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Monodispersed strontium containing bioactive glass nanoparticles (Sr-BGNPs) with two compositions were synthesised, through a modified sol-gel Stöber process, wherein silica ...nanoparticles (SiO2-NPs) were formed prior to incorporation of calcium and strontium, with diameters of 90 ± 10 nm. The osteogenic response of a murine preosteoblast cell line, MC3T3-E1, was investigated in vitro for a nanoparticle concentration of 250 µg/mL with compositions of 87 mol% SiO2, 7 mol% CaO, 6 mol% SrO and 83 mol% SiO2, 3 mol% CaO, 14 mol% SrO. Dissolution studies in minimum essential media (α-MEM) at pH 7.4 and artificial lysosomal fluid (ALF) at pH 4.5 showed that the particles dissolved and that Sr2+ ions were released from Sr-BGNPs in both environments. Both particle compositions and their ionic dissolution products enhanced the alkaline phosphatase (ALP) activity of the cells and calcium deposition. Immunohistochemistry (IHC) staining of Col1a1, osteocalcin (OSC) and osteopontin (OSP) showed that these proteins were expressed in the MC3T3-E1 cells following three weeks of culture. In the basal condition, the late osteogenic differentiation markers, OSC and OSP, were more overtly expressed by cells cultured with Sr-BGNPs with 14 mol% SrO and their ionic release products than in the control condition. Col1a1 expression was only slightly enhanced in the basal condition, but was enhanced further by the osteogenic supplements. These data demonstrate that Sr-BGNPs accelerate mineralisation without osteogenic supplements. Sr-BGNPs were internalised into MC3T3-E1 cells by endocytosis and stimulated osteogenic differentiation of the pre-osteoblast cell line. Sr-BGNPs are likely to be beneficial for bone regeneration and the observed osteogenic effects of these particles can be attributed to their ionic release products.
We report, for the first time, that monodispersed bioactive glass nanoparticles (∼90 nm) are internalised into preosteoblast cells by endocytosis but by unspecific mechanisms. The bioactive nanoparticles and their dissolution products (without the particles present) stimulated the expression of osteogenic markers from preosteoblast cells without the addition of other osteogenic supplements.
Incorporating Sr into the bioactive glass nanoparticle composition, in addition to Ca, increased the total cation content (and therefore dissolution rate) of the nanoparticles, even though nominal total cation addition was constant, without changing size or morphology.
Increasing Sr content in the nanoparticles and in their dissolution products enhanced osteogenesis in vitro. The particles therefore have great potential as an injectable therapeutic for bone regeneration, particularly in patients with osteoporosis, for which Sr is known to be therapeutic agent.
Mineralization is a ubiquitous process in the animal kingdom and is fundamental to human development and health. Dysfunctional or aberrant mineralization leads to a variety of medical problems, and ...so an understanding of these processes is essential to their mitigation. Osteoblasts create the nano-composite structure of bone by secreting a collagenous extracellular matrix (ECM) on which apatite crystals subsequently form. However, despite their requisite function in building bone and decades of observations describing intracellular calcium phosphate, the precise role osteoblasts play in mediating bone apatite formation remains largely unknown. To better understand the relationship between intracellular and extracellular mineralization, we combined a sample-preparation method that simultaneously preserved mineral, ions, and ECM with nano-analytical electron microscopy techniques to examine osteoblasts in an in vitro model of bone formation. We identified calcium phosphate both within osteoblast mitochondrial granules and intracellular vesicles that transported material to the ECM. Moreover, we observed calcium-containing vesicles conjoining mitochondria, which also contained calcium, suggesting a storage and transport mechanism. Our observations further highlight the important relationship between intracellular calcium phosphate in osteoblasts and their role in mineralizing the ECM. These observations may have important implications in deciphering both how normal bone forms and in understanding pathological mineralization.
