ESTABLISHING A PROTOCOL TO QUANTIFY LEAFLET FIBROBLAST RESPONSES TO PHYSIOLOGICAL FLOW-THROUGH A VIABLE HEART-VALVE

Mechanical stresses are thought to affect the metabolism of a variety of cell types. Little quantitative data exist regarding heart valve le aflet fibroblast activity after dynamic loading. The goal of this stud y was to examine leaflet fibroblast function and differentiation in re sponse to flow through an intact valve. This requires the development of a flow system capable of reproducing the valve's native environment , as well as assay protocols to analyze cellular viability and protein and collagen synthesis. As a tool to expose viable tissue valves to p hysiologic flow, a sterilizable pulsatile flow system has been develop ed to recreate the dynamic flow environment of the aortic valve while preventing contamination from room air. Physiologic flow conditions [f requency 70 bpm, aortic pressure 129/82 mmHg (systolic/diastolic), car diac output 2.3 L/min] were sustained for 71 hr without microbiologic contamination. Analytic tools for assessment of fibroblast function in clude a viability assay, which demonstrated that leaflet viability dec reases after prolonged exposure to antibiotics. Proline incorporation studies revealed that 11 times more protein is retained by leaflet tis sue than is released into the medium, and 27% of this protein is colla gen. Polyacrylamide gel electrophoresis clearly resolved collagen Type s I and III from both prepared standards as well as leaflet extracts. In ongoing work, the sterile flow loop will be used to expose fresh po rcine aortic valves to defined flow conditions, and the viability and protein/collagen biosynthetic activity of leaflet fibroblasts in respo nse to flow will be quantified. These experiments will provide a basel ine by which to design and evaluate future tissue engineered substitut es.