Bovine Dentin Research Articles

Chitosan Enhances the Anti-Biofilm Activity of Biodentine against an Interkingdom Biofilm Model.

Endodontic infection is a biofilm disease that is difficult to irradicate with current treatment protocols, and as such, persistent micro-organisms may lead to ongoing or recurrent disease. The potential for the use of enhanced filling materials to modify biofilm regrowth is a promising strategy. This current study aimed to evaluate the anti-biofilm efficacy of calcium silicate cements modified with chitosan. The development of mono-species and multi-species biofilms on ProRoot MTA, Biodentine and bovine dentine discs were explored using quantitative microbiology analysis. The effect on regrowth of biofilms was assessed following the addition of chitosan to each cement. In comparison to a dentine substrate, both materials did not show the ability to inhibit biofilm regrowth. Biodentine incorporated with chitosan displayed a dose-dependent reduction in multi-species biofilm regrowth, unlike MTA. Notably, interkingdom biofilms were shown to enhance bacterial tolerance in the presence of chitosan. This study demonstrates the potential to enhance the antimicrobial properties of Biodentine. The findings highlight the need for appropriate model systems when exploring antimicrobial properties of materials in vitro so that interspecies and interkingdom interactions that modify tolerance are not overlooked while still supporting the development of innovative materials.

In vitro Evaluation of Surface Free Energy of Dentin after Treatment with Sodium Trimetaphosphate Associated or Not with Fluoride, Exposed or Not to Calcium

It has been stated that sodium trimetaphosphate (TMP) promotes a more anionic dentin surface inducing greater calcium (Ca) and phosphate precipitation. The aim of the present study was to evaluate in vitro the surface free energy (γ_s) of dentin after treatment with TMP associated or not with fluoride (F), exposed or not to Ca, as well as the adsorption of TMP, F, and Ca by dentin. Bovine dentin blocks (n = 12 blocks/group) were treated with solutions containing TMP at 0, 1, 3, or 9 (w/v) followed or not by the application of Ca. These solutions were or were not associated to 1,100 ppm F. F, Ca, and TMP were determined in the solutions before and after the treatment to calculate the adsorption by dentin. To analyze the γ_s of dentin, the apolar (γ_s^LW), and polar (γ_s^AB), components were determined by contact angle measurement. Data were submitted to 2-way ANOVA followed by the Student-Newman-Keuls test (p < 0.05). TMP reduces γ_s of dentin and increases electron donor sites (γ_s⁻). Higher values of γ_s⁻ led to higher adsorption of Ca (p < 0.001). The F/TMP association did not change γ_s or γ_s^LW and reduced the values of γ_s⁻, but the adsorption of Ca was higher. There was correlation between the adsorption of TMP and γ_s⁻ (Pearson’s r = 0.801; p < 0.001) and F (Pearson’s r = 0.871, p < 0.001). It is possible to conclude that TMP increased γ_s⁻ and Ca adsorption, and reduced γ_s. The association with F increased the adsorption of TMP without rising γ_s⁻; however, there was higher adsorption of Ca.

Green Tea Extract Reduces the Erosive Dentine Wear Caused by Energy Drinks In Vitro.

To investigate the effect of energy drinks supplementation with green tea extract on the erosive dentine wear. Six groups of bovine dentine samples (n = 15) were subjected to four cycles erosive attacks (10 min, 25 °C) and remineralisation (artificial saliva, 60 min, 37°C) using the following formulas: tap water; green tea extract; Red Bull; Red Bull supplemented with green tea extract; Red Bull Light; Red Bull Light supplemented with green tea extract. The erosive dentine wear - ie, the irreversible dentine loss - was measured using a stylus profilometer (µm, accuracy = 40 nm). Median and interquartile range (IQR) of erosive dentine wear for the tested energy drinks before and after the supplementation with green tea extract were calculated as follows: Red Bull (before: 3.3 µm (1.0)); after: 1.2 µm (0.6)); Red Bull Light (before: 3.3 µm (0.9)); after: 2.0 µm (0.4)). The difference between the groups before and after the supplementation was statistically significant (P ˂0.05). The erosive dentine wear for the tap water group was calculated at 0.4 µm (0.6) and for the green tea extract group at -1.0 µm (1.3). Supplementation of energy drinks with green tea extract could reduce the erosive dentine wear caused by energy drink in vitro.

