Evaluation of the remineralizing capacity of silver diamine fluoride on demineralized dentin under pH-cycling conditions

Abstract Objective (1) to determine the effects of the silver diamine fluoride (SDF) and sodium fluoride (NaF) in demineralized dentin exposed to an acid challenge by pH-cycling, (2) to evaluate the remineralizing capacity of SDF/NaF products based on the physicochemical and mechanical properties of the treated dentin surfaces. Methodology In total, 57 human molars were evaluated in different stages of the experimental period: sound dentin - negative control (Stage 1), demineralized dentin - positive control (Stage 2), and dentin treated with SDF/NaF products + pH-c (Stage 3). Several commercial products were used for the SDF treatment: Saforide, RivaStar, and Cariestop. The mineral composition and crystalline and morphological characteristics of the dentin samples from each experimental stage were evaluated by infrared spectroscopy (ATR-FTIR), X-ray diffraction, and electron microscopy (SEM-EDX) analytical techniques. Moreover, the mechanical response of the samples was analyzed by means of the three-point bending test. Statistics were estimated for ATR-FTIR variables by Wilcoxon test, while the mechanical data analyses were performed using Kruskal-Wallis and Mann Whitney U tests. Results Regarding the chemical composition, we observed a higher mineral/organic content in the SDF/NaF treated dentin + pH-c groups (Stage 3) than in the positive control groups (Saforide p=0.03; Cariestop p=0.008; RivaStar p=0.013; NaF p=0.04). The XRD results showed that the crystallite size of hydroxyapatite increased in the SDF/NaF treated dentin + pH-c groups (between +63% in RivaStar to +108% in Saforide), regarding the positive control. SEM images showed that after application of the SDF/NaF products a crystalline precipitate formed on the dentin surface and partially filled the dentin tubules. The flexural strength (MPa) values were higher in the dentin treated with SDF/NaF + pH-c (Stage 3) compared to the positive control groups (Saforide p=0.002; Cariestop p=0.04; RivaStar p=0.04; NaF p=0.02). Conclusions The application of SDF/NaF affected the physicochemical and mechanical properties of demineralized dentin. According to the results, the use of SFD/NaF had a remineralizing effect on the dentin surface even under acid challenge.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Comparative Evaluation of MTA Sealers Biomineralization Ability: an in Vivo Study / Avaliação Comparativa da Capacidade de Biomineralização de Cimentos MTA: Estudo in Vivo

This research aimed to evaluate the biomineralization induced by different concentrations of MTA Flow® and compare it to MTA Angelus®. Fifteen male Wistar rats received subcutaneous implants containing the materials to be tested (MTA Flow® at putty, thick, and thin consistencies and MTAAngelus®) and empty tubes (control). After 7, 40 and 90 days, the animals were euthanized, and the implants were removed with the surrounding tissue. The presence of biomineralization was analyzed in light microscope by Von Kossa technique. The statistica l differences were considered for p < 0.05. Calcification areas were present in all the MTA Flow® and MTA Angelus® groups. In the control group, no mineralized areas were observed. MTA Angelus® and thin-MTA Flow® showed significant reduction in calcification as time went by. A significant increase in areas with calcification, proportional to the exposure time, was observed in putty-MTA Flow® and thick-MTA Flow®. MTA Angelus® and thin-MTA Flow® showed significantly higher calcification than thick-MTA Flow® in the shortest exposure time. Analysis of putty-MTA Flow® showed significantly higher calcification areas than MTA Angelus® and thin-MTA Flow® in the longest exposure time. MTA Flow® stimulated mineralization, which has varied according to the concentration. Besides, in longer periods, MTA Flow® biomineralization performance was higher than MTAAngelus®, especially in highest concentration. (AU)

Esse estudo teve como objetivo avaliar a biomineralização induzida por diferentes concentrações do MTA Flow® e compará-la ao MTA Angelus®. Quinze ratos Wistar machos receberam implantes subcutâneos contendo os materiais a serem testados (MTA Angelus® e MTA Flow® nas consistências pastosa, espessa e fluida) e tubos vazios (controle). Após 7, 40 e 90 dias, os animais foram eutanasiados e os implantes foram removidos juntamente com o tecido circundante. A presença de biomineralização foi analisada em microscópio de luz pela técnica de Von Kossa. Diferenças estatísticas foram consideradas para valores de p < 0,05. Áreas de calcificação estavam presentes em todos os grupos do MTA Flow® e MTA Angelus®. No grupo controle não foram observadas áreas mineralizadas. O MTA Angelus® e o MTA Flow® fluido apresentaram redução significativa na quantidade de calcificação ao longo do tempo. Um aumento significativo na quantidade de áreas calcificadas, proporcional ao tempo de exposição, foi observado no MTA Flow® pastoso e no MTA Flow® espesso. O MTA Angelus® e o MTA Flow® fluido apresentaram calcificação significativamente maior do que o MTA Flow® espesso no menor período de exposição. Análises contento o MTA Flow® pastoso demonstraram áreas de calcificação significativamente maior do que o MTA Angelus® e MTA Flow® fluido no maior tempo de exposição. O MTA Flow® induziu a formação de áreas mineralizadas, que variou de acordo com a concentração do cimento. Em períodos mais longos, o MTA Flow® apresentou desempenho superior ao MTA Angelus®, principalmente quando utilizado na maior concentração. (AU)

