Vincenzo D'Antò

Effect of Mineral Trioxide Aggregate on Mesenchymal Stem Cells

INTRODUCTION Mineral trioxide aggregate (MTA) is known to stimulate the hard tissue repair process. The purpose of this study was to evaluate the ability of MTA to support the adhesion, proliferation, and migration of human bone marrow-derived mesenchymal stem cells (hMSCs). METHODS White ProRoot MTA and white Portland cement were mixed and left to set 24 hours. MSCs were cultured on the samples and observed after 24 hours by confocal laser scanning microscopy (CLSM) by using the cytoskeleton marker CellTracker. Cell proliferation was evaluated by means of alamar blue assay in the presence and absence of differentiation medium during a period of 28 days, and cells seeded on polystyrene culture wells were the control. To assess the effect on migratory ability of hMSCs, a transwell migration assay was performed for 18 hours, positioning MTA and Portland cement in 6-well plates and the cells in 8-μm pore inserts. RESULTS hMSCs observed under CLSM showed attachment and spread activity on the upper surface of the MTA. Cell proliferation was significantly higher on MTA than on Portland cement. A rate proliferation increase of the MTA group compared with the control was observed after 14 days in presence of basic medium, whereas the same effect was reached after 21 days in presence of differentiation medium. Moreover, MTA was able to enhance cell migration significantly more than Portland cement. CONCLUSIONS Our findings suggest that MTA was able to assist hMSC adhesion, growth, and migration.

Effect of 2-Hydroxyethyl Methacrylate on Human Pulp Cell Survival Pathways ERK and AKT

Previous investigations have revealed that dental monomers could affect intracellular pathways leading to cell survival or cell death. Mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) might mediate cell responses as well as cell survival and apoptosis. The purpose of this study was to evaluate the effects of 2-hydroxyethyl methacrylate (HEMA) on the ERK1/2 and AKT pathways in human primary pulp fibroblasts (HPCs). HPCs were treated with various concentrations of HEMA, after which viability and reactive oxygen species levels were determined by flow cytometry with Annexin V-PI staining and 2,7-dichlorofluorescine diacetate, respectively. Whole-cell extracts were immunoblotted with anti-P-Akt or anti-P-ERK1/2. Cell viability decreased in a dose-dependent manner after HEMA exposure, showing a significant decrease with 10 mmol/L HEMA (p < .05). HEMA treatment resulted in a 4-fold increase in reactive oxygen species formation (p < .05). A short HEMA exposure (30-90 minutes) increased ERK1/2 phosphorylation, whereas a decrease in the AKT phosphorylation was observed. Selective inhibitors of the ERK (PD98059) and AKT (LY294002) pathways amplified HPC cell damage after HEMA exposure. Our findings demonstrated that HEMA exposure modulates the ERK and AKT pathways in different manners, and that in turn, they function in parallel to mediate pro-survival signaling in pulp cells subjected to HEMA cytotoxicity.

Evaluation of surface roughness of orthodontic wires by means of atomic force microscopy

OBJECTIVE To compare the surface roughness of different orthodontic archwires. MATERIALS AND METHODS Four nickel-titanium wires (Sentalloy(®), Sentalloy(®) High Aesthetic, Titanium Memory ThermaTi Lite(®), and Titanium Memory Esthetic(®)), three β-titanium wires (TMA(®), Colored TMA(®), and Beta Titanium(®)), and one stainless-steel wire (Stainless Steel(®)) were considered for this study. Three samples for each wire were analyzed by atomic force microscopy (AFM). Three-dimensional images were processed using Gwiddion software, and the roughness average (Ra), the root mean square (Rms), and the maximum height (Mh) values of the scanned surface profile were recorded. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukeys post hoc test (P < .05). RESULTS The Ra, Rms, and Mh values were expressed as the mean ± standard deviation. Among as-received archwires, the Stainless Steel (Ra  =  36.6 ± 5.8; Rms  =  48 ± 7.7; Mh  =  328.1 ± 64) archwire was less rough than the others (ANOVA, P < .05). The Sentalloy High Aesthetic was the roughest (Ra  =  133.5 ± 10.8; Rms  =  165.8 ± 9.8; Mh  =  949.6 ± 192.1) of the archwires. CONCLUSIONS The surface quality of the wires investigated differed significantly. Ion implantation effectively reduced the roughness of TMA. Moreover, Teflon(®)-coated Titanium Memory Esthetic was less rough than was ion-implanted Sentalloy High Aesthetic.

