a) The six-layer tissue graft engineered by the double-step procedure was removed from the bioreactor system and transplanted in the neck of a nude rat with anastomosis between the protruding femoral vessels of the graft and the connectable neck blood vessels of the host rat. ( b) The six-layer graft was transplanted without the vessel anastomosis. ( c) The six-layer cell sheets (CS) without the vascular bed (VB) were simply transplanted. ( d– f) In vivo bioluminescent imaging of each graft is shown. Obvious signals were observed in the graft with vessel anastomoses ( d). The intensity was low in the graft without anastomosis ( e) and in the graft without a vascular bed ( f). ( g) The intensity was significantly the highest in the graft with vessel anastomosis, compared with the other two grafts. Data are shown as mean±s.d. (* P<0.05) ( n=4). ( h) The vascularized graft with blood vessel anastomoses also maintained its vascular structure at 2 weeks after the procedure. Dotted line indicates the area of the graft with the vascular bed and arrows indicate the area of the cell sheet layers. ( i) Immunostaining for CD31 (green: ECs) and calponin (red: smooth muscle cell) shows the uniform distribution of micro blood vessels in the cell sheet construct. ( j) The detail in the white box of i illustrates the characteristics of a matured blood vessels including red blood cells.