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Abstract
This study aimed to investigate the effectiveness of two-photon induced collagen cross-linking (CXL) using femtosecond lasers in human corneal stroma. An 800-nm femtosecond laser optical path for CXL was established. Corneal samples that received two-photon induced CXL and ultraviolet-A (UVA) CXL underwent uniaxial stretching experiments, proteolytic resistance assays and observation of collagen fiber structure changes. Two-photon induced CXL can achieve corneal stiffening effects comparable to UVA CXL and showed better advantages at low strains. The cornea after two-photon induced CXL exhibited high enzymatic resistance and tight collagen fiber arrangement. Two-photon induced CXL promises to be a new option for keratoconus.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Keratoconus (KC) is characterized by the thinning and steepening of the central and/or paracentral cornea and a subsequent high degree of irregular astigmatism and/or myopia that can lead to blindness in severe cases [1,2]. Morphological changes associated with KC may underlie its biomechanical changes [3–7]. Studies have shown that KC is associated with increased proteolytic enzyme expression, decreased protease inhibitor expression, and fibrillar misalignment, and these changes lead to a decrease in corneal thickness and biomechanics in KC [6–10]. Ultraviolet-A-riboflavin (UVA) corneal collagen cross-linking (CXL) has been shown to slow progression of KC by increasing the mechanical strength and resistance to proteolysis [11–15].
Conventional UVA CXL (Dresden CXL) is the most common form of CXL and has been extensively studied to prove its effectiveness in treating KC [16–19]. However, it has some limitations. Firstly, there is growing evidence that KC begins in the posterior and progresses to the anterior stroma [20–22]. However, the stiffening effect occurs mainly in the anterior 200–250 µm of the corneal stroma in conventional UVA CXL [23–25]. Secondly, the Dresden CXL procedure takes time and is poorly tolerated by patients [14,26]. High-intensity rapid UVA CXL has been used in the clinic; however, a recent meta-analysis concluded that the improvement in mechanical strength is less after rapid CXL than after Dresden CXL [27,28]. Thirdly, neither Dresden CXL nor accelerated UVA CXL is indicated for patients with KC with a corneal thickness <400 µm because of the increased risk of corneal endothelial damage [29,30]. Finally, the homogeneous irradiation of the Dresden CXL resulted in no substantial differences in the optical properties of the conical and non-conical corneal areas, resulting in no significant refractive improvement [31]. Given these issues, more effective CXL methods with shorter CXL durations and better lateral and axial control would be beneficial for treating KC.
Femtosecond (FS) lasers are proven to be effective in activating photosensitizers in small focal volumes through non-linear optics and multiphoton absorption processes [32–34]. Since FS lasers cannot be absorbed by optically transparent tissues, they can be focused on tissues of varying depths to provide high lateral and axial control [35,36], and their ultrafast nature can significantly reduce the procedure times [37,38]. This is promising for addressing some of the limitations of conventional UVA CXL.
Herein, we report two-photon induced CXL using an 800-nm femtosecond laser in ex vivo human corneal stroma and compare it with conventional UVA CXL in terms of corneal biomechanical properties, corneal resistance to enzymatic degradation, and structural alteration of collagen fibers, thereby further determining the effectiveness of two-photon induced CXL in human corneal stroma. This study provides an experimental basis for accurate and effective in vivo crosslinking with a controlled crosslinking range in the future.
2. Methods
2.1 Experimental light path construction
The experimental setup for performing two-photon induced CXL in this study is shown in Fig. 1. The FS laser pulse (central wavelength of 800 nm, pulse width of 120 fs, and repetition frequency of 1 kHz) from a Ti-sapphire chirped pulse amplification laser system (Spitfire; Spectra-Physics, Milpitas, CA) was used as the excitation source. The FS laser beam was focused on a human corneal stroma sample using an LSM54-850 scanning lens (Thorlabs, Newton, NJ). The sample was placed on a slide and positioned on a 3-Axis XYZ Stage (Thorlabs) for precise positional control. During two-photon induced CXL, the human corneal stroma sample and scanning lens were stationary, and the scanning path and speed of the FS laser were controlled by a two-dimensional scanning galvanometer (Thorlabs).
