Final Report Summary - ENGVASC (Engineering Vascularized Tissues)
When engineering tissue implants for regenerative medicine applications, self-assembly of vessel networks can be induced in vitro within three-dimensional (3D) tissue constructs by means of multicellular culturing of endothelial cells (EC), vascular mural cells and cells specific to the tissue of interest. This approach both supports formation of endothelial vessels and promotes EC and tissue-specific cell interactions. The resulting EC-dependent, tube-like openings may also form the basis for improved media penetration to the inner regions of thick 3D constructs, allowing for enhanced construct survival and for effective engineering of large complex tissues in the lab. It has also been shown that implanted pre-vascularized engineered constructs can anastomose with host vasculature and form functional blood vessels in vivo. However, the mechanisms underlying vascularization of engineered constructs and implant-host vessel integration remain unclear. In this research, we aimed to uncover the mechanisms governing in vitro vessel network formation in engineered 3D tissues and to elucidate the process of engineered-host vessel network integration and implant vessel-stimulated promotion of neovascularization in vivo.
We found that in–vitro vascular network formation in engineered 3D constructs involves a series of distinct vessel network maturity levels. The dynamics of the process were defined and a novel mechanism of vascular node assembly was uncovered. We show that endothelial junctions are formed by two mechanisms: anastomosis and cluster thinning. The maturity of the vasculature formed within the scaffolds was characterized and we found that immature vessels are clogged and un-functioning while mature vessels appear open and well-functioning. The effect of the scaffold biomaterial on the formation of the engineered vasculature was characterized, and several biomaterials (PLLA-PLGA with fibrin or Tropoelastin, Alginate ferrogel, collagen, and more) were found to induce good vasculature formation.
We showed that direct flow-induced shear stress can affect vessel tube formation by increasing the vessels formation, maturity and extracellular matrix (ECM) secretion, and that tensile forces affect the orientation of the engineered vascular network created within the constructs. We also identified dominant elements responsible for angiogenesis under tensile force conditions and showed that such oriented engineered vasculature significantly improved vessel integration upon in-vivo implantation.
The dynamics of vascular integration of grafts with the host vasculature were followed and we found that the degree of maturity of the vascularized graft directly influences host vasculature penetration and the rate at which it will integrate with graft vascular networks. Graft-host vessel integration was also characterized by way of abdominal implantation of engineered muscle flap into muscle defect in a mouse model. The engineered flaps showed potential to speed recovery and limit scarring, presenting a significant breakthrough in the field of tissue engineering, particularly with respect to cases of large soft tissue trauma, common following serious injury or cancer surgery. To gain molecular insight into the formation and fate of engineered networks both in-vitro and in-vivo, we identified specific angiogenic factors involved in these processes. These findings may lead to improved vascularization efficiency and vascularized tissue engineered construct characteristics.
We studied the impact of construct pre-vascularization on implant function in two disease models:
ischemic heart model- cardiac patch and diabetic mouse islet implantation. We established iPSC differentiation protocol to create well-functioning cardiomyocyte cells, and started establishing the ischemic heart model and the vascularized patch implantation. We found that sequential seeding of murine islets on pre-vascularized scaffolds is advantageous in diabetic mouse islet implantation. We also extended the diabetic research and established a total pancreatectomy rat model in which we implanted islets into pre-vascularized scaffolds. This implantation led to the remission of the hyperglycemia.
We found that in–vitro vascular network formation in engineered 3D constructs involves a series of distinct vessel network maturity levels. The dynamics of the process were defined and a novel mechanism of vascular node assembly was uncovered. We show that endothelial junctions are formed by two mechanisms: anastomosis and cluster thinning. The maturity of the vasculature formed within the scaffolds was characterized and we found that immature vessels are clogged and un-functioning while mature vessels appear open and well-functioning. The effect of the scaffold biomaterial on the formation of the engineered vasculature was characterized, and several biomaterials (PLLA-PLGA with fibrin or Tropoelastin, Alginate ferrogel, collagen, and more) were found to induce good vasculature formation.
We showed that direct flow-induced shear stress can affect vessel tube formation by increasing the vessels formation, maturity and extracellular matrix (ECM) secretion, and that tensile forces affect the orientation of the engineered vascular network created within the constructs. We also identified dominant elements responsible for angiogenesis under tensile force conditions and showed that such oriented engineered vasculature significantly improved vessel integration upon in-vivo implantation.
The dynamics of vascular integration of grafts with the host vasculature were followed and we found that the degree of maturity of the vascularized graft directly influences host vasculature penetration and the rate at which it will integrate with graft vascular networks. Graft-host vessel integration was also characterized by way of abdominal implantation of engineered muscle flap into muscle defect in a mouse model. The engineered flaps showed potential to speed recovery and limit scarring, presenting a significant breakthrough in the field of tissue engineering, particularly with respect to cases of large soft tissue trauma, common following serious injury or cancer surgery. To gain molecular insight into the formation and fate of engineered networks both in-vitro and in-vivo, we identified specific angiogenic factors involved in these processes. These findings may lead to improved vascularization efficiency and vascularized tissue engineered construct characteristics.
We studied the impact of construct pre-vascularization on implant function in two disease models:
ischemic heart model- cardiac patch and diabetic mouse islet implantation. We established iPSC differentiation protocol to create well-functioning cardiomyocyte cells, and started establishing the ischemic heart model and the vascularized patch implantation. We found that sequential seeding of murine islets on pre-vascularized scaffolds is advantageous in diabetic mouse islet implantation. We also extended the diabetic research and established a total pancreatectomy rat model in which we implanted islets into pre-vascularized scaffolds. This implantation led to the remission of the hyperglycemia.