>Magister Scientiae - MSc / Rapid industrialization growth without appropriate emission standards for discharging environmental pollutants had exposed the ecosystem to detrimental effect. Due to their bioaccumulation propensity in organisms, toxicity and non-biodegradable aspect, heavy metal spoliation is currently a significant ecological obligation. Owing to their natural abundance and extensive use, cadmium (Cd) and lead (Pb), among different heavy metals, are the predominant toxic metals. The intrusion of Cd and Pb metal ions into the ecosystem is accomplished by widely accepted methods such as anticorrosive coating of steel, electronic circuit and batteries. / 2023

Conventionally, adherent cells are cultured in vitro using flat 2D cell culture trays. However the 2D cell culture method is tedious, unreliable and does not replicate the complexity of the 3D dynamic environment of native tissue. Nowadays 3D scaffolds can be used to culture cells. However a number of challenges still exist, including the need for destructive enzymes to release confluent cells. Poly(Nisopropylacrylamide) (PNIPAAm), a temperature responsive polymer, has revolutionised the cell culture fraternity by providing a non-invasive means of harvesting adherent cells, whereby confluent cells can be spontaneously released by simply cooling the cell culture medium and without requiring enzymes. While PNIPAAm monolayer cell culturing is a promising tool for engineering cell sheets, the current technology is largely limited to the use of flat 2D substrates, which lacks structural and organisational cues for cells. The aim of this project was to develop a 3D PNIPAAm scaffold which could be used efficiently for non-invasive 3D culture of adherent cells. This project was divided into three phases: Phase 1 (preliminary phase) involved development and characterisation of cross-linked PNIPAAm hydrogels; Phase 2 involved development and characterisation of PNIPAAm grafted 3D non-woven scaffolds, while Phase 3 focused on showing proof of concept for non-invasive temperature-induced cell culture from the 3D PNIPAAm grafted scaffolds. In Phase 1, PNIPAAm was cross-linked with N,N’-methylene-bis-acrylamide (MBA) using solution free-radical polymerisation to form P(PNIPAAm-co-MBA) hydrogels. A broad cross-link density (i.e. 1.1 - 9.1 Mol% MBA) was investigated, and the effect of using mixed solvents as the co-polymerisation medium. The P(PNIPAAm-co-MBA) gels proved unsuitable as a robust cell culture matrix, due to poor porosity, slow swelling/deswelling and poor mechanical properties. Subsequently, in Phase 2, polypropylene (PP), polyethylene terephthalate (PET), and nylon fibers were processed into highly porous non-woven fabric (NWF) scaffolds using a needle-punching technology. The NWF scaffolds were grafted with PNIPAAm using oxyfluorination-assisted graft polymerisation (OAGP). The OAGP method involved a 2 step process whereby the NWF was first fluorinated (direct fluorination or oxyfluorination) to introduce new functional groups on the fibre surface. The functionalised NWF scaffolds were then graft-polymerised with NIPAAm in an aqueous medium using ammonium persulphate as the initiator. Following oxyfluorination, new functional groups were detected on the surface of the NWF scaffolds, which included C-OH; C=O; CH2-CHF, and CHF-CHF. PP and nylon were both easily modified by oxyfluorination, while PET displayed very little changes to its surface groups. Improved wetting and swelling in water was observed for the oxyfluorinated polymers compared to pure NWF scaffolds. PP NWF showed the highest graft yield followed by nylon and then PET. PNIPAAm graft yield on the PP NWF was ~24 ±6 μg/cm2 on grafted pre-oxyfluorinated NWF when APS was used; which was found to be significantly higher compared to when pre-oxyfluorinated NWF was used without initiator (9 ±6 μg/cm2, p= 1.7x10-7); or when grafting was on pure PP with APS (2 ±0.3 μg/cm2, p = 8.4x10-12). This corresponded to an average PNIPAAm layer thickness of ~220 ±54 nm; 92 ± 60 nm; and 19 ± 3 nm respectively. Scanning electron microscopy (SEM) revealed a rough surface morphology and confinement of the PNIPAAm graft layer to the surface of the fibers when oxyfluorinated NWF scaffolds were used, however when pure NWF scaffolds were used during grafting, homopolymerisation was observed as a loosely bound layer on the NWF surface. The OAGP method did not affect the crystalline phase of bulk PP as was determined by X-ray diffraction (XRD), however, twin-melting thermal peaks were detected from DSC for the oxyfluorinated PP and PP-g-PNIPAAm NWF which possibly indicated crystal defects. Contact angle studies and microcalorimetric DSC showed that the PP-g-PNIPAAm NWF scaffolds exhibited thermoresponsive behaviour. Using the 2,2-Diphenyl-1-1-picrylhydrazyl (DPPH) radical method and electron-spin resonance (ESR), peroxides, as well as trapped long-lived peroxy radicals were identified on the surface of the oxyfluorinated PP NWF, which are believed to be instrumental in initiating graft polymerisation from the NWF. A free radical mechanism which is diffusion controlled was proposed for the OAGP method with initiation via peroxy radicals (RO•), as well as SO4•- and OH• radicals, whereby the latter result from decomposition of APS. In Phase 3 of this study, proof-of-concept is demonstrated for use of the PNIPAAm grafted NWF scaffolds in non-invasive culture of hepatocytes. Studies demonstrated that hepatocyte cells attached onto the 3D PNIPAAm scaffolds and remained viable in culture over long periods. The cells were released spontaneously and non-destructively as 3D multi-cellular constructs by simply cooling the cell culture medium from 37°C to 20°C, without requiring destructive enzymes. The PP-g- PNIPAAm NWF scaffolds performed the best in 3D cell culture. Additionally the CSIR is developing a thermo responsive 3D (T3D) cell culturing device, whereby the 3D thermo responsive NWF scaffolds are used in the bioreactor for cell culture. Temperature-induced cell release was also verified from the 3D Thermo responsive scaffolds in the bioreactor. This technology could lead to significant advances in improving the reliability of the in vitro cell culture model. Please cite as follows: Chetty, AS 2012, Thermoresponsive 3D scaffolds for non-invasive cell culture, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06112013-151344/ > D13/4/713/ag / Thesis (PhD)--University of Pretoria, 2012. / Chemical Engineering / unrestricted

