Angiogenesis in Free-Standing Two-Vasculature-Embedded Scaffold Extruded by Two-Core Laminar Flow Device

Chanh Trung Nguyen, Van Thuy Duong, Chang Ho Hwang, Kyo-in Koo

Article ID: 557
Vol 8, Issue 3, 2022, Article identifier:

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Rapid construction of pre-vascular structure is highly desired for engineered thick tissue. However, angiogenesis in free-standing scaffold has been rarely reported because of limitation in growth factor (GF) supply into the scaffold. This study, for the 1st time, investigated angiogenic sprouting in free-standing two-vasculature-embedded scaffold with three different culture conditions and additional GFs. A two-core laminar flow device continuously extruded one vascular channel with human umbilical vein endothelial cells (HUVECs) and a 3 mg/ml type-1 collagen, one hollow channel, and a shell layer with 2% w/v gelatin-alginate (70:30) composite. Under the GF flowing condition, angiogenic sprouting from the HUVEC vessel had started since day 1 and gradually grew toward the hollow channel on day 10. Due to the medium flowing, the HUVECs showed elongated spindle-like morphology homogeneously. Their viability has been over 80% up to day 10. This approach could apply to vascular investigation, and drug discovery further, not only to the engineered thick tissue.


Angiogenesis; Pre-vascularized tissue; Two-core vasculature; Gelatin-alginate; Free standing; Culture condition

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Vajda J, Milojević M, Maver U, et al., 2021, Microvascular Tissue Engineering a Review. Biomedicines, 9:589.

Meng X, et al. Rebuilding the Vascular Network: In Vivo and In Vitro Approaches. Front Cell Dev Biol Rev, 9:639299.

Sarker MD, Naghieh S, Sharma NK, et al., 2018, 3D Biofabrication of Vascular Networks for Tissue Regeneration: A Report on Recent Advances. J Pharm Anal, 8:277–96.

Bae H, Puranik AS, Gauvin R, et al., 2012, Building Vascular Networks. Sci Transl Med, 4:160ps23.

Torre-Muruzabal A, Daelemans L, Van Assche G, et al., 2016, Creation of a Nanovascular Network by Electrospun Sacrificial Nanofibers for Self-healing Applications and its Effect on the Flexural Properties of the Bulk Material. Polymer Testing, 54:78–83.

Kinstlinger IS, Saxton SH, Calderon GA, et al., 2020, Generation of Model Tissues with Dendritic Vascular Networks Via Sacrificial Laser-sintered Carbohydrate Templates. Nat Biomed Eng, 4:916–32.

Arakawa CK, Badeau BA, Zheng Y, et al., 2017, Multicellular Vascularized Engineered Tissues through User-Programmable Biomaterial Photodegradation. Adv Mater, 29:1703156.

Duong VT, Dang TT, Nguyen T, et al., 2018, Cell Attachment on Inside-Outside Surface and Cell Encapsulation in Wall of Microscopic Tubular Scaffolds for Vascular Tissue-Like Formation. Hawaii, USA: EMBC.

Duong VT, Koo K, 2019, Over-Five-Millimeter Diameter Alginate-Collagen Endothelialized Tubular Scaffold Formation. Basel, Switzerland: MicroTAS.

Duong VT, Jong PK, Kim K, et al., 2018, Three-dimensional Bio-printing Technique: Trend and Potential for High Volume Implantable Tissue Generation. Korean Soc Med Biomed Eng, 39:188–207.

Koo KI, Lenshof A, Huong LT, et al., 2021, Acoustic Cell Patterning in Hydrogel for Three-Dimensional Cell Network Formation. Micromachines, 12:3.

Sekine H, Okano T, 2021, Capillary Networks for Bio-Artificial Three-Dimensional Tissues Fabricated Using Cell Sheet Based Tissue Engineering. Int J Mol Sci, 22:92.