Biomineralization of lithium nanoparticles by Li-resistant Pseudomonas rodhesiae isolated from the Atacama salt flat

BACKGROUND: The Atacama salt flat is located in northern Chile, at 2300 m above sea level, and has a high concentration of lithium, being one of the main extraction sites in the world. The effect of lithium on microorganism communities inhabiting environments with high concentrations of this metal has been scarcely studied. A few works have studied the microorganisms present in lithium-rich salt flats (Uyuni and Hombre Muerto in Bolivia and Argentina, respectively). Nanocrystals formation through biological mineralization has been described as an alternative for microorganisms living in metal-rich environments to cope with metal ions. However, bacterial lithium biomineralization of lithium nanostructures has not been published to date. In the present work, we studied lithium-rich soils of the Atacama salt flat and reported for the first time the biological synthesis of Li nanoparticles. RESULTS: Bacterial communities were evaluated and a high abundance of Cellulomonas, Arcticibacter, Mucilaginibacter, and Pseudomonas were determined. Three lithium resistant strains corresponding to Pseudomonas rodhesiae, Planomicrobium koreense, and Pseudomonas sp. were isolated (MIC > 700 mM). High levels of S2− were detected in the headspace of P. rodhesiae and Pseudomonas sp. cultures exposed to cysteine. Accordingly, biomineralization of lithium sulfide-containing nanomaterials was determined in P. rodhesiae exposed to lithium salts and cysteine. Transmission electron microscopy (TEM) analysis of ultrathin sections of P. rodhesiae cells biomineralizing lithium revealed the presence of nanometric materials. Lithium sulfide-containing nanomaterials were purified, and their size and shape determined by dynamic light scattering and TEM. Spherical nanoparticles with an average size < 40 nm and a hydro-dynamic size ~ 44.62 nm were determined. CONCLUSIONS: We characterized the bacterial communities inhabiting Li-rich extreme environments and reported for the first time the biomineralization of Li-containing nanomaterials by Li-resistant bacteria. The biosynthesis method described in this report could be used to recover lithium from waste batteries and thus provide a solution to the accumulation of batteries.

Ex Vivo Analysis of MTA FLOW® Biomineralization and Push-Out Strength: A Pilot Study / Análisis ex vivo del proceso de biomineralización y resistencia al desplazamiento del MTA FLOW®: Estudio piloto

Abstract: This study evaluated the biomineralization processes and push-out strength of MTA Flow® with radicular dentine in three different consistencies. The push-out test was performed on an ex vivo model, using 2mm thick dentin discs from the middle third of the root with standardized cavities of 1.5 mm. Samples were filled with MTA-Angelus (Angelus Dental, Brazil), Biodentine (Septodont, France), MTA Flow® Putty (Ultradent, USA), MTA Flow® Thick or MTA Flow® Thin. The samples were divided into 3 groups: subgroup 1 (n=5), analysis of the biomineralization process; 2 (n=20), evaluation of the bonding strength and push-out resistance; and 3 (n=5), evaluation of the cement/ dentin interface. The samples filled with Biodentine had a higher precipitation of carbonate apatite. However, there was no significant difference between MTA-Angelus, MTA Flow® Putty, or Thick (p=0.0536), but there was a significant difference in the Thin group (P<0.05). The samples with Biodentine displayed the greatest release of calcium ions. The formation of a partially carbonated intermediate apatite layer was observed in all groups. Zones of biomineralization were observed at the interface but were not continuous. After 72 hours, a significant difference was found between the Biodentine and MTA Flow® Thin groups (p=0.0090) in the push-out test. The samples submerged in phosphate-buffered saline (PBS) for 15 days showed a significant difference between all groups and MTA Flow® Thin (p=0.0147). Putty or Thick consistencies presented a similar bonding strength to MTA-Angelus and Biodentine. MTA Flow® Putty and Thick consistencies show a good adaptation to dentin, similar to MTA-Angelus. However, the thickness of the interface was lower compared to that of Biodentine. MTA Flow® Thin, despite their tubular infiltration, results in gaps and a defective peripheral seal.Therefore, MTA Flow®, in Putty or Thick consistencies, presents a biomineralization process and push-out strength similar to MTA Angelus and Biodentine, however, both characteristics decreases considerably in Thin consistency.

