Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Engineering access control for distributed enterprise applications

Access control (AC) is a necessary defense against a large variety of security attacks on the resources of distributed enterprise applications. However, to be effective, AC in some application domains has to be fine-grain, support the use of application-specific factors in authorization decisions, as well as consistently and reliably enforce organization-wide authorization policies across enterprise applications. Because the existing middleware technologies do not provide a complete solution, application developers resort to embedding AC functionality in application systems. This coupling of AC functionality with application logic causes significant problems including tremendously difficult, costly and error prone development, integration, and overall ownership of application software. The way AC for application systems is engineered needs to be changed. ^ In this dissertation, we propose an architectural approach for engineering AC mechanisms to address the above problems. First, we develop a framework for implementing the role-based access control (RBAC) model using AC mechanisms provided by CORBA Security. For those application domains where the granularity of CORBA controls and the expressiveness of RBAC model suffice, our framework addresses the stated problem. ^ In the second and main part of our approach, we propose an architecture for an authorization service, RAD, to address the problem of controlling access to distributed application resources, when the granularity and support for complex policies by middleware AC mechanisms are inadequate. Applying this architecture, we developed a CORBA-based application authorization service (CAAS). Using CAAS, we studied the main properties of the architecture and showed how they can be substantiated by employing CORBA and Java technologies. Our approach enables a wide-ranging solution for controlling the resources of distributed enterprise applications. ^

Design and performance assessment of aortic heart valve for tissue engineering

Current artificial heart valves are classified as mechanical and bioprosthetic. An appealing pathway that promises to overcome the shortcomings of commercially available heart valves is offered by the interdisciplinary approach of cardiovascular tissue engineering. However, the mechanical properties of the Tissue Engineering Heart Valves (TEHV) are limited and generally fail in the long-term use. To meet this performance challenge novel biodegradable triblock copolymer poly(ethylene oxide)-polypropylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO or F108) crosslinked to Silk Fibroin (F108-SilkC) to be used as tri-leaflet heart valve material was investigated. ^ Synthesis of ten polymers with varying concentration and thickness (55 µm, 75 µm and 100 µm) was achieved via a covalent crosslinking scheme using bifunctional polyethylene glycol diglycidyl ether (PEGDE). Static and fatigue testing were used to assess mechanical properties of films, and hydrodynamic testing was performed to determine performance under a simulated left ventricular flow regime. The crosslinked copolymer (F108-Silk C) showed greater flexibility and resilience, but inferior ultimate tensile strength, by increasing concentration of PEGDE. Concentration molar ratio of 80:1 (F108: Silk) and thickness of 75 µm showed longer fatigue life for both tension-tension and bending fatigue tests. Four valves out of twelve designed satisfactorily complied with minimum performance requirement ISO 5840, 2005. ^ In conclusion, it was demonstrated that the applicability of a degradable polymer in conjugation with silk fibroin for tissue engineering cardiovascular use, specifically for aortic valve leaflet design, met the performance demands. Thinner thicknesses (t<75 µm) in conjunction with stiffness lower than 320 MPa (80:1, F108: Silk) are essential for the correct functionality of proposed heart valve biomaterial F108-SilkC. Fatigue tests were demonstrated to be a useful tool to characterize biomaterials that undergo cyclic loading. ^

Regulation of Bone Marrow Stem Cells through Oscillatory Shear Stresses - A Heart Valve Tissue Engineering Perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range.

Engineering Analysis in Imprecise Geometric Models

Engineering analysis in geometric models has been the main if not the only credible/reasonable tool used by engineers and scientists to resolve physical boundaries problems. New high speed computers have facilitated the accuracy and validation of the expected results. In practice, an engineering analysis is composed of two parts; the design of the model and the analysis of the geometry with the boundary conditions and constraints imposed on it. Numerical methods are used to resolve a large number of physical boundary problems independent of the model geometry. The time expended due to the computational process are related to the imposed boundary conditions and the well conformed geometry. Any geometric model that contains gaps or open lines is considered an imperfect geometry model and major commercial solver packages are incapable of handling such inputs. Others packages apply different kinds of methods to resolve this problems like patching or zippering; but the final resolved geometry may be different from the original geometry, and the changes may be unacceptable. The study proposed in this dissertation is based on a new technique to process models with geometrical imperfection without the necessity to repair or change the original geometry. An algorithm is presented that is able to analyze the imperfect geometric model with the imposed boundary conditions using a meshfree method and a distance field approximation to the boundaries. Experiments are proposed to analyze the convergence of the algorithm in imperfect models geometries and will be compared with the same models but with perfect geometries. Plotting results will be presented for further analysis and conclusions of the algorithm convergence

Engineering access control for distributed enterprise applications

Access control (AC) is a necessary defense against a large variety of security attacks on the resources of distributed enterprise applications. However, to be effective, AC in some application domains has to be fine-grain, support the use of application-specific factors in authorization decisions, as well as consistently and reliably enforce organization-wide authorization policies across enterprise applications. Because the existing middleware technologies do not provide a complete solution, application developers resort to embedding AC functionality in application systems. This coupling of AC functionality with application logic causes significant problems including tremendously difficult, costly and error prone development, integration, and overall ownership of application software. The way AC for application systems is engineered needs to be changed. In this dissertation, we propose an architectural approach for engineering AC mechanisms to address the above problems. First, we develop a framework for implementing the role-based access control (RBAC) model using AC mechanisms provided by CORBA Security. For those application domains where the granularity of CORBA controls and the expressiveness of RBAC model suffice, our framework addresses the stated problem. In the second and main part of our approach, we propose an architecture for an authorization service, RAD, to address the problem of controlling access to distributed application resources, when the granularity and support for complex policies by middleware AC mechanisms are inadequate. Applying this architecture, we developed a CORBA-based application authorization service (CAAS). Using CAAS, we studied the main properties of the architecture and showed how they can be substantiated by employing CORBA and Java technologies. Our approach enables a wide-ranging solution for controlling the resources of distributed enterprise applications.

Regulation of bone marrow stem cells through oscillatory shear stresses - A heart valve tissue engineering perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range. ^