Silicone Dishes Research Articles

A 3D cell culture system for bioengineering human neuromuscular junctions to model ALS.

The signals that coordinate and control movement in vertebrates are transmitted from motoneurons (MNs) to their target muscle cells at neuromuscular junctions (NMJs). Human NMJs display unique structural and physiological features, which make them vulnerable to pathological processes. NMJs are an early target in the pathology of motoneuron diseases (MND). Synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is the starting point of the pathophysiological cascade leading to MN death. Therefore, the study of human MNs in health and disease requires cell culture systems that enable the connection to their target muscle cells for NMJ formation. Here, we present a human neuromuscular co-culture system consisting of induced pluripotent stem cell (iPSC)-derived MNs and 3D skeletal muscle tissue derived from myoblasts. We used self-microfabricated silicone dishes combined with Velcro hooks to support the formation of 3D muscle tissue in a defined extracellular matrix, which enhances NMJ function and maturity. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the function of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and found a decrease in neuromuscular coupling and muscle contraction in co-cultures with MNs harboring ALS-linked SOD1 mutation. In summary, the human 3D neuromuscular cell culture system presented here recapitulates aspects of human physiology in a controlled in vitro setting and is suitable for modeling of MND.

Cyclic tensile stress of human annulus fibrosus cells induces MAPK activation: involvement in proinflammatory gene expression

To study the role of mitogen-activated protein kinases (MAPKs) in human annulus fibrosus (AF) cells subjected to cyclic tensile stress (CTS). An invitro system for CTS studies was established using AF cultures on fibronectin-coated silicone dishes. MAPK phosphorylation was studied by western analysis, while gene expression was followed by qRT-PCR. DNA synthesis was assessed by both tritiated thymidine incorporation and flow cytometry, and collagen synthesis using tritiated proline incorporation and the protease-free collagenase method. All three MAPKs studied, i.e., ERK, SAPK/JNK, and p38 were found to be phosphorylated immediately after CTS application within physiological range. A second wave of phosphorylation appeared at later time points. MAPK activation was elevated at higher CTS magnitudes, but independent of the frequency. CTS did not stimulate DNA synthesis neither extracellular matrix turnover, but it stimulated the proinflammatory genes, COX-2, IL-6, and IL-8. This stimulation was more intense at the highest magnitude (8%) testedand at the median frequency (1Hz) and time interval (12h). Blocking of ERK, SAPK/JNK, and p38 MAPK inhibited the CTS-induced stimulation of COX-2 and IL-8, while IL-6 expression was mediated only by SAPK/JNK and p38 MAPK. We have described for the first time the activation of MAPKs in human AF cells in response to CTSand showed that it drives an inflammatory reaction. These observations shed light on the mechanisms of intervertebral disc (IVD) cell responses to mechanical stress, contributing to the understanding of disc pathophysiologyand possibly to the design of novel therapeutic interventions.

