Development and Application of a 3-D Perfusion Bioreactor Cell Culture System for Bone Tissue Engineering

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.Ph.D.Committee Chair: Guldberg, Robert; Committee Member: Fyhrie, David; Committee Member: Garcia, Andres; Committee Member: Neitzel, Paul; Committee Member: Wick, Ti

A realist evaluation of the management of a well- performing regional hospital in Ghana

<p>Abstract</p> <p>Background</p> <p>Realist evaluation offers an interesting approach to evaluation of interventions in complex settings, but has been little applied in health care. We report on a realist case study of a well performing hospital in Ghana and show how such a realist evaluation design can help to overcome the limited external validity of a traditional case study.</p> <p>Methods</p> <p>We developed a realist evaluation framework for hypothesis formulation, data collection, data analysis and synthesis of the findings. Focusing on the role of human resource management in hospital performance, we formulated our hypothesis around the high commitment management concept. Mixed methods were used in data collection, including individual and group interviews, observations and document reviews.</p> <p>Results</p> <p>We found that the human resource management approach (the actual intervention) included induction of new staff, training and personal development, good communication and information sharing, and decentralised decision-making. We identified 3 additional practices: ensuring optimal physical working conditions, access to top managers and managers' involvement on the work floor. Teamwork, recognition and trust emerged as key elements of the organisational climate. Interviewees reported high levels of organisational commitment. The analysis unearthed perceived organisational support and reciprocity as underlying mechanisms that link the management practices with commitment.</p> <p>Methodologically, we found that realist evaluation can be fruitfully used to develop detailed case studies that analyse how management interventions work and in which conditions. Analysing the links between intervention, mechanism and outcome increases the explaining power, while identification of essential context elements improves the usefulness of the findings for decision-makers in other settings (external validity). We also identified a number of practical difficulties and priorities for further methodological development.</p> <p>Conclusion</p> <p>This case suggests that a well-balanced HRM bundle can stimulate organisational commitment of health workers. Such practices can be implemented even with narrow decision spaces. Realist evaluation provides an appropriate approach to increase the usefulness of case studies to managers and policymakers.</p

Is Vasomotion in Cerebral Arteries Impaired in Alzheimer’s Disease?

A substantial body of evidence supports the hypothesis of a vascular component in the pathogenesis of Alzheimer’s disease (AD). Cerebral hypoperfusion and blood-brain barrier dysfunction have been indicated as key elements of this pathway. Cerebral amyloid angiopathy (CAA) is a cerebrovascular disorder, frequent in AD, characterized by the accumulation of amyloid-β (Aβ) peptide in cerebral blood vessel walls. CAA is associated with loss of vascular integrity, resulting in impaired regulation of cerebral circulation, and increased susceptibility to cerebral ischemia, microhemorrhages, and white matter damage. Vasomotion— the spontaneous rhythmic modulation of arterial diameter, typically observed in arteries/arterioles in various vascular beds including the brain— is thought to participate in tissue perfusion and oxygen delivery regulation. Vasomotion is impaired in adverse conditions such as hypoperfusion and hypoxia. The perivascular and glymphatic pathways of Aβ clearance are thought to be driven by the systolic pulse. Vasomotion produces diameter changes of comparable amplitude, however at lower rates, and could contribute to these mechanisms of Aβ clearance. In spite of potential clinical interest, studies addressing cerebral vasomotion in the context of AD/CAA are limited. This study reviews the current literature on vasomotion, and hypothesizes potential paths implicating impaired cerebral vasomotion in AD/CAA. Aβ and oxidative stress cause vascular tone dysregulation through direct effects on vascular cells, and indirect effects mediated by impaired neurovascular coupling. Vascular tone dysregulation is further aggravated by cholinergic deficit and results in depressed cerebrovascular reactivity and (possibly) impaired vasomotion, aggravating regional hypoperfusion and promoting further Aβ and oxidative stress accumulation

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Noninvasive image analysis of 3D construct mineralization in a perfusion bioreactor

10.1016/j.biomaterials.2007.01.013Biomaterials28152525-2533BIMA