Chemical Cross-Linking of Corneal Tissue to Reduce Progression of Loss of Sight in Patients With Keratoconus

Atikah Haneef, Ramprasad Obula Giridhara Gopalan, Divya T Rajendran, Jessica Nunes, Dharmalingam Kuppamuthu, Naveen Radhakrishnan, Tai-Horng Young, Hao-Ying Hsieh, Namperumalsamy Venkatesh Prajna, Colin Willoughby, Rachel Williams

Research output: Contribution to journalSpecial issuepeer-review

2 Citations (Scopus)
64 Downloads (Pure)


Purpose: We aimed to develop a novel chemical cross-linker treatment for keratoconus by reacting dicarboxylic acid spacer molecules and amine functional groups on protein structure of the tissue using carbodi-imide chemistry. We propose this as an alternative to conventional cross-linking treatment for keratoconus.

Methods: The study involved optimization of the cross-linker formulation. Mechanical stiffness of ex vivo porcine and human corneas after application of the cross-linker was measured. Histochemical analysis was performed to record changes in gross morphology after cross-linker treatment on ex vivo porcine and human and in vivo rabbit corneas. Terminal deoxynucleotidyl transferase–mediated dUTP-X nick-end-labeling (TUNEL) staining was performed to study apoptotic effects of cross-linker. Cytotoxicity potential of cross-linker was evaluated by studying explant cultures for cellular outgrowth and immunostaining assays on porcine and human corneas after treatment.

Results: We demonstrated a clinically relevant increase in stiffness in ex vivo experiments using porcine and human cornea without removal of corneal epithelium. Histological analysis showed no change in gross morphology of cornea and no evidence of apoptosis. In vivo treatment of rabbit eyes demonstrated initial thinning of corneal epithelium that recovered after seven days although with abnormal regularity of cells. Cellular outgrowth from corneal explant cultures after treatment further confirmed cell survival after treatment.

Conclusions: This chemical cross-linking of corneal tissue has potential advantages over current therapeutic options including lower cytotoxicity to stromal cells than ultraviolet A treatment.

Translational Relevance: The cross-linker has potential to become a treatment for keratoconus because it overcomes the need for procedures using specialized equipment and ensures accessibility to large populations.
Original languageEnglish
Article number6
Number of pages11
JournalTranslational Vision Science & Technology
Issue number5
Publication statusPublished (in print/issue) - 29 Apr 2021

Bibliographical note

Funding Information:
Supported by the EPSRC through a Translational Alliance Platform Grant (EP/N022807/1) and a contribution from University of Liverpool GCRF Pump-Priming Award (EP/P510981/1).

Publisher Copyright:
© 2021 The Authors.


  • keratoconus
  • Cross-linking
  • cornea
  • Carbodi-imide chemistry
  • Cornea
  • Keratoconus
  • Collagen
  • Cross-Linking Reagents
  • Rabbits
  • Keratoconus/drug therapy
  • Humans
  • Riboflavin
  • Animals
  • Ultraviolet Rays
  • Swine


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