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Heart transplantation is, for certain types of cardiovascular disease, the only form of treatment resulting in patient survival. Its clinical application is, however, limited by the shortage of donor organs. This shortage is largely due to the inability to consistently preserve adequate myocardial function over prolonged ischemic periods. It is the goal of this research to provide information which may contribute to techniques for heart preservation, thus improving graft survival following preservation and transplantation. Current methods for myocardial preservation generally involve the arrest and immersion of the heart in cold cardioplegic solution, the composition of which is designed to provide for the reduced metabolic demands of the cold, arrested muscle. These methods have extended the preservation period to approximately 6 hours; however, hearts cannot be held longer than this period because, although metabolism has been slowed by hypothermia, alterations take place which compromise functional recovery upon reperfusion. A variety of perfusates and perfusion techniques have been developed to protect the myocardium from the damage thought to occur as a consequence of ischemic storage of the isolated heart. However, a consistently successful technique for long-term preservation of the heart remains undefined. A growing body of knowledge has led to the hypothesis that injury to the microcirculation may result in myocardial ischemia during preservation and decreased contractile function following preservation. To test this hypothesis, standard Langendorff techniques for the measurement of left ventricular function were combined with biochemical, histological and morphological techniques to determine: (1) whether loss of microvascular function occurs in isolated hearts hypothermically perfused with an oxygenated solution; (2) the impact of microvascular dysfunction during the preservation period on the functional recovery of hearts; and (3) which mechanisms contribute to decreased microvascular function during preservation. This experimental approach will allow for characterization of the role of the microvasculature in decreased contractility of preserved hearts and will provide information regarding the contribution of specific mechanisms to the compromised contractility of preserved hearts. Systematic evaluation of mechanisms thought to be responsible for decreased contractility of isolated hearts could contribute to improved myocardial preservation techniques that can be applied to clinical transplantation.

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Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2006. / Wick, Tim, Committee Member ; Neitzel, Paul, Committee Member ; Fyhrie, David, Committee Member ; Garcia, Andres, Committee Member ; Guldberg, Robert, Committee Chair. Vita.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 17, 2007) Vita. Includes bibliographical references.

MR allows non-invasive means of evaluating the non-mineralized components of bone, particularly the bone marrow. This thesis focuses on potential changes occurring in bone marrow perfusion and marrow fat in osteoporosis, - changes which may improve our understanding of osteoporosis pathophysiology. We know from histology studies that as osteoporosis develops and bone tissue is lost, it is replaced by fat filling the vacated marrow space. MR allows non-invasive quantification of this fat component. Although fat content may be increasing, it is not known whether any change in fat composition occurs with osteoporosis i.e. does the type of fat within bone change. Epidemiological studies have indicated a link between arterial disease and osteoporosis. It is not known, however, whether any changes occur in bone perfusion in osteoporosis and how these may be related to increasing fat within the marrow. / The hypothesis to be tested is that: Advanced magnetic resonance (MR) techniques can be applied to investigate the non-mineralised components of bone tissue in osteoporosis thereby providing more information on bone physiology both in health and disease / This thesis is based on a series of eight studies designed to study the relationship between bone marrow perfusion, bone marrow fat content, bone marrow fat composition and bone mineral density. These studies showed that as bone mineral density decreased, bone marrow perfusion decreased. A strong reciprocal relationship was found between decreasing bone marrow perfusion and increasing marrow fat. The reduction in perfusion occurred only with bone and did not affect the extra-osseous tissues alongside bone with the same arterial supply. This indicates that the reduction in bone perfusion is not simply a reflection of a more generalized circulatory impairment in subjects with osteoporosis. This same effect is seen in both males and