Corneal Cross-Linking (CXL): Leaving the Epithelium On

Leaving the corneal epithelium intact during CXL (‘epi-on’ CXL) had long been desired, pushing researchers to quickly develop two main approaches to getting riboflavin through the epithelial cell tight junctions and into the stroma: iontophoresis^1,2 (where an electrical current is applied to electrostatically force riboflavin molecules through the epithelium) and the use of penetration enhancers such as ethylenediaminetetraacetic acid (EDTA) or trometamol that degrade the tight junctions and allow riboflavin to pass between the epithelial cells.³

Once again, these approaches resulted in lower cross-linking efficacy than Dresden protocol CXL (sometimes to quite an extent)⁴ and left efficacy on the table. The reason was simple: not only does an intact epithelium act as a barrier to oxygen diffusion into the stroma—it also absorbs up to a fifth of the UV fluence administered.⁵ Again, approaches like supplemental oxygen were used to try to optimize that aspect of the CXL procedure, and again, the delivery of higher UV fluences helped to close the efficacy gap.⁶

Customized and combination CXL: personalizing treatment

Laser refractive surgery involves the removal of stromal tissue, which weakens the cornea’s biomechanical strength. Most corneas have more than enough strength in reserve for this surgery to be performed safely, but in thin corneas or ones with forme fruste ectasias, the procedure’s safety can be compromised. This is why comprehensive screening is necessary before undertaking any refractive laser procedure.

CXL strengthens the cornea and may allow the laser procedure to be performed with a higher degree of confidence a corneal ectasia will not develop later (these are called ‘Xtra’ procedures).⁷ However, recent long-term follow-up studies suggest no additional benefit in visual or safety outcomes achieved by prophylactically cross-linking corneas undergoing laser refractive surgery.^8,9

Therapeutic laser surgery is also possible in ectatic corneas, where the aim is not perfect refractive correction (which would be unachievable) but to regularize the cross-linked and therefore strengthened cornea to reduce certain higher-order aberrations and improve visual quality, often in combination with special contact lenses. The first examples of these procedures were the combinations of CXL and topography-guided PRK (Athens^10,11 and Cretan protocols¹²), and these approaches appear to bring sustained visual acuity and astigmatism reduction benefits.^11,13–15 Further attempts to optimize (such as Miguel Reichichi’s STARE-X¹⁶ and Rohit Shetty’s TREK¹⁷) aim to achieve these benefits while minimizing the amount of stromal tissue ablated by the excimer laser.

Customized CXL

CXL alone typically causes, on average, around 1 dioptre (D) of corneal flattening; the issue is the amount of flattening is highly variable and unpredictable.¹⁸ But in ectatic corneas, any flattening of the cone is of value as it partially rehabilitates the shape of the cornea and, with it, patients’ vision.

Topography-guided CXL, which attempts to selectively flatten the thinnest or steepest areas of the ectatic cornea, was developed. This approach uses specialized equipment to deliver higher UV fluences to these regions, along with an eye tracker to accurately target the UV energy.¹⁹ This resulted in corneal flattening responses of approximately 2–4 D,^20–22 even when supplemental oxygen was used.²³

A second-generation customized CXL technique, PTK-assisted customized epi-on CXL (PACE), has since been developed.²⁴ PACE involves three steps. First, an epithelial map-driven phototherapeutic keratectomy (PTK) is performed to remove a limited region of the epithelium over the cone while sparing stromal tissue. This makes PACE a partial epi-on/epi-off procedure, which means when riboflavin is applied in the next step, it generates a concentration gradient that peaks over the cone. Finally, the cone region receives a higher total fluence than the rest of the cornea.

Preliminary results demonstrate a more pronounced flattening effect compared to first-generation customized CXL, with observed reductions in corneal asymmetry of up to 12 D within the first month following the procedure.²⁴ This larger flattening enables a greater amount of visual rehabilitation.

Both first- and second-generation customized CXL approaches offer the advantage that, after corneal stabilization, PTK, PRK, intracorneal ring segment surgery, or a combination thereof, can further enhance corneal symmetry and reduce aberrations.

PACK-CXL and beyond: expanding indications

CXL’s potential extends beyond corneal ectasias. As mentioned above, CXL kills keratocytes in the stroma; it also kills pathogens. The ROS generated by the UV-riboflavin reaction directly attacks and damages pathogen cell membranes (leading to lysis) and intercalates with pathogen nucleic acids, inhibiting replication.^25,26 This is the same method blood banks use to sterilize blood products.

