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3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation.
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Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.
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Células Epiteliales Alveolares , Fibrosis Pulmonar , Humanos , Células Epiteliales Alveolares/metabolismo , Pulmón/metabolismo , Alveolos Pulmonares , Fibrosis Pulmonar/metabolismo , Células Epiteliales/metabolismo , Geles/metabolismoRESUMEN
Tendon exhibits the capacity to be stretched and to return to its original length without suffering structural damage in vivo, a capacity known as elastic recoil. Collagen fibres are aligned longitudinally and elastin fibres mostly run parallel to collagen fibres in tendon. However, their interactions and contributions to tendon elastic behaviours are not well understood. The present study examined functional roles of collagen and elastin in tendon elastic behaviours using a variety of mechanical tests. We prepared three types of fascicle specimens from mouse tail tendon: fascicles freshly isolated, those digested with elastase in PBS to selectively remove elastin, and those incubated in PBS without elastase. A quasi-static tensile test demonstrated that elastase-treated fascicles had higher tangent moduli and strength compared to fresh and PBS fascicles. Cyclic stretching tests showed that fresh and PBS fascicles could withstand cyclic strain at both small and large amplitudes, but elastase-treated fascicles could only behave elastically to a limited degree. Fibre-sliding analysis revealed that fresh fascicles could be elongated both through stretching of collagen fibers and through movement of the fibres. However, elastase-treated fascicles could be stretched only via fibre stretching. This evidence suggests that normal tendons can be extended through both fibre stretching and fibre sliding, whereas tendons without elastin can only extend as much as collagen fibers can withstand. Accordingly, collagen fibres mainly contribute to tendon elastic behaviours by furnishing rigidity and elasticity, whereas elastin provides tendon viscoelasticity and also enables sliding of collagen fibres during elastic behaviours. STATEMENT OF SIGNIFICANCE: The present study revealed distinct mechanical functions of collagen and elastin fibres in elastic behaviours of mouse tail tendon fascicle using a variety of mechanical tests at both microscopic and macroscopic levels. It was demonstrated that collagen mainly governs tendon fascicle rigidity and elasticity, but only possesses limited extensibility, whereas elastin contributes to viscoelasticity and collagen fibre sliding, enabling elastic recoil behaviour against relatively large deformation. By their interactions, tendon can be elongated without suffering major structural damage and withstand a large magnitude of tensile force in response to mechanical loading. Such information should be particularly useful in designing collagen-based biomaterials such as artificial tendons, in that previous studies have merely considered collagen without incorporation of elastin.
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Colágeno , Elastina , Ratones , Animales , Colágeno/metabolismo , Elastina/metabolismo , Matriz Extracelular/metabolismo , Elastasa Pancreática/análisis , Elastasa Pancreática/metabolismo , Tendones/fisiología , Estrés MecánicoRESUMEN
Adult mammals are known for their poor ability to regenerate tissues, including tendons. On the other hand, urodeles have become an important model in regenerative studies for their remarkable ability to regenerate various body parts and organs throughout life, such as limbs, retinas, or even the brain. However, little is known about their capacity to regenerate injured tendons. If newts can also repair tendons without scar formation, they may be a suitable animal model for tendon regeneration studies in other adult vertebrates. Therefore, the present study used Iberian ribbed newts to characterize mechanical and structural regeneration of tendons following transection, using tensile tests and multiphoton microscopy. A digital flexor tendon in a hindlimb was transected either partially or completely, and regenerated tendon was examined 6 and 12 weeks after the operation. Tensile strength of regenerated tendons was significantly less than normal at 6 weeks, but was remarkably recovered at 12 weeks, reaching levels comparable to those of uninjured tendons. On the other hand, mouse tendons demonstrated poor recovery of strength even after 12 weeks. Multiphoton microscopy revealed that tendon-like collagenous tissue bridges residual tendon stubs in newts, but disorganized scar-like tissue filled the injured location in mice. These findings highlight the remarkable capacity of newts to recover from tendon injury and confirm the utility of newts as a model to study tendon regeneration.
