Research Article: Collagen cross-linking impact on keratoconus extracellular matrix

Date Published: July 18, 2018

Publisher: Public Library of Science

Author(s): Rabab Sharif, Ben Fowler, Dimitrios Karamichos, Rajiv R. Mohan.


Keratoconus (KC) is a common multifactorial ectatic corneal disease with unknown onset. KC most commonly appears in adolescence and affects approximately 1:400 people worldwide. Treatment options, for advanced KC cases, are collagen cross-linking (CXL) and corneal transplants. CXL is a new KC treatment that helps arrest the disease. Unfortunately, only a fraction of KC patients will qualify for CXL treatment. Our goal, in this study, was to begin to understand how CXL affects the corneal microenvironment and pave the way towards a more patient-driven CXL treatment.

Primary human corneal fibroblasts from healthy and KC donors were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. At 4 weeks, riboflavin was added followed by UVA irradiation. Transmission Electron Microscopy (TEM) and western blots were used to assess the effect of CXL on the extracellular matrix (ECM) and the resident cells, pre- and post CXL.

Data shows CXL improved lamellar organization showing more organized collagen fibrils decorated with proteoglycans (PGs). The distribution of the collagen fibrils and interfibrillar spacing was also visibly improved, post-CXL. Lumican, mimecan, and decorin were the dominant PGs and were significantly upregulated in post-CXL cultures. ECM degradation proteins, matrix metalloproteinases (MMPs), MMP-1, -3, and -9, but not MMP-2, were significantly downregulated post-CXL. TIMP-1 and -2 were not modulated by CXL.

The unknown effects of CXL on the human corneal microenvironment have hampered our ability to make CXL available to all KC patients. Our current study provides a deeper understanding on CXL activity, using our unique 3D in vitro model.

Partial Text

Keratoconus (KC) is a common ectatic corneal disease which impairs vision by causing corneal thinning, bulging and scarring [1]. Clinical findings include discomfort, visual disturbance, and possible blindness if left untreated [2]. KC is known to affect approximately 1:400 to 1:2000 people worldwide [3]. Initially, spectacles and rigid gas permeable lenses are used to correct the vision of KC patients [4]. As the disease progresses and refraction correction is no longer possible, the treatment for KC has traditionally been penetrating keratoplasty (PK) [5].

CXL or else known as the Dresden protocol was introduced in 2003 [16] and is currently considered the standard KC treatment. CXL requires removal of the epithelial layer followed by 30 minutes riboflavin application and 30 minutes UVA irradiation [16, 31]. CXL also requires that the riboflavin solution is applied every 3–5 minutes for the duration of the irradiation. While CXL is accepted as the standard protocol for CXL, the need for corneal epithelial debridement (for maximum riboflavin penetration) is accompanied by pain and discomfort [32, 33]. Epithelial debridement also leads to total corneal thickness reduction, with potential extremely unwanted clinical problems in thin corneas [34, 35].

Our 3D cell-based CXL model is unique in that it gives us the opportunity to determine and quantify the effect of CXL at the cellular and molecular level. The most critical outcomes from the current work are: 1) CXL is effective in strengthening and organizing the collagenous ECM, as shown by our TEM data, 2) Not all PGs respond the same to CXL, highlighting the need for delineating their role in KC, 3) CXL is selectively modulating MMPs in order to arrest ECM degradation, as indicated by the MMP/TIMP data.