Argelia Almaguer-Flores

Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro

OBJECTIVES The aim of this study was to analyse the influence of the microtopography and hydrophilicity of titanium (Ti) substrates on initial oral biofilm formation. MATERIALS AND METHODS Nine bacterial species belonging to the normal oral microbiota, including: Aggregatibacter actinomycetemcomitans, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus sanguinis were tested on Ti surfaces: pretreatment (PT [R(a) <0.2 μm]), acid-etched (A [R(a) <0.8 μm]), A modified to be hydrophilic (modA), sand-blasted/acid-etched (SLA [R(a) =4 μm]), and hydrophilic SLA (modSLA). Disks were incubated for 24 h in anaerobic conditions using a normal culture medium (CM) or human saliva (HS). The total counts of bacteria and the proportion of each bacterial species were analysed by checkerboard DNA-DNA hybridization. RESULTS Higher counts of bacteria were observed on all surfaces incubated with CM compared with the samples incubated with HS. PT, SLA, and modSLA exhibited higher numbers of attached bacteria in CM, whereas SLA and modSLA had a significant increase in bacterial adhesion in HS. The proportion of the species in the initial biofilms was also influenced by the surface properties and the media used: SLA and modSLA increased the proportion of species like A. actinomycetemcomitans and S. sanguinis in both media, while the adhesion of A. israelii and P. gingivalis on the same surfaces was affected in the presence of saliva. CONCLUSIONS The initial biofilm formation and composition were affected by the microtopography and hydrophilicity of the surface and by the media used.

Oral bacterial adhesion on amorphous carbon and titanium films: effect of surface roughness and culture media.

Implant infections can cause severe problems from malfunctioning to dangerous sepsis affecting the health of the patient. For many years, titanium has been the most common material used on dental implants due to their mechanical and biocompatibility properties. Recent studies suggest that amorphous carbon (a-C) films can be possible candidates for coating dental implants, improving some important features like biocompatibility and bone formation. In the oral cavity, the risk of an implant infection is high due to multiple species are capable to colonize this site and these biofilm infections can limit the use of these medical devices. The purpose of this study was to evaluate the influence of the surface chemistry, roughness, and culture media in the bacterial colonization process. To achieve this, a-C and Ti films were deposited on rough and smooth surfaces and cultured with different microorganisms belonging to the oral microbiota with mycoplasma medium (MM) or human saliva (HS). Samples were incubated for 24 h, after this, samples were sonicated and the number of attached bacteria was determined by counting the colony-forming units (CFUs) from each sample. The proportion of the species in the biofilms was determined using checkerboard DNA-DNA hybridization. Data were analyzed by Students t test using Bonferronis modification of Students t test and differences on the proportion of the bacterial species attached to each surface were determined using the Mann-Whitney test. Results show an increased number of CFUs on rough surfaces, especially on the a-C surfaces. The incubation media were an important factor on the adhesion of certain taxa, whereas other species were more sensitive to surface chemistry and others to surface roughness.

Role of integrin subunits in mesenchymal stem cell differentiation and osteoblast maturation on graphitic carbon-coated microstructured surfaces

Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra < 0.4 μm], rough [Ra ≥ 3.4 μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition.

The effect of simulated inflammatory conditions on the surface properties of titanium and stainless steel and their importance as biomaterials.

This work compares the surface modifications induced by the immersion in solutions that simulate inflammatory conditions of pure titanium (cpTi) and medical grade stainless steel (SS). The inflammatory conditions were simulated using a mixture of Hartman solution and 50mM of hydrogen peroxide (H2O2) at pH=5.2. The samples were immersed by 7days refreshing the solution every day to keep the reactivity of the H2O2. The surface characteristics that were investigated were: elemental composition by X-ray photoelectron spectroscopy (XPS); topography by atomic force microscopy (AFM) and profilometry; wettability and surface energy by sessile drop contact angle and point of zero charge by titration. Moreover, the variations in the electrochemical response were evaluated by open circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization (PP) performed before and after the treatment using the Hartman solution as the electrolyte. The XPS results indicated that for both metallic samples, oxidation of the surface was promoted and/or the oxide layer was thicker after the immersion. The roughness and the solid-liquid surface energy were increased; the samples showed a more hydrophilic character after the treatment. However, the surface energy of the solid estimated using the Van Oss-Chaudhury-Good approach showed different trends between the cpTi and the SS surfaces; the polar component decreased for cpTi, while it increased for SS. Finally, the electrochemical results indicated that the corrosion resistance (Rcor) and the pore resistance (Rpo) significantly decreased for cpTi, while both resistances were not significantly different for the SS. This is indicative of a higher dissolution of the cpTi compared to SS and the lower Rpo means that the species are easily transported through the surface layer, which can be explained in terms of the formation of a porous TiOx layer, not observed on the SS. The cpTi surface suffered from a dissolution/oxidation process that allows its integration with the surrounding media, while the SS remained completely passive and this different response might be related to their distinguished clinical outcome.

