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.

Mild acids facilitate functional dentin remineralization under thermo-mechanical stimuli.

PURPOSE: To evaluate if mechanical and thermal cycling promote remineralization at the resin-dentin interface after bonding with three different adhesive approaches. METHODS: Dentin surfaces were subjected to three different treatments: demineralization (1) by 37% phosphoric acid followed by application of an etch-and-rinse dentin adhesive Single Bond (Adper Single Bond) (SB); (2) by 0.5 M ethylenediaminetetraacetic acid (EDTA) followed by SB; (3) application of a self-etch dentin adhesive: Clearfil-SEB (Clearfil SE Bond). Bonded interfaces were stored during 24 hours and then submitted for 3 months to: (1) storage at 37ºC, (2) load cycling, (3) thermocycling, and (4) thermo+load cycling. One section was extracted from each tooth, monthly. Resin-dentin interfaces were analyzed by AFM nano-indentation, Raman spectroscopy/cluster analysis and Masson's trichrome staining at 24 hours, 1, 2 and 3 months, to determine remineralization at the interface. RESULTS: Thermo+load cycling promoted the highest biomimetic remineralization at the hybrid layer formed with EDTA+SB and Clearfil-SEB, at the 1 month time point. A narrow mineral-depleted zone was observed after thermo+load cycling with EDTA+ SB, and at those specimens bonded with Clearfil-SEB. Thermo+load cycling remineralized the dentin interface treated with EDTA+SB and Clearfil-SEB, after 1 month of study period, providing bioactivity and maturity of formed minerals. CLINICAL SIGNIFICANCE: In vitro challenging (thermo+load cycling) favors dentin remineralization at the resin-dentin bonded interfaces promoted with mild conditioning acids.

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.

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.

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.

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.

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.

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.

Dexamethasone and doxycycline functionalized nanoparticles enhance osteogenic properties of titanium surfaces.

OBJECTIVES: To evaluate the effect of doxycycline and dexamethasone doped nanoparticles covering titanium surfaces, on osteoblasts proliferation and differentiation. METHODS: Doxycycline and dexamethasone doped polymeric nanoparticles were applied on titanium discs (Ti-DoxNPs and Ti-DexNPs). Undoped NPs and uncovered Ti discs were used as control. Human MG-63 osteoblast-like cells were cultured. Osteoblasts proliferation was tested by MTT assay. Alkaline phosphatase activity was analyzed. Differentiation gene expression was assessed by real-time quantitative polymerase chain reaction. Scanning Electron Microscopy was performed to assess osteoblasts morphology. Mean comparisons were conducted by ANOVA and Wilcoxon or Tukey tests (p < 0.05). RESULTS: No differences in osteoblasts proliferation were found. Osteoblasts grown on Ti-DoxNPs significantly increased alkaline phosphatase activity. Doxycycline and dexamethasone nanoparticles produced an over-expression of the main osteogenic proliferative genes (TGF-ß1, TGF-ßR1 and TGF-ßR2). The expression of Runx-2 was up-regulated. The osteogenic proteins (AP, OSX and OPG) were also overexpressed on osteoblasts cultured on Ti-DoxNPs and Ti-DexNPs. The OPG/RANKL ratio was the highest when DoxNPs were present (75-fold increase with respect to the control group). DexNPs also produced a significantly higher OPG/RANKL ratio with respect to the control (20 times higher). Osteoblasts grown on titanium discs were mainly flat and polygonal in shape, with inter-cellular connections. In contrast, osteoblasts cultured on Ti-DoxNPs or Ti-DexNPs were found to be spindle-shaped and had abundant secretions on their surfaces. SIGNIFICANCE: DoxNPs and DexNPs were able to stimulate osteoblasts differentiation when applied on titanium surfaces, being considered potential inducers of osteogenic environment when performing regenerative procedures around titanium dental implants.

Effect of doxycycline doped nanoparticles on osteogenic/cementogenic and anti-inflammatory responses of human cells derived from the periodontal ligament.

OBJECTIVES: This work aimed to evaluate if doxycycline-doped polymeric nanoparticles possessed any anti-inflammatory effect and promote osteogenic/cementogenic differentiation of stem cells from human periodontal ligament (PDLSCs). METHODS: The polymeric nanoparticles (NPs) were produced by a polymerization/precipitation process and doped with doxycycline (Dox-NPs). PDLSCs were cultured in the presence or absence of the NPs under osteogenic medium or IL-1ß treatment. Cells' differentiation was assessed by gene expression analysis of osteogenic/cementogenic markers alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). An anti-inflammatory effect was also ascertained by analyzing IL-1ß gene expression. Adipogenic and chondrogenic differentiation was used to confirm the multipotency of PDLSCs. RESULTS: Gene expression of ALP and RUNX2 in PDLSCs was significantly upregulated by the osteogenic medium (ALP: p<0.001; RUNX2: p = 0.005) while Dox-NPs further enhanced ALP gene expression of PDLSCs treated with the osteogenic medium. Furthermore, Dox-NPs suppressed the up-regulation of IL-1ß when cells were subjected to an inflammatory challenge. CONCLUSIONS: Dox-NPs enhanced PDLSCs differentiation into osteoblasts/cementoblasts lineages while providing an anti-inflammatory effect. CLINICAL SIGNIFICANCE: Due to their biocompatibility as well as anti-inflammatory and osteogenic/cementogenic effects, Dox-NPs are potential candidates for being used in periodontal regeneration.

