Flow-Induced Vascular Network Formation and Maturation in Three

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Flow-Induced Vascular Network Formation and Maturation in Three-Dimensional Engineered Tissue Barak Zohar, Yaron Blinder, David J. Mooney, and Shulamit Levenberg ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00025 • Publication Date (Web): 11 Apr 2017 Downloaded from http://pubs.acs.org on April 13, 2017

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ACS Biomaterials Science & Engineering

Flow-Induced Vascular Network Formation and Maturation in Three-Dimensional Engineered Tissue 1

Barak Zohar*, 1,3Yaron Blinder*, 2,3David J Mooney and 1Shulamit Levenberg**

1

Department of Biomedical Engineering, Technion-Israel Institute of Technology 2

3

School of Engineering and Applied Sciences, Harvard University

Wyss Institute for Biologically Inspired Engineering at Harvard University *These authors contributed equally to this work **Corresponding author: [email protected]

FULL MAILING ADDRESS OF AUTHORS Barak Zohar [email protected]

Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa 3200003 ROOM 161

Dr. Yaron Blinder [email protected]


1 ACS Paragon Plus Environment


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Prof. David J Mooney [email protected]

School of Engineering and Applied Sciences, Harvard University, 319 Pierce Hall

Prof. Shulamit Levenberg [email protected]


KEYWORDS: engineered tissue, vascular networks, endothelial cells, flow bioreactors, and fluid shear stress 2 ACS Paragon Plus Environment

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ABSTRACT: Engineered three-dimensional (3D) constructs have received much attention as in vitro tools for the study of cell-cell and cell-matrix interactions, and have been explored for potential use as experimental models or therapeutic human tissue substitutes. Yet, due to diffusion limitations, lack of a stable and perfusable blood vessel networks, jeopardizes cell viability once the tissue dimensions extend beyond several hundred microns. Direct perfusion of 3D scaffold cultures has been shown to enhance oxygen and nutrient availability. Additionally, flow-induced shear stress at physiologically relevant levels, positively impacted endothelial cell migration and alignment in various two-dimensional (2D) culture models and promoted angiogenic sprouting in microfluidic systems. However, little is known about the effect of flow on vascularization in implantable 3D engineered tissue models. The present study investigated the effect of direct flowinduced shear stress on vascularization in implantable 3D tissue. The differential effect of various levels of shear stress, applied while maintaining constant culture conditions, on vascular parameters was measured. Samples grown under direct flow conditions showed significant increases (>100%) in vessel network morphogenesis parameters and increases in vessel and extracellular matrix (ECM) protein depth distribution, as compared to those grown under static conditions. Enhanced vascular network morphogenesis parameters and higher colocalization of alpha-smooth muscle actin (α-SMA) with endothelial vessel networks characterized the specific contribution of direct flow to vessel network complexity and maturation. These observations suggest that flow conditions promote 3D neovascularization, and may be advantageous in attempts to create large-volume, clinically relevant tissue substitutes.



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INTRODUCTION Engineered implantable constructs generally contain cells seeded on three-dimensional (3D) polymeric scaffolds. These scaffolds are designed to support tissue formation and mimic the cellcell and cell-extracellular matrix (ECM) interactions of naturally occurring 3D niches1,2. Currently, construct thickness, limited by inadequate nutrient perfusion, is one of the main obstacles in tissue engineering3–5. Following transplantation, insufficient blood perfusion further threatens construct integrity, and can result in failed integration and collapse of the engineered tissue6. Co-culture of endothelial cells (ECs) with supportive cells, such as fibroblasts, skeletal muscle cells7 or cardiomyocytes,8 on 3D scaffolds has been shown to vascularize the engineered tissue in vitro and consequently facilitate its survival and viability upon implantation7. This approach relies on natural mechanisms, such as vasculogenesis and angiogenesis, which drive the de novo formation of blood vessel networks9-11. These mechanisms are highly regulated by a variety of chemical molecules12– 14

and mechanical forces, such as strain15-16 and shear stress17. Fluid shear stress has been

demonstrated to play a crucial role in vessel formation, maturation and stabilization both in in vitro9,18-20 and in vivo5,21 models, presumably by triggering mechanical stimulation and enhancing nutrient transport. Flow-induced shear stress applied to 2D monolayer “flow-over” in vitro models 22,23

has been shown to regulate endothelial migration, apoptosis, proliferation and alignment.

