Oxygen-supplemented and topography-guided epithelium-on corneal crosslinking with pulsed irradiation for progressive keratoconus.

PURPOSE: To investigate the effects of customized topography-guided epithelium-on crosslinking (epi-on CXL) with oxygen supplementation on procedural efficacy and corrected distance visual acuity (CDVA) in patients with progressive keratoconus (KC) at 1 year. SETTING: Private eye clinic, Brisbane, Australia. DESIGN: Retrospective, single-center, nonrandomized case series. METHODS: Topography-guided epi-on CXL using the Mosaic system was performed on patients with progressive KC. Oxygen goggles; transepithelial riboflavin; and pulsed, high UV-A irradiance (1 second on, 1 second off; 30 mW/cm2) were applied to enhance oxygen kinetics and bioavailabilities of riboflavin and UV-A. Guided by baseline topography, a higher UV-A dose (15 J/cm2) was applied to the area of steepest anterior curvature with decreasing fluence (as low as 7.2 J/cm2) toward the outer 9 mm. Postoperative CDVA and maximum keratometry (Kmax) were evaluated. RESULTS: 102 eyes (80 patients) were followed for 11.5 ± 4.8 months. At the latest follow-up, mean CDVA (logMAR), mean K, and Kmax (diopters [D]) improved from 0.18 ± 0.28, 46.2 ± 3.8, and 53.0 ± 5.67 at baseline to 0.07 ± 0.18, 45.8 ± 3.7, and 51.9 ± 5.56, respectively (P < .001). 3 eyes (3%) lost more than 1 CDVA line, and another 3 eyes (3%) had increased Kmax greater than 2 D. 43 eyes were followed for at least 12 months (n = 43): mean CDVA, mean K, and Kmax improved from 0.19 ± 0.33 logMAR, 46.5 ± 3.5 D, and 53.6 ± 5.67 D to 0.07 ± 0.17 logMAR, 46.0 ± 3.5 D, and 52.33 ± 5.49 D, respectively (P ≤ .002). No complications were observed. CONCLUSIONS: Tailoring oxygen-supplemented epi-on CXL with differential UV-A energy distributions, guided by baseline topography, in patients with KC seems to be safe and effective. At 1 year, study reports sustained improved CDVA and corneal stabilization.

Novel Correlation between TGF-ß1/-ß3 and Hormone Receptors in the Human Corneal Stroma.

This study investigated the interplay between transforming growth factor beta (TGF-ß1/T1 and TGF-ß3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 106 cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα) and beta (ERß), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERα, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERα, ERß, and GnRHR. T3 caused significant upregulation in expressions PR, ERα, ERß, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERα, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-ß isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.

Transepithelial corneal crosslinking with oxygen enhancement and pulsed light for progressive ectasia: one-year results.

PURPOSE: To evaluate the efficacy and safety of transepithelial accelerated crosslinking (TE-ACXL) using pulsed light and supplemental oxygen. METHODS: Thirty eyes of 30 consecutive patients with progressive keratoconus or post-LASIK ectasia were enrolled in a prospective non-comparative study conducted at the Magrabi Eye Center (Jeddah, Saudi Arabia). All eyes underwent TE-ACXL with supplemental oxygen. Primary outcome measures were the mean change in corrected distance visual acuity (CDVA) (logMAR) and maximum keratometry (max K) from preoperatively to 12 months postoperatively. Secondary outcome measures included change in manifest refractive spherical equivalent (MRSE), refractive cylinder, keratometry, symmetry index (SI), center-surrounding index (CSI) and ectasia index (EI) of the anterior and posterior corneal surfaces, corneal and epithelial thickness at corneal vertex and thinnest location, corneal densitometry, corneal high order aberrations (HOA) and endothelial cell density (ECD). RESULTS: Mean age was 29.6 ± 8.2 years. At 1 year, the follow up rate was 93.3%. CDVA improved statistically significantly at 12 months (p = 0.027). Measures of corneal keratometry or pachymetry did not change significantly (p < 0.05). Postoperatively, a demarcation line was documented in 78.6% eyes at 1 month, and in 12 (42.9%) eyes at 12 months. The mean depth of the demarcation line was 341.9 ± 49.4 µm. Corneal densitometry increased significantly at 1- and 3-months (p < 0.05) and returned to normal levels at 6- and 12-months postoperatively. CONCLUSION: TE-ACXL with oxygen supplement is effective at halting the progression of corneal ectasia for at least 1 year and can be a refractive neutral procedure.

