Advertisement

Encapsulated Neural Stem Cell Neuronal Differentiation in Fluorinated Methacrylamide Chitosan Hydrogels

Abstract

Neural stem/progenitor cells (NSPCs) are able to differentiate into the primary cell types (neurons, oligodendrocytes and astrocytes) of the adult nervous system. This attractive property of NSPCs offers a potential solution for neural regeneration. 3D implantable scaffolds should mimic the microstructure and dynamic properties found in vivo, enabling the natural exchange of oxygen, nutrients, and growth factors for cell survival and differentiation. We have previously reported a new class of materials consisting of perfluorocarbons (PFCs) conjugated to methacrylamide chitosan (MAC), which possess the ability to repeatedly take-up and release oxygen at beneficial levels for favorable cell metabolism and proliferation. In this study, the neuronal differentiation responses of NSPCs to fluorinated methacrylamide chitosan (MACF) hydrogels were studied for 8 days. Two treatments, with oxygen reloading or without oxygen reloading, were performed during culture. Oxygen concentration distributions within cell-seeded MACF hydrogels were found to have higher concentrations of oxygen at the edge of the hydrogels and less severe drops in O2 gradient as compared with MAC hydrogel controls. Total cell number was enhanced in MACF hydrogels as the number of conjugated fluorines via PFC substitution increased. Additionally, all MACF hydrogels supported significantly more cells than MAC controls (p < 0.001). At day 8, MACF hydrogels displayed significantly greater neuronal differentiation than MAC controls (p = 0.001), and among MACF groups methacrylamide chitosan with 15 fluorines per addition (MAC(Ali15)F) demonstrated the best ability to promote NSPC differentiation.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

References

  1. 1.

    Aiedeh, K., E. Gianasi, I. Orienti, and V. Zecchi. Chitosan microcapsules as controlled release systems for insulin. J. Microencapsul. 14(5):567–576, 1997.

  2. 2.

    Arkudas, A., J. P. Beier, K. Heidner, J. Tjiawi, E. Polykandriotis, S. Srour, M. Sturzl, R. E. Horch, and U. Kneser. Axial prevascularization of porous matrices using an arteriovenous loop promotes survival and differentiation of transplanted autologous osteoblasts. Tissue Eng. 13(7):1549–1560, 2007.

  3. 3.

    Bagheri-Khoulenjani, S., S. M. Taghizadeh, and H. Mirzadeh. An investigation on the short-term biodegradability of chitosan with various molecular weights and degrees of deacetylation. Carbohydr. Polym. 78(4):773–778, 2009.

  4. 4.

    Campos, L. S. Beta1 integrins and neural stem cells: making sense of the extracellular environment. BioEssays 27(7):698–707, 2005.

  5. 5.

    Castro, C. I., and J. C. Briceno. Perfluorocarbon-based oxygen carriers: review of products and trials. Artif. Organs 34(8):622–634, 2010.

  6. 6.

    Cha, C., S. Y. Kim, L. Cao, and H. Kong. Decoupled control of stiffness and permeability with a cell-encapsulating poly(ethylene glycol) dimethacrylate hydrogel. Biomaterials 31(18):4864–4871, 2010.

  7. 7.

    Chandler, D. Structures of molecular liquids. Annu. Rev. Phys. Chem. 29:441–471, 1978.

  8. 8.

    Chang, T. M. S. Blood Substitutes: Principles, Methods, Products, and Clinical Trials. Tissue Engineering. New York: Basel Karger Landes Systems, 1997.

  9. 9.

    Chin, K., S. F. Khattak, S. R. Bhatia, and S. C. Roberts. Hydrogel-perfluorocarbon composite scaffold promotes oxygen transport to immobilized cells. Biotechnol. Prog. 24(2):358–366, 2008.

  10. 10.

    Chubb, C. Reversal of the endocrine toxicity of commercially produced perfluorochemical emulsion. Biol. Reprod. 33(4):854–858, 1985.

  11. 11.

    Chubb, C., and P. Draper. Steroid-secretion by rat testes perfused with perfluorochemicals as oxygen carriers. Am. J. Physiol. 248(4):E432–E437, 1985.

  12. 12.

    Csete, M. Oxygen in the cultivation of stem cells. Ann. N. Y. Acad. Sci. 1–8:2005, 1049.

  13. 13.

    De Filippis, L., and D. Delia. Hypoxia in the regulation of neural stem cells. Cell. Mol. Life Sci. 68(17):2831–2844, 2011.

