This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.

Aortic valve disease (AVD) affects millions of people of all ages around the world. Current treatment for AVD consists of valvular replacement with a non-living prosthetic valve, which is incapable of growth, self-repair, or remodeling. While tissue engineering has great promise to develop a living heart valve alternative, success in animal models has been limited. This may be attributed to the fact that understanding of valvular cell biology has not kept pace with advances in biomaterial development. Aortic valve leaflets are exposed to a complex and dynamic mechanical environment unlike any in the vasculature, and it is likely that native endothelial and interstitial cells respond to mechanical forces differently from other vascular cells. The objective of this thesis was to compare valvular cell phenotype to vascular cell phenotype, and assess the influence of steady shear stress on valvular cell biology. This thesis demonstrates that valvular endothelial cells respond differently to shear than vascular endothelial cells, by aligning perpendicular to the direction of steady shear stress, and by the differential regulation of hundreds of genes in both static and fluid flow environments. Valvular interstitial cells expressed a combination of contractile and synthetic phenotypes not mimicked by vascular smooth muscle cells. Two three-dimensional leaflet models were developed to assess cellular interactions and the influences of steady laminar shear stress. Valvular co-culture models exhibited a physiological response profile, while interstitial cell-only constructs behaved more pathologically. Steady shear stress enhanced physiological functions of valvular co-cultures, but increased pathological response of interstitial cell-only constructs. These results showed that valvular cells, whether cultured separately or together, behaved distinctly different from vascular cells. It was also determined that shear stress alone cannot induce tissue remodeling to more resemble native valve leaflets. The leaflet models developed in this thesis can be used in future experiments to explore valvular cell biology, assess the progression of certain forms AVD, and develop targeted diagnostic and therapeutic strategies to hopefully eliminate the need for valvular replacement entirely.

Bileaflet mechanical heart valves (BMHVs) are prone to thrombus formation in the hinge region due to high shear stress combined with stagnation regions. This thesis research addresses the hypothesis that models that isolate and mimic BMHV hinge geometries can be used to quantitatively characterize procoagulant potential using a novel in vitro blood flow system. Furthermore, these results can be correlated with digital particle image velocimetry (DPIV) measurements detailing flow fields for the same models. The significant findings were that: 1) recalcification of recirculating citrated blood markedly increases the magnitude of thrombus forming reactions and the sensitivity for their detection; 2) platelet activation, and the presence of adequate platelet numbers are essential for the activation of coagulation under conditions of high shear; and 3) thrombin formation can be inhibited by blocking the platelet receptors that facilitate platelet aggregation. The DPIV studies give some insight into why different channel geometries resulted in varying propensities for coagulation. The channel geometries with abrupt changes in diameter induced significantly higher levels of TAT and also formed jets that were subject to increased entrainment of the stagnant fluid in the chamber. This entrainment enables more mixing of the shear-activated platelets with the surrounding flow, which can propagate the coagulation cascade, thus increasing the chance for thrombus formation. The influence of abrupt changes in diameter was also evident in the BMHV human blood studies. The MP valve, which has a tortuous hinge pathway, induced significantly more TAT formation than the SJM Standard valve with a smoother hinge channel. Thus, BMHV hinge geometry should be as smooth and free of diameter changes as possible to eliminate stagnation regions that enable activated platelets to congregate and propagate the coagulation cascade. Leakage gap width also had a significant effect not only on procoagulant potential but also on platelet activation. Both the low and high leaker prototype valves had significantly higher levels of platelet activation compared to the SJM Standard valve, but only the low leaker valve demonstrated a higher propensity for coagulation. Thus, to minimize both platelet activation and thromboemboli formation, an optimal gap width should be maintained for BMHVs.

