Next-generation antibacterial nanopolymers for treating oral chronic inflammatory diseases of bacterial origin.

BACKGROUND: 'Periodontitis' refers to periodontal destruction of connective tissue attachment and bone, in response to microorganisms forming subgingival biofilms on the root surface, while 'apical periodontitis' refers to periapical inflammatory processes occurring in response to microorganisms within the root canal system. The treatment of both diseases is based on the elimination of the bacterial challenge, though its predictability depends on the ability of disrupting these biofilms, what may need adjunctive antibacterial strategies, such as the next-generation antibacterial strategies (NGAS). From all the newly developed NGAS, the use of polymeric nanotechnology may pose a potential effective approach. Although some of these strategies have only been tested in vitro and in preclinical in vivo models, their use holds a great potential, and therefore, it is relevant to understand their mechanism of action and evaluate their scientific evidence of efficacy. OBJECTIVES: To explore NGAS based on polymeric nanotechnology used for the potential treatment of periodontitis and apical periodontitis. METHOD: A systemic search of scientific publications of adjunctive antimicrobial strategies using nanopolymers to treat periodontal and periapical diseases was conducted using The National Library of Medicine (MEDLINE by PubMed), The Cochrane Oral Health Group Trials Register, EMBASE and Web of Science. RESULTS: Different polymeric nanoparticles, nanofibres and nanostructured hydrogels combined with antimicrobial substances have been identified in the periodontal literature, being the most commonly used nanopolymers of polycaprolactone, poly(lactic-co-glycolic acid) and chitosan. As antimicrobials, the most frequently used have been antibiotics, though other antimicrobial substances, such as metallic ions, peptides and naturally derived products, have also been added to the nanopolymers. CONCLUSION: Polymeric nanomaterials containing antimicrobial compounds may be considered as a potential NGAS. Its relative efficacy, however, is not well understood since most of the existing evidence is derived from in vitro or preclinical in vivo studies.

Doxycycline-doped collagen membranes accelerate in vitro osteoblast proliferation and differentiation.

OBJECTIVE: The aim of the study was to evaluate the effect of doxycycline- and dexamethasone-doped collagen membranes on the proliferation and differentiation of osteoblasts. BACKGROUND: Collagen barrier membranes are frequently used to promote bone regeneration and to boost this biological activity their functionalization with antibacterial and immunomodulatory substances has been suggested. METHODS: The design included commercially available collagen membranes doped with doxycycline (Dox-Col-M) or dexamethasone (Dex-Col-M), as well as undoped membranes (Col-M) as controls, which were placed in contact with cultured MG63 osteoblast-like cells (ATCC). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and differentiation by measuring the alkaline phosphatase (ALP) activity using spectrophotometry. Real-time quantitative polymerase chain reaction was used to study the expression of the genes: Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3. Scanning electron microscopy was used to study osteoblast morphology. Data were assessed using one-way analysis of variance or Kruskal-Wallis tests, once their distribution normality was assessed by Kolmogorov-Smirnov tests (p > .05). Bonferroni for multiple comparisons were carried out (p < .05). RESULTS: Osteoblast proliferation was significantly enhanced in the functionalized membranes as follows: (Col-M < Dex-Col-M < Dox-Col-M). ALP activity was significantly higher on cultured osteoblasts on Dox-Col-M. Runx-2, OSX, ALP, OSC, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3 were overexpressed, and RANKL was down-regulated in osteoblasts cultured on Dox-Col-M. The osteoblasts cultured in contact with the functionalized membranes demonstrated an elongated spindle-shaped morphology. CONCLUSION: The functionalization of collagen membranes with Dox promoted an increase in the proliferation and differentiation of osteoblasts.

Novel non-resorbable polymeric-nanostructured scaffolds for guided bone regeneration.

