Effect Of Cyclic Mechanical Strain Research Articles

Cyclic tensile strain promotes the osteogenic differentiation of a bone marrow stromal cell and vascular endothelial cell co-culture system

Mechanical stimuli and neovascularization are closely coupled to osteogenic differentiation and new bone formation. The purpose of present study was to detect the effect of cyclic mechanical strain on a co-culture system of bone marrow stromal cells (BMSCs) and vascular endothelial cells (VECs) and to clarify the related mechanisms. Primary BMSCs and VECs were isolated from Sprague-Dawley rats and co-cultured at various ratios (1:0, 1:2, 1:4, 4:1, 2:1, 1:1, and 0:1). To determine optimized loading conditions, the cells were then subjected to various cyclic tensile strains (0%, 3%, 6% and 9%) using a Flexcell 5000 mechanical loading system. A protocol of 6% strain on the co-cultured cells at a 1:1 ratio was selected as the optimized culture conditions based on the best osteogenic effects, which included increased ALP activity, matrix mineralization and the expressions of VEGF, Runx-2 and Col-1. The VEGF-R inhibitor tivozanib was used to analyze the paracrine role of VEGF, and the osteogenesis-promoting effects of 6% tensile strain were abrogated in the co-cultured cells treated with tivozanib. These results demonstrate that cyclic tensile strain promotes osteogenic differentiation in BMSC/VEC co-culture systems, possibly via a VEC-mediated paracrine effect of VEGF on BMSCs.

Cyclic mechanical strain induces TGFβ1-signalling in dermal fibroblasts embedded in a 3D collagen lattice.

Many tissues are constantly exposed to mechanical stress, e.g. shear stress in vascular endothelium, compression forces in cartilage or tensile strain in the skin. Dermal fibroblasts can differentiate into contractile myofibroblasts in a process requiring the presence of TGFβ1 in addition to mechanical load. We aimed at investigating the effect of cyclic mechanical strain on dermal fibroblasts grown in a three-dimensional environment. Therefore, murine dermal fibroblasts were cultured in collagen gels and subjected to cyclic tension at a frequency of 0.1 Hz (6 cycles/min) with a maximal increase in surface area of 10 % for 24 h. This treatment resulted in a significant increase in active TGFβ1 levels, leaving the amount of total TGFβ1 unaffected. TGFβ1 activation led to pSMAD2-mediated transcriptional elevation of downstream mediators, such as CTGF, and an auto-induction of TGFβ1, respectively.

Uniaxial cyclic strain of human adipose–derived mesenchymal stem cells and C2C12 myoblasts in coculture

Tissue engineering skeletal muscle in vitro is of great importance for the production of tissue-like constructs for treating tissue loss due to traumatic injury or surgery. However, it is essential to find new sources of cells for muscle engineering as efficient in vitro expansion and culture of primary myoblasts are problematic. Mesenchymal stem cells may be a promising source of myogenic progenitor cells and may be harvested in large numbers from adipose tissue. As skeletal muscle is a mechanically dynamic tissue, we have investigated the effect of cyclic mechanical strain on the myogenic differentiation of a coculture system of murine C2C12 myoblasts and human adipose–derived mesenchymal stem cells. Fusion of mesenchymal stem cells with nascent myotubes and expression of human sarcomeric proteins was observed, indicating the potential for myogenic differentiation of human mesenchymal stem cells. Cyclic mechanical strain did not affect the fusion of mesenchymal stem cells, but maturation of myotubes was perturbed.

