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Cyclic compression and tension regulate differently the metabolism of chondrocytes
Abstract
Articular cartilage is the primary structure in joints which is responsible for withstanding compressive loading; however, it is possible that cartilage also experiences localized tensile loading under various loading and boundary conditions. To date, little is known as to whether chondrocyte functions are dependent on the loading variation, especially when loading is switched from compression to tension. The purpose of this study is to examine the metabolism of chondrocytes under both compressive and tensile loadings in vitro. Our results suggest that synthesis of both collagen II and aggrecan is regulated differently by compression and tension at the mRNA level. Tensile loading significantly inhibited the mRNA expression of both collagen II and aggrecan. Since poor content of collagen II and proteoglycan has been considered a detrimental factor in the integrity of cartilage matrix, cartilage degradation and the possible formation of osteoarthritis may be the consequence of loading patterns switching from compression to tension.
... Results demonstrated that the total hydroxyproline production (Fig. 5 (c)), and the total DNA contents ( Fig. 5 (d)) were not significantly influenced by the tensile loading (ANOVA, p>0.05), within the experimental conditions investigated. Some reports in literature have revealed the adverse effect of tensile loading on the collagen synthesis of articular chondrocytes (Lee et al. 2005;Huang et al. 2007;Mawatari et al. 2010) while some other researches demonstrated its positive role (Huang et al. 2007;Hirano et al. 2008;Ueki et al. 2008). The possible causes to these inconsistencies are complex, and may be due to the use of loading frequency (Ueki et al. 2008), magnitude (Fan and Waldman 2010), and duration (Huang et al. 2007) as these factors have been revealed to influence the collagen synthesis of chondrocytes. ...
Mechanical compression plays an important role in modulating cell physiology. To precisely investigate the cellular response to compression, a stable culture condition is required. In this study, we developed a microbioreactor platform capable of providing dynamic compressive loading to the 3-D cell culture constructs under a steady environment. The mechanism of generating compression stimulation to cells is based on the pneumatically-driven deformation of a polydimethylsiloxane (PDMS) membrane, which then exerts compressive loading to the cultured cells through a micropillar. By modulating the magnitude and frequency of the applied pneumatic pressure, the compressive loading can be generated in a tunable manner. In this study, the quantitative relationship between the applied pneumatic pressure and the generated compressive strain on the culture construct was established. Moreover, the effects of compression on the cell viability and the metabolic and biosynthetic activities of articular chondrocytes were investigated. The results disclosed that the dynamic compressive loading (51.3% strain, 1 Hz) might up-regulate the metabolic activity and glycosaminoglycan biosynthesis of articular chondrocytes after 5 day culture. Overall, this study presents a micro cell culture device that is capable of exploring the effects of compressive loading on cell physiology in a precise, high-throughput, low-cost, and user-friendly manner.
... Results demonstrated that the total hydroxyproline production (Fig. 5 (c)), and the total DNA contents ( Fig. 5 (d)) were not significantly influenced by the tensile loading (ANOVA, p>0.05), within the experimental conditions investigated. Some reports in literature have revealed the adverse effect of tensile loading on the collagen synthesis of articular chondrocytes (Lee et al. 2005;Huang et al. 2007;Mawatari et al. 2010) while some other researches demonstrated its positive role (Huang et al. 2007;Hirano et al. 2008;Ueki et al. 2008). The possible causes to these inconsistencies are complex, and may be due to the use of loading frequency (Ueki et al. 2008), magnitude (Fan and Waldman 2010), and duration (Huang et al. 2007) as these factors have been revealed to influence the collagen synthesis of chondrocytes. ...
