Standardization of antimicrobial testing of dental devices.

OBJECTIVE: Dental device is a very broad term that can be used to include any foreign material or product that is introduced in the host oral cavity to replace missing tissues. These devices are subjected to different environments which include dental hard tissues, tissue fluids, blood and saliva. All dental devices are continuously challenged microbiologically and a number of failures in clinical management are related to microbial colonization. Thus, the assessment of the antimicrobial properties of dental devices are extremely important. In this paper, a classification of dental devices is being proposed. This classification distinguishes the devices based on whether they are implantable or not, and also sub-classified based on their specific application and the substrate receiving the device. METHODS AND RESULTS: A literature search was conducted to identify how dental devices have been tested with relation to the microbial strains used and whether the testing has been performed in isolation or reported with other relevant tests such as material characterization and biological activity. The results of the literature review were analyzed and recommendations for antimicrobial testing of dental devices are proposed. These recommendations include the need for the setting up of pre-testing parameters such as ageing and the details of the pre-testing sterilization procedures, as these may affect the material chemistry and the specification for antimicrobial testing to be done with specific single strains or polymicrobial that are native to the region where the device is located are also suggested. Testing can be undertaken in vitro, ex vivo and in vivo. Since the antimicrobial and biological activities influence/condition one another and the material chemistry may affect both the antimicrobial and biological testing this document also makes recommendations regarding biological assessment which can be carried out in isolation or integrated with the microbiological testing and also material testing methods including chemical and physical characterization of bulk, surface, eluted and degraded materials as well as physical characterization methods. SIGNIFICANCE: The level of standardization of antimicrobial testing for the dental devices needs to be based on the device location and host interaction in order to increase the clinical applicability of the mentioned tests.

The NATO project: nanoparticle-based countermeasures for microgravity-induced osteoporosis.

Recent advances in nanotechnology applied to medicine and regenerative medicine have an enormous and unexploited potential for future space and terrestrial medical applications. The Nanoparticles and Osteoporosis (NATO) project aimed to develop innovative countermeasures for secondary osteoporosis affecting astronauts after prolonged periods in space microgravity. Calcium- and Strontium-containing hydroxyapatite nanoparticles (nCa-HAP and nSr-HAP, respectively) were previously developed and chemically characterized. This study constitutes the first investigation of the effect of the exogenous addition of nCa-HAP and nSr-HAP on bone remodeling in gravity (1 g), Random Positioning Machine (RPM) and onboard International Space Station (ISS) using human bone marrow mesenchymal stem cells (hBMMSCs). In 1 g conditions, nSr-HAP accelerated and improved the commitment of cells to differentiate towards osteoblasts, as shown by the augmented alkaline phosphatase (ALP) activity and the up-regulation of the expression of bone marker genes, supporting the increased extracellular bone matrix deposition and mineralization. The nSr-HAP treatment exerted a protective effect on the microgravity-induced reduction of ALP activity in RPM samples, and a promoting effect on the deposition of hydroxyapatite crystals in either ISS or 1 g samples. The results indicate the exogenous addition of nSr-HAP could be potentially used to deliver Sr to bone tissue and promote its regeneration, as component of bone substitute synthetic materials and additive for pharmaceutical preparation or food supplementary for systemic distribution.

Polyvinyl alcohol/chitosan hydrogels with enhanced antioxidant and antibacterial properties induced by lignin nanoparticles.

Polyvinyl alcohol/chitosan (PVA/Ch) hydrogels containing 1 and 3wt% of lignin nanoparticles (LNPs) were prepared through a freezing-thaw procedure. Results from microstructural, thermal and mechanical characterization of LNPs based PVA/Ch demonstrated that the lowest amount of LNPs (1wt%) was beneficial, whereas the presence of agglomerates at higher LNP content limited the effect. Moreover, a different swelling behaviour was observed for hydrogels containing LNPs with respect of PVA/Ch, due to the formation of a porous honeycomb-like structure. A synergic effect of Ch and LNPs was revealed in terms of antioxidative response by DPPH (1,1-Diphenyl-2-picryl-hydrazyl) activity of migrated substances, whereas results from antimicrobial tests confirmed LNPs as effective against Gram negative bacteria (E. coli) when compared to Gram positive (S.aureus and S. epidermidis) strains. The obtained results suggested the possible use of produced PVA/Ch hydrogels incorporating LNPs in many different sectors, such as drug delivery, food packaging, wound dressing.

