Corneal crosslinking - A review

Purpose: To review cross-linking the cornea using riboflavin and ultraviolet A light, which has been widely adopted, refined and applied in a range of corneal surgeries and pathologies where the strength of the cornea might be compromised. Recent findings: A large number of clinical trials have been carried out, most of which have demonstrated that standard cross-linking is a successful method to halt the progression of keratoconus or even aid regression. Summary: This review describes our current understanding of the technique, focussing on how cross-linking works, how the treatment is being optimised, the clinical results that have been reported to date and the potential use of the therapy in the treatment of other corneal disorders

An investigation into corneal enzymatic resistance following epithelium-off and epithelium-on corneal cross-linking protocols

The aim of this study was to investigate corneal enzymatic resistance following epithelium off and on riboflavin/UVA cross-linking (CXL). One hundred and fourteen porcine eyes were divided into four non-irradiated control groups and seven CXL groups. The latter comprised; (i) epithelium-off, 0.1% iso-osmolar riboflavin, 9 mW UVA irradiation for 10 min, (ii) disrupted epithelium, 0.1% hypo-osmolar riboflavin, 9 mW UVA for 10 min, (iii) epithelium-on, 0.25% hypo-osmolar riboflavin with 0.01% benzylalkonium chloride (BACS), 9 mW UVA for 10 min, (iv) epithelium-on, 5 min iontophoresis at 0.1 mA for 5 min with 0.1% riboflavin solution, 9 mW UVA for 10 min or (v) 12.5 min, (vi) epithelium-on, prolonged iontophoresis protocol of 25 min with 1.0 mA for 5 min and 0.5 mA for 5 min with 0.25% riboflavin with 0.01% BACS, 9 mW UVA for 10 min or (vii) 12.5 min. Enzymatic resistance was assessed by daily measurement of a corneal button placed in pepsin solution and measurement of corneal button dry weight after 11 days of digestion. This study revealed that the enzymatic resistance was greater in CXL corneas than non-irradiated corneas (p < 0.0001). Epithelium-off CXL showed the greatest enzymatic resistance (p < 0.0001). The prolonged iontophoresis protocol was found to be superior to all other trans-epithelial protocols (p < 0.0001). A 25% increase in UVA radiance significantly increased corneal enzymatic resistance (p < 0.0001). In conclusion, although epithelium-on CXL appears to be inferior to epithelium-off CXL in terms of enzymatic resistance to pepsin digestion, the outcome of epithelium-on CXL may be significantly improved through the use of higher concentrations of riboflavin solution, a longer duration of iontophoresis and an increase in UVA radiance

An X-ray diffraction investigation of corneal structure in lumican-deficient mice

PURPOSE. The corneas of mice homozygous for a null mutation in lumican, a keratan sulfate–containing proteoglycan, are not as clear as normal. In the present study, mutant corneas were examined by synchrotron x-ray diffraction to see what structural changes might lie behind the loss of transparency. METHODS. X-ray diffraction patterns were obtained from the corneas of 6-month-old and 2-month-old lumican-null and wild-type mice. Measured in each cornea were the average collagen fibril diameter, average collagen fibril spacing, and the level of order in the collagen array. RESULTS. The x-ray reflection arising from regularly packed collagen was well-defined on all x-ray patterns from 6-month-old wild-type corneas. Patterns from 6-month-old lumican-deficient corneas, however, contained interfibrillar reflections that were measurably more diffuse, a fact that points to a widespread alteration in the way the collagen fibrils are configured. The same distinction between mutant and wild-type corneas was also noted at 2-months of age. Average collagen fibril spacing was marginally higher in corneas of 6-month-old lumican-null mice than in corneas of normal animals. Unlike x-ray patterns from wild-type corneas, patterns from lumican-deficient corneas of both ages registered no measurable subsidiary x-ray reflection, evidence of a wider than normal range of fibril diameters. CONCLUSIONS. The spatial arrangement of stromal collagen in the corneas of lumican-deficient mice is in disarray. There is also a considerable variation in the diameter of the hydrated collagen fibrils. These abnormalities, seen at 2 months as well as 6 months of age, probably contribute to the reduced transparency

