María Cecilia Cortizo

Metallic dental material biocompatibility in osteoblastlike cells

Ions released from metallic dental materials used in orthodontic appliances could induce undesirable effects on cells and tissues. This study evaluates the biocompatibility of two of the most labile components of metallic dental alloys on osteoblastlike cells. The influence of protein and ions on metal dissolution properties is also investigated using different electrolyte solutions. Morphological alterations, cell growth, and differentiation of osteoblasts were assessed after exposure to pure metals (Ag, Cu, Pd, Au) and Ni−Ti alloy and correlated with the kinetics of elements released into the culture media. Results showed that Cu and Ag were the most cytotoxic elements and the other metals were biocompatible with the osteoblasts. The parameters of biocompatibility were correlated with the levels of ions detected into the culture media. Metal ions induced cell death through early mitosis arrest, apoptotic phenomena, and necrotic processes. Voltammograms showed that anions and proteins interfered in the corrosion process. Fetal bovine serum (FBS) strongly affected the electrochemical process, decreasing the oxidation rate of the metals. In conclusion, copper and silver ions showed a time-dependent low biocompatibility, which correlated with the concentration of released ions. The dissolution of the metallic materials was dependent on the composition of the simulated biological media.

Influence of the nano-micro structure of the surface on bacterial adhesion

Biomaterials failures are frequently associated to the formation of bacterial biofilms on the surface. The aim of this work is to study the adhesion of non motile bacteria streptococci consortium and motile Pseudomonas fluorescens. Substrates with micro and nanopatterned topography were used. The influence of surface characteristics on bacterial adhesion was investigated using optical and epifluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Results showed an important influence of the substratum nature. On microrough surfaces, initial bacterial adhesion was less significant than on smooth surfaces. In contrast, nanopatterned samples showed more bacterial attachment than the smooth control. It was also noted a remarkable difference in morphology, orientation and distribution of bacteria between the smooth and the nanostructured substrate. The results show the important effect of substratum nature and topography on bacterial adhesion which depended on the relation between roughness characteristics dimensions and bacterial size.

Electrochemical behaviour of titanium in fluoride-containing saliva

The effect of fluoride on the electrochemical behaviour of titanium was studied. Open circuit potentials, breakdown potentials (Eb) and potentiostatic transient currents were measured in synthetic salivas of different compositions. Optical and scanning electron microscopic observations were also made. Results show that the growth rate of Ti oxide layer is affected by fluoride anions and tensile stresses are developed. The OCP/time relationship of Ti immersed in salivas A and B obeys a logarithmic law which depends on the saliva composition. The Eb value is influenced by the thickness of the oxide layer, by the composition of the saliva (including fluoride concentration), and by the technique utilised for its evaluation. Thus, results reported in the literature, which seem to be contradictory, could be explained taking into account the experimental conditions assayed. A careful control of the titanium-containing dental materials should be made after long treatments with fluoride-containing prophylactic products or when fluoride-releasing restorative materials are present in the vicinity.

Cytotoxicity of copper ions released from metal: variation with the exposure period and concentration gradients.

The aim of this work is to contribute to the elucidation of the cytotoxic process caused by the copper ions released from the biomaterials. Clonal cell lines UMR106 were used in the experiments. Copper ions were obtained from two different sources: copper salts and metal dissolution. Experiments carried out with constant ion concentrations (copper salts) were compared with those with concentrations that vary with time and location (dissolution of the metal). Present results and others previously reported could be interpreted through mathematical models that describe: (1) the variation of concentration of copper ions with time and location within a biofilm and (2) the variation of the killing rate with the concentration of the toxic ion and time. The large number of dead cells found near the copper sample with an average ion concentration below the toxic limit could be interpreted bearing in mind that these cells should be exposed to a local concentration higher than this limit. A logarithmic dependence between the number of cells and exposure time was found for nearly constant ion concentrations. Apparent discrepancies, observed when these results and those of different researchers were contrasted, could be explained considering the dissimilar experimental conditions such as the source of the ions and their local concentration at real time.

Chlorhexidine delivery system from titanium/polybenzyl acrylate coating: Evaluation of cytotoxicity and early bacterial adhesion

OBJECTIVES The formation of biofilms on titanium dental implants is one of the main causes of failure of these devices. Streptococci are considered early colonizers that alter local environment favouring growing conditions for other colonizers. Chlorhexidine (CHX) is so far the most effective antimicrobial treatment against a wide variety of Gram-positive and Gram-negative organisms as well as fungi. This study was designed to develop a CHX delivery system appropriate for healing caps and abutments, with suitable drug release rate, effective as antimicrobial agent, and free of cytotoxic effects. METHODS Polybenzyl acrylate (PBA) coatings with and without CHX (Ti/PBA and Ti/PBA-CHX, respectively) and different drug loads (0.35, 0.70, and 1.40%, w/w) were assayed. The cytotoxic effect of CHX released from the different substrates on UMR106 cells was tested by alkaline phosphatase specific activity (ALP), and microscopic evaluation of the cells. Non-cytotoxic drug load (0.35%, w/w) was selected to evaluate the antimicrobial effectiveness of the system using a microbial consortium of Streptococcus species. RESULTS The kinetic profile of CHX delivered by Ti/PBA-CHX showed an initial fast release rate followed by a monotonic increase of delivered mass over 48 h. The number of attached bacteria decreased in the following order: Ti>Ti/PBA>Ti/PBA-0.35. CONCLUSIONS PBA-0.35 coating is effective to inhibit the adhesion of early colonizers on Ti without any cytotoxic effect on UMR-106 cells.

