Micro tensile tester measurement of biomechanical properties and adhesion force of microtubule-polymerization-inhibited cancer cells.

Both mechanical and adhesion properties of cancer cells are complex and reciprocally related to migration, invasion, and metastasis with large cell deformation. Therefore, we evaluated these properties for human cervical cancer cells (HeLa) simultaneously using our previously developed micro tensile tester system. For efficient evaluation, we developed image analysis software to modify the system. The software can analyze the tensile force in real time. The modified system can evaluate the tensile stiffness of cells to which a large deformation is applied, also evaluate the adhesion strength of cancer cells that adhered to a culture substrate and were cultured for several days with their adhesion maturation. We used the modified system to simultaneously evaluate the stiffness of the cancer cells to which a large deformation was applied and their adhesion strength. The obtained results revealed that the middle phase of tensile stiffness and adhesion force of the microtubule-depolymerized group treated with colchicine (an anti-cancer drug) (stiffness, 13.4 ± 7.5 nN/%; adhesion force, 460.6 ± 258.2 nN) were over two times larger than those of the control group (stiffness, 5.0 ± 3.5 nN/%; adhesion force, 168.2 ± 98.0 nN). Additionally, the same trend was confirmed with the detailed evaluation of cell surface stiffness using an atomic force microscope. Confocal fluorescence microscope observations showed that the stress fibers (SFs) of colchicine-treated cells were aligned in the same direction, and focal adhesions (FAs) of the cells developed around both ends of the SFs and aligned parallel to the developed direction of the SFs. There was a possibility that the microtubule depolymerization by the colchicine treatment induced the development of SFs and FAs and subsequently caused an increment of cell stiffness and adhesion force. From the above results, we concluded the modified system would be applicable to cancer detection and anti-cancer drug efficacy tests.

Actin crosslinking by α-actinin averts viscous dissipation of myosin force transmission in stress fibers.

Contractile force generated in actomyosin stress fibers (SFs) is transmitted along SFs to the extracellular matrix (ECM), which contributes to cell migration and sensing of ECM rigidity. In this study, we show that efficient force transmission along SFs relies on actin crosslinking by α-actinin. Upon reduction of α-actinin-mediated crosslinks, the myosin II activity induced flows of actin filaments and myosin II along SFs, leading to a decrease in traction force exertion to ECM. The fluidized SFs maintained their cable integrity probably through enhanced actin polymerization throughout SFs. A computational modeling analysis suggested that lowering the density of actin crosslinks caused viscous slippage of actin filaments in SFs and, thereby, dissipated myosin-generated force transmitting along SFs. As a cellular scale outcome, α-actinin depletion attenuated the ECM-rigidity-dependent difference in cell migration speed, which suggested that α-actinin-modulated SF mechanics is involved in the cellular response to ECM rigidity.

Partial endothelial-to-mesenchymal transition mediated by HIF-induced CD45 in neointima formation upon carotid artery ligation.

AIMS: Endothelial-to-mesenchymal transition (EndMT) is a fundamental process in vascular remodelling. However, the precise regulatory mechanism of vascular remodelling during neointima formation and the source of neointima cells are not entirely understood. METHODS AND RESULTS: To investigate the origin of neointima cells and their relevance to vascular wall remodelling, we used an endothelial cell (EC)-specific lineage tracing system [VE-Cadherin (Cdh5)-BAC-CreERT2 mice] and carotid artery ligation model and showed evidence that resident ECs transdifferentiate into neointima cells with the expression of CD45. During the early stages of neointima formation, ECs transiently expressed CD45, a haematopoietic marker, accompanied by a host of EndMT markers, and CD31 and αSMA were prominently expressed in developing neointima. In vitro, CD45-positive EndMT was induced by stabilization of HIF1α with cobalt chloride or with a VHL inhibitor in human primary ECs, which mimicked the hypoxic condition of the ligated artery, and promoted the formation of an integrin α11-shank-associated RH domain-interacting protein (SHARPIN) complex. Notably, a CD45 phosphatase inhibitor disrupted this integrin α11-SHARPIN complex, thereby destabilizing cell-cell junctions. Deletion of Hif1α in ECs suppressed expression of CD45 and EndMT markers and ameliorated neointima formation. CONCLUSION: These results suggest that the HIF-induced CD45 expression is normally required for the retention of an EC fate and cell-cell junctions, CD45-positive EndMT (termed as 'partial EndMT') contributes to neointima formation and vascular wall remodelling.

