High hydrostatic pressure induces ERK and PI3 kinase phosphorylation in human HCS-2/8 chondrosarcoma cells.

High continuous hydrostatic pressure has been shown to affect many cellular functions within the pressurised cells, for instance, accumulation of heat shock protein 70 occurs during pressurisation. Various signal transduction pathways are likely to mediate these changes, however, at the present time our knowledge of the pathways involved is rather limited. The aim of this study was to investigate whether some of the well known transduction pathways are activated by the exposure of human chondrosarcoma cells to 15-30 MPa hydrostatic pressure. The results showed an increased presence of the active, phosphorylated forms of extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K) in cells exposed to 15 and 30 MPa continuous hydrostatic pressure, while 0.5 Hz cyclic loading had weaker effects. Inhibition of ERK-pathway with UO126 did not prevent the accumulation of heat shock protein 70. No activation of c-Jun N-terminal protein kinase (JNK) or p38 could be noticed in pressurised cells. In conclusion, we could identify at least two different signal transduction pathways that are activated under high continuous hydrostatic pressure. Accumulation of heat shock protein 70 was independent of ERK-activation.     

Hydrostatic pressure modulates mRNA expressions for matrix proteins in human meniscal cells

There have been few reports describing the effects of mechanical loading on the metabolism of meniscal cells. The aim of this study was to investigate the effects of hydrostatic pressure on meniscal cell metabolism. Human meniscal cells were cultured in alginate beads for 3 days. They were then subjected to 4 MPa hydrostatic pressure for 4 hours in either a static or cyclic (1 Hz) mode using a specially designed and constructed system. Immediately after the pressure application, the messenger RNA levels for aggrecan, type I collagen, matrix metalloproteinases (MMP) -1, -3, -9, -13 and tissue inhibitors of metalloproteinases (TIMP) -1 and -2 were measured. It was found that the application of static hydrostatic pressure caused a significant decrease in mRNA expression for MMP-1 and -13 (p<0.05). In contrast, the application of cyclic hydrostatic pressure was associated with a significant increase in type I collagen (p<0.01), TIMP-1 and -2 mRNA expression (p<0.01). These results would suggest that hydrostatic pressure in isolation can modulate mRNA expressions for matrix proteins in meniscal cells.     

The role of microtubules in the regulation of proteoglycan synthesis in chondrocytes under hydrostatic pressure

Chondrocytes of the articular cartilage sense mechanical factors associated with joint loading, such as hydrostatic pressure, and maintain the homeostasis of the extracellular matrix by regulating the metabolism of proteoglycans (PGs) and collagens. Intermittent hydrostatic pressure stimulates, while continuous high hydrostatic pressure inhibits, the biosynthesis of PGs. High continuous hydrostatic pressure also changes the structure of cytoskeleton and Golgi complex in cultured chondrocytes. Using microtubule (MT)-affecting drugs nocodazole and taxol as tools we examined whether MTs are involved in the regulation of PG synthesis in pressurized primary chondrocyte monolayer cultures. Disruption of the microtubular array by nocodazole inhibited [(35)S]sulfate incorporation by 39-48%, while MT stabilization by taxol caused maximally a 17% inhibition. Continuous hydrostatic pressure further decreased the synthesis by 34-42% in nocodazole-treated cultures. This suggests that high pressure exerts its inhibitory effect through mechanisms independent of MTs. On the other hand, nocodazole and taxol both prevented the stimulation of PG synthesis by cyclic 0. 5 Hz, 5 MPa hydrostatic pressure. The drugs did not affect the structural and functional properties of the PGs, and none of the treatments significantly affected cell viability, as indicated by the high level of PG synthesis 24-48 h after the release of drugs and/or high hydrostatic pressure. Our data on two-dimensional chondrocyte cultures indicate that inhibition of PG synthesis by continuous high hydrostatic pressure does not interfere with the MT-dependent vesicle traffic, while the stimulation of synthesis by cyclic pressure does not occur if the dynamic nature of MTs is disturbed by nocodazole. Similar phenomena may operate in cartilage matrix embedded chondrocytes.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

Effect of hydrostatic pressure on morphological and ultrastructural aspects of normal and osteoarthritic human articular chondrocytes.

