Double crosslinking decellularized bovine pericardium of dialdehyde chondroitin sulfate and zwitterionic copolymer for bioprosthetic heart valves with enhanced antithrombogenic, anti-inflammatory and anti-calcification properties.

Due to the increasing aging population and the advancements in transcatheter aortic valve replacement (TAVR), the use of bioprosthetic heart valves (BHVs) in patients diagnosed with valvular disease has increased substantially. Commercially available glutaraldehyde (GA) cross-linked biological valves suffer from reduced durability due to a combination of factors, including the high cell toxicity of GA, subacute thrombus, inflammation and calcification. In this study, oxidized chondroitin sulfate (OCS), a natural polysaccharide derivative, was used to replace GA to cross-link decellularized bovine pericardium (DBP), carrying out the first crosslinking of DBP to obtain OCS-BP. Subsequently, the zwitterion radical copolymerization system was introduced in situ to perform double cross-linking to obtain double crosslinked BHVs with biomimetic modification (P(APM/MPC)-OCS-BP). P(APM/MPC)-OCS-BP presented enhanced mechanical properties, collagen stability and enzymatic degradation resistance due to double crosslinking. The ex vivo AV-shunt assay and coagulation factors test suggested that P(APM/MPC)-OCS-BP exhibited excellent anticoagulant and antithrombotic properties due to the introduction of P(APM/MPC). P(APM/MPC)-OCS-BP also showed good HUVEC-cytocompatibility due to the substantial reduction of its residual aldehyde group. The subcutaneous implantation also demonstrated that P(APM/MPC)-OCS-BP showed a weak inflammatory response due to the anti-inflammatory effect of OCS. Finally, in vivo and in vitro results revealed that P(APM/MPC)-OCS-BP exhibited an excellent anti-calcification property. In a word, this simple cooperative crosslinking strategy provides a novel solution to obtain BHVs with good mechanical properties, and HUVEC-cytocompatibility, anti-coagulation, anti-inflammatory and anti-calcification properties. It might be a promising alternative to GA-fixed BP and exhibited good prospects in clinical applications.

A comparison of conventional and accelerated corneal crosslinking: corneal epithelial remodeling and in vivo confocal microscopy analysis.

PURPOSE: To evaluate the effect of conventional and accelerated corneal crosslinking (CXL) on visual acuity, corneal topography, corneal epithelial thickness, and subbasal nerve morphology in progressive keratoconus patients. METHODS: In this prospective and randomized study, twenty eyes of 20 patients were treated with conventional CXL (3 mW/cm2, 30 min, C-CXL) and 19 eyes of 19 patients were treated with accelerated CXL (9 mW/cm2, 10 min, A-CXL). The spherical equivalent, uncorrected visual acuity, best-corrected visual acuity, keratometric measurements, demarcation line measurement and epithelial thickness mapping analyses, and subbasal nerve morphology with in vivo confocal microscopy (IVCCM) were evaluated at baseline and at postoperative months 1, 3 and 6. RESULTS: At postoperative 6 months, a significant improvement was observed in all keratometric values in both treatment groups (p < 0.05). All epithelial thickness indices, except central, temporal, and inferotemporal thickness, were reduced at 1 month postoperatively in both treatment groups. The epithelial map uniformity indices (standard deviation and difference between min-max thickness) were significantly lower than the baseline values at all time points after CXL in both treatment groups (p < 0.001). Compared with the preoperative values, there was a significant decrease in all IVCCM parameters at 1 month postoperatively (p < 0.05). At 6 months postoperatively, corneal nerve fiber density and corneal nerve branch density recovered to preoperative values in the A-CXL group, whereas corneal nerve regeneration was not complete in the C-CXL group. CONCLUSION: Both conventional and accelerated CXL treatments appear to be effective in halting the progression of KC. Corneal epithelial irregularity slightly improves after CXL. The regeneration of subbasal nerves is faster after A-CXL treatment.

A wireless battery-free eye modulation patch for high myopia therapy.