Comparação entre os resultados do implante de um segmento de arco longo versus dois segmentos de arco tradicional no tratamento de pacientes com ceratocone / Comparison between the results of implanting a long-arch segment versus two standard arch segments for treatment of keratoconus patients

RESUMO Objetivo: Analisar retrospectivamente as alterações na ceratometria e no astigmatismo corneano obtidas após cirurgia de implante de anel intraestromal, comparando o uso de um segmento de arco longo versus o implante de dois segmentos de comprimento de arco tradicional. Métodos: A partir de um estudo transversal, obtivemos os dados de 94 olhos de pacientes diagnosticados com ceratocone, que foram submetidos ao implante de anel corneano. Eles foram divididos em dois grupos, dependendo do tipo de implante recebido: Grupo A, um segmento de arco longo; Grupo B, dois segmentos tradicionais. Todos os segmentos implantados possuíam 250µ de espessura. Os dados do pré-operatório dos dois grupos foram comparados, para garantir que as amostras eram similares (as diferenças encontradas entre os dois grupos não eram estatisticamente significativas). As variáveis analisadas no pré e no pós-operatório foram acuidade visual com correção, ceratometria, astigmatismo corneano e refração. Resultados: A amostra que recebeu apenas um segmento de arco longo (Grupo A) obteve redução da ceratometria média de 4,42D (8,7%) e do astigmatismo corneano de 2,43D (40,4%). Já na amostra dos olhos que receberam dois segmentos de arco tradicional (Grupo B), houve redução média de 2,66D (5,1%) em relação à ceratometria média e redução média de 2,11D (34,8%) em relação ao astigmatismo corneano. A redução obtida na ceratometria média no Grupo A foi maior que a obtida no Grupo B (diferença estatisticamente significativa). A redução obtida no astigmatismo do Grupo A não foi estatisticamente significante, se comparada com o resultado obtido no Grupo B (considerando p≤0,05). Conclusão: Foi demonstrado que o uso de um segmento de arco longo possui maior capacidade de aplanação corneana, se comparado com o uso de dois segmentos com comprimento de arco tradicional. Em relação à redução do astigmatismo, os dois grupos mostraram resultados equivalentes.

ABSTRACT Purpose: To retrospectively analyze the changes in corneal keratometry and astigmatism after intrastromal ring surgery, comparing the use of one long arch segment versus two traditional arc length segments. Methods: A cross-sectional study obtained data from 94 eyes of patients diagnosed with keratoconus that underwent surgical treatment with corneal ring implant. They were divided into two groups according to the type of implant received: one long-arch segment (Group A) or two traditional segments (Group B), both 250 microns thick. Preoperative data from the two groups were compared to ensure that the samples were similar (the differences between the two groups were not statistically significant). The variables (pre and post-operatively) analyzed were: best corrected visual acuity, keratometry, corneal astigmatism and refraction. Results: Group A, which received one long arch segment, showed a Km decrease of 4.42D (8.7%) and a corneal astigmatism reduction of 2.43D (40.4%). Group B, where the eyes acquired two traditional arch segments, showed an average Km decrease of 2.66D (5.1%) and corneal astigmatism reduction of 2.11D (34.8%). The mean keratometry (Km) reduction obtained was statistically significant (p≤0.05) when comparing both groups (A and B). The mean corneal astigmatism reduction was not statistically significant (p≤0.05) when comparing both groups (A and B). Conclusions: One long-arch segment has been demonstrated to have a greater capacity to reduce corneal curvature when compared to the use of two traditional-sized arch segments. No significant differences were found regarding the reduction of corneal astigmatism after comparing the results obtained in both groups.