In this work we studied the morphological and ultrastructural aspects of normal and osteoarthritic (OA) human articular chondrocytes cultivated in alginate gel for 48 hours. After this period the chondrocytes in Petri dishes were exposed to cyclic pressurization (minimum pressure 1 MPa and maximum pressure 5 MPa) at 0.25 Hz frequency for three hours. In other loading procedures the cells were exposed to continuous pressure (24 MPa) for three hours. Some dishes were not pressurised and these served as controls. The cells were then fixed for transmission electron microscopy (T.E.M.) and for scanning electron microscopy (S.E.M.). No ultrastructural changes were observed in normal chondrocytes exposed at physiological pressure. OA cells placed under physiological pressure showed a partial recovery on morphological and ultrastructural aspects. Normal and OA samples exposed to continuous pressure (24 MPa) showed a morphological worsening in both T.E.M. and S.E.M. studies.     

Influence of hydrostatic and distortional stress on chondroinduction

Undifferentiated connective tissue that arises during embryonic development and some healing processes contains pluripotent mesenchymal stem cells. It is becoming increasingly evident that the mechanical environment is an important differentiation factor for these cells. In our laboratory, we have focused on the potential for mechanical signals to induce chondrogenic differentiation of mesenchymal stem cells. Using C3H10T1/2 cells as a model, we have investigated the influence of hydrostatic pressure, equibiaxial contraction, and centrifugal pressure on chondroinduction. Cells responded to cyclic hydrostatic compression (5 MPa at 1 Hz) and cyclic contractile strain (15% at 1 Hz) by upregulating aggrecan and collagen type II gene expression. In addition, a preliminary study of the effects of centrifugal pressure (4.1 MPa for 30 min) suggests that it may increase cell proliferation and stimulate proteoglycan and collagen type II production. We speculate that compression, whether it is distortional or hydrostatic in nature, applied to undifferentiated connective tissue triggers differentiation toward a chondrocyte-like phenotype and production of a less permeable extracellular matrix which is capable of sustaining increasingly higher hydrostatic fluid pressure for compressive load support.     

周期性机械应力对髓核细胞增殖和细胞外基质表达的影响

目的:探讨周期性机械应力对髓核细胞增殖和细胞外基质表达的影响。方法:对兔髓核细胞进行体外细胞培养,对细胞施加周期性机械应力(0.25Mpa,0.1Hz)。实验分为2组,不加压组和加压组,不加压组置于单纯旋转式生物反应器内,加压组每天置于周期性机械应力场内2小时。分别于3天,7天检测细胞数目以及聚集蛋白聚糖(aggrecan)和Ⅱ型胶原的基因表达。结果:髓核细胞的增殖和聚集蛋白聚糖、Ⅱ型胶原基因的表达水平与周期性压力密切相关,在周期性机械应力刺激下髓核细胞增殖明显,细胞外基质的分泌增加,组织工程髓核细胞的活性显著提高。结论:周期性机械应力能够显著促进髓核细胞增殖,同时上调聚集蛋白聚糖、Ⅱ型胶原的基因的表达。     

High pressure effects on cellular expression profile and mRNA stability. A cDNA array analysis

