Abstract
Proper vascularization remains critical to the clinical application of engineered tissues. To engineer microvessels in vitro, we and others have delivered endothelial cells through preformed channels into patterned extracellular matrix-based gels. This approach has been limited by the size of endothelial cells in suspension, and results in plugging of channels below ~30 µm in diameter. Here, we examine physical and chemical signals that can augment direct seeding, with the aim of rapidly vascularizing capillary-scale channels. By studying tapered microchannels in type I collagen gels under various conditions, we establish that stiff scaffolds, forward pressure, and elevated cyclic AMP levels promote endothelial stability and that reverse pressure promotes endothelial migration. We applied these results to uniform 20-µm-diameter channels and optimized the magnitudes of pressure, flow, and shear stress to best support endothelial migration and vascular stability. This vascularization strategy is able to form millimeter-long perfusable capillaries within 3 days. Our results indicate how to manipulate the physical and chemical environment to promote rapid vascularization of capillary-scale channels within type I collagen gels.
Similar content being viewed by others
References
Auger, F. A., L. Gibot, and D. Lacroix. The pivotal role of vascularization in tissue engineering. Annu. Rev. Biomed. Eng. 15:177–200, 2013.
Badylak, S. F., D. Taylor, and K. Uygun. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu. Rev. Biomed. Eng. 13:27–53, 2011.
Bogorad, M. I., J. DeStefano, J. Karlsson, A. D. Wong, S. Gerecht, and P. C. Searson. In vitro microvessel models. Lab Chip 15:4242–4255, 2015.
Califano, J. P., and C. A. Reinhart-King. Exogenous and endogenous force regulation of endothelial cell behavior. J. Biomech. 43:79–86, 2010.
Chan, K. L. S., A. H. Khankhel, R. L. Thompson, B. J. Coisman, K. H. K. Wong, J. G. Truslow, and J. Tien. Crosslinking of collagen scaffolds promotes blood and lymphatic vascular stability. J. Biomed. Mater. Res. A. 102:3186–3195, 2014.
Chan, J. M., I. K. Zervantonakis, T. Rimchala, W. J. Polacheck, J. Whisler, and R. D. Kamm. Engineering of in vitro 3D capillary beds by self-directed angiogenic sprouting. PLoS ONE 7:e50582, 2012.
Chrobak, K. M., D. R. Potter, and J. Tien. Formation of perfused, functional microvascular tubes in vitro. Microvasc. Res. 71:185–196, 2006.
Dewey, C. F., S. R. Bussolari, M. A. Gimbrone, and P. F. Davies. The dynamic response of vascular endothelial cells to fluid shear stress. J. Biomech. Eng. 103:177–185, 1981.
Galie, P. A., D. H. Nguyen, C. K. Choi, D. M. Cohen, P. A. Janmey, and C. S. Chen. Fluid shear stress threshold regulates angiogenic sprouting. Proc. Natl. Acad. Sci. USA 111:7968–7973, 2014.
Hsu, P. P., S. Li, Y. S. Li, S. Usami, A. Ratcliffe, X. Wang, and S. Chien. Effects of flow patterns on endothelial cell migration into a zone of mechanical denudation. Biochem. Biophys. Res. Commun. 285:751–759, 2001.
Kim, S., H. Lee, M. Chung, and N. L. Jeon. Engineering of functional, perfusable 3D microvascular networks on a chip. Lab Chip 13:1489–1500, 2013.
Kiosses, W. B., N. H. McKee, and V. L. Kalnins. Evidence for the migration of aortic endothelial cells towards the heart. Arterioscler. Thromb. Vasc. Biol. 2891:2891–2896, 1997.
Koike, N., D. Fukumura, O. Gralla, P. Au, J. S. Schechner, and R. K. Jain. Creation of long-lasting blood vessels. Nature 428:138–139, 2004.
Leung, A. D., K. H. K. Wong, and J. Tien. Plasma expanders stabilize human microvessels in microfluidic scaffolds. J. Biomed. Mater. Res. A. 100:1815–1822, 2012.