Resumen: El objetivo de este estudio fue evaluar, en un modelo ex vivo, el proceso de biomineralización y fuerza de adhesión del MTA Flow® en sus tres diferentes consistencias por medio de la prueba de resistencia al desplazamiento (Push-out). Se utilizaron discos de dentina de 2mm de espesor del tercio medio radicular con cavidades estandarizadas de 1.5mm de diámetro, las cuales se obturaron con diferentes materiales entre ellos: MTA Angelus (Angelus Dental, Brasil), Biodentine (Septodont, Francia), MTA Flow® Consistencia Putty (Ultradent, E.E.U.U), MTA Flow® Consistencia Thick (Ultradent, E.E.U.U) y MTA Flow® Consistencia Thin (Ultradent, E.E.U.U). Las muestras se sometieron al proceso de biomineralización y a pruebas de Push-out. Las muestras obturadas con Biodentine promovieron una mayor precipitación de apatita carbonatada, sin embargo, no se presentó diferencia estadística significativa con respecto al MTA Angelus, MTA Flow® Putty ni Thick (p=0.0536). No obstante, si presentó una diferencia significativa con respecto al grupo de MTA Flow® consistencia Thin (P<0.05). Las muestras con Biodentine presentaron la mayor liberación de iones calcio. De acuerdo a las pruebas de resistencia al desplazamiento, a las 72 horas post-obturación, solamente se encontró diferencia significativa entre las muestras obturadas con Biodentine y las correspondientes al MTA Flow consistencia Thin (p=0.0090), sin embargo las muestras sumergidas 15 días en PBS presentaron diferencia significativa entre todos los grupos con respecto al MTA Flow Thin (p=0.0147). En general se observaron zonas de biomineralización en la interface, sin embargo, no fueron continuas. Se concluye que el MTA Flow en consistencia Putty o Thick presenta un proceso de biomineralización y una resistencia al desplazamiento similar al MTA Angelus y al Biodentine, sin embargo, esta última disminuye considerablemente en presentación Thin.

Influence of the Vehicle on the Tissue Reaction and Biomineralization of Fast Endodontic Cement

ABSTRACT Objective: To investigate the tissue response and the biomineralization ability of CER prepared with epoxy resin or water compared to Mineral Trioxide Aggregate (MTA). Material and Methods: Polyethylene tubes containing materials or empty tubes for control were inserted into the subcutaneous tissues of 30 rats. After 7, 15, 30, 60, and 90 days, the rats were killed and the tubes were removed for analysis using hematoxylin-eosin staining, von Kossa staining, and under polarized light. Inflammation was graded through a score system; the thickness of the fibrous capsule was classified as thin or thick; the biomineralization ability was recorded as present or absent. The results were statistically analyzed using the Kruskal-Wallis test (p<0.05). Results: Histologic analysis performed after 7 and 15 days for CER prepared with epoxy resin or water and for MTA showed moderate inflammation and a thick fibrous capsule (p>0.05). After 30, 60, and 90 days, mild inflammation, and a thin fibrous capsule were observed in all groups (p>0.05). Conclusion: All materials had structures positive for von Kossa and birefringent to polarized light. CER epoxy resin showed biocompatibility and biomineralization similar to CER water and MTA.

Biocompatibility, biomineralization, and maturation of collagen by rtr®, bioglass and dm bone® materials

Abstract This study evaluated the biocompatibility, biomineralization, and collagen fiber maturation induced by Resorbable Tissue Replacement (RTR®; β-tricalcium phosphate [TCP]), Bioglass (BIOG; bioactive glass), and DM Bone® (DMB; hydroxyapatite and β-TCP) in vivo. Sixty-four polyethylene tubes with or without (control group; CG) materials (n=8/group/period) were randomly implanted in the subcutaneous tissue of 16 male Wistar rats (four per rat), weighting 250 to 280 g. The rats were killed after 7 and 30 days (n=8), and the specimens were removed for analysis of inflammation using hematoxylin-eosin; biomineralization assay using von Kossa (VK) staining and polarized light (PL); and collagen fiber maturation using picrosirius red (PSR). Nonparametric data were statistically analyzed by Kruskal-Wallis and Dunn tests, and parametric data by one-way ANOVA test (p<0.05). At 7 days, all groups induced moderate inflammation (p>0.05). At 30 days, there was mild inflammation in the BIOG and CG, and moderate inflammation in the RTR and DMB groups, with a significant difference between the CG and RTR (p<0.05). The fibrous capsule was thick at 7 days and predominantly thin at 30 days in all groups. All materials exhibited structures that stained positively for VK and PL. Immature collagen fibers were predominant at 7 and 30 days in all groups (p>0.05), although DMB exhibited more mature fibers than BIOG at 30 days (p<0.05). RTR, BIOG, and DMB were biocompatible, inducing inflammation that reduced over time and biomineralization in the subcutaneous tissue of rats. DMB exhibited more mature collagen fibers than BIOG over a longer period.