Tissue Engineering for Intervertebral Disc Repair and Regeneration

Introduction Tissue engineering aims to combine isolated cells, engineered materials and small molecule biochemical factors to generate neotissues with specific form and function to replace or repair damaged or degenerate native tissue. Intervertebral disc (IVD) degeneration directly causes back pain, and understanding how to regenerate this tissue may help alleviate this costly global problem. Several studies have combined isolated nucleus pulposus (NP) disc cells with novel scaffold materials to produce promising therapeutics toward IVD repair. We have taken three avenues toward IVD tissue engineering: culturing isolated IVD cells on continuous expansion surfaces to enhance cell phenotypes, utilizing novel injectable hydrogels for cell delivery to intact discs, and production of 3D printed scaffold geometries enhancing disc matrix production by seeded NP cells. Material and Methods Bovine NP cells were isolated from caudal discs of freshly slaughtered steer (∼ 24 months) obtained from a local slaughterhouse. Bovine NP cells are expanded on either tissue culture plastic with two passages, or on highly elastic silicone rubber dishes which slowly expands the surface from 12 to 78 cm2 and marker gene expression is compared. Isolated cells are labeled with fluorescent membrane binding dye, and cells are mixed with an injectable, chitosan/hyaluronic acid hydrogel. Approximately 100 µL of cell/gel (106 cells/µL of gel) mixtures is injected into isolated whole bovine discs (obtained as described earlier, with vertebral bone drilled down to the cartilage end plate). Discs are then placed into a custom bioreactor, and dynamic diurnal loading is applied to the injected discs. After 14 days, localization and viability of injected cells is assessed by confocal microscopy. For 3D printed constructs, 10 × 10 × 0.3 mm plastic scaffolds were printed using a desktop 3D printer, with pore size of approximately 700 µm. Overall, 5 × 105 bovine NP cells were seeded on scaffolds, and cell viability, DNA content, proteoglycan content, and collagen type II content was assessed after 21 days in culture. Results When cultured on elastic silicone dishes, continuous expansion of bovine NP cells preserve cell phenotype and promotes higher aggrecan and collagen type II expression compared with tissue culture plastic controls. NP cells embedded in a hydrogel and injected into whole discs were localized throughout the discs. Cells also showed high viability after 14 days in culture. Bovine NP cells seeded on 3D printed orthogonal plastic scaffolds grow extremely well and fill in the large 700 µm pores over 21 days culture. The cultured constructs have high viability and produce ample aggrecan and collagen type II protein. Conclusion By combining three different approaches we show the following: (1) individual NP cells can be engineered to maintain superior phenotypic qualities compared with standard culture procedures, (2) combination of expanded cells with injectable hydrogels can be delivered to intact discs for potential repair, and (3) simple 3D printed scaffolds can promote NP matrix production by seeded cells. Combination of these avenues may further enhance IVD regenerative capacities.

Mechanical Injury to Human Intervertebral Discs and Isolated Cells Initiates Events Implicated in Degeneration and Pain

Introduction Painful degenerating intact human intervertebral discs (IVDs) produce elevated levels of Toll-like receptors (TLRs), cytokines, and neuropeptides but events regulating their expression are poorly understood. Mechanical overload has been proposed as one potential initiating mechanism. The aim of this study was to investigate the cellular responses in IVDs and isolated disc cells exposed to mechanical overload or strain. Methods Lumbar spine segments were harvested from consenting donors via the Transplant Quebec organ donation program in Montreal, Quebec, Canada. A total of nine discs from six donors were used in this study. Intact IVDs were prepared and cells isolated using a standard technique. Intact discs with cartilaginous endplates were loaded with a single ramp compression peaking at either approximately 0.2 MPa (noninjurious) or about 1.5 MPa (injurious) stress applied at a rate of 30% strain per second. Injurious compression protocol consistently yields endplate fracture. Discs were immediately put in low serum containing media after loading. For isolated cell experiments, 2 × 105 nucleus pulposus (NP) and annulus fibrosus (AF) cells were seeded on flexible or static silicone dishes. The flexible dishes were exposed to a 20% magnitude cyclic mechanical strain at 0.0001 Hz was applied to the cultures for 8 h/d over 2 days. Conditioned media was collected and RNA was isolated. Quantitative RT-PCR was performed to assess TLR, NGF, and TNF gene expression. Conditioned media from intact discs and isolated cells was applied to PC12 cells and neurite outgrowth was assessed. Commercially available cytokine arrays were used to investigate protein levels of cytokines released from intact discs and isolated cells. Results Elevated levels of proteoglycan, inflammatory cytokines, and pain mediators were released from intact discs exposed to injurious loading. The highest levels were found 3 days postinjury and the conditioned media caused significantly induced neurite outgrowth in PC12 cells compared with that of the control group. About 50% of the cells in the IVDs died as a result of the inflicted trauma. Isolated NP and AF cells were exposed to a high-magnitude strain at low frequency to preserve cell attachments and maintain cell viability. The protocol was applied to evaluate if the adverse mechanical stimulation of the cells or the induced cell death caused by the injurious loading of the intact IVDs was responsible for the increased expression of inflammatory cytokines and pain mediators. Our data showed that high-magnitude low-frequency strain is sufficient to induce expression inflammatory cytokines and pain mediators in the absence of cell death. We also found a small but significant upregulation of TLR 2 and 4 in both NP and AF cells. Conclusion Cell death and cytokine production combined with loss of glycosaminoglycan indicate that mechanical injury to intact IVDs and isolated cells initiates cellular processes leading to disc degeneration. Once the proteoglycan content in the matrix has been degraded, elevated levels of NGF could lead to neuronal ingrowth directly causing pain. Increased TLR expression induced by high strain may prime cells toward a degenerate phenotype and could be a novel therapeutic target for degenerative disc disease.

Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels

Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications.

Using Functional Tissue Engineering and Bioreactors to Mechanically Stimulate Tissue-Engineered Constructs

Bioreactors precondition tissue-engineered constructs (TECs) to improve integrity and hopefully repair. In this paper, we use functional tissue engineering to suggest criteria for preconditioning TECs. Bioreactors should (1) control environment during mechanical stimulation; (2) stimulate multiple constructs with identical or individual waveforms; (3) deliver precise displacements, including those that mimic in vivo activities of daily living (ADLs); and (4) adjust displacement patterns based on reaction loads and biological activity. We apply these criteria to three bioreactors. We have placed a pneumatic stimulator in a conventional incubator and stretched four constructs in each of five silicone dishes. We have also programmed displacement-limited stimuli that replicate frequencies and peak in vivo patellar tendon (PT) strains. Cellular activity can be monitored from spent media. However, our design prevents direct TEC force measurement. We have improved TEC stiffness as well as PT repair stiffness and shown correlations between the two. We have also designed an incubator to fit within each of two electromagnetic stimulators. Each incubator provides cell viability like a commercial incubator. Multiple constructs are stimulated with precise displacements that can mimic ADL strain patterns and record individual forces. Future bioreactors could be further improved by controlling and measuring TEC displacements and forces to create more functional tissues for surgeons and their patients.

047 Stretching Magnitude-Dependent Inflammatory Response of Human Tendon Fibroblasts

Previous studies have shown that cyclic mechanical stretching exerts anti-inflammatory effects on rabbit chondrocytes. But whether mechanical stretching has similar effects on human tendon fibroblasts are not known. This study therefore aimed to test the hypothesis that cyclic mechanical stretching regulates IL-1β induced COX-2 gene expression in a stretching magnitude-dependent manner. In custom-made silicone dishes, human patellar tendon fibroblasts (HPTFs) were grown on microgrooved culture surfaces, with which the shape and organization of HPTFs in vivo were closely mimicked. To induce inflammatory responses in HPTFs, 10 pM of IL-1β was added to the culture medium. A 4% or 8% cyclic uniaxial stretching was then applied to silicone dishes for 4 hrs using a custom-design stretching apparatus. After the end of stretching, total RNA of stretched and nonstretched tendon fibroblasts was collected, and RT-PCR was performed for measuring COX-2 mRNA expression levels. We found that tendon fibroblasts subjected to 4% stretching decreased COX-2 mRNA expression level by 20%(p = 0.0002; n = 6) compared to that of nonstretched, IL-1β-treated cells. However, cells subjected to 8% stretching showed a 39% increase (p = 0.007, n = 6) in COX-2 mRNA expression. Thus, the results of this study suggest that small stretching (4%) has anti-inflammatory effects on HPTFs in a mildly inflammatory environment such as that induced by 10 pM of IL-1β. In contrast, large stretching (8%) may amplify inflammatory responses of tendon fibroblasts. Therefore, physical therapy with low levels of tendon stretching may be beneficial in reducing mild tendon inflammation. Supported by the Arthritis Investigator Award and NIHAR049921 (JHW)

Mechanical Stretch Induced Interleukin‐18 (IL‐18) Expression through Angiotensin Subtype 1 Receptor (AT1R) and Endothelin‐1 in Cardiomyocytes

