Outlining cell interaction and inflammatory cytokines on UV-photofunctionalized mixed-phase TiO2 thin film.

Photofunctionalization mediated by ultraviolet (UV) light seems to be a promising approach to improve the physico-chemical characteristics and the biological response of titanium (Ti) dental implants. Seeing that photofunctionalization is able to remove carbon from the surface, besides to promote reactions on the titanium dioxide (TiO2) layer, coating the Ti with a stable TiO2 film could potentialize the UV effect. Thus, here we determined the impact of UV-photofunctionalized mixed-phase (anatase and rutile) TiO2 films on the physico-chemical properties of Ti substrate and cell biology. Mixed-phase TiO2 films were grown by radiofrequency magnetron sputtering on commercially pure titanium (cpTi) discs, and samples were divided as follow: cpTi (negative control), TiO2 (positive control), cpTi UV, TiO2 UV (experimental). Photofunctionalization was performed using UVA (360 nm - 40 W) and UVC (250 nm - 40 W) lamps for 48 h. Surfaces were analyzed in terms of morphology, topography, chemical composition, crystalline phase, wettability and surface free energy. Pre-osteoblastic cells (MC3T3E1) were used to assess cell morphology and adhesion, metabolism, mineralization potential and cytokine secretion (IFN-γ, TNF-α, IL-4, IL-6 and IL-17). TiO2-coated surfaces exhibited granular surface morphology and greater roughness. Photofunctionalization increased wettability (p < 0.05) and surface free energy (p < 0.001) on both surface conditions. TiO2-treated groups featured normal cell morphology and spreading, and greater cellular metabolic activity at 2 and 4 days (p < 0.05), whereas UV-photofunctionalized surfaces enhanced cell metabolism, cell adhered area, and calcium deposition (day 14) (p < 0.05). In general, assessed proteins were found slightly affected by either UV or TiO2 treatments. Altogether, our findings suggest that UV-photofunctionalized TiO2 surface has the potential to improve pre-osteoblastic cell differentiation and the ability of cells to form mineral nodules by modifying Ti physico-chemical properties towards a more stable context. UV-modified surfaces modulate the secretion of key inflammatory markers.

Cementocyte alterations associated with experimentally induced cellular cementum apposition in Hyp mice.

BACKGROUND: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology. METHODS: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp). RESULTS: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43. CONCLUSION: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation.

Orthodontic tooth movement alters cementocyte ultrastructure and cellular cementum proteome signature.

Cementum is a mineralized tissue that covers tooth roots and functions in the periodontal attachment complex. Cementocytes, resident cells of cellular cementum, share many characteristics with osteocytes, are mechanoresponsive cells that direct bone remodeling based on changes in loading. We hypothesized that cementocytes play a key role during orthodontic tooth movement (OTM). To test this hypothesis, we used 8-week-old male Wistar rats in a model of OTM for 2, 7, or 14 days (0.5 N), whereas unloaded contralateral teeth served as controls. Tissue and cell responses were analyzed by high-resolution micro-computed tomography, histology, tartrate-resistant acid phosphatase staining for odontoclasts/osteoclasts, and transmission electron microscopy. In addition, laser capture microdissection was used to collect cellular cementum, and extracted proteins were identified by liquid chromatography coupled to tandem mass spectrometry. The OTM model successfully moved first molars mesially more than 250 µm by 14 days introducing apoptosis in a small number of cementocytes and areas of root resorption on mesial and distal aspects. Cementocytes showed increased nuclear size and proportion of euchromatin suggesting cellular activity. Proteomic analysis identified 168 proteins in cellular cementum with 21 proteins found only in OTM sites and 54 proteins only present in control samples. OTM-down-regulated several extracellular matrix proteins, including decorin, biglycan, asporin, and periostin, localized to cementum and PDL by immunostaining. Furthermore, type IV collagen (COL14A1) was the protein most down-regulated (-45-fold) by OTM and immunolocalized to cells at the cementum-dentin junction. Eleven keratins were significantly increased by OTM, and a pan-keratin antibody indicated keratin localization primarily in epithelial remnants of Hertwig's epithelial root sheath. These experiments provide new insights into biological responses of cementocytes and cellular cementum to OTM.

Targeting Pathogenic Biofilms: Newly Developed Superhydrophobic Coating Favors a Host-Compatible Microbial Profile on the Titanium Surface.

