Collagen fibre-mediated mechanical damage increases calcification of bovine pericardium for use in bioprosthetic heart valves.

In cases of aortic stenosis, bioprosthetic heart valves (BHVs), with glutaraldehyde-fixed bovine pericardium leaflets (GLBP), are often implanted to replace the native diseased valve. Widespread use of BHVs, however, is restricted due to inadequate long-term durability, owing specifically to premature leaflet failure. Mechanical fatigue damage and calcification remain the primary leaflet failure modes, where glutaraldehyde treatment is known to accelerate calcification. The literature in this area is limited, with some studies suggesting mechanical damage increases calcification and others that they are independent degenerative mechanisms. In this study, specimens which were non-destructively pre-sorted according to collagen fibre architecture and uniaxially cyclically loaded until failure or 1 million cycles, were placed in an in vitro calcification solution. The weakest specimen group (those with fibres aligned perpendicular to the load) had statistically significantly higher volumes of calcification when compared to those with a high fatigue life. Moreover, SEM imaging revealed that ruptured and damaged fibres presented calcium binding sites; resulting in 4 times more calcification in fractured samples in comparison to those which did not fail by fatigue. To the authors' knowledge, this study quantifies for the first time, that mechanical damage drives calcification in commercial-grade GLBP and that calcification varies spatially according to localised damage levels. These findings illustrate that not only is calcification of GLBP exacerbated by fatigue damage, but that both failure phenomena are underpinned by the collagen fibre organisation. Consequently, controlling for GLBP collagen fibre architecture in leaflets could minimise the progression of these primary failure modes in patient BHVs. STATEMENT OF SIGNIFICANCE: Mechanical damage and calcification are the primary premature failure modes of glutaraldehyde-fixed bovine pericardial (GLBP) leaflets in bioprosthetic heart valves. In this study, commercial-grade GLBP specimens which were uniaxially cyclically loaded to failure or 1 million cycles, were placed in an in vitro calcification solution. MicroCT and SEM analysis showed that localised calcification levels varied spatially according to damage, where ruptured fibres offered additional calcium binding sites. Furthermore, specimens with a statistically significant lower fatigue life were associated with statistically significant higher calcification. This study revealed that mechanical damage drives calcification of GLBP. Non-destructive pre-screening of collagen fibres demonstrated that both the fatigue life and calcification potential of commercial-grade GLBP, are underpinned by the collagen fibre architecture.

Enhancing medial layer recellularization of tissue-engineered blood vessels using radial microchannels.

Aim: Cell repopulation of tissue-engineered vascular grafts (TEVGs) from decellularized arterial scaffolds is limited by dense concentric tunica media layers which impede cells migrating radially between the layers. We aimed to develop and validate a new microneedle device to modify decellularized carotid arteries with radial microchannels to enhance medial layer repopulation. Material & methods: Modified decellularized porcine arteries were seeded with rat mesenchymal stem cells using either standard longitudinal injection, or a dual vacuum-perfusion bioreactor. Mechanical tests were used to assess the arterial integrity following modification. Results & conclusion: The method herein achieved radial recellularization of arteries in vitro without significant loss of mechanical integrity, Thus, we report a novel method for successful radial repopulation of decellularized carotid artery-based tissue-engineered vascular grafts.

A bioresorbable biomaterial carrier and passive stabilization device to improve heart function post-myocardial infarction.

The limited regenerative capacity of the heart after a myocardial infarct results in remodeling processes that can progress to congestive heart failure (CHF). Several strategies including mechanical stabilization of the weakened myocardium and regenerative approaches (specifically stem cell technologies) have evolved which aim to prevent CHF. However, their final performance remains limited motivating the need for an advanced strategy with enhanced efficacy and reduced deleterious effects. An epicardial carrier device enabling a targeted application of a biomaterial-based therapy to the infarcted ventricle wall could potentially overcome the therapy and application related issues. Such a device could play a synergistic role in heart regeneration, including the provision of mechanical support to the remodeling heart wall, as well as providing a suitable environment for in situ stem cell delivery potentially promoting heart regeneration. In this study, we have developed a novel, single-stage concept to support the weakened myocardial region post-MI by applying an elastic, biodegradable patch (SPREADS) via a minimal-invasive, closed chest intervention to the epicardial heart surface. We show a significant increase in %LVEF 14 days post-treatment when GS (clinical gold standard treatment) was compared to GS + SPREADS + Gel with and without cells (p ≤ 0.001). Furthermore, we did not find a significant difference in infarct quality or blood vessel density between any of the groups which suggests that neither infarct quality nor vascularization is the mechanism of action of SPREADS. The SPREADS device could potentially be used to deliver a range of new or previously developed biomaterial hydrogels, a remarkable potential to overcome the translational hurdles associated with hydrogel delivery to the heart.

