Treatment of Keratoconus by Collagen Cross-Linking with Riboflavin (C3R)

Dr. Neera Agrawal, Neera Eye Centre, Darya Ganj, New Delhi

Keratoconus is a bilateral non-inflammatory corneal ectasia (fig.1) with an incidence of approximately 1 per 2,000 in the general population. It normally presents in early teens or twenties and is known to be progressive. Despite intensive clinical and laboratory investigation, the aetiology of keratoconus remains unclear. Thinning of the corneal stroma, breaks in Bowman’s layer, and deposition of iron in the basal layers of the corneal epithelium comprise a triad of the classical histopathologic features found in keratoconus.

Fig. 1- Natural Progression of Keratoconus in a patient over 1.5 years

Till date, accepted methods of treatment have been contact lenses, intra-stromal corneal rings (INTACS), Photo-refractive keratectomy and cornea transplant. Cornea transplant is reserved for advanced cases in which vision can not be improved with contact lenses. Unfortunately none of these modalities prevent the progression of the disease. Now with the advent of Collagen Cross-Linking with Riboflavin and UVA, there is scope to arrest the progression of disease in these patients. Moreover, it is technically simple and less invasive than all other surgical therapies proposed for Keratoconus.

Mechanism of action

Similar to photo polymerization of polymers, Collagen Cross-Linking of Cornea using ultraviolet light and the Photo-sensitizer Riboflavin was developed to treat corneal thinning and ectasia by increasing the biomechanical strength of the tissue. This procedure is also addressed by various eponyms such as C3R, CXL and CCL.

First studies in Photobiology began in 1990’s, with attempts to identify biological glues that could be activated by heat or light to increase resistance of stromal collagen (2). It was discovered that the gluing effect was mediated by oxidative mechanism associated with hydroxyl radical release. A similar mechanism of natural hardening and thickening of collagen fibres has been demonstrated in corneal aging (3). Similarly, young diabetics never develop progressive keratoconus due to natural cross linking effect of glucose, which increases corneal resistance.

Fig. 2- Mechanism of action : C3R

Fig. 3- Mechanism of action : C3R

Collagen cross linking results in an increase in inter-fibrillar covalent bonds by photosensitized oxidation (fig.4), and causes bio-mechanical stabilization of cornea. A significant increase of 328.9% in the biomechanical rigidity of human corneas has been documented after collagen cross linking (5).

Fig.4- Effect of Collagen Cross Linking on Corneal Collagen

Fig. 5 – Effect of Cross-Linking on Cornea

Riboflavin plays a dual role in the procedure; it not only acts as a photo sensitizer for the photo-oxidative cross linking process but also has a barrier effect. It acts like a shield and prevents UV induced collateral damage to sensitive ocular structures like Corneal Endothelium, Lens and Retina.

Quality of Beam

All safety considerations regarding the Cross-Linking procedure assume an optically homogenous irradiation of the cornea. Optical in-homogeneities can lead to damage to corneal endothelium, which represents most endangered structure. Thus, having homogenizing optics and as well as pre-compensation for corneal curvature in the system is important (fig.6).

Fig. 6: Quality of beam for C3R


Most common indications are –

1. Mild to moderate Keratoconus – In a case of Keratoconus, the selection criteria for C3R are as follows -
· There should not be corneal scarring.
· There should be reasonably good acuity with glasses or contact lenses.
· Corneal thickness should be more than 450 microns (400 microns after epithelium removal) at thinnest point. This is based on the fact that UV light can penetrate cornea up to a thickness of 350 microns and will damage endothelium if enough corneal thickness is not there. That is why some people advocate doing specular microscopy before and after the procedure.
· Age should more than 16 yrs.
· Maximum K readings should be < 60Diopters.
· Keratoconus must be documented to be progressive. Progression of Keratoconus is ideally determined by Kaplan Meier or Fourier analysis which are based on Spherical equivalent power, Decentration component and Irregular astimatism component and calculation their yearly rate of progression. It is generally accepted that 1 dioptre increase in the power of cone per year is an indicator of progression of Keratoconus. It can also be seen as steepening of Keratometry readings (17)

2. Post Lasik Ectasia – Good results have been reported in treatment of post Lasik ectasia (13).

3. Progressive Hyperopia post RK – good results have been reported (12).

4. Pellucid Marginal Degeneration

5. Bullous Keratopathy – reduces Corneal oedema by increasing stromal compaction after Cross-linking. It may be combined with Intra-stromal administration of 0.1% Riboflavin with the help of Femtosecond laser (Can also be staged i.e. anterior and posterior separately)(16).