Effects on collagen orientation in the cornea after trephine injury

Purpose: Structural changes are well known to occur in the cornea after injury. The aim of this study was to investigate collagen orientation changes in the cornea during a short-term wound healing process. Methods: Seven bovine corneas were injured using a penetrating 5 mm biopsy punch and were subsequently organ cultured for up to two weeks. Six uninjured corneas acted as controls. The trephine wounded samples were snap frozen in liquid nitrogen either immediately after injury (0 h) or after 1 or 2 weeks in culture. Control/uninjured samples were snap frozen on arrival (0 h) or after 1 or 2 weeks in culture. Wide angle X-ray diffraction data were collected from each cornea at the UK Synchrotron Radiation Source or at the European Synchrotron Radiation Facility. Data analysis revealed information about collagen orientation and distribution in the corneal stroma during wound healing. For histology, two trephine wounded corneas at 0 h and 1 week and one control/uninjured cornea at 0 h were fixed in 10% neutral buffered formalin and processed for wax embedding. Wax sections were subsequently counterstained with haematoxylin and eosin to observe tissue morphology and the time course of complete re-epithelialization. Results: Immediately after injury, collagen organization was altered in a small area inside the wound but remained similar to the control/uninjured sample in the remainder of the tissue. After one week, the trephine wounded corneas showed complete re-epithelialization and evidence of swelling while collagen adopted a radial arrangement inside and outside the wound. Conclusions: Remarkable changes in collagen fibril orientation were observed in trephine wounded corneas. Orientation changes immediately after wounding are likely to be due to the mechanical deformation of the tissue during the wounding process. However, tissue swelling and changes in collagen orientation at later stages probably reflect the processes of tissue repair. These differences will determine corneal stability and strength following trauma and possibly refractive surgery

Mapping collagen organization in the human cornea: left and right eyes are structurally distinct

PURPOSE. Aspects of the biomechanics and surface topography of fellow human corneas are known to exhibit midline symmetry, but the structural basis of these observations is poorly understood. The mechanical performance of the cornea is strongly influenced by the organization of stromal collagen fibrils. The present study was designed to examine and compare the organization of collagen fibrils in the corneal stroma of left and right eyes. METHODS. Wide-angle x-ray scattering was used to map in detail the orientation and distribution of fibrillar collagen across the cornea, limbus, and adjacent sclera of three normal human eyes, including a fellow pair, and the central 9-mm corneal region of a further four eyes. RESULTS. Fibrillar collagen in the human cornea and limbus is arranged anisotropically, and in a highly specific manner. Left and right corneas are structurally distinct. In general, the mass distribution of preferentially aligned fibrils in the cornea appears to exhibit a degree of midline symmetry between left and right eyes. CONCLUSIONS. Structural information, such as that presented herein, will enable a better understanding of corneal biomechanics and shape. Midline symmetry in the distribution of aligned, mechanically reinforcing collagen fibrils between left and right eyes may relate to the biomechanical and topographical enantiomorphism reported in the literature

Elastic microfibril distribution in the cornea: Differences between normal and keratoconic stroma

The optical and biomechanical properties of the cornea are largely governed by the collagen-rich stroma, a layer that represents approximately 90% of the total thickness. Within the stroma, the specific arrangement of superimposed lamellae provides the tissue with tensile strength, whilst the spatial arrangement of individual collagen fibrils within the lamellae confers transparency. In keratoconus, this precise stromal arrangement is lost, resulting in ectasia and visual impairment. In the normal cornea, we previously characterised the three-dimensional arrangement of an elastic fiber network spanning the posterior stroma from limbus-to-limbus. In the peripheral cornea/limbus there are elastin-containing sheets or broad fibers, most of which become microfibril bundles (MBs) with little or no elastin component when reaching the central cornea. The purpose of the current study was to compare this network with the elastic fiber distribution in post-surgical keratoconic corneal buttons, using serial block face scanning electron microscopy and transmission electron microscopy. We have demonstrated that the MB distribution is very different in keratoconus. MBs are absent from a region of stroma anterior to Descemet's membrane, an area that is densely populated in normal cornea, whilst being concentrated below the epithelium, an area in which they are absent in normal cornea. We contend that these latter microfibrils are produced as a biomechanical response to provide additional strength to the anterior stroma in order to prevent tissue rupture at the apex of the cone. A lack of MBs anterior to Descemet's membrane in keratoconus would alter the biomechanical properties of the tissue, potentially contributing to the pathogenesis of the disease

Standard versus accelerated riboflavin/ultraviolet corneal cross-linking: Resistance against enzymatic digestion

Purpose To examine the effect of standard and accelerated corneal collagen crosslinking (CXL) on corneal enzymatic resistance. Setting School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom. Design Experimental study. Methods Sixty-six enucleated porcine eyes (with corneal epithelium removed) were assigned to 6 groups. Group 1 remained untreated, group 2 received dextran eyedrops, and groups 3 to 6 received riboflavin/dextran eyedrops. Group 4 had standard CXL (3 mW/cm2 ultraviolet-A for 30 minutes), whereas groups 5 and 6 received accelerated CXL (9 mW/cm2 for 10 minutes and 18 mW/cm2 for 5 minutes, respectively). Trephined central 8.0 mm buttons from each cornea underwent pepsin digestion. Corneal diameter was measured daily, and the dry weight of 5 samples from each group was recorded after 12 days of digestion. Results All CXL groups (4 to 6) took longer to digest and had a greater dry weight at 12 days (P accelerated CXL 9 mW > accelerated CXL 18 mW (P < .0001). Conclusions Standard and accelerated CXL both increased corneal enzymatic resistance; however, the amount of CXL might be less when accelerated CXL is used. The precise amount of CXL needed to prevent disease progression is not yet know