The effect of cyclic mechanical strain on activation of dendritic cells cultured on adhesive substrates

Dendritic cells (DCs), key regulators of tolerance and immunity, have been found to reside in mechanically active tissues such as the interior layers of the arterial wall, which experience cyclic radial wall strain due to pulsatile blood flow. Although experimentally difficult to determine in vivo, it is reasonable to postulate DCs experience the mechanical forces in such mechanically active tissues. However, it is currently unknown how DCs respond to cyclic mechanical strain. In order to explore the hypothesis that DCs are responsive to mechanical strain, DCs were cultured in vitro on pre-adsorbed adhesive proteins (e.g., laminin, collagen, fibrinogen) and 1 Hz cyclic strain was applied for various durations and strain magnitudes. It was determined that a strain magnitude of 10% and 24 h duration adversely affected DC viability compared to no-strain controls, but culture on certain adhesive substrates provided modest protection of viability under this harsh strain regime. In contrast, application of 1 h of 1 Hz cyclic 3% strain did not affect DC viability and this strain regime was used for the remaining experiments for quantifying DC activation and T-cell priming capability. Application of 3% strain increased expression of stimulatory (MHC-II) and costimulatory molecules (CD86, CD40), and this effect was generally increased by culture on pre-coated adhesive substrates. Interestingly, the cytokine secretion profile of DCs was not significantly affected by strain. Lastly, strained DCs demonstrated increased stimulation of allogeneic T-cell proliferation, in a manner that was independent of the adhesive substrate. These observations indicate generation of a DC consistent with what has been described as a semi-mature phenotype. This work begins elucidating a potential role for DCs in tissue environments exposed to cyclic mechanical forces.

The effect of cyclic mechanical strain on the expression of adhesion‐related genes by periodontal ligament cells in two‐dimensional culture

Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6-24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12 h to a 2.49-fold downregulation of COL11A1 at 24 h. The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases.

Synergistic Effect of Cyclic Mechanical Strain and Activated Macrophage Cells on Nitinol Corrosion

Several previous studies have documented that self-expanding (SE) Nitinol stents implanted in highly mobile peripheral arteries exhibit a fracture rate as high as 30%. The hypothesis of this study is that repetitive mechanical stresses interact synergistically with the atherosclerotic lesion environment to induce corrosive events that ultimately lead to nitinol stent material failure. Using a novel in vitro chamber, electro-polished nitinol coupons were subjected to cyclic defined stress regimens in the presence or absence of adherent acetylated lipoprotein-activated macrophage-like (THP-1) cells over a 5 day period. Nitinol surface corrosion was monitored in real time using open circuit potential (OCP) measurements. Changes in surface chemistry and topography were measured post-stress exposure using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The combination of defined bending in the presence of adherent activated macrophages resulted in a significant increase in nitinol surface corrosion as compared to nitinol subjected to either treatment or media exposure alone. Further, a deterioration of the protective oxide layer and an increase in the surface roughness were observed in nitinol specimens exposed to the combined influence of surface-adherent macrophages and repetitive loading. These results demonstrate a significant interaction between the biological and mechanical environment relative to nitinol corrosion potential and provide a basis for design of future studies to improve the long-term performance of peripheral nitinol stents.

Effect of cyclic mechanical strain on glycosaminoglycan and proteoglycan synthesis by heart valve cells

Heart valves are presumed to remodel their extracellular matrix upon application of mechanical strains. In this study, we investigated the effect of cyclic tensile strain on valvular interstitial cells’ synthesis of glycosaminoglycans (GAGs) and proteoglycans (PGs), which are altered during myxomatous degeneration. Interstitial cells were isolated from mitral valve leaflets and chordate, and seeded separately within three-dimensional collagen gels. Cell-seeded collagen gels were then subjected to cyclic strains of 2%, 5% or 10% at 1.16Hz for 48h using a custom-built stretching device. The application of cyclic strains reduced the total GAGs retained within collagen gels in a magnitude-dependent manner for both leaflet and chordal cells. With increasing strain magnitude, however, secretion of total GAGs into the medium was reduced for leaflet cells and elevated for chordal cells. Retention of 4-sulfated GAGs increased with increasing strain magnitude for both cell types; for the chordal samples, retention of 6-sulfated GAGs was reduced at higher strain magnitudes. Compared to statically constrained or unconstrained conditions, the application of cyclic strain reduced the secretion of 6-sulfated GAGs by both cell types, and elevated secretion of 4-sulfated GAGs by leaflet cells only. Retention of the PG biglycan and secretion of the PG decorin was significantly reduced at 10% strain compared to 2% strain. In addition, there were numerous differences in the strain-dependent retention and secretion of GAGs and PGS within the leaflet and chordal groups. These results demonstrate that GAG and PG synthesis by VICs is regulated by cyclic stretching conditions.