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Cicatriz , Tendones , Animales , Ratones , Cicatriz/patología , Tendones/patología , Regeneración , Modelos Animales de Enfermedad , Salamandridae , Fenómenos Biomecánicos , MamíferosRESUMEN
FRET-based sensors are utilized for real-time measurements of cellular tension. However, transfection of the sensor gene shows low efficacy and is only effective for a short period. Reporter mice expressing such sensors have been developed, but sensor fluorescence has not been measured successfully using conventional confocal microscopy. Therefore, methods for spatiotemporal measurement of cellular tension in vivo or ex vivo are still limited. We established a reporter mouse line expressing FRET-based actinin tension sensors consisting of EGFP as the donor and mCherry as the acceptor and whose FRET ratio change is observable with confocal microscopy. Tension-induced changes in FRET signals were monitored in the aorta and tail tendon fascicles, as well as aortic smooth muscle cells isolated from these mice. The pattern of FRET changes was distinctive, depending on tissue type. Indeed, aortic smooth muscle cells exhibit different sensitivity to macroscopic tensile strain in situ and in an isolated state. This mouse strain will enable novel types of biomechanical investigations of cell functions in important physiological events.
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Actinina , Transferencia Resonante de Energía de Fluorescencia , Ratones , Animales , Transferencia Resonante de Energía de Fluorescencia/métodos , Actinina/metabolismo , Línea Celular , Transfección , Microscopía ConfocalRESUMEN
In recent years, three-dimensional (3D) cell culture has been attracting attention as a cell culture model that mimics an environment closer to that of a living organism. It is known that there is a close relationship between cell nuclear shape and cellular function, which highlights the importance of cell nucleus shape analysis in the 3D culture. On the other hand, it is difficult to observe the cell nuclei inside the 3D culture models because the penetration depth of the laser light under a microscope is limited. In this study, we adopted an aqueous iodixanol solution to the 3D osteocytic spheroids derived from mouse osteoblast precursor cells to make the spheroids transparent for 3D quantitative analysis. With a custom-made image analysis pipeline in Python, we found that the aspect ratio of the cell nuclei near the surface of the spheroid was significantly greater than that at the center, suggesting that the nuclei on the surface were deformed more than those at the center. The results also quantitatively showed that the orientation of nuclei in the center of the spheroid was randomly distributed, whereas those on the surface of the spheroid were oriented parallel to the surface of the spheroid. Our 3D quantitative method with an optical clearing technique will contribute to the 3D culture models including various organoid models to elucidate the nuclear deformation during the development of the organs. Insight box Although 3D cell culture has been a powerful tool in the fields of fundamental biology and tissue engineering, it raises the demand for quantification techniques for cell nuclear morphology in the 3D culture model. In this study, we attempted to optically clear a 3D osteocytic spheroid model using iodixanol solution for the nuclear observation inside the spheroid. Moreover, using a custom-made image analysis pipeline in Python, we successfully quantified the nuclear morphology regarding aspect ratio and orientation. Our quantitative method with the optical clearing technique will contribute to the 3D culture models such as various organoid models to elucidate the nuclear deformation during the development of the organs.
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Técnicas de Cultivo de Célula , Núcleo Celular , Animales , Ratones , Procesamiento de Imagen Asistido por Computador , LuzRESUMEN
The deformation of the cell nucleus may cause dispersion of chromatin and eventually enhance transcription, translation, and protein expression. If this happens in the hypertensive artery, an excessive stretch of smooth muscle cell (SMC) nuclei caused by hypertension may provoke wall thickening. Here, we measured deformation of SMC nuclei in rabbit thoracic aortas stretched in different directions. Thin 0.2-mm-thick specimens were sliced in the direction perpendicular to their axial and circumferential directions, and stretched in the circumferential and axial directions, respectively. The deformation of the actin filament (AF) network was similar to that of the whole tissue, whereas the deformation of the nucleus was significantly smaller than the others. Notably, the nucleus seldom deformed when the tissue was stretched in the axial direction. A novel cell model in which the nucleus is connected to the extracellular matrix via the AF network successfully explained the relative unresponsiveness of the nucleus to the axial stretch. It has been pointed out that stress is maintained constant in the circumferential direction but not in the axial direction in the artery wall during hypertension. The relative unresponsiveness of the nucleus to the axial stretch represented in this study explains this phenomenon.
