Establishing a Protocol to Quantify Leaflet Fibroblast Responses to Physiologic Flow Through a Viable Heart Valve

Abstract

Mechanical stresses are thought to affect the metabolism of a variety of cell types. Little quantitative data exist regarding heart valve leaflet fibroblast activity after dynamic loading. The goal of this study was to examine leaflet fibroblast function and differentiation in response 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 physiologic flow, a sterilizable pulsatile flow system has been developed to recreate the dynamic flow environment of the aortic valve while preventing contamination from room air. Physiologic flow conditions [frequency 70 bpm, aortic pressure 129/82 mmHg (systolic/diastolic), cardiac output 2.3 L/min] were sustained for 71 hr without microbiologic contamination. Analytic tools for assessment of fibroblast function include a viability assay, which demonstrated that leaflet viability decreases after prolonged exposure to antibiotics. Proline incorporation studies revealed that 11 times more protein is retained by leaflet tissue than is released into the medium, and 27% of this protein is collagen. Polyacrylamide gel electrophoresis clearly resolved collagen Types 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 porcine aortic valves to defined flow conditions, and the viability and protein/collagen biosynthetic activity of leaflet fibroblasts in response to flow will be quantified. These experiments will provide a baseline by which to design and evaluate future tissue engineered substitutes.