Osmotic Loading Research Articles

Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading

SummaryObjectivesSOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9.MethodsEquine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK–ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured using qRT-PCR. De novo glycosaminoglycan synthesis of explants was determined with 35S sulphate during static hyperosmolar loading.ResultsMEK–ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition.ConclusionsThe response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype. This variation may be important in disease progression.

Transient receptor potential vanilloid 4 channel as an important modulator of chondrocyte mechanotransduction of osmotic loading

Transient receptor potential vanilloid 4 channel as an important modulator of chondrocyte mechanotransduction of osmotic loading

Influence of the Partitioning of Osmolytes by the Cytoplasm on the Passive Response of Cells to Osmotic Loading

Due to the dense organization of organelles, cytoskeletal elements, and protein complexes that make up the intracellular environment, it is likely that membrane-permeant solutes may be excluded from a fraction of the interstitial space of the cytoplasm via steric restrictions, electrostatic interactions, and other long-range intermolecular forces. This study investigates the hypothesis that the intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery in response to hyperosmotic loading, based on prior theoretical and biomimetic experimental studies. Osmotic loading experiments are performed on immature bovine chondrocytes using culture conditions where regulatory volume responses are shown to be insignificant. Osmotic loading with membrane-permeant glycerol (92 Da) and urea (60 Da) are observed to produce partial volume recoveries consistent with the proposed hypothesis, whereas loading with 1,2-propanediol (76 Da) produces complete volume recovery. Combining these experimental results with the previous theoretical framework produces a measure for the intracellular partition coefficient of each of these solutes. At 1000 mOsm, 1,2-propanediol is the only osmolyte to yield a partition coefficient not statistically different from unity, κ p i = 1.00 ± 0.02. For glycerol, the partition coefficient increases with osmolarity from κ p i = 0.48 ± 0.19 at 200 mOsm to κ p i = 0.80 ± 0.07 at 1000 mOsm; urea exhibits no such dependence, with an average value of κ p i = 0.87 ± 0.07 for all osmolarities from 200 to 1000 mOsm. The finding that intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery under osmotic loading offers a simple method for characterizing the partition coefficient. These measurements suggest that significant partitioning may occur even for small membrane-permeant osmolytes. Furthermore, a positive correlation is observed, suggesting that a solute's cytoplasmic partition coefficient increases with increasing hydrophobicity.

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 5–60min 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.

Modeling the Matrix of Articular Cartilage Using a Continuous Fiber Angular Distribution Predicts Many Observed Phenomena

Cartilage is a hydrated soft tissue whose solid matrix consists of negatively charged proteoglycans enmeshed within a fibrillar collagen network. Though many aspects of cartilage mechanics are well understood today, most notably in the context of porous media mechanics, there remain a number of responses observed experimentally whose prediction from theory has been challenging. In this study the solid matrix of cartilage is modeled with a continuous fiber angular distribution, where fibers can only sustain tension, swelled by the osmotic pressure of a proteoglycan ground matrix. It is shown that this representation of cartilage can predict a number of observed phenomena in relation to the tissue's equilibrium response to mechanical and osmotic loading, when flow-dependent and flow-independent viscoelastic effects have subsided. In particular, this model can predict the transition of Poisson's ratio from very low values in compression (approximately 0.02) to very high values in tension (approximately 2.0). Most of these phenomena cannot be explained when using only three orthogonal fiber bundles to describe the tissue matrix, a common modeling assumption used to date. The main picture emerging from this analysis is that the anisotropy of the fibrillar matrix of articular cartilage is intimately dependent on the mechanism of tensed fiber recruitment, in the manner suggested by our recent theoretical study (Ateshian, 2007, ASME J. Biomech. Eng., 129(2), pp. 240-249).