females and in the proximal femora as well as the spine. / In animal-based studies, we found that reduction in bone perfusion was apparent as little as two weeks after orchidectomy or oorphorectomy and closely paralleled features of impaired endothelial function as well as decreased bone mineral density and a hitherto unrecognized reduction in red marrow fraction within the medullary canal. Nitric oxide synthase, produced by the endothelium, is a potent stimulator of angiogenesis and osteoblastic activity. Mesenchymal stem cell differentiation may switch from osteoblastogenesis to adipocytogenesis under hypoxic conditions, while haematopoetic stem cells also supply endothelial stem cells. Potentially endothelial dysfunction, mesenchymal stem cell differentiation and haematopoetic stem cell maturation may be implemented in the development of osteoporosis. / In normal subjects, blood perfusion was markedly reduced in the femoral head compared to the femoral neck. In osteoporotic subjects, a further reduction in blood perfusion occurred in both areas. Overall perfusion indices reduced relatively more in the femoral neck than the femoral head region. These changes in bone perfusion help explain why subjects with osteoporosis have impaired healing of femoral neck fractures though do not seem to be at increased risk of avascular necrosis. At a micro-architectural level, reduced bone perfusion may also help explain the impaired healing of microfractures seen in subjects with osteoporosis, a feature likely to contribute to reduce bone strength, microfracture accumulation and eventually clinical insufficiency fracture. / Marrow fat was increased in subjects with osteoporosis. Our studies showed that percentage marrow fat content increased even allowing for the quantitative effect increased marrow fat has on the bone densitometry measurements. This effect was shown to be of negligible clinical significance. We found a strong reciprocal relationship between increasing fat and decreasing bone perfusion in both the proximal femur and vertebral body. Although fat content increased, very little difference in marrow fat composition was apparent between normal subjects and those with osteoporosis. We found no difference was apparent in either the N3/N6 marrow fat ratio or the spectrum of individual fatty acids in the marrow of subjects with either normal bone mineral density or osteoporosis. This suggests that alternation of marrow fat composition is not likely to be a direct contributory factor in osteoporosis. Also marrow fat increase was shown to be due to an increase in number rather than size of marrow fat cells. This suggests that marrow fat increases as a result of a switch in mesenchymal cell maturation to adipocytes rather than osteoblasts. / Below average perfusion indices in the acetabulum and adductor muscle is predictive of more pronounced bone loss at the femoral neck over the ensuing four years. Perfusion indices can also predict between fast and slow losers with a high sensitivity / To summarise, in the eight studies presented, it was shown that osteoporosis is associated with a significant reduction in bone perfusion and a reciprocal increase in marrow fat content though no change in marrow fat composition. Reduction in bone perfusion is most likely due to an accompanying reduction in functioning marrow fraction. Marrow fat increase is most likely the result of a switch in mesenchymal cell maturation to adipocytes rather than osteoblasts. The studies present in this thesis confirmed the initial hypothesis that “Advanced magnetic resonance techniques can be applied to investigate the non-mineralised components of bone tissue in osteoporosis thereby providing more information on bone physiology both in health and disease. / Griffith, James Frances. / "June 2009." / Thesis (M.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 227-250). / Appendix includes Chinese. / PREFACE AND DECLARATION --- p.1 / DEDICATION --- p.2 / ACKNOWLEDGEMENT --- p.3 / PRECIS --- p.4 / PUBLICATIONS AND PRESENTATIONS OF STUDIES RELATED TO THIS THESIS --- p.8 / INTRODUCTION --- p.16 / Chapter STUDY 1 --- What is the relationship between bone perfusion, marrow fat and bone mineral density? --- p.76 / Chapter STUDY 2 --- Vertebral marrow fat content, molecular diffusion, and perfusion indices in women with varying bone density, including osteoporosis: MR evaluation --- p.94 / Chapter STUDY 3 --- Could the results of Study 1 and Study 2 be spurious due to the effect of increasing marrow fat lowering BMD estimation by DEXA? --- p.111 / Chapter STUDY 4 --- Are the same changes in perfusion and marrow fat seen in the proximal femur as were seen in the lumbar spine (Study 1 & Study 2)? --- p.128 / Chapter STUDY 5 --- What is the reproducibility of MR perfusion studies and 1H spectroscopy of bone marrow? --- p.150 / Chapter STUDY 6 --- Marrow fat content increases but does the composition of marrow fat change in osteoporosis? --- p.159 / Chapter STUDY 7 --- Likely causes of reduced bone perfusion in osteoporosis: novel findings in an ovariectomy rat model --- p.180 / Chapter STUDY 8 --- Do perfusion indices or marrow fat content predict rate of bone loss? --- p.204 / SUMMARY --- p.222 / REFERENCES --- p.227 / ABBREVIATION LIST --- p.251 / APPENDIX --- p.253

