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Abstract: Cyclic strain evokes the expression of the small leucine-rich proteoglycans decorin and biglycan in 2D cultures and native tissues. However, strain-dependent expression of these proteoglycans has not been demonstrated in engineered tissues. We hypothesized that the absence of decorin may compromise the effect of cyclic strain on the development of engineered tissues. Thus, we investigated the contribution of decorin to tissue organization in cyclically strained collagen gels relative to statically cultured controls. Decorin null (Dcn −/−) and wild-type murine embryonic fibroblasts were seeded within collagen gels and mechanically conditioned using a Flexcell® Tissue Train® culture system. After 8 days, the cyclically strained samples demonstrated greater collagen fibril density, proteoglycan content, and material strength for both cell types. On the other hand, increases in cell density, collagen fibril diameter, and biglycan expression were observed only in the cyclically strained gels seeded with Dcn −/− cells. Although cyclic strain caused an elevation in proteoglycan expression regardless of cell type, the type of proteoglycan differed between groups: the Dcn −/− cell-seeded gels produced an excess of biglycan not found in the wild-type controls. These results suggest that decorin-mediated tissue organization is strongly dependent upon tissue type and mechanical environment. [Copyright &y& Elsevier]

Repetitive deformation due to vilous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/mm. Phosphorylation of Src-Tyr418, FAK-Tyr397-Tyr576-Tyr925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 μmol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr397 and Tyr576 but not FAK-Tyr925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr925-dependent path- way that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting. [ABSTRACT FROM AUTHOR]

ABSTRACT Using biodegradable scaffold and a biomimetic perfusion system, our lab has successfully engineered small-diameter vessel grafts using endothelial cells (ECs) and smooth muscle cells (SMCs) obtained from vessels in various species. However, translating this technique into humans has presented tremendous obstacles due to species and age differences. SMCs from elderly persons have limited proliferative capacity and a reduction in collagen production, which impair the mechanical strength of engineered vessels. As an alternative cell source, adult human bone marrow-derived mesenchymal stem cells (hMSCs) were studied for their ability to differentiate into SMCs in culture plates as well as in a bioreactor system. In the former setting, immunofluorescence staining showed that MSCs, after induction for 14 days, expressed smooth muscle α-actin (SMA) and calponin, early and mid-SMC phenotypic markers, respectively. In the latter setting, vessel walls were constructed with MSCderived SMCs. Various factors (i.e., matrix proteins, soluble factors, and cyclic strain) in the engineering system were further investigated for their effects on hMSC cell proliferation and differentiation into SMCs. Based on a screening of multiple factors, the engineering system was optimized by dividing the vessel culture into proliferation and differentiation phases. The vessel walls engineered under the optimized conditions were examined histologically and molecularly, and found to be substantially similar to native vessels. In conclusion, bone marrow-derived hMSCs can serve as a new cell source of SMCs in vessel engineering. Optimization of the culture conditions to drive SMC differentiation and matrix production significantly improved the quality of the hMSC-derived engineered vessel wall. [ABSTRACT FROM AUTHOR]

Abstract: Cardiomyocytes in the body are subjected to cyclic mechanical strain induced by the rhythmic heart beating. In this study, we tested the hypothesis that cyclic strain promotes cardiomyogenesis of embryonic stem cell-derived cardiomyocytes (ESCs). ESCs cultured on elastic polymer [poly(lactide-co-caprolactone), PLCL] scaffolds subjected to cyclic strain in vitro displayed elevated cardiac gene expression compared to unstrained controls. Six weeks after implantation into infarcted rat myocardium, the elastic cardiac patches (ESC-seeded PLCL scaffolds) showed reduced fibrotic tissue formation, likely due to a combination of lower apoptotic activity, higher vascular endothelial growth factor (VEGF) expression, and more extensive angiogenesis in the strained versus unstrained control [ESC-seeded, non-elastic poly(lactide-co-glycolide) scaffolds] patches. Importantly, cardiac gene expression was upregulated in the elastic patches compared to control, with evidence for cardiomyocyte-specific microstructures including myofibrillar bundles and Z-lines. This study shows that the use of an elastic polymer scaffold designed to permit mechanical strain transduction as a cell transplantation vehicle significantly increases cardiomyogenesis of the implanted ESCs. [Copyright &y& Elsevier]

