There are two classifications of carbon nanotubes (CNT), firstly single walled carbon nanotubes (SWCNT) and secondly multiwall carbon nanotubes (MWCNT). CNTs have the ability of absorbing near infra-red light and generating heat along with the ability to deliver drugs to the site of action. Different application of CNT has so far studied on the in vitro and in vivo models. This thesis is looking into the application of SWCNT on different in vitro models. These models include human breast cancer cell line (MCF7), human colorectal cancer cell line (HT29 and SW480), human pancreatic cancer cell lines (PANC-1) and mouse fibroblast cell line (3T3). In this research, the functionalization techniques of SWCNT have been investigated. In addition, a detailed observation on the application of SWCNT for thermal treatment and the delivery of anti-cancer drugs to the site of action has been obtained. The thesis also looks into conjugation techniques of other materials to the surface of SWCNT for the purpose of both directing the SWCNT to the site of action and tracking their movement through the cells. Finally yet importantly, a detailed investigation has been performed into the effect of SWCNT size and the conjugation of different functional groups to the SWCNT’s surface on its toxicity. From the results, it can be concluded that treating SWCNT in acid solution and further functionalization with OctaAmmonium-POSS will significantly increase the biocompatibility of the SWCNT. Using functionalized SWCNT could be used for the delivery of anticancer drugs to the site of action and thermal treatment of cancer. It has been demonstrated that attachment of QDs to the SWCNT’s surface can be used for tracking the SWCNT’s movement. Finally, it can be concluded that the smaller length SWCNT with a higher coating concentration of OctaAmmonium-POSS will decrease the toxicity of the SWCNT.

BACKGROUND. Coronary artery bypass graft surgery is a commonly performed procedure. The internal thoracic or mammary artery is gaining widespread preference as the bypassing conduit. Synthetic grafts used for large diameter substitutes are successful but have dismal patency as small diameter (< 6mm) grafts due to compliance mismatch and thrombogeniecity. To overcome this, cell adhesion to synthetic scaffolds is used to construct tissue engineered grafts. For this to be successful a precise understanding of the behaviour of cells at the synthetic graft surface is required under static and haemodynamic conditions. The aim of this research was to investigate gene expression of seeded human umbilical vein endothelial cells (HUVEC) on the novel compliance conduit under physiological flow condition; furthermore various physiological shear stress preconditioning was used to investigate adhesion of HUVEC on the conduit. METHODS. HUVEC seeding of a novel polymer nanocomposite was undertaken. An optimal method for extracting mRNA from HUVEC seeded onto conduits was then validated. The optimal seeding conditions for the conduits were delineated. Haemodynamic conditions were applied to the seeded conduits and gene expression was investigated using polymerase chain reaction (PCR). Shear stress was used to assess the ideal preconditioning environment. RESULTS. Studies of nanocomposite graft material and cultured HUVEC proved that the novel nanocomposite polymer was non-toxic to cells and supported good rates of growth. To provide useful flow studies an extrusion-phase inversion method was used to reproducibly fabricate conduits of this nanocomposite with compliance similar to the native artery. The optimal seeding density of the conduits was found to be 1.2 x 104 cells/cm2. It was demonstrated that RNA can be extracted from seeded conduits and I succeeded in showing the optimal technique. This study culminates in the combination of all these techniques when the gene expression of HUVEC under flow was studied after physiological shear stress was applied on the conduits. Genes significantly upregulated included TGF-β1, COL-1 and PECAM-1. Low shear stress demonstrated the optimal preconditioning environment with increasing expression of VEGFR-1 and VEGFR-2 genes. CONCLUSION. This thesis demonstrated that novel nanocomposite small diameter bypass graft can be seeded with human endothelial cells.