Iontophoresis Corneal Cross-linking With Oxygen Supplementation in Ovine Eyes.

PURPOSE: To evaluate the biomechanical changes and advanced oxidation protein products (AOPP) production after different corneal cross-linking (CXL) protocols with or without oxygen supplementation. METHODS: Ovine eyes in the study were equally distributed to five groups as control, standard Dresden protocol, diluted alcohol- and iontophoresis-assisted CXL (DAI-CXL), and 0.1% and 0.2% riboflavin-mediated iontophoresis-assisted CXL with oxygen supplementation (I-CXL). Corneas that received CXL were divided into two equal parts, one part was used for uniaxial tensiometry and one part was used for AOPP measurement. RESULTS: All treatment groups showed higher Young's modulus and stiffness compared to the control group (P < .05). Both oxygen-assisted I-CXL groups with 0.1% and 0.2% riboflavin concentrations had higher corneal Young's modulus (P = .009 and .006, respectively) and stiffness (P = .009) values, whereas the DAI-CXL group had lesser Young's modulus and stiffness values (P = .032) compared to the Dresden protocol group. All treatment groups showed higher AOPP concentrations compared to the control group (P < .05). DAI-CXL and I-CXL groups showed similar AOPP formation compared to the Dresden protocol (P = .673). CONCLUSIONS: When the epithelium is intact, the desired increase in corneal stiffness might not be achieved. However, increasing the oxygen in the environment might provide a sufficient increase in stiffness in cases undergoing epitheliumon I-CXL, which might be promising in terms of shortening the CXL therapy and decreasing the complications. [J Refract Surg. 2022;38(10):674-681.].

The Yin and Yang of Mesenchymal Cells in the Corneal Stromal Fibrosis Response to Injury: The Cornea as a Model of Fibrosis in Other Organs.

Mesenchymal cells (keratocytes, corneal fibroblasts, and myofibroblasts), as well as mesenchymal progenitor bone marrow-derived fibrocytes, are the major cellular contributors to stromal fibrosis after injury to the cornea. Corneal fibroblasts, in addition to being major progenitors to myofibroblasts, also have anti-fibrotic functions in (1) the production of non-basement membrane collagen type IV that binds activated transforming growth factor (TGF) beta-1 and TGF beta-2 to downregulate TGF beta effects on cells in the injured stroma, (2) the production of chemokines that modulate the entry of bone marrow-derived cells into the stroma, (3) the production of hepatocyte growth factor and keratinocyte growth factor to regulate corneal epithelial healing, (4) the cooperation with the epithelium or corneal endothelium in the regeneration of the epithelial basement membrane and Descemet's membrane, and other functions. Fibrocytes also serve as major progenitors to myofibroblasts in the corneal stroma. Thus, mesenchymal cells and mesenchymal cell progenitors serve Yin and Yang functions to inhibit and promote tissue fibrosis depending on the overall regulatory milieu within the injured stroma.

Comparison of Continuous versus Pulsed Mode in Accelerated Corneal Collagen Cross-linking for Keratoconus.

