A system to reproduce and quantify the biomechanical environment of the cell

An in vitro system that permits application of a uniform biomechanical stimulus to a population of cells with great precision has been developed. The device is designed to subject living cells to reproducible and quantifiable biaxial strains from 0 to 10% at rates from quasi-static to 1 s-1 and frequencies from 0 to 5 Hz. Equations for determining the strain in the substrate upon which the cells are grown, based on easily measured parameters, are derived and validated experimentally. The mechanical properties of the substrate are determined, and it is demonstrated that cells can easily be cultured in the apparatus. By use of the system, cloned bovine pulmonary artery endothelial cell clones are subjected to 5% biaxial strains applied at a peak strain rate of 0.5 s-1 and a frequency of 1 Hz for 7 h with cell viability greater than 84% and cell detachment less than 8%. We demonstrate that cells must be attached to the substrate for them to be stretched and that cell strain and substrate strain are not equal. With the use of fluorescently labeled beads as cell surface markers to measure the actual strain produced in the cells as a result of the deformation of the substrate, cell elongation was found to be approximately 60% of the strain in the substrate. This constant appeared to be affected by both in vitro cell age and morphology.     

Leukotrienes and tyrosine phosphorylation mediate stretching-induced actin cytoskeletal remodeling in endothelial cells

We studied actin cytoskeletal remodeling and the role of leukotrienes and tyrosine phosphorylation in the response of endothelial cells to different types of cyclic mechanical stretching. Human aortic endothelial cells were grown on deformable silicone membranes subjected to either cyclic one-directional (strip) stretching (10%, 0.5 Hz), or biaxial stretching. After 1 min of either type of stretching, actin cytoskeletons of the stretched cells were already disrupted. After stretching for 10 and 30 min, the percentage of the stretched cells that had disrupted actin cytoskeletons were significantly increased, compared with control cells without stretching. Also, at these two time points, biaxial stretching consistently produced higher frequencies of actin cytoskeleton disruption. At 3 h, strip stretching caused the formation of stress fiber bundles, which were oriented nearly perpendicular to the stretching direction. With biaxial stretching, however, actin cytoskeletons in many stretched cells were remodeled into three-dimensional actin structures protruding outside the substrate plane, within which cyclic stretching was applied. In both stretching conditions, actin filaments were formed in the direction without substrate deformation. Moreover, substantially inhibiting either leukotriene production with nordihydroguaiaretic acid or tyrosine phosphorylation with tyrphostin A25 did not block the actin cytoskeletal remodeling. However, inhibiting both leukotriene production and tyrosine phosphorylation completely blocked the actin cytoskeletal remodeling. Thus, the study showed that the remodeling of actin cytoskeletons of the stretched endothelial cells include rapid disruption first and then re-formation. The resulting pattern of the actin cytoskeleton after remodeling depends on the type of cyclic stretching applied, but under either type of cyclic stretching, the actin filaments are formed in the direction without substrate deformation. Finally, leukotrienes and tyrosine phosphorylation are necessary for actin cytoskeletal remodeling of the endothelial cells in response to mechanical stretching.     

Cyclic mechanical stretch induces VEGF and FGF-2 expression in pulmonary vascular smooth muscle cells

Vascular endothelial growth factor (VEGF) and basic (b) fibroblast growth factor (FGF-2/bFGF) are involved in vascular development and angiogenesis. Pulmonary artery smooth muscle cells express VEGF and FGF-2 and are subjected to mechanical forces during pulsatile blood flow. The effect of stretch on growth factor expression in these cells is not well characterized. We investigated the effect of cyclic stretch on the expression of VEGF and FGF-2 in ovine pulmonary artery smooth muscle cells. Primary confluent cells from 6-wk-old lambs were cultured on flexible silicon membranes and subjected to cyclic biaxial stretch (1 Hz; 5-25% stretch; 4-48 h). Nonstretched cells served as controls. Expression of VEGF and FGF-2 was determined by Northern blot analysis. Cyclic stretch induced expression of both VEGF and FGF-2 mRNA in a time- and amplitude-dependent manner. Maximum expression was found at 24 h and 15% stretch (VEGF: 1.8-fold; FGF-2: 1.9-fold). These results demonstrate that mechanical stretch regulates VEGF and FGF-2 gene expression, which could play a role in pulmonary vascular development or in postnatal pulmonary artery function or disease.     

