A reproducible in vitro model of mechanically injured (scalpel cut) articular cartilage was developed in this work utilising bovine and human osteochondral tissue. Using fluorescence-mode confocal laser scanning microscopy (CLSM), the model allowed (1) spatial and temporal quantification of in situ (within the matrix) chondrocyte viability following a full thickness cartilage injury and (2) serial evaluation of three chondroprotective strategies in injured bovine and human articular cartilage: (a) medium osmolarity (b) medium calcium concentration and, (c) subchondral bone attachment to articular cartilage. Medium osmolarity significantly influenced superficial zone chondrocyte death in injured (scalpel cut) bovine and human articular cartilage. Greatest percentage cell death occurred at 0 mOsm (distilled water). Conversely, a raised medium osmolarity (600 mOsm) was chondroprotective. The majority of in situ cell death occurred within 2.5 hours of the experimental injury, with no further increase over 7 days. Exposure of articular cartilage to calcium-free media significantly decreased superficial zone chondrocyte death in injured (scalpel cut) articular cartilage compared with exposure to calcium-rich media (2-20 mM). In calcium-rich media, the extent of percentage cell death increased with increasing medium calcium concentration but remained localised to the superficial zone of injured articular cartilage over 7 days. However, in calcium-free media, there was an increase in percentage cell death within deeper zones of injured articular cartilage over 7 days. Excision of subchondral bone from injured (scalpel cut) articular cartilage resulted in an increase in chondrocyte death at 7 days that occurred in the superficial zone of injured as well as the adjacent uninjured regions of articular cartilage. However, the presence of subchondral bone in the culture medium prevented this increase in chondrocyte death within the superficial zone. Subchondral bone may have interacted with articular cartilage via soluble mediator(s) that influenced chondrocyte survival. In human articular cartilage, healthy subchondral bone also interacted with articular cartilage in explant culture and promoted in situ chondrocyte survival, while sclerotic subchondral bone was detrimental to chondrocyte viability. These findings are of translational relevance to fluid management systems used during open and arthroscopic articular surgery, clinical and experimental research into cartilage injury, repair and degeneration as well as current techniques of tissue engineering.

<p>This thesis considers the effect of manufacturing conditions and polymer ratio on water permeability and morphology of free-films. A novel spray method for producing ethyl cellulose (EC) and hydroxypropyl cellulose (HPC) free-films was developed where several process parameters was controlled. The process was optimised by pre-spraying solvent until the system reached a steady-state temperature. This minimised the variation of outlet air temperature to < 2.5 °C. Coating time was approximately 4 minutes excluding drying.</p><p>Free-films were produced using 94 wt% solvent (95 %-ethanol) and 6 wt% polymer. The amount of HPC in the films was varied (wt% HPC defined as HPC/(HPC+EC)*100). Films with 30-40-50-57 wt% HPC were studied. Phase diagrams was constructed to study the phase transformation of polymer mixtures. Results show that all polymer mixtures with HPC content above 30 wt% were phase separated prior to film manufacturing. Temperature had an effect on the polymer phase transformation. In the phase diagram, the 2-phase area was larger for temperatures above 40 °C.</p><p>The investigated manufacturing conditions were outlet air temperature (°C) and spray rate (g/min). Outlet air temperature was controlled by adjusting the inlet air temperature. The films were characterized by measuring water permeability (m²/s). Cross section structure of the films was analyzed with confocal laser scanning microscopy (CLSM). FITC-HPC was added for enhanced contrast between the domains.</p><p>Higher outlet air temperature gave higher water permeability of the film whereas higher spray rate gave lower water permeability. The outlet air temperature had an impact on evaporation rate. The evaporation rate together with spray rate affected the solidification and hence the structure of the film. Images show that longer solidification time smeared the domains into larger domains. Lower water permeability was caused by less connectivity between the pores.</p><p>In conclusion, experiments show that water permeability of EC/HPC free-films was highly dependent on the manufacturing conditions.</p><p></p>