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Aorta , Hipertensión , Animales , Anisotropía , Aorta/fisiología , Núcleo Celular , Conejos , Estrés MecánicoRESUMEN
The media of aortic wall is characterized by altering layers of elastin and smooth muscle cells (SMCs), along with collagen fibers in both layers, and plays a central role in functional and pathological remodeling such as hypertension and atherosclerosis. Because the arterial function is linked closely to the arterial wall internal structure, it is essential to investigate the alteration of the arterial microstructure during macroscopic deformation to understand cardiovascular pathologies. The present study adopted a tissue clearing method in three-dimensional mechanical characterization of rat thoracic aorta, and successfully observed changes in the structure of each of the three primary components of the aorta under intraluminal pressurization while maintaining tissue mechanical integrity and flexibility. Layers of elastic fibers and SMCs deformed greater on the intimal side than those on the adventitial side. Furthermore, there was a structural agreement in the alignment angle between SMC nuclei and elastic fibers on their intimal side, but not on the adventitial side. This is the first study that changes in the microstructure of three primary components of the aorta were visualized and evaluated through the aorta. The method established here would also be useful to understand tissue mechanics of other load-bearing soft tissues.
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Aorta Torácica , Aorta , Adventicia , Animales , Aorta/patología , Núcleo Celular , Elastina , Miocitos del Músculo Liso , Ratas , Estrés MecánicoRESUMEN
Artificial tissue replacement is a promising strategy for better healing outcomes for tendon and ligament injuries, due to the very limited self-regeneration capacity of these tissues in mammals, including humans. Because clinically available synthetic and biological scaffolds for tendon repair have performed more poorly than autografts, both biological and mechanical compatibility need to be improved. Here we propose a rapid fabrication method for tendon-like structure from collagen hydrogel, simultaneously achieving collagen fibre alignment and intermolecular cross-linking. Collagen gel, 24 h after polymerization, was subjected to mechanical loading in the presence of the chemical cross-linker, genipin, for 24 or 48 h. Mechanical loading during gel incubation oriented collagen fibres in the loading direction and made chemical cross-linking highly effective in a loading magnitude-dependent manner. Gel incubated with 4 g loading in the presence of genipin for 48 h possessed tensile strength of 4 MPa and tangent modulus of 60 MPa, respectively, which could fulfill the minimum biomechanical requirement for artificial tendon. Although mechanical properties of gels fabricated using the present method can be improved by using a larger amount of collagen in the starting material and through optimisation of mechanical loading and cross-linking, the method is a simple and effective for producing highly aligned collagen fibrils with excellent mechanical properties.
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Colágeno , Tendones , Animales , Colágeno/química , Humanos , Hidrogeles , Mamíferos , Resistencia a la Tracción , Cicatrización de HeridasRESUMEN
Tendons exhibit a hierarchical collagen structure, wherein higher-level components, such as collagen fibres and fascicles, are elongated, slid, and rotated during macroscopic stretching. These mechanical behaviours of collagen fibres play important roles in stimulating tenocytes, imposing stretching, compression, and shear deformation. It was hypothesised that a lack of local fibre behaviours in healing tendon tissue may result in a limited application of mechanical stimuli to cells within the tissue, leading to incomplete recovery of tissue structure and functions in regenerated tendons. Therefore, the present study aimed to measure the microscopic strain field in the healing tendon tissue. A central third defect was created in the patellar tendon of mice, and the regenerated tissue in the defect was examined by tensile testing, collagen fibre analysis, and local strain measurement using confocal microscopy at 3 and 6 weeks after surgery. Healing tissue at 3 weeks exhibited a significantly lower strength and disorganised collagen fibre structure compared with the normal tendon. These characteristics at 6 weeks remained significantly different from those of the normal tendon. Moreover, the magnitude of local shear strain in the healing tissue under 4% tissue strain was significantly smaller than that in the normal tendon. Differences in the local strain field may be reflected in the cell nuclear shape and possibly the amount of mechanical stimuli applied to the cells during tendon deformation. Accordingly, restoration of a normal local mechanical environment in the healing tissue may be key to a better healing outcome of tendon injury.