Solutions and Gels Containing a Sugarcane-Derived Cystatin (CaneCPI-5) Reduce Enamel and Dentin Erosion in vitro

The effect of solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) on the protection against enamel and dentin erosion in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 135 and 153/group for enamel and dentin, respectively) that were treated with solutions or chitosan gels containing 0.1 or 0.25 mg/mL CaneCPI-5. The positive controls for solutions and gels were Elmex Erosion Protection™ solution and NaF gel (12,300 ppm F), respectively. Deionized water and chitosan gel served as controls, respectively. The solutions were first applied on the specimens for 1 min and the gels for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.1% citric acid pH 2.5/90 s, artificial saliva/2 h, and artificial saliva overnight). The solutions and gels were applied again during pH cycling, 2 times/day for 1 min and 4 min, respectively, after the first and last erosive challenges. Enamel and dentin losses (µm) were assessed by contact profilometry. Data were analyzed by 2-way ANOVA and Tukey’s test (p < 0.05). All the treatments significantly reduced enamel and dentin loss in comparison with controls. Both CaneCPI-5 concentrations had a similar protective effect against enamel erosion, but only the higher concentration was as effective against dentin erosion as the positive control. Regarding the vehicles, only the 0.1 mg/mL gel performed worse than the positive control for dentin. CaneCPI-5 reduced enamel and dentin erosion to a similar extent as the fluoride-containing vehicles. However, dentin requires higher CaneCPI-5 concentrations, in the case of gels. Solutions or gels containing CaneCPI-5 might be a new approach to protect against dental erosion.

Ultrastructural changes of bovine tooth surfaces under erosion in presence of biomimetic hydroxyapatite

Enamel and dentin are susceptible to acids from food sources leading to dental erosion, a global problem affecting millions of individuals. Particulate hydroxyapatite (HAP) on the tooth surface can influence the effects of acid attacks. Standardized bovine enamel and dentin samples with artificial saliva are used in an in vitro cyclic demineralization–remineralization protocol to analyze the structural changes experienced by tooth surfaces using high-resolution scanning electron microscopy and to evaluate the potential of a HAP-based oral care gel in the protection of teeth from erosive attacks. The interfaces between HAP particle and enamel HAP crystallites are investigated using focused ion beam preparation and transmission electron microscopy. The results show that erosion with phosphoric acid severely affects enamel crystallites and dentin tubules, while artificial saliva leads to remineralization effects. The HAP-gel forms a microscopic layer on both enamel and dentin surfaces. Upon acid exposure, this layer is sacrificed before the native tooth tissues are affected, leading to significantly lower degrees of demineralization compared to the controls. This demonstrates that the use of particulate HAP as a biomaterial in oral care formulations can help protect enamel and dentin surfaces from erosive attacks during meals using a simple and effective protection principle.

In vitro Cell Proliferation Assay of Demineralized Dentin Material Membrane in Osteoblastic MC3T3-E1 Cells.

AimDemineralized dentin material membrane (DDMM) is a novel bioresorbable guided bone regeneration (GBR) which is derived from the demineralization process of bovine dentin. This material/process could be an alternative to resolve musculoskeletal dysfunction that harms the quality of human life.PurposeTo evaluate the cytotoxic effect of DDMM as GBR membrane on MC3T3-E1 osteoblast cell line.MethodsCytotoxic effect was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteoblast MC3T3-E1 cell culture was used as a parameter of cell viability after reacting with GBR materials. The absorbance values were examined at each treatment to determine the percentage of cell viability. There were four groups created in the present study: two treatment groups and two control groups. The treatment groups consisted of a DDMM group and a bovine pericardium collagen membrane (BPCM) group. The control groups comprised a group containing cell culture medium as a negative control group and another positive control group that contained cell cultures.ResultsThe results revealed no significant difference in MC3T3-E1 cell viability between the treatment and control groups (p < 0.05). Moreover, as observed in the DDMM group, there was an increase in the number of osteoblast cells.ConclusionDDMM is a suitable alternative biomaterial for GBR as it is non-cytotoxic and could potentially increase the rate of repair of craniofacial defects.