Interleukin‐18 (IL‐18) is a proinflammatory cytokine with multiple biological functions. We and others have demonstrated that an increased level of circulating IL‐18 is one of the risk factors for cardiovascular diseases. Endothelin‐1 (ET‐1) has been reported to be a potent hypertrophy‐promoting factor through RhoA and Rho‐Kinase. Mechanical stretch induces a hypertrophic response, partly through the production of ET‐1 through Endothelin A receptor (ETAR). Moreover, it has also been reported that mechanical stretch induces cardiac hypertrophy through Angiotensin subtype 1 receptor (AT1R). However, the mechanism by which the IL‐18 gene expression is regulated in cardiomyocytes has not yet been fully understood. This study was designed to elucidate the functional significance of IL‐18 gene expression in response to mechanical stretch. Neonatal rat cardiomyocytes cultured on silicone dishes were subjected to stretch. The moderate 20% mechanical stretch resulted in the elevation of IL‐18 expression in a time‐dependent manner with the maximal level achieved 36 hours after the stretch. Olmesartan, AT1R antagonist inhibited stretch‐induced IL‐18 expression. ETAR blockade BQ123 inhibited stretch‐induced IL‐18 expression. However, the Endothelin B receptor (ETBR) receptor blockade BQ788 did not inhibit this reaction. ET‐1 induced IL‐18 expression, with a peak induction after 4 hours of incubation. These results might suggest that stretch stimulation of cardiomyocytes induced ET‐1 and, subsequently, ET‐1 up‐regulated the IL‐18 expression. Furthermore, Fasudil, a Rho‐Kinase inhibitor, and Simvastatin, a HMG‐CoA reductase inhibitor, led to a significant reduction in mechanical stretch‐induced IL‐18 expression. These results indicated, for the first time, that IL‐18 expression is induced by mechanical stretch in cardiomyocytes via the ETAR, AT1R, and the Rho/Rho‐K pathways. The induction of IL‐18 from cardiomyocytes by mechanical stress might cause the deterioration of cardiac functions in autocrine and paracrine fashion. The inhibition of IL‐18 expression induced by mechanical stress might be one of the mechanisms that account for the beneficial cardiovascular effects of AT1R antagonist, ETAR blockade, Statin, and Rho‐Kinase inhibitor.

Cyclic strain induces FosB and initiates osteogenic differentiation of mesenchymal cells

Mechanical loading is crucial for bone remodeling and osteoblast differentiation. FosB belongs to the AP-1 family of transcription factors, a group of proteins known to regulate osteoblast differentiation and bone formation. In mice, FosB is rapidly induced by mechanical stress at the transcriptional level. The aim of this study was to determine the effect of different mechanical stretch patterns on FosB gene expression and on osteogenic differentiation of human osteoblast precursor cells. Human bone-marrow-derived mesenchymal precursor cells were grown in flexible silicone dishes and stimulated by a daily application of three rounds of 2 h of cyclic stretch of either 2% or 8% elongation at 1 Hz on 3 consecutive days using a special motor-driven apparatus. By real-time PCR, we quantified FosB mRNA and the expression of genes involved in osteoblast differentiation such as Runx2 and collagen 1 to determine the osteogenic effect of mechanical stretch. Stretching induced FosB transcription and the expression of osteoblast markers in partly committed human mesenchymal precursor cells in a stretch- and time-dependent manner. We conclude that cyclic stretch-induced FosB expression and the upregulation of osteoblast genes plays a role in osteogenic differentiation of human mesenchymal precursor cells.

Mechanical Stimulation of Tissue Engineered Tendon Constructs: Effect of Scaffold Materials