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Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection against the consequences of ischaemia/reperfusion injury. Insulin-induced improvements in cardiac functions are widely investigated in models of ischaemia and reperfusion. It has been shown that many signalling pathways may be involved in the cardioprotection properties of insulin under those conditions. These pathways include PI3-K, PKB/Akt, p70S6k, ERK and many others. However, little data exists on the effects of insulin on the heart under normoxic condition. Some evidence has been presented that insulin has a positive inotropic effect on the normoxic perfused rat heart, but no precise cellular mechanism has been investigated or described in this regard. We believe that an investigation into the effects of insulin on cardiac function and pathways involved under normoxic conditions may help us to better understand the mechanisms of insulin-induced cardioprotection. Aims: To determine a suitable dose of insulin at which a positive inotropic response could be detectable under normoxic conditions, to investigate the possible mechanisms involved in insulin-induced increases in contractility with specific reference to the vasculature and the coronary flow and to investigate a possible involvement of PI3-K and its downstream effectors on the insulin effects on cardiac functions under normoxic conditions. Materials and methods: Isolated rat hearts were perfused retrogradely using the Langendorff technique. After 10 minutes of stabilization hearts were perfused for 30 minutes either with standard perfusion solution i.e. Krebs-Henseleit buffer + glucose gassed with 95%O2, 5%CO2 (control hearts), or with standard perfusion solution plus insulin alone or insulin together with the nitric oxide synthase inhibitor L-NAME or the PI3-K inhibitor wortmannin. Left ventricular developed pressure (LVDevP), heart rate (HR) and coronary flow (CF) as well as phosphorylated PI3-K and PKB/Akt in heart were measured. Results: Administration of insulin alone at physiological concentrations showed improved cardiac function compared to hearts in the control group. Hearts that received insulin+L-NAME showed a significant decrease in function compared to the control hearts and the hearts that received insulin alone (p<0.05). Phosphorylated PKB/Akt (Thr308) was increased in hearts that received insulin alone and insulin+L-NAME compared to the control hearts. Phosphorylated PI3-K tended to be higher in hearts where insulin was administered alone compared to the hearts that received insulin+L-NAME or insulin+wortmannin. Conclusion: This study confirmed that physiological concentrations of insulin exert positive inotropic effects on cardiac function in normoxic perfused rat hearts as seen with the improved LVDevP. Inhibition of PI3-K by wortmannin induced a decrease in phosphorylated PKB/Akt in hearts that received insulin+wortmannin and administration of L-NAME impaired the beneficial effects of insulin on cardiac functions. Therefore these results may indicate that nitric oxide may have a role in the positive effect of insulin on cardiac function in the healthy heart perfused under normoxic conditions. L-NAME as well as wortmannin reversed the positive inotropic effects of insulin. Both inhibitors also unmasked effects of insulin via nitric oxide and PI3-K on heart rate and coronary flow. / AFRIKAANSE OPSOMMING: Inleiding: Dit is welbekend dat toediening van insulien die hart beskerm teen ischemie/reperfusie-beserings, wat lei tot verbeterde hartfunksie. Hierdie effek word wyd ondersoek in modelle van ischemie en reperfusie. Dit is bewys dat ‘n verskeidenheid seintransduksie paaie, insluitend PI3-K, PKB/Akt, p70S6k en ERK, betrokke is by hierdie beskermende effek van insulien op die hart. Baie min data is egter beskikbaar rakende die effek van insulien tydens normoksiese toestande. Alhoewel dit bekend is dat insulien ’n inotropiese effek op die normale geperfuseerde hart het, is die presiese sellulêre meganismes wat dit bewerkstellig nog nie nagevors nie. Om dus ‘n beter begrip van hierdie meganismes te verkry is dit dus noodsaaklik om die effekte van insulien onder normoksiese perfusie toestande na te vors. Doelstellings: Om ‘n geskikte dosis, waarby insulien sy positiewe inotropiese effek onder normale toestande het, vas te stel, om die moontlike meganismes betrokke by insulien-geïnduseerde verbetering in hartsametrekbaarheid te bestudeer, met