Human Patellar Tendon Fibroblasts Research Articles

Cyclic Mechanical Stretching Induces Autophagic Cell Death in Tenofibroblasts Through Activation of Prostaglandin E2 Production

Background/Aims: Autophagic cell death has recently been implicated in the pathophysiology of tendinopathy. Prostaglandin E2 (PGE2), a known inflammatory mediator of tendinitis, inhibits tenofibroblast proliferation in vitro; however, the underlying mechanism is unclear. The present study investigated the relationship between PGE2 production and autophagic cell death in mechanically loaded human patellar tendon fibroblasts (HPTFs) in vitro. Methods: Cultured HPTFs were subjected to exogenous PGE2 treatment or repetitive cyclic mechanical stretching. Cell death was determined by flow cytometry with acridine orange/ethidium bromide staining. Induction of autophagy was assessed by autophagy markers including the formation of autophagosomes and autolysosomes (by electron microscopy, AO staining, and formation of GPF-LC3-labeled vacuoles) and the expression of LC3-II and BECN1 (by western blot). Stretching-induced PGE2 release was determined by ELISA. Results: Exogenous PGE2 significantly induced cell death and autophagy in HPTFs in a dose-dependent manner. Blocking autophagy using inhibitors 3-methyladenine and chloroquine, or small interfering RNAs against autophagy genes Becn-1 and Atg-5 prevented PGE2-induced cell death. Cyclic mechanical stretching at 8% and 12% magnitudes for 24 h significantly stimulated PGE2 release by HPTFs in a magnitude-dependent manner. In addition, mechanical stretching induced autophagy and cell death. Blocking PGE2 production using COX inhibitors indomethacin and celecoxib significantly reduced stretching-induced autophagy and cell death. Conclusion: Taken together, cyclic mechanical stretching induces autophagic cell death in tenofibroblasts through activation of PGE2 production.

Repetitive mechanical stretching modulates transforming growth factor-β induced collagen synthesis and apoptosis in human patellar tendon fibroblasts

The cellular and molecular mechanisms underlying the development of tendinopathy are not clear, but inflammatory mediators produced by tendon fibroblasts in response to repetitive mechanical loading may be an important factor for this illness. In this study, we explored the effect of cyclic mechanical stretching on collagen synthesis and apoptosis of human patellar tendon fibroblasts (HPTFs). The role of a candidate inflammatory mediator, transforming growth factor-β1 (TGFβ1), which we identified in a cytokine antibody array, in collagen synthesis and apoptosis during repetitive mechanical stretching was also investigated. Our results showed that there was a significant increase in collagen type I synthesis at 4% and 8% stretch. Significantly, enhancement of apoptosis may account for the observed decrease in fibroblast numbers after 8% stretching. Furthermore, the exogenous addition of an anti-TGFβ1 antibody or gene silencing by si-TGFβ1 eliminated the increase in collagen type I production and activities of caspases during apoptosis under cyclic uniaxial stretching conditions. These results suggest that TGFβ1 may take part in the increase of cellular production of collagen type I and apoptosis during the development of tendinopathy. Furthermore, caspase 8 mediates activation of caspase 3 and poly ADP-ribose polymerase (PARP) cleavage during TGFβ1-induced apoptosis in stretching HPTFs.

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)

Leukotriene B 4 at low dosage negates the catabolic effect of prostaglandin E 2 in human patellar tendon fibroblasts

Tendinopathy often involves inflammation and matrix degeneration. The inflammatory mediators such as prostaglandin E 2 (PGE 2) and leukotriene B 4 (LTB 4) are implicated in the development of tendinopathy. Therefore, the purpose of this study was to determine the effect of PGE 2 and LTB 4 on the proliferation of human patellar tendon fibroblasts (HPTFs), the gene expression of collagen type I, MMP-1 and MMP-3, as well as the protein secretion of these gene products by the cells. The results showed that LTB 4 at low doses (0.1 and 1 nM) significantly increased cell proliferation compared to controls and LTB 4 at 0.1 nM negated the PGE 2-induced decrease in cell proliferation. In addition, PGE 2 at 100 ng/ml significantly increased the expression of MMP-1 and MMP-3 at both mRNA and protein levels. These stimulatory effects were significantly diminished by co-treatment with LTB 4 at 0.1 nM. Finally, neither PGE 2 nor LTB 4 treatment affected collagen type I gene expression. These results suggest that low levels of LTB 4 counterbalance the negative effects mediated by PGE 2 on tendon fibroblast proliferation and MMP production, which may lead to matrix degradation. Thus, our findings suggest that although LTB 4 is generally thought to be pathogenic, low levels of LTB 4 are actually beneficial in maintaining tendon tissue homeostasis.

