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Corneal scarring is a leading cause of worldwide blindness. Human mesenchymal stem cells (MSC) have been reported to promote corneal wound healing through secreted exosomes. This study investigated the wound healing and immunomodulatory effects of MSC-derived exosomes (MSC-exo) in corneal injury through an established rat model of corneal scarring. After induction of corneal scarring by irregular phototherapeutic keratectomy (irrPTK), MSC exosome preparations (MSC-exo) or PBS vehicle as controls were applied to the injured rat corneas for five days. The animals were assessed for corneal clarity using a validated slit-lamp haze grading score. Stromal haze intensity was quantified using in-vivo confocal microscopy imaging. Corneal vascularization, fibrosis, variations in macrophage phenotypes, and inflammatory cytokines were evaluated using immunohistochemistry techniques and enzyme-linked immunosorbent assays (ELISA) of the excised corneas. Compared to the PBS control group, MSC-exo treatment group had faster epithelial wound closure (0.041), lower corneal haze score (p = 0.002), and reduced haze intensity (p = 0.004) throughout the follow-up period. Attenuation of corneal vascularisation based on CD31 and LYVE-1 staining and reduced fibrosis as measured by fibronectin and collagen 3A1 staining was also observed in the MSC-exo group. MSC-exo treated corneas also displayed a regenerative immune phenotype characterized by a higher infiltration of CD163+, CD206+ M2 macrophages over CD80+, CD86+ M1 macrophages (p = 0.023), reduced levels of pro-inflammatory IL-1β, IL-8, and TNF-α, and increased levels of anti-inflammatory IL-10. In conclusion, topical MSC-exo could alleviate corneal insults by promoting wound closure and reducing scar development, possibly through anti-angiogenesis and immunomodulation towards a regenerative and anti-inflammatory phenotype.

Femtosecond lasers have revived the possibility of stromal keratophakia or tissue additive keratoplasty, a technique originally introduced by Prof. Jose Ignacio Barraquer in the 1960s. The surgical technique offers a unique solution to treat keratoconus. In the current study, we reviewed and performed a meta-analysis of the clinical outcomes of the femtosecond laser-assisted stromal keratophakia in the treatment of keratoconus. This is a systematic review and meta-analysis of the estimated outcome difference between pre- and post-lenticule implantations. A total of related 10 studies were found in the literature. No studies reported adverse events, such as persistent haze or graft rejection, at last patients’ visits. We further narrowed down the article selection in accordance to our inclusion criteria to report the composite outcomes (9 studies) and meta-analysis (4 studies). In the composite analysis, we demonstrated that lenticule implantation in keratoconus and post-LASIK ectasia patients appeared to expand the stromal volume of the thin corneas, flattened the cones, and significantly improved uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA) and spherical equivalent (SE). The meta-analysis showed that the random estimated UCVA, BCVA, SE and mean keratometry (Km) differences following the lenticule implantation was −0.214 (95% CI: −0.367 to 0.060; p = 0.006), −0.169 (−0.246 to 0.091; p

Corneal blindness due to scarring is conventionally treated by corneal transplantation, but the shortage of donor materials has been a major issue affecting the global success of treatment. Pre-clinical and clinical studies have shown that cell-based therapies using either corneal stromal stem cells (CSSC) or corneal stromal keratocytes (CSK) suppress corneal scarring at lower levels. Further treatments or strategies are required to improve the treatment efficacy. This study examined a combined cell-based treatment using CSSC and CSK in a mouse model of anterior stromal injury. We hypothesize that the immuno-regulatory nature of CSSC is effective to control tissue inflammation and delay the onset of fibrosis, and a subsequent intrastromal CSK treatment deposited collagens and stromal specific proteoglycans to recover a native stromal matrix. Using optimized cell doses, our results showed that the effect of CSSC treatment for suppressing corneal opacities was augmented by an additional intrastromal CSK injection, resulting in better corneal clarity. These in vivo effects were substantiated by a further downregulated expression of stromal fibrosis genes and the restoration of stromal fibrillar organization and regularity. Hence, a combined treatment of CSSC and CSK could achieve a higher clinical efficacy and restore corneal transparency, when compared to a single CSSC treatment.

The majority of clinical corneal prostheses (KPros) adopt a core-skirt configuration. This configuration is favored owing to the optic core (generally a cylindrical, acrylic-based material, such as PMMA), that not only provides a clear window for the patients' vision, but also confers resistance to biodegradability. The surrounding skirt (typically a biological material, such as corneal tissue) allows for host tissue integration. However, due to poor biointegration between the dissimilar core and skirt materials, it results in a weak adhesion at the interface, giving rise to clinical complications, such as bacterial infections in the tissue-PMMA interface and device extrusion. Here, we physically immobilized nano-hydroxyapatite (nHAp) on a PMMA cylinder via a dip-coating technique, to create a bioactive surface that improved biointegration in vivo. We established that the nHAp coating was safe and stable in the rabbit cornea over five weeks. More importantly, we found that apoptotic, wound healing and inflammatory responses to nHAp-coated PMMA were substantially milder than to non-coated PMMA. More mature collagen, similar to the non-operated cornea, was maintained in the corneal stroma adjacent to the nHAp-coated implant edge. However, around the non-coated cylinder, an abundant new and loose connective tissue formed, similar to bone tissue response to bioinert scaffolds. As a result of superior biointegration, tissue adhesion with nHAp-coated PMMA cylinders was also significantly enhanced compared to non-coated cylinders. This study set a precedent for the future application of the nHAp coating on clinical KPros. STATEMENT OF SIGNIFICANCE: Currently, all clinical corneal prostheses utilize as-manufactured, non-surface modified PMMA optic cylinder. The bioinert cylinder, however, has poor biointegration and adhesion with the surrounding biological tissue, which can give rise to postoperative complications, such as microbial invasion in the tissue-PMMA loose interface and PMMA optic cylinder extrusion. In the current study, we showed that surface modification of the PMMA cylinder with bioactive nano-hydroxyapatite (nHAp) significantly enhanced its biointegration with corneal stromal tissue in vivo. The superior biointegration of the nHAp-coated PMMA was signified by a more attenuated corneal wound healing, inflammatory and fibrotic response, and better tissue apposition, as well as a significantly improved corneal stromal tissue adhesion when compared to the non-coated PMMA.