Mammalian cells are sensitive to extracellular microenvironments. In order to precisely explore the physiological responses of cells to tensile loading, a stable and well-defined culture condition is required. In this study, a high-throughput perfusion-based microbioreactor platform capable of providing dynamic equibiaxial tensile loading to the cultured cells under a steady culture condition was proposed. The mechanism of generating tensile stimulation to cells is based on the pneumatically-driven deformation of an elastic polydimethylsiloxan (PDMS) membrane which exerts tensile loading to the attached cells. By modulating the magnitude and frequency of the applied pneumatic pressure, various tensile loading can be generated in a controllable manner. In this study, the microbioreactor platform was designed with the aid of the experimentally-validated finite element (FE) analysis to ensure the loading of tensile strain to cells is uniform and definable. Based on this design, the quantitative relationship between the applied pneumatic pressure and the generated tensile strain on the PDMS membrane was established via FE analysis. Results demonstrated that the proposed device was able to generate the tensile strain range (0~0.12), which covers the physiological condition that articular chondrocytes experience tensile strain under human walking condition. In this study, moreover, the effect of tensile loading on the metabolic, biosynthetic and proliferation activities of articular chondrocytes was investigated. Results disclosed that the dynamic tensile loading of 0.12 strain at 1 Hz might significantly up-regulate the synthesis of glycosaminoglycans while such stimulation was found no significant influence on the metabolic activity, the synthesis of collagen, and the proliferation of chondrocytes. Overall, this study has presented a high throughput perfusion micro cell culture device that is suitable for precisely exploring the effect of tensile loading on cell physiology.
Sol–gel with microwave heating was employed to prepare fine particles Sr2SiO4:Tb phosphor. X-ray diffractometer was used to characterize the structural of the samples. The Scanning Electron Microscope image shows that the particle size is about 300nm. The phosphor particles have several advantages in the morphology, such as excellent surface quality, spherical shape, and narrow size distribution with no aggregation. The VUV luminescence measurements indicate that the phosphor presents an intense excitation band at 173nm. Because the wavelength of excitation source in PDP is mainly at 147 and 172nm, it makes Sr2SiO4:Tb a potential candidate for green emitting phosphor for plasma display panel (PDP) application. Photoluminescence (PL) measurements indicate that the Sr2SiO4:Tb particles present excellent green emission at 542 and 547nm excitated at 236 and 172nm, respectively.
Eu-doped Y2O3 particles with spherical shape and fine size were prepared by spray pyrolysis. The cathodoluminescence of Y2O3:Eu3+ powder was optimized by substituting small amount of zinc atoms in place of yttrium sites. As a result, the optimized (Y, Zn)2O3:Eu3+ phosphor showed 60% improved cathodoluminescence compared with Y2O3:Eu3+ particles. The prepared (Y, Zn)2O3:Eu3+ phosphor had spherical shape and 0.726 μm in mean size. Using these particles, the thickness of the phosphor film was controlled by varying the phosphor loading. The brightness and luminous efficiency of phosphor films prepared were monitored with varying the accelerating voltage ranges from 4 to 14 kV. The dependency of the luminous efficiency on the accelerating voltage was very sensitive to the phosphor loading. As increasing the accelerating voltage from 4 to 14 kV, the brightness of phosphor films prepared was monotonically increased from 200 to 1085 cd/cm2, but the saturation in the luminous efficiency appeared at 10 kV. The highest efficiency was achieved when the number of phosphor-particles layer was about 3. More details about the luminous efficiency and brightness were discussed with changing the phosphor loading.