Abstract: Cellular alignment studies have shown that cell orientation has a large effect on the expression and behavior of cells. Cyclic strain and substrate microtopography have each been shown to regulate cellular alignment. This study examined the combined effects of these two stimuli on the alignment of bovine vascular smooth muscle cells (VSMCs). Cells were cultured on substrates with microgrooves of varying widths oriented either parallel or perpendicular to the direction of an applied cyclic tensile strain. We found that microgrooves oriented parallel to the direction of the applied strain limited the orientation response of VSMCs to the mechanical stimulus, while grooves perpendicular to the applied strain enhanced cellular alignment. Further, the extent to which parallel grooves limited cell alignment was found to be dependent on the groove width. It was found that for both a small (15μm) and a large (70μm) groove width, cells were better able to reorient in response to the applied strain than for an intermediate groove width (40μm). This study indicates that microtopographical cues modulate the orientation response of VSMCs to cyclic strain. The results suggest that there is a range of microgroove dimensions that is most effective at maintaining the orientation of the cells in the presence of an opposing stimulus induced by cyclic strain. [Copyright &y& Elsevier]

Abstract: Silicon will continue to be the critical structure material for micromechanical components for years to come so that reliability must be a key concern. Consequently, in order to ensure reliability design approaches must account for fatigue behavior. This work is aimed at studying the fatigue of single crystal silicon (SCS) thin films by a specially designed method. The films were tested using cantilever microbeam deflection with respect to the effect of loading conditions. To conduct a fatigue test under cyclic strain would be more realistic because many MEMS applications, such as micro-accelerometers and micro-filters, consist of beams vibrating in the same mode. A micro-force testing machine with a micro-probe and specially designed fixtures is used to contact and load the beams under the cyclic strain. Before the fatigue test, the failure strain ɛ f of beams in the flexural test is achieved as the testing criterion. In fatigue testing, various percentages of failure strain ɛ f, two times of the strain amplitude, are adopted. First of all, fatigue strain–life cycle (S/N) curve is achieved. Further, the curve of fatigue force detected on the SCS versus fatigue life is measured. SEM is also employed to observe the fracture modes of films under fatigue. Based on the SEM observation and force/life curve, the failure mechanism of the fatigued SCS films under the cyclic strain is proposed. This provides a viable method to evaluate the reliability of the SCS. [Copyright &y& Elsevier]

Phenotype transformation of vascular smooth muscle cells (VSMCs) is known to be modulated by mechanical strain. The present study was designed to investigate how different frequencies of mechanical strain affected VSMC phenotype. VSMCs were subjected to the strains of 10% elongation at 0, 0.5, 1 and 2 Hz for 24 h using a Flexercell strain unit. VSMC phenotype was assessed by cell morphology, measurement of two-dimensional cell area, Western blotting for protein and RT-PCR for mRNA expression of differentiation markers. Possible protein kinases involved were evaluated by Western blotting with their specific antibodies. The strains at certain frequencies could induce a contractile morphology in VSMC with almost perpendicular alignment to the strain direction. The strains also regulated protein and mRNA expression of several differentiation markers, as well as the activation of extracellular signal-regulated kinases (ERKs), p38 MAP kinase and protein kinase B (Akt) in a frequency-dependent manner. Furthermore, the inhibition of the p38 pathway could block the frequency-induced phenotype modulation of VSMCs, but not inhibition of ERK or Akt pathways. These results indicate that the frequency of cyclic strain can result in the differentiated phenotype of VSMCs, and it is mediated at least partly by the activation of the p38 pathway. [ABSTRACT FROM AUTHOR]

Abstract: Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport. [Copyright &y& Elsevier]

Abstract: In structures subjected to extreme loading, plate steels are relied upon to plastically deform under large repeated strains. Nevertheless, limited data exists on the hysteretic stress–strain response and low-cycle fatigue life of these steels. In the study described in this paper, axial coupons were tested for five types of plate steel, including conventional and high performance A709 steel as well as specialty low yield point steels, to investigate the material response under repeated inelastic demands of constant amplitude between 1% and 7% strain. Parameters for analytical models were established for cyclic stress–strain and low-cycle fatigue life relationships. Based on this study, it was found that the low-cycle fatigue life of the different types of plate steels was similar and that the effect of increased strain rate was minimal. However, the maximum cyclic stress was found to exceed the yield strength by a factor of 2.0 for the conventional grade steel and up to 4.8 times for the low yield point steels, within just a few reversals in the considered strain ranges. The maximum cyclic stress behavior was not related to the yield strength as much as it was to the manufacturing specifications. This behavior should be considered when using these materials in the limit state design of structures for extreme events such as earthquakes. [Copyright &y& Elsevier]

