Interactions between Dental MSCs and Biomimetic Composite Scaffold during Bone Remodeling Followed by In Vivo Real-Time Bioimaging.

Oral-maxillofacial tumor removal can generate critical bone defects and major problems for patients, causing dysfunctionalities and affecting oral competencies such as mastication, swallowing, and breathing. The association of novel biomaterials and cell therapies in tissue engineering strategies could offer new strategies to promote osteomucosa healing. This study focused on the development of a bioengineered construct loaded with human dental follicle cells (MSCs). To increase the bioconstruct integration to the surrounding tissue, a novel and comprehensive approach was designed combining an injectable biomimetic hydrogel and dental stem cells (hDFMSCs) expressing luminescence/fluorescence for semi-quantitative tissue imaging in live animals. This in vivo model with human MSCs was based on an intramembranous bone regeneration process (IMO). Biologically, the biocomposite based on collagen/nanohydroxyapatite filled with cell-loaded osteopontin-fibrin hydrogel (Coll/nanoHA OPN-Fb) exhibited a high cellular proliferation rate, increased bone extracellular matrix deposition (osteopontin) and high ALP activity, indicating an early osteogenic differentiation. Thus, the presence of human OPN enhanced hDFMSC adhesion, migration, and spatial distribution within the 3D matrix. The developed 3D bioconstruct provided the necessary pro-regenerative effect to modulate the biological response, precisely fitting the bone defect with fine-tuned adjustment to the surrounding original structure and promoting oral osteomucosa tissue regeneration. We were also able to track the cells in vivo and evaluate their behavior (migration, proliferation, and differentiation), providing a glimpse into bone regeneration and helping in the optimization of patient-specific therapies.

One sample fits all: a microfluidic-assisted methodology for label-free isolation of CTCs with downstream methylation analysis of cfDNA in lung cancer.

Lung cancer (LC) is a major cause of mortality. Late diagnosis, associated with limitations in tissue biopsies for adequate tumor characterization contribute to limited survival of lung cancer patients. Liquid biopsies have been introduced to improve tumor characetrization through the analysis of biomarkers, including circulating tumour cells (CTCs) and cell-free DNA (cfDNA). Considering their availability in blood, several enrichment strategies have been developed to augment circulating biomarkers for improving diagnostic, prognostic and treament efficacy assessment; often, however, only one biomarker is tested. In this work we developed and implemented a microfluidic chip for label-free enrichment of CTCs with a methodology for subsequent cfDNA analysis from the same cryopreserved sample. CTCs were successfully isolated in 38 of 42 LC patients with the microfluidic chip. CTCs frequency was significantly higher in LC patients with advanced disease. A cut-off of 1 CTC per mL was established for diagnosis (sensitivity = 76.19%, specificity = 100%) and in patients with late stage lung cancer, the presence of ≥5 CTCs per mL was significantly associated with shorter overall survival. MIR129-2me and ADCY4me panel of cfDNA methylation performed well for LC detection, whereas MIR129-2me combined with HOXA11me allowed for patient risk stratification. Analysis of combinations of biomarkers enabled the definition of panels for LC diagnosis and prognosis. Overall, this study demonstrates that multimodal analysis of tumour biomarkers via microfluidic devices may significantly improve LC characterization in cryopreserved samples, constituting a reliable source for continuous disease monitoring.

Can measurements be physically conditioned by thought? Further observations following a focused intention experiment.

Background The biofield is a controversial concept among the scientific community. Some aspects of this phenomenon relate to measurable factors of mainstream science such as electromagnetics, while others, involving nonlocal interventions, intention, and consciousness, seem to produce physical changes through mechanisms that are still unknown, making the outcomes of many studies not fully explained by our current state of scientific understanding. This study explored the hypothetical effects of intention on the conditioning of a pH system with continuous data acquisition for real-time measurements. As a follow-up study, those effects were related to changes in the physicochemical properties of water samples chosen as a target in a previous large-scale intervention of focused intention. Methods The intention experiments were conducted under controlled conditions from the 8th to September 11, 2015. During this period, 286 qualified biofield therapy practitioners meditated at a distance with the intention of changing the vibrational state of the water molecules contained in specific flasks. Several variables were evaluated, including the magnetic field and ultraviolet-visible (UV-VIS) radiation from 175 to 954 nm near the experimental spot, as well as Raman spectra, pH, and electrical conductivity of all the water samples. After this period, real-time pH measurements of water samples were taken with the same equipment and under the same experimental conditions, except for the practitioners' awareness of those experiments, and without their focused intention. Real-time pH, electrical conductivity and the concentration of cations and anions measured by ion chromatography in the water samples were used to test the proposed hypothesis. Results Real-time pH was responsive during the intention experiments and after this period. Further continuous measurements performed after the 11th September showed that the pH variations overtime kept a systematic and consistent tendency similar to the one observed during the experimental activities involving focused intention. After the replacement of the electrode internal electrolyte, this behavior was no longer verified, and the pH was stable as the initial tests to evaluate the equipment sensitivity. Conclusions After the experimental period involving focused intention, the pH system maintained a systematic and consistent behavior while measuring the pH of new water samples. An eventual intention-mediated conditioning of the pH measurement system occurred because of changes in the properties of the electrode internal electrolyte.

MobilityAnalyser: A novel approach for automatic quantification of cell mobility on periodic patterned substrates using brightfield microscopy images.