PURPOSE: To compare efficacy and safety between the two modes of energy delivery-pulsed and continuous, in accelerated corneal collagen cross-linking (KXL) to stop the keratoconus advancement through topographical, visual, and refractive endpoints. METHODS: It was a prospective, comparative, randomized, interventional trial. Patients with bilateral progressive keratoconus were subjected to pulsed mode KXL (P-KXL) in the right and continuous mode KXL (C-KXL) treatment in the left eye. In both methods, additional supplemental oxygen was delivered to corneal surface using a nasal cannula connected to an oxygen supply. Uncorrected distance visual acuity (UDVA) and corrected distance visual acuity, posttreatment manifest spherical equivalent and astigmatism, and corneal topography were studied. The deformation amplitude index (DAI) was measured by Corvis-ST. Postoperative follow-up was done. RESULTS: One hundred eyes of 50 patients underwent P-KXL in the right eye and C-KXL in the left eye. The average follow-up was 12.1 ± 1.2 months. At 1 year posttreatment, the UDVA had a mean change of 0.11 ± 0.14 logMAR in P-KXL and 0.18 ± 0.14 logMAR in C-KXL groups. The line of demarcation was observed at 251.13 ± 18.28 µ and 245.28 ± 28.26 µ deep, respectively, in P-KXL- and C-KXL-treated eyes at 6 months' follow-up. The DAI as measured by Corvis-ST showed a significant decrease from pretreatment values of 1.12 ± 0.13 mm to 0.84 ± 0.14 mm (P < 0.01) in P-KXL eyes and from 1.04 ± 0.14 mm to 0.85 ± 0.15 mm (P = 0.03) in C-KXL eyes. Both the groups did not show any statistically significant endothelial cell loss posttreatment. CONCLUSION: C-KXL can give similar functional outcomes as P-KXL with the help of supplemental oxygen delivery with the added advantage of a shortened procedure time in comparison to pulsed mode.

The anti-scarring role of Lycium barbarum polysaccharide on cornea epithelial-stromal injury.

PURPOSE: Cornea epithelial-stromal scarring is related to the differentiation of fibroblasts into opaque myofibroblasts. Our study aims to assess the effectiveness of Lycium barbarum polysaccharide (LBP) solution as a pre-treatment in minimizing corneal scarring. METHODS: Human corneal fibroblasts were cultured in a three-dimensional collagen type I-based hydrogel in an eye-on-a-chip model. Fibroblasts were pre-treated with 2 mg/mL LBP for 24 h, followed by another 24-h incubation with 10 ng/mL transforming growth factor-beta 1 (TGF-ß1) to induce relevant physiological events after stromal injury. Intracellular pro-fibrotic proteins, extracellular matrix proteins, and pro-inflammatory cytokines that involved in fibrosis, were assessed using immunocytochemistry and enzyme-linked immunosorbent assays. RESULTS: Compared to the positive control TGF-ß1 group, LBP pre-treated cells had a significantly lower expression of alpha-smooth muscle actin, marker of myofibroblasts, vimentin (p < 0.05), and also extracellular matrix proteins both collagen type II and type III (p < 0.05) that can be found in scar tissues. Moreover, LBP pre-treated cells had a significantly lower secretion of pro-inflammatory cytokines interleukin-6 and interleukin-8 (p < 0.05). The cell-laden hydrogel contraction and stiffness showed no significant difference between LBP pre-treatment and control groups. Fibroblasts pretreated with LBP as well had reduced angiogenic factors expression and suppression of undesired proliferation (p < 0.05). CONCLUSION: Our results showed that LBP reduced both pro-fibrotic proteins and pro-inflammatory cytokines on corneal injury in vitro. We suggest that LBP, as a natural Traditional Chinese Medicine, may potentially be a novel topical pre-treatment option prior to corneal refractive surgeries with an improved prognosis.

Diffuse Lamellar Keratitis in a Patient Undergoing Collagen Corneal Cross-Linking 18 Years After Laser In Situ Keratomileusis Surgery.

PURPOSE: To report a case of diffuse lamellar keratitis (DLK) after corneal collagen cross-linking in an eye with a remote history of laser in situ keratomileusis (LASIK) surgery. METHODS: This is a case report and literature review. RESULTS: This report describes the development of unilateral stage IV DLK in a patient who underwent bilateral corneal cross-linking for corneal ectasia 18 years after LASIK surgery. The patient was treated with high-dose topical steroids that were tapered over 1 month and multiple flap lifts. The ultimate best-corrected visual outcome was 20/60. CONCLUSIONS: DLK is a potential sight-threatening complication of refractive surgery that can occur at any time in the postoperative period, even years after the procedure. Undergoing a subsequent corneal procedure that may disrupt or promote inflammation within the surgical flap-stromal interface, such as corneal collagen cross-linking, is a recognized risk factor for the development of DLK. This case suggests that patients with any history of LASIK surgery undergoing corneal cross-linking or other lamellar corneal surgeries may benefit from closer follow-up (eg, daily) than patients with no history of LASIK.