Biomaterials and biophysical stimuli for bone regeneration.

First, we review basic concepts of Tissue Engineering, that is, how the tensegrity is able to modulate the cell behavior. Then, we review our experimental results regarding the bone tissue engineering via biomaterials and bioreactors.

Heterogeneous and self-organizing mineralization of bone matrix promoted by hydroxyapatite nanoparticles.

The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At the molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using scanning micro X-ray diffraction and scanning micro X-ray fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit heterogeneous and self-organizing dynamics. At the same time the possibility of controlling the differentiation kinetics, through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine.

Silver nanoparticles synthesized and coated with pectin: An ideal compromise for anti-bacterial and anti-biofilm action combined with wound-healing properties.

The synthesis of Ag nanoparticles from Ag+ has been investigated, with pectin acting both as reductant and coating.∼100% Ag+ to Ag(0) one-pot conversion was obtained, yielding p-AgNP, i.e. an aqueous solution of pectin-coated spherical Ag nanoparticles (d=8.0±2.6nm), with a<1ppm concentration of free Ag+ cation. Despite the low free Ag+ concentration and low Ag+ release with time, the nature of the coating allows p-AgNP to exert excellent antibacterial and antibiofilm actions, comparable to those of ionic silver, tested on E. coli (Gram-) and S. epidermidis (Gram+) both on planctonic cells and on pre- and post-biofilm formation conditions. Moreover, p-AgNP were tested on fibroblasts: not only p-AgNP were found to be cytocompatible but also revealed capable of promoting fibroblasts proliferation and to be effective for wound healing on model cultures. The antibacterial activity and the wound healing ability of silver nanoparticles are two apparently irreconcilable properties, as the former usually requires a high sustained Ag+ release while the latter requires low Ag+ concentration. p-AgNP represents an excellent compromise between opposite requirements, candidating as an efficient medication for repairing wounds and/or to treat vulnerable surgical site tissues, including the pre-treatment of implants as an effective prophylaxis in implant surgery.

Cellulose nanocrystals as templates for cetyltrimethylammonium bromide mediated synthesis of Ag nanoparticles and their novel use in PLA films.

In the present paper, we reported how cellulose nanocrystals (CNC) from microcrystalline cellulose have the capacity to assist in the synthesis of metallic nanoparticles chains. A cationic surfactant, cetyltrimethylammonium bromide (CTAB), was used as modifier for CNC surface. Silver nanoparticles were synthesized on CNC, and nanoparticle density and size were optimized by varying concentrations of nitrate and reducing agents, and the reduction time. The experimental conditions were optimized for the synthesis and the resulting Ag grafted CNC (Ag-g-CNC) were characterized by means of TGA, SEM, FTIR and XRD, and then introduced in PLA matrix. PLA nanocomposite containing silver grafted cellulose nanocrystals (PLA/0.5Ag-g-1CNC) was characterized by optical and thermal analyses and the obtained data were compared with results from PLA nanocomposites containing 1% wt. of CNC (PLA/1CNC), 0.5% wt. of silver nanoparticles (PLA/0.5Ag) and hybrid system containing CNC and silver in the same amount (PLA/1CNC/0.5Ag). The results demonstrated that grafting of silver nanoparticles on CNC positively affected the thermal degradation process and cold crystallization processes of PLA matrix. Finally, the antibacterial activity of the different systems was studied at various incubation times and temperatures, showing the best performance for PLA/1CNC/0.5Ag based nanocomposite.

Wool fibril sponges with perspective biomedical applications.

