Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.     

Osmotic loading of in situ chondrocytes in their native environment

Changes in the osmotic environment cause changes in volume of isolated cells and cells in tissue explants, and the osmotic environment becomes hypotonic in cartilage diseases such as osteoarthritis (OA). However, it is not known how cells respond to a hypotonic osmotic challenge when situated in the fully intact articular cartilage. A confocal laser scanning microscope was used to image chondrocytes of intact rabbit patellae in an isotonic (300 mOsm) and hypotonic (172 mOsm) immersion medium. Cell volumes were calculated before and 5, 15, 60, 120 and 240 minutes after the change in saline concentration. Local tissue strains and swelling of the entire tissue were estimated from the relative movements of cells and displacements of single cells, respectively. Cell volumes increased rapidly (< or = 5 minutes, p<0.05) by approximately 22%, after which they remained constant for an hour (p>0.05). However, two and four hours post the hypotonic challenge, cell volumes were statistically greater (p<0.05) than those at all earlier time points, and swelling of the entire tissue continued throughout the four hour loading period. The results of our study suggest that osmotic loading induced volume changes of in situ chondrocytes in their native environment occur quickly and continue for hours. Understanding the behaviour of cells in their native environment provides novel insigth into the cell mechanics in ostearthritic joints and so may help understand the onset and progression of this disease.     

The osmotic sensitivity of rat growth plate chondrocytes in situ; clarifying the mechanisms of hypertrophy

Bone elongation is predominantly driven by the volume expansion of growth plate chondrocytes. This mechanism was initially believed to be "hypertrophy", describing a proportional increase of cell water and organelles. However, morphometrical analysis subsequently assumed the increase to be "swelling", resulting in a disproportionate increase of cell water (osmotically active fraction). Histological approaches were performed on fixed tissue, and for the "swelling" assumption to be valid, the osmotic sensitivity of living cells before and during volume increase should differ. To test this, analysis of images acquired by 2-photon laser scanning microscopy (2PLSM) were used to determine the osmotic sensitivity, and osmotically active/inactive proportions of in situ chondrocytes from 15 living rat growth plates exposed to varying media osmolarities ( approximately 0-580 mOsm). The dimensions of cell volume swelling in hypotonic media were different to the preferential lengthening seen in vivo, confirming the complexity of directional cell volume increase. Boyle-van't Hoff analysis of cell volume over the range of media osmolarity indicated no significant difference (Student's t-test) in the osmotically inactive fraction, 39.5 +/- 2.9% and 47.0 +/- 4.3% (n = 13) for proliferative and hypertrophic zones, respectively, or the sensitivity of volume to changes in media osmolarity (proliferative 15.5 +/- 0.8 and hypertrophic zone 15.5 +/- 1.2%volume . Osm). The osmotic fractions did not change as chondrocytes progress from proliferative to hypertrophic regions of the growth plate. Our data suggest cell volume increase by hypertrophy may play a greater role in cell enlargement than swelling, and should be re-evaluated as a mechanism responsible for growth plate chondrocyte volume increase and hence bone elongation.     

Regulation of matrix synthesis rates by the ionic and osmotic environment of articular chondrocytes

Chondrocytes in cartilage are embedded in a matrix containing a high concentration of proteoglycans and hence of fixed negative charges. Their extracellular ionic environment is thus different from that of most cells, with extracellular Na⁺ being 250–350 mM and extracellular osmolality 350–450 mOsm. When chondrocytes are isolated from the matrix and incubated in standard culture medium (DMEM; osmolality 250–280 mOsm), their extracellular environment changes sharply. We incubated isolated bovine articular chondrocytes and cartilage slices in DMEM whose osmolity was altered over the range 250–450 mOsm by Na⁺ or sucrose addition. ³⁵S-sulphate and ³H-proline incorporation rates were at a maximum when the extracellular osmolality was 350–400 mOsm for both freshly isolated chondrocytes and for chondrocytes in cartilage. The incorporation rate per cell of isolated chondrocytes was only 10% that of chondrocytes in situ both 4 and 24 hours after isolation. For freshly isolated chondrocytes, the rate increased 30–50% in DMEM to which NaCl or sucrose had been added to the increase osmolality. In chondrocytes incubated overnight in DMEM, the rate was greatest in DMEM of normal osmolality and fell from the maximum in proportion to the change in osmolality. The effects of surcrose addition on incorporation rates were similar but not identical to those of Na⁺ addition. Changes in cell volume might be linked to changes in synthesis rates since the cell volume of chondrocytes (measured by Coulter-counter) increased 30–40% when the cells are removed from their in situ environment into DMEM. Synthesis rates can thus be partly regulated by changes in extracellular osmolality, which in cartilage is controlled by proteoglycan concentration. This provides a mechanism by which the chondrocytes can rapidly respond to changes in extracellular matrix composition. © 1993 Wiley-Liss, Inc.     

