Eijiro Maeda

Differential regulation of gene expression in isolated tendon fascicles exposed to cyclic tensile strain in vitro

Mechanical stimulus is a regulator of tenocyte metabolism. The present study investigated temporal regulation of the expression of selected genes by tenocytes in isolated fascicles subjected to tensile strain in vitro. Cyclic tensile strain with a 3% amplitude superimposed on a 2% static strain was provided for 10 min, followed by either an unstrained period or continuous cyclic strain until the end of a 24-h incubation period. mRNA expression of selected anabolic and catabolic genes were evaluated with quantitative PCR at 10 min, 1 h, 6 h, and 24 h. The application of 6-h cyclic strain significantly upregulated type III collagen mRNA expression in strained fascicles compared with unstrained controls, but no alterations were observed in mRNA expression of type I collagen and biglycan. Significant downregulation in the expression of the decorin core protein was observed in fascicles subjected to 24-h cyclic strain. MMP3 and MMP13 expression levels were upregulated by the application of 10 min of cyclic strain, followed by a progressive downregulation until the end of the incubation period in both the absence and the presence of the continuing cyclic strain. Accordingly, alterations in the expression of anabolic genes were limited to the upregulation of type III collagen by prolonged exposure to cyclic strain, whereas catabolic genes were upregulated by a small number of strain cycles and downregulated by a prolonged cyclic strain. These findings demonstrate distinctive patterns of mechanoregulation for anabolic and catabolic genes and help our understanding of tenocyte response to mechanical stimulation.

Cytoskeletal tension modulates MMP-1 gene expression from tenocytes on micropillar substrates

Actin cytoskeletons, aggregated with myosin II, generate intracellular cytoskeletal tension, which is induced to cell attaching substrate as cell traction forces. It is thought that cytoskeletal tension links closely to cell functions. The present study examined quantitative relationships between cytoskeleton tension and the balance of cell metabolism of tenocytes. Using micromachining techniques, micropillar substrates were prepared with polydimethylsiloxane, having three different values of substrate elasticity (6, 18 and 33 kPa) by changing the micropillar height. After 24h incubation of bovine tenocytes on these micropillar substrates, cell traction forces were determined. Gene expressions for type I collagen (anabolic marker) and MMP-1 (catabolic marker) from tenocytes on micropillars were also analyzed with qPCR. In addition, effects of an inhibition of myosin II activity on tenocyte cytoskeletal tension and metabolism were examined using the inhibitor, blebbistatin. It was exhibited that cell traction forces were significantly larger in tenocytes on 33 kPa substrates compared to those on 6 kPa substrates. This was associated with significant lower expression of MMP-1 mRNA on 33 kPa substrates. Cell traction forces were decreased significantly by the supplementation of blebbistatin in a dose-dependent manner. Indeed, there were significant smaller traction forces and higher expression for MMP-1 mRNA from tenocytes treated with 10 μM blebbistatin compared to their corresponding controls. Accordingly, tenocyte responses to substrate stiffness are associated with alterations in intracellular tension and catabolic gene expression. On the other hand, tenocyte anabolism, as measured by type I collagen mRNA expression, was not altered with substrate stiffness.

Functional analysis of tenocytes gene expression in tendon fascicles subjected to cyclic tensile strain