Protein phosphorylation in cultured endothelial cells

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.     

Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells

We have shown previously that the tumor promoter phorbol myristate acetate (PMA) induces capillary endothelial cells grown on the surface of three-dimensional collagen gels to invade the underlying matrix as capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell 42:469, 1985). To establish whether the potential to invade the extracellular matrix as capillary-like sprouts is restricted to microvascular endothelial cells or is also shared by large vessel endothelium, we have examined the response to PMA of endothelial cells isolated from the human umbilical vein and the calf pulmonary artery. The results of these experiments show that both types of macrovascular endothelial cells are able to penetrate into collagen gels as vessel-like tubes following treatment with PMA. This demonstrates that endothelial cells derived from large vessels can, in response to appropriate signals, express invasive properties thought to be associated specifically with capillary endothelial cells in vivo.     

Specificity of endothelial cell reorientation in response to cyclic mechanical stretching

Evidence suggests that cellular responses to mechanical stimuli depend specifically on the type of stimuli imposed. For example, when subjected to fluid shear stress, endothelial cells align along the flow direction. In contrast, in response to cyclic stretching, cells align away from the stretching direction. However, a few aspects of this cell alignment response remain to be clarified: (1) Is the cell alignment due to actual cell reorientation or selective cell detachment? (2) Does the resulting cell alignment represent a response of the cells to elongation or shortening, or both? (3) Does the cell alignment depend on the stretching magnitude or rate, or both? Finally, the role of the actin cytoskeleton and microtubules in the cell alignment response remains unclear. To address these questions, we grew human aortic endothelial cells on deformable silicone membranes and subjected them to three types of cyclic stretching: simple elongation, pure uniaxial stretching and equi-biaxial stretching. Examination of the same cells before and after stretching revealed that they reoriented. Cells subjected to either simple elongation or pure uniaxial stretching reoriented specifically toward the direction of minimal substrate deformation, even though the directions for the two types of stretching differed by only about 20°. At comparable stretching durations, the extent of cell reorientation was more closely related to the stretching magnitude than the stretching rate. The actin cytoskeleton of the endothelial cell subjected to either type of stretching was reorganized into parallel arrays of actin filaments (i.e., stress fibers) aligned in the direction of the minimal substrate deformation. Furthermore, in response to equi-biaxial stretching, the actin cytoskeleton was remodeled into a “tent-like” structure oriented out of the membrane plane—again towards the direction of the minimal substrate deformation. Finally, abolishing microtubules prevented neither the formation of stress fibers nor cell reorientation. Thus, endothelial cells respond very specifically to the type of deformation imposed upon them.     

Mechano-chemical control of human endothelium orientation and size

Human umbilical vein endothelial cells (EC) were grown on elastic silicone membranes subjected to cyclic stretch, simulating arterial wall motion. Stretching conditions (20% amplitude, 52 cycle/min) stimulated stress fiber formation and their orientation transversely to the strain direction. Cell bodies aligned along the same axis after the actin cytoskeleton. EC orientation response was inhibited by the adenylate cyclase activator, forskolin (10(-5) M), which caused stress fiber disassembly and the redistribution of F-actin to the cortical cytoplasm. Preoriented EC depleted of stress fibers by forskolin treatment retained their aligned state. Thus, stress fibers are essential for the process of EC orientation induced by repeated strain, but not for the maintenance of EC orientation. The monolayer formed by EC grown to confluence in conditions of intermittent strain consisted of uniform elongated cells and was resistant to deformation. In contrast, the monolayer assembled in stationary conditions was less compliant and exposed local denudations on initiation of stretching. When stretched in the presence of 10(-5) M forskolin it rapidly (3-4 h) reestablished integrity but gained a heterogeneous appearance since denuded areas were covered by giant cells. The protective effect of forskolin was because of the stimulation of EC spreading. This feature of forskolin was demonstrated while studying its action on EC spreading and repair of a scratched EC monolayer in conventional culture. Thus mechanical deformation and adenylate cyclase activity may be important factors in the control of endothelium morphology in human arteries.     

