Tuning Alginate Bioink Stiffness and Composition for Controlled Growth Factor Delivery and to Spatially Direct MSC Fate within Bioprinted Tissues

Alginate is a commonly used bioink in 3D bioprinting. Matrix stiffness is a key determinant of mesenchymal stem cell (MSC) differentiation, suggesting that modulation of alginate bioink mechanical properties represents a promising strategy to spatially regulate MSC fate within bioprinted tissues. In this study, we define a printability window for alginate of differing molecular weight (MW) by systematically varying the ratio of alginate to ionic crosslinker within the bioink. We demonstrate that the MW of such alginate bioinks, as well as the choice of ionic crosslinker, can be tuned to control the mechanical properties (Young’s Modulus, Degradation Rate) of 3D printed constructs. These same factors are also shown to influence growth factor release from the bioinks. We next explored if spatially modulating the stiffness of 3D bioprinted hydrogels could be used to direct MSC fate inside printed tissues. Using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, these spatially varying microenvironments were found to have a significant effect on the fate of MSCs within the alginate bioinks, with stiffer regions of the bioprinted construct preferentially supporting osteogenesis over adipogenesis.

[1]  W. Hennink,et al.  Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing. , 2007, Tissue engineering.

[2]  Ralph Müller,et al.  Tunable hydrogel composite with two-step processing in combination with innovative hardware upgrade for cell-based three-dimensional bioprinting. , 2014, Acta biomaterialia.

[3]  David J Mooney,et al.  The tensile properties of alginate hydrogels. , 2004, Biomaterials.

[4]  David J Mooney,et al.  Alginate hydrogels as biomaterials. , 2006, Macromolecular bioscience.

[5]  Fergal J O'Brien,et al.  Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells. , 2014, Acta biomaterialia.

[6]  和田 八三久.  Materials science. , 1973, Science.

[7]  Kenneth M. Yamada,et al.  Taking Cell-Matrix Adhesions to the Third Dimension , 2001, Science.

[8]  David J. Mooney,et al.  Harnessing Traction-Mediated Manipulation of the Cell-Matrix Interface to Control Stem Cell Fate , 2010, Nature materials.

[9]  Vladimir Mironov,et al.  Organ printing: computer-aided jet-based 3D tissue engineering. , 2003, Trends in biotechnology.

[10]  K. Draget,et al.  Alginic acid gels: the effect of alginate chemical composition and molecular weight , 1994 .

[11]  Anja Lode,et al.  Three‐dimensional plotting of a cell‐laden alginate/methylcellulose blend: towards biofabrication of tissue engineering constructs with clinically relevant dimensions , 2017, Journal of tissue engineering and regenerative medicine.

[12]  Jos Malda,et al.  Strategies for zonal cartilage repair using hydrogels. , 2009, Macromolecular bioscience.

[13]  Robert Langer,et al.  In vivo engineering of organs: the bone bioreactor. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D J Mooney,et al.  Release from alginate enhances the biological activity of vascular endothelial growth factor. , 1998, Journal of biomaterials science. Polymer edition.

[15]  D. Mooney,et al.  Alginate: properties and biomedical applications. , 2012, Progress in polymer science.

[16]  Eben Alsberg,et al.  FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[17]  P. Dubruel,et al.  The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. , 2014, Biomaterials.

[18]  Yujiang Fan,et al.  Adsorption and Release Behaviors of Vascular Endothelial Growth Factor on Porous Hydroxyapatite Ceramic Under Competitive Conditions , 2014 .

[19]  F. O'Brien,et al.  Chondrogenically primed mesenchymal stem cell-seeded alginate hydrogels promote early bone formation in critically-sized defects , 2015 .

[20]  Wouter J A Dhert,et al.  Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture. , 2014, Journal of controlled release : official journal of the Controlled Release Society.

[21]  Eben Alsberg,et al.  Engineering growing tissues , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  F. Guilak,et al.  Control of stem cell fate by physical interactions with the extracellular matrix. , 2009, Cell stem cell.

[23]  D. Hutmacher,et al.  Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects. , 2011, Bone.

[24]  David J Mooney,et al.  Effects of VEGF temporal and spatial presentation on angiogenesis. , 2010, Biomaterials.

