For years, corneal cross-linking (CXL) has followed a well-defined biochemical script: riboflavin, UV-A light, and—critically—oxygen. Remove oxygen from the equation, and you disrupt the very reaction that stiffens the cornea. That much has been established. But CXL does more than just stiffen the cornea—it also increases resistance to enzymatic digestion, and that’s where things get interesting.
Why CXL Research into Enzymatic Resistance Matters
Inflammation, a key player in keratoconus, can elevate the levels of proteolytic enzymes in the cornea, potentially accelerating its degradation. In infectious keratitis, the problem is even more direct—pathogens secrete proteases to break down corneal tissue as a food source. Fortunately, CXL research has shown that cross-linking enhances corneal resistance to enzymatic breakdown. But is oxygen required for this effect?
A new study, led by ELZA’s M. Enes Aydemir and published in Translational Vision Science & Technology, provides a striking answer—no, it does not. This groundbreaking CXL research reveals that corneal resistance to enzymatic digestion can be enhanced without oxygen.
Oxygen in CXL: An Assumption Challenged
This flies in the face of conventional wisdom. The standard Dresden protocol for CXL has always been oxygen-dependent. The process relies on a photochemical reaction where UV-A excites riboflavin, generating reactive oxygen species (ROS) that drive the cross-linking process. Previous CXL research, such as the 2013 study by Richoz et al., firmly established that oxygen is essential for biomechanical stiffening. The assumption was clear: no oxygen, no cross-linking.
But Aydemir et al. have introduced a major shift in this model. Their study tested three different CXL protocols:
- Traditional riboflavin/UV-A CXL (Dresden protocol)
- Rose bengal/green light CXL
- A combined approach using both riboflavin/UV-A and rose bengal/green light
The researchers then exposed the corneas to enzymatic digestion and measured degradation time.
Breakthrough Findings in CXL Research
The key results:
- All three CXL protocols significantly increased corneal resistance to enzymatic digestion compared to untreated controls.
- The rose bengal/green light CXL alone was effective in enhancing resistance—without requiring oxygen.
- The combination protocol (riboflavin/UV-A + rose bengal/green light) further enhanced resistance, suggesting a synergistic effect between the two methods.
- The traditional riboflavin/UV-A method, as expected, remained oxygen-dependent for its enzymatic resistance effects.
This CXL research suggests that enzymatic resistance and biomechanical stiffening may be driven by different mechanisms. While traditional riboflavin/UV-A CXL requires oxygen for structural reinforcement, rose bengal-based cross-linking does not, opening new possibilities for treatment approaches.
How This CXL Research Informs Clinical Practice
The implications of this CXL research could be profound. Oxygen dependency has long been a limiting factor in corneal cross-linking, requiring strict environmental control and restricting treatment availability in certain settings, such as high-altitude regions or low-resource clinics.
If rose bengal-based CXL sidesteps this requirement while still enhancing enzymatic resistance, it could make the procedure more accessible and robust against external variables. However, further research is needed.
- Does oxygen-independent CXL confer the same long-term structural reinforcement?
- Can it match or exceed the durability of traditional riboflavin-based protocols?
- What is the precise mechanism behind enzymatic resistance in oxygen-independent CXL?
These are the questions that will define the next chapter in CXL research.
CXL Research: What’s Next?
One thing is certain: this study doesn’t just tweak the formula—it forces us to reexamine the chemistry of CXL from the ground up. If oxygen isn’t essential in some forms of cross-linking, what other biochemical assumptions are waiting to be rewritten?
This CXL research opens the door to new cross-linking strategies that could reshape keratoconus treatment and corneal therapy. The next challenge? Understanding how to translate these findings into real-world clinical practice.
Reference: Aydemir ME, Hafezi NL, Lu NJ, et al. Corneal resistance to enzymatic digestion after rose bengal and combined rose bengal/riboflavin cross-linking is oxygen independent. Trans Vis Sci Tech. 2025;14(3):1. Link