Resumo Este estudo avaliou a biocompatibilidade, biomineralização e maturação das fibras de colágeno induzidas por Resorbable Tissue Replacement (RTR®; fosfato β-tricálcico [TCP]), Bioglass (BIOG; vidro bioativo) e DM Bone® (DMB; hidroxiapatita e β-TCP) in vivo. Sessenta e quatro tubos de polietileno com ou sem (grupo controle; GC) os materiais (n=8/grupo/período) foram implantados aleatoriamente em tecido subcutâneo de 16 ratos machos Wistar (quatro por rato), pesando entre 250 a 280g. Os ratos foram mortos após 7 e 30 dias (n=8), e as amostras foram removidas para análise da inflamação utilizando hematoxilina-eosina; avaliação da biomineralização utilizando coloração de von Kossa (VK) e luz polarizada (LP); e maturação das fibras colágenas, utilizando picrosirius red (PSR). Os dados não-paramétricos foram analisados pelos testes de Kruskal-Wallis e Dunn, e os paramétricos pelo teste de one-way ANOVA (p<0.05). Aos 7 dias, todos os grupos induziram inflamação moderada (p>0,05). Aos 30 dias, houve inflamação leve nos grupos BIOG e GC, e inflamação moderada nos grupos RTR e DMB, com diferença significativa entre os GC e RTR (p<0,05). A cápsula fibrosa foi espessa aos 7 dias, e predominantemente fina aos 30 dias em todos os grupos. Todos os materiais exibiram estruturas positivas para VK e LP. Fibras colágenas imaturas foram predominantes aos 7 e 30 dias em todos os grupos (p>0,05), embora o DMB exibiu fibras mais maduras do que o BIOG aos 30 dias (p<0,05). RTR, BIOG e DMB foram biocompatíveis, induzindo inflamação que reduziu com o tempo, e biomineralização no tecido subcutâneo de ratos. O DMB exibiu mais fibras colágenas maduras do que o BIOG em período mais longo.

Ex Vivo Evaluation of a Restoration Protocol for Teeth with Simulated Incomplete Rhizogenesis / Evaluación ex vivo de un protocolo de restauración para dientes con rizogénesis incompleta simulada

Abstract: The use of phosphate buffered saline (PBS) as an intracanal medication triggers a biomineralization process within mineral trioxide aggregate (MTA) apical plugs during the apexification process in teeth with incomplete rhizogenesis. However, no consensus is available in the literature regarding a restorative protocol for this type of treatment. Thus, the objective of this study was to use scanning electron microscopy (SEM) to evaluate the processes of biomineralization and adhesion in a restorative protocol for teeth with simulated incomplete rhizogenesis. Methodology: Root sections with a thickness of 2mm and cavities with a diameter of 2mm were used. The sections were randomly prepared and filled with the following materials: Group 1 (n=12), ProRoot MTA; and Group 2 (n=12): MTA Exp. Subsequently, the samples were immersed in PBS for 35 days. Every 5 days, the PBS was replaced, and the precipitates were collected, dried, and weighed. Two samples from each group were analyzed by SEM. Moreover, 24 single-rooted teeth were standardized, incomplete rhizogenesis was simulated, and 5-mm-long apical plugs were created with Pro Root MTA. As an intracanal medication, PBS was used for different periods of time: Group 1:48 h; Group 2:7 days; and Group 3:15 days. Then, fiberglass posts were cemented with the REBILDA® Post System. The samples were prepared and analyzed by SEM. Results: ProRoot MTA and MTA Exp effectively promoted the formation of carbonated apatite precipitates and biomineralization with dentin. ProRoot MTA yielded more carbonated apatite precipitates compared to MTA Exp (p=0.0536). The use of PBS as an intracanal medication for 7 and 15 days promoted intratubular mineralization (MIT), and treatment for 15 days was more effective (p < 0.05). The REBILDA® Post System effectively promoted the microimbrication of the adhesive system and the formation of resinous tags with lateral adhesive branches. Conclusion: Apexification with MTA associated with the use of PBS as an intracanal medication for 15 days, in addition to the use of the REBILDA® Post System, seems to be a feasible restorative protocol.

