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Quercitrin Nanocoated Implant Surfaces Reduce Osteoclast Activity In Vitro and In Vivo.

ABSTRACT: In this study, the effect on osteoclast activity in vitro and in vivo of titanium implants that were coated with quercitrin was evaluated. Titanium surfaces were covalently coated with the flavonoid quercitrin. The effect of the surfaces on osteoclastogenesis was first tested in vitro on RAW264.7 cells that were supplemented with receptor activator of nuclear factor kappa-B ligand (RANKL) to generate osteoclast-like cells by tartrate-resistant acid phosphatase (TRAP) inmunostaining after five days of culture, and by analysis of the mRNA expression levels of markers related to bone resorption after seven days of culture. A rabbit tibial model was used to evaluate the in vivo biological response to the implant surfaces after eight weeks of healing, analyzing the lactate dehydrogenase (LDH) and the alkaline phosphatase (ALP) activities in the wound fluid that were present at the implant interface and the peri-implant bone mRNA expression levels of several markers related to inflammation, bone resorption and osteoblast-osteoclast interaction. No differences between groups and control surfaces were found in the wound fluid analyses. Moreover, quercitrin implant surfaces significantly decreased the expression of osteoclast related genes in vitro (Trap, CalcR, Ctsk, H?ATPase, Mmp9) and in vivo (Ctsk, H?ATPase, Mmp9) as well as the expression of RankL in vivo. Moreover, quercitrin surfaces were not cytotoxic for the cells. Thus, quercitrin implant surfaces were biocompatible and decreased osteoclastogenesis in vitro and in vivo. This could be used to improve the performance of dental implants.

SUBMITTER: Cordoba A

PROVIDER: S-EPMC6274788 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Quercitrin Nanocoated Implant Surfaces Reduce Osteoclast Activity In Vitro and In Vivo.

Córdoba Alba A Manzanaro-Moreno Nahuel N Colom Carme C Rønold Hans J HJ Lyngstadaas Staale P SP Monjo Marta M Ramis Joana M JM

International journal of molecular sciences 20181025 11

In this study, the effect on osteoclast activity in vitro and in vivo of titanium implants that were coated with quercitrin was evaluated. Titanium surfaces were covalently coated with the flavonoid quercitrin. The effect of the surfaces on osteoclastogenesis was first tested in vitro on RAW264.7 cells that were supplemented with receptor activator of nuclear factor kappa-B ligand (RANKL) to generate osteoclast-like cells by tartrate-resistant acid phosphatase (TRAP) inmunostaining after five da ...[more]

PMID: 30366383

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Project description:IntroductionMicro- or nano-topography can both provide antimicrobial properties and improve osseointegration of dental implant titanium surfaces. Laser treatment is one of the best surface microtexturing techniques. The aim of this study was to evaluate in vitro and in situ biofilm formation on a laser-treated titanium surface, comparing it with two conventional surfaces, machined and grit-blasted.MethodsFor the in vitro experiment, an oral microcosm biofilm model was developed on the surface of titanium disks and reference human enamel using a bioreactor for 48 h. For the in situ experiment, titanium implants with laser-treated, machined and grit-blasted surfaces were mounted on intraoral trays and worn by ten volunteers for 48 h. Biofilm formation was quantitatively evaluated, and surfaces were analyzed using confocal laser scanning microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy.Results–in vitro studyBiofilm structures with a prevalence of viable cells covered most of the machined, grit-blasted and human enamel surfaces, whereas less dense biofilm structures with non-confluent microcolonies were observed on the laser-treated titanium. Laser-treated titanium showed the lowest biofilm formation, where microorganisms colonized the edges of the laser-created pits, with very few or no biofilm formation observed inside the pits.Results–in situ studyThe biofilm formation pattern observed was similar to that in the in vitro experiment. Confocal laser scanning microscopy showed complete coverage of the implant threads, with mostly viable cells in grit-blasted and machined specimens. Unexpectedly, laser-treated specimens showed few dead microbial cells colonizing the bottom of the threads, while an intense colonization was found on the threading sides.ConclusionThis data suggests that laser-created microtopography can reduce biofilm formation, with a maximum effect when the surface is blasted orthogonally by the laser beam. In this sense the orientation of the laser beam seems to be relevant for the biological interaction with biofilms.