  14. 14.

    de Vos, P., M. M. Faas, B. Strand, and R. Calafiore. Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials 27(32):5603–5617, 2006.

  15. 15.

    Dias, A. M. A., C. M. B. Goncalves, J. L. Legido, J. A. P. Coutinho, and I. M. Marrucho. Solubility of oxygen in substituted perfluorocarbons. Fluid Phase Equilib. 238(1):7–12, 2005.

  16. 16.

    Dominici, M., K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop, and E. Horwitz. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8(4):315–317, 2006.

  17. 17.

    Drury, J. L., and D. J. Mooney. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 24(24):4337–4351, 2003.

  18. 18.

    Dunn, J. C., W. Y. Chan, V. Cristini, J. S. Kim, J. Lowengrub, S. Singh, and B. M. Wu. Analysis of cell growth in three-dimensional scaffolds. Tissue Eng. 12(4):705–716, 2006.

  19. 19.

    Engler, A. J., S. Sen, H. L. Sweeney, and D. E. Discher. Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689, 2006.

  20. 20.

    Enzmann, V., R. M. Howard, Y. Yamauchi, S. R. Whittemore, and H. J. Kaplan. Enhanced induction of RPE lineage markers in pluripotent neural stem cells engrafted into the adult rat subretinal space. Invest. Ophthalmol. Vis. Sci. 44(12):5417–5422, 2003.

  21. 21.

    Fitzpatrick, C. M., and J. D. Kerby. Blood substitutes: hemoglobin-based oxygen carriers. Oral Maxillofac. Surg. Clin. N. Am. 17(3):261–266, v–vi, 2005.

  22. 22.

    Flaim, S. F. Pharmacokinetics and side-effects of perfluorocarbon-based blood substitutes. Artif. Cells Blood Substit. Immobil. Biotechnol. 22(4):1043–1054, 1994.

  23. 23.

    Gao, W., J. C. Lai, and S. W. Leung. Functional enhancement of chitosan and nanoparticles in cell culture, tissue engineering, and pharmaceutical applications. Front Physiol. 3:321, 2012.

  24. 24.

    Gattas-Asfura, K. M., C. A. Fraker, and C. L. Stabler. Perfluorinated alginate for cellular encapsulation. J. Biomed. Mater. Res. A 100(8):1963–1971, 2012.

  25. 25.

    Goh, F., J. D. Gross, N. E. Simpson, and A. Sambanis. Limited beneficial effects of perfluorocarbon emulsions on encapsulated cells in culture: experimental and modeling studies. J. Biotechnol. 150(2):232–239, 2010.

  26. 26.

    Greenburg, A. G., and H. W. Kim. Hemoglobin-based oxygen carriers. Crit. Care 8(Suppl 2):S61–S64, 2004.

  27. 27.

    Gustafsson, M. V., X. Zheng, T. Pereira, K. Gradin, S. Jin, J. Lundkvist, J. L. Ruas, L. Poellinger, U. Lendahl, and M. Bondesson. Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev. Cell 9(5):617–628, 2005.

  28. 28.

    Harrison, B. S., D. Eberli, S. J. Lee, A. Atala, and J. J. Yoo. Oxygen producing biomaterials for tissue regeneration. Biomaterials 28(31):4628–4634, 2007.

  29. 29.

    Huang, Y., B. Zhang, G. Xu, and W. Hao. Swelling behaviours and mechanical properties of silk fibroin–polyurethane composite hydrogels. Compos. Sci. Technol. 84:15–22, 2013.

  30. 30.

    Ifkovits, J. L., and J. A. Burdick. Review: photopolymerizable and degradable biomaterials for tissue engineering applications. Tissue Eng. 13(10):2369–2385, 2007.

  31. 31.

    Johnson, A. S., R. J. Fisher, G. C. Weir, and C. K. Colton. Oxygen consumption and diffusion in assemblages of respiring spheres: performance enhancement of a bioartificial pancreas. Chem. Eng. Sci. 64(22):4470–4487, 2009.

  32. 32.

    Ju, L. K., J. F. Lee, and W. B. Armiger. Enhancing oxygen-transfer in bioreactors by perfluorocarbon emulsions. Biotechnol. Prog. 7(4):323–329, 1991.

  33. 33.

    Kanichai, M., D. Ferguson, P. J. Prendergast, and V. A. Campbell. Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: a role for AKT and hypoxia-inducible factor (HIF)-1alpha. J. Cell. Physiol. 216(3):708–715, 2008.

  34. 34.