Current designs of bileaflet mechanical heart valves (BMHVs) and trileaflet polymeric heart valves(TPHVs) are plagued by unacceptable levels of hemolysis and thrombus formation in critical areas thereby producing mediocre clinical performance. The objective of this study is: (1) to investigate the influence of BMHV designs on hinge flow characteristics, (2) to quantify the influence of hinge gap width tolerance in a BMHV design, and (3) to investigate the influence of TPHV design on flow characteristics. St. Jude Medical (SJM) provided four transparent mitral BMHVs: one 23 mm CarboMedics (CM), one 27 mm SJM Standard and two 27 mm prototype BHMVs with altered hinge gap widths. Aortech Inc. provided three 23 mm aortic prototype TPHVs. Laser Doppler velocimetry and Particle Image velocimetry were used to measure flow velocity inside these valve prostheses. The flows through the valves were maintained within physiological limits. All valves revealed Reynolds shear stress (RSS) levels greater than 200 Pa far exceeding the threshold for platelet activation and hemolysis. MHV hinge flows in the mitral position were characterized by a strong recirculation during ventricular diastole while leakage jets over and adjacent to leaflets were prominent during ventricular systole. CM hinge flow had higher RSS than in the SJM hinge. The large gap width hinge had the largest leakage jet size and highest RSS (>400 Pa) during ventricular diastole. The Standard gap width hinge showed better washout during systole and provided optimum hemodynamic performance than the prototype designs. In aortic prototype PHVs, elevated RSS conducive to hemolysis was observed along the central jet during systole and the leakage jet at the high central region inside the valve during diastole. This study showed that hinge geometry designs and hinge gap width tolerance governed the success of the bileaflet MHV design. Also the performance of the three aortic PHVs is dependent on commissural designs and leaflet thicknesses. Owing to the critical nature flow fields on clinical outcomes studies such as the current study should be conducted in the pre-clinical evaluation phase for all new MHV or PHVs.

Mechanical forces are important regulators of cell function in many tissues including, for example, bone and components of the cardiovascular system. The endothelial lining of blood vessels has been shown to respond in an atheroprotective manner to unidirectional, laminar flow-induced shear stress and in an atherogenic manner to oscillating and low levels of shear. We have developed a cone and plate shear apparatus to simulate fluid shear stress on endothelial cells in vitro. The significant feature of this apparatus is that, unlike other in vitro flow systems, it accurately produces varying levels of shear stress, consistent with those created in vivo during the cardiac cycle. Flow characteristics of this system were verified by computational fluid dynamics (CFD) and laser Doppler velocimetry (LDV). Cellular responses were monitored by cell morphology and protein expression. These responses are consistent with in vivo responses as well as previous work using other in vitro flow systems.

Collecting microlymphatics play a vital role in promoting lymph flow from the initial lymphatics in the interstitial spaces to the large transport lymph ducts. In most tissues, the primary mechanism for producing this flow is the spontaneous contractions of the lymphatic wall. Individual units, known as lymphangion, are separated by valves that help prevent backflow when the vessel contracts, thus promoting flow through the lymphatic network. Lymphatic contractile activity is inhibited by flow in isolated lymphatics, however there are virtually no in situ measurements of lymph flow in these vessels. Initially, a high speed imaging system was set up to image in situ preparations at 500 fps. These images were then manually processed to extract information regarding lymphocyte velocity (-4 to 10 mm/sec), vessel diameter (25 to 165 um), and particle location. Fluid modeling was performed to obtain reasonable estimates of wall shear stress (-8 to 17 dynes/cm2). One of the difficulties encountered was the time consuming methods of manual particle tracking. Using previously captured images, an image correlation method was developed to automate lymphatic flow measurements and to track wall movements as the vessel contracts. Using this method the standard error of prediction for velocity measurements was 0.4 mm/sec and for diameter measurements it was 7.0 µm. It was found that the actual physical quantity being measured through this approach is somewhere between the spatially averaged velocity and the maximum velocity of a Poiseuille flow model.

Scour around various structures obstructing flow in an open channel is a common problem; therefore a better understanding of how turbulent flow affects sediment transport is needed. Additionally, is it the mean flow or the turbulence properties that are more important in contributing to bed shear stress? To this end, an experimental study was conducted in a fixed-bed flume containing a series of dikes. Turbulence intensities and Reynold's stresses were calculated from 3-D velocity measurements gathered with a microADV. Results showed that the maximum shear stress was nearly 12-20 times that of the approach flow, while maximum turbulence intensities were about 3-5 times those of the incoming flow. Highest magnitudes of both were seen at the tip of the second dike in the three-dike series. The mean velocity appeared to have no relation to the formation of scour near the tips of the dikes but the turbulence intensities did.

