Effects of a functional food supplemented with probiotics on biological factors related to dental caries in children: a pilot study.

PURPOSE: The aim of this study was to evaluate the effects of a functional food supplemented with probiotics on biological factors related to dental caries in children aged 3-5 years. METHODS: A repeated measures pilot study was conducted with children who have consumed a commercial milk containing two lactic acid bacteria as probiotics (WP milk) for a period of 3 months and another period of 3 months consuming a milk without probiotics (NP milk). Salivary pH, plaque index, pH variation before and after a sugar rinse, quantification of Streptococcus mutans in saliva and demineralisation of the carious lesions were determined at the beginning and at the end of both milk ingestion periods. RESULTS: Regarding WP milk, a non-significant decrease in terms of the concentration of S. mutans and pH variation (p > 0.05), a significant decrease (i.e. acidification) in salivary pH (p < 0.01) and a remineralisation of 39.4% of the caries were found. On the other hand, for NP milk, a non-significant increase in terms of the concentration of S. mutans, pH variation, salivary pH (p > 0.05) and a remineralisation of 64.2% were found. CONCLUSIONS: Lactic acid probiotics can contribute to the decrease in the number of cariogenic microorganisms. However, the appropriate selection of the bacteria type with regard to its acidogenicity is fundamental to avoid the generation of an effect contrary to that expected, e.g. a significant decrease in salivary pH.

A novel approach to create an antibacterial surface using titanium dioxide and a combination of dip-pen nanolithography and soft lithography.

Soft lithography and Dip-Pen Nanolithography (DPN) are techniques that have been used to modify the surface of biomaterials. Modified surfaces play a role in reducing bacterial adhesion and biofilm formation. Also, titanium dioxide has been reported as an antibacterial substance due to its photocatalytic effect. This work aimed at creating patterns on model surfaces using DPN and soft lithography combined with titanium dioxide to create functional antibacterial micropatterned surfaces, which were tested against Streptococcus mutans. DPN was used to create a master pattern onto a model surface and microstamping was performed to duplicate and transfer such patterns to medical-grade stainless steel 316L using a suspension of TiO2. Modified SS316L plates were subjected to UVA black light as photocatalytic activator. Patterns were characterized by atomic force microscopy and biologically evaluated using S. mutans. A significant reduction of up to 60% in bacterial adhesion to TiO2 -coated and -micropatterned surfaces was observed. Moreover, both TiO2 surfaces reduced the viability of adhered bacteria after UV exposure. TiO2 micropatterned demonstrated a synergic effect between physical and chemical modification against S. mutans. This dual effect was enhanced by increasing TiO2 concentration. This novel approach may be a promising alternative to reduce bacterial adhesion to surfaces.

Self-Assembled Monolayers for Dental Implants.

Implant-based therapy is a mature approach to recover the health conditions of patients affected by edentulism. Thousands of dental implants are placed each year since their introduction in the 80s. However, implantology faces challenges that require more research strategies such as new support therapies for a world population with a continuous increase of life expectancy, to control periodontal status and new bioactive surfaces for implants. The present review is focused on self-assembled monolayers (SAMs) for dental implant materials as a nanoscale-processing approach to modify titanium surfaces. SAMs represent an easy, accurate, and precise approach to modify surface properties. These are stable, well-defined, and well-organized organic structures that allow to control the chemical properties of the interface at the molecular scale. The ability to control the composition and properties of SAMs precisely through synthesis (i.e., the synthetic chemistry of organic compounds with a wide range of functional groups is well established and in general very simple, being commercially available), combined with the simple methods to pattern their functional groups on complex geometry appliances, makes them a good system for fundamental studies regarding the interaction between surfaces, proteins, and cells, as well as to engineering surfaces in order to develop new biomaterials.

Soft Lithography and Minimally Human Invasive Technique for Rapid Screening of Oral Biofilm Formation on New Microfabricated Dental Material Surfaces.

INTRODUCTION: Microfabrication offers opportunities to study surface concepts focused to reduce bacterial adhesion on implants using human minimally invasive rapid screening (hMIRS). Wide information is available about cell/biomaterial interactions using eukaryotic and prokaryotic cells on surfaces of dental materials with different topographies, but studies using human being are still limited. OBJECTIVE: To evaluate a synergy of microfabrication and hMIRS to study the bacterial adhesion on micropatterned surfaces for dental materials. MATERIALS AND METHODS: Micropatterned and flat surfaces on biomedical PDMS disks were produced by soft lithography. The hMIRS approach was used to evaluate the total oral bacterial adhesion on PDMS surfaces placed in the oral cavity of five volunteers (the study was approved by the University Ethical Committee). After 24 h, the disks were analyzed using MTT assay and light microscopy. RESULTS: In the present pilot study, microwell structures were microfabricated on the PDMS surface via soft lithography with a spacing of 5 µm. Overall, bacterial adhesion did not significantly differ between the flat and micropatterned surfaces. However, individual analysis of two subjects showed greater bacterial adhesion on the micropatterned surfaces than on the flat surfaces. SIGNIFICANCE: Microfabrication and hMIRS might be implemented to study the cell/biomaterial interactions for dental materials.