6. Infective Keratitis – to prevent Corneal Perforation by increasing resistance to the effect of collagen digesting enzymes.(16)

7. Scleral CXL for Glaucoma and Pathological Myopia (16)

Types of C3R

C3R surgery can be of several types depending on the technique used –

1. C3R Epi-off – this is the most commonly used technique where you remove the epithelium mechanically. Commonly follows Dresden Protocol which means that you instill Riboflavin for every 2 min for 30 min and cross link for 30 min at 3 MW energy. It needs min Corneal thickness of 400 microns after epithelium removal. It has been there for longest time and has proven its efficacy.

2. Trans-epithelial C3R or epi-on C3R – here we use special Riboflavin which can penetrate through epithelium and do not remove epithelium, rest of the protocol is same as above. However, I have noticed that despite leaving the epithelium in situ, healing is much slower after this procedure compared to epi-off C3R.

3. Rapid C3R – here Riboflavin can be of any of the above type but machine setting used are 9 MW for 10 min or 18 MW for 5 min. The efficacy and safety data on this type is still awaited.

4. Lasik Extra – In this treatment C3R is combined with Lasik surgery. After making the flap Riboflavin is directly put on the flap and cross linking is done with short duration of settings like 5 min or 2 min. It is said to be useful for suspected ectasia cases where you would like to do Lasik. However, there are no studies to prove its efficacy. In my opinion, it may help in preventing ectasia but will significantly alter the refractive result by causing flattening.

Types of Riboflavin in C3R-

1. Iso-tonic Riboflavin
2. Hypo-tonic Riboflavin
3. Trans-epithelial Riboflavin
4. Rapid Ribo-flavin
5. Riboflavin for Lasik Extra

Iso-tonic Riboflavin

It is of two types –1. Riboflavin in Dextran
2. Riboflavin in HPMC

Iso-tonic Riboflavin with Dextran

It contains Riboflavin 0.1% in 20% Dxtran 500. It is time tested and gives predictable results. It causes better Corneal staining. Unfortunately not many companies produce this.

Iso-tonic Riboflavin with HPMC

Fig 7: Riboflavin with HPMC

Contains 0.1% Riboflavin with 1.1% HPMC.

Causes poor Corneal staining. I have noticed higher incidence of post operative Corneal oedema and Descemets folds after this.

Hypo-tonic RiboflavinIt is used for Corneas which are thin for Iso-osmolar Ribo-flavin. It is used after use of Iso-osmolar Riboflavin for 30 min. Therefore, you will need both types of Riboflavins for one case. It can swell Corneas upto 60 microns but the flip side is that effect lasts only for 10-30 minutes. You will keep repeating its use after repeating the per-op Pachymetry. That is why its use has gone down specially with advent of Trans-epithelial Riboflavin.

Trans-epithelial Riboflavin

Contains EDTA or BAK, which loosen the bonds in epithelium and allow Riboflavin to penetrate. Different companies have different composition.

Fig 8 :Trans-epithelial Riboflavin

Fig 9:Trans-epithelial Riboflavin

Trans-epithelium Riboflavin with IontophoresisIs a new modality. It reduces the diffusion time to 5 min.

Main advantage is that it is less toxic to the epithelium. It substantially reduces post operative pain and debility to the patient.

Rapid Riboflavin

Contains Riboflavin 0.1%, Saline and HPMC
Has a diffusion rate twice of normal Riboflain
Used only as epi-off .

Fig 10:Rapid Riboflavin

Riboflavin for Lasik Extra
Contains Riboflavin 0.22% in Saline and is applied to Stromal Bed.

Fig 11: Riboflavin for Lasik Extra

Technique ( Dresden Protocol)The procedure is conducted in an operating room under sterile conditions. Topical anaesthesia is given by Proparacaine Hydrochloride (0.5%) eye drops & Xylocaine 4% drops. Central 7-8 mm of corneal epithelium is removed by mechanical debridement (gentle scraping with hockey stick knife) or Alcohol. My personal preference is mechanical debridement.