OBJECTIVE: The aim of this study was to evaluate the bone-regeneration efficiency of novel polymeric nanostructured membranes and the effect of zinc, calcium, titanium, and bone morpho-protein loading on membranes, through an in vivo rabbit model. MATERIAL AND METHODS: Nanostructured membranes of methylmethacrylate were loaded with zinc, calcium, TiO2 nanoparticles, and bone-morphogenetic protein (BMP). These membranes covered the bone defects prepared on the skulls of six rabbits. Animals were sacrificed 6 weeks after surgery. Micro computed tomography was used to evaluate bone architecture through BoneJ pluging and ImageJ script. Three histological processing of samples, including von Kossa silver nitrate, toluidine blue, and fluorescence by the deposition of calcein were utilized. RESULTS: Zn-membranes (Zn-Ms) promoted the highest amount of new bone and higher bone perimeter than both unloaded and Ti-membranes (Ti-Ms). Ca-membranes (Ca-Ms) attained higher osteoid perimeter and bone perimeter than Zn-Ms. The skeleton analysis showed that Zn-Ms produced more branches and junctions at the trabecular bone than BMP-loaded membranes (BMP-Ms). Samples treated with Ti-Ms showed less bone formation and bony bridging processes. Both Zn-Ms and Ca-Ms achieved higher number of osteoblasts than the control group. BMP-Ms and Ca-Ms originated higher number of blood vessels than Ti-Ms and control group. CONCLUSIONS: Zn incorporation in novel nanostructured membranes provided the highest regenerative efficiency for bone healing at the rabbit calvarial defects. CLINICAL RELEVANCE: Zn-Ms promoted osteogenesis and enhanced biological activity, as mineralized and osteoid new bone with multiple interconnected ossified trabeculae appeared in close contact with the membrane.

The mineralizing effect of zinc oxide-modified hydroxyapatite-based sealer on radicular dentin.

OBJECTIVE: The aim of this study was to evaluate the remineralization ability of three endodontic sealer materials at different root dentin regions. MATERIAL AND METHODS: Cervical, medial, and apical root dentin surfaces were treated with two experimental hydroxyapatite-based cements, containing sodium hydroxide (calcypatite) or zinc oxide (oxipatite); an epoxy resin-based canal sealer, AH Plus; and gutta-percha. Remineralization, at the inner and outer zones of dentin disk surfaces, was studied by nanohardness (Hi) and Raman analysis. Nanoroughness and collagen fibrils width measurements were performed. Numerical data, at 24 h or 12 m, were analyzed by ANOVA and Student-Newman-Keuls (p < 0.05). RESULTS: At the outer and inner zones of the cervical dentin treated with oxipatite, the highest Hi after 12 m of immersion was achieved. The same group showed the highest intensity of phosphate peak, markers for calcification and crystallinity. Nanoroughness was lower and fibril diameter was higher at the inner zone of the dentin treated with oxipatite. Dentin mineralization occurred in every region of the root dentin treated with oxipatite and calcypatite, especially at the inner zone of the dentin after 12 m. CONCLUSIONS: Oxipatite reinforced the inner root zone at any third of the radicular dentin, by increasing both nanohardness and remineralization. When using calcypatite, the highest nanohardness was found at the apical third of the inner root dentin, but the lowest mechanical performance was obtained at the cervical and the medial thirds of the roots. Therefore, application of oxipatite as sealing cement of root canals is recommended. CLINICAL RELEVANCE: Oxipatite, when used as an endodontic sealing material, strengthens radicular dentin.

Zn-containing polymer nanogels promote cervical dentin remineralization.

OBJECTIVE: Nanogels designing for effective treatment of eroded cervical dentin lesions. MATERIALS AND METHODS: Polymethylmetacrylate-based nanoparticles (NPs) were doxycycline (D), calcium, or zinc loaded. They were applied on eroded cervical dentin. Treated surfaces were characterized morphologically by atomic force and scanning electron microscopy, mechanically probed by a nanoindenter to test nanohardness and Young's modulus, and chemically analyzed by Raman spectroscopy at 24 h and 7 days of storage. Data were submitted to ANOVA and Student-Newman-Keuls multiple comparisons tests. RESULTS: Dentin treated with Zn-NPs attained the highest nanomechanical properties, mineralization, and crystallinity among groups. Nanoroughness was lower in Zn-treated surfaces in comparison to dentin treated with undoped gels. Dentin treated with Ca-NPs created the minimal calcification at the surface and showed the lowest Young's modulus at peritubular dentin. Intertubular dentin appeared remineralized. Dentinal tubules were empty in samples treated with D-NPs, partially occluded in cervical dentin treated with undoped NPs and Ca-NPs, and mineral covered when specimens were treated with Zn-NPs. CONCLUSIONS: Zn-loaded NPs permit functional remineralization of eroded cervical dentin. Based on the tested nanomechanical and chemical properties, Zn-based nanogels are suitable for dentin remineralization. CLINICAL RELEVANCE: The ability of zinc-loaded nanogels to promote dentin mineralization may offer new strategies for regeneration of eroded cervical dentin and effective treatment of dentin hypersensitivity.

