On proper linearization, construction and analysis of the Boyle–van’t Hoff plots and correct calculation of the osmotically inactive volume

Katkov, I.

doi:10.1016/j.cryobiol.2011.02.006

Cited by 19 publications

(16 citation statements)

References 14 publications

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“…As Figure 1B shows, chondrocytes increased its volume as a function of external media, as predicted by the BoyleVan't Hoff law [40], this result indicates that freshly isolated rat chondrocytes are osmotically sensitive over the osmolar range tested. It was observed that not all cells were osmotically sensitive, as some did not swell when challenged.…”

Section: Regulatory Volume Decrease Response From Rat Articular Chondsupporting

confidence: 52%

The Role of Swelling-Activated Chloride Currents (I_CL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

Ponce¹,

Jimenez-Peña²,

Tejeda-Guzmán³

2012

Cell Physiol Biochem

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Background/Aims: Articular chondrocytes dwell in an environment that is continuously changing its osmolarity as a consequence of mechanical loading, yet their volume regulation capabilities (RVD) are not fully understood. This work aimed to determine the osmotic sensitivity of freshly isolated rat chondrocytes, their RVD capabilities and to study the properties of any anion currents associated. Methods: Cell volume responses were determined by microscopy. Whole cell patch clamp was used to record ion currents. Results: Chondrocytes showed to be osmotically sensitive and capable of RVD in a size-dependent manner. RVD was accompanied by activation of outwardly rectifying chloride currents, featuring time and voltage independent activation and inactivation at most depolarizing voltage levels, with an anion selectivity sequence of: SCN->I-≅NO3->Br->Cl->F->> Gluconate-> Methanesulphonate-, corresponding to Eisenman’s sequence I. They were also permeable to taurine. These currents were blocked by DIDS, SITS, 9-AC and NPPB and by drugs that block ICl,swell such as fluoxetine, phloretin, DCPIB and tamoxifen. RT-PCR assays show the presence of mRNA for CLC-3 and TMEM16A, that had been proposed as molecular determinants of ICl,swell currents. Conclusions: These findings indicate that freshly isolated rat articular chondrocytes have ICl,swell. The further finding that ICl,swell blockers alter the RVD response suggest that ICl,swell is involved in the RVD response of rat articular chondrocytes.

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Section: Regulatory Volume Decrease Response From Rat Articular Chondsupporting

confidence: 52%

The Role of Swelling-Activated Chloride Currents (I_CL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

Ponce¹,

Jimenez-Peña²,

Tejeda-Guzmán³

2012

Cell Physiol Biochem

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“…To determine the applicability of the BVH relation to both cell types, the following equations, taken from Katkov (2011), were utilized to calculate the osmotically inactive volume, ν b , and plot the true BVH line (Katkov, 2011). The average normalized volume at the given osmolarity was found using:

{\bar{v}}_{i} = \frac{\sum_{j = 1}^{S_{i}} \frac{V_{i j}}{V_{iso, j}}}{N_{i}}

where V iso,j is the iso-osmotic volume obtained at a particular j th replicate.…”

Section: Methodsmentioning

confidence: 99%

Applied osmotic loading for promoting development of engineered cartilage

Sampat

Dermksian

Oungoulian

et al. 2013

Journal of Biomechanics

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This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300 mOsM) or isotonic (330 mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400 mOsM media from day 0 to day 49 yielded a Young’s modulus of 513±89 kPa and GAG content of 7.39±0.52% ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young’s modulus, reaching 487±187 kPa and 6.77±0.54%ww (GAG) for the 400 mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle–van’t Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.

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“…The difference is small because here the measured volume of the isotonic cell is close to the value calculated from the best-fit line. Katkov [14] has argued that the standard method of calculating b is wrong on mathematical grounds. He states that the correct regression line must pass through the measured cell volume in isotonic media.…”

Section: Implications Of Volume Regulation For Boyle Van’t Hoff Plotsmentioning

confidence: 99%

Regulatory volume decrease in COS-7 cells at 22 °C and its influence on the Boyle van’t Hoff relation and the determination of the osmotically inactive volume

Peckys

Mazur

2012

Cryobiology

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Cryobiological analyses assume that the direction and rate of water movements across cell membranes and equilibrium cell volumes are determined solely by differences in the chemical potentials of intra- and extracellular water. A consequence of this assumption is that cells obey the Boyle van’t Hoff (BvH) law which states that cell volumes are a linear function of reciprocal osmolality. Extrapolation of the BvH plot to infinite osmolality yields a quantity b, the fractional volume of the cell occupied by solids. In many cells, however, a cell volume excursion above the isotonic volume initiates an energy-requiring response that causes the swollen cells to shrink back to or towards isotonic volume. It is referred to as Regulatory Volume Decrease (RVD). We have observed a strong RVD in COS-7 cells. If not eliminated by keeping exposure times short, this RVD produces a b that is 60% too high (0.48 vs. 0.30) These results indicate the importance of examining cells for volume regulatory mechanisms before performing measurements to determine their osmotic parameters.

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On proper linearization, construction and analysis of the Boyle–van’t Hoff plots and correct calculation of the osmotically inactive volume

Cited by 19 publications

References 14 publications

The Role of Swelling-Activated Chloride Currents (I_CL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

The Role of Swelling-Activated Chloride Currents (I_CL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

Applied osmotic loading for promoting development of engineered cartilage

Regulatory volume decrease in COS-7 cells at 22 °C and its influence on the Boyle van’t Hoff relation and the determination of the osmotically inactive volume

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On proper linearization, construction and analysis of the Boyle–van’t Hoff plots and correct calculation of the osmotically inactive volume

Cited by 19 publications

References 14 publications

The Role of Swelling-Activated Chloride Currents (ICL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

The Role of Swelling-Activated Chloride Currents (ICL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

Applied osmotic loading for promoting development of engineered cartilage

Regulatory volume decrease in COS-7 cells at 22 °C and its influence on the Boyle van’t Hoff relation and the determination of the osmotically inactive volume

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The Role of Swelling-Activated Chloride Currents (I_CL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes

The Role of Swelling-Activated Chloride Currents (I_CL,swell) in the Regulatory Volume Decrease Response of Freshly Dissociated Rat Articular Chondrocytes