Project description:Sealing ring formation is a requirement for osteoclast function. We have recently identified the role of an actin-bundling protein L-plastin in the assembly of nascent sealing zones (NSZs) at the early phase of sealing ring formation in osteoclasts. TNF-α signaling regulates this actin assembly by the phosphorylation of L-plastin on serine -5 and -7 residues at the amino-terminal end. These NSZs function as a core for integrin localization and coordinating integrin signaling required for maturation into fully functional sealing rings. Our goal is to elucidate the essential function of L-plastin phosphorylation in actin bundling, a process required for NSZs formation. The present study was undertaken to determine whether targeting serine phosphorylation of cellular L-plastin would be the appropriate approach to attenuate the formation of NSZs. Our approach is to use TAT-fused small molecular weight amino-terminal L-plastin peptides (10 amino acids) containing phospho- Ser-5 and Ser-7. We used peptides unsubstituted (P1) and substituted (P2- P4) at serine-to-alanine residues. Immunoblotting, actin staining, and dentine resorption analyses were done to determine cellular L-plastin phosphorylation, NSZ or sealing ring formation, and osteoclast function, respectively. Immunoblotting for bone formation markers, Alizarin red staining and alkaline phosphatase activity assay have been done to determine the effect of peptides on the mineralization process mediated by osteoblasts. Transduction of unsubstituted (P1) and substituted peptides at either Serine 5 or Serine 7 with Alanine (P3 and P4) demonstrated variable inhibitory effects on the phosphorylation of cellular L-plastin protein. Peptide P1 reduces the following processes substantially: 1) cellular L-plastin phosphorylation; 2) formation of nascent sealing zones and sealing rings; 3) bone resorption. Substitution of both Serine-5 and -7 with Alanine (P2) had no effects on the inhibitory activities described above. Furthermore, either the L-plastin (P1-P5) or (P6) control peptides had a little or no impact on the a) assembly/disassembly of podosomes and migration of osteoclasts; b) mineralization process mediated by osteoblasts in vitro. Small molecular weight peptidomimetics of L-plastin inhibits bone resorption by osteoclasts via attenuation of NSZ and sealing ring formation but not bone formation by osteoblasts in vitro. The L-plastin may be a valuable therapeutic target to treat and prevent diseases associated with bone loss without affecting bone formation.

Project description:Background and aimBacterial biofilms play a major role in the etiology of periodontal and peri-implant diseases. The aim of the study was to evaluate the removal of bacterial biofilms and attachment of epithelial cells (EC), gingival fibroblasts (GF) and osteoblast-like cells (OC) to dentin and titanium surfaces after Er:YAG laser (Er:YAG) in comparison with other treatment methods.Material and methodsMulti-species bacterial biofilms were grown on standardized dentin and titanium specimens with a sand-blasted and acid etched (SLA) surface for 3.5 d. Thereafter, the specimens were placed into artificially-created pockets. The following methods for biofilm removal were used: 1) Gracey (dentin) or titanium curettes (CUR), 2) Er:YAG, 3) photodynamic therapy (PDT) and 4) CUR with adjunctive PDT (CUR/PDT). Colony forming units (CFUs) of the remaining biofilms and attachment of EC, GF and OC were determined. Statistical analysis was performed by means of ANOVA with post-hoc LSD.ResultsAll treatment methods decreased statistically significantly (p<0.001) total CFUs in biofilms compared with untreated dentin and titanium surfaces respectively. On dentin, Er:YAG was equally efficient as CUR and PDT but inferior to CUR/PDT (p = 0.005). On titanium, surfaces, the use of Er:YAG resulted in statistically significantly superior biofilm removal compared to the 3 other treatments (each p<0.001). Counts of attached EC, GF and OC were the lowest on untreated contaminated dentin and titanium surfaces each. After CUR/PDT higher EC counts were found on dentin (p = 0.006). On titanium, all decontamination methods statistically significantly increased (p<0.001) the counts of attached EC without differences between groups. Statistically significantly higher counts of GF (p = 0.024) and OC (p<0.001) were observed after Er:YAG decontamination compared with untreated surfaces.ConclusionAblation of subgingival biofilms and in particular decontamination of titanium implant surfaces with an Er:YAG laser seem to be a promising approach and warrants further investigations.

Project description:ObjectivesTotal joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings.Materials and methodsFor initial surface adhesion, human primary osteoblasts (hOB) were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R®, and PALACOS R® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04), and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU). The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces.ResultsAfter 2 days, we observed approximately 104 CFU/ml biofilm-bound S. epidermidis (103 CFU/ml initial population) on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 105 CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10% of its initial value compared to 150% in the mono-culture of hOB.ConclusionWe developed a co-culture setup that serves as a more comprehensive in vitro model for the onset of implant-associated infections and provides a test method for antimicrobial implant materials and coatings. We demonstrate that observations can be made that are unavailable from mono-culture experiments.

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Quercitrin Nanocoated Implant Surfaces Reduce Osteoclast Activity In Vitro and In Vivo.

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Quercitrin Nanocoated Implant Surfaces Reduce Osteoclast Activity In Vitro and In Vivo.

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