The development of single-walled carbon nanotubes for various biomedical applications is an area of great promise. However, the contradictory data on the toxic effects of single-walled carbon ...nanotubes highlight the need for alternative ways to study their uptake and cytotoxic effects in cells. Single-walled carbon nanotubes have been shown to be acutely toxic in a number of types of cells, but the direct observation of cellular uptake of single-walled carbon nanotubes has not been demonstrated previously due to difficulties in discriminating carbon-based nanotubes from carbon-rich cell structures. Here we use transmission electron microscopy and confocal microscopy to image the translocation of single-walled carbon nanotubes into cells in both stained and unstained human cells. The nanotubes were seen to enter the cytoplasm and localize within the cell nucleus, causing cell mortality in a dose-dependent manner.
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While bioactive glass and ions released during its dissolution are known to stimulate osteoblast cells, the effect bioactive glass has on human stem cells is not clear. Here, we show ...that spherical monodispersed strontium containing bioactive nanoparticles (Sr-BGNPs) of composition 90.6 mol% SiO2, 5.0 mol% CaO, 4.4% mol% SrO (4.4%Sr-BGNPs) and 88.8 mol% SiO2, 1.8 mol% CaO, and 9.4 mol% SrO (9.4%Sr-BGNPs) stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without osteogenic supplements. The particles were synthesised using a modified Stӧber process and had diameters of 90 ± 10 nm. Previous work on similar particles that did not contain Sr (80 mol% SiO2, 20 mol% CaO) showed stem cells did not differentiate when exposed to the particles. Here, both compositions of the Sr-BGNPs (up to concentration of 250 μg/mL) stimulated the early-, mid-, and late-stage markers of osteogenic differentiation and accelerated mineralisation in the absence of osteogenic supplements. Sr ions play a key role in osteogenic stem cell differentiation. Sr-BGNP dissolution products did not adversely affect hMSC viability and no significant differences in viability were measured between each particle composition. Confocal and transmission electron microscopy (TEM) demonstrated that monodispersed Sr-BGNPs were internalised and localised within vesicles in the cytoplasm of hMSCs. Degradation of particles inside the cells was observed, whilst maintaining effective cations (Ca and Sr) in their silica network after 24 h in culture. The uptake of Sr-BGNPs by hMSCs was reduced by inhibitors of specific routes of endocytosis, indicating that the Sr-BGNPs uptake by hMSCs was probably via mixed endocytosis mechanisms. Sr-BGNPs have potential as injectable therapeutic devices for bone regeneration or treatment of conditions such as osteoporosis, because of their ability deliver a sustained release of osteogenic inorganic cations, e.g. calcium (Ca) or and strontium (Sr), through particle degradation locally to cells.
Here, we show that 90 nm spherical strontium containing bioactive nanoparticles of stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without the use of osteogenic supplements. While bioactive glass and its dissolution products are known to promote excellent bone regeneration in vivo and to stimulate osteoblast cells to produce bone matrix in vitro, their effect on human stem cells is not clear. Previously our nanoparticles that contained only SiO2 and CaO did not provoke human bone marrow or adipose derived stem cell differentiation.
Osteopontin (OPN) is a non-collagenous protein involved in biomineralization of bone tissue. Beyond its role in biomineralization, we show that osteopontin is essential to the quality of collagen ...fibrils in bone. Transmission electron microscopy revealed that, in Opn−/− tissue, the organization of the collagen fibrils was highly heterogeneous, more disorganized than WT bone and comprised of regions of both organized and disorganized matrix with a reduced density. The Opn−/− bone tissue also exhibited regions in which the collagen had lost its characteristic fibrillar structure, and the crystals were disorganized. Using nanobeam electron diffraction, we show that damage to structural integrity of collagen fibrils in Opn−/- bone tissue and their organization causes mineral disorganization, which could ultimately affect its mechanical integrity.
