Skip to main content

Alginate-Chitosan Hydrogels Provide a Sustained Gradient of Sphingosine-1-Phosphate for Therapeutic Angiogenesis

Abstract

Sphingosine-1-phosphate (S1P), a bioactive lipid, is a potent candidate for treatment of ischemic vascular disease. However, designing biomaterial systems for the controlled release of S1P to achieve therapeutic angiogenesis presents both biological and engineering challenges. Thus, the objective of this study was to design a hydrogel system that provides controlled and sustained release of S1P to establish local concentration gradients that promote neovascularization. Alginate hydrogels have been extensively studied and characterized for delivery of proangiogenic factors. We sought to explore if chitosan (0, 0.1, 0.5, or 1%) incorporation could be used as a means to control S1P release from alginate hydrogels. With increasing chitosan incorporation, hydrogels exhibited significantly denser pore structure and stiffer material properties. While 0.1 and 0.5% chitosan gels demonstrated slower respective release of S1P, release from 1% chitosan gels was similar to alginate gels alone. Furthermore, 0.5% chitosan gels induced greater sprouting and directed migration of outgrowth endothelial cells (OECs) in response to released S1P under hypoxia in vitro. Overall, this report presents a platform for a novel alginate-chitosan hydrogel of controlled composition and in situ gelation properties that can be used to control lipid release for therapeutic applications.

This is a preview of subscription content, access via your institution.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

References

  1. 1.

    Anderson, E. M., and D. J. Mooney. The combination of vascular endothelial growth factor and stromal cell-derived factor induces superior angiogenic sprouting by outgrowth endothelial cells. J. Vasc. Res. 52:62–69, 2015.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Baysal, K., A. Z. Aroguz, Z. Adiguzel, and B. M. Baysal. Chitosan/alginate crosslinked hydrogels: Preparation, characterization and application for cell growth purposes. Int. J. Biol. Macromol. 59:342–348, 2013.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Bencherif, S. A., R. Warren Sands, O. A. Ali, W. A. Li, S. A. Lewin, T. M. Braschler, T. Y. Shih, C. S. Verbeke, D. Bhatta, G. Dranoff, and D. J. Mooney. Injectable cryogel-based whole-cell cancer vaccines. Nat. Commun. 6:7556, 2015.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Bidarra, S. J., C. C. Barrias, K. B. Fonseca, M. A. Barbosa, R. A. Soares, and P. L. Granja. Injectable in situ crosslinkable rgd-modified alginate matrix for endothelial cells delivery. Biomaterials 32:7897–7904, 2011.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Binder, B., P. Williams, E. Silva, and J. K. Leach. Lysophosphatidic acid and sphingosine-1-phosphate: A concise review of biological function and applications for tissue engineering. Tissue Eng. Part B Rev. 21:531–542, 2015.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Boontheekul, T., H. J. Kong, and D. J. Mooney. Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. Biomaterials 26:2455–2465, 2005.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Bronckaers, A., P. Hilkens, Y. Fanton, T. Struys, P. Gervois, C. Politis, W. Martens, and I. Lambrichts. Angiogenic properties of human dental pulp stem cells. PLoS One 8:e71104, 2013.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Das, R. K., N. Kasoju, and U. Bora. Encapsulation of curcumin in alginate-chitosan-pluronic composite nanoparticles for delivery to cancer cells. Nanomedicine 6:153–160, 2010.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Gaserod, O., O. Smidsrod, and G. Skjak-Braek. Microcapsules of alginate-chitosan—I. A quantitative study of the interaction between alginate and chitosan. Biomaterials 19:1815–1825, 1998.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Hao, X., E. A. Silva, A. Mansson-Broberg, K. H. Grinnemo, A. J. Siddiqui, G. Dellgren, E. Wardell, L. A. Brodin, D. J. Mooney, and C. Sylven. Angiogenic effects of sequential release of vegf-a165 and pdgf-bb with alginate hydrogels after myocardial infarction. Cardiovasc. Res. 75:178–185, 2007.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Huguet, M. L., and E. Dellacherie. Calcium alginate beads coated with chitosan: Effect of the structure of encapsulated materials on their release. Process Biochem. 31:745–751, 1996.

    CAS  Article  Google Scholar 

  12. 12.

    Ingram, D. A., N. M. Caplice, and M. C. Yoder. Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells. Blood. 106:1525–1531, 2005.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Ingram, D. A., L. E. Mead, H. Tanaka, V. Meade, A. Fenoglio, K. Mortell, K. Pollok, M. J. Ferkowicz, D. Gilley, and M. C. Yoder. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood. 104:2752–2760, 2004.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Khong, T. T., O. A. Aarstad, G. Skjak-Braek, K. I. Draget, and K. M. Varum. Gelling concept combining chitosan and alginate-proof of principle. Biomacromolecules. 14:2765–2771, 2013.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Kong, H. J., E. Wong, and D. J. Mooney. Independent control of rigidity and toughness of polymeric hydrogels. Macromolecules. 36:4582–4588, 2003.

    CAS  Article  Google Scholar 

  16. 16.