Bacterial adhesion on amorphous and crystalline metal oxide coatings.

Several studies have demonstrated the influence of surface properties (surface energy, composition and topography) of biocompatible materials on the adhesion of cells/bacteria on solid substrates; however, few have provided information about the effect of the atomic arrangement or crystallinity. Using magnetron sputtering deposition, we produced amorphous and crystalline TiO2 and ZrO2 coatings with controlled micro and nanoscale morphology. The effect of the structure on the physical-chemical surface properties was carefully analyzed. Then, we studied how these parameters affect the adhesion of Escherichia coli and Staphylococcus aureus. Our findings demonstrated that the nano-topography and the surface energy were significantly influenced by the coating structure. Bacterial adhesion at micro-rough (2.6 μm) surfaces was independent of the surface composition and structure, contrary to the observation in sub-micron (0.5 μm) rough surfaces, where the crystalline oxides (TiO2>ZrO2) surfaces exhibited higher numbers of attached bacteria. Particularly, crystalline TiO2, which presented a predominant acidic nature, was more attractive for the adhesion of the negatively charged bacteria. The information provided by this study, where surface modifications are introduced by means of the deposition of amorphous or crystalline oxide coatings, offers a route for the rational design of implant surfaces to control or inhibit bacterial adhesion.

Influence of the Periodontal Status on the Initial-Biofilm Formation on Titanium Surfaces.

BACKGROUND Dental implants will be exposed to a complex ecosystem once they are placed in the oral cavity. The bacterial colonization and biofilm formation on these devices will depend not only on the physicochemical surface implant properties but also on the periodontal health conditions of the patients, as these devices are exposed. PURPOSE The aim of this study was to correlate the subgingival microbial profile with the composition of initial biofilm formed on different microstructured titanium (Ti) surfaces. MATERIALS AND METHODS Ten periodontitis and 10 periodontally healthy subjects were included in this study. The subjects wore a removable acrylic device with four different fixed Ti surfaces for 48 hours. Microbial samples of subgingival plaque and the biofilm formed on each Ti surface were individually analyzed by the checkerboard DNA-DNA hybridization technique. RESULTS Despite the roughness or hydrophilicity of the Ti surfaces, a characteristic pattern of bacterial adhesion was observed on each of the study groups. However, significant differences in the proportion of the species that colonized the Ti surfaces were found between the periodontitis and periodontally healthy groups. Treponema denticola, Neisseria mucosa, Eikenella corrodens, and Tannerella forsythia were detected in higher proportions on the Ti disks placed in the periodontitis subjects, while significant higher proportions of Capnocytophaga sputigena, Fusobacterium periodonticum, Prevotella melaninogenica, and Streptococcus mitis were detected on the Ti disks placed in the periodontally healthy group. CONCLUSIONS The results obtained in this study shows that the composition and the proportion of the species that initially colonize Ti surfaces are highly influenced by the periodontal status more than the surface characteristics of the Ti implant.

Amelogenin Peptide Extract Increases Differentiation and Angiogenic and Local Factor Production and Inhibits Apoptosis in Human Osteoblasts

Enamel matrix derivative (EMD), a decellularized porcine extracellular matrix (ECM), is used clinically in periodontal tissue regeneration. Amelogenin, EMD’s principal component, spontaneously assembles into nanospheres in vivo, forming an ECM complex that releases proteolytically cleaved peptides. However, the role of amelogenin or amelogenin peptides in mediating osteoblast response to EMD is not clear. Human MG63 osteoblast-like cells or normal human osteoblasts were treated with recombinant human amelogenin or a 5 kDa tyrosine-rich amelogenin peptide (TRAP) isolated from EMD and the effect on osteogenesis, local factor production, and apoptosis assessed. Treated MG63 cells increased alkaline phosphatase specific activity and levels of osteocalcin, osteoprotegerin, prostaglandin E2, and active/latent TGF-β1, an effect sensitive to the effector and concentration. Primary osteoblasts exhibited similar, but less robust, effects. TRAP-rich 5 kDa peptides yielded more mineralization than rhAmelogenin in osteoblasts in vitro. Both amelogenin and 5 kDa peptides protected MG63s from chelerythrine-induced apoptosis. The data suggest that the 5 kDa TRAP-rich sequence is an active amelogenin peptide that regulates osteoblast differentiation and local factor production and prevents osteoblast apoptosis.