Mechanical effects of surgical procedures on osteochondral grafts elucidated by osmotic loading and real-time ultrasound

IntroductionOsteochondral grafts have become popular for treating small, isolated and full-thickness cartilage lesions. It is recommended that a slightly oversized, rather than an exact-sized, osteochondral plug is transplanted to achieve a tight fit. Consequently, impacting forces are required to insert the osteochondral plug into the recipient site. However, it remains controversial whether these impacting forces affect the biomechanical condition of the grafted articular cartilage. The present study aimed to investigate the mechanical effects of osteochondral plug implantation using osmotic loading and real-time ultrasound.MethodsA full-thickness cylindrical osteochondral defect (diameter, 3.5 mm; depth, 5 mm) was created in the lateral lower quarter of the patella. Using graft-harvesting instruments, an osteochondral plug (diameter, 3.5 mm as exact-size or 4.5 mm as oversize; depth, 5 mm) was harvested from the lateral upper quarter of the patella and transplanted into the defect. Intact patella was used as a control. The samples were monitored by real-time ultrasound during sequential changes of the bathing solution from 0.15 M to 2 M saline (shrinkage phase) and back to 0.15 M saline (swelling phase). For cartilage sample assessment, three indices were selected, namely the change in amplitude from the cartilage surface (amplitude recovery rate: ARR) and the maximum echo shifts from the cartilage surface and the cartilage-bone interface.ResultsThe ARR is closely related to the cartilage surface integrity, while the echo shifts from the cartilage surface and the cartilage-bone interface are closely related to tissue deformation and NaCl diffusion, respectively. The ARR values of the oversized plugs were significantly lower than those of the control and exact-sized plugs. Regarding the maximum echo shifts from the cartilage surface and the cartilage-bone interface, no significant differences were observed among the three groups.ConclusionsThese findings demonstrated that osmotic loading and real-time ultrasound were able to assess the mechanical condition of cartilage plugs after osteochondral grafting. In particular, the ARR was able to detect damage to the superficial collagen network in a non-destructive manner. Therefore, osmotic loading and real-time ultrasound are promising as minimally invasive methods for evaluating cartilage damage in the superficial zone after trauma or impact loading for osteochondral grafting.

Interleukin-1 inhibits osmotically induced calcium signaling and volume regulation in articular chondrocytes

Articular chondrocytes respond to osmotic stress with transient changes in cell volume and the intracellular concentration of calcium ion ([Ca(2+)](i)). The goal of this study was to examine the hypothesis that interleukin-1 (IL-1), a pro-inflammatory cytokine associated with osteoarthritis, influences osmotically induced Ca(2+) signaling. Fluorescence ratio imaging was used to measure [Ca(2+)](i) and cell volume in response to hypo- or hyper-osmotic stress in isolated porcine chondrocytes, with or without pre-exposure to 10-ng/ml IL-1alpha. Inhibitors of IL-1 (IL-1 receptor antagonist, IL-1Ra), Ca(2+) mobilization (thapsigargin, an inhibitor of Ca-ATPases), and cytoskeletal remodeling (toxin B, an inhibitor of the Rho family of small GTPases) were used to determine the mechanisms involved in increased [Ca(2+)](i), F-actin remodeling, volume adaptation and active volume recovery. In response to osmotic stress, chondrocytes exhibited transient increases in [Ca(2+)](i), generally followed by decaying oscillations. Pre-exposure to IL-1 significantly inhibited regulatory volume decrease (RVD) following hypo-osmotic swelling and reduced the change in cell volume and the time to peak [Ca(2+)](i) in response to hyper-osmotic stress, but did not affect the peak magnitudes of [Ca(2+)](i) in those cells that did respond. Co-treatment with IL-1Ra, thapsigargin, or toxin B restored these responses to control levels. The effects were associated with alterations in F-actin organization. IL-1 alters the normal volumetric and Ca(2+) signaling response of chondrocytes to osmotic stress through mechanisms involving F-actin remodeling via small Rho GTPases. These findings provide further insights into the mechanisms by which IL-1 may interfere with normal physiologic processes in the chondrocyte, such as the adaptation or regulatory responses to mechanical or osmotic loading.