Sheep's wool was used as a natural source to prepare keratin microfibril sponges for scaffolding, by disruption of the histological structure of the fibres through mild alkali treatment, followed by ultrasonication, casting and salt-leaching. The wool sponges showed highly interconnected porosity (93%) and contain intrinsic sites of cellular recognition that mimic the extracellular matrix (ECM). They displayed good thermal and water stability due to the conversion of disulphide cystine bonds into shorter monosulphide lanthionine intermolecular bonds, but significantly swelled in water, because of the high hydrophilicity and porosity, with a volume increasing up to 38%. Nevertheless, sponges were stable in water without structural changes, with a neutral pH in aqueous media, and showed excellent resilience to repeated compression stresses. According to in vitro biocompatibility assays, wool fibril sponges showed a good cell adhesion and proliferation as proved by MTT, FDA assays and SEM observations. The unique structure of the cortical cell network made by wool keratin proteins with controlled-size macro-porosity suitable for cell guesting, and nutrient feeding, provides an excellent scaffold for future tissue engineering applications.

Data in support of Gallium (Ga(3+)) antibacterial activities to counteract E. coli and S. epidermidis biofilm formation onto pro-osteointegrative titanium surfaces.

This paper contains original data supporting the antibacterial activities of Gallium (Ga(3+))-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in "The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii" (Cochis et al. 2016) [1]. In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga(3+) enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga(3+) activity were compared to Silver.

The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii.

Implant-related infection of biomaterials is one of the main causes of arthroplasty and osteosynthesis failure. Bacteria, such as the rapidly-emerging Multi Drug Resistant (MDR) pathogen Acinetobacter Baumannii, initiate the infection by adhering to biomaterials and forming a biofilm. Since the implant surface plays a crucial role in early bacterial adhesion phases, titanium was electrochemically modified by an Anodic Spark Deposition (ASD) treatment, developed previously and thought to provide osseo-integrative properties. In this study, the treatment was modified to insert gallium or silver onto the titanium surface, to provide antibacterial properties. The material was characterized morphologically, chemically, and mechanically; biological properties were investigated by direct cytocompatibility assay, Alkaline Phosphatase (ALP) activity, Scanning Electron Microscopy (SEM), and Immunofluorescent (IF) analysis; antibacterial activity was determined by counting Colony Forming Units, and viability assay. The various ASD-treated surfaces showed similar morphology, micrometric pore size, and uniform pore distribution. Of the treatments studied, gallium-doped specimens showed the best ALP synthesis and antibacterial properties. This study demonstrates the possibility of successfully doping the surface of titanium with gallium or silver, using the ASD technique; this approach can provide antibacterial properties and maintain high osseo-integrative potential.

Nano-biocomposite films with modified cellulose nanocrystals and synthesized silver nanoparticles.

Ternary nano-biocomposite films based on poly(lactic acid) (PLA) with modified cellulose nanocrystals (s-CNC) and synthesized silver nanoparticles (Ag) have been prepared and characterized. The functionalization of the CNC surface with an acid phosphate ester of ethoxylated nonylphenol favoured its dispersion in the PLA matrix. The positive effects of the addition of cellulose and silver on the PLA barrier properties were confirmed by reductions in the water permeability (WVP) and oxygen transmission rate (OTR) of the films tested. The migration level of all nano-biocomposites in contact with food simulants were below the permitted limits in both non-polar and polar simulants. PLA nano-biocomposites showed a significant antibacterial activity influenced by the Ag content, while composting tests showed that the materials were visibly disintegrated after 15 days with the ternary systems showing the highest rate of disintegration under composting conditions.

Ternary PVA nanocomposites containing cellulose nanocrystals from different sources and silver particles: part II.

Cellulose nanocrystals (CNC) extracted from three different sources, namely flax, phormium, and commercial microcrystalline cellulose (MCC) have been used in a polyvinyl alcohol (PVA) matrix to produce anti-bacterial films using two different amounts of silver nanoparticles (0.1 wt% and 0.5 wt%). In general, CNC confer an effect of reinforcement to PVA film, the best values of stiffness being offered by composites produced using phormium fibres, whilst for strength those produced using flax are slightly superior. This was obtained without inducing any particular modification in transition temperatures and in the thermal degradation patterns. As regards antibacterial properties, systems with CNC from flax proved slightly better than those with CNC from phormium and substantially better than those including commercial MCC. Dynamic mechanical thermal analysis (DMTA) has only been performed on the ternary composite containing 0.1 wt% Ag, which yielded higher values of Young's modulus, and as a whole confirmed the above results.