Plastics are synthetic materials made from organic polymers that are ubiquitous in daily living and are especially important in the healthcare setting. However, recent advances have revealed the pervasive nature of microplastics, which are formed by degradation of existing plastic products. Although the impact on human health has yet to be fully characterised, there is increasing evidence that microplastics can trigger inflammatory damage, microbial dysbiosis, and oxidative stress in humans. Although there are limited studies investigating their effect on the ocular surface, studies of microplastics on other organs provide some insights. The prevalence of plastic waste has also triggered public outcry, culminating in the development of legislation aimed at reducing microplastics in commercial products. We present a review outlining the possible sources of microplastics leading to ocular exposure, and analyse the possible mechanisms of ocular surface damage. Finally, we examine the utility and consequences of current legislation surrounding microplastic regulation.

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs. Ministry of Health (MOH) Published version This study was supported by the NMRC Clinician Scientist Award-Senior Category (MOH000197-00).

With the expected rise in patients undergoing refractive lenticule extraction worldwide, the number of discarded corneal stromal lenticules will increase. Therefore, establishing a lenticule bank to collect, catalog, process, cryopreserve, and distribute the lenticules (for future therapeutic needs) could be advantageous. In this study, we validated the safety of lenticule banking that involved the collection of human lenticules from our eye clinic, transportation of the lenticules to a Singapore Ministry of Health-licensed lenticule bank, processing, and cryopreservation of the lenticules, which, after 3 months or, a longer term, 12 months, were retrieved and transported to our laboratory for implantation in rabbit corneas. The lenticule collection was approved by the SingHealth Centralised Institutional Review Board (CIRB). Both short-term and long-term cryopreserved lenticules, although not as transparent as fresh lenticules due to an altered collagen fibrillar packing, did not show any sign of rejection and cytotoxicity, and did not induce haze or neovascularization for 16 weeks even when antibiotic and steroidal administration were withdrawn after 8 weeks. The lenticular transparency progressively improved and was mostly clear after 4 weeks, the same period when we observed the stabilization of corneal hydration. We showed that the equalization of the collagen fibrillar packing of the lenticules with that of the host corneal stroma contributed to the lenticular haze clearance. Most importantly, no active wound healing and inflammatory reactions were seen after 16 weeks. Our study suggests that long-term lenticule banking is a feasible approach for the storage of stromal lenticules after refractive surgery. Impact statement Since 2011, close to 3 million refractive lenticule extraction procedures have been performed. The majority of the extracted lenticules are discarded. The lenticules could have been cryopreserved and retrieved at a later date for therapeutic or refractive applications. Therefore, establishing a lenticule bank to collect, catalog, process, cryopreserve, and distribute the lenticules could be advantageous. In this study, we simulated a lenticule banking service in a validated health authority-licensed facility and showed that long-term cryopreservation of the lenticules in the facility was safe and feasible

Bacterial infection following implantation of an artificial corneal scaffold is a serious complication. Conventional antibiotic prophylaxis, which includes topical vancomycin application, is limited by low bioavailability, high dosing requirement, and poor patient compliance. The ideal option to overcome these issues is an antibiotic-eluting corneal prosthesis that sustains the local release of drug. In this study, we incorporated vancomycin in thick 15% collagen hydrogels to create an artificial corneal scaffold with anti-infective capability. The incorporation of vancomycin did not significantly alter the Young's modulus, transparency and refractive index of the vancomycin-loaded hydrogel (VH), which were 0.79 ± 0.04 MPa (p = 0.233 compared to blank hydrogel), 94.3 ± 2.3% (p = 0.115) and 1.346 ± 0.005 (p = 0.264), respectively. In vitro, the drug elution was sustained for up to 7 days. The VH was subsequently implanted intrastromally in rabbit corneas, replacing stromal tissue that was removed following femtosecond laser-assisted small incision lenticule extraction procedure. In vivo, the vancomcyin could be detected in the aqueous humor for up to 10 days. We then created a corneal infectious keratitis model by intrastromal injection of 1 × 108 CFU/ml of Staphylococcus aureus inoculate on day 2 postimplantation. On day 3 postinfection, the VH-implanted corneas were clear and nonedematous and showed a substantial reduction of log 2.5 in S. aureus compared to the blank hydrogel-implanted corneas, which appeared hazy, edematous, and had excessive inflammation. Immunohistochemistry of inflammatory marker, CD18, demonstrated a significant reduction in inflammatory cells in VH-implanted corneas (49 ± 9 cells/unit area) compared to blank hydrogel-implanted corneas (523 ± 15 cells/unit area) (p < 0.001). In conclusion, we have demonstrated the efficacy of a drug-eluting corneal implant in preventing perioperative bacterial infections.