Cell sheet technology: Influence of culture conditions on in vitro-cultivated corneal stromal tissue for regenerative therapies of the ocular surface.

The in vitro reconstruction of stromal tissue by long-term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L-glutamine, components of collagen such as L-proline, L-4-hydroxyproline and glycine, and TGF-ß1, bFGF, IGF-2, PDGF-BB and insulin. After long-term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small-angle X-ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF-ß1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold-free, potentially autologous, in vitro-generated construct by culturing fibroblasts with extracellular matrix synthesis-stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.

Comparison of Visual, Tomographic, and Biomechanical Outcomes of 360 Degrees Intracorneal Ring Implantation With and Without Corneal Crosslinking for Progressive Keratoconus: A 5-Year Follow-up.

PURPOSE: To evaluate the long-term (5 years) results of 360 degrees intracorneal ring (ICR) implantation with and without corneal crosslinking (CXL) in patients with progressive keratoconus (KCN). METHOD: This historical cohort study was performed on 35 eyes with progressive KCN, which was randomly divided into 2 groups. Fourteen patients were implanted only with ICR, and 21 patients with KCN were treated with ICR plus CXL simultaneously. Uncorrected distance visual acuity (UCDVA), corrected distance visual acuity (CDVA), and refractive components were collected from patients' medical records. The biomechanical properties of patients were measured using the Corvis ST (Oculus, Inc, Weltzar, Germany). Corneal topography and aberrometry parameters were also recorded from the Pentacam HR device (Oculus). RESULTS: Both UCVA and CDVA increased in both groups after ICR implantation; however, this improvement was more significant in the ICR plus CXL group (P = 0.002 and P = 0.001, respectively). The mean improvement of CDVA in patients with ICR implantation with and without CXL was 0.56 ± 0.67 and 0.33 ± 0.61, respectively. A comparison of the long-term postoperative Tomographic and Biomechanical Index between 2 groups showed a better result in the ICR plus CXL group (P = 0.012). Topographic findings of the anterior corneal surface (flat-K, steep-K, mean-K, and astigmatism) after surgery were significantly better than before surgery in both groups (P < 0.05). CONCLUSIONS: Although ICR implantation alone might halt the KCN progression with acceptable visual, topographic, and biomechanical outcomes, the combination of ICR and CXL has an adjuvant and synergistic effect, especially in long-term follow-up.

L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes.

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

Therapeutic Effect of Corneal Crosslinking on Fungal Keratitis: Efficacy of Corneal Collagen Crosslinking as an Adjuvant Therapy for Fungal Keratitis in a Tertiary Eye Hospital in South India.

PURPOSE: To evaluate the efficacy of CXL in treating fungal keratitis as an adjuvant therapy. METHODS: Detailed clinical examination microbiological investigation was performed. Twenty fungal keratitis patients were recruited and randomized into two groups: group 1 (n= 11, standard antifungal), group 2 (n=9, corneal collagen crosslinking with standard antifungal). Corneal scraping and tear samples collected were subjected to real-time PCR targeting ITS, TLR analysis and cytokine analysis. RESULTS: The mean time for complete resolution of ulcer for group 2 was significantly shorter compared to group 1 and the final mean BCVA was better for group 2. Expression of IL-1ß, IL-8, IFN-γ significantly decreased immediately post CXL in group 2 patients. Significant downregulation of TLR 6, TLR-3, TLR-4 was observed 3-days post CXL compared to group 1 patients. CONCLUSION: Adjuvant effect of CXL was significant in treating fungal keratitis compared to standalone antifungal treatment.