Reference gene selection for real‐time polymerase chain reaction in human lung cells subjected to cyclic mechanical strain

The respiratory system is constantly exposed to mechanical forces that influence cellular phenotype in health and disease. Quantitative real-time PCR (qPCR) is widely used to determine gene expression. The validity of qPCR depends on using stable reference genes for normalization. The effect of cyclic mechanical strain on reference gene expression by lung epithelial, fibroblast and endothelial cells has not been studied systematically. The stability of expression of fourteen potential reference genes in response to six different regimens of cyclic mechanical strain was ranked using the geNorm tool in human lung epithelial cell lines (A549 and H441), human fetal lung fibroblasts (HFL-1), human lung microvascular endothelial cells, primary human lung fibroblasts and primary human alveolar type 2 (hAT2) cells. The expression variation of these reference genes was also screened in unstimulated whole human lung. The stability of the selected reference genes varied within and between cell types, the variation in expression being greatest in primary cultures of hAT2. Correspondingly, the effect of expressing message for the stretch responsive gene IL-8 normalized to the 14 reference genes was greatest in the hAT2 cells, there being an almost fivefold difference in mRNA relative change comparing different reference genes in the same samples. The minimum number of genes required to derive a reliable normalization factor for experiments on single lung cell types undergoing mechanical strain was two and for whole human lung it was four. These results demonstrate that the optimal reference genes for lung cells subjected to CMS are cell type specific.

Mechanical stretch decreases migration of alveolar epithelial cells through mechanisms involving Rac1 and Tiam1

Mechanical ventilation can overdistend the lungs or generate shear forces in them during repetitive opening/closing, contributing to lung injury and inflammation in patients with acute respiratory distress syndrome (ARDS). Repair of the injured lung epithelium is important for restoring normal barrier and lung function. In the current study, we investigated the effects of cyclic mechanical strain (CS), constant distention strain (CD), and simulated positive end-expiratory pressure (PEEP) on activation of Rac1 and wound closure of rat primary alveolar type 2 (AT2) cells. Cyclic stretch inhibited the migration of wounded AT2 cells in a dose-dependent manner with no inhibition occurring with 5% CS, but significant inhibition with 10% and 15% CS. PEEP conditions were investigated by stretching AT2 cells to 15% maximum strain (at a frequency of 10 cycles/min) with relaxation to 10% strain. AT2 cells were also exposed to 20% CD. All three types of mechanical strain inhibited wound closure of AT2 cells compared with static controls. Since lamellipodial extensions in migrating cells at the wound edge were significantly smaller in stretched cells, we measured Rac1 activity and found it to be decreased in stretched cells. We also demonstrate that Tiam1, a Rac1-specific guanine nucleotide exchange factor, was expressed mainly in the cytosol of AT2 cells exposed to mechanical strain compared with membrane localization in static cells. Downregulation of Tiam1 with 100 microM NSC-23766 inhibited activation of Rac1 and migration of AT2 cells, suggesting its involvement in repair mechanisms of AT2 cells subjected to mechanical strain.