Hedge-hog (Hh) signaling has recently been shown to be both responsive to mechanical loading in vitro and to control vascular development in vivo. We investigated the role of cyclic strain and pulsatile flow in modulating Hh signaling and growth of adult rat vascular smooth muscle cells (SMC) in culture. Exposure of SMC to defined equibiaxial cyclic strain (0% and 10% stretch, 60 cycles/min, for 24 h) significantly decreased sonic hedgehog (Shh) and patched 1 (Ptc1) expression while concurrently inhibiting Gli2-dependent promoter activity and mRNA expression, respectively. Cyclic strain significantly decreased SMC proliferation (cell counts and proliferating cell nuclear antigen expression) concomitant with a marked increase in SMC apoptosis (fluorescence-activated cell sorter analysis, acridine orange staining of apoptotic nuclei and Bax/Bcl-xL ratio). These strain-induced changes in proliferation and apoptosis were significantly attenuated following addition of either recombinant Shh (3.5 μg/ml) or overexpression of the Notch 3 intracellular domain (Notch IC). Further studies using a perfused transcapillary culture system demonstrated a significant decrease in Hh signaling in SMC following exposure of cells to increased pulsatile flow concomitant with a decrease in proliferation and an increase in apoptosis. Finally, the pulsatile flow-induced decreases in Hh signaling were validated in vivo following flow-induced rat carotid arterial remodeling after 28 days. These data suggest that Hh expression is diminished by biomechanical stimulation in vitro and in vivo and thus may play a fundamental role in arterial remodeling and atherogenesis in vivo. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of the mechanical cyclic strain on the extracellular matrix (ECM) production by cultivated human limbal epithelial cells (HLECs) in vitro. Methods: HLECs were repetitively stretched and relaxed by 20% of their original length. Morphology of HLECs was observed, and concentrations of fibronectin and laminin V were measured. Results: The strained HLECs were elongated and aligned perpendicular to the direction of force. Fibronectin and laminin V were highly expressed in the strain group compared with the control. Conclusions: Cyclic strain induces the synthesis of fibronectin and laminin V in cultivated HLECs. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24–96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-α, α-smooth muscle actin (α-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-α and α-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-α and α-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system. [ABSTRACT FROM AUTHOR]

Abstract: Rapid induction of matrix production and mechanical strengthening is essential to the development of bio-artificial constructs for repair and replacement of load-bearing connective tissues. Toward this end, we describe the development of a mechanical bioreactor and its application to investigate the influence of cyclic strain on fibroblast proliferation, matrix accumulation, and the mechanical properties of fibroblast-seeded polyurethane constructs (FSPC). Human fibroblasts were cultured in 10% serum-containing conditions within three-dimensional, porous elastomeric substrates under static conditions and a model regime of cyclic strain (10% strain, 0.25Hz, 8h/day), with and without ascorbic acid supplementation. After one week, the combination of cyclic strain and ascorbic acid resulted in significantly increased construct elastic modulus (>110%) relative to either condition alone. In contrast, cyclic strain alone was sufficient to stimulate significant increases in fibroblast proliferation. Mechanical strengthening of FSPCs was accompanied by increased type I collagen and fibronectin matrix accumulation and distribution, and significantly increased gene expression for type I collagen, TGFβ-1, and CTGF. These results suggest that strain-induced conditioning in vitro leads to mechanical strengthening of fibroblast/material constructs, most likely resulting from increased collagen matrix deposition, secondary to strain-induced increases in cytokine production. [Copyright &y& Elsevier]

Abstract: We showed that cyclic strain (CS) of osteoblastic cells induced tyrosine phosphorylation of two homologous tyrosine kinases FAK and PYK2, and of two homologous adaptor proteins paxillin and Hic5, with similar kinetics. Immunostaining showed that all four proteins were localized to focal contacts in controls. In contrast, the dynamics of their subcellular localization observed after CS differed. While FAK and paxillin remained at the focal contact, Hic-5 and PYK2 translocated outside ventral focal contacts as early as 30min after CS and were sequestered by the cytoskeleton. Co-immunoprecipitation showed that the association of PYK2/Hic-5 and PYK2/FAK increased with time after strain while that of paxillin and Hic-5 decreased. Altogether these results suggested that CS regulates focal contact activity in osteoblasts by modulating PYK2-containing complexes in particular by shuttling out of the focal contact the adaptor Hic-5 and favoring the anchorage of FAK within contacts. [Copyright &y& Elsevier]

