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Larissa Rocha Bertelli Cabral,1 Lucas Novaes Teixeira,1 Rodrigo Pinto Gimenez,2 Ana Paula Dias Demasi,1 Rui Barbosa de Brito Junior,1 Vera Cavalcanti de Araújo,1 Elizabeth Ferreira Martinez1 1Division of Cell Biology and Oral Pathology, Faculdade São Leopoldo Mandic, Campinas, São Paulo, Brazil; 2Division of Plastic Surgery, Faculdade São Leopoldo Mandic, Campinas, São Paulo, BrazilCorrespondence: Elizabeth Ferreira MartinezDivision of Cell Biology and Oral Pathology, Faculdade São Leopoldo Mandic, Rua José Rocha Junqueira, 13, Campinas CEP 13045-755, São Paulo, BrazilTel/Fax +55 19 3211 3600Email dr.efmartinez@gmail.comPurpose: Skin ageing is marked by structural and functional changes in epidermis and dermis, which result clinically in wrinkles, loss of elasticity, and rough-textured appearance. In this context, different dermal fillers have been used to overcome these negative effects associated with skin ageing, such as hyaluronic acid (HA) and poly-L-lactic acid (PLLA). Despite their low immunogenicity, these materials can cause an inflammatory reaction after application.Materials and Methods: Considering high demand of HA and PLLA as filler material, this study aimed to evaluate their in vitro and in vivo effects. For the in vitro study, human dermal fibroblast cell cultures were supplemented with HA or PLLA for 24, 48, and 72 h. The following parameters were assayed: 1) cell proliferation, 2) cell viability, and 3) quantification of type I collagen by ELISA. For the in vivo study, HA or PLLA was injected in the dermis of Wistar rats and the tissues were collected after 15, 30, and 60 days for histologic evaluation and for quantification of type I collagen by Western blotting. The quantitative data were statistically analyzed using an ANOVA two-way. The significance level was set at 5%.Results: At 72 h, high cell proliferation was observed for HA compared to control (p< 0.05). Cultures exposed to PLLA exhibited a reduction in both cell proliferation and viability compared to control in all time points (p< 0.05). Type I collagen expression was greater in cultures exposed to HA or PLLA compared to control (p< 0.05). Histologic analysis showed the presence of multinucleated cells only in the PLLA group in all experimental time points. Western blotting analysis revealed high content of type I collagen in HA compared to PLLA (p< 0.05).Conclusion: The present study addresses a potentially unfavorable effect of dermal PLLA filler on the fibroblast phenotype, with possible clinical complications, unlike HA.Keywords: cell culture, extracellular matrix, ageing, skin

José Carlos Elias Mouchrek Júnior,1 Cristina Gomes Macedo,2 Henrique Ballassini Abdalla,2 Ana Karina Saba,1 Lucas Novaes Teixeira,1 Adriana Quinzeiro e Silva Mouchrek,3 Marcelo Henrique Napimoga,1 Juliana Trindade Clemente-Napimoga,1 Alvaro Henrique Borges,4 Mateus Rodrigues Tonetto,4 Shelon Cristina Souza Pinto,5 Matheus Coelho Bandeca,3 Elizabeth Ferreira Martinez1 1Laboratory of Cell and Molecular Biology, São Leopoldo Mandic Institute and Research Center, Campinas, 2Physiological Sciences, Piracicaba Dental School, University of Campinas, Campinas, São Paulo, 3Department of Dentistry, CEUMA University, São Luis, Maranhão, 4Department of Integrated Dental Science, University of Cuiaba, Cuiabá, Mato Grosso, 5Department of Dentistry, Ponta Grossa State University, Ponta Grossa, Paraná, Brazil Purpose: The aim of this study was to evaluate the effect of simvastatin on the synthesis of cytokines TNF-α and IL-10 and metalloproteinase (MMPs) 2 and 9 in a rat model of ligature-induced periodontitis.Materials and methods: Twenty Wistar rats were used, and a cotton ligature was place in a subgingival position encircling the entire cervix of the first molar of the left (ipsilateral) side of the mandible. The right (contralateral) side of the mandible had no ligature placed and was used as control. After the ligature placement, animals were randomly assigned to two experimental groups (n=10): 1) rats with ligature + vehicle (saline; 10 mL/kg; orally) and 2) rats with ligature + simvastatin (25 mg/kg; orally). After 14 days of treatment, the animals were euthanized by anesthetic overdose and the gingival tissue was removed and homogenized in appropriate buffer. MMP-2 and -9 release as well as the IL-10 and TNF-α levels were detected by enzyme-linked immunosorbent assay. Statistical comparison was performed by unpaired Student’s t-test, with p0.05). However, IL-10 was upregulated in simvastatin-treated animals (1.8-fold increase) in comparison with the vehicle-treated group (p

Introduction: Fragile X syndrome (FXS) is the most common cause of hereditary genetic disorder in a single gene characterised by intellectual disability. Behavioural features such as autism, hyperactivity and anxiety disorder may be present. Biofilm development and pathogenicity of Streptococcus mutans may be altered because FXS renders the dental approach and oral hygiene more complex., Objectives: The purpose of this study was to compare the levels of transcripts for VicRK and CovR of S. mutans isolated from FXS patients with the levels of transcripts for VicRK and CovR of standard strain ATCC, using a quantitative polymerase chain reaction (qPCR)., Methods: The caries experience index was assessed by the International Caries Detection and Assessment System (ICDAS), Periodontal Condition Index (PCI) and Invasive Dental Treatment Need Index (INI)., Results: The clinical index findings revealed a high rate of caries cavities and bleeding on probing of FXS patients. When VicRK and CovR transcript levels were compared with the reference strain, Fragile X patients were found to have significantly higher values., Conclusion: The present study demonstrated that FXS patients have more adverse clinical conditions, with increased biofilm accumulation and virulence. When combined with behavioural abnormalities, these patients become even more vulnerable to dental caries., (© 2024 John Wiley & Sons and MENCAP.)

The present study aimed to evaluate the effect of photobiomodulation therapy (PBM) on different stages of osteogenesis in vitro. For this, osteoblastic-like cells (Saos-2 cell lineage) were irradiated in two different periods: during the Proliferation phase (PP; from the second to the fourth day) and during the Differentiation phase (DP; from the seventh to the ninth day). The energy density used in the study was 1.5 J/ cm 2 . The following parameters were evaluated: 1) quantification of collagen type 1 (COL 1), osteopontin (OPN), and bone morphogenetic protein 2 (BMP-2); 2) quantification of alkaline phosphatase (ALP) activity; and 3) quantification of  extracellular matrix (ECM) mineralization. Non-irradiated cultures were used as controls. The data were analyzed using the Student's t-test or one-way ANOVA, considering a significance level of 5%. The results indicated that COL 1 and BMP-2 quantification was higher in Saos-2 irradiated during the DP in relation to the control group at day 10 (p < 0.05). No differences were observed for other comparisons at this time point (p > 0.05). OPN expression was greater in PP compared with the other experimental groups at day 10 (p < 0.05). Irradiation did not affect ALP activity in Saos-2 regardless of the exposure phase and the time point evaluated (p > 0.05). At day 14, ECM mineralization was higher in Saos-2 cultures irradiated during the DP in relation to the PP (p < 0.05). In conclusion, the results suggested that the effects of PBM on osteoblastic cells may be influenced by the stage of cell differentiation., (© 2024. The Author(s), under exclusive licence to the European Photochemistry Association, European Society for Photobiology.)