N-acetyl cysteine directed detoxification of 2-hydroxyethyl methacrylate by adduct formation

Cytotoxicity of the dental resin monomer 2-hydroxyethyl methacrylate (HEMA) and the protective effects of N-acetyl cysteine (NAC) on monomer-induced cell damage are well demonstrated. The aim of our study was to analyze the hypothesis that the protection of NAC from HEMA cytotoxicity might be due to direct NAC adduct formation. To this end, using HPLC we first measured the actual intracellular HEMA concentrations able to cause toxic effects on 3T3-fibroblasts and then determined the decrease in intracellular and extracellular HEMA levels in the presence of NAC. In addition, by capillary electrophoresis coupled with mass spectrometry analysis (CE-MS), we evaluated NAC-HEMA adduct formation. HEMA reduced 3T3 cell vitality in a dose- and time-dependent manner. The concentration of HEMA inside the cells was 15-20 times lower than that added to the culture medium for cell treatment (0-8 mmol/L). In the presence of 10 mmol/L NAC, both intracellular and extracellular HEMA concentrations greatly decreased in conjunction with cytotoxicity. NAC-HEMA adducts were detected both in the presence and absence of cells. Our findings suggest that the in vitro detoxification ability of NAC against HEMA-induced cell damage occurs through NAC adduct formation. Moreover, we provide evidence that the actual intracellular concentration of HEMA able to cause cytotoxic effects is at least one magnitude lower than that applied extracellularly.

The influence of Ni(II) on surface antigen expression in murine macrophages.

Biomedical alloys may release nickel ions during corrosion phenomena and, in addition to their interaction with oral tissues, these ions may also influence characteristic properties of the immune system cells. The aim of this study was to evaluate the effect of nickel chloride on the expression of functionally distinct surface antigens in murine RAW macrophages. The expression of the surface antigens CD14, CD40, MHC class I, MHC class II, CD80, CD86, CD54 was analyzed by flow cytometry. The bacterial endotoxin lipopolysaccharide (LPS) was used as a positive control to induce antigen expression. Cells were stimulated with NiCl(2) (0.1 and 0.5mm) in the presence and absence of LPS (0.1 or 25 microg/ml). After exposure periods of 6, 24 and 48 h, LPS caused a time- and dose-dependent increase in the expression of all surface antigens. CD14 expression was up-regulated by 0.1 microg/ml LPS by about 10-fold after 24h and 100-fold after 48 h. After 48 h, NiCl(2) alone up-regulated the expression of all surface antigens between 2- and 4-fold, while in cells stimulated by LPS, 0.1mm NiCl(2) was effective only on CD14, CD40 and MHC class I. Moreover, 0.5mm NiCl(2) even inhibited the LPS-induced expression of all surface antigens, except for CD54, which was still significantly up-regulated. These results show that nickel chloride is able to induce an up-regulation of surface antigen expression, but a high concentration may impair essential functions of macrophages stimulated by LPS.

Effects of intraoral aging on surface properties of coated nickel-titanium archwires

OBJECTIVE To evaluate the effects of intraoral aging on surface properties of esthetic and conventional nickel-titanium (NiTi) archwires. MATERIALS AND METHODS Five NiTi wires were considered for this study (Sentalloy, Sentalloy High Aesthetic, Superelastic Titanium Memory Wire, Esthetic Superelastic Titanium Memory Wire, and EverWhite). For each type of wire, four samples were analyzed as received and after 1 month of clinical use by an atomic force microscope (AFM) and a scanning electronic microscope (SEM). To evaluate sliding resistance, two stainless steel plates with three metallic or three monocrystalline brackets, bonded in passive configuration, were manufactured; four as-received and retrieved samples for every wire were pulled five times at 5 mm/min for 1 minute by means of an Instron 5566, recording the greatest friction value (N). Data were analyzed by one-way analysis of variance and by Students t-test. RESULTS After clinical use, surface roughness increased considerably. The SEM images showed homogeneity for the as-received control wires; however, after clinical use esthetic wires exhibited a heterogeneous surface with craters and bumps. The lowest levels of friction were observed with the as-received Superelastic Titanium Memory Wire on metallic brackets. When tested on ceramic brackets, all the wires exhibited an increase in friction (t-test; P < .05). Furthermore, all the wires, except Sentalloy, showed a statistically significant increase in friction between the as-received and retrieved groups (t-test; P < .05). CONCLUSION Clinical use of the orthodontic wires increases their surface roughness and the level of friction.

Catechol-O-methyltransferase (COMT) gene polymorphisms as risk factor in temporomandibular disorders patients from Southern Italy.

Objectives:To evaluate the role of COMT gene variants as potential risk factors in a group of patients affected with chronic temporomandibular disorder (TMD) pain. Methods:We sequenced COMT gene in 182 Italian subjects (50 affected by TMD and 132 controls). The study population consisted of patients affected by myogenous and/or arthrogenous pain (RDC/TMD: Ia, Ib, IIIa, IIIb diagnostic categories). Results:We detected 40 single nucleotide polymorphisms (SNPs) variants (18 novel). Three SNPs, all located in the promoter regions, were more frequently present in cases than in controls (rs 4646310 P=0.018, rs165656 P=0.001, rs 165722 P=0.007). After the False Discovery Rate (FDR) correction rs165656 remained significantly associated with TMD (P=0.049). In addition, the rs 4646310 (AG vs GG, P=0.015) and rs 165656 (GG vs CC, P=0.001) were at binary logistic regression analysis independently associated with TMD, conferring a risk disease of 2.6 (CI= 1.2-5.6) and of 5.3 (CI= 2.0-13.7) respectively. Discussion:Our data extend the number of SNPs present in the promoter region that could play a regulatory role in COMT gene and suggest that the genetic polymorphisms rs 165656 and rs 4646310 exert a role in TMD susceptibility.

