Rachael W. Quinn, PhD, Arthur A. Bert, MD, Gabriel L

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Performance of allogeneic bioengineered replacement pulmonary valves in rapidly growing young lambs Rachael W. Quinn, PhD, Arthur A. Bert, MD, Gabriel L. Converse, PhD, Eric E. Buse, MS (BME), Stephen L. Hilbert, PhD, MD, William B. Drake, MD, MS, Richard A. Hopkins, MD The Journal of Thoracic and Cardiovascular Surgery Volume 152, Issue 4, Pages 1156-1165.e4 (October 2016) DOI: 10.1016/j.jtcvs.2016.05.051 Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 A, Schematic of the coaptation index (% coaptation) measured in the long-axis view in diastole. Curvilinear distance: x - y = coaptation length; x - z = radial length of the diastolically loaded semilunar leaflet. Lune meridians lengths are proportional to surface areas; thus, (x−yx−z)×100=% coaptation area. B, Scatterplot of regurgitation scores (x-axis) and % coaptation (y-axis). As regurgitation severity score increased, the % coaptation decreased. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Representative images depicting recellularization of cusp base and sub-base regions of BEPVS (A and B) compared with CryoVs (C and D) at explant (all Movat pentachrome, 100×). In the BEPVs, in-migrating cells from the sinus (A, ***) and sub-base regions (A, **) extend into the cusp base (A, *) and fibrous sheath (A, line). Note, in A, that leaflet folded back on itself. Recellularization of the proximal cusp is apparent but incomplete (B, **). Cusp re-endothelialization (B, *) and transmural recellularization of the PA wall (B, ***) are common. CryoVs demonstrate hypocellularity in the sub-base region (C, *) and cusp base (C, **), as well as incomplete transmural PA cell retention (D, **) and virtually acellular cusp matrix (D, *). The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Histology and immunohistochemistry of BEPVs explanted after 6 months. Representative images from BEPVs depicting transmural recellularization of cusp base and sub-base regions as well as sinus and PA walls by cells consistent with the myofibroblast VIC phenotype. Positive α-SMA staining suggests contractile and migratory functionality, whereas HSP-47+ suggests active collagen synthesis and PCNA+ indicates proliferative capacity in the recellularized population. Mid to distal cusp portions have VEC restoration of blood-exposed surfaces but minimal VIC restoration. A-D, Histological sections (H&E of the sub-base region (A), cusp base (B), distal cusp (C, magnification 200×), and distal conduit (D) demonstrating recellularization of the cusp base and conduit). E-H, HSP-47+ (green) cells at the base of the cusp (E), in the sub-base region (F), in the pannus at the mid-cusp (G), and in the sinus (H) and vWF+ (red) cells within the pannus (G) and in the sinus (H). I and J, α-SMA+ (green) cells and vimentin+ (red) cells in the cusp sub-base (I) and base (J) regions. K and L, PCNA+ (red) cells in the cusp base (K) and sub-base (L) regions. Nuclear DNA is demonstrated by 4′,6-diamidino-2-phenylindole staining (blue); elastic fibers are autofluorescent. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Immunohistochemistry of bioengineered heart valve leaflet base explanted at 6 months. Asterisk marks the upper left sinus wall junction. Arrow is the interstitial confluence of leaflet spongiosa with sinus wall. Blue = Dapi nuclear stain of viable cells recellularizing the initially acellular valve conduit. Green = Heat-shock protein 47, a valve interstitial cell marker. Red = von Willebrand factor, a valve endothelial cell marker. Note that leaflet is in “open position abutting the sinus wall” (100×). The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure E1 Anatomic diagram of a pulmonary valved conduit with cross-sectional arrows indicating the measurement locations: RVOT (A), annulus/proximal suture line (B), mid-sinus (C), sinotubular junction (D), mid-conduit (E), and prebifurcation pulmonary artery/distal suture line (F). Diameters are measured at peak expansion. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure E2 A, Longitudinal section of a sinus of a pulmonary valved conduit with boxes indicating areas sampled for decellularization verification (1) and cell density (2, pulmonary artery; 4, sinus; 5, sub-base; 6, cusp base; 7, mid-cusp; 8, distal cusp). Sinotubular junction (3) is indicated for anatomic reference. B, Representative decellularized, unimplanted ovine pulmonary valve (H&E, 25×) demonstrating adequate cell removal and anatomic structures. C, Explanted ovine BEPV (H&E, 25×) demonstrating recellularization of the sub-base and cusp base regions, with progressively decreasing cellularity toward the distal portion of the cusp. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure E4 A, In a CryoV, note fibrous sheath (pannus) formation on the outflow portion of the sinus (thin arrow) and cusp base, as well as the inflow portion of the cusp and subvalvular lumen (thick arrow). B, In a BEPV, pannus is observed on the inflow portion of the cusp base (thick arrow), but not in the outflow region. Note the recellularization of the BEPV sinus wall (thin arrow). *Cusp origin. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Figure E3 Macroscopic images of the CryoVs (A-C) and BEPVs (D-J) at explant. Cusps are thin and transparent, with minor fibrous sheathing and minimal thrombus/fibrin stranding. One CryoV cusp presented with a small nodule, likely calcified thrombus (no organisms visible in micrograph), in addition to visible calcified plaques on the sinus walls (C). This likely represents initial stages of Cryo calcific degradation. K and L, Unimplanted BEPV images are included for reference. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

BEPV recellularization at 6 months after surgery: ↓ sub-base BEPV recellularization at 6 months after surgery: ↓ sub-base. *Sinus wall–leaflet junction. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions

Video 1 Video capture during TEE of the PV (long axis) in diastole, demonstrating the measurements taken to determine % coaptation and sinus and conduit wall retained compliance (pulsatility). Video available at http://www.jtcvsonline.org/article/S0022-5223(16)30489-5/addons. The Journal of Thoracic and Cardiovascular Surgery 2016 152, 1156-1165.e4DOI: (10.1016/j.jtcvs.2016.05.051) Copyright © 2016 The American Association for Thoracic Surgery Terms and Conditions