Superb K. Misra

Comparison of nanoscale and microscale bioactive glass on the properties of P(3HB)/Bioglass composites.

This study compares the effects of introducing micro (m-BG) and nanoscale (n-BG) bioactive glass particles on the various properties (thermal, mechanical and microstructural) of poly(3hydroxybutyrate) (P(3HB))/bioactive glass composite systems. P(3HB)/bioactive glass composite films with three different concentrations of m-BG and n-BG (10, 20 and 30 wt%, respectively) were prepared by a solvent casting technique. The addition of n-BG particles had a significant stiffening effect on the composites, modulus when compared with m-BG. However, there were no significant differences in the thermal properties of the composites due to the addition of n-BG and m-BG particles. The systematic addition of n-BG particles induced a nanostructured topography on the surface of the composites, which was not visible by SEM in m-BG composites. This surface effect induced by n-BG particles considerably improved the total protein adsorption on the n-BG composites compared to the unfilled polymer and the m-BG composites. A short term in vitro degradation (30 days) study in simulated body fluid (SBF) showed a high level of bioactivity as well as higher water absorption for the P(3HB)/n-BG composites. Furthermore, a cell proliferation study using MG-63 cells demonstrated the good biocompatibility of both types of P(3HB)/bioactive glass composite systems. The results of this investigation confirm that the addition of nanosized bioactive glass particles had a more significant effect on the mechanical and structural properties of a composite system in comparison with microparticles, as well as enhancing protein adsorption, two desirable effects for the application of the composites in tissue engineering.

The complexity of nanoparticle dissolution and its importance in nanotoxicological studies.

Dissolution of nanoparticles (NPs) is an important property that alters their abundance and is often a critical step in determining safety of nanoparticles. The dissolution status of the NPs in exposure media (i.e. whether they remain in particulate form or dissolve - and to what extent), strongly affects the uptake pathway, toxicity mechanisms and the environmental compartment in which NPs will have the highest potential impact. A review of available dissolution data on NPs demonstrates there is a range of potential outcomes depending on the NPs and the exposure media. For example two nominally identical nanoparticles, in terms of size and composition, could have totally different dissolution behaviours, subject to different surface modifications. Therefore, it is imperative that toxicological studies are conducted in conjunction with dissolution of NPs to establish the true biological effect of NPs and hence, assist in their regulation.

Biomedical applications of polyhydroxyalkanoates, an overview of animal testing and in vivo responses

Polyhydroxyalkanoates (PHAs) have been established as biodegradable polymers since the second half of the twentieth century. Altering monomer composition of PHAs allows the development of polymers with favorable mechanical properties, biocompatibility and desirable degradation rates, under specific physiological conditions. Hence, the medical applications of PHAs have been explored extensively in recent years. PHAs have been used to develop devices, including sutures, nerve repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone-marrow scaffolds, tissue engineered cardiovascular devices and wound dressings. So far, various tests on animal models have shown polymers, from the PHA family, to be compatible with a range of tissues. Often, pyrogenic contaminants copurified with PHAs limit their pharmacological application rather than the monomeric composition of the PHAs and thus the purity of the PHA material is critical. This review summarizes the animal testing, tissue response, in vivo molecular stability and challenges of using PHAs for medical applications. In future, PHAs may become the materials of choice for various medical applications.

Silver Bioaccumulation Dynamics in a Freshwater Invertebrate after Aqueous and Dietary Exposures to Nanosized and Ionic Ag

We compared silver (Ag) bioavailability and toxicity to a freshwater gastropod after exposure to ionic silver (Ag(+)) and to Ag nanoparticles (Ag NPs) capped with citrate or with humic acid. Silver form, exposure route, and capping agent influence Ag bioaccumulation dynamics in Lymnaea stagnalis. Snails efficiently accumulated Ag from all forms after either aqueous or dietary exposure. For both exposure routes, uptake rates were faster for Ag(+) than for Ag NPs. Snails efficiently assimilated Ag from Ag NPs mixed with diatoms (assimilation efficiency (AE) ranged from 49 to 58%) and from diatoms pre-exposed to Ag(+) (AE of 73%). In the diet, Ag NPs damaged digestion. Snails ate less and inefficiently processed the ingested food, which adversely impacted their growth. Loss rates of Ag were faster after waterborne exposure to Ag NPs than after exposure to dissolved Ag(+). Once Ag was taken up from diet, whether from Ag(+) or Ag NPs, Ag was lost extremely slowly. Large Ag body concentrations are thus expected in L. stagnalis after dietborne exposures, especially to citrate-capped Ag NPs. Ingestion of Ag associated with particulate materials appears as the most important vector of uptake. Nanosilver exposure from food might trigger important environmental risks.

Poly(3-hydroxybutyrate) multifunctional composite scaffolds for tissue engineering applications

Poly(3-hydroxybutyrate) (P(3HB)) foams exhibiting highly interconnected porosity (85% porosity) were prepared using a unique combination of solvent casting and particulate leaching techniques by employing commercially available sugar cubes as porogen. Bioactive glass (BG) particles of 45S5 Bioglass grade were introduced in the scaffold microstructure, both in micrometer ((m-BG), <5 microm) and nanometer ((n-BG), 30 nm) sizes. The in vitro bioactivity of the P(3HB)/BG foams was confirmed within 10 days of immersion in simulated body fluid and the foams showed high level of protein adsorption. The foams interconnected porous microstructure proved to be suitable for MG-63 osteoblast cell attachment and proliferation. The foams implanted in rats as subcutaneous implants resulted in a non-toxic and foreign body response after one week of implantation. In addition to showing bioactivity and biocompatibility, the P(3HB)/BG composite foams also exhibited bactericidal properties, which was tested on the growth of Staphylococcus aureus. An attempt was made at developing multifunctional scaffolds by incorporating, in addition to BG, selected concentrations of Vitamin E or/and carbon nanotubes. P(3HB) scaffolds with multifunctionalities (viz. bactericidal, bioactive, electrically conductive, antioxidative behaviour) were thus produced, which paves the way for next generation of advanced scaffolds for bone tissue engineering.

Cellular Internalization of Silver Nanoparticles in Gut Epithelia of the Estuarine Polychaete Nereis diversicolor

Silver nanoparticles (AgNPs) are widely used which may result in environmental impacts, notably within aquatic ecosystems. As estuarine sediments are sinks for numerous pollutants, but also habitat and food for deposit feeders such as Nereis diversicolor, ingested sediments must be investigated as an important route of uptake for NPs. N. diversicolor were fed sediment spiked with either citrate capped AgNPs (30 ± 5 nm) or aqueous Ag for 10 days. Postexposure AgNPs were observed in the lumen of exposed animals, and three lines of evidence indicated direct internalization of AgNPs into the gut epithelium. With TEM, electron-dense particles resembling AgNPs were observed associated with the apical plasma membrane, in endocytotic pits and in endosomes. Energy dispersive X-ray analysis (EDX) confirmed the presence of Ag in these particles, which were absent in controls. Subcellular fractionation revealed that Ag accumulated from AgNPs was predominantly associated with inorganic granules, organelles, and the heat denatured proteins; whereas dissolved Ag was localized to the metallothionein fraction. Collectively, these results indicate separate routes of cellular internalization and differing in vivo fates of Ag delivered in dissolved and NP form. For AgNPs an endocytotic pathway appears to be a key route of cellular uptake.

Effect of nanoparticulate bioactive glass particles on bioactivity and cytocompatibility of poly(3-hydroxybutyrate) composites

This work investigated the effect of adding nanoparticulate (29 nm) bioactive glass particles on the bioactivity, degradation and in vitro cytocompatibility of poly(3-hydroxybutyrate) (P(3HB)) composites/nano-sized bioactive glass (n-BG). Two different concentrations (10 and 20 wt %) of nanoscale bioactive glass particles of 45S5 Bioglass composition were used to prepare composite films. Several techniques (Raman spectroscopy, scanning electron microscopy, atomic force microscopy, energy dispersive X-ray) were used to monitor their surface and bioreactivity over a 45-day period of immersion in simulated body fluid (SBF). All results suggested the P(3HB)/n-BG composites to be highly bioactive, confirmed by the formation of hydroxyapatite on material surfaces upon immersion in SBF. The weight loss and water uptake were found to increase on increasing bioactive glass content. Cytocompatibility study (cell proliferation, cell attachment, alkaline phosphatase activity and osteocalcin production) using human MG-63 osteoblast-like cells in osteogenic and non-osteogenic medium showed that the composite substrates are suitable for cell attachment, proliferation and differentiation.