Zinc and silica are active components to efficiently treat in vitro simulated eroded dentin.

OBJECTIVES: Biomaterials for treating dentin hypersensitivity and dentin wear were evaluated to efficiently occlude the dentinal tubules and to increase dentin resistance to abrasion. MATERIALS AND METHODS: Twenty-four dentin surfaces were treated with EDTA to expose dentinal tubules and were (1) non-brushed, (2) brushed with distilled water, or with pastes containing (3) monetite, (4) brushite, (5) Zn-monetite, (6) Zn-brushite, (7) Silica-brushite, and (8) NovaMin®. Topographical, nanomechanical, and chemical analysis were assessed on dentin surfaces (n = 3) after artificial saliva immersion for 24 h, and after citric acid challenge. Twenty-one further dentin specimens were created to evaluate dentin permeability after brushing, saliva storage, and acid application (n = 3). ANOVA, Student-Newman-Keuls (p < 0.05), and Student t test (p < 0.001) were used. RESULTS: Particles containing major proportion of silica attained intratubular occlusion by carbonate crystals (Raman carbonate peak heights 15.17 and 19.24 au; complex modulus 110 and 140 GPa, at intratubular dentin). When brushing with pastes containing higher proportion of silica or zinc, phosphate calcium compounds were encountered into tubules and over dentin surfaces (Raman intratubular phosphate peak heights 49 to 70 au, and at the intertubular dentin 78 to 92). The formed carbonated apatite and calcium phosphate layer were resistant to citric acid application. Zinc compounds drastically increased tubule occlusion, decreased dentin permeability (up to 30%), and augmented mechanical properties at the intertubular dentin (90-130 GPa); it was maintained after acid challenging. CONCLUSIONS: Zinc-containing pastes occluded dentinal tubules and improved dentin mechanical properties. CLINICAL RELEVANCE: Using zinc as an active component to treat eroded dentin is encouraged.

Oral Function Improves Interfacial Integrity and Sealing Ability Between Conventional Glass Ionomer Cements and Dentin.

The aim of this study was to investigate if load cycling affects interfacial integrity of glass ionomer cements bonded to sound- or caries-affected dentin. A conventional glass ionomer, Ketac Bond, and a resin-modified glass ionomer (Vitrebond Plus), were applied to dentin. Half of the specimens were load cycled. The interfaces were submitted to dye-assisted confocal microscopy evaluation. The unloaded specimens of sound and carious dentin were deficiently hybridized when Ketac Bond was used. Ketac Bond samples showed an absorption layer and an adhesive layer that were scarcely affected by fluorescein penetration (nanoleakage), in sound dentin. Nevertheless, a higher degree of micropermeability was found in carious dentin. In Ketac Bond specimens, load cycling improves the sealing capability and remineralization at the cement-dentin interface as porosity and nanoleakage was reduced. In contrast, samples treated with Vitrebond Plus exhibited a Rhodamine B-labeled absorption layer with scarce nanoleakage in both sound and carious unloaded dentin. The adhesive layer was affected by dye sorption throughout the porous cement-dentin interface. Samples treated with Vitrebond Plus had significant increases in nanoleakage and cement-dye sorption after load cycling. Within the limitations of an in vitro study, it is expected that conventional glass ionomers will provide major clinical efficacy when applied to carious-affected or sound dentin.

Novel potential scaffold for periodontal tissue engineering.