The vascular endothelium is a single layer of cells that lines the interior surface of our blood vessels. The endothelium plays a key role in vasoprotection and vasoregulation and its proper function is therefore essential to the maintenance of vascular health. The endothelial cells respond to the frictional force (shear stress (SS)) that occurs with an increase in blood flow. As a response, vasoactive substances are released, causing the artery to dilate, this is termed flow-mediated dilation (FMD). Endothelial cell function can be assessed by measuring the vasodilatory response to an increase in SS. Currently however, our ability to interpret the results of FMD assessment in order to make accurate judgements regarding arterial health is hindered by an incomplete understanding of the “dose-response” relationship between SS and FMD. The dose-response relationship is characterized by 1) the SS stimulus required to elicit an FMD response (threshold), 2) the magnitude of dilation for a given increase in SS (the slope of the SS-FMD relationship), and 3) the point at which further increases in SS no longer elicit dilation (the ceiling). The primary purpose of the current study was to characterize the magnitude and day-to-day variability of the parameters described above. N=20 males (mean 22-years). Brachial artery diameter (BAD) and blood velocity (BV) were assessed with echo and Doppler ultrasound. SS was estimated as shear rate (SR=BV/BAD). Subjects performed 2 incremental handgrip exercise trials on two separate visits (V1 and V2). CV=co-efficient of variation. The SS-FMD relationship was characterized by a shallow slope followed by an inflection point (threshold (T1)) and a steeper slope (pre vs. post T1 slope p=0.002). There was no difference between V1 vs. V2 in the SR-FMD slope or threshold (p>0.05), but there was considerable within-subject variability in the SR-FMD parameters: pre-T1 slope CV = 47.0 ± 33.1%; post-T1 slope CV = 55.3 ± 40.7%; T1 CV = 25.6 ± 6.3%. In conclusion, %FMD did not plateau with increasing SR, therefore no ceiling was identified. The inflection in slope may indicate the involvement of different or additional vasodilator mechanisms post-threshold. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2011-09-15 20:17:11.582

This thesis presents a computational analysis of multi-element guard-heated sensors designed to overcome the most severe limitation of conventional thermal sensors for wall shear stress (WSS) measurement in turbulent flows –that of indirect heat conduction through the substrate. The objectives of this thesis are the study of guard-heated sensors {i} to quantify the reduction, over conventional single-element sensors, of substrate heat conduction losses and resultant errors over a range of applied shear and {ii} to examine a range of values of guard heater geometric parameters, in two common fluids, air and water and identify the best designs. Wall-turbulence, the turbulent flow in the vicinity of solid boundaries, has proved difficult to model accurately, due to the lack of accurate WSS measurements. Examples of areas of impact are drag force reduction on transport vehicles in land, sea, air, which today largely translate to reduced fossil fuel use and dependence; aerodynamic noise and control for flight and for wind energy conversion; atmospheric and oceanic transport studies for weather, climate and for pollutant transport; riverbank erosion. Constant-temperature anemometry with MEMS devices, flush-mounted hot-film thermal sensors, is non-intrusive, affords the best temporal resolution and is well-established. However, these hot-film probes suffer from unwanted heat transport to the fluid through the substrate, with errors and nonlinearity large enough to overwhelm quantitative utility of the data. Microfabrication techniques have enabled multi-element guard-heated prototypes to be fabricated. Our results show that errors in sensing-element signals, contributing to spectral distortion, are sensitive to sensor location within the guard heater. These errors can be reduced to below 1% of the signal with proper location of the sensor. Guard heating also reduces the large variation in spatial averaging due to substrate conduction. This makes them suitable for turbulent flows with a large range of fluctuations. / Graduate

During inflammation, leukocytes modulate α4β1(VLA-4) integrin avidity in order to rapidly stabilize nascent adhesive contacts to VCAM-1-expressing endothelial cells and resist detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization under conditions of fluid flow remains poorly understood. We modeled leukocyte (U937 cell, mouse lymphocyte and human monocyte) arrest and adhesion stabilization through the use of a parallel plate flow chamber and visualized cells by phase contrast or fluorescent confocal microscopy. Live cell imaging with Lifeact-transfected U937 cells revealed that mechanical forces imparted by fluid flow induced formation of upstream tension-bearing anchors attached to the VCAM-1-coated surface. Scanning electron microscopy confirmed that flow-induced mechanical force culminates in the formation of structures that anchor monocyte adhesion. These structures are critical for adhesion stabilization, since disruption of actin polymerization dramatically inhibited VLA-4-dependent resistance to detachment, but did not affect VLA-4 expression, affinity modulation, and clustering or constitutive linkage to F-actin. Transfection of dominant-negative constructs and inhibition of kinase function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. Rap1 was shown to be critical for resistance to flow-induced detachment and accumulated in its GTP form at the sites of anchor formation. A key mediator of force-induced Rac activation and actin polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension-bearing anchors and blockade of PI3K or deficiency of PI3Kγ isoform reproduced the adhesion defect produced by inhibition of actin polymerization. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces; these included activation of Rap1, phosphoinositide 3-kinase γ-isoform and Rac, but not Cdc42.