This is followed by instillation of 0.1% Riboflavin (3 mg Riboflavin - 5 Phosphate in 3 ml of Dextran-T-500 20% solution) eye drops at every 2 minute interval for 30 minutes (15 times).

To confirm adequate penetration of Riboflavin into the cornea, the patient may be examined on slit lamp. A greenish flare, similar to that seen with fluorescein dye instillation, may be seen in anterior chamber, signifying penetration through the thickness of cornea.

Fig.12 - C3R being done in a patient

There are studies available which say that epithelial debridement is not necessary. However, it’s mandatory to demonstrate the penetration of the riboflavin in the corneal stroma when performing the treatment without removing the epithelium.

Following this, the cornea is subjected to Ultraviolet A radiation (365nm) from a distance of 5 cm (Peschke Meditrade UVX system was used) for the next 30 minutes (fig.6). During this period, topical Riboflavin drops are again instilled at 5 minute intervals, to complete photo-sensitization and provide photo-protection by the ‘barrier’ effect. Thereby, a dose of 3 mW/ Sq cm (+ 0.3 Mw) (5.4 J/Sq cm) UV-A is delivered. The UV-A radiation lamp is checked for calibration with a UV- meter before and after the treatment. On completion of the treatment, eye is patched with antibiotic eye ointment or sterile bandage lens is applied.

Post-operative Care

Topical antibiotics along with lubricants and viscous tear substitute, mild steroids are prescribed 4-5 times a day for 3-4 weeks. Complete re-epithelization usually occurs in 3-4 days. Patients may experience mild pain and discomfort for first 2 days, for which oral analgesics are also required to keep them comfortable.

A mild epithelial haze with transient, mild stromal oedema may be seen in some cases after C3R, which usually disappears completely in a few weeks. There is no damage to the corneal endothelium.

Histopathological changes in cornea post C3R

C3R treatment leads to a dose-dependent keratocyte apoptosis (1) that can be expected in human corneas to a depth of 300 microns from the anterior surface utilising a surface UV-A dose of 5.4 J/ sq cm. In the first few weeks after the procedure, a ‘vertical transition line’ may be visible on the slit lamp, delineating the anterior cross-linked zone with the posterior unaffected stroma (fig.13).

Repopulation of corneal stroma may take up to 6 months. It is well documented that the corneal epithelium attains a regular morphology and density within 5 days after C3R. Initially, disappearance of the sub-epithelial stromal nerve fibres was observed in the treated area and initial re-innervation was seen one month after the procedure. Complete recolonization of the anterior sub-epithelial stroma by the keratocytes was observed in 6 months after C3R, with restoration of corneal sensitivity.

Figure 13- Effect of Cross-Linking on cornea

Histopathologically, it has been demonstrated that there is a significant increase in collagen fibre diameter after collagen cross linking. After Collagen Cross-Linking, the cornea shows an increase in thermo-mechanical stability as well as a markedly increased resistance to collagen digesting enzymes (5).


· C3-R appears to stabilize corneas up to six months after treatment.
· In a 5 year study, it was noted that in all treated eyes, the progression of keratoconus was at least stopped. About 70% eyes show regression with a reduction of the maximal keratometry readings by 2 dioptres and of the refractive error by 1 D was found (14). Best-corrected visual acuity improved by 1.4 lines. I have personally experienced similar results in my 7 years experience with the procedure.
· The addition of C3-R to the INTACS procedure results in greater keratoconus improvements compared to INTACS alone (10).
· There are reports available that surface ablation procedures could be used to correct refractive errors following C3R in Keratoconus patients but conclusive evidence is lacking.

Fig 14:About 5 D Corneal Flattening after C3R

Fig.15: About 4 D Corneal Flattening after C3R

Fig 16: Corneal Flattening after C3R in PMD


During corneal Cross-Linking, 95% of UVA energy is absorbed by only 350 microns of Riboflavin soaked Cornea (fig.17). As long as the treated cornea has a minimum thickness of 400 microns (after removal of epithelium), the corneal endothelium will not experience damage, nor will deeper structures such as lens and retina.

Fig. 17 - Absorption of UVA by Cornea

The light source should provide a homogenous irradiance, avoiding hot spots.

