Effects of the mechanical properties of collagen gel on the in vitro formation of microvessel networks by endothelial cells.

Vascularization by endothelial cells (ECs) is an essential element in tissue-engineering of organoids. Morphogenesis of these cells is regulated not only by the biochemical properties of the extracellular matrix (ECM) but also by its mechanical properties. Here, we investigated the effect of substrate mechanical properties on the formation of capillary-like networks by ECs; in particular, we examined the three-dimensional (3D) configurations of the resulting networks. Bovine pulmonary microvascular ECs (BPMECs) were cultured on a series of collagen gels of different stiffness but the same collagen concentration. Imaging techniques revealed that cells cultured in rigid and flexible gels formed 3D networks via different processes; cells formed dense, thin networks in the flexible gel, whereas thicker and deeper networks were formed in the rigid gel. Cross-sections of the networks revealed that those formed within the rigid gel had large lumens composed of multiple cells, whereas those formed within the flexible gel had small, intracellular vacuoles. The expression of vinculin, a focal adhesion protein, appeared to change with the mechanical properties of collagen gel. Our results indicate that the mechanical properties of adhesion substrates play an important role in regulating 3D network formation.

[1]  D E Ingber,et al.  Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer. , 2000, Biochemical and biophysical research communications.

[2]  K. Todoki,et al.  Co-treatment with basic fibroblast growth factor and 17beta-estradiol in the presence of dexamethasone accelerates bone formation by rat bone marrow stromal cell culture. , 2008, Nihon Hotetsu Shika Gakkai zasshi.

[3]  L. Griffith,et al.  Tissue Engineering--Current Challenges and Expanding Opportunities , 2002, Science.

[4]  L Orci,et al.  In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices , 1983, The Journal of cell biology.

[5]  C M Lapiere,et al.  In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. , 2001, Cardiovascular research.

[6]  Benjamin Geiger,et al.  Exploring the Neighborhood Adhesion-Coupled Cell Mechanosensors , 2002, Cell.

[7]  Dai Fukumura,et al.  Engineering vascularized tissue , 2005, Nature Biotechnology.

[8]  E. Sage,et al.  Between molecules and morphology. Extracellular matrix and creation of vascular form. , 1995, The American journal of pathology.

[9]  Kazuo Tanishita,et al.  Reconstruction of 3D stacked‐up structures by rat small hepatocytes on microporous membranes , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

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

[11]  F. Rosso,et al.  From Cell–ECM interactions to tissue engineering , 2004, Journal of cellular physiology.

[12]  Donald E Ingber,et al.  Intact vinculin protein is required for control of cell shape, cell mechanics, and rac-dependent lamellipodia formation. , 2002, Biochemical and biophysical research communications.

[13]  R. Wells.  The role of matrix stiffness in regulating cell behavior , 2008, Hepatology.

[14]  J. K. Leach,et al.  Proangiogenic Potential of a Collagen/Bioactive Glass Substrate , 2008, Pharmaceutical Research.

[15]  N. Kawazoe,et al.  Collagen-based scaffolds , 2008 .

[16]  Mitsuo Umezu,et al.  Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces , 2002, Circulation research.

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

[18]  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.

[19]  Shinsuke Yuasa,et al.  A new method for manufacturing cardiac cell sheets using fibrin-coated dishes and its electrophysiological studies by optical mapping. , 2005, Artificial organs.

[20]  J. Hubbell,et al.  Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering , 2005, Nature Biotechnology.

[21]  K J Gooch,et al.  The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro. , 2004, Experimental cell research.

[22]  L. Orci,et al.  Basic fibroblast growth factor induces angiogenesis in vitro. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Chau-Zen Wang,et al.  Rigidity of Collagen Fibrils Controls Collagen Gel-induced Down-regulation of Focal Adhesion Complex Proteins Mediated by α2β1 Integrin* , 2003, Journal of Biological Chemistry.

[24]  M. Dembo,et al.  Substrate flexibility regulates growth and apoptosis of normal but not transformed cells. , 2000, American journal of physiology. Cell physiology.

[25]  D. Kohane,et al.  Engineering vascularized skeletal muscle tissue , 2005, Nature Biotechnology.

[26]  Donald E Ingber,et al.  Cell tension, matrix mechanics, and cancer development. , 2005, Cancer cell.

[27]  M. Iruela-Arispe,et al.  Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. , 1992, Laboratory investigation; a journal of technical methods and pathology.

[28]  F H Silver,et al.  Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties. , 2000, Matrix biology : journal of the International Society for Matrix Biology.

[29]  G. Breier,et al.  The role of vascular endothelial growth factor in blood vessel formation. , 1996, Trends in cell biology.

[30]  K. Tanishita,et al.  Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model. , 2004, American journal of physiology. Heart and circulatory physiology.

[31]  M. Sheetz,et al.  Forces on adhesive contacts affect cell function. , 1998, Current opinion in cell biology.

[32]  Michael P. Sheetz,et al.  The relationship between force and focal complex development , 2002, The Journal of cell biology.

[33]  D. Ingber.  Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. , 2002, Circulation research.

[34]  E. Sage,et al.  A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices. , 1999, Microvascular research.

[35]  J. Groopman,et al.  Extracellular matrix regulates endothelial functions through interaction of VEGFR‐3 and integrin α5β1 , 2005 .