<p>Additive manufacturing (AM) of hydrogels has gained increasing interest across various fields. Drop-on-demand (DOD) printing (also known as inkjet printing) shows the great potential to construct 3D hydrogels with spatially controlled properties and compositions. However, a limited mechanistic understanding of the behavior of printed polymer drops makes it challenging to design and optimize DOD printing protocols for a wide variety of hydrogels. Here, we have demonstrated an extensive and in-depth study from the theoretical and experimental research of drop-wise structure to the development of functional materials by DOD printing of polymer inks. First, computational and experimental studies are performed to establish a mechanism of the water-matrix interaction within printed polymer drops. The results ultimately enabled a dimensionless parameter that characterizes water transport during the dehydration process of printed polymer drops. Next, as particles are suspended in polymer inks to add functionality, this dimensionless parameter was further extended to characterize particle movement and distribution patterns in the printed particle-laden hydrogels. By correlating the intra-drop particle distribution to the similarity parameter, a scaling law is confirmed to guide ink formulation and printing protocol that enables advanced materials with spatially digitized functionality (i.e., digital hydrogels). Finally, cells that serve as active particles are embedded in the hydrogels to mimic the native tissues. A "digital cell printing" method based on DOD printing of "two colors" cell-laden (i.e., cancer cells and CAFs) polymer inks is developed to rapidly (< 1 day) create 3D tumor models with tumor-stroma interface (i.e., tumoroids) and high cell density (~108 cells/cm3) that closely recapitulate the tumor microenvironment in vivo. Overall, DOD printing of particulate-laden polymer inks showed the great potential to construct 3D functional hydrogels with spatially controlled properties and compositions.</p>