    Lai, H. L., A. Abu’Khalil, and D. Q. Craig. The preparation and characterisation of drug-loaded alginate and chitosan sponges. Int. J. Pharm. 251:175–181, 2003.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Lee, B. H., B. Li, and S. A. Guelcher. Gel microstructure regulates proliferation and differentiation of mc3t3-e1 cells encapsulated in alginate beads. Acta Biomater. 8:1693–1702, 2012.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Lee, K. Y., and D. J. Mooney. Alginate: Properties and biomedical applications. Prog. Polym. Sci. 37:106–126, 2012.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Liu, J., A. Hsu, J. F. Lee, D. E. Cramer, and M. J. Lee. To stay or to leave: Stem cells and progenitor cells navigating the s1p gradient. World J. Biol. Chem. 2:1–13, 2011.

    Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Madan, M., A. Bajaj, S. Lewis, N. Udupa, and J. A. Baig. In situ forming polymeric drug delivery systems. Indian J. Pharm. Sci. 71:242–251, 2009.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Maghami, G. G., and G. A. F. Roberts. Studies on the adsorption of anionic dyes on chitosan. Macromol. Chem. Phys. 189:2239–2243, 1988.

    CAS  Article  Google Scholar 

  22. 22.

    Maia, F. R., K. B. Fonseca, G. Rodrigues, P. L. Granja, and C. C. Barrias. Matrix-driven formation of mesenchymal stem cell-extracellular matrix microtissues on soft alginate hydrogels. Acta Biomater. 10:3197–3208, 2014.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Meng, X., F. Tian, J. Yang, C. N. He, N. Xing, and F. Li. Chitosan and alginate polyelectrolyte complex membranes and their properties for wound dressing application. J. Mater. Sci. Mater. Med. 21:1751–1759, 2010.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Mi, F.-L., H.-W. Sung, and S.-S. Shyu. Drug release from chitosan–alginate complex beads reinforced by a naturally occurring cross-linking agent. Carbohydr. Polym. 48:61–72, 2002.

    CAS  Article  Google Scholar 

  25. 25.

    Nakatsu, M. N., R. C. Sainson, J. N. Aoto, K. L. Taylor, M. Aitkenhead, S. Perez-del-Pulgar, P. M. Carpenter, and C. C. Hughes. Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (huvec) in fibrin gels: The role of fibroblasts and angiopoietin-1. Microvasc. Res. 66:102–112, 2003.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Naor, M. M., M. D. Walker, J. R. Van Brocklyn, G. Tigyi, and A. L. Parrill. Sphingosine 1-phosphate pka and binding constants: Intramolecular and intermolecular influences. J. Mol. Graph. Model. 26:519–528, 2007.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Neves, S. C., D. B. Gomes, A. Sousa, S. J. Bidarra, P. Petrini, L. Moroni, C. C. Barrias, and P. L. Granja. Biofunctionalized pectin hydrogels as 3d cellular microenvironments. J. Mater. Chem. B. 3:2096–2108, 2015.

    CAS  Article  Google Scholar 

  28. 28.

    Ogle, M. E., L. S. Sefcik, A. O. Awojoodu, N. F. Chiappa, K. Lynch, S. Peirce-Cottler, and E. A. Botchwey. Engineering in vivo gradients of sphingosine-1-phosphate receptor ligands for localized microvascular remodeling and inflammatory cell positioning. Acta Biomater. 10:4704–4714, 2014.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Oyama, O., N. Sugimoto, X. Qi, N. Takuwa, K. Mizugishi, J. Koizumi, and Y. Takuwa. The lysophospholipid mediator sphingosine-1-phosphate promotes angiogenesis in vivo in ischaemic hindlimbs of mice. Cardiovasc. Res. 78:301–307, 2008.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Peppas, N. A., J. Z. Hilt, A. Khademhosseini, and R. Langer. Hydrogels in biology and medicine: From molecular principles to bionanotechnology. Adv. Mater. 18:1345–1360, 2006.

    CAS  Article  Google Scholar 

  31. 31.

    Qi, X., Y. Okamoto, T. Murakawa, F. Wang, O. Oyama, R. Ohkawa, K. Yoshioka, W. Du, N. Sugimoto, Y. Yatomi, N. Takuwa, and Y. Takuwa. Sustained delivery of sphingosine-1-phosphate using poly(lactic-co-glycolic acid)-based microparticles stimulates akt/erk-enos mediated angiogenesis and vascular maturation restoring blood flow in ischemic limbs of mice. Eur. J. Pharmacol. 634:121–131, 2010.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Sefcik, L. S., C. E. Petrie Aronin, K. A. Wieghaus, and E. A. Botchwey. Sustained release of sphingosine 1-phosphate for therapeutic arteriogenesis and bone tissue engineering. Biomaterials. 29:2869–2877, 2008.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Sezer, A. D., and J. Akbuga. Release characteristics of chitosan treated alginate beads: II. Sustained release of a low molecular drug from chitosan treated alginate beads. J. Microencapsul. 16:687–696, 1999.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Sibaja, B., E. Culbertson, P. Marshall, R. Boy, R. M. Broughton, A. A. Solano, M. Esquivel, J. Parker, L. De La Fuente, and M. L. Auad. Preparation of alginate-chitosan fibers with potential biomedical applications. Carbohydr. Polym. 134:598–608, 2015.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Silva, E. A., E. S. Kim, H. J. Kong, and D. J. Mooney. Material-based deployment enhances efficacy of endothelial progenitor cells. Proc. Natl. Acad. Sci. USA 105:14347–14352, 2008.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Silva, E. A., and D. J. Mooney. Spatiotemporal control of vascular endothelial growth factor delivery from injectable hydrogels enhances angiogenesis. J. Thromb. Haemost. 5:590–598, 2007.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Silva, E. A., and D. J. Mooney. Effects of vegf temporal and spatial presentation on angiogenesis. Biomaterials 31:1235–1241, 2010.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Silva, C. M., A. J. Ribeiro, M. Figueiredo, D. Ferreira, and F. Veiga. Microencapsulation of hemoglobin in chitosan-coated alginate microspheres prepared by emulsification/internal gelation. AAPS J. 7:E903–E913, 2005.

