Osteogenic lithium-doped brushite cements for bone regeneration.

This study investigated the osteogenic performance of new brushite cements obtained from Li+-doped ß-tricalcium phosphate as a promising strategy for bone regeneration. Lithium (Li+) is a promising trace element to encourage the migration and proliferation of adipose-derived stem cells (hASCs) and the osteogenic differentiation-related gene expression, essential for osteogenesis. In-situ X-ray diffraction (XRD) and in-situ 1H nuclear magnetic resonance (1H NMR) measurements proved the precipitation of brushite, as main phase, and monetite, indicating that Li+ favored the formation of monetite under certain conditions. Li+ was detected in the remaining pore solution in significant amounts after the completion of hydration. Isothermal calorimetry results showed an accelerating effect of Li+, especially for low concentration of the setting retarder (phytic acid). A decrease of initial and final setting times with increasing amount of Li+ was detected and setting times could be well adjusted by varying the setting retarder concentration. The cements presented compressive mechanical strength within the ranges reported for cancellous bone. In vitro assays using hASCs showed normal metabolic and proliferative levels. The immunodetection and gene expression profile of osteogenic-related markers highlight the incorporation of Li+ for increasing the in vivo bone density. The osteogenic potential of Li-doped brushite cements may be recommended for further research on bone defect repair strategies.

Modulation of inflammation by anti-TNF α mAb-dendrimer nanoparticles loaded in tyramine-modified gellan gum hydrogels in a cartilage-on-a-chip model.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease characterized by joint inflammation. Since the inflammatory condition plays an important role in the disease process, it is important to develop and test new therapeutic approaches that specifically target and treat joint inflammation. In this study, a human 3D inflammatory cartilage-on-a-chip model was established to test the therapeutic efficacy of anti-TNFα mAb-CS/PAMAM dendrimer NPs loaded-Tyramine-Gellan Gum in the treatment of inflammation. The results showed that the proposed therapeutic approach applied to the human monocyte cell line (THP-1) and human chondrogenic primary cells (hCH) cell-based inflammation system revealed an anti-inflammatory capacity that increased over 14 days. It was also possible to observe that Coll type II was highly expressed by inflamed hCH upon the culture with anti-TNF α mAb-CS/PAMAM dendrimer NPs, indicating that the hCH cells were able maintain their biological function. The developed preclinical model allowed us to provide more robust data on the potential therapeutic effect of anti-TNF α mAb-CS/PAMAM dendrimer NPs loaded-Ty-GG hydrogel in a physiologically relevant model.

Physicochemical properties and cytocompatibility assessment of non-degradable scaffolds for bone tissue engineering applications.

Bone is a dynamic tissue with an amazing but yet limited capacity of self-healing. Bone is the second most transplanted tissue in the world and there is a huge need for bone grafts and substitutes which lead to a decrease in bone banks donors. In this study, we developed three-dimensional scaffolds based on Ti6Al4V, ZrO2 and PEEK targeting bone tissue engineering applications. Experimental mechanical compressive tests and finite element analyses were carried out to study the mechanical performance of the scaffolds. Overall, the scaffolds presented different hydrophilicity properties and a reduced elastic modulus when compared with the corresponding solid materials which can in some extension minimize the phenomenon of stress shielding. The ability as a scaffold material for bone tissue regeneration applications was evaluated in vitro by seeding human osteosarcoma (SaOS-2) cells onto the scaffolds. Then, the successful culture of SaOS-2 cells on developed scaffolds was monitored by assessment of cell's viability, proliferation and alkaline phosphatase (ALP) activity up to 14 days of culturing. The in vitro results revealed that Ti6Al4V, ZrO2 and PEEK scaffolds were cytocompatible allowing the successful culture of an osteoblastic cell line, suggesting their potential application in bone tissue engineering. Statement of Significance. The work presented is timely and relevant since it gathers both the mechanical and cellular study of non-degradable cellular structures with the potential to be used as bone scaffolds. This work allow to investigate three possible bone scaffolds solutions which exhibit a significantly reduced elastic modulus when compared with conventional solid materials. While it is generally accepted that the Ti6Al4V, ZrO2 and PEEK are candidates for such applications a further study of their features and their comparison is extremely important for a better understanding of their potential.