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Ligamento Rotuliano , Animales , Ratones , Rótula , Tendones , Resistencia a la Tracción , Cicatrización de HeridasRESUMEN
Tendon cells, tenocytes, are constantly subjected to mechanical stress in vivo, which maintains a level of cellular tension. When a tendon is subjected to overloading, local rupture of collagen fibers are induced, which deprives tenocytes of mechanical stress, lowers their cellular tension level and upregulates their catabolism. In addition, leukocytes are attracted to the rupture sites and produce interleukin-1ß (IL-1ß), and this exogenous IL-1ß also stimulates tenocyte catabolism. We tested a hypothesis that catabolic tenocytes with low cellular tension at the rupture sites excessively respond to the exogenous IL-1ß and further upregulate matrix metalloproteinase 1 (MMP-1) gene expression. Tenocytes from rabbit Achilles tendon were cultured on the following substrates: glass or polydimethylsiloxane micropillar substrates with a height of 2, 4, or 8 µm. Following a 3-day IL-1ß stimulation at a concentration of 0, 1, 10, or 100 pM, the effects of IL-1ß stimulation on cell morphology and MMP-1 gene expression was analysed with fluorescent microscopy and fluorescence in situ hybridization, respectively. In addition, the effects of IL-1ß stimulation on cell membrane fluidity were examined. It was demonstrated that the cells on 8-µm-height micropillars exhibited a greater response than those on rigid substrates with flat (glass) and topologically the same surface (2-µm-height micropillars) to IL-1ß when supplied at the same concentration. Besides this, membrane fluidity was lower in the cells on micropillars. Therefore, it appears that cellular attachment to softer substrates lowers the cellular actin cortex tension, reducing the membrane fluidity and possibly elevating the sensitivity of IL-1 receptors to ligand binding. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:150-159, 2020.
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Interleucina-1beta/farmacología , Metaloproteinasa 1 de la Matriz/genética , Tenocitos/patología , Animales , Células Cultivadas , Expresión Génica , Hibridación Fluorescente in Situ , Masculino , Fluidez de la Membrana , Conejos , Estrés Mecánico , Tenocitos/efectos de los fármacos , Tenocitos/enzimologíaRESUMEN
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 1â¯Hz for 7â¯days for cell shape analysis and for 3â¯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.
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Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Animales , Células de la Médula Ósea/fisiología , Forma de la Célula , Células Cultivadas , Fémur/citología , Ratones , Músculo Liso Vascular , Osteogénesis , Estrés MecánicoRESUMEN
Glycosphingolipids (GSLs) are ubiquitous membrane components that play an indispensable role in maintaining chondrocyte homeostasis. To gain better insight into roles of GSLs, we studied the effects of GSL-deletion on the physiological responses of chondrocytes to mechanical stress. Mice lacking Ugcg gene (Ugcg-/-) were genetically generated to obtain GSL-deficient mice, and their chondrocytes from the joints were used for functional analyses in vitro culture experiments. The cells were seeded in a three-dimensional collagen gel and subjected to 5%, 10% or 16% cyclic tensile strain for either 3 or 24â¯h. The gene expressions of chondrocyte anabolic and catabolic factors, and the induction of Ca2+ signaling were analyzed. Our results revealed that chondrocytes derived from GSL-deficient mice exhibited an elevation in the expression of catabolic factors (ADAMTS-5, MMP-13) following the exposure to strain with amplitudes of 10%. Likewise, applying cyclic tensile strain with these amplitudes resulted in an increased Ca2+ oscillation ratio in chondrocytes from GSL-deficient as compared to the ratio from control mice. These results demonstrated that deletion of GSL stimulated the catabolic responses of chondrocytes to mechanical stress via the augmentation of the sensitivity to mechanical stress that may lead to the cartilage deterioration. These findings suggest that the regulation of the physiological responses of chondrocytes by GSLs could be a potential target in a therapeutic intervention in osteoarthritis.
