Blood Vessel Model using Tissue Modules with on-demand Stimuli

Artificial vascularization of tissue has been a major barrier in the upscaling of tissue engineering. Achieving angiogenesis from a pre-existing vessel in a controlled manner is a possible solution to prevascularize tissue. Microfluidic approaches do not allow yet the creation of a complex hierarchical tissue construct that can be manipulated and removed from the creation template.Thus the challenge is to simulate angiogenesis in a 1:1 scale. We aim to assemble a blood vessel module that will include: on-demand flow, through a tubular structure comprised of endothelial cells, fibroblasts and smooth muscle cells suspended in a hydrogel environment functionalized with growth factors

The role of mechanical environment in regulating vascular network formation

Introduction: The integration of engineered tissues after implantation is limited due to the lack of a vascular network. When vascular networks are included, they generally are not organized, or lose their initial organization fast1. Due to the ability of cells to sense their environment by mechanotransduction, signaling, maturation, organization and cell survival is regulated. The objective of this study is to analyze the combinatory effect of substrate stiffness and fluid flow on vascular organization and maturation to find the basic parameters for pre-vascularization of engineered tissue. Methods A microfluidic PDMS framed system was used, which makes it possible to investigate different hydrogel compositions in parallel. Pre-glycation by D-(-)-Ribose allowed for the use of one type of hydrogel in the same concentration but with different modulated mechanical properties. During the polymerization process of the hydrogels, needles were integrated into the gels to create hollow channels by their removing afterwards. The down and top side of the system were sealed with thin cover glasses to ensure the visibility of the inner system. The fluid-flow channels were coated with 0.1% Gelatin to improve the cell attachment and seeded with Smooth muscle cells. Afterwards Human Umbilical Vein Endothelial Cells (HUVECs) were seeded on top of the smooth muscle cells to mimic the physiological blood vessel structure. An additional channel was filled with VEGF (50 ng/mL), which is known as one of the main angiogenic factors which diffuse into the hydrogel over time2. Different fluid-flow profiles were applied to the cell seeded channels. The newly formed capillary network was analyzed by ImageJ. Results: The pre-glycation by incubation of different concentrations of D-(-)-Ribose resulted in an increase of stiffness of the same type of hydrogel by additionally crosslinking of the different hydrogels components. The use of different modulated hydrogels allowed for the simultaneous analysis of the effect of fluid flow on the vascular sprouting into the hydrogels triggered by the diffused VEGF. Different mechanical properties in combination with different fluid flow patterns affected the ability of HUVEC to migrate and organize into the hydrogels and show differences in the sprouting morphology. Outlook: To mimic the physiological state, different Endothelial cell types (e.g. HUVECs, HMECs, HIAEC) will be integrated into the fluid flow channels. This will allow us to see if different endothelial cell origins leads to a different sprouting behavior or if the already described endothelial plasticity leads to similar results

Effect of fluid flow on vascular network organization in a multi-structural in vitro model