Objectives: To assess the impact of different concentrations TiO 2 -nt incorporated into a glass ionomer cement on the proliferation, mitochondrial metabolism, morphology, and pro- and anti-inflammatory cytokine production of cultured fibroblasts (NIH/3T3), whether or not stimulated by lipopolysaccharides (LPS-2 μg/mL, 24 h)., Methods: TiO 2 -nt was added to KM (Ketac Molar EasyMix™, 3 %, 5 %, 7 % in weight); unblended KM was used as the control. The analyses included: Cell proliferation assay (n = 6; 24/48/72h); Mitochondrial metabolism assay (n = 6; 24/48/72h); Confocal laser microscopy (n = 3; 24/48/72h); Determination of biomarkers (IL-1β/IL-6/IL-10/VEGF/TNF) by using both multiplex technology (n = 6; 12/18 h) and the quantitative real-time PCR assay (q-PCR) (n = 3, 24/72/120 h). The data underwent analysis using both the Shapiro-Wilk and Levene tests, and by generalized linear models (α = 0.05)., Results: It demonstrated that cell proliferation increased over time, regardless of the presence of TiO 2 -nt or LPS, and displayed a significant increase at 72 h; mitochondrial metabolism increased (p < 0.05), irrespective of exposure to LPS (p = 0.937); no cell morphology changes were observed; TiO 2 -nt reverted the impact of KM on the secreted levels of the evaluated proteins and the gene expressions in the presence of LPS (p < 0.0001)., Conclusions: TiO 2 -nt did not adversely affect the biological behavior of fibroblastic cells cultured on GIC discs., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Kamila Rosamilia Kantovitz reports financial support was provided by State of Sao Paulo Research Foundation. Pedro Viel Gogolla reports was provided by State of Sao Paulo Research Foundation. Joao Pedro Coelho Rangel reports financial support was provided by State of Sao Paulo Research Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Objective: Guided tissue regeneration (GTR) is based on the use of different membranes that function as sealants and barriers in specific clinical situations. Among the several tissue production methods and origins, resorbable porcine-derived membranes are the most commonly used. Because these membranes are so diverse, and have several different clinical applications, doubts linger as to their effect in stimulating osteogenesis. The objective of this study was to make an in vitro evaluation of the viability and differentiation of osteoblastic cells cultured on the surface of the following collagen membranes: Jason® (Botiss Biomaterials), Collprotect® (Botiss Biomaterials), and Bio-Gide® (Geistlich)., Material and Methods: Fragments of the 3 resorbable collagen membranes (5 × 5 mm) were used, and pre-osteoblastic SAOS-2 cells (ATCC, USA) were plated on their porous surfaces. Evaluation of the membranes was performed at 3, 5 and 7 days, considering the following parameters: (1) topographic analysis of the different surfaces by scanning electron microscope; (2) cellular viability by MTT, (3) quantification of type I collagen and osteopontin by Elisa. The quantitative analyses were carried out using a significance level of 5%., Results: Collprotect® and Jason® membranes presented a rough surface with an irregular aspect on both sides, while double-layered Bio-Gide® had one layer with a smooth surface and the other with a rough surface along each respective length. The viability assays revealed that the cells cultured the cells grown on Collprotect® showed higher viability than those grown in Bio-Gide® or Jason®, especially after 5 and 7 days. After 3 and 5 days, evaluation of type I collagen showed that the cells plated on the Jason® and Collprotect® surfaces had greater collagen secretion than those plated on BioGide®. After 7 days, an increase in osteopontin levels was observed when the cells were plated on all the experimental membranes, compared with the control group., Conclusion: All the tested membranes were suitable for use in GTR clinical procedures. Their indication in specific regenerative cases depends on the mechanical and biological properties of their originating tissues, thus enabling better results and assertive choices by dental professionals., Competing Interests: Declaration of Competing Interest The authors state no conflicts of interest., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Aim: This study evaluated cytotoxicity and antioxidant gene expression of resin cements on human gingival fibroblasts (hGF)., Materials and Method: RelyX Ultimate™(RXU), Variolink™II(VLII), and RelyXU200™(RXU200) resin cements were incubated with culture medium for 24 h to obtain eluates. Then, the eluates were applied over hGF to assess cell viability at 24 h, 48 h, and 72 h and antioxidant gene expression at 24 h. hGF cultures non-exposed to the eluates were used as Control. Data were submitted to ANOVA and Bonferroni tests (α≤0.05)., Results: RXU and RXU200 reduced the number of viable cells in 24 h. Longer exposure to cement extracts caused cell death. Gene expression showed peroxiredoxin 1 (PRDX1) induction by all resin cement types, and superoxide dismutase 1 (SOD1) induction by RXU200 and VLII. Moreover, RXU200 induced not only PRDX1 and SOD1, but also glutathione peroxidase 1 (GPX1), catalase (CAT), and glutathione synthetase (GSS)., Conclusions: All resin cements showed toxicity, and induced antioxidant genes in hGF. Antioxidant gene induction is at least partly associated with cytotoxicity of tested cements to oxidative stress experience., Competing Interests: The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.

Background: This study aimed to investigate the differentiation of ameloblastic-like cells and the nature of the secreted eosinophilic materials in adenomatoid odontogenic tumors., Methods: We studied histological and immunohistochemical characteristics of 20 cases using: cytokeratins 14 and 19, amelogenin, collagen I, laminin, vimentin, and CD34., Results: Rosette cells differentiated into ameloblastic-like cells positioned face-to-face, displaying collagen I-positive material between them. Epithelial cells of the rosettes can differentiate into ameloblastic-like cells. This phenomenon probably occurs due to an induction phenomenon between these cells. The secretion of collagen I is probably a brief event. Amelogenin-positive areas were interspersed by epithelial cells in the lace-like areas, outside the rosettes and distant from the ameloblastic-like cells., Conclusions: There are at least two types of eosinophilic material in different areas within the tumor, one in the rosette and solid areas and another in lace-like areas. The secreted eosinophilic material in the rosettes and solid areas is probably a product of well-differentiated ameloblastic-like cells. It is positive for collagen I and negative for amelogenin, whereas some eosinophilic materials in the lace-like areas are positive for amelogenin. We hypothesize that the latter eosinophilic material could be a product of odontogenic cuboidal epithelial or intermediate stratum-like epithelial cells., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This study evaluated the influence of different implant diameters, insertion torques, and transmucosal heights on the loosening of abutments installed on short implants, after mechanical cycling. The Morse taper connection implants (n = 96) tested were 5 mm high, divided according to the platform diameter: 4 or 6 mm. A universal abutment was coupled to each implant (with different transmucosal heights: 1 or 5 mm). The sets were subdivided into 20- and 32-Ncm torque. After the cycle fatigue test, the detorque values were measured with a digital torque indicator. After mechanical cycling, the mean detorque values obtained for the abutment with 20-Ncm insertion torque were lower than for implants with 32-Ncm insertion torque, regardless of the platform diameter or transmucosal height. In the 20-Ncm torque group, there was no statistically significant difference in the detorque values between platform diameters or transmucosal heights. Otherwise, for 32-Ncm sets, a smaller platform diameter (4 mm), and a longer transmucosal height (5 mm) showed the lowest detorque values. In conclusion, implants placed with 32-Ncm insertion torque and abutments with 1 mm transmucosal height and a 6 mm implant diameter demonstrated the highest detorque values., (© 2023 Scandinavian Division of the International Association for Dental Research. Published by John Wiley & Sons Ltd.)

