Morphologic and mechanical characteristics of engineered bovine arteries Laura E. Niklason, MD,a PhD, William Abbott, MDb, Jinming Gao, PhDc, Brian Klagges,

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Morphologic and mechanical characteristics of engineered bovine arteries Laura E. Niklason, MD,a PhD, William Abbott, MDb, Jinming Gao, PhDc, Brian Klagges, BSd, Karen K. Hirschi, PhDe, Kezban Ulubayram, PhDf, Nancy Conroy, BSg, Rosemary Jones, PhDb, Ami Vasanawala, BSh, Seema Sanzgiri, BSi, Robert Langer, DSci Journal of Vascular Surgery Volume 33, Issue 3, Pages 628-638 (March 2001) DOI: 10.1067/mva.2001.111747 Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 1 Bioreactor and cell seeding. A, SMCs are seeded onto PGA scaffold by direct pipetting of concentrated cell suspension. Bioreactors are assembled with PGA scaffold and polyester fiber (Dacron) sleeves, which allow formation of fluid-tight connection between vascular tissue and bioreactor. Silicone tubing extends through lumen of vessel and through side arms of bioreactor to connect with perfusion system. B, After removal of silicone tubing, EC suspension is injected into engineered vessel via side arm, and bioreactor is slowly rotated to allow EC seeding in lumen. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 2 Engineered vessel and PGA scaffolding. A, “Top view” of engineered vessel after lid has been removed and culture medium has been partially drained from bioreactor. Extent of vessel is indicated by bracket. Polyester vinyl (Dacron) sleeves are visible at either end of vessel. B, Sample of 2-mm PGA mesh on left and rolled into tube with 3.1-mm internal diameter on right. Mesh is visibly porous, reflecting its high void volume. Ruler is in centimeters, applies to A and B. C, Higher magnification view of engineered vessel secured to plastic connector for compliance testing. Bracket indicates vessel; scale bar is 1 mm. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 3 Vessel morphology and culture time. Bovine SMCs seeded onto PGA mesh and cultured under pulsatile conditions for 11 days (A) , 21 days (B) , 5 weeks (C) , and 8 weeks (D). All were stained with hematoxylin and eosin, all original magnification ×4. (Defect in lower left of panel C is processing artifact.) Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 4 Endothelial layer of engineered vessels. A, Scanning electron micrograph of luminal surface of nonendothelialized engineered vessel. Collagenous extracellular matrix forms sheet on inner lumen, with little evidence of underlying SMCs. B, Scanning electron micrograph of ECs after 3 days of luminal perfusion of vessel. Cells are dense, but not as confluent as native endothelium. Scale bars in A and B are 10 μ. C, Immunoperoxidase staining for bovine collagen type I, hematoxylin counterstain. Positive brown staining on vessel lumen (arrows ) confirms presence of collagen type I; EC nuclei stain purple. Nonspecific staining of polymer remnants is visible deeper in the vessel wall. Original magnification, ×400. D, Platelet-endothelial cell adhesion molecule immunoperoxidase staining confirms EC identity on luminal surface. Original magnification, ×200. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 5 Masson's trichrome staining reveals extensive collagen deposition and polymer fragments. A, Nonpulsed vessel cultured for 8 weeks on 2-mm mesh. B, Pulsed vessel cultured for 8 weeks on 2-mm mesh. C, Pulsed vessel cultured for 8 weeks on 1-mm mesh. External vessel surface on top, luminal surface on bottom of figure. Collagen stains blue, PGA residuals are nonstaining and appear white in this preparation. Although some PGA is evident in C , it is much reduced compared with A and B. Original magnification, ×100. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig 6 TEM of vessel wall. Pulsed vessel cultured for 8 weeks. SMCs are indicated by asterisks. Extensive Golgi apparatus noted in lowermost cell. Arrow indicates lysosome-containing phagocytosed polymer fragment. SMCs in vessel wall are separated by layers of collagen fibrils that have alternating orthogonal relative orientations (for example, area indicated by bracket ). Original magnification, ×16,700. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 7 Distribution of SMC differentiation markers. Immunoperoxidase staining for SM α-actin: nonpulsed, 2-mm mesh (A) and pulsed, 1-mm mesh (B). Immunoperoxidase staining for calponin: nonpulsed, 2-mm mesh C and pulsed, 1-mm mesh (D). External vessel surface on top , luminal surface on bottom of figure. Cells near polymer fragments are essentially nonstaining. Arrows indicate extent of nonstaining polymer regions. Some artifactual staining of PGA fragments noted in C. Original magnification, ×100. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 8 Response to endothelin-1. Engineered vessel segments (n = 3) were suspended in organ bath and exposed to increasing concentrations of endothelin-1, a potent vasoconstrictor. Contraction is shown as a percentage of maximal contraction at 10–6 mol/L. Absolute magnitudes of contractile forces were 0.3 g; native rabbit aorta controls generated 1.7 g force in response to 10–6 mol/L endothelin-1. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 9 Mechanical characteristics at physiologic pressures. A, Vessel diameter for 5-week (n = 4, open circles ) and 8-week (n = 3, closed circles ) vessels as a function of pressure. Baseline diameter was diameter measured at 25 mm Hg intraluminal pressure. Error bars are SD. B, Representative strip-chart recording of pulsatile pressure ranges applied to engineered vessel. Pulse rate of pump was 90 bpm. C, Static (closed circles ) and dynamic (open circles ) compliances of 5-week vessels. Mean pressure calculated from: {(2 × diastolic + systolic)/3}. Curves are exponential best-fit. D, Static (closed circles ) and dynamic (open circles ) compliances of 8-week vessels. Curves are exponential best-fit. Journal of Vascular Surgery 2001 33, 628-638DOI: (10.1067/mva.2001.111747) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions