Direct 3D bioprinting of perfusable vascular constructs using a blend bioink.

Despite the significant technological advancement in tissue engineering, challenges still exist towards the development of complex and fully functional tissue constructs that mimic their natural counterparts. To address these challenges, bioprinting has emerged as an enabling technology to create highly organized three-dimensional (3D) vascular networks within engineered tissue constructs to promote the transport of oxygen, nutrients, and waste products, which can hardly be realized using conventional microfabrication techniques. Here, we report the development of a versatile 3D bioprinting strategy that employs biomimetic biomaterials and an advanced extrusion system to deposit perfusable vascular structures with highly ordered arrangements in a single-step process. In particular, a specially designed cell-responsive bioink consisting of gelatin methacryloyl (GelMA), sodium alginate, and 4-arm poly(ethylene glycol)-tetra-acrylate (PEGTA) was used in combination with a multilayered coaxial extrusion system to achieve direct 3D bioprinting. This blend bioink could be first ionically crosslinked by calcium ions followed by covalent photocrosslinking of GelMA and PEGTA to form stable constructs. The rheological properties of the bioink and the mechanical strengths of the resulting constructs were tuned by the introduction of PEGTA, which facilitated the precise deposition of complex multilayered 3D perfusable hollow tubes. This blend bioink also displayed favorable biological characteristics that supported the spreading and proliferation of encapsulated endothelial and stem cells in the bioprinted constructs, leading to the formation of biologically relevant, highly organized, perfusable vessels. These characteristics make this novel 3D bioprinting technique superior to conventional microfabrication or sacrificial templating approaches for fabrication of the perfusable vasculature. We envision that our advanced bioprinting technology and bioink formulation may also have significant potentials in engineering large-scale vascularized tissue constructs towards applications in organ transplantation and repair.

[1]  Ivan Donati,et al.  Effect of Ca2+, Ba2+, and Sr2+ on alginate microbeads. , 2006, Biomacromolecules.

[2]  Wim E Hennink,et al.  25th Anniversary Article: Engineering Hydrogels for Biofabrication , 2013, Advanced materials.

[3]  Brendon M. Baker,et al.  Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues , 2012 .

[4]  Brendon M. Baker,et al.  Rapid casting of patterned vascular networks for perfusable engineered 3D tissues , 2012, Nature materials.

[5]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[6]  Noo Li Jeon,et al.  Diffusion limits of an in vitro thick prevascularized tissue. , 2005, Tissue engineering.

[7]  Ali Khademhosseini,et al.  Hydrogel Templates for Rapid Manufacturing of Bioactive Fibers and 3D Constructs , 2015, Advanced healthcare materials.

[8]  A. Khademhosseini,et al.  Cell-laden microengineered gelatin methacrylate hydrogels. , 2010, Biomaterials.

[9]  H. DeLisser,et al.  Platelet endothelial cell adhesion molecule (CD31). , 1993, Current topics in microbiology and immunology.

[10]  A. Hoffman.  Hydrogels for Biomedical Applications , 2001, Advanced drug delivery reviews.

[11]  A. Khademhosseini,et al.  Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold. , 2013, Acta biomaterialia.

[12]  A. Khademhosseini,et al.  Composite Living Fibers for Creating Tissue Constructs Using Textile Techniques , 2014, Advanced functional materials.

[13]  A. Khademhosseini,et al.  Micro fl uidic techniques for development of 3 D vascularized tissue , 2015 .

[14]  Anthony Atala,et al.  3D bioprinting of tissues and organs , 2014, Nature Biotechnology.

[15]  Liang Ma,et al.  Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery. , 2015, Biomaterials.

[16]  A. Khademhosseini,et al.  Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low‐Viscosity Bioink , 2016, Advanced materials.

[17]  S. Yoo,et al.  Creating perfused functional vascular channels using 3D bio-printing technology. , 2014, Biomaterials.

