Effect of Nano-to Micro-Scale Surface Topography on the Orientation of Endothelial Cells

The effect of grating textures on the alignment of cell shape and intracellular actin cytoskeleton has been investigated in bovine aortic endothelial cells (BAECs) cultured on a model cross-linked poly(dimethylsiloxane) (PDMS). Grating-textured PDMS substrates, having a variation in channel depths of 200 nm, 500 nm, 1 µm and 5 µm, were coated with fibronectin (Fn) to promote endothelial cell adhesion and cell orientation. As cells adhered to the Fn-coated surface, the underlying grating texture has shown to direct the alignment of cell shape, F-actin and focal contacts parallel to the channels. Cell alignment was observed to increase with increasing channel depths, reaching the maximum orientation where most cells aligned parallel to channels on 1-µm textured surface. Immunofluorescence studies showed that F-actin stress fibers and vinculin at focal contacts also aligned parallel to the channels. Cell proliferation was found to be independent of grating textures and the alignment of cell shape was maintained at confluence

Functionalized Nanodiamond Reinforced Biopolymers

The present invention includes a composition for implantation in a patient, comprising surface-functionalized nanodiamonds and at least one biodegradable biocompatible polymer. The present invention also includes a surgical fixation device for use in a patient

A novel real-time system to monitor cell aggregation and trajectories in rotating wall vessel bioreactors

Journal of Biotechnology, 125(3): pp. 416-424.Rotating wall vessel bioreactors (RWVs) constitute dynamic suspension culture venues for tissue engineering. Quantitative real-time assessment of the kinetics of cell–cell aggregation in RWVs can yield mechanistic information about the initial steps leading to the assembly of individual cells into tissue-like constructs. In our imaging system, fluorescently labeled cells suspended in a HARV-type RWV were irradiated by a laser-beam. Emission was recorded by a camera mounted at 90° to the excitation plane. Using macro lenses, the system identified 5 μm particles from a 5 cm working distance, distinguished aggregated 20 μm microspheres from larger (45 and 90 μm) microspheres, and plotted local trajectories of microspheres and cells. Sizes of PC12 cells assessed by our system matched conventional measurements. We validated the system's ability to follow HepG2 and PC12 aggregation in real time over 24 h of RWV culture. Taken together, our system provides the means to measure and analyze in real time the processes that lead to the 3D tissue-like assembly of diverse cell types into spheroids. Future studies include development of intelligent feedback algorithms, allowing automatic control over RWV rotational speed required to maintain aggregating cells and nascent tissue in continual free fall

The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein

AbstractThe effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001–0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, γ-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-α-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of Gi-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca2+ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive Gi-protein

Development of a flow cytometry based assay for defective T cell signal transduction in the rotating wall vessel bioreactor

Poster presented at Biomedical Technology Showcase 2006, Philadelphia, PA. Retrieved 18 Aug 2006 from http://www.biomed.drexel.edu/new04/Content/Biomed_Tech_Showcase/Poster_Presentations/Lelkes_5.pdf.Dysfunction of T cell signal transduction is responsible for many immune-mediated diseases as well as in aging. Rotating wall vessel (RWV) bioreactors offer a unique environment, in which pathologies due to signaling dysfunction can be mimicked non-pharmacologically. There is an urgent un-met clinical need for developing a flow cytometric assay to rapidly measure signal transduction by T cells cultured in an RWV bioreactor as a means for developing meaningful therapeutic approaches. Our goal is to develop and optimize such an assay in for of a user-friendly kit as a high throughput method for testing immune-ameliorative drugs in the RWV

Tissue engineering technologies for cardiac and neuronal applications: intelligent scaffolds made by electrospinning, lyophilization, and critical point drying