OBJECTIVE: The objective of the study is characterization of novel calcium and zinc-loaded electrospun matrices to be used for periodontal regeneration. MATERIALS AND METHODS: A polymethylmetacrylate-based membrane was calcium or zinc loaded. Matrices were characterized morphologically by atomic force and scanning electron microscopy and mechanically probed by a nanoindenter. Biomimetic calcium phosphate precipitation on polymeric tissues was assessed. Cell viability tests were performed using oral mucosa fibroblasts. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests or by ANOVA and Student-Newman-Keuls multiple comparisons. RESULTS: Zinc and calcium loading on matrices did not modify their morphology but increased nanomechanical properties and decreased nanoroughness. Precipitation of calcium and phosphate on the matrix surfaces was observed in zinc-loaded specimens. Matrices were found to be non-toxic to cells in all the assays. Calcium- and zinc-loaded scaffolds presented a very low cytotoxic effect. CONCLUSIONS: Zinc-loaded membranes permit cell viability and promoted mineral precipitation in physiological conditions. Based on the tested nanomechanical properties and scaffold architecture, the proposed membranes may be suitable for cell proliferation. CLINICAL RELEVANCE: The ability of zinc-loaded matrices to promote precipitation of calcium phosphate deposits, together with their observed non-toxicity and its surface chemistry allowing covalent binding of proteins, may offer new strategies for periodontal regeneration.

Zinc-Containing Restorations Create Amorphous Biogenic Apatite at the Carious Dentin Interface: A X-Ray Diffraction (XRD) Crystal Lattice Analysis.

The aim of this research was to assess the ability of amalgam restorations to induce amorphous mineral precipitation at the caries-affected dentin substrate. Sound and caries-affected dentin surfaces were subjected to both Zn-free and Zn-containing dental amalgam restorations. Specimens were submitted to thermocycling (100,000 cycles/5°C-55°C, 3 months). Dentin surfaces were studied by atomic force microscopy (nanoroughness), X-ray diffraction, field emission scanning electron microscopy, and energy-dispersive analysis, for physical and morphological surface characterization. Zn-containing amalgam placement reduced crystallinity, crystallite size, and grain size of calcium phosphate crystallites at the dentin surface. Both microstrain and nanoroughness were augmented in caries-affected dentin restored with Zn-containing amalgams. Caries-affected dentin showed the shortest mineral crystallites (11.04 nm), when Zn-containing amalgams were used for restorations, probably leading to a decrease of mechanical properties which might favor crack propagation and deformation. Sound dentin restored with Zn-free amalgams exhibited a substantial increase in length of grain particles (12.44 nm) embedded into dentin crystallites. Zn-containing amalgam placement creates dentin mineralization and the resultant mineral was amorphous in nature. Amorphous calcium phosphate provides a local ion-rich environment, which is considered favorable for in situ generation of prenucleation clusters, promotong further dentin remineralization.

Zinc-modified nanopolymers improve the quality of resin-dentin bonded interfaces.

INTRODUCTION: Demineralized collagen fibers at the hybrid layer are susceptible to degradation. Remineralization may aid to improve bond longevity. OBJECTIVES: The aim of the present study was to infiltrate zinc and calcium-loaded polymeric nanoparticles into demineralized dentin to facilitate hybrid layer remineralization. MATERIALS AND METHODS: Zinc or calcium-loaded polymeric nanoparticles were infiltrated into etched dentin, and Single Bond Adhesive was applied. Bond strength was tested after 24 h and 6 months storage. Nanomechanical properties, dye-assisted confocal laser microscopy, and Masson's trichrome staining evaluation were performed to assess for the hybrid layer morphology, permeability, and remineralization ability after 24 h and 3 months. Data were analyzed by ANOVA and Student-Newman-Keuls multiple comparisons tests (p < 0.05). RESULTS: Immediate bond strength was not affected by nanoparticles infiltration (25 to 30 MPa), while after 6 months, bond strengths were maintained (22 to 24 MPa). After 3 months, permeability occurred only in specimens in which nanoparticles were not infiltrated. Dentin remineralization, at the bottom of the hybrid layer, was observed in all groups. After microscopy analysis, zinc-loaded nanoparticles were shown to facilitate calcium deposition throughout the entire hybrid layer. Young's modulus at the hybrid layer increased from 2.09 to 3.25 GPa after 3 months, in specimens with zinc nanoparticles; meanwhile, these values were reduced from 1.66 to 0.49 GPa, in the control group. CONCLUSION: Infiltration of polymeric nanoparticles into demineralized dentin increased long-term bond strengths. Zinc-loaded nanoparticles facilitate dentin remineralization within the complete resin-dentin interface. CLINICAL RELEVANCE: Resin-dentin bond longevity and dentin remineralization at the hybrid layer were facilitated by zinc-loaded nanoparticles.