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.

Downregulation of Human Type III Collagen Gene Expression by Antisense Oligodeoxynucleotide

The amount of type III collagen is increased during the early healing stage following ligament and tendon injury. Concomitantly, the mechanical properties of the healing tissues are abnormal and the fibril diameters are homogeneously small. It is therefore thought that downregulating type III collagen gene expression after injury may be helpful in improving the quality of healing tissue. In the current study, the efficacy of using antisense oligodeoxynucleotides (ODNs) to downregulate type III collagen gene expression in human patellar tendon fibroblasts (HPTFs) was tested, with Lipofectamine reagent used to deliver the ODN. It was shown that the majority of HPTFs can efficiently uptake antisense ODN from as early as 1 h to as long as 3 days after delivery; also, one selected ODN can consistently inhibit human type III collagen gene expression at both the mRNA and protein levels. Reverse transcriptase-polymerase chain reaction results showed that the inhibitory effects by this ODN were significant at 1 day, as the type III collagen mRNA level was 38.9 +/- 19.6 and 42.8 +/- 28.1% of missense and sense controls, respectively (p < 0.05). At 3 days, these differences could no longer be observed (p >0.05), but the amount of type III collagen protein was significantly less than for missense and sense controls (31.7 +/- 5.5 and 25.3 +/- 5.3%, respectively; p < 0.05). At 5 days after the delivery, these differences in protein were no longer observed (p > 0.05). Immunohistochemical staining of the type III collagen confirmed these results. The findings of this study demonstrate that antisense ODN can downregulate type III collagen gene expression of tendon fibroblasts. Therefore, this approach offers the potential to explore the effect of the reduction of type III collagen in healing ligaments and tendons as a means to improve their mechanical properties.

Bone morphogenetic protein 13 stimulates cell proliferation and production of collagen in human patellar tendon fibroblasts

Background Recombinant human (rh) bone morphogenetic protein 13 (BMP13) has been shown to induce the formation of tendon and ligament tissues in animal experiments. The role of BMP13 in tissue regeneration in human tendons remains unexplored, however.Material and methods We collected healthy human patellar tendon samples for histological examination and tendon fibroblast culture. The cultured cells were incubated in the presence and absence of rhBMP13 and the effect of the protein on cell proliferation was measured using 5-bromo-2’-deoxyuridine uptake.Results BMP13 was detectable by immunohisto-chemical staining in healthy patellar tendon samples, and was located exclusively in active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of proliferating cell nuclear antigen (PCNA) and pro-collagen type I showed a similar distribution. In vitro studies showed that rhBMP13 can increase proliferation of tendon fibroblasts and increase the gene expression of pro-collagen type I in tendon fibroblast culture.Interpretation Our findings indicate that BMP13 may be involved in the matrix remodeling process in adult tendon, and that it may play a role in tissue regeneration in tendons.

Controlling Cell Responses to Cyclic Mechanical Stretching

In most cell culture studies, cells are grown on smooth culture surfaces. Using microfabrication technology, we have developed microgrooved silicone surfaces to grow cells and subject them to repetitive mechanical stretching. When human patellar tendon fibroblasts were plated on these microgrooved surfaces, the cells had an elongated shape and underwent cyclic uniaxial stretching parallel to their long axes, all of which closely mimic conditions of tendon fibroblasts in vivo. Also, when fibroblasts were grown on microgrooves oriented at 45 and 90 degrees with respect to stretching direction, they did not change alignment or shape under cyclic mechanical stretching. Furthermore, compared to nonstretched cells, 8% cyclic stretching of tendon fibroblasts oriented at 0 (i.e., parallel to stretching direction), 45, and 90 degrees was found to increase alpha-SMA protein expression level by 46, 31, and 14%, respectively. In addition, 8% cyclic stretching tendon fibroblasts for 4 and 8 h oriented parallel to stretching direction increased alpha-SMA protein expression level by 25 and 57%, respectively. Thus, the results of this study showed that alpha-SMA protein expression levels of tendon fibroblasts depend on cell orientation with respect to stretching direction and stretching duration. We suggest that microgrooved silicone substrates can be used to study biological responses of tendon or ligament fibroblasts to repetitive mechanical stretching conditions in a more controlled manner.

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.