The aim of this study was to evaluate the effects of photobiomodulation (PBM) on cell migration and alkaline phosphatase (ALP), type I collagen (Col-1), runt-related transcription factor 2 (RUNX-2), and Osterix (OSX) gene expression in a cementoblast culture (OCCM-30), in a microenvironment mimicking an injury on the cementoblast layer, such as it occurs during root resorption. For this, OCCM-30 cells were cultured in 6-well plates and the following parameters were assayed: (1) migration by scratch assay and ALP, Col-1, Runx2, and Osx by real-time PCR. PBM was performed in two protocols using a LED device emitting light at 660 nm (± 30 nm). OCCM-30 cementoblasts were grown and divided into four groups: (1) negative control; (2) positive control (scratch); (3) scratch + PBM with a total energy of 36 J and energy density 1.6 J/cm 2 ; and (4) scratch + PBM with a total energy of 72 J and energy density of 3.2 J/cm 2 . Data were statistically analyzed, with the level of significance set at 5%. Cementoblasts migrated from the edge of the scratch toward the center, and the wound closed after 24 h, with the PBM3.2J/cm 2 group showing the higher cell migration compared with the other groups at 2 h, 6 h, 8 h, and 13 h (p < 0.05). The control and PBM1.6J/cm 2 groups showed similar levels of cell migration, with no significant differences (p > 0.05). PBM3.2J/cm 2 group exhibited greater ALP, Col-1, OSX, and RUNX2 in comparison with the other experimental groups (p < 0.05). Similar levels of all genes evaluated were observed between the PBM1.6J/cm 2 group and the positive control group (p > 0.05). In conclusion, our findings support the effectiveness of photobiomodulation on cementoblast migration and gene expression, which may contribute to the formation of a new cementum layer., (© 2023. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

Tissue engineering focuses on wound healing and tissue regeneration. Platelet-rich fibrin (PRF) is a fibrin matrix containing cytokines, growth factors and cells that are gradually released into the wound over time. This study aimed to evaluate the effect of PRF membranes on wound repair and microbial control in infected wounds. Skin wounds were performed on the dorsum of rats using a 6 mm diameter metal punch. The defects were randomly assigned into four groups (n = 12/each) accordingly to the treatment: G1, noninfected wound filled only with clot; G2, noninfected wound with PRF; G3, infected wound (S. aureus) without PRF; G4, infected wound (S. aureus) with PRF. After 7 and 14 days, macroscopic and histological analyses of the wounds were performed. Furthermore, the quantification of β-defensin in PRF was measured by ELISA. At 14 days, the groups with PRF (G2 and G4) had wound sizes significantly smaller than the original defects (6 mm) (p < 0.05) and significantly smaller than those not treated with PRF, in both the infected and noninfected groups (p < 0.05). Furthermore, the groups with infected wounds (G3 and G4) demonstrated a significantly lower inflammation score in the PRF group than in the noninfected groups (p < 0.05). In vitro analysis of β-defensin was performed in all PRF membrane groups, and the median value was 1.444 pg/mL. PRF in the wounds of both control and infected rats played an important role in the modulation of tissue healing, most notably in infected sites., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: The aim of this study was to evaluate the effect of ozone therapy on new bone formation and inflammation modulation in defects of rat calvaria filled with autogenous bone., Material and Methods: Critical size defects were created in the calvaria of 24 male Wistar rats. The animals were randomly divided into four groups according to the treatment: G1: clot; G2: clot and covered with xenogenic membrane; G3: particulate autogenous bone graft; G4: autogenous bone graft and application of 3 mL O 2 /O 3 gas mixture (10 µg/ml). The defects were filled immediately after surgery with a bilateral retroauricular application, in the region immediately above the incision. After 21 days, the animals were euthanized, and the samples were processed for morphometric evaluations designed to measure both the intensity of the inflammatory infiltrate, and the presence of new bone formation in the defect., Results: The results showed a lower inflammation score and higher mean of newly formed bone in the region of the defect for the group associated with ozone therapy (G4). The bone formed in the region of the defect could be observed as being more lamellar and mineralized in the case of associated ozone therapy., Conclusion: Ozone therapy represents a promising adjuvant therapy to accelerate tissue regeneration., Competing Interests: Declaration of Competing Interest The authors state no conflicts of interest., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Objective: Guided bone regeneration (GBR) is a technique that involves the placement of mechanical barriers to protect the blood clot, and create an isolated space to prevent competition from epithelial and connective tissues in bone augmentation treatments. Collagen membranes stand out from other materials available for performing regenerative surgeries, and are widely used because of their ability to promote cell adhesion and homeostasis, and their biocompatibility, ease of handling, and low immunogenicity. In this context, researchers have investigated xenogenic membranes/barriers that cost less and have slower resorption rates. The current study aimed to assess the osteogenic potential induced by a crosslinked, synthesized xenogenic membrane 100 µm thick when applied in vivo to critical defects in rat calvaria., Material and Methods: Critical size defects were created in the calvaria of thirty male Wistar rats, and randomly divided into the following two groups: G1 - clot covered with a commercial xenogenic membrane (Lumina-Coat®, Criteria, Brazil), and G2 - clot covered with a synthesized xenogenic membrane. The animals were euthanized after 7, 15 and 30 days, and samples of calvaria were processed to perform morphometric evaluations to measure bone neoformation in the defect region. In addition, ultrastructural characterization of the collagen membranes was performed by scanning electron microscope. The quantitative analyses were carried out by adopting a significance level of 5%., Results: The ultrastructural characterization revealed that the synthesized membrane had thicker collagen fibers and a more cohesive surface, compared with the Lumina-Coat® collagen membrane (G1). There was no significant difference in bone neoformation between the membranes (p>0.05), at any of the time periods analyzed. The bone quantification area increased significantly over time for both membranes (p<0.05)., Conclusion: The synthesized membrane exhibited morphological characteristics similar to those of the commercial membrane evaluated, allowed potentially active participation in the bone neoformation process, and served as a low-cost alternative for GBR procedures., Competing Interests: Declaration of Competing Interest The authors state no conflicts of interest., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Objective: To define the potential of polycaprolactone (PCL) scaffold for cementoblast delivery., Background: Dental cementum is critical for tooth attachment and position, and its regenerative capabilities remain unpredictable., Methods: PCL scaffolds were manufactured by the electrospinning technique at 10% and 20% (w/v) and seeded with cementoblasts (OCCM-30). Scaffolds were characterized for their morphology and biological performance by scanning electron microscopy (SEM), confocal and conventional histology, cytocompatibility (PrestoBlue assay), gene expression (type I collagen - Col1; bone sialoprotein - Bsp; runt-related transcription factor 2 - Runx-2; alkaline phosphatase - Alpl; osteopontin - Opn; osteocalcin - Ocn, osterix - Osx), and the potential to induce extracellular matrix deposition and mineralization in vitro., Results: Overall, data analysis showed that PCL scaffolds allowed cell adhesion and proliferation, modulated the expression of key markers of cementoblasts, and led to enhanced extracellular matrix deposition and calcium deposition as compared to the control group., Conclusion: Altogether, our findings allow concluding that PCL scaffolds are a viable tool to culture OCCM-30 cells, leading to an increased potential to promote mineralization in vitro. Further studies should be designed in order to define the clinical relevance of cementoblast-loaded PCL scaffolds to promote new cementum formation., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This study evaluated in vitro the biologic profile of manually polished surfaces of pressed lithium disilicate (LD) ceramic compared to zirconia (Zir) in human gingival fibroblasts. Samples with a 10-mm diameter and 3-mm thickness were used. After manual polishing, the average roughness (Ra) of the samples was measured. The cell proliferation and viability of gingival fibroblasts on the surfaces were assessed at 24, 48, and 96 hours. Additionally, the morphology, cell adhesion, and type III collagen (COLIII) and vimentin (VIM) expression by fibroblasts plated onto these surfaces was analyzed. Polystyrene (Pol) was used as control for all assays. The mean Ra was 0.261 ± 0.053 μm for Zir and 0.345 ± 0.130 μm for LD. Both surfaces presented similar cell proliferation and viability (P > .05). The cell morphology demonstrated that, for both surfaces, the cells were occasionally spindle-shaped, parallel to the direction of the grooves. Compared to Pol, an upregulation of COLIII and VIM gene expression was observed by fibroblasts cultured on Zir and LD at all time points (P < .05). The characteristics presented by LD and Zir surfaces after manual polishing protocol were similar and had biologically favorable performances, thus suggesting LD as a suitable alternative to Zir in the peri-implant region for esthetic purposes.