Femtosecond lasers have revived the possibility of stromal keratophakia or tissue additive keratoplasty, a technique originally introduced by Prof. Jose Ignacio Barraquer in the 1960s. The surgical technique offers a unique solution to treat keratoconus. In the current study, we reviewed and performed a meta-analysis of the clinical outcomes of the femtosecond laser-assisted stromal keratophakia in the treatment of keratoconus.This is a systematic review and meta-analysis of the estimated outcome difference between pre- and post-lenticule implantations.A total of related 10 studies were found in the literature. No studies reported adverse events, such as persistent haze or graft rejection, at last patients' visits. We further narrowed down the article selection in accordance to our inclusion criteria to report the composite outcomes (9 studies) and meta-analysis (4 studies). In the composite analysis, we demonstrated that lenticule implantation in keratoconus and post-LASIK ectasia patients appeared to expand the stromal volume of the thin corneas, flattened the cones, and significantly improved uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA) and spherical equivalent (SE). The meta-analysis showed that the random estimated UCVA, BCVA, SE and mean keratometry (KFemtosecond laser-assisted stromal keratophakia is a feasible technique to correct the refractive aberrations, expand corneal volume and regularize corneal curvature in patients with keratoconus. However, there is a need to standardize the technique (e.g., whether to crosslink or not or to use convex or concave lenticules) and to formulate a mathematical model that accounts for the long-term epithelial thickness changes and stromal remodeling to determine the shape or profile of the lenticules, in order to improve the efficacy of the keratophakia further.

Bacterial biofilm on medical devices is difficult to eradicate. Many have capitalized the anti-infective capability of silver ions (Ag+) by incorporating nano-silver (nAg) in a biodegradable coating, which is then laid on polymeric medical devices. However, such coating can be subjected to premature dissolution, particularly in harsh diseased tissue microenvironment, leading to rapid nAg clearance. It stands to reason that impregnating nAg directly onto the device, at the surface, is a more ideal solution. We tested this concept for a corneal prosthesis by immobilizing nAg and nano-hydroxyapatite (nHAp) on poly(methyl methacrylate), and tested its biocompatibility with human stromal cells and antimicrobial performance against biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Three different dual-functionalized substrates&mdash, high Ag (referred to as 75:25 HAp:Ag), intermediate Ag (95:5 HAp:Ag), and low Ag (99:1 HAp:Ag) were studied. The 75:25 HAp:Ag was effective in inhibiting biofilm formation, but was cytotoxic. The 95:5 HAp:Ag showed the best selectivity among the three substrates, it prevented biofilm formation of both pathogens and had excellent biocompatibility. The coating was also effective in eliminating non-adherent bacteria in the culture media. However, a 28-day incubation in artificial tear fluid revealed a ~40% reduction in Ag+ release, compared to freshly-coated substrates. The reduction affected the inhibition of S. aureus growth, but not the P. aeruginosa. Our findings suggest that Ag+ released from surface-immobilized nAg diminishes over time and becomes less effective in suppressing biofilm formation of Gram-positive bacteria, such as S. aureus. This advocates the coating, more as a protection against perioperative and early postoperative infections, and less as a long-term preventive solution.

Stromal keratophakia was first performed by Jose Ignacio Barraquer in the 1960s. The refractive lamellar keratoplasty technique was intensely pursued in the 1980s as a method to alter corneal refractive power. However, because sculpting of the donor stromal lenticule and lamellar keratectomy of the recipient's cornea were performed with a mechanical microkeratome, the quality of the cut was inconsistent. Consequently, the refractive outcomes of the lenticule implantation were poor. In addition, epithelial ingrowth, interface scarring, and induced astigmatism were common due to the manual resection. With the advancements of femtosecond laser, we are now able to optically sculpt a refractive lenticule and create an intrastromal pocket for implantation, with greater accuracy and precision compared to manual incisions. The lenticule can be decellularized, cryopreserved, and implanted on a later date to correct hyperopia and presbyopia, as well as to treat corneal ectasia and perforations. In this article, we will review the history of stromal keratophakia and the shortcomings of the previous attempts that led to its abandonment. We will then discuss the reinvigoration of stromal keratophakia with the emergence of advanced femtosecond laser technologies, including the basic science and clinical applications of femtosecond laser-assisted stromal keratophakia, methods to decellularize, cryopreserve and transport the refractive lenticule, lenticule banking, and regulatory framework that oversees the distribution and clinical translation of stromal lenticule implantation.

An artificial cornea or keratoprosthesis requires high mechanical strength, good biocompatibility, and sufficient wear and corrosion resistance to withstand the hostile environment. We report a reduced graphene oxide-reinforced titania-based composite for this application. Graphene oxide nanoparticles (GO) and liquid crystalline graphene oxide (LCGO) were the graphene precursors and mixed with titanium dioxide (TiO2) powder. The composites reinforced with reduced GO or LCGO were produced through spark plasma sintering (SPS). The mechanical properties (Young's modulus and hardness), wear behaviour and corrosion resistance were studied using nanoindentation, anoidic polarization, long-term corrosion assay in artificial tear fluid and tribology assay in corroboration with atomic force microscopy and scanning electron microscopy. Biocompatibility was assessed by human corneal stromal cell attachment, survival and proliferation, and DNA damages. Sintered composites were implanted into rabbit corneas to assess for in vivo stability and host tissue responses. We showed that reduced graphene/TiO2 hybrids were safe and biocompatible. In particular, the 1% reduced LCGO/TiO2 (1rLCGO/TiO2) composite was mechanically strong, chemically stable, and showed better wear and corrosion resistance than pure titania and other combinations of graphene-reinforced titania. Hence the 1rLCGO/ TiO2 bioceramics can be a potential skirt biomaterial for keratoprosthesis to treat end-stage corneal blindness. STATEMENT OF SIGNIFICANCE: The osteo-odonto-keratoprosthesis (OOKP) is an artificial cornea procedure used to restore vision in end-stage corneal diseases, however it is contraindicated in young subjects, patients with advanced imflammatory diseases and posterior segment complications. Hence, there is a need of an improved keratoprosthesisskirt material with high mechanical and chemical stability, wear resistance and tissue integration ability. Our study characterized a reduced graphene oxide-reinforced titania-based biomaterial, which demonstrated strong mechanical strength, wear and corrosion resistance, and was safe and biocompatible to human corneal stromal cells. In vivo implantation to rabbit corneas did not cause any immune and inflammation outcomes. In conclusion, this invention is a potential keratoprosthesis skirt biomaterial to withstand the hostile environment in treating end-stage corneal blindness.