Elk‐1 is a novel protein‐binding partner for FAK, regulating phagocytosis in medfly hemocytes

Focal adhesion kinase (FAK) and its downstream signaling targets, mitogen-activated protein kinase (MAPKs), are implicated in the process of phagocytosis by insect hemocytes. The goal of this study was to explore further the signaling pathways underlining the process of phagocytosis. The combination of bioinformatics, biochemical, and immunofluorescence approaches strongly support the expression of Elk-1-like protein in medfly hemocytes. Elk-1 is phosphorylated in E. coli or latex beads-challenged hemocytes and osmotic loading experiments as well as flow cytometry analysis demonstrated that Elk-1-like protein regulates the uptake of bacteria. RNA interference (RNAi) and pharmacological inhibitors show that the signaling for Elk-1 phosphorylation is transmitted via FAK/Src and MAPKs pathways. Furthermore, confocal analysis clearly shows that FAK and the phosphorylated FAK at Y397 are localized in the nucleus and cytoplasm, whereas, the phosphorylated Elk-1-like protein is exclusively localized in the nucleus. Finally, co-immunoprecipitation and reciprocal co-immunoprecipitation analysis demonstrated the association of low molecular weight protein bands recognized by FAK antibodies, with Elk-1 or phospho-Elk-1 at ser 383 and confocal microscopy specifies that this association occurs only in the nucleus. These results are strongly supporting that Elk-1-like protein is a novel protein-binding partner for FAK, a finding that significantly broadens the potential functioning of FAK and Elk-1 generally. Evidently, the complex participates in the process of phagocytosis in medfly hemocytes.

Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes

Articular chondrocytes have a well-developed osmoregulatory system that enables cells to survive in a constantly changing osmotic environment. However, osmotic loading exceeding that occurring under physiological conditions severely compromises chondrocyte function and leads to degenerative changes. The aim of the present study was to investigate the form of cell death and changes in apoptotic signaling pathways under hyperosmotic stress using a primary chondrocyte culture. Cell viability and apoptosis assays performed with annexin V and propidium iodide staining showed that a highly hyperosmotic medium (600 mOsm) severely reduced chondrocyte viability and led mainly to apoptotic cell death, while elevating osmotic pressure within the physiological range caused no changes compared to isosmotic conditions. Western blot analysis revealed that a 600 mOsm hyperosmotic environment induced the activation of proapoptotic members of the mitogen-activated protein kinase family such as c-Jun N-terminal kinase (JNK) and p38, and led to an increased level of extracellular signal regulated kinase (ERK1/2). Hyperosmotic stress also induced the activation of caspase-3. In summary, our results show that hyperosmotic stress leads to mainly apoptotic cell death via the involvement of proapoptotic signaling pathways in a primary chondrocyte culture.

Necrotic and apoptotic volume changes of red blood cells investigated by low-angle light scattering technique

A low-angle light scattering technique, which has been applied previously to studies of blood platelets and Ehrlich ascite tumor cells, revealed differences in the dynamics of necrotic and apoptotic red blood cell death. Under hypotonic loading or in ammonia medium, red blood cells (RBC) swelled to a critical size (diameter approximately 13μm) prior to hemolysis (necrosis). Under acidic loading, hemolysis occurred with less pronounced swelling of cells (diameter approximately 10μm). Apoptosis induced by a calcium ionophore resulted in initial formation of echinocytes, followed by development of rounded red blood cells with uneven membrane, capable of agglomeration. In such a way, RBC aggregation can precede the final stages of the RBC apoptosis when small cellular fragments are generated. On the basis of erythrograms of the cells hemolysing in ammonia medium, the echinocytic (preapoptotic) and stomatocytic (prenecrotic) RBC were discerned due to the very high resistance of apoptotic RBC to osmotic (ammonia) loading.

Osmotic Loading of Spherical Gels: A Biomimetic Study of Hindered Transport in the Cell Protoplasm

Osmotic loading of cells has been used to investigate their physicochemical properties as well as their biosynthetic activities. The classical Kedem-Katchalsky framework for analyzing cell response to osmotic loading, which models the cell as a fluid-filled membrane, does not generally account for the possibility of partial volume recovery in response to loading with a permeating osmolyte, as observed in some experiments. The cell may be more accurately represented as a hydrated gel surrounded by a semi-permeable membrane, with the gel and membrane potentially exhibiting different properties. To help assess whether this more elaborate model of the cell is justified, this study investigates the response of spherical gels to osmotic loading, both from experiments and theory. The spherical gel is described using the framework of mixture theory. In the experimental component of the study alginate is used as the model gel, and is osmotically loaded with dextran solutions of various concentrations and molecular weight, to verify the predictions from the theoretical analysis. Results show that the mixture framework can accurately predict the transient and equilibrium response of alginate gels to osmotic loading with dextran solutions. It is found that the partition coefficient of dextran in alginate regulates the equilibrium volume response and can explain partial volume recovery based on passive transport mechanisms. The validation of this theoretical framework facilitates future investigations of the role of the protoplasm in the response of cells to osmotic loading.