In vitro biocompatibility of 45S5 Bioglass-derived glass-ceramic scaffolds coated with poly(3-hydroxybutyrate).

The aim of this work was to study the in vitro biocompatibility of glass–ceramic scaffolds based on 45S5 Bioglass®, using a human osteosarcoma cell line (HOS‐TE85). The highly porous scaffolds were produced by the foam replication technique. Two different types of scaffolds with different porosities were analysed. They were coated with a biodegradable polymer, poly(3‐hydroxybutyrate) (P(3HB)). The scaffold bioactivity was evaluated by soaking in a simulated body fluid (SBF) for different durations. Compression strength tests were performed before and after immersion in SBF. These experiments showed that the scaffolds are highly bioactive, as after a few days of immersion in SBF a hydroxyapatite‐like layer was formed on the scaffolds surface. It was also observed that P(3HB)‐coated samples exhibited higher values of compression strength than uncoated samples. Biocompatibility assessment was carried out by qualitative evaluation of cell morphology after different culture periods, using scanning electron microscopy, while cell proliferation was determined by using the AlamarBlue™ assay. Alkaline phosphatase (ALP) and osteocalcin (OC) assays were used as quantitative in vitro indicators of osteoblast function. Two different types of medium were used for ALP and OC tests: normal supplemented medium and osteogenic medium. HOS cells were seeded and cultured onto the scaffolds for up to 2 weeks. The AlamarBlue assay showed that cells were able to proliferate and grow on the scaffold surface. After 7 days in culture, the P(3HB)‐coated samples had a higher number of cells on their surfaces than the uncoated samples. Regarding ALP‐ and OC‐specific activity, no significant differences were found between samples with different pore sizes. All scaffolds containing osteogenic medium seemed to have a slightly higher level of ALP and OC concentration. These experiments confirmed that Bioglass®/P(3HB) scaffolds have potential as osteoconductive tissue engineering substrates for maintenance and normal functioning of bone tissue. Copyright

Isotopically Modified Nanoparticles for Enhanced Detection in Bioaccumulation Studies

This work presents results on synthesis of isotopically enriched (99% (65)Cu) copper oxide nanoparticles and its application in ecotoxicological studies. (65)CuO nanoparticles were synthesized as spheres (7 nm) and rods (7 × 40 nm). Significant differences were observed between the reactivity and dissolution of spherical and rod shaped nanoparticles. The extreme sensitivity of the stable isotope tracing technique developed in this study allowed determining Cu uptake at exposure concentrations equivalent to background Cu concentrations in freshwater systems (0.2-30 μg/L). Without a tracer, detection of newly accumulated Cu was impossible, even at exposure concentrations surpassing some of the most contaminated water systems (>1 mg/L).

Effects of sediment-associated copper to the deposit-feeding snail, Potamopyrgus antipodarum: a comparison of Cu added in aqueous form or as nano- and micro-CuO particles.

Increasing use of engineered nanoparticles (NPs) is likely to result in release of these particles to the aquatic environment where the NPs may eventually accumulate in sediment. However, little is known about the potential ecotoxicity of sediment-associated engineered NPs. We here consider the case of metal oxide NPs using CuO to understand if the effects of NPs differ from micron-sized particles of CuO and aqueous Cu (CuCl₂). To address this issue, we compared effects of copper added to the sediment as aqueous Cu, nano- (6 nm) and micro- (<5 μm) CuO particles on the deposit-feeding snail, Potamopyrgus antipodarum. Effects were assessed as mortality, specific growth rate, feeding rate, reproduction, and bioaccumulation after 8 weeks of exposure to nominal concentrations of 0, 30, 60, 120 and 240 μg Cu/g dry weight sediment. The results demonstrate that copper added to sediment as nano-CuO had greater effects on growth, feeding rate, and reproduction of P. antipodarum than copper added as micro-CuO or aqueous Cu. P. antipodarum accumulated more copper in the nano-CuO treatment than in aqueous Cu or micro-CuO treatments, indicating that consideration of metal form may be important when assessing risks of metals to the aquatic environment.