Microstructural characteristics of thin biofilms through optical and scanning electron microscopy

The combination of a conventional optical microscope with a specially designed glass flow cell was used to visualize ‘in situ’ biofilms formed on opaque thin biomaterials through a simple non-invasive way (optical microscopy of thin biofilms, OMTB). Comparisons of OMTB with scanning electron microscopy (SEM) images were made. Thin metallic dental biomaterials were used as substrata. They were immersed in a synthetic saliva and in a modified Mitis–Salivarius medium inoculated with a consortium of oral microorganisms. To study the effect of bacterial motility, Pseudomonas fluorescens cultures were also used. The processes which give rise to the formation of the biofilm were monitored through OMTB. Biofilm microstructures like pores, water channels, streamers and chains of Streptococci, attached to the surface or floating in the viscous interfacial environment, could be distinguished. Thickness and roughness of the biofilms formed on thin substrata could also be evaluated. Distortions introduced by pretreatments carried out to prepare biological materials for SEM observations could be detected by comparing OMTB and SEM images. SEM images (obtained at high magnification but ex situ, not in real time and with pretreatment of the samples) and OMTB images (obtained in situ, without pretreatments, in real time but at low magnification) in combination provided complementary information to study biofilm processes on thin substrata.

Biodeterioration of dental materials: Influence of bacterial adherence

The aim of this work was to carry out a comparative study of microbial adhesion on dental alloys and glass ionomers that release fluoride. The action of NaF on the early stages of biofilm development and on the corrosion of the metallic dental materials was analysed. Open circuit potential measurements and potentiostatic electrochemical techniques with different perturbation programs as well as SEM observations, and optical and epifluorescence microscopy were employed. A notable effect of topography and the nature of the substratum on bacterial distribution was observed. In addition, changes in the density and thickness of microbial colonies were noticed when fluoride was present. The results show that the antimicrobial effect of fluoride was significant against planktonic but not against sessile microorganisms. Fluoride released by glass ionomers did not impede bacterial adhesion to the surface. With respect to corrosion, fluoride did not alter significantly the passivity of the dental metallic biomaterials assayed, except for Cu‐Al alloy. Titanium dissolution could occur at high fluoride concentrations (8gl‐1) during oxide layer formation. Consequently, bacterial adherence was influenced by the nature and topography of the substratum and by the presence of fluoride which could also affect the electrochemical behaviour of some metallic substrata.

Preliminary characterization of thin biofilms by optical microscopy

A simple non‐invasive technique has been used that employs conventional optical microscopy and a glass flow cell to observe biofilms formed on opaque thin substrata. The technique allows the roughness of the biofilm and the substratum to be evaluated, and the biofilm thickness to be easily measured. The biofilm density may be quantified through colour gradients. In addition, some details of biofilm growth processes like the formation of water channels and pores, and interactions between planktonic and sessile cells can be visualized. Results related to the development of thin biofilms and their response to the environment under different conditions are reported. Pure and mixed microbial cultures and different solid substrata were assessed.

Corrosion protection of AZ31 alloy and constrained bacterial adhesion mediated by a polymeric coating obtained from a phytocompound

The prevention of microbial biofilm formation on a biomaterial surface is crucial in avoiding implants failures and the development of antibiotic resistant bacteria. It was reported that biodegradable Mg alloys may show antimicrobial effects due to the alkalinization of the corroding area. However, this issue is controversial and deserves a detailed study, since the processes occurring at the [biodegradable metal/biological medium] interface are complex and varied. Results showed that bacterial adhesion on AZ31 was lower than that of the titanium control and revealed that was dependent on surface composition, depicting some preferential sites for bacterial attachment (C-, P-, O-containing corrosion products) and others that are particularly avoided (active corrosion sites). As a key challenge, a strategy able to improve the performance of Mg alloys by both, reducing the formation of corrosion products and inhibiting bacterial adhesion was subsequently developed. A polymeric layer (polyTOH) was obtained by electropolymerization of thymol (TOH), a phytophenolic compound. The polyTOH can operate as a multifunctional film that improves the surface characteristics of the AZ31 Mg alloy by enhancing corrosion resistance (ions release was reduced to almost the half during the first days) and create an anti-adherent surface (bacterial attachment was 30-fold lower on polyTOH-AZ31 than on non-coated Mg alloy and 200-fold lower than Ti control and was constrained to specific regions). This anti-adherent property implies an additional advantage: enhancement of the efficacy of antibiotic treatments.