Faculty of Health Sciences (ETDs)

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    Design and development of a bioactive-loaded polymer-engineered neural device for potential application in reducing neurological deficits after spinal cord injuries and neuro-regeneration
    (University of the Witwatersrand, Johannesburg, 2017) Kumar, Pradeep; Choonara, Yahya Essop; Modi, Girish; Naidoo, Dinesh; Pillay, Viness
    Traumatic Spinal Cord Injuries (SCI), due to their devastating nature, present several interventional challenges (extensive inflammation, axonal tethering, scar formation, neuronal degeneration and functional loss) that need to be addressed before even a slight neuronal recovery can be achieved. Recent post-TSCI investigational approaches include support strategies capable of providing scaffold architecture to allow axonal growth and conformal repair. This research provided detailed insight towards the development and fabrication of six specialized Polymer-Engineered Neural Devices (PENDs): 1) poly(lactic-co-glycolic acid)-gliadin (PLGA-GLDN) nanofibrous mats, 2) polyacrylamidated chitosan (PAAm-g-HT) scaffold, 3) functionalized chitosan methoxypolyethylene glycol (CHT-mPEG) cryosponges, 4) polyacrylonitrile-elastin-collagen (PANi-EC) neurosponge, 5) methylcellulose-alginate-polyethylene glycol (MAP) thermogel, and 6) chitosan-luronic F127-β glycerophosphate (CHT-PF127-βGP) composite thermogel for potential restriction, repair, regeneration, restoration and reorganization post-SCI. The latest trends in biomaterials-based SCI intervention were reviewed, discussed and analyzed in detail throughout the thesis. The research also involved an in silico analytico mathematical interpretation of multi(biomed)material assemblies wherein quantification of energy surfaces and molecular attributes via atomistic, dynamic, and docking simulations was carried out. The in silico experimentation additionally confirmed the potential of curcumin as a bioactive of choice for SCI intervention. Curcumin and dexamethasone were incorporated into the compact scaffolds and the bioactive release was determined over a period extending up to 60 days. The PLGA-GLDN nanofibrous mats demonstrated the formation of a compatible blend among the component polymers at equal weight ratios (PG55) as confirmed by quantitative physicochemical characterizations. Image processing analysis (DiameterJ plug-in of ImageJ) was performed on the SEM images of nanofibers to quantify the size, porosity, and orientation of the samples. Nanofibers within the size range of 10nm and 250nm were obtained in case of compatible blend and the nano stack was used for in vivo implantation post-SCI. Polyacrylamidated chitosan (PAAm-g-CHT) was synthesized via a unique persulfate-mediated polymer slicing and complexation as determined by static lattice atomistic simulations. The graft copolymer so obtained was fabricated into an anisotropic neurodurable scaffold. The CHT/mPEG cryosponges showed unique morphological features such as fringe thread-like structures (CHT alone); hemispherical, pebble-like structures (CHT-mPEG); curved quartz crystal-like or crystal-flower-like structures (CHT-mPEG-CHO); and grouped, congealed, steep-sided canyon-like structures (CHT-mPEG COOH). A novel image processing protocol involving DiameterJ and ND plugins of ImageJ software was employed for analyses of the SEM micrographs in terms of % porosity, pore wall thickness and % xiiehaviorxii of the porous scaffolds. The PANi-EC interpenetrating polymer network neurosponges were synthesized employing free radical polymerization under acidic conditions wherein first-in-the-world spinomimetic scaffolds were obtained. The unique feature of the PANi-EC neurosponge was the formation of a fibrous neurotunnel architecture mimicking the native spinal cord. The physicochemical characterization revealed that the secondary structure of the peptide molecules (elastin and collagen) rearranged in the presence of PANi to their native extracellular matrix (ECM) form confirming the self-assembling nature of the polymer-peptide architecture. Furthermore, the PANi-EC neurosponge provided a perfect balance of matrix resilience and matrix hardness similar to the native collagen-elastin complex in vivo. Two very interesting tri-component thermogels were reported here viz. a simple blend thermogel comprising methylcellulose, sodium alginate and poly(ethylene glycol) and a complex thermogel incorporating chitosan, Pluronic F127 and