    Khattak, S. F., K. S. Chin, S. R. Bhatia, and S. C. Roberts. Enhancing oxygen tension and cellular function in alginate cell encapsulation devices through the use of perfluorocarbons. Biotechnol. Bioeng. 96(1):156–166, 2007.

  35. 35.

    Kimelman-Bleich, N., G. Pelled, D. Sheyn, I. Kallai, Y. Zilberman, O. Mizrahi, Y. Tal, W. Tawackoli, Z. Gazit, and D. Gazit. The use of a synthetic oxygen carrier-enriched hydrogel to enhance mesenchymal stem cell-based bone formation in vivo. Biomaterials 30(27):4639–4648, 2009.

  36. 36.

    Kratz, G., C. Arnander, J. Swedenborg, M. Back, C. Falk, I. Gouda, and O. Larm. Heparin-chitosan complexes stimulate wound healing in human skin. Scand. J. Plast. Reconstr. Surg. Hand Surg. 31(2):119–123, 1997.

  37. 37.

    Kresie, L. Artificial blood: an update on current red cell and platelet substitutes. Proc. (Bayl Univ. Med. Cent.) 14(2):158–161, 2001.

  38. 38.

    Kumar, G., P. J. Smith, and G. F. Payne. Enzymatic grafting of a natural product onto chitosan to confer water solubility under basic conditions. Biotechnol. Bioeng. 63(2):154–165, 1999.

  39. 39.

    Langer, R., and J. P. Vacanti. Tissue engineering. Science 260(5110):920–926, 1993.

  40. 40.

    Leipzig, N. D., and M. S. Shoichet. The effect of substrate stiffness on adult neural stem cell behavior. Biomaterials 30(36):6867–6878, 2009.

  41. 41.

    Leipzig, N. D., R. G. Wylie, H. Kim, and M. S. Shoichet. Differentiation of neural stem cells in three-dimensional growth factor-immobilized chitosan hydrogel scaffolds. Biomaterials 32(1):57–64, 2011.

  42. 42.

    Leung, R., D. Poncelet, and R. J. Neufeld. Enhancement of oxygen transfer rate using microencapsulated silicone oils as oxygen carriers. J. Chem. Technol. Biotechnol. 68(1):37–46, 1997.

  43. 43.

    Lewis, M. C., B. D. Macarthur, J. Malda, G. Pettet, and C. P. Please. Heterogeneous proliferation within engineered cartilaginous tissue: the role of oxygen tension. Biotechnol. Bioeng. 91(5):607–615, 2005.

  44. 44.

    Li, H., A. Wijekoon, and N. D. Leipzig. 3D differentiation of neural stem cells in macroporous photopolymerizable hydrogel scaffolds. PLoS ONE 7(11):e48824, 2012.

  45. 45.

    Lovett, M., K. Lee, A. Edwards, and D. L. Kaplan. Vascularization strategies for tissue engineering. Tissue Eng. B Rev. 15(3):353–370, 2009.

  46. 46.

    Lowe, K. C. Fluorinated blood substitutes and oxygen carriers. J. Fluorine Chem. 109(1):59–65, 2001.

  47. 47.

    Lutolf, M. P., J. L. Lauer-Fields, H. G. Schmoekel, A. T. Metters, F. E. Weber, G. B. Fields, and J. A. Hubbell. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. Proc. Natl Acad. Sci. U.S.A. 100(9):5413–5418, 2003.

  48. 48.

    Madihally, S. V., and H. W. Matthew. Porous chitosan scaffolds for tissue engineering. Biomaterials 20(12):1133–1142, 1999.

  49. 49.

    Miyazaki, S., K. Ishii, and T. Nadai. The use of chitin and chitosan as drug carriers. Chem. Pharm. Bull. 29(10):3067–3069, 1981.

  50. 50.

    Mohyeldin, A., T. Garzon-Muvdi, and A. Quinones-Hinojosa. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell 7(2):150–161, 2010.

  51. 51.

    Morrison, S. J., M. Csete, A. K. Groves, W. Melega, B. Wold, and D. J. Anderson. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J. Neurosci. 20(19):7370–7376, 2000.

  52. 52.

    Oh, S. H., C. L. Ward, A. Atala, J. J. Yoo, and B. S. Harrison. Oxygen generating scaffolds for enhancing engineered tissue survival. Biomaterials 30(5):757–762, 2009.

  53. 53.

    Panchision, D. M. The role of oxygen in regulating neural stem cells in development and disease. J. Cell. Physiol. 220(3):562–568, 2009.