The role of BKCa channels on hyperpolarization mediated by hyperosmolarity in human articular chondrocytes

Chondrocytes, the only cell in cartilage, are subjected to hyperosmotic challenges continuously since extracellular osmolarity in articular cartilage increases in response to mechanical loads during joint movement. Hyperosmolarity can affect membrane transport, and it is possible that load modulates matrix synthesis through alterations in intracellular composition. In the present study, the effects of hyperosmotic challenges were evaluated using the whole-cell patch clamp technique, whole cell mode on freshly isolated human and bovine articular chondrocytes. In human chondrocytes, hypertonicity induced the activation of outward Ca(2+)-sensitive K(+) currents, which were inhibited by iberiotoxin and TEA-Cl. The current induced by hypertonic switching (osmolarity from 300 to 400 mOsm/l) caused cell hyperpolarization (from -39 mV to -70 mV) with a reversal potential of -96 ± 7 mV. These results suggest a role for Ca(2+)-activated K(+) channels in human articular chondrocytes, leading to hyperpolarization as a consequence of K(+) efflux through these channels. These channels could have a role in the articular chondrocyte's response to a hyperosmotic challenge and matrix metabolism regulation by load.     

Effects of osmotic challenges on membrane potential in human articular chondrocytes from healthy and osteoarthritic cartilage

Changes in external osmolarity arise from variations in mechanical loads on joints and may affect the homeostasis of chondrocytes, which are the only cell type responsible for matrix turnover. Accordingly, variations in membrane potential may affect cartilage production. The present study assessed the effects of variations in external osmolarity on membrane potential and the possible mechanisms responsible for this response. Membrane potential was measured by the patch clamp whole-cell technique using human articular chondrocytes freshly isolated from healthy and osteoarthritic cartilage. The membrane potential was -39±4 mV in articular human chondrocytes from healthy cartilage and -26±4 mV in those from osteoarthritic cartilage. Increasing the osmolarity produced a reversible hyperpolarization mediated by K+ efflux through BKCa channels in both groups of chondrocytes, but the response in osteoarthritic cells was significantly reduced; no other K+ pathways were involved in this effect. Alternatively, decreasing the osmolarity elicited depolarization in healthy chondrocytes but did not produce any response in chondrocytes from osteoarthritic cartilage. The depolarization was dependent on Na+ influx through Gd3+-sensitive stretch-activated cation channels and was independent of external Ca2+. The differential responses observed in chondrocytes from osteoarthritic cartilage suggest that disregulation on the responses to external osmolarity may be involved in the process that leads to the alterations in the cartilage structure observed in osteoarthritis.     

Hypotonic challenge modulates cell volumes differently in the superficial zone of intact articular cartilage and cartilage explant

The objective of this study was to evaluate the effect of sample preparation on the biomechanical behaviour of chondrocytes. We compared the volumetric and dimensional changes of chondrocytes in the superficial zone (SZ) of intact articular cartilage and cartilage explant before and after a hypotonic challenge. Calcein-AM labelled SZ chondrocytes were imaged with confocal laser scanning microscopy through intact cartilage surfaces and through cut surfaces of cartilage explants. In order to clarify the effect of tissue composition on cell volume changes, Fourier Transform Infrared microspectroscopy was used for estimating the proteoglycan and collagen contents of the samples. In the isotonic medium (300 mOsm), there was a significant difference (p < 0.05) in the SZ cell volumes and aspect ratios between intact cartilage samples and cartilage explants. Changes in cell volumes at both short-term (2 min) and long-term (2 h) time points after the hypotonic challenge (180 mOsm) were significantly different (p < 0.05) between the groups. Further, proteoglycan content was found to correlate significantly (r ² = 0.63, p < 0.05) with the cell volume changes in cartilage samples with intact surfaces. Collagen content did not correlate with cell volume changes. The results suggest that the biomechanical behaviour of chondrocytes following osmotic challenge is different in intact cartilage and in cartilage explant. This indicates that the mechanobiological responses of cartilage and cell signalling may be significantly dependent on the integrity of the mechanical environment of chondrocytes.     