Effect of long-term hypoxia on cultured aortic and pulmonary arterial endothelial cells

In a previous study, we found a marked difference in the release of a cytokine, neutrophil chemoattractant activity (NCA), from cultured endothelial cells exposed to acute decreases in ambient oxygen, depending on the vascular bed of origin. In the current study, we used this cytokine to evaluate the effect of long-term exposure to decreased oxygen on endothelial cell function. We found that, in aortic and pulmonary arterial endothelial cells maintained for months in decreased ambient oxygen (10 or 3% oxygen), exposure to acute decreases in ambient oxygen caused a change in the pattern of NCA release; however, the differential response between the two cell types persisted. Aortic endothelial cells release NCA when exposed acutely to a level of oxygen below that in which they have been chronically maintained. In contrast, pulmonary arterial endothelial cells release NCA only when exposed to 0% oxygen acutely, but only if grown chronically in 10% oxygen; otherwise there was no release of NCA. As another indicator of endothelial cell function, we evaluated the effects of acute hypoxic exposure on prostacyclin production by endothelial cells maintained in 21 or 3% oxygen. If grown in 21% oxygen, both cell types decreased prostacyclin production upon exposure to 0% oxygen. However, when grown in 3% oxygen, only aortic endothelial cells decreased prostacyclin production when exposed acutely to 0% oxygen; pulmonary arterial endothelial cell prostacyclin production did not change. This study demonstrating the persistence of a differential pattern of NCA release and the appearance of a differential pattern of prostacyclin production after a long-term decrease in environmental oxygen suggests that the capacity of certain vascular endothelial cells to respond to decreases in oxygen concentration is carried by the cell throughout its existence. Thus, in certain situations, vascular endothelial cells may be important in sensing acute decreases in ambient oxygen.     

Orientation response of arterial smooth muscle cells to mechanical stimulation

Arterial smooth muscle cells from rabbit aortic media in primary culture and subculture were grown on hydrophilized and collagen-coated silicone membranes which were then subjected to cyclic and directional stretches and relaxations at a frequency of 60 times/min. The membranes were stretched with various amplitudes ranging from 2% to 20%. Smooth muscle cells on unstretched membranes in the same incubation chamber served as controls. In long-term experiments the stretching and relaxing of the membranes was continued for several days. While the smooth muscle cells grown on unstretched membranes remained in random orientation in all experiments, the cells which underwent mechanical stimulation showed a high degree of orientation. The angle of cell orientation varied in direct relation to the stretching amplitude and became steeper in correlation to the intensity of the mechanical stimulus. The angle of cell orientation was reversible, as preoriented cells changed their orientation when another stretching amplitude was applied. To study the role of cytoskeleton in the process of cell orientation, we examined the behaviour of the intracellular actin filament system. In short-term experiments the smooth muscle cells were exposed for 3 to 12 h to cyclic and directional stretches and relaxations with an amplitude of 10%. We observed a rearrangement of the intracellular actin filament system prior to the orientation of the whole cell bodies. The present study provides evidence that stretching the artery wall by blood pulsation may result in an orientation response of the intracellular actin cytoskeleton and in the orientation of the smooth muscle cells within the media of artery walls.     

Cyclical strain effects on production of vasoactive materials in cultured endothelial cells.