Our group has shown that numerous factors can influence how tissue engineered tendon constructs respond to in vitro mechanical stimulation. Although one study showed that stimulating mesenchymal stem cell (MSC)-collagen sponge constructs significantly increased construct linear stiffness and repair biomechanics, a second study showed no such effect when a collagen gel replaced the sponge. While these results suggest that scaffold material impacts the response of MSCs to mechanical stimulation, a well-designed intra-animal study was needed to directly compare the effects of type-I collagen gel versus type-I collagen sponge in regulating MSC response to a mechanical stimulus. Eight constructs from each cell line (n=8 cell lines) were created in specially designed silicone dishes. Four constructs were created by seeding MSCs on a type-I bovine collagen sponge, and the other four were formed by seeding MSCs in a purified bovine collagen gel. In each dish, two cell-sponge and two cell-gel constructs from each line were then mechanically stimulated once every 5 min to a peak strain of 2.4%, for 8 h/day for 2 weeks. The other dish remained in an incubator without stimulation for 2 weeks. After 14 days, all constructs were failed to determine mechanical properties. Mechanical stimulation significantly improved the linear stiffness (0.048+/-0.009 versus 0.015+/-0.004; mean+/-SEM (standard error of the mean ) N/mm) and linear modulus (0.016+/-0.004 versus 0.005+/-0.001; mean+/-SEM MPa) of cell-sponge constructs. However, the same stimulus produced no such improvement in cell-gel construct properties. These results confirm that collagen sponge rather than collagen gel facilitates how cells respond to a mechanical stimulus and may be the scaffold of choice in mechanical stimulation studies to produce functional tissue engineered structures.

Effect of length of the engineered tendon construct on its structure–function relationships in culture

Constructs containing autogenous mesenchymal stem cells (MSCs) seeded in collagen gels have been used by our group to repair rabbit central patellar tendon defect injuries. Although these cell–gel composites exhibit improved repair biomechanics compared to natural healing, they can be difficult to handle at surgery and lack the necessary stiffness to resist peak in vivo forces early thereafter. MSCs are typically suspended in collagen gels around two posts in the base of a well in a specially designed silicone dish. The distance between posts is approximately the length of the tendon wound site. MSCs contract the gel around the posts prior to removal of the construct for implantation at surgery. We hypothesized that in vitro construct alignment and stiffness might be enhanced in the midregion of the longer construct where the end effects of the posts on the bulk material (St. Venant effects) could be minimized. Rabbit MSCs were seeded in purified bovine collagen gel at 0.04 M cells/mg collagen. The cell–gel mixture was pipetted into silicone dishes having two post-to-post lengths (short: 11 mm and long: 51 mm) but equivalent well widths and depths and post diameters. After 14 days of incubation, tensile stiffness and modulus of the constructs were measured using equivalent grip-to-grip lengths. Collagen fiber orientation index or OI (which measures angular dispersion of fibers) was quantified using small angle light scattering (SALS). Long constructs showed significantly lower angular dispersion vs. short constructs (OI of 41.24°±1.57° vs. 48.43°±1.27°, mean±SEM, p<0.001) with significantly higher linear modulus (0.064±0.009 MPa vs. 0.024±0.004 MPa, p = 0.0022 ) and linear stiffness (0.031±0.005 MPa vs. 0.018±0.004 N/mm, mean±SEM, respectively, p = 0.0404 ). We now plan to use principles of functional tissue engineering to determine if repairs containing central regions of longer MSC-collagen constructs improve defect repair biomechanics after implantation at surgery.

Effects of Cell-to-Collagen Ratio in Stem Cell-Seeded Constructs for Achilles Tendon Repair

The objective of the present study was to test the hypotheses that implantation of cell-seeded constructs in a rabbit Achilles tendon defect model would 1) improve repair biomechanics and matrix organization and 2) result in higher failure forces than measured in vivo forces in normal rabbit Achilles tendon (AT) during an inclined hopping activity. Autogenous tissue-engineered constructs were fabricated in culture between posts in the wells of silicone dishes at four cell-to-collagen ratios by seeding mesenchymal stem cells (MSC) from 18 adult rabbits at each of two seeding densities (0.1 x 10(6) and 1 x 10(6) cell/mL) in each of two collagen concentrations (1.3 and 2.6 mg/mL). After 5 days of contraction, constructs having the two highest ratios (0.4 and 0.8 M/mg) were damaged by excessive cell traction forces and could not be used in subsequent in vivo studies. Constructs at the lower ratios (0.04 and 0.08 M/mg) were implanted in bilateral, 2 cm long gap defects in the rabbit's lateral Achilles tendon. At 12 weeks after surgery, both repair tissues were isolated and either failed in tension (n = 13) to determine their biomechanical properties or submitted for histological analysis (n = 5). No significant differences were observed in any structural or mechanical properties or in histological appearance between the two repair conditions. However, the average maximum force and maximum stress of these repairs achieved 50 and 85% of corresponding values for the normal AT and exceeded the largest peak in vivo forces (19% of failure) previously recorded in the rabbit AT. Average stiffness and modulus were 60 and 85% of normal values, respectively. New constructs with lower cell densities and higher scaffold stiffness that do not excessively contract and tear in culture and that further improve the repair stiffness needed to withstand various levels of expected in vivo loading are currently being investigated.