Influence of Different Application Methods on the Bonding Effectiveness of Universal Adhesives to Dentin in the Early Phase.

To investigate the changes in the dentin bond strengths of universal adhesives during the early phase and evaluate the effect of a double-layer adhesive application on the performance of the dentin bond. Three universal adhesives and a two-step self-etch adhesive were employed to ascertain the shear bond strengths (SBS) of specimens to bovine dentin with the use of the etch-and-rinse or self-etch mode. The specimens were further divided into two groups based on adhesive application in a single or a double layer. The bonded specimens were stored in distilled water at 37°C for 5 min or 1, 6, 12, or 24 h prior to SBS measurement and the adhesives' Knoop hardness number (KHN). All the adhesives showed increased SBS with prolonged storage periods regardless of the adhesive layer (single or double) or etching mode. Most universal adhesives in the double adhesive layer groups showed significantly higher SBS than single adhesive layer groups for the same storage period. All the adhesives also showed increased KHN with increased storage period. The SBS and KHN values of the adhesives increased with increasing storage duration over a 24-h period. Double adhesive layer application mediated increased dentin bond strength in the early phase.

Wear of conventional and pre-polymerized composite materials under erosive/abrasive conditions.

The aim of this study was to examine the wear of three different composite materials usable for vertical bite reconstruction under erosive/abrasive conditions and to compare them with the bovine dental hard tissues enamel and dentin in-vitro. The composite materials Filtek Supreme XTE, CeraSmart and Brilliant Crios and bovine enamel and dentin specimens were evenly allocated to five groups (n=10). Samples were firstly exposed to hydrochloric acid for 1 min, then stored for 30 min in artificial saliva. In the second step, the samples were brushed for 1 min with 100 brushing strokes each and an applied force of 2.5 N in an automated brushing device. After 60 of these erosive/abrasive cycles, wear of the samples was determined by contact profilometry. Filtek Supreme XTE showed significantly less wear (mean ± standard deviation; 0.15 ± 0.11 μm) compared to the other two composite materials (p < 0.05). No significant difference (p > 0.05) could be found between CeraSmart (0.25 ± 0.03 μm) and Brilliant Crios (0.24 ± 0.04 μm). The two bovine dental hard tissues, enamel (13.70 ± 0.94 μm) and dentin (50.08 ± 4.46 μm), each showed a significantly higher amount of wear than the three restorative materials (p <0.05). In conclusion, this study exhibited that the three composite materials, Filtek Supreme XTE, CeraSmart and Brilliant Crios, were more resistant under erosive/abrasive conditions compared to bovine enamel and bovine dentin, respectively.

Effect of TiF4/NaF and chitosan solutions on biofilm formation and prevention of dentin demineralization

ObjectiveThis study evaluated the effect of experimental solutions containing TiF4/NaF and chitosan on bacterial species of microcosm biofilm and on dentin demineralization. DesignMicrocosm biofilm was produced from human saliva mixed with McBain medium (0.2% sucrose) on bovine dentin for 5 days, under 5% CO2 and 37 °C. From the 2nd day to 5th day, the treatments were applied (1×60s/day) as following: (1) NaF (500 ppm F−, positive control); (2) TiF4 and NaF (TiF4: 190 ppm Ti4+ and 300 ppm F-; NaF: 190 ppm F−); (3) similar to 2 plus 0.5% chitosan (Ch 500 mPa.s, 75% deacetylation); (4) phosphate buffer solution (negative control); and (5) 0.5% chitosan (Ch 500 mPa.s, 75% deacetylation). CFU counting was performed for total microorganism, total streptococci, total lactobacilli and mutans streptococci. Dentin demineralization was measured by transverse microradiography-TMR. The data were compared using ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p < 0.05). ResultsNo differences were found between the treatments with respect to CFU counting (p > 0.05). Dentin treated with TiF4/NaF plus chitosan solution presented the lowest demineralization compared to the negative control and pure chitosan solution. On the other hand, this experimental solution did not significantly differ from TiF4/NaF solution, being both able to significantly reduce mineral loss. ConclusionTiF4/NaF plus chitosan solution, at suitable pH to be clinically applicable, had no antimicrobial effect, but it was able to reduce dentin caries development under this model.