Cyclic Mechanical Strain Induces Interleukin-6 Expression via Prostaglandin E2 Production by Cyclooxygenase-2 in MC3T3-E1 Osteoblast-like Cells

Interleukin (IL)-6, produced by osteoblasts, is one of the key cytokines that promote bone resorption. This study examined the effect of cyclic mechanical strain on the expression of IL-6 mRNA and protein, and the induction mechanism of IL-6 in MC3T3-E1 osteoblast-like cells using RT-PCR and ELISA. MC3T3-E1 cells were cultured in α-MEM with 10% FBS on flexible, collagen I-coated membranes and subjected to cyclic mechanical strain at 6 cycles/min. The production of IL-6 protein was increased at 3 h, reached a peak at 6 h, and was maintained up to 24 h by the cyclic mechanical strain. The strain increased the production of IL-6 protein in a force-dependent manner. The expression of IL-6 mRNA was increased by the cyclic mechanical strain. Prostaglandin(PG)E2 production was induced at 1 h, dramatically increased to 3 h, and was gradually increased from 3 to 24 h by the cyclic mechanical strain. The strain increased PGE2 production in a force-dependent manner. Treatment with PGE2 increased the production of IL-6 protein from MC3T3-E1 cells. The cyclic mechanical strain promoted cyclooxygenase (COX)-2 but not COX-1 mRNA expression. Pretreatment with indomethacin and NS-398 prevented PGE2 production and partly inhibited the production of IL-6 protein induced by the cyclic mechanical strain. These findings suggest that cyclic mechanical strain increases IL-6 expression in osteoblasts, the induction of which is in part mediated via PGE2 production by COX-2.

The Motogenic Effects of Cyclic Mechanical Strain on Intestinal Epithelial Monolayer Wound Closure Are Matrix Dependent

Complex deformation during normal digestion due to peristalsis or villous motility may be trophic for the intestinal mucosa. Because tissue fibronectin is increased in inflammatory states that may accompany mucosal injury, we evaluated the effects of cyclic mechanical strain and fibronectin on intestinal epithelial monolayer wound closure in Caco-2 and IEC-6 intestinal epithelial cells. Wounds created in intestinal epithelial monolayers were subjected to cyclic deformation. Wound closure was assessed by morphometry using microscopic imaging. Cell signals were assessed by Western blot and confocal microscopy. Mechanical strain stimulated wound closure on fibronectin but inhibited closure on collagen in Caco-2 and IEC-6 cells. The effect was independent of proliferation or cell spreading. Myosin light chain (MLC) and extracellular signal-regulated kinase (ERK) were phosphorylated in response to strain in confluent monolayers on both collagen and fibronectin. Blocking MLC or ERK phosphorylation inhibited the motogenic effect of strain on fibronectin. Although phosphorylated MLC was redistributed to the leading edge of migrating cells following 6 hours of strain on collagen and fibronectin, phosphorylated ERK was redistributed to the lamellipodial edge only on fibronectin. Strain promotes intestinal epithelial wound closure by a pathway requiring ERK and MLC kinase. Fibronectin-dependent ERK redistribution in response to strain in confluent migrating cells may explain the matrix dependence of the motogenic effect. Repetitive deformation stimulates intestinal epithelial proliferation on a collagen substrate, but not fibronectin. Deformation may exert matrix-dependent effects on intestinal epithelial cells, promoting epithelial restitution in fibronectin-rich tissue and proliferation in fibronectin-poor mucosa.

Time‐dependent Modulation of Alignment and Differentiation of Smooth Muscle Cells Seeded on a Porous Substrate Undergoing Cyclic Mechanical Strain

The orientation of cellular alignment in smooth muscle tissue engineering is directly related to optimal movement of engineered tissue when it is transplanted in vivo. Cyclic mechanical strain has been applied to modulate the alignment, proliferation, and differentiation of smooth muscle cells. This study was conducted to investigate the effects of cyclic mechanical strain on primary cultured myofibroblasts seeded onto three-dimensional polymeric scaffolds, and to determine the optimal mechanical treatment time required to produce artificial smooth muscle. The cells were primary cultured from rabbit esophageal smooth muscle layer, and a self-designed stretching chamber was used to modulate the cells on porous polyurethane (PU) scaffolds with 10% strain at a frequency of 1 Hz. The applied cyclic strain induced cellular alignment. In particular, cellular alignment perpendicular to the direction of strain was generated in the condition strained over 18 h. In terms of proliferation, the strained groups differed significantly from the statically cultured group, but no difference was observed between groups that were subjected to straining for different lengths of time. Quantitative analysis of alpha-smooth muscle actin (SMA) showed that differentiation was significantly promoted at 18 h of strain. Penetration of primary cultured cells into the pores of PU scaffolds was shown after cyclic strain application, especially in 18 and 24 h of strain. Consequently, it is expected that myofibroblast/scaffold hybrids, cyclically strained in the defined time course, could be practically applied to organize functional smooth muscle tissues having consistent cell alignment and up-regulated SMA.