Changes in the intra- and extra-mechanical environment of the nucleus in Saos-2 osteoblastic cells during bone differentiation process: Nuclear shrinkage and stiffening in cell differentiation.

Osteogenic differentiation has been reportedly regulated by various mechanical stresses, including fluid shear stress and tensile and compressive loading. The promotion of osteoblastic differentiation by these mechanical stresses is accompanied by reorganization of the F-actin cytoskeleton, which is deeply involved in intracellular forces and the mechanical environment. However, there is limited information about the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be directly involved in various cellular functions. Here, we investigated the changes in the intracellular force applied to the nucleus and the effect on nuclear morphology and mechanical properties during osteogenic differentiation in human osteoblast-like cells (Saos-2). We carried out cell morphological analyses with confocal fluorescence microscopy, nuclear indentation test with atomic force microscopy (AFM), and fluorescence recovery after photobleaching (FRAP) for intranuclear DNA. The results revealed that a significant reorganization of the F-actin cytoskeleton from the nuclear surfaces to the cell periphery occurred in the osteogenic differentiation processes, simultaneously with the reduction of compressive forces to the nucleus. Such changes also facilitated nuclear shrinkage and stiffening, and further intranuclear chromatin compaction. The results indicate that the reduction of the intracellular compressive force due to reorganization of the F-actin cytoskeleton affects the intra- and extra-mechanical environment of the nucleus, and this change may affect gene expression and DNA replication in the osteogenic differentiation process.

Polarized light retardation analysis allows for the evaluation of tension in individual stress fibers.

The tension in the stress fibers (SFs) of cells plays a pivotal role in determining biological processes such as cell migration, morphological formation, and protein synthesis. Our previous research developed a method to evaluate the cellular contraction force generated in SFs based on photoelasticity-associated retardation of polarized light; however, we employed live cells, which could have caused an increase in retardation and not contraction force. Therefore, the present study aimed to confirm that polarized light retardation increases inherently due to contraction, regardless of cell activity. We also explored the reason why retardation increased with SF contractions. We used SFs physically isolated from vascular smooth muscle cells to stop cell activity. The retardation of SFs was measured after ATP administration, responsible for contracting SFs. The SFs were imaged under optical and electron microscopes to measure SF length, width, and retardation. The retardation of isolated SFs after ATP administration was significantly higher than before. Thus, we confirmed that retardation increased with elevated tension in individual SFs. Furthermore, the SF diameter decreased while the SF length remained almost constant. Thus, we conclude that a contraction force-driven increase in the density of SFs is the main factor for the rise in polarized light retardation.

C9orf72-Derived Proline:Arginine Poly-Dipeptides Modulate Cytoskeleton and Mechanical Stress Response.

Proline:arginine (PR) poly-dipeptides from the GGGGCC repeat expansion in C9orf72 have cytotoxicity and bind intermediate filaments (IFs). However, it remains unknown how PR poly-dipeptides affect cytoskeletal organization and focal adhesion (FA) formation. Here, we show that changes to the cytoskeleton and FA by PR poly-dipeptides result in the alteration of cell stiffness and mechanical stress response. PR poly-dipeptides increased the junctions and branches of the IF network and increased cell stiffness. They also changed the distribution of actin filaments and increased the size of FA and intracellular calcium concentration. PR poly-dipeptides or an inhibitor of IF organization prevented cell detachment. Furthermore, PR poly-dipeptides induced upregulation of mechanical stress response factors and led to a maladaptive response to cyclic stretch. These results suggest that the effects of PR poly-dipeptides on mechanical properties and mechanical stress response may serve as a pathogenesis of C9orf72-related neurodegeneration.