N-cadherin signals through Rac1 determine the localization of connexin 43 in cardiac myocytes

It has been proposed that the formation of gap junction is influenced by adherens junction in cardiac myocytes. To examine whether signals through N-cadherin are involved in the distribution of connexin 43 (Cx43), the distribution of cell-cell adhesion molecules, N-cadherin and Cx43, was analyzed in aligned cardiac myocytes. To induce cell orientation running in parallel to tension direction, neonatal rat cardiac myocytes were plated for 3 hours and exposed to 20% cyclic stretch for 24 hours on silicone dishes. The aligned cells cultured for 0-5 days were immunostained with anti- N-cadherin or anti-Cx43 antibody. After cultivation for 3-5 days, following the accumulation of N-cadherin, Cx43 was localized at the longitudinal cell termini. Adenoviral gene transfer of dominant negative N-cadherin significantly attenuated the localization of Cx43 at the longitudinal cell termini, suggesting that Cx43 localization is regulated downstream of N-cadherin. In the process of Cx43 localization, Rho family proteins, RhoA and Rac1, were activated, but not Cdc42. RhoA and Rac1 activation was inhibited by the transfection of dominant negative N-cadherin, indicating that RhoA and Rac1 were activated by N-cadherin in the oriented cardiac myocytes. The inhibition of Rho family proteins by Rho GDI significantly attenuated the accumulation of Cx43, but not that of N-cadherin. Furthermore, the translocation of Cx43 to longitudinal cell termini was prevented by the inhibition of Rac1, but not RhoA. Collectively, these findings suggest that the localization of Cx43 was determined through the Rac1 pathway downstream of N-cadherin in cardiac myocytes.

Repetitive mechanical stretching modulates IL-1β induced COX-2, MMP-1 expression, and PGE 2 production in human patellar tendon fibroblasts

While mechanical loading is known to be essential in maintaining tendon homeostasis, repetitive mechanical loading has also been implicated in the etiology of tendon overuse injuries. The purpose of this study was to determine whether cyclic mechanical stretching regulates inflammatory responses induced by interleukin-1β (IL-1β) treatment in human patellar tendon fibroblasts (HPTFs). HPTFs were grown in microgrooved silicone dishes, where they became elongated in shape and aligned with the microgrooves, which is similar to the shape and organization of tendon fibroblasts in vivo. Cyclic uniaxial stretching was then applied to silicone culture dishes with a 4% or 8% stretch at a stretching frequency of 0.5 Hz for a duration of 4 h in the presence or absence of 10 pM IL-1β treatment. Non-stretched cells in the presence or absence of IL-1β were used for controls, respectively. The expression of cyclooxygenase-2 (COX-2), matrix metalloproteinase-1 (MMP-1), and the production of prostaglandin E 2 (PGE 2) were measured. In the absence of stretching, it was found that 10 pM of IL-1β markedly induced higher levels of COX-2, MMP-1 gene expression, and PGE 2 production than non-treated cells. Furthermore, cells with 4% stretching decreased the COX-2 and MMP-1 gene expression and PGE 2 production that were stimulated by IL-1β, whereas cells with 8% stretching further increased these gene products and/or expression levels in addition to the effects of IL-1β stimulation. Thus, the results suggest that repetitive, small-magnitude stretching is anti-inflammatory, whereas large-magnitude stretching is pro-inflammatory. Therefore, moderate exercise may be beneficial to reducing tendon inflammation.