Cross-Linking Assisted Infection Reduction (CLAIR): A Randomized Clinical Trial Evaluating the Effect of Adjuvant Cross-Linking on Bacterial Keratitis.

PURPOSE: To determine whether there is a benefit to adjuvant corneal cross-linking (CXL) for bacterial keratitis. METHODS: This is an outcome-masked, randomized controlled clinical trial. Consecutive patients presenting with a smear-positive bacterial ulcer at Aravind Eye Hospitals at Madurai, Pondicherry, and Coimbatore in India were enrolled. Study eyes were randomized to topical moxifloxacin 0.5% or topical moxifloxacin 0.5% plus CXL. The primary outcome of the trial was microbiological cure at 24 hours on repeat culture. Secondary outcomes included best spectacle corrected visual acuity at 3 weeks and 3 months, percentage of study participants with epithelial healing at 3 weeks and 3 months, infiltrate and/or scar size at 3 weeks and 3 months, 3-day smear and culture, and adverse events. RESULTS: Those randomized to CXL had 0.60 decreased odds of culture positivity at 24 hours (95% confidence interval [CI]: 0.10-3.50; P = 0.65), 0.9 logarithm of the minimum angle of resolution lines worse visual acuity (95% CI: -2.8 to 4.6; P = 0.63), and 0.41-mm larger scar size (95% CI: -0.48 to 1.30; P = 0.38) at 3 months. We note fewer corneal perforations or need for therapeutic penetrating keratoplasty in the CXL group. CONCLUSIONS: We were unable to confirm a benefit to adjuvant CXL in the primary treatment of moderate bacterial keratitis. However, CXL may reduce culture positivity and complication rates; therefore, a larger trial to fully evaluate this is warranted. TRIAL REGISTRATION: NCT02570321.

Oxygen Kinetics During Corneal Cross-linking With and Without Supplementary Oxygen.

PURPOSE: To measure and simulate oxygen kinetics during corneal cross-linking at different irradiances with and without supplementary oxygen. DESIGN: Experimental, laboratory study. METHODS: In de-epithelialized porcine eyes, a femtosecond-laser-generated tunnel was used to place a fiber probe in corneal depths of 100, 200, and 300 µm to measure the local oxygen concentration. After riboflavin imbibition, the corneas were irradiated at 3, 9, 18, and 30 mW/cm2 while the oxygen concentration was measured. All experiments were performed under normoxic (21%) and hyperoxic (>95%) conditions. The obtained data were used to identify parameters of a numerical model for oxygen consumption and diffusion. RESULTS: The equilibrium stromal oxygen concentration under atmospheric oxygen at 3 mW/cm2 was 2.3% in 100 µm decreasing to <1% in 300 µm. With 9, 18, and 30 mW/cm2, no oxygen was available in 200 µm, respectively, 100 µm or deeper. Using a hyperoxic environment, the concentration was 50% using 3 mW/cm2 in 100 µm, decreasing to 40% in 300 µm. At 9 mW/cm2, the concentrations were 5%, 3%, and 1% in 100, 200 and 300 µm, respectively. Using 18 and 30 mW/cm2, all oxygen was depleted at 100 µm; however, oxygen half-lives were longer at 18 mW/cm2 than at 30 mW/cm2. The oxygen model was able to reproduce the experiments and indicated an exponential decay with increasing distance to the anterior surface. CONCLUSION: Supplementary oxygen increases the oxygen availability during corneal cross-linking. At higher irradiances, supplementary oxygen is beneficial and eliminates the bottleneck of oxygen allowing a potentially more efficient cross-linking. The calibrated numerical model can quantify the spatial oxygen concentration related to different scenarios such as irradiance or environmental oxygen concentration.

Customized corneal crosslinking for treatment of progressive keratoconus: Clinical and OCT outcomes using a transepithelial approach with supplemental oxygen.

