC was calculated for each sample. Later on, the cross-sectional slices were processed by a 3-dimensional (3D) software, and 3D images of each sample were used for descriptive analysis and display the microtopography and macrotopography. Results:The wide-diameter short implant (4.5 × 6 mm) showed the higher PBIC (210.89 mm2) value followed by the standard (178.07 mm2 and 185.37 mm2) and short implants (130.70 mm2 and 110.70 mm2). Conclusions:Wide-diameter short implants show a surface area comparable with standard implants. Micro-CT analysis is a promising technique to evaluate surface area in dental implants with different macrodesign, microdesign, and surface features.

Novel hydroxyapatite biomaterial covalently linked to raloxifene

Since raloxifene, a drug used in osteoporosis therapy, inhibits osteoclast, but not osteoblast functions, it has been suggested to improve recovery during implant surgery. The present paper describes an effective method to link raloxifene, through a covalent bond, to a nano-Hydroxyapatite-based biomaterial by interfacing with (3-aminopropyl)-Triethoxysilane as assessed by Infra Red-Fourier Transformed (IR-FT) spectroscopy and Scanning Electron Microscope (SEM). To evaluate the safety of this modified new material, the vitality of osteoblast-like cells cultured with the new biomaterial was then investigated. Raloxifene-conjugated HA-biomaterial has been shown to be a safe material easy to obtain which could be an interesting starting point for the use of a new functional biomaterial suitable in bone regeneration procedures.