Not only does this property of CXL support its use in an office-based setting (such as at the slit lamp),²⁷ it is also a valuable tool in treating infectious keratitis and a method that sidesteps the issues of pathogen antimicrobial drug prophylaxis. A phase III trial has shown a single treatment with this ‘photoactivated chromophore for keratitis-CXL’ (PACK-CXL) procedure can be as effective as standard-of-care antimicrobial drug therapy for treating infectious keratitis of bacterial, fungal, or mixed bacterial/fungal origin.²⁸

Unlike with CXL for ectasia, it also appears increasing the UV intensity does not significantly compromise its efficacy; like CXL for ectasia, higher UV fluences increase efficacy.^29,30 The main reason is corneas with infectious keratitis are typically opaque, and UV transmission reduces significantly with ulcer depth.³¹ This also means higher fluences can be used safely concerning the corneal endothelium under these circumstances.

It is worth noting that other chromophore/light combinations are under investigation for PACK-CXL, principally rose Bengal and 532 nm green light (as seen in the image with this article). These have also shown good pathogen-killing efficacy and can be combined with riboflavin/UV CXL.³²

Conclusion: the future of CXL

CXL has gone from a single-indication ectasia treatment (keratoconus) to one used to treat several conditions, including iatrogenic post-refractive laser surgery ectasia, Terrien³³ and pellucid marginal degenerations,³⁴ and even keratoglobus.³⁵ Customized CXL protocols like PACE mean CXL can now be used not only to arrest ectasia progression but also to rehabilitate the shape of the cornea and improve patients’ vision.

PACK-CXL is a valuable addition to cornea specialists’ arsenal for treating infectious keratitis. As PACK-CXL can often succeed after a single treatment, it can hold particular value in low- to middle-income countries (LMICs), where physician and treatment costs are high and patients often lack the resources for a first (let alone a second) medical appointment. Indeed, the pathogen-killing nature of CXL, which enables it to be performed as safely in a doctor’s office as an operating room, is a cost-saving feature that can be particularly beneficial to LMICs.²⁷

The future of CXL probably encompasses further optimization of existing techniques, either when used alone or in combination with other procedures. One exciting prospect may be two-photon CXL, in which a femtosecond laser can be used to selectively cross-link parts of the cornea at specific points in three-dimensional space.^36–38 What value this might have—perhaps refractive, as a treatment for myopia and astigmatism in general, or rehabilitating irregular corneas—remains to be seen. But CXL still appears to have a great deal of potential yet to be revealed.

Part one of this series appeared in the November 2024 issue; part two appeared in the December issue.

Farhad Hafezi MD, PhD, FARVO is the medical director of the ELZA Institute in Zurich, Switzerland, and is an ESCRS board member.

Mark Hillen PhD is the communications director of the ELZA Institute.

Emilio Torres-Netto MD, PhD, FWCRS is a cornea, cataract, and refractive surgeon at the ELZA Institute.

1. Vinciguerra R, Legrottaglie EF, Tredici C, Mazzotta C, Rosetta P, Vinciguerra P. “Transepithelial Iontophoresis-Assisted Cross Linking for Progressive Keratoconus: Up to 7 Years of Follow Up,” J Clin Med, 28 Jan 2022; 11(3). doi:10.3390/jcm11030678

2. Mastropasqua L, Nubile M, Calienno R, et al. “Corneal cross-linking intrastromal riboflavin concentration in iontophoresis-assisted imbibition versus traditional and transepithelial techniques,” Am J Ophthalmol, Mar 2014; 157(3): 623–630 e1. doi:10.1016/j.ajo.2013.11.018

3. Hafezi F. “Corneal Cross-Linking: Epi-On,” Cornea, 2022; 41(10): 1203–1204. doi:10.1097/ICO.0000000000003075

4. Soeters N, Wisse RP, Godefrooij DA, Imhof SM, Tahzib NG. “Transepithelial versus epithelium-off corneal cross-linking for the treatment of progressive keratoconus: a randomized controlled trial,” Am J Ophthalmol, 2015; 159(5): 821–828 e3. doi:10.1016/j.ajo.2015.02.005

5. Torres-Netto EA, Kling S, Hafezi N, Vinciguerra P, Randleman JB, Hafezi F. “Oxygen Diffusion May Limit the Biomechanical Effectiveness of Iontophoresis-Assisted Transepithelial Corneal Cross-linking,” J Refract Surg, 2018; 34(11): 768–774. doi:10.3928/1081597X-20180830-01

6. Mazzotta C, Pandolfi A, Ferrise M. “Progressive high-fluence epithelium-on accelerated corneal crosslinking: a novel corneal photodynamic therapy for early progressive keratoconus,” Front Med (Lausanne), 2023; 10: 1198246. doi:10.3389/fmed.2023.1198246

7. Rajpal RK, Wisecarver CB, Williams D, et al. “LASIK Xtra^® Provides Corneal Stability and Improved Outcomes,” Ophthalmol Ther, 2015; 4(2): 89–102. doi:10.1007/s40123- 015-0039-x