The proper axial length of the eye is crucial for achieving emmetropia. In this study, we present a wireless battery-free eye modulation patch designed to correct high myopia and prevent relapse. The patch consists of piezoelectric transducers, an electrochemical micro-actuator, a drug microneedle array, µ-LEDs, a flexible circuit, and biocompatible encapsulation. The system can be wirelessly powered and controlled using external ultrasound. The electrochemical micro-actuator plays a key role in precisely shortening the axial length by driving the posterior sclera inward. This ensures accurate scene imaging on the retina for myopia eye. The drug microneedle array delivers riboflavin to the posterior sclera, and µ-LEDs' blue light induces collagen cross-linking, reinforcing sclera strength. In vivo experiments demonstrate that the patch successfully reduces the rabbit eye's axial length by ~1217 µm and increases sclera strength by 387%. The system operates effectively within the body without the need for batteries. Here, we show that the patch offers a promising avenue for clinically treating high myopia.

Scleral collagen cross linkage in progressive myopia.

High myopia is often associated with local ectasia and scleral thinning. The progression of myopia depends upon scleral biochemical and biomechanical properties. Scleral thinning is associated with decreased collagen fiber diameter, defective collagen fibrillogenesis, and collagen cross-linking. Reversing these abnormalities may make the sclera tougher and might serve as a treatment option for myopic progression. Collagen cross-linking is a natural process in the cornea and sclera, which makes the structure stiff. Exogenous collagen cross-linkage is artificially induced with the help of external mediators by using light and dark methods. In this systematic review, we discussed existing literature available on the internet on current evidence-based applications of scleral collagen cross-linking (SXL) by using different interventions. In addition, we compared them in tabular form in terms of their technique, mechanisms, cytotoxicity, and the stage of transition from preclinical to clinical development. Furthermore, we discussed the in-vivo technique to evaluate the post-SXL scleral biomechanical property and outcome in the human eye.

Long-term outcomes of tailored stromal expansion with refractive lenticule for crosslinking thin corneas.

PURPOSE: To assess the long-term safety and stability of visual outcomes following the modified technique of collagen crosslinking (CXL) using refractive lenticule in eyes with thin corneas (<400 µm) and progressive keratoconus. SETTING: A tertiary eye care hospital in India. DESIGN: Prospective, interventional case series. METHODS: Eyes with progressive keratoconus and thin corneas (<400 µm) underwent CXL with intraoperative stromal augmentation using a refractive lenticule obtained from small-incision lenticule extraction (SMILE). Preoperative and postoperative evaluation (3 months, and then yearly thereafter) included corneal tomography (Oculus Pentacam), uncorrected and corrected distance visual acuity (UDVA and CDVA, respectively), manifest refraction, and endothelial cell count (specular microscopy), and adverse events, if any, were noted. The patients were followed up for a period of 5 years. RESULTS: Seven eyes were included in the analysis. Mean corneal flattening of -4.29 D was noted from preoperative maximum keratometry (P = 0.018). An improvement in UDVA and CDVA of 0.38 logarithm of minimum angle of resolution (logMAR) and 0.36 logMAR, respectively, was noted at 5 years postoperative visit. Four eyes demonstrated a gain of two lines in CDVA. Mean spherical equivalent improved from -6.85 D preoperatively to -6.05 D at 5 years postoperatively. Clear demarcation line was noted between 230 to 270 µm on anterior segment optical coherence topography. No significant endothelial cell loss was noted postoperatively. CONCLUSION: Long-term outcomes demonstrated safety and disease stability following lenticule-assisted CXL.

Collagen crosslinking impacts stromal wound healing and haze formation in a rabbit phototherapeutic keratectomy model.