Tenocytes are known to be mechanoresponsive and the present study tests the hypothesis that distinct mechanical stimulation regimes, associated with the short-term and extended application of cyclic tensile strain, alters the balance between anabolic and catabolic processes. Microarray technology has been used to provide a comprehensive analysis of alterations in gene expression within isolated tendon fascicles in response to cyclic tensile strain using a well-established model system. Isolated rat tail tendon fascicles were subjected to cyclic tensile strain (3% amplitude superimposed on a 2% static strain) for 1 or 24 hr. Messenger RNA expression level was assessed using Illumina microarray. The number of genes significantly altered in strained fascicles from the level of unstrained control fascicles was greater at 24 hr than 1 hr. The expression levels of many extracellular matrix components remained unchanged at both time points; however, a number of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with a thrombospondin (ADAMTS) families were significantly downregulated at 24 hr. Functional annotation revealed that upregulated genes were significantly associated with the regulation of transcription at 1 hr and translation at 24 hr. Downregulated genes were associated with inflammatory responses at 1 hr, and genes inhibited at 24 hr were significantly associated with cell apoptosis and a variety of metabolic functions. The present results suggest that the metabolic balance was shifted in favor of catabolism by the application of a small number of tensile strain cycles, whereas an extended number stimulates strong anti-catabolic effects.

A new experimental system for simultaneous application of cyclic tensile strain and fluid shear stress to tenocytes in vitro

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 Ca2+ 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.

Effects of stress shielding and subsequent restressing on mechanical properties of regenerated and residual tissues in rabbit patellar tendon after resection of its central one-third

Central third of patellar tendon (PT) is used as an autograft for anterior cruciate ligament (ACL) reconstruction. Previous studies investigated temporal changes in material properties of healing tissues in PT after resection of the central third. However, no study has been performed on effects of stress shielding (SS) and restressing (RS) on the properties of healing tissues. The present study hypothesised that SS adversely affects the mechanical integrity of healing tissues, which is recovered by subsequent RS. An entire rectangular defect was created in the central third of rabbit PT. Operated PTs were subjected to either SS or no stress shielding (NSS). A subgroup of stress-shielded PTs was followed by the resumption of normal loading, namely RS. Tensile properties of tissues regenerated in the defect and residual tendons were evaluated. Regenerated tissues of SS for 3 weeks resulted in significantly lower strength than NSS, which was recovered to NSS level by 3 weeks of RS. Strength of residual tissues in RS reversed SS effects, leading to the strength at NSS level after 12 weeks. However, tangent modulus of residual tissues in RS was still significantly lower than that of NSS at 12 weeks. Therefore, SS induces detrimental effects on the mechanical integrity of healing PTs, and the response to RS was different between regenerate and residual tissues, the latter of which took longer period to reach NSS level.

Effects of maturation on the mechanical properties of regenerated and residual tissues in the rabbit patellar tendon after resection of its central one-third

BACKGROUND The central one-third portion of the patellar tendon is commonly used as a graft for the reconstruction of the anterior cruciate ligament. Although several studies have been carried out on mechanical properties of healing tendons in mature animals, there have been no studies on regenerated and residual tissues in the immature patellar tendon after the removal of its central portion. METHODS An entire one-third defect was made in the patellar tendon of 2-, 3- and 6-month-old rabbits. After 3 weeks, the tissue regenerated in the defect and the residual tissue were biomechanically and histologically evaluated. FINDINGS The length of patellar tendons in 6-month-old animals after the resection of its central one-third was significantly longer than that in age-matched controls. The cross-sectional area of all operated tendons was significantly larger compared to age-matched controls. There were no significant effects of maturation on the mechanical properties of regenerated and residual tissues in operated tendons, although tensile strength and tangent modulus of normal tendons were significantly greater in 6-month rabbits than in immature ones. The histology of each of regenerated and residual tissues was similar in the three groups. INTERPRETATION There were no remarkable effects of maturation on regenerated and residual tissues after the removal of the central one-third tendon. However, the strength and the modulus of normal tendons are significantly lower in immature patients than in mature ones. Therefore, surgeons should take account of the inferior mechanical properties of the tendon in skeletally immature patients at the time of surgeries for the reconstruction of the anterior cruciate ligament.

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 β-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.

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.

2D15 Temporal characterization of gap junction communication between tenocytes under tensile strain in vitro

Tendons are mostly composed of collagen fibers and transmit forces from muscle to bone. Tenocytes are cells in tendon that produce main constituent of tendon such as type I collagen, and are linked each other via gap junctions at cell-cell junctions. Most cells in normal tissues generally communicate via gap junction. For example, ion and small molecule movements through the gap junction channels occur by passive diffusion. It has been shown that gap junction intercellular communication (GJIC) is essential in mechanosensitive response of tenocytes (1) .