Fig. 1. Femtosecond laser two-photon induced collagen cross-linking optical path diagram. FS, femtosecond laser; PC, computer.
2.2 Specimen preparation and experimental study design
In this study, corneal lenticules were produced during small incision lenticule extraction (SMILE), and were obtained from the Refractive Surgery Centre of Tianjin Eye Hospital, Tianjin Medical University. The study was approved by the Ethics Committee of Tianjin Eye Hospital, and all patients voluntarily underwent the procedure after signing an informed consent form. The inclusion criteria were: (1) patients aged 18 to 25 years old, and (2) spherical equivalent of -5.00 to -8.00 diopters (D) with cylinder within 1.00 D. (3) Patients have no history of ocular disease (e.g., severe dry eye, corneal disease, cataracts, glaucoma, retinal detachment, etc.), history of ocular surgery and presence of chronic systemic disease. All procedures were performed by the same refractive surgeon with extensive surgical experience. The Visual Max FS laser system (Carl Zeiss Meditec, Jena, Germany) was used to perform the FS laser portion of the SMILE procedure. Afterward, the corneal lenticule was bluntly separated from the cornea using a microlens separator and removed with microforceps to assess the integrity of the corneal lenticule. After removal, the corneal lenticule was marked with gentian violet at 12 o’clock, immediately placed in 20% T-500 dextran solution prepared with 0.01 M phosphate-buffered saline (PBS) [39,40], and stored at 4°C.
The corneal lenticules obtained during SMILE were randomly allocated into two groups; two-photon induced CXL (n = 37) and conventional UVA CXL (n = 37). Each group of corneal lenticules was cut with a blade into two 2-mm wide corneal strips along the 12 to 6 o’clock position. One strip was used for two-photon induced CXL or conventional UVA CXL and the other as a control for the corresponding CXL method. The interval between removing the lenticules and the end of the experiment was no more than 24 h. The corneal strips were subsequently cross-linked and were subjected to uniaxial stretching tests (n = 18 for each group), the microstructure of the corneal collagen fibers was observed using light (n = 8 for each group) and electron microscopy (n = 8 for each group), and the resistance to enzymatic degradation was tested (n = 3 for each group). The experimental steps are shown in Fig. 2.
Fig. 2. Schematic illustration of the experimental design. Step Ι: Specimen preparation. Step II: Corneal cross-linking protocol. Step III: (1) uniaxial tensile tests; (2) histological examination; (3) transmission electron microscope observation; (4) testing of corneal resistance to enzymatic degradation. RF, 0.1% riboflavin; CXL, corneal cross-linking; FS, femtosecond; SMILE, small incision lenticule extraction; TEM, transmission electron microscope; UVA, ultraviolet-A.
2.3 Corneal cross-linking protocol
All corneal strips were first immersed in 0.1% riboflavin solution (VibeX Rapid riboflavin; Simovision, Overijse, Belgium) for 30 min. In the two-photon induced CXL group, the FS laser beam was focused on the corneal strips using the constructed optical path, and the entire corneal strip was raster-scanned at 10 µm scan intervals (Fig. 2). The single-pulse energy was set at 4.5 µJ, the scanning speed was 2 mm/s, and the scanning time was approximately 15 min, with a 0.1% riboflavin solution dropped every 2 min during the scanning process. In the conventional UVA CXL group, the corneal strips were irradiated for 30 min using an Avedro KXL UV crosslinker (wavelength of 365 nm, intensity of 3 mW/cm2, and total energy of 5.4 J/cm2; Avedro, Waltham, MA), with a 0.1% riboflavin solution added dropwise every 2 min during irradiation. To prevent CXL of the corneal strips with riboflavin by UV radiation from daylight, the corneal strips in the control group were placed in a black box until the end of the experiment.