Development and validation of an enzyme linked immunosorbent assay for palivizumab serum determination.

Palivizumab (Synagis) is a humanized monoclonal antibody (IgG1K) composed of 95 percent human and 5 percent murine sequences. It is directed to an epitope in the A antigenic site of the F protein of respiratory syncytial virus (RSV). Palivizumab is used for prevention of serious lower respiratory tract disease caused by RSV in pediatric patients who are at increased risk of severe disease and is administered intramuscularly (IM) for a total of 5 monthly doses. Herein, we report on the development and validation of a very sensitive enzyme-linked immunosorbent assay (ELISA) to measure serum concentrations of palivizumab by a rabbit polyclonal antibody specifically produced against the murine sequence. The method was developed and validated according to the guidelines "Guidance for Industry" (1998) and has proved suitable for the determination of palivizumab serum levels in the target infant population. The ELISA assay was successfully applied to test the serum samples in an infant population who received palivizumab intramuscularly; thus, the assay could be used to determine serum levels in palivizumab-treated infants to optimize dosing and scheduling and to study the relationship between dose and clinical response.

High-Frequency Vibration Treatment of Human Bone Marrow Stromal Cells Increases Differentiation toward Bone Tissue.

In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30 Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient.

Electromagnetic stimulation to optimize the bone regeneration capacity of gelatin-based cryogels.

One of the key challenges in reconstructive bone surgery is to provide living constructs that possess the ability to integrate in the surrounding host tissue. Bone graft substitutes and biomaterials have already been widely used to heal critical-size bone defects due to trauma, tumor resection and tissue degeneration. In the present study, gelatin-based cryogels have been seeded with human SAOS-2 osteoblasts followed by the in vitro culture of the cells. In order to overcome the drawbacks associated with static culture systems, including limited diffusion and in homogeneous cell-matrix distribution, the present work describes the application of a bioreactor to physically enhance the cell culture in vitro using an electromagnetic stimulus. The results indicate that the physical stimulation of cell-seeded gelatin-based cryogels upregulates the bone matrix production. We anticipate that the scaffolds developed consisting of human bone proteins and cells could be applied for clinical purposes related to bone repair.

Effects of electromagnetic stimulation on osteogenic differentiation of human mesenchymal stromal cells seeded onto gelatin cryogel.

Bone tissue engineering typically uses biomaterial scaffolds, osteoblasts or cells that can become osteoblasts, and biophysical stimulations to promote cell attachment and differentiation. In this study, we investigated the effects of an electromagnetic wave on mesenchymal stromal cells isolated from the bone marrow and seeded upon gelatin cryogel disks. In comparison with control conditions without electromagnetic stimulus, the electromagnetic treatment (magnetic field, 2 mT; frequency, 75 Hz) increased the cell proliferation and differentiation and enhanced the biomaterial surface coating with bone extracellular matrix proteins. Using this tissue-engineering approach, the gelatin biomaterial, coated with differentiated cells and their extracellular matrix proteins, may be used in clinical applications as an implant for bone defect repair.

The differentiation of human adipose-derived stem cells (hASCs) into osteoblasts is promoted by low amplitude, high frequency vibration treatment.

Several studies have demonstrated that tissue culture conditions influence the differentiation of human adipose-derived stem cells (hASCs). Recently, studies performed on SAOS-2 and bone marrow stromal cells (BMSCs) have shown the effectiveness of high frequency vibration treatment on cell differentiation to osteoblasts. The aim of this study was to evaluate the effects of low amplitude, high frequency vibrations on the differentiation of hASCs toward bone tissue. In view of this goal, hASCs were cultured in proliferative or osteogenic media and stimulated daily at 30Hz for 45min for 28days. The state of calcification of the extracellular matrix was determined using the alizarin assay, while the expression of extracellular matrix and associated mRNA was determined by ELISA assays and quantitative RT-PCR (qRT-PCR). The results showed the osteogenic effect of high frequency vibration treatment in the early stages of hASC differentiation (after 14 and 21days). On the contrary, no additional significant differences were observed after 28days cell culture. Transmission Electron Microscopy (TEM) images performed on 21day samples showed evidence of structured collagen fibers in the treated samples. All together, these results demonstrate the effectiveness of high frequency vibration treatment on hASC differentiation toward osteoblasts.