Bond strength and bioactivity of Zn-doped dental adhesives promoted by load cycling.

The purpose of this study was to evaluate if mechanical loading influences bioactivity and bond strength at the resin-dentin interface after bonding with Zn-doped etch-and-rinse adhesives. Dentin surfaces were subjected to demineralization by 37% phosphoric acid (PA) or 0.5 M ethylenediaminetetraacetic acid (EDTA). Single bond (SB) adhesive­3M ESPE­SB+ZnO particles 20 wt% and SB+ZnCl2 2 wt% were applied on treated dentin to create the groups PA+SB, SB+ZnO, SB+ZnCl2, EDTA+SB, EDTA+ZnO, and EDTA+ZnCl2. Bonded interfaces were stored in simulated body fluid for 24 h and tested or submitted to mechanical loading. Microtensile bond strength (MTBS) was assessed. Debonded dentin surfaces were studied by high-resolution scanning electron microscopy. Remineralization of the bonded interfaces was assessed by atomic force microscope imaging/nanoindentation, Raman spectroscopy/cluster analysis, and Masson's trichrome staining. Load cycling (LC) produced reduction in MTBS in all PA+SB, and no change was encountered in EDTA+SB specimens, regardless of zinc doping. LC increased the mineralization and crystallographic maturity at the interface; a higher effect was noticed when using ZnO. Trichrome staining reflected a narrow demineralized dentin matrix after loading of dentin surfaces that were treated with SB-doped adhesives. This correlates with an increase in mineral platforms or plate-like multilayered crystals in PA or EDTA-treated dentin surfaces, respectively.

Improved Sealing and Remineralization at the Resin-Dentin Interface After Phosphoric Acid Etching and Load Cycling.

The purpose of this study was to investigate micro-morphology of the resin-dentin inter-diffusion zone using two different single-bottle self-etching dentin adhesives with and without previous acid-etching, after in vitro mechanical loading stimuli. Extracted human third molars were sectioned to obtain dentin surfaces. Two different single-bottle self-etching dentin adhesives, Futurabond U and Experimental both from VOCO, were applied following the manufacturer's instructions or after 37% phosphoric acid application. Resin-dentin interfaces were analyzed with dye assisted confocal microscopy evaluation (CLSM), including the calcium-chelation technique, xylenol orange (CLSM-XO). CLSM revealed that resin-dentin interfaces of unloaded specimens were deficiently resin-hybridized, in general. These samples showed a Rhodamine B-labeled hybrid complex and adhesive layer completely affected by fluorescein penetration (nanoleakage) through the porous resin-dentin interface, but thicker after PA-etching. Load cycling promoted an improved sealing of the resin-dentin interface at dentin, a decrease of the hybrid complex porosity, and an increment of dentin mineralization. Load cycled specimens treated with the XO technique produced a clearly outlined fluorescence due to consistent Ca-mineral deposits within the bonding interface and inside the dentinal tubules, especially when the experimental adhesive was applied.

Effect of zinc-doping in physicochemical properties of dental adhesives.

PURPOSE: To evaluate changes in the physicochemical properties, water sorption (WS), solubility (SO), modulus of elasticity (E), ultimate tensile strength (UTS), and microhardness (MH) tests were undertaken in zinc-doped dental adhesives. METHODS: Two bonding resins, Adper Single Bond Plus (SB) and Clearfil SE Bond (SEB), were zinc-doped by mixing them with 5, 10 or 20 wt% of ZnO powder, or with 1 or 2 wt% ZnCl2. Resin disks were made of each adhesive blend for the evaluation of WS, SO, and MH, and dumbbell-shaped specimens were prepared for E and UTS testing. RESULTS: An increase in WS and SO was observed for adhesives doped with ZnCl2. A reduction in WS was observed for the adhesive blends containing 10% or 20 wt% ZnO, while the SO was not altered in any of the ZnO-doped adhesives. An increase in E values was observed only for the SB adhesive doped with ZnCl2. For SEB-blends, the incorporation of zinc compounds did not alter the E values. UTS values decreased when SEB was doped with ZnO. SB-blends doped with 20 wt% ZnO significantly increased their MH, and the addition of zinc to the SEB-blends augmented the MH values in all cases.