Purpose: The purpose of this study was to evaluate the elastic modulus, keratocyte-fibroblast-myocyte transformation, and haze formation of the corneal stroma following combined phototherapeutic keratectomy (PTK) and epithelium-off UV-A/riboflavin corneal collagen crosslinking (CXL) using an in vivo rabbit model. Methods: Rabbits underwent PTK and CXL, PTK only, or CXL 35 days before PTK. Rebound tonometry, Fourier-domain optical coherence tomography, and ultrasound pachymetry were performed on days 7, 14, 21, 42, 70, and 90 post-operatively. Atomic force microscopy, histologic inflammation, and immunohistochemistry for α-smooth muscle actin (α-SMA) were assessed post-mortem. Results: Stromal haze formation following simultaneous PTK and CXL was significantly greater than in corneas that received PTK only and persisted for more than 90 days. No significant difference in stromal haze was noted between groups receiving simultaneous CXL and PTK and those receiving CXL before PTK. Stromal inflammation did not differ between groups at any time point, although the intensity of α-SMA over the number of nuclei was significantly greater at day 21 between groups receiving simultaneous CXL and PTK and those receiving CXL before PTK. The elastic modulus was significantly greater in corneas receiving simultaneous CXL and PTK compared with those receiving PTK alone. Conclusions: We showed that stromal haze formation and stromal stiffness is significantly increased following CXL, regardless of whether it is performed at or before the time of PTK. Further knowledge of the biophysical cues involved in determining corneal wound healing duration and outcomes will be important for understanding scarring following CXL and for the development of improved therapeutic options.

Use of Nanoindentation in Determination of Regional Biomechanical Properties of Rabbit Cornea After UVA Cross-Linking.

Purpose: To evaluate the regional effects of different corneal cross-linking (CXL) protocols on corneal biomechanical properties. Methods: The study involved both eyes of 50 rabbits, and the left eyes were randomized to the five intervention groups, which included the standard CXL group (SCXL), which was exposed to 3-mW/cm2 irradiation, and three accelerated CXL groups (ACXL1-3), which were exposed to ultraviolet-A at irradiations of 9 mW/cm2, 18 mW/cm2, and 30 mW/cm2, respectively, but with the same total dose (5.4 J/cm2). A control (CO) group was not exposed to ultraviolet-A. No surgery was done on the contralateral eyes. The corneas of each group were evaluated by the effective elastic modulus (Eeff) and the hydraulic conductivity (K) within a 7.5-mm radius using nanoindentation measurements. Results: Compared with the CO group, Eeff (in regions with radii of 0-1.5 mm, 1.5-3.0 mm, and 3.0-4.5 mm) significantly increased by 309%, 276%, and 226%, respectively, with SCXL; by 222%, 209%, and 173%, respectively, with ACXL1; by 111%, 109%, and 94%, respectively, with ACXL2; and by 59%, 41%, and 37%, respectively, with ACXL3 (all P < 0.05). K was also significantly reduced by 84%, 81%, and 78%, respectively, with SCXL; by 75%, 74%, and 70%, respectively, with ACXL1; by 64%, 62%, and 61%, respectively, with ACXL2; and by 33%, 36%, and 32%, respectively, with ACXL3 (all P < 0.05). For the other regions(with radii between 4.5 and 7.5 mm), the SCXL and ACXL1 groups (but not the ACXL2 and ACXL3 groups) still showed significant changes in Eeff and K. Conclusions: CXL had a significant effect on corneal biomechanics in both standard and accelerated procedures that may go beyond the irradiated area. The effect of CXL in stiffening the tissue and reducing permeability consistently decreased with reducing the irradiance duration.

Fabrication and In vitro Evaluation of Carbopol/Polyvinyl Alcohol-based pH-sensitive Hydrogels for Controlled Drug Delivery.

