Chloride channels and the reactions of cells to topography.

The reactions of rat epitenon cells to substratum topography on the micrometric and nanometric scale such as groove-ridge structures include cell extension, elongation and orientation reactions. In this paper we report that stretch-sensitive chloride channels may be involved in the earliest stages of these reactions in epitenon fibroblast-like cells. We report that rat epitenon-cells can develop appreciable lateral mechanical tension that could stretch both the force generating cells themselves and those nearby. We show that cells in medium in which more than 80% of the chloride has been replaced by nitrate show little reaction to topography. Spreading of the cells takes place but is much reduced along the direction of the groove-ridge topography but enhanced across the topography. The chloride channel inhibitors NPPB (5-Nitro-2- (3phenylpropylamino) benzoicacid) 4,4'-disothiocyanostilbene-2, 2' sulphonic acid (DIDS) and Chlorotoxin produce similar results which are further accentuated when these inhibitors are presented in low chloride medium. An antibody against ClC3, which has close homology to ClC5/6 also, blocked reaction to topography. These treatments have no significant effect on cell spreading on planar surfaces nor do they lead to changes in internal pH in the cells. There is a slight inhibition of rates of cell movement. Experiments using antisense oligoribonucleotides to ClC-5 or ClC-6 channel m-RNA also inhibit topographic reactions, which provides further confirmation of the hypothesis. Since the ClC-3,4 and 5 share considerable sequence similarities in the genes and in their proteins it has not been possible to make an unambigous determination of which precise chloride channel(s) is (are) involved.

Dynamic in vitro calcification of porcine aortic valves.

Previous work in our laboratory has demonstrated a simple, dynamic in vitro calcification method for studying bovine pericardial heart valves. The calcification produced closely resembled that found in clinical explant valves. The current study extends this technique to the porcine aortic bioprosthesis. Five Carpentier-Edwards porcine aortic bioprostheses were calcified in vitro in a modified wear tester. All valves calcified to a similar degree as bovine pericardial valves. Calcification predominated on the ribbed tissue structures near the commissures on the outflow surfaces. The same calcification pattern was seen in clinical explant valves. A number of anti-calcification modifications of porcine aortic valves were also investigated. These had all previously inhibited calcification of bovine pericardium in a rat subdermal implant model but had failed to reduce calcification in whole pericardial valves calcified in vitro under dynamic conditions. The modified porcine valves produced similar results: no modification achieved reduction of calcification on exposure to the functional valve calcification model. The dynamic in vitro calcification test has been shown to be useful for the study of both main types of bioprostheses, bovine pericardial and porcine aortic valves, and for the assessment of alterations to these biomaterials.