Abstract Calcium aluminate cement (CAC) has been highlighted as a promising alternative for endodontic use aiming at periapical tissue repair. However, its effects on dental pulp cells have been poorly explored. Objective: This study assessed the impact of calcium chloride (CaCl2) and bismuth oxide (Bi2O3) or zinc oxide (ZnO) additives on odontoblast cell response to CAC. Methodology: MDPC-23 cells were exposed for up to 14 d: 1) CAC with 2.8% CaCl2 and 25% ZnO (CACz); 2) CAC with 2.8% CaCl2 and 25% Bi2O3 (CACb); 3) CAC with 10% CaCl2 and 25% Bi2O3 (CACb+); or 4) mineral trioxide aggregate (MTA), placed on inserts. Non-exposed cultures served as control. Cell morphology, cell viability, gene expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), and dentin matrix protein 1 (DMP-1), ALP activity, and extracellular matrix mineralization were evaluated. Data were compared using ANOVA (α=5%). Results: Lower cell density was detected only for MTA and CACb+ compared with Control, with areas showing reduced cell spreading. Cell viability was similar among groups at days one and three (p>0.05). CACb+ and MTA showed the lowest cell viability values at day seven (p>0.05). CACb and CACb+ promoted higher ALP and BSP expression compared with CACz (p<0.05); despite that, all cements supported ALP activity. Matrix mineralization were enhanced in CACb+ and MTA. Conclusion: In conclusion, CAC with Bi2O3, but not with ZnO, supported the expression of odontoblastic phenotype, but only the composition with 10% CaCl2 promoted mineralized matrix formation, rendering it suitable for dentin-pulp complex repair.

The radiopacity of CAC-based compositions containing additives (ZnO, Ag and Bi2O3) was evaluated by optical density measurements, cold crushing strength, apparent porosity, pore size distribution, in vitro apatite-formation ability (bioactivity) and cell viability. The bioactivity was assessed via pH and calcium concentration measurements in simulated body fluid (SBF) for samples previously treated or not with sodium silicate (SS) solution. The surface of the samples after treatment was analyzed by scanning electron microscopy, energy dispersive X-ray analysis and confocal Raman spectroscopy. Cell viability was evaluated by MTT and alkaline phosphatase (ALP) activity. Radiopacity results confirmed that compositions containing ZnO-Ag (25:1 wt%) and ZnO-Bi2O3 (15:10 wt%) presented suitable values of radiopacity (> 3 mmAl). The identification of calcium and mainly phosphorus by EDX, highlighted the generation of an apatite-like layer on the compositions' surface after immersion in SBF or SBF/SS. CAC-blend containing 25 wt% ZnO, ZnO-Ag and ZnO-Bi2O3 presented slightly lower cell viability compared to the positive control.

Objective : To investigate the influence of a three-dimensional cell culture model on the expression of osteoblastic phenotype in human periodontal ligament fibroblast (hPDLF) cultures.Material and Methods : hPDLF were seeded on bi-dimensional (2D) and three-dimensional (3D) collagen type I (experimental groups) and and on a plastic coverslip (control) for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity were performed. Also, cell morphology and immunolabeling for alkaline phosphatase (ALP) and osteopontin (OPN) were assessed by epifluorescence and confocal microscopy. The expression of osteogenic markers, including alkaline phosphatase, osteopontin, osteocalcin (OC), collagen I (COL I) and runt-related transcription factor 2 (RUNX2), were analyzed using real-time polymerase chain reaction (RT-PCR). Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay.Results : Experimental cultures produced an increase in cell proliferation. Immunolabeling for OPN and ALP in hPDLF were increased and ALP activity was inhibited by three-dimensional conditions. OPN and RUNX2 gene expression was significantly higher on 3D culture when compared with control surface. Moreover, ALP and COL I gene expression were significantly higher in three-dimensional collagen than in 2D cultures at 7 days. However, at 14 days, 3D cultures exhibited ALP and COL I gene expression significantly lower than the control, and the COL I gene expression was also significantly lower in 3D than in 2D cultures. Significant calcium mineralization was detected and quantified by alizarin red assay, and calcified nodule formation was not affected by tridimensionality.Conclusion : This study suggests that the 3D cultures are able to support hPDLF proliferation and favor the differentiation and mineralized matrix formation, which may be a potential periodontal regenerative therapy.