Inflammatory Response of Human Tendon Fibroblasts to Cyclic Mechanical Stretching

Background The cellular and molecular mechanisms for the development of tendinopathy are not clear, but inflammatory mediators produced by tendon fibroblasts in response to repetitive mechanical loading may be an important factor. Hypotheses (1) Cyclic stretching of tendon fibroblasts affects the production of leukotriene B4 and the expression of 5-lipoxygenase; and (2) the production level of leukotriene B4 is inversely related to that of prostaglandin E2. Study Design Controlled laboratory study. Methods Human patellar tendon fibroblasts were uniaxially stretched in the presence of indomethacin (25 μM) or MK-886 (10 μM). After stretching for 4 hours, followed by 4 hours rest, levels of prostaglandin E2, leukotriene B4, and expression of 5-lipoxygenase were measured. Results Stretched tendon fibroblasts increased the levels of leukotriene B4 but did not appreciably change the expression of 5-lipoxygenase. Indomethacin decreased the cellular production of prostaglandin E2 but caused increased leukotriene B4 levels. MK-886 caused decreased production of leukotriene B4 but increased production of prostaglandin E2. Conclusions Cyclic stretching of human tendon fibroblasts increases the production of prostaglandin E2 and leukotriene B4. Blocking prostaglandin E2 production leads to increased leukotriene B4 levels and vice versa. Clinical Relevance The use of nonsteroidal anti-inflammatory drugs for the treatment of tendon inflammation might increase the levels of leukotriene B4 within the tendon, potentially contributing to the development of tendinopathy.

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.

Effect of Agkistroden blomhoffi (mamushi) on the proliferation of human fibroblasts

Objectives: The purpose of this study is to investigate the effect of Agkistroden blomhoffi (mamushi) aqueous extract on human patellar tendon cells in vitro, to pharmacologically explain the natural medicine's healing effect on tendon, bone and muscle injuries. Design and methods: Human patellar tendon fibroblasts (HPTF) were incubated in media containing different concentrations of mamushi aqueous extract. Cell proliferation was studied by microscopic observations and total protein, actin, collagen I, and cyclooxygenase-2 (Cox 2) expressions. Results: Mamushi aqueous extract enhanced HPTF proliferation when its concentration was lower than 333 μg/ml. Cells cultured in manushi-containing medium showed developed intercellular structure and increased protein production. However, mamushi extract higher than 500 μg/ml oppressed cell growth. At 667 μg/ml, mamushi induced Cox 2 production, a sign of cytotoxicity. Conclusion: A. blomhoffi aqueous extract was found to directly stimulate the proliferation and protein production, particularly collagen I synthesis, of HPTF in a dose-dependent manner.

Fibroblast responses to cyclic mechanical stretching depend on cell orientation to the stretching direction

Fibroblasts in intact tendons align with stretching direction, but they tend to orient randomly in healing tendons. Therefore, a question arises: Do fibroblast responses to mechanical stretching depend on their orientation? To address this question, human patellar tendon fibroblasts were grown in custom-made silicone dishes that possess microgrooved culture surfaces. The direction of the microgrooves was either parallel or normal to the direction of cyclic uniaxial stretching. Fibroblasts grown in these microgrooves had a polar morphology and oriented along the direction of the microgrooves regardless of the stretching conditions. Tendon fibroblasts expressed higher levels of α-smooth muscle actin when they were oriented parallel to the stretching direction than when they were oriented normal to the stretching direction. Also, cyclic stretching of the fibroblasts perpendicular to their orientation induced a higher activity level of secretory phospholipase A 2 compared with stretching of the cells parallel to their orientation. Thus, these results show that fibroblast responses to mechanical stretching depend on cell orientation to the stretching direction.

Paper #156 The effects of bone morphogenetic proteins 13 on human patellar tendon fibroblasts