Bertlein S, Hikimoto D, Hochleitner G, et al., 2018, Development of Endothelial Cell Networks in 3D Tissues by Combination of Melt Electrospinning Writing with Cell-Accumulation Technology. Small,


Wang Z, Mithieux SM, Weiss AS, 2019, Fabrication Techniques for Vascular and Vascularized Tissue Engineering. Adv Healthc Mater, 8:1900742.

van Duinen V, Zhu D, Ramakers C, et al., 2019, Perfused 3D Angiogenic Sprouting in a High-throughput In Vitro Platform. Angiogenesis, 22:157–65.

Del Amo C, Borau C, Gutiérrez R, et al., 2016, Quantification of Angiogenic Sprouting under Different Growth Factors in a Microfluidic Platform. J Biomech, 49:1340–6.

Farahat WA, Wood LB, Zervantonakis IK, et al., 2012, Ensemble Analysis of Angiogenic Growth in Three dimensional Microfluidic Cell Cultures. PLoS One, 7:e37333.

Duong VT, Dang TT, Hwang CH, et al., 2020, Coaxial Printing of Double-layered and Free-standing Blood Vessel Analogues without Ultraviolet Illumination for High-volume Vascularised Tissue. Biofabrication, 12:045033.

Gao G, Park JY, Kim BS, et al., 2018, Coaxial Cell Printing of Freestanding, Perfusable, and Functional In Vitro Vascular Models for Recapitulation of Native Vascular Endothelium Pathophysiology. Adv Healthc Mater, 7:e1801102.

Duong VT, Nguyen T, Phan L, et al., 2018, Multi-Lumen Tubular Calcium-Alginate Cell-Laden Scaffold Formation for 3D Bioprinting. Taiwan: Presented at the MicroTAS, Kaohsiung, Taiwan.

Nguyen DH, Stapleton SC, Yang MT, et al., 2013, Biomimetic Model to Reconstitute Angiogenic Sprouting Morphogenesis In Vitro. Proc Natl Acad Sci U S A, 110:6712–7.

Iruela-Arispe ML, Davis GE, 2009, Cellular and Molecular Mechanisms of Vascular Lumen Formation. Dev Cell, 16:222–31.

Sugihara K, Yamaguchi Y, Usui A, et al., “A New Perfusion Culture Method with a Self-organized Capillary Network. PLoS One, 15:e0240552.

Duong VT, Dang TT, Kim JP, et al., 2019, Twelve-day Medium Pumping into Tubular Cell-laden Scaffold Using a Lab-made PDMS Connector. Eur Cell Mater, 38:1–13.

Duong VT, Nguyen T, Choi M, et al., 2017, Twenty-Day Culturing of Tubular Scaffolds Using Micro-Connector with HeartMimicking Medium Pumping for Blood Vessel Modeling. Georgia, USA: MicroTAS Georgia, USA.

Fisher AB, Chien S, Barakat AI, et al., 2001, Endothelial Cellular Response to Altered Shear Stress. Am J Physiol Lung Cell Mol Physiol, 281:L529–33.

Sarker B, Singh R, Silva R, et al., 2014, Evaluation of Fibroblasts Adhesion and Proliferation on Alginate-gelatin Crosslinked Hydrogel. PLoS One, 9:e107952.

Sarker B, Papageorgiou DG, Silva R, et al., 2014, Fabrication of Alginate-gelatin Crosslinked Hydrogel Microcapsules and Evaluation of the Microstructure and Physico-chemical Properties. J Mater Chem B, 2:1470–82.

Jiang T, Munguia-Lopez J, Flores-Torres S, et al., 2018, Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation. J Vis Exp, 137:57826.

Rouwkema J, Koopman B, Blitterswijk C, et al., 2010, Supply of Nutrients to Cells in Engineered Tissues. Biotechnol Genet Eng Rev, 26:163–78.

Krogh A, 1919, The Supply of Oxygen to the Tissues and the Regulation of the Capillary Circulation. J Physiol, 52:457–74.