Hydrostatic pressure has a profound effect on cartilage tissue and chondrocyte metabolism. Depending on the type and magnitude of pressure various responses can occur in the cells. The mechanisms of mechanotransduction at cellular level and the events leading to specific changes in gene expression are still poorly understood. We have previously shown that induction of stress response in immortalized chondrocytes exposed to high static hydrostatic pressure increases the stability of heat shock protein 70 mRNA. In this study, our aim was to examine the effect of high pressure on gene expression profile and to study whether stabilization of mRNA molecules is a general phenomenon under this condition. For this purpose a cDNA array analysis was used to compare mRNA expression profile in pressurized vs. non-pressurized human chondrosarcoma cells (HCS 2/8). mRNA stability was analyzed using actinomycin-treated and nontreated samples collected after pressure treatment. A number of immediate-early genes, and genes regulating cell cycle and growth were up-regulated due to high pressure. Decrease in osteonectin, fibronectin, and collagen types VI and XVI mRNAs was observed. Also bikunin, cdc37 homologue and Tiam1, genes linked with hyaluronan metabolism, were down-regulated. In general, stability of down-regulated mRNA species appeared to increase. However, no increase in mRNA above control level due to stabilization was noticed in the genes available in the array. On the other hand, mRNAs of certain immediate-early genes, like c-jun, jun-B and c-myc, became destabilized under pressure treatment. Increased accumulation of mRNA on account of stabilization under high pressure conditions seems to be a tightly regulated, specific phenomenon.     

Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading

Articular cartilage in vivo experiences the effects of both cell-regulatory proteins and mechanical forces. This study has addressed the hypothesis that the frequency of intermittently or continuously applied mechanical loads is a critical parameter in the regulation of chondrocyte collagen biosynthesis. Cyclic compressive pressure was applied intermittently to bovine articular cartilage explants by using a sinusoidal waveform of 0.1–1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5–20 s followed by a load-free period of 10–1,000 s. These loading protocols were repeated for a total duration of 6 days. In separate experiments, cyclic loading was continuously applied by using a sinusoidal waveform of 0.001–0.5 Hz frequency and a peak stress of 1.0 MPa for a period of 3 days. Unloaded cartilage discs of the same condyle were cultured in identically constructed loading chambers and served as controls. We report quantitative data showing that (1) no correlation exists between the relative rate of collagen synthesis expressed as the proportion of newly synthesized collagen among newly made proteins and either the frequency of intermittently or continuously applied loads or the overall time cartilage is actively loaded, and (2) individual protocols of intermittently applied loads can reduce the relative rate of collagen synthesis and increase the water content, whereas (3) continuously applied cyclic loads always suppress the relative rate of collagen synthesis compared with that of unloaded control specimens. The results provide further experimental evidence that collagen metabolism is difficult to manipulate by mechanical stimuli. This is physiologically important for the maintainance of the material properties of collagen in view of the heavy mechanical demands made upon it. Moreover, the unaltered or reduced collagen synthesis of cartilage explants might reflect more closely the metabolism of normal or early human osteoarthritic cartilage.This work was supported by the Federal Ministry of Education and Research (BMBF no. 0311058) and by the foundation S.E.T.     

Using changes in hydrostatic and osmotic pressure to manipulate metabolic function in chondrocytes

Articular cartilage has distinct histological depth zones. In each zone, chondrocytes are subject to different hydrostatic (HP) and osmotic pressure (OP) due to weight-bearing and joint-loading. Previous in vitro studies of regeneration and pathophysiology in cartilage have failed to consider the characteristics of histological heterogeneity and the effects of combinations of changes in HP and OP. Thus, we have constructed molecular, biochemical, and histological profiles of anabolic and catabolic molecules produced by chondrocytes from each depth zone isolated from bovine articular cartilage in response to changes in HP and OP. We cultured the chondrocytes with combinations of loading or off-loading of HP at 0-0.5 MPa, 0.5 Hz, and changes in OP of 300-450 mosM over 1 wk, and evaluated mRNA expression and immunohistology of both anabolic and catabolic molecules and amounts of accumulated sulfated glycosaminoglycan. Any changes in HP and OP upregulated mRNA of anabolic and catabolic molecules in surface-, middle-, and deep-zone cells, in descending order of magnitude. Off-loading HP maintained the anabolic and reduced the catabolic mRNA; high OP retained upregulation of catabolic mRNA. These molecular profiles were consistent with immunohistological and biochemical findings. Changes in HP and OP are essential for simulating chondrocyte physiology and useful for manipulating phenotypes.     