Microanalysis of thermal-induced changes at the resin-dentin interface.

The purpose of this study was to evaluate the ability of two dentin adhesive systems to induce remineralization in the bonded dentin interface after in vitro thermo-cycling. Dentin surfaces were treated with two different adhesive approaches: (1) 37% phosphoric acid (PA) plus an "etch-and-rinse" dentin adhesive (single bond, SB) (PA+SB) or (2) application of a "self-etch" dentin adhesive (Clearfil SE bond, SEB). Three groups were established: (i) 24 h or (ii) 3 m storage, and (iii) specimens submitted to thermal cycling (100,000 cy/5 and 55ºC). Atomic force microscopy imaging/nanoindentation, Raman spectroscopy/cluster analysis with dye-assisted confocal laser scanning microscopy (CLSM) evaluation and Masson's trichrome staining assessments were implemented for characterization. Thermo-cycling increased nanohardness in PA+SB at the hybrid layer (HL) and in SEB at the bottom of the HL (BHL). Young's modulus increased at both the HL and BHL in SEB and at the HL in PA+SB, after thermal stress. Cluster analysis demonstrated an augmentation of the mineral-matrix ratio in thermo-cycled specimens. CLSM showed a decrease of both micropermeability and nanoleakage after thermo-cycling in PA+SB, and were completely absent in SEB. Trichrome staining reflected a scarce demineralized front in PA+SB after thermo-cycling and total remineralization in SEB.

Surface microanalysis and chemical imaging of early dentin remineralization.

This study reports physical and chemical changes that occur at early dentin remineralization stages. Extracted human third molars were sectioned to obtain dentin discs. After polishing the dentin surfaces, three groups were established: (1) untreated dentin (UD), (2) 37% phosphoric acid application for 15 s (partially demineralized dentin-PDD), and (3) 10% phosphoric acid for 12 h at 25° C (totally demineralized dentin-TDD). Five different remineralizing solutions were used: chlorhexidine (CHX), artificial saliva (AS), phosphate solution (PS), ZnCl2, and ZnO. Wettability (contact angle), ζ potential and Raman spectroscopy analysis were determined on dentin surfaces. Demineralization of dentin resulted in a higher contact angle. Wettability decreased after immersion in all solutions. ζ potential analysis showed dissimilar performance ranging from -6.21 mV (TDD + AS) up to 3.02 mV (PDD + PS). Raman analysis showed an increase in mineral components after immersing the dentin specimens, in terms of crystallinity, mineral content, and concentration. This confirmed the optimal incorporation and deposition of mineral on dentin collagen. Organic content reflected scarce changes, except in TDD that appeared partially denatured. Pyridinium, as an expression of cross-linking, appeared in all spectra except in specimens immersed in PS.

Effect of functionalized titanium particles with dexamethasone-loaded nanospheres on macrophage polarization and activity.

OBJECTIVE: The aim of this study was to determine the effect of titanium micro particles (TiP) previously functionalized with nanoparticles doped with dexamethasone (Dex) and doxycycline (Dox), on macrophage polarization and activity. METHODS: Macrophages RAW264.7 were cultured in the presence TiP loaded with dexamethasone -NPs (Dex)- and doxycycline -NPs (Dox)-, and as control, TiP with or without doped NPs. Cells were tested with and without previous bacterial lipopolysaccharide endotoxin (LPS) stimulation. Their morphology, proliferation, cytotoxicity, phenotypic change, and cytokines release were assessed by LIVE/DEAD, DNA release, metabolic activity, brightfield and scanning electron microscopy. The test Kruskall-Wallis was used for comparisons, while the cytokine expression profiles were examined by hierarchical clustering (p < 0.05). RESULTS: Upon exposure with TiP macrophages were activated and polarized to M1, but without depicting cytotoxic effects. The particles were phagocytised, and vacuolized. When exposed to functionalised TiP with NPs(Dex) and NPs(Dox), the ratio M1/M2 was up to forty times lower compared to TiP alone. When exposed to LPS, TiP reduced cell viability in half. Functionalised TiP with NPs(Dex) inhibited the cytokine release exerted by TiP on macrophages. When macrophages were exposed to functionalised TiPs with NPs(Dex) with and without LPS, the effect of TiP on cytokine secretion was inhibited. SIGNIFICANCE: Functionalised TiPs with NPs(Dex) and NPs(Dox) may potentially have beneficial effects on modulating titanium and LPS-related inflammatory reactions.