Furthermore, in various microfluidic assemblies,24–28 physiologically relevant shear stress (1-10 dyne/cm2) has been shown to regulate morphological processes such as angiogenic sprouting. In line with these findings, direct flow stimulated vascular morphogenesis and angiogenesis-related gene expression in 3D collagen and alginate constructs embedded with endothelial cells29,30.


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The effect of flow has proven beneficial in culture of engineered tissues such as cardiac31,32, hepatic 33,34

cartilage35 and bone tissue36. However, the effect of shear stress on vascularization in 3D

engineered constructs remains questionable, as the process can also be affected by accelerated mass transport. In addition, the effect of flow-induced shear stress on vessel network complexity, maturation and stabilization in implantable 3D engineered tissue remains to be established. Herein, we assessed the biomechanical impact of flow-induced shear stress on vascularization of 3D implantable engineered tissue, by applying various flow regimens while maintaining the medium circulation rate. The effect of constant flow-induced shear stress was compared to that of higher, intermittent flow-induced shear stress. The effects elicited by direct versus indirect perfusion were also compared.

MATERIALS AND METHODS 2.1 Scaffold Preparation. Poly-L-lactic acid (PLLA) (Polysciences, Warrington) and Poly-L-glycolic acid ( PLGA) (Boehringer Ingelhein) (1:1) were dissolved in chloroform to yield a 5% polymer solution. The solution (0.24mL) was loaded into molds containing 0.4g sodium chloride particles with a size distribution ranging between 200-600µm (isolated by sieve size-exclusion). The solvent was allowed to evaporate, and the sponges were subsequently immersed, for 8h, in distilled water (replaced every hour), to leach the salt and create an interconnected pore structure. The sponges were sliced to ~28 mm3 circles (diameter 6mm, width 1mm). 2.2 Cell Culture and Scaffold Seeding. Zs-green-expressing human adipose microvascular endothelial cells (HAMECs; ScienceCell), isolated from human adipose tissue, Zs-greenexpressing human umbilical vein endothelial cells (HUVECs; Lonza) and neonatal human dermal 5 ACS Paragon Plus Environment


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fibroblasts (HNDFs; Lonza) were grown separately on standard tissue culture plates. HNDFs were cultivated in DMEM (Gibco), supplemented with 10% fetal bovine serum (FBS) (Hyclone), 1% non-essential amino acids, 0.2% -mercaptoethanol and 1% penstrep (Sigma Aldrich). HAMECs were cultivated in endothelial cell medium (ScienceCell), supplemented with 5% FBS (ScienceCell) and endothelial cell growth supplement (ScienceCell) and HUVECs were cultivated in EGM-2 medium, supplemented with a bullet kit containing FBS, hydrocortisone, hFGF-, VEGF, R3-IGF-1, hEGF, GA-1000 and heparin (Lonza). Cells were cultured in a 5% CO2 humidified incubator at 37°C and harvested during passages 5–8. Then, ECs and HNDFs were mixed at a ratio of 5:1 (total 6 × 105 cells per scaffold) in 14μL human fibrin gel, prepared from a 1:1 mixture of thrombin solution (15 mg/mL, Johnson & Johnson Medical, Israel) and human fibrinogen solution (5 mU/mL, Johnson & Johnson Medical), and then rapidly pipetted and seeded upon the PLLA/PLGA scaffolds. Scaffolds were then incubated (30 min, 37 °C, 5 % CO2) on a 12-well non-tissue culture plate. Co-culture medium (a 1:1 mixture of the two respective cell media) was added (1-3 mL per scaffold) and replaced every 2-3 days. Scaffolds were cultured under static conditions (37 °C, 5 % CO2) in a 12-well non-tissue culture plate for 5 days before being transferred to the flow bioreactors. 2.3 Flow Bioreactor Configuration. The flow bioreactor contained flow chambers and a perfusion system. Flow chambers were prepared from commercially available sterile scaffold holders (P6D, Ebers Medical) (Figure 1A) or in-house developed poly(methyl methacrylate) (PMMA) chambers that were design to test the effect of direct flow compared to indirect perfusion (control) conditions (Figure 1C). The perfusion system was composed of a glass reservoir bottle, silicone tubes and a multi-channel peristaltic pump (Ismatec or EBERS TEB1000 pumps) (Figure 1B). Before use, the flow bioreactor was cleaned with 1% Liquinox ™ detergent and distilled water, washed with PBS