Objectives: Pneumatization of the maxillary sinus can make it difficult, if not impossible, to install osseointegrated implants, and undertake their eventual functional rehabilitation, which may ultimately require regenerative techniques to achieve. This randomized controlled study proposed conducting a histological evaluation of the behavior of different graft materials in wide maxillary sinuses, at a height of 8 to 10 mm from the alveolar ridge, combined with bone remnants less than 3mm., Materials and Methods: Thirty-six patients underwent a sinus elevation procedure through the lateral window. The sinuses were randomly filled with the following materials (n=12/group): group 1, xenogenic bone + autogenous bone (ratio 70:30, respectively); group 2, xenogenic bone + L-PRF; and group 3, xenogenic bone. At 8 months, bone biopsies of engrafted sites were harvested and analyzed histomorphometrically in order to quantify newly formed bone tissue., Results: The results showed a greater area of newly formed bone for G1, averaging 2678.37 (1116.40) μm 2 , compared with G2 at 984.87 (784.27) μm 2 , and G3 at 480.66 (384.76) μm 2 (p < 0.05). Additionally, fewer xenogenic bone particles and a large amount of connective tissue were observed in G2., Conclusions: In maxillary sinuses with large antral cavities, autogenous bone combined with xenogenic bone seems to demonstrate better graft remodeling and improve bone formation, compared with the addition of L-PRF., Clinical Relevance: L-PRF produces few advantages regarding new bone formation in the wide maxillary sinuses., Trial Registration: Brazilian Clinical Trials Registry (REBEC) number RBR-2pbbrvg., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study evaluated the influence of Libidibia ferrea (Lf) extract used as dentin pretreatment on the resin-dentin bond strength stability and dentin endogenous enzymatic activity. The phytochemical profile (PP) of the Lf extract was evaluated by liquid chromatography; particle size, polydispersity index (PdI), and zeta potential (ZP) were evaluated by dynamic light scattering. The tested groups were ER-Scotchbond Universal (SBU) in the etch-and-rinse (ER) mode; ERLf-SBU in the ER mode + Lf after etching; SE- SBU in the self-etch (SE) mode; and LfSE-Lf before SBU in the SE mode. Sticks were obtained for microtensile bond strength tests and failure mode (24 hr and 12 months). The hybrid layer was evaluated using scanning electron microscopy. The endogenous enzymatic activity of the underlying dentin was analyzed by in situ zymography with the same treatments. The PP showed the presence of quercetin (2.6% w/w). Lf particles were considered large after the analysis of the PdI. The ZP remained stable over time. The ER and ERLf groups had lower bond strength after 12 months, but SE and LfSE remained stable. The predominant failure mode was adhesive for both times. ER and ERLf had longer resin tags and a thicker hybrid layer. The ER and LfSE groups showed higher enzymatic activity than the ERLf and SE groups after 12 months. The Lf extract may contribute to inhibit the dentin endogenous enzymatic activity when associated with an adhesive system in the ER mode., (© 2021 Wiley Periodicals LLC.)

Objective: The aim of this study was to evaluate the effects of caffeic acid phenethyl ester (CAPE) on osteoblast-like cell cultures (SAOS-2)., Methods: SAOS-2 were exposed to CAPE at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM. Non-exposed cultures were used as control. The following parameters were assayed: 1) cell viability at 1, 3, and 7 days; 2) alkaline phosphatase (ALP) activity at 5 and 10 days; 3) matrix mineralization at 14 days; and 4) Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (SPP1), and osteocalcin (BGLAP) gene expression at 5 and 10 days. The data were analyzed by ANOVA two-way or Kruskal-Wallis (α = 5%)., Results: At day 1, cell viability was similar among all groups (p > 0.05). At days 3 and 7, cultures exposed to CAPE at 10 μM exhibited a significant reduction in cell viability compared with the others groups (p < 0.05). At day 5, ALP activity was similar among all experimental groups; at day 10, however, the stain intensity was higher in cultures exposed to CAPE at 100 nM and 10 nM in comparison with the other groups (p < 0.05). At days 5 and 10, RUNX2, ALP, SPP1, and BGLAP gene expression was greater in cultures exposed to CAPE in comparison with the control (p < 0.05). At day 14, matrix mineralization was similar in cultures exposed to CAPE at 1 nM and 10 nM (p > 0.05), but superior to those ones observed in the other experimental groups (p < 0.05)., Conclusion: CAPE at low concentrations can positively module the osteogenesis in vitro., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Although odontogenic lesions have been extensively described and studied, anomalous, challenging cases occasionally come to the attention of the pathologist. Here, we report the clinical and microscopic characteristics of an unusual cystic lesion of odontogenic origin. A 16-year-old male presented with swelling and pain to palpation of the right mandible as well as numbness of the right lower lip. Radiographically, the corresponding lesion was well-defined and radiolucent with internal radiopaque foci. It extended from the right first premolar posteriorly, approaching the angle of the mandible, and involved the mandibular first molar which was impacted and displaced. The second and third right mandibular molars were also impacted and displaced. The patient was treated by excisional biopsy under general anesthesia. The histopathologic examination revealed the presence of multicystic areas lined by a thin, non-keratinizing squamous epithelium that resembled the epithelial lining of a dentigerous cyst. In continuity with the cystic lining, areas of myxoid tissue reminiscent of dental papilla were observed. The myxoid tissue formed structures that were surfaced by an epithelium comprising a basal layer of ameloblast-like cells with reverse polarity of the nuclei. Above the basilar cells, additional layers of epithelial cells composed a structure resembling the enamel organ. Subjacent to the basilar ameloblast-like cells, a condensation of mesenchymal cells with polarized nuclei opposite to the ameloblast-like cells was present. These mesenchymal cells resembled odontoblasts. In addition, numerous mineralized structures amongst the odontogenic epithelial tissue were present. To date, the patient remains well and without evidence of recurrence after 36 months of follow-up.