  54. 54.

    Park, H., C. D. Vecitis, J. Cheng, W. Choi, B. T. Mader, and M. R. Hoffmann. Reductive defluorination of aqueous perfluorinated alkyl surfactants: effects of ionic headgroup and chain length. J. Phys. Chem. A 113(4):690–696, 2009.

  55. 55.

    Park, I. K., J. Yang, H. J. Jeong, H. S. Bom, I. Harada, T. Akaike, S. Kim, and C. S. Cho. Galactosylated chitosan as a synthetic extracellular matrix for hepatocytes attachment. Biomaterials 24(13):2331–2337, 2003.

  56. 56.

    Pistollato, F., H. L. Chen, P. H. Schwartz, G. Basso, and D. M. Panchision. Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes. Mol. Cell. Neurosci. 35(3):424–435, 2007.

  57. 57.

    Portner, R., S. Nagel-Heyer, C. Goepfert, P. Adamietz, and N. M. Meenen. Bioreactor design for tissue engineering. J. Biosci. Bioeng. 100(3):235–245, 2005.

  58. 58.

    Powers, D. E., J. R. Millman, R. B. Huang, and C. K. Colton. Effects of oxygen on mouse embryonic stem cell growth, phenotype retention, and cellular energetics. Biotechnol. Bioeng. 101(2):241–254, 2008.

  59. 59.

    Quijano, G., S. Revah, M. Gutierrez-Rojas, L. B. Flores-Cotera, and F. Thalasso. Oxygen transfer in three-phase airlift and stirred tank reactors using silicone oil as transfer vector. Process Biochem. 44(6):619–624, 2009.

  60. 60.

    Richardson, S. C. W., H. J. V. Kolbe, and R. Duncan. Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. Int. J. Pharm. 178(2):231–243, 1999.

  61. 61.

    Riess, J. G. Reassessment of criteria for the selection of perfluorochemicals for 2nd-generation blood substitutes—analysis of structure property relationships. Artif. Organs 8(1):44–56, 1984.

  62. 62.

    Riess, J. G. Oxygen carriers (“blood substitutes”)—Raison d’Etre, chemistry, and some physiology. Chem. Rev. 101(9):2797–2919, 2001.

  63. 63.

    Riess, J. G. Understanding the fundamentals of perfluorocarbons and perfluorocarbon emulsions relevant to in vivo oxygen delivery. Artif. Cells Blood Substit. Immobil. Biotechnol. 33(1):47–63, 2005.

  64. 64.

    Riess, J. G. Perfluorocarbon-based oxygen delivery. Artif. Cells Blood Substit. Immobil. Biotechnol. 34(6):567–580, 2006.

  65. 65.

    Riva, R., H. Ragelle, A. des Rieux, N. Duhem, C. Jerome, and V. Preat. Chitosan and chitosan derivatives in drug delivery and tissue engineering. Chitosan Biomater. II. 244:19–44, 2011.

  66. 66.

    Rouwkema, J., N. C. Rivron, and C. A. van Blitterswijk. Vascularization in tissue engineering. Trends Biotechnol. 26(8):434–441, 2008.

  67. 67.

    Saha, K., J. Kim, E. Irwin, J. Yoon, F. Momin, V. Trujillo, D. V. Schaffer, K. E. Healy, and R. C. Hayward. Surface creasing instability of soft polyacrylamide cell culture substrates. Biophys. J. 99(12):L94–L96, 2010.

  68. 68.

    Santilli, G., G. Lamorte, L. Carlessi, D. Ferrari, L. Rota Nodari, E. Binda, D. Delia, A.L. Vescovi, and L. De Filippis. Mild hypoxia enhances proliferation and multipotency of human neural stem cells. PLoS One 5(1):e8575, 2010.

  69. 69.

    Sashiwa, H., and S. I. Aiba. Chemically modified chitin and chitosan as biomaterials. Prog. Polym. Sci. 29(9):887–908, 2004.

  70. 70.

    Schroeder, J. L., J. M. Highsmith, H. F. Young, and B. E. Mathern. Reduction of hypoxia by perfluorocarbon emulsion in a traumatic spinal cord injury model. J. Neurosurg. Spine 9(2):213–220, 2008.

  71. 71.

    Simon, M. C., and B. Keith. The role of oxygen availability in embryonic development and stem cell function. Nat. Rev. Mol. Cell Biol. 9(4):285–296, 2008.

  72. 72.

    Spiess, B. D. Perfluorocarbon emulsions as a promising technology: a review of tissue and vascular gas dynamics. J. Appl. Physiol. 106(4):1444–1452, 2009.

  73. 73.

    Spiess, B. D., and R. P. Cochran. Perfluorocarbon emulsions and cardiopulmonary bypass: a technique for the future. J. Cardiothorac. Vasc. Anesth. 10(1):83–89; quiz 89–90, 1996.

  74. 74.

    Subramanian, A., U. M. Krishnan, and S. Sethuraman. Development of biomaterial scaffold for nerve tissue engineering: biomaterial mediated neural regeneration. J. Biomed. Sci. 16:108, 2009.

  75. 75.

    Tremper, K. K., and S. T. Anderson. Perfluorochemical emulsion oxygen-transport fluids—a clinical review. Annu. Rev. Med. 36:309–313, 1985.

  76. 76.

    Turturro, M. V., M. C. Christenson, J. C. Larson, D. A. Young, E. M. Brey, and G. Papavasiliou. MMP-sensitive PEG diacrylate hydrogels with spatial variations in matrix properties stimulate directional vascular sprout formation. PLoS ONE 8(3):e58897, 2013.

  77. 77.

    Volkmer, E., I. Drosse, S. Otto, A. Stangelmayer, M. Stengele, B. C. Kallukalam, W. Mutschler, and M. Schieker. Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone. Tissue Eng. A 14(8):1331–1340, 2008.

  78. 78.

    Wang, J., Y. Zhu, H. K. Bawa, G. Ng, Y. Wu, M. Libera, H. C. van der Mei, H. J. Busscher, and X. Yu. Oxygen-generating nanofiber cell scaffolds with antimicrobial properties. ACS Appl. Mater. Interfaces 3(1):67–73, 2011.

  79. 79.

    White, J. C., W. L. Stoppel, S. C. Roberts, and S. R. Bhatia. Addition of perfluorocarbons to alginate hydrogels significantly impacts molecular transport and fracture stress. J. Biomed. Mater. Res. A 101A(2):438–446, 2013.

  80. 80.

    Wijekoon, A., N. Fountas-Davis, and N.D. Leipzig. Fluorinated methacrylamide chitosan hydrogel systems as adaptable oxygen carriers for wound healing. Acta Biomater. 9(3):5653–5664, 2012.

  81. 81.

    Yu, L. M., K. Kazazian, and M. S. Shoichet. Peptide surface modification of methacrylamide chitosan for neural tissue engineering applications. J. Biomed. Mater. Res. A 82(1):243–255, 2007.

Download references

Acknowledgments

We are grateful for funding from the University of Akron that supported this work. The authors would also like to thank Dr. Rebecca Willits for allowing us to perform rheological measurements in her laboratory as well as assistance with interpreting mechanical and swelling results.

Author information

Correspondence to Nic D. Leipzig.

Additional information

Associate Editor Michael S. Detamore oversaw the review of this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 2 (AVI 25322 kb)

Supplementary material 3 (AVI 25665 kb)

Supplementary material 4 (AVI 25322 kb)

Supplementary material 5 (AVI 25322 kb)

Supplementary material 6 (AVI 19913 kb)

Supplementary Fig. S1 Representative images of scaffolds with no oxygen resupplementation obtained by confocal microscope at day 8 at the center of scaffolds, each picture represents a stack of 50 images with a spacing of 200 μm. MAC(Ali15)F shows the highest cell number indicated by nuclear Hoechst 33342 stain. Corresponding 3D reconstruction movies generated from these images are presented as supplemental material (Movies 5–8). Scale bar equals 100 μm (PDF 736 kb)

Supplementary material 2 (AVI 25322 kb)

Supplementary material 3 (AVI 25665 kb)

Supplementary material 4 (AVI 25322 kb)

Supplementary material 5 (AVI 25322 kb)

Supplementary material 6 (AVI 19913 kb)

Supplementary material 7 (AVI 19439 kb)

Supplementary material 8 (AVI 19439 kb)

Supplementary material 9 (AVI 19439 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, H., Wijekoon, A. & Leipzig, N.D. Encapsulated Neural Stem Cell Neuronal Differentiation in Fluorinated Methacrylamide Chitosan Hydrogels. Ann Biomed Eng 42, 1456–1469 (2014) doi:10.1007/s10439-013-0925-0

Download citation

Keywords

  • Chitosan
  • Oxygen
  • Hydrogel
  • Perfluorocarbons
  • Neural stem cells
  • Neuronal differentiation