Dependence of zonal chondrocyte water transport properties on osmotic environment

Abstract

OBJECTIVE: The increasing concentration of proteoglycans from the surface to the deep zone of articular cartilage produces a depth-dependent gradient in fixed charge density, and therefore extracellular osmolarity, which may vary with loading conditions, growth and development, or disease. In this study we examine the relationship between in situ variations in osmolarity on chondrocyte water transport properties. Chondrocytes from the depth-dependent zones of cartilage, effectively preconditioned in varying osmolarities, were used to probe this relationship. DESIGN: First, depth variation in osmolarity of juvenile bovine cartilage under resting and loaded conditions was characterized using a combined experimental/theoretical approach. Zonal chondrocytes were isolated into two representative "baseline" osmolarities chosen from this analysis to reflect in situ conditions. Osmotic challenge was then used as a tool for determination of water transport properties at each of these baselines. Cell calcium signaling was monitored simultaneously as a preliminary examination of osmotic baseline effects on cell signaling pathways. RESULTS: Osmotic baseline exhibits a significant effect on the cell membrane hydraulic permeability of certain zonal subpopulations but not on cell water content or incidence of calcium signaling. CONCLUSIONS: Chondrocyte properties can be sensitive to changes in baseline osmolarity, such as those occurring during OA progression (decrease) and de novo tissue synthesis (increase). Care should be taken in comparing chondrocyte properties across zones when cells are tested in vitro in non-physiologic culture media.

Figure 1

Schematic of the study design, with study labels A, B1, and B2 corresponding to expanded descriptions in the Materials and Methods section.

Figure 2

Percent water content (A) and fixed charge density (B) through the juvenile bovine cartilage tissue depth (0=cartilage surface, 1=bone) as determined by biochemical assaying. Solid lines represent (A) exponential decay and (B) third-degree polynomial curve fits through the total data set. Symbols represent average values (accompanied by standard deviation bars) obtained from binning data in 0.025 increments for normalized tissue depth positions 0–0.1; 0.05 increments were used thereafter.

Figure 3

Results of finite element modeling of juvenile cartilage tissue osmolarity under resting and strained conditions. Solid line represents tissue osmolarity under resting conditions. Dotted line represents osmolarity when tissue is under 25% engineering strain applied in 0.5 seconds. Dashed line represents osmolarity under 25% engineering strain applied in nominal increments, where tissue was allowed to equilibrate after each increment.

Figure 4

Baseline cell volume of zonal cells isolated during digestion into 300 or 400 mOsM media (*p<0.05 with other zones, same baseline; **p<0.05 with MZC at 300 mOsM baseline; n=70–100 cells per zone, each baseline).

Figure 5

Zonal cell water content at each osmotic baseline, obtained from application of Kedem-Katchalsky equations to transient change in cell volume response to step osmotic loading. (all groups average 61 ± 1%, n=37–65 cells per zone, each baseline).

Figure 6

Zonal cell hydraulic membrane permeability, obtained from application of Kedem-Katchalsky equations to cell size change response to step osmotic loading. (*p<0.01 with same zone at 300 mOsM baseline; n=37–65 cells per zone, each baseline).

Figure 7

Percent zonal cells exhibiting at least one intracellular calcium peak upon ±100 mOsM step osmotic load from 300 or 400 mOsM baseline media (*p<0.05 with −100 mOsM group, same zone same baseline; n=84–119 cells per zone, each baseline).