A new experimental system for the extended application of cyclic hydrostatic pressure to cell culture

Mechanical forces have been shown to be important stimuli for the determination and maintenance of cellular phenotype and function. Many cells are constantly exposed in vivo to cyclic pressure, shear stress, and/or strain. Therefore, the ability to study the effects of these stimuli in vitro is important for understanding how they contribute to both normal and pathologic states. While there exist commercial as well as custom-built devices for the extended application of cyclic strain and shear stress, very few cyclic pressure systems have been reported to apply stimulation longer than 48 h. However, pertinent responses of cells to mechanical stimulation may occur later than this. To address this limitation, we have designed a new cyclic hydrostatic pressure system based upon the following design variables: minimal size, stability of pressure and humidity, maximal accessibility, and versatility. Computational fluid dynamics (CFD) was utilized to predict the pressure and potential shear stress within the chamber during the first half of a 1.0 Hz duty cycle. To biologically validate our system, we tested the response of bone marrow progenitor cells (BMPCs) from Sprague Dawley rats to a cyclic pressure stimulation of 120/80 mm Hg, 1.0 Hz for 7 days. Cellular morphology was measured using Scion Image, and cellular proliferation was measured by counting nuclei in ten fields of view. CFD results showed a constant pressure across the length of the chamber and no shear stress developed at the base of the chamber where the cells are cultured. BMPCs from Sprague Dawley rats demonstrated a significant change in morphology versus controls by reducing their size and adopting a more rounded morphology. Furthermore, these cells increased their proliferation under cyclic hydrostatic pressure. We have demonstrated that our system imparts a single mechanical stimulus of cyclic hydrostatic pressure and is capable of at least 7 days of continuous operation without affecting cellular viability. Furthermore, we have shown for the first time that BMPCs respond to cyclic hydrostatic pressure by alterations in morphology and increased proliferation.     

Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure

Gene expression patterns in response to hydrostatic pressure were determined by whole genome microarray hybridization. Functional classification of the 274 genes affected by pressure treatment of 200 MPa for 30 min revealed a stress response expression profile. The majority of the >2-fold upregulated genes were involved in stress defense and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. Furthermore, uncharacterized genes were among the 10 highest expressed sequences and represented 45% of the total upregulated genes. The results of this study revealed a hydrostatic pressure-specific stress response pattern and suggested interesting information about the mechanisms involved in adaptation of cells to a high-pressure environment.     

周期性机械应力对髓核细胞增殖和细胞外基质表达的影响

目的：探讨周期性机械应力对髓核细胞增殖和细胞外基质表达的影响。方法：对兔髓核细胞进行体外细胞培养,对细胞施加周期性机械应力（0.25Mpa,0.1Hz）。实验分为2组,不加压组和加压组,不加压组置于单纯旋转式生物反应器内,加压组每天置于周期性机械应力场内2小时。分别于3天,7天检测细胞数目以及聚集蛋白聚糖（aggrecan）和Ⅱ型胶原的基因表达。结果：髓核细胞的增殖和聚集蛋白聚糖、Ⅱ型胶原基因的表达水平与周期性压力密切相关,在周期性机械应力刺激下髓核细胞增殖明显,细胞外基质的分泌增加,组织工程髓核细胞的活性显著提高。结论：周期性机械应力能够显著促进髓核细胞增殖,同时上调聚集蛋白聚糖、Ⅱ型胶原的基因的表达。     

Effects of the piezo-tolerance of cultured deep-sea eel cells on survival rates, cell proliferation, and cytoskeletal structures

We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366–2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.     

Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells

For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.