Quantitative ultrasound imaging of cell-laden hydrogels and printed constructs

In the present work we have revisited the application of quantitative ultrasound imaging (QUI) to cellular hydrogels, by using the reference phantom method (RPM) in combination with a local attenuation compensation algorithm. The investigated biological samples consisted of cell-laden collagen hydrogels with PC12 neural cells. These cell-laden hydrogels were used to calibrate the integrated backscattering coefficient (IBC) as a function of cell density, which was then used to generate parametric images of local cell density. The image resolution used for QUI and its impact on the relative IBC error was also investigated. Another important contribution of our work was the monitoring of PC12 cell proliferation. The cell number estimates obtained via the calibrated IBC compared well with data obtained using a conventional quantitative method, the MTS assay. Evaluation of spectral changes as a function of culture time also provided additional information on the cell cluster size, which was found to be in close agreement with that observed by microscopy. Last but not least, we also applied QUI on a 3D printed cellular construct in order to illustrate its capabilities for the evaluation of bioprinted structures. Statement of Significance: While there is intensive research in the areas of polymer science, biology, and 3D bio-printing, there exists a gap in available characterisation tools for the non-destructive inspection of biological constructs in the three-dimensional domain, on the macroscopic scale, and with fast data acquisition times. Quantitative ultrasound imaging is a suitable characterization technique for providing essential information on the development of tissue engineered constructs. These results provide a detailed and comprehensive guide on the capabilities and limitations of the technique

Towards an in vitro innervated model of the cornea

Visual impairment due to corneal disease is a global health concern with few FDA-approved pharmaceuticals being developed. In the cornea, the interactions between the various cell types present are essential for its functioning. In particular, innervation through sensory nerves is crucial for optimal functioning of this tissue. However, the mechanisms underlying these interactions are poorly understood, and representative 3D innervated in vitro cornea models could be used as systems to model the native situation. Therefore, an innervated model of the cornea is proposed and initiated. Electrocompacted collagen constructs serve as a basis for mimicking the cornea, and its mechanical, optical, and degradative properties are shown to be favorable. Furthermore, three dimensional extrusion- based printing has been employed to print methacrylated gelatin, and this scaffold was shown to support neuronal cell survival (83.4% viability 1 day after printing). A sustained release of neural growth factor to induce differentiation was established through incorporation of growth- factor loaded microparticles within the electrocompacted collagen. Additionally, the bioactivity was confirmed through an in vitro PC12 cell assay. The two biomaterials have been interfaced to fabricate a model to guide neuronal innervation. The current model shows potential in mimicking the complex structure of the cornea, but some optimization is required for neurite outgrowth. In the future, a viable in vitro corneal model could be used to provide fundamental insight into the process of corneal innervation and corneal diseases, as well as pre-clinical toxicity testing of new ocular drugs

Preparation of oriented neuronal supports

Tese de Mestrado. Engenharia Biomédica. Faculdade de Engenharia. Universidade do Porto. 201

Investigation and Characterization of PC12 Cells Adhesion, Proliferation, and Regeneration on Aerogels as a function of Topography and Stiffness.

Limitations with existing methods and materials used for nerve repair shows the significant interest to keep researching new materials for nerve implants. Recent studies show the importance of material properties to cell behavior. This body of work focuses on understanding the effect of material properties on the behavior of PC12 neurons. The first part of the work attemepts to quantify the relationships between cell parameters and substrate properties. Based on this study, random topographies with surface roughness of 0.5 µm and Young’s modulus of 2MPa are the optimum substrates for neurite outgrowth in PC12 cells. The second part investigates the feasibility of creating precise patterns on aerogel susbtrates for alignment by means of plasmonic photopatterning techniques. Those results have shown a degree of alignment in neurites due to the continuous topography

Interfacing graphene with peripheral neurons: influence of neurite outgrowth and NGF axonal transport

Graphene displays properties that make it appealing for neuroregenerative medicine, yet the potential of large-scale highly-crystalline graphene as a conductive peripheral neural interface has been scarcely investigated. In particular, pristine graphene offers enhanced electrical properties that can be advantageous for nervous system regeneration applications. In this work, we investigate graphene potential as peripheral nerve interface. First, we perform an unprecedented analysis aimed at revealing how the typical polymeric coatings for neural cultures distribute on graphene at the nanometric scale. Second, we examine the impact of graphene on the culture of two established cellular models for peripheral nervous system: PC12 cell line and primary embryonic rat dorsal root ganglion (DRG) neurons, showing a better and faster axonal elongation using graphene. We then observe that the axon elongation in the first days of culture correlates to an altered nerve growth factor (NGF) axonal transport, with a reduced number of retrogradely moving NGF vesicles in favor of stalled vesicles. We thus hypothesize that the axon elongation observed in the first days of culture could be mediated by this pool of NGF vesicles locally retained in the medial/distal parts of axons. Furthermore, we investigate electrophysiological properties and cytoskeletal structure of peripheral neurons. We observe a reduced neural excitability and altered membrane potential together with a reduced inter-microtubular distance on graphene and correlate these electrophysiological and structural reorganizations of axon physiology to the observed vesicle stalling. Finally, the potential of another 2D material as neural interface, tungsten disulfide, is explored

An implantable nano-enabled bio-robotic intracranial device for targeted and prolonged drug delivery

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctor of PhilosophyAlzheimer’s disease (AD) is the most prevalent and progressive neurodegenerative disorder (ND). It is characterized by a progressive decline of cognitive function, complete loss of memory, deterioration of visual capacity and the inability to function independently. According to the World Health Organization (WHO) it is estimated that about 26 million people suffer with AD worldwide. Although the etiology of AD is not fully understood, the aggregation of β-amyloidal (A) peptides that are associated with the formation of extracellular neurotoxin senile plaques and neurofibrillary tangles comprising hyperphosphorylated tau proteins have been recognized as the primary constituents that play a crucial role in AD. Several potential neurotherapeutic agents that can improve the management of AD such as metal chelators and alkaloid drugs have been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). Metal chelators [e.g. histidine, Ethylenediaminetetraacetic acid (EDTA) and zinc acetate (ZnAc)] are the main therapy used for modulating Aβ peptide aggregation with biological metals (such as zinc and copper ions) which is associated with promoting neurotoxicity in AD. While alkaloid drugs, such as donepezil, galantamine and rivastigmine, are used to inhibit the enzyme acetylcholinesterase (AChE); memantine is used to block the N-methyl-D-aspartate (NMDA) receptors associated with pathological activation. Despite the availability of these indispensable drugs, the clinical utility of these drugs is hampered by their poor retention and difficulty in bypassing the highly restrictive Blood Brain Barrier (BBB). Therefore this study aimed at developing novel nanoliposomes (NLPs) surface-engineered with chelating and synthetic peptides that are capable of crossing the BBB thus improving delivery efficacy and modulating the extracellular neurotoxicity associated with β-Amyloid aggregates of AD. Furthermore, since this system was designed for a chronic condition, a temporary depot-based polymeric system was integrated for further enhancement of the liposomal half-life, storage and prolonged drug delivery over a period of 50 days. The surface-engineered NLPs produced were spherical in shape, 100-149±28nm ~ size, with a zeta potential range of -9.59 to -37.3mV and a polydispersity index (PdI) of 0.02-0.2. A Box-Behnken experimental design was employed for maximizing the ligand coupling efficiency (40-78%) and drug entrapment efficiency (DEE) that ranged from 42-79%. The optimized peptide-based ligand NLP formulation showed sustained drug release (30% of drug released within 48 hours). Chelating ligands on the surface of NLPs showed 50-68% modulation of neurotoxicity on PC12 neuronal cells induced by ZnAβ (1-42) or CuAβ (1-42) aggregates. When drug-loaded functionalized NLPs were embedded within the temporal hydrophilic hydrogel network/scaffold as an implantable nano-enabled bio-robotic intracranial device (BICD), the physicomechanical and physicochemical dynamics showed improvement of liposomal structure such as the stability, and homogeneity in distribution of the liposomes within the internal core of the hydrogel networks and post-lyophilized scaffold. In vitro studies in simulated cerebrospinal fluid (CSF) showed prolonged release behavior of the drug-loaded functionalized NLPs from the BICD with 50-70% released over 50 days. Scanning Electron Microscopy (SEM) and confocal microscopy confirmed intact liposomal structures within the temporal polymeric scaffold/depot post-fixation and post-lyophilization. Ex vivo studies confirmed cell proliferation and a low level of lactate dehydrogenase (LDH), which is associated with cell membrane damage/injury, after PC12 neuronal cells were exposed to the BICD. In addition, when PC12 neuronal cells were exposed to the BICD high accumulation of galantamine (GAL) into these PC12 neuronal cells was observed post-cultivation. This outcome indicated that the released drug-loaded functionalized NLPs from the BICD were still in their intact form and capable of serving as bio-robotic markers for the delivery of GAL into the neuronal cells in response to AD. Furthermore, intracellular activity validated that the synthetic peptide has the potency for targeted delivery of the drug-loaded NLPs post-release of the BICD in ex vivo studies. Overall, results from this study revealed that the BICD device had superior cytocompatibility and may be suitable for application as a prolonged and targeted delivery system for GAL into neuronal cells to treat AD

Diamond-based sensors for in vitro cellular radiobiology: Simultaneous detection of cell exocytic activity and ionizing radiation

The investigation of secondary effects induced by ionizing radiation represents a new and ever-growing research field in radiobiology. This new paradigm cannot be investigated only using standard instrumentation and methodologies, but rather requires novel technologies to achieve significant progress. In this framework, we developed diamond-based sensors that allow simultaneous real-time measurements with a high spatial resolution of the secretory activity of a network of cells cultured on the device, as well as of the dose at which they are exposed during irradiation experiments. The devices were functionally characterized by testing both the above-mentioned detection schemes, namely: amperometric measurements of neurotransmitter release from excitable cells (such as dopamine or adrenaline) and dosimetric evaluation using different ionizing particles (alpha particle and X-ray photons). Finally, the sensors were employed to investigate the effects induced by X-rays on the exocytotic activity of PC12 neuroendocrine cells by monitoring the modulation of the dopamine release in real-time