Polymicrobial infections are one of the most common reasons for inflammation of surrounding tissues and failure of implanted biomaterials. Because microorganism adhesion is the first step for biofilm formation, physical-chemical modifications of biomaterials have been proposed to reduce the initial microbial attachment. Thus, the use of superhydrophobic coatings has emerged because of their anti-biofilm properties. However, these coatings on the titanium (Ti) surface have been developed mainly by dual-step surface modification techniques and have not been tested using polymicrobial biofilms. Therefore, we developed a one-step superhydrophobic coating on the Ti surface by using a low-pressure plasma technology to create a biocompatible coating that reduces polymicrobial biofilm adhesion and formation. The superhydrophobic coating on Ti was created by the glow discharge plasma using Ar, O2, and hexamethyldisiloxane gases, and after full physical, chemical, and biological characterizations, we evaluated its properties regarding oral biofilm inhibition. The newly developed coating presented an increased surface roughness and, consequently, superhydrophobicity (contact angle over 150°) and enhanced corrosion resistance (p < 0.05) of the Ti surface. Furthermore, proteomic analysis showed a unique pattern of protein adsorption on the superhydrophobic coating without drastically changing the biologic processes mediated by proteins. Additionally, superhydrophobic treatment did not present a cytotoxic effect on fibroblasts or reduction of proliferation; however, it significantly reduced (≈8-fold change) polymicrobial adhesion (bacterial and fungal) and biofilm formation in vitro. Interestingly, superhydrophobic coating shifted the microbiological profile of biofilms formed in situ in the oral cavity, reducing by up to ≈7 fold pathogens associated with the peri-implant disease. Thus, this new superhydrophobic coating developed by a one-step glow discharge plasma technique is a promising biocompatible strategy to drastically reduce microbial adhesion and biofilm formation on Ti-based biomedical implants.

Tailoring the synthesis of tantalum-based thin films for biomedical application: Characterization and biological response.

The aim of this study was to tailor the deposition parameters of magnetron sputtering to synthetize tantalum oxide (TaxOy) films onto commercially pure titanium (cpTi) surface. The structural and optical properties, morphology, roughness, elemental chemical composition and surface energy were assessed. The impact of TaxOy films on initial Streptococcus sanguinis adhesion was investigated. The morphology and spreading of pre-osteoblastic (MC3T3-E1) cells on a crystalline tantalum oxide film were evaluated. TaxOy films with estimated thickness of 600 nm and different structures (amorphous or crystalline) were produced depending on the various oxygen flow rates and parameters used. X-ray diffraction analysis revealed that the 8 O2 sccm (600 °C/400 W) group showed crystallization corresponding to the ß-Ta2O5 phase. Optical analysis showed that the 4 O2 sccm (200 °C 300 W) to 8 O2 sccm (600 °C 300 W) groups and 10 O2 sccm (200 °C 300 W) group presented regular and large-amplitude interference oscillations, suggesting high optical homogeneity of the films. The crystalline ß-Ta2O5 coating showed higher roughness and surface energy values than the other groups (P < .05) and was biocompatible. Compared with cpTi, the amorphous and crystalline tantalum oxide films did not increase bacterial adhesion (P > .05). By tailoring the deposition parameters, we synthetized a crystalline ß-Ta2O5 coating that improved titanium surface properties and positively affected cell spreading and morphology, making it a promising surface treatment for titanium-based implants.

Three-species biofilm model onto plasma-treated titanium implant surface.

In this study, titanium (Ti) was modified with biofunctional and novel surface by micro-arc oxidation (MAO) and glow discharge plasma (GDP) and we tested the development of a three-species periodontopatogenic biofilm onto the treated commercially-pure titanium (cpTi) surfaces. Machined and sandblasted surfaces were used as control group. Several techniques for surface characterizations and monoculture on bone tissue cells were performed. A multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum was developed onto cpTi discs for 16.5h (early biofilm) and 64.5h (mature biofilm). The number of viable microorganisms and the composition of the extracellular matrix (proteins and carbohydrates) were determined. The biofilm organization was analyzed by scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM). In addition, MC3T3-E1 cells were cultured on the Ti surfaces and cell proliferation (MTT) and morphology (SEM) were assessed. MAO treatment produced oxide films rich in calcium and phosphorus with a volcano appearance while GDP treatment produced silicon-based smooth thin-film. Plasma treatments were able to increase the wettability of cpTi (p<0.05). An increase of surface roughness (p<0.05) and formation of anatase and rutile structures was noted after MAO treatment. GDP had the greatest surface free energy (p<0.05) while maintaining the surface roughness compared to the machined control (p>0.05). Plasma treatment did not affect the viable microorganisms counts, but the counts of F. nucleatum was lower for MAO treatment at early biofilm phase. Biofilm extracellular matrix was similar among the groups, excepted for GDP that presented the lowest protein content. Moreover, cell proliferation was not significantly affected by the experimental, except for MAO at 6days that resulted in an increased cell proliferative. Together, these findings indicate that plasma treatments are a viable and promising technology to treat bone-integrated dental implants as the new surfaces displayed improved mechanical and biological properties with no increase in biofilm proliferation.