An artificial cornea or keratoprosthesis requires high mechanical strength, good biocompatibility, and sufficient wear and corrosion resistance to withstand the hostile environment. We report a novel reduced graphene oxide-reinforced titania-based composite for this application. Graphene oxide nanoparticles (GO) and liquid crystalline graphene oxide (LCGO) were the graphene precursors and mixed with titanium dioxide (TiO2) powder. The composites reinforced with reduced GO or LCGO were produced through spark plasma sintering (SPS). The mechanical properties (Young's modulus and hardness), wear behaviour and corrosion resistance were studied using nanoindentation, anoidic polarization, long-term corrosion assay in artificial tear fluid and tribology assay in corroboration with atomic force microscopy, scanning and transmission electron microscopies. Biocompatibility was assessed by human corneal stromal cell attachment, survival and proliferation, and DNA damages. Sintered composites were implanted into rabbit corneas to assess for in vivo stability and host tissue responses. We showed that reduced graphene/TiO2 hybrids were safe and biocompatible. In particular, the 1% reduced LCGO/TiO2 (1rLCGO/TiO2) composite was mechanically strong, chemically stable, and showed better wear and corrosion resistance than pure titania and other combinations of graphene-reinforced titania. Hence the novel 1rLCGO/ TiO2 bioceramics can be a potential skirt biomaterial for keratoprosthesis to treat end-stage corneal blindness.

Implantation of biological corneal inlays, derived from small incision lenticule extraction, may be a feasible method for surgical management of refractive and corneal diseases. However, the refractive outcome is dependent on stromal remodelling of both the inlay and recipient stroma. This study aimed to investigate the refractive changes and tissue responses following implantation of 2.5-mm biological inlays with or without corneal collagen crosslinking (CXL) in a rabbit model. Prior to implantation, rotational rheometry demonstrated an almost two-fold increase in corneal stiffness after CXL. After implantation, haze gradually subsided in the CXL-treated inlays (p = 0.001), whereas the untreated inlays preserved their clarity (p = 0.75). In-vivo confocal microscopy revealed reduced keratocyte cell count at the interface of the CXL inlays at week 8. Following initial steepening, regression was observed in anterior mean curvature from week 1 to 12, being most prominent for the non-CXL subgroups (non-CXL: −12.3 ± 2.6D vs CXL: −2.3 ± 4.4D at 90 μm depth, p = 0.03; non-CXL: −12.4 ± 8.0D vs CXL: −5.0 ± 4.0D at 120 μm depth, p = 0.22). Immunohistochemical analysis revealed comparable tissue responses in CXL and untreated subgroups. Our findings suggest that CXL of biological inlays may reduce the time before refractive stabilization, but longer postoperative steroid treatment is necessary in order to reduce postoperative haze. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

The only nonthermal method of depositing a bioceramic-based coating on polymeric substrates is by incubation in liquid, e.g., simulated body fluid to form an apatite-like layer. The drawbacks of this method include the long processing time, the production of low scratch resistant coating, and an end product that does not resemble the intended bioceramic composition. Techniques, such as plasma spraying and magnetron sputtering, involving high processing temperature are unsuitable for polymers, e.g., PMMA. Here, we introduce a nonthermal coating method to immobilize hydroxyapatite (HAp) and TiO2 nanoparticles on PMMA via a simple and fast dip coating method. Cavities that formed on the PMMA, induced by chloroform, appeared to trap the nanoparticles which accumulated to form layers of bioceramic coating only after 60 s. The resulting coating was hydrophilic and highly resistant to delamination. In the context of our research and to address the current clinical need, we demonstrate that the HAp-coated PMMA, which is intended to be used as a visual optic of a corneal prosthetic device, improves its bonding and biointegration with collagen, the main component of a corneal stroma. The HAp-coated PMMA resulted in better adhesion with the collagen than untreated PMMA in artificial tear fluid over 28 days. Human corneal stromal fibroblasts showed better attachment, viability, and proliferation rate on the HAp-coated PMMA than on untreated PMMA. This coating method is an innovative solution to immobilize various bioceramic nanoparticles on polymers and may be used in other biomedical implants. NMRC (Natl Medical Research Council, S’pore) Accepted version

The study of corneal stromal keratocytes is motivated by its strong association with corneal health and visual function. They play a dominant role in the maintenance of corneal homeostasis and transparency through the production of collagens, proteoglycans and corneal crystallins. Trauma-induced apoptosis of keratocytes and replacement by fibroblasts and myofibroblasts disrupt the stromal matrix organization, resulting in corneal haze formation and vision loss. It is, therefore, important to understand the biology and behaviours of keratocytes and the associated stromal cell types (like fibroblasts, myofibroblasts, stromal stem cells) in wound healing, corneal pathologies (including keratoconus, keratitis, endothelial disorders) as well as different ophthalmic situations (such as collagen crosslinking/photodynamic treatment, keratoplasty and refractive surgery, and topical medications). The recent development of ex vivo propagation of keratocytes and stromal stem cells, and their translational applications, either via stromal injection or incorporated in bioscaffold, have been shown to restore the corneal transparency and regenerate native stromal tissue in animal models of corneal haze and other disorders.