Bicarbonate-dependent pH(i) regulation by chondrocytes within the superficial zone of bovine articular cartilage

Control of chondrocyte pH (pH(i)) determines articular cartilage matrix metabolism. However, the transporters of chondrocytes in situ throughout cartilage zones are unclear, and we tested the hypothesis that chondocytes within the superficial zone (SZ) utilise a HCO(3) (-)-dependent system absent from other zones. Imaging of single BCECF-labelled cells was used to monitor the pH(i) of in situ chondrocytes within the cartilage zones, and also that of cells isolated from the SZ or full depth (FD) explants. Resting pH(i) and intrinsic buffering power (beta(i)) in HEPES-buffered saline was not different between SZ and DZ cells, however the pH(i) of SZ chondrocytes was lower in HCO(3) (-) saline. Ammonium pre-pulse was used to acid-load cells and pH(i) recovery by in situ or isolated SZ chondrocytes shown to be totally dependent on HCO(3) (-). pH(i) recovery rate was significantly (P < 0.05) greater for in situ cells, suggesting that isolation damaged the HCO(3) (-)-dependent system. Recovery of pH(i) by in situ cells was blocked by the anion transport inhibitor DIDS, and partially inhibited by EIPA probably non-specifically. Recovery of pH(i) by acidified MZ or DZ cells or those isolated from FD explants was not affected by HCO(3) (-) (P > 0.05). Na(+)-dependent HCO(3) (-)-(NBC) transporters were identified in SZ chondrocytes by fluorescence immunohistochemistry suggesting that this system might account for the HCO(3) (-)-dependent recovery of pH(i). Bovine articular cartilage chondrocytes possess a HCO(3) (-)-dependent transporter which plays a key role in pH(i) regulation in cells in the SZ, but not in chondrocytes within deeper cartilage zones.     

Calciumdependent mechanisms in vasopressin regulation of osmotic water permeability in the mouse kidney collecting duct cells

In the study, the role of PKC and Ca++ in vasopressin regulation of the plasma membrane water permeability was studied in the cells of the mouse kidney collecting duct. Coefficient of osmotic water permeability of total cell surface (Pf) was calculated from the initial rate of cell swelling following the osmotic shock caused by changing the medium osmolarity from isotonic to hypotonic (300 mOsm to 200 mOsm). Desmopressin (dDAVP 1 nM) increased the Pf in hydrated mice from 168.4 +/- 11.8 microm/s up to 231.3 +/- 14.7 microm/s. The Ca++ chelator BAPTA prevented the desmopressin-induced increase in water permeability. Inhibition of PKC (Ro-31-8220 0.1 microM) also abolished the desmopressin-stimulated increase of plasma membrane water permeability, whereas inhibitor of PKC alone did not suppress the stimulation of the water permeability by db-cAMP. The PKC activity and calciumdependent second messengers seem to be important for regulation of water permeability by vasopressin.     

Cryopreservation of articular cartilage. Part 2: mechanisms of cryoinjury

Although isolated chondrocytes can be cryopreserved by standard methods, at the present time there is no satisfactory method that will preserve living chondrocytes in situ in surgical grafts, between the time of procurement or manufacture and actual use; survival of living chondrocytes in situ is inadequate at best and is also very variable. The first step in identifying the cause of this discrepancy was to establish that the cryoprotectants we had chosen to use, dimethyl sulphoxide and propylene glycol, do actually penetrate into the tissue rapidly. They do. Moreover, chondrocytes were shown to tolerate 10 or 20% Me2SO and were not unusually susceptible to osmotic stress. An experiment in which the effects of freezing with 10% Me2SO to -50 degrees C were separated from the effects of the concomitant rise in solute concentration showed that injury was associated with the formation of ice as such. Freeze substitution microscopy showed that large ice crystals were formed within the chondron, some at least within chondrocytes, even when the cooling rate was optimal for isolated chondrocytes. It is proposed that the nucleation and preferential growth of ice within the chondron (rather than the surrounding acellular matrix) is responsible for the very poor survival of chondrocytes in situ when current methods of cartilage cryopreservation are used.     