The HOX genes are expressed, in vivo, in human tooth germs: In vitro cAMP exposure of dental pulp cells results in parallel HOX network activation and neuronal differentiation

Homeobox‐containing genes play a crucial role in odontogenesis. After the detection of Dlx and Msx genes in overlapping domains along maxillary and mandibular processes, a homeobox odontogenic code has been proposed to explain the interaction between different homeobox genes during dental lamina patterning. No role has so far been assigned to the Hox gene network in the homeobox odontogenic code due to studies on specific Hox genes and evolutionary considerations. Despite its involvement in early patterning during embryonal development, the HOX gene network, the most repeat‐poor regions of the human genome, controls the phenotype identity of adult eukaryotic cells. Here, according to our results, the HOX gene network appears to be active in human tooth germs between 18 and 24 weeks of development. The immunohistochemical localization of specific HOX proteins mostly concerns the epithelial tooth germ compartment. Furthermore, only a few genes of the network are active in embryonal retromolar tissues, as well as in ectomesenchymal dental pulp cells (DPC) grown in vitro from adult human molar. Exposure of DPCs to cAMP induces the expression of from three to nine total HOX genes of the network in parallel with phenotype modifications with traits of neuronal differentiation. Our observations suggest that: (i) by combining its component genes, the HOX gene network determines the phenotype identity of epithelial and ectomesenchymal cells interacting in the generation of human tooth germ; (ii) cAMP treatment activates the HOX network and induces, in parallel, a neuronal‐like phenotype in human primary ectomesenchymal dental pulp cells. J. Cell. Biochem. 97: 836–848, 2006.

Dental and skeletal effects of palatal expansion techniques: a systematic review of the current evidence from systematic reviews and meta-analyses

The aim was to assess the quality and to summarise the findings of the Systematic Reviews (SRs) and Meta-Analyses (MAs) on the dental and skeletal effects of maxillary expansion. Electronic and manual searches have been independently conducted by two investigators, up to February 2015. SRs and MAs on the dentoalveolar and skeletal effects of fixed expanders were included. The methodological quality was assessed using the AMSTAR (A Measurement Tool to Assess Systematic Reviews). The design of the primary studies included in each SR/MA was assessed with the LRD (Level of Research Design scoring). The evidence for each outcome was rated applying a pre-determined scale. Twelve SRs/MAs were included. The AMSTAR scores ranged from 4 to 10. Two SRs/MAs included only RCTs. The current findings from SRs/MAs support with high evidence a significant increase in the short-term of maxillary dentoalveolar transversal dimensions after Rapid Maxillary Expansion (RME). The same effect is reported with moderate evidence after Slow Maxillary Expansion (SME). However, there is moderate evidence of a non-significant difference between the two expansion modalities concerning the short-term dentoalveolar effects. With both RME and SME, significant increase of skeletal transversal dimension in the short-term is reported, and the skeletal expansion is always smaller than the dentoalveolar. Even though dental relapse to some extent is present, long-term results of the dentoalveolar effects show an increase of the transversal dimension, supported by moderate evidence for RME and low evidence for SME. Skeletal long-term effects are reported only with RME, supported by very low evidence.

Deregulated HOX genes in ameloblastomas are located in physical contiguity to keratin genes

The expression of the HOX gene network in mid‐stage human tooth development mostly concerns the epithelial tooth germ compartment and involves the C and D HOX loci. To further dissect the HOX gene implication with tooth epithelium differentiation we compared the expression of the whole HOX network in human ameloblastomas, as paradigm of epithelial odontogenic tumors, with tooth germs. We identified two ameloblastoma molecular types with respectively low and high number of active HOX C genes. The highly expressing HOX C gene ameloblastomas were characterized by a strong keratinized phenotype. Locus C HOX genes are located on chromosome 12q13–15 in physical contiguity with one of the two keratin gene clusters included in the human genome. The most posterior HOX C gene, HOX C13, is capable to interact with hair keratin genes located on the other keratin gene cluster in physical contiguity with the HOX B locus on chromosome 17q21–22. Inside the HOX C locus, a 2.2 kb ncRNA (HOTAIR) able to repress transcription, in cis, along the entire HOX C locus and, in trans, at the posterior region of the HOX D locus has recently been identified. Interestingly both loci are deregulated in ameloblastomas. Our finding support an important role of the HOX network in characterizing the epithelial tooth compartment. Furthermore, the physical contiguity between locus C HOX and keratin genes in normal tooth epithelium and their deregulation in the neoplastic counterparts suggest they may act on the same mechanism potentially involved with epithelial tumorigenesis. J. Cell. Biochem. 112: 3206–3215, 2011.