Abstract: Angiogenesis is the formation of new blood vessels from preexisting capillaries or venules. It occurs in a mechanically dynamic environment due to blood flow, but the role of hemodynamic forces in angiogenesis remains poorly understood. We have developed a unique in vitro system for the investigation of angiogenesis under cyclic strain. In this system, tubulogenesis of vascular endothelial cells in 3D collagen gels occurs under well-defined cyclic strain, which mimics blood-pressure-induced stretch. Using this system, we demonstrate that cyclic strain results in alignment of endothelial-cord-like structures perpendicular to the principal axis of stretch. Such preferential orientation was the most evident in deep and long cord-like structures. This in vitro system, along with the novel findings of strain-modulated endothelial tube morphology, enables the formation of an experimental basis for understanding the role of cyclic strain in the regulation of angiogenesis. [Copyright &y& Elsevier]

Vascular smooth muscle cell (VSMC) phenotypic modulation is a key factor in vascular pathology. We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of a-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-Jκ (RBP-Jκ) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of α-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF-1/RBP-Jκ-dependent signaling by coexpression of RPMS-1 (Epstein-Barr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split (HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/mm, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP-Jκ- dependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/ RBP-Jκ-dependent pathways in vitro and contributes to the phenotypic response of VSMCs to cyclic strain-induced changes in VSMC differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Hemodynamic forces, including shear stress and cyclic strain, have been recognised as important modulators of vascular cell morphology and function. However, the mechanism by which vascular cells sense and transduce the extracellular mechanical signals into the cell nucleus has not yet been clarified. The purpose of our study was to assess the involvement of the signal transducer and activator of transcription-3 (STAT-3) in the signaling pathway mediating the response of vascular smooth muscle cells (SMC) to cyclic strain. Embryonic A7r5 SMC derived from thoracic aortas of DB1X rats were seeded on flexible collagen I-coated plates. Cells were subjected to 10% average strain at 60cycles/min for various time periods. Activation of STAT-3, p38, extracellular signal-regulated kinase (ERK) 1/2 and Src was assessed by immunoblotting using phosphospecific antibodies. The interactions between STAT-3 phosphorylation and p38, ERK1/2, phosphatidylinositol-3 (PI3K), mammalian target of rapamycin (mTOR), Janus kinase (JAK) 2 and Src were evaluated by pretreating the cells with specific inhibitors including SB202190, PD98059, LY294002, wortmannin, rapamycin, AG490 and PP1. Serine phosphorylation of STAT-3 was increased by 2-fold after 15min of cyclic strain, while tyrosine phosphorylation was increased by 2.3-fold after 60min. Inhibition of ERK1/2 by PD98059 prevented serine phosphorylation of STAT-3, whereas inhibition of Src by PP1 prevented STAT-3 tyrosine phosphorylation. Pretreating the cells with SB202190, a specific inhibitor of p38, resulted in an increase in basal phosphorylation of ERK1/2 and a subsequent increase in basal serine phosphorylation of STAT-3. In conclusion, both serine and tyrosine phosphorylation of STAT-3 are involved in the signaling pathway mediating the effects of cyclic strain on vascular SMC. Serine phosphorylation of STAT-3 is mediated by ERK1/2, while tyrosine phosphorylation is mediated by Src. A negative feedback loop was also found between p38 and ERK1/2. [Copyright &y& Elsevier]