Introduction: Engineered tissues offer a great promise to the field of medicine as an alternative for donor tissues for which the supply is not meeting the demands. However, the integration of engineered tissues after implantation is limited due to the lack of a vascular network. Currently, strategies to include vascular networks rely on the spontaneous organization of vascular cells, or on the patterning of these cells. However, this results in either vascular networks that are not organized, or networks that lose their initial organization fast.1-3 In this project we will use interstitial flow as one of the main cues to control vascular organization and maturation in hydrogel-based tissues. Aim: To develop a microfluidic system to evaluate the effect of fluid flow profiles on vascular organization and maturation. Materials and Methods: We use a microfluidic 5-channel PDMS system that was developed in our group. The hydrogel channels are flanked by media channels and PDMS pillars to contain the Collagen I (5 mg/mL) (see Scheme 1). Additionally both hydrogel channels possess together four different diameters to analyze the effect of hydrogel thickness on endothelial cell sprouting. The media channels are coated with 0.1% Collagen I to improve the cell attachment and seeded with Human Umbilical Vein Endothelial Cells (HUVECs). One channel is filled with VEGF (50 ng/mL), which is known as one of the main angiogenic factors.4 Different fluid-flow profiles are applied to the cell seeded channels 24 hours later. The newly formed capillary network are analysed by ImageJ. Results and Conclusions: The Geltrex® (soluble form of basement membrane extracted from murine Engelbreth-Holm-Swarm tumors) based hydrogel channels shrink rapidly during the polymerization process, which further led to the formation of deep pores between the pillars. Due to the presence of the pores, the formation of a smooth HUVEC monolayer is disturbed. Therefore, it is better to use Collagen I hydrogel instead of Geltrex®, which could reduce the shrinking phenomenon during polymerization. Based on various fluid-flow profiles, hydrogel thicknesses and diffusion of VEGF within the hydrogel, different sprouting of HUVECs into the Collage I hydrogel channel is observed. Future Plans: Gradients of stiffness of different hydrogels (Collagen I, Geltrex®) will be generated and used in a designed mold with a 3-Channel system. To mimic the physiological state, different Endothelial cell types (e.g. HUVECs, HMECs, HIAEC) will be integrated into the fluid flow channels. This will allow us to see if different endothelial cell origins leads to a different sprouting behaviour or if the already described endothelial plasticity leads to similar results. References: 1. Levenberg et. al., Engineering vascularized skeletal muscle tissue. Nat Biotechnol, 2005. 23(7): 879-84. 2. Rivron et. al., Tissue deformation spatially modulates VEGF signaling and angiogenesis. Proc Natl Acad Sci U S A, 2012. 109(18): 6886-91. 3. Rivron et. al., Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation. Proc Natl Acad Sci U S A, 2012. 109(12): 4413-8. 4. Shibuya M., Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases. J Biochem, 2013. 153(1):13-9. Acknowledgements This work is supported by an ERC Consolidator Grant under grant agreement n

Improved endothelialization by silicone surface modification and fluid hydrodynamics modulation: Implications for oxygenator biocompatibility

Klein Nulend, J. [Promotor]Amoabediny, G. [Promotor]Zandieh Doulabi, B. [Copromotor]Shokrgozar, M.A. [Copromotor

Flow-driven vascular network organization in a chorioallantoic membrane model

Introduction Adding vascular network to engineered tissues is an important step for the clinical application of these tissues. Recent studies have shown that vascular cells can display plasticity with respect to local cues, meaning that the organization is influenced by the environment and not just by genetics [1, 2]. Since it is clear that vascular cells are programmed to respond to hydrodynamic signals, and by considering the challenging nature of internal fluid flow control within vessels, we hypothesize that fluid flow shear stress on the outside of a vascular network can also guide vascular organization. As a natural highly vascularized model, the chick embryo and its chorioallantoic membrane (CAM) is applied in this project. Methods An artificial cubic eggshell is designed in a way that allows oxygen passage into the chick embryo as well as high observability. A patterned membrane fabricated by microfabrication techniques is used in one of the walls of the artificial cubic eggshell to exert external fluid shear stress on CAM. The shear stress profile in the designed pattern is modeled using COMSOL Multiphysics software tool. The spreading vascular network is visualized using optical color imaging and the degree of vasculature revealed by the vessels-occupied area are calculated by using ImageJ software. Results The results of this project show how external fluid flow shear stress affects vascular development and organization. When the hypothesis holds true, the results of this project will provide us with an extra tool to control vascular organization in engineered tissues. Discussion and Conclusions If the external fluid flow shear stresses could control vascular organization, the blood vessels can be designed and reorganized without disturbing blood flow within the vascular network. This method can open new horizons in developing a functional vascular network within engineered tissues, which is a key challenge in tissue engineering. Acknowledgements This work is supported by an ERC Consolidator Grant under grant agreement no 724469. NSN is supported by a NWO Grant under grant agreement no OCENW.XS.021. References [1] Le Noble F, Moyon D, Pardanaud L, Yuan L, Djonov V, et al. Flow regulates arterial-venous differentiation in the chick embryo yolk sac. Development 2004;131(2):361-375. [2] Huang W, Itayama M, Arai F, Furukawa KS, Ushida T, et al. An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane. Plos One 2017; 12(4)