Crosslinking Evidences In-Vitro and In-Vivo

Keratoconus is an ectatic disease of the cornea characterized by biochemical and biomechanical instability of stromal collagen leading to a reduction of corneal thickness [1], variation in posterior and anterior corneal curvatures and progressive deterioration of visual acuity due to irregular astigmatism [1, 2]. The recent advent of corneal collagen cross-linking in the panorama of ophthalmology of the last decade [3, 4] has transformed the conventional therapy of keratoconus, including in the best case scenario, rigid contact lens wearing for a lifetime or, at worst, corneal transplant. Conservative treatment has improved and thus reduced the necessity of a lamellar and penetrating corneal graft. Riboflavin UV-A induced corneal collagen cross-linking (CXL) demonstrated its efficacy in the conservative treatment of progressive keratoconus [3, 4] and secondary corneal ectasia [5] due to its ability to increase biomechanical corneal resistance [3, 4] and intrinsic anti-collagenase activity [6]. The physiochemical basis of cross-linking lies in the photo-dynamic type I-II reactions [7], induced by the interaction between 0.1% riboflavin molecules absorbed in the corneal tissue, and UV-A rays delivered at 3 mW/cm2 for 30 min (5.4 J/cm2 energy dose) releasing reactive oxygen species (ROS) that mediate cross-link formation between and within collagen fibers [8, 9]. The conventional epithelium-off cross-linking procedure (CXL) demonstrated its long-term efficacy stabilizing progressive keratoconus and secondary ectasia in different clinical trials [10–14]. Conventional CXL requires a long treatment time (1 h) [15]. A novel approach called Accelerated cross-linking (ACXL), based on the physical concept of photochemical reactions stated in the Bunsen–Roscoe’s law of reciprocity [16–18], has been recently proposed to shorten treatment time while maintaining the same efficacy. This theory [16] demonstrated that the photochemical process behind cross-linking depends on the absorbed UV-A energy, and its biological effect is proportional to the total energy dose delivered in the tissue [16–19]. Indeed, according to the “equal-dose” [18, 19] physical principle, 9 mW/cm2 for 10 min, 30 mW/cm2 for 3 min, 18 mW/cm2 for 5 min, 45 mW/cm2 for 2 min, at a constant energy dose of 5.4 J/cm2, have the same photochemical impact of conventional 3 mW/cm2 for 30 min [18, 19]. Moreover, an energy dose of 7.2 J/cm2 was demonstrated to be effective both in terms of corneal strengthening and anti-enzyme activity compared with the standard dose of 5.4 J/cm2, respectively tested by biaxial corneal extensiometry and papain digestion [20]. Previous histological reports in literature on conventional and accelerated corneal cross-linking [20, 21] demonstrated keratocyte damage and repopulation of the corneal stroma by proliferating cells, and an increase in collagen fiber diameter. These modifications are the morphological correlate of the process leading to increased corneal biomechanical stability. These observations were confirmed by in vivo studies in humans provided by Mazzotta et al. with scanning laser confocal microscopy which demonstrated the reduction in anterior and intermediate stromal keratocytes followed by gradual repopulation [22]. Long term keratoconus stability after conventional cross-linking treatment was correlated in vivo to increased cross-links formation, synthesis of restructured collagen and new lamellar interconnections [23].