Computational simulations predict a key role for oscillatory fluid shear stress in de novo valvular tissue formation

Previous efforts in heart valve tissue engineering demonstrated that the combined effect of cyclic flexure and steady flow on bone marrow derived stem cell-seeded scaffolds resulted in significant increases in engineered collagen formation [Engelmayr et al. Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: Implications for engineered heart valve tissues. Biomaterials 2006; 27(36): 6083–95]. Here, we provide a new interpretation for the underlying reason for this observed effect. In addition, another related investigation demonstrated the impact of fluid flow on DNA content and quantified the fluid-induced shear stresses on the engineered heart valve tissue specimens [Engelmayr et al. A Novel Flex-Stretch-Flow Bioreactor for the Study of Engineered Heart Valve Tissue Mechanobiology]. Annals of Biomedical Engineering 2008, 36, 1–13]. In this study, we performed more advanced CFD analysis with an emphasis on oscillatory wall shear stresses imparted on specimens when mechanically conditioned by a combination of cyclic flexure and steady flow. Specifically, we hypothesized that the dominant stimulatory regulator of the bone marrow stem cells is fluid-induced and depends on both the magnitude and temporal directionality of surface stresses, i.e., oscillatory shear stresses (OSS) acting on the developing tissues. Therefore, we computationally quantified the (i) magnitude of fluid-induced shear stresses as well as (ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter. Noting that sample cyclic flexure induces a high degree of OSS, we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: (1) No Flow, No Flexure (control group), (2) Steady Flow-alone, (3) Cyclic Flexure-alone and (4) Combined Steady flow and Cyclic Flexure environments. Indeed we found that the coexistence of both OSS and appreciable shear stress magnitudes explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress.     

Bioreactor studies of native and tissue engineered cartilage

Functional tissue engineering of cartilage involves the use of bioreactors designed to provide a controlled in vitro environment that embodies some of the biochemical and physical signals known to regulate chondrogenesis. Hydrodynamic conditions can affect in vitro tissue formation in at least two ways: by direct effects of hydrodynamic forces on cell morphology and function, and by indirect flow-induced changes in mass transfer of nutrients and metabolites. In the present work, we discuss the effects of three different in vitro environments: static flasks (tissues fixed in place, static medium), mixed flasks (tissues fixed in place, unidirectional turbulent flow) and rotating bioreactors (tissues dynamically suspended in laminar flow) on engineered cartilage constructs and native cartilage explants. As compared to static and mixed flasks, dynamic laminar flow in rotating bioreactors resulted in the most rapid tissue growth and the highest final fractions of glycosaminoglycans and total collagen in both tissues. Mechanical properties (equilibrium modulus, dynamic stiffness, hydraulic permeability) of engineered constructs and explanted cartilage correlated with the wet weight fractions of glycosaminoglycans and collagen. Current research needs in the area of cartilage tissue engineering include the utilization of additional physiologically relevant regulatory signals, and the development of predictive mathematical models that enable optimization of the conditions and duration of tissue culture.     

Differentiation and Distribution of Marrow Stem Cells in Flex-Flow Environments Demonstrate Support of the Valvular Phenotype

For treatment of critical heart valve diseases, prosthetic valves perform fairly well in most adults; however, for pediatric patients, there is the added requirement that the replacement valve grows with the child, thus extremely limiting current treatment options. Tissue engineered heart valves (TEHV), such as those derived from autologous bone marrow stem cells (BMSCs), have the potential to recapitulate native valve architecture and accommodate somatic growth. However, a fundamental pre-cursor in promoting directed integration with native tissues rather than random, uncontrolled growth requires an understanding of BMSC mechanobiological responses to valve-relevant mechanical environments. Here, we report on the responses of human BMSC-seeded polymer constructs to the valve-relevant stress states of: (i) steady flow alone, (ii) cyclic flexure alone, and (iii) the combination of cyclic flexure and steady flow (flex-flow). BMSCs were seeded onto a PGA: PLLA polymer scaffold and cultured in static culture for 8 days. Subsequently, the aforementioned mechanical conditions, (groups consisting of steady flow alone—850ml/min, cyclic flexure alone—1 Hz, and flex-flow—850ml/min and 1 Hz) were applied for an additional two weeks. We found samples from the flex-flow group exhibited a valve-like distribution of cells that expressed endothelial (preference to the surfaces) and myofibroblast (preference to the intermediate region) phenotypes. We interpret that this was likely due to the presence of both appreciable fluid-induced shear stress magnitudes and oscillatory shear stresses, which were concomitantly imparted onto the samples. These results indicate that flex-flow mechanical environments support directed in vitro differentiation of BMSCs uniquely towards a heart valve phenotype, as evident by cellular distribution and expression of specific gene markers. A priori guidance of BMSC-derived, engineered tissue growth under flex-flow conditions may serve to subsequently promote controlled, engineered to native tissue integration processes in vivo necessary for successful long-term valve remodeling.     

