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Review Article

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

Zhang, Fengju MD, PhD; Lai, Lingbo MD

Author Information
Asia-Pacific Journal of Ophthalmology: March-April 2021 - Volume 10 - Issue 2 - p 161-166
doi: 10.1097/APO.0000000000000340
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Abstract

THE THEORY AND TYPES OF COLLAGEN CROSS-LINKING ON THE SCLERA

Collagen cross-linking (CXL) in the cornea and sclera tissue is a natural phenomenon, which accumulates with age and enhances the biomechanical properties of the tissue.1–3 Researchers have used riboflavin as a photosensitizer with ultraviolet A (UVA) irradiation on the cornea to generate artificial cross-linking reactions, which can improve the tissue biomechanical strength, and resist the ECM degradation by matrix metalloproteinase degradation.4 Consequently, the corneal CXL with riboflavin-UVA has been widely applied in the clinical treatment of keratoconus5,6 and keratectasia after corneal refractive surgery.7

Myopia is one of the most common eye diseases,8 which is characterized by a mismatch in refractive power and axial length. Researchers have found excessive ocular axial elongation, weakened scleral biomechanics, and changes in the scleral composition and structure in both human myopic eyes and animal myopia models.9 This process is scleral remodeling. In terms of the scleral compositions, researchers found that the dry weight of the sclera was reduced in the human sclera, and the synthesis of type I collagen and proteoglycan decreased.10,11 In terms of scleral structure, researchers found that the scleral thickness decreased in highly myopic human eyes,12 and this phenomenon has been confirmed in animal models.13 A significant diameter thinning of scleral collagen fibrils in both human myopia and animal myopia models have been observed.13–15

In view of the pathological changes in the sclera of high myopia, researchers hope to inhibit progressive myopia with scleral collagen cross-linking. Nowadays, scleral CXL is generally divided into physical and chemical CXL methods. The physical CXL method mainly contains riboflavin-UVA induced CXL and riboflavin/blue light CXL, which use excitation light (370 nm UVA or 460 nm blue light) to activate riboflavin into triplet, which in turn produces reactive oxygen species. The CXL is used to form a chemical bridge between collagen protein and other molecules after chemical reactions. When riboflavin as a photosensitizer absorbs the energy from UVA or blue light, the riboflavin excited end exchange from singlet state to triplett state. After that, type I and type II reactions both take place to generate new chemical bonds.16–18 New chemical bonds also increase the mechanical strength of the sclera by connecting individual collagen fibrils of the sclera.19

Chemical CXL can enhance the stability of scleral by CXL reagents containing reactive groups (amino, thiol) that can react with proteins or other molecules to form new covalent bonds between scleral molecules. In 2004, Wollensak et al20 compared the improvement in scleral stiffness after different CXL methods including physical CXL by riboflavin–UVA and chemical CXL by incubation with glucose, ribose, glyceraldehyde, and glutaraldehyde solutions. After CXL scleral stripes of human postmortem and pig eyes, the stress–strain measurements were performed and the result showed a statistically significant increase in scleral stiffness after CXL with riboflavin-UVA, with an elevation in stress in treated porcine (157%) and human (29%) sclera, and after treatment with glyceraldehyde, with a rise in stress in treated porcine (487%) and human (34%) sclera, and with glutaraldehyde, with increasing stress in treated porcine (817%) and human sclera (122%) at 8% strain. The other reagents showed ineffectiveness. As for CXL using genipin, it could increase the biomechanical strength in the porcine sclera and the effect depends on the concentration and treatment time.21 Levy et al found that the scleral CXL with genipin can inhibit the profound cyclic softening effect in the sclera of tree shrew myopia model.22 Metzler et al23 demonstrated that the decorin core protein could produce stiffer biomechanical properties and higher elastic modulus of human and porcine corneas in vitro study.