An injectable alginate/extra cellular matrix (ECM) hydrogel towards acellular treatment of heart failure.

As treatments for myocardial infarction (MI) continue to improve, the population of people suffering from heart failure (HF) is rising significantly. Novel treatment strategies aimed at achieving long-term functional stabilisation and improvement in heart function post MI include the delivery of biomaterial hydrogels and myocardial matrix-based therapies to the left ventricle wall. Individually alginate hydrogels and myocardial matrix-based therapies are at the most advanced stages of commercial/clinical development for this potential treatment option. However, despite these individual successes, the potential synergistic effect gained by combining the two therapies remains unexplored. This study serves as a translational step in evaluating the minimally invasive delivery of dual acting alginate-based hydrogels to the heart. We have successfully developed new production methods for hybrid alginate/extracellular matrix (ECM) hydrogels. We have identified that the high G block alginate/ECM hybrid hydrogel has appropriate rheological and mechanical properties (1.6 KPa storage modulus, 29 KPa compressive modulus and 14 KPa dynamic modulus at day 1) and can be delivered using a minimally invasive delivery device. Furthermore, we have determined that these novel hydrogels are not cytotoxic and are capable of enhancing the metabolic activity of dermal fibroblasts in vitro (p < 0.01). Overall these results suggest that an effective minimally invasive HF treatment option could be achieved by combining alginate and ECM particles.

Advanced Material Catheter (AMCath), a minimally invasive endocardial catheter for the delivery of fast-gelling covalently cross-linked hyaluronic acid hydrogels.

Injectable hydrogels that aim to mechanically stabilise the weakened left ventricle wall to restore cardiac function or to deliver stem cells in cardiac regenerative therapy have shown promising data. However, the clinical translation of hydrogel-based therapies has been limited due to difficulties injecting them through catheters. We have engineered a novel catheter, Advanced Materials Catheter (AMCath), that overcomes translational hurdles associated with delivering fast-gelling covalently cross-linked hyaluronic acid hydrogels to the myocardium. We developed an experimental technique to measure the force required to inject such hydrogels and determined the mechanical/viscoelastic properties of the resulting hydrogels. The preliminary in vivo feasibility of delivering fast-gelling hydrogels through AMCath was demonstrated by accessing the porcine left ventricle and showing that the hydrogel was retained in the myocardium post-injection (three 200 µL injections delivered, 192, 204 and 183 µL measured). However, the mechanical properties of the hydrogels were reduced by passage through AMCath (≤20.62% reduction). We have also shown AMCath can be used to deliver cardiopoietic adipose-derived stem cell-loaded hydrogels without compromising the viability (80% viability) of the cells in vitro. Therefore, we show that hydrogel/catheter compatibility issues can be overcome as we have demonstrated the minimally invasive delivery of a fast-gelling covalently cross-linked hydrogel to the beating myocardium.

The development and mechanical characterisation of a novel reinforced venous conduit that mimics the mechanical properties of an arterial wall.

Venous grafts have been used to bypass stenotic arteries for many decades. However, this "gold standard" treatment is far from optimal, with long-term vein graft patency rates reported to be as low as 50% at >15 years. These results could be a result of the structural and functional differences of veins compared to arteries. In this study we developed a new protocol for manufacturing reinforced fresh veins with a decellularized porcine arterial scaffold. This novel method was designed to be replicated easily in a surgical setting, and manufactured reinforced constructs were robust and easier to handle than the veins alone. Furthermore, we demonstrate that these Reinforced Venous-Arterial Conduits have comparable mechanical properties to native arteries, in terms of ultimate tensile strength (UTS) (2.36 vs. 2.24MPa) and collagen dominant phase (11.04 vs. 12.26MPa). Therefore, the Reinforced Venous-Arterial Conduit combines the benefits of using the current gold standard homogenous venous grafts composed of a confluent endothelial surface, with an "off-the-shelf" decellularized artery to improve the mechanical properties to closely mimic those of native arteries, while maintaining the self-repairing characteristics of native tissue. In conclusion in this study we have produced a construct and a new technique that combines the mechanical properties of both a natural vein and a decellularized artery to produce a reinforced venous graft that closely mimics the mechanical response of an arterial segment.