Purpose: To evaluate the feasibility of excimer laser reshaping of biological lenticules available after small incision lenticule extraction (SMILE). Methods: Fresh and cryopreserved SMILE-derived human lenticules underwent excimer laser ablation for stromal reshaping. The treatment effects in the lasered group were compared with the nonlasered group with respect to changes in surface functional groups (by Fourier transform infrared spectroscopy [FTIR]) and surface morphology (by scanning electron microscopy [SEM] and atomic force microscopy [AFM]). Ten SMILE-derived porcine lenticules, five nonlasered (107-μm thick, −6 diopter [D] spherical power) and five excimer lasered (50% thickness reduction), were implanted into a 120-μm stromal pocket of 10 porcine eyes. Corneal thickness and topography were assessed before and after implantation. Results: FTIR illustrated prominent changes in the lipid profile. The collagen structure was also affected by the laser treatment but to a lesser extent. SEM exhibited a more regular surface for the lasered lenticules, confirmed by the lower mean Rz value (290.1 ± 96.1 nm vs. 380.9 ± 92.6 nm, P = 0.045) on AFM. The lasered porcine lenticules were thinner than the nonlasered controls during overhydration (132 ± 26 μm vs. 233 ± 23 μm, P < 0.001) and after 5 hours in a moist chamber (46 ± 3 μm vs. 57 ± 3 μm, P < 0.001). After implantation, the nonlasered group showed a tendency toward a greater increase in axial keratometry (6.63 ± 2.17 D vs. 5.60 ± 3.79 D, P = 0.613) and elevation (18.6 ± 15.4 vs. 15.2 ± 5.5, P = 0.656) than the lasered group. Conclusions: Excimer laser ablation may be feasible for thinning and reshaping of SMILE-derived lenticules before reimplantation or allogenic transplantation. However, controlled lenticule dehydration before ablation is necessary in order to allow stromal thinning. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version

Patients with advanced corneal disease do poorly with conventional corneal transplantation and require a keratoprosthesis (KPro) for visual rehabilitation. The most widely used KPro is constructed using poly(methyl methacrylate) (PMMA) in the central optical core and a donor cornea as skirt material. In many cases, poor adherence between the PMMA and the soft corneal tissue is responsible for device "extrusion" and bacterial infiltration. The interfacial adhesion between the tissue and the PMMA was therefore critical to successful implantation and device longevity. In our approach, we modified the PMMA surface using oxygen plasma (plasma group); plasma followed by calcium phosphate (CaP) coating (p-CaP); dopamine followed by CaP coating (d-CaP); or plasma followed by coating with (3-aminopropyl)triethoxysilane (3-APTES). To create a synthetic KPro model, we constructed and attached 500 μm thick collagen type I hydrogel on the modified PMMA surfaces. Surface modifications produced significantly improved interfacial adhesion strength compared to untreated PMMA (p < 0.001). The p-CaP group yielded the best interfacial adhesion with the hydrogel (177 ± 27 mN/cm(2)) followed by d-CaP (168 ± 31 mN/cm(2)), 3-APTES (145 ± 12 mN/cm(2)), and plasma (119 ± 10 mN/cm(2)). Longer-term stability of the adhesion was achieved by d-CaP, which, after 14 and 28 days of incubation in phosphate buffered saline, yielded 164 ± 25 mN/cm(2) (p = 0.906 compared to adhesion at day 1) and 131 ± 20 mN/cm(2) (p = 0.053), respectively. In contrast, significant reduction of adhesion strength was observed in p-CaP group over time (p < 0.001). All surface coatings were biocompatible to human corneal stromal fibroblasts, except for the 3-APTES group, which showed no live cells at 72 h of culture. In contrast, cells on d-CaP surface showed good anchorage, evidenced by the expression of focal adhesion complex (paxillin and vinculin), and prominent filopodia protrusions. In conclusion, d-CaP can not only enhance and provide stability to the adhesion of collagen hydrogel on the PMMA surface but also promote biointegration.

Biointegration of a keratoprosthesis (KPro) is critical for the mitigation of various long-term postoperative complications. Biointegration of a KPro occurs between the haptic skirt (corneal graft) and the central optic [poly(methyl methacrylate) (PMMA)]. Various studies have highlighted common problems associated with poor bonding and biointegration between these 2 incompatible biomaterials. Resolution of these issues could be achieved by surface modification of the inert material (PMMA). A calcium phosphate (CaP) coating deposited on dopamine-activated PMMA sheets by simulated body fluid incubation (d-CaP coating) was shown to improve adhesion to collagen type I (main component of corneal stroma) compared with untreated PMMA and PMMA with other surface modifications. However, the d-CaP coating could easily undergo delamination, thereby reducing its potential for modification of KPro optical cylinders. In addition, the coating did not resemble the Ca and P composition of hydroxyapatite (HAp). A novel dip-coating method that involves the creation of cavities to trap and immobilize HAp nanoparticles on the PMMA surface was introduced to address the problems associated with the d-CaP coating. The newly obtained coating offered high hydrophilicity, resistance to delamination, and preservation of the Ca and P composition of HAp. These advantages resulted in improved adhesion strength by more than 1 order of magnitude compared with untreated PMMA. With respect to biointegration, human corneal stromal fibroblasts were able to adhere strongly and proliferate on HAp-coated PMMA. Furthermore, the new coating technique could be extended to immobilization of HAp nanoparticles on 3-mm-diameter PMMA cylinders, bringing it closer to clinical application.