Abstract: Hemodynamic forces exerted by blood flow (cyclic strain, shear stress) affect the initiation and progression of angiogenesis; however, the precise signaling mechanism(s) involved are unknown. In this study, we examine the role of cyclic strain in regulating bovine aortic endothelial cell (BAEC) migration and tube formation, indices of angiogenesis. Considering their well-documented mechanosensitivity, functional inter-dependence, and involvement in angiogenesis, we hypothesized roles for matrix metalloproteinases (MMP-2/9), RGD-dependent integrins, and urokinase plasminogen activator (uPA) in this process. BAECs were exposed to equibiaxial cyclic strain (5% strain, 1Hz for 24h) before their migration and tube formation was assessed by transwell migration and collagen gel tube formation assays, respectively. In response to strain, both migration and tube formation were increased by 1.83±0.1- and 1.84±0.1-fold, respectively. Pertussis toxin, a Gi-protein inhibitor, decreased strain-induced migration by 45.7±32% and tube formation by 69.8±13%, whilst protein tyrosine kinase (PTK) inhibition with genistein had no effect. siRNA-directed attenuation of endothelial MMP-9 (but not MMP-2) expression/activity decreased strain-induced migration and tube formation by 98.6±41% and 40.7±31%, respectively. Finally, integrin blockade with cRGD peptide and siRNA-directed attenuation of uPA expression reduced strain-induced tube formation by 85.7±15% and 84.7±31%, respectively, whilst having no effect on migration. Conclusions: Cyclic strain promotes BAEC migration and tube formation in a Gi-protein-dependent PTK-independent manner. Moreover, we demonstrate for the first time a putative role for MMP-9 in both strain-induced events, whilst RGD-dependent integrins and uPA appear only to be involved in strain-induced tube formation. [Copyright &y& Elsevier]

Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38 ± 6%), an inhibitory effect which could be completely reversed by targeted siRNA ‘knock-down’ of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 μM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration. [Copyright &y& Elsevier]

Hemodynamic forces, including shear stress and cyclic strain, have been recognized as important modulators of vascular cell morphology and function. The mechanism by which vascular cells sense and transduce the extracellular mechanical signals into the cell nucleus has only recently begun to come to light. Integrins, ion channels, platelet-derived growth factor receptors, and G proteins have been recognized as mechanosensors, converting the mechanical stimuli into chemical signals. Activation of second messengers, including mitogen-activated protein kinases, protein kinase C, and Akt, follows, leading to an increase in the activity of transcription factors such as activator protein (AP)-1, AP-2, cAMP-responsive element (CRE), early growth response (Egr)-1, and nuclear factor (NF)-n B. Binding of these factors to the DNA leads to activation of numerous genes that regulate cell proliferation, apoptosis, differentiation, morphology, migration, and secretory function. Understanding of these responses has provided new insights in the pathogenesis and treatment of vascular diseases, such as atherosclerosis and intimal hyperplasia. [ABSTRACT FROM AUTHOR]

The development of PSAs suitable for specific applications requires an optimization of the two most essential properties: sufficient bond strength and clean removability. The latter is conveniently obtained by controlling the mode of debonding to an interfacial failure, yet the issue of bond strength is challenging since the optimum bond strength must be available in the same failure mode. More complication arises from the fact that the methods, such as peel and creep tests, used to evaluate the bond strength are often qualitative and the results obtained may not accurately reveal the effects of various PSA design parameters. In the present study, a new method, named cyclic strain application (CSA) test, is devised to evaluate the durability of PSAs. A key feature of this test is that by applying a controlled cyclic strain on a PSA/substrate interface we can quantitatively monitor the temporal stress variation as well as the time for interfacial failure (t[sub f]). As a result, the effects of various PSA design parameters, such as molar mass, blending with low molar mass fractions, adhesion promoters, and the level of crosslinking on PSA durability can be clearly seen. Other factors being equal, higher molar mass samples showed greater t[sub f], while, blending with low molar mass fractions led to a reduced t[sub f]. The use of a strong adhesion promoter, as anticipated, increased t[sub f]. However, it should be noted that excessive bond strength may lead to a cohesive failure, which is detrimental in the design of good PSAs. From a comparison of t[sub f] with peel strength, it was clearly shown that t[sub f], the key parameter obtained from the CSA test, had a strong correlation with peel strength. In addition, some other information, such as the detailed influences of the level of crosslinking and molar mass on PSA durability was also obtained from the CSA test. Lastly, some important PSA design concepts and other examples utilizing the CSA test are presented. [ABSTRACT FROM AUTHOR]

