In situ hybridization: A new technique to determine the origin of fibroblasts in cryopreserved aortic homograft valve explants

Tissue degeneration reduces the durability of cryopreserved homografts. Earlier studies indicated that the presence of fibroblasts in homograft leaflets may contribute to increased valve longevity, These fibroblasts may be of recipient origin or represent surviving donor cells. We developed a method, based on in situ hybridization, to determine the origin of fibroblasts in homograft explants. In young pigs we performed aortic valve replacement with a cryopreserved porcine aortic homograft, A male homograft was implanted in a female pig, whereas two male recipients received a female homograft. After 3 to 4 months the homografts were explanted, Frozen sections were made and alternately examined with hematoxylin-eosin staining and in situ hybridization. With a biotinylated porcine Y chromosome-specific deoxyribonucleic acid probe, male fibroblasts could be clearly distinguished from female fibroblasts. In all leaflets we observed both donor and recipient fibroblasts. The distribution of these populations was marked in schematic drawings. Recipient fibroblasts mostly spread onto the leaflet surface but also penetrated the leaflet tissue. Remaining donor fibroblasts did not show morphologic signs of decreased viability on hematoxylin-eosin staining. In situ hybridization may become a useful technique in homograft research, In this porcine model, the fibroblasts in the aortic homograft explants were of both donor and recipient origin.