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and sterilized in an autoclave (121˚C, 30 min). The bioreactor was then incubated with relevant culture media for at least 24 h before scaffolds were inserted. After verifying the integrity of the system, flow chambers were loaded with pre-seeded scaffolds and plugged into the perfusion system. Flow bioreactors were placed inside the designated EBERS incubator (EBERS TEB1000, highly humidified 37 °C, 5 % CO2) for further culturing under different flow conditions. Constant and intermittent flow profiles were programmed using the EBERS TEB1000 software. 2.4 Shear stress estimation using a pre-designed computational fluid dynamics model. A computational fluid dynamics (CFD) model for direct flow through the complex 3D microstructure of a porous scaffold was designed37 to predict physiological shear stress levels sensed by endothelial cells. The model considered the accumulation of cells and ECM proteins in a scaffold 7 days post-seeding, by introducing geometrical modifications to differences in cell layer thicknesses. Flow-associated parameters were extracted from the model (Table 1). Both intermittent and constant flow rates were set to generate identical medium circulation rate of 3 scaffold volume replacements in a minute. The direct intermittent flow was set as 0.6 mL/min, corresponding to a mean shear stress of 4.5 dyne/cm2 in a duty cycle of 16.7% (10 seconds), applied for 1 minute. The constant flow was set at 0.1mL/min for both direct and indirect perfusion (control) conditions, corresponding to a mean shear stress of 0.75 dyne/cm2 for the direct flow conditions (Table 1, Figure 2A). Flow velocity was characterized inside the direct flow and control chambers by CFD model using the FLUENT software to evaluate media circulation rates and to assure negligible shear stress under indirect perfusion (control) conditions (Figure 1D). Medium circulation rates were estimated by the CFD model to be 3 and 0.3 scaffold volume replacements in a minute for direct flow and control conditions respectively.



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Cells exposed to constant and intermittent flow were cultured for 2 days and those exposed to direct flow and indirect perfusion (control), were cultured for 6 days. Table 1. Mean velocity and shear stress through a porous 3D scaffold, at the tested flow rates, as estimated by the CFD model.

Flow Profile

Flow rate (mL/min)

Constant Intermittent

0.1 0.6

Mean Mean/Maximum velocity shear stress (cm/sec) (dyne/cm2) 0.093 0.75/2 0.55 4.5/13

2.5 Scaffold Sectioning and Immunohistochemical Staining. Constructs were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), and then embedded in an agar gel (5% low melting temperature SeaPlaque© agarose in distilled water, Lonza). Sections (200µm-thick) were obtained from the agar-embedded constructs using a vibratome (VT1000S, Leica), and then permeabilized with 0.5% Triton X-100 (Bio Lab Ltd), rinsed several times with PBS and blocked with 5% bovine serum albumin (BSA) (w/v, Millipore). Subsequently, samples were incubated with primary monoclonal rabbit anti-human von Willebrand factor (vWF) antibody (1:150; Abcam) or CD31 (1:50, Cell Marque), monoclonal mouse anti-human Alpha-smooth muscle actin (α-SMA) (1:50, Dako) antibody, or with collagen I (1:200; Abcam) and collagen IV (1:200;Dako) antibodies overnight. Samples were then rinsed several times with PBS, and then incubated (3hr, room temperature) with Cy3-conjugated goat anti-mouse (1:100, Jackson Immuno-research laboratory) and Alexa 488-conjugated anti-rabbit (1:800, Thermo Fisher Scientific) antibodies, diluted in staining buffer and then stored in PBS until imaging.