The effects of cyclic mechanical strain and tumor necrosis factor alpha on the response of cells of the meniscus

Cells of the knee meniscus respond to changes in their biochemical and biomechanical environments with alterations in the biosynthesis of matrix constituents and inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine that is involved in the pathogenesis of both osteoarthritis and rheumatoid arthritis, but its influence on meniscal physiology or mechanobiology is not fully understood. The objectives of this study were to examine the hypothesis that cyclic mechanical strain of meniscal cells modulates the biosynthesis of matrix macromolecules and pro-inflammatory mediators, and to determine if this response is altered by TNF-alpha. Cells were isolated from the inner two-thirds of porcine medial menisci and subjected to biaxial tensile strain of 5-15% at a frequency of 0.5Hz. The synthesis of proteoglycan, protein, nitric oxide (NO), and prostaglandin E(2) were determined. Cyclic tensile strain increased the production of nitric oxide through the upregulation of nitric oxide synthase 2 (NOS2) and also increased synthesis rates of prostaglandin E(2), proteoglycan, and total protein in a manner that depended on strain magnitude. TNF-alpha increased the production of NO and total protein, but inhibited proteoglycan synthesis rates. TNF-alpha prevented the mechanical stimulation of proteoglycan synthesis, and this effect was not dependent on NOS2. These findings indicate that pro-inflammatory cytokines can modulate the responses of meniscal cells to mechanical signals, suggesting that both biomechanical and inflammatory factors could contribute to the progression of joint disease as a consequence of altered loading of the meniscus.

Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells.

The aim of the study was to investigate the effect of cyclic mechanical strain on differentiation markers in the presence or absence of dexamethasone. Human bone marrow stromal cells (BMSC) from seven donors (32.5+/-6.2 years) were cultivated with (D+) or without (D-) dexamethasone. A cyclic mechanical strain with an elongation of 2% (D+2; D-2) or 8% (D+8; D-8) was applied for three days with a stimulation time of three times two hours each day. Levels of alkaline phosphatase (ALP) and osteocalcin (OC) were compared after time intervals of four and seven days. mRNA expression of Collagen I, III and Cbfa1 was investigated after one, four, and seven days. ALP levels were significantly increased in the D+8 group after four and seven days (147.1+/-6.3%; p<0.05 and 168.6+/-6,5%; p<0.03) and in the D-8 group after 7 days (197.4+/-10.4; p<0.04). Cyclic strain had a significant influence on ALP-secretion (F=7.5; p<0.01). In the D-8 group there was a significant increase in OC secretion after 4 days (140.9+/-12.5%; p<0.05).; p<0.01). The effect of stretching was significantly stronger than that of dexamethasone (F=17.2 vs. 1.8). Collagen I (Col I) expression was upregulated in D+8 cultures after 4 days (215.0+/-53.3 p<0.04) and after seven days (166.7+/-55.7; p<0.04). Collagen III (Col III) expression was upregulated in D+2 and D+8 cultures after 4 days (200.7+/-16.3 and 185.9+/-12.7; p<0.04) and after seven days (154.4+/-10.1 and 118.8+/-16.4; p<0.04). There was a significant increase of Cbfa1 expression in D+8 cultures at all investigated time intervals (day 1: 105.5+/-3.7%; day 4: 104.7+/-3.0%; day 7: 104.4+/-2.1%; p<0.03). Stretching (F=20.0; p<0.01) was a stronger contributor to Cbfa-1 expression than dexamethasone (F=12.1; p<0.01). Cyclical mechanical stimulation with 8% elongation increases ALP and OC levels and upregulates Col I and III synthesis and Cbfa1 expression. In the short term, cyclical stretching is a stronger differentiation factor than dexamethasone. Cyclical stretching and dexamethasone both enhance the osteogenic commitment of hBMSC.