β-glycerophosphate. Both the thermogels solidified at physiological temperature confirming their applicability in vivo. The outstanding feature of MAP thermogels was the formation of hydrogen bonded O-H…C=O which only formed in the tripolymeric blend while the bipolymeric blends showing no such interaction. We proposed that the MAP thermogel self-assembled into a repeating network structure represented by “PEG400-ALG-hydrophillicMChydrophobic}-{hydrophobicMC-hydrophilic}-ALG-PEG400” and the physical “compression” might have led to the formation of hydrogen bonded O-H…C=O among MC/alginate or PEG/alginate in the presence of PEG or MC, respectively. In case of the complex CHT/PF127/βGP thermogel, a unique triphasic gel-sol-gel transition xiiehavior was observed with the thermogel forming a gel-phase at lower temperatures (T<20°C), a sol-phase at intermediate temperatures (20°C35°C). The MTT proliferation studies indicated that all polymer engineered neural devices (PANi-alone matrix) were capable of efficiently supporting the growth of PC12 cells compared to the control over a period of 72 hours. The fundamental objective of this thesis was to test the applicability and capability of various biomaterial composites towards the repair and regeneration of neuronal tissue after traumatic spinal cord injury. Although drug-loaded scaffolds were also developed, only drug-free scaffolds (PLGA-Gliadin 5:5 electrospun nanofibers; PANi-Elastin-Collagen neurosponge; and Chitosan/Pluronic F127/β-glycerophosphate thermogel) were tested in vivo for the proof-of-concept. The 21-point scale BBB locomotor rating analysis demonstrated that PEND I (14), PEND II (19) and PEND III (18) provided significant motor recovery as compared to the lesion-control (5) group 28 days post-SCI and –implantation. The immunohistochemistry confirmed that reparative changes were accompanied by marked upregulation of iNOS, a notable influx of ED1-positive chronic inflammatory cells, the appearance of multinucleate cells characteristic of presumptive regenerative neuroblasts and near-complete loss of GFAP and NF-200 protein/structural integrity. Almost complete functional and neurostructural recovery was observed with post-SCI implantation of PEND II and III. In conclusion, the composite scaffolds tested in this research demonstrated immense potential in improving the neurological, neurochemical, and behavioral outcome after implantation post-SCI.
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    Surgical aortopulmonary shunts - a thirty-seven year experience in a South African tertiary institution
    (University of the Witwatersrand, Johannesburg, 2019-11) Dladla-Mukansi, Nontobeko Charity; Cilliers, Antoinette; Mammen, Vijay; Vanderdonk, Kathy
    Introduction: The surgical aortopulmonary shunt is a valuable palliative procedure in the management of congenital heart diseases. There is a paucity of data regarding aortopulmonary shunts in the developing world, including South Africa. Objectives: The primary objective was to describe the demographic, clinical and echocardiographic characteristics of children between ages 0 and 14 years that underwent surgical aortopulmonary shunts. The secondary objectives were to describe trends in aortopulmonary shunt designs, outcomes in terms of morbidity and mortality, progression to definitive surgery and to assess patency of shunts. Material and Methods: A retrospective clinical audit of patient files who underwent an aortopulmonary shunt between 01 January 1980 to 30 December 2016 was undertaken at Chris Hani Baragwanath Academic Hospital (CHBAH) in Soweto, Johannesburg. The study period was divided into 3 stages and for descriptive purposes as follows: 1980-1991 refers to period 1, 1992-2003 refers to period 2 and 2004-2016 refers to period 3. Results: A total of 177 aortopulmonary shunts were done over the 37-year study period. Of these 177 patients, 165 (93.2%) patient files were available. Fifty-six percent of the patients included in the study were male. The majority of patients were from the Gauteng Province (76.8%). The four most common diagnoses across the entire study period were tricuspid atresia (26.0%), pulmonary atresia with VSD (23.7%), tetralogy of Fallot (23.2%) and complex cardiac lesions (16.9%), with no particular trend in the proportion of these diagnoses presenting across this study period. There was no statistical difference between period 1 and 2 (p-value a=0,328) and between period 1 and 3 (p-value b=0,548). The total