BACKGROUND: Diclofenac sodium has a short half-life (about 1.5 hours), requiring repeated administration, and as a result, serious complications, such as GI bleeding, peptic ulcer, and kidney and liver dysfunction, are generated. Hence, a sustained/controlled drug delivery system is needed to overcome the complications caused by the administration of diclofenac sodium. AIMS: This study aimed to fabricate and evaluate carbopol/polyvinyl alcohol-based pH-sensitive hydrogels for controlled drug delivery. OBJECTIVE: pH-sensitive carbopol/polyvinyl alcohol graft-poly(acrylic acid) hydrogels (Cp/PVA-g-PAa hydrogels) were developed for the controlled delivery of diclofenac sodium. METHODS: The combination of carbopol/polyvinyl alcohol, acrylic acid, and ethylene glycol dimethacrylate was used as polymer, monomer, and cross-linker, respectively. The effects of the formulation's composition on porosity, swelling index, and release pattern of diclofenac sodium from the developed hydrogels were investigated. RESULTS: An increase in porosity and swelling was observed with the increasing amounts of carbopol and acrylic acid, whereas polyvinyl alcohol showed the opposite effect. Due to the formation of a highly viscous system, the drug release decreased with the increasing concentrations of carbopol and polyvinyl alcohol while increased with increasing acrylic acid concentration. The pH-responsive properties of the fabricated hydrogels were demonstrated by dynamic swelling and drug release studies at three different pH values. Higher dynamic swelling and diclofenac sodium (model drug) release were found at high pH values compared to low pH values, i.e., pH 7.4 > 4.6 > 1.2, respectively. Cytotoxicity studies reported no toxic effect of the prepared hydrogels, thus indicating that the prepared hydrogels are safe to be used on clinical basis. CONCLUSION: The prepared carbopol/polyvinyl alcohol crosslinked hydrogel can be used as a promising carrier for the controlled release of drugs.

Intraocular pressure after combined photorefractive keratectomy and corneal collagen cross-linking for keratoconus.

PURPOSE: The purpose of this prospective study was to evaluate the effect of combined photorefractive keratectomy (PRK) and corneal collagen cross-linking (CXL) on intraocular pressure (IOP) in patients with keratoconus (KC). METHODS: We included 64 eyes of 34 patients (19 males and 15 females; age: 19-40y) with stages 1-2 keratoconus which had undergone combined wavefront-optimized photorefractive keratectomy and corneal collagen cross linking. Two other groups of patients were added as controls: the PRK group including 110 eyes of 57 patients (23 males and 34 females; age: 18-44y) which had undergone wavefront-optimized photorefractive keratectomy for myopic refractive errors, and the CXL group including 36 eyes of 23 patients (14 males and 9 females; age: 12-38y) with keratoconus, not filling the inclusion criteria for combined PRK and CXL, which had undergone corneal collagen cross-linking. IOP was recorded preoperatively and postoperatively at 3, 6 and 12 months follow-up visits. RESULTS: Preoperative IOP in both CXL (12.1 ± 2.53 mmHg) and PRK + CXL (13.2 ± 2.50 mmHg) groups was significantly lower than PRK group (15.8 ± 3.10 mmHg) (F = 30.505, p < 0.001). At 3 months postoperatively, IOP showed no statistically significant difference between the three studied groups (F = 1.821, p = 0.164). At 6 months postoperatively, IOP in the CXL group (14.6 ± 2.64 mmHg) was significantly higher than both PRK (13.4 ± 2.27 mmHg) and PRK + CXL (13.3 ± 2.62 mmHg) groups (F = 3.721, p = 0.026). At 12 months postoperatively, IOP in the CXL group (14.3 ± 2.69 mmHg) was significantly higher than the PRK group (13.2 ± 2.23 mmHg) and was higher than PRK + CXL group (13.3 ± 2.59 mmHg) although not statistically significant (F = 3.393, p = 0.035). Regarding the percent of change from preoperative IOP, a statistically significant difference between the three studied groups was detected at 3, 6 and 12 months postoperatively (H = 117.459, 109.303, 122.694 respectively, p < 0.001). The median percent of change from preoperative IOP in the PRK group was -16.7%, -15%, and -16.7%, in the CXL group was + 14.3%, + 19.4%, and + 19.1%, while in PRK + CXL group was 0% at 3, 6 and 12 months postoperatively. (Post-hoc power analysis 75%). CONCLUSIONS: Combined PRK and CXL in patients with KC shows no significant effect on IOP, in contrast to either procedure performed separately.