Background: There has been great interest recently in the mechanisms of cell-to-cell communication through microvesicles (MV). These structures are produced by many different cell types and can modulate cellular activity by induction of epigenetic alterations. These vesicles may promote tumor mass increase either by stimulating cell proliferation via growth factors or by inhibiting apoptosis, which reinforces the role of such vesicles as important modulators of tumor progression., Methods: The present in vitro study aimed to characterize MV derived from malignant neoplastic epithelial cell cultures (EP) and their effect on the expression of apoptosis/autophagy and invasion related genes of benign myoepithelial (Myo) cell cultures., Results: The results revealed round structures with a mean size of 153.6 (±0.2) nm, with typical MV morphology. CD63 quantification indicated that EP cell culture at 70%-80% confluence secreted 3.088 × 10 8 MV/mL. Overall, Myo exposed to MVs derived from EP showed both up- and downregulation of tumorigenesis promoting genes. MVs from EP cells promoted cell death of Myo cells and positively modulate BAX, SURVIVIN, LC3B, MMP-2, and MMP-9 expression. Furthermore, an increasing of MMP-2 and MMP-9 secretion by Myo was observed after MV exposure., Conclusions: These findings suggest that MVs from EP modulate autophagy of Myo cells, which may, in part, explain the disappearance of these cells in in situ areas of invasive carcinoma ex-pleomorphic adenoma. Additionally, the overexpression of MMPs contributes to the development of an invasive phenotype of Myo cells, which could favor the dissolution of the basement membrane during tumorigenesis process., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: This study evaluated the ability of lithium chloride (LiCl) to increase bone filling (BF) around threaded titanium implants inserted in estrogen-deficient rats and, thein-vitro effects of this drug on osteoblast-like cell viability, proliferation, mineralization and expression of bone-related markers., Design: In vivo: Rats received sham surgery plus water (Estrogen-sufficient group), ovariectomy plus water (Estrogen-deficient group) or ovariectomy plus LiCl (150 mg/kg/every other day) (LiCl/estrogen-deficient group). On the 21 st day after ovariectomy/sham surgeries, a threaded titanium implant was inserted in the rat tibia. BF and the number of TRAP + cells were assessed at 10, 20 and 30 days after implant placement. In vitro: Osteosarcoma SAOS-2 cells were exposed to 0, 0.01, 0.05, and 0.1 mM of LiCl; cell proliferation, viability, mineralization (alizarin red staining) and gene expressions of RUNX-2, OCN, OPN, BSP and ALP (Real Time PCR) were estimated in the cultures., Results: In vivo: The estrogen-sufficient and LiCl/estrogen-deficient groups demonstrated higher percentages of BF, within the limits of implant threads, than the estrogen-deficient group at 20 and 30 days (p < 0.05). The number of TRAP + cells was lower in LiCl/estrogen-deficient than in the estrogen-deficient group at all experimental times (p < 0.05). In vitro: Cell cultures exposed to LiCl (0.01 or 0.05 mM) exhibited larger areas of mineralized matrix than the non-exposed cultures (p < 0.05) and demonstrated the highest expressions of the genes investigated., Conclusion: LiCl treatment improved BF around threaded titanium implants inserted in estrogen-deficient rats and stimulated matrix mineralization and overexpression of bone-formation markers in osteoblastic cells in culture., Competing Interests: Declaration of Competing Interest The authors explicitly state that they have no conflicts of interest in connection with this article., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: To investigate the long-term effect of 0.05% or 0.1% caffeic acid phenethyl ester (CAPE) on dentin matrix stability and hybrid layer stability, using an etch-and-rinse (Adper Scotchbond Multipurpose/ASB) or a self-etch adhesive (Clearfil SE Bond/CSE)., Materials and Methods: Dentin matrix specimens were assigned to five groups: 0.05% or 0.1% CAPE, green tea (GT), and the controls distilled water (DW) and dimethyl sulfoxide (DMSO). Following immersion of specimens for 1 h, modulus of elasticity (ME) and dentin mass change (MG) were determined at 3 post-treatment time points: immediately afterwards and at 3 and 6 months. Collagen solubilization (CS) was estimated by hydroxyproline (HYP) quantification. Resin-dentin interfaces with both adhesives were assessed with in situ zymography tests to evaluate gelatinolytic activity (GA). The dentin pretreatments were actively applied for 60 s. The sealing ability of aged resin-bonded slices was assessed by nanoleakage tests., Results: GT increased immediate ME, which decreased significantly after 3 months (p < 0.0001). The CAPE groups did not differ from the control groups. GT provided a significant increase in dentin matrix mass after treatment (p < 0.0001). No significant differences regarding MG were observed for CAPE 0.1%, CAPE 0.05%, DW, and DMSO groups after 3 and 6 months. Cumulative HYP release revealed that CAPE groups and GT were statistically similar to DW and DMSO; the GT group exhibited statistically significantly less HYP release than did CAPE groups (p = 0.0073). Treatment with 0.05% or 0.1% CAPE presented lower GA when applied to ASB before acid conditioning (p < 0.05), but no differences were detected when the CAPE groups were applied to CSE. CAPE at 0.1% significantly reduced nanoleakage for CSE, and 0.05% CAPE with CSE presented levels of nanoleakage similar to those of the CSE control group., Conclusion: CAPE at 0.05% or 0.01% did not influence ME, MG, or CS, but reduced GA when applied to ASB before acid conditioning. CAPE at 0.1% with CSE promoted adhesive layer integrity.

Objectives: This study aimed to revisit benign odontogenic ghost cell lesions (BOGCL) by hematoxylin and eosin staining and immunohistochemistry., Materials and Methods: Thirty cases of calcifying odontogenic cyst (COC) and 6 cases of dentinogenic ghost cell tumor (DGCT) were selected for histopathological and immunohistochemical analysis. Sections stained for cytokeratin (K) 14, K-19, amelogenin, collagen type 1 (COL-1), and dentin matrix acidic phosphoprotein 1 (DMP-1) were evaluated using qualitative analysis. Sections stained for Ki-67 and minichromosome maintenance protein-2 (MCM-2) were evaluated using semi-quantitative analysis., Results: A morphologic overlap was noticed in all BOGCL. Moreover, no differences were detected in the expression of K-14 and K-19. The expression of proliferative markers Ki-67 and MCM-2 was similar between cystic and tumor lesions (p > .05). The presence of COL-1 and absence of amelogenin in the so-called dysplastic dentin, associated with its histologic pattern, suggest that this is in fact an enameloid-like tissue., Conclusions: The dysplastic dentin should be considered an enameloid-like tissue in these lesions., Clinical Relevance: The similarity in histology, protein expression, and proliferative marker indices between COC and DGCT suggest that they are a sole entity and likely represent types of the same neoplasia.