Biodegradable microgrooved polymeric surfaces obtained by photolithography for skeletal muscle cell orientation and myotube development.

During tissue formation, skeletal muscle precursor cells fuse together to form multinucleated myotubes. To understand this mechanism, in vitro systems promoting cell alignment need to be developed; for this purpose, micrometer-scale features obtained on substrate surfaces by photolithography can be used to control and affect cell behaviour. This work was aimed at investigating how differently microgrooved polymeric surfaces can affect myoblast alignment, fusion and myotube formation in vitro. Microgrooved polymeric films were obtained by solvent casting of a biodegradable poly-l-lactide/trimethylene carbonate copolymer (PLLA-TMC) onto microgrooved silicon wafers with different groove widths (5, 10, 25, 50, 100microm) and depths (0.5, 1, 2.5, 5microm), obtained by a standard photolithographic technique. The surface topography of wafers and films was evaluated by scanning electron microscopy. Cell assays were performed using C2C12 cells and myotube formation was analysed by immunofluorescence assays. Cell alignment and circularity were also evaluated using ImageJ software. The obtained results confirm the ability of microgrooved surfaces to influence myotube formation and alignment; in addition, they represent a novel further improvement to the comprehension of best features to be used. The most encouraging results were observed in the case of microstructured PLLA-TMC films with grooves of 2.5 and 1microm depth, presenting, in particular, a groove width of 50 and 25microm.

Photodynamic action of merocyanine 540 on Staphylococcus epidermidis biofilms.

Photodynamic treatment (PDT) has been proposed as a new approach for inactivation of biofilms associated with medical devices that are resistant to chemical additives or biocides. In this study, we evaluated the antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye that is used for purging malignant cells from autologous bone marrow grafts, against Staphylococcus epidermidis biofilms. Effect of the combined photodynamic action of MC 540 and 532 nm laser was investigated on the viability and structure of biofilms of two Staphylococcus epidermidis strains, RP62A and 1457. Significant inactivation of cells was observed when biofilms were exposed to MC 540 and laser simultaneously. The effect was found to be light dose-dependent but S. epidermidis 1457 biofilm proved to be slightly more susceptible than S. epidermidis RP62A biofilm. Furthermore, significant killing of both types of cells was attained even when a fixed light dose was delivered to the biofilms. Confocal laser scanning microscope (CLSM) analysis indicated damage to bacterial cell membranes in photodynamically treated biofilms, while disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM).

Combating implant infections. Remarks by a women's team.

Research on implant infections requires cooperative efforts and integration between basic and clinical expertises. An international group of women scientists is acting together in this field. The main research topics of the participants of this group are described. Formation of bacterial biofilms, antibiotic resistance and production of virulence factors like adhesins and toxins are investigated. New biomaterials, coatings and drugs designed to inhibit microbial adhesion are evaluated, and infection-resistant biomaterials are under study, such as a novel heparinizable polycarbonate-urethane (Bionate) or incorporation of diamino-diamide-diol (PIME) to reduce bacterial attachment. The correlation between biofilm production and the accessory-gene-regulator (agr) is investigated in Staphylococcus aureus. The ability to form biofilm has also been shown to be one of the important virulence factors of Enterococcus faecalis, favouring colonization of inert and biological surfaces. The study of quorum sensing has led to the discovery of a quorum sensing inhibitor termed RIP that suppresses staphylococcal biofilm and infections. The immune response and the local defence mechanisms of the host against implant-associated infections, activation and infiltration of immunocompetent cells into the sites of infection have been studied in patients with implant-associated osteomyelitis. Production of monoclonal antibodies (mAbs) as possible vaccines against the staphylococcal collagen-binding MSCRAMMs is in progress.