44
shown to regulate and enhance angiogenic processes. Several groups have studied
the effects of flow-induced shear stress on endothelial cells in vitro, using a parallel
plate flow chamber [5, 18, 51]. This experimental setup is simple to model and provides better control over the applied shear stress. However, it does not closely mimic
the 3D nature of interstitial flow, as the medium flows over the cells rather than
through the scaffold. Moreover, endothelial cell response is limited as the system
does not support 3D sprouting and network formation. In an alternative 3D model,
consisting of 3D collagen and alginate gel plugs embedded with endothelial cells,
direct flow simulation resulted in enhanced vascular morphogenesis and
angiogenesis- related gene expression [29, 46]. Furthermore, in our recently published work, we demonstrated the effect of flow-induced shear stress on vascular
formation and maturation in an implantable 3D engineered construct. Direct flow
conditions resulted in a significant increase (>100%) in vessel network morphogenesis parameters, EC and ECM protein depth distribution, and colocalization incidence of alpha-smooth muscle actin (α-SMA) with endothelial vessel networks.
These findings suggest that flow conditions promote 3D neovascularization and
vascular network maturation in a 3D engineered tissue (Fig. 2.3b) [63]. In
microfluidic- based platforms, luminal flow through a self-assembled microvasculature was applied to generate shear stress stimulation inside microvessels [23, 57].
The microvasculature patency was demonstrated by tracking flowing FITC-dextran
and fluorescent microbeads (Fig. 2.3c) [23, 57]. The self-assembled microvessels
possessed morphological and biochemical markers characteristic of natural blood
vessels and exhibited strong barrier function, long-term stability, cytoskeleton rearrangements, and increased nitric oxide synthesis (Fig. 2.3c) [23].
However, the true nature of the effect of shear stress on vessel formation and
behavior is still poorly understood [1]. Many molecular mechanisms and signal
transduction pathways are suggested to bear shear-sensing or mechanotransductive
roles. These mechanisms involve cell-matrix and cell-cell adhesion and junction
molecules, membranous receptors and ion channels, the extra-membranous glycocalyx complex, and cytoskeleton proteins among others [1, 33, 44]. For example, shear
stress has been shown to mediate changes in endothelial cell phenotype, by increasing RhoA (a cytoskeletal regulator) activity and promoting the formation of stress
fibers [8]. Additionally, a direct connection between flow-induced shear stress and
VEGF signaling during angiogenesis has been shown to be mediated by an endothelial cell-specific microRNA, called mir-126, which is regulated by the mechanosensitive zinc finger transcription factor klf2a [11, 40]. This microRNA has a
pro-angiogenic function, by naturally repressing VEGF inhibitors ispred1 and pik3r2.
2.4 Conclusions and Future Perspectives
Creation of vascularized 3D engineered tissue requires a multidisciplinary approach.
This involves integrating knowledge of vascular biology together with biomaterials
engineering and microfluidic design, to recapitulate a microenvironment in which
B. Zohar et al.
shown to regulate and enhance angiogenic processes. Several groups have studied
the effects of flow-induced shear stress on endothelial cells in vitro, using a parallel
plate flow chamber [5, 18, 51]. This experimental setup is simple to model and provides better control over the applied shear stress. However, it does not closely mimic
the 3D nature of interstitial flow, as the medium flows over the cells rather than
through the scaffold. Moreover, endothelial cell response is limited as the system
does not support 3D sprouting and network formation. In an alternative 3D model,
consisting of 3D collagen and alginate gel plugs embedded with endothelial cells,
direct flow simulation resulted in enhanced vascular morphogenesis and
angiogenesis- related gene expression [29, 46]. Furthermore, in our recently published work, we demonstrated the effect of flow-induced shear stress on vascular
formation and maturation in an implantable 3D engineered construct. Direct flow
conditions resulted in a significant increase (>100%) in vessel network morphogenesis parameters, EC and ECM protein depth distribution, and colocalization incidence of alpha-smooth muscle actin (α-SMA) with endothelial vessel networks.
These findings suggest that flow conditions promote 3D neovascularization and
vascular network maturation in a 3D engineered tissue (Fig. 2.3b) [63]. In
microfluidic- based platforms, luminal flow through a self-assembled microvasculature was applied to generate shear stress stimulation inside microvessels [23, 57].
The microvasculature patency was demonstrated by tracking flowing FITC-dextran
and fluorescent microbeads (Fig. 2.3c) [23, 57]. The self-assembled microvessels
possessed morphological and biochemical markers characteristic of natural blood
vessels and exhibited strong barrier function, long-term stability, cytoskeleton rearrangements, and increased nitric oxide synthesis (Fig. 2.3c) [23].
However, the true nature of the effect of shear stress on vessel formation and
behavior is still poorly understood [1]. Many molecular mechanisms and signal
transduction pathways are suggested to bear shear-sensing or mechanotransductive
roles. These mechanisms involve cell-matrix and cell-cell adhesion and junction
molecules, membranous receptors and ion channels, the extra-membranous glycocalyx complex, and cytoskeleton proteins among others [1, 33, 44]. For example, shear
stress has been shown to mediate changes in endothelial cell phenotype, by increasing RhoA (a cytoskeletal regulator) activity and promoting the formation of stress
fibers [8]. Additionally, a direct connection between flow-induced shear stress and
VEGF signaling during angiogenesis has been shown to be mediated by an endothelial cell-specific microRNA, called mir-126, which is regulated by the mechanosensitive zinc finger transcription factor klf2a [11, 40]. This microRNA has a
pro-angiogenic function, by naturally repressing VEGF inhibitors ispred1 and pik3r2.
2.4 Conclusions and Future Perspectives
Creation of vascularized 3D engineered tissue requires a multidisciplinary approach.
This involves integrating knowledge of vascular biology together with biomaterials
engineering and microfluidic design, to recapitulate a microenvironment in which
B. Zohar et al.