Compared to physical scleral CXL, the chemical scleral CXL can improve the scleral stiffness more effectively. Besides, we can perform the chemical scleral CXL by sub-Tenons injection, which is less invasive than opening the conjunctiva for the sclera exposure in riboflavin-UVA or -blue light sCXL. Although the chemical CXL showed these advantages, convincing evidence of safety is still lacking. Chemical CXL reagents are fluidity, and their CXL area is difficult to control. Especially glyceraldehyde induces the formation of advanced glycosylation products, which can increase the scleral stiffness and reduce the elasticity of lamina cribrosa, which may be a risk factor for glaucoma optic nerve damage.24 Furthermore, Kimball et al25 found CXL by glyceraldehyde in a mouse model decreased scleral permeability and produced a steeper pressure, increasing susceptibility to retinal ganglion cell (RGC) damage. There was no histological damage found on the retina or choroid in guinea pigs after cross-linked with genipin.26

EXPLORATION OF THE PHYSICAL SCLERAL COLLAGEN CROSS-LINKING PROCEDURE

As early as 2004, Wollensak et al20 applied CXL with riboflavin-UVA and rose bengal/white-light irradiation on the scleras of human postmortem eyes and porcine eyes. A significant rise in stress up to 157% in the human sclera and 29% in porcine sclera were achieved after CXL with riboflavin-UVA, which suggested that the scleral biomechanical property can be enhanced after crosslinking with riboflavin-UVA but cannot be enhanced with bengal/white-light. In 2005, Wollensak et al19 performed CXL on rabbit scleras in vivo using riboflavin-UVA. Six New Zealand rabbit eyes were treated in vivo using a UVA double diode with 4.2 mW/cm2 UVA and applying 0.1% riboflavin for 30 minutes. In 1 day postoperatively, the ultimate stress was increased by 227.9%, Young's modulus by 464.7%, and the ultimate strain decreased by 31.07%, which demonstrated that the proposed method induces an improvement of the mechanical properties of the sclera. On the other hand, histopathological results showed that retinal photoreceptors, outer nuclear layer cells, and retinal pigment epithelium cells were almost completely lost. They speculated that toxic side-effects on retina might be related to the irradiance of UVA. In 2009, they modified the CXL method and treated rabbit eyes in vivo using UVA with a lower surface irradiance of 3 mW/cm2 for 30 minutes.27 Biomechanical stress–strain measurements performed 3 days, 4 months and 8 months postoperatively showed that Young's modulus increased by 320.4% after 3 days, 277.6% after 4 months, and 502% after 8 months; ultimate stress increased by 341.4% after 3 days, 131% after 4 months, and 213.8% after 8 months; and the decrease in ultimate strain were 24.1%, 33.2%, and 44.8%. There was no significant damage on the retina postoperatively by histology examination. This study demonstrated that scleral CXL with riboflavin-UVA can significantly increase scleral stiffness and maintain at least 8 months without side-effects on the structure of the retinal cells. Interestingly, in the study of the safety evaluation of scleral CXL by riboflavin-UVA, Wang and Zhang et al28 drew a different conclusion from Wollensak, with the identical CXL procedure in 2015. They found that compared with the control eyes, the electroretinography (ERG) amplitudes appeared to be statistically reduced and the retina of irradiated regions showed histopathological damage after scleral CXL in New Zealand rabbits.

Researchers investigated the effect of different regions of the sclera to find the suitable therapy area. In 2012, Lv et al29 performed scleral CXL on equatorial and posterior scleras of guinea pigs with riboflavin-UVA. Biomechanical stress-strain measurements performed 1 month postoperatively showed that ultimate stress of an equatorial sclera increased by 147%, Young's modulus increased by 193%, and the decrease in ultimate strain was 21.9%; ultimate stress of posterior sclera increased by 108%, Young's modulus increased by 191%, and the decrease in ultimate strain was 40.42%. Furthermore, Wang et al30 evaluated the biomechanical difference of different cross-linked regions (equatorial and posterior sclera) of the human cadaver by riboflavin-UVA. The Young's modulus at 8% strain of equatorial and posterior sclera showed no statistical difference. They also suggested that 20 minutes of riboflavin infiltration before CXL is recommended. This conclusion was verified by Zhang et al,31 who found riboflavin reached a saturation level in the sclera of human cadavers and rabbits after 20 minutes.

Lately, Gawargious et al32 used scleral strips in different regions of the human cadaver eyes to compare the effect of scleral CXL with riboflavin-UVA to improve mechanical properties. The result demonstrated that the scleral Young's modulus increased more in the equatorial area than the posterior sclera, and most in the lateral equatorial sclera (Table 1).

