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ANGIOGENESIS IN TISSUE ENGINEERING
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 Aim: The purpose of this study was to investigate the influence of the naturally occurring fibrinogen variants, HMW- and LMW-fibrinogen, on in vitro and in vivo angiogenesis. Methods and Results: Both fibrinogen variants were fractionated and purified by (NH4)2SO4 precipitation followed by dialysis. Fibrin formation and clot structure were established by turbidity and scanning electron microscopy. In an in vitro angiogenesis assay using human microvascular endothelial cells (hMVEC) cultured on a three-dimensional fibrin matrix, made of unfractionated, HMW-, or LMW-fibrinogen, it was shown that the usage of the respective fibrinogen variants influences the invasion of hMVEC and the formation of tube-like structures in fibrin matrices. HMW-fibrinogen favors cell growth and leads to an increased cell and vessel ingrowth, compared with unfractionated and LMW-fibrinogen. In addition, confirmative results were obtained in vivo by testing the different forms of fibrinogen in a mouse model for angiogenesis. Conclusions: The heterogeneity in naturally occurring fibrinogen variants, HMW- and LMW-fibrinogen, influences the angiogenic capacity of blood vessels in fibrin matrices. The different effects of the two fibrinogen variants provide further insight in the structural features required for angiogenesis in a fibrinous environment.
P514 A NEW TECHNOLOGY FOR FULLY DESIGNED CAPILLARY FORMATION IN VITRO. Kobayashi Akiko, Hiruma Yuko, Onodera Mitsue, Miyake Hideyuki, Hattori Hideshil, Nakamura Makoto, Akiyoshi Kazunaril, Takeda Satoru, Morita Ikuo. Saitama Medical School, Kawogoe-shi. Saitama. Japan, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan, Dai Nippon Printing Co.Ltd Kashiwa-shi, Chiba, Japan Objectives: Regulation of angiogenesis is one of the important factors for the development of tissue engineering and regenerative medicine. A new approach for in-vitro fully designed neovascularizaticm was invented by combining photolithographic and cell-culture techniques. Methods: The glass substrates for cell culture were prepared using a modified photolithographic technique. Fluoroalkylsilane (FAS) was spin coated on glass substrates to obtain hydrophobic surfaces. The FAS coatedsubstrate was faced to TiO2-coated lithographic mask in which specific lines (width= 50~m) were drawn and then UV was irradiated through the mask. UV-irradiated region of the FAS coated-substrate was oxidized and changed to hydrophilic surface by photo-catalytic reaction of TiO2. Bovine endothelial cells were seeded on the prepared substrate with 5 % FBSMEM and incubate 24~36 hours. The pattering endothelial cells on the hydrophilic region were then turn over MATRIGEL and the cells were transferred. The capillary formation of endothelial cells was confirmed by VE-cadherin immunostaining and electron microscopic observation. Results: We cultured bovine endothelial cells on the special glass plate as described above. In this manner, guided and distributed cells were obtained on the hydrophilic region of the substrate. This result indicates that anisotropic cell culture surfaces patterned with FAS and hydroxyl part successfully guided cell distribution. The transferred cells to MATRIGEL formed tube like structure along the fully designed lines (or pattern) for l – 2 days. By immunochemical study, VE-cadherin was observed along intercellular borders of endothelial cells. Moreover, electron microscopic study demonstrated the presence of a central luminal space within a reorganized capillary tube. Conclusion: The molecular control of blood vessel formation has been a major topic of investigation for some diseases and tissue regeneration over the past several decades. The present technique will be a new concept for application to a wide variety of transplantations and diseases. And this method is also useful for elucidation of the vasculogenesis mechanism.