Resumen: El uso de solución salina tamponada con fosfato (PBS) como medicamento intracanal desencadena un proceso de biomineralización en los plugs apicales con agregado de trióxido mineral (MTA) durante el proceso de apexificación en dientes con rizogénesis incompleta. Sin embargo, no hay consenso disponible en la literatura sobre un protocolo restaurador para este tipo de tratamiento. Por lo tanto, el objetivo de este estudio fue utilizar microscopía electrónica de barrido (MEB) para evaluar los procesos de biomineralización y adhesión en un protocolo restaurador para dientes con rizogénesis incompleta simulada. Metodología: Se utilizaron secciones de raíz con un espesor de 2mm y se realizaron cavidades con un diámetro de 2 mm. Las cavidades en las secciones se obturaron con: Grupo 1 (n=12), ProRoot MTA; y Grupo 2 (n=12): MTA Exp. Posteriormente, las muestras se sumergieron en PBS durante 35 días. Cada 5 días, se reemplazó el PBS y se recogieron los precipitados, se secaron y pesaron. Dos muestras de cada grupo fueron analizadas por MEB. Además, se estandarizaron 24 dientes de raíz única, se simuló la rizogénesis incompleta y se crearon tapones apicales de 5mm de longitud con Pro Root MTA. Como medicamento intracanal, se utilizó PBS durante diferentes períodos de tiempo: Grupo 1:48 h; Grupo 2:7 días; y Grupo 3:15 días. Posteriormente, los postes de fibra de vidrio se cementaron con el sistema de postes REBILDA®. Las muestras fueron preparadas y analizadas por MEB. Resultados: ProRoot MTA y MTA Exp promovieron efectivamente la formación de precipitados de apatita carbonatada y la biomineralización con dentina. ProRoot MTA produjo más precipitados de apatita carbonatada en comparación con MTA Exp (p=0.0536). El uso de PBS como medicamento intracanal durante 7 y 15 días promovió la mineralización intratubular (MIT), siendo el tratamiento durante 15 días más efectivo (p <0.05). El sistema de postes REBILDA® promovió efectivamente la microimbricación del sistema adhesivo y la formación de tags resinosos. Conclusión: La apexificación con MTA asociada con el uso de PBS como medicación intracanal durante 15 días, además del uso del sistema de postes REBILDA®, parece ser un protocolo factible y eficaz en este tipo de tratamientos.

Effect of Using Biomimetic Analogs on Dentin Remineralization with Bioactive Cements

Abstract This study evaluated the impact of using biomimetic analogs (poly-acrylic acid and sodium tri-meta-phosphate) on dentin remineralization using two cement materials, the first is calcium silicate based and the second is calcium hydroxide based materials. Two standardized occlusal cavities (mesial and distal) were prepared within dentin after removal of occlusal enamel. Artificial demineralized dentin was induced through pH cycling (8 h in demineralizing and 16 h in remineralizing solutions). Demineralized cavities were divided into four groups; two groups received cement materials. The other groups were first treated with biomimetic analogs then restored with pulp cement materials. Teeth were sectioned buccolingually into two halves. Treated cavities with analogs were stored in simulated body fluid containing poly-acrylic acid. Untreated cavities were stored in simulated body fluid only. Ground unstained sections of demineralized dentin were examined using light microscope. Specimens were examined after 1, 6 and 12 weeks of storage using energy dispersive X-ray Spectroscopy (EDX) and Vickers microhardness was evaluated. Two-way ANOVA was used to analyze data statistically. Calcium silicate-based cement group with biomimetic analogs showed the highest statistically significant calcium and phosphorous wt% in addition to highest surface hardness values after 12 weeks of storage. Demineralized dentin ground sections showed increase in light zones after total period of storage. Calcium silicate-based cement showed the best ability to enrich the artificial carious dentin with ions for remineralization. Using biomimetic analogs had a significant impact on demineralized dentin surface hardness improvement.

Resumo Este estudo avaliou o impacto do uso de análogos biomiméticos (ácido poli-acrílico e tri-meta-fosfato de sódio) na remineralização da dentina usando dois materiais de cimento, o primeiro à base de silicato de cálcio e o segundo à base de hidróxido de cálcio. Duas cavidades oclusais padronizadas (mesial e distal) foram preparadas na dentina após a remoção do esmalte oclusal. A dentina desmineralizada artificial foi induzida por ciclagem de pH (8 h em soluções desmineralizantes e 16 h em soluções remineralizantes). Cavidades desmineralizadas foram divididas em quatro grupos; dois grupos receberam materiais de cimento. Os outros grupos foram tratados primeiro com análogos biomiméticos e depois restaurados com materiais de cimento de celulose. Os dentes foram seccionados bucolingualmente em duas metades. As cavidades tratadas com análogos foram armazenadas em fluido corporal simulado contendo ácido poli-acrílico. As cavidades não tratadas foram armazenadas apenas em fluido corporal simulado. Secções não coradas de dentina desmineralizada foram examinadas usando microscópio óptico. As amostras foram examinadas após 1, 6 e 12 semanas de armazenamento usando Espectroscopia de Raios-X dispersiva por energia (EDX) e microdureza Vickers. ANOVA a dois fatores foi usada para analisar os dados estatisticamente. O grupo de cimento à base de silicato de cálcio com análogos biomiméticos apresentou os maiores percentagens em peso estatisticamente significantes de cálcio e fósforo, além dos maiores valores de dureza superficial após 12 semanas de armazenamento. Seções desmineralizadas de dentina mostraram aumento nas zonas de luz após o período total de armazenamento. O cimento à base de silicato de cálcio mostrou a melhor capacidade de enriquecer a dentina cariada artificial com íons para remineralização. O uso de análogos biomiméticos teve um impacto significativo na melhoria da dureza superficial da dentina desmineralizada.

