Structural and functional properties of astrocytes on PCL based electrospun fibres.

Increasing evidences are demonstrating that structural and functional properties of non-neuronal brain cells, called astrocytes, such as those of cytoskeleton and of ion channels, are critical for brain physiology. Also, changes in astrocytes structure and function concur to and might determine the outcome of neuronal damage in acute neurological conditions or of chronic disease. Thus, the design and engineering of biomaterials that can drive the structural and functional properties of astrocytes is of growing interest for neuroregenerative medicine. Poly-ɛ-caprolactone (PCL), is FDA-approved polyester having excellent mechanical and chemical properties that can be tailored to obtain neural implants for regenerative purposes. However, the study on the use of PCL substrates for neuroregenerative purposes are mainly aimed at investigating the interaction of the material with neurons. Here, we report on the long-term viability, morphology, structural and functional properties of primary astrocytes grown on electrospun fibres of PCL (-GEL) and on blending of PCL and Gelatin protein (+GEL). We found that topography and morphological features of the substrate are the properties that mainly drives astrocytes adhesion and survival, over the long term, while they do not alter the cell function. Specifically, aligned PCL fibres induced in astrocytes a dramatic actin-cytoskeletal rearrangement as well as focal adhesion point number and distribution. Interestingly, structural changes observed in elongated astrocytes are not correlated with alterations in their electrophysiological properties. Our results indicated that PCL electrospun fibres are a permissive substrate that can be tuned to selectively alters astrocytes structural components while preserving astrocytes function. The results open the view for the use of PCL based electrospun fibres to target astrocytes for the treatment of brain dysfunction such as injuries or chronical disease.

Indentation probe with optical fibre array-based optical coherence tomography for material deformation.

We present a new optomechanical probe for mechanical testing of soft matter. The probe consists of a micromachined cantilever equipped with an indenting sphere, and an array of 16 single-mode optical fibres, which are connected to an optical coherence tomography (OCT) system that allows subsurface analysis of the sample during the indentation stroke. To test our device and its capability, we performed indentation on a PDMS-based phantom. Our findings demonstrate that Common Path (CP)-OCT via lensed optical fibres can be successfully combined with a microindentation sensor to visualise the phantom's deformation profile at different indentation depths and locations in real time. LAY DESCRIPTION: This work presents a new approach to simultaneously perform micro-indentation experiments and OCT imaging. An optical fiber array-based sensor is used to develop a new hybrid tool where micro-indentation is combined with optical coherence tomography. The sensor is therefore capable of compressing a sample with a small force and simultaneously collecting OCT depth profiles underneath and around the indentation point. This method offers the opportunity to characterize the mechanical properties of soft materials and simultaneously visualize their deformation profile. The ability to integrate OCT imaging with indentation technology is promising for the non-invasive and precise characterization of different soft materials.

In vivo characterization of chick embryo mesoderm by optical coherence tomography-assisted microindentation.

Embryos are growing organisms with highly heterogeneous properties in space and time. Understanding the mechanical properties is a crucial prerequisite for the investigation of morphogenesis. During the last 10 years, new techniques have been developed to evaluate the mechanical properties of biological tissues in vivo. To address this need, we employed a new instrument that, via the combination of micro-indentation with Optical Coherence Tomography (OCT), allows us to determine both, the spatial distribution of mechanical properties of chick embryos, and the structural changes in real-time. We report here the stiffness measurements on the live chicken embryo, from the mesenchymal tailbud to the epithelialized somites. The storage modulus of the mesoderm increases from (176 ± 18) Pa in the tail to (716 ± 117) Pa in the somitic region (mean ± SEM, n = 12). The midline has a mean storage modulus of (947 ± 111) Pa in the caudal (PSM) presomitic mesoderm (mean ± SEM, n = 12), indicating a stiff rod along the body axis, which thereby mechanically supports the surrounding tissue. The difference in stiffness between midline and presomitic mesoderm decreases as the mesoderm forms somites. This study provides an efficient method for the biomechanical characterization of soft biological tissues in vivo and shows that the mechanical properties strongly relate to different morphological features of the investigated regions.

Integrating Microstructured Electrospun Scaffolds in an Open Microfluidic System for in Vitro Studies of Human Patient-Derived Primary Cells.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

Investigating the Effects of Mechanical Stimulation on Retinal Ganglion Cell Spontaneous Spiking Activity.

Mechanical forces are increasingly recognized as major regulators of several physiological processes at both the molecular and cellular level; therefore, a deep understanding of the sensing of these forces and their conversion into electrical signals are essential for studying the mechanosensitive properties of soft biological tissues. To contribute to this field, we present a dual-purpose device able to mechanically stimulate retinal tissue and to record the spiking activity of retinal ganglion cells (RGCs). This new instrument relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with high-density multi-electrode array (HD-MEAs) to simultaneously record the spontaneous activity of the retina. In this paper, we introduce this instrument, describe its technical characteristics, and present a proof-of-concept experiment that shows how RGC spiking activity of explanted mice retinas respond to mechanical micro-stimulations of their photoreceptor layer. The data suggest that, under specific conditions of indentation, the retina perceive the mechanical stimulation as modulation of the visual input, besides the longer time-scale of activation, and the increase in spiking activity is not only localized under the indentation probe, but it propagates across the retinal tissue.

Micro-indentation and optical coherence tomography for the mechanical characterization of embryos: Experimental setup and measurements on chicken embryos.