number of all surgeries done over the entire study period was 2145, of which 8.3% were aortopulmonary shunts. Period 1 had the highest percentage [35 (10.9%)] of aortopulmonary shunts compared to the total number of surgeries performed. There was a decline in the number of aortopulmonary shunts performed over the study periods 1-3. With no statistical difference across periods as shown in table 1 with p-value a and b. Of the different types of aortopulmonary shunts, most patients [157 (88.7%)] had a modified Blalock-Taussig shunt (BTS). The remainder of the shunts included 3 (1.7%) classic BTS, 12 (6.8%) central shunts and 5 (2.8%) unknown BTS. The percentage of modified BTS done increased from 80% in period 1 to 87.3% in period 2 and to 95.2% in period 3. Period 1 had the most complications (28.6%) compared to 11.4% in period 2 and 19.1% in period 3. Sepsis as a complication following surgery increased over the study period from 2.9% in period 1 to 3.8% and 7.9% in periods 2 and 3 respectively. Early mortality was 17.1%, 26.6% and 25.4% from periods 1-3 respectively. Late mortality declined from 17.0% in period 1 to 11.4% and 0% in periods 2 and 3 respectively. Only 37 (20.9%) patients were documented to have further surgery after the initial aortopulmonary shunt. Across all three study periods, no blocked shunts were documented. Conclusions: This study describes the characteristics and outcomes of aortopulmonary shunts over a 37-year period in a tertiary care resource limited low to middle income country setting. The commonest cardiac lesions for which aortopulmonary shunts are performed are tricuspid atresia, pulmonary atresia with VSD, tetralogy of Fallot and other complex cyanotic cardiac lesions. The frequency of aortopulmonary shunts compared to total surgeries has corrective surgery for these cardiac lesions. The modified BTS is the most frequently performed aortopulmonary shunt used for palliative surgery in our setting, which is a similar trend in developed countries. The morbidity and mortality in this study is higher than developed countries, with sepsis being the most common complication. Attention to infection control practises need to be emphasized peri- and post-operatively in our hospitals.
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    Mathematical representation and analysis of articular surfaces: application to the functional anatomy and palaeo-anthropology of the ankle joint
    (University of the Witwatersrand, Johannesburg, 1990) Webb, Christie Peter; Tobias, Phillip Vallentine
    This thesis is a study of quantifiable variation in the geometric shape of the superior articular surface of the talus of higher primates, with special reference to fossil tali of Plio-Pleistocene hominids. (Abbreviation abstract).
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    The role of increased gastrointestinal alcohol production in patients with the metabolic syndrome: Implications for the pathogenesis of non-alcoholic fatty liver disease
    (University of the Witwatersrand, Johannesburg, 2006) Menezes, Colin Nigel; Immelman, Ronnie; Raal, Derick
    Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease with hepatic histology that resembles alcoholic liver disease. It is a frequent cause of chronic liver disease and is attracting increasing scientific attention worldwide. I explored the possibility that increased gastrointestinal alcohol production may have a role as a “second hit” in the pathogenesis of NAFLD in study subjects with the metabolic syndrome. In an attempt to investigate this hypothesis, this study looked at blood, urine and breath levels of alcohol in patients with the metabolic syndrome versus matched age and ethnic group healthy controls. Of the twenty study subjects, 80% had dyslipidaemia, 60% had hypertension and 70% had type 2 diabetes mellitus. Their mean BMI was 35.1±8.2 kg/m² (mean ± SD, P < 0.0001 versus controls). The serum aminotransferases were significantly elevated in the study subjects, their ALT levels being 57.4±44.79 U/L versus 17.4±4.60 U/L in the controls (95% CI 18.02 – 61.42, P < 0.001), and their AST levels 52.5±36.21 U/L versus 23.4±4.86 U/L in the controls (95% CI 11.99 – 46.20, P < 0.01). Seventy five percent of the study group had sonar features suggestive of fatty liver disease. Two adipocytokines, adiponectin and leptin, mediators of insulin resistance, an important factor in the development and progression of NAFLD, were also measured. Adiponectin levels were