Place TL, Domann FE, Case AJ, 2017, Limitations of Oxygen Delivery to Cells in Culture: An Underappreciated Problem in Basic and Translational Research. Free Radic Biol Med, 113:311–22.

Cao X, Maharjan S, Ashfaq R, et al., 2021, Bioprinting of Small-Diameter Blood Vessels. Engineering, 7:832–44.

Yan J, Liu X, Liu J, et al., 2021, A Dual-layer Cell-laden Tubular Scaffold for Bile Duct Regeneration. Mater Des, 212:110229.

Bombaldi de Souza FC, Camasão DB, Bombaldi de Souza RF, et al., 2020, A Simple and Effective Approach to Produce Tubular Polysaccharide-based Hydrogel Scaffolds. J Appl Polym Sci, 137:48510.

Akkouch A, Yu Y, Ozbolat IT, 2015, Microfabrication of Scaffold-free Tissue Strands for Three-dimensional Tissue Engineering. Biofabrication, 7:031002.

Fayol D, Le Visage C, Ino J, et al., 2013, Design of Biomimetic Vascular Grafts with Magnetic Endothelial Patterning. Cell Transplantation, 22:2105–18.

Ju YM, Ahn H, Arenas-Herrera J, et al., 2017, Electrospun Vascular Scaffold for Cellularized Small Diameter Blood Vessels: A Preclinical Large Animal Study. Acta Biomater, 59:58–67.

Daum R, Visaser D, Wild C, et al., 2020, Fibronectin Adsorption on Electrospun Synthetic Vascular Grafts Attracts Endothelial Progenitor Cells and Promotes Endothelialization in Dynamic In Vitro Culture. Cells, 9:778.

Hossain KM, Zhu C, Felfel RM, et al., 2015, Tubular Scaffold with Shape Recovery Effect for Cell Guide Applications. J Funct Biomater, 6:564–84.

Alessandrino A, et al., 2019, Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to In Vivo Pilot Tests. Front Bioeng Biotechnol, 7:356.

Li MX, Li L, Zhou SY, et al., 2021, A Biomimetic Orthogonal bilayer Tubular Scaffold for the Co-culture of Endothelial Cells and Smooth Muscle Cells. RSC Adv, 11:31783–90.

Niu Y, Galluzzi M, 2021, Hyaluronic Acid/Collagen Nanofiber Tubular Scaffolds Support Endothelial Cell Proliferation, Phenotypic Shape and Endothelialization. Nanomaterials, 11:2334.

Niu Y, Galluzzi M, Fu M, et al., 2021, In Vivo Performance of Electrospun Tubular Hyaluronic Acid/Collagen Nanofibrous Scaffolds for Vascular Reconstruction in the Rabbit Model. J Nanobiotechnol, 19:349.

Hu Q, Su C, Zeng Z, et al., 2020, Fabrication of Multilayer Tubular Scaffolds with Aligned Nanofibers to Guide the Growth of Endothelial Cells. J Biomater Appl, 35:553–66.

Hong S, Kim JS, Jung B, et al., 2019, Coaxial Bioprinting of Cell-laden Vascular Constructs Using a Gelatin-Tyramine Bioink. Biomater Sci, 7:4578–87.

Wang X, Li X, Dai X, et al., 2018, Coaxial Extrusion Bioprinted Shell-core Hydrogel Microfibers Mimic Glioma Microenvironment and Enhance the Drug Resistance of Cancer Cells. Colloids Surf B Biointerfaces, 171:291–9.

Abkarian M, Viallat A, 2005, Dynamics of Vesicles in a Wall- Bounded Shear Flow. Biophys J, 89:1055–66.

Zhu C, Yago T, Lou J, et al., 2008, Mechanisms for Flow enhanced Cell Adhesion. Ann Biomed Eng, 36:604–21.

Park S, Joo YK, Chen Y, 2018, Dynamic Adhesion Characterization of Cancer Cells under Blood Flow-mimetic Conditions: Effects of Cell Shape and Orientation on Drag Force. Microfluid Nanofluid, 22:108.