The cross-linking and protective effect of artemisinin and its derivatives on collagen fibers of demineralized dentin surface.

OBJECTIVE: To investigate the cross-linking and protective effect of artemisinin (ART), dihydroartemisinin (DHA), and artesunate (AST) on collagen fibers of demineralized dentin surface. METHODS: Molecular docking was used to predict potential interactions of ART, DHA, and AST with dentin type I collagen. Human third molars without caries were completely demineralized and treated with different solutions for 1 min. The molecular interactions and cross-linking degree of ART and its derivatives with dentin collagen were evaluated by FTIR spectroscopy, total extractable protein content, and a ninhydrin assay. Scanning electron microscopy, hydroxyproline release, and ultimate microtensile strength tests (µUTS) were employed to confirm the mechanical properties and anti-collagenase degradation properties of dentin collagen fibers. RESULTS: ART, DHA, and AST combined with dentin type I collagen mainly through hydrogen bonding and hydrophobic interactions, and the cross-linking reaction sites were mainly C=O and CN functional groups. Compared to the control group, ART and its derivatives significantly increased the degree of cross-linking. Additionally, significant increases were observed in resistance to enzymatic digestion and mechanical properties of the artemisinin and its derivatives group. CONCLUSION: ART, DHA, and AST could cross-link with demineralized dentin collagen, through improving the mechanical properties and anti-collagenase degradation properties. CLINICAL SIGNIFICANCE: The study endorses the potential use of ART and its derivatives as a prospective collagen cross-linking agent for degradation-resistant and long-period dentin bonding in composite resin restorations.

A review of the epithelial and stromal effects of corneal collagen crosslinking.

Induced corneal collagen crosslinking and mechanical stiffening via ultraviolet-A photoactivation of riboflavin (UVA CXL) is now a common treatment for corneal ectasia and Keratoconus. Some effects of the procedure such as induced mechanical stiffening, corneal flattening, and cellular toxicity are well-known, but others remain more controversial. Authors report a variety of contradictory effects, and provide evidence based on individual results and observations. A full understanding of the effects of and mechanisms behind this procedure are essential to predicting its outcome. A growing interest in modifications to the standard UVA CXL protocol, such as transepithelial or accelerated UVA CXL, makes analyzing the literature as a whole more urgent. This review presents an analysis of both the agreed-upon and contradictory results reported and the various methods used to obtain them.

Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

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.

H2 O2 -Inducible DNA Cross-linking Agents Capable of Releasing Multiple DNA Alkylators as Anticancer Prodrugs.

Three compounds with arylboronate esters conjugated with two equivalent nitrogen mustards [bis(2-chloroethyl)methylamine, HN2] have been synthesized and characterized. These inactive small molecules selectively react with H2 O2 to produce multiple DNA cross-linkers, such as two HN2 molecules alongside a bisquinone methide (bisQM), leading to efficient DNA ICL formation. In comparison to other amine functional groups, using HN2 as a leaving group greatly improves the DNA cross-linking efficiency of these arylboronate esters as well as cellular activity. The introduction of HN2 in these arylboronate ester analogues favored the generation of bisQM that can directly cross-link DNA. Two equivalents of HN2 are also generated from these compounds upon treatment with H2 O2 , which directly produces DNA ICL products. The cumulative effects of HN2 and bisQM on DNA cross-linking makes these molecules highly effective H2 O2 -inducible DNA ICL agents. The three compounds with HN2 as a leaving group showed greatly enhanced cytotoxicity towards cancer cells in comparison to those containing trimethyl amine as a leaving group. This provides an effective strategy for further design of novel potential ROS-activated anticancer prodrugs.

The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment.

The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.

Imaging the cellular response to an antigen tagged interstrand crosslinking agent.