Epithelial membrane antigen (EMA) and DOG1 are used as marker of epithelial cells, particularly the luminal cells, of salivary gland tumours. The aim of this study was to compare the EMA and DOG1 expression in tumours of minor salivary glands. Cases of pleomorphic adenoma (PA), basal cell adenoma (BCA), canalicular adenoma (CA), adenoid cystic carcinoma (ACC), polymorphous adenocarcinoma (PAC), mucoepidermoid carcinoma (MEC) and epithelial-myoepithelial carcinoma (EMC) were submitted to immunohistochemistry for EMA and DOG1. In PA and BCA, EMA and DOG1 were observed in luminal cells, while in CA the tumour cells were negative for both proteins. The EMA and DOG1 pattern expression detected in EMC was similar to that one observed in benign tumours. In ACC, both myoepithelial e epithelial expressed EMA and DOG-1. PAC tumour cells were only positive for DOG1, whereas MEC were only positive for EMA. In conclusion, EMA and DOG1 expression in benign salivary gland tumours was similar to normal salivary gland tissue and can be used as good marker of tumoral cells derived from intercalated ducts or its progenitor cells, while in malignant salivary gland tumours EMA expression is, however, better used as an indicator of aggressive behavior than a marker of luminal cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Adenoid cystic carcinoma (AdCC) and polymorphous adenocarcinoma (PAC) are included among the most common salivary gland cancers. They share clinical and histological characteristics, making their diagnosis challenging in specific cases. MicroRNAs (miRNA) are short, non-coding RNA sequences of 19-25 nucleotides in length that are involved in post-transcriptional protein expression. They have been shown to play important roles in neoplastic and non-neoplastic processes and have been suggested as diagnostic and prognostic markers., Methods: This study, using quantitative RT-PCR, investigated miR-150, miR-455-3p and miR-375 expression, in order to identify a possible molecular distinction between AdCC and PAC., Results: miRNA-150 and miRNA-375 expression was significantly decreased in AdCC and PAC compared with salivary gland tissue controls, whilst miRNA-455-3p showed significantly increased expression in AdCC when compared to PAC, (P < 0.05). miR-150, miR-357 and miR-455-3p expression in AdCC, PAC and control was not associated with age, gender nor with anatomic site (major and minor salivary glands) (P > 0.05)., Conclusion: MiR-455-3p could be used as a complimentary tool in the diagnosis of challenging AdCC cases., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The present study aimed to compare the expression of p63/p40 with smooth muscle actin (SMA) and vimentin (VIM) by myoepithelial cells in minor salivary gland tumors. Fifty-two formalin-fixed paraffin-embedded samples of minor salivary gland tumors derived from intercalated duct (pleomorphic adenoma [PA], adenoid cystic carcinoma [ACC], epithelial-myoepithelial carcinoma [EMC], polymorphous adenocarcinoma [PAC], and secretory carcinoma [SC]) and 3 samples of minor salivary gland tumors derived from excretory duct (mucoepidermoid carcinoma [MEC]) were evaluated by means of immunohistochemistry. The data were analyzed qualitatively. The results indicated that p63 and p40 expression were detected in myoepithelial cells present in PA, ACC, and EMC. However, both proteins were also observed in squamous areas of PA and all cases of MEC. SMA were noticed in some myoepithelial cells of PA, ACC, and EMC. Expression of SMA was negative in the other salivary gland tumors evaluated. VIM was constantly expressed by myoepithelial cells in PA, ACC, and EMC. VIM was also observed in cells of PAC and SC, but not in squamous areas of PA and MEC. In conclusion, p63 expression is almost comparable with VIM in detecting myoepithelial cells, an immunolabeling pattern not followed by p40, and consequently, caution has to be taken during the interpretation of salivary gland tumor exhibiting an p63/p40 phenotype in order to avoid a misdiagnosis.

Objective: Investigate the long-term effect of dentin pretreatment with 0.05 or 0.1% caffeic acid phenethyl ester (CAPE) on (1) bond strength of resin composite to dentin by a three-step etch-and-rinse (Adper Scotchbond Multipurpose/ ASB) or a two-step self-etch adhesive system (Clearfil SE Bond/ CSE), (2) their fracture mode, (3) the micromorphological features of the hybrid layer formed; and (4) the level of MMP-2 in dentin (after application, using a correlative immunoexpression/quantification approach)., Design: Composite resin blocks were fabricated on 48 third molars (n = 6), according to the type of adhesive and treatment (control, CAPE 0.05% and CAPE 0.1%). Slices were obtained for scanning electron microscopy (SEM) evaluation, and sticks were fabricated for microtensile tests (24 h and 1 year). Aliquots of dentin powder were distributed (n = 12) according to the treatment and the MMP-2 concentration was determined by ELISA., Results: Tukey test showed that ASB groups presented higher BS in 24 h than CSE groups. ASB presented a reduction in BS values after 1-year. ASB and CSE presented no significant differences in BS after 1-year. CAPE had no effect on BS for both adhesive systems. The predominant failure mode for the ASB groups were adhesive; when 0.1% CAPE was applied there was a predominance of mixed fractures. Regarding the CSE group, 0.05% CAPE led to more adhesive failures, and the 0.1% concentration resulted in a higher number of cohesive failures in dentin. Higher MMP-2 concentrations were detected for the groups that did not undergo demineralization treatment, and the lowest values for the ASB groups treated with CAPE. SEM analysis showed no influence of pretreatment with CAPE., Conclusions: CAPE did not influence the BS of the adhesives tested, or the micromorphology of the hybrid layer, irrespective of concentration or storage time. CAPE affected the fracture pattern at 24 h, depending on the concentration and the adhesive system used. Immunoassay analysis showed that CAPE 0.1% reduced the MMP-2 concentration in the ASB adhesive without affecting bond strength to dentin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aim: To evaluate the effects of 2.8% or 10% calcium chloride (CaCl 2 ) in calcium aluminate cement (CAC) with either bismuth oxide (Bi 2 O 3 ) or zinc oxide (ZnO) as radiopacifiers on the progression of osteogenic cell cultures., Methodology: Rat calvaria-derived cells were grown on Thermanox ® coverslips for 24 h and exposed to samples of (i) CACb: with 2.8% CaCl 2 and 25% Bi 2 O 3 ; (ii) CACb+: with 10% CaCl 2 and 25% Bi 2 O 3 ; (iii) CACz: with 2.8% CaCl 2 and 25% ZnO; or (iv) CACz+: with 10% CaCl 2 and 25% ZnO, placed on inserts. Nonexposed cultures served as the control. Calcium and phosphorus contents in culture media were quantified. The effects of the cements on cell apoptosis, cell viability and acquisition of the osteogenic cell phenotype were evaluated. Data were compared by Kruskal-Wallis test (α = 5%)., Results: CACb+ promoted the highest levels of calcium in the culture media; CACz+, the lowest levels of phosphorus (P < 0.05). CACz+ and CACb increased cell apoptosis (P < 0.05). CACb reduced cell viability (P < 0.05) and the expression of the osteoblastic phenotype. CACz+ and CACb+ promoted greater cell differentiation and matrix mineralization compared to CACz and CACb (P < 0.05)., Conclusion: For CAC with the lower CaCl 2 content, the use of Bi 2 O 3 was detrimental for osteoblastic cell survival and differentiation compared to ZnO, while CAC with the higher CaCl 2 content supported the acquisition of the osteogenic cell phenotype in vitro regardless of the radiopacifier used. Thus, CAC with 10% CaCl 2 would potentially promote bone repair in the context of endodontic therapies., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Barrier membranes for guided bone regeneration (GBR) mainly promote mechanical maintenance of bone defect space and induce osteopromotion. Additionally, biopolymer-based membranes may provide greater bioactivity and biocompatibility due to their similarity to extracellular matrix (ECM). In this study, biopolymers-based membranes from bacterial cellulose (BC) and collagen (COL) associated with osteogenic growth peptide (OGP(10-14)) were evaluated to determine in vitro osteoinductive potential in early osteogenesis; moreover, histological study was performed to evaluate the BC-COL OGP(10-14) membranes on bone healing after GBR in noncritical defects in rat femur. The results showed that the BC-COL and BC-COL OGP(10-14) membranes promoted cell proliferation and alkaline phosphatase activity in osteoblastic cell cultures. However, ECM mineralization was similar between cultures grown on BC OGP(10-14) and BC-COL OGP(10-14) membranes. In vivo results showed that all the membranes tested, including the peptide-free BC membrane, promoted better bone regeneration than control group. Furthermore, the BC-COL OGP(10-14) membranes induced higher radiographic density in the repaired bone than the other groups at 1, 4 and 16 weeks. Histomorphometric analyses revealed that the BC-COL OGP(10-14) induced higher percentage of bone tissue in the repaired area at 2 and 4 weeks than others membranes. In general, these biopolymer-based membranes might be potential candidates for bone regeneration applications.

