Freeze drying of cardiac valves in preparation for cellular repopulation

When freeze-dried cardiac valves have been implanted they remained acellular. This study is the initial step in the development of a method designed to repopulate the substance of the freeze-dried valve with fibroblasts and the lumenal surface with endothelial cells. In this scheme, the freeze-drying process performs three functions; it provides a porous matrix, it kills the donor cells, and it preserves the collagen structure and hence the mechanical strength of the valve. This paper describes the production of appropriate porosity in freeze-dried porcine pulmonary valve leaflets. We found that Tg' for this material is -83 degrees C, which made it impracticable to freeze-dry exclusively from the glassy state. Uncontrolled freeze-drying produced a variable structure with most of the pores considerably smaller than the desired size and a dense layer, apparently devoid of perforations, on the surface. Compacted layers also occurred within the substance of the leaflets. These appearances suggested that extensive collapse had occurred during the drying process. Variation of the cooling rate, the primary drying temperature, and the warming rate during secondary drying enabled us to identify the following conditions that provided satisfactory internal porosity: cooling at 5 degrees C/min, vacuum drying for 6 h at -20 degrees C, and secondary drying for 10 h during rewarming at 0.06-0.08 degrees C/min. The internal cavities measured 100-350 microns2 by ca. 400 microns2, which is adequate to provide access for the fibroblasts (cross-sectional area ca. 150-200 microns2 when rounded but fusiform when attached. However, the internal porous structure rarely communicated with the surface and mechanical perforation was required to provide continuity between the surface and the internal sponge. The resulting method provides a basis for studies of cell colonization.