Ischaemia of the liver followed by reperfusion results in endothelial and parenchymal injury through a complex cascade of events. This often occurs in human liver transplantation as well as with major liver resections and is referred to as Ischaemia Reperfusion (IR) Injury. Bucillamine is a low molecular weight thiol antioxidant that is capable of rapidly entering cells. This thesis evaluates the effect of Bucillamine on both the early and late phases of liver warm IR injury with the hypothesis that beneficial effects are induced could be due to its action as a free radical scavenger. The drug was evaluated in an in vivo lobar liver ischemia reperfusion model as previously described. Male Sprague –Dawley rats were subjected to 45 mins of partial hepatic (70 %) ischaemia followed by 3 hrs of reperfusion to investigate the early phase of hepatic IR and 24 hrs of reperfusion to study the late phase of hepatic IR. Changes to the microcirculation, leucocyte adherence and apoptosis were assessed by intra-vital microscopy. Hepatocellular injury was assessed by standard liver function tests. Expression of pro and antiapoptotic gene expression was studied by RT-PCR. Oxidative stress was assessed by measuring plasma and hepatic F2 isoprostane levels and tissue glutathione levels. Cytokine response was assessed by measuring serum CINC-1 levels. Bucillamine improved liver sinusoidal perfusion, reduced leukocyte adherence and apoptosis in both the early and late phases of IR injury. Hepatocellular injury was reduced. There was no difference in the level of tissue glutathione or tissue and plasma F2 isoprostane levels. This study shows that the hepato protective effect of Bucillamine in warm Liver ischemia reperfusion injury is not by direct replenishment of Glutathione level; however, it is through decreased neutrophil activation and recruitment. A clinical trial could hence be undertaken in the future to study its efficacy.

Facial transplantation has emerged as the next step on the reconstructive ladder for severe facial disfigurement. Clinical issues surrounding facial tissue donation are examined, comprising pre-transplant facial vessel delineation; pre-operative aesthetic matching; and attitudes towards donation. An anatomical study of 200 consecutive facial and transverse facial vessels was performed using colour Doppler ultrasound. Facial vessels were measured at three landmarks and their branching pattern documented. The facial artery main branch was detected at the lower mandibular border in 99.5% of cases, the accompanying facial vein in 97.5%. The transverse facial artery was present in 75.5% of cases, the vein found in 58%. When the facial artery was undetectable, there was transverse facial artery dominance. When the facial vein was absent it was replaced with a transverse facial vein. This provides valuable pre-operative information regarding vessel status. A quantitative eleven point skin tonal matching scheme is described using digital analysis of facial imagery. Attitudes towards tonal mismatch in facial and hand transplantation are examined in two representative skin types. There was more scope for skin tonal mismatching in skin tone 2 (slightly tanned white) than in skin tone 6 (light golden brown) participants. Tonal mismatches were more tolerated in facial than in hand transplant simulations in both groups. More acceptable donor tonal groups exist for males than females. Targeted matching of skin tone is thus required. Attitudes and beliefs of 170 transplant professionals were examined. Areas of concern included the organ retrieval process; impact on the retrieval team and donor family. In-depth analysis of a transplant donor focus group was performed; provision of information, posttransplant contact, and post-retrieval donor facial appearance was deemed important. A method of fabricating a donor-specific artificial prosthesis within the time frame of facial graft retrieval is described. Finally, a method of framing the informed consent process is described.

As humans ascend to high altitude, the resulting hypobaric hypoxia necessitates adaptation in order continue functioning. Whilst much is known about changes that facilitate a restoration of systemic oxygen delivery during ascent, less is know about alterations in the peripheral microcirculation and how these affect acclimatisation and performance at altitude. The limit of human adaptation to hypoxia is also undefined. Using data derived from a number of studies conducted on healthy volunteers at high altitude, this thesis explores changes in skeletal muscle oxygenation and sublingual microcirculatory blood flow, and how these may relate to the process of acclimatisation. In addition, exercise was used to perturbate the relationship between oxygen delivery and utilisation at altitude, and experiments at extreme altitude sought to define the limits of human tolerance to hypoxia. Data relating to four subjects resting just below the summit of Mount Everest (at 8400m), demonstrated a degree of systemic hypoxia never before reported in humans. Given sufficient time, arterial oxygen content remained steady during ascent to 7100m. Sublingual microcirculatory blood flow declined at altitude, whilst the density of blood vessels increased. At altitude, absolute skeletal muscle oxygenation declined; the response to a brief ischaemic episode was a reduction in rate of subsequent muscle reoxygenation; and the rate of muscle desaturation during exercise increased. Systemic oxygen extraction during exercise at altitude remained unchanged from that observed at sea level. These results support the hypothesis that a significant barrier to oxygen flux exists within tissues that is heightened at altitude. Restoration of convective oxygen delivery does little to improve tissue oxygenation, and adaptations within the microcirculation may differentiate phenotypic responses observed on prolonged exposure to hypoxia. Graded ascent to altitude by healthy volunteers provides valid data that may herald further research in the clinical arena, bringing about improved outcome in critically ill patients.