TABLE 1 - Changes of Scleral Biomechanical Parameters After Scleral Cross-Linking in Different Studies
Study Cross-linking Method Species Young's Modulus (Mpa)
Wollensak et al.,20 2004 Ex vivo: Riboflavin-rose bengal/white lightGlucoseRiboseRibo in-UVA (3.0 mW/cm2, 30 min)GlyceraldehydeGlutaraldehyde Human and porcine cadaver eyes Human vs. porcine:IneffectiveIneffectiveIneffective+ 29% vs. + 157%+ 34% vs. + 487%+ 122% vs. + 817%
Wollensak et al.,19 2005 In vivo: Riboflavin-UVA (4.2 mW/cm2, 30 min) Rabbits + 464.7% (posterior - equatorial sclera)
Wollensak et al.,27 2009 In vivo:Riboflavin-UVA (3.0 mW/cm2, 30 min) Rabbits Postoperation:3 days: + 320.4%4 months: + 277.6%8 months: + 502%
Lv et al.,29 2012 In vivo:Riboflavin-UVA (3.0 mW/cm2, 30 min) Rabbits Equatorial sclera vs. posterior sclera:+ 193% vs. + 191%
Wang et al.,30 2012 Ex vivo:Riboflavin-UVA (3.0 mW/cm2, 5 to 30 min) Human cadaver eyes Intervention duration (equatorial sclera vs. posterior sclera):5 min: + 130% vs. + 132%10 min: + 150% vs. + 149%20 min: + 173% vs. + 181%30 min: + 185% vs. + 201%
Wang et al.,28 2015 In vivo:Riboflavin-UVA (3.0 mW/cm2, 30 min) Rabbits + 179% (equatorial sclera)
Gawargious et al.,32 2019 Ex vivo:Riboflavin-UVA (6.0 mW/cm2, 30 min) Human cadaver eyes Equatorial sclera vs. posterior sclera:139 ± 43% vs. 46 ± 15% (superolateral)68 ± 27% vs. 32 ± 11% (superomedial)143 ± 92% vs. 67 ± 20% (inferolateral)68 ± 14% vs. 53 ± 11% (inferomedial)

Zhang et al explored the optimum duration of irradiation on vitro rabbit sclera using 3 mW/cm2 UVA at 365 nm and 0.1% riboflavin.33 The results included that the biomechanical properties of the sclera were not statistically different from control eyes after 10 or 20 minutes irradiation (P > 0.05). Irradiation for more than 30 minutes had a significant increase in physiological modulus. Irradiated for more than 40 minutes increased significantly in ultimate stress, Young's modulus, and the physiological modulus (P < 0.05). More than 50 minutes can result in retinal damage (retinal epithelial cells and retinal pigment epithelial cells present abnormal morphology and nuclear edema). Thus, this study provided the optimum therapy time was 40 minutes. Besides, they compared the biomechanical properties of vitro porcine, rabbit, and human sclera before and after riboflavin-UVA CXL (3 mW/cm2, 40 minutes, 5.4 J/cm2).34 According to the values of stress, and Young's modulus, human sclera was 4 times stiffer than the porcine sclera and 3 times stiffer than the rabbit sclera. After CXL, the ultimate stress increased significantly in rabbit and porcine sclera, and Young's modulus was only significantly increased in rabbit sclera, which indicated that with the same irradiation dose, riboflavin-UVA CXL increases the biomechanical stiffness of rabbit sclera but not porcine or human sclera.

Above all, it has been proved that CXL with 3.0 mW/cm2 UVA (370 nm) for 30 minutes can markedly enhance the biomechanical strength of sclera. However, the safety of the CXL treatment plan was still controversial among different research teams, and the identical CXL procedure showed a significant difference in different animal models. To explore this discrepancy, Zhang's research team firstly used rhesus monkeys as an animal model, since the physiological structure of their eyes is much closer to human.35 They investigated ocular safety in rhesus monkeys after CXL with riboflavin and UVA for 1 year by testing in vivo biological parameters like diopter, ocular axis length, the thickness of retina and choroids, the flow density of retinal superficial vascular networks, and ERG. It is demonstrated that the differences of intraocular pressure, refractive state, total axial length, and axial dimensions of the anterior chamber, crystalline lens, vitreous chamber, and central corneal thickness were not statistically significant between the control and cross-linked specimens in different periods (each P > 0.05), which indicated the scleral CXL with riboflavin-UVA would not interfere the normal ocular growth. No obvious changes in the waveform of the standard full-field ERG were observed in the control and cross-linked specimens. There were no statistically significant differences between the control and cross-linked specimens in the dark-adapted 0.01 ERG, the dark-adapted 3.0 ERG, the light-adapted 3.0 ERG, and the amplitudes of the a-wave and b-wave for different periods (each P > 0.05).36 Though the choroidal thickness close to the fovea center of CXL eyes revealed a transient change in the early period of postoperation, it recovered from 1 month postoperatively; histopathological examination showed scleral CXL had no significant influence on ocular wall organization.37 Moreover, this technique enhanced scleral biomechanical strength which can be maintained at least 12 months.