An in vitro investigation to assess procedure parameters for injecting therapeutic hydrogels into the myocardium.

Localized delivery of stem cells is potentially a promising therapeutic strategy for regenerating damaged myocardium. Many studies focus on limiting the biologic component of cell loss, but few address the contribution of mechanical factors. This study investigates optimal parameters for retaining the largest volume of cell loaded hydrogels post intramyocardial injection, without compromising cell viability. In vitro, hydrogel was injected into porcine hearts using various needle designs. Hydrogel retention and distribution pattern was then determined. The two most promising needles were then investigated to understand the effect of needle geometry on stem cell viability. The needle to best impact cell viability was then used to investigate the effect of differing hydrogels on retention and distribution. Three-dimensional experimental modeling revealed needles with smaller diameter's to have greater poloxamer 407 hydrogel retention. No difference in retention existed among various needle designs of similar gauge, despite differences in bolus geometries. When hMSC's, embedded in fibrin hydrogel, were injected through helical and 26G bevel needles no difference in the percent of live cells was seen at 48 h. However, the helical group had almost half the metabolic activity of the 26G bevel group at both time points, and had a significant decline in the percent of live cells from 24 to 48 h. Varying gel type resulted in significantly more alginate being retained in the tissue in comparison to fibrin or poloxamer hydrogels. In conclusion, mechanical properties of injected hydrogels, and the diameter of the needle used, highly influences the volume of hydrogel retained. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2618-2629, 2017.

Nanomedicines for advanced cancer treatments: Transitioning towards responsive systems.

The development of nanomedicines for the treatment of cancer focuses on the local targeted delivery of chemotherapeutic drugs to enhance drug efficacy and reduce adverse effects. The nanomedicines which are currently approved for clinical use are mainly successful in terms of improved bioavailability and tolerability but do not necessarily increase drug performance. Therefore, there is a need for improved drug carrier systems which are able to deliver high doses of anti-cancer drugs to the tumor. Stimuli responsive carriers are promising candidates since drug release can be triggered locally in the tumor via internal (i.e. pH, redox potential, metabolite or enzyme concentration) or external (i.e. heat, ultrasound, light, magnetic field) stimuli. This review summarizes the recent progress in the transition towards stimuli responsive nanomedicines (i.e. liposomes, polymeric micelles, nanogels and mesoporous silica nanoparticles) and other therapy modalities that are currently developed in the fight against cancer like the application of ultrasound, tumor normalization and phototherapy. Furthermore, the potential role of image guided drug delivery in the development of new nanomedicines and its clinical application is discussed.

The effects of decellularization and cross-linking techniques on the fatigue life and calcification of mitral valve chordae tendineae.

In cases of severely diseased mitral valves (MV), the required treatment is often valve replacement. Bioprosthetic and stentless replacement valves are usually either fully or partially composed of animal derived tissue treated with a decellularization process, a cross-linking process, or both. In this study, we analysed the effects of these treatments on the fatigue properties of porcine MV chordae tendineae (CT), as well as on the calcification of the CT using an in vitro technique. CT were tested in 4 groups; (1) native, (2) decellularized (DC), (3) decellularized and cross-linked with glutaraldehyde (DC-GTH), and (4) decellularized and cross-linked with 1-ehtyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)(DC-EDC). CT were tested in both uniaxial tension, and in fatigue at 10MPa peak stress (1Hz). The cycles to failure (mean±SD) for the four groups are as follows; Native- 53,397±55,798, DC- 28,013±30,634, DC-GTH- 97,665±133,556, DC-EDC- 318,601±322,358. DC-EDC CT were found to have a slightly longer fatigue life than the native and DC groups. The DC-EDC group also had a marginally lower dynamic creep rate, meaning those CT elongate more slowly. After in vitro calcification, X-ray microtomography was used to determine relative levels of calcification. The DC-EDC and DC-GTH groups had the lowest volume of calcific deposits. Under uniaxial testing, the ultimate tensile strength (UTS) of the DC-GTH CT was statistically significantly reduced after calcification, while the UTS was relatively unchanged for the DC-EDC group. Overall, these results indicate that a treatment of decellularization plus cross-linking with EDC may improve the fatigue life of porcine CT, reduce the rate of elongation, and help the CT resist the negative effects of calcification. This may be a preferable treatment in the preparation of porcine MVs for the replacement of diseased MVs.