[25]  GeunHyung Kim,et al.  Cells (MC3T3-E1)-laden alginate scaffolds fabricated by a modified solid-freeform fabrication process supplemented with an aerosol spraying. , 2012, Biomacromolecules.

[26]  M. Dembo,et al.  Cell movement is guided by the rigidity of the substrate. , 2000, Biophysical journal.

[27]  David J Mooney,et al.  Quantitative assessment of scaffold and growth factor‐mediated repair of critically sized bone defects , 2007, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[28]  Jos Malda,et al.  Biofabrication of osteochondral tissue equivalents by printing topologically defined, cell-laden hydrogel scaffolds. , 2012, Tissue engineering. Part C, Methods.

[29]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[30]  Werner E. G. Müller,et al.  Effect of Bioglass on Growth and Biomineralization of SaOS-2 Cells in Hydrogel after 3D Cell Bioprinting , 2014, PloS one.

[31]  Eben Alsberg,et al.  Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells. , 2004, Bone.

[32]  David J Mooney,et al.  An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects. , 2011, Biomaterials.

[33]  Adam J. Engler,et al.  Myotubes differentiate optimally on substrates with tissue-like stiffness , 2004, The Journal of cell biology.

[34]  Eben Alsberg,et al.  Calcium phosphate-DNA nanoparticle gene delivery from alginate hydrogels induces in vivo osteogenesis. , 2009, Journal of biomedical materials research. Part A.

[35]  Melinda Larsen,et al.  Extracellular matrix dynamics in development and regenerative medicine , 2008, Journal of Cell Science.

[36]  Luca Gasperini,et al.  Natural polymers for the microencapsulation of cells , 2014, Journal of The Royal Society Interface.

[37]  D J Mooney,et al.  Regulating Bone Formation via Controlled Scaffold Degradation , 2003, Journal of dental research.

[38]  D. Mooney,et al.  Controlling rigidity and degradation of alginate hydrogels via molecular weight distribution. , 2004, Biomacromolecules.

[39]  P. Ma,et al.  Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties. , 2001, Biomaterials.

[40]  D. Kelly,et al.  A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage , 2016, Biofabrication.

[41]  Andrés J. García,et al.  Effects of protein dose and delivery system on BMP-mediated bone regeneration. , 2011, Biomaterials.

[42]  Dinorath Olvera,et al.  Mechanical Testing of Cartilage Constructs. , 2015, Methods in molecular biology.

[43]  Wenmiao Shu,et al.  Three-dimensional bioprinting of complex cell laden alginate hydrogel structures , 2015, Biofabrication.

[44]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

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

[46]  P. R. van Weeren,et al.  Hyaluronic acid and dextran-based semi-IPN hydrogels as biomaterials for bioprinting. , 2011, Biomacromolecules.

[47]  Harrie Weinans,et al.  Sustained Release of BMP-2 in Bioprinted Alginate for Osteogenicity in Mice and Rats , 2013, PloS one.

[48]  David J Mooney,et al.  Mechanical confinement regulates cartilage matrix formation by chondrocytes , 2017, Nature materials.

[49]  D. E. Discher,et al.  Matrix elasticity directs stem cell lineage — Soluble factors that limit osteogenesis , 2009 .

[50]  H. Hakimzadeh,et al.  Part 1 , 2011 .

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

[52]  P. Janmey,et al.  Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. , 2009, Tissue engineering. Part A.

[53]  N. Balaban,et al.  Adhesion-dependent cell mechanosensitivity. , 2003, Annual review of cell and developmental biology.

[54]  Hod Lipson,et al.  Direct Freeform Fabrication of Seeded Hydrogels in Arbitrary Geometries , 2022 .

[55]  Michele Marcolongo,et al.  Characterization of cell viability during bioprinting processes. , 2009, Biotechnology journal.

[56]  D. Mooney,et al.  Designing alginate hydrogels to maintain viability of immobilized cells. , 2003, Biomaterials.

[57]  T Ahlfeld,et al.  Development of a clay based bioink for 3D cell printing for skeletal application , 2017, Biofabrication.

[58]  David J Mooney,et al.  Comparison of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in SCID mice. , 2003, Journal of controlled release : official journal of the Controlled Release Society.

[59]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[60]  David J Mooney,et al.  Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. , 2005, Biomaterials.