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CG collagen scaffold recruits effectively the MSCs to the center of full-thickness cartilage defect and enhances regeneration of the full-thickness cartilage defect.

Articular chondrocytes are exposed in vivo to the continually changing osmotic environment and thus require volume regulatory mechanisms. The present study was designed to investigate (i) the functional role of the swelling-activated Cl − current (I Cl,swell ) in the regulatory volume decrease (RVD) and (ii) the regulatory role of tyrosine phosphorylation in I Cl,swell , in isolated rabbit articular chondrocytes. Whole-cell membrane currents were recorded from chondrocytes in isosmotic, hyposmotic and hyperosmotic external solutions under conditions where Na + , K + and Ca 2+ currents were minimized. The cell surface area was also measured using microscope images from a separate set of chondrocytes and was used as an index of cell volume. The isolated chondrocytes exhibited a RVD during sustained exposure to hyposmotic solution, which was mostly inhibited by the I Cl,swell blocker 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl)oxobutyric acid (DCPIB) at 20 μm. Exposure to a hyposmotic solution activated I Cl,swell , which was also largely inhibited by 20 μm DCPIB. I Cl,swell in rabbit articular chondrocytes had a relative taurine permeability (P tau /P Cl ) of 0.21. Activation of I Cl,swell was significantly reduced by the protein tyrosine kinase (PTK) inhibitor genistein (30 μm) but was only weakly affected by its inactive analogue daidzein (30 μm). Intracellular application of protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate (250 and 500 μm) resulted in a gradual activation of a Cl − current even in isosmotic solutions. This Cl − current was almost completely inhibited by 4,4 -diisothiocyanatostilbene-2,2 -disulfonate (DIDS, 500 μm) and was also largely suppressed by exposure to hyperosmotic solution, thus indicating a close similarity to I Cl,swell . Pretreatment of chondrocytes with genistein significantly prevented the activation of the Cl − current by sodium orthovanadate, suggesting that the basal activity of endogenous PTK is required for the activation of this Cl − current. Our results provide evidence to indicate that activation of I Cl,swell is involved in RVD in isolated rabbit articular chondrocytes and is facilitated by tyrosine phosphorylation.

Abstract. Articular chondrocytes play an important role in maintaining the structure and function of the cartilage in synovial joints, which is closely influenced by mechanical or osmotic stress. In the present study, isolated rabbit articular chondrocytes were examined during hyposmotic stress using the whole-cell patch-clamp method. When exposed to hyposmotic external solutions (approximately 5% or 32% decrease in osmolarity), isolated rabbit articular chondrocytes exhibited hyposmotic cell swelling, accompanied by the activation of the swellingactivated Cl − current (I Cl,swell ). I Cl,swell was practically time-independent at potentials negative to +50 mV but exhibited rapid inactivation at more positive potentials. I Cl,swell was potently inhibited by the Cl − channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and tamoxifen, but was little affected by pimozide. I Cl,swell was also found to be acutely inhibited by arachidonic acid in a concentration-dependent manner with an IC 50 of 0.81 μM. The maximal effect (approximately 100% block) was obtained with 10 μM arachidonic acid. The arachidonic acid metabolites prostaglandin E 2 , leukotriene B 4 , and leukotriene D 4 had no appreciable effect on I Cl,swell , suggesting that the inhibitory effect of arachidonic acid did not require its metabolism. The present study thus reveals the presence of I Cl,swell in rabbit articular chondrocytes that exhibits high sensitivity to direct inhibition by arachidonic acid.

BACKGROUND AND PURPOSEChondrocyte apoptosis contributes to disruption of cartilage integrity in osteoarthritis. Recent evidence suggested that the volume-sensitive organic osmolyte/anion channel [volume-sensitive (outwardly rectifying) Cl -current (ICl,vol)] plays a functional role in the development of cell shrinkage associated with apoptosis (apoptotic volume decrease) in several cell types. In this study, we investigated the cellular effects of 17b-oestradiol on doxorubicin-induced apoptotic responses in rabbit articular chondrocytes. EXPERIMENTAL APPROACHWhole-cell membrane currents and cross-sectional area were measured from chondrocytes using a patch-clamp method and microscopic cell imaging, respectively. Caspase-3/7 activity was assayed as an index of apoptosis. KEY RESULTSAddition of doxorubicin (1 mM) to isosmotic bath solution rapidly activated the Cl -current with properties similar to those of ICl,vol in chondrocytes. Doxorubicin also gradually decreased the cross-sectional area of chondrocytes, followed by enhanced caspase-3/7 activity; both of these responses were totally abolished by the ICl,vol blocker DCPIB (20 mM). Pretreatment of chondrocytes with 17b-oestradiol (1 nM) for short (approximately 10 min) and long (24 h) periods almost completely prevented the doxorubicin-induced activation of ICl,vol and subsequent elevation of caspase-3/7 activity. These effects of 17b-oestradiol were significantly attenuated by the oestrogen receptor blocker ICI 182780 (10 mM), as well as the phosphatidyl inositol-3-kinase (PI3K) inhibitors wortmannin (100 nM) and LY294002 (20 mM). Testosterone (10 nM) had no effect on the doxorubicin-induced Cl -current. CONCLUSIONS AND IMPLICATIONS17b-Oestradiol prevents the doxorubicin-induced cell shrinkage mediated through activation of ICl,vol and subsequent induction of apoptosis signals, through a membrane receptor-dependent PI3K pathway in rabbit articular chondrocytes. AbbreviationsAVD, apoptotic volume decrease; Cm, cell membrane capacitance; DCPIB, 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxybutyric