Advertisement

Human Corneal Keratocyte Response to Micro- and Nano-Gratings on Chitosan and PDMS

  • Stephanie Koo
  • Sang Joon Ahn
  • Hao Zhang
  • Jenn C. Wang
  • Evelyn K. F. Yim
Article

Abstract

Corneal stroma accounts for major refractive power and the transparency of cornea. This transparency is contributed mainly by the highly ordered arrangement of the extracellular matrix (ECM), particularly the collagen fibrils. Shortage and complications of cornea transplantation have remained an issue for decades. Attempts to produce tissue-engineered cornea are met with challenges, one of which is to induce alignment of stromal ECM produced by keratocytes. In this study, human corneal keratocyte response toward substrate topography on two different materials was examined. Primary human keratocytes were cultured on chitosan and polydimethylsiloxane (PDMS) surface patterned with anisotopic topography of gratings in various widths. Cell response in the form of alignment, elongation and proliferation were analyzed. Collagen I deposition was imaged and the gene expression analysis of keratocytes cultured on PDMS were studied. On both chitosan and PDMS, keratocytes were found to be aligned and elongated in grating direction. Proliferation rate was reduced as grating width decreased. Aldehyde-3-dehydrogenase (ALDH3) expression was increased on nanogratings. Deposited collagen I followed the keratocyte orientation, which was aligned along the direction of gratings. In conclusion, keratocytes responded similarly to topographical cues when presented on materials with different surface chemistry and stiffness and nanogratings were observed to be more efficient in inducing these responses than microgratings.

Keywords

Nanotopography Corneal stroma Keratocyte Extracellular matrix alignment Response to topography Chitosan 

Notes

Acknowledgments

This work is supported by National Medical Research Council Singapore NMRC/NIG/0037/2008, and partly supported by the Mechanobiology Institute Singapore for SK. The authors would like to thank E. L. S. Fong for her assistance and helpful discussions.

Supplementary material

12195_2011_186_MOESM1_ESM.doc (62 kb)
Supplementary material 1 (DOC 62 kb)

