Introduction:In situ tissue engineering (TE) of heart valves uses readily available acellular synthetic biodegradable scaffolds that transform in vivo in autologous living valves. This study hypothesized that scaffold fiber orientation that resembles native collagen alignment (i.e. anisotropic) results in superior valve function, mechanical properties, and matrix formation during in situ TE.Methods: Trileaflet heart valve scaffolds of biodegradable ureidopyrimidinone (UPy)-polymers with anisotropic (n=10; fibers in circumferential direction) or isotropic (n=10) fiber orientation were produced and implanted in the pulmonary position in sheep. Functional evaluation with echocardiography was performed. Explants (1, 6 and 12 months) were analyzed to evaluate cellularity, extracellular matrix formation and organization, and mechanical propertiesResults: Seventeen animals survived the entire follow-up time and showed functional valves. Consistently higher-pressure gradients were observed for valves in the anisotropic group (not significant at 12 months). Macroscopic analysis revealed pliable leaflets, with no evident differences between the groups. In both groups, fiber resorption was not completed after 12 months and most pronounced at the cell-rich base. Matrix organization did not show apparent differences between the groups, nor in quantity nor in orientation. Notably, anisotropic valves were stiffer in circumferential direction prior to implantation, which already was negated after 1 month of implantation.Conclusions:In situ TE of pulmonary valves demonstrated sustained functionality up to 12 months. Surprisingly, the predefined scaffold fiber organization did not result in significant differences in the newly formed matrix orientation. Apparently, other cues seem to overrule the effect of predefined fiber organization, limiting our possibilities to guide matrix formation by changing scaffold fiber orientation.