The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber  S.O.P. Hofer, G.M. Mitchell, A.J. Penington, W.A. Morrison, R. RomeoMeeuw, E. Keramidaris, J. Palmer, K.R. Knight  British Journal of Plastic Surgery  Volume 58, Issue 8, Pages 1104-1114 (December 2005) DOI: 10.1016/j.bjps.2005.04.033 Copyright © 2005 The British Association of Plastic Surgeons Terms and Conditions

Figure 1 (A) The polycarbonate chamber. (B) The AV loop sitting on the base of the chamber prior to the chamber lid being closed over the top. A: recipient artery, V: recipient vein, VG: vein graft, C: chamber base. Note in this specimen no BM gel has been added so that the AV loop can be clearly visualised. British Journal of Plastic Surgery 2005 58, 1104-1114DOI: (10.1016/j.bjps.2005.04.033) Copyright © 2005 The British Association of Plastic Surgeons Terms and Conditions

Figure 2 (A) Fibroblast culture after 4h hypoxia, most cells have labelled with hypoxyprobe-1 as indicated by the brown labelling. One of these cells is arrowed. Scale bar=100μm. (B) Fibroblast culture after 4h normoxia and subsequent hypoxyprobe-1 labelling. Note no cells label in normoxia. Scale bar=100μm. (C) Negative control of 4h hypoxic cells with hypoprobe-1 protocol applied without the primary antibody. Note no labelling. Scale bar=100μm. British Journal of Plastic Surgery 2005 58, 1104-1114DOI: (10.1016/j.bjps.2005.04.033) Copyright © 2005 The British Association of Plastic Surgeons Terms and Conditions

Figure 3 Low power micrographs of vertical sections through the AV loop chamber at different time points. Five micron thick haematoxylin and eosin stained sections. (A) The construct at 3 days consists primarily of a fibrin clot (F) mixed in some areas with BM gel. Cross sections of the recipient artery (A) and vein (V) surrounded by a narrow rim of new connective tissue (CT) are evident. Scale bar=100μm. (B) At 7 days a much wider area of vascularised connective tissue (CT) is present around the recipient artery (A) and vein (V). Fibrin clot (F) is still present. Scale bar=100μm. (C) After 28 days fibrin clot is no longer evident and the construct consists of vascularised connective tissue (CT) which at this level surrounds two profiles of the vein graft (VG) portion of the AV loop. Scale bar=100μm. British Journal of Plastic Surgery 2005 58, 1104-1114DOI: (10.1016/j.bjps.2005.04.033) Copyright © 2005 The British Association of Plastic Surgeons Terms and Conditions

Figure 4 (A) Cross section of a vein graft in an AV loop 3 days after insertion. Bracket extends from vein graft endothelium to outer edge of proliferative/migratory zone growth. Surrounding matrix (*) is a fibrin clot/BM gel mixture. Five micron thick paraffin section, stained with haematoxylin and eosin. Scale bar=100μm. (B) Higher power of vein graft wall (VG) in another AV loop 3 days after insertion. Bracket encompasses distance from vein graft luminal surface to the outer limit of proliferative/migratory zone growth. Cells in this zone include endothelial cells or endothelial cell precursors, fibroblast-like cells, macrophages and polymorphs. Outside the proliferative/migratory zone fibrin clot (*) forms the supporting matrix. Five micron thick paraffin section, stained with haematoxylin and eosin. Scale bar=100μm. (C) Adjacent paraffin section to 4b. This section has been stained immunohistochemically with hypoxyprobe-1 Mab. Note the positive staining cells within the proliferative/migratory zone (arrows). The vein graft wall (VG) did not stain. Intensely stained macrophages further out in the fibrin clot (arrowhead) were also observed. Scale bar=100μm. (D) Cross section of a vein graft wall in an AV loop that had not generated a proliferative/migratory cuff at 3 days post-insertion. The section has been stained with hypoxyprobe-1 Mab. The vein graft wall (VG) is surrounded by BM gel (*) into which large numbers of polymorphs (arrowheads) have migrated. No immunopositive cells are apparent. Scale bar=100μm. (E) Cross section of a vein graft wall in an AV loop 7 days post-insertion. The proliferative/migratory zone is now wider (compare with Fig. 4(B) and (C)), and stains strongly with hypoxyprobe-1 Mab. Bracket indicates distance from luminal surface of vein graft to outer limit of proliferative/migratory zone. Arrows indicate numerous intensely stained new blood vessels within the proliferative zone. Scale bar=100μm. (F) Cross section of vein graft from AV loop in a 7 day specimen, where the proliferative/migratory zone is wider than 0.7mm (compare with Fig. 4(A)). Bracket indicates distance from vein graft luminal surface to outer limit of proliferative zone. The new tissue is a vascularised cellular connective tissue (*). Boxed area is shown at higher magnification in Fig. 4(G). Five micron thick paraffin section stained with haematoxylin and eosin. Scale bar=100μm. (G) Higher power micrograph of the area outlined by the box in Fig. 4(F). This adjacent tissue section has been stained for hypoxyprobe-1 Mab (VG) vein graft wall. The vascularised cellular connective tissue displays weak staining in occasional capillaries and individual cells (arrows). This staining when compared to Fig. 4(C) and (E) would appear to be nonspecific. Scale bar=100μm. (H) Cross section through vein graft wall (VG) and surrounding mature vascularised connective tissue (*) in a 2 week chamber. Section has been stained with hypoxyprobe-1 Mab, however, there is no positive staining. Scale bar=100μm. British Journal of Plastic Surgery 2005 58, 1104-1114DOI: (10.1016/j.bjps.2005.04.033) Copyright © 2005 The British Association of Plastic Surgeons Terms and Conditions

Figure 5 Five micron thick, rat endothelial cell specific lectin—Bandeiraea simplicifolia stained (brown material) paraffin sections. (A) A 7 days construct. A few new blood vessels (arrows) are visible. Scale bar=100μm. (B) Many more new blood vessels (arrows) are evident in a cross section at 28 days. Scale bar=100μm. British Journal of Plastic Surgery 2005 58, 1104-1114DOI: (10.1016/j.bjps.2005.04.033) Copyright © 2005 The British Association of Plastic Surgeons Terms and Conditions