References

  1. 1.
    Agnihotri, S. A., N. N. Mallikarjuna, and T. M. Aminabhavi. Recent advances on chitosan-based micro- and nanoparticles in drug delivery. J. Control. Release 100(1):5–28, 2004.CrossRefGoogle Scholar
  2. 2.
    Armani, D., C. Liu, and N. Aluru. Re-configurable fluid circuits by PDMS elastomer micromachining. In: Proceedings of the IEEE Micro Electro Mechanical Systems (MEMS), 1999, pp. 222–227.Google Scholar
  3. 3.
    Benedek, G. B. Theory of Transparency of the Eye. Appl. Opt. 10(3):459–473, 1971.CrossRefGoogle Scholar
  4. 4.
    Billmeyer, F. W. Textbook of Polymer Science (3rd ed.). New York: Wiley, 1984.Google Scholar
  5. 5.
    Birk, D. E., J. M. Fitch, and T. F. Linsenmayer. Organization of collagen types I and V in the embryonic chicken cornea. Invest. Ophthalmol. Vis. Sci. 27(10):1470–1477, 1986.Google Scholar
  6. 6.
    Birk, D. E., et al. Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter. J. Cell Sci. 95(4):649–657, 1990.MathSciNetGoogle Scholar
  7. 7.
    Borene, M. L., V. H. Barocas, and A. Hubel. Mechanical and cellular changes during compaction of a collagen-sponge-based corneal stromal equivalent. Ann. Biomed. Eng. 32(2):274–283, 2004.CrossRefGoogle Scholar
  8. 8.
    Chung, H. J., and T. G. Park. Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering. Adv. Drug Deliv. Rev. 59(4–5):249–262, 2007.CrossRefGoogle Scholar
  9. 9.
    Clyne, A. M. Thermal processing of tissue engineering scaffolds. J. Heat Transfer 133(3):034001, 2011.CrossRefGoogle Scholar
  10. 10.
    de Britto, D., and O. B. G. de Assis. Synthesis and mechanical properties of quaternary salts of chitosan-based films for food application. Int. J. Biol. Macromol. 41(2):198–203, 2007.CrossRefGoogle Scholar
  11. 11.
    Discher, D. E., P. Janmey, and Y. L. Wang. Tissue cells feel and respond to the stiffness of their substrate. Science 310(5751):1139–1143, 2005.CrossRefGoogle Scholar
  12. 12.
    Engler, A. J., et al. Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689, 2006.CrossRefGoogle Scholar
  13. 13.
    Fini, M. E. Keratocyte and fibroblast phenotypes in the repairing cornea. Prog. Retin. Eye Res. 18(4):529–551, 1999.CrossRefGoogle Scholar
  14. 14.
    Foster, C. S., D. T. Azar, and C. H. Dohlman (eds.). Smolin and Thoft’s The Cornea Scientific Foundations and Clinical Practice (4th ed.). Philadelphia: Lippincott Williams and Wilkins, p. 1295, 2005.Google Scholar
  15. 15.
    Fu, J., et al. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 7(9):733–736, 2010.CrossRefGoogle Scholar
  16. 16.
    Fuard, D., et al. Optimization of poly-di-methyl-siloxane (PDMS) substrates for studying cellular adhesion and motility. Microelectron. Eng. 85(5–6):1289–1293, 2008.CrossRefGoogle Scholar
  17. 17.
    Guilak, F., et al. Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5(1):17–26, 2009.CrossRefGoogle Scholar
  18. 18.
    Hahnel, C. P. D., et al. The keratocyte network of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy. Cornea 19(2):185–193, 2000.CrossRefGoogle Scholar
  19. 19.
    Hassell, J. R., and D. E. Birk. The molecular basis of corneal transparency. Exp. Eye Res. 91(3):326–335, 2010.CrossRefGoogle Scholar
  20. 20.
    Hu, W., et al. Effects of nanoimprinted patterns in tissue-culture polystyrene on cell behavior. J. Vac. Sci. Technol. B: Microelectron. Nanomet. Struct. 23(6):2984–2989, 2005.CrossRefGoogle Scholar
  21. 21.
    Jester, J. V., et al. Corneal keratocytes: in situ and in vitro organization of cytoskeletal contractile proteins. Invest. Ophthalmol. Vis. Sci. 35(2):730–743, 1994.Google Scholar
  22. 22.
    Jester, J., et al. The cellular basis of corneal transparency: evidence for ‘corneal crystallins’. J. Cell Sci. 112(5):613–622, 1999.Google Scholar
  23. 23.
    Khalid, M. N., et al. Water state characterization, swelling behavior, thermal and mechanical properties of chitosan based networks. Eur. J. Pharm. Sci. 15(5):425–432, 2002.CrossRefGoogle Scholar
  24. 24.
    Krachmer, J., M. Mannis, and E. Holland. Cornea Fundamentals, Diagnosis, and Management (2nd ed.). St. Louis, MO: Mosby, pp. 3–26, 2005.Google Scholar
  25. 25.
    Maurice, D. M. The structure and transparency of the cornea. J. Physiol. 136(2):263–286, 1957.Google Scholar
  26. 26.
    Moller-Pedersen, T. Keratocyte reflectivity and corneal haze. Exp. Eye Res. 78(3):553–560, 2004.CrossRefGoogle Scholar
  27. 27.
    Muller, L. J., L. Pels, and G. F. J. M. Vrensen. Novel aspects of the ultrastructural organization of human corneal keratocytes. Invest. Ophthalmol. Vis. Sci. 36(13):2557–2567, 1995.Google Scholar
  28. 28.
    Nishida, T., et al. Interactions of extracellular collagen and corneal fibroblasts: morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix. In Vitro Cell. Dev. Biol. 24(10):1009–1014, 1988.CrossRefGoogle Scholar
  29. 29.
    Pei, Y., R. Y. Reins, and A. M. McDermott. Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes. Exp. Eye Res. 83(5):1063–1073, 2006.CrossRefGoogle Scholar
  30. 30.
    Pot, S. A., et al. Nanoscale topography–induced modulation of fundamental cell behaviors of rabbit corneal keratocytes, fibroblasts, and myofibroblasts. Invest. Ophthalmol. Vis. Sci. 51(3):1373–1381, 2010.CrossRefGoogle Scholar
  31. 31.
    Qazi, Y., et al. Corneal transparency: genesis, maintenance and dysfunction. Brain Res. Bull. 81(2–3):198–210, 2010.CrossRefGoogle Scholar
  32. 32.
    Rickett, T. A., et al. Rapidly photo-cross-linkable chitosan hydrogel for peripheral neurosurgeries. Biomacromolecules 12(1):57–65, 2011.CrossRefGoogle Scholar
  33. 33.
    Saez, A., et al. Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates. Proc. Natl Acad. Sci. USA 104(20):8281–8286, 2007.CrossRefGoogle Scholar
  34. 34.
    Tatsuno, I., A. Hirai, and Y. Saito. Cell-anchorage, cell cytoskeleton, and Rho-GTPase family in regulation of cell cycle progression. Prog. Cell Cycle Res. 4:19–25, 2000.CrossRefGoogle Scholar
  35. 35.
    Toworfe, G. K., et al. Fibronectin adsorption on surface-activated poly(dimethylsiloxane) and its effect on cellular function. J. Biomed. Mater. Res. A 71(3):449–461, 2004.CrossRefGoogle Scholar
  36. 36.
    Trinkaus-Randall, V., and M. A. Nugent. Biological response to a synthetic cornea. J. Control. Release 53(1–3):205–214, 1998.CrossRefGoogle Scholar
  37. 37.
    Xia, W., et al. Biological activities of chitosan and chitooligosaccharides. Food Hydrocolloids 25(2):170–179, 2011.CrossRefGoogle Scholar
  38. 38.
    Yeung, T., et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskeleton 60(1):24–34, 2005.CrossRefGoogle Scholar
  39. 39.
    Yim, E. K. F., S. W. Pang, and K. W. Leong. Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage. Exp. Cell Res. 313:1820–1829, 2007.CrossRefGoogle Scholar
  40. 40.
    Yim, E. K. F., et al. Nanopattern-induced changes in morphology and motility of smooth muscle cells. Biomaterials 26(26):5405–5413, 2005.CrossRefGoogle Scholar
  41. 41.
    Yim, E. K. F., et al. Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells. Biomaterials 31:1299–1306, 2010.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2011

Authors and Affiliations

  • Stephanie Koo
    • 1
    • 2
  • Sang Joon Ahn
    • 2
  • Hao Zhang
    • 2
  • Jenn C. Wang
    • 4
    • 5
  • Evelyn K. F. Yim
    • 1
    • 2
    • 3
  1. 1.Mechanobiology Institute SingaporeNational University of SingaporeSingaporeSingapore
  2. 2.Division of BioengineeringNational University of SingaporeSingaporeSingapore
  3. 3.Department of SurgeryNational University of SingaporeSingaporeSingapore
  4. 4.Cornea, Cataract and Refractive ServiceNational University Hospital SystemSingaporeSingapore
  5. 5.Cataract and Comprehensive ServiceSingapore National Eye CentreSingaporeSingapore

Personalised recommendations