The corneal functions (transparency, refractivity and mechanical strength) deteriorate in many corneal diseases but can be restored after corneal transplantation (penetrating and lamellar keratoplasties). However, the global shortage of transplantable donor corneas remains significant and patients are subject to life-long risk of immune response and graft rejection. Various studies have shown the differentiation of multipotent mesenchymal stem cells (MSCs) into various corneal cell types. With the unique properties of immunomodulation, anti-angiogenesis and anti-inflammation, they offer the advantages in corneal reconstruction. These effects are widely mediated by MSC differentiation and paracrine signaling via exosomes. Besides the cell-free nature of exosomes in circumventing the problems of cell-fate control and tumorigenesis, the vesicle content can be genetically modified for optimal therapeutic affinity. The pharmacology and toxicology, xeno-free processing with sustained delivery, scale-up production in compliant to Good Manufacturing Practice regulations, and cost-effectiveness are the current foci of research. Routes of administration via injection, topical and/or engineered bioscaffolds are also explored for its applicability in treating corneal diseases. NRF (Natl Research Foundation, S’pore) Published version

Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11(-/-) mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.

PURPOSE: To compare four different Circle patterns for flap creation after small incision lenticule extraction (SMILE). METHODS: SMILE was performed on six rabbits. Twenty-eight days after the initial procedure, corneal flaps were created using Circle patterns. Rabbits were divided into four groups (patterns A, B, C, and D). Pattern A creates a circular side cut to meet the cap cut within the clearance zone (outside of the optical zone). Patterns B, C, and D create a lamellar ring posterior, anterior, and at the same depth, respectively, to the cap to meet the cap cut in the clearance zone with the help of a junction cut. Difficulty of flap lift was graded from 1 (easiest) to 5 (most difficult). The bed quality was assessed by scanning electron microscopy. RESULTS: Flaps created by patterns A and D were the easiest to lift (grade 2). The resulting flap bed was smooth and undisrupted. However, pattern A resulted in a reduced re-treatment area. Flaps created by pattern B were the most difficult to lift (grade 4). The stromal dissection was difficult in an attempt to ascertain the original optical zone from the lamellar ring, placed posterior. Flaps produced by pattern C were easy to lift, with minor intrastromal resistance experienced during the lifting process (grade 3). The transition between the lamellar ring and cap cut was hardly discernible in pattern C-treated corneas. CONCLUSIONS: Pattern D, a lamellar ring adjacent to the cap cut, was the most optimal to be used for flap creation in cases of SMILE re-treatment. [ J Refract Surg. 2013;29(4):236–244.]

Aim Keratoprosthesis (KPro) devices are prone to long-term corrosion and microbiological assault. The authors aimed to compare the inflammatory response and material dissolution properties of two candidate KPro skirt materials, hydroxyapatite (HA) and titania (TiO 2 ) in a simulated in vitro cornea inflammation environment. Methods Lipopolysaccharide-stimulated cytokine secretions were evaluated with human corneal fibroblasts on both HA and TiO 2 . Material specimens were subjected to electrochemical and long-term incubation test with artificial tear fluid (ATF) of various acidities. Topography and surface roughness of material discs were analysed by scanning electron microscopy and atomic force microscopy. Results There were less cytokines secreted from human corneal fibroblasts seeded on TiO 2 substrates as compared with HA. TiO 2 was more resistant to the corrosion effect caused by acidic ATF in contrast to HA. Moreover, the elemental composition of TiO 2 was more stable than HA after long-term incubation with ATF. Conclusions TiO 2 is more resistant to inflammatory degradation and has a higher corrosion resistance as compared with HA, and in this regard may be a suitable material to replace HA as an osteo-odonto-keratoprosthesis skirt. This would reduce resorption rates for KPro surgery.

PURPOSE To investigate gene and protein expression profiles of neural receptors found in the mouse meibomian gland. RNA and protein levels were determined for neuropeptide Y (NPY) receptor, vasoactive intestinal peptide (VIP) receptor, substance P (SP) receptor, and muscarinic cholinergic receptor (mAChR) subtypes M1-M5 in the mouse meibomian gland. METHODS Frozen sections of Balb/c mouse eyelids were subjected to laser capture microdissection to isolate pure samples of meibomian gland ductal and acinar cells. Real-time polymerase chain reaction, immunolabeling, and Western blot analysis for SP receptor, VIP receptor, NPY receptor, and mAChR subtypes M1-M5 were performed on meibomian gland ductal and acinar cells. RESULTS Expression of NPY1 receptor, VIP receptor 1, SP receptor, and all 5 mAChR subtypes was found in all meibomian gland ductal and acinar cells analyzed by real-time polymerase chain reaction. Immunolabeling and Western blot analysis confirmed the presence of NPY1 receptor, VIP receptor 1, SP receptor, and all 5 mAChR subtypes in the meibomian gland. The levels were variable with the duct showing greater levels of NPY1 receptor, SP receptor, and mAChRs 1, 2, 4, and 5 than with the gland. CONCLUSIONS VIP receptor 1, SP receptor, NPY1 receptor, and mAChR subtypes may be involved in the regulation of meibomian gland secretion. Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying meibomian gland cells about which relatively little is known at the molecular level.