Reproductive phenology of Melastomataceae species with contrasting reproductive systems: contemporary and historical drivers.

Flowering and fruiting are key events in the life history of plants, and both are critical to their reproductive success. Besides the role of evolutionary history, plant reproductive phenology is regulated by abiotic factors and shaped by biotic interactions with pollinators and seed dispersers. In Melastomataceae, a dominant Neotropical family, the reproductive systems vary from allogamous with biotic pollination to apomictic, and seed dispersal varies from dry (self-dispersed) to fleshy (animal-dispersed) fruits. Such variety in reproductive strategies is likely to affect flowering and fruiting phenologies. In this study, we described the reproductive phenology of 81 Melastomataceae species occurring in two biodiversity hotspots: the Atlantic rain forest and the campo rupestre. We aim to disentangle the role of abiotic and biotic factors defining flowering and fruiting times of Melastomataceae species, considering the contrasting breeding and seed dispersal systems, and their evolutionary history. In both vegetation types, pollinator-dependent species had higher flowering seasonality than pollinator-independent ones. Flowering patterns presented phylogenetic signal regardless of vegetation type. Fruiting of fleshy-fruited species was seasonal in campo rupestre but not in Atlantic rain forest; the fruiting of dry-fruited species was also not seasonal in both vegetation types. Fruiting showed a low phylogenetic signal, probably because the influence of environment and dispersal agents on fruiting time is stronger than the phylogenetic affinity. Considering these ecophylogenetic patterns, our results indicate that flowering may be shaped by the different reproductive strategies of Melastomataceae lineages, while fruiting patterns may be governed mainly by the seed dispersal strategy and flowering time, with less phylogenetic influence.

Apomixis does not affect visitation to flowers of Melastomataceae, but pollen sterility does.

Apomixis is an asexual seed reproduction mechanism thorough which embryos are originated from material tissues inside the ovules, without precedent fertilisation. It allows plants to colonise new habitats, even in places where flower visitors are scarce or where plants are isolate. Apomixis seems to be related to pollen sterility and, in species with flowers that offer pollen as a reward for pollinators, the amount or quality of the pollen offered by these species may influence the amount of the visits and specific composition of the visitors. In order to test this hypothesis, we studied breeding systems of 16 species of Melastomataceae and their flower visitors, evaluating composition and abundance of the visits to apomictic and sexual species. Apomictic plants with no viable pollen or with pollen with low viability did not receive visits from pollinators, and consequently probably produce strictly apomictic fruits. On the other hand, apomictic and sexual plants with high pollen viability do receive visits; in this case, apomictic plants may produce fruits and seeds through both sexual and apomictic methods. The species composition of insects visiting Melastomataceae with high pollen viability was similar, regardless of whether the plants were apomictic or not. It seems that pollen viability levels are important to determine visits to the flowers irrespective of breeding system.

The linac coherent light source single particle imaging road map.

Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.

The extraction of single-particle diffraction patterns from a multiple-particle diffraction pattern.

The structures of biological molecules may soon be determined with X-ray free-electron lasers without crystallization by recording the coherent diffraction patterns of many identical copies of a molecule. Most analysis methods require a measurement of each molecule individually. However, current injection methods deliver particles to the X-ray beam stochastically and the maximum yield of single particle measurements is 37% at optimal concentration. The remaining 63% of pulses intercept no particles or multiple particles. We demonstrate that in the latter case single particle diffraction patterns can be extracted provided the particles are sufficiently separated. The technique has the potential to greatly increase the amount of data available for three-dimensional imaging of identical particles with X-ray lasers.

Impact of wave front and coherence optimization in coherent diffractive imaging.