Polymorphous adenocarcinoma (PAC) is a malignant epithelial neoplasm that affects almost exclusively the minor salivary glands, generally described as having a relatively good prognosis. Aberrant nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) activation in tumor cells has been associated with induction of antioxidant enzymes, such as peroxiredoxin I (Prx I) and increased matrix metalloproteinase (MMP) expression. In this context, the aim of the present study was to evaluate the expression of Nrf2 and correlate it with Prx I and MMP-2 secretion in PAC. Thirty-one cases of PAC from oral biopsies were selected and immunohistochemically analyzed for Nrf2 and Prx I. MMP-2 quantification was performed on primary cell cultures derived from PAC. Oral squamous cell carcinoma (OSCC) cell cultures were used as control. A high immunoexpression of Nrf2 was observed in both the cytoplasm and the nucleus of neoplastic cells from PAC. Nuclear staining for Nrf2 suggested its activation in the majority of the PAC cells, which was confirmed by the high expression of its target gene, Prx I. Quantification of MMP-2 secretion showed lower levels in PAC cell cultures when compared to OSCC cell cultures (p < 0.05). In conclusion, although Nrf2 overexpression has been frequently associated with high-grade malignancies, such relationship is not infallible and, in fact, the opposite may occur in low-grade tumors, such as PAC of minor salivary glands.

Objective: The aim of the present study was to establish a primary cell culture derived from polymorphous low grade adenocarcinoma (PLGA)., Design: The neoplastic cells were derived from a 57-year-old female patient diagnosed with PLGA. A fragment of the tumor was collected and submitted to enzymatic digestion followed by centrifugation on a Percoll gradient. The cell population was characterized by means of immunofluorescence and detection of PRKD1 gene mutations., Results: Epifluorescence analysis of the primary culture revealed that the malignant epithelial cells were predominantly polygonal in shape and positive for cytokeratin 7, vimentin, and S100. The doubling time of the cell culture was 86.73h. The restriction digestion assay showed that the neoplastic cells possess PRKD1 gene mutations., Conclusion: The establishment of primary cell culture derived from PLGA should be considered a useful tool for molecular analysis of this salivary gland tumor., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Despite advances in the field of biomaterials for bone repair/regeneration, some challenges for developing an ideal bone substitute need to be overcome. Herein, this study synthesized and evaluated in vitro a nanocomposite based on bacterial cellulose (BC), collagen (COL), apatite (Ap) and osteogenic growth peptide (OGP) or its C-terminal pentapeptide [OGP(10-14)] for bone regeneration purposes. The BC-COL nanocomposites were successfully obtained by carbodiimide-mediated coupling as demonstrated by spectroscopy analysis. SEM, FTIR and 31 P NMR analyses revealed that in situ synthesis to apatite was an effective route for obtaining of bone-like apatite. The OGP-containing (BC-COL)-Ap stimulated the early development of the osteoblastic phenotype. Additionally, the association among collagen, apatite, and OGP peptides enhanced cell growth compared with OGP-containing BC-Ap. Furthermore, none of the nanocomposites showed cytotoxic, genotoxic or mutagenic effects. These promising results suggest that the (BC-COL)-Ap associated with OGP peptides might be considered a potential candidate for bone tissue engineering applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of the present study was to analyze the in vitro effect of various doses of epidermal growth factor (EGF; 5 and 10 ng/ml) on matrix metalloproteinase-2 (MMP-2) secretion and E-cadherin/β-catenin expression by co-cultured cells that mimic an in situ carcinoma ex-pleomorphic adenoma, where benign myoepithelial cells from a pleomorphic adenoma surround malignant epithelial cells. EGF was supplemented in various doses and the effects were evaluated following four days of cell culture. ELISA was performed to determine MMP-2 secretion levels. Gene expression for E-cadherin and β-catenin was analyzed using quantitative polymerase chain reaction. The results revealed that E-cadherin expression decreased when the cells were supplemented with 5 ng/ml EGF. ELISA results indicated that MMP-2 secretion increased when EGF was supplemented at concentrations of 5 and 10 ng/ml. The present findings demonstrated that EGF may be involved in the epithelial-mesenchymal transition process via altering the E-cadherin/β-catenin complex and increasing MMP-2 secretion, which may then favor the dissolution of the basement membrane to the benefit of malignant cell clusters, contributing to the development of an invasive phenotype in this in vitro model of tumorigenesis.