Fluid shear stress exerts numerous effects on gene expression in endothelial cells. To investigate the regulatory mechanisms involved, we designed oligonucleotides composed of a 20-bp core containing the classical shear stress response element (SSRE+) or of a 20-bp core, in which base pairs flanking the SSRE were mutated (SSRE-). Hexamers of the oligonucleotides were cloned in front of reporter genes, transfected in bovine aortic endothelial cells (BAECs), and subjected to either a continuous low shear stress (3 dynes cm -2 ) or to a stepwise increase in shear stress from 3 dynes cm -2 to 12 dynes cm -2 (16/4 h). Shear stress increased reporter gene activity in cells transfected with pSSRE-, but not with pSSRE+. Cyclic strain (6%, 1 Hz, 4 h) did not significantly affect reporter gene activity. In gel retardation assays, more proteins bound to SSRE- than to SSRE+ in response to high shear stress. In competition experiments, a cAMP response element-binding (CREB) protein-specific oligonucleotide suppressed protein binding to the SSRE-; however, an antibody directed against CREB itself did not affect protein binding to the SSRE+. Our results indicate that the sequence ACC G / T AGACCAG represents a novel SSRE and that a protein that binds a CREB-specific oligonucleotide is part of the complex implicated in response to shear stress. [ABSTRACT FROM AUTHOR]

To determine the effect of cyclic strain amplitude and frequency on MMP-1 (interstitial collagenase) expression in tendon cells, rat tail tendons (RTT) were immobilized or cyclically displaced to various amplitudes (1, 3, or 6% strain at 0.017 Hz) or frequencies (1% strain at 0.017, 0.17, or 1.0 Hz) for 24 hr. Stress-deprivation for 24 hr resulted in a marked upregulation in MMP-1 expression. Cyclic tensile loading at 0.017 Hz was found to significantly inhibit, but not completely eliminate, MMP-1 expression at 1% strain. MMP-1 expression was completely eliminated at 3 and 6% strain. Increasing the frequency of application of the 1% strain to 0.17 or 1.0 Hz completely eliminated MMP-1 expression. Disruption of the actin cytoskeleton with cytochalasin D abolished all inhibitory effects of cyclic strain on MMP-1 expression. The results of our study demonstrate that MMP-1 expression in tendon cells can be modulated by varying amplitudes and frequencies of cyclic tensile strain, presumably through a cytoskeletally based mechanotransduction pathway. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) are exposed to hemodynamic forces such as shear stress (SS) and cyclic strain (CS) in vivo. Alterations in these forces may stimulate EC growth and intimal hyperplasia, possibly by promotion of cell survival through inhibition of apoptosis. The authors examined the effect of SS and CS on inhibition of apoptosis and phosphorylation of Akt and its downstream target Bad in bovine aortic ECs in vitro. Arterial levels of laminar SS (14 dyne/cm 2 ) or CS (10%) suppressed apoptosis due to serum withdrawal in EC; this suppression due to SS or CS was completely inhibited by phosphatidylinositol 3′-kinase (PI3K) inhibition. Phosphorylation of Akt in EC exposed to SS or CS was time dependent but with maximal stimulation at 30 min (SS) or 5 min (CS); SS- or CS-induced Akt phosphorylation was inhibited in the presence of PI3K inhibition. SS-induced, but not CS-induced, phosphorylation of Bad was inhibited by PI3K inhibition. These results suggest that hemodynamic forces suppress apoptosis in ECs via phosphorylation of Akt and that SS and CS differentially activate the downstream phosphorylation of Bad, possibly by stimulating an alternate pathway. This suggests an additional mechanism by which hemodynamic forces can differentially regulate transcription in ECs, and thereby possibly maintain the viability of normal endothelium. [ABSTRACT FROM AUTHOR]

Mechanical stimulation is critical for the maintenance of bone architecture and bone mass. These effects are dependent on the magnitude, duration, and rate of the mechanical stimuli. The goals of the present study were to develop and optimize a micromechanical-testing device for in vitro mechanical stimulation of whole viable bones, and to identify the physical parameters of loading that elicit maximal anabolic responses. The model was the 7–8-day-old neonatal CD-1 mouse tibia. A range of cyclic strain magnitudes [500–7000 microstrain (me)] and frequencies [0.2–30 hertz (Hz)] were applied to the neonatal bones. Incremental cyclic compression tests showed that the bones were nonlinearly viscoelastic. Bone stiffness and hysteresis energy dissipation were dependent on the maximum load magnitude. DNA and protein synthesis were significantly enhanced in bones that were cyclically loaded at 0.5 Hz/1000 me, 0.5 Hz/2000 me, or l Hz/1000 me, compared to nonloaded controls. Anabolic responses were maximal at a peak load of 100 mN at l Hz/1000 me. Autoradiography of the bones loaded under these conditions showed proliferation of cells at periosteal surfaces. Hysteresis energy per cycle was greatest at loads that caused the largest anabolic responses. The parameters of strain and load that elicit optimal effects on the neonatal bones are comparable to those in other systems, validating the use of the instrumentation for studying the mechanisms of the anabolic responses. The findings also suggest that hysteresis energy per cycle may be a determinant of the anabolic response of bones to mechanical stimulation. [ABSTRACT FROM AUTHOR]