We present single shot nanoscale imaging using a table-top femtosecond soft X-ray laser harmonic source at a wavelength of 32 nm. We show that the phase retrieval process in coherent diffractive imaging critically depends on beam quality. Coherence and image fidelity are measured from single-shot coherent diffraction patterns of isolated nano-patterned slits. Impact of flux, wave front and coherence of the soft X-ray beam on the phase retrieval process and the image quality are discussed. After beam improvements, a final image reconstruction is presented with a spatial resolution of 78 nm (half period) in a single 20 fs laser harmonic shot.

Fractal morphology, imaging and mass spectrometry of single aerosol particles in flight.

The morphology of micrometre-size particulate matter is of critical importance in fields ranging from toxicology to climate science, yet these properties are surprisingly difficult to measure in the particles' native environment. Electron microscopy requires collection of particles on a substrate; visible light scattering provides insufficient resolution; and X-ray synchrotron studies have been limited to ensembles of particles. Here we demonstrate an in situ method for imaging individual sub-micrometre particles to nanometre resolution in their native environment, using intense, coherent X-ray pulses from the Linac Coherent Light Source free-electron laser. We introduced individual aerosol particles into the pulsed X-ray beam, which is sufficiently intense that diffraction from individual particles can be measured for morphological analysis. At the same time, ion fragments ejected from the beam were analysed using mass spectrometry, to determine the composition of single aerosol particles. Our results show the extent of internal dilation symmetry of individual soot particles subject to non-equilibrium aggregation, and the surprisingly large variability in their fractal dimensions. More broadly, our methods can be extended to resolve both static and dynamic morphology of general ensembles of disordered particles. Such general morphology has implications in topics such as solvent accessibilities in proteins, vibrational energy transfer by the hydrodynamic interaction of amino acids, and large-scale production of nanoscale structures by flame synthesis.

Femtosecond dark-field imaging with an X-ray free electron laser.

The emergence of femtosecond diffractive imaging with X-ray lasers has enabled pioneering structural studies of isolated particles, such as viruses, at nanometer length scales. However, the issue of missing low frequency data significantly limits the potential of X-ray lasers to reveal sub-nanometer details of micrometer-sized samples. We have developed a new technique of dark-field coherent diffractive imaging to simultaneously overcome the missing data issue and enable us to harness the unique contrast mechanisms available in dark-field microscopy. Images of airborne particulate matter (soot) up to two microns in length were obtained using single-shot diffraction patterns obtained at the Linac Coherent Light Source, four times the size of objects previously imaged in similar experiments. This technique opens the door to femtosecond diffractive imaging of a wide range of micrometer-sized materials that exhibit irreproducible complexity down to the nanoscale, including airborne particulate matter, small cells, bacteria and gold-labeled biological samples.

Cryptotomography: reconstructing 3D Fourier intensities from randomly oriented single-shot diffraction patterns.

We reconstructed the 3D Fourier intensity distribution of monodisperse prolate nanoparticles using single-shot 2D coherent diffraction patterns collected at DESY's FLASH facility when a bright, coherent, ultrafast x-ray pulse intercepted individual particles of random, unmeasured orientations. This first experimental demonstration of cryptotomography extended the expansion-maximization-compression framework to accommodate unmeasured fluctuations in photon fluence and loss of data due to saturation or background scatter. This work is an important step towards realizing single-shot diffraction imaging of single biomolecules.

Single-shot diffractive imaging with a table-top femtosecond soft x-ray laser-harmonics source.

Coherent x-ray diffractive imaging is a powerful method for studies on nonperiodic structures on the nanoscale. Access to femtosecond dynamics in major physical, chemical, and biological processes requires single-shot diffraction data. Up to now, this has been limited to intense coherent pulses from a free electron laser. Here we show that laser-driven ultrashort x-ray sources offer a comparatively inexpensive alternative. We present measurements of single-shot diffraction patterns from isolated nano-objects with a single 20 fs pulse from a table-top high-harmonic x-ray laser. Images were reconstructed with a resolution of 119 nm from the single shot and 62 nm from multiple shots.