Levenberg, S., J. Rouwkema, M. Macdonald, E. S. Garfein, D. S. Kohane, D. C. Darland, R. Marini, C. A. van Blitterswijk, R. C. Mulligan, P. A. D’Amore, and R. Langer. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol. 23:879–884, 2005.
Lovett, M., K. Lee, A. Edwards, and D. L. Kaplan. Vascularization strategies for tissue engineering. Tissue Eng. B. 15:353–370, 2009.
Mancuso, M. R., R. Davis, S. M. Norberg, S. O’Brien, B. Sennino, T. Nakahara, V. J. Yao, T. Inai, P. Brooks, B. Freimark, D. R. Shalinsky, D. D. Hu-Lowe, and D. M. McDonald. Rapid vascular regrowth in tumors after reversal of VEGF inhibition. J. Clin. Invest. 116:2610–2621, 2006.
Morgan, J. P., P. F. Delnero, Y. Zheng, S. S. Verbridge, J. Chen, M. Craven, N. W. Choi, A. Diaz-Santana, P. Kermani, B. Hempstead, J. A. Lopez, T. N. Corso, C. Fischbach, and A. D. Stroock. Formation of microvascular networks in vitro. Nat. Protoc. 8:1820–1836, 2013.
Morin, K. T., A. O. Smith, G. E. Davis, and R. T. Tranquillo. Aligned human microvessels formed in 3D fibrin gel by constraint of gel contraction. Microvasc. Res. 90:12–22, 2013.
Moya, M. L., Y. H. Hsu, A. P. Lee, C. C. Hughes, and S. C. George. In vitro perfused human capillary networks. Tissue Eng. C. 19:730–737, 2013.
Nichol, J. W., S. T. Koshy, H. Bae, C. M. Hwang, S. Yamanlar, and A. Khademhosseini. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 31:5536–5544, 2010.
Ostrowski, M. A., N. F. Huang, T. W. Walker, T. Verwijlen, C. Poplawski, A. S. Khoo, J. P. Cooke, G. G. Fuller, and A. R. Dunn. Microvascular endothelial cells migrate upstream and align against the shear stress field created by impinging flow. Biophys. J. 106:366–374, 2014.
Ott, H. C., B. Clippinger, C. Conrad, C. Schuetz, I. Pomerantseva, L. Ikonomou, D. Kotton, and J. P. Vacanti. Regeneration and orthotopic transplantation of a bioartificial lung. Nat. Med. 16:927–933, 2010.
Petersen, T. H., E. A. Calle, L. Zhao, E. J. Lee, L. Gui, M. B. Raredon, K. Gavrilov, T. Yi, Z. W. Zhuang, C. Breuer, E. Herzog, and L. E. Niklason. Tissue-engineered lungs for in vivo implantation. Science 329:538–541, 2010.
Pettersson, A., J. A. Nagy, L. F. Brown, C. Sundberg, E. Morgan, S. Jungles, R. Carter, J. E. Krieger, E. J. Manseau, V. S. Harvey, I. A. Eckelhoefer, D. Feng, A. M. Dvorak, R. C. Mulligan, and H. F. Dvorak. Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab. Invest. 80:99–115, 2000.
Price, G. M., K. M. Chrobak, and J. Tien. Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes. Microvasc. Res. 76:46–51, 2008.
Price, G. M., K. H. K. Wong, J. G. Truslow, A. D. Leung, C. Acharya, and J. Tien. Effect of mechanical factors on the function of engineered human blood microvessels in microfluidic collagen gels. Biomaterials 31:6182–6189, 2010.
Ren, X., P. T. Moser, S. E. Gilpin, T. Okamoto, T. Wu, L. F. Tapias, F. E. Mercier, L. Xiong, R. Ghawi, D. T. Scadden, D. J. Mathisen, and H. C. Ott. Engineering pulmonary vasculature in decellularized rat and human lungs. Nat. Biotechnol. 33:1097–1102, 2015.