Jung and Choi et al38,39 examined the effect of CXL with riboflavin-UVA irradiation on the chemical bonds and ultrastructural changes of human sclera tissues using Raman spectroscopy and atomic force microscopy. The study showed after riboflavin-UVA treatment, the atomic force microscopy image revealed interlocking arrangements of collagen fibrils instead of a regular parallel arrangement of normal collagen fibrils. The observed changes in the surface topography of the collagen fibrils, as well as in their chemical bonds in the sclera tissue, support the formation of interfibrilar cross-links in sclera tissues. This research team further quantitatively investigated the in vitro ultrastructural effects of riboflavin-UVA CXL in human corneo-scleral collagen fibrils. Such treatment led to an increase in the area, density, and diameters of both corneal (110%, 112%, and 103%) and scleral (133%, 133%, and 127%) stromal collagens, as well as increases in corneal (107%) and scleral (105%) thickness. It inferred that CXL with riboflavin-UVA may cause structural property changes in the collagen fibril network of the cornea and sclera as a result of stromal edema and interfibrillar spacing narrowing. These changes were particularly prominent in the sclera. Their studies provided ultrastructural evidence to confirm the effectiveness of CXL with riboflavin-UVA. Previous studies have shown riboflavin (0.5%) with blue light (465 nm) can strengthen scleral biomechanical rigidity.40 Blue light also corresponds with an absorption maximum (peak) of riboflavin, but with a longer wavelength, and the potential for biological damage is lower.41 However, the safety of scleral CXL with blue light is still the focus of ongoing research. Karl et al42 investigated the safety of different irradiation intensities (200 mW/cm2, 400 mW/cm2, 650 mW/cm2) with transmission electron microscope. They reported that treatment with blue light at 200 mW/cm2 did not induce ultrastructural changes in cells or collagen fibrils in rabbit scleral stroma. Zhang et al42 investigated the scleral biomechanical properties of blue light scleral CXL and demonstrated that greater scleral stiffening in refresh humans cadaver eyes was achieved with riboflavin (0.5%) and blue light (460 nm) at 22.5 mW/cm2 than at 26 mW/cm2. They further investigated retinal and choroidal biological changes associated with scleral CXL with riboflavin and blue light (22.5 mW/cm2) application on rhesus eyes in vivo and found there were no significant changes in retinal thickness, vessel density of retinal superficial capillary plexus, and choroid thickness after blue light scleral CXL. However, f-ERG parameters reductions and retinal ultrastructural changes at the early stage, which indicated blue light scleral CXL could cause transient retina damage,43 and the safety in the long-term should be investigated further compared with sclera CXL by UVA.

SCLERAL COLLAGEN CROSS-LINKING CONTROLS THE PROGRESSION OF MYOPIA

Based on the validity and safety of riboflavin-UVA, scleral CXL is gradually widely confirmed in laboratory researches. Scientific researchers around the world started focusing on the intervention and prevention of pathologic myopia progression by riboflavin-UVA scleral CXL. In 2004, Dotan et al44 used New Zealand white rabbits as a model. The right eyes underwent scleral CXL using riboflavin and UVA radiation, and every quadrant had either 2 or 6 scleral irradiation zones. The eyelids of the right eyes were sutured after therapy to establish the myopic model. The ocular axial length was measured and the result showed that scleral CXL with riboflavin-UVA effectively prevents occlusion-induced axial elongation, and the size of the treatment area would not affect the efficiency in a rabbit model. After that, Zeugolis et al45 established a lens-induced myopia model in guinea pigs to develop methods of CXL in the sclera for the treatment of progressive myopia. The result indicated that scleral CXL using riboflavin-UVA irradiation effectively prevents the progression of myopia by increasing scleral biomechanical strength in a guinea pig model. Additionally, scleral collagen fiber arrangements of the cross-linked eyes were denser and more regularly distributed than the myopic eyes.

