Effects of riboflavin/ultraviolet-A(UVA) scleral crosslinking on the mechanical behavior of the scleral fibroblasts of lens-induced myopia Guinea pigs.

Myopia is becoming increasingly severe, and studies have shown that the cellular mechanics of scleral fibroblasts are altered following myopia. Scleral UVA-Riboflavin Collagen Crosslinking（sCXL） is a promising treatment for myopia prevention and control of axial growth. Understanding the mechanical properties of scleral fibroblasts is crucial, as it influences the cellular response and limits the extent of molecular deformation triggered. Thus, our study aimed to investigate the effect of mechanical properties of scleral fibroblasts in a lens-induced myopic guinea pig model following sCXL. For this purpose, we performed the 0.1% riboflavin/UVA scleral crosslinking (365 nm,3 mW/cm2,30 min) in the right eyes of guinea pigs in Group CXL. In Group LIM, the right eyes were only administrated negative lens for 6 weeks. No treatment was performed in both eyes of the guinea pigs in group Control. The scleral fibroblasts were isolated and cultured from the scleral tissue at the cross-linking area in Group CXL and the corresponding area in Group LIM and control. The curve of the length of microtubules inhaled by cells under negative pressure was measured by a microaspiration-based isolation technique, and the equilibrium Young's modulus and apparent viscosity of scleral fibroblasts were calculated by formula fitting. The equilibrium Young's modulus of scleral fibroblasts in group CXL was significantly lower than that in the LIM group (P < 0.01, two-sample t-test between pairs）, and there was no significant difference between groups CXL and control. The results show that sCXL can effectively moderate the phenomenon that scleral fibroblasts are not easy to deform after myopia. The apparent viscosity modulus in the CXL group was higher than the groups' control and LIM. Taken together, our data demonstrate the biomechanics of the scleral fibroblasts altered after Riboflavin/UVA scleral collagen cross-linking in a lens-induced myopia model.

Corneal biomechanical characteristics following small incision lenticule extraction for myopia and astigmatism with 3 different cap thicknesses.

BACKGROUND: The design of cap thickness for small incision lenticule extraction (SMILE) plays a role in post-laser vision correction (post-LVC) corneal biomechanics. This study aimed to compare the corneal biomechanical characteristics following SMILE with different cap thicknesses of 110 µm, 120 µm, and 130 µm for myopia and myopic astigmatism correction. METHODS: Seventy-five patients (146 eyes) who underwent SMILE with designed cap thickness of 110 µm, 120 µm, and 130 µm were recruited at the Eye Center of Beijing Tongren Hospital between August 2020 and November 2021. Visual acuity, refraction, and corneal biomechanical parameters were measured preoperatively, 1 week and 1, 3, 6 months postoperatively. One-way analysis of variances (ANOVA) with Bonferroni correction or Kruskal-Wallis test was performed to compare the parameters among different groups. Repeated-measures analysis of variance with Bonferroni correction or Friedman test was applied for comparing the parameters within different follow-up times. RESULTS: Uncorrected distance visual acuity of 110-µm group was better only at 1-week and 1-month postoperatively (P = 0.012, 0.037). There were no significant differences in spherical equivalent, nor in Corvis biomechanical index-laser vision correction (CBI-LVC). All the parameters reached stability at 3-month postoperatively. Integrated radius (IR) and deformation amplitude ratio 2 mm (DA ratio 2 mm) in 120-µm and 130-µm groups were higher than 110-µm group at 1-month postoperatively (P = 0.019, 0.002). So was Ambrósio relational thickness (ARTh) at 6-month postoperatively (P = 0.011). Stiffness parameter at applanation A1 (SP-A1), stress-strain index (SSI), biomechanically corrected intraocular pressure (bIOP) and central corneal thickness (CCT) were highest in 130-µm group, followed by 120-µm group, then 110-µm group at 3-month (P<0.001, P = 0.030, P = 0.027, P = 0.008) and 6-month (P<0.001, P = 0.002, P = 0.0023, P = 0.001) postoperatively. CONCLUSIONS: The corneal stiffness following SMILE was greatest with 130-µm cap, followed by 120-µm cap, then 110-µm cap. 130-µm cap might have advantages in terms of corneal biomechanics and retreatment option. The SMILE-designed protocol should be customized in practice.

Effects of riboflavin/ultraviolet-A scleral collagen cross-linking on regional scleral thickness and expression of MMP-2 and MT1-MMP in myopic guinea pigs.