Cell type-specific orientation and migration responses for a microgrooved surface with shallow grooves.

BACKGROUND: Directional cell migration due to mechanosensing for in vivo microenvironment, such as microgrooved surfaces, is an essential process in tissue growth and repair in both normal and pathological states. Cell migration responses on the microgrooved surfaces might be reflected by the cell type difference, which is deeply involved in cellular physiological functions. Although the responses are implicated in focal adhesions (FAs) of cells, limited information is available about cell migration behavior on the microgrooved surfaces whose dimensions are comparable with the size of FAs. OBJECTIVE: In the present study, we investigated the cell orientation and migration behavior of normal vascular smooth muscle cells (VSMCs) and cervical cancer HeLa cells on the microgrooved surface. METHOD: The cells were cultured on the PDMS substrate comprising shallow grooves with 2-µm width and approximately 150-nm depth, which indicates the same order of magnitude as that of the horizontal and vertical size of FAs, respectively. The cell migration and intracellular structures were analyzed by live cell imaging and confocal fluorescence microscopy. The intracellular tension was also assessed using atomic force microscopy (AFM). RESULTS: VSMCs presenting well-aligned actin stress fibers with mature FAs revealed marked cell elongation and directional migration on the grooves; however, HeLa cells with nonoriented F-actin with smaller FAs did not. The internal force of the actin fibers was significantly higher in VSMCs than that in HeLa cells, and the increase or decrease in the cytoskeletal forces improved or diminished the sensing ability for shallow grooves, respectively. The results strongly indicated that directional cell migration should be modulated by cell type-specific cytoskeletal arrangements and intracellular traction forces. The differences in cell type-specific orientation and migration responses can be emphasized on the microgrooves as large as the horizontal and vertical size of FAs. CONCLUSION: The microgoove structure in the size range of the FA protein complex is a powerful tool to clarify subtle differences in the intracellular force-dependent substrate mechanosensing.

A Loss of Nuclear-Cytoskeletal Interactions in Vascular Smooth Muscle Cell Differentiation Induced by a Micro-Grooved Collagen Substrate Enabling the Modeling of an In Vivo Cell Arrangement.

Vascular smooth muscle cells (VSMCs) remodel vascular walls actively owing to mechanical cues and dedifferentiate to the synthetic phenotype from contractile phenotype in pathological conditions. It is crucial to clarify the mechanisms behind the VSMC phenotypic transition for elucidating their role in the vascular adaptation and repair and for designing engineered tissues. We recently developed novel micro-grooved collagen substrates with "wavy wrinkle" grooves to induce cell-substrate adhesion, morphological polarization, and a tissue-like cell arrangement with cytoskeletal rearrangements similar to those in vascular tissue in vivo. We found that cultivation with this micro-grooved collagen significantly induced VSMC contractile differentiation. Nonetheless, the detailed mechanism underlying the promotion of such VSMC differentiation by micro-grooved collagen has not been clarified yet. Here, we investigated the detailed mechanism of the cell arrangement into a tissue and contractile-differentiation improvement by our micro-grooved collagen substrates in terms of nuclear-cytoskeletal interactions that possibly affect the nuclear mechanotransduction involved in the activation of transcription factors. We found that VSMCs on micro-grooved collagen manifested significant cell arrangement into a tissue and nucleus slimming with a volume reduction in response to the remodeling of the actin cytoskeleton, with consequent inhibition of nuclear shuttling of a transcriptional coactivator, Yes-associated protein (YAP), and improved contractile differentiation. Furthermore, VSMC nuclei rarely deformed during macroscopic cell stretching and featured a loss of nesprin-1-mediated nuclear-cytoskeletal interactions. These results indicate that our micro-grooved collagen induces a cell alignment mimicking in vivo VSMC tissue and promotes contractile differentiation. In such processes of contractile differentiation, mechanical interaction between the nucleus and actin cytoskeleton may diminish to prevent a nuclear disturbance from the excess mechanical stress that might be essential for maintaining vascular functions.