2.4 Uniaxial tensile tests
Uniaxial stretching of the corneal strips was performed using the Biotester planar biaxial testing system (CellScale, Waterloo, Canada) (Fig. 3(A)). Corneal strips with a width of 2 mm were fixed by loading hooks, with a spacing of 3 mm between the two hooks, which corresponded to an effective test area of 2 × 3 mm (Fig. 3(B)). The strips were immersed in a 0.9% physiological saline bath at approximately 37°C to simulate the physiological conditions of the cornea during the stretching process (Fig. 3(C)) [41].
Fig. 3. Uniaxial tensile tests. (A) Biotester planar biaxial testing system (CellScale, Waterloo, Canada). (B) Corneal strips with 2-mm widths were fixed by loading hooks, with a spacing of 3 mm between the two hooks. (C) Corneal strips are immersed in 0.9% physiological saline at approximately 37°C. (D) Stress-strain curve of the corneal strip. MPa, megapascals.
Displacement loading was used for this tensile experiment. The loading and unloading curves differ due to the viscosity of the cornea. Therefore, a loading speed of 0.04 mm/s was used for preconditioning in this study, and three cycles until the loading and unloading curves of the corneal strips nearly coincided. Subsequently, the corneal strips were stretched uniaxially at 0.01 mm/s until they fractured. Given that the cornea is a biologically soft tissue, its stress-strain relationship is non-linear. Figure 3(D) shows the cornea stress-strain curve, which has been divided into the toe zone (OA segment), heel zone (AB segment), linear zone (BC segment), and fracture point D [42]. Due to the large fluctuations in the OA segment of the corneal strip stress-strain curve, we used the AB and BC segments to calculate the elastic modulus (E) and make comparisons. Since the thickness of the corneal lenticules was not uniform, the ablation equation method previously investigated by our team was used to further correct the average thickness of the experimental area of the corneal strips to improve the accuracy of the stress-strain curve [16]. The elastic modulus is the tangent slope of the stress-strain curve; the stress in the corneal strip was calculated as $\sigma = \frac{F}{{W \times D}}$ and strain was calculated as $\varepsilon = \frac{{\varDelta L}}{{{L_0}}}$, where $\sigma $ was the stress, F was the load sensed by the machine during stretching, W was the corneal stroma strip width (in this study W = 2 mm), D was the corrected corneal stroma strip mean thickness, $\varepsilon $ was the strain, $\varDelta L$ was the magnitude of change in displacement during stretching, and L0 was the initial length of the corneal stroma strip before stretching began. The elastic modulus (E) of the cornea was calculated as $= \frac{\sigma }{\varepsilon }$.
Based on the constitutive model of the corneal stroma [42], the tensile data of the AB and BC segments were extracted. Additionally, an exponential function was fitted to the AB segment (see Equation [1]), a linear function was fitted to the BC segment (see Equation [2]), and the derivatives were used to calculate the elastic moduli E6% (see Equation [3]) and Ebc:
E6% was the elastic modulus value of the corneal strip at 6% strain, Ebc was the elastic modulus value of the corneal strip in the BC segment, and A, R and b were constants.
To reduce the heterogeneity of the corneal strips, we used the multiples of the change in the relative elastic modulus of the corneal strips ($\varDelta {E_m}$) for the comparison of the groups, calculated in Equation [4]:
Etreated is the elastic modulus of the corneal strips in the cross-linked group and Econtrol is the elastic modulus of the corneal strips in the blank control group.
2.5 Testing of corneal resistance to enzymatic digestion
After the CXL experiment, the corneal strips from the treatment groups were placed in 2 mg/mL of type I collagenase solution at room temperature, and their complete dissolution durations were observed and recorded.
2.6 Histological examination
The corneal strips were immediately placed in 4% paraformaldehyde and left overnight at 4°C, followed by paraffin-embedded samples. The sections were made on the sagittal plane, followed by hematoxylin and eosin (H&E) and Masson staining to observe the corneal tissue structure. Three randomly selected sections of each group of samples were photographed under a light microscope (Olympus, Tokyo, Japan) to observe structural changes in the corneal collagen fibrous laminae. The sample central part was selected for the proportion analysis of the voids between the collagen fibrous laminae of the corneal strips to the total section sample area, and the images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
2.7 Transmission electron microscopy
After the CXL experiments, the corneal strips from different treatment groups were immediately placed in 2.5% glutaraldehyde fixative at 4°C overnight. Electron microscopic biospecimens with thicknesses of 60-70 nm were obtained. Under transmission electron microscopy (TEM, HT7700; Hitachi, Tokyo, Japan), three different fields of viewwere randomly selected for each group of samples to be photographed, and cross-sections of the collagen fibers were selected for analysis and measurements of diameter and interfibrillar spacing [43]. TEM images were processed using ImageJ (National Institutes of Health).