表皮細胞における伸展刺激の影響

Mechanical stress has profound influences on human body. Muscle training strengthens the muscles, and high blood pressure thickens the vessel walls. Intense studies have been made by various investigators on how mechanical stress influences the human tissues. Epidermal keratinocytes are continuously exposed to mechanical forces. The human skin surface can be thickened and enlarged by various stress such as tissue expander or abrasive pressure. Keratinocytes were plated on flexible silicone dishes, and they were continuously stretched. Stretching of keratinocytes caused up-regulation of 5-bromo-2'-deoxyuridine (BrdU)-positive cells and activation of extracellular signal-regulated kinases (ERK) 1/2. EGF receptor, calcium channel, and ERK were involved in stretch-induced BrdU incorporation. Stretching also induced keratin K6, which is expressed in activated and proliferating keratinocytes, and suppressed keratin K10, which is expressed in differentiated keratinocytes, and their regulation was inhibited by MEK1/2 inhibitor. EGF receptor as well as adhesion molecules have been reported to be involved in transducing mechanical stresses. Several diseases involving heart and palmoplantar skin are caused by the mutation in desmosomal proteins, indicating that adhesion molecules play an important part in sustaining normal structure of skin under the mechanical stress.

N-cadherin-mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes

Mechanical stretch has been implicated as the growth stimuli in the heart. Physiologically, mechanical stretch is reported to contribute to the orientation of cardiomyocytes, though the molecular mechanism remains to be elucidated. This study was designed to make clear functional significances of N-cadherin in plasticity of cell alignment in response to mechanical stretch. Neonatal rat cardiomyocytes, cultured on silicone dishes, were subjected to artificial uniaxial cyclic stretch. Mechanical stretch was started at certain times (3–75 h) after seeding and continued for 24 h. Stretch stimulation in 3 h after cultivation promoted cell orientation running parallel to tension direction. In contrast, cardiac myocytes fail to align when exposed to stretch 24–75 h after cultivation. To address the importance of N-cadherin in the responsiveness to stretch, the expression and distribution of N-cadherin were analyzed. Immediately after seeding, N-cadherin showed dispersed distributions. During cultivation, N-cadherin localized to cell–cell contacts accompanied by the upregulation of its protein. Next, to investigate influence of cell–cell adhesion, cardiomyocytes cultured for 72 h were replated by trypsin treatment and exposed to stretch 3 h after replating. The cardiomyocytes replated by trypsinization were oriented in parallel to tension direction by mechanical stretch. Finally, adenoviral transfection of dominant-negative N-cadherin recovered the ability to exhibit cell orientation in response to stretch. Our results suggested that N-cadherin was involved in the oriented responses of cardiomyocytes induced by mechanical stretch.

051 Superoxide Regulates α‐SMA via p‐38 MAPK

This study was to investigate the effect of cyclic mechanical stretching on superoxide and nitric oxide production in human patellar tendon fibroblasts (HPTFs) and to establish the role of superoxide and p38 MAPK in stretching‐induced α‐smooth muscle actin (α‐SMA) expression. When HPTFs were grown in deformable silicone dishes and subjected to 8% cyclic, uniaxial stretching, it was found that HPTFs markedly increased the production of superoxide and nitrite but not nitric oxide. And, cyclic stretching of HPTFs increased phosphorylation of p38 MAPK with increasing stretching magnitude. In addition, stretching duration also influenced the expression of phosphorylated p38 MAPK, where the dependence followed a pattern of cellular response in p38 MAPK activation consistent with previous studies using human mesangial cells and rat 3Y1 fibroblasts. For short stretching durations (∼15 min), the expression of phosphorylated p38 MAPK increased rapidly but decreased for longer stretching durations. Furthermore, in HPTFs treated with superoxide dismutase (SOD), which converts superoxide anions to hydrogen peroxide and an oxygen molecule, levels of stretching‐induced p38 MAPK phosphorylation and α‐SMA expression decreased compared to stretched fibroblasts not treated with SOD. Similarly, cells treated with SB202190, which specifically inhibits p38 MAPK activation, also decreased α‐SMA expression levels. Therefore, these results suggest that superoxide regulates stretching‐induced α‐SMA expression via p38 MAPK activation in HPTFs subjected to cyclic stretching conditions.

Mechanical Stretching In Vitro Regulates Signal Transduction Pathways and Cellular Proliferation in Human Epidermal Keratinocytes

Epidermal keratinocytes are continuously exposed to mechanical forces. The human skin surface can be thickened and enlarged by various stresses such as tissue expander or abrasive pressure. To investigate the mechanism of epidermal hyperproliferation by mechanical stress, keratinocytes were plated on flexible silicone dishes, which were continuously stretched by +20%. Stretching of cells for 24 h caused upregulation of 5-bromo-2'-deoxyuridine (BrdU)-positive cells to 200%-220% and activation of extracellular signal-regulated kinases (ERK)1/2. Inhibition of mitogen and ERK with U0126 and phosphoinositide 3-OH kinase attenuated BrdU incorporation and ERK1/2 activation. The EGF receptor kinase inhibitor and the calcium channel inhibitor also inhibited BrdU incorporation and the activation of ERK1/2. Twenty-four hours of stretching stimulated reporter activity driven by activator protein 1 (AP-1), induction of K6, and suppression of K10, which were inhibited by U0126. Our results indicate that mechanical stretching induces proliferative signals on human keratinocytes via induction of calcium influx, phosphorylation of epidermal growth factor receptor (EGFR), and ERK1/2. These mechanisms may contribute to the hyperproliferative nature of the epidermis, which is mechanically stretched by various stimuli.

Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions

We studied the effect of cyclic mechanical stretching on the proliferation and collagen mRNA expression and protein production of human patellar tendon fibroblasts under serum-free conditions. The role of transforming growth factor- β1 (TGF- β1) in collagen production by cyclically stretched tendon fibroblasts was also investigated. The tendon fibroblasts were grown in microgrooved silicone dishes, where the cells were highly elongated and aligned with the microgrooves. Cyclic uniaxial stretching with constant frequency and duration (0.5 Hz, 4 h) but varying magnitude of stretch (no stretch, 4%, and 8%) was applied to the silicone dishes. Following the period of stretching, the cells were rested for 20 h in stretching-conditioned medium to allow for cell proliferation. In separate experiments, the cells were stretched for 4 h and then rested for another 4 h. Samples of the medium, total cellular RNA and protein were used for analysis of collagen and TGF- β1 gene expression and production. It was found that there was a slight increase in fibroblast proliferation at 4% and 8% stretch, compared to that of non-stretched fibroblasts, where at 8% stretch the increase was significant. It was also found that the gene expression and protein production of collagen type I and TGF- β1 increased in a stretching-magnitude-dependent manner. And, levels of collagen type III were not changed, despite gene expression levels of the protein being slightly increased. Furthermore, the exogenous addition of anti-TGF- β1 antibody eliminated the increase in collagen type I production under cyclic uniaxial stretching conditions. The results suggest that mechanical stretching can modulate proliferation of human tendon fibroblasts in the absence of serum and increase the cellular production of collagen type I, which is at least in part mediated by TGF- β1.

Cyclic strain influences the expression of the vascular endothelial growth factor (VEGF) and the hypoxia inducible factor 1 alpha (HIF-1α) in tendon fibroblasts

Neovascularization is involved in beneficial and detrimental processes of tendon pathology. We investigated the influence of repetitive motion on the expression of the most important angiogenic factor, the vascular endothelial growth factor (VEGF) in the 3T3 NIH fibroblast cell line and in cultures of rat Achilles tendon fibroblasts. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic uniaxial movement. Strain was applied over 24 h varying the frequency (0.5–1 Hz). Fibroblasts (3T3 fibroblasts and rat Achilles tendon cultures) cultivated without the application of cyclic strain released measurable VEGF amounts into their culture supernatants. Cyclic stretching of the cells with a frequency of 1 Hz resulted in an increased expression of VEGF. A low frequency (0.5 Hz) reduced VEGF expression to control levels. RT PCR revealed VEGF 121 and VEGF 165 as the only splice forms that were induced by cyclic stretching. Western blot experiments could further show that cyclic stretching induced activation of the transcription factor HIF-1α. These results demonstrate that mechanical factors are involved in the regulation of VEGF expression in tendon tissue.