Mechanical forces due to fluid flow and cyclical strain can alter endothelial cell morphology and function, including the release of vasoactive materials endothelin, prostacyclin (PGI2), and tissue plasminogen activator (t-PA). In this study, effects of cyclical strain were modeled by culturing bovine aortic endothelial cells on fibronectin-coated elastic membranes of silicone rubber (Silastic) or poly-etherurethane urea (Mitrathane). After growing to confluence under static conditions of 37 degrees C in humidified air with 5% CO2, cells were strained cyclically at membrane elongations of 5% or 10% for 24 hours at 1 Hz. Controls were maintained under static conditions or were exposed to fluid motions similar to the strained cells but without stretching. Secretion rates were constant throughout experiments in the strain chamber with no initial burst in metabolism associated with the initiation of strain. Secretion rates were not altered by choice of elastic membrane. At a physiological level of 10% cyclical strain, prostacyclin and endothelian secretion rates were increased by 2.5-fold and 1.7-fold, respectively, above stationary controls. Endothelin production demonstrated a dose-dependent response with cyclical strain, while PGI2 appeared to require a threshold strain before an increase in secretion occurred. No significant differences in t-PA levels were seen in cyclically strained cells compared with controls. These results indicate that endothelial cells respond metabolically to cyclical strain and suggest that mechanical strain may modulate secretion of selective vasoactive materials by vascular endothelial cells.     

Pyk2/CAKbeta tyrosine kinase activity-mediated angiogenesis of pulmonary vascular endothelial cells.

Endothelial cell spreading, migration, and morphogenesis are essential for angiogenesis, the formation of new blood vessels. In the present study, we explored roles of tyrosine kinase Pyk2 in angiogenesis of pulmonary endothelial cells. We found that tyrosine kinase Pyk2 was particularly enriched in pulmonary vascular endothelial cells and lung, a major organ site for tumor metastasis. By using adenovirus-mediated expression of various Pyk2 mutants, we demonstrated that Pyk2 tyrosine kinase activity was essential for the pulmonary vascular endothelial cell spreading, migration, morphogenesis, as well as pulmonary vein and artery angiogenesis ex vivo. We further showed that Pyk2 kinase activity was required for the expression of focal adhesion kinase, p130Crk-associated substrate, and its homologue human enhancer of filamentation 1, thus regulating formation of focal adhesions and cytoskeletal reorganization. These results indicate that Pyk2 plays a crucial role in the pulmonary endothelial cell motility such as spreading and migration necessary for angiogenesis.     

Confocal microscopy-based three-dimensional cell-specific modeling for large deformation analyses in cellular mechanics

This study introduces a new confocal microscopy-based three-dimensional cell-specific finite element (FE) modeling methodology for simulating cellular mechanics experiments involving large cell deformations. Three-dimensional FE models of undifferentiated skeletal muscle cells were developed by scanning C2C12 myoblasts using a confocal microscope, and then building FE model geometries from the z-stack images. Strain magnitudes and distributions in two cells were studied when the cells were subjected to compression and stretching, which are used in pressure ulcer and deep tissue injury research to induce large cell deformations. Localized plasma membrane and nuclear surface area (NSA) stretches were observed for both the cell compression and stretching simulation configurations. It was found that in order to induce large tensile strains (>5%) in the plasma membrane and NSA, one needs to apply more than ～15% of global cell deformation in cell compression tests, or more than ～3% of tensile strains in the elastic plate substrate in cell stretching experiments. Utilization of our modeling can substantially enrich experimental cellular mechanics studies in classic cell loading designs that typically involve large cell deformations, such as static and cyclic stretching, cell compression, micropipette aspiration, shear flow and hydrostatic pressure, by providing magnitudes and distributions of the localized cellular strains specific to each setup and cell type, which could then be associated with the applied stimuli.     