    CAS  Article  Google Scholar 

  39. 39.

    Singh, S., B. M. Wu, and J. C. Dunn. Delivery of vegf using collagen-coated polycaprolactone scaffolds stimulates angiogenesis. J. Biomed. Mater. Res. A 100:720–727, 2012.

    Article  PubMed  Google Scholar 

  40. 40.

    Staton, C. A., M. W. Reed, and N. J. Brown. A critical analysis of current in vitro and in vivo angiogenesis assays. Int. J. Exp. Pathol. 90:195–221, 2009.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Tengood, J. E., K. M. Kovach, P. E. Vescovi, A. J. Russell, and S. R. Little. Sequential delivery of vascular endothelial growth factor and sphingosine 1-phosphate for angiogenesis. Biomaterials 31:7805–7812, 2010.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Thomas, A. M., and L. D. Shea. Polysaccharide-modified scaffolds for controlled lentivirus delivery in vitro and after spinal cord injury. J. Control Release. 170:421–429, 2013.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Venkatesan, J., I. Bhatnagar, and S. K. Kim. Chitosan-alginate biocomposite containing fucoidan for bone tissue engineering. Mar. Drugs. 12:300–316, 2014.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Wacker, B. K., E. A. Scott, M. M. Kaneda, S. K. Alford, and D. L. Elbert. Delivery of sphingosine 1-phosphate from poly(ethylene glycol) hydrogels. Biomacromolecules. 7:1335–1343, 2006.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Walter, D. H., U. Rochwalsky, J. Reinhold, F. Seeger, A. Aicher, C. Urbich, I. Spyridopoulos, J. Chun, V. Brinkmann, P. Keul, B. Levkau, A. M. Zeiher, S. Dimmeler, and J. Haendeler. Sphingosine-1-phosphate stimulates the functional capacity of progenitor cells by activation of the cxcr4-dependent signaling pathway via the s1p3 receptor. Arterioscler. Thromb. Vasc. Biol. 27:275–282, 2007.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Williams, P. A., and E. A. Silva. The role of synthetic extracellular matrices in endothelial progenitor cell homing for treatment of vascular disease. Ann. Biomed. Eng. 43(10):2301–2313, 2015.

    Article  PubMed  Google Scholar 

  47. 47.

    Williams, P. A., R. S. Stilhano, V. P. To, L. Tran, K. Wong, and E. A. Silva. Hypoxia augments outgrowth endothelial cell (oec) sprouting and directed migration in response to sphingosine-1-phosphate (s1p). PLoS One. 10:e0123437, 2015.

    Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Yan, X.-L., E. Khor, and L.-Y. Lim. Chitosan-alginate films prepared with chitosans of different molecular weights. J. Biomed. Mater. Res. 58:358–365, 2001.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Yoder, M. C., L. E. Mead, D. Prater, T. R. Krier, K. N. Mroueh, F. Li, R. Krasich, C. J. Temm, J. T. Prchal, and D. A. Ingram. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood. 109:1801–1809, 2007.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the American Heart Association (15BGIA25730057 and 15PRE22930044) and the Hellman Family for the funding support for this work. We also acknowledge Dr. J. Kent Leach and Dr. Scott Simon for the use of their equipment in acquiring this data. We thank Fred Hayes and the UC Davis Advanced Materials Characterization and Testing (AMCAT) facility for guidance with SEM imaging.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Eduardo A. Silva.

Additional information

Associate Editor Michael Gower oversaw the review of this article.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Williams, P.A., Campbell, K.T., Gharaviram, H. et al. Alginate-Chitosan Hydrogels Provide a Sustained Gradient of Sphingosine-1-Phosphate for Therapeutic Angiogenesis. Ann Biomed Eng 45, 1003–1014 (2017). https://doi.org/10.1007/s10439-016-1768-2

Download citation

Keywords

  • Composite hydrogel
  • Controlled release
  • Lipid
  • Outgrowth endothelial cell
  • Homing
  • Proangiogenic factors
  • Sphingosine-1-phosphate (S1P)