The anatomic forms of diarthrodial joints are important structural features which provide and limit the motions required for the joint. Typically, the length scale of topographic variation of anatomic forms ranges from 0.5 to 15 cm. Articular cartilage is the thin layer of hydrated soft tissue (0.5–5.0 mm thick) covering the articulating bony ends in diarthrodial joints. This tissue has a set of unique mechanical and physicochemical properties which are responsible for its load-carrying capabilities and near-frictionless qualities. The mechanical properties of articular cartilage are determined at the tissue-scale level and these properties depend on the composition of the tissue, mainly collagen and proteoglycan, and their molecular and ultrastructural organization (ultra-scale: 10−8–10−6m). Because proteoglycans possess a high density of fixed negative charges, articular cartilage exhibits a significant Donnan osmotic pressure effect. This physicochemically derived osmotic pressure is an important component of the total swelling pressure; the other component of the total swelling pressure stems from the charge-to-charge repulsive force exerted by the closely spaced (1–1.5 nm) negative charge groups along the proteoglycan molecules. Thus these interactions take place at a nano-scale level: 10−10–10−9 m. Finally, cartilage biochemistry and organization are maintained by the chondrocytes which exist at a micro-scale level (10−7–10−6 m). Significant mechanoelectro-chemical transduction occurs within the extracellular matrix at the micro-scale level which affects and modulates cellular anabolic and catabolic activities. At present, the exact details of these transduction mechanisms are unknown. In this review, we present a summary of the hierarchical features for articular cartilage and diarthrodial joints and tables of known material properties for cartilage. Also we summarize how the multi-scale interactions in articular cartilage provide for its unique material properties and tribological characteristics.
Cultured bovine articular cartilage was subjected to 50 ms, 0.5-1.0 MPa compressions repeated at intervals of 2-60 s for 1.5 h and simultaneously labeled with 35SO4. The compression was delivered with a 4-mm-diameter nonporous loading head on an 8-mm-diameter cartilage explant. This method created directly compressed (central) and uncompressed (border) areas within the tissue. Analysis of the whole explant under a 0.5 MPa load showed significantly increased 35SO4 incorporation by compression repeated at 2- and 4-s but not at 20- and 60-s intervals. When the incorporation was studied separately in the border and central areas, a statistically significant stimulation was noticed in the central area with a 4-s cycle, while the border area was stimulated with a 2-s cycle. Autoradiography of the central area showed that the stimulation with 0.5 MPa and a 4-s cycle occurred through the whole depth of the cartilage, while raising the pressure to 1 MPa or the frequency to 2 s reduced the stimulation, particularly in the superficial cartilage. In the border area the stimulation with 0.5 MPa and a 2-s cycle was noted in the superficial zone only. The stimulation of proteoglycan synthesis is thus limited to certain loading frequencies and pressures and occurs in specific areas under and around the loaded site. Its rapid appearance suggests enhanced glycosylation or sulfation of core proteins or enhanced speed of posttranslational processing.
The direct effects of hydrostatic pressure on matrix synthesis in articular cartilage can be studied independently of the other factors that change during loading. We have found that the influence of hydrostatic pressure on incorporation rates of 35SO4 and [3H]proline into adult bovine articular cartilage slices in vitro depends on the pressure level and on the time at pressure. Pressures in the "physiological" range (5-15 MPa) applied for 20 s or for 5 min could stimulate tracer incorporation (30-130%) during the following 2 h, but higher pressures (20-50 MPa) had no effect on incorporation rates. The degree of stimulation in cartilage obtained from different animals was found to vary; in some animals none was seen. Stimulation also varied with position along the joint. Physiological pressures (5-10 MPa) applied continuously for the 2-h incubation period also stimulated incorporation rates, but pressures greater than 20 MPa always produced a decrease that was related to the applied pressure and that was reversible. These results suggests that the hydrostatic pressure that occurs during loading is a signal that can stimulate matrix synthesis rates in articular cartilage.
A pressure-measuring Moore-type endoprosthesis was implanted in a seventy-three-year-old patient who had sustained a displaced fracture of the femoral neck. The measurement and telemetry of contact pressures in the hip began in the operating room, and data were acquired periodically for more than thirty-six months. Unexpectedly high localized contact pressures between the acetabular cartilage and the prosthesis were recorded. Early in the period of recovery, activities such as using a bedpan or performing isometric exercise produced pressures that were close to those recorded during normal walking. The highest pressure, eighteen megapascals, was recorded one year postoperatively, while the patient was rising from a chair. High pressures occurred in the superior and posterior aspects of the acetabulum.