The flexural rigidity of the aortic valve leaflet in the commissural region

Flexure is a major deformation mode of the aortic valve (AV) leaflet, particularly in the commissural region where the upper portion of the leaflet joins the aortic root. However, there are no existing data known on the mechanical properties of leaflet in the commissural region. To address this issue, we quantified the effective stiffness of the commissural region using a cantilever beam method. Ten specimens were prepared, with each specimen flexed in the direction of natural leaflet motion (forward) and against the natural motion (reverse). At a flexure angle (phi) of 30 degrees , the effective forward direction modulus E was 42.63+/-4.44 kPa and the reverse direction E was 75.01+/-14.53 kPa (p=0.049). Further, E-phi response was linear (r(2) approximately 0.9) in both flexural directions. Values for dE/dphi were -2.24+/-0.6 kPa/ degrees and -1.90+/-0.3 kPa/ degrees in the forward and reverse directions, respectively (not statistically different, p=0.424), indicating a consistent decrease in stiffness with increased flexure. In comparison, we have reported that the effective tissue stiffness of AV leaflet belly region was 150-200 kPa [Merryman, W.D., Huang, H.Y.S., Schoen, F.J., Sacks, M.S. (2006). The effects of cellular contraction on AV leaflet flexural stiffness. Journal of Biomechanics 39 (1), 88-96], which was also independent of direction and amount of flexure. Histological studies of the commissure region indicated that tissue buckling was a probable mechanism for decrease in E with increasing flexure. The observed change in E with flexural angle in the commissural region is a subtle aspect of valve function. From a valve design perspective, these findings can be used as design criteria in fabricating prosthetic devices AV resulting in better functional performance.     

Do low oxygen environments facilitate marine invasions? Relative tolerance of native and invasive species to low oxygen conditions

Biological invasions are one of the biggest threats to global biodiversity. Marine artificial structures are proliferating worldwide and provide a haven for marine invasive species. Such structures disrupt local hydrodynamics, which can lead to the formation of oxygen‐depleted microsites. The extent to which native fauna can cope with such low oxygen conditions, and whether invasive species, long associated with artificial structures in flow‐restricted habitats, have adapted to these conditions remains unclear. We measured water flow and oxygen availability in marinas and piers at the scales relevant to sessile marine invertebrates (mm). We then measured the capacity of invasive and native marine invertebrates to maintain metabolic rates under decreasing levels of oxygen using standard laboratory assays. We found that marinas reduce water flow relative to piers, and that local oxygen levels can be zero in low flow conditions. We also found that for species with erect growth forms, invasive species can tolerate much lower levels of oxygen relative to native species. Integrating the field and laboratory data showed that up to 30% of available microhabitats within low flow environments are physiologically stressful for native species, while only 18% of the same habitat is physiologically stressful for invasive species. These results suggest that invasive species have adapted to low oxygen habitats associated with manmade habitats, and artificial structures may be creating niche opportunities for invasive species.     

Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting Endoplasmic Reticulum (ER) Stress Induced by Disturbed Flow

Disturbed blood flow with low-oscillatory shear stress (OSS) is a predominant atherogenic factor leading to dysfunctional endothelial cells (ECs). Recently, it was found that disturbed flow can directly induce endoplasmic reticulum (ER) stress in ECs, thereby playing a critical role in the development and progression of atherosclerosis. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid, has long been used to treat chronic cholestatic liver disease and is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, its role in atherosclerosis remains unexplored. In this study, we demonstrated the anti-atherogenic activity of UDCA via inhibition of disturbed flow-induced ER stress in atherosclerosis. UDCA effectively reduced ER stress, resulting in a reduction in expression of X-box binding protein-1 (XBP-1) and CEBP-homologous protein (CHOP) in ECs. UDCA also inhibits the disturbed flow-induced inflammatory responses such as increases in adhesion molecules, monocyte adhesion to ECs, and apoptosis of ECs. In a mouse model of disturbed flow-induced atherosclerosis, UDCA inhibits atheromatous plaque formation through the alleviation of ER stress and a decrease in adhesion molecules. Taken together, our results revealed that UDCA exerts anti-atherogenic activity in disturbed flow-induced atherosclerosis by inhibiting ER stress and the inflammatory response. This study suggests that UDCA may be a therapeutic agent for prevention or treatment of atherosclerosis.     

Microfluidic co-culture platform for investigating osteocyte-osteoclast signalling during fluid shear stress mechanostimulation

Bone cells exist in a complex environment where they are constantly exposed to numerous dynamic biochemical and mechanical stimuli. These stimuli regulate bone cells that are involved in various bone disorders, such as osteoporosis. Knowledge of how these stimuli affect bone cells have been utilised to develop various treatments, such as pharmaceuticals, hormone therapy, and exercise. To investigate the role that bone loading has on these disorders in vitro, bone cell mechanotransduction studies are typically performed using parallel plate flow chambers (PPFC). However, these chambers do not allow for dynamic cellular interactions among different cell populations to be investigated. We present a microfluidic approach that exposes different cell populations, which are located at physiologically relevant distances within adjacent channels, to different levels of fluid shear stress, and promotes cell-cell communication between the different channels. We employed this microfluidic system to assess mechanically regulated osteocyte-osteoclast communication. Osteoclast precursors (RAW264.7 cells) responded to cytokine gradients (e.g., RANKL, OPG, PGE-2) developed by both mechanically stimulated (fOCY) and unstimulated (nOCY) osteocyte-like MLO-Y4 cells simultaneously. Specifically, we observed increased osteoclast precursor cell densities and osteoclast differentiation towards nOCY. We also used this system to show an increased mechanoresponse of osteocytes when in co-culture with osteoclasts. We envision broad applicability of the presented approach for microfluidic perfusion co-culture of multiple cell types in the presence of fluid flow stimulation, and as a tool to investigate osteocyte mechanotransduction, as well as bone metastasis extravasation. This system could also be applied to any multi-cell population cross-talk studies that are typically performed using PPFCs (e.g. endothelial cells, smooth muscle cells, and fibroblasts).     

GENETIC ISOLATION BY ENVIRONMENT OR DISTANCE: WHICH PATTERN OF GENE FLOW IS MOST COMMON?

Gene flow among populations can enhance local adaptation if it introduces new genetic variants available for selection, but strong gene flow can also stall adaptation by swamping locally beneficial genes. These outcomes can depend on population size, genetic variation, and the environmental context. Gene flow patterns may align with geographic distance (IBD—isolation by distance), whereby immigration rates are inversely proportional to the distance between populations. Alternatively gene flow may follow patterns of isolation by environment (IBE), whereby gene flow rates are higher among similar environments. Finally, gene flow may be highest among dissimilar environments (counter‐gradient gene flow), the classic “gene‐swamping” scenario. Here we survey relevant studies to determine the prevalence of each pattern across environmental gradients. Of 70 studies, we found evidence of IBD in 20.0%, IBE in 37.1%, and both patterns in 37.1%. In addition, 10.0% of studies exhibited counter‐gradient gene flow. In total, 74.3% showed significant IBE patterns. This predominant IBE pattern of gene flow may have arisen directly through natural selection or reflect other adaptive and nonadaptive processes leading to nonrandom gene flow. It also precludes gene swamping as a widespread phenomenon. Implications for evolutionary processes and management under rapidly changing environments (e.g., climate change) are discussed.     