Characterisation of the fatigue life, dynamic creep and modes of damage accumulation within mitral valve chordae tendineae.

Mitral valve prolapse is often caused by either elongated or ruptured chordae tendineae (CT). In many cases, rupture is spontaneous, meaning there is no underlying cause. We hypothesised that spontaneous rupture may be due to mechanical fatigue. To investigate this hypothesis, we tested porcine marginal CT: in uniaxial tension, and in fatigue at a range of peak stresses (n=12 at 15, 10 and 7.5MPa respectively, n=6 at 5MPa). The rupture surfaces of failed CT were observed histologically, under polarised light microscopy, and SEM. The cycles to failure for 15, 10, 7.5 and 5 MPa peak stresses were: (average±SD): 5077±4366, 49513±56414, 99927±108908, 197099±69103. A Weibull plot was constructed and from this, the number of cycles at 50% probability of failure was established in order to approximate the fatigue life, which was found to be 2.43MPa at 10 million cycles. The rate of creep increases exponentially with increasing peak stress. Under histological examination it was observed that CT which have been fatigued at low stress partially lose their organised collagen structure and can sustain micro-cracks that can be linked to increases in the creep rate. Furthermore our SEM images closely matched descriptions from the literature of spontaneous in vivo rupture. In conclusion, we believe that the mechanical test results we present strongly suggest that spontaneous chordal rupture and chordal elongation in vivo can be caused by mechanical fatigue.

The effect of a thermal renal denervation cycle on the mechanical properties of the arterial wall.

The aim of this study was to determine the effect that a thermal renal denervation cycle has on the mechanical properties of the arterial wall. Porcine arterial tissue specimens were tested in three groups: native tissue, decellularized tissue, decellularized with collagen digestion (e.g. elastin only). One arterial specimen was used as an unheated control specimen while another paired specimen was subjected to a thermal cycle of 70°C for 120s (n=10). The specimens were subjected to tensile loading and a shrinkage analysis. We observed two key results: The mechanical properties associated with the elastin extracellular matrix (ECM) were not affected by the thermal cycle. The effect of the thermal cycle on the collagen (ECM) was significant, in both the native and decellularized groups the thermal cycle caused a statistically significant decrease in stiffness, and failure strength, moreover the native tissue demonstrated a 27% reduction in lumen area post exposure to the thermal cycle. We have demonstrated that a renal denervation thermal cycle can significantly affect the mechanical properties of an arterial wall, and these changes in stiffness and failure strength were associated with alterations to the collagen rather than the elastin extracellular matrix component.

An experimental investigation of the effect of mechanical and biochemical stimuli on cell migration within a decellularized vascular construct.

The goal of this study was to promote rapid repopulation of the medial layer of decellularized tissues for use as vascular grafts. We utilized a combined approach of biochemical and mechanical stimuli to enhance repopulation of decellularized porcine arterial tissue. Chitosan ß-glycerophosphate loaded with hepatocyte growth factor (HGF) was injected into a channel in the artery wall while rat mesenchymal stem cells (rMSCs) were injected in two channels located 120° to this channel. In a second group rMSCs were injected into channels located at intervals of 120°. Both groups were subjected to 7 days mechanical stimuli in comparison to non-dynamically conditioned static controls. The combined effect of the biochemical and mechanical stimuli demonstrated that the repopulation zone was significantly enhanced, maximum migration achieved was 1.8 times more than that of the static HGF cultured control and 10 times higher than the average migration for statically cultured scaffolds without biochemical stimulus. Human umbilical vein endothelial cells were also successfully adhered to the scaffold and dynamically cultured. The response of medially injected cells to the biomechanically and biochemically altered environment demonstrated that enhanced circumferential scaffold repopulation could be achieved.

The application of a thermoresponsive chitosan/ß-GP gel to enhance cell repopulation of decellularized vascular scaffolds.