Effects of occlusal splint therapy on opposing tooth tissues, filling materials and restorations.

BackgroundLittle is known about the effect of the type of splint material, heat‐cured PMMA (HC) or chemical‐cured PMMA (CC) on the wear of opposing tooth surfaces.ObjectiveThe aim of this in vitro study was to evaluate two‐body wear of dentin, enamel, glass‐ceramic or one of four resin composites when opposing splint materials, namely ProBase HC and CC.MethodsThe two‐body wear of bovine dentine, bovine enamel, glass‐ceramic IPS e.max CAD (EMAX) and four composites (Filtek Z250 [Z250], Clearfil AP‐X [AP‐X], Clearfil Majesty Posterior [CMP], Filtek Supreme XTE [FSE]) opposing three antagonists (HC and CC and stainless steel as control) were evaluated in the ACTA wear machine. In addition, all the surfaces were evaluated with scanning electron microscopy.ResultsThe highest average wear was observed in the case of dentin. The lowest average wear was found EMAX. In every case—except for EMAX—the wear rate was higher with HC than with CC (all differences being statistically significant).ConclusionsThe level of wear of enamel, dentin and various resin composites was higher in contact with HC than in CC, the wear of dentin being the highest. In the case of a patient with no or little tooth wear or whose teeth are restored with composite material or glass‐ceramic, the splint HC might be preferred because of its better durability. However, when the splint is in contact with opposing dentin preservation of the dentin, CC might be the best choice.

In vitro study on how cold plasma affects dentin surface characteristics

PurposeStudies evaluating different features of cold plasma action on dentin surface characteristics are lacking. Thus, this in vitro study aimed to determine the effect of cold plasma under different protocols of exposure time, distance to plasma source, and the association of argon gas with distinct concentrations of oxygen on the wettability, surface energy, total free interaction energy, surface roughness, morphology and chemical composition of dentin. Material and methodsOne hundred and twenty-five bovine dentin samples were used and divided into twenty-five groups according to the exposure time to plasma (15, 30, or 60 s); distance between plasma source and dentin surface (3 or 6 mm); argon gas without plasma generation; and plasma generated by argon gas and association of argon gas with distinct concentrations of oxygen (2 % or 3 %) (n = 5). Contact angle (θ), surface energy (γs) and total free interaction energy (ΔG) were measured using a goniometer (Krüss), while surface roughness (Ra) was evaluated by a profilometer (Mitutoyo). Representative samples were submitted to scanning electron microscopy (JEOL) to ilustrate the morphology and chemical composition of dentin. Data comparing control group with all experimental groups were submitted to ANOVA followed by Tukey's test (α = .05). Data comparing oxygen gas action at different concentrations and argon gas on dentin characteristics were submitted to non-parametric Kruskal–Wallis test, followed by Dunn test for comparison between the groups and methods (α = 0.05). ResultsIn general, argon gas without plasma generation promoted no significant difference on dentin surface characteristics compared to control group (P > .05), differently for the cold plasma that significantly reduced contact angle values and increased total free interaction energy of dentin surface (P < .05). Overall, feeding of oxygen at distinct concentrations promoted significant difference on dentin surface characteristics compared to control group (P < .05). Exposure time and distance protocols interfered with contact angle, surface energy and total free interaction energy analyses for each gas. There was no significant difference on surface roughness (P > .05), morphology and chemical composition of dentin submitted to argon gas, cold plasma, and distinct concentrations of oxygen. ConclusionIn conclusion, plasma generated by argon gas and its feeding with 2 % and 3 % oxygen gas improved the dentin surface characteristics about wettability, surface energy and total free interaction energy. Such treatments preserved the surface roughness, morphology and chemical composition of dentin. The protocols of groups Ar-6mm-15sec, ArO2-3mm-30sec and ArO3-3mm-15sec are recommended for improvement of dentin surface characteristics.

