An “off the shelf” vascular allograft supports angiogenic growth in three-dimensional tissue engineering  Johann M. Zdolsek, MD, Wayne A. Morrison, MB BS, MD, Aaron M. Dingle, B Sc (Hons), Jason A. Palmer, B Sc (Hons), Anthony J. Penington, MB BS, Geraldine Margaret Mitchell, PhD  Journal of Vascular Surgery  Volume 53, Issue 2, Pages 435-444 (February 2011) DOI: 10.1016/j.jvs.2010.08.019 Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 1 a, Tissue engineering chambers, volume 1.7 mL. Left: chamber with perforations, right: smooth chamber without perforations. b, Arteriovenous loops (AVLs) construct with autologous vein graft inserted (arrow) placed on a perforated chamber base. c, AVL construct with cold-stored vein-graft (arrow) inserted, sitting on a smooth unperforated chamber base. d, e, f, and g, Macroscopic appearance of constructs (arrows) at 6 weeks. Note smaller amount of vascularized (red) tissue and larger amount of fibrin (yellow material), not yet vascularized in (d and f) (smooth chambers). In (e and g), note the well-vascularized tissue with knobs of new tissue originating from tissue growth from outside the chamber coming through the chamber perforations into the fibrin surrounding the AVL. h, Weight, and (i), volume of constructs at harvest at 2 or 6 weeks (mean values ± SEM). *Weight of perforated chamber constructs significantly greater than smooth chamber constructs (P = .015). #Volume of perforated chamber constructs significantly greater than smooth chamber constructs (P = .006). Journal of Vascular Surgery 2011 53, 435-444DOI: (10.1016/j.jvs.2010.08.019) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 2 Autologous vein graft constructs. a, Autologous vein in arteriovenous loops (AVLs) in a smooth chamber at 2 weeks. Note that new blood vessel/connective tissue (CT) growth into the fibrin (F) from the AVL (A) is not complete. Asterisk indicates hemorrhage into the fibrin. b, Autologous vein in AVL (A) at 6 weeks. The vascularized CT growth has completely replaced the fibrin. c, The autograft vein wall at 2 weeks, lined with endothelium (arrow). No branches occurred from the graft, and new blood vessels and CT surround the autologous graft. d, Autologous vein graft in the AVL (A) in a perforated chamber at 2 weeks. Note the “knobs” (arrows) of new tissue that originate from the perforations in the chamber. e, Autologous vein graft in the AVL (A) in a perforated chamber at 6 weeks. Arrows indicate the ‘knobs’ of tissue originating from chamber perforations. f, The autologous vein wall (asterisk) surrounded by intensely angiogenic tissue (note numerous lectin labeled blood vessels outside the autograft vein, arrows indicate two of these new blood vessels). g, Autograft vein AVL construct in a perforated chamber at 2 weeks. Note that knob-like (arrows) ingrowths of vascularized tissue (VT) from outside the chamber via the perforations into fibrin (F) surrounding the AVL. At this stage, the “knobs” of tissue are yet to unite with the vascularized connective tissue growing around the AVL (not shown). h, Lectin labeled low power figure of an autograft vein AVL construct in a perforated chamber at 6 weeks demonstrating a tract of blood vessels (arrow) extending from the knob of tissue formed via the perforation into the vascularized tissue around the AVL (A). i, The femoral vein portion of an occluded AVL demonstrating dilated branches (arrow) bringing blood from the new microcirculatory bed into the femoral vein. a-e, g, and i, Hematoxylin and eosin stained 5μm thick paraffin sections. f and h, Lectin labeling of 5μm thick paraffin sections. Scale bars in: a, b, d, e, and g, = 1000 μm. Scale bars in: c and f = 200 μm, scale bar in h = 500 μm. Journal of Vascular Surgery 2011 53, 435-444DOI: (10.1016/j.jvs.2010.08.019) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 3 Cold-stored allograft vein constructs. a, Cold-stored allograft vein in arteriovenous loops (AVLs) construct in a smooth chamber at 2 weeks. The AVL (A) is surrounded by some vascularized connective tissue (CT), but hemorrhage and fibrin still exist in the chamber (asterisk). b, Cold-stored allograft vein in an AVL (A) construct in a smooth chamber at 6 weeks. The AVL is surrounded by vascularized CT and the fibrin and hemorrhage have disappeared. c, A cold-stored allograft vein wall at 2 weeks, it is acellular (asterisk) and does not have an endothelial lining (arrow) and is surrounded by fibrin (F). d, A cold-stored allograft vein at 2 weeks in a perforated chamber. The AVL (A) is still largely surrounded by fibrin/hemorrhage (asterisks) and new vascularized CT growth is minimal. Arrow indicates “knob” of tissue entering construct via chamber perforations. e, A cold-stored allograft vein in an AVL (A) at 6 weeks in a perforated chamber. The amount of vascularized CT has greatly increased compared to 2 weeks and little fibrin can be seen. Arrow indicates “knob” of tissue entering construct via chamber perforations. f, A cold-stored allograft vein in an AVL construct at 6 weeks. An endothelial lining is evident (arrow), the graft wall is still largely acellular (asterisk), and the graft is surrounded by new vascularized CT. g, Lectin labeled cold-stored allograft vein wall at 6 weeks (asterisk) surrounded by numerous lectin-positive new capillaries (two capillaries indicated by arrows). h, Lectin labeled allograft wall at 6 weeks at an anastomosis indicating a lack of endothelial lining (lectin-negative, arrow) and neointima formation (N). a, b, c, d, e, and f, Hematoxylin and eosin stained 5 μm thick paraffin sections. g and h, Lectin labeled 5 μm thick paraffin sections. Scale bars a, b, and e = 1000 μm, c and d, the scale bars = 500 μm, f and g, the scale bars = 200 μm, and in (h) the scale bar = 50 μm. Journal of Vascular Surgery 2011 53, 435-444DOI: (10.1016/j.jvs.2010.08.019) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 4 a-f, phosphotungstic acid-hematoxylin (PTAH) labeling of constructs. a, Autologous vein construct at 2 weeks. The arrows indicate peripheral fibrin. b, High power view of fibrin areas from (a). c, Autologous vein construct at 6 weeks, the peripheral fibrin (arrow) is reduced. d, Cold-stored vein construct at 2 weeks. The arrow indicates fibrin. e, High power view of fibrin areas from (d). f, Cold-stored vein construct at 6 weeks, demonstrating less fibrin (arrow) than at 2 weeks. g-h, Masson's trichrome staining of constructs. g, Autologous vein construct at 2 weeks. Blue areas (arrow) indicate new collagen around the arteriovenous loop (AVL). h, High power view of the autologous vein wall with a neointimal covering (arrow). The surrounding new tissue includes many capillaries in a collagenous connective tissue (blue areas). i, Cold stored vein allograft construct at 2 weeks. Blue areas (arrow) indicate new collagen around the AVL (similar to g). j, High power view of the cold stored vein wall (arrow) surrounded by new vascularized connective tissue including much collagen (blue areas). k-m, Giemsa staining of the graft wall. k, Autologous vein wall (bracket) at 2 weeks. Note cells in wall and endothelium (arrow) on luminal surface. l, Cold-stored vein wall at 2 weeks. Note a small accumulation of lymphocytes outside the vein wall (arrow) - this was not common. There are few cells in the graft wall (bracket) and the internal elastic lamina covers the luminal surface. m, Cold-stored vein wall at 2 weeks. Similar to l, but there are no lymphocytes in or near the graft wall. n, High power view of graft wall (bracket; PTAH staining). Note the lack of cells in the graft wall and the broken shreds of internal elastic lamina (arrow) on the graft luminal surface - note there is no endothelium. Scale bars: a, c, d, f, g, and i, scale bar = 2000 μm. b, e, h, and j, scale bars = 200 μm. k, l, m, and n, scale bar = 50 μm. Journal of Vascular Surgery 2011 53, 435-444DOI: (10.1016/j.jvs.2010.08.019) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 5 a, The percent volume of arteriovenous loop (AVL) construct tissue components AVL, fibrin, new connective tissue, and blood in four groups at 6 weeks: A, autograft vein; CS, cold-stored allograft vein; P, perforated chambers; S, smooth chambers. There was no difference in the tissue composition in constructs with autograft veins or cold-stored allograft veins. However, in relation to chamber type, there was a significant increase in new connective tissue in perforated chambers (regardless of graft type) compared to smooth chambers (*P = .001). b, The percent vascular volume of new blood vessels in four groups at 6 weeks. There was no difference in percent vascular volume related to graft or chamber type. c, The absolute vascular volume of AVL construct components in four groups at 6 weeks. The only significant difference was a significant increase in new connective tissue absolute volume in perforated chambers (#) regardless of whether autograft or cold-stored allograft veins were used (#P = .01). d, The absolute vascular volume of all four groups at 6 weeks. Although it seems that vascular volume was increased in perforated chambers, this was not significant. There was no difference in vascular volume generated related to graft type. Journal of Vascular Surgery 2011 53, 435-444DOI: (10.1016/j.jvs.2010.08.019) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 6 a, Comparison of the new percent vascular volume generated in patent and occluded autologous vein constructs and cold-stored vein constructs. There was no significant difference in the percent vascular volume generated based on patency status. b, Comparison of the percent connective tissue volume generated in patent and occluded autologous vein constructs and cold-stored vein constructs. There was no significant difference in the percent connective tissue generated based on patency status. c, Comparison of the percent fibrin volume generated in patent and occluded autologous vein constructs and cold-stored vein constructs. There was no significant difference in the percent fibrin volume based on patency status. AVLs, Arteriovenous loops. Journal of Vascular Surgery 2011 53, 435-444DOI: (10.1016/j.jvs.2010.08.019) Copyright © 2011 Society for Vascular Surgery Terms and Conditions