Glucocorticoids known to be such powerful agents that cell growth, differentiation and cell death are influenced in the brain of mammals throughout life. Despite this, relatively little toxicological information regarding prenatal exposure is available. The aim of this study was to determine the effect of prenatal hydrocortisone exposure on cell viability and cell morphology in chick embryonic neurons. Four different histological staining techniques namely, Hematoxylin and Eosin (H&E), Cresyl Fast Violet, Silver impregnation and a combination of Gold Chloride and Toluidine Blue were used to evaluate chick embryo neural tissue exposed to 0.137ƒÝM or 0.685ƒÝM hydrocortisone on day 3.75 (Carnegie stage 16) and day 5.5 (Carnegie stage 18) of development. Histological processing was optimized and neural tissue evaluated for any changes in neuron morphology and cell number. Specific ultrastructural changes to membraneous structures were evaluated by transmission electron microscopy (TEM). Fixation procedures that resulted in little to no disruption of these structures were optimized and used in studies evaluating the effect of hydrocortisone on neuron morphology. Primary chick embryonic neuronal cultures were prepared and increasing concentrations of hydrocortisone (26.3nM, 0.16ƒÝM, 0.63ƒÝM, 3.8ƒÝM, and 22.8ƒÝM) added. Fluorescence microscopy was applied to the in vitro hydrocortisone exposed primary neuronal cultures. A combination of fluorescein diacetate (FDA) and propidium iodide (PI) was used to evaluate the effect of hydrocortisone on cell viability, whereas dichlorodihydrofluorescein diacetate (DCH2FDA) was used to visualize reactive oxygen species (ROS) generation in neurons. Histological evaluation of the neural tissue of chick embryos exposed to 0.137ƒÝM and 0.685ƒÝM hydrocortisone showed reduced neuron density and morphological changes associated with cell death. Glutaraldehyde with added magnesium chloride (MgCl2) as stabilizing chemical and potassium permangenate were two fixatives that caused minimal disruption to neural tissue. These two fixating methods were applied to control neural tissue as well as tissues exposed to 0.137ƒÝM and 0.685ƒÝM hydrocortisone. When evaluated by TEM, the control tissue appeared to be intact with no displacement. Exposure of neurons to 0.137ƒÝM hydrocortisone appeared to have severe effects on the morphology of the mitochondria, endoplasmic reticulum (ER), nuclear and plasma membranes. More extensive damage was noted with 0.685ƒÝM hydrocortisone, leaving almost no cellular structure. Both concentrations of hydrocortisone indicated cell death associated with apoptosis and necrosis. In vitro studies using primary cultures of chick neurons indicated that hydrocortisone is non-toxic at low concentrations (26.3nM ¡V 3.8ƒÝM) with the percentage viability ranging between 73% and 88%. A more toxic effect was seen at high concentrations (22.8ƒÝM). Cell death at the higher concentrations (22.8ƒÝM and 3.8ƒÝM) of hydrocortisone occurred due to ROS generation, as indicated by DCH2FDA fluorescence In conclusion, hydrocortisone indicated neurotoxicity at high concentrations of exposure. Although cell death could be detected, the exact mechanism (apoptosis or necrosis) still needs to be investigated. Since the developing brain is so susceptible to chemical insults care should be taken when administering this drug to pregnant mothers or young children. / Dissertation (MSc (Cell Biology)))--University of Pretoria, 2007. / Anatomy / unrestricted

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

Tissue engineering (TE) uses a combination of biomaterial scaffolds, cells, and drug delivery systems (DDS) to create tissues that resemble the human physiology. Such engineered tissues could be used to treat, repair, replace, and augment damaged tissues or organs, for disease modeling, and drug screening purposes. This work describes the development and use of novel strategies for engineering neural tissue using a combination of drug delivery systems (DDS), human induced pluripotent stem cells (hiPSCs), and bioprinting technologies for the generation of a drug screening tool to be used in the process of drug discovery and development. The DDS consisted of purmorphamine (puro) loaded microspheres that were fabricated using an oil-in-water single emulsion with 84% encapsulation efficiency and showed the slow release of puro for up to 46 days in vitro. Puro and retinoic acid (RA)-loaded microspheres were combined with hiPSCs-derived neural aggregates (NAs) that differentiated into neural tissues expressing βT-III and showed increased neural extension. hiPCS-derived neural progenitor cells (NPCs) were bioprinted on a layer-by-layer using a fibrin based-bioink and extrusion based- bioprinting. The bioprinted structures showed >81% cellular viability after 7 days of culture in vitro and the expression of the mature motor neuron (MN) markers HB9 and CHAT. Lastly, hiPCS-derived NPCs were bioprinted in combination with puro and RA-loaded microspheres and cultured for 45 days in vitro. The microspheres slowly released the drug and after 30 and 45 days the tissues contained mature neurons, astrocytes and oligodendrocytes expressing CHAT, GFAP, and O4, respectively. Changes in membrane potential indicated tissue responsiveness to different types of treatments such as acetylcholine and gamma-aminobutyric acid (GABA). In the future the bioprinted tissues could contain localized regions of varied drug releasing microspheres using a concentration gradient to promote differentiation into specific cell types in order to create more complex tissues. Moreover, these tissues will benefit from the presence of a neurovascular unit (NVU). Upon validation, the engineered tissues could be used as preclinical tools to test potential drugs and be used for personalized medicine by using patient specific hiPSCs. / Graduate / 2020-11-19

Despite tremendous progress in medicine, injuries of the adult central neural system remain without satisfactory solution. Regenerative medicine employs tissue engineering, cellular therapies, medical devices, gene therapy, or growth factors with the aim to bridge the lesion, re-establish lost connections and enhance endogenous repair in order to restore neural function. The aim of my thesis was to evaluate therapeutic potential of two approaches, transplantation of human mesenchymal stromal cells (hMSCs) and biological scaffolds derived from extracellular matrix (ECM) for neural regeneration, particularly in models of spinal cord injury (SCI). First, hMSCs from various sources - bone marrow (BM), adipose tissue (AT) and Wharton's jelly (WJ) - were isolated and characterized in vitro. All cell types met the minimal criteria for MSC phenotype and displayed similar properties in terms of their surface marker expression, differentiation potential, migratory capacity, and secretion of cytokines and growth factors. On the other hand, the cell yield from WJ and AT was significantly higher, and MSCs isolated from these tissues proliferated better than from BM. Therapeutic effect of intrathecal application of hWJ-MSCs was then evaluated in SCI compression model in rats. The effect of low (0.5 million) and...