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2.6 Confocal Imaging and Analysis Flow-oriented sides and cross-sections of the scaffolds were scanned with an LSM700 confocal microscope (Zeiss). The depth of imaging was 300–500µm, split into at least 20 z-stacks. Threedimensional (XYZ) confocal z-stacks were converted to 2D TIFF stacks, by performing highintensity z-projections using the NIH ImageJ software. Stacks were then prepared for morphological analysis by contrast enhancement (stack histogram equalization and normalization, 0.4% saturated pixels). Each frame was processed using the Angiotool® interface, as described in the online user manual (see http://ccrod.cancer.gov/confluence/display/ROB2/Quick+Guide), to quantify vessel percentage area, total number of junctions, junction density, total and average vessel lengths, total number of vessel endpoints and lacunarity. EC bulk distribution was quantified by isolating and masking 30µm-thick layers at increasing distances from the exterior edge of each cross-section image and quantifying the degree of endothelial area coverage by applying an image analysis algorithm (MATLAB code). Colocalization of vWF (ECs) and α-SMA was quantified by the Colocalization Threshold plugin (ImageJ), as described in the online user manual (see http://imagej.net/Colocalization_Analysis#Colocalization_Threshold). Colocalization percentage is presented as the percentage of above-threshold vWF (ECs) intensity colocalized with abovethreshold α-SMA intensity. Auto-threshold was determined using the Costes method. 2.7 Statistics Measurements were performed in triplicates, at minimum, and images were scanned, processed and analyzed using an identical setup each time. For streamline vascular parameters presentation, data were normalized to the relevant control treatment. Normalized means were plotted, with error bars representing the standard error of the mean (SEM). Statistical comparisons were performed



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using the Student’s t test with a 95% confidence limit (two-tailed and unequal variance). Differences with a p-value ˂ 0.05 were considered statistically significant.

Figure 1. Flow bioreactor configurations. (A) Schematic drawing of a commercially available scaffold holder for direct perfusion (Ebers Medical). (B) Schematic view of a typical perfusion bioreactor in which culture medium flows directly through a porous cellular scaffold (Reproduced with permission from ref 37). (C) Schematic drawing of the in-house-designed direct flow and control chambers. (D) Flow velocity characterization by CFD model (FLUENT software) in the direct flow and control chambers while applying flow rate of 0.1 ml/min.

RESULTS


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3.1 The impact of constant and intermittent flow on vascularization. Scaffolds seeded with fluorescence-labeled ECs and HNDFs and cultured under static conditions, displayed ECs that had migrated and typically formed multicellular clusters by day 5. After subjecting the scaffolds to direct intermittent or constant flow conditions for two days, clusters began to exhibit enhanced outward sprouting, resulting in formation of branched endothelial networks, which were rarely observed in samples cultured under static conditions (Figure 2B). Constant flow-cultured constructs exhibited a statistically significant (n=4, p < 0.05) decrease in mean lacunarity and increase in vessel area and density (>100%), junction count and density (~ 200%), overall vessel length (>100%), average vessel length and number of vessel endpoints (~100%) as compared to static-cultured constructs (Figure 2C). Although intermittent flow-cultured constructs showed higher vascular measures, as compared to static-cultured constructs, the mean differences were all smaller than those measured for constant flow-cultured constructs.



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Figure 2. Vessel network formation in constructs cultured for 5 days under static conditions, followed by 2 days under static, constant (0.1mL/min) or intermittent (0.6mL/min) flow conditions. (A) Shear stress profiles corresponding to constant and intermittent volumetric flow rates, as estimated by CFD model. (B) EC epifluorescence (green) and respective Angiotool segmentation (green = EC epifluorescence, red = segmented vessels, light blue = segmented junctions) (scale bar - 500µm). (C) Normalized mean vessel network morphological measures in


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the explant area, vessel area and density, junction count and density, overall vessel length, average vessel length, number of vessel endpoints, and mean E lacunarity as determined by Angiotool (n = 4). The results were normalized to measurements made under static conditions. Error bars represent standard error of the mean (SEM). * indicates statistical significance (*p ˂ 0.05) 3.2 Vessel depth-distribution and ECM protein structures. Examination of scaffold cross-sections revealed a clear difference in vascular and ECM protein structures in constructs cultured under static versus flow conditions. Flow-cultured constructs displayed higher EC, collagen I and collagen IV epifluorescence intensities within the scaffold (Figure 3A, C). High-magnification images demonstrated the higher colocalization of collagens I and IV with endothelial vascular structures formed following exposure to flow versus static conditions (Figure 3B, D). In addition, constructs cultured under static conditions showed high endothelial densities in the exterior 60µm of the construct, which decreased at increasing distances from the scaffold perimeter. Conversely, constructs cultured under flow conditions, showed a lower endothelial density in the outermost layers, but a higher and relatively constant density at greater depths (Figure 4B).