Bare-metal stents (BMS) and drug-eluting stents (DES) are the two main categories of FDA-approved coronary stents in the market for treating atherosclerosis. Problems associated with BMS include in-stent restenosis due to intimal hyperplasia, leading to stent failure, while DES harbours a life-threatening complication called late-stent thrombosis due to drug-polymer hypersensitivity and impaired re-endothelialization. One approach to overcoming the above-mentioned problems could be using covered stents. Covered stents have an additional layer of membrane spanning the stent struts, and can be considered hybrid stent-grafts. Due to the added protection that the membrane affords, covered stents are currently used for vessel perforations and aneurysms. They can act as a physical barrier to inhibit smooth muscle cell in-growth and intimal hyperplasia formation. The most commonly used membrane for covered stents is expanded polytetrafluoethylene (ePTFE). However, its non-compliant and thrombogenic nature prevents it from being suitable for use in small-diameter vessels, resulting in an unmet clinical need for a haemocompatible covered stent for this application. A polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) nanocomposite polymer was developed by our group, and has already been used in three first-in-man studies as a bypass graft, lacrimal duct, and the world’s first synthetic trachea. Hence we sought to assess the feasibility of using POSS-PCU as a membrane for covered stents. Results indicate that POSS-PCU was haemocompatible, and was able to support the growth and proliferation of endothelial cells, compared to controls. Mechanical tests on membranes revealed that POSS-PCU was superior to ePTFE. Furthermore, it was also found that integration of POSS-PCU membrane onto stents did not adversely affect stent mechanics. In summary, the overall biomechanical performance of POSS-PCU indicates that it has the potential to function as a viable membrane material for covered stents in small diameter vessels.

Morbidity following major surgery is a growing healthcare problem. Previous studies have shown associations between preoperative lymphopenia and adverse postoperative outcomes, albeit in patient groups with multiple confounding factors. Lymphopenia is also associated with poor outcomes in a range of chronic and acute disease states including cardiac failure and sepsis, where lymphocyte dysfunction and excess lymphocyte apoptosis is observed. Cellular bioenergetic failure has been observed in these disease states and is proposed as a key pathophysiological mediator. The core hypothesis of this thesis is that preoperative lymphopenia is associated with increased morbidity after a standardized physiological insult due to underlying bioenergetic dysfunction. A large observational cohort study in patients undergoing elective lower limb arthroplasty showed preoperative lymphopenia to be a common phenomenon independently associated with increased postoperative morbidity and length of hospital stay. Further analysis of a subset of this cohort showed lymphopenia to be a chronic feature, not associated with common diagnoses known to cause lymphopenia. Immunophenotyping using flow cytometry confirmed lymphopenia affecting all lymphocyte subsets, but with normal monocyte and neutrophil number and function. Lymphocyte function was further explored using ex-vivo culture experiments in normal and lymphopenic subjects. In lymphopenic patients, lymphocyte apoptosis in response to a variety of insults was increased and ex-vivo stimulated lymphocyte proliferation was reduced. A novel experimental model was developed to assess lymphocyte bioenergetic function, using respirometry to assess glycolysis and mitochondrial function in freshly isolated cells. Lymphocytes from lymphopenic patients had global reductions in bioenergetic function and intracellular ATP content. In summary, chronic lymphopenia is common in the elective orthopaedic population and is robustly associated with adverse postoperative outcomes. The data here characterise a patient phenotype at increased perioperative risk. The underlying cellular dysfunction described may have important implications in a range of acute and chronic illnesses.