Fracture resistance of teeth obturated with two different types of mineral trioxide aggregate cements / Resistência à fratura de dentes obturados com dois tipos diferentes de cimentos agregados de trióxido minera

Objective: Endodontically obturated teeth have lower fracture resistance depending on the obturating material and technique. The purpose of this study was therefore to evaluate the influence of ProRoot MTA (Dentsply Sirona, Tulsa Division) and OrthoMTA III (BioMTA, Daejeon, Korea) as an obturating material on the fracture resistance of endodontically treated teeth. Material and Methods: Thirty extracted human maxillary central incisors were decoronated and instrumented using Protaper instruments (size F5). Irrigation was performed with 2.5% sodium hypochlorite between each instrument change followed by 7% maleic acid for one minute. Finally, canals were flushed with 5 ml of PBS solution for one minute. Samples were then divided into three groups. Group I- positive control (no root canal filling); Group II- obturation with ProRoot MTA; Group III- obturation with OrthoMTA III. Ten teeth were randomly selected as a negative control in which no treatment was performed. All the specimens were then subjected to fracture strength testing using universal testing machine. For evaluation of biomineralization, six maxillary central incisors were divided into two groups. Group I obturated with ProRoot MTA and group II obturated with OrthoMTA III. These samples were subjected to SEM analysis. Results: Positive control group demonstrated the least fracture resistance, while OrthoMTA III group showed the highest fracture resistance. There was no significant difference between negative control group and ProRoot MTA groups (p=0.821). OrthoMTA III group showed better tubular biomineralization when compared to ProRoot MTA. Conclusions: Root canals obturated with OrthoMTA III had better fracture resistance and increased tubular biomineralization compared to ProRoot MTA. Since root canals obturated with OrthoMTA III had better fracture resistance, it can be used as a promising obturating material.(AU)

Objetivo: Dentes obturados endodonticamente apresentam menor resistência à fratura, dependendo do material e da técnica de obturação. Portanto, o objetivo deste estudo foi avaliar a influência do ProRoot MTA (Dentsply Sirona, Tulsa Division) e OrthoMTA III (BioMTA, Daejeon, Coréia) como material obturador na resistência à fratura de dentes tratados endodonticamente. Material e Métodos: Trinta incisivos centrais superiores humanos extraídos foram decoronados e instrumentados com instrumentos Protaper (tamanho F5). A irrigação foi realizada com hipoclorito de sódio a 2,5% entre cada troca de instrumento, seguida por ácido maleico a 7% por um minuto. Finalmente, os canais foram lavados com 5 ml de solução de PBS por um minuto. As amostras foram então divididas em três grupos. Grupo I - controle positivo(sem preenchimento do canal radicular); Grupo II - obturação com ProRoot MTA; Grupo III -obturação com OrthoMTA III. Dez dentes foram selecionados aleatoriamente como controle negativo, no qual nenhum tratamento foi realizado. Todas as amostras foram então submetidas atestes de resistência à fratura usando uma máquina de teste universal. Para avaliação da biomineralização, seis incisivos centrais superiores foram divididos em dois grupos: grupo Iobturado com ProRoot MTA e grupo II obturado com OrthoMTA III. Essas amostras foram submetidas à análise SEM. Resultados: O grupo controle positivo demonstrou a menor resistência à fratura, enquanto o grupo OrthoMTA III apresentou a maior resistência à fratura. Não houve diferença significativa entre o grupo controle negativo e os grupos ProRoot MTA (p= 0,821). O grupo OrthoMTA III apresentou melhor biomineralização tubular quando comparado ao ProRoot MTA. Conclusões: Os canais radiculares obturados com OrthoMTA III apresentaram melhor resistência à fratura e maior biomineralização tubular em comparação como ProRoot MTA. Como os canais radiculares obturados com OrthoMTA III apresentaram melhor resistência à fratura, podendo ser utilizado como um material obturador promissor.(AU)

Design of drug-like hepsin inhibitors against prostate cancer and kidney stones

Hepsin, a transmembrane serine protease abundant in renal endothelial cells, is a promising therapeutic target against several cancers, particularly prostate cancer. It is involved in the release and polymerization of uromodulin in the urine, which plays a role in kidney stone formation. In this work, we design new potential hepsin inhibitors for high activity, improved specificity towards hepsin, and promising ADMET properties. The ligands were developed through a novel hierarchical pipeline. This pipeline explicitly accounts for off-target binding to the related serine proteases matriptase and HGFA (human hepatocyte growth factor activator). We completed the pipeline incorporating ADMET properties of the candidate inhibitors into custom multi-objective optimization functions. The ligands designed show excellent prospects for targeting hepsin the blood stream and the urine and thus enable key experimental studies. The computational pipeline proposed is remarkably cost-efficient and can be easily adapted for designing inhibitors against new drug targets.

Mining and engineering of microbial carbonic anhydrases for biomimetic carbon dioxide sequestration / 生物工程学报

The increasing atmospheric carbon dioxide levels have been correlated with global warming. Carbonic anhydrases (CA) are the fastest among the known enzymes to improve carbon capture. The capture of carbon dioxide needs high temperature and alkaline condition, which is necessary for CaCO₃ precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential, and polyextremophilic microbes are one of the important sources of these enzymes. The current review focuses on both those isolated by thermophilic organisms from the extreme environments and those obtained by protein engineering techniques, and the industrial application of the immobilized CAs is also briefly addressed. To reduce the greenhouse effect and delay global warming, we think further research efforts should be devoted to broadening the scope of searching for carbonic anhydrase, modifying the technology of protein engineering and developing highly efficient immobilization strategies.