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Condrocitos/metabolismo , Glicoesfingolípidos/metabolismo , Mecanotransducción Celular , Animales , Señalización del Calcio , Cartílago Articular/metabolismo , Células Cultivadas , Glucosiltransferasas/genética , Ratones , Ratones Noqueados , Osteoartritis/metabolismo , Estrés MecánicoRESUMEN
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 24â¯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 â¼3%, 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.
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Calcificación Fisiológica , Colágeno/metabolismo , Fenómenos Mecánicos , Tibia/fisiología , Animales , Fenómenos Biomecánicos , Pollos , Matriz Extracelular/metabolismo , Estrés Mecánico , Tibia/citología , Tibia/metabolismoRESUMEN
Elevation of tendon core temperature during severe activity is well known. However, its effects on tenocyte function have not been studied in detail. The present study tested a hypothesis that heat stimulation upregulates tenocyte catabolism, which can be modulated by the inhibition or the enhancement of gap junction intercellular communication (GJIC). Tenocytes isolated from rabbit Achilles tendons were subjected to heat stimulation at 37 °C, 41 °C or 43 °C for 30 min, and changes in cell viability, gene expressions and GJIC were examined. It was found that GJIC exhibited no changes by the stimulation even at 43 °C, but cell viability was decreased and catabolic and proinflammatory gene expressions were upregulated. Inhibition of GJIC demonstrated further upregulated catabolic and proinflammatory gene expressions. In contrast, enhanced GJIC, resulting from forced upregulation of connexin 43 gene, counteracted the heat-induced upregulation of catabolic and proinflammatory genes. These findings suggest that the temperature rise in tendon core could upregulate catabolic and proinflammatory activities, potentially leading to the onset of tendinopathy, and such upregulations could be suppressed by the enhancement of GJIC. Therefore, to prevent tendon injury at an early stage from becoming chronic injury, tendon core temperature and GJIC could be targets for post-activity treatments.
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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.
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The present study has been performed on temporal changes in gap junctional intercellular communication (GJIC) between tenocytes under static tensile strain with the magnitude of 0% (no strain), 4% (physiological magnitude) or 8% (overloading magnitude) during a 24-h culture period. Tenocytes were isolated from rabbit Achilles tendon and seeded on a stretchable microgroove substrate. GJIC was evaluated as intercellular diffusion coefficient of calcein (DGJ) using fluorescence loss in photobleaching (FLIP) protocol accompanied with a mathematical model of molecular diffusion both within the cell and between the cells. It was exhibited that the application of 4% strain for 1 h increased DGJ significantly. The increased level was maintained for 6 h, followed by returning to the pre-strain level at 24 h. This was associated with a transient increase in connexin 43 (Cx43) gene expression and protein localisation at 1 h, suggesting the increased GJIC may have involved new synthesis of gap junctions. By contrast, the application of 8% static strain reduced DGJ to the similar or lower level from 0% strain group for 6 h, associated with inhibited Cx43 gene expression. However, Cx43 protein localisation was not changed much, and thus, there seem no direct interactions among changes in GJIC, Cx43 gene expression and Cx43 localisation. The present findings highlight the differences in mechanical regulation of GJIC between physiological and non-physiological loadings, and thus the increase or the decrease in GJIC may affect tenocyte functions in different ways.
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Comunicación Celular , Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Tenocitos/metabolismo , Tendón Calcáneo/metabolismo , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Modelos Teóricos , Fotoblanqueo , Conejos , Estrés Mecánico , Tenocitos/citología , Resistencia a la Tracción , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Large magnitudes of mechanical strain applied to tendon cells induce catabolic and inflammatory responses, whereas a moderate level of strain promotes anabolism. Gap junction intercellular communication (GJIC) plays an essential role in these responses, however direct regulation of GJIC by mechanical loading has not been characterised in detail. Here, we show that the GJIC between tenocytes are enhanced or inhibited depending on the magnitude of the tensile strain. The GJIC was analysed using fluorescence loss in photobleaching (FLIP), combined with a molecular diffusion model. Intercellular and intracellular transport of fluorescence tracer molecules, calcein, across multiple cells through the gap junctions was evaluated by determining the intercellular and intracellular diffusion coefficients of calcein. It was demonstrated that the intercellular diffusion coefficient was significantly higher when the cells were subjected to a physiological static tensile strain (4%) for 1 h, but significantly lower when subjected to a strain with non-physiological amplitude (8%). The intracellular diffusion coefficient was not altered by the application of static strain at any level. Connexin 43 proteins were localised within cytoplasm and at cell-cell boundaries in no strained state and were also localised near cell nuclei by the 4% strain, but the localisation was reduced by the 8% strain. The findings suggest that the increase in GJIC in response to 4% strain involves opening of gap junction pores via mechanotransduction events of tenocytes, whereas the inhibition in response to 8% strain involves mechanical disruption of the junctions.