Exercise training is well recognised as an effective treatment for intermittent claudication. The mechanism underlying exercise induced improvements is multi-factorial but remains poorly understood. Low angiotensin-converting enzyme (ACE) activity has been associated with enhanced responses to endurance training. Specifically, low ACE activity has been associated with improved muscle metabolism, endothelial function, and suppressed inflammatory responses; processes linked with exercise training benefits in claudicants. Furthermore, pharmacological inhibition of ACE has been associated with enhanced angiogenesis in animal models of ischaemia, secondary to increases in vascular endothelial growth factor (VEGF). In this study, 11 claudicants were randomised to 8 weeks of supervised exercise training (n=6) or exercise advice (n=5). Walking ability was recorded before and after this period, and blood samples taken. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the effects of exercise training on ACE, VEGF and VEGF receptor (VEGFR) gene expression, and enzyme-linked immunosorbant assays (ELISA) measured changes in ACE and VEGF protein levels. In another experiment, a cell culture model of hypoxia, utilising ECV 304 cells and diethylenetriamine-nitric oxide (DETA-NO), was used to study the effects of the ACE inhibitor ramiprilat on ACE and VEGF responses to hypoxia, using RT-PCR and ELISA. Supervised exercise improved claudication distance by 105 metres (p < 0.05) and maximum walking distance by 141 metres (p < 0.05). ACE mRNA expression increased 30%, VEGF121 expression 43% and VEGF165 expression 70% (all p < 0.05). Soluble VEGFR-1 mRNA expression increased by 63% and VEGFR-2 72% (both p < 0.05). ACE and VEGF protein levels remained comparatively stable. In the cell culture experiments, ramiprilat increased VEGF protein levels in hypoxia. Although a lack of experimental runs prevented statistical analysis, the results also suggest that ramiprilat has a stimulatory effect on ACE mRNA expression in hypoxia. Improvements in walking ability after exercise training are associated with increases in both VEGF and VEGF receptor expression. ACE inhibitors could play a role in improving claudication by potentiating increases in VEGF in addition to their known action of suppressing ACE activity.

Mobilisation of injured tendons results in improved tendon healing and reduced adhesions. The precise mechanism for the effect on adhesions is unknown. This thesis presents the development of a series of techniques to examine the hypotheses: that mobilisation exerts its favourable effects by modifying local strain responses to applied stress by altering cell attachment to the extracellular matrix; that selectively blocking cell-matrix attachment can mimic these effects. Attachment of tendon-synovial complex cells and mobilised and immobilised adhesion cells to collagen and fibronectin was examined in vitro. Attachment of intrinsic cells and mobilised adhesion cells was higher than that of extrinsic cells and immobilised adhesion cells. These data suggest that mobilisation favours intrinsic cell attachment to the matrix and their contribution to healing. An in vivo model system was developed in the injured flexor tendon-synovial complex, enabling quantitative hierarchical mechanical assessment of mobilised and immobilised adhesions. Three-fold higher local strain values and increased heterogeneity of local strain values were seen in mobilised adhesions. Mobilisation may result in localised mechanical failure due to altered local strain patterns. A novel biomaterial was investigated in vitro and in vivo. Reduction in restrictive adhesion parameters was observed, with an associated decrease in adhesion cellularity without compromising tendon cellularity. Inhibition of fibroblast attachment resulted in a mimicking of the effects of mobilisation.