We tested the hypothesis whether the number of applied load cycles and the frequency of uniaxial strain have an effect on proliferation of human bone derived osteoblast-like cells. A new approach was developed in order to differentiate between the effects of frequency and the effects of cycle number and strain duration. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along there longitudinal axes. Strain was applied over 2 days varying the number of applied load cycles (4–3600) at a constant frequency (1 Hz) or varying the frequency (0.1–30 Hz) at a constant number of applied cycles (1800) or at a constant strain duration (5 min). At a constant frequency, proliferative response increases (103%) with the number of applied cycles until a cycle number maximum (1800 cycles) was reached. 3600 cycles reduced cell number (43%) in contrast to the maximum. The variation of the frequency of applied strain tended to result in slight differences with regard to cell proliferation when cycle number was left constant. However, combined with an appropriate number of cycles there was an optimal frequency (1 Hz) as stimulus for bone cell proliferation (84%). A higher frequency (30 Hz) in combination with a high cycle number (9000) reduced cell number to control level (4%). This study demonstrates a frequency and cycle number dependent proliferative response of human osteoblast-like cells. It could be shown that effects of the frequency should not be considered separately from the effects of the cycle number. [Copyright &y& Elsevier]

Excessive mechanical load induces harmful outcomes for joint diseases, such as osteoarthritis and rheumatoid arthritis, but physical stimuli at appropriate intensity are essential for growth and maintenance of bone and articular cartilage. Using a fibroblast-like synoviocyte cell line derived from a patient with rheumatoid arthritis, we examined the effects of gentle cyclic strain, focusing on the expression and activity of matrix metalloproteinase-1 (MMP-1) and MMP-13. Synovial cells were cultured on a collagen-coated agar block and exposed to 2% cyclic strain at 6 rev./min for 1 h. Expression of MMP-1 and MMP-13 was assayed using semi-quantitative and real-time PCR, as well as immunoblotting. Their activity was measured using spectrofluorometry and zymography. The results showed that the cyclic strain reduced the mRNA and protein levels of MMP-1 and MMP-13, and that both collagenase and gelatinase activity was decreased under the strain. The reduction in MMP activity by the cyclic strain was not achieved by the transcriptional inhibitor, actinomycin D. In the presence of proinflammatory cytokines, such as IL-1β and TNF-α, the strain reduced the cytokine-induced expression and activities of MMPs. Interestingly, the strain elevated the mRNA level of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. These results support a potential role of mechanical strain in down-regulating the cytokine-mediated proteolytic processes in synoviocytes. [Copyright &y& Elsevier]

Tissue engineering offers the opportunity to develop vascular substitutes that mimic the responsive nature of native arteries. A good blood vessel substitute should be able to remodel its matrix in response to mechanical stimulation, as imposed by the hemodynamic environment. We have developed a novel method of studying the influence of mechanical strain on the remodeling of cell-seeded collagen gel blood vessel analogs. We assessed the remodeling capacity by examining the effect of mechanical conditioning upon the expression of enzymes which remodel the extracellular matrix, called matrix metalloproteinases (MMPs), and upon the mechanical properties of the constructs. We found that subjecting collagen constructs to a 10% cyclic radial distention, over a course of 4 days, resulted in an overall increase in the production of MMP-2. Cyclic mechanical strain also stimulated enzymatic activation of latent MMP-2. We found that cyclic strain also significantly increased the mechanical strength and material modulus, as indicated by an increase in circumferential tensile properties of the constructs. These observations suggested that MMP-2-dependent remodeling affects the material properties of vascular tissue analogs. To further investigate this possible connection we examined the effects of dynamic conditioning in the presence of two nonspecific inhibitors of MMP activity. Interestingly, we found that nonspecific inhibition of MMP ablated the benefits of mechanical conditioning upon mechanical properties. Our observations suggest that a better understanding of the complex relation between mechanical stimulation and construct remodeling is key for the proper design of tissue-engineered blood vessel substitutes. © 2001 Biomedical Engineering Society. PAC01: 8719Rr, 8714Ee, 8717-d [ABSTRACT FROM AUTHOR]