OBJECTIVE: To investigate the effects of scleral collagen cross-linking (SXL) using riboflavin and ultraviolet A (UVA) light on the scleral thickness of different regions and expression of matrix metalloproteinase 2 (MMP-2) and membrane-type MMP-1 (MT1-MMP) in guinea pigs with lens-induced myopia. METHODS: Forty-eight 4-week-old guinea pigs were assigned to three groups (n = 16 per group): SXL group, lens-induced myopia (LIM) group, and control group. The sclera of the right eye of the guinea pig in the SXL group was surgically exposed, riboflavin was dropped on the treatment area for 10 minutes before the 30-minute UVA irradiation. The same surgical procedure was performed in the LIM group without UVA irradiation. The -10.00 D lenses were then placed on the right eyes of guinea pigs in the SXL and LIM groups for six weeks. The control group received no treatment. The left eyes were untreated in all groups. The ocular axial length (AXL) and refraction were measured at 4 weeks and 10 weeks of age. 10-week-old guinea pigs were sacrificed, and the right eyes were enucleated and evenly divided for preparation of hematoxylin and eosin (HE) stained sections, quantitative real-time polymerase chain reaction (qPCR) and western blotting. The scleral thickness of different regions was measured on HE stained sections. The temporal half of the sclera was harvested to measure the expression of MMP-2 and MT1-MMP by qPCR and western blotting. RESULTS: The AXL was significantly shorter, and the degree of myopic refraction was significantly lower in the SXL group than those in the LIM group at 10 weeks of age. The scleral thickness of the cross-linked area was significantly greater in the SXL group than that of the corresponding area in the LIM group, while the scleral thickness of the untreated nasal side was not significantly different between the SXL group and the LIM group. The expression of MMP-2 and MT1-MMP of the cross-linked sclera was significantly downregulated compared with that of the corresponding area in the LIM group. CONCLUSION: Riboflavin/UVA SXL could slow myopia progression and thicken the cross-linked sclera in guinea pigs, which might be related to the downregulation of MMP-2 and MT1-MMP expression during the scleral remodeling process.

Scleral Remolding-Related Gene Expression After Scleral Collagen Cross-Linking Using Ultraviolet A and Riboflavin in Myopic Guinea Pig Model.

PURPOSE: This study was conducted to evaluate scleral remolding-related gene expression after scleral collagen cross-linking (SCXL) using ultraviolet A (UVA) and riboflavin in lens-induced myopia (LIM) guinea pigs. METHODS: A total of 100 4-week-old pigmented guinea pigs were randomly divided into five groups (n = 20): SCXL + LIM, LIM, SCXL, Sham, and Control. Refraction, anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) were measured using streak retinoscope and A-scan ultrasonography. SCXL was performed using 0.1% riboflavin solution and 365 nm UVA irradiation. Lens-induced myopia was achieved by wearing -10 D concave lenses. Quantitative real-time PCR (qPCR) and western blot were used to measure mRNA and protein levels, respectively. RESULTS: Myopia was successfully induced in the LIM group, while myopic refraction was higher and ACD and AL were shorter in SCXL + LIM compared with LIM, suppressing myopia progression. The scleral COL1A1 mRNA levels were significantly decreased and MMP2 and ACTA2 mRNA levels were significantly increased in LIM compared with other groups, while COL1A1 mRNA levels were increased and MMP2 and ACTA2 mRNA levels were decreased in SCXL + LIM compared with LIM. The scleral COL1A1 protein levels were significantly increased at 1 week and 4 weeks and MMP2 protein levels were significantly decreased at 1 week in SCXL compared with SCXL + LIM, LIM and Control. MMP2 protein levels were significantly decreased in SCXL + LIM and SCXL compared with LIM at 4 weeks. The differences in TGFB1, BMP2, CCN2, ITGA2, and ITGB1 mRNA levels and ACTA2 protein levels between the five groups were not significantly different. CONCLUSION: SCXL using UVA and riboflavin could influence the expression of scleral remolding-related genes, including COL1A1, MMP2, TIMP2, and ACTA2, and thus contribute to improving collagen synthesis and reducing collagen degradation and might have an effect on slowing myopia progression.

Comparing the Differences in Slowing Myopia Progression by Riboflavin/Ultraviolet A Scleral Cross-linking before and after Lens-induced Myopia in Guinea Pigs.