Purpose: To investigate the repeatability and reproducibility of the Visante Omni topography in obtaining topography measurements of rabbit corneas and to develop a post-surgical model of corneal ectasia. Methods: Eight rabbits were used to study the repeatability and reproducibility by assessing the intra- and interobserver bias and limits of agreement. Another nine rabbits underwent different diopters (D) of laser in situ keratosmileusis (LASIK) were used for the development of ectasia model. All eyes were examined with the Visante Omni, and corneal ultrastructure were evaluated with transmission electron microscopy (TEM). Results: There was no significant intra- or interobserver difference for mean steep and flat keratometry (K) values of simulated K, anterior, and posterior elevation measurements. Eyes underwent -5 D LASIK had a significant increase in mean amplitude of astigmatism and posterior surface elevation with time (P for trend < 0.05). At 2 and 3 months, the -5 D LASIK group had significant greater mean amplitude of astigmatism (P = 0.036; P = 0.027) and posterior surface elevation (both P < 0.005) compared with control group. On TEM, the mean collagen fibril diameter and interfibril distance in the -5 D LASIK eyes were significantly greater than in controls at 3 months (P = 0.018; P < 0.001). Conclusions: The Visante Omni provided imaging of the rabbit cornea with good repeatability and reproducibility. Application of -5 D LASIK treatment produced a rabbit model of corneal ectasia that was gradual in development and simulated the human condition. Translational Relevance: The results provide the foundations for the future evaluation of novel treatment modalities for post-surgical ectasia and keratoconus. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

With any refractive correction, including Small Incision Lenticule Extraction (SMILE), there may be a residual refractive error that requires a retreatment. Here, we investigated the tissue responses following various retreatment procedures in a rabbit model of SMILE. All rabbits underwent a -6.00D correction with SMILE. Two weeks later, they underwent -1.00D enhancement by: (i) VisuMax Circle, followed by excimer ablation (S+C); (ii) secondary SMILE anterior to the primary procedure (S+SE); or (iii) surface ablation (S+P), and were examined for 28 days. S+P induced corneal edema and haze, and more CD11b- (23±6 cells) and TUNEL-positive (36±4 cells) cells in the central stromal superficial layers early post-operatively (p

Over the last decade, femtosecond lasers have emerged as an important tool to perform accurate and fine dissections with minimal collateral damage in biological tissue. The most common surgical procedure in medicine utilizing femtosecond laser is LASIK. During the femtosecond laser dissection process, the corneal collagen fibers inevitably undergo biomechanical and thermal changes on a sub-micro- or even a nanoscale level, which can potentially lead to post-surgical complications. In this study, we utilized helium ion microscopy, complemented with transmission electron microscopy to examine the femtosecond laser-induced collagen fibrillar damage in ex vivo human corneas. We found that the biomechanical damage induced by laser etching, generation of tissue bridges, and expansion of cavitation bubble and its subsequent collapse, created distortion to the surrounding collagen lamellae. Femtosecond laser-induced thermal damage was characterized by collapsed collagen lamellae, loss of collagen banding, collagen coiling, and presence of spherical debris. Our findings have shown the ability of helium ion microscopy to provide high resolution images with unprecedented detail of nanoscale fibrillar morphological changes in order to assess a tissue damage, which could not be resolved by conventional scanning electron microscopy previously. This imaging technology has also given us a better understanding of the tissue-laser interactions in a nano-structural manner and their possible effects on post-operative wound recovery.

Titanium (Ti) is a promising candidate biomaterial for an artificial corneal skirt. Antimicrobial peptide (AMP) immobilization may improve the bactericidal effect of the Ti substrate. In this study, we tested the bactericidal efficacy of a functionalized Ti surface in a rabbit keratitis model. A corneal stromal pocket was created by a femtosecond laser. The Ti films were then inserted into the pocket, and Staphylococcus aureus or Pseudomonas aeruginosa was inoculated into the pocket above the implant films. The corneas with Ti-AMP implants were compared with the corneas implanted with unprotected Ti by slit lamp observation and anterior segment optical coherence tomography (AS-OCT). Inflammatory responses were evaluated by bacterium counting, hematoxylin-eosin staining, and immunostaining. There was a lower incidence and a lesser extent of infection on rabbit corneas with Ti-AMP implants than on those with unprotected Ti implants. The bactericidal effect of AMP against S. aureus was comparable to that of postoperative prophylactic antibiotic treatment; hence, SESB2V AMP bound to the Ti implant provided functional activity in vivo , but its efficacy was greater against S. aureus than against P. aeruginosa . This work suggests that SESB2V AMP can be successfully functionalized in a rabbit keratitis model to prevent perioperative corneal infection.

Purpose We compared early corneal nerve changes after small incision lenticule extraction (SMILE) and laser in situ keratomileusis (LASIK). Methods A total of 12 rabbits underwent LASIK in one eye and SMILE in the fellow eye. Baseline and follow-up evaluations at 1, 2, and 4 weeks postoperatively were performed with in vivo confocal microscopy to evaluate 5 different areas within the treated zone: center, superior, inferior, nasal, and temporal. Cryosections of the corneas and whole mount of the extracted SMILE lenticules were analyzed with immunostaining of βIII-tubulin. Results One week after SMILE and LASIK, a decrease in nerve length and density was observed in all evaluated areas. A trend toward greater subbasal nerve length and density (SLD), more eyes with subbasal nerves (ESN), more eyes with subbasal nerves longer than 200 μm (SNL), and higher mean number of subbasal nerves by frame (NSN) in SMILE than in LASIK groups was observed at subsequent follow-up time points. Only the SMILE group showed a recovery of SLD, ESN, and NSN by week 4 (P > 0.05). A trend toward more eyes with sprouting subbasal nerves and greater mean number of sprouting nerves was observed in LASIK than in SMILE, indicating that more subbasal nerves were disrupted and undergoing regeneration after LASIK. Immunostaining at postoperative week 4 revealed a faster stromal nerve recovery in post-SMILE eyes compared to post-LASIK eyes. Conclusions Our findings suggest that SMILE results in less nerve damage and faster nerve recovery than LASIK.