Effect of temperature on the fracture toughness of compact bone

Bone is a composite composed mainly of organics, minerals, and water. Many researchers have studied effects such as crack velocity, density, orientation, storage media, porosity, and age on the fracture toughness (K(C), also called critical stress intensity factor) of compact bone. Most of these studies were conducted at room temperature. Considering that the body temperature of animals is greater than room temperature, and that bone has a large volumetric percentage of organics and water (generally, 55-65%), it is hypothesized that temperature has a significant effect on the fracture toughness of compact bone. Single-edge V-notched (SEVN) specimens were prepared to measure the fracture toughness of bovine femur and manatee rib in water at 0, 10, 23, 37, and 50 degrees C in four-point flexure. The fracture toughness values of bovine femur and manatee rib were found to decrease from 7.0 to 4.3MPam(1/2) and from 5.5 to 4.0MPam(1/2), respectively, as temperature increased over a temperature range of 50 degrees C. The results support the hypothesis that temperature has a significant effect on the fracture toughness of compact bone. Therefore, we suggest that study on fracture toughness of bone should be done at physiologically relevant temperatures.     

A fully coupled fluid-structure interaction model of the secondary lymphatic valve

Abstract

The secondary lymphatic valve is a bi-leaflet structure frequent throughout collecting vessels that serves to prevent retrograde flow of lymph. Despite its vital function in lymph flow and apparent importance in disease development, the lymphatic valve and its associated fluid dynamics have been largely understudied. The goal of this work was to construct a physiologically relevant computational model of an idealized rat mesenteric lymphatic valve using fully coupled fluid-structure interactions to investigate the relationship between three-dimensional flow patterns and stress/deformation within the valve leaflets. The minimum valve resistance to flow, which has been shown to be an important parameter in effective lymphatic pumping, was computed as 268?g/mm⁴?s. Hysteretic behavior of the lymphatic valve was confirmed by comparing resistance values for a given transvalvular pressure drop during opening and closing. Furthermore, eddy structures were present within the sinus adjacent to the valve leaflets in what appear to be areas of vortical flow; the eddy structures were characterized by non-zero velocity values (up to ～4?mm/s) in response to an applied unsteady transvalvular pressure. These modeling capabilities present a useful platform for investigating the complex interplay between soft tissue motion and fluid dynamics of lymphatic valves and contribute to the breadth of knowledge regarding the importance of biomechanics in lymphatic system function.     

The dynamic viscoelasticity and plasticizer leachability of tissue conditioners

doi: 10.1111/j.1741‐2358.2012.00639.x
The dynamic viscoelasticity and plasticizer leachability of tissue conditioners Objectives: Dynamic viscoelasticity is one of the most important characteristics of tissue conditioners. Leaching of plasticizer from tissue conditioners may cause changes in the dynamic viscoelasticity of these materials. The purpose of this study was to determine the dynamic viscoelasticity and plasticizer leachability of commercial tissue conditioners. Materials and methods: Five commercial tissue conditioners were used in this study: COE Comfort (CC), Fit Softer (FS), Hydro‐Cast (HC), Soft Conditioner (SC) and Visco‐Gel (VG). Five specimens of each material were stored in 37°C distilled water. The dynamic viscoelasticity and plasticizer leaching of each specimen were measured at 0, 1, 3, 7 and 14 days after immersion using a dynamic mechanical analyzer and high performance liquid chromatography. Data were analyzed using one‐way anova and Student–Newman–Keuls test (α = 0.05). Results: Significant differences in dynamic viscoelasticity and plasticizer leaching were found among the materials. The dynamic viscoelasticity of all materials increased or decreased significantly with time. Rapid changes in dynamic viscoelasticity were evident on day 1 after water immersion. The material CC recorded the highest level of plasticizer leaching. Conclusion: These results suggest that the dynamic viscoelasticity of commercial tissue conditioners is affected by plasticizer leaching. However, these effects are limited.     

