Segmentation of each valve, and as well as each constituent leaflet, from the raw µCT images was done using Synopsys’ Simpleware ScanIP (Version 2018.12-SP2; Synopsys, Inc., Mountain View, USA). The PV was first highlighted from the whole structure (including part of the myocardium, aorta, and PA). Next, the three leaflets of the PV were separated and identified as the anterior (A), the left (L), and the right (R) leaflets. Subsequently, we identified the commissure points as the three points (i.e., P1, P2, P3) on the interface of the segmented PV and PA at which the leaflets made contact (Fig. 2a). The basal attachment (blue curve) and free edge (red curve) of each leaflet were then defined as part of the leaflet edge between the commissure points that was or was not attached to the PA, respectively. Lastly, we defined the annulus (ANL) plane of the PV as the plane that passed through the mid-points of the basal attachments of all leaflets (e.g., P5 in Fig. 2a), and the sino-tubular junction (STJ) plane as the plane that passed through the commissure points.

Analysis of the µCT image of individual leaflets. (a) schematic for the geometry of a murine PV and key geometric quantities of interest, (b) segmented µCT image of a single leaflet, (c) NURBS fitted mid-surface of the leaflet and cross sections in the circumferential and radial directions. The curves c2 and r2 represent the central cross sections of the leaflet in which r2 are chosen to pass through the mid-point of the free edge P4.

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