Poster presented at Biomedical Technology Showcase 2006, Philadelphia, PA. Retrieved 18 Aug 2006 from http://www.biomed.drexel.edu/new04/Content/Biomed_Tech_Showcase/Poster_Presentations/Lelkes_1.pdf.Tissue engineering is a rapidly growing area that aims to create, repair and/or replace tissues and organs by using combinations of cells, biomaterials, and/or biologically active molecules. Tissue engineering strategies promise to advance current therapies for numerous clinical applications, such as repair of spinal cord injury, or of irreversible myocardial damage, heart failure, and significantly improve the quality of life for millions of patients. Amongst the most challenging goals in the field of neuronal and also of cardiovascular tissue engineering is the creation of engineered functional task-specific tissue constructs. In this study, we summarized the tissue engineering scaffolds that could be used to grow functional tissue constructs for several clinical applications

Dynamic hi-resolution horizontal microscope for monitoring and manipulating real-time tissue assembly

Poster presented at Biomedical Technology Showcase 2006, Philadelphia, PA. Retrieved 18 Aug 2006 from http://www.biomed.drexel.edu/new04/Content/Biomed_Tech_Showcase/Poster_Presentations/Lelkes_4.pdf.There is an un-met clinical and technological need in regenerative medicine and tissue engineering for developing a non-invasive technique for monitoring and manipulating tissue assembly. Our goal is to create an integrated hardware/ software system to automatically diagnose, monitor, and control tissue growth in dynamic bioreactors in real-time. For this we propose to engineer and test a versatile, hi-resolution horizontal microscope that can intelligently aid in the on-line optimization of tissue growth parameters within a bioreactor. Having developed a functional prototype, we succeeded in compacting and significantly reducing the costs for the 2nd generation system, which now needs further validation

Engineered tissue models for drug development: the lung as a paradigm

Poster presented at Biomedical Technology Showcase 2006, Philadelphia, PA. Retrieved 18 Aug 2006 from http://www.biomed.drexel.edu/new04/Content/Biomed_Tech_Showcase/Poster_Presentations/Lelkes_6.pdf.There is an un-met clinical and technological need for developing high-fidelity tissue models for high-throughput drug discovery. Tissue engineering is a viable means for engineering functional tissue equivalents, which hold the promise of revolutionizing the pharmacological industry by providing novel venues for high throughput drug testing. Working at the interface between applied developmental biology/medicine and tissue engineering we propose to generate such functional tissue equivalents, using the distal lung as our prime paradigm. With such model tissue we will demonstrate physiological responses, induce pathological conditions, and test therapeutics in disease models, such as pulmonary hypertension and emphysema

A highthroughput production of composite breast tumoroids: a tool for investigation of cellular heterogeneity and drug delivery

Poster presented at Biomedical Technology Showcase 2006, Philadelphia, PA. Retrieved 18 Aug 2006 from http://www.biomed.drexel.edu/new04/Content/Biomed_Tech_Showcase/Poster_Presentations/Tozeren.pdf.Breast tumors are typically heterogeneous and contain diverse subpopulations of tumor cells with differing phenotypic properties. This study has developed an in vitro co-culture-based three-dimensional breast tumor model that studies the effects of mixing heterogeneous tumor cell populations. Breast cancer cell lines of different phenotypes (MDAMB231, MCF7 and ZR751) were co-cultured in a rotating wall vessel (RWV) bioreactor to form a large number of heterogeneous tumoroids. Prior to each experiment, cells were labeled with cell tracker dyes to allow for time-course fluorescence microscopy to monitor cell aggregation. Histological sections of the tumor spheroids were stained with hematoxylin and eosin (HE), progesterone receptor (PR), E-cadherin (E-cad) and proliferation marker, ki67. Results showed that heterogeneous tumoroids reflecting the composition of the growth rate, invasion potential, and spatial distributions of heterogeneous tumor spheroids were highly dependent on cell composition. A suitable in vitro model for studying tumor-cell heterogeneity and reciprocal interactions will accelerate understanding of tumor cell phenotype population dynamics