Calcium chloride-enriched calcium aluminate cement promotes in vitro osteogenesis.

AIM: To evaluate the effects of 2.8% or 10% calcium chloride (CaCl2 ) in calcium aluminate cement (CAC) with either bismuth oxide (Bi2 O3 ) 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% CaCl2 and 25% Bi2 O3 ; (ii) CACb+: with 10% CaCl2 and 25% Bi2 O3 ; (iii) CACz: with 2.8% CaCl2 and 25% ZnO; or (iv) CACz+: with 10% CaCl2 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 CaCl2 content, the use of Bi2 O3 was detrimental for osteoblastic cell survival and differentiation compared to ZnO, while CAC with the higher CaCl2 content supported the acquisition of the osteogenic cell phenotype in vitro regardless of the radiopacifier used. Thus, CAC with 10% CaCl2 would potentially promote bone repair in the context of endodontic therapies.

Influence of early mineral deposits of silicate- and aluminate-based cements on push-out bond strength to root dentine.

AIM: To evaluate mineral trioxide aggregate (MTA), Biodentine and several formulations of calcium aluminate cements (CACb) in terms of their ability to release calcium ions (Ca2+ ) and form apatite-like precipitates after short-term immersion in phosphate-buffered saline (PBS) and its influence on the bond strength to the root-end cavity. METHODOLOGY: Ten samples of MTA, Biodentine, CACb and calcium-enriched aluminate cement (CACb+) were placed in contact with PBS or deionized water for 14 days. The cement surfaces were analysed using SEM, EDS-X and FTIR. Eighty standardized root-end cavities filled with the cements (ten samples of each cement) were immersed in PBS or deionized water for 14 days, and the bond strengths were measured. Data from the push-out test were analysed using two-way ANOVA and Tukey's tests (α = 0.05). RESULTS: A gradual decrease was observed in Ca2+ concentrations and pH of all solutions. FTIR bands of different phases of hydroxyapatite were identified. Crystalline formation was observed on the surface of all cements after immersion in PBS. No significant difference was observed in the bond strength of the test materials (P > 0.05); however, all cements without contact with the solution revealed significantly lower bond strength values than those in contact with the solution (P < 0.05). CONCLUSION: MTA, Biodentine, CACb e CACb+ were associated with precipitation of crystals after being in contact with PBS for 14 days, indicated by different phases of hydroxyapatite crystalline formation, which also increased dislodgment resistance of the material from root-end cavities. The CACb+ had similar bond strengths and precipitation of crystals to existing materials.

Osteogenic cell response to calcium aluminate-based cement.

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.

Nanotopography drives stem cell fate toward osteoblast differentiation through α1ß1 integrin signaling pathway.

The aim of our study was to investigate the osteoinductive potential of a titanium (Ti) surface with nanotopography, using mesenchymal stem cells (MSCs) and the mechanism involved in this phenomenon. Polished Ti discs were chemically treated with H2 SO4 /H2 O2 to yield nanotopography and rat MSCs were cultured under osteogenic and non-osteogenic conditions on both nanotopography and untreated polished (control) Ti surfaces. The nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of key bone markers of cells grown under both osteogenic and non-osteogenic conditions. Additionally, the gene expression of α1 and ß1 integrins was higher in cells grown on Ti with nanotopography under non-osteogeneic condition compared with control Ti surface. The higher gene expression of bone markers and Alp activity induced by Ti with nanotopography was reduced by obtustatin, an α1ß1 integrin inhibitor. These results indicate that α1ß1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. This finding highlights a novel mechanism involved in nanosurface-mediated MSCs fate and may contribute to the development of new surface modifications aiming to accelerate and/or enhance the process of osseointegration.

Effects of blood and root-dentin cleaning on the porosity and bond strength of a collagen bioceramic material.

To assess the effect of cleaning protocols on dentin contaminated with blood in reparative endodontic materials, bovine root samples were divided: no contamination (N); contamination (P); contamination and cleaning with saline (S), 2.5% NaOCl+saline (Na) or 2.5% NaOCl+17% EDTA+saline (NaE) and filled with: mineral trioxide aggregate (MTA), calcium-aluminate-cement (C), or C+collagen (Ccol) (n=13). The samples were evaluated for porosity, chemical composition, and bond strength. MTA porosity was lower than C (p=0.02) and higher than Ccol (p<0.001). P and NaE were similar (p=1.00), but higher than the other groups (p<0.001). MTA bond strength was similar to Ccol (p=0.777) and lower than C (p=0.028). P presented lower bond strength than the N (p<0.001); S and Na were similar to each other (p=0.969), but higher than P and lower than N (p<0.001). It was observed a predominance of mixed and cohesive failures. None of the samples showed Ca/P ratio values similar to human hydroxyapatite. This study showed that contamination with blood increased the materials porosity, but dentin cleaning with 2.5% NaOCl reduced this effect, and the collagen additive reduced the material porosity. Furthermore, blood contamination reduced the materials bond strength, and cleaning with saline or 2.5% NaOCl diminished this effect.