Scarritt, M. E., N. C. Pashos, and B. A. Bunnell. A review of cellularization strategies for tissue engineering of whole organs. Front. Bioeng. Biotechnol. 3:43, 2015.
Tang, M. D., A. P. Golden, and J. Tien. Molding of three-dimensional microstructures of gels. J. Am. Chem. Soc. 125:12988–12989, 2003.
Teichmann, J., A. Morgenstern, J. Seebach, H. J. Schnittler, C. Werner, and T. Pompe. The control of endothelial cell adhesion and migration by shear stress and matrix-substrate anchorage. Biomaterials 33:1959–1969, 2012.
Truslow, J. G., G. M. Price, and J. Tien. Computational design of drainage systems for vascularized scaffolds. Biomaterials 30:4435–4443, 2009.
Vickerman, V., and R. D. Kamm. Mechanism of a flow-gated angiogenesis switch: early signaling events at cell–matrix and cell–cell junctions. Integr. Biol. 4:863–874, 2012.
Whisler, J. A., M. B. Chen, and R. D. Kamm. Control of perfusable microvascular network morphology using a multiculture microfluidic system. Tissue Eng. C. 20:543–552, 2014.
Wong, K. H. K., J. M. Chan, R. D. Kamm, and J. Tien. Microfluidic models of vascular functions. Annu. Rev. Biomed. Eng. 14:205–230, 2012.
Wong, K. H. K., J. G. Truslow, A. H. Khankhel, K. L. S. Chan, and J. Tien. Artificial lymphatic drainage systems for vascularized microfluidic scaffolds. J. Biomed. Mater. Res. A. 101:2181–2190, 2013.
Wong, K. H. K., J. G. Truslow, A. H. Khankhel, and J. Tien. Biophysical mechanisms that govern the vascularization of microfluidic scaffolds. In: vascularization, edited by E. M. Brey. Boca Raton: CRC Press, 2014, pp. 109–124.
Wong, K. H. K., J. G. Truslow, and J. Tien. The role of cyclic AMP in normalizing the function of engineered human blood microvessels in microfluidic collagen gels. Biomaterials 31:4706–4714, 2010.
Yevick, H. G., G. Duclos, I. Bonnet, and P. Silberzan. Architecture and migration of an epithelium on a cylindrical wire. Proc. Natl. Acad. Sci. USA 112:5944–5949, 2015.
Zheng, Y., J. Chen, M. Craven, N. W. Choi, S. Totorica, A. Diaz-Santana, P. Kermani, B. Hempstead, C. Fischbach-Teschl, J. A. Lopez, and A. D. Stroock. In vitro microvessels for the study of angiogenesis and thrombosis. Proc. Natl. Acad. Sci. USA 109:9342–9347, 2012.
Acknowledgments
We thank Cliff Brangwynne and Marina Feric for access to their pipette puller, and Aimal Khankhel for assistance with experiments. This work was supported by Boston University through a Dean’s Catalyst Award (J.T.), a Lutchen Fellowship (R.M.L.), and awards from the Undergraduate Research Opportunities Program (R.M.L., N.F.B., G.C.). R.M.L. thanks Mr. and Mrs. William Felder for support through a Summer Term Alumni Research Scholarship at Boston University.
Conflicts of interest
Raleigh M. Linville, Nelson F. Boland, Gil Covarrubias, Gavrielle M. Price, and Joe Tien declare that they have no conflict of interest.
Ethical Standards
No human or animal studies were carried out by the authors for this article.
Author information
Authors and Affiliations
Corresponding author
Additional information
Associate Editor Michael R. King oversaw the review of this article.
Rights and permissions
About this article
Cite this article
Linville, R.M., Boland, N.F., Covarrubias, G. et al. Physical and Chemical Signals That Promote Vascularization of Capillary-Scale Channels. Cel. Mol. Bioeng. 9, 73–84 (2016). https://doi.org/10.1007/s12195-016-0429-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12195-016-0429-8