Investigation of the effect of Tideglusib on the hydroxyapatite formation, crystallinity and elasticity of conditioned resin-dentin interfaces.

OBJECTIVES: To investigate the effect of dentin infiltration with polymeric nanoparticles (NPs) doped with tideglusib (TDg) (TDg-NPs) on hydroxyapatite formation, crystallinity and elasticity of conditioned resin-dentin interfaces. METHODS: Dentin conditioned surfaces were infiltrated with NPs or TDg-NPs. Bonded interfaces were created, stored for 24 h and submitted to mechanical and thermal challenging. Resin-dentin interfaces were evaluated through nanoindentation to determine the modulus of elasticity, X-ray diffraction and transmission electron microscopy through selected area diffraction and bright-filed imaging. RESULTS: TDg-NPs provoked peaks narrowing after the diffraction-intensity analysis that corresponded with high crystallinity, with an increased modulus of Young after load cycling in comparison with the samples treated with undoped NPs. New minerals, in the group of TDg-NPs, showed the greatest both deviation of line profile from perfect crystal diffraction and dimension of the lattice strain, i.e., crystallite, grain size and microstrain and 002 plane-texture. The new minerals generated after TDg-NPs application and mechanical loading followed a well defined lineation. Undoped NPs mostly produced small hydroxyapatite crystallites, non crystalline or amorphous in nature with poor maturity. CONCLUSIONS: Tideglusib promoted the precipitation of hydroxyapatite, as a major crystalline phase, at the intrafibrillar compartment of the collagen fibrils, enabling functional mineralization. TDg-NPs facilitated nucleation of crystals randomly oriented, showing less structural variation in angles and distances that improved crystallographic relative order of atoms and maturity. Nanocrystals inducted by TDg-NPs were hexagonal prisms of submicron size. Thermal challenging of dentin treated with TDg-NPs have provoked a decrease of functional mineralization and crystallinity, associated to immature hydroxyapatite. CLINICAL SIGNIFICANCE: New polycrystalline lattice formation generated after TDg-NPs infiltration may become correlated with high mechanical performance. This association can be inferred from the superior crystallinity that was obtained in presence of tideglusib. Immature crystallites formed in dentin treated with undoped NPs will account for a high remineralizing activity.

Mitigating lipopolysaccharide-induced impairment in human dental pulp stem cells with tideglusib-doped nanoparticles: Enhancing osteogenic differentiation and mineralization.

OBJECTIVE: Drug-loaded non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for pulp regenerative strategies. The present in vitro investigation aimed to evaluate the effectiveness of tideglusib-doped nanoparticles (TDg-NPs) in mitigating the adverse effects of bacterial lipopolysaccharide endotoxin (LPS) on the viability, morphology, migration, differentiation and mineralization potential of human dental pulp stem cells (hDPSCs). METHODS: Cell viability, proliferation, and differentiation were assessed using a MTT assay, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining and expression of the odontogenic related genes by a real-time quantitative polymerase chain reaction (RT-qPCR) were also performed. Cells were tested both with and without stimulation with LPS at various time points. One-way ANOVA and Tukey's test were employed for statistical analysis (p < 0.05). RESULTS: Adequate cell viability was encountered in all groups and at every tested time point (24, 48, 72 and 168 h), without differences among the groups (p > 0.05). The analysis of cell cytoskeleton showed nuclear alteration in cultures with undoped NPs after LPS stimulation. These cells exhibited an in blue diffuse and multifocal appearance. Some nuclei looked fragmented and condensed. hDPSCs after LPS stimulation but in the presence of TDg-NPs exhibited less nuclei changes. LPS induced down-regulation of Alkaline phosphatase, Osteonectin and Collagen1 gene markers, after 21d. LPS half-reduced the cells production of calcium deposits in all groups (p < 0.05), except in the group with TDg-NPs (decrease about 10 %). SIGNIFICANCE: LPS induced lower mineral deposition and cytoskeletal disorganization in hDPSCs. These effects were counteracted by TDg-NPs, enhancing osteogenic differentiation and mineralization.