Extraction of Mechanical Properties of Articular Cartilage From Osmotic Swelling Behavior Monitored Using High Frequency Ultrasound

Articular cartilage is a biological weight-bearing tissue covering the bony ends of articulating joints. Negatively charged proteoglycan (PG) in articular cartilage is one of the main factors that govern its compressive mechanical behavior and swelling phenomenon. PG is nonuniformly distributed throughout the depth direction, and its amount or distribution may change in the degenerated articular cartilage such as osteoarthritis. In this paper, we used a 50 MHz ultrasound system to study the depth-dependent strain of articular cartilage under the osmotic loading induced by the decrease of the bathing saline concentration. The swelling-induced strains under the osmotic loading were used to determine the layered material properties of articular cartilage based on a triphasic model of the free-swelling. Fourteen cylindrical cartilage-bone samples prepared from fresh normal bovine patellae were tested in situ in this study. A layered triphasic model was proposed to describe the depth distribution of the swelling strain for the cartilage and to determine its aggregate modulus H(a) at two different layers, within which H(a) was assumed to be linearly dependent on the depth. The results showed that H(a) was 3.0+/-3.2, 7.0+/-7.4, 24.5+/-11.1 MPa at the cartilage surface, layer interface, and deep region, respectively. They are significantly different (p<0.01). The layer interface located at 70%+/-20% of the overall thickness from the uncalcified-calcified cartilage interface. Parametric analysis demonstrated that the depth-dependent distribution of the water fraction had a significant effect on the modeling results but not the fixed charge density. This study showed that high-frequency ultrasound measurement together with triphasic modeling is practical for quantifying the layered mechanical properties of articular cartilage nondestructively and has the potential for providing useful information for the detection of the early signs of osteoarthritis.

Pathogenesis of osteoarthritis‐like changes in the joints of mice deficient in type IX collagen

To examine the pathogenetic mechanisms of osteoarthritis (OA)-like changes in Col9a1-/- mice, which are deficient in type IX collagen. Knee joints and temporomandibular joints (TMJs) from Col9a1-/- mice and their wild-type (Col9a1+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, degraded type II collagen, and the discoidin domain receptor 2 (DDR-2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle. Histologic analysis showed age-dependent OA-like changes in the knee and TMJs of Col9a1-/- mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP-13 and DDR-2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1-/- mice than in their wild-type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1-/- mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1-/- mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites. Lack of type IX collagen in Col9a1-/- mice results in age-dependent OA-like changes in the knee joints and TMJs.

Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading

While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (-200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli.

Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole‐organ intervertebral disc model

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.

Ultrasound elastomicroscopy using water jet and osmosis loading: Potentials for assessment for articular cartilage

Research in elasticity imaging typically relies on 1–10 MHz ultrasound. Elasticity imaging at these frequencies can provide strain maps with a resolution in the order of millimeters, but this is not sufficient for applications to skin, articular cartilage, or other fine structures. In this paper, we introduced two methods of ultrasound elastomicroscopy using water jet and osmosis loading for imaging the elasticity of biological soft tissues with high resolutions. In the first system, the specimens were compressed using water jet compression. A water jet was used to couple a focused 20 MHz ultrasound beam into the specimen and meanwhile served as a “soft” indenter. Because there was no additional attenuation when propagating from the ultrasound transducer to the specimen, the ultrasound signal with high signal-to-noise ratio could be collected from the specimens simultaneously with compressing process. The compression was achieved by adjusting the water flow. The pressure measured inside the water pipe and that on the specimen surface was calibrated. This system was easily to apply C-scan over sample surfaces. Experiments on the phantoms showed that this water jet indentation method was reliable to map the tissue stiffness distribution. Results of 1D and 2D scanning on phantoms with different stiffness are reported. In the second system, we used osmotic pressure caused by the ion concentration change in the bathing solutions for the articular cartilage to deform them. When bovine articular cartilage specimens were immerged in solutions with different salt concentration, a 50 MHz focused ultrasound beam was used to monitor the dynamic swelling or shrinkage process. Results showed that the system could reliably map the strain distribution induced by the osmotic loading. We extract intrinsic layered material parameters of the articular cartilage using a triphasic model. In addition to biological tissues, these systems have potential applications for the assessment of bioengineered tissues, biomaterials with fine structures, or some engineering materials. Further studies are necessary to fully realize the potentials of these two new methods.