Roux E, Bougaran P, Dufourcq P, et al., 2020, Fluid Shear Stress Sensing by the Endothelial Layer. Front Physiol, 11:861–1.

del Álamo JC, Norwich GN, Li YS, et al., 2008, Anisotropic Rheology and Directional Mechano transduction in Vascular Endothelial Cells. Proc Natl Acad Sci, 105:15411–6.

Krüger-Genge A, Blocki A, Franke RP, et al., 2019, Vascular

Endothelial Cell Biology: An Update. Int J Mol Sci, 20:4411.

Chistiakov DA, Orekhov AN, Bobryshev YV, 2017, Effects of Shear Stress on Endothelial Cells: Go with the Flow. Acta Physiologica, 219:382–408.

Malek AM, Izumo S, 1996, Mechanism of Endothelial Cell Shape Change and Cytoskeletal Remodeling in Response to Fluid Shear Stress. J Cell Sci, 109 Pt 4:713–26.

Ballermann BJ, Dardik A, Eng E, et al., 1998, Shear Stress and the Endothelium. Kidney Int, 54:S100–8.

Tsuji-Tamura K, Ogawa M, 2018, Morphology Regulation in Vascular Endothelial Cells. Inflamm Regen, 38:25.

Bai C, Hou L, Zhang M, et al., 2012, Characterization of Vascular Endothelial Progenitor Cells from Chicken Bone Marrow. BMC Vet Res, 8:54.

Fletcher DA, Mullins RD, 2010, Cell Mechanics and the Cytoskeleton. Nature, 463:485–92.

Inglebert M, Mullins RD, 2020, The Effect of Shear Stress Reduction on Endothelial Cells: A Microfluidic Study of the Actin Cytoskeleton. Biomicrofluidics, 14:024115.

Osborn EA, Rabodzey A, Dewey JC, et al., 2006, Endothelial Actin Cytoskeleton Remodeling during Mechano stimulation with Fluid Shear Stress. Am J Physiol Cell Physiol, 290:C444–52.

Pasini A, et al., 2021, Perfusion Flow Enhances Viability and Migratory Phenotype in 3D-Cultured Breast Cancer Cells. Ann Biomed Eng, 49:2103-13.

Cavey M, Lecuit T, 2009, Molecular Bases of Cell-cell Junctions Stability and Dynamics. Cold Spring Harb Perspect Biol, 1:a002998.

Coffman CR, 2003, Cell Migration and Programmed Cell Death of Drosophila Germ Cells. Ann N Y Acad Sci, 995:117–26.

Ye F, Song J, Wang Y, et al., 2018, Proliferation Potential-Related Protein Promotes the Esophageal Cancer Cell Proliferation, Migration and Suppresses Apoptosis by Mediating the Expression of p53 and Interleukin-17. Pathobiology, 85:322–31.

Gorelick-Ashkenazi A, Weiss R, Sapozhnikov L, et al., 2018, Caspases Maintain Tissue Integrity by an Apoptosis independent Inhibition of Cell Migration and Invasion. Nat Commun, 9:2806.

Sailon AM, Allori AC, Davidson EH, et al., 2009, A Novel Flow-Perfusion Bioreactor Supports 3D Dynamic Cell Culture. J Biomed Biotechnol, 2009:873816.

Ashton RS, Banerjee A, Punyani S, et al., 2007, Scaffolds Based on Degradable Alginate Hydrogels and Poly(Lactideco-glycolide) Microspheres for Stem Cell Culture. Biomaterials, 28:5518–25.

Melly L, Boccardo S, Eckstein F, et al., 2012, Cell and Gene Therapy Approaches for Cardiac Vascularization. Cells, 1:961–75.

Shin Y, Jeon JS, Han S, et al., 2011, In Vitro 3D Collective Sprouting Angiogenesis under Orchestrated ANG-1 and VEGF Gradients. Lab Chip, 11:2175–81.



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