Interactions of boron released from surface pre-reacted glass ionomer with enamel/dentin and its effect on pH

This study investigated the interaction of borate ions released from surface pre-reacted glass ionomer (S-PRG) fillers with the biological apatites of enamel and dentin using solid-state (SS) magic-angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy analysis. We further evaluated the effect of borate ions on the pH change. Bovine enamel and dentin powder were submerged in S-PRG eluate (with borate ion concentration of 100 mM) for 3 h, then washed with deionized water (DW). The dried enamel and dentin specimens were used for 11B SS-NMR and Fourier transform infrared spectroscopy (FT-IR) analysis. Enamel and dentin blocks were treated with three solutions: DW (control), S-PRG eluent and borate solution (100 mM). The treated samples were submerged in the demineralization solution and the pH was measured using a pH meter daily for 6 days. The surfaces of the enamel and dentin blocks were then observed using field emission scanning electron microscopy (FE-SEM). SS-NMR analysis revealed the presence of adsorbed borate ions in the enamel and dentin samples in a tetra-coordinated form. The pH results demonstrated an increase in pH values in the S-PRG and borate groups. SEM images showed that the surfaces of the control group were demineralized, whereas the surfaces of the S-PRG and borate groups were intact. These results concluded that borate ions could be adsorbed to enamel and dentin in the tetra-coordinated form. Borate ions possess a buffer capacity which may help to protect the tooth structure against acid attacks and promote remineralization.

Effects of desensitizer containing fluoroaluminocalciumsilicate glass nanoparticles on remineralization of root dentin subsurface lesions in vitro.

We investigated the remineralization effects of Nanoseal (NS) dentin desensitizer on demineralized root dentin. Baseline lesion specimens prepared from bovine root dentin were immersed in artificial saliva (AS) or deionized water (DW) after treatment with NS or fluoride-free Nanoseal (NS(-)). Treatment and control groups comprised: 1, AS; 2, NS/AS; 3, NS(-)/AS; 4,NS/DW; 5, NS(-)/DW; and 6, baseline demineralization. Integrated mineral loss (IML) and lesion depth (LD) were determined by transverse microradiography. Fluoride concentrations in the immersion solutions were measured. AS, NS/AS and NS(-)/AS showed higher mineral volume % at the surface and lesion body than did other groups. NS/AS showed significantly lower IML than did AS. There was no significant difference in IML between NS/AS and NS(-)/AS. The highest concentration of fluoride was in the NS/AS immersion solution. The findings suggest Nanoseal facilitated remineralization of demineralized root dentin, and fluoride and other ions included may have contributed to this effect.

Effect of toothbrush bristle stiffness and toothbrushing force on the abrasive dentine wear.