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide.

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices.

The effect of immobilization of thrombin inhibitors onto self-assembled monolayers on the adsorption and activity of thrombin.

Thrombus formation is the major problem associated with biomaterials for blood contact medical devices. The immobilization of inhibitors to thrombin, a serine protease that plays a central role on the coagulation system, on the surface of biomaterials should be a good strategy to avoid blood clotting and increase their hemocompatibility. The aim of this work is the design of a nanostructured surface with capacity to adsorb and inactivate thrombin. The pentapeptide sequence d-Phenylalanine-Proline-Arginine-Proline-Glycine (fPRPG), that was described as a thrombin inhibitor, was immobilized onto tetra(ethylene glycol) terminated self-assembled monolayers (EG4-SAMs). Surface containing different amounts of fPRPG were prepared using different concentrations of N,N'-Carbonyldiimidazole (CDI) during immobilization. The efficiency of fPRPG immobilization was followed using ellipsometry, contact angle measurements, Infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS). Thrombin adsorption was quantified using radiolabelled thrombin and its activity in solution and after adsorption on the developed surfaces was assessed using a chromogenic assay. It was found that, although the immobilization of fPRPG on to EG4-SAMs does not increase its selectivity to thrombin, the activity of the adsorbed thrombin was inhibited in a peptide concentration dependent way. We concluded that SAMs with fPRPG immobilized in high amounts can be used as thrombin-inhibitor surfaces, which is a good step on the development of new surfaces for blood contact devices.

Adhesion of human leukocytes on mixtures of hydroxyl- and methyl-terminated self-assembled monolayers: effect of blood protein adsorption.

The adhesion of human leukocytes to nanostructured surfaces with different chemical properties and the effect of protein adsorption were investigated. Self-assembled monolayers (SAMs) prepared with mixtures of methyl- and hydroxyl-terminated alkanethiols in different percentages on gold were used. The surfaces were pre-immersed in distinct protein solutions (human serum albumin, human fibrinogen, and autologous plasma). Adherent leukocytes were analyzed both by light and SEM. SAMs submitted to pre-immersion in plasma presented higher numbers of adherent leukocytes in the pure OH-terminated SAM, whereas methyl-terminated surfaces accounted for the lowest number of adherent cells. We observed a general increase in the number of adherent human leukocytes as the percentage of OH groups on the surface of the SAMs increased for all the pre-immersion conditions investigated. The number of adherent human leukocytes is highly influenced by the pre-immersion conditions used, and this observation is particularly relevant in the case of the methyl-terminated SAMs. The results obtained demonstrate that surface chemistry has a major influence in leukocyte adhesion to biomaterials, and that pre-immersion in protein solutions has a determinant effect in leukocyte adhesion.

Molecularly designed surfaces for blood deheparinization using an immobilized heparin-binding peptide.

Systemic heparinization, used during haemodialysis to prevent blood clotting on the extracorporeal circuit, leads to a high incidence of hemorrhagic complications. The adverse reactions associated with heparin neutralization using protamine sulphate justify the development of an alternative system for blood deheparinization. The main objective of this work is to design nanostructured surfaces with the capacity to bind heparin from blood in a selective way. A heparin-binding polypeptide, composed of L-lysine and L-leucine (pKL), was synthesized and immobilized, in different concentrations, onto self-assembled monolayers (SAMs) terminated with tetra(ethylene-glycol) (EG4 SAMs). Immobilization was performed using a fixed concentration of pKL after surface activation to different degrees using a range of CDI (N,N'-carbonyldiimidazole) concentrations. Results demonstrated that the presence of pKL increases heparin adsorption to EG4-SAMs, independently of the pKL concentration and the way of immobilization (adsorption or covalent bound). Selectivity towards heparin was successfully achieved on SAMs with low concentrations of immobilized pKL (9-17% of pKL). Surfaces were characterized using ellipsometry, contact angle measurements, Fourier transform infrared reflection absorption spectroscopy (IRAS), atomic force microscopy, and X-ray photoelectron spectroscopy. Heparin adsorption was assessed using IRAS and N-sulphonate-(35)S-heparin. Therefore, this study could give a good contribution for the design of blood deheparinization devices.