8. Hira S, Klein Heffel K, Mehmood F, Sehgal K, Felix De Farias Santos AC, Steuernagel Del Valle G. “Comparison of refractive surgeries (SMILE, LASIK, and PRK) with and without corneal crosslinking: systematic review and meta-analysis,” J Cataract Refract Surg, 2024; 50(5): 523–533. doi:10.1097/j.jcrs.0000000000001405

9. Dong R, Zhang Y, Yuan Y, Liu Y, Wang Y, Chen Y. “A prospective randomized self-controlled study of LASIK combined with accelerated cross-linking for high myopia in Chinese: 24-month follow-up,” BMC Ophthalmol, 2022; 22(1): 280. doi:10.1186/s12886-022-02491-y

10. Kontadakis GA, Kankariya VP, Tsoulnaras K, Pallikaris AI, Plaka A, Kymionis GD. “Long-Term Comparison of Simultaneous Topography-Guided Photorefractive Keratectomy Followed by Corneal Cross-linking versus Corneal Cross-linking Alone,” Ophthalmology, 2016; 123(5): 974–983. doi:10.1016/j.ophtha.2016.01.010

11. Kanellopoulos AJ. “Management of progressive keratoconus with partial topography-guided PRK combined with refractive, customized CXL—a novel technique: the enhanced Athens protocol,” Clin Ophthalmol, 2019; 13: 581–588. doi:10.2147/OPTH.S188517

12. Kymionis GD, Grentzelos MA, Karavitaki AE, et al. “Transepithelial Phototherapeutic Keratectomy Using a 213-nm Solid-State Laser System Followed by Corneal Collagen Cross-Linking with Riboflavin and UVA Irradiation,” J Ophthalmol, 2010; 146543. doi:10.1155/2010/146543

13. Grentzelos MA, Liakopoulos DA, Siganos CS, Tsilimbaris MK, Pallikaris IG, Kymionis GD. “Long-term Comparison of Combined t-PTK and CXL (Cretan Protocol) Versus CXL with Mechanical Epithelial Debridement for Keratoconus,” J Refract Surg, 2019; 35(10): 650–655. doi:10.3928/108 1597X-20190917-01

14. Nattis A, Donnenfeld ED, Rosenberg E, Perry HD. “Visual and keratometric outcomes of keratoconus patients after sequential corneal crosslinking and topography-guided surface ablation: Early United States experience,” J Cataract Refract Surg, 2018; 44(8): 1003–1011. doi:10.1016/j. jcrs.2018.05.020

15. Nattis AS, Rosenberg ED, Donnenfeld ED. “One-year visual and astigmatic outcomes of keratoconus patients following sequential crosslinking and topography-guided surface ablation: the TOPOLINK study,” J Cataract Refract Surg, 2020; 46(4): 507–516. doi:10.1097/j. jcrs.0000000000000110

16. Rechichi M, Mazzotta C, Oliverio GW, et al. “Selective transepithelial ablation with simultaneous accelerated corneal crosslinking for corneal regularization of keratoconus: STARE-X protocol,” J Cataract Refract Surg, 2021; 47(11): 1403–1410. doi:10.1097/j.jcrs.0000000000000640

17. Shetty R, Vunnava K, Khamar P, Choudhary U, Sinha Roy A. “Topography-Based Removal of Corneal Epithelium for Keratoconus: A Novel and Customized Technique,” Cornea, 2018; 37(7): 923–925. doi:10.1097/ICO.0000000000001580

18. Randleman JB, Khandelwal SS, Hafezi F. “Corneal cross-linking,” Surv Ophthalmol, 2015; 60(6): 509–523. doi:10.1016/j.survophthal.2015.04.002

19. Kanellopoulos AJ, Dupps WJ, Seven I, Asimellis G. “Toric topographically customized transepithelial, pulsed, very high-fluence, higher energy and higher riboflavin concentration collagen cross-linking in keratoconus,” Case Rep Ophthalmol, 2014; 5(2): 172–180. doi:10.1159/000363371

20. Seiler TG, Fischinger I, Koller T, Zapp D, Frueh BE, Seiler T. “Customized Corneal Cross-linking: One-Year Results,” Am J Ophthalmol, 2016; 166: 14–21. doi:10.1016/j.ajo.2016.02.029

21. Nordstrom M, Schiller M, Fredriksson A, Behndig A. “Refractive improvements and safety with topography-guided corneal crosslinking for keratoconus: 1-year results,” Br J Ophthalmol, 2017; 101(7): 920–925. doi:10.1136/bjophthalmol-2016-309210

22. Cassagne M, Pierne K, Galiacy SD, Asfaux-Marfaing MP, Fournie P, Malecaze F. “Customized Topography-Guided Corneal Collagen Cross-linking for Keratoconus,” J Refract Surg, 2017; 33(5): 290–297. doi:10.3928/1081597X-20170201-02

23. Mazzotta C, Sgheri A, Bagaglia SA, Rechichi M, Di Maggio A. “Customized corneal crosslinking for treatment of progressive keratoconus: Clinical and OCT outcomes using a transepithelial approach with supplemental oxygen,” J Cataract Refract Surg, 2020; 46(12): 1582–1587. doi:10.1097/j.jcrs.0000000000000347

24. Hafezi F. “PACE (2nd generation customized CXL),” presented at International CXL Experts’ Meeting, 10 Dec 2022; Zurich, Switzerland.