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Figure 3. Colocalization of collagens I and IV with EC structures in scaffold cross-sections. (A) A cross-section of a scaffold cultured for 7 days under static conditions (scale bar - 500µm). (B) A higher magnification of the ECM structure of the cross-sectioned scaffold sample shown in (A) (scale bar - 100µm). (C) A cross-section of a scaffold cultured for 5 days under static conditions, followed by 2 days under direct constant flow conditions (scale bar - 500µm). (D) A higher magnification of the ECM structure of the cross-sectioned scaffold sample shown in (C) (scale bar - 100µm).

Figure 4. The effect of direct flow on vessel distribution in 3D scaffolds. (A) Endothelial cell epifluorescence, as observed in static- and indirect-flow-cultured scaffold cross-sections (scale bar - 500µm). (B) The percentage of epifluorescence area at increasing distances (30µm intervals) from the surface of scaffolds subjected to static versus flow conditions ((n = 4, different scaffolds for each condition). 3.3 The effect of direct flow on vessel formation and maturation. To isolate the effect of flowinduced shear stress on vascularization, we compared vessel formation and maturation under direct flow versus indirect perfusion (control) conditions (Figure 1C), while applying identical perfusion rates in both flow chambers. At 11 days post-seeding, endothelial network branching was observed


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for both flow-culture regimens (Figure 5A). Direct flow-cultured constructs displayed significantly lower mean lacunarity and significantly higher vascular measures, such as vessel area and density, junction count and density (>50% increase) and overall vessel length, when compared to the control (Figure 5B). Although the increase in average vessel length was statistically insignificant, it was 2-fold higher following exposure to direct flow versus control conditions (Figure 5B). In addition, vessel network maturity, as reflected by α-SMA expression and its colocalization with endothelial vascular structures, were greatest in vascularized areas of direct flow-cultured samples (Figure 6E) and were considerably low in static- or control-cultured samples (Figure 6C, D). More specifically, ~70% of the ECs in constructs subjected to direct flow, colocalized with α-SMA, while only ~40% colocalized with α-SMA following exposure to control or static conditions (n=3, p < 0.05 ).



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Figure 5. Vessel network formation in constructs cultured for 5 days under static conditions, followed by 6 days under direct flow (0.1mL/min) or control conditions. (A) EC epifluorescence (green) and respective Angiotool segmentation (green = EC epifluorescence, red = segmented vessels, light blue = segmented junctions) (scale bar - 500µm). (B) Normalized mean vessel network morphological measures of the explant area, vessel area and density, junction count and density, overall vessel length, average vessel length, number of vessel endpoints, and mean E lacunarity, as determined by Angiotool (n = 3). The results were normalized to the measures


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obtained following exposure to control conditions. Error bars represent standard error of the mean (SEM). * indicates statistical significance (*p ˂ 0.05)

Figure 6. vWF (ECs) and α-SMA colocalization in scaffolds subjected to direct flow (0.1mL/min) versus static and control culture conditions. (A) Top and cross-sectional views of ECs in a scaffold cultured for 6 days under direct flow. (B) Top view in higher magnification (scale bar - 50µm) (CE) Cross-sectional view of scaffolds cultured for 6 days under direct flow, control or static conditions (scale bar - 100µm). (F) vWF (ECs) and α-SMA colocalization percentage, as determined from cross-sections of scaffolds subjected to direct flow, control or static culture



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conditions (n = 3, different scaffolds for each condition). Error bars represent standard error of the mean (SEM). * indicates statistical significance (*p ˂ 0.05 and **p ˂ 0.01)