This study presents new evidence about the role of osteopontin (OPN) – a non-collagenous protein – on the structure and organization of the organic and mineral matrix in bone. In previous work, osteopontin has been suggested to regulate the nucleation and growth of bone mineral crystals and to form sacrificial bonds between mineralized collagen fibrils to enhance bone's toughness. Our findings show that OPN plays a crucial role before mineralization, during the formation of the collagen fibrils. OPN-deficient bones present a lower collagen content compared to wild type bone and, at the tissue level, collagen fibrils organization can be significantly altered in the absence of OPN. Our results suggest that OPN is critical for the formation and/or remodeling of bone collagen matrix. Our findings could lead to the development of new therapeutic strategies of bone diseases affecting collagen formation and remodeling.
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The Stӧber process is commonly used for synthesising spherical silica particles. This article reports the first comprehensive study of how the process variables can be used to obtain ...monodispersed particles of specific size. The modal particle size could be selected within in the range 20–500nm. There is great therapeutic potential for bioactive glass nanoparticles, as they can be internalised within cells and perform sustained delivery of active ions. Biodegradable bioactive glass nanoparticles are also used in nanocomposites. Modification of the Stӧber process so that the particles can contain cations such as calcium, whilst maintaining monodispersity, is desirable. Here, whilst calcium incorporation is achieved, with a homogenous distribution, careful characterisation shows that much of the calcium is not incorporated. A maximum of 10mol% CaO can be achieved and previous reports are likely to have overestimated the amount of calcium incorporated.
Gold nanostars (AuNSs) are receiving increasing attention for their potential applications in bionanotechnology because of their unique optical properties related to their complex branched ...morphology. Their sharp features allow strong localized surface plasmon resonances, tunable in the near-infrared (NIR) region, and large enhancements of local electromagnetic fields. Here, the application of AuNSs in metal-enhanced fluorescence (MEF) in the NIR and second NIR (NIR-II) biological windows is explored for the first time. NIR/NIR-II fluorophores are incorporated onto monolayers of AuNSs with tunable plasmonic responses. Over 320-fold fluorescence enhancement is achieved in the NIR, confirming that AuNS substrates are promising NIR-MEF platforms for the development of ultrasensitive biosensing applications. Using fluorescence lifetime measurements to semiquantitatively deconvolute the excitation enhancement from emission enhancement, as well as modeling to simulate the electric field enhancement, we show that a combination of enhanced excitation and an increased radiative decay rate, accompanied by an increase in the quantum yield, contribute to the observed large enhancement. AuNSs with different morphological features exhibit significantly different excitation enhancements, indicating the important role of the particle morphology on the magnitude of electromagnetic field enhancement and the resulting enhancement factor. Importantly, enhancement factors of up to 50-fold are also achieved in the NIR-II region, suggesting that this system holds promise for the future development of bright probes for NIR/NIR-II biosensing and bioimaging.
Particulate matter (PM) is a crucial health risk factor for respiratory and cardiovascular diseases. The smaller size fractions, ≤2.5 μm (PM2.5; fine particles) and ≤0.1 μm (PM0.1; ultrafine ...particles), show the highest bioactivity but acquiring sufficient mass for in vitro and in vivo toxicological studies is challenging. We review the suitability of available instrumentation to collect the PM mass required for these assessments. Five different microenvironments representing the diverse exposure conditions in urban environments are considered in order to establish the typical PM concentrations present. The highest concentrations of PM2.5 and PM0.1 were found near traffic (i.e. roadsides and traffic intersections), followed by indoor environments, parks and behind roadside vegetation. We identify key factors to consider when selecting sampling instrumentation. These include PM concentration on-site (low concentrations increase sampling time), nature of sampling sites (e.g. indoors; noise and space will be an issue), equipment handling and power supply. Physicochemical characterisation requires micro- to milli-gram quantities of PM and it may increase according to the processing methods (e.g. digestion or sonication). Toxicological assessments of PM involve numerous mechanisms (e.g. inflammatory processes and oxidative stress) requiring significant amounts of PM to obtain accurate results. Optimising air sampling techniques are therefore important for the appropriate collection medium/filter which have innate physical properties and the potential to interact with samples. An evaluation of methods and instrumentation used for airborne virus collection concludes that samplers operating cyclone sampling techniques (using centrifugal forces) are effective in collecting airborne viruses. We highlight that predictive modelling can help to identify pollution hotspots in an urban environment for the efficient collection of PM mass. This review provides guidance to prepare and plan efficient sampling campaigns to collect sufficient PM mass for various purposes in a reasonable timeframe.