O objetivo do presente estudo foi avaliar a progressão de cultura de células osteogênicas expostas a um novo cimento de aluminato de cálcio (CAC+) em comparação ao agregado de trióxido mineral (MTA). As células foram obtidas por digestão enzimática de calvária de ratos recém-nascidos, plaqueadas sobre lamínulas de vidro contendo em sua área central discos de CAC+ ou MTA e crescidas em condições osteogênicas por até 10 dias. Durante a cultura primária, observou-se o arredondamento das bordas das amostras de cimento apenas para MTA. Embora ambos os cimentos tenham permitido a adesão, o espraiamento e a proliferação celulares, as culturas crescidas em contato com CAC+ exibiram valores maiores de número total de células em 3 e 7 dias, e de conteúdo de proteína total e atividade de fosfatase alcalina em 10 dias. Os resultados indicam que a exposição ao CAC+ permite o desenvolvimento de uma proporção maior de células em estágios mais avançados da diferenciação osteoblástica, quando comparado ao MTA. Deve-se considerar em futuros estudos experimentais a utilização do CAC+ como um material alternativo ao MTA especialmente quando um dos objetivos do tratamento endodôntico é o de reparação dos tecidos mineralizados da região periapical.

The present study evaluated the progression of osteogenic cell cultures exposed to a novel calcium aluminate cement (CAC+) in comparison with the gold standard mineral trioxide aggregate (MTA). Cells were enzimatically isolated from newborn rat calvarial bone, plated on glass coverslips containing either CAC+ or a control MTA samples in the center, and grown under standard osteogenic conditions. Over the 10-day culture period, roundening of sample edges was clearly noticed only for MTA group. Although both cements supported osteogenic cell adhesion, spreading, and proliferation, CAC+-exposed cultures showed significantly higher values in terms of total cell number at days 3 and 7, and total protein content and alkaline phosphatase activity at day 10. The present in vitro results indicate that the exposure to CAC+ supports a higher differentiation of osteogenic cells compared with the ones exposed to MTA. Further experimental studies should consider CAC+ as a potential alternative to MTA when the repair of mineralized tissues is one of the desired outcomes in endodontic therapy.

A interface osso-implante é caracterizada pela presença de uma camada de matriz extracellular (MEC) afibrilar rica em proteínas não-colágenas, incluindo osteopontina (OPN), cujas funções no tecido ósseo estão relacionadas à adesão celular e ao controle do processo de mineralização da MEC (crescimento de cristais). Aspectos físicos e químicos das superfícies de biomateriais podem afetar as interações célula-MEC-substrato. O objetivo do presente estudo foi demonstrar a capacidade de aspectos nanotopográficos de superfície de titânio (Ti) de controlar a deposição extracelular de OPN in vitro. Discos de Ti foram tratados quimicamente por solução de H2SO4/H2O2 durante 30 min [Nano(30') Ti] ou 4 h [Nano(4h) Ti]. Superfícies de Ti não tratadas foram usadas como controle. Células osteogênicas primárias derivadas de calvárias de ratos recém-nascidos foram plaqueadas sobre os discos de Ti e cultivadas em condições osteogênicas por até 7 dias. Microscopia eletrônica de varredura de alta resolução revelou que os discos de Ti tratados quimicamente exibiam superfície nanoporosa, com áreas de nanoporos maiores para Nano(4h) Ti. Apenas para esse grupo detectavam-se acúmulos extensos de OPN extracelular, os quais se distribuíam em áreas adjacentes a células OPN-positivas, com aspectos morfológicos típicos de células em migração. Em conclusão, a nanoestruturação química de superfície de Ti pode favorecer o aumento da deposição extracelular de OPN endógena por células osteogênicas in vitro, dependendo do tempo de condicionamento utilizado, o que deve ser considerado no desenvolvimento de estratégias para funcionalizar superfícies de implantes com moléculas com reconhecido efeito no processo de adesão celular.

The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30') Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30') Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity.