25. Tabibian D, Mazzotta C, Hafezi F. “PACK-CXL: Corneal cross-linking in infectious keratitis,” Eye Vis (Lond), 2016; 3: 11. doi:10.1186/s40662-016-0042-x

26. Raiskup F, Herber R, Lenk J, Pillunat LE, Spoerl E. “Crosslinking with UV-A and riboflavin in progressive keratoconus: From laboratory to clinical practice—developments over 25 years,” Prog Retin Eye Res, 2024: 101276. doi:10.1016/j.preteyeres.2024.101276

27. Hafezi F, Richoz O, Torres-Netto EA, Hillen M, Hafezi NL. “Corneal Cross-linking at the Slit Lamp,” J Refract Surg, 2021; 37(2): 78–82. doi:10.3928/1081597X-20201123-02

28. Hafezi F, Hosny M, Shetty R, et al. “PACK-CXL vs. antimicrobial therapy for bacterial, fungal, and mixed infectious keratitis: a prospective randomized phase 3 trial,” Eye Vis (Lond), 2022; 9(1): 2. doi:10.1186/s40662-021-00272-0

29. Lu NJ, Koliwer-Brandl H, Gilardoni F, et al. “The Antibacterial Efficacy of High-Fluence PACK Cross-Linking Can Be Accelerated,” Transl Vis Sci Technol, 2023; 12(2): 12. doi:10.1167/tvst.12.2.12

30. Lu NJ, Koliwer-Brandl H, Hillen M, Egli A, Hafezi F. “High-Fluence Accelerated PACK-CXL for Bacterial Keratitis Using Riboflavin/UV-A or Rose Bengal/Green in the Ex Vivo Porcine Cornea,” Transl Vis Sci Technol, 2023; 12(9): 14. doi:10.1167/tvst.12.9.14

31. Hafezi F, Torres-Netto EA, Hillen MJP. “Re: Prajna et al.: Cross-Linking-Assisted Infection Reduction: a randomized clinical trial evaluating the effect of adjuvant cross-linking on outcomes in fungal keratitis (Ophthalmology, 2020; 127: 159–166),” Ophthalmology, Jan 2021; 128(1): e6. doi:10.1016/j.ophtha.2020.07.011

32. Hafezi F, Messerli J, Lu N, Aydemir E, Hillen M. “Successful Combination of Riboflavin/UV-A and Rose Bengal/ Green Light PACK Cross-Linking in Acanthamoeba Keratitis,” presented at European Society of Cataract and Refractive Surgeons Annual Meeting, 8–12 Sept 2023; Vienna, Austria.

33. Hafezi F, Gatzioufas Z, Seiler TG, Seiler T. “Corneal collagen cross-linking for Terrien marginal degeneration,” J Refract Surg, 2014; 30(7): 498–500. doi:10.3928/108159 7X-20140527-02

34. Spadea L. “Corneal collagen cross-linking with riboflavin and UVA irradiation in pellucid marginal degeneration,” J Refract Surg, 2010; 26(5): 375–377. doi:10.3928/108159 7X-20100114-03

35. Hafezi F, Torres-Netto EA, Randleman JBH, Nikki L, Mazzotta C, Ambrósio R, Kollros L. “Corneal Cross-linking for Keratoglobus Using Individualized Fluence,” Journal of Refractive Surgery Case Reports, 2021; 1(1). doi:10.3928/ jrscr-20210527-01

36. Kwok SJJ, Kuznetsov IA, Kim M, Choi M, Scarcelli G, Yun SH. “Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy,” Optica, 2016; 3(5): 469–472. doi:10.1364/OPTICA.3.000469

37. Batista A, Breunig HG, Hager T, Seitz B, Konig K. “Early evaluation of corneal collagen crosslinking in ex-vivo human corneas using two-photon imaging,” Sci Rep, 2019; 9(1): 10241. doi:10.1038/s41598-019-46572-3

38. Chang L, Zhang L, Cheng Z, et al. “Effectiveness of collagen cross-linking induced by two-photon absorption properties of a femtosecond laser in ex vivo human corneal stroma,” Biomed Opt Express, 2022; 13(9): 5067–5081. doi:10.1364/BOE.468593