Osmotic loading of articular cartilage modulates cell deformations along primary collagen fibril directions

Osmotic loading is known to modulate chondrocyte (cell) height, width and volume in articular cartilage. It is not known how cartilage architecture, especially the collagen fibril orientation, affects cell shape changes as a result of an osmotic challenge.Intact patellae of New Zealand white rabbits (n=6) were prepared for fluorescence imaging. Patellae were exposed to a hypotonic osmotic shock and cells were imaged before loading and 5–60 min after the osmotic challenge. Cell volumes and aspect ratios (height/width) were analyzed. A fibril-reinforced poroelastic swelling model with realistic primary collagen fibril orientations, i.e. horizontal, random and vertical orientation in the superficial, middle and deep zones, respectively and cells in different zones was used to estimate cell aspect ratios theoretically.As the medium osmolarity was reduced, cell aspect ratios decreased and volumes increased in the superficial zone of cartilage both experimentally (p<0.05) and theoretically. Theoretically determined aspect ratios of middle zone cells remained virtually constant, while they increased for deep zone cells as osmolarity was reduced.Findings of this study suggest that osmotic loading modulates chondrocyte shapes in accordance with the primary collagen fibril directions in articular cartilage.     

Hyper-osmotic stress induces volume change and calcium transients in chondrocytes by transmembrane, phospholipid, and G-protein pathways

Mechanical compression of cartilage is associated with a rise in the interstitial osmotic pressure, which can alter cell volume and activate volume recovery pathways. One of the early events implicated in regulatory volume changes and mechanotransduction is an increase of intracellular calcium ion ([Ca²⁺]_i). In this study, we tested the hypothesis that osmotic stress initiates intracellular Ca²⁺ signaling in chondrocytes. Using laser scanning microscopy and digital image processing, [Ca²⁺]_i and cell volume were monitored in chondrocytes exposed to hyper-osmotic solutions. Control experiments showed that exposure to hyper-osmotic solution caused significant decreases in cell volume as well as transient increases in [Ca²⁺]_i. The initial peak in [Ca²⁺]_i was generally followed by decaying oscillations. Pretreatment with gadolinium, a non-specific blocker of mechanosensitive ion channels, inhibited this [Ca²⁺]_i increase. Calcium-free media eliminated [Ca²⁺]_i increases in all cases. Pretreatment with U73122, thapsigargin, or heparin (blockers of the inositol phosphate pathway), or pertussis toxin (a blocker of G-proteins) significantly decreased the percentage of cells responding to osmotic stress and nearly abolished all oscillations. Cell volume decreased with hyper-osmotic stress and recovered towards baseline levels throughout the duration of the control experiments. The peak volume change with 550 mOsm osmotic stress, as well as the percent recovery of cell volume, was dependent on [Ca²⁺]_i. These findings indicate that osmotic stress causes significant volume change in chondrocytes and may activate an intracellular second messenger signal by inducing transient increases in [Ca²⁺]_i.     

Hypotonic swelling activates the Na*/H* exchange in rat brain synaptosomes

The influence of hypotonic swelling and hypertonic shrinking on cytosolic pH in synaptosomes was investigated. It was shown that decreasing the osmolarity of incubation medium to 230 mOsm leads to alkalization and increasing the osmolarity of incubation medium to 810 mOsm leads to acidification. Alkalization was inhibited by amiloride, indicating the involvement of the Na+/H+ exchanger. The acidification of cytosol upon hypertonic shrinking was insensitive, to amiloride and the inhibitor of Na+, K+, Cl- cotransport bumetanide. Thus, the Na+/H+ exchange in synaptosomes is activated by hypotonic swelling but not hypertonic shrinking, in contrast with erythrocytes and lymphocytes, which have been investigated earlier.     

Dynamic osmotic loading of chondrocytes using a novel microfluidic device

Many cells exhibit disparate responses to a mechanical stimulus depending on whether it is applied dynamically or statically. In this context, few studies have examined how cells respond to dynamic changes of the extracellular osmolality. In this study, we hypothesized that the cell size change response of cultured articular chondrocytes would be dependent on the frequency of applied osmotic loading. To test this hypothesis, we developed a novel microfluidic device, to apply hydrostatic pressure-driven dynamic osmotic loading by applying composition modulated flow, adapted from Tang and co-workers. This microfluidic device was used to study osmotic loads of +/-180 mOsm at a frequency up to 0.1 Hz with a constant minimal fluid-shear stress, and permit real-time monitoring of cell responses. Bovine articular chondrocytes were observed to exhibit increasing changes in cell volume with decreasing osmotic loading frequency. When the cell volume response was modeled by an exponential function, chondrocytes exhibited significantly different volume change responses to dynamic osmotic loading at 0.0125 Hz and static osmotic loading applied for a period of four minutes (Delta = +/-180 mOsm relative to the isotonic 360 mOsm). The intracellular calcium response at 0.0125 Hz was also monitored and compared with the response to static loading. Coupled with phenomenological or constitutive models, this novel approach could yield new information regarding cell material properties in response to dynamic loading that may contribute new insights into mechanisms of cellular homeostasis and mechanotransduction.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Osmolarity regulates chondrogenic differentiation potential of synovial fluid derived mesenchymal progenitor cells