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited. Consequently, DDR factor accumulation often serves as a surrogate for damage, without reporting the actual inducing structure. This limitation has practical implications given the importance of the response to DNA reactive drugs such as those used in cancer therapy. These include interstrand crosslink (ICL) forming compounds which are frequently employed clinically. Among them are the psoralens, natural products that form ICLs upon photoactivation and applied therapeutically since antiquity. However, despite multiple attempts, antibodies against psoralen ICLs have not been developed. To overcome this limitation, we developed a psoralen tagged with an antigen for which there are commercial antibodies. In this report we describe our application of the tagged psoralen in imaging experiments, and the unexpected discoveries they revealed.

Characterization of the Immediate and Delayed Biomechanical Response to UV-A Crosslinking of Human Corneas.

PURPOSE: Keratoconus leads to visual deterioration due to irregular astigmatism and corneal thinning. Riboflavin-based corneal UV-A crosslinking (CXL) induces novel intramolecular and intermolecular links resulting in corneal tissue stiffening, thereby halting disease progression. The purpose of this study was to analyze the immediate and delayed biomechanical responses of human donor corneas to CXL. METHODS: CXL was performed according to the Dresden protocol to corneas not suitable for transplantation. Biomechanical properties were subsequently monitored by measuring the Young modulus using nanoindentation. The immediate tissue response was determined after 0, 1, 15, and 30 minutes of irradiation. Delayed biomechanical effects were investigated with follow-up measurements immediately and 1, 3, and 7 days after CXL. RESULTS: Young's modulus indicated a linear trend in direct response to increasing irradiation times (mean values: total 61.31 kPa [SD 25.53], 0 minutes 48.82 kPa [SD 19.73], 1 minute 53.44 kPa [SD 25.95], 15 minutes 63.56 kPa [SD 20.99], and 30 minutes 76.76 kPa [SD 24.92]). The linear mixed model for the elastic response of corneal tissue was 49.82 kPa + (0.91 kPa/min × time [minutes]); P < 0.001. The follow-up measurements showed no significant delayed changes in the Young modulus (mean values: total 55,28 kPa [SD 15.95], immediately after CXL 56,83 kPa [SD 18.74], day 1 50.28 kPa [SD 14.15], day 3 57.08 kPa [SD 14.98], and day 7 56.83 kPa [SD 15.07]). CONCLUSIONS: This study suggests a linear increase of corneal Young modulus as a function of CXL timing. No significant short-term delayed biomechanical changes posttreatment were observed.

Corneal crosslinking with riboflavin using sunlight.

PURPOSE: To assess whether sunlight might be used to induce a biomechanical stiffening effect in riboflavin-soaked corneas similar to the effect observed in corneal crosslinking (CXL) using riboflavin and UV-A light. SETTING: Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland. DESIGN: Experimental study. METHODS: 52 porcine eyes were assayed. The concentration of riboflavin in the corneal stroma was estimated using UV-A transmission in a preliminary experiment. Then, the duration of sunlight exposure to achieve a fluence of 7.2/cm 2 was calculated. Finally, de-epithelialized corneas were divided equally into 3 groups and soaked with riboflavin 0.1% (control group and Group 1) or 0.5% (Group 2). Eyes from Groups 1 and 2 were then exposed to sunlight. The elastic modulus was calculated as an indicator of stiffness. RESULTS: Riboflavin concentration in Group B was higher by a factor of 2.8 than Group A. According to live illuminance measurements and stromal riboflavin concentration, the sunlight exposure duration varied between 16 minutes and 45 minutes. Groups 1 and 2 had higher elastic modulus than controls ( P < .0001) but did not differ between them ( P = .194). The stiffening effect was 84% and 55%, respectively. CONCLUSIONS: Sunlight exposure of ex vivo corneas soaked in both riboflavin 0.1% and 0.5% resulted in increased corneal stiffness. Specifically, riboflavin 0.1% with longer UV-A exposure showed a trend for a greater stiffening effect, which might open new alleys for the use of oral riboflavin and fractioned sunlight exposure as less invasive CXL techniques.