ObjectiveTo investigate the influence of toothbrush bristle stiffness and applied brushing force on the resulting abrasive dentine wear in vitro.MethodsOne hundred sixty bovine dentine samples were randomly allocated in eight groups (n = 20). Groups one to four were brushed with a soft‐bristle toothbrush with soft bristles applying 1, 2, 3 and 4 N brushing force, respectively. Groups five to eight were brushed with a medium‐bristle toothbrush applying the same aforementioned brushing forces (120 strokes/min, abrasive slurry (RDA = 121), 25 min). Profiles were recorded before and after the brushing sequence, and the median and interquartile range of abrasive dentine wear were calculated and compared using two‐way ANOVA and pairwise tests corrected after Tukey (α = 0.05).ResultsAt 1, 2 and 3 N brushing force, the tested toothbrushes caused no significantly different abrasive dentine wear. At 4 N brushing force, the medium‐bristle toothbrush caused statistically significantly higher abrasive force than the soft‐bristle toothbrush. Using the medium‐bristle toothbrush, abrasive dentine wear statistically significantly increased with increasing brushing force from 1 to 3 N. However, increasing the brushing force to 4 N did not result in statistically significantly higher wear. Using the soft‐bristle toothbrush, abrasive dentine wear statistically significantly increased with increasing brushing force from 1 to 2 N. However, increasing the brushing force to 3 or 4 N did not result in statistically significantly higher wear.ConclusionThe soft‐bristle toothbrush tends to cause less abrasive wear with increasing brushing force than the medium‐bristle toothbrush.

Physicochemical properties and antimicrobial activity of calcium hydroxide pastes in association with other compounds

Aim: This study aimed to evaluate the pH, Ca2+ release, solubility, and antimicrobial activity of calcium hydroxide (Ca(OH)2) pastes in association with different substances. Methods: Sixty acrylic teeth (n=10) were filled with pastes that associated Ca(OH)2 with the following substances: benzalkonium chloride 5% (G1) and 50% (G2) both in propylene glycol, arnica glycolic extract (G3), green tea glycolic extract (G4), Calen/PMCC™ (G5), and Calen™ (G6). In the group G1 to G4 were used 1g of Ca(OH)2 powder with 0,8g of vehicle. pH and Ca2+ release was measured after 7, 15, and 30 days. For solubility, micro-CT was used immediately and at the periods of 7, 15, and 30 days. For the antimicrobial analysis, a biofilm of E. faecalis was induced in vitro on bovine dentin discs. Live/dead viability dye and confocal scanning microscopy were used. Results: The highest pH values occurred on the first 7 days, and the G6, G1, G3, and G5 presented the highest pH values at this period (P &lt;0.05). Ca2+ release was higher in all groups at 7 days, with the highest values observed in G1, G5, and G6. The volume of all pastes showed no significant difference in the intragroup analysis at 7 and 15 days (P &lt;0.05). G1 and G2 showed the highest antimicrobial action (P &lt;0.05). For the biovolume, there was difference between the G6 and the other groups (P &gt;0.05) with G1 presenting the lowest values. Conclusion: Benzalkonium chloride 5% increases the antimicrobial action of the Ca(OH2), without impairs physicochemical properties.

Physicochemical Properties and Antibiofilm Activity of Tricalcium Silicate Cement and its Association with Cetrimide

Cetrimide (CTR) is a cationic surfactant detergent with antimicrobial and antibiofilm activity. The aim of this study was to evaluate setting time, pH, solubility and antibiofilm activity of tricalcium silicate cement (TSC) with zirconium oxide (ZrO2) and its association with 0.2 and 0.4% cetrimide. Initial and final setting times (IST and FST) were assessed based on ISO-6876. pH was evaluated at periods of 1,3,7,14 and 21 days. Solubility was analyzed by weight loss. A modified direct contact test (MDCT) on the biofilm of Enterococcus faecalis formed on bovine root dentin blocks was performed, after 6 hours of manipulation and 15 hours of contact time. The analysis was performed by UFC mL ­¯¹ counting. The data were analyzed by ANOVA and Tukey’s tests (α=0.05). Higher IST was observed for TSC/ZrO2+CTR in both concentrations than for TSC/ZrO2 and lower FST for TSC/ZrO2+0.4% CTR (p&lt;0.05). On day 1, TSC/ZrO2 showed lower pH than the associations with CTR (p&lt;0.05). During the other periods, TSC/ZrO2 and associations promoted similar alkalinization (p&gt;0.05). All materials exhibited increased mass. TSC/ZrO2+CTR 0.4% had lower mass gain than the other materials (p&lt;0.05). The highest antibiofilm activity was observed for TSC/ZrO2+CTR in both concentrations, when compared with the positive control (p&lt;0.05). In conclusion, CTR exhibited potential to promote greater antibiofilm activity to tricalcium silicate cement, without harming its physicochemical properties of setting time, pH and solubility.