The investigation of the mechanical properties of embryos is expected to provide valuable information on the phenomenology of morphogenesis. It is thus believed that, by mapping the viscoelastic features of an embryo at different stages of growth, it may be possible to shed light on the role of mechanics in embryonic development. To contribute to this field, we present a new instrument that can determine spatiotemporal distributions of mechanical properties of embryos over a wide area and with unprecedented accuracy. The method relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with Optical Coherence Tomography, which can reveal changes in tissue morphology and help the user identify the indentation point. To prove the working principle, we have collected viscoelasticity maps of fixed and live HH11-HH12 chicken embryos. Our study shows that the instrument can reveal correlations between tissue morphology and mechanical behavior. STATEMENT OF SIGNIFICANCE: Local mechanical properties of soft biological tissue play a crucial role in several biological processes, including cell differentiation, cell migration, and body formation; therefore, measuring tissue properties at high resolution is of great interest in biology and tissue engineering. To provide an efficient method for the biomechanical characterization of soft biological tissues, we introduce a new tool in which the combination of non-invasive Optical Coherence Tomography imaging and depth-controlled indentation measurements allows one to map the viscoelastic properties of biological tissue and investigate correlations between local mechanical features and tissue morphology with unprecedented resolution.

70 µm diameter optical probe for common-path optical coherence tomography in air and liquids.

We investigate and validate a novel method to fabricate ultrathin optical probes for common-path optical coherence tomography (CP-OCT). The probes are obtained using a 65 µm barium titanate microsphere inserted into an inward concave cone chemically etched at the end of a single-mode fiber. We demonstrate that the high refractive index (n=1.95) of the barium titanate microspheres allows one to maintain high sensitivity even while imaging in liquids, reaching a sensitivity of 83 dB. Due to its low cost, flexibility, and ease of use, the probe holds promise for the development of a new generation of ultrathin needle-based OCT systems.

Short-Term Degradation of Bi-Component Electrospun Fibers: Qualitative and Quantitative Evaluations via AFM Analysis.

Electrospun polymeric fibers are currently used as 3D models for in vitro applications in biomedical areas, i.e., tissue engineering, cell and drug delivery. The high customization of the electrospinning process offers numerous opportunities to manipulate and control surface area, fiber diameter, and fiber density to evaluate the response of cells under different morphological and/or biochemical stimuli. The aim of this study was to investigate-via atomic force microscopy (AFM)-the chemical and morphological changes in bi-component electrospun fibers (BEFs) during the in vitro degradation process using a biological medium. BEFs were fabricated by electrospinning a mixture of synthetic-polycaprolactone (PCL)-and natural polymers (gelatin) into a binary solution. During the hydrolytic degradation of protein, no significant remarkable effects were recognized in terms of fiber integrity. However, increases in surface roughness as well as a decrease in fiber diameter as a function of the degradation conditions were detected. We suggest that morphological and chemical changes due to the local release of gelatin positively influence cell behavior in culture, in terms of cell adhesion and spreading, thus working to mimic the native microenvironment of natural tissues.

Atomic Force Microscopy: A Powerful Tool to Address Scaffold Design in Tissue Engineering.

Functional polymers currently represent a basic component of a large range of biological and biomedical applications including molecular release, tissue engineering, bio-sensing and medical imaging. Advancements in these fields are driven by the use of a wide set of biodegradable polymers with controlled physical and bio-interactive properties. In this context, microscopy techniques such as Atomic Force Microscopy (AFM) are emerging as fundamental tools to deeply investigate morphology and structural properties at micro and sub-micrometric scale, in order to evaluate the in time relationship between physicochemical properties of biomaterials and biological response. In particular, AFM is not only a mere tool for screening surface topography, but may offer a significant contribution to understand surface and interface properties, thus concurring to the optimization of biomaterials performance, processes, physical and chemical properties at the micro and nanoscale. This is possible by capitalizing the recent discoveries in nanotechnologies applied to soft matter such as atomic force spectroscopy to measure surface forces through force curves. By tip-sample local interactions, several information can be collected such as elasticity, viscoelasticity, surface charge densities and wettability. This paper overviews recent developments in AFM technology and imaging techniques by remarking differences in operational modes, the implementation of advanced tools and their current application in biomaterials science, in terms of characterization of polymeric devices in different forms (i.e., fibres, films or particles).

Optimization of fully aligned bioactive electrospun fibers for "in vitro" nerve guidance.

Complex architecture of natural tissues such as nerves requires the use of multifunctional scaffolds with peculiar topological and biochemical signals able to address cell behavior towards specific events at the cellular (microscale) and macromolecular (nanoscale) level. In this context, the electrospinning technique is useful to generate fiber assemblies having peculiar fiber diameters at the nanoscale and patterned by unidirectional ways, to facilitate neurite extension via contact guidance. Following a bio-mimetic approach, fully aligned polycaprolactone fibers blended with gelatin macromolecules have been fabricated as potential bioactive substrate for nerve regeneration. Morphological and topographic aspects of electrospun fibers assessed by SEM/AFM microscopy supported by image analyses elaboration allow estimating an increase of fully aligned fibers from 5 to 39% as collector rotating rate increases from 1,000 to 3,000 rpm. We verify that fully alignment of fibers positively influences in vitro response of hMSC and PC-12 cells in neurogenic way. Immunostaining images show that the presence of topological defects, i.e., kinks--due to more frequent fiber crossing--in the case of randomly organized fiber assembly concurs to interfere with proper neurite outgrowth. On the contrary, fully aligned fibers without kinks offer a more efficient contact guidance to direct the orientation of nerve cells along the fibers respect to randomly organized ones, promoting a high elongation of neurites at 7 days and the formation of bipolar extensions. So, this confirms that the topological cue of fully alignment of fibers elicits a favorable environment for nerve regeneration.