Use of the MicroSiM (µSiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier.

The microSiM (µSiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the µSiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between 'blood' and 'brain' chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the µSiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues

Ultrathin Silicon Membranes for Wearable Dialysis

The development of wearable or implantable technologies that replace center-based hemodialysis (HD) hold promise to improve outcomes and quality of life for patients with ESRD. A prerequisite for these technologies is the development of highly efficient membranes that can achieve high toxin clearance in small-device formats. Here we examine the application of the porous nanocrystalline silicon (pnc-Si) to HD. pnc-Si is a molecularly thin nanoporous membrane material that is orders of magnitude more permeable than conventional HD membranes. Material developments have allowed us to dramatically increase the amount of active membrane available for dialysis on pnc-Si chips. By controlling pore sizes during manufacturing, pnc-Si membranes can be engineered to pass middle-molecular-weight protein toxins while retaining albumin, mimicking the healthy kidney. A microfluidic dialysis device developed with pnc-Si achieves urea clearance rates that confirm that the membrane offers no resistance to urea passage. Finally, surface modifications with thin hydrophilic coatings are shown to block cell and protein adhesion

The Effects of Topical Heating for Therapeutic Uses

This item is not available.The application of topical heat for therapeutic purposes has become commonplace in America. It is used by professionals including physical therapists and physicians to treat their patients as well as by individuals within their home or at work. Using computer simulation of two-dimensional heat transfer through the outer tissue layers of the body, the process of heat transfer and temperature gradients within the tissues can be predicted. The objective of this study was to determine the temperature gradient of the muscle layer after applications of heat for less than one-half hour. Finite energy heat sources such as a hot water bottle as well as electric heat constant-temperature sources were evaluated. Applied temperatures were maintained at 50 C or less so not to irritate the skin surface. It was determined in all cases that the temperature of the muscle did not significantly increase within our time frame and actually began to cool after fifteen minutes with the hot water bottle case studies. On the other hand, the temperature of the epidermal-dermal layer, where nerve endings exist, remained at an elevated state of above 40 C for an extended period of time. It is inferred that a heat stimulated response of the neurons may be the cause of muscle relaxation and pain relief when a topical heat source is applied

Therapeutic Potential of Extracellular Vesicles in Degenerative Diseases of the Intervertebral Disc

Extracellular vesicles (EVs) are lipid membrane particles carrying proteins, lipids, DNA, and various types of RNA that are involved in intercellular communication. EVs derived from mesenchymal stem cells (MSCs) have been investigated extensively in many different fields due to their crucial role as regeneration drivers, but research for their use in degenerative diseases of the intervertebral disc (IVD) has only started recently. MSC-derived EVs not only promote extracellular matrix synthesis and proliferation in IVD cells, but also reduce apoptosis and inflammation, hence having multifunctional beneficial effects that seem to be mediated by specific miRNAs (such as miR-233 and miR-21) within the EVs. Aside from MSC-derived EVs, IVD-derived EVs (e.g., stemming from notochordal cells) also have important functions in IVD health and disease. This article will summarize the current knowledge on MSC-derived and IVD-derived EVs and will highlight areas of future research, including the isolation and analysis of EV subpopulations or exposure of MSCs to cues that may enhance the therapeutic potential of released EVs.ISSN:2296-418

Quantitative methods for understanding physical mechanisms of neutrophil adhesion

Thesis (Ph. D.)--University of Rochester. Dept. of Biomedical Engineering, 2008.During inflammation, neutrophils first roll and then firmly adhere to activated endothelium in a sequence known as the adhesion cascade. Knowing the mobility and localization of the receptors that mediate binding between endothelium and neutrophils is key to building an accurate biophysical model of the adhesion cascade. The objective of this thesis is to characterize the mobility and localization of the main neutrophil adhesion receptors involved in the rolling and firm arrest stages of neutrophil recruitment To measure the natural movements of neutrophil membrane receptors, we built a novel fluorescence recovery after photobleaching (FRAP) experimental system and developed a set of complementary analytical tools to interpret data. Experiments examined non-activated neutrophils in their normal, spherical morphology. Prior studies have been performed on artificially flattened cells to accommodate the requirements of established methods for measuring diffusion of membrane proteins. Further, our studies are the first to measure the mobility of all four neutrophil receptors involved in early adhesion: the rolling associated adhesion molecules L-selectin and PSGL-1, and the β2 integrins LFA-1 and Mac-1. On resting neutrophils we found that β2 integrins have mobilities 3-7x greater than rolling associated adhesion molecules, but that mobilities within these classes was indistinguishable. We also found that β2 integrin mobility (LFA-1, D = 1.2 x 10-10 cm2/sec; 37oC, Fab) approached the expected value for free protein diffusion and was not affected by cytoskeletal poisons. These results suggest any cytoskeletal hindrance does not limit bulk integrin diffusion in resting neutrophils over distances and times important for adhesive plaque formation. Another important physical parameter in adhesion regulation is localization on the ruffled neutrophil surface. Total internal reflection fluorescence microscopy confirms for the first time on live resting neutrophils that L-selectin is localized to the microvilli tips, while LFA-1 is restricted to the cell body, away from potential contact surfaces. All four key adhesion receptors were observed to be localized to dynamic, mobile domains that corresponded to this topological positioning. We adapted image correlation microscopy (ICM) to wide-field use with the goal of measuring lateral mobility of these domains. We find that domain lateral mobility is similar to measurements made with FRAP, suggesting domain movement is a significant component of adhesion molecule lateral mobility in the membrane. Our results help elucidate how adhesion receptors move on the neutrophil surface and how adhesive plaques can form and strengthen following rolling. Beyond adding insight and building intuition, the quantitative results will aid computer simulations of the recruitment process, helping predict behavior and test theorems based on empirical evidence