Bovine and human articular cartilage segments exhibit variable alterations in metabolism following in vitro exposure to hydrostatic pressure. A decrease in incorporation of the labeled substrates 35SO4, 3H-glycine and 3H-uridine to values less than 50% of non-pressurized tissue results from exposure to pressures between 75 and 300 psi. A pressure of 375 psi consistently results in a 10-15% increase in cartilage synthetic activity in the presence or absence of 10% fetal calf serum. Dialyzed fetal calf serum increases the metabolic response at 375 psi from 10% to 55%. The recovery phase following exposure to pressure includes a release (rebound) phenomenon whereby a burst of metabolic activity elevates the metabolic rate to normal levels when the tissues are inhibited (75-300 psi) and accelerates the metabolic rate by 60% in tissues whose metabolism was elevated (375 psi). These data suggest that articular cartilage chondrocytes have the capacity to rapidly and differentially transform mechanical signals derived from application of hydrostatic pressure into metabolic events. The direction of the response is apparently dictated by the magnitude of the applied force and presence of dialyzable components in serum. Although the force applied only partially mimics in vivo forces, the observed responses to pressure support the thesis that pressure modulation of metabolic activity in articular cartilage may be an important factor in its maintenance.
Primary chondrocyte cell cultures and explants of bovine articular cartilage were subjected to cyclic hydrostatic pressure in a novel computer-controlled pressure chamber designed for this purpose. The cultures were labeled with 5 microCi/ml 35SO4 and simultaneously pressurized with 5 MPa load for 1.5 or 20 h with pressure cycles of 0.0167, 0.05, 0.25, and 0.5 Hz. The chondrocyte cell cultures were also subjected to 0.0082 and 0.0034 Hz cycles. Sulfate incorporation was significantly inhibited in cell cultures subjected to the 0.5, 0.25, or 0.05 Hz cyclic loads for 1.5 h, but stimulated in explant cultures with a 0.5 Hz cyclic 1.5-h load. Chondrocyte cultures subjected to longer (20 h) loading showed a stimulation of sulfate incorporation with 0.5 and 0.25 Hz cycles, but an inhibition with 0.0167 Hz. The results indicate that cyclic hydrostatic pressures of presumably physiological magnitude have significant influences on proteoglycan synthesis in articular cartilage chondrocytes. Comparison of the cell and explant cultures under identical pressure conditions suggested that chondrocyte interactions with extracellular matrix are involved in this regulation by cyclic hydrostatic pressure. The responses of the chondrocytes to pressurization also varied according to the total length of the treatment, a finding compatible with the idea of multiple metabolic steps in chondrocytes, both pre- and post-translational, controlled by the ambient hydrostatic pressure.
An elastostatic model of rapidly loaded articular cartilage is presented. It is assumed that the cartilage experiences little volumetric change or interstitial fluid flow while loaded instantaneously. Subchondral bone compliance and articular surface friction are incorporated. Integral representations of the stress distributions within cartilage are derived using Fourier transform techniques and the integrals are solved numerically. Localized tensile stresses are found and occur in regions close to the cartilage-bone interface as well as at the articular surface, outside the embrace of the load. The qualitative similarity between the results and those of previous investigations is explained by an elementary equilibrium analysis. The stress distributions suggest that the splits and cracks observed in diseased cartilage may be initiated, or propagated, by tensile stress.
Osteoarthrosis, a common pathway of joint deterioration, is caused by mechanical stress loaded on articular cartilage. We previously demonstrated the involvement of protein kinase C (PKC) in the development of osteoarthritis in vitro. In this study, we examined the effect of mechanical stress on chondrocyte metabolism and the activity of PKC in vitro. Low frequency and magnitude of cyclic tensile stretch loaded on chondrocytes increased proteoglycan synthesis. However, high frequency and magnitude of stress decreased its synthesis. In this condition, activity of PKC was reduced. These results suggest an involvement of PKC in the stress-mediated inhibition of proteoglycan synthesis.