2.8 Statistical analysis
SPSS software (version 25.0; IBM, Armonk, NY) was used for statistical analysis of the relevant data. The Kolmogorov-Smirnov method was used to test the normality of the distribution of the data. Normally distributed data were analyzed using a T-test, and a non-parametric test was used for those that did not conform to a normal distribution. Differences were considered statistically significant at P < 0.05. The data in this study are expressed as mean ± standard deviation (SD).
3. Results
3.1 FS laser light path photoactivation riboflavin test
An 800-nm FS laser with a single-pulse energy of 4.5 µJ was focused in a cuvette containing a 0.1% riboflavin solution in a dark room (Fig. 4(A)), and the spectrum was recorded with a spectrum analyzer. The results are shown in Fig. 4(B). The spectrum analyzer recorded a spectrum of approximately 520 nm, confirming that the 800-nm FS laser can activate the photosensitizer riboflavin through its two-photon absorption property. The FS laser can penetrate riboflavin to produce a two-photon focal volume of 170 µm in the axial direction and 10 µm in the lateral direction. In contrast, UV-A irradiation of riboflavin can only start from the surface layer, and the light intensity of UV-A decreased with increasing depth (Fig. 4(A)).
Fig. 4. (A) Schematic diagram of the femtosecond laser and ultraviolet-A focused on riboflavin. (B) Results of spectral recording. RF, 0.1% riboflavin.
3.2 Elastic modulus
The elastic modulus of the corneal strips increased after both two-photon induced CXL and conventional UVA CXL treatment with a strain of 6% in the BC segment, with statistically significant differences (P < 0.0001, Table 1, Figs. 5(A) and 5(B)). At a 6% strain, the elastic modulus of the corneal strips improved by factors of 1.44 ± 0.41 after two-photon induced CXL and 0.59 ± 0.27 after conventional UVA CXL, with two-photon induced CXL showing a better outcome (P < 0.0001, Fig. 5(C)). In the BC segment, the corneal strip elastic modulus increased by factors of 0.42 ± 0.28 after two-photon induced CXL and 0.55 ± 0.31 after conventional UVA CXL, with no significant difference between the two groups (P = 0.131, Fig. 5(C)).
Fig. 5. Uniaxial tensile test results. (A) Comparison of stress-strain experimental data from uniaxial tensile tests of corneal strips in the two-photon induced CXL and control groups. (B) Comparison of stress-strain experimental data from uniaxial tensile tests of corneal strips in the conventional UVA CXL and control groups. (C) Bar chart comparing the multiplicative increments in the elastic modulus after the cross-linking of corneal strips. ***, P < 0.0001. n.s, non-significant. CXL, corneal crosslinking; UVA, ultraviolet-A.
Table 1. Elastic modulus values for different treatment groups
3.3 Resistance to enzymatic digestion
The time required for the control group to be completely solubilized by collagenase type I was 111.67 ± 7.64 min for the two-photon induced CXL group and 111.33 ± 4.04 min for the conventional UVA CXL group (P < 0.05, Fig. 6). The time required to dissolve the corneal strips increased to 180 ± 15 min after two-photon induced CXL and 163.67 ± 14.01 min after conventional UVA CXL (P < 0.05), with no significant difference between the two groups (P = 0.102, Fig. 6).
Fig. 6. Bar chart comparing the durations for the complete dissolution of corneal strips by protease. *, P < 0.05. n.s, non-significant. CXL, corneal crosslinking; UVA, ultraviolet-A.