Macroscopic and microscopic analysis of the mechanical properties and adhesion force of cells using a single cell tensile test and atomic force microscopy: Remarkable differences in cell types.

Many experimental techniques have been reported to provide knowledge of the mechanical behavior of cells from biomechanical viewpoints, however, it is unclear how the intercellular structural differences influence macroscopic and microscopic mechanical properties of cells. The aim of our study is to clarify the comprehensive mechanical properties and cell-substrate adhesion strength of cells, and the correlation with intracellular structure in different cell types. We developed an originally designed micro tensile tester, and performed a single cell tensile test to estimate whole cell tensile stiffness and adhesion strength of normal vascular smooth muscle cells (VSMCs) and cervical cancer HeLa cells: one half side of the specimen cell was lifted up by a glass microneedle, then stretched until the cell detached from the substrate, while force was simultaneously measured. The tensile stiffness and adhesion strength were 49 ± 10 nN/% and 870 ± 430 nN, respectively, in VSMCs (mean ± SD, n = 8), and 19 ± 17 nN/% and 320 ± 160 nN, respectively, in HeLa cells (n = 9). The difference was more definite in the surface elastic modulus map obtained by atomic force microscopy, indicating that the internal tension of the actin cytoskeleton was significantly higher in VSMCs than in HeLa cells. Structural analysis with confocal microscopy revealed that VSMCs had a significant alignment of F-actin cytoskeleton with mature focal adhesion, contrary to the randomly oriented F-actin with smaller focal adhesion of HeLa cells, indicating that structural arrangement of the actin cytoskeleton and their mechanical tension generated the differences in cell mechanical properties and adhesion forces. The finding strongly suggests that the mechanical and structural differences in each cell type are deeply involved with their physiological functions.

Moderate substrate stiffness induces vascular smooth muscle cell differentiation through cellular morphological and tensional changes.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are one of the main components of arterial walls and actively remodel the arterial walls in which they reside through biomechanical signals applied to themselves. Contractile or differentiated VSMCs have been observed in normal blood vessels. In pathological vascular conditions, they become dedifferentiated from contractile to non-contractile or synthetic cells, and a similar change is observed when VSMCs are placed in culture conditions. The mechanisms regulating VSMC differentiation remain unclear at this stage. OBJECTIVE: In this paper we investigated the effects of substrate stiffness on the morphology, intercellular tension, and differentiation of VSMCs. METHODS: Rat VSMCs were cultured on polyacrylamide (PA) gels, with elastic moduli of 15 kPa, 40 kPa, and 85 kPa, and PDMS substrate with elastic modulus of 1 MPa, and their morphology, intercellular tension, and contractile differentiation were assessed. RESULTS: Using fluorescence microscope image-based analysis and nano-indentation imaging with atomic force microscopy, we found that cell spreading and stiffening were induced by substrate stiffening in VSMCs. Interestingly, VSMCs on PA gel substrates with medium stiffness (40 kPa) showed significant elongation and shape polarization, and their 𝛼-SMA with F-actin cytoskeleton expression ratio was significantly higher than those of cells on other substrates. CONCLUSION: The results indicate an existing optimal substrate stiffness for promoting VSMC differentiation, and also indicate that cell shape polarization might be a key factor for VSMC differentiation.

Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling.

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix-cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvß1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin ß1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.

Cyclic stretch-induced mechanical stress to the cell nucleus inhibits ultraviolet radiation-induced DNA damage.