[18]  G. Owens,et al.  Transforming growth factor- (cid:1) 1 signaling contributes to development of smooth muscle cells from embryonic stem cells , 2022 .

[19]  B. Duan,et al.  3D bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels. , 2013, Journal of biomedical materials research. Part A.

[20]  M. Hosoya,et al.  Renal expression of alpha-smooth muscle actin and c-Met in children with Henoch–Schönlein purpura nephritis , 2008, Pediatric Nephrology.

[21]  P. Kasten,et al.  Culture media for the differentiation of mesenchymal stromal cells. , 2011, Acta biomaterialia.

[22]  F. Guilak,et al.  Advanced Material Strategies for Tissue Engineering Scaffolds , 2009, Advanced materials.

[23]  Ali Khademhosseini,et al.  Synthesis and characterization of tunable poly(ethylene glycol): gelatin methacrylate composite hydrogels. , 2011, Tissue engineering. Part A.

[24]  Ibrahim T. Ozbolat,et al.  In Vitro Study of Directly Bioprinted Perfusable Vasculature Conduits. , 2015, Biomaterials science.

[25]  H. Inoue,et al.  Vascular endothelial growth factor principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis. , 2003, Journal of biochemistry.

[26]  Alessandro Tocchio,et al.  Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor. , 2015, Biomaterials.

[27]  J. Jansen,et al.  Oppositely Charged Gelatin Nanospheres as Building Blocks for Injectable and Biodegradable Gels , 2011, Advanced materials.

[28]  Jason A. Burdick,et al.  Hyaluronic Acid Hydrogels for Biomedical Applications , 2011, Advanced materials.

[29]  Thomas Eschenhagen,et al.  Physiological aspects of cardiac tissue engineering. , 2012, American journal of physiology. Heart and circulatory physiology.

[30]  Jason P. Gleghorn,et al.  Microfluidic scaffolds for tissue engineering. , 2007, Nature materials.

[31]  Tal Dvir,et al.  Nanotechnological strategies for engineering complex tissues. , 2020, Nature nanotechnology.

[32]  Ruei-Zeng Lin,et al.  Bioengineering Human Microvascular Networks in Immunodeficient Mice , 2011, Journal of visualized experiments : JoVE.

[33]  Glenn D Prestwich,et al.  Bioprinting vessel-like constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetracrylates. , 2010, Biomaterials.

[34]  Shoji Takeuchi,et al.  Metre-long cell-laden microfibres exhibit tissue morphologies and functions. , 2013, Nature materials.

[35]  Ibrahim T. Ozbolat,et al.  Direct Bioprinting of Vessel-Like Tubular Microfluidic Channels , 2013 .

[36]  Ibrahim T. Ozbolat,et al.  Characterization of printable cellular micro-fluidic channels for tissue engineering , 2013, Biofabrication.

[37]  G. Owens,et al.  Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells. , 2004, American journal of physiology. Cell physiology.

[38]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[39]  Gulden Camci-Unal,et al.  Synthesis and characterization of hybrid hyaluronic acid-gelatin hydrogels. , 2013, Biomacromolecules.

[40]  Mary E Dickinson,et al.  Biomimetic hydrogels with pro-angiogenic properties. , 2010, Biomaterials.

[41]  Ali Khademhosseini,et al.  Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels , 2012, Advanced functional materials.

[42]  A. Khademhosseini,et al.  Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. , 2015, Biomaterials.

[43]  Matthias P. Lutolf,et al.  Designing materials to direct stem-cell fate , 2009, Nature.

[44]  Holger Gerhardt,et al.  Endothelial-pericyte interactions in angiogenesis , 2003, Cell and Tissue Research.

[45]  Liliang Ouyang,et al.  3D printing of HEK 293FT cell-laden hydrogel into macroporous constructs with high cell viability and normal biological functions , 2015, Biofabrication.

[46]  C. De Bari,et al.  The regulation of differentiation in mesenchymal stem cells. , 2010, Human gene therapy.