Relationship between streaming potential and compressive stress in bovine intervertebral tissue

The intervertebral disc is formed by the nucleus pulposus (NP) and annulus fibrosus (AF), and intervertebral tissue contains a large amount of negatively charged proteoglycan. When this tissue becomes deformed, a streaming potential is induced by liquid flow with positive ions. The anisotropic property of the AF tissue is caused by the structural anisotropy of the solid phase and the liquid phase flowing into the tissue with the streaming potential. This study investigated the relationship between the streaming potential and applied stress in bovine intervertebral tissue while focusing on the anisotropy and loading location. Column-shaped specimens, 5.5 mm in diameter and 3 mm thick, were prepared from the tissue of the AF, NP and the annulus–nucleus transition region (AN). The loading direction of each specimen was oriented in the spinal axial direction, as well as in the circumferential and radial directions of the spine considering the anisotropic properties of the AF tissue. The streaming potential changed linearly with stress in all specimens. The linear coefficients k_e of the relationship between stress and streaming potential depended on the extracted positions. These coefficients were not affected by the anisotropy of the AF tissue. In addition, these coefficients were lower in AF than in NP specimens. Except in the NP specimen, the k_e values were higher under faster compression rate conditions. In cyclic compression loading the streaming potential changed linearly with compressive stress, regardless of differences in the tissue and load frequency.     

Design of an ex vivo culture system to investigate the effects of shear stress on cardiovascular tissue

Mechanical forces are known to affect the biomechanical properties of native and engineered cardiovascular tissue. In particular, shear stress that results from the relative motion of heart valve leaflets with respect to the blood flow is one important component of their mechanical environment in vivo. Although different types of bioreactors have been designed to subject cells to shear stress, devices to expose biological tissue are few. In an effort to address this issue, the aim of this study was to design an ex vivo tissue culture system to characterize the biological response of heart valve leaflets subjected to a well-defined steady or time-varying shear stress environment. The novel apparatus was designed based on a cone-and-plate viscometer. The device characteristics were defined to limit the secondary flow effects inherent to this particular geometry. The determination of the operating conditions producing the desired shear stress profile was streamlined using a computational fluid dynamic (CFD) model validated with laser Doppler velocimetry. The novel ex vivo tissue culture system was validated in terms of its capability to reproduce a desired cone rotation and to maintain sterile conditions. The CFD results demonstrated that a cone angle of 0.5 deg, a cone radius of 40 mm, and a gap of 0.2 mm between the cone apex and the plate could limit radial secondary flow effects. The novel cone-and-plate permits to expose nine tissue specimens to an identical shear stress waveform. The whole setup is capable of accommodating four cone-and-plate systems, thus concomitantly subjecting 36 tissue samples to desired shear stress condition. The innovative design enables the tissue specimens to be flush mounted in the plate in order to limit flow perturbations caused by the tissue thickness. The device is capable of producing shear stress rates of up to 650 dyn cm(-2) s(-1) (i.e., maximum shear stress rate experienced by the ventricular surface of an aortic valve leaflet) and was shown to maintain tissue under sterile conditions for 120 h. The novel ex vivo tissue culture system constitutes a valuable tool toward elucidating heart valve mechanobiology. Ultimately, this knowledge will permit the production of functional tissue engineered heart valves, and a better understanding of heart valve biology and disease progression.     

The passive properties of muscle fibers are velocity dependent

The passive properties of skeletal muscle play an important role in muscle function. While the passive quasi-static elastic properties of muscle fibers have been well characterized, the dynamic visco-elastic passive behavior of fibers has garnered less attention. In particular, it is unclear how the visco-elastic properties are influenced by lengthening velocity, in particular for the range of physiologically relevant velocities. The goals of this work were to: (i) measure the effects of lengthening velocity on the peak stresses within single muscle fibers to determine how passive behavior changes over a range of physiologically relevant lengthening rates (0.1–10L_o/s), and (ii) develop a mathematical model of fiber viscoelasticity based on these measurements. We found that passive properties depend on strain rate, in particular at the low loading rates (0.1–3L_o/s), and that the measured behavior can be predicted across a range of loading rates and time histories with a quasi-linear viscoelastic model. In the future, these results can be used to determine the impact of viscoelastic behavior on intramuscular stresses and forces during a variety of dynamic movements.     