significantly lower (6875 ng/L versus 15475 ng/L; median value, P < 0.01), and leptin concentration levels significantly higher (13.56 ng/L versus 3.05 ng/L; median value, P < 0.05) in the study subjects than in the control group. Alcohol was detected in 60% of the study subjects, of which 35% tested positive for ethanol, 55% tested positive for methanol, and 30% tested positive for both ethanol and methanol. This was a statistically significant result, as none of the control group tested positive for any of the alcohols. The ethanol concentration in the study subjects’ blood was 7.14±3.28 mg% (mean ± SD), in their urine 3.71± 12.87 mg% (mean ± SD) whilst none was detected in their breath. The methanol concentration in the study subjects’ blood was 16.17±17.95 mg% (mean ± SD), in their urine 6.8± 13.58 mg% (mean ± SD) while their breath level was 2.05±3.19 mg (mean ± SD). This study therefore suggests that endogenous alcohol production may be indeed be involved in the pathogenesis of NAFLD in subjects with the metabolic syndrome. Not only ethanol but also methanol was detected in the subjects tested. endogenous alcohol may therefore be responsible for the ‘second hit’ theory in the pathogenesis of NAFLD, and it is likely that formaldehyde, the metabolite of methanol may be a more potent toxin of the patocyte injury as opposed to acetaldehyde, the metabolite of ethanol. The most likely source of the alcohol is from intestinal bacterial flora. These findings provide further insight into the pathogenesis of NALFD, suggesting other therapeutic alternatives such as the use of antibiotics and probiotics as a potential treatment strategy for NAFLD.
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    The effect of all-trans retinoic acid on the migration of avian neural crest cells in vitro an in vivo
    (University of the Witwatersrand, Johannesburg, 2007-02-15T11:43:45Z) Tshabalala, Vincent Abie Thabiso
    Retinoic acid, the active metabolite of Vitamin A is known to play a major role in embryonic growth and differentiation during development. It has been shown that either excess or deficiency of retinoic acid during embryogenesis can be teratogenic. In order to study the teratogenic effects of retinoic acid, the aim of the present study was therefore to investigate the effect of all-trans retinoic acid on the migration and fate of neural crest cells in vitro and in vivo. In addition, the study investigated the effect of retinoic acid on the cytoskeletal elements of neural crest cells and on Rac and Rho, two members of the Rho family of GTPases. The neural tubes containing neural crest cells of quail embryos were removed at cranial levels and cultured on fibronectin as a substrate. The neural tubes were cultured in either Dulbecco’s minimal essential medium (DMEM) or in DMEM+Dimethylsulphoxide (DMSO) as controls. In order to test the effect of retinoic acid, the neural tubes were cultured in 10⁻⁵M all-trans retinoic acid (RA) which was reconstituted in DMSO. The distance of migration of the cultured quail neural crest cells was measured and compared between the controls and the experimentals. To study the effect of RA on the cell actin cytoskeleton in vitro, cultured neural crest cells were stained with rhodamine phalloidin. In addition, following 24 hours of culture, the quail neural crest cells were brought into suspension and micro-injected into 36 hour-old chick hosts. While the migration of neural crest cells was extensive in the control cultures in vitro, migration was inhibited in the retinoic acid-treated neural crest cells. In addition, retinoic-acid treated neural crest cells showed pigmentation and neuronal processes earlier than did the control neural crest cells. Retinoic acid-treated neural crest cells showed a disarray of the cytoskeletal elements as they were devoid of stress fibres and focal adhesions. In addition, retinoic acid appears to decrease the expression of Rac and Rho of cultured quail neural crest cells. Following micro-injection of cultured control and RA-treated quail neural crest into the cranial region of chick hosts, the control cells populated the beak area, whereas the retinoic acid-treated quail neural crest cells migrated to the retina of the eye, a region they normally do not populate. These results suggest that retinoic acid disturbs the migration of neural crest cells. It appears to do this by affecting the cytoskeletal elements of neural crest cells and the genes that are involved in forming these elements.