July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Mechanical properties of collagen gels crosslinked by copper-free click chemistry and their effects on encapsulated keratocytes
Author Affiliations & Notes
  • Hyun Jong Lee
    Ophthalmology, Stanford University, Palo Alto, California, United States
  • Gabriella Fernandes-Cunha
    Ophthalmology, Stanford University, Palo Alto, California, United States
  • Sarah Heilshorn
    Materials Science and Engineering, Stanford University, Palo Alto, California, United States
  • David Myung
    Ophthalmology, Stanford University, Palo Alto, California, United States
  • Footnotes
    Commercial Relationships   Hyun Jong Lee, None; Gabriella Fernandes-Cunha, None; Sarah Heilshorn, None; David Myung, None
  • Footnotes
    Support  National Eye Institute/NIH K08 EY028176; Stanford SPARK Translational Research Program; National Institutes of Health P30 Core Grant; Research to Prevent Blindness Core Grant
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2268. doi:
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      Hyun Jong Lee, Gabriella Fernandes-Cunha, Sarah Heilshorn, David Myung; Mechanical properties of collagen gels crosslinked by copper-free click chemistry and their effects on encapsulated keratocytes. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2268.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : We are developing a bioengineered substitute for corneal stroma composed of keratocytes within a crosslinked collagen scaffold. In this study, bio-orthogonal copper-free click chemistry was used to form three-dimensional collagen matrices with the intent of minimizing any cytotoxic effects of covalent crosslinking during gel synthesis.

Methods : To adjust the mechanical properties of the gels, the azide/alkyne ratio and collagen concentration were manipulated. The keratocytes were encapsulated and we observed their behavior in modulus-controlled collagen gels. Collagen was conjugated with azide and dibenzocyclooctyne (DBCO) groups by reaction with N-hydroxysuccinimide (NHS) esters.
To fabricate crosslinked collagen hydrogels, azide-collagen and DBCO-collagen were mixed and incubated for 15 min. The ratio and concentration of azide-collagen and DBCO-collagen were modulated to alter the gels’ mechanical properties.
To assess the effect of the mechanical properties on the cells, primary rabbit keratocytes were encapsulated using different gelation conditions. The cell viability and proliferation were measured as a function of the various gel conditions.

Results : Azide and DBCO conjugation efficiency was about 70%. The gelation was completed within 900 s at room temperature, and the storage modulus of the crosslinked gels was 2.6-fold higher than that of non-crosslinked gels.
The crosslinked collagen gel exhibited storage moduli that depended on the ratio of azide and DBCO groups, with the 1:1 ratio showing the highest storage modulus. Also, the storage moduli were correlated to the concentration of collagen.
The keratocytes were cultured in the collagen gels. A live/dead assay showed that showed excellent cell viability, and cell morphologies were highly correlated with modulus. The cells spread widely with polygonal and spindle shapes in low modulus. In high modulus gels, the cells tended to maintain a rounded morphology.
Cell density in low modulus gels increased steadily for 8 days, while there were no significant changes in cell density for 8 days in high modulus gels.

Conclusions : We observed that the mechanical properties of encapsulating gels can control the cellular behavior of keratocytes. Copper-free click chemistry appears to provide a useful way to encapsulate cultured keratocytes and control their phenotype without impacting their viability.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.


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