An in vitro method has been developed of cultivating anatomically intact articular cartilage (humeral head of dog) while excluding both bone tissue and other connective tissues with a condom, which controls changes in hydration and minimizes loss of proteoglycans. Compressive loading experiments were carried out with the condom-covered humeral head, to which contact stresses of 2.1 MPa, 3.3 MPa and 6.4 MPa, respectively, were applied with a rubber disc of a simple loading apparatus. The model makes it possible, to compare experimentally loaded regions with experimentally unloaded regions in both the same joint area and the contralateral control joint. Intermittent pressure loading (4-s-on/16-s-off-cycle) during 2 hours of pulse-experiment and 18 hours of chase-experiment resulted in a 40% increase in proteoglycan synthesis rate at 2.1 MPa, an unaltered synthesis rate at 3.3 MPa, and a 45% decrease in proteoglycan synthesis rate at 6.4 MPa, as measured by 35S-sulfate incorporation. No significant increase in degradation rate of proteoglycans was noted during the various loading experiments.
This study describes the effect of load magnitude, frequency and duration on proteoglycan (PG) biosynthesis and loss in mature bovine articular cartilage explants. Cultured full thickness cartilage discs were subjected to a continuously applied, uniaxial compressive cyclic load. The loads were applied using a sinusoidal waveform of 0.001, 0.01, 0.1 or 0.5 Hz-frequency and a peak stress of 0.1, 1.0, 2.5, or 5.0 MPa for a period of 1, 3 or 6 days. Increasing the load magnitude, as well as the duration of loading, reduced the PG biosynthesis. Reducing the load frequency abolished the inhibitory effect of a given load magnitude on PG biosynthesis, even though explants were more compressed. Increasing the load magnitude stimulated the release of newly synthesized PGs from explants, whereas an elevated duration of loading significantly decreased the release of endogenous PGs. Explants loaded for 1 or 3 days were viable as determined biochemically, whereas 6 days of loading resulted in a slightly diminished viability of explants. This study demonstrates that the duration and intensity of loading influences the inhibition of PG biosynthesis, while PG loss is only modulated by the magnitude and duration of loading.
The effects of short- and long-term load-controlled compression on the levels of aggrecan mRNA have been determined. Results show that a compressive stress of 0.1 MPa on bovine articular cartilage explants for 1, 4, 12, and 24 h produces a transient up-regulation of aggrecan mRNA synthesis. At 1 h, aggrecan mRNA levels in loaded explants were increased 3.2-fold compared to control explants. At longer times (>/=4 h), the levels of aggrecan mRNA returned to baseline values or stayed slightly higher. There is a dose dependence in the response of the explant to increasing levels of compressive stress (0-0.5 MPa) for 1 h. Aggrecan mRNA levels increased 2- to 3-fold at 0-0.25 MPa. At 0.5 MPa, the level of aggrecan mRNA was lower than those at 0.1 and 0.25 MPa. This dose-dependent effect suggests a reversal of the stimulatory effects of compression on aggrecan gene expression at higher loads. After 24 h of compression, the levels of aggrecan mRNA in explants subjected to any of the stress levels were not significantly different from those in control explants. The stimulatory effect of 0.1 MPa compressive stress on aggrecan mRNA levels was blocked by Rp-cAMP and U-73122, indicating the involvement of the classical signal transduction pathways in the mechanical modulation of aggrecan gene expression. The responses of link protein mRNA to compression paralleled those of aggrecan, while there was no significant change in expression of the gene for the housekeeping protein elongation factor-1 alpha. The results indicate that articular cartilage chondrocytes can respond to short-term compressive loads by transiently up-regulating expression of the aggrecan gene. The fact that long-term compression did not significantly alter aggrecan mRNA levels suggests that previously observed inhibitory effects of prolonged static compression on proteoglycan synthesis in articular cartilage may be, for the most part, mediated through mechanisms other than suppression of aggrecan mRNA levels.