Ultraviolet (UV) radiation exerts adverse effects on genome stability, alters the normal state of life, and causes several diseases by inducing DNA damage. Although mechanical stimulation such as stretching has significant effects on the prevention and treatment of diseases, its influence on nuclear morphology and/or intranuclear functions involving resistance to DNA damage remains unknown. Here, we investigated the effects of mechanical stimulation by cyclic stretching on nuclear morphology and resistance of DNA to UV damage in NIH3T3 fibroblasts. Adherent cells on silicone elastic membranes were subjected to ~ 10% cyclic uniaxial stretch at a frequency of 0.5 Hz for 12 h. As a result, the intracellular actin cytoskeleton and nucleus were found to be elongated and aligned in the direction of zero normal strain (~ 62° with respect to the stretch direction) in an actomyosin tension-dependent manner. The nuclei of the stretched cells were dramatically compressed by the reorganized actin stress fibers located on their apical and both sides, and a significant increase in the intranuclear DNA density was observed. Intercellular tension, as assessed with live cell atomic force microscopy imaging, also increased following exposure to cyclic stretch. The UV radiation-induced DNA damage, estimated from the fluorescence intensity of the phospho-histone γ-H2AX, significantly decreased in these stretched cells. These results indicate that the cyclic stretch-induced morphological changes in the nucleus may improve the UV radiation resistance of cells, probably owing to the intracellular force-induced condensation of chromatin. To our knowledge, this is the first study to demonstrate the inhibition of the UV radiation-induced DNA damage by mechanical stimulation.

Shape-dependent regulation of differentiation lineages of bone marrow-derived cells under cyclic stretch.

Multipotent stem cells are considered as a key material in regenerative medicine, and the understanding of the heterogeneity in the differentiation potentials of bone marrow-derived cells is important in the successful regenerative tissue repair. Therefore, the present study has been performed to investigate how the differentiation of post-harvest, native bone marrow-derived cells is regulated by cyclic stretch in vitro. Bone marrow-derived cells were obtained from mouse femur of both hind limbs and categorized into the following five categories: amebocytes, round cells, spindle cells, stellate cells and others. The cells were seeded on a silicone-made stretch chamber, and subjected to cyclic stretch with an amplitude of 10% at a frequency of 1 Hz for 7 days for cell shape analysis and for 3 days for the analysis of the expression of marker proteins of osteogenic (osteocalcin), vascular smooth muscle (α-smooth muscle actin and smooth muscle myosin heavy chain) and neurogenic (neurofilament) differentiation. When disregarding the differences in the cell shapes, there was an overall trend that the application of 10% cyclic stretch inhibited osteogenic and neurogenic differentiation, but enhanced smooth muscle differentiation. Close examinations revealed that round cells were influenced the most by cyclic stretch (significant up- or down-regulation in all the four marker protein expressions) while amebocytes and spindle cells were only influenced by cyclic stretch for vascular smooth muscle and/or neurogenic differentiation. As far as the authors know, this is the first study reporting the shape-related differences in the fate decision criteria for mechanical strain in bone marrow-derived cells.

A novel micro-grooved collagen substrate for inducing vascular smooth muscle differentiation through cell tissue arrangement and nucleus remodeling.

Vascular smooth muscle cells (SMCs) actively remodel arterial walls through biomechanical signals and dedifferentiate from the contractile to the synthetic state under pathological conditions. It is important to determine the differentiation mechanism of SMCs to understand their pathophysiology in disease. Previously, we found that the F-actin cytoskeleton in dedifferentiated SMCs on dishes was firmly connected to the nucleus, and that internal mechanical signals in SMCs are transmitted directly to the nucleus, indicating that nuclear-cytoskeletal interactions could be associated with SMC differentiation. However, mechanical environments in vivo are quite different from those of cultured cells: SMCs in vivo show an elongated shape and form a tissue that aligns with the circumferential direction of the walls. Thus, in the present study, we established a simple technique to fabricate a novel micro-grooved native collagen substrate that mimics the elongated cell shapes and alignment observed in vivo. The substrates had "wavy wrinkle" grooves with a width of ~5 µm and a Young's modulus of ~500 kPa, which were quite similar to those of the elastic lamina in vascular tissues. Using confocal microscopy image-based analysis, and nano-indentation imaging with atomic force microscopy, we found that SMCs on the micro-grooved collagen formed significant cell tissue arrangement, and changed their nuclear morphology to a "slim ellipsoid" in response to the force-reduction caused by F-actin remodeling, which consequently improved SMC differentiation. These findings indicated that this type of intracellular force-reduction around the nucleus has a crucial effect on SMC differentiation. Our micro-grooved collagen substrate is a powerful tool to investigate the mechanism of vascular SMC mechanotransduction.