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Conexina 43/metabolismo , Fibroblastos/metabolismo , Uniones Comunicantes/metabolismo , Mecanotransducción Celular , Tendones/metabolismo , Animales , Transporte Biológico , Comunicación Celular , Conexina 43/genética , Difusión , Fibroblastos/citología , Fluoresceínas , Recuperación de Fluorescencia tras Fotoblanqueo , Colorantes Fluorescentes , Uniones Comunicantes/ultraestructura , Expresión Génica , Masculino , Conejos , Tendones/citología , Resistencia a la Tracción/fisiologíaRESUMEN
A double-network (DN) gel, which was composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly(N,N'-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated the biomechanical and biological responses of chondrogenic progenitor ATDC5 cells cultured on the DN gel. ATDC5 cells were cultured on a polystyrene surface without insulin (Culture 1) and with insulin (Culture 2), and on the DN gel without insulin (Culture 3). The cultured cells were evaluated using micropipette aspiration for cell Young's modulus and qPCR for gene expression of chondrogenic and actin organization markers on days 3, 7 and 14. On day 3, the cells in Culture 3 formed nodules, in which the cells exhibited an actin cortical layer inside them, and gene expression of type-II collagen, aggrecan, and SOX9 was significantly higher in Culture 3 than Cultures 1 and 2 (p<0.05). Young's modulus in Culture 3 was significantly higher than that in Culture 1 throughout the testing period (p<0.05) and that in Culture 2 on day 14 (p<0.01). There was continuous expression of actin organization markers in Culture 3. This study highlights that the cells on the DN gel increased the modulus and mRNA expression of chondrogenic markers at an earlier time point with a greater magnitude compared to those on the polystyrene surface with insulin. This study also demonstrates a possible strong interrelation among alteration of cell mechanical properties, changes in actin organization and the induction of chondrogenic differentiation.
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Acrilamidas/química , Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Módulo de Elasticidad/efectos de los fármacos , Insulina/farmacología , Polímeros/química , Polímeros/farmacología , Ácidos Sulfónicos/química , Ácidos Sulfónicos/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Biomarcadores/metabolismo , Línea Celular Tumoral , Condrocitos/citología , Condrocitos/efectos de los fármacos , Geles , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Tenocyte mechanotransduction has been of great interest to researchers in tendon mechanobiology and biomechanics. In vivo, tenocytes are subjected to tensile strain and fluid shear stress, but most studies of tenocyte mechanobiology have been to understand how tenocytes regulate their functions in response to tensile strain. Thus, there is still much to know about tenocyte responses to fluid shear stress, partly due to the difficulty of devising a suitable experimental set-up and understanding the exact magnitude of imposed fluid shear stress. Therefore, this study was performed to test a new experimental system, which is suitable for the application of tensile strain and fluid shear stress to tenocytes in vitro. It was experimentally and numerically confirmed that tenocytes could maintain their in situ morphology within microfabricated microgrooves; also, physiological tensile strain and a wide range of fluid shear stress magnitudes can be applied to these cells. Indeed, it was demonstrated that the combined stimulation of cyclic tensile strain and oscillatory fluid shear stress induced a greater synergetic effect on tenocyte calcium response and significantly increased the percentage of tenocyte exhibiting increases in intracellular Ca(2+) concentration compared to the solo applications of these two modes of mechanical stimulation. The experimental system presented here is suitable for research of tenocyte mechanobiology, particularly mechanotransduction events, which were difficult to study using previous experimental models like explants and cell monolayers.