The goal of this study was to examine the simultaneous effects of mechanical compression of chondrocytes on mRNA expression and macromolecular synthesis of aggrecan and type-II collagen. Bovine cartilage explants were exposed to different magnitudes and durations of applied mechanical compression, and levels of aggrecan and type-IIa collagen mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase were measured and quantified by Northern blot analysis. Synthesis of aggrecan and type-II collagen protein was measured by radiolabel incorporation of [35S]sulfate and [3H]proline into macromolecules. The results showed a dose-dependent decrease in mRNA levels for aggrecan and type-II collagen, with increasing compression relative to physiological cut thickness applied for 24 hours. Radiolabel incorporation into glycosaminoglycans and collagen also decreased with increasing compression in a dose-related manner similar to the changes seen in mRNA expression. The modulation of aggrecan and type-II collagen mRNA and protein synthesis were dependent on the duration of the compression. Aggrecan and type-II collagen mRNA expression increased during the initial 0.5 hours of static compression; however, 4-24 hours after compression was applied total mRNA levels had significantly decreased. The synthesis of aggrecan and collagen protein decreased more rapidly than did mRNA levels after the application of a step compression. Together, these results suggest that mechanical compression rapidly alters chondrocyte aggrecan and type-II collagen gene expression on application of load. However, our results indicate that the observed decreases in biosynthesis may not be related solely to changes in mRNA expression. The mechanisms by which mechanical forces affect different segments of the biosynthetic pathways remain to be determined.
Physiologic loading of the intervertebral disc may lead to changes in the osmotic pressure experienced by the resident cells. In this study, changes in gene expression levels for extracellular matrix and cytoskeletal proteins were quantified in disc cells subjected to hypo-osmotic (255 mOsm) or hyper-osmotic conditions (450 mOsm), relative to iso-osmotic conditions (293 mOsm). Important differences were observed in osmolarity and between cells of different regions, corresponding to the transition zone and nucleus pulposus. Under hypo-osmotic conditions, gene expressions for aggrecan and type II collagen were up-regulated in the transition zone, but not in the nucleus pulposus cells. Genes for the small proteoglycans, biglycan, and decorin, but not lumican, were up-regulated in transition zone cells following incubation in either hypo- or hyper-osmotic media. The same genes were down-regulated in nucleus pulposus cells under either hypo- or hyper-osmotic conditions. Differences in the response to altered osmolarity between cells of the intervertebral disc may relate to their different cytoskeletal structures or embryological origins.
Monolayer cell cultures and cartilage tissue fragments have been used to examine the effects of hydrostatic fluid pressure (HFP) on the anabolic and catabolic functions of chondrocytes. In this study, bovine articular chondrocytes (bACs) were grown in porous three-dimensional (3-D) collagen sponges, to which constant or cyclic (0.015 Hz) HFP was applied at 2.8 MPa for up to 15 days. The effects of HFP were evaluated histologically, immunohistochemically, and by quantitative biochemical measures. Metachromatic matrix accumulated around the cells within the collagen sponges during the culture period. There was intense intracellular, pericellular, and extracellular immunoreactivity for collagen type II throughout the sponges in all groups. The incorporation of [(35)S]-sulfate into glycosaminoglycans (GAGs) was 1.3-fold greater with constant HFP and 1.4-fold greater with cyclic HFP than in the control at day 5 (P < 0.05). At day 15, the accumulation of sulfated-GAG was 3.1-fold greater with constant HFP and 2.7-fold with cyclic HFP than the control (0.01). Quantitative immunochemical analysis of the matrix showed significantly greater accumulation of chondroitin 4-sulfate proteoglycan (C 4-S PG), keratan sulfate proteoglycan (KS PG), and chondroitin proteoglycan (chondroitin PG) than the control (P < 0.01). With this novel HFP culture system, 2.8 MPa HFP stimulated synthesis of cartilage-specific matrix components in chondrocytes cultured in porous 3-D collagen sponges.