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•PM mass collection for toxicological studies in various microenvironments is reviewed.•Lower amounts of PM mass limit the workflows for toxicity assessments.•Portable samplers with high flow rates are suitable for both indoor and outdoor measurement.•Cyclone sampling technique effectively collects viruses in all environments.•Predictive modelling aids in identifying hotspots for particle mass collection.
Background Diesel exhaust particles (DEPs) are a major component of particulate matter in Europe's largest cities, and epidemiologic evidence links exposure with respiratory symptoms and asthma ...exacerbations. Respiratory reflexes are responsible for symptoms and are regulated by vagal afferent nerves, which innervate the airway. It is not known how DEP exposure activates airway afferents to elicit symptoms, such as cough and bronchospasm. Objective We sought to identify the mechanisms involved in activation of airway sensory afferents by DEPs. Methods In this study we use in vitro and in vivo electrophysiologic techniques, including a unique model that assesses depolarization (a marker of sensory nerve activation) of human vagus. Results We demonstrate a direct interaction between DEP and airway C-fiber afferents. In anesthetized guinea pigs intratracheal administration of DEPs activated airway C-fibers. The organic extract (DEP-OE) and not the cleaned particles evoked depolarization of guinea pig and human vagus, and this was inhibited by a transient receptor potential ankyrin-1 antagonist and the antioxidant N-acetyl cysteine. Polycyclic aromatic hydrocarbons, major constituents of DEPs, were implicated in this process through activation of the aryl hydrocarbon receptor and subsequent mitochondrial reactive oxygen species production, which is known to activate transient receptor potential ankyrin-1 on nociceptive C-fibers. Conclusions This study provides the first mechanistic insights into how exposure to urban air pollution leads to activation of guinea pig and human sensory nerves, which are responsible for respiratory symptoms. Mechanistic information will enable the development of appropriate therapeutic interventions and mitigation strategies for those susceptible subjects who are most at risk.
High-aspect ratio ZnO nanowires have become one of the most promising products in the nanosciences within the past few years with a multitude of applications at the interface of optics and ...electronics. The interaction of zinc with cells and organisms is complex, with both deficiency and excess causing severe effects. The emerging significance of zinc for many cellular processes makes it imperative to investigate the biological safety of ZnO nanowires in order to guarantee their safe economic exploitation. In this study, ZnO nanowires were found to be toxic to human monocyte macrophages (HMMs) at similar concentrations as ZnCl2. Confocal microscopy on live cells confirmed a rise in intracellular Zn2+ concentrations prior to cell death. In vitro, ZnO nanowires dissolved very rapidly in a simulated body fluid of lysosomal pH, whereas they were comparatively stable at extracellular pH. Bright-field transmission electron microscopy (TEM) showed a rapid macrophage uptake of ZnO nanowire aggregates by phagocytosis. Nanowire dissolution occurred within membrane-bound compartments, triggered by the acidic pH of the lysosomes. ZnO nanowire dissolution was confirmed by scanning electron microscopy/energy-dispersive X-ray spectrometry. Deposition of electron-dense material throughout the ZnO nanowire structures observed by TEM could indicate adsorption of cellular components onto the wires or localized zinc-induced protein precipitation. Our study demonstrates that ZnO nanowire toxicity in HMMs is due to pH-triggered, intracellular release of ionic Zn2+ rather than the high-aspect nature of the wires. Cell death had features of necrosis as well as apoptosis, with mitochondria displaying severe structural changes. The implications of these findings for the application of ZnO nanowires are discussed.
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