Cartilage is one of few tissues where adult stem/progenitor cells have not been putatively identified. Recent studies have provided strong evidence that a sub-population of mesenchymal progenitor cells (MPCs) derived from the synovial fluid may be able to affect some degree of cartilage repair both in vivo and in vitro/ex vivo, however this does not appear to be the case in patients with arthritis. Previously, it has been found that synovial fluid osmolarity is decreased in patients with osteoarthritis (OA) or Rheumatoid arthritis (RA) and these changes in osmolarity have been linked to changes in chondrocyte gene regulation. However, it is yet unknown if changes in osmolarity regulate the gene expression in synovial fluid MPCs (sfMPCs), and by extension, chondrogenesis of this cell population. In the present study we have collected synovial fluid samples from normal, OA and RA knee joints, quantified the osmolarity of the fluid and modified the culture/differentiation media to span a range of osmolarities (264-375 mOsm). Chondrogenesis was measured with Alcian blue staining of cultures in addition to quantitative PCR (qPCR) using probes to Sox9, ACAN and Col2A1. Overall, sfMPCs from arthritic joints demonstrated decreased chondrogenic potential compared to sfMPCs isolated from normal synovial fluid. Furthermore, the sfMPCs retained increased chondrogenic potential if differentiated under the same osmolarity conditions for which they were initially derived within. In conclusion, it does appear the synovial fluid osmolarity regulates the chondrogenic potential of sfMPCs, however, further study is required to elucidate the mechanism by which the changes in osmolarity are sensed by the cells and regulate chondrogenic gene expression.     

Osmotic control of luminescence and growth in Photobacterium leiognathi from ponyfish light organs

Osmolarity was found to control the luminescence and growth of Photobacterium leiognathi strain LN-1a isolated from the light organ of the ponyfish Leiognathus nuchalis (family Leiognathidae). Low osmolarity (ca. 400 mOsm) stimulated luminescence per cell 80 to 100-fold to a level (ca. 2.0×10⁴ quanta·s^-1·cell^-1) equal to that of bacteria taken directly from the light organ and increased the level of luciferase per cell 8 to 10-fold compared to high osmolarity (ca. 800 mOsm). Conversely, high osmolarity stimulated oxygen uptake and growth rate 2 to 4-fold compared to low osmolarity. Of 21 additional tested strains of P. leiognathi from light organs of 9 other ponyfish species, all responded similarly. Low osmolarity may be a host control factor that functions to stimulate the luminescence and restrict the growth of ponyfish light organ bacteria in situ.     

Changes in the lipid dynamics of liposomal membranes induced by poly(ethylene glycol): free volume alterations revealed by inter- and intramolecular excimer-forming phospholipid analogs.

Influence of osmotic shrinkage, swelling, and dehydration on large unilamellar liposomes (LUVs) of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) was investigated using the fluorescent lipid probes 1-palmitoyl-2-[10-(pyren-1-yl)]-decanoyl-sn-glycero-3-phosphocholi ne (PPDPC) and 1,2-bis[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC). Increasing concentrations of poly(ethylene glycol) (PEG, average molecular weight of 6000) producing osmotic gradients delta omega up to 250 mOsm/kg were first added to the outside of LUV labeled with 0.1 mol% of either of the above fluorescent phospholipids. The resulting osmotic shrinkage was accompanied by a progressive reduction in the lateral diffusion of the membrane-incorporated PPDPC, evident as a decrease in the rate of its intermolecular excimer formation. In contrast, under the same conditions the rate of intramolecular excimer formation by bisPDPC increased. Notably, signals opposite to those described above were observed for both of the fluorescent probes upon osmotic swelling of DOPC liposomes with encapsulated PEG. The lateral diffusion of PPDPC became progressively reduced upon membrane dehydration due to increasing concentrations of symmetrically distributed PEG (with equal polymer concentrations inside and outside of the liposomes) when neither shrinkage nor swelling occurs while enhanced excimer formation by bisPDPC was evident. The later results were interpreted in terms of osmotically induced changes in the hydration of lipids. In brief, the removal of water from the phospholipid hydration shell diminishes the effective size of the polar headgroup, which subsequently allows for an enhanced lateral packing of the phospholipid acyl chains. Our findings are readily compatible with membrane free volume Vf changes due to osmotic forces under three different kinds of stress (shrinkage, swelling, and dehydration) applied on the lipid bilayers.