Influence of silicon dioxide capping layers on pore characteristics in nanocrystalline silicon membranes

Abstract Porous nanocrystalline silicon (pnc-Si) membranes are a new class of membrane material with promising applications in biological separations. Pores are formed in a silicon film sandwiched between nm thick silicon dioxide layers during rapid thermal annealing. Controlling pore size is critical in the size-dependent separation applications. In this work, we systematically studied the influence of the silicon dioxide capping layers on pnc-Si membranes. Even a single nm thick top oxide layer is enough to switch from agglomeration to pore formation after annealing. Both the pore size and porosity increase with the thickness of the top oxide, but quickly reach a plateau after 10 nm of oxide. The bottom oxide layer acts as a barrier layer to prevent the a-Si film from undergoing homo-epitaxial growth during annealing. Both the pore size and porosity decrease as the thickness of the bottom oxide layer increases to 100 nm. The decrease of the pore size and porosity is correlated with the increased roughness of the bottom oxide layer, which hinders nanocrystal nucleation and nanopore formation

Porous Substrates Promote Endothelial Migration at the Expense of Fibronectin Fibrillogenesis

Porous substrates have gained increased usage in cell studies and tissue mimetic applications because they can partition distinct cell types while still allowing important biochemical crosstalk. In the presented work, we investigated how porous substrates with micron and submicron features influence early cell migration and the associated ECM establishment, which can critically affect the rate of cell coverage on the substrate and the ensuing tissue organization. We showed through time-lapse microscopy that cell speed and migratory distance on membranes with 0.5 μm pores were nearly 2-fold of those observed on nonporous membranes, while values on membranes with 3.0 μm pores fell in between. Although the cell directionality ratio and the persistence time was unaffected by the presence of pores, the cells did exhibit directionality preferences based on the hexagonal pore patterning. Fibronectin fibrillogenesis exhibited a distinct inverse relationship to cell speed, as the fibrils formed on the nonporous control were significantly longer than those on both types of porous substrates. We further confirmed on a per cell basis that there is a negative correlation between fibronectin fibril length and cell speed. The observed trade-off between early cell coverage and ECM establishment thus warrants consideration in the selection or the engineering of the ideal porous substrate for tissue mimetic applications and may help guide future cell studies

Membrane Pore Spacing Can Modulate Endothelial Cell–Substrate and Cell–Cell Interactions

Mechanical cues and substrate interaction affect the manner in which cells adhere, spread, migrate and form tissues. With increased interest in tissue-on-a-chip and coculture systems utilizing porous membranes, it is important to understand the role of disrupted surfaces on cellular behavior. Using a transparent glass membrane with defined pore geometries, we investigated endothelial fibronectin fibrillogenesis and formation of focal adhesions as well as development of intercellular junctions. Cells formed fewer focal adhesions and had shorter fibronectin fibrils on porous membranes compared to nonporous controls, which was similar to cell behavior on continuous soft substrates with Young’s moduli 7 orders of magnitude lower than glass. Additionally, porous membranes promoted enhanced cell–cell interactions as evidenced by earlier formation of tight junctions. These findings suggest that porous membranes with discontinuous surfaces promote reduced cell–matrix interactions similarly to soft substrates and may enhance tissue and barrier formation

Membrane Mobility of β2 Integrins and Rolling Associated Adhesion Molecules in Resting Neutrophils

The mobilities of transmembrane adhesion proteins are key underlying physical factors that contribute to neutrophil adhesion and arrest during inflammation. Here we present a novel (to our knowledge) fluorescence recovery after photobleaching system and a complementary analytical model to measure the mobility of the four key receptors involved in the adhesion cascade: L-selectin, PSGL-1, Mac-1, and LFA-1 for resting, spherical, and human neutrophils. In general, we find that β2 integrins (Mac-1, LFA-1) have mobilities 3–7 times faster than rolling associated molecules (L-selectin; PSGL-1), but that the mobilities within each of these groups are indistinguishable. Increasing temperature (room temperature versus 37°C) results in increased mobility, in all cases, and the use of a bivalent antibody label (mAb versus Fab) decreases mobility, except in the case of rolling associated molecules at room temperature. Disrupting the actin cytoskeleton increased mobility except that the highest mobilities measured for integrins (D = 1.2 × 10−9 cm2/s; 37°C, Fab) are not affected by actin poisons and approach the expected value for free diffusion. Although evidence of cytoskeletal hindrance of integrin mobility has been found in other systems, our data suggest such hindrance does not limit bulk integrin diffusion in resting neutrophils over distances and times important for adhesive plaque formation