A matriz dérmica acelular (MDA) foi introduzida na Periodontia como um substituto para enxertos autógenos, os quais se tornaram restritos devido à quantidade limitada de tecido doador. O objetivo deste estudo foi verificar, in vitro, a distribuição, proliferação e viabilidade de fibroblastos gengivais humanos cultivados em MDA. Fibroblastos gengivais foram cultivados sobre MDA por até 21 dias. Os seguintes parâmetros foram avaliados: distribuição, proliferação e viabilidade celular. Os resultados revelaram que, aos 7 dias, os fibroblastos estavam aderidos e espraiados na superfície da MDA, e estavam distribuídos de forma desigual, formando uma camada celular descontínua; aos 14 dias, uma monocamada confluente de fibroblastos revestindo a superfície da MDA foi observada. Aos 21 dias, a monocamada celular exibiu uma redução na densidade celular. Aos 7 dias, cerca de 90% das células aderidas na superfície da MDA estavam no ciclo celular, enquanto que aos 14 e 21 dias esse número reduziu significativamente. Uma maior proporção de células viáveis foi detectada na superfície da MDA tanto aos 14 quanto aos 21 dias. Os resultados sugerem que fibroblastos cultivados sobre a MDA por 14 dias permitem boas condições de adesão e espraiamento das células sobre a matriz, porém, a migração de células para o interior da matriz foi limitada.

The acellular dermal matrix (ADM) was introduced in periodontology as a substitute for the autogenous grafts, which became restricted because of the limited source of donor's tissue. The aim of this study was to investigate, in vitro, the distribution, proliferation and viability of human gingival fibroblasts seeded onto ADM. ADM was seeded with human gingival fibroblasts for up to 21 days. The following parameters were evaluated: cell distribution, proliferation and viability. Results revealed that, at day 7, fibroblasts were adherent and spread on ADM surface, and were unevenly distributed, forming a discontinuous single cell layer; at day 14, a confluent fibroblastic monolayer lining ADM surface was noticed. At day 21, the cell monolayer exhibited a reduction in cell density. At 7 days, about to 90% of adherent cells on ADM surface were cycling while at 14 and 21 days this proportion was significantly reduced. A high proportion of viable cell was detected on AMD surface both on 14 and 21 days. The results suggest that fibroblast seeding onto ADM for 14 days can allow good conditions for cell adhesion and spreading on the matrix; however, migration inside the matrix was limited.

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

O objetivo desse estudo foi avaliar a resposta do tecido ósseo à superfície de titânio (Ti) enriquecida com Ca e P obtida por anodização (BSP-AK). Três cilindros de Ti (4,1 x 12 mm) BSP-AK ou usinado (controle) foram implantados bilateralmente nos úmeros de dois cães de raça indefinida. Oito semanas após a implantação, os fragmentos ósseos contendo os implantes foram removidos e processados para análises histológica e histomorfométrica. A formação óssea foi observada na região cortical e no canal medular até aproximadamente um terço da extensão do implante. Na maioria dos casos, feixes de fibras colágenas dispostos paralelamente à superfície do implante foram observados na região medular. Nessa região observaram-se também áreas focais de formação de matriz mineralizada e osteoblastos ativos. Os implantes do grupo BSP-AK apresentaram média de contato osso-implante 2,3%, com medidas variando de 0,0 a 7,2% e os do grupo controle tiveram média 0,4%, com medidas variando de 0,0 a 1,3%. Apesar do teste de Mann-Whitney não mostrar diferença estatisticamente significante entre os grupos, nossos resultados indicaram uma tendência para a ocorrência de osteogênese de contato na superfície BSP-AK em um modelo experimental que resulta em baixos valores de contato osso-implante.

This study evaluated bone response to a Ca- and P- enriched titanium (Ti) surface treated by a multiphase anodic spark deposition coating (BSP-AK). Two mongrel dogs received bilateral implantation of 3 Ti cylinders (4.1 x 12 mm) in the humerus, being either BSP-AK treated or untreated (machined - control). At 8 weeks postimplantation, bone fragments containing the implants were harvested and processed for histologic and histomorphometric analyses. Bone formation was observed in cortical area and towards the medullary canal associated to approximately 1/3 of implant extension. In most cases, in the medullary area, collagen fiber bundles were detected adjacent and oriented parallel to Ti surfaces. Such connective tissue formation exhibited focal areas of mineralized matrix lined by active osteoblasts. The mean percentages of bone-to-implant contact were 2.3 (0.0-7.2 range) for BSP-AK and 0.4 (0.0-1.3 range) for control. Although the Mann-Whitney test did not detect statistically significant differences between groups, these results indicate a trend of BSP-AK treated surfaces to support contact osteogenesis in an experimental model that produces low bone-to-implant contact values.