Enhancing the osteoblastic differentiation through nanoscale surface modifications

Human mesenchymal stem cells (MSCs) showed larger differentiation into osteoblasts on nanoscale amorphous titanium oxide (TiO2 ) coatings in comparison to polycrystalline TiO2 coatings or native oxide layers. In this article, we showed that the subtle alterations in the surface properties due to a different atomic ordering of titanium oxide layers could substantially modify the osteoblastic differentiation of MSCs. Amorphous (a) and polycrystalline (c) TiO2 coatings were deposited on smooth (PT) and microstructured sandblasted/acid-etched (SLA) Ti substrates using a magnetron sputtering system. The surface roughness, water contact angle, structure, and composition were measured using confocal microscopy, drop sessile drop, X-ray diffraction, and X-ray photoelectron spectroscopy, respectively. The ∼70-nm-thick coatings presented a well-passivated and uniform TiO2 (Ti4+ ) surface composition, while the substrates (native oxide layer) showed the presence of Ti atoms in lower valence states. The polycrystalline TiO2 -coated surfaces (cPT and cSLA) showed the same cell attachment as the uncoated metallic surfaces (PT and SLA), and in both cases, it was lower on the rough than on the smooth surfaces. However, attachment and differentiation were significantly increased on the amorphous TiO2 -coated surfaces (aPT and aSLA). The amorphous coated Ti surfaces presented the highest expression of integrins and production of osteogenic proteins in comparison to the uncoated and crystalline-coated Ti surfaces.

Antibacterial Effect of Biodegradable Magnesium Alloys Modified By Biocompatible Transitions Metals

Magnesium (Mg) alloys can be use as biodegradable medical devices, eliminating the need for a second operation for implant removal. An important feature on biomedical devices is to avoid the bacterial adhesion and subsequent biofilm formation that cause most of the implant-failures. The aim of this study was to analyze the differences on bacterial adhesion and biofilm development on Magnesium alloys (Mg-Al-Zn) modified by different transition metals; Tantalum, Niobium and Titanium. Nine oral bacterial strains ( Aggregatibacter actinomycetemcomitans serotipe b, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens , Fusobacterium nucleatum , Parvimonas micra , Porphyromonas gingivalis , Prevotella intermedia and Streptococcus sanguinis) were incubated on the different alloys and commercial medical grade stainless steel (AISI 316L) was used as a control. The initial bacterial adhesion was determined after 24 hours using a counting plate technique and the subsequent biofilm development at 1, 3, 7 days was studied using the Scanning Electron Microscopy. Significant differences were determined using t-test. The results showed that on the magnesium-alloys, the number of bacteria attached after 24 hours was two orders of magnitude lower than the stainless steel. On the other hand, bacterial colonies were not observed by electron microscopy in any of the days of incubation, even though in the control surface clear colonies and biofilm development were observed. This study showed that magnesium alloys inhibits the bacterial adhesion and the subsequent biofilm development.

Fabrication and in vitro behavior of dual-function chitosan/silver nanocomposites for potential wound dressing applications

We report the synthesis and in vitro evaluation of dual-function chitosan-silver nanoparticles (CTS-AgNPs) films with potential applications as wound dressings. We attempted to formulate nanocomposite films with appropriate AgNPs concentrations to simultaneously display antibacterial activity and suitability for cell culture. Nanocomposites were obtained by CTS-mediated in situ chemical reduction of AgNO3. Circular-shape AgNPs (sizes ca. 7-50 nm) well distributed within the CTS matrices were obtained in concentrations from 0.018 to 0.573 wt%. Efficacy (bacteriostatic and bactericidal properties) of CTS-AgNPs films to decrease planktonic and biofilm bacterial growth was AgNPs concentration- and bacteria strain-dependent. Films showed significant antibacterial activity against Gram-negative E. coli and P. aeruginosa and Gram-positive S. aureus. Antibacterial activity against S. epidermidis was moderated. Films suitability for cell culture was characterized using primary human fibroblasts (HF). HF displayed cell viability higher than 90% and the characteristic fusiform morphology of adhered fibroblast upon culture on films with AgNPs concentration ≤ 0.036 wt%. HF cultured on these films also showed positive expression of tropoelastin, procollagen type I and Ki-67, characteristic proteins of extracellular matrix and proliferative cells, respectively. In vitro assays demonstrated that cytocompatibility/antibacterial properties decreased/increased as silver concentration increased, suggesting that CTS-AgNPS nanocomposite films with ≈0.04-0.20 wt% might be considered as potential temporary dual-function wound dressings.