3.4 Histological changes
Figure 7(A) shows the representative results of the histopathological examination of the sagittal surface of the corneal strips in the cross-linked and blank control groups (H&E staining and Masson staining, ×400). The corneal collagen fibrous layer showed pink bands on H&E staining and green bands on Masson staining arranged in a wavy pattern. In the control group, the collagen lamellae of the corneal strips were loosely arranged with large interlaminar voids, whereas in the cross-linked group, the corneal strips after both two-photon induced CXL and conventional UVA CXL showed tightly arranged collagen lamellae with reduced interlaminar voids. The interlaminar voids were reduced by 13.15 ± 0.04% and 15.49 ± 0.03% after two-photon induced CXL (P < 0.0001) and conventional UVA CXL (P < 0.0001), respectively, with no statistical difference between the two CXL methods (P = 0.296, Fig. 7(B)).
Fig. 7. Histological examination. (A) Representative results of histopathological examination of corneal strips in the cross-linked and control groups (H&E and Masson staining, ×400, scale bar: 25 µm). (B) Bar chart showing the proportion of the void area between the corneal fiber laminae in the different treatment groups. ***, P < 0.0001. n.s, non-significant. H&E, hematoxylin and eosin; CXL, corneal crosslinking; UVA, ultraviolet-A.
3.5 Transmission electron microscopy imaging
The corneal strips showed intertwined collagen fibers under TEM. Figure 8(A) shows representative TEM images (×20k) of the sagittal plane of the corneal strips in the cross-linked and control groups. The interfibrillar spacing and fiber diameters of the control corneal strips were 46.73 ± 4.96 nm and 24.77 ± 1.08 nm in the two-photon induced CXL group and 44.81 ± 6.16 nm and 24.51 ± 1.08 nm in the conventional UVA CXL group, respectively. The corneal strips after both two-photon induced CXL and conventional UVA CXL showed reduced interfibrillar spacing and increased fiber diameter (Figs. 8(B) and 8(C)). The interfibrillar spacing and fiber diameter of the corneal strips were 23.69 ± 3.46 nm and 26.13 ± 1.58 nm after two-photon induced CXL and 21.85 ± 2.95 nm and 25.19 ± 0.82 nm after conventional UVA CXL, respectively. These changes after the two CXL methods were not significantly different (Figs. 8(B) and 8(C)).
Fig. 8. Transmission electron microscope observation. (A) Comparison of longitudinal and transverse sections of the collagen fibers in the corneal strips of the different treatment groups (×20k, scale bar: 100 nm). (B) Bar chart showing the interfibrillar spacing. (C) Bar chart showing the fiber diameters. *, P < 0.05. ***, P < 0.0001. n.s, non-significant. CXL, corneal crosslinking; UVA, ultraviolet-A.
4. Discussion
This study successfully constructed an optical path that allows two-photon induced CXL using an 800-nm FS laser, and this optical path can limit the riboflavin photoactivation to a controlled depth and range of light-focused volume. This study applied two-photon induced CXL to human corneal stroma for the first time and investigated the corneal stiffening effect of two-photon induced CXL, and the two-photon induced CXL effect on corneal microstructure and enzymatic resistance. The comparison of two-photon induced CXL and conventional UVA CXL suggested that two-photon induced CXL may be a new option for KC treatment with the potential to address some of the limitations of conventional UVA CXL.
For human corneal stroma, two-photon induced CXL produced mechanical enhancement comparable to that of conventional UVA CXL and showed better corneal stiffening at low strains. The changes in biomechanical properties are usually based on corresponding changes in microscopic morphology [44–47]. Light microscopy showed that corneal collagen fibers after two-photon induced CXL were more closely arranged and had fewer interlaminar voids and increased resistance to deformation, which were consistent with the trend of alterations in collagen fibers after conventional UVA CXL. These changes may be related to the increased covalent connections between collagen fibers after CXL [48]. TEM showed an increase in corneal fiber diameter and a decrease in the interfibrillar spacing after two-photon induced CXL, which were also consistent with the changes in CFs after conventional UVA CXL and were consistent with those reported by previous studies [43,48–50], and proteoglycans in the cornea may be associated with the changes in collagen fibers in this study [51,52]. Studies have shown that keratin sulfate proteoglycans are involved in regulating the diameter of collagen protofibrils and keratin sulfate proteoglycans are involved in controlling the spacing of protofibrils and the laminar adhesion properties of collagen fibers [53,54]. For UVA CXL, proteoglycans were linked to collagen fibers, in addition to covalent linkages between collagen fibers [55]. The potential effect of CXL on proteoglycans and the increased covalent linkage between proteoglycans and collagen fibers caused by CXL may be responsible for the shortened interfibril spacing and increased fiber diameter after CXL [56,57].