The impact of UV cross-linking on corneal stromal cell migration, differentiation and patterning.

Previous studies have demonstrated that UV cross-linking (CXL) increases stromal stiffness and produces alterations in extracellular matrix (ECM) microstructure. In order to investigate how CXL impacts both keratocyte differentiation and patterning within the stroma, and fibroblast migration and myofibroblast differentiation on top of the stroma, we combined CXL with superficial phototherapeutic keratectomy (PTK) in a rabbit model. Twenty-six rabbits underwent a 6 mm diameter, 70 µm deep phototherapeutic keratectomy (PTK) with an excimer laser to remove the epithelium and anterior basement membrane. In 14 rabbits, standard CXL was performed in the same eye immediately after PTK. Contralateral eyes served as controls. In vivo confocal microscopy through focusing (CMTF) was used to analyze corneal epithelial and stromal thickness, as well as stromal keratocyte activation and corneal haze. CMTF scans were collected pre-operatively, and from 7 to 120 days after the procedure. A subset of rabbits was sacrificed at each time point, and corneas were fixed and labeled in situ for multiphoton fluorescence microscopy and second harmonic generation imaging. In vivo and in situ imaging demonstrated that haze after PTK was primarily derived from a layer of myofibroblasts that formed on top of the native stroma. Over time, this fibrotic layer was remodeled into more transparent stromal lamellae, and quiescent cells replaced myofibroblasts. Migrating cells within the native stroma underneath the photoablated area were elongated, co-aligned with collagen, and lacked stress fibers. In contrast, following PTK + CXL, haze was derived primarily from highly reflective necrotic "ghost cells" in the anterior stroma, and fibrosis on top of the photoablated stroma was not observed at any time point evaluated. Cells formed clusters as they migrated into the cross-linked stromal tissue and expressed stress fibers; some cells at the edge of the CXL area also expressed α-SM actin, suggesting myofibroblast transformation. Stromal thickness increased significantly between 21 and 90 days after PTK + CXL (P < 0.001) and was over 35 µm higher than baseline at Day 90 (P < 0.05). Overall, these data suggest that cross-linking inhibits interlamellar cell movement, and that these changes lead to a disruption of normal keratocyte patterning and increased activation during stromal repopulation. Interestingly, CXL also prevents PTK-induced fibrosis on top of the stroma, and results in long term increases in stromal thickness in the rabbit model.

Analysis of Biomechanical Response After Corneal Crosslinking with Different Fluence Levels in Porcine Corneas.

PURPOSE: To evaluate corneal stiffening of porcine corneas induced by corneal crosslinking (CXL) with constant irradiance as a function of total fluence. METHODS: Ninety corneas from freshly enucleated porcine eyes were divided into five groups of 18 eyes. Groups 1-4 underwent epi-off CXL using a dextran-based riboflavin solution and an irradiance of 18 mW/cm2, group 5 served as the control group. Groups 1 to 4 were treated with a total fluence of 20, 15, 10.8, and 5.4 J/cm2, respectively. Thereafter, biomechanical measurements were performed on 5 mm wide and 6 mm long strips using an uniaxial material tester. Pachymetry measurements were performed on each cornea. RESULTS: At 10% strain, the stress was 76, 56, 52, and 31% higher in groups 1-4, respectively compared to the control group. The Young's modulus was 2.85 MPa for group 1, 2.53 MPa for group 2, 2.46 MPa for group 3, 2.12 MPa for group 4, and 1.62 MPa for the control group. The difference between groups 1 to 4 and the control group 5 were statistically significant (p = <0.001; p = <0.001; p = <0.001; p = 0.021). In addition, group 1 showed significantly more stiffening than group 4 (p = <0.001), no other significant differences were found. Pachymetry measurements revealed no statistically significant differences among the five groups. CONCLUSION: Additional mechanical stiffening can be achieved by increasing the fluence of the CXL. There was no threshold detected up to 20 J/cm2. A higher fluence could compensate the weaker effect of accelerated or epi-on CXL procedures.