101 Effect Of Hyperthermia On Tenocyte Physiological Functions

Introduction It is known that core temperature in Achilles tendon rises up to around 45°C during intensive exercises in both animal and human models. It may indicate that both overloading and hyperthermia are involved in the onset of tendinopathy. Although a large number of studies have been performed on effects of overloading on tenocyte functions, only a few studies have been performed on effects of hyperthermia on tenocyte activities. In these studies, lethal effects of hyperthermia on tenocytes were mainly reported. However, little has been known about effects of hyperthermia on tenocyte functions, in particular regulation of catabolism. Gap junctions are known to be involved in mechano-response of tenocytes. It is demonstrated that gap junction and associated intercellular communication (GJIC) are regulated by mechanical loading.1 However, how GJIC in tenocytes is changed under hyperthermia remains unknown. Therefore, the present study was performed to evaluate changes in tenocyte physiological functions and GJIC. It was hypothesised that hyperthermia upregulates tenocyte catabolism and downregulates GJIC. Methods Tenocytes were isolated from rabbit Achilles tendon and cultured in a microgroove device2 for 24 hours, at cell density of 2000 cells/cm.2 To evaluate cell viability and mRNA expression analysis, tenocytes were maintained at 37, 41 and 43°C for 30 min and then incubated at 37°C for 24 h. Cell viability assay was performed by staining viable cells with calcein-AM and dead cells by ethidium homodimer. qPCR was performed to measure mRNA expressions for COL1 (type I collagen), MMP-1 (Matrix metalloproteinase-1), IL-1β (Interleukin-1β) and CASP3 (Caspase-3). For determination of GJ diffusivity, tenocytes were stained by calcein-AM and incubated at 37, 43°C for 30 min followed by being subjected to FLIP experiment(1) modified for the present study. Laser photobleaching to a target cell was repeated 100 times with the laser power of 70 μW. Results Average cell viability at 37, 41, 43°C were 95.5, 76.0 and 60.5% respectively, although the changes were not statistically significant. Fig 1 shows results from mRNA expression analysis. Expressions of MMP-1 and IL-1β were elevated at 43°C compared to that 37°C. Meanwhile, expression of COL1 and CASP3 decreased from 37 to 43°C. Diffusion coefficient of gap junction at 37 and 43°C was 1.57 and 2.18 µm2/s, respectively. Intracellular diffusion coefficients at 37 and 43°C was 15.68 and 33.55 µm2/s, respectively. Discussion The observed tend of the decreased cell viability by hyperthermia is in an agreement with a previous report(3). Increases in expressions of MMP-1 and IL-1β and decrease in expressions of COL1 indicate that hyperthermia upregulates tenocyte catabolism and downregulates anabolism. As MMP-1 and IL-1β are markers for inflammation, results suggest that inflammation is triggered by hyperthermia. As expression of CASP3 is a marker of cell apoptosis, decreased expression of CASP3 by hyperthermia indicates that cell death induced by hyperthermia is not due to apoptosis. Gap junction diffusion coefficient increased at 43ºC, suggests that GJIC is enhanced under hyperthermia, possibly to protect tenocytes from high temperature and/or to recover the damage by heat stress. Abstract 101 Figure 1 Relative expression ratio of COL1, MMP-1, IL-1β and CASP3 mRNA from tenocytes with heat treatment at 37, 41 and 43°C References 1 Maeda E, et al. Biomechanics and Modeling in Mechanobiology. 2012;11:439–447 2 Maeda E, et al. Biomedical Microdevices. 2013;15:1067–1075 3 Birch HL, et al. The Journal of Experimental Biology. 1997;200:1703–1708