Metastatic cancer is more dangerous and difficult to treat than pre-metastatic cancer. Ninety percent of cancer-related deaths are caused by metastatic cancer. When cells go through metastases, they go through changes that allow them to break away from the primary tumor and invade secondary tissues. These changes, in lipid membrane composition and cellular glycocalyx, make the cell more resistant to therapeutics. Actin cytoskeleton contractility plays a major role in these changes, as increased contractility has been linked to upregulation of phosphoinositides and production of glycoproteins. Light induced molecular adsorption of proteins (LIMAP) was used to control the actin arrangement and cell shape in order to mimic and study metastatic cells. Negatively charged proteins electrostatically adhere to the surface in order to create patterns for the cells to stick. Neutravidin was conjugated to poly(glutamic acid) to improve attachment to the surface. We observed differences in cell shape and phosphoinositide behavior based on LIMAP patterning. Additionally, expression of key glycoproteins related to cancer metastasis increased with increased actin contractility. The actin cytoskeleton was the main driver of changes to the cell membrane and glycocalyx.

Phononic crystals are structures composed of periodically arranged scatterers in a background medium that affect the transmission of elastic waves. They have garnered much interest in recent years for their macro-scale properties that can be modulated by the micro-scale components. The elastic properties of the composite materials, the contrast in the elastic properties of the composite materials, and the material arrangement all directly affect how an elastic wave will behave as it propagates through the sonic structure. The behavior of an elastic wave in a periodic structure is revealed in its transmission bandstructure, and modification of any the elastic parameters will result in tuning of the band structure. In this dissertation, a phononic crystal with properties that can be modulated using electromagnetic radiation, and more specifically, radio-frequency (RF) light will be presented.

Adenosine triphosphate (ATP) is the major carrier of chemical energy in cells. The diffusion of ATP in hydrogels, which have a structural resemblance to the natural extracellular matrix, is therefore of great importance to understand many biological processes. In continuation of our recent studies of ATP diffusion in poly(ethylene glycol) diacrylate (PEG-DA) hydrogels by pulsed field gradient nuclear magnetic resonance (PFG-NMR), we present precise diffusion measurements of ATP in aqueous solutions of PEG-DA polymers, which are not cross-linked to a three-dimensional network. The dependence of the ATP diffusion on the polymer volume fraction in the hydrogels, φ, was found to be consistent with the predictions of a modified obstruction model or the free volume theory in combination with the sieving behavior of the polymer chains. The present measurements of ATP diffusion in aqueous solutions of the polymers revealed that the diffusion coefficient is determined by φ only, regardless of whether the polymers are cross-linked or not. These results seem to be inconsistent with the free volume model, according to which voids are formed by a statistical redistribution of surrounding molecules, which is expected to occur more frequently in the case of not cross-linked polymers. The present results indicate that ATP diffusion takes place only in the aqueous regions of the systems, with the volume fraction of the polymers, including a solvating water layer, being blocked for the ATP molecules. The solvating water layer increases the effective volume of the polymers by 66%. This modified obstruction model is most appropriate to correctly describe the ATP diffusion in PEG-DA hydrogels.

New approaches for the treatment of SCI use advances in the fields of nanotechnology, biomaterial science and cell therapy. The results presented in this thesis showed that superparamagnetic iron oxide nanoparticles coated with a stable dopamine-hyaluronane associate can be used for the safe and effective labeling of MSC. Cell labeling efficiency, viability and the relaxivity of the tested particles were significantly better than those obtained with the commercial particles Endorem®. The DPA-HA coated nanoparticles can be used for the noninvasive monitoring of cell therapy using MRI. Furthermore, we showed that SPION can be used for the targeted delivery of MSC to the site of a spinal cord lesion. The labeled cells can be concentrated in the lesion area by means of a magnetic implant. The process of cell targeting depends on the physical characteristics of the magnetic implant as well as on the biological features of the cells and nanoparticles, as we described with a proposed mathematical model. It is possible to modify the properties of the magnetic system, e.g. by changing the shape or size of the magnet, thus tuning the magnetic force distribution and the gradient of the magnetic field necessary for effective cell targeting. A promising therapeutic strategy for the treatment of spinal cord injury is the...