PURPOSE: To evaluate the outcomes of customized corneal crosslinking (CXL) for treatment of progressive keratoconus (KC) using a transepithelial approach with supplemental oxygen. SETTING: Siena Crosslinking Center, Siena, Italy. DESIGN: Prospective interventional case series. METHODS: Twenty-seven eyes of 24 patients (mean age 29.3 ± 7.3 years) with progressive KC underwent customized corneal CXL using a transepithelial approach with supplemental oxygen. Ultraviolet (UV)-A irradiation of 365 nm wavelength was delivered in an accelerated (30 mW/cm) pulsed-light UV light exposure in a 2-zone elliptical pattern. A total dose of 10 J/cm was delivered at the KC apex, surrounded by a broadbeam spot of 7.2 J/cm. After 0.25% riboflavin corneal soaking, the UV-A irradiation was initiated in the presence of additional oxygen (≥90% concentration) delivered through special goggles connected to an oxygen delivery system (flow-rate 2.5 liters per minute). Key outcome measures included corrected distance visual acuity (CDVA), keratometry (AK, K1, K2, and K-average), corneal higher-order aberrations, topographic and manifest cylinder, corneal optical coherence tomography (OCT) demarcation line, and endothelial cell count. RESULTS: Of the 27 eyes studied, a significant improvement of CDVA was recorded at 6-month follow-up visits, from baseline 0.19 ± 0.06 logarithm of the minimum angle of resolution (logMAR) to 0.11 ± 0.04 logMAR (P < .05). Significant flattening of steep keratometry (K2) was reported with mean change of -1.9 diopters (D) (P < .05), and coma values improved from 0.47 ± 0.28 µm to 0.28 ± 0.16 µm (P < .05). OCT revealed 2 demarcation lines at mean depths of 218.23 ± 43.32 µm and 325.71 ± 39.70 µm. CONCLUSIONS: In this series, customized CXL using a transepithelial approach with intraoperative supplemental oxygen resulted in clinically meaningful improvements in corneal curvature and CDVA without significant adverse events.

Decellularized human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblasts (HCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets [CS]) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure, and cell toxicity of the decellularized CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free, and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however, future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.

Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique.

To overcome the serious shortage of donor corneas for transplantation, alternatives based on tissue engineering need to be developed. Decellularized corneas are one potential alternative, but their densely packed collagen architecture inhibits recellularization in vitro. Therefore, a new rapid method of recellularizing these constructs to ensure high cellularity throughout the collagen scaffold is needed. In this study, we developed a novel method for fabricating corneal constructs by using decellularized porcine corneal sheets assembled using a bottom-up approach by layering multiple sheets between cell-laden collagen I hydrogel. Corneal lenticules were cut from porcine corneas by cryosectioning, then decellularized with detergents and air-dried for storage as sheets. Human corneal stromal cells were encapsulated in collagen I hydrogel and cast between the dried sheets. Constructs were cultured in serum-free medium supplemented with ascorbic acid and insulin for 2 weeks. Epithelial cells were then seeded on the surface and cultured for an additional week. Transparency, cell viability, and phenotype were analyzed by qPCR, histology, and immunofluorescence. Constructs without epithelial cells were sutured onto an ex vivo porcine cornea and cultured for 1 week. Lenticules were successfully decellularized, achieving dsDNA values of 13 ± 1.2 ng/mg dry tissue, and were more resistant to degradation than the collagen I hydrogels. Constructs maintained high cell viability with a keratocyte-like phenotype with upregulation of keratocan, decorin, lumican, collagen I, ALDH3A1, and CD34 and the corneal epithelial cells stratified with a cobblestone morphology. The construct was amenable to surgical handling and no tearing occurred during suturing. After 7 days ex vivo, constructs were covered by a neoepithelium from the host porcine cells and integration into the host stroma was observed. This study describes a novel approach toward fabricating anterior corneal substitutes in a simple and rapid manner, obtaining mature and suturable constructs using only tissue-derived materials.

Photorefractive intrastromal corneal crosslinking for treatment of low myopia: clinical outcomes using the transepithelial approach with supplemental oxygen.