Activation of guanylate cyclase and inhibition of platelet aggregation by endothelium-derived relaxing factor released from cultured cells

The aggregation of gel-filtered rabbit platelets by 50 microM ADP was inhibited by a labile factor produced by suspensions of cultured bovine pulmonary artery endothelial cells. Inhibition of aggregation occurred when indomethacin-treated endothelial cells (6.10(5) per ml) and rabbit platelets (3.2.10(8) per ml) were incubated together. This anti-aggregatory activity was characterized as similar to endothelium-derived relaxing factor (EDRF) in that it was unstable at neutral pH and by its inhibition by hemoglobin. The activity was unaffected by treatment of the platelets and endothelial cells with the cyclooxygenase inhibitor, indomethacin, and by the lipoxygenase inhibitor, BW755c. In association with the anti-aggregatory activity, the levels of cyclic GMP were elevated 4-fold. The effect of the EDRF-like product on the levels of cyclic nucleotides was mimicked by treatment of platelets with sodium nitroprusside, an activator of soluble guanylate cyclase; sodium nitroprusside had no measurable effect on the levels of cyclic nucleotides of endothelial cells. We conclude that a factor with the properties of EDRF inhibits platelet aggregation, and that this is associated with an activation of guanylate cyclase as in smooth muscle. Thus, EDRF may exert an inhibitory effect on platelets in a manner analogous to its actions on vascular smooth muscle.     

Effects of Cyclic Intermittent Hypoxia on ET-1 Responsiveness and Endothelial Dysfunction of Pulmonary Arteries in Rats

Obstructive sleep apnoea (OSA) is a risk factor for cardiovascular disorders and in some cases is complication of pulmonary hypertension. We simulated OSA by exposing rats to cyclic intermittent hypoxia (CIH) to investigate its effect on pulmonary vascular endothelial dysfunction. Sprague-Dawley Rats were exposed to CIH (FiO₂ 9% for 1 min, repeated every 2 min for 8 h/day, 7 days/wk for 3 wk), and the pulmonary arteries of normoxia and CIH treated rats were analyzed for expression of endothelin-1 (ET-1) and ET receptors by histological, immunohistochemical, RT-PCR and Western Blot analyses, as well as for contractility in response to ET-1. In the pulmonary arteries, ET-1 expression was increased, and ET-1 more potently elicited constriction of the pulmonary artery in CIH rats than in normoxic rats. Exposure to CIH induced marked endothelial cell damage associated with a functional decrease of endothelium-dependent vasodilatation in the pulmonary artery. Compared with normoxic rats, ET_A receptor expression was increased in smooth muscle cells of the CIH rats, while the expression of ET_B receptors was decreased in endothelial cells. These results demonstrated endothelium-dependent vasodilation was impaired and the vasoconstrictor responsiveness increased by CIH. The increased responsiveness to ET-1 induced by intermittent hypoxia in pulmonary arteries of rats was due to increased expression of ET_A receptors predominantly, meanwhile, decreased expression of ET_B receptors in the endothelium may also participate in it.     

Nebivolol: a selective beta(1)-adrenergic receptor antagonist that relaxes vascular smooth muscle by nitric oxide- and cyclic GMP-dependent mechanisms.

Nebivolol is a highly selective beta(1)-adrenergic receptor antagonist that also possesses vasodilator properties that are attributed largely to nitric oxide (NO). The objective of the present study was to elucidate in more detail the mechanisms by which nebivolol relaxes vascular smooth muscle. In the canine species, nebivolol caused relaxation of isolated precontracted rings of coronary artery and pulmonary artery largely by endothelium-dependent, NO-dependent, and cyclic GMP-dependent mechanisms. Vasorelaxation was inhibited by N(G)-methylarginine, and this inhibition was reversed by addition of excess L-arginine. Moreover, the vasorelaxant responses to nebivolol were markedly inhibited by oxyhemoglobin, methylene blue, and 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), whereas vasorelaxation was enhanced by zaprinast. Rat aortic ring preparations, however, relaxed in response to nebivolol by both endothelium-dependent and endothelium-independent mechanisms, both involving NO, and cyclic GMP. Endothelium-dependent and endothelium-independent vasorelaxation were inhibited by oxyhemoglobin, methylene blue, and ODQ. However, only endothelium-dependent vasorelaxation in response to nebivolol was inhibited by N(G)-methylarginine. Additional experiments ruled out other endothelium-independent vasorelaxant mechanisms. In conclusion, the vasodilator responses to nebivolol involve NO and cyclic GMP in both vascular endothelial and smooth muscle cells.     

