Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

AbstractThe cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system

Biologically modified microelectrode sensors provide enhanced sensitivity for detection of nucleic acid sequences from Mycobacterium tuberculosis

This paper describes improved sensitivity when using biosensors based on microfabricated microelectrodes to detect DNA, with the goal of progressing towards a low cost and mass manufacturable assay for antibiotic resistance in tuberculosis (TB). The microelectrodes gave a near 20 times improvement in sensitivity compared to polycrystalline macroelectrodes. In addition, experimental parameters such as redox mediator concentration and experimental technique were investigated and optimised. It was found that lower concentrations of redox mediator gave higher signal changes when measuring hybridisation events and, at these lower concentrations, square wave voltammetry was more sensitive and consistent than differential pulse voltammetry. Together, this paper presents a quantifiable comparison of macroelectrode and microelectrode DNA biosensors. The final assay demonstrates enhanced sensitivity through reduction of sensor size, reduction of redox mediator concentration and judicious choice of detection technique, therefore maintaining manufacturability for incorporation into point of care tests and lab-on-a-chip devices

A textile platform using mechanically reinforced hydrogel fibres towards engineering tendon niche

INTRODUCTION: Tendon injuries can result from tendon overuse or trauma, resulting in substantial pain and disability. Given that natural or surgical repair of tendons lead to a poor outcome in terms of mechanical properties and functionality, there is a great need for tissue engineering strategies. Textile platforms enable the generation of biomimetic constructs [1]. Therefore, the main goal of this study is the development of cell-laden hybrid hydrogel fibers reinforced with a mechanically robust core fiber and their assembly into braided constructs towards replicating tendon mechanical properties and architecture. METHODS: To fabricate mechanically reinforced hydrogel fibres, a commercially available suture was coated using a cell-hydrogel mixture of methacryloyl gelatine (GelMA) and alginate. Composite fibres (CFs) were obtained by ionic crosslinking of alginate followed by photocrosslinking of GelMA. CFs were assembled using braiding technique and the mechanical properties of single fibres and braided constructs were evaluated. Different cells were encapsulated in the hydrogel layer, including MC-3T3, mesenchymal stem cells (MSCs) and human tendon-derived cells (TDCs). Cell viability and metabolic activity were evaluated by LIVE/DEAD staining and presto blue assay of metabolic activity. The expression of tendon-related markers and matrix deposition were also investigated. RESULTS: CFs were fabricated with a GelMA:alginate hydrogel layer and using multifilament twisted cotton or biodegradable suturing threads. The biocompatibility of this system was evaluated on encapsulated cells (Fig.1a). Cells (MC-3T3, MSCs and TDCs) were homogeneously distributed along the hydrogel layer, being viable up to 14 days in culture. In addition, TDCs were spreading inside the hydrogel after less than 48 h. Moreover, to further improve the mechanical properties of CFs, braided constructs were generated (Fig. 1b). Braiding CFs together enhanced their tensile strength and the process did not affect the viability of encapsulated cells.DISCUSSION & CONCLUSIONS: CFs were generated with a load bearing core and a hydrogel layer towards mimicking both mechanical properties and the matrix-rich microenvironment of tendon tissue. Accordingly, cell behaviour can be further modulated by modifying the hydrogel composition or, ultimately, through the addition of bioactive cues. Finally, braiding CFs together allows tuning the mechanical properties of developed constructs to match those of native tendon tissues.Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, the PhD grant SFRH/BD/96593/2013 of R.C-

Impedance testing of porous Si3N4 scaffolds for skeletal implant applications

Si3N4 ceramics show excellent characteristics of mechanical and chemical resistance in combination with good biocompatibility, antibacterial property and radiolucency. Therefore, they are intensively studied as structural materials in skeletal implant applications. Despite their attractive properties, there are limited data in the field about in vitro studies of cellular growth on ceramic implant materials. In this study, the growth of bone cells was investigated on porous Silicon Nitride (Si3N4) ceramic implant by using electrochemical impedance spectroscopy (EIS). Partial sintering was performed at 1700 °C with limited amount of sintering additive for the production of porous Si3N4 scaffolds. All samples were then sterilized by using ethylene oxide followed by culturing MG-63 osteosarcoma cells on the substrates for in vitro assays. At 20 and 36 hours, EIS was performed and results demonstrated that magnitude of the impedance as a result of the changes in the culture media increased after incubation with osteosarcoma cells. The changes are attributed to the cellular uptake of charged molecules from the media. Si3N4 samples appear to show large impedance magnitude changes, especially between 100 Hz and 1 Hz. Impedance changes were also correlated with WST-1 measurements (36 hr) and DAPI results

Machine learning based prediction of squamous cell carcinoma in ex vivo confocal laser scanning microscopy

Image classification with convolutional neural networks (CNN) offers an unprecedented opportunity to medical imaging. Regulatory agencies in the USA and Europe have already cleared numerous deep learning/machine learning based medical devices and algorithms. While the field of radiology is on the forefront of artificial intelligence (AI) revolution, conventional pathology, which commonly relies on examination of tissue samples on a glass slide, is falling behind in leveraging this technology. On the other hand, ex vivo confocal laser scanning microscopy (ex vivo CLSM), owing to its digital workflow features, has a high potential to benefit from integrating AI tools into the assessment and decision-making process. Aim of this work was to explore a preliminary application of CNN in digitally stained ex vivo CLSM images of cutaneous squamous cell carcinoma (cSCC) for automated detection of tumor tissue. Thirty-four freshly excised tissue samples were prospectively collected and examined immediately after resection. After the histologically confirmed ex vivo CLSM diagnosis, the tumor tissue was annotated for segmentation by experts, in order to train the MobileNet CNN. The model was then trained and evaluated using cross validation. The overall sensitivity and specificity of the deep neural network for detecting cSCC and tumor free areas on ex vivo CLSM slides compared to expert evaluation were 0.76 and 0.91, respectively. The area under the ROC curve was equal to 0.90 and the area under the precision-recall curve was 0.85. The results demonstrate a high potential of deep learning models to detect cSCC regions on digitally stained ex vivo CLSM slides and to distinguish them from tumor-free skin

Developing an Optical Microlever for Stable and Unsupported Force Amplification

— Optical micromachines have the potential to im prove the capabilities of optical tweezers by amplifying forces and allowing for indirect handling and probing of specimens. However, systematic design and testing of micromachine per formance is still an emerging field. In this work we have designed and tested an unsupported microlever, suitable for general-purpose optical tweezer studies, that demonstrates stable trapping performance and repeatable doubling of applied forces. Stable trapping was ensured by analysing images to monitor focus shift when levers oscillated repeatedly, before the best-performing design was selected for force amplification. This study also shows that direct measurement of trap stiffness using the equipartition theorem appears to be a valid method for measuring applied forces on the spherical handles of microlevers