It can be seen this treatment has positive significance for prevention and control of myopia. Scientists further studied to optimize the exact therapy procedure accordingly. Xiao et al46 used a specially made light emitting diode light source that was inserted through a minimally invasive conjunctival incision to gain close contact with the sclera for irradiation and investigated the efficacy of minimally invasive repetitive scleral CXL with riboflavin-UVA. The study suggested that repetitive minimally invasive UVA irradiation with riboflavin significantly increased biomechanical strength of the sclera in the irradiated area, and biomechanical strength increased with repeated times of irradiation.

Rong et al47 modified the scleral riboflavin-UVA CXL procedure with an iontophoresis-assisted drug delivery system and accelerated UVA irradiation (10 mW/cm2, 9 minutes) on New Zealand white rabbits, which have developed relatively stable myopia by visual deprivation. They found that the strength of sclera was significantly increased and maintained 3 months. The abnormal elongation of the myopic eye was effectively controlled for 1 month and even almost halted for 3 months after the treatment. The histochemical assay revealed no notable postsurgery damage or apoptosis in the retina and choroid. Vigorous collagen synthesis was observed in scleral fibroblasts of the cross-linked samples. Furthermore, the expression of the collagen gene and protein altered in treated eyes also indicated that new collagen metabolism may be triggered by CXL.

Recently, researchers have investigated the effect of oral administration of riboflavin combined with whole-body UVA irradiation on the biochemical and biomechanical properties of the sclera in a lens-induced myopic guinea pig model.48 Oral administration of riboflavin with whole-body UVA irradiation could increase the strength and stiffness of sclera by altering the biochemical and biomechanical properties, and decreases in axial elongation and myopic diopter are greater in the guinea pig myopic model.

Zhang's team is currently investigating the key points to administrate scleral CXL with riboflavin and UVA in guinea pigs with and without defocusing myopic eyes. The result until now suggested the effect of scleral CXL before myopic modeling was quite better than after myopic modeling on suppressing ocular axial length increasing and myopia diopter development, which may be meaningful to clinical myopic prevention strategy further.

Although the effectiveness of increasing scleral stiffness in chemical CXL has been confirmed, the effectiveness of chemical CXL in halting axial elongation and preventing progressive myopia still has controversy. In the form-deprived myopia model of guinea pigs, the posterior sub-Tenon injection of 0.5 M/L glyceraldehyde did not suppress the progression of myopia.49

Interestingly, Lin et al used the identical method but injected 7 times and successfully suppressed the experimental myopia progression in rabbits.50 Wang et al reported that sub-Tenon injections of genipin can effectively block the myopia progression.26 Besides, Xue et al used genipin-crosslinked donor sclera for posterior scleral contraction/ reinforcement surgery, and effectively halted eyeball elongation in young patients during 2 to 3 years follow-up period.51

SUMMARY AND EXPECTATION

The animal experiments suggested that the modified scleral CXL procedure may be a potential method to control the pathologic process of myopia, even though the safety of scleral CXL still needs further investigation. Scleral CXL by riboflavin-UVA is a minimally invasive procedure without allograft material implantation and a noninvasive procedure on the eyeball wall, which makes it possible to reduce complications such as infection and rejection. Its effectiveness, stability, and safety make it hopeful to arrest progressive myopia or to inhibit the over-expansion of the sclera. It is also expected to be used for preventing myopia, and the appropriate time for treatment is the urge to be investigated. What is more, this procedure has not been directly applied to human eyes in vivo and potential problems should be investigated further for clinical application in the future, including long-term safety and stability of CXL, and the nomogram parameters of CXL in human eyes in vivo such as exact position and suitable area, amount of energy, and exposure time, and so forth.

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Keywords:

blue light; cross-linking; myopia; sclera; UVA

Copyright © 2021 Asia-Pacific Academy of Ophthalmology. Published by Wolters Kluwer Health, Inc. on behalf of the Asia-Pacific Academy of Ophthalmology.