Cellular disposition of transported polyamines in hypoxic rat lung and pulmonary arteries

The polyamines putrescine, spermidine (SPD), and spermine are a family of low-molecular-weight organic cations essential for cell growth and differentiation and other aspects of signal transduction. Hypoxic pulmonary vascular remodeling is accompanied by depressed lung polyamine synthesis and markedly augmented polyamine uptake. Cell types in which hypoxia induces polyamine transport in intact lung have not been delineated. Accordingly, rat lung and rat main pulmonary arterial explants were incubated with [(14)C]SPD in either normoxic (21% O(2)) or hypoxic (2% O(2)) environments for 24 h. Autoradiographic evaluation confirmed previous studies showing that, in normoxia, alveolar epithelial cells are dominant sites of polyamine uptake. In contrast, hypoxia was accompanied by prominent localization of [(14)C]SPD in conduit, muscularized, and partially muscularized pulmonary arteries, which was not evident in normoxic lung tissue. Hypoxic main pulmonary arterial explants also exhibited substantial increases in [(14)C]SPD uptake relative to control explants, and autoradiography revealed that enhanced uptake was most evident in the medial layer. Main pulmonary arterial explants denuded of endothelium failed to increase polyamine transport in hypoxia. Conversely, medium conditioned by endothelial cells cultured in hypoxic, but not in normoxic, environments enabled hypoxic transport induction in denuded arterial explants. These findings in arterial explants were recapitulated in rat cultured main pulmonary artery cells, including the enhancing effect of a soluble endothelium-derived factor(s) on hypoxic induction of [(14)C]SPD uptake in smooth muscle cells. Viewed collectively, these results show in intact lung tissue that hypoxia enhances polyamine transport in pulmonary artery smooth muscle by a mechanism requiring elaboration of an unknown factor(s) from endothelial cells.     

外源性硫化氢对家兔内毒素休克诱发肺动脉高压的干预作用

目的:探讨外源性硫化氢(H₂S)对家兔内毒素休克(ES)诱发肺动脉血管反应性的影响。方法:本实验采用家兔经颈静脉注射脂多糖(LPS,8 mg/0.8 ml/kg)复制家兔ES模型,并提前15 min腹腔注射H₂S供体硫氢化钠(NaHS,28μmol/kg)进行干预。随机将新西兰大耳白家兔分为4组(n=8):溶剂对照组、LPS组、LPS+NaHS组和NaHS组。监测平均动脉压(MAP)和平均肺动脉压(MPAP)的变化;应用血管环张力测定技术,检测各组家兔离体肺动脉张力变化;应用光镜和扫描电镜分别观察肺动脉管壁结构及肺动脉内皮细胞超微结构变化。结果:(1)注射LPS后家兔出现MAP降低、MPAP升高,成功复制家兔ES模型;与LPS组相比,LPS+NaHS组家兔MPAP在各个时间点均显著降低(P均﹤0.05);(2)与正常对照组相比,LPS组家兔肺动脉对苯肾上腺素(PE)的收缩反应增强,对乙酰胆碱(ACh)的舒张反应降低(P均﹤0.01);与LPS组相比,LPS+NaHS组家兔肺动脉对PE的收缩反应降低,而对ACh的舒张反应增强(P均﹤0.05)。...     