PURPOSE: To evaluate the safety and efficacy of photorefractive intrastromal corneal crosslinking (PiXL) for the treatment of low myopia using the epithelium-on approach with supplemental oxygen. SETTING: The Eye Foundation, Coimbatore, India. DESIGN: Prospective interventional case series. METHODS: Myopic nonectatic eyes underwent PiXL using the Mosaic system. Ultraviolet-A (UV-A) irradiation of 365 nm wavelength was delivered in an accelerated (30 mW/cm) pulsed approach to provide a total fluence of 15 J/cm. Supplemental oxygen (concentration greater than 95%) was provided to increase the efficacy of the epithelium-on approach during the UV-A irradiation. Efficacy was determined by improvement in mean refractive spherical equivalent (MRSE), uncorrected distance visual acuity (UDVA), and keratometric flattening. Safety was determined by loss of lines of corrected distance visual acuity, endothelial cell loss, and adverse events. RESULTS: Fifty eyes of 26 patients with a mean age of 22.73 ± 3.74 years were included. A significant improvement in UDVA from baseline (logarithm of the minimum angle of resolution [logMAR] 0.63 ± 0.25) was noted at the 3-month (logMAR 0.08 ± 0.15) and 6-month (logMAR 0.13 ± 0.18) follow-up visits (P < .001). Significant keratometric flattening from baseline was noted at all follow-up visits (P < .001). No significant endothelial cell loss or adverse effects were noted. A significant correlation was noted between the change in MRSE and preoperative corneal biomechanics (deformation amplitude ratio, P = .029). CONCLUSIONS: Transepithelial PiXL with supplemental oxygen might be a safe and effective approach for reduction of myopia. The change in MRSE and keratometric flattening was greater in comparison with earlier protocols, including the epithelium-off approach.

Optimization of Oxygen Dynamics, UV-A Delivery, and Drug Formulation for Accelerated Epi-On Corneal Crosslinking.

Purpose: Corneal collagen crosslinking (CXL) through an intact epithelium (epi-on) at high irradiance could potentially improve patient comfort, visual recovery, and clinical workflow compared to conventional epi-off CXL. However, intact epithelium limits stromal delivery of the oxygen, photosensitizer, and ultraviolet-A (UV-A) radiation needed to drive CXL. This ex vivo study evaluated three different epi-on CXL protocols compared to positive and negative controls, specifically focusing on the impact of supplemental oxygen. Endpoints included stromal oxygen levels, stiffness of crosslinked tissue, and acute flattening of whole eyes.Materials & Methods: Ex vivo porcine eyes were held in a custom environmental chamber. Intrastromal oxygen levels were continuously measured before, during, and after UV illumination by a fiberoptic probe inserted into a laser-cut flap. Accelerated, high irradiance, epi-on CXL protocols using riboflavin formulated with benzalkonium chloride (BAC) were studied, with and without supplemental oxygen. These were compared to an alternate, low irradiance, epi-on protocol using riboflavin formulated with sodium iodide. Both negative (no CXL) and positive (epi-off modified Dresden protocol) controls were performed. Post-CXL elastic modulus was measured using extensiometry and anterior tangential curvature was measured using a Scheimpflug tomographer.Results: Protocols including supplemental oxygen resulted in an approximately 5-fold increase in stromal oxygen levels prior to CXL. During epi-on, high-irradiance UV-A delivery under hyperoxic conditions, an aerobic state was maintained. Conversely, under normoxic conditions, stromal oxygen rapidly depleted to 0-5% for all other protocols. The combination of supplemental oxygen, BAC formulation, and high-irradiance UV-A resulted in the largest biomechanical changes and most pronounced flattening effects of the three epi-on protocols.Conclusions: Ex vivo analysis of stromal oxygen levels, corneal stiffness, and acute anterior curvature change indicates that simultaneous optimization of the oxygen environment, riboflavin formulation, and UV-A protocol can significantly increase the effects of corneal collagen crosslinking.