Properties of engineered vascular constructs made from collagen, fibrin, and collagen–fibrin mixtures

Vascular constructs were formed by embedding rat aortic smooth muscle cells in three-dimensional matrices of Type I collagen, fibrin, or a mixture of collagen and fibrin in a 1:1 ratio, at total matrix protein concentrations of 2 and 4 mg/ml. Morphological and mechanical properties were evaluated after 6 days in culture, and the effect of cyclic mechanical strain on collagen–fibrin mixture constructs was also studied. Constructs made with the lower protein concentration compacted to the greatest degree, and fibrin was found to enhance gel compaction. Each matrix type exhibited a characteristic stress–strain profile. Pure collagen had the highest linear modulus and pure fibrin had the lowest. The ultimate tensile stress was strongly dependent on the degree of gel compaction, and collagen–fibrin mixtures at 2 mg/ml total protein content exhibited the highest values. Application of cyclic mechanical strain to collagen–fibrin mixture constructs caused a significant increase in gel compaction and a decrease in cell proliferation. The linear modulus, ultimate tensile stress and toughness of the constructs were all augmented by mechanical strain. These results demonstrate that the properties of engineered vascular tissues can be modulated by the combination of selected extracellular matrix components, and the application of mechanical stimulation.

Induction of SM-alpha-actin expression by mechanical strain in adult vascular smooth muscle cells is mediated through activation of JNK and p38 MAP kinase.

Mechanical forces have direct effects on the growth and differentiation of vascular smooth muscle. The goal of this study was to examine the effects of cyclic mechanical strain on expression of smooth muscle-alpha-actin (SM-alpha-actin), a marker for the differentiated state of vascular smooth muscle, in cultured rat aortic smooth muscle cells (VSMC). Cells grown on dishes coated with either laminin or pronectin were subjected to mechanical strain and effects on expression of SM-alpha-actin were evaluated using the Flexercell Strain Unit. Application of mechanical strain to cells in full media increased SM-alpha-actin protein expression and promoter activity. This was not associated with any effect on growth. Mechanical strain increased activity of all three members of the MAP kinase family (ERKs, JNKs, and p38 MAP kinase), with similar kinetics. Inhibition of either JNKs or p38 MAP kinase blocked the strain-induced increase in SM-alpha-actin promoter activity, and expression of constitutively active forms of JNK or MKK6, a p38 kinase, increased promoter activity. These studies indicate that in adult VSMC, mechanical strain leads to increased expression of smooth muscle markers, resulting in a more contractile phenotype.

Effect of cyclical stretch on matrix synthesis of human patellar tendon cells

The significance of mobilization and loading for healing ligaments and tendons is generally accepted today. Local deformation of cells thereby represents the key stimulus for the cellular response. Less is known, however, about the effects of cyclic strain on the cellular and molecular level. The aim of the in vitro investigation was to determine the effect of cyclic mechanical strain on collagen type I and III and on fibronectin formation in human patellar tendon derived fibroblasts. Human patellar tendon derived fibroblasts from 5 donors (mean age 29.2 years) were cultured under standard conditions. Monolayers of subconfluently grown 3rd passage cells were stretched in rectangular silicone dishes with cyclic movement along their longitudinal axes. Cyclic strain (5%, 1 Hz) was applied for 30 min and 60 min, respectively. Carboxyterminal procollagen type I propeptide (P-I-CP) and aminoterminal procollagen type III propeptide (P-III-NP) release was measured by a radio-immunoassay 6 h and 12 h after stretching. The release of fibronectin was measured by nephelometry following immunoreaction with a specific antiserum. Cells from each donor without any cyclic stretching served as controls. Compared with the controls, only cyclic stretching for 60 min resulted in a significantly increased release of P-I-CP and fibronectin after 6 h. The release of P-III-NP was significantly increased 12 h after 30 min of cyclic stretching as well as 6 h and 12 h after 60 min of cyclic stretching, respectively. We conclude that cyclic stretching causes a time-dependent, differential regulation of formation of fibronectin and collagen type I and III. This may effect the quality and thus the mechanical properties of healing tendon and ligament tissues. In order to improve current treatment protocols and to enlarge our knowledge of tissue healing, it is necessary to understand the cellular response to cyclic strain.

