A novel in vitro loading system to produce supraphysiologic oscillatory fluid shear stress

A multi-well fluid loading (MFL) system was developed to deliver oscillatory subphysiologic to supraphysiologic fluid shear stresses to cell monolayers in vitro using standard multi-well culture plates. Computational fluid dynamics modeling with fluid-structure interactions was used to quantify the squeeze film fluid flow between an axially displaced piston and the well plate surface. Adjusting the cone angle of the piston base modulated the fluid pressure, velocity, and shear stress magnitudes. Modeling results showed that there was near uniform fluid shear stress across the well with a linear drop in pressure across the radius of the well. Using the MFL system, RAW 264.7 osteoclastic cells were exposed to oscillatory fluid shear stresses of 0, 0.5, 1.5, 4, 6, and 17 Pa. Cells were loaded 1 h per day at 1 Hz for two days. Compared to sub-physiologic and physiologic levels, supraphysiologic oscillatory fluid shear induced upregulation of osteoclastic activity as measured by tartrate-resistant acid phosphatase activity and formation of mineral resorption pits. Cell number remained constant across all treatment groups.     

Modeling fluid flow through irregular scaffolds for perfusion bioreactors

Direct perfusion of 3D tissue engineered constructs is known to enhance osteogenesis, which can be partly attributed to enhanced nutrient and waste transport. In addition flow mediated shear stresses are known to upregulate osteogenic differentiation and mineralization. A quantification of the hydrodynamic environment is therefore crucial to interpret and compare results of in vitro bioreactor experiments. This study aims to deal with the pitfalls of numerical model preparation of highly complex 3D bone scaffold structures and aims to provide more accurate wall shear stress (WSS) estimates. µCT imaging techniques were used to reconstruct the geometry of both a titanium (Ti) and a hydroxyapatite scaffold, starting from 430 images with a resolution of 8 µm. To tackle the tradeoff between model size and mesh resolution we selected two concentric regions of interest (cubes with a volume of 1 and 3.375 mm³, respectively) for both scaffolds. A flow guidance in front of the real inlet surface of the scaffold was designed to mimic realistic inlet conditions. With a flow rate of 0.04 mL/min perfused through a 5 mm diameter scaffold at an inlet velocity of 33.95 µm/s we obtained average WSSs of 1.10 and 1.46 mPa for the 1 mm³ and the 3.375 mm³ model of the hydroxyapatite scaffold compared to 1.40 and 1.95 mPa for the 1 mm³ model and the 3.375 mm³ model of the Ti scaffold, showing the important influence of the scaffold micro‐architecture heterogeneity and the proximity of boundaries. To assess that influence we selected cubic portions, of which the WSS data were analyzed, with the same size and the same location within both 1 and 3.375 mm³ cubic models. Varying the size of the inner portions simultaneously in both model selections gives a quantification of the sensitivity to boundary neighborhood. This methodology allows to get more insight in the complex concept of tissue engineering and will likely help to understand and eventually improve the fluid‐mechanical aspects. Biotechnol. Bioeng. 2009;103: 621–630. © 2009 Wiley Periodicals, Inc.     

Allometric and nonallometric components of Drosophila wing shape respond differently to developmental temperature

Phenotypic plasticity of wing size and shape of Drosophila simulans was analyzed across the entire range of viable developmental temperatures with Procrustes geometric morphometric method. In agreement with previous studies, size clearly decreases when temperature increases. Wing shape variation was decomposed into its allometric (24%) and nonallometric (76%) components, and both were shown to involve landmarks located throughout the entire wing blade. The allometric component basically revealed a progressive, monotonous variation along the temperature. Surprisingly, nonallometric shape changes were highly similar at both extremes of the thermal range, suggesting that stress, rather than temperature per se, is the key developmental factor affecting wing shape.     

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.     

Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and l-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation.     

Two Mediterranean annuals feature high within-population trait variability and respond differently to a precipitation gradient

Intraspecific trait variability plays an important role in species adaptation to climate change. However, it still remains unclear how plants in semi-arid environments respond to increasing aridity. We investigated the intraspecific trait variability of two common Mediterranean annuals (Geropogon hybridus and Crupina crupinastrum) with similar habitat preferences. They were studied along a steep precipitation gradient in Israel similar to the maximum predicted precipitation changes in the eastern Mediterranean basin (i.e. −30% until 2100). We expected a shift from competitive ability to stress tolerance with decreasing precipitation and tested this expectation by measuring key functional traits (canopy and seed release height, specific leaf area, N- and P-leaf content, seed mass). Further, we evaluated generative bet-hedging strategies by different seed traits. Both species showed different responses along the precipitation gradient. C. crupinastrum exhibited only decreased plant height towards aridity, while G. hybridus showed strong trends of generative adaptation to aridity. Different seed trait indices suggest increased bet-hedging of G. hybridus in arid environments. However, no clear trends along the precipitation gradient were observed in leaf traits (specific leaf area and leaf N-/P-content) in both species. Moreover, variance decomposition revealed that most of the observed trait variation (≫50%) is found within populations. The findings of our study suggest that responses to increased aridity are highly species-specific and local environmental factors may have a stronger effect on intraspecific trait variation than shifts in annual precipitation. We therefore argue that trait-based analyses should focus on precipitation gradients that are comparable to predicted precipitation changes and compare precipitation effects to effects of local environmental factors.     