This thesis considers the effect of manufacturing conditions and polymer ratio on water permeability and morphology of free-films. A novel spray method for producing ethyl cellulose (EC) and hydroxypropyl cellulose (HPC) free-films was developed where several process parameters was controlled. The process was optimised by pre-spraying solvent until the system reached a steady-state temperature. This minimised the variation of outlet air temperature to &lt; 2.5 °C. Coating time was approximately 4 minutes excluding drying. Free-films were produced using 94 wt% solvent (95 %-ethanol) and 6 wt% polymer. The amount of HPC in the films was varied (wt% HPC defined as HPC/(HPC+EC)*100). Films with 30-40-50-57 wt% HPC were studied. Phase diagrams was constructed to study the phase transformation of polymer mixtures. Results show that all polymer mixtures with HPC content above 30 wt% were phase separated prior to film manufacturing. Temperature had an effect on the polymer phase transformation. In the phase diagram, the 2-phase area was larger for temperatures above 40 °C. The investigated manufacturing conditions were outlet air temperature (°C) and spray rate (g/min). Outlet air temperature was controlled by adjusting the inlet air temperature. The films were characterized by measuring water permeability (m2/s). Cross section structure of the films was analyzed with confocal laser scanning microscopy (CLSM). FITC-HPC was added for enhanced contrast between the domains. Higher outlet air temperature gave higher water permeability of the film whereas higher spray rate gave lower water permeability. The outlet air temperature had an impact on evaporation rate. The evaporation rate together with spray rate affected the solidification and hence the structure of the film. Images show that longer solidification time smeared the domains into larger domains. Lower water permeability was caused by less connectivity between the pores. In conclusion, experiments show that water permeability of EC/HPC free-films was highly dependent on the manufacturing conditions.

The membrane aerated bioreactor (MABR) is a unique technological innovation where a gas permeable membrane is applied to biological processes. In an MABR, oxygen and other substrates diffuse from the opposite directions into a biofilm, and thus simultaneous chemical oxygen demand (COD) and nitrogen removal can be achieved. However, controlling biofilm thickness, stability, and attachment is challenging. The objectives of this research were to study the effect of oxygen partial pressure on process performance with respect to nitrogen removal and examine the biomass properties in MABRs at different oxygen partial pressures and COD loadings. The conditions within the bioreactors were based on a low hydrodynamic condition (average fluid velocity 22 cm/min along the membrane surface), with the intention of minimizing the impact of the hydrodynamic shear on biomass properties. Simultaneous nitrification and denitrification were achieved in the reactors, and increasing oxygen partial pressure enhanced the total nitrogen removal. The biomass at the membrane-biofilm interface was more porous at a loading of 11.3 kg COD/1000 m2/day (areal porosity about 0.9) as compared with a loading of 22.6 kg COD/1000 m2/day (areal porosity about 0.7), indicating carbon substrate was limiting near the membrane. Long-term (over 30 days) experimental results showed that at the loading of 11.3 kg COD/1000 m2/day, the oxygen partial pressures of 0.59 atm and 0.88 atm caused over 80% of the biomass to become suspended in the bulk phase while at 0.25 atm and 0.41 atm oxygen over 97% of the biomass was immobilized on the membrane. There is a critical oxygen partial pressure that can sustain the biofilm, which increases with an increasing COD loading. The nitrifying population in the reactors was examined by applying fluorescence in situ hybridization (FISH). At the loading of 22.6 kg COD/1000 m2/day, there were 12% beta-proteobacterial ammonia oxidizing bacteria (AOB) and 17%Nitrobacter in homogenized biofilm biomass at 0.59 atm oxygen while there were 7% beta-proteobacterial AOB and 4% Nitrobacter at 0.25 atm oxygen. The ratio of protein to carbohydrate in extracellular polymeric substances (EPS) of the homogenized biomass in the reactor decreased with increasing oxygen partial pressure. Surface characterization of the biomass revealed that the higher the oxygen partial pressure, the lower the biomass hydrophobicity and surface charge. The ratio of EPS protein to carbohydrate in a membrane aerated biofilm decreased when approaching the membrane-biofilm interface. The distribution of nitrifiers and dissolved oxygen profiles inside the biofilm suggested that dual substrate limitations exist, and it was concluded that the membrane aerated biofilm had an aerobic region in the inner layer and an anoxic region in the outer layer. It is proposed that the loss of EPS due to secondary substrate consumption, especially the loss of EPS proteins, at the bottom of the biofilm was responsible for biofilm detachment subjected to a critical oxygen partial pressure.

