O21	Blood vessel growth and remodelling in in vivo tissue engineering.
Z.Lokmic, A.Messina, R.Idrizi, F.Stillaert, A.Penington, E.Thompson, W.Morrison, G.Mitchell
Bernard O'Brien Institute of Microsurgery and University of Melbourne, Department of Surgery at St Vincent's Hospital, Melbourne, AU.

In vivo tissue engineering constructs rely on extrinsic vascularization from surrounding tissues. The capacity of this mode of vascularization to support new tissue is thought to be limited. As an alternative we have developed a novel model utilizing intrinsic vascularization whereby a new microcirculatory network with supporting tissue is spontaneously generated when a functional macrovascular loop is placed in a non-collapsible space. When a rat arteriovenous loop (AVL) based on the femoral vessels is inserted into a rigid plastic chamber it spontaneously generates a highly angiogenic connective tissue. The stability of this microcirculatory system is unknown. In this study morphological and morphometric assessment of angiogenic growth and remodelling in the rat femoral arteriovenous chamber model has been characterized for up 16 weeks. A large fibrin clot surrounds the AVL at 3 days. Extensive capillary sprouting from the venous side was evident after 7 days, whilst arteriolar sprouting from the artery was observed at 10 days. These developments created a proliferative zone around the AVL and as new tissue matured this zone migrated outwards through the fibrin clot. Percent vascular volume (PVV) in newly formed connective tissue peaked at 10 days (23.2+/-3.14%, Mean +/-S.E.M.)and remained at slightly lower levels until 16 weeks (13.73+/-2.14%). Hypoxia indicated by hypoxyprobe-1 labelling was maximal at 7 days. The majority of cells invading the construct were alpha smooth cell actin positive. Their numbers decreased after 6 weeks. Less numerous were large VEGFR-2 positive cells initially observed in the fibrin clot at 3 days and capillary sprouts at 7 days. This intrinsic vascularization model is being investigated for its ability to support specific tissue growth from seeded precursor/stem cells or differentiated tissue. To date skeletal muscle, adipose tissue and liver have been grown in these constructs. Tissue engineering employing intrinsic vascularization derived from a macrovascular loop in a non-collapsible chamber provides spontaneous and permanent microcirculatory development in a defined, conserved space which permits tissue growth.

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