Aim: To evaluate the effect of a calcium aluminate-based cement (CAC+) on the development of the osteogenic phenotype in vitro., Methodology: Rat calvaria-derived cells were grown on Thermanox ® coverslips for 24 h and then exposed to either samples (4-h set) of CAC+ or mineral trioxide aggregate (MTA) placed on Transwell ® inserts for periods of up to 14 days. Nonexposed cultures were used as the controls. The comparisons were made using the nonparametric Kruskal-Wallis test, followed by the Student-Newman-Keuls post hoc test when appropriate., Results: The results showed that proximity to MTA or CAC+ samples inhibited cell growth, whereas at a distance, viable and proliferative cells adhered to and spread on the Thermanox ® , expressing osteoblast differentiation markers prior to mineralization of the extracellular matrix. Compared with MTA, the osteogenic cell cultures exposed to CAC+ exhibited significantly greater cell viability, alkaline phosphatase (ALP) activity and expression of runt-related transcription factor 2, osterix, ALP, bone sialoprotein and osteocalcin (P < 0.05 for all). For the osteogenic cell cultures exposed to CAC+, the quantification of matrix mineralization was not altered (P > 0.05)., Conclusions: CAC+ supported the acquisition of the osteogenic cell phenotype in vitro, rendering this novel material a potential alternative to MTA in endodontic procedures. Further in vivo studies are needed to verify if the beneficial in vitro effects of CAC+ on osteoblastic cells correspond to an increase and/or acceleration of bone repair in the periapical region., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

This study evaluated the temporal expression of osteopontin (OPN) in co-cultures of human osteoblastic cells (SAOS-2) and oral squamous cell carcinoma (OSCC)-derived cells (SCC9) and examined the effects of osteoblast-derived OPN on the neoplastic cell phenotype. Additionally, the effects of these co-cultures on subsequent osteoclastic activity were explored. SCC9 cells were plated on Transwell® membranes that were either coated or not coated with Matrigel and were then co-cultured with SAOS-2 cells during the peak of OPN expression. SCC9 cells exposed to OPN-silenced SAOS-2 cultures and SCC9 cells cultured alone served as controls. SCC9 cells were quantitatively evaluated for cell adhesion, proliferation, migration, and invasion into Matrigel. The impact of co-culturing SAOS-2 and SCC9 cells on the resorptive capacity of U-937-derived osteoclastic cells was also investigated. Furthermore, a reciprocal induction of SAOS-2 and SCC9 cells in terms of OPN expression over the co-culture interval was identified. SAOS-2-secreted OPN altered the SCC9 cell phenotype, leading to enhanced cell adhesion and proliferation and higher Matrigel invasion. This invasion was also enhanced, albeit to a lesser degree, by co-culture with OPN-silenced SAOS-2 cells. Cell migration was not affected. Co-culture with SAOS-2 cells-mainly during the period of peak OPN expression-promoted over-expression of IL-6 and IL-8 by SCC9 cells and enhanced the resorptive capacity of osteoclastic cells. Taken together, these results suggest that osteoblast-derived OPN affects the interactions among OSCC-derived epithelial cells, osteoblasts, and osteoclasts, which could contribute to the process of bone destruction during bone invasion by OSCC.

Objective: Bisphosphonates (BPs) have been widely used in the treatment of bone disorders due to their ability to modulate bone turnover. The biological mechanisms through BFs exert their effects on osteoclasts are well established. However, the role of BFs on the osteoblasts is controversial. The present study aimed to evaluate the effects of risedronate on osteoblastic cells., Design: MC3TE-E1 cells were exposed to risedronate at 0, 10(-8), 10(-6), 10(-4), and 10(-3)M. The following parameters were assayed: (1) cell proliferation by hemocytometer counting after 24, 48 and 72h, (2) cell viability by MTT assay after 24, 48 and 72h, (3) Type I Collagen quantification by ELISA after 24, 48 and 72h, (3) alkaline phosphatase activity after 7 and 10days and (4) matrix mineralization after 14days., Results: After 24h, risedronate did not affect both cell proliferation and viability (p>0.05). However, after 48 and 72h, a decrease in cell proliferation and viability was detected in osteoblastic cultures exposed to risedronate at 10(-4) and 10(-3)M (p<0.05). After 48 and 72h, Type I Collagen synthesis was stimulated by risedronate at 10(-4)M (p<0.05). High levels of ALP activity were detected in cultures exposed to risedronate at 10(-4)M after 7 and 10days (p<0.05). After 14day, high calcium content was observed in cultures exposed to risedronate at 10(-4)M (p>0.05)., Conclusion: These results indicated that risedronate can promote osteoblast differentiation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Strontium ranelate (SR) is the first generation of a new class of medication for osteoporosis, which is capable of inducing bone formation and, to a certain extent, inhibiting bone resorption. The aim of this study was to evaluate the in vitro effects of SR on osteoblastic cell cultures. MC3TE-E1 cells were seeded in 24-well plates at a density of 2×10(4) cells/well and exposed to SR at 0.05, 0.1, and 0.5mM. The following parameters were assayed: 1) Cell proliferation by hemocytometer counting after 24, 48 and 72h, 2) Cell viability by MTT assay after 24, 48 and 72h, 3) Type I Collagen and Osteopontin (OPN) quantification by Western Blotting, ELISA, and Real Time PCR after 48h, 3) Immunolocalization of fibronectin (FN) by epifluorescence, and 4) matrix mineralization by Alizarin Red staining after 14days. After 24, 48 and 72h, the cell proliferation and viability were not affected by SR at 0.05 and 0.1mM (p>0.05). However, cell cultures exposed to SR at 0.5mM exhibited a decrease in both cell proliferation and cell viability in all time points assayed (p<0.05). High levels of protein and mRNA for Type I Collagen and OPN were detected in cultures exposed to SR, particularly at 0.5mM (p<0.05). SR allowed the expression of FN in osteoblastic cell cultures as observed by epifluorescence analysis. The mineralized bone-like nodule formation was affected in a concentration-dependent manner by SR, with large bone-like nodules being detected in osteoblastic cell cultures exposed to SR at 0.5mM. In conclusion, these results suggest that SR can accelerate acquisition of the osteoblastic phenotype, which explains, at least in part, the rebalancing of bone turnover in favor of bone formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study was designed to investigate the influence of mesenchymal stem cells (MSCs) on osteoblast (OB) differentiation. Rat bone marrow MSCs were cultured either in growth medium that maintained a MSC phenotype or in osteogenic medium that induced differentiation into OBs. Then, cells were grown in two different culture conditions: indirect co-culture of MSCs and OBs and OBs cultured in MSC-conditioned medium. As a control culture condition, OBs were grown in osteogenic medium without the influence of MSCs. We evaluated cell proliferation, the gene expression of key bone markers, alkaline phosphatase (ALP) activity, bone sialoprotein (BSP) expression, and extracellular matrix mineralization. The results showed that, regardless of whether OBs were indirectly co-cultured with MSCs or cultured in MSC-conditioned medium, MSCs repressed OB differentiation, as evidenced by the downregulation of all evaluated bone marker genes, decreased ALP activity, inhibition of BSP protein expression, and reduced extracellular matrix mineralization. Taken together, these results indicate that despite the key role of both MSCs and OBs in the osteogenic process, the repressive effect of MSCs on OB differentiation in an osteogenic environment may represent a barrier to the strategy of using them together in cell-based therapies to induce bone repair., (© 2015 Wiley Periodicals, Inc.)

The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass-based materials are due to altered mRNA and protein levels. Primary rat-derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real-time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals., (© 2015 Wiley Periodicals, Inc.)

Mucoepidermoid carcinoma (MEC) is the most common primary salivary gland malignancy in both adults and children. It has a slight female predilection and usually presents as a painless, rubber-like or soft mass, which may be fixed or mobile. Histologically, MEC is comprised of a mixture of cell types including mucous, epidermoid, and intermediate cells that can be arranged in solid nests or cystic structures. In the oral cavity, it most frequently occurs at the palate or buccal mucosa. The present paper aimed to describe an unusual case of MEC arising in the palatine tonsil.