Molecular characterization of CHST11 and its potential role in nacre formation in pearl oyster Pinctada fucata martensii

Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.

Efecto del Tween ® 80 sobre la bioprecipitación de carbonato de calcio por Bacillus cereus / Effect of Tween ® 80 on calcium carbonate bioprecipitation mediated by Bacillus cereus

Resumen Se llevaron a cabo procesos de biosíntesis de carbonato de calcio, empleando una cepa de Bacillus cereus, aislada de los jardines de la Universidad Pontificia Bolivariana (Medellín, Colombia). Se evaluó el efecto disgregante del Tween® 80 a tres concentraciones: 0.00% p/v, 0.25% p/v y 0.50% p/v. Los experimentos se monitorizaron por 6 días con mediciones de pH y análisis mineralógicos a los precipitados finales por microscopía electrónica de barrido, difracción de rayos X y espectroscopia de infrarrojo con transformada de Fourier. El Tween® 80 tuvo un papel importante como desaglomerante de las estructuras de carbonato formadas por los microorganismos. Aunque los ensayos con el surfactante presentaron menor formación de precipitado, las concentraciones evaluadas no inhibieron el crecimiento bacteriano. Adicionalmente, este compuesto favoreció la formación de vaterita incrementando su proporción en comparación con la calcita (alrededor del 98%, utilizando Tween® 80 al 0.50% p/v).

Abstract This research presents the sprinkling effect of Tween® 80 for three concentrations (0.00% w, 0.25% w y 0.50% w) in a processes of calcium carbonate biosynthesis. The assays used a culture of Bacillus cereus, isolated from the gardens of the Universidad Pontificia Bolivariana (Medellin, Colombia). The experiments were monitored for 6 days measuring pH as well as through mineralogical analyses for precipitates using scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. Tween® 80 had an important role as deagglomerate, decreasing the precipitation of biomineralized-carbonate structures (generated by bacteria). It was also found that the evaluated concentrations did not inhibit the bacterial growth. Additionally, Tween® 80 favored the production of vaterite, increasing its ratio in comparison to calcite (around 98% by using 0.50% w of Tween® 80).

Mechanism and Applications of Bio-mineralization Induced by Sporosarcina pasteurii and Related Microorganisms / 中国生物工程杂志

As known so far,Sporosarcina pasteurii,or formally termed as Bacillus pasteurii,was considered as one of the most efficient biosystem which is capable of inducing biological mineralization through breaking down urea.Taking advantage of the ‘super power’ of bio-mineralization,Sporosarcina pasteurii has been successfully utilized in application of solidifying sand as a novel biological construction technology,termed as ‘bio-cementation’ Due to the nature of Sporosarcina pasteurii isolated from soil,non-pathogenicity has been observed,it was considered as a very environmentally friendly method.Recently,Sporosarcina pasteurii has been further applied into fields including environmental improvement and biomedicine.However,the mechanism under the strong Sporosarcina pasteurii mediated bio-mineralization is still not well understood.Here,the knowledge and the up-to-date studies about the biological mechanism of Sporosarcina pasteurii mediated bio-mineralization,and the utilization in construction,environment,and biomedicine are reviewed.

Investigation on Properties of Collagen Nanowires Quasiepitaxially Grown on Mica Lattice Plane / 分析化学

Collagen fibrils and hydroxyapatite might recognize each other at the mesoscale by multiple cooperative interactions due to their intrinsically repetitive structured surfaces, and thus effectively directing the biomineralization, a biological process involving regulating the growth of bones, teeth and other organs. In this work, we developed a simple technique to prepare nanowire arrays of biological macromolecules by reversely using the biomineralization mechanism, with results similar to the hot wall epitaxy, a molecular beam deposition technique under vacuum. With this technique, we successfully cultured 5-10 μg/mL rat tail type I collagen monomer solutions into collagen nanowire arrays on the mica (001) lattice plane along one unique direction across the whole cleavage surface. The atomic force microscope experiments indicated that the nanowires in the arrays became more crowded with higher monomer concentration, but their width and height remained unchanged, about 60.0 nm and 1.5 nm, respectively. The collagen nanowire coating enhanced the hydrophilicity of the mica surface, reducing the contact angle from 25.8° to 9.5°. Based on the characterization results of electron back scattering diffraction and transmission electron microscope, the collagen nanowires were most likely to be oriented along the mica [110] direction, which validated the quasiepitaxial growth mechanism in more details.