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The use of autografts is the gold standard for peripheral nerve regeneration (PNR) while biomedical engineering made some contributions to improve PNR. A next generation of nerve guidance conduits (NGC) is required to transmit topographical and biochemical signals towards severed nerves. In this thesis, the gelatin hydrolyzate Collagel® (COL) and anhydride-containing cross-linkers (oPNMA, oPDMA) were used to fabricate crosslinked hydrogels (cGEL) for PNR. At first, established cGEL formulations were adjusted towards an injection-molding tool with static mixer. Therefore, the gelation kinetic was modified by variation of the gelation base. Hence, high reactive oPNMA was available for fabrication of robust cGEL based NGC. Secondly, novel cGEL and molding technique were adapted towards the fabrication of cGEL-based filler for polymer-derived braided NGC. Shear-thinning filler was developed that allowed direct application inside the conduit lumen with minimal mechanical stiffness but sufficient scaffolding properties. Besides pristine filler, chemically modified filler was designed with a small mimetic of the nerve growth factor, LM11A-31, that was grafted to oPNMA. In a rat sciatic nerve model, the performance of this derivatized filler was comparable to the control and underlined the potential of chemical cues in PNR. A number of small diamines were further integrated into oPNMA and oPDMA to modify cGEL bulk. In addition to chemical feasibility, the cytocompatibility and cellular response were tested on L929 mouse fibroblasts and human adipose-derived stem cells. The functionalization showed an impact on the cell behavior with differences in cell proliferation, migration and spreading. Finally, modified oPNMA-derived hydrogels were tested on neonatale Schwann cells. The cell viability and extension was maintained in all hydrogels while the impact of LM11A-31 was not as pronounced. This thesis emphasizes the potential of cGEL hydrogels in nerve implants as fillers or conduits and, thus, is a promising building block for a new generation of NGC.

The building blocks of human tissues are cells. The cells interact and respond to the characteristics of their local microenvironment. The cellular microenvironment is formed by three main components, the extracellular matrix, neighbouring cells and signalling molecules. Particularly, the extracellular matrix and neighbouring cells impose boundary conditions that limits the cell volume and cell spreading. However, these characteristics are often not present in traditional in vitro models, where cells experience a stiff and vast environment.   An approach to improve in vitro models is to use hydrogels, soft and highly hydrated polymers. Through chemical modifications, polymers naturally found in the extracellular matrix can be functionalized to form crosslinked hydrogels. Moreover, these functionalities can also be used to prepare micropatterns, micrometre sized cell adhesive areas on the hydrogels. These micropatterns guide the cell shape and permit the study of the cell response to these changes in shape, which has been observed in e.g. endothelial cells from various origins.   Taken all together, the aim of this work was to develop a hydrogel-based cell culture scaffold that permits the control of the spatial adhesion of brain endothelial cells in order to study the morphological effects on these cells and contribute to the understanding of the function of brain endothelial cells in health and disease.   This thesis demonstrates the functionalization of hyaluronic acid, a naturally occurring extracellular matrix polymer, to prepare photocrosslinkable hydrogels. Furthermore, through photolithography, micropatterns of cell adhesive peptides were prepared on these hydrogels. Brain microvascular endothelial cells, a highly specialized type of endothelial cells, adhered to the micropatterns, and the effect on their alignment depending on the micropatterned sized was studied. Furthermore, changes in their alignment were also observed when exposed to different glucose concentration.