Decellularized arterial scaffolds have achieved success in advancing towards clinical use for small diameter vascular graft applications. Issues remain with effectively cell seeding these scaffolds, which may result in slow remodeling in vivo and reduced patency rates. This study aims to efficiently bulk load decellularized arterial scaffolds with cells and/or growth factors to promote cell migration within the scaffold. A chitosan/ß-glycerophosphate (ß-GP) hydrogel was used as a delivery vehicle for scaffold repopulation with rat mesenchymal stem cells (rMSCs) and/or murine hepatocyte growth factor (HGF). The thermoresponsivity of the chitosan/ß-GP HGF gel was determined by rheological analysis and the release of HGF by ELISA. The chitosan/ß-GP gel was fully injectable with gelation at 30°C in ∼30 min and demonstrated sustained controlled HGF release up to 28 days. Encapsulation of rMSCs allowed for consistent efficient cell delivery within the scaffold. Directional migration toward the released HGF was demonstrated within the medial layer of the scaffold up to a distance of 3 mm. The gel/cell/growth factor combination demonstrates a means of accelerating scaffold repopulation, without costly excessive in vitro maturation times.

Determination of the tensile mechanical properties of the segmented mitral valve annulus.

The mitral valve annulus is a complex and irregular component of the mitral valve apparatus, serving both a structural and sphincteric role. We have sought to determine the mechanical properties of the mitral valve annulus segmentally. Twenty porcine hearts were dissected to isolate the annulus. The annulus was segmented into four sections: anterior, posterior, and left and right commissural sections. Ten of these were tensile tested to failure as control samples. The remaining ten were digested in order to fully isolate the annulus from the myocardium, and subsequently tensile tested to failure. Histological samples of each segment were analysed to determine collagen/annular content. Whole segments of muscular annulus were tensile tested to failure; the stress and strain at failure and location of failure were determined in these larger specimens. Our results demonstrated that the anterior annulus is stiffer than the posterior segment by a factor of approximately 27 at a 2% strain level, and approximately 13 at a 6% strain. There is a trend in the results that identifies that the muscular annulus is stiffest at the right commissural segment, while the posterior segment tends to be the least stiff. The stiffness of the samples can be correlated with the area associated with the dense collagen annulus using histological analysis. Finally, the weakest section of the mitral valve annulus was identified as the intersection of the right commissural segment and the posterior segment.

Optimum parameters for freeze-drying decellularized arterial scaffolds.

Decellularized arterial scaffolds have achieved success in advancing toward clinical use as vascular grafts. However, concerns remain regarding long-term preservation and sterilization of these scaffolds. Freeze drying offers a means of overcoming these concerns. In this study, we investigated the effects of various freeze-drying protocols on decellularized porcine carotid arteries and consequently, determined the optimum parameters to fabricate a stable, preserved scaffold with unaltered mechanical properties. Freeze drying by constant slow cooling to two final temperatures ((Tf), -10 °C and -40 °C) versus instant freezing was investigated by histological examination and mechanical testing. Slow cooling to Tf= -10 °C produced a stiffer and less distensible response than the non freeze-dried scaffolds and resulted in disruption to the collagen fibers. The mechanical response of Tf= -40 °C scaffolds demonstrated disruption to the elastin network, which was confirmed with histology. Snap freezing scaffolds in liquid nitrogen and freeze drying to Tf= -40 °C with a precooled shelf at -60 °C produced scaffolds with unaltered mechanical properties and a histology resembling non-freeze-dried scaffolds. The results of this study demonstrate the importance of optimizing the nucleation and ice crystal growth/size to ensure homogenous drying, preventing extracellular matrix disruption and subsequent inferior mechanical properties. This new manufacturing protocol creates the means for the preservation and sterilization of decellularized arterial scaffolds while simultaneously maintaining the mechanical properties of the tissue.

HUVEC ICAM-1 and VCAM-1 synthesis in response to potentially athero-prone and athero-protective mechanical and nicotine chemical stimuli.