Doped Electrospinned Material-Guides High Efficiency Regional Bone Regeneration.

The main target of bone tissue engineering is to design biomaterials that support bone regeneration and vascularization. Nanostructured membranes of (MMA)1-co-(HEMA)1/(MA)3-co-(HEA)2 loaded with 5% wt of SiO2-nanoparticles (Si-M) were doped with zinc (Zn-Si-M) or doxycycline (Dox-Si-M). Critical bone defects were effectuated on six New Zealand-bred rabbit skulls and then they were covered with the membranes. After six weeks, a histological analysis (toluidine blue technique) was employed to determine bone cell population as osteoblasts, osteoclasts, osteocytes, M1 and M2 macrophages and vasculature. Membranes covering the bone defect determined a higher count of bone cells and blood vessels than in the sham group at the top regions of the defect. Pro-inflammatory M1 appeared in a higher number in the top regions than in the bottom regions, when Si-M and Dox-Si-M were used. Samples treated with Dox-Si-M showed a higher amount of anti-inflammatory and pro-regenerative M2 macrophages. The M1/M2 ratio obtained its lowest value in the absence of membranes. On the top regions, osteoblasts were more abundant when using Si-M and Zn-Si-M. Osteoclasts were equally distributed at the central and lateral regions. The sham group and samples treated with Zn-Si-M attained a higher number of osteocytes at the top regions. A preferential osteoconductive, osteoinductive and angiogenic clinical environment was created in the vicinity of the membrane placed on critical bone defects.

Biomimetic Collagen Membranes as Drug Carriers of Geranylgeraniol to Counteract the Effect of Zoledronate.

To counteract the effect of zoledronate and decrease the risk of osteonecrosis of the jaw (BRONJ) development in patients undergoing guided bone regeneration surgery, the use of geranylgeraniol (GGOH) has been proposed. Collagen membranes may act as biomimetical drug carriers. The objective of this study was to determine the capacity of collagen-based membranes doped with GGOH to revert the negative impact of zoledronate on the growth and differentiation of human osteoblasts. MG-63 cells were cultured on collagen membranes. Two groups were established: (1) undoped membranes and (2) membranes doped with geranylgeraniol. Osteoblasts were cultured with or without zoledronate (50 µM). Cell proliferation was evaluated at 48 h using the MTT colorimetric method. Differentiation was tested by staining mineralization nodules with alizarin red and by gene expression analysis of bone morphogenetic proteins 2 and 7, alkaline phosphatase (ALP), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), type I collagen (Col-I), osterix (OSX), osteocalcin (OSC), osteoprotegerin (OPG), receptor for RANK (RANKL), runt-related transcription factor 2 (Runx-2), TGF-ß1 and TGF-ß receptors (TGF-ßR1, TGF-ßR2, and TGF-ßR3), and vascular endothelial growth factor (VEGF) with real-time PCR. One-way ANOVA or Kruskal-Wallis and post hoc Bonferroni tests were applied (p < 0.05). Scanning electron microscopy (SEM) observations were also performed. Treatment of osteoblasts with 50 µM zoledronate produced a significant decrease in cell proliferation, mineralization capacity, and gene expression of several differentiation markers if compared to the control (p < 0.001). When osteoblasts were treated with zoledronate and cultured on GGOH-doped membranes, these variables were, in general, similar to the control group (p > 0.05). GGOH applied on collagen membranes is able to reverse the negative impact of zoledronate on the proliferation, differentiation, and gene expression of different osteoblasts' markers.

Antibiotic-Loaded Polymeric Barrier Membranes for Guided Bone/Tissue Regeneration: A Mini-Review.

Polymeric membranes are frequently used for bone regeneration in oral and periodontal surgery. Polymers provide adequate mechanical properties (i.e., Young's modulus) to support oral function and also pose some porosity with interconnectivity to permit for cell proliferation and migration. Bacterial contamination of the membrane is an event that may lead to infection at the bone site, hindering the clinical outcomes of the regeneration procedure. Therefore, polymeric membranes have been proposed as carriers for local antibiotic therapy. A literature search was performed for papers, including peer-reviewed publications. Among the different membranes, collagen is the most employed biomaterial. Collagen membranes and expanded polytetrafluoroethylene loaded with tetracyclines, and polycaprolactone with metronidazole are the combinations that have been assayed the most. Antibiotic liberation is produced in two phases. A first burst release is sometimes followed by a sustained liberation lasting from 7 to 28 days. All tested combinations of membranes and antibiotics provoke an antibacterial effect, but most of the time, they were measured against single bacteria cultures and usually non-specific pathogenic bacteria were employed, limiting the clinical relevance of the attained results. The majority of the studies on animal models state a beneficial effect of these antibiotic functionalized membranes, but human clinical assays are scarce and controversial.