Alien and native plant life‐forms respond differently to human and climate pressures

Aim To investigate whether differences in the elevational trend in native and alien species richness were dependent on climate or human pressures. Specifically we tested whether life‐form and/or alien/native status modifies the response of plant species richness to human population and temperature along: (1) a complete elevational gradient, and (2) within separate elevational bands that, by keeping temperature within a narrow range, elucidate the effects of human pressures more clearly. Location Two provinces (c. 7507 km²) on the southern border of the European Alps (Italy), subdivided into 240 contiguous sampling cells (c. 35.7 km²). Methods We used an extensive dataset on alien and native species richness across an elevation gradient (20–2900 m a.s.l.). Richness of natives and naturalized aliens were separately related to temperature, human population and Raunkiaer life‐form using general linear mixed models. Life‐form describes different plant strategies for survival during seasons with adverse cold/arid conditions. Results The relationship between species richness and temperature for natives was strongly dependent on life‐form, while aliens showed a consistent positive trend. Similar trends across alien and native life‐forms were found for the relationship between species richness and human population along the whole gradient and within separate elevational bands. Main conclusions The absence of life‐form‐dependent responses amongst aliens supports the hypothesis that the distribution of alien plant species richness was more related to propagule pressure and availability of novel niches created by human activities than to climatic filtering. While climate change will potentially contribute to relaxing species thermal constraints, the response of alien species to future warming will also be contingent on changes in anthropogenic pressures.     

Common and rare plant species respond differently to fertilisation and competition, whether they are alien or native

Plant traits associated with alien invasiveness may also distinguish rare from common native species. To test this, we grew 23 native (9 common, 14 rare) and 18 alien (8 common, 10 rare) herbaceous species in Switzerland from six plant families under nutrient-addition and competition treatments. Alien and common species achieved greater biomass than native and rare species did overall respectively. Across alien and native origins, common species increased total biomass more strongly in response to nutrient addition than rare species did and this difference was not confounded by habitat dissimilarities. There was a weak tendency for common species to survive competition better than rare species, which was also independent of origin. Overall, our study suggests that common alien and native plant species are not fundamentally different in their responses to nutrient addition and competition.     

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress

Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.     

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell‐culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell‐carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1002–1012, 2013     

Research of smooth muscle cells response to fluid flow shear stress by hyaluronic acid micro-pattern on a titanium surface

The morphology of vascular smooth muscle cells (SMCs) in the normal physiological state depends on cytoskeletal distribution and topology beneath, and presents vertical to the direction of blood flow shear stress (FFSS) although SMCs physiologically are not directly exposed to the shear conditions of blood flow. However, this condition is relevant for arteriosclerotic plaques and the sites of a vascular stent, and little of this condition in vitro has been studied and reported till now. It is unclear what will happen to SMC morphology, phenotype and function when the direction of the blood flow changed. In this paper, the distribution of SMCs in a specific area on Ti surface was regulated by micro-strips of hyaluronic acid (HA). Cell morphology depended on the distribution of the cytoskeleton extending along the micrographic direction. Simulated vascular FFSS was perpendicular or parallel to the direction of the cytoskeleton distribution. Based on investigating the morphology, apoptotic number, phenotypes and functional factors of SMCs, it was obtained that SMCs of vertical groups showed more apoptosis, expressed more contractile types and secreted less TGF-β1 factor compared with SMCs of parallel groups, the number of ECs cultured by medium from SMCs of parallel groups was larger than vertical groups. This study could help to understand the effect of direction change of FFSS on patterned SMC morphology, phenotype and function.     