Operant sensation seeking requires metabotropic glutamate receptor 5 (mGluR5)

Pharmacological and genetic studies have suggested that the metabotropic glutamate receptor 5 (mGluR5) is critically involved in mediating the reinforcing effects of drugs of abuse, but not food. The purpose of this study was to use mGluR5 knockout (KO), heterozygous (Het), and wildtype (WT) mice to determine if mGluR5 modulates operant sensation seeking (OSS), an operant task that uses varied sensory stimuli as a reinforcer. We found that mGluR5 KO mice had significantly reduced OSS responding relative to WT mice, while Het mice displayed a paradoxical increase in OSS responding. Neither KO nor Het mice exhibited altered operant responding for food as a reinforcer. Further, we assessed mGluR5 KO, Het and WT mice across a battery of cocaine locomotor, place preference and anxiety related tests. Although KO mice showed expected differences in some locomotor and anxiety measures, Het mice either exhibited no phenotype or an intermediate one. In total, these data demonstrate a key role for mGluR5 in OSS, indicating an important role for this receptor in reinforcement-based behavior.     

Pulsatile spiral blood flow through arterial stenosis

Pulsatile spiral blood flow in a modelled three-dimensional arterial stenosis, with a 75% cross-sectional area reduction, is investigated by using numerical fluid dynamics. Two-equation k-ω model is used for the simulation of the transitional flow with Reynolds numbers 500 and 1000. It is found that the spiral component increases the static pressure in the vessel during the deceleration phase of the flow pulse. In addition, the spiral component reduces the turbulence intensity and wall shear stress found in the post-stenosis region of the vessel in the early stages of the flow pulse. Hence, the findings agree with the results of Stonebridge et al. (2004). In addition, the results of the effects of a spiral component on time-varying flow are presented and discussed along with the relevant pathological issues.     

Determinants of RNA quality from FFPE samples

The large archives of formalin-fixed paraffin-embedded (FFPE) tissue specimens that exist are a highly valuable source of sample material for molecular biological analysis, including gene expression profiling. However, current data on adverse effects of standard pathological practice on the usefulness of biomolecular analytes obtained from such archived specimens is largely anecdotal. Here, we present a systematic examination of the most relevant parameters for integrity and useability of RNA obtained from FFPE samples, including storage time and conditions, fixation time, and specimen size. The results are particularly relevant for any application relying on cDNA synthesis as an initial step of the procedure, such as RT-PCR, and microarray analysis.     

The Oxygen-Scavenging System Protecting Nitrogenase from Oxygen in Cells of Blue-Green Algae

There is a heat stable oxygen-scavenging system (OSS) associated with membrane which reduces oxygen endogenously in cells of blue-green algae. Addition of the OSS to cell suspension of heterocystous oxygen sensitive Anabaena mutant and non-heterocystous Pleetonema boryanum led to an increase in their nitrogenase activity by 10–100-fold higher than those under microaerobic condition and also could restore effectively their acetylene reduction activity at higher oxygen concentration since the oxygen presented was reduced effectively. The results suggest that the OSS possesses a function protecting nitrogenase from oxygen in cells. Furthermore, it was found that the efficiency of reducing oxygen of OSS from the Anabaena mutant and Plectonema was lower than those from Anabaena wild and Gloeocapsa in atm. oxygen level. This may be ralated with the susceptibility of nitrogen fixation to oxygen in the cells of Anabaena mutant and Plectonema. The present study firstly indicades the relationship between the heat stable OSS associated with membrane and the mechanism of protecting nitrogenase from oxygen in cells of blue-green algae. Activities of catalase, peroxidase and superoxide dismutase do not show obvious difference in cellfree extract of Anabaena wild and mutant. Methyl viologen can induce nitrogenase activity of Anabaena mutant by subverting a portion of electon flow to accelerate oxygen reduction.