PURPOSE: To compare the effectiveness and differences in slowing myopia progression in Guinea pigs by riboflavin/ultraviolet A (UVA) scleral cross-linking (sCXL) before and after lens-induced myopia (LIM). METHODS: Forty 4-week-old Guinea pigs were randomly divided into four groups (n = 10 per group): CXL-A, CXL-B, LIM, and Control groups. The right eyes in CXL-A, CXL-B, LIM groups were treated with -10.00 D lenses from 4 to 10-week old and the left eyes were untreated. In CXL-A and CXL-B groups, riboflavin/UVA sCXL was performed on the right eyes at 4 weeks and 8 weeks of age, respectively. Both eyes were untreated in Control group. The intraocular pressure (IOP), the axial length (AXL), and the refraction were measured in vivo at 4, 8, and 10 weeks of age. At 10 weeks of age, the right eyes were enucleated for the tensile test and transmission electron microscopy observations. RESULTS: The myopia has been successfully induced in LIM and CXL-B groups during 4-8 weeks. In CXL-A group, the growth rate of AXL and myopic refraction was markedly inhibited during 4-8 weeks and the inhibitory effects diminished during 8-10 weeks. During 8-10 weeks, the growth rate of AXL and myopic refraction in CXL-B were marked suppressed. At 10 weeks of age, the myopia refraction was lower and the AXL was shorter in CXL-A group in comparison to CXL-B group. The IOP was not significantly different among the 4 groups of eyes at 4, 8, and 10 weeks of age. The scleral stiffness, the fibril diameters, and the fibril density of the sclera were significantly increased in CXL-A and CXL-B groups compared to LIM group. CONCLUSION: Riboflavin/UVA sCXL administrated before and after the myopia modeling could both slow the myopia progression in Guinea pigs. The before-myopia preventative sCXL showed lower myopic refraction in the same age comparison between the cross-linked groups. The effect of riboflavin/UVA sCXL might reduce over time and the long-term effect should be further investigated. This sCXL intervention might control the ultrastructure alterations of the sclera during the myopia remodeling.

Advanced Research in Scleral Cross-Linking to Prevent From Progressive Myopia.

ABSTRACT: Riboflavin-ultraviolet A (UVA) collagen cross-linking (CXL) has been applied in clinical settings to prevent the progression of keratoconus and corneal dilatation caused by other reasons in past decades. As CXL with riboflavin-UVA can enhance the stiffness of collagen-rich tissues, this technique has been further used on sclera to investigate as a safe and effective myopia prevention treatment. Despite the riboflavin-UVA scleral CXL is still in the animal and in vitro experimental phases and the mechanism is not very clear, it is promising to control myopia development clinically. In this article, researches on the laboratory experiments of riboflavin-UVA scleral CXL on scheme exploration and mechanism were reviewed in order to provide more laboratory evidence for scleral CXL in clinical myopia prevention and control in the future.

Safety and Long-term Scleral Biomechanical Stability of Rhesus Eyes after Scleral Cross-linking by Blue Light.

Purpose: To assess the safety and long-term scleral biomechanical stability of rhesus eyes after blue light scleral CXL by investigating the biomechanical and microstructural changes.Methods: Seven rhesus monkeys (14 eyes) were observed in this study. All right eyes received blue light scleral CXL at the superior temporal equatorial sclera, and the left eyes served as controls. Biological ocular parameters were followed up to 1 year after scleral CXL. Stress-strain measurements of three rhesus sclera were measured, three rhesus retinas were examined histologically by H&E and TUNEL staining. And the microstructure of both the sclera and retina were observed by transmission electron microscopy at 1 year.Results: As for the retinal thickness, choroidal thickness, flow density of retinal superficial vascular networks and flash electroretinography (f-ERG) results, no significant differences were observed between the paired eyes at 1 year (P >.05). At the same time, the scleral collagen fibril distribution was much tighter, and the scleral biomechanical properties were significantly increased in the experimental eyes. However, apoptotic cells and retinal ultrastructural changes could still be found in the retina of the experimental eyes.Conclusion: This study demonstrates that blue light scleral CXL could effectively increase the scleral stiffness of the rhesus eye for at least 1 year, but ultrastructural change was still observed in the retina of scleral CXL eye. Therefore, the long-term intraocular safety of the blue light scleral CXL technique for preventing myopia progression should be investigated further.