Internal fluid flow increases cellular interconnects between Medial Collateral Ligament fibroblasts and cellular extensions within three-dimensional collagen matrixes

The interconnectivity of fibroblasts within the ligamentous extracellular matrix has been largely overlooked. Studies on the cell-to-cell contacts with their neighbors via gap junctions in ligament fibroblasts, and works on the ability of fibroblasts to generate interconnected networks in vivo, suggest interfibroblastic interactions play an important role in fundamental biological processes, including homeostasis and wound healing. The current study examines how fluidic shear stresses imposed by internal flow can be used to mediate the formation of three-dimensional, interconnected fibroblast networks within collagen solutions. Several fibroblast-collagen solutions were exposed to shear stresses via Poiselle Flow. The consequent changes in cell networking, interconnections, and cell morphology within collagen matrixes exhibited by cells derived from Bovine Medial Collateral Ligaments were analyzed. Results illustrate that higher imposed stresses generate cells with more dendritic and/or branched morphologies, which form more visible three-dimensional networks within collagen matrixes than fibroblast-collagen solutions that were unexposed to shear stress.     

Drosophila melanogaster males respond differently at the behavioural and genome‐wide levels to Drosophila melanogaster and Drosophila simulans females

Drosophila melanogaster are found in sympatry with Drosophila simulans, and matings between the species produce nonfertile hybrid offspring at low frequency. Evolutionary theory predicts that females choose mates, so males should alter their behaviour in response to female cues. We show that D. melanogaster males quickly decrease courtship towards D. simulans females. Courtship levels are reduced within 5 min of exposure to a heterospecific female, and overall courtship is significantly lower than courtship towards conspecific females. To understand changes at the molecular level during mate choice, we performed microarray analysis on D. melanogaster males that courted heterospecific D. simulans females and found nine genes have altered expression compared with controls. In contrast, males that court conspecific females alter expression of at least 35 loci. The changes elicited by conspecific courtship likely modulate nervous system function to reinforce positive conspecific signals and dampen the response to heterospecific signals.     

Regulation of VE-cadherin linkage to the cytoskeleton in endothelial cells exposed to fluid shear stress

Endothelial cells exposed to shear stress realigned and elongated in the direction of flow through the coordinated remodeling of their adherens junctions and actin cytoskeleton. The elaborate networks of VE-cadherin complexes in static cultures became more uniform and compact in response to shear. In contrast, the cortical actin present in static cultures was reorganized into numerous stress fiber bundles distributed parallel to the direction of flow. Exposure to shear did not significantly alter the expression of the junctional proteins VE-cadherin, beta-catenin, and alpha-catenin, but the composition of the junctional complexes did change. We detected a marked decrease in the alpha-catenin associated with VE-cadherin complexes in endothelial monolayers subjected to shear. This loss of alpha-catenin, the protein that links beta-catenin-bound cadherin to the actin cytoskeleton, was not due to decreased quantities of beta-catenin associated with VE-cadherin. Instead, the loss of alpha-catenin from the junctional complexes coincided with the increased tyrosine phosphorylation of beta-catenin associated with VE-cadherin. The change in beta-catenin phosphorylation closely correlated with the shear-induced loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes. Thus, the functional interaction of alpha-catenin with VE-cadherin-bound beta-catenin is regulated by the extent of tyrosine phosphorylation of beta-catenin. This, concomitantly, is regulated by SHP-2 associated with VE-cadherin complexes.     

Species richness and phylogenetic diversity of native and non‐native species respond differently to area and environmental factors

Aim

To test whether native and non‐native species have similar diversity–area relationships (species–area relationships [SARs] and phylogenetic diversity–area relationships [PDARs]) and whether they respond similarly to environmental variables.

Location

United States.

Methods

Using lists of native and non‐native species as well as environmental variables for >250 US national parks, we compared SARs and PDARs of native and non‐native species to test whether they respond similarly to environmental conditions. We then used multiple regressions involving climate, land cover and anthropogenic variables to further explore underlying predictors of diversity for plants and birds in US national parks.

Results

Native and non‐native species had different slopes for SARs and PDARs, with significantly higher slopes for native species. Corroborating this pattern, multiple regressions showed that native and non‐native diversity of plants and birds responded differently to a greater number of environmental variables than expected by chance. For native species richness, park area and longitude were the most important variables while the number of park visitors, temperature and the percentage of natural area were among the most important ones for non‐native species richness. Interestingly, the most important predictor of native and non‐native plant phylogenetic diversity, temperature, had positive effects on non‐native plants but negative effects on natives.

Main conclusions

SARs, PDARs and multiple regressions all suggest that native and non‐native plants and birds responded differently to environmental factors that influence their diversity. The agreement between diversity–area relationships and multiple regressions with environmental variables suggests that SARs and PDARs can be both used as quick proxies of overall responses of species to environmental conditions. However, more importantly, our results suggest that global change will have different effects on native and non‐native species, making it inappropriate to apply the large body of knowledge on native species to understand patterns of community assembly of non‐native species.

     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.