spesifieke verwysing na die bloedvoorsiening en koronêre vloei, en om die moontlike betrokkenheid van die PI3-K pad en sy teiken effektore onder normale suurstof-toestande te ondersoek. Materiaal en metodes: Geïsoleerde rotharte is geperfuseer deur gebruik te maak van die Langendorff tegniek. Na ‘n stabilisasie periode van 10 minute is rotharte blootgestel aan 30 minute perfusie met een van vier oplossings: ‘n standaard perfusie oplossing (Krebs-Henseleit buffer met glukose onder spesifieke gaskondisies van 95% O2, 5% CO2 – kontrole harte); standaard perfusie oplossing en insulien; standaard perfusie oplossing met insulien en die stikstofoksied sintase inhibitor L-NAME, of standaard perfusie oplossing, met insulien en die PI3-K inhibitor wortmannin. Met verloop van die perfusie protokol, is ontwikkelde linker ventrikulêre druk (LVDevP), harttempo (HR) en koronêre vloei (CF), sowel as PI3-K en PKB/Akt fosforilasie, gemeet. Resultate: Toediening van insulien teen fisiologiese konsentrasies het ‘n verbeterde hartfunksie tot gevolg, in vergelyking met harte in die kontrole groep. In teenstelling hiermee het harte wat insulien+L-NAME ontvang het ‘n betekenisvolle verlaagde funksie getoon in vergelyking met die kontrole harte en harte wat slegs insulien ontvang het (p<0.05). Harte wat slegs insulien, of insulien+L-NAME ontvang het, het ‘n verhoging in gefosforileerde PKB/Akt (Thr308) getoon in vergelyking met kontrole harte. Gefosforileerde PI3-K het ook geneig om hoër te wees in harte wat insulien+L-NAME of insulien+wortmannin ontvang het, as in harte wat slegs insulien ontvang het. Gevolgtrekking: Hierdie studie bewys dat fisiologiese konsentrasies van insulien, onder normale suurstof-toestande, ‘n positiewe inotropiese effek op hartfunksie uitoefen, soos gesien in die verbeterde LVDevP. Wortmannin-geïnduseerde inhibering van die PI3-K pad het ‘n verlaagde PKB/Akt fosforilasie tot gevolg gehad in harte wat insulien+wortmannin ontvang het, terwyl die toediening van L-NAME die voordelige effekte van insulien op hartfunksie onderdruk het. Hierdie resultate dui dus aan dat stikstofoksied ‘n rolspeler is in die positiewe inotropsiese effek van insulien op hartfunksie tydens normoksiese toestande, aangesien beide inhibitore hierdie effek onderdruk het. Beide inhibitore het ook die betrokkenheid van stikstofoksied en die PI3-K pad by die effek van insulien op harttempo en koronêre vloei onthul.

Tissue engineering strategies that combine porous biomaterial scaffolds with cells capable of osteogenesis or bioactive proteins have shown promise as effective bone graft substitutes. Attempts to culture bone tissue-engineering constructs thicker than 1mm in vitro often result in a shell of viable cells and mineralized matrix surrounding a necrotic core. To address this limitation, we developed a perfusion bioreactor system that improves mass transport throughout large cell-seeded constructs. Additionally, we established and validated 3-D computational methods to model flow and shear stresses within the microporosity of perfused constructs. Micro-CT scanning and analysis techniques were used to non-destructively monitor mineral development over time in culture. CFD modeling of axial perfusion through cylindrical scaffolds with a regular microarchitecture revealed a uniform flow field distributed throughout the scaffold. Perfusion resulted in a 140-fold increase in mineral deposition at the interior of 3 mm thick polymer scaffolds seeded with rat bone marrow stromal cells. The total detected mineral volume tripled as the construct length was increased from 3 to 9 mm. Increasing scaffold length to 9 mm did not affect the mineral volume fraction (MVF) within the full volume of each construct. Mineral volume, spatial distribution, density, particle size and particle number were then quantified on cell-seeded constructs in 5 different culture environments. The effect of time varying flow conditions was compared with continuous perfusion as well as two different control cell culture methods in an attempt to enhance mineralized matrix within the constructs. Intermittent elevated perfusion and dynamic culture in an orbital rocker plate produced the greatest amount of mineral within 9 mm long constructs compared to low continuous flow and high continuous flow cases. Together, these studies indicate that dynamic culture conditions enhance construct development with regards to cell viability, mineralized matrix deposition, growth rate, and distribution. Furthermore, these techniques provide a rational approach to selecting perfusion culture conditions that optimize the amount and distribution of mineralized matrix production. Finally, the established perfusion bioreactor, in combination with micro-CT analysis, provides a foundation for evaluating new scaffolds and cell types that may be useful for the development of effective bone graft substitutes.