A unique and biocompatible corneal collagen crosslinking in vivo

Abstract

Corneal crosslinking (CXL) is a widely applied technique to halt the progression of ectatic diseases through increasing the thickness and mechanical stiffness of the cornea. This study investigated the biocompatibility and efficiency of a novel CXL procedure using ruthenium and blue light in rat corneas and evaluated parameters important for clinical application. To perform the CXL procedure, the corneal epithelium of rats was removed under anaesthesia, followed by the application of a solution containing ruthenium and sodium persulfate (SPS). The corneas were then exposed to blue light at 430 nm at 3 mW/cm² for 5 min. Rat corneas were examined and evaluated for corneal opacity, corneal and limbal neovascularization, and corneal epithelial regeneration on days 0, 1, 3, 6, 8, and 14. On day 28, the corneas were isolated for subsequent tissue follow-up and analysis. CXL with ruthenium and blue light showed rapid epithelial healing, with 100% regeneration of the corneal epithelium and no corneal opacity on day 6. The ruthenium group also exhibited significantly reduced corneal (p < 0.01) and limbal neovascularization (p < 0.001). Histological analysis revealed no signs of cellular damage or apoptosis, which further confirms the biocompatibility and nontoxicity of our method. Confocal and scanning electron microscopy (SEM) images confirmed high density of collagen fibrils, indicating efficient crosslinking and enhanced structural integrity. This study is unique that demonstrates in vivo safety, biocompatibility, and functionality of ruthenium and blue light CXL. This approach can prevent toxicity caused by UV-A light and can be an immediate alternative compared to the existing crosslinking procedures that have side effects and clinical risks for the patients.

Keywords: Blue light; Collagen; Cornea; Crosslinking; In vivo; Ruthenium.

Fig. 1

A metal ring with an opening of 7 mm in diameter was positioned on the ocular surface to limit UV-A irradiation to the peripheral cornea.

Fig. 2

Corneal examination after the crosslinking procedure. (A) Eye images of the groups were taken under an ophthalmic microscope at different time intervals after the crosslinking procedure. Quantitative analysis of the scores calculated for each time interval. (B) Corneal opacity increased in all groups until day 3. The ruthenium group had a clear cornea from day 6 to day 14. Despite the improvement observed in the riboflavin group after day 3, there was still a noticeable and statistically significant difference in corneal opacity between the ruthenium-treated group and the riboflavin-treated group until day 14. (C) There was less corneal neovascularization in the ruthenium group than in the riboflavin group after day 6. Compared with the riboflavin group, the ruthenium group showed no corneal neovascularization, whereas the riboflavin group exhibited significantly greater corneal neovascularization scores on days 8 and 14. (D) The limbal neovascularization score showed that the ruthenium group had low vascularity in the cornea, and this effect was not detected after day 8. The data are expressed as the mean ± SEM (n = 4–6 rats/group), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3

Epithelial wound healing rate with fluorescein staining after corneal crosslinking. (A) Fluorescein-stained images of the control, riboflavin, and ruthenium groups were taken 0, 1, 3, 6, 8, and 14 days after the crosslinking procedure. The green color shows the stained areas, which indicate the absence of epithelial cells on the wounds. (B) The percentage of epithelial wound closure was calculated and compared at each time point. Wound closure in the ruthenium group was greater than that in the riboflavin group on days 1 and 3. The data are expressed as the means ± SEMs (n = 4–6 rats/group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4

Histological examination of corneal sections. At the end of 28 days, the enucleated eyes were fixed and sectioned for staining. (A) The results of hematoxylin and eosin staining showed that the integrity of the cornea was preserved in all groups. Compared with the control group, the ruthenium group showed no visible alterations in tissue structure. Scale bars represent 20 µm. (B) TUNEL assay indicating corneal crosslinking-induced apoptosis. DAPI (blue) shows cell nuclear immunofluorescence staining, and TUNEL (red) shows DNA fragmentation. (C) TUNEL-positive cells. Compared to those in the control and ruthenium groups, the number of TUNEL-positive cells throughout the cornea in the riboflavin group was greater. However, the number of TUNEL-positive cells in the control and ruthenium groups was not significantly different (p > 0.05). Scale bars represent 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5

Expression of corneal-specific cell markers in tissue sections. (A) CD31 is used as a marker for angiogenesis and inflammation. In a normal healthy cornea, CD31 expression is not detected. However, during wound healing, corneal neovascularization occurs, and CD31 is expressed in the central cornea. CD31 expression in the Riboflavin group is indicated by white arrows. (B) CK12 is a highly expressed intermediate filament in epithelial cells that maintains corneal structural integrity. All groups had similar CK12-labeled epithelial cells showing wound healing after 28 days of treatment. Scale bars represent 50 µm.

Fig. 6

Ultrastructure of corneal collagen fibers. (A) Collagen type-I staining showing the organization of collagen fibers and their integrity in the corneal stroma in all the groups. Red arrows indicate the collagen fibers in experimental groups. Scale bars represent 50 µm and 100 µm. (B) Scanning electron microscopy images of the cornea. Both the ruthenium and riboflavin groups exhibited a greater density of collagen fibrils and narrower interstitial spaces between the collagen layers compared to the control group, as shown in the magnified figure (right).