Bone morphogenetic proteins (BMPs) are a family of proteins that can induce bone and cartilage formation during embryogenesis and tissue repair. Human recombinant (hr) BMP13 has been recently cloned and proved to promote the formation of tendon and ligament tissues in in vivo experiments. But the roles of BMP13 on tissue regeneration in tendons remain unexamined. In the present study, human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, chiefly located on active tenoblasts and perivascular mesenchymal cells, with a similar distribution as proliferation cell nuclear antigens (PCNA), pro-collagen I and decorin. In vitro studies on tendon fibroblast culture showed that hrBMP13 could increase cell proliferation and the gene expression of pro-collagen I and biglycan, but the gene expression of decorin was not affected. Our findings suggest that BMP13 may play a role in tendon healing. Bone morphogenetic proteins (BMPs) are a family of proteins that can induce bone and cartilage formation during embryogenesis and tissue repair. Human recombinant (hr) BMP13 has been recently cloned and proved to promote the formation of tendon and ligament tissues in in vivo experiments. But the roles of BMP13 on tissue regeneration in tendons remain unexamined. In the present study, human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, chiefly located on active tenoblasts and perivascular mesenchymal cells, with a similar distribution as proliferation cell nuclear antigens (PCNA), pro-collagen I and decorin. In vitro studies on tendon fibroblast culture showed that hrBMP13 could increase cell proliferation and the gene expression of pro-collagen I and biglycan, but the gene expression of decorin was not affected. Our findings suggest that BMP13 may play a role in tendon healing.

Antisense Oligonucleotides Reduce Synthesis of Procollagen α1 (V) Chain in Human Patellar Tendon Fibroblasts: Potential Application in Healing Ligaments and Tendons

Antisense Oligonucleotides Reduce Synthesis of Procollagen α1 (V) Chain in Human Patellar Tendon Fibroblasts: Potential Application in Healing Ligaments and Tendons

The roles of bone morphogenetic protein (BMP) 12 in stimulating the proliferation and matrix production of human patellar tendon fibroblasts

Recombinant human (rh) bone morphogenetic protein 12 (BMP12) is proved to induce the formation of tendon and ligament tissues in animal experiments. But the roles of BMP12 on tissue regeneration in human tendons remain unexplored. In the present study, healthy human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, only located on active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of PCNA and procollagen type I also exhibited a similar distribution. It indicates that BMP12 may be involved in matrix remodeling process in adult tissues. In vitro studies showed that rhBMP12 could increase proliferation of tendon fibroblasts and increase the gene expression of procollagen type I and type III, but decrease the gene expression of decorin in tendon fibroblasts culture. Our findings suggest that BMP12 may play a role in early phases of tissue regeneration in tendons.

Cyclic mechanical strain induces NO production in human patellar tendon fibroblasts – a possible role for remodelling and pathological transformation

The mechanism by which tendon fibroblasts can detect strain forces and respond to them is fairly unknown. Nitric oxide (NO) is a messenger molecule that among others can respond to shear stress in endothelial cells. Therefore, it was investigated whether cyclic mechanical strain induces NO in vitro in human patellar tendon fibroblasts. Human patellar tendon fibroblasts were cultured from remnants of patellar tendon transplants after reconstructive surgery. Fibroblasts were cultured on elastic silicone dishes. The cells were longitudinally strained (5%, 1 Hz) for 15' or 60'. As a control, no strain was applied. The experiments were finished after 0', 5', 15', and 30'. NO was determined using the Griess reaction. 15' strain showed at 0' and 5' 200% activation, which thereafter at 15' and 30' returned to normal levels. 60' strain showed a biphasic pattern. At 5' and 30', NO levels were increased to 175%. At 15', NO measurement displayed 120% increased levels. Mechanical strain induces NO production by tendon fibroblasts. Therefore, NO produced by tendon fibroblasts, as a response to alteration in their mechanical microenvironment, could modulate fibroblast function. The results of our study suggests that strain-related adaptive changes may, at least in part, be controlled by a process in which strain-related NO production from the fibroblast network may play a pivotal role. Moreover, these are basic findings that are important for further unravelling pathophysiology of tendon diseases.

Antisense Oligonucleotides Reduce Synthesis of Procollagen α1 (V) Chain in Human Patellar Tendon Fibroblasts: Potential Application in Healing Ligaments and Tendons

Many ligaments and tendons will heal after injury. However, they heal with poor mechanical properties when compared with the native tissue and show no improvement of these properties with time. Although the mechanisms that lead to this process are poorly understood, the presence of uniformly smaller collagen fibrils is believed to play a major role. Quantitatively minor when compared with type I collagen, type V collagen was found to be significantly elevated in healing medial collateral ligament of the rabbit knee. Previous studies have shown that type V collagen plays a role in regulating the diameter of type I collagen fibrils and reducing its level may lead to the formation of larger collagen fibrils in healing ligaments. Hence, type V collagen antisense gene therapy may be an approach to obtain this goal. In this study, our objective was to find specific antisense oligonucleotide sequences for type V procollagen f 1 chain to elucidate the feasibility of type V collagen antisense gene therapy in ligaments or tendons. We hypothesized that antisense oligonucleotides that selectively target the type V procollagen f 1 chain mRNA could partially reduce the synthesis of type V procollagen f 1 chain in human tendon fibroblasts. Western blotting analysis showed that antisense oligonucleotides (AS-V1 and AS-V2) significantly reduced synthesis of type V procollagen f 1 chain. In addition, reverse transcription polymerase chain reaction revealed that both antisense oligonucleotides partially reduced type V procollagen f 1 chain mRNA expression. This experiment identified two sequences within the type V procollagen coding region that are susceptible to antisense suppression, and thus provide the basis to explore the effects of antisense oligonucleotides on type V collagen synthesis, collagen fibril diameter, and mechanical properties of healing tendons and ligaments.