This dissertation presents techniques of three-dimensional microscopy. First, an economical method of microscopic image formation that employs a raster-scanning laser beam focused on a sample, while non-imaging detector receives the scattered light is presented. The images produced by this method are analogous to the scanning electron microscopy with visible effects of shadowing and reflection. Compared to a conventional wide-field imaging system, the system allows for a greater flexibility, as the variety of optical detectors, such as PMT and position-sensitive quadrant photodiode can be used to acquire images. The system demonstrates a simple, low-cost method of achieving the resolution on the order of a micron. A further gain in terms of resolution and the depth of focus by using Bessel rather than Gaussian beams is discussed. Then, a phase-imaging technique to quantitatively study the three-dimensional structure of reflective and transmissive microscopic samples is presented. The method, based on the simultaneous dual-wavelength digital holography, allows for higher axial range at which the unambiguous phase imaging can be performed. The technique is capable of nanometer axial resolution. The noise level, which increases as a result of using two wavelengths, is then reduced to the level of a single wavelength. The method compares favorably to software unwrapping, as the technique does not produce non-existent phase steps. Curvature mismatch between the reference and object beams is numerically compensated. The 3D images of porous coal samples and SKOV-3 ovarian cancer cells are presented.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Low back pain represents a significant concern in the United States, with 70% of individuals experiencing symptoms at some point in their lifetime. Although the specific cause of low back pain remains unclear, symptoms have been strongly associated with degeneration of the intervertebral disc. Insufficient nutritional supply to the disc is believed to be a major mechanism for tissue degeneration. Understanding nutrients' transport in intervertebral disc is crucial to elucidate the mechanisms of disc degeneration, and to develop strategies for tissue repair (in vivo), and tissue engineering (in vitro). Transport in intervertebral disc is complex and involves a series of electromechanical, chemical and biological coupled events. Despite of the large amount of studies performed in the past, transport phenomena in the disc are still poorly understood. This is partly due to the limited number of available experimental techniques for investigating transport properties, and the paucity of theoretical or numerical methods for systematically predicting the mechanisms of solute transport in intervertebral disc. In this dissertation, a theoretical and experimental approach was taken in order to investigate the mechanisms of solute transport and binding interactions in intervertebral disc. New imaging techniques were developed for the experimental determination of diffusive and binding parameters in biological tissues. The techniques are based on the principle of fluorescence recovery after photobleaching, and allow the determination of the anisotropic diffusion tensor, and the rates of binding and unbinding of a solute to the extracellular matrix of a biological tissue. When applied to the characterization of transport properties of intervertebral disc, these methods allowed the establishment of a relationship between solute anisotropic and inhomogeneous diffusivity and the unique morphology of human lumbar annulus fibrosus. A mixture theory for charged hydrated soft tissues was presented as a framework for theoretical investigations on solute transport and binding interactions in cartilaginous tissues. Based on this theoretical framework and on experimental observations, a finite element model was developed to predict solute diffusive-convective-reactive transport in cartilaginous tissues. The numerical model was applied to simulate the effect of mechanical loading on solute transport and binding interactions in cartilage explants and intervertebral disc.

Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community. / <p>Paid Open Access</p>

Purpose: To locate protein sorption on the surface and inside the matrix of soft contact lens materials and intraocular lenses (IOL). Methods: The proteins albumin and lysozyme were investigated as they are highly abundant in blood serum and tears, respectively. Proteins were conjugated with organic fluorescent probes and using confocal laser scanning microscopy (CLSM) the sorption profile to contact lenses and IOL could be determined. Radiolabeled protein was used for quantification purposes. • Albumin sorption to etafilcon A and lotrafilcon B was determined (Chapter 3) • Different fluorescent probes were used for conjugation and the impact on albumin sorption behaviour was investigated (Chapter 4) • Lysozyme sorption to nine different pHEMA-based and silicone hydrogel contact lenses was determined using two fluorescent probes (Chapter 5) • The efficiency of protein removal from contact lenses using contact lens care regimens was investigated (Chapter 6) • Albumin sorption to IOL materials was quantified and imaged using a modified CLSM technique (Chapter 7) Results: Albumin and lysozyme sorption profiles differed between materials, and were influenced by the fluorescent probes used for conjugation. After one day of incubation, both proteins could be located within all contact lens materials, except for lotrafilcon A and lotrafilcon B, which primarily allowed deposition on the lens surface. An increase in protein accumulation was found for most materials over the maximum investigated period of 14 days, using CLSM and radiolabel techniques. The efficiency of contact lens care regimens to remove lysozyme and albumin depended on the lens material, care regimen and protein type investigated. PMMA and silicone IOLs showed protein exclusively on the surface, while a hydrophilic acrylic IOL allowed penetration into the lens matrix over time. Despite the albumin penetration depth into hydrophilic acrylic, the highest albumin levels were determined for the silicone IOL. Conclusions: CLSM provides detailed information that can describe the protein distribution in transparent biomaterials, with scanning depths up to a few hundred microns. However, the CLSM data are primarily of qualitative value, which necessitates a quantitative technique (e.g. radiolabeling) to determine the total protein content.

Purpose: To locate protein sorption on the surface and inside the matrix of soft contact lens materials and intraocular lenses (IOL). Methods: The proteins albumin and lysozyme were investigated as they are highly abundant in blood serum and tears, respectively. Proteins were conjugated with organic fluorescent probes and using confocal laser scanning microscopy (CLSM) the sorption profile to contact lenses and IOL could be determined. Radiolabeled protein was used for quantification purposes. • Albumin sorption to etafilcon A and lotrafilcon B was determined (Chapter 3) • Different fluorescent probes were used for conjugation and the impact on albumin sorption behaviour was investigated (Chapter 4) • Lysozyme sorption to nine different pHEMA-based and silicone hydrogel contact lenses was determined using two fluorescent probes (Chapter 5) • The efficiency of protein removal from contact lenses using contact lens care regimens was investigated (Chapter 6) • Albumin sorption to IOL materials was quantified and imaged using a modified CLSM technique (Chapter 7) Results: Albumin and lysozyme sorption profiles differed between materials, and were influenced by the fluorescent probes used for conjugation. After one day of incubation, both proteins could be located within all contact lens materials, except for lotrafilcon A and lotrafilcon B, which primarily allowed deposition on the lens surface. An increase in protein accumulation was found for most materials over the maximum investigated period of 14 days, using CLSM and radiolabel techniques. The efficiency of contact lens care regimens to remove lysozyme and albumin depended on the lens material, care regimen and protein type investigated. PMMA and silicone IOLs showed protein exclusively on the surface, while a hydrophilic acrylic IOL allowed penetration into the lens matrix over time. Despite the albumin penetration depth into hydrophilic acrylic, the highest albumin levels were determined for the silicone IOL. Conclusions: CLSM provides detailed information that can describe the protein distribution in transparent biomaterials, with scanning depths up to a few hundred microns. However, the CLSM data are primarily of qualitative value, which necessitates a quantitative technique (e.g. radiolabeling) to determine the total protein content.