Biomineralization of electrospun polycaprolactone-guided bone regeneration membrane / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the biomineralization of the tissue-engineering electrospun polycaprolactone (PCL) scaffold and its potential use for guided bone regeneration (GBR) membranes.</p><p><b>METHODS</b>PCL ultrafinefiber scaffolds were fabricated by electrospinning and then immersed in supersaturated calcification solution (SCS) for biomineralization investigation. The electrospun PCL scaffolds and the calcium phosphate coating were identified by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Water-contact angles were also measured to evaluate the hydrophilicity of the modified surface. The biocompatibility of the composite was investigated by culturing osteoblasts on the scaffolds. Cell behavior was observed by SEM.</p><p><b>RESULTS</b>The electrospun PCL scaffold was composed of ultrafine fibers and well-interconnected pores. The deposits on the fibers grew in number and size as the biomineralization time increased. Then, the covering of the whole PCL film was identified as dicalcium phosphate dehydrate and apatite. Good cell attachment and proliferation behavior were observed on the membranes.</p><p><b>CONCLUSIONS</b>The quick apatite formation on the surface of the PCL electrospun scaffold indicated that SCS biomineralization may be an effective approach for obtaining PCL/calcium phosphate composites. The cellular biocompatibility of the composite scaffold was preliminarily confirmed by the in vitro culture of osteoblasts on the scaffold. As such, the composite scaffold is a promising biomimetic extracellular matrix biomaterial for bone tissue engineering and GBR membranes.</p>

Biophysical Methods for the Elucidation of the S-Layer Proteins/Metal Interaction.

Surface-layers (S-layers) are macromolecular paracrystalline arrays of proteins or glycoproteins that can self-assemble into 2-dimensional semi-permeable meshworks to overlay the cell surface of many bacteria and archaea. They usually assemble into lattices with oblique, square or hexagonal symmetry and serve as an interface between the bacterial cell and the environment. Isolated S-layers can recrystallize into two-dimensional regular arrays in suspension or on various surfaces, thus being an appropriate material for several bionanotechnological purposes. Promising applications of S-layers include their use as biotemplates for the capture of metal ions or the synthesis of metal nanoclusters.

Over-expression of carbonic anhydrase 1 is involved in bio-mineralization process / 中华风湿病学杂志

Objective Carbonic anhydrase 1 (CA1) not only enhances the hydration reaction but also promotes the formation of CaCO3,which is an essential step for new bone formation in vitro.However,there is no direct evidence to demonstrate the involvement of CA1 in bio-mineralization in cells and tissues.This study is aimed to evaluate the important role of CA1 in bio-mineralization and ossification in cultured cells.Methods Calcification in Saos-2 cells was induced using osteogenic medium (OM) and the calcification was determined by Alizarin Red-S staining.The expressions of ossification protein marker Runt-related transcription factor-2 (Runx2),osterix (OSX),alkaline phosphatase (ALP),osteocalcin (OCN) and bone sialoprotein (BSP) were detected in the process of bone formation by real-time PCR.The expression of CA 1 in the calcified cells were measured using real-time PCR and Western blotting.We also detected calcification in Saos-2 cells in the presence of acetazolamide,an anti-carbonic anhydrase drug to CA1,to determine the role of CA1 in biomineralization in culture cells.T test analysis was used to compare the two groups,M-ANOVA of repeated measurs was conducted for different time point.Results Following the stimulation of OM,Saos-2 cells produced a great amount of calcium-rich deposits [0.68±0.03 vs 2.76±0.13,P＜0.01].Increased transcriptions of ossification protein markers were also detected in these stimulated Saos-2 cells,indicating that the OM launched the process of bone formation in the cells.CA1 had a significantly increased expression during this process [0.25±0.03 vs 0.94±0.06,P＜0.01].Following treatment with acetazolamide,the expression of CA1 evidently declined [1.09±0.05 vs 0.55±0.07,P＜0.05],and the mineralized nodule formation was declined [2.76±0.13 vs 2.19±0.07,P＜0.01].Conclusion These findings indicate that CA1 participates in the biomineralization and ossification,and may play an important roles in bone formation.

Promotion of calcium oxalate crystallization after human renal tubular epithelial cell injury / 中华泌尿外科杂志

Objective To investigate the injury caused by hydrogen peroxide (H2O2) on human renal tubular epithelial cell (HKC) and its effect on calcium oxalate (CaOxa) crystal crystallization time before and after the injury. Methods The injury degree of HKC by H2O2 was measured by detecting the cell survival rate and the concentration change of malonaldehyde (MDA). CaOxa crystallization was investigated by scanning electron microscopy (SEM). Results Control cells induced only a small amount of calcium oxalate dihydrate (COD) crystals, while the injured cells not only induced calcium oxalate monohydrate (COM) crystals, but also increased the number and aggregation of CaOxa crystals. After incubating with CaOxa supersaturated solution, the control group HKC cells could be injured as well. Conclusions H2O2 can cause oxidative damage on HKC. The injured HKC promotes the nucleation and aggregation of COM crystals. In the body environment, the long-term presence of crystals in urinary tract is a risk factor for stone formation.