S181
P515 ANGIOGENESIS IN TISSUE ENGINEERING. Zerina Lokmic, Erik W. Thompson, Wayne A. Morrison, Geraldine M. Mitchell. Bernard O’Brien Institute of Microsurgery and University of Melbourne Department of Surgery, St Vincent’s Hospital, Melbourne, Australia. Background/Aim: One of the major challenges in tissue engineering (TE) is the generation of large three-dimensional tissues with an adequate blood supply. To resolve this problem, an understanding and characterisation of blood vessel development from proliferation and migration to maturation, remodelling and regression is necessary. Once the molecular mechanisms governing these processes are identified, we can then manipulate them so to develop a vascular supply within an organ what will survive transplantation and resume normal function. Our aim in this study was to characterize angiogenesis in a rat in vivo chamber model of tissue engineering and compare this to angiogenesis in a rat skin wound using morphometric techniques. Methods: The chamber model involves creating an arterio-venous (AV) loop on rat femoral vessels by microsurgically inserting a femoral vein graft harvested from the opposite leg. The AV loop is inserted into a semi-sealed polycarbonate chamber in the rat groin and incubated for periods of 3 days to 16 weeks. Cutaneous incisional wounds were also created and harvested at identical time points from the same rats. Wound tissue and the tissue spontaneously generated on the loop were examined by standard light microscopic, immunohistochemical and morphometric techniques. Results: Three days after AV chamber implantation, a fibrin exudate filled the chamber providing a scaffold for white blood cells and spindle-shaped cells (which are either endothelial or mesenchymal precursor cell) to migrate into the chamber. At 7 days these spindle-shaped cells either gave rise to angiogenic sprouts or secreted collagen matrix which leads to a formation of a more stable scaffold. In the subsequent weeks the formation of vascularised connective tissue is accompanied by fibrinolysis and by 16 weeks the fibrin clot is completely replaced by a mature vascularized connective tissue. Vascular volume density peaks of 32% and 26% occurred at 10 and 42 days respectively and were higher than the vascular volume density in healing rat skin wounds harvested at identical time points (19% and 9% respectively). Conclusion: By identifying the patterns of vascular development in an in vivo model of TE, we hope to identify the crucial time-points at which the angiogenic development could be encouraged to develop further and not undergo remodelling and regression. St Vincent’s Hospital Research Grant 2003/48
P516 OSTEOPROTEGERIN PROMOTES ENDOTHELIAL CELL SURVIVAL AND ANGIOGENESIS. Joseph McGonigle, Cecilia Giachelli, Marta Scatena. Department of Bioengineering, University of Washington, Seattle, WA. The success of tissue engineering based therapies as well as the performance of implanted biomaterials is highly dependent upon the ability to establish a neovasculature within and immediately surrounding the implanted construct. Growth factors such as VEGF and FGF are commonly used to promote angiogenesis in such situations, but there is a need for a greater understanding of all of the mechanisms and molecules involved in blood vessel formation. We are particularly interested in osteoprotegerin (OPG), a member of the tumor necrosis factor receptor (TNFR) superfamily initially found to play a role in bone metabolism by acting as a soluble decoy receptor for receptor activator of nuclear factor-kB ligand (RANKL). Recently, OPG has been found to play a role in the vascular system where it has been shown to prevent arterial calcification and promote the survival of endothelial cells. Rat aortic rings embedded in collagen gels generate an angiogenic response and provide a convenient
P514 A NEW TECHNOLOGY FOR FULLY DESIGNED CAPILLARY FORMATION IN VITRO. Kobayashi Akiko, Hiruma Yuko, Onodera Mitsue, Miyake Hideyuki, Hattori Hideshil, Nakamura Makoto, Akiyoshi Kazunaril, Takeda Satoru, Morita Ikuo. Saitama Medical School, Kawogoe-shi. Saitama. Japan, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan, Dai Nippon Printing Co.Ltd Kashiwa-shi, Chiba, Japan Objectives: Regulation of angiogenesis is one of the important factors for the development of tissue engineering and regenerative medicine. A new approach for in-vitro fully designed neovascularizaticm was invented by combining photolithographic and cell-culture techniques. Methods: The glass substrates for cell culture were prepared using a modified photolithographic technique. Fluoroalkylsilane (FAS) was spin coated on glass substrates to obtain hydrophobic surfaces. The FAS coatedsubstrate was faced to TiO2-coated lithographic mask in which specific lines (width= 50~m) were drawn and then UV was irradiated through the mask. UV-irradiated region of the FAS coated-substrate was oxidized and changed to hydrophilic surface by photo-catalytic reaction of TiO2. Bovine endothelial cells were seeded on the prepared substrate with 5 % FBSMEM and incubate 24~36 hours. The pattering endothelial cells on the hydrophilic region were then turn over MATRIGEL and the cells were transferred. The capillary formation of endothelial cells was confirmed by VE-cadherin immunostaining and electron microscopic observation. Results: We cultured bovine endothelial cells on the special glass plate as described above. In this manner, guided and distributed cells were obtained on the hydrophilic region of the substrate. This result indicates that anisotropic cell culture surfaces patterned with FAS and hydroxyl part successfully guided cell distribution. The transferred cells to MATRIGEL formed tube like structure along the fully designed lines (or pattern) for l – 2 days. By immunochemical study, VE-cadherin was observed along intercellular borders of endothelial cells. Moreover, electron microscopic study demonstrated the presence of a central luminal space within a reorganized capillary tube. Conclusion: The molecular control of blood vessel formation has been a major topic of investigation for some diseases and tissue regeneration over the past several decades. The present technique will be a new concept for application to a wide variety of transplantations and diseases. And this method is also useful for elucidation of the vasculogenesis mechanism.