It has been seen that corneal and lens transparency, endothelial cell density, and intraocular pressure remained unchanged after the procedure (9).


C3R for ectatic Disorders of Cornea has revolutionised the treatment of Keratoconus and other ectatic disorders. Cornea Collagen Cross-linking with Riboflavin is a simple, safe and effective procedure in the management of early progressive ecstatic disorders of the cornea.

Different types of Riboflavin and machines have made this techniques useful in more number of patients.


1. Apoptosis After Corneal Collagen Cross-linking using Riboflavin/UVA treatment. Wollensak G. et al. Cornea 2004; 23: 43-49
2. Photodynamic biologic tissue glue. J. Khaderm, T. Truong, J.T. Ernest. Cornea 1994;13: 406-410
3. Collagen fibrils in the Human corneal stroma . structure and aging. A. Dover, K. Misof et al. Invest. Ophthalmol. Vis. Sc. 1998; 39: 644-648
4. Stress-strain measurements of human and porcine corneas after Riboflavin- ultraviolet-A induced cross-linking. Wollensak G.,Spoerl E., Seiler T. J Cataract Refract Surg. 2003;29:1780-1785
5. Increased resistance of cross-linked cornea against enzymatic digestion. Seiler T., Wollensak G., Spoerl E. Curr. Eye Res. 2004 July; 29(1): 35-40.
6. Thermo-mechanical behaviour of collagen cross linked porcine cornea. Spoerl E., Wollensak G., Dittert D.D., Seiler T., Ophthalmologica.2004, Mar-Apr; 218(2):136-140
7. C3-R Treatment Opens New Frontiers for Keratoconus and Corneal Ectasia. Roberto Pinelli, MD. Eyeworld 2007, 34-36
8. Safety of C3-R UVA at the Retinal Level and Compared to Outdoor UVA Exposure Leonard Yuen, MD and Brian S. Boxer Wachler, MD
9. Safety of UVA-Riboflavin Cross-Linking of the Cornea Eberhard Spoerl, PhD, Michael Mrochen, PhD, David Sliney PhD, Stephen Trokel, MD, Theo Seiler, MD, PhD, Cornea 2007; 26:385-389
10. The Effect of Inferior Segment Intacs with and without Corneal Collagen Crosslinking with Riboflavin (C3-R) on Keratoconus. Colin C.K. Chan, MD, FRANZCO, Munish Sharma, MD, Brian S. Boxer Wachler MD, J Cataract Refract Surg 2007;33:75-80
11. No Progression of Keratoconus 5 Years after C3-R. Wollensak G. Crosslinking treatment of progressive keratoconus: new hope. Curr Opin Ophthalmol. 2006 Aug; 17:356-60.
12. C3-R for Stabilization of Progressive Hyperopia (Farsightedness) after Radial Keratotomy and Laser Ablation. Presented at the American Academy of Ophthalmology Annual Meeting, 2006
13. Visual rehabilitation and outcomes of ectasia after corneal refractive surgery. Woodward MA, Randleman JB, Russel B, Lynn MJ, Ward MA, Stulting RD - J. cataract Ref. surg. 2008 mar; 34(3): 383-8
14. Riboflavin/UVA induced collagen cross-linking for treatment of Keratoconus. G. Wollensak, MD et al, Am. J. Ophthal. 2003 May; 135(2) : 620-627
15. Staged Intra-stromal Delivery of Riboflavin with UV-A Cross-linking in Advanced Bullous Keratopathy. Krueger RR, Ramos-Esteban JC, Kannelopoulos J – J. Refract. Surg. 2008; 730-736
16. Corneal Cross-linking for Different Corneal Diseases. Aylin Kilic Ertan – Cataract & Refractive Surgery Today Europe. April 2009; 25-28
17. Progression of Keratoconus Assessed by Fourier Analysis of Videokeratography Data. Tetsuro Oshika, Tatsuro Tanabe, Atsuo Tomidokoro, Shiro Amano – Ophthalmology 2002; 109:339-342

For Reprints request to -

Dr. Neera Agrawal
Neera Eye Centre
B-99, Bharat Ram Road
Opposite Commercial School
Darya Ganj
New Delhi- 110002
Phone: 23270775, 9810134653

No comments

Powered by Blogger.