Skeletal muscle overexpression of sAnk1.5 in transgenic mice does not predispose to type 2 diabetes.

Genome-wide association studies (GWAS) and cis-expression quantitative trait locus (cis-eQTL) analyses indicated an association of the rs508419 single nucleotide polymorphism (SNP) with type 2 diabetes (T2D). rs508419 is localized in the muscle-specific internal promoter (P2) of the ANK1 gene, which drives the expression of the sAnk1.5 isoform. Functional studies showed that the rs508419 C/C variant results in increased transcriptional activity of the P2 promoter, leading to higher levels of sAnk1.5 mRNA and protein in skeletal muscle biopsies of individuals carrying the C/C genotype. To investigate whether sAnk1.5 overexpression in skeletal muscle might predispose to T2D development, we generated transgenic mice (TgsAnk1.5/+) in which the sAnk1.5 coding sequence was selectively overexpressed in skeletal muscle tissue. TgsAnk1.5/+ mice expressed up to 50% as much sAnk1.5 protein as wild-type (WT) muscles, mirroring the difference reported between individuals with the C/C or T/T genotype at rs508419. However, fasting glucose levels, glucose tolerance, insulin levels and insulin response in TgsAnk1.5/+ mice did not differ from those of age-matched WT mice monitored over a 12-month period. Even when fed a high-fat diet, TgsAnk1.5/+ mice only presented increased caloric intake, but glucose disposal, insulin tolerance and weight gain were comparable to those of WT mice fed a similar diet. Altogether, these data indicate that sAnk1.5 overexpression in skeletal muscle does not predispose mice to T2D susceptibility.

Effects of blood and root-dentin cleaning on the porosity and bond strength of a collagen bioceramic material

Abstract To assess the effect of cleaning protocols on dentin contaminated with blood in reparative endodontic materials, bovine root samples were divided: no contamination (N); contamination (P); contamination and cleaning with saline (S), 2.5% NaOCl+saline (Na) or 2.5% NaOCl+17% EDTA+saline (NaE) and filled with: mineral trioxide aggregate (MTA), calcium-aluminate-cement (C), or C+collagen (Ccol) (n=13). The samples were evaluated for porosity, chemical composition, and bond strength. MTA porosity was lower than C (p=0.02) and higher than Ccol (p<0.001). P and NaE were similar (p=1.00), but higher than the other groups (p<0.001). MTA bond strength was similar to Ccol (p=0.777) and lower than C (p=0.028). P presented lower bond strength than the N (p<0.001); S and Na were similar to each other (p=0.969), but higher than P and lower than N (p<0.001). It was observed a predominance of mixed and cohesive failures. None of the samples showed Ca/P ratio values similar to human hydroxyapatite. This study showed that contamination with blood increased the materials porosity, but dentin cleaning with 2.5% NaOCl reduced this effect, and the collagen additive reduced the material porosity. Furthermore, blood contamination reduced the materials bond strength, and cleaning with saline or 2.5% NaOCl diminished this effect.

Resumo As amostras de raízes bovinas foram divididas em: sem contaminação (N); contaminação sanguínea (P); contaminação sanguínea e limpeza com soro fisiológico (S), 2,5% NaOCl+soro fisiológico (Na) ou 2,5% NaOCl+17%EDTA+soro fisiológico (NaE) e preenchido com: agregado de trióxido mineral (MTA), cimento de aluminato de cálcio (C), ou C+colágeno (Ccol) (n=13). A porosidade do MTA foi menor que C (p=0,02) e maior que Ccol (p<0,001). P e NaE foram semelhantes (p=1,00), mas superiores aos demais grupos (p<0,001). A resistência de união do MTA foi semelhante ao Ccol (p=0,777) e inferior ao C (p=0,028). P apresentou menor resistência de união que o N (p<0,001); S e Na foram semelhantes entre si (p=0,969), porém maiores que P e menores que N (p<0,001). Este estudo mostrou que a contaminação com sangue aumentou a porosidade dos cimentos, mas a limpeza da dentina com NaOCl 2,5% reduziu esse efeito, e o aditivo de colágeno reduziu a porosidade dos cimentos. Além disso, a contaminação sanguínea reduziu a resistência de união dos cimentos e a limpeza com solução salina ou NaOCl 2,5% diminuiu esse efeito.