A Mechano-chemical Model for the Passive Swelling Response of an Isolated Chondron under Osmotic Loading

The chondron is a distinct structure in articular cartilage that consists of the chondrocyte and its pericellular matrix (PCM), a narrow tissue region surrounding the cell that is distinguished by type VI collagen and a high glycosaminoglycan concentration relative to the extracellular matrix. We present a theoretical mechano-chemical model for the passive volumetric response of an isolated chondron under osmotic loading in a simple salt solution at equilibrium. The chondrocyte is modeled as an ideal osmometer and the PCM model is formulated using triphasic mixture theory. A mechano-chemical chondron model is obtained assuming that the chondron boundary is permeable to both water and ions, while the chondrocyte membrane is selectively permeable to only water. For the case of a neo-Hookean PCM constitutive law, the model is used to conduct a parametric analysis of cell and chondron deformation under hyper- and hypo-osmotic loading. In combination with osmotic loading experiments on isolated chondrons, model predictions will aid in determination of pericellular fixed charge density and its relative contribution to PCM mechanical properties.

A mixture theory analysis for passive transport in osmotic loading of cells

The theory of mixtures is applied to the analysis of the passive response of cells to osmotic loading with neutrally charged solutes. The formulation, which is derived for multiple solute species, incorporates partition coefficients for the solutes in the cytoplasm relative to the external solution, and accounts for cell membrane tension. The mixture formulation provides an explicit dependence of the hydraulic conductivity of the cell membrane on the concentration of permeating solutes. The resulting equations are shown to reduce to the classical equations of Kedem and Katchalsky (Biochem. Biophys. Acta 27 (1958) 229, J. Gen. Physiol. 45 (1961) 143) in the limit when the membrane tension is equal to zero and the solute partition coefficient in the cytoplasm is equal to unity. Numerical simulations demonstrate that the concentration-dependence of the hydraulic conductivity is not negligible; the volume response to osmotic loading is very sensitive to the partition coefficient of the solute in the cytoplasm, which controls the magnitude of cell volume recovery; and the volume response is sensitive to the magnitude of cell membrane tension. Deviations of the Boyle–van’t Hoff response from a straight line under hypo-osmotic loading may be indicative of cell membrane tension.

Subfornical organ disconnection alters Fos expression in the lamina terminalis, supraoptic nucleus, and area postrema after intragastric hypertonic NaCl

The lamina terminalis was severed by a horizontal knife cut through the anterior commissure to determine the effects of a disconnection of the subfornical organ (SFO) on drinking and Fos-like immunoreactivity (Fos-ir) in the rat brain in response to an intragastric load of hypertonic saline (5 ml/kg of 1.5 M NaCl by gavage). After an initial load, knife-cut rats drank significantly less water than sham-cut rats, thus confirming a role for the SFO in osmotic drinking. After a second load at least 1 wk later, the rats were not allowed to drink after the gavage and were perfused for analysis of Fos-ir at 90 min. Compared with sham-cut rats, the knife-cut rats displayed significantly elevated Fos-ir in the main body of the SFO, in the dorsal cap of the organum vasculosum laminae terminalis, and in the ventral median preoptic nucleus after the hypertonic load. The knife cut significantly decreased Fos-ir in the supraoptic nucleus. Fos-ir was expressed mainly in the midcoronal and caudal parts of the area postrema of sham-cut rats, and this expression was greatly reduced in knife-cut rats. These findings strengthen the case for the presence of independently functioning osmoreceptors within the SFO and suggest that the structures of the lamina terminalis provide mutual inhibition during hypernatremia. They also demonstrate that the Fos-ir in the area postrema after intragastric osmotic loading is heavily dependent on the intact connectivity of the SFO.

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 ±180mOsm at a frequency up to 0.1Hz 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 0.0125Hz and static osmotic loading applied for a period of four minutes (Δ=±180mOsm relative to the isotonic 360mOsm). The intracellular calcium response at 0.0125Hz 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.