Influence of substrate stiffness on circulating progenitor cell fate

In situ vascular tissue engineering (TE) aims at regenerating vessels using implanted synthetic scaffolds. An envisioned strategy is to capture and differentiate progenitor cells from the bloodstream into the porous scaffold to initiate tissue formation. Among these cells are the endothelial colonies forming cells (ECFCs) that can differentiate into endothelial cells and transdifferentiate into smooth muscle cells under biochemical stimulation. The influence of mechanical stimulation is unknown, but relevant for in situ vascular TE because the cells perceive a change in mechanical environment when captured inside the scaffold, where they are shielded from blood flow induced shear stresses. Here we investigate the effects of substrate stiffness as one of the environmental mechanical cues to control ECFC fate within scaffolds. ECFCs were seeded on soft (3.58±0.90 kPa), intermediate (21.59±2.91 kPa), and stiff (93.75±18.36 kPa) fibronectin-coated polyacrylamide gels, as well as on glass controls, and compared to peripheral blood mononuclear cells (PBMC). Cell behavior was analyzed in terms of adhesion (vinculin staining), proliferation (BrdU), phenotype (CD31, αSMA staining, and flow cytometry), and collagen production (col I, III, and IV). While ECFCs adhesion and proliferation increased with substrate stiffness, no change in phenotype was observed. The cells produced no collagen type I, but abundant amounts of collagen type III and IV, albeit in a stiffness-dependent organization. PBMCs did not adhere to the gels, but they did adhere to glass, where they expressed CD31 and collagen type III. Addition mechanical cues, such as cyclic strains, should be studied to further investigate the effect of the mechanical environment on captured circulating cells for in situ TE purposes.     

Microstructural strain near osteocyte lacuna in cortical bone in vitro

Mechanical factors affect bone remodeling such that increased mechanical demand results in net bone formation, whereas decreased demand results in net bone resorption. Two proposed mechanical signals are stress-generated fluid flow forces acting on cells and bone matrix deformation itself. A prominent current theory is that bone cells are more responsive to fluid flow than to mechanical strain. Recent experiments support this conclusion: bone cells increase their production of osteopontin (OPN) mRNA, prostaglandin (PGE(2)), and nitric oxide (NO) in response to fluid flow in contrast to cells stimulated by mechanical strain levels similar to those measured in vivo. However, when cells are subjected to substrate strains levels many times greater than those measured in vivo, increased biological activity again results. We assert that it is neither fluid flow nor matrix deformation per se, but rather the resulting cell deformation that causes cell biological response. Machined specimens of undamaged bovine cortical bone were subjected to increasing levels of macroscopic strain while observed under an optical microscope at 220X. Continuum level strain was measured using a standard foil strain gauge attached to the back of the specimen and ranged from 500 to 6,000 microstrain. Images of the specimen surface at each strain level were captured. To determine the level of osteocyte deformation that results from fluid flow in vitro, MLO-Y4 cells were cultured on collagen coated 190 cm2 plastic sheets and subjected to steady fluid flow at 16 dynes/cm(2). Images representing the initial undisturbed cell configuration and the configuration of the cells after ten minutes of fluid flow were acquired from a videotape of the flow experiment. The captured unloaded vs. loaded image pairs were analyzed to determine the local deformation and strain fields using a digital stereoimaging system. When subjected to a nominal continuum strain level approximately equal to that measured in humans in vivo during rigorous activity (2,000 microstrain), the local, osteocyte level strains can be as high as 12,000 to 15,000 microstrain (1.2% to 1.5%). Average osteocyte strains due to fluid flow in vitro increase from 7,972 microstrains after 16 seconds of flow to 22,856 microstrains after 64 seconds of flow. In contrast, maximum strains measured in vivo are approximately 1,800 microstrain in humans and up to 3,000 microstrain in other species. These data may help to explain why bone cells are more sensitive to fluid flow than substrate strain; fluid forces result in cell deformations much higher than those considered to be "physiological".