DISCUSSION AND CONCLUSIONS Flow-induced, physiologically relevant levels of shear stress have been shown to influence endothelial alignment in 2D “flow-over” models and to promote vessel sprouting in microfabricated systems. Thus, we hypothesized that exposure of 3D engineered tissue constructs to direct flow conditions would have similar effects. To assess the effect of direct flow on vessel network morphology, we designed two experiments in which we applied different perfusion approaches. 3D engineered constructs cultured under constant flow conditions exhibited significant increases (100-200%, p < 0.05) in almost all vascular measures, as compared to those grown under static conditions. Direct flow through the 3D construct triggered a significantly different bulk distribution of vascular structures and ECM protein expression, as compared to static conditions. More specifically, endothelial structures in static-cultured constructs were distributed more closely to the exterior construct edges, with a substantial drop in endothelial presence within the scaffold core. Conversely, flow-cultured constructs exhibited slightly lower endothelial density at the scaffold exterior, but increased and uniform distribution deeper within the scaffold volume. These observations demonstrated that medium convection through the 3D scaffold promotes cell viability and vitality, ECM protein secretion and vessel network formation at depths beyond diffusion limits (~150 µm). A relatively low constant flow of 0.1mL/min, inducing an estimated shear stress of 0.75 dyne/cm2, applied for only 48 hours, was sufficient to positively impact angiogenic processes. The reduced vascular measures recorded in intermittent versus constant 18 ACS Paragon Plus Environment

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flow-cultured constructs, may be the result of a positive effect of continuous shear when in a physiological range. In both cases, there was identical minutely medium exchange, thus, the differences between set-ups were unlikely to be due to major differences in mass transport. The positive effect of flow on vessel formation can be ascribed to the perfusion system that improves mass transport by generating better mixing of media near the scaffold; and/or media convection through the scaffold that increases shear stress stimuli and accelerates mass transport within the scaffold. To better define how its effect is mediated, we compared the effect of direct flow to indirect perfusion (control) conditions while maintaining similar flow rate. Our results indicate that direct flow contributed (20-50%, p < 0.05) to vessel formation. Moreover, direct flow-culture constructs showed significant higher EC and α-SMA colocalization as compared to control-cultured and static-cultured constructs (~75%, p < 0.05 and p < 0.01 respectively), indicative of enhanced vascular maturation and stabilization. In conclusion, this work demonstrated that direct flow through implantable engineered 3D tissue induces vessel network formation, increases vessel and ECM depth-distribution and enhances vessel maturation and stabilization. The positive effect correlated with the convection of media through the scaffold in direct-perfusion mode. Our findings provide a deeper understanding of vascularization processes that are induced and maintained by flow. These observations are expected to promote judicious utilization of flow stimuli in tissue engineering, toward creation of real-scale, vascularized artificial tissues that may function better and survive longer in vivo.

AUTHOR INFORMATION 19 ACS Paragon Plus Environment


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Corresponding Author **E-mail: [email protected]. Tel:+972-48294810. Fax: +972-48294809 Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This research was supported by FP7 European Research Council Grant 281501, ENGVASC (to S.L.)

REFERENCES (1) Langer, R.; Vacanti, J. P. Tissue engineering. Science 1993, 260 (5110), 920–926. (2) Levenberg, S.; Langer, R. Advances in tissue engineering. Curr. Top. Dev. Biol. 2004, 61, 113–134. (3) Novosel, E. C.; Kleinhans, C.; Kluger, P. J. Vascularization is the key challenge in tissue engineering. Adv. Drug Deliv. Rev. 2011, 63 (4-5), 300–311. (4) Nomi, M.; Atala, A.; Coppi, P. D.; Soker, S. Principals of neovascularization for tissue engineering. Mol. Aspects Med. 2002, 23 (6), 463–483. (5) Jain, R. K.; Au, P.; Tam, J.; Duda, D. G.; Fukumura, D. Engineering vascularized tissue. Nat. Biotechnol. 2005, 23 (7), 821–823. (6) Rouwkema, J.; Rivron, N. C.; van Blitterswijk, C. A. Vascularization in tissue engineering. Trends Biotechnol. 2008, 26 (8), 434–441. (7) Levenberg, S.; Rouwkema, J.; Macdonald, M.; Garfein, E. S.; Kohane, D. S.; Darland, D. C.; Marini, R.; van Blitterswijk, C. A.; Mulligan, R. C.; D’Amore, P. A.; et al. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol. 2005, 23 (7), 879–884.