[47]  P. Wakeley,et al.  Synthesis , 2013, The Role of Animals in Emerging Viral Diseases.

[48]  Deok‐Ho Kim,et al.  Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink , 2014, Nature Communications.

[49]  Alexandra L. Rutz,et al.  A Multimaterial Bioink Method for 3D Printing Tunable, Cell‐Compatible Hydrogels , 2015, Advanced materials.

[50]  Ali Khademhosseini,et al.  Microengineered hydrogels for tissue engineering. , 2007, Biomaterials.

[51]  Ali Khademhosseini,et al.  Microfluidic techniques for development of 3D vascularized tissue. , 2014, Biomaterials.

[52]  A. Khademhosseini,et al.  Hydrogels in Regenerative Medicine , 2009, Advanced materials.

[53]  Milica Radisic,et al.  Medium perfusion enables engineering of compact and contractile cardiac tissue. , 2004, American journal of physiology. Heart and circulatory physiology.

[54]  A. Khademhosseini,et al.  Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs. , 2014, Lab on a chip.

[55]  Ali Khademhosseini,et al.  Advancing Tissue Engineering: A Tale of Nano-, Micro-, and Macroscale Integration. , 2016, Small.

[56]  Didier Letourneur,et al.  Concentrated collagen hydrogels as dermal substitutes. , 2010, Biomaterials.

[57]  L. Moreland.  Platelet-endothelial cell adhesion molecule-1 , 2004 .

[58]  L. Niklason,et al.  Scaffold-free vascular tissue engineering using bioprinting. , 2009, Biomaterials.

[59]  Holger Weber,et al.  Vascular endothelial growth factor (VEGF‐A) expression in human mesenchymal stem cells: Autocrine and paracrine role on osteoblastic and endothelial differentiation , 2005, Journal of cellular biochemistry.

[60]  S. Rizzi,et al.  Elucidating the role of matrix stiffness in 3D cell migration and remodeling. , 2011, Biophysical journal.

[61]  J. West,et al.  Vascularization of engineered tissues: approaches to promote angio-genesis in biomaterials. , 2008, Current topics in medicinal chemistry.

[62]  Tadashi Sasagawa,et al.  Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering. , 2010, Biomaterials.

[63]  Younan Xia,et al.  Multiple facets for extracellular matrix mimicking in regenerative medicine. , 2015, Nanomedicine.

[64]  James J. Yoo,et al.  A 3D bioprinting system to produce human-scale tissue constructs with structural integrity , 2016, Nature Biotechnology.

[65]  P. Bártolo,et al.  Additive manufacturing of tissues and organs , 2012 .

[66]  Feng Zhao,et al.  Increasing Mechanical Strength of Gelatin Hydrogels by Divalent Metal Ion Removal , 2014, Scientific Reports.

[67]  S. Hofmann,et al.  Controlled Positioning of Cells in Biomaterials—Approaches Towards 3D Tissue Printing , 2011, Journal of functional biomaterials.

[68]  A. Khademhosseini,et al.  Building Vascular Networks , 2012, Science Translational Medicine.

[69]  J. Lewis,et al.  3D Bioprinting of Vascularized, Heterogeneous Cell‐Laden Tissue Constructs , 2014, Advanced materials.

[70]  B. Geiger,et al.  Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells , 1994, The Journal of cell biology.

[71]  Ali Khademhosseini,et al.  Directed Differentiation of Size‐Controlled Embryoid Bodies Towards Endothelial and Cardiac Lineages in RGD‐Modified Poly(Ethylene Glycol) Hydrogels , 2013, Advanced healthcare materials.

[72]  Lisa E. Freed,et al.  Accordion-Like Honeycombs for Tissue Engineering of Cardiac Anisotropy , 2008, Nature materials.

[73]  Fan Yang,et al.  Effects of the poly(ethylene glycol) hydrogel crosslinking mechanism on protein release. , 2016, Biomaterials science.