Mechanical strain stimulates a mitogenic response in coronary vascular smooth muscle cells via release of basic fibroblast growth factor

Mechanical strain has been shown to induce mitogenesis in a rat neonatal vascular smooth muscle (VSM) cell line in a response mediated predominantly by transcription, expression, and release of platelet-derived growth factor (PDGF). We examined the effect of cyclic mechanical strain and growth factor production on mitogenic response in ovine coronary artery smooth muscle cells. Vascular smooth muscle cells were cultured from explants of left anterior descending (LAD) coronary arteries from young sheep. Cells for experiments were grown on wells with silicone-elastomer bottoms, and subjected to strain (60 cycles/min) using a vacuum actuated strain device. Tritiated thymidine incorporation was used as a measure of DNA synthesis. Cell membrane damage was assessed with differentially permeable nuclear staining dyes. We observed an increase in tritiated thymidine incorporation in response to strain with a temporal response identical to that observed in response to exogenous growth factors (PDGF-BB and basic fibroblast growth factor [bFGF]). Supernatant medium obtained from stretched cells induced a twofold increase in DNA synthesis in unstretched cells. The mitogenic response was abolished by monoclonal antibodies to bFGF, but not by antibodies to PDGF-AB. Studies of fluorescent dye exclusion indicated the stretching protocol caused no cell membrane damage. Thus, mechanical strain is an important stimulus for growth factor release in coronary VSM cells. The mitogenic response is mediated by release of bFGF.

A novel three-dimensional tissue equivalent model to study the combined effects of cyclic mechanical strain and wear particles on the osteolytic potential of primary human macrophages in vitro.

The effects of cyclic mechanical strain and challenge with physiologically relevant doses of submicrometre-size polyethylene (PE) particles on the osteolytic potential of primary human mononuclear phagocytes were investigated. Cells were seeded into a three-dimensional tissue matrix and co-cultured with particles (mean size 0.21 microm) at particle volume to cell number ratios of 7.5, 15, 30 and 100 microm3/cell. Matrices were then either cultured statically or subjected to 20 per cent cyclic compressional strain in the 'ComCell' for 16 h prior to the assessment of cell viability and quantification of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazdium bromide) assay was shown to be too insensitive to detect changes in cell viability. However, when quantified by the adenosine triphosphate (ATP) assay, cell viability was demonstrated to be reduced following exposure to cyclic strain. Macrophages cultured in the static three-dimensional tissue equivalent model produced very high levels of TNFalpha in response to submicrometre PE particles at a ratio of 100 microm3/cell. Cyclic strain in the absence of particles gave only a small increase in TNFa production. However, the combined effects of strain and particle stimulation at a ratio of 30 microm3/cell resulted in the secretion of significantly more TNFalpha than was produced by macrophages subjected to strain alone, or the cells-only control. This synergy between cyclic strain and PE particle stimulation was only evident when the volume of particles was reduced below the volume that maximally stimulated cells. These results suggest that while cyclic strain may not be the primary factor responsible for macrophage activation and periprosthetic osteolysis, at low particle load, it may contribute significantly to the osteolytic potential of macrophages in vitro or in vivo.

Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF.

The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to alpha-thrombin (from 15- to 33-fold), but not to PDGF. As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF-A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75% the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation. Thus, the mechanism of strain-induced growth appears to involve the intermediary action of secreted PDGF.