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(8) Caspi, O.; Lesman, A.; Basevitch, Y.; Gepstein, A.; Arbel, G.; Habib, I. H. M.; Gepstein, L.; Levenberg, S. Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circ. Res. 2007, 100 (2), 263–272. (9) Carmeliet, P. Angiogenesis in health and disease. Nat. Med. 2003, 9 (6), 653–660. (10) Tepper, O. M.; Capla, J. M.; Galiano, R. D.; Ceradini, D. J.; Callaghan, M. J.; Kleinman, M. E.; Gurtner, G. C. Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow–derived cells. Blood 2005, 105 (3), 1068–1077. (11) Blinder, Y. J.; Freiman, A.; Raindel, N.; Mooney, D. J.; Levenberg, S. Vasculogenic dynamics in 3D engineered tissue constructs. Sci. Rep. 2015, 5, 17840. (12) Wieghaus, K. A.; Capitosti, S. M.; Anderson, C. R.; Price, R. J.; Blackman, B. R.; Brown, M. L.; Botchwey, E. A. Small molecule inducers of angiogenesis for tissue engineering. Tissue Eng. 2006, 12 (7), 1903–1913. (13) Zhu, X. H.; Tabata, Y.; Wang, C.-H.; Tong, Y. W. Delivery of basic fibroblast growth factor from gelatin microsphere scaffold for the growth of human umbilical vein endothelial cells. Tissue Eng. Part A 2008, 14 (12), 1939–1947. (14) Wong, V. W.; Crawford, J. D. Vasculogenic cytokines in wound healing. Biomed Res. Int. 2013, 2013, 190486. (15) Matsumoto, T.; Yung, Y. C.; Fischbach, C.; Kong, H. J.; Nakaoka, R.; Mooney, D. J. Mechanical strain regulates endothelial cell patterning in vitro. Tissue Eng. 2007, 13 (1), 207–217. (16) Rosenfeld, D.; Landau, S.; Shandalov, Y.; Raindel, N.; Freiman, A.; Shor, E.; Blinder, Y.; Vandenburgh, H. H.; Mooney, D. J.; Levenberg, S. Morphogenesis of 3D vascular networks is regulated by tensile forces. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (12), 3215–3220. (17) Jain, R. K. Molecular regulation of vessel maturation. Nat. Med. 2003, 9 (6), 685–693. (18) Davies, P. F. Spatial Microstimuli in Endothelial Mechanosignaling. Circ. Res. 2003, 92 (4), 359–370. (19) Resnick, N.; Yahav, H.; Shay-Salit, A.; Shushy, M.; Schubert, S.; Zilberman, L. C. M.; Wofovitz, E. Fluid shear stress and the vascular endothelium: for better and for worse. Prog. Biophys. Mol. Biol. 2003, 81 (3), 177–199. 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