One of the lessons to be learned from the tragic Gujrat earthquakes is the importance of damage control of our civil structures. Many structures that escaped total collapse still sustained significant structural and non-structural damages, causing a threat to the occupants and necessitating costly repairs. The need for more positive control of damage has been raised, and improved performance of reinforcing bars by using continuous casting and thermo-mechanically steel is shown to provide a potential solution. [ABSTRACT FROM AUTHOR]

The effects of cyclic mechanical strain and challenge with physiologically relevant doses of submicrometre-size polyethylene (PE) particles on the osteolytic potential of primary human mononuclear phagocytes were investigated. Cells were seeded into a three-dimensional tissue matrix and co-cultured with particles (mean size 0.21 microm) at particle volume to cell number ratios of 7.5, 15, 30 and 100 microm3/cell. Matrices were then either cultured statically or subjected to 20 per cent cyclic compressional strain in the 'ComCell' for 16 h prior to the assessment of cell viability and quantification of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazdium bromide) assay was shown to be too insensitive to detect changes in cell viability. However, when quantified by the adenosine triphosphate (ATP) assay, cell viability was demonstrated to be reduced following exposure to cyclic strain. Macrophages cultured in the static three-dimensional tissue equivalent model produced very high levels of TNFalpha in response to submicrometre PE particles at a ratio of 100 microm3/cell. Cyclic strain in the absence of particles gave only a small increase in TNFa production. However, the combined effects of strain and particle stimulation at a ratio of 30 microm3/cell resulted in the secretion of significantly more TNFalpha than was produced by macrophages subjected to strain alone, or the cells-only control. This synergy between cyclic strain and PE particle stimulation was only evident when the volume of particles was reduced below the volume that maximally stimulated cells. These results suggest that while cyclic strain may not be the primary factor responsible for macrophage activation and periprosthetic osteolysis, at low particle load, it may contribute significantly to the osteolytic potential of macrophages in vitro or in vivo. [ABSTRACT FROM AUTHOR]

Dynamic mechanical conditioning is investigated as a means of improving the mechanical properties of tissue-engineered blood vessel constructs composed of living cells embedded in a collagen-gel scaffold. This approach attempts to elicit a unique response from the embedded cells so as to reorganize their surrounding matrix, thus improving the overall mechanical stability of the constructs. Mechanical conditioning, in the form of cyclic strain, was applied to the tubular constructs at a frequency of 1 Hz for 4 and 8 days. The response to conditioning thus evinced involved increased contraction and mechanical strength, as compared to statically cultured controls. Significant increases in ultimate stress and material modulus were seen over an 8 day culture period. Accompanying morphological changes showed increased circumferential orientation in response to the cyclic stimulus. We conclude that dynamic mechanical conditioning during tissue culture leads to an improvement in the properties of tissue-engineered blood vessel constructs in terms of mechanical strength and histological organization. This concept, in conjunction with a proper biochemical environment, could present a better model for engineering vascular constructs. © 2000 Biomedical Engineering Society. PAC00: 8719Rr, 8714Ee, 8718-h, 8768+z [ABSTRACT FROM AUTHOR]

Cyclic strain generated at the cell-material interface is critical for the engraftment of biomaterials. Mechanosensitive immune cells, macrophages regulate the host-material interaction immediately after implantation by priming the environment and remodeling ongoing regenerative processes. This study investigated the ability of mechanically active scaffolds to modulate macrophage function in vitro and in vivo . Remotely actuated magnetic scaffolds enhance the phenotype of murine classically activated (M1) macrophages, as shown by the increased expression of the M1 cell-surface marker CD86 and increased secretion of multiple M1 cytokines. When scaffolds were implanted subcutaneously into mice and treated with magnetic stimulation for 3 days beginning at either day 0 or day 5 post-implantation, the cellular infiltrate was enriched for host macrophages. Macrophage expression of the M1 marker CD86 was increased, with downstream effects on vascularization and the foreign body response. Such effects were not observed when the magnetic treatment was applied at later time points after implantation (days 12-15). These results advance our understanding of how remotely controlled mechanical cues, namely, cyclic strain, impact macrophage function and demonstrate the feasibility of using mechanically active nanomaterials to modulate the host response in vivo .