Aplidin (Plitidepsin) Regulates Viability and Function of Myeloma Cells and Bone Cells in Combination with Other Anti-MM Drugs

In multiple myeloma (MM) the clonal expansion of malignant plasma cells within the bone marrow results in the development of lytic bone lesions due to exacerbated bone resorption. Despite recent progress in its treatment, MM remains incurable and the associated bone disease persists even after complete remission. Thus, identification of new therapeutic approaches that suppress MM cell growth and protect the bone is an unmet need. Aplidin, a novel anti-cancer compound isolated from the marine tunicate Aplidium albicans, exhibits anti-MM activity even in MM cells resistant to conventional chemotherapy and is currently in phase III clinical trials in Europe. However, the effects of Aplidin on bone cells and MM-induced bone disease are unknown. The goal of the current study was to examine the effect of Aplidin, alone or in combination with other anti-MM drugs, on myeloma cells and bone cells (osteocytes, osteoblasts, and osteoclasts) using in vitro approaches.Aplidin decreased the number of human JJN3 and murine 5TGM1 MM cells with an EC50~10nM, determined by MTT assay, due to decreased cell viability, assessed by Trypan blue exclusion, reaching a 20% increase in MM cell death after 48h. Treatment with glucocorticoids (GC) (10-5M) or Bortezomib (3nM) also decreased MM viability and enhanced the anti-MM activity of Aplidin by 1.6-fold and 2.5-fold, respectively. In addition, Aplidin at >10nM induced MLO-A5 osteocytic dead up to 15% and in combination with GC up to 50%; whereas, in contrast, Bortezomib did not affect osteocyte viability and partially prevented the increase in MLO-A5 cell death induced by Aplidin. Further, 1nM Aplidin increased OB-6 osteoblastic cell death up to 20% and decreased their matrix mineralization capacity, quantified by Alizarin red. These effects were enhanced when Aplidin was combined with GC, but partially prevented by combination of Aplidin with Bortezomib. Cells of the osteoclastic lineage exhibited higher sensitivity to Aplidin in comparison to MM cells or osteoblastic cells, and thus it was tested at lower concentrations. Specifically, 50 pM Aplidin decreased the number of hematopoietic precursors by 15-20%, as quantified by enumerating GM-CSF colonies in cultures established from murine bone marrow. Moreover, Aplidin, at concentrations as low as 0.01 pM, reduced osteoclast precursor commitment and differentiation by 10%, as quantified by enumerating the number of osteoclast precursors maintained in the presence of M-CSF for 3 days with or without Aplidin. In addition, Aplidin decreased by 40% the number of TRAP positive osteoclasts generated after 4 days in the presence of M-CSF and sRANKL; and blunted the rapid (5-15min) increased in ERK phosphorylation induced by Rankl, but did not affect RANKL-induced p38 phosphorylation, suggesting that Aplidin inhibited RANKL-induced differentiation of osteoclast precursors by preventing ERK activation. Lastly, to test the effects of Aplidin on osteoclasts function, mature osteoclasts generated in the presence of M-CSF/sRANKL were detached by trypsinization, replated on bovine dentin slides and exposed to Aplidin for 3 and 7 days. 10pM Aplidin reduced the number of mature osteoclasts by 60% and markedly decreased the number of resorption pits.In summary, Aplidin (1nM and up) decreases MM cell viability and GC or Borteozmib enhances its anti-MM activity. In addition, Aplidin (10nM and up) decreases osteocytic cell viability and this effect is exacerbated by GC but partially prevented by Bortezomib. Moreover, Aplidin (1nM and up) induces osteoblastic cell death and decreases their mineral production, and these effects are increased by GC, but partially prevented by Bortezomib. Furthermore, Aplidin (0.01pM and up) reduces osteoclast precursor commitment and differentiation, and inhibits mature osteoclast resorption activity. Taken together these findings suggest that combining Aplidin with proteosomal inhibitors may be a novel therapy for MM to enhance anti-tumor responses, decrease bone destruction by inhibiting bone resorption, and prevent potential side effects of Aplidin on cells of the osteoblastic lineage. DisclosuresDelgado-Calle:PharmaMar: Research Funding. Galmarini:PharmaMar: Employment. Roodman:PharmaMar: Research Funding. Bellido:PharmaMar: Research Funding.