(20) Ichioka, S.; Shibata, M.; Kosaki, K.; Sato, Y.; Harii, K.; Kamiya, A. Effects of shear stress on woundhealing angiogenesis in the rabbit ear chamber. J. Surg. Res. 1997, 72 (1), 29–35. (21) Nasu, R.; Kimura, H.; Akagi, K.; Murata, T.; Tanaka, Y. Blood flow influences vascular growth during tumour angiogenesis. Br. J. Cancer 1999, 79 (5-6), 780–786. (22) Anisi, F.; Salehi-Nik, N.; Amoabediny, G.; Pouran, B.; Haghighipour, N.; Zandieh-Doulabi, B. Applying shear stress to endothelial cells in a new perfusion chamber: hydrodynamic analysis. J. Artif. Organs 2014, 17 (4), 329–336. (23) Walshe, T. E.; dela Paz, N. G.; D’Amore, P. A. The Role of Shear-Induced Transforming Growth FactorSignaling in the Endothelium. Arterioscler. Thromb. Vasc. Biol. 2013, 33 (11), 2608–2617. (24) Galie, P. A.; Nguyen, D.-H. T.; Choi, C. K.; Cohen, D. M.; Janmey, P. A.; Chen, C. S. Fluid shear stress threshold regulates angiogenic sprouting. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (22), 7968–7973. (25) Jeon, J. S.; Bersini, S.; Whisler, J. A.; Chen, M. B.; Dubini, G.; Charest, J. L.; Moretti, M.; Kamm, R. D. Generation of 3D functional microvascular networks with human mesenchymal stem cells in microfluidic systems. Integr. Biol. 2014, 6 (5), 555–563. (26) Vickerman, V.; Kamm, R. D. Mechanism of a flow-gated angiogenesis switch: early signaling events at cell--matrix and cell--cell junctions. Integr. Biol. 2012, 4 (8), 863–874. (27) Miller, J. S.; Stevens, K. R.; Yang, M. T.; Baker, B. M.; Nguyen, D.-H. T.; Cohen, D. M.; Toro, E.; Chen, A. A.; Galie, P. A.; Yu, X.; et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat. Mater. 2012, 11 (9), 768–774. (28) Bellan, L. M.; Singh, S. P.; Henderson, P. W.; Porri, T. J.; Craighead, H. G.; Spector, J. A. Fabrication of an artificial 3-dimensional vascular network using sacrificial sugar structures. Soft Matter 2009, 5 (7), 1354. (29) Lee, E. J.; Niklason, L. E. A novel flow bioreactor for in vitro microvascularization. Tissue Eng. Part C Methods 2010, 16 (5), 1191–1200. (30) Rotenberg, M. Y.; Ruvinov, E.; Armoza, A.; Cohen, S. A multi-shear perfusion bioreactor for investigating shear stress effects in endothelial cell constructs. Lab Chip 2012, 12 (15), 2696–2703.


Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(31) Sekine, H.; Shimizu, T.; Sakaguchi, K.; Dobashi, I.; Wada, M.; Yamato, M.; Kobayashi, E.; Umezu, M.; Okano, T. In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels. Nat. Commun. 2013, 4, 1399. (32) Dvir, T.; Benishti, N.; Shachar, M.; Cohen, S. A novel perfusion bioreactor providing a homogenous milieu for tissue regeneration. Tissue Eng. 2006, 12 (10), 2843–2852. (33) Shvartsman, I.; Dvir, T.; Harel-Adar, T.; Cohen, S. Perfusion cell seeding and cultivation induce the assembly of thick and functional hepatocellular tissue-like construct. Tissue Eng. Part A 2009, 15 (4), 751–760. (34) Powers, M. J.; Domansky, K.; Kaazempur-Mofrad, M. R.; Kalezi, A.; Capitano, A.; Upadhyaya, A.; Kurzawski, P.; Wack, K. E.; Stolz, D. B.; Kamm, R.; et al. A microfabricated array bioreactor for perfused 3D liver culture. Biotechnol. Bioeng. 2002, 78 (3), 257–269. (35) Rath, S. N.; Cohn, D.; (Henley), P. D. W. H. P. (nus) M. Comparison of chondrogenesis in static and dynamic environments using a SFF designed and fabricated PCL-PEO scaffold. Virtual Phys. Prototyp. 2008, 3 (4), 209–219. (36) Gardel, L. S.; Correia-Gomes, C.; Serra, L. A.; Gomes, M. E.; Reis, R. L. A novel bidirectional continuous perfusion bioreactor for the culture of large-sized bone tissue-engineered constructs. J. Biomed. Mater. Res. B Appl. Biomater. 2013, 101 (8), 1377–1386. (37) Lesman, A.; Blinder, Y.; Levenberg, S. Modeling of flow-induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering. Biotechnol. Bioeng. 2010, 105 (3), 645–654.



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For Table of Contents Use Only

Flow-Induced Vascular Network Formation and Maturation in Three-Dimensional Engineered Tissue 1

Barak Zohar*, 1,3Yaron Blinder*, 2,3David J Mooney and 1Shulamit Levenberg**

1

Department of Biomedical Engineering, Technion-Israel Institute of Technology 2

3

School of Engineering and Applied Sciences, Harvard University

Wyss Institute for Biologically Inspired Engineering at Harvard University *These authors contributed equally to this work **Corresponding author


Flow-Induced Vascular Network Formation and Maturation in Three

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