Direct application of mechanical stimulation to cell adhesion sites using a novel magnetic-driven micropillar substrate.

Cells change the traction forces generated at their adhesion sites, and these forces play essential roles in regulating various cellular functions. Here, we developed a novel magnetic-driven micropillar array PDMS substrate that can be used for the mechanical stimulation to cellular adhesion sites and for the measurement of associated cellular traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 µm, respectively. Sufficient quantities of iron particles were successfully embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field. We established two methods to apply magnetic fields to the micropillars (Suresh 2007). Applying a uniform magnetic field of 0.3 T bent all of the pillars by ~4 µm (Satcher et al. 1997). Creating a magnetic field gradient in the vicinity of the substrate generated a well-defined local force on the pillars. Deflection of the micropillars allowed transfer of external forces to the actin cytoskeleton through adhesion sites formed on the pillar top. Using the magnetic field gradient method, we measured the traction force changes in cultured vascular smooth muscle cells (SMCs) after local cyclic stretch stimulation at one edge of the cells. We found that the responses of SMCs were quite different from cell to cell, and elongated cells with larger pre-tension exhibited significant retraction following stretch stimulation. Our magnetic-driven micropillar substrate should be useful in investigating cellular mechanotransduction mechanisms.

Ex-vivo observation of calcification process in chick tibia slice: Augmented calcification along collagen fiber orientation in specimens subjected to static stretch.

Bone formation through matrix synthesis and calcification in response to mechanical loading is an essential process of the maturation in immature animals, although how mechanical loading applied to the tissue increases the calcification and improves mechanical properties, and which directions the calcification progresses within the tissue are largely unknown. To address these issues, we investigated the calcification of immature chick bone under static tensile stretch using a newly developed real-time observation bioreactor system. Bone slices perpendicular to the longitudinal axis obtained from the tibia in 2- to 4-day-old chick legs were cultured in the system mounted on a microscope, and their calcification was observed up to 24 h while they were stretched in the direction parallel to the slice. Increase in the calcified area, traveling distance and the direction of the calcification and collagen fiber orientation in the newly calcified region were analyzed. There was a significant increase in calcified area in the bone explant subjected to tensile strain over ∼3%, which corresponds to the threshold strain for collagen fibers showing alignment in the direction of stretch, indicating that the fiber alignment may enhance tissue calcification. The calcification progressed to a greater distance to the stretching direction in the presence of the loading. Moreover, collagen fiber orientation in the calcified area in the loaded samples was coincided with the progression angle of the calcification. These results clearly show that the application of static tensile strain enhanced tissue calcification, which progresses along collagen fibers aligned to the loading direction.

A Finite Element Bendo-Tensegrity Model of Eukaryotic Cell.