Self-etch Approach of Universal Adhesives as an Alternative to Minimize Bond Degradation on Sound Dentin vs Caries-affected Dentin over Time.

To investigate the influence of the etching strategy of universal adhesives on bond degradation to sound and artificially-induced caries-affected dentin. The universal adhesives (Scotchbond Universal Adhesive; All-Bond Universal; Prime&Bond Elect) and adhesives used as controls (Adper Single Bond 2 and Clearfil SE Bond) were applied to sound and artificially-induced caries-affected bovine dentin. Microtensile bond strength was evaluated immediately (24 h) and after one year of water storage (1 year). Representative specimens were also prepared to assess nanoleakage. Bond strength data (MPa) were analyzed using three-way ANOVA and post-hoc Tukey's test (α = 0.05), considering each substrate separately. Bonding degradation was observed for all universal adhesives on caries-affected dentin, irrespective of the etching strategy. On sound dentin, bonding degradation was observed when adhesives were used on the etch-and-rinse strategy. The universal adhesives were not capable of maintaining bond stability over time on caries-affected dentin. The self-etch strategy seems better able to maintain the durability of adhesive interfaces created on sound dentin.

Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization.

To analyze the effect of a sugarcane cystatin (CaneCPI-5) on the microbial profile and viability, as well as on the prevention of dentin demineralization using a microcosm biofilm model. Ninety bovine dentine specimens were divided into five experimental groups according with the solution they were treated for 60s: (1) PBS (negative control), (2) 0.12% chlorhexidine (positive control), (3) Fluoride (500ppm F, as NaF), (4) 0.025mg/ml CaneCPI-5, and (5) 0.05mg/ml CaneCPI-5. Specimens were incubated with inoculum (McBain's saliva plus human saliva) in the first 8h, and from then on, they were exposed to McBain saliva containing sucrose and daily treated (60s) with the solutions for 5days. Resazurin and colony-forming unit counting assays were performed. Dentin demineralization was measured by transverse micro-radiography (TMR). 0.12% chlorhexidine significantly reduced the metabolic activity of the microcosm biofilm in relation to the negative control and treated groups (p < 0.01). CHX and F significantly reduced the counts of total microorganisms, mutans group streptococci, and lactobacilli when compared with the negative control. None of the treatments was able to significantly reduce dentin demineralization in comparison with the negative control. In the model evaluated, CaneCPI-5 neither altered the microcosm biofilm profile and viability nor protected dentin against demineralization.

The influence of the resin-based cement layer on ceramic-dentin bond strength.

The purpose of the study was to measure the cement thickness obtained when ceramic rods were luted to dentin and to analyze the relation between cement thickness and the previously published tensile bond strength of similar test specimens. In addition, the ISO standard 4049:2019 method was used to determine the film thickness of the used cements. Zirconia (n=100) and lithium disilicate (n=50) rods were cemented to bovine dentin using one of five different resin-based cements. The ceramic-dentin test specimens were cut into two slices and the cement thickness was measured using a scanning electron microscope and compared to the bond strength values of similar specimens already published. The mean cement thickness recorded for ceramic rods cemented to dentin was in the range 20-40μm, which was larger than the cement film thickness found by the ISO method. The cement film thickness determined according to ISO standard methods did not concur with the results obtained when cementing ceramic rods to dentin. For cementing ceramic restorations, a cement thickness in the range 25-35μm seems to be favorable for the bond strength.