Background: 33 million people worldwide are infected with HIV, a complex disease that affects many of the processes involved in wound and fracture healing. There is little evidence available to guide acute management of open fractures in these patients and fears of acute and delayed sepsis often inhibit the use of surgical fixation, which may be the most effective way of achieving union. This study addresses the hypothesis that the presence of either HIV or advanced HIV (CD4 count <=350) leads to an increased risk of complications in patients with open fractures treated with surgical stabilization. South Africa has one of the highest rates of both HIV and high energy trauma in the world, so was deemed an appropriate place for the study of this interaction. Methods: This prospective observational study compared surgical fixation of open fractures in HIV positive and negative patients. 133 patients with 135 open fractures fulfilled the inclusion criteria. 86 fractures were in HIV negative and 33 in HIV positive patients. The remaining 16 patients refused HIV tests. 12 HIV positive patients had advanced disease (CD4 <=350), 14 had early disease (CD4 >350), 7 refused CD4 count testing. This cohort was three times larger (number of HIV positive patients) than any similar previously published study. There was no randomised allocation; the treatment of these patients was based on locally developed protocols and was dependent on; fracture type, location and the grade of wound. Patients were followed up either till union had been achieved or for 6 months in tibia/femur fractures, and 3 months in other fractures. The primary outcome was acute wound infection, secondary outcomes tested were fracture union and pin site sepsis. The analysis of the binary nominal data was done using the Chi squared test. In cases where the expected value was less than 5, then the Fisher’s exact test was used. In the assessment of multiple potential risk factors, binary logistic regression was used. Results: Analysis of background characteristics showed that HIV positive and negative populations were broadly similar with regard to demographics, injury type/location and grade of wound. In the analysis of the primary outcome, the risk of wound infection was marginally higher in patients without HIV (22%) as compared to patients with HIV (15%). This difference was small and did not reach statistical significance (n=135, Risk Ratio 0.7, p value 0.40). However, as hypothesized, the infection risk was higher in patients with advanced HIV (26%), compared to patients with early HIV (5%). The numbers, however, were small and this did not reach statistical significance (n=33, Risk Ratio=4.8, P value= 0.12). Sub group analyses, conceived prior to the study, provided strong evidence that patients with Gustilo Anderson grade 1 injuries had a higher risk of wound infection in patients with advanced HIV than controls (HIV negative and early HIV) (n=46, Risk Ratio=6.3, P value =0.02). Of note, departmental guidelines meant that patients with grade 1 injury were not prioritised for theatre and had, on average, a delay of 3.5 days to surgery. The average delay was similar in both HIV positive and negative groups. Analysis of the secondary outcome, nonunion, provided strong evidence that the risk of nonunion was higher in HIV positive than HIV negative patients (n=115, Risk Ratio=4.1, P value=0.04). Interestingly, the patients with advanced HIV had a slightly lower nonunion risk (13%) than patients with early HIV (20%). However the numbers were small and the difference was not statistically significant (n=33, Risk Ratio=0.8, P value=1). The incidence of nonunion was not correlated with the presence of wound infection. The risk of mild pin site sepsis in fractures treated with external fixation was similar in both HIV positive (60%) and negative (67%) patients (n=31, Risk Ratio=0.9, P value=1). An increased risk of severe pin site sepsis was noted in patients with advanced HIV (50%), compared to controls (25%). Although the difference is large, the numbers are small and the difference was not statistically significant (n=28, Risk Ratio=2, P value= 0.31). It would require 160 patients to prove a difference of this size. Conclusions: Data from this study appears to dispute the conclusion of previous studies that suggest that all patients with HIV are at higher risk of wound infection, and therefore internal fixation should be considered with caution. In this study it was only the patients with advanced HIV that showed a small increase in the risk of wound infection. Based on this study the author suggests that early HIV should not be a contraindication to either internal or external fixation in open fractures, due to concerns of wound infection. However, advanced HIV should continue to be considered a relative contraindication to internal fixation, until further data becomes available. Since this finding applied equally to grade 1 (Gustilo Anderson) injuries, the data suggests that any theatre delays in patients with advanced HIV may be detrimental to outcomes. This is contrary to published data that suggests that grade 1 injuries do not need to be prioritised. The data provides strong evidence that HIV leads to an increased risk of non unions. Interestingly, the risk of non union is less in patients with advanced HIV. This may fit with recently published laboratory studies suggesting that the absence of lymphocytes is beneficial to bone healing. Based on this evidence the author suggests that in patients with HIV treatment strategies should be aimed at achieving union, rather than on potentially unfounded concerns of preventing infection. In patients treated with external fixation, the data provides weak evidence of an increased risk of severe pin site sepsis in advanced HIV. This observation may be due to an increased susceptibility to infection, or to problems with bone healing in these patients. Based on this evidence, and the evidence that patients with HIV may be at increased risk of non union, the author suggests that HIV positive patients being treated with external fixators, should be considered for treatment strategies that will prolong the life of the pin bone interface. These may include additional pins, wires and/or the use of hydroxyapatite coated half pins.