S181
P515 ANGIOGENESIS IN TISSUE ENGINEERING. Zerina Lokmic, Erik W. Thompson, Wayne A. Morrison, Geraldine M. Mitchell. Bernard O’Brien Institute of Microsurgery and University of Melbourne Department of Surgery, St Vincent’s Hospital, Melbourne, Australia. Background/Aim: One of the major challenges in tissue engineering (TE) is the generation of large three-dimensional tissues with an adequate blood supply. To resolve this problem, an understanding and characterisation of blood vessel development from proliferation and migration to maturation, remodelling and regression is necessary. Once the molecular mechanisms governing these processes are identified, we can then manipulate them so to develop a vascular supply within an organ what will survive transplantation and resume normal function. Our aim in this study was to characterize angiogenesis in a rat in vivo chamber model of tissue engineering and compare this to angiogenesis in a rat skin wound using morphometric techniques. Methods: The chamber model involves creating an arterio-venous (AV) loop on rat femoral vessels by microsurgically inserting a femoral vein graft harvested from the opposite leg. The AV loop is inserted into a semi-sealed polycarbonate chamber in the rat groin and incubated for periods of 3 days to 16 weeks. Cutaneous incisional wounds were also created and harvested at identical time points from the same rats. Wound tissue and the tissue spontaneously generated on the loop were examined by standard light microscopic, immunohistochemical and morphometric techniques. Results: Three days after AV chamber implantation, a fibrin exudate filled the chamber providing a scaffold for white blood cells and spindle-shaped cells (which are either endothelial or mesenchymal precursor cell) to migrate into the chamber. At 7 days these spindle-shaped cells either gave rise to angiogenic sprouts or secreted collagen matrix which leads to a formation of a more stable scaffold. In the subsequent weeks the formation of vascularised connective tissue is accompanied by fibrinolysis and by 16 weeks the fibrin clot is completely replaced by a mature vascularized connective tissue. Vascular volume density peaks of 32% and 26% occurred at 10 and 42 days respectively and were higher than the vascular volume density in healing rat skin wounds harvested at identical time points (19% and 9% respectively). Conclusion: By identifying the patterns of vascular development in an in vivo model of TE, we hope to identify the crucial time-points at which the angiogenic development could be encouraged to develop further and not undergo remodelling and regression. St Vincent’s Hospital Research Grant 2003/48
P516 OSTEOPROTEGERIN PROMOTES ENDOTHELIAL CELL SURVIVAL AND ANGIOGENESIS. Joseph McGonigle, Cecilia Giachelli, Marta Scatena. Department of Bioengineering, University of Washington, Seattle, WA. The success of tissue engineering based therapies as well as the performance of implanted biomaterials is highly dependent upon the ability to establish a neovasculature within and immediately surrounding the implanted construct. Growth factors such as VEGF and FGF are commonly used to promote angiogenesis in such situations, but there is a need for a greater understanding of all of the mechanisms and molecules involved in blood vessel formation. We are particularly interested in osteoprotegerin (OPG), a member of the tumor necrosis factor receptor (TNFR) superfamily initially found to play a role in bone metabolism by acting as a soluble decoy receptor for receptor activator of nuclear factor-kB ligand (RANKL). Recently, OPG has been found to play a role in the vascular system where it has been shown to prevent arterial calcification and promote the survival of endothelial cells. Rat aortic rings embedded in collagen gels generate an angiogenic response and provide a convenient