Cyclic Mechanical Stretching of Human Tendon Fibroblasts Increases the Production of Prostaglandin E 2 and Levels of Cyclooxygenase Expression: A Novel In Vitro Model Study

Prostaglandin E 2 (PGE 2 ) is a known inflammatory mediator of tendinitis, for which mechanical loading on tendons is believed to be one of the most prominent causation factors. Previous in vitro studies have shown that cyclic mechanical stretching of cells can cause changes in cell morphology and alteration of both DNA and protein syntheses. In our study, a novel system was used whereby tendon fibroblasts are cultured on microgrooved silicone surfaces and are subjected to cyclic uniaxial stretching along their long axes to mimic in vivo conditions. Using this unique model system, the cell shape and alignment can be controlled. Further, this study was designed to test the hypotheses that PGE 2 production increases in a stretching magnitude-dependent manner and that cyclooxygenase (COX) is responsible for the increased PGE 2 production in tendon fibroblasts. Human patellar tendon fibroblasts were cultured on the microgrooved silicone membranes and cyclically stretched at 4%, 8%, or 12% of nominal dish length for 24 hr. PGE 2 production was found to be increased 1.7-fold at 8% cyclic stretching and 2.2-fold at 12% cyclic stretching compared with nonstretched controls. In addition, human tendon fibroblasts had increased expression of both COX-1 and COX-2 for all three applied stretching magnitudes, with the exception of COX-1 at 4% cyclic stretching. Also, cellular PGE 2 production, after 8% cyclic stretching, was significantly decreased with the addition of indomethacin (25 w M), a COX competitive inhibitor, compared with stretched cells without indomethacin treatment. These findings suggest that the increase in PGE 2 production by the human tendon fibroblasts is stretching magnitude-dependent, and that the increase in COX expression contributes to the increased production of PGE 2 after cyclic stretching. As PGE 2 is a known inflammatory mediator of tendinitis, the contribution of COX-1 and COX-2 to PGE 2 production and their roles in tendon inflammation are clearly indicated.

Cyclic mechanical stretching of human patellar tendon fibroblasts: activation of JNK and modulation of apoptosis.

Accelerated rehabilitation after tendon, ligament and bone injuries is widely accepted to avoid adverse effects of immobilization. However, progressive rehabilitation may also lead to an excessive inflammatory soft tissue response and often leads to structural and functional problems such as excessive scarring. The equivalent at the molecular/cellular level is in part the regulation of the sensitive homeostasis between proliferation and apoptosis. However, little attention has been paid to this aspect of tendon pathogenesis. This study investigated the response profile of human tendon fibroblasts in terms of apoptosis and anticipated alteration of Jun N-terminal kinase (JNK) activation to cyclic mechanical stretching. Human tendon fibroblasts of six patients were stretched for 15 or 60 min with 1 Hz and an elongation of 5%. Activation of stress-activated protein kinase (SAPK)/JNK was measured by western blot analysis. Apoptotic cells were determined in the stretched cells and in controls by annexin-V staining and detection by flow cytometry. Additionally DNA laddering was determined by ligation-mediated (LM) polymerase chain reaction (PCR). Application of 15 and 60 min stretch increased activation of SAPK/JNK at a maximum after 60 min. However, JNK activation after the longer stretch period 50% less than after the shorter stretch period (15 min). The apoptosis rate was correspondingly increased after short stretch application but not after longer stretch. This might be caused by an inactivation of the activated JNKs by cell protection mechanisms. The findings suggest that mechanical stretching directly activates intracellular signaling pathways, which in turn induce apoptosis. The longer stretch period resulted in a decreased apoptotic rate due to development of stress tolerance. This might be caused by heat-shock protein mediated suppression of JNK activation. This novel observation is an important issue, as defined mechanical stretching, depending on its duration, modulates apoptosis and thus affects tendon remodeling.