Mechanical interaction of cell with extracellular environment affects its function. The mechanisms by which mechanical stimuli are sensed and transduced into biochemical responses are still not well understood. Considering this, two finite element (FE) bendo-tensegrity models of a cell in different states are proposed with the aim to characterize cell deformation under different mechanical loading conditions: a suspended cell model elucidating the global response of cell in tensile test simulation and an adherent cell model explicating its local response in atomic force microscopy (AFM) indentation simulation. The force-elongation curve obtained from tensile test simulation lies within the range of experimentally obtained characteristics of smooth muscle cells (SMCs) and illustrates a nonlinear increase in reaction force with cell stretching. The force-indentation curves obtained from indentation simulations lie within the range of experimentally obtained curves of embryonic stem cells (ESCs) and exhibit the influence of indentation site on the overall reaction force of cell. Simulation results have demonstrated that actin filaments (AFs) and microtubules (MTs) play a crucial role in the cell stiffness during stretching, whereas actin cortex (AC) along with actin bundles (ABs) and MTs are essential for the cell rigidity during indentation. The proposed models quantify the mechanical contribution of individual cytoskeletal components to cell mechanics and the deformation of nucleus under different mechanical loading conditions. These results can aid in better understanding of structure-function relationships in living cells.

Analysis of the effect of the size of three-dimensional micro-geometric structures on physical adhesion phenomena using microprint technique.

Thrombus formation on biomaterial surfaces with microstructures is complex and not fully understood. We have studied the micro-secondary flow around microstructures that causes components of blood to adhere physically in a low Reynolds number region. The purpose of this study was to investigate the effect of micro-column size on the adhesion phenomena and show a quantitative relationship between the micro-secondary flow and physical adhesion phenomena, considering microstructures of various sizes. The flow simulation and quantitative assessment of adhesion rates around micro-columns was conducted using four sizes of micro-columns. This study also calculated the vectors of micro-secondary flow and average shear rate around a micro-column using a computational fluid dynamics analysis. The simulation showed the micro-secondary flow toward the bottom surface at upstream side and low shear rate distribution generated around a micro-column. Furthermore, physical adhesion tests were conducted using microbeads and a perfusion circuit to examine the size effect of the micro-columns on the physical adhesion. The results showed that the average adhesion rate around the micro-column increases with the associated size increase of the micro-column. Our results indicate that quantification of micro-secondary flow on a material surface with microstructures of several sizes and shapes (such as in a rough surface) is important for the evaluation of the adhesion phenomenon even though the surface roughness value on the material surface is small.

A novel patterned magnetic micropillar array substrate for analysis of cellular mechanical responses.

Traction forces generated at cellular focal adhesions (FAs) play an essential role in regulating various cellular functions. These forces (1-100 nN) can be measured by observing the local displacement of a flexible substrate upon which cells have been plated. Approaches employing this method include using microfabricated arrays of poly(dimethylsiloxane) (PDMS) micropillars that bend by cellular traction forces. A tool capable of applying a force to FAs independently, by actively moving the micropillars, should become a powerful tool to delineate the cellular mechanotransduction mechanisms. Here, we developed a patterned magnetic micropillar array PDMS substrate that can be used for the mechanical stimulation of cellular FAs and the measurement of associated traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 µm, respectively. Iron particles were embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field, which also controlled their location on the substrate. Applying a magnetic field of 0.3 T bent the pillars by ∼4 µm and allowed transfer of external forces to the actin cytoskeleton through FAs formed on the pillar top. Using this approach, we investigated the traction force changes in cultured aortic smooth muscle cells (SMCs) after local compressive stimuli to release cell pretension. The mechanical responses of SMCs were roughly classified into two types: almost a half of the cells showed a little decrease of traction force at each pillar following compressive stimulation, although cell area increased significantly; and the rest showed the opposite, with increased forces and a simultaneous decrease in area. The traction forces of SMCs fluctuated markedly during the local compression. The root mean square of traction forces significantly increased during the compression, and returned to the baseline level after its release. These results suggest that the fluctuation of forces may be caused by active reorganization of the actin cytoskeleton and/or its dynamic interaction with myosin molecules. Thus, our magnetic micropillar substrate would be useful in investigating the mechanotransduction mechanisms of cells.

Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.

Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3-4 cycles/min, in the presence of ß-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to the loading direction. The ex vivo loading system established here can be further applied to study responses to mechanical loading in osteogenesis as well as callus maturation for better understanding of factors to consider in successful bone regeneration with mechanical factors.