NP-12 peptide functionalized nanoparticles counteract the effect of bacterial lipopolysaccharide on cultured osteoblasts.

OBJECTIVE: To evaluate whether nanoparticles (NPs) functionalized with Tideglusib (TDg, NP-12), and deposited on titanium surfaces, would counteract the effect of bacterial lipopolysaccharide (LPS) on osteoblasts. METHODS: Experimental groups were: (a) Titanium discs (TiD), (b) TiD covered with undoped NPs (Un-NPs) and (c) TiD covered with TDg-doped NPs (TDg-NPs). Human primary osteoblasts were cultured onto these discs, in the presence or absence of bacterial LPS. Cell proliferation was assessed by MTT-assay and differentiation by measuring the alkaline phosphatase activity. Mineral nodule formation was assessed by the alizarin red test. Real-time quantitative polymerase chain reaction was used to study the expression of Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3 genes. Osteoblasts morphology was studied by Scanning Electron Microscopy. One-way ANOVA or Kruskal-Wallis and Bonferroni multiple comparisons tests were carried out (p < 0.05). RESULTS: TDg-NPs enhanced osteoblasts proliferation. Similarly, this group increased ALP production and mineral nodules formation. TDg-NPs on titanium discs resulted in overexpression of the proliferative genes, OSC and OSX, regardless of LPS activity. In the absence of LPS, TDg-NPs up-regulated Runx2, COL-I, ALP, BMP2 and BMP7 genes. OPG/RANKL gene ratios were increased about 2500 and 4,000-fold by TDg-NPs, when LPS was added or not, respectively. In contact with the TDg-NPs osteoblasts demonstrated an elongated spindle-shaped morphology with extracellular matrix production. SIGNIFICANCE: TDg-NPs on titanium discs counteracted the detrimental effect of LPS by preventing the decrease on osteoblasts proliferation and mineralization, and produced an overexpression of proliferative and bone-promoting genes on human primary osteoblasts.

Effect of the anti-Alzheimer drug GSK-3ß antagonist on numerical modeling of the energy dissipation through the resin-dentin interface.

OBJECTIVES: The aim of this study was to determine the viscoelastic performance and energy dissipation of conditioned dentin infiltrated with polymeric nanoparticles (NPs) doped with tideglusib (TDg) (TDg-NPs). METHODS: Dentin conditioned surfaces were infiltrated with NPs and TDg-NPs. Bonded interfaces were created, stored for 24 h and submitted to mechanical and thermal challenging. Resin-dentin interfaces were evaluated through nano-DMA/complex-loss-storage moduli-tan delta assessment and atomic force microscopy (AFM) analysis. RESULTS: Dentin infiltrated with NPs and load cycled attained the highest complex modulus at hybrid layer and bottom of hybrid layer. Intertubular dentin treated with undoped NPs showed higher complex modulus than peritubular dentin, after load cycling, provoking energy concentration and breakdown at the interface. After infiltrating with TDg-NPs, complex modulus was similar between peri-intertubular dentin and energy dissipated homogeneously. Tan delta at intertubular dentin was higher than at peritubular dentin, after using TDg-NPs and load cycling. This generated the widest bandwidth of the collagen fibrils and bridge-like mineral structures that, as sight of energy dissipation, fastened active dentin remodeling. TDg-NPs inducted scarce mineralization after thermo-cycling, but these bridging processes limited breakdown zones at the interface. SIGNIFICANCE: TDg-based NPs are then proposed for effective dentin remineralization and tubular seal, from a viscoelastic approach.