Studies have suggested that, in the AB segment of the corneal stress-strain curve, the coiled CFs within the cornea are in a process of being stretched gradually until the collagen fibers in the BC segment are completely straightened [42,58]. In the AB segment, the increase in covalent linkages between collagen fibers and between proteoglycans and collagen fibers may increase the resistance of the fibers during the process from curling to being straightened, resulting in an increase in the elastic modulus [59]. Christiansen et al. investigated the relationship between the assembly of type I collagen and its mechanical properties [60], and found that lateral fusion of protofibril subunits increased fiber diameter, which was positively correlated with low strain elastic modulus. The mean percentage increase in the fiber diameter after two-photon induced CXL was higher than that of conventional UVA CXL, and although the difference was not statistically significant, this may be a reason why the corneal strips in the two-photon induced CXL group had a higher multiple increase in the elastic modulus at 6% strain than the conventional UVA CXL group. Two-photon induced CXL may have a better stiffening effect on corneas with KC under physiological intraocular pressure because the elastic modulus in the low-strain region is associated with the biomechanical properties of the cornea in the physiological state [61]. In the BC segment of the stress-strain curve, the collagen fibers are straightened and have high linear elasticity [58,59]. The shortening of the interfibrillar spacing increases the number of collagen fibers in each region of the corneal strip, which may account for the increase in corneal elastic modulus in the BC segment after CXL [62].
The corneal strips treated with two-photon induced CXL and conventional UVA CXL were significantly more resistant to enzymatic degradation than the controls. The increased corneal resistance to enzymatic degradation after two-photon induced CXL may be as important as increased collagenase activity, which has been found in the KC [9,63]. It may be related to the large molecules produced in the CXL. Wollensak et al. found the presence of giant molecules with molecular weights of at least 1000 kD in cross-linked porcine corneas [64]. It has also been found that CXL can lead to the production of macromolecular polymers by linkages between corneal matrix proteoglycans, and the production of these macromolecular polymers improves the resistance of the cornea to protease hydrolysis [55,65].
We recognize some limitations of the current study. Firstly, the FS laser used in this study had a low re-frequency (1 kHz); therefore, the two-photon induced CXL duration was not reduced to a great extent. Secondly, due to the scarcity of human corneal samples, corneal lenticules produced during SMILE were used in this study for experiments. Corneal lenticules are mainly components of the anterior corneal stroma and do not fully represent the morphological and mechanical characteristics of the whole cornea [47]. The effectiveness of two-photon induced CXL in the posterior corneal stroma is unclear and needs to be further explored. Thirdly, this study neglected the effect of corneal thickness alterations by CXL on the accuracy of the calculation of the elastic modulus. The effects of different CXL methods on corneal thickness need to be further explored in the future.
5. Conclusion
In conclusion, two-photon induced CXL demonstrated human corneal stiffening effects comparable to those of conventional UVA CXL and better outcomes at low strains. The corneal stroma after two-photon induced CXL also showed trends of alterations in enzymatic resistance and microstructure consistent with conventional UVA CXL. Utilizing the ability of photoactivated riboflavin to be laterally and axially controllable, two-photon induced CXL is expected to allow safe deep stromal CXL, thin corneal CXL, and topography-guided personalized CXL for KC.
Funding
National Natural Science Foundation of China (Grant No. 81873684).
Acknowledgments
The authors thank Yi Song for technical assistance, Yuchuan Wang for his technical support, and Tianjin Eye Hospital Research Institute for experimental facilities.
Disclosures
The authors declare that they have no competing interests.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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