Enzymatic resistance of corneas crosslinked using riboflavin in conjunction with low energy, high energy, and pulsed UVA irradiation modes

Purpose: To investigate the effect of various riboflavin/ultraviolet light (UVA) crosslinking (CXL) protocols on corneal enzymatic resistance. Methods: A total of 66 enucleated porcine eyes, with the corneal epithelium removed, were divided into 6 groups. Group 1 remained untreated. Groups 2 to 6 received riboflavin/dextran for 30 minutes. Group 3 underwent standard CXL (SCXL) with 3 mW/cm2 UVA for 30 minutes (total energy dose 5.4 J/cm2). Groups 4 and 5 underwent high intensity CXL (HCXL) using 30 mW/cm2 UVA for 3 minutes (5.4 J/cm2) and 30 mW/cm2 for 4 minutes (7.2 J/cm2), respectively. Group 6 was exposed to 8 minutes of 30 mW/cm2 UVA in a 10-second on/10-second off pulsed-radiation mode (p-HCXL; 7.2 J/cm2). A central 8-mm disk from each cornea was submerged in pepsin digest solution at 23°C and measured daily. After 13 days, the dry weight was recorded from 5 samples in each group. Results: The CXL-treated corneas took longer to digest than nonirradiated corneas (P < 0.0001). Differences in digestion time also were observed between CXL groups, such that, HCXL (5.4 J/cm2) < SCXL (5.4 J/cm2) < HCXL (7.2 J/cm2) < p-HCXL (7.2 J/cm2; P < 0.0001). The dry weight of the SCXL (5.4 J/cm2) group was higher than the HCXL (5.4 and 7.2 J/cm2; P < 0.001) and p-HCXL 7.2 J/cm2 (P <0.05) groups. No difference was detected between the HCXL and p-HCXL 7.2 J/cm2 groups. Conclusions: The intensity and distribution of the crosslinks formed within the cornea vary with different UVA protocols. The precise location and amount of crosslinking needed to prevent disease progression is unknown

Collagen organization in the secondary chick cornea during development

PURPOSE. The latter stages of morphogenesis in the embryonic chick cornea are instrumental in the establishment of a properly formed corneal stroma. This study was designed to provide better appreciation of collagen reorganization in the avian corneal stroma during the latter stages of embryogenesis. METHODS. High-angle synchrotron x-ray diffraction patterns were obtained from 47 developing chick corneas daily at developmental days 13 through 18 (n = 7 or 8 at each time point) and analyzed to establish collagen molecular spacing and fibril orientation. RESULTS. Collagen intermolecular x-ray reflections were of approximately constant intensity between days 13 and 15 of development, but thereafter became progressively more intense, suggesting that extra collagen is deposited in embryonic chick corneas after day 16 of development. At all times, the mean collagen intermolecular spacing measured approximately 1.43 nm. X-ray intensity was not uniform around the intermolecular x-ray reflections at earlier time points. Rather, a fourfold symmetry was evident, indicative of an orthogonal array of collagen fibrils. An index of this symmetry was essentially unchanged between developmental days 13 and 15, but thereafter diminished considerably. CONCLUSIONS. The lateral spacing of fibril-forming collagen molecules does not change as the chick cornea develops between days 13 and 18. An orthogonal array of collagen fibrils is present in the corneas of developmental day-13 to -18 chicks, but starting at developmental day 16, additional collagen is deposited in a less well-oriented manner and thus acts to obscure the overall orthogonality, with implications for the biomechanical strength and shape of the cornea

Changes in corneal collagen architecture during mouse postnatal development

Purpose. To characterize changes in corneal collagen arrangement during mouse postnatal development. Methods. Small-angle X-ray scatter patterns were gathered from the centers of 32 excised mice corneas aged between postnatal days 10 (before eye opening) and 28 (onset of sexual maturity). These were analyzed to produce measurements of the average separation distance between corneal collagen fibrils. Changes in the predominant orientation of corneal collagen and its relative distribution during the same developmental period were determined using wide-angle X-ray scatter data collected at 0.2-mm intervals over the entire cornea and limbal region of each specimen. Results. Collagen interfibrillar spacing decreased in the days leading up to eye opening (61.3 ± 2.9 nm at day 10 to 45.5 ± 4.5 nm at day 14), after which it remained constant. However, changes in collagen orientation and distribution occurred throughout the entire developmental period. After eye opening at day 12, collagen alignment gradually increased in the peripheral cornea and limbus. By day 28, an annulus of highly aligned collagen surrounded the cornea. Conclusions. Changes in corneal thickness before and after eye opening are not caused by widespread alterations in the collagen fibrillar array but are more likely caused by expansion and contraction of regions devoid of regularly arranged collagen. The postnatal development of a corneal annulus of collagen, thought to play a role in stabilizing the curvature of the cornea, may be triggered by visual factors