BACKGROUND AND OBJECTIVE: Surface topography of biomaterials has been shown to have an effect on cells behaviour. Cell-material interactions can be visually characterized by assessing both cell shape and spreading at initial time-points and, its migration patterns, as a response to the underlying topography. Whilst many have reported the study of cell migration and shape with fluorescence labelling, the focus on evaluating cells response to surface topography is to observe, under real-time conditions, interactions between cells and surfaces. In this manuscript we present a novel approach to automatically detect and remove periodic background patterns in brightfield microscopy images in order to perform automatic cell mobility analysis. METHODS: The developed software, MobilityAnalyser, performs automatic tracking of unmarked cells and allows the user to manually correct any incorrectly detected or tracked cell. Human Mesenchymal Stem Cells (hMSCs) trajectory, migration distance, velocity and persistence were evaluated over line and pillar micropatterned SiO2 films and on a flat SiO2 control substrate. RESULTS: The developed software proved to be effective in automatically removing background patterns of both line and pillar shapes and in performing cell detection and tracking. MobilityAnalyser accurately measured cell mobility in a fraction of the time required for manual analysis and eliminated user subjectivity. The results obtained with the software confirmed how different topographies affect cell trajectory, migration pathways and velocities, with a statistically significant increase for micropatterned surfaces, when compared with the flat control. The persistence parameter also proved the influence of both patterns on the directionality of cell movement. CONCLUSIONS: MobilityAnalyser is an automatic tool that removes periodic background patterns, detects and tracks cells, and provides cell mobility parameters that characterize the response of cells to different surface topographies. The software is freely available at: https://drive.google.com/open?id=1Fbb321ogLD19SlRjceMETNUqDHgpeBPl.

Instrumental Measurements of Water and the Surrounding Space During a Randomized Blinded Controlled Trial of Focused Intention.

The main goal of this work was the assessment of measurable interactions induced by focused intention, frequently used in biofield practices such as Healing Touch and Reiki. Water, as the main component of the human body, was chosen as a model. Intention experiments were performed over 4 different days at a scheduled interval, during which 286 trained biofield practitioners from several countries were instructed to meditate with the intention to change the molecular vibrational state of water samples selected by a blinded operator. The experimental protocol was randomized, blinded, and controlled; the measured variables included Raman spectra and the pH and electrical conductance of the water, as well as the magnetic field and UV-VIS (ultraviolet-visible) radiation near the experimental spot. Although a direct causal relationship cannot be established, some measurements of the water samples, as well as the magnetic field and radiation near the experimental spot, were responsive during the experimental period.

Enhanced biosafety of silica coated gadolinium based nanoparticles.

One of the most important and novel approaches of biomedical engineering is the development of new, effective and non-invasive medical diagnosis abilities, and treatments that have such requirements as advanced technologies for tumor imaging. Gadolinium (Gd) compounds can be used as MRI contrast agents, however the release of Gd3+ ions presents some adverse side effects such as renal failure, pancreatitis or local necrosis. The main aim of the work was the development and optimization of Gadolinium based nanoparticles coated with silica to be used as bioimaging agent. Gd based nanoparticles were prepared through a precipitation method and afterwards, these nanoparticles were covered with silica, using Stöber method with ammonia and functionalized with 3-Aminopropyltriethoxysilane (APTES). Results showed that nanoparticles were homogeneous regarding chemical composition, silica layer thickness, total size and morphology. Also, silica coating was successfully not degraded after 4 weeks at pH 5.5, 6.0 and 7.4, contrary to GdOHCO3 nanoparticles that degraded. Regarding the in vitro cell tests, very good cell proliferation and viability were observed. In conclusion, the results showed that Gd based nanoparticles coated with silica for imaging applications were successfully obtained under a well-controlled method. Furthermore, silica coating may enhance magnetic nanoparticles biosafety because it avoids GdOHCO3 degradation into harmful products (such as Gd3+ ions) at physiological conditions.

Biodegradation, biocompatibility, and osteoconduction evaluation of collagen-nanohydroxyapatite cryogels for bone tissue regeneration.

Designing biomimetic biomaterials inspired by the natural complex structure of bone and other hard tissues is still a challenge nowadays. The control of the biomineralization process onto biomaterials should be evaluated before clinical application. Aiming at bone regeneration applications, this work evaluated the in vitro biodegradation and interaction between human bone marrow stromal cells (HBMSC) cultured on different collagen/nanohydroxyapatite cryogels. Cell proliferation, differentiation, morphology, and metabolic activity were assessed through different protocols. All the biocomposite materials allowed physiologic apatite deposition after incubation in simulated body fluid and the cryogel with the highest nanoHA content showed to have the highest mechanical strength (DMA). The study clearly showed that the highest concentration of nanoHA granules on the cryogels were able to support cell type's survival, proliferation, and individual functionality in a monoculture system, for 21 days. In fact, the biocomposites were also able to differentiate HBMSCs into osteoblastic phenotype. The composites behavior was also assessed in vivo through subcutaneous and bone implantation in rats to evaluate its tissue-forming ability and degradation rate. The cryogels Coll/nanoHA (30 : 70) promoted tissue regeneration and adverse reactions were not observed on subcutaneous and bone implants. The results achieved suggest that scaffolds of Coll/nanoHA (30 : 70) should be considered promising implants for bone defects that present a grotto like appearance with a relatively small access but a wider hollow inside. This material could adjust to small dimensions and when entering into the defect, it could expand inside and remain in close contact with the defect walls, thus ensuring adequate osteoconductivity.