Previous mechano-transduction studies have investigated the endothelial cell (EC) morphological response to mechanical stimuli; generally consisting of a wall shear stress (WSS) and a cyclic tensile hoop strain (THS). More recent studies have investigated the EC biochemical response (intercellular adhesion molecule, ICAM-1, and vascular cellular adhesion molecule, VCAM-1, expression) to idealized mechanical stimuli. However, current literature is lacking in the area of EC biochemical response to combinations of physiological WSS and THS mechanical stimuli. The objective of this study is to investigate the EC response to physiological WSS and THS stimuli and to compare this response to that of ECs exposed to idealized steady WSS and cyclic THS of the same magnitudes. This study also investigated the EC response to a nicotine chemical stimulus combined with a suspected athero-prone physiological mechanical stimulus. A bioreactor was designed to apply a range of combinations of physiological WSS and THS waveforms. The bioreactor was calibrated and validated using computational fluid dynamics and video extensometry techniques. The bioreactor was used to investigated the biochemical response exhibited by human umbilical vein endothelial cells (HUVECs) exposed to physiological athero-protective (first bioreactor test case, pulsatile WSS combined with pulsatile THS) and athero-prone (second bioreactor test case, oscillating WSS combined with pulsatile THS) mechanical environments. The final testing environment (third bioreactor test case) combined a nicotine chemical stimulus with the mechanical stimuli of the second bioreactor test case. In first and second bioreactor test cases, the addition of a pulsatile THS to the WSS resulted in opposite trends of ICAM-1 down-regulation and up-regulation, respectively. This outcome suggests that the effect of the additional pulsatile THS depends on the state of the applied WSS waveform. Similarly, in first and second bioreactor test cases, the addition of a pulsatile THS to the WSS resulted in a VCAM-1 up-regulation. However, it has been previously shown that the addition of a cyclic THS to a high- or low-steady WSS resulted in a VCAM-1 down-regulation, indicating that the EC response to idealized mechanical stimuli (steady WSS and cyclic THS) is not comparable to physiological mechanical stimuli (unsteady WSS and pulsatile THS), even though in both situations the average magnitude of WSS and THS applied were similar. In third bioreactor test case, a nicotine chemical stimulus induced a substantial VCAM-1 up-regulation and a moderate ICAM-1 up-regulation. The addition of the mechanical stimuli of the second bioreactors test case resulted in a greater VCAM-1 up-regulation than what was expected, considering the observations of the previous second bioreactor test case alone. This study found that the EC biochemical response to physiological mechanical stimuli is not comparable to the previously observed EC response to idealized mechanical stimuli, even though in both environments the mechanical stimuli were of a similar magnitude. Also, the level of VCAM-1 expressed by the nicotine stimulated ECs showed an elevated level of sensitivity to the athero-prone mechanical stimuli.

Multi-axial mechanical stimulation of HUVECs demonstrates that combined loading is not equivalent to the superposition of individual wall shear stress and tensile hoop stress components.

Over the past 25 years, many laboratory based bioreactors have been used to study the cellular response to hemodynamic forces. The vast majority of these studies have focused on the effect of a single isolated hemodynamic force, generally consisting of a wall shear stress (WSS) or a tensile hoop strain (THS). However, investigating the cellular response to a single isolated force does not accurately represent the true in vivo situation, where a number of forces are acting simultaneously. This study used a novel bioreactor to investigate the cellular response of human umbilical vein endothelial cells (HUVECs) exposed to a combination of steady WSS and a range of cyclic THS. HUVECs exposed to a range of cyclic THS (0-12%), over a 12 h testing period, expressed an upregulation of both ICAM-1 and VCAM-1. HUVECs exposed to a steady WSS (0 dynes/cm2 and 25 dynes/cm2), over a 12 h testing period, also exhibited an ICAM-1 upregulation but a VCAM-1 downregulation, where the greatest level of WSS stimulus resulted in the largest upregulation and downregulation of ICAM-1 and VCAM-1, respectively. A number of HUVEC samples were exposed to a high steady WSS (25 dynes/cm2) combined with a range of cyclic THS (0-4%, 0-8%, and 0-12%) for a 12 h testing period. The initial ICAM-1 upregulation, due to the WSS alone, was downregulated with the addition of a cyclic THS. It was observed that the largest THS (0-12%) had the greatest reducing effect on the ICAM-1 upregulation. Similarly, the initial VCAM-1 downregulation, due to the high steady WSS alone, was further downregulated with the addition of a cyclic THS. A similar outcome was observed when HUVEC samples were exposed to a low steady WSS combined with a range of cyclic THS. However, the addition of a THS to the low WSS did not result in an expected ICAM-1 downregulation. In fact, it resulted in a trend of unexpected ICAM-1 upregulation. The unexpected cellular response to the combination of a steady WSS and a cyclic THS demonstrates that such a response could not be determined by simply superimposing the cellular responses exhibited by ECs exposed to a steady WSS and a cyclic THS that were applied in isolation.