Background The rabbit is a common animal model for ophthalmic research, especially corneal research. Ocular structures grow rapidly during the early stages of life. It is unclear when the rabbit cornea becomes mature and stabilized. We investigated the changes of keratometry, refractive state and central corneal thickness (CCT) with age. In addition, we studied the intra- and inter-observer reproducibility of anterior chamber depth (ACD) and anterior chamber width (ACW) measurements in rabbits using anterior segment-optical coherence tomography (AS-OCT). Results The growth of New Zealand White rabbits (n = 16) were monitored from age 1 to 12 months old. Corneal keratometric and refractive values were obtained using an autorefractor/keratometer, and CCT was measured using an AS-OCT. Keratometry and CCT changed rapidly from 1 to 7 months and appeared to be stabilizing after 8 months. The reduction of corneal curvature was approximately 1.36 diopter (D)/month from age 1 to 7 months, but the change decelerated to 0.30 D/month from age 8 to 12 months. An increase of 10 μm/month in CCT was observed from age 1 to 7 months, but the gain was reduced to less than 1 μm/month from age 8 to 12 months. There was a hyperopic shift over the span of 12 months, albeit the increase in spherical equivalent was slow and gradual. Rabbits of random age were then selected for 2 repeated ACD and ACW measurements by 2 independent and masked observers. Bland-Altman plots revealed a good agreement of ACD and ACW measurements inter- and intra-observer and the ranges of 95% limit of agreement were acceptable from a clinical perspective. Conclusions Corneal keratometry, spherical equivalent refraction and CCT changed significantly during the first few months of life of rabbits. Young rabbits have been used in a large number of eye research studies. In certain settings, the ocular parametric changes are an important aspect to note as they may alter the findings made in a rabbit experimental model. In this study, we have also demonstrated for the first time a good between observer reproducibility of measurements of ocular parameters in an animal model by using an AS-OCT.

PURPOSE To characterize the clinical profile of femtosecond lenticule extraction (FLEx) correlated with ultrastructural analysis of the corneal interface and in vivo real-time intraocular pressure (IOP). METHODS Prospective clinical case series with experimental studies; consecutive patients underwent FLEx at a single tertiary center over 10 months with postsurgical follow-up of 3 months. The patients were divided into three groups according to spherical equivalence (SE) (A, < -5.0 diopters [D]; B, ≥ -5.00 D and < -9.00 D; and C, ≥ -9.0 D). Twelve human cadaveric eyes analyzed using scanning electron microscopy after receiving FLEx; 40 rabbit eyes received FLEx with in vivo IOP measurements. The main outcome measures were refractive outcomes from study subjects; with corneal interface and IOP in experimental studies. RESULTS Thirty-three subjects (22 females, 66.7%) underwent FLEx in both eyes (66 eyes). Mean age was 32 years (range, 21 to 46 years). Preoperative mean SE was -5.77 ± 2.04 D with astigmatism of -1.03 ± 0.72 D. There was a slight hyperopic shift (mean SE 0.14 ± 0.53 D); 94% achieved uncorrected visual acuity ≥20/25 3 months postoperatively. Refractive stability was achieved within 1 month (P < 0.001). Ultrastructurally, the smoothness of the corneal interface was independent of ablation depth (mean irregularity scores A, B, C: 8.8 ± 0.6, 10.3 ± 0.4, 8.7 ± 0.6, respectively; P = 0.88). The increase in IOP during FLEx was similar to that in femtosecond (FS)-LASIK, albeit a twofold duration of raised IOP in FLEx (P < 0.001). CONCLUSIONS These results suggest that FLEx is predictable and effective in treating myopia and myopic astigmatism. Experimental studies support the early clinical results and safety of this procedure.

PURPOSE To compare the early corneal wound repair and inflammatory responses after refractive lenticule extraction (ReLEx) and LASIK. METHODS Eighteen rabbits underwent ReLEx and another 18 underwent LASIK. Each group was divided into three subgroups of six rabbits each and these were subjected to refractive corrections of -3.00 diopters (D), -6.00 D, and -9.00 D. Slit lamp photography, anterior segment optical coherence tomography (AS-OCT), corneal topography, and in vivo confocal microscopy were performed 1 day after surgery. After euthanatization, the corneas were subjected to immunofluorescent staining for fibronectin, CD11b, Ki-67, and TUNEL assay. RESULTS On slit lamp microscopy, all corneas appeared clear pre- and postoperatively in both ReLEx and LASIK eyes. Corneal topography showed a more significant corneal flattening after LASIK than after ReLEx as the degree of correction was increased (P = 0.916 after -3.00 D correction to P = 0.097 after -9.00 D correction). In vivo confocal microscopy showed less light-scattering particles at the flap interface after ReLEx compared with LASIK. Immunostaining of fibronectin showed a less abundant expression in corneas that underwent ReLEx than LASIK. The differences became more marked as the power of correction was increased. Similar trend was seen in the number of CD11b-positive cells (P = 0.476 after -3.00 D correction to P < 0.001 after -9.00D correction). There was no marked disparity observed in cell death and proliferation between post-ReLEx and -LASIK eyes. CONCLUSIONS This study has shown that the ReLEx procedure may result in less topographic changes, inflammation, and early extracellular matrix deposition than LASIK, especially at high refractive correction.

Purpose To study the expression and cellular distribution of multiple S100A genes and proteins in normal corneal-limbal epithelium and ocular surface squamous cell carcinoma (SCC) tissue. Methods Normal corneal-limbal tissue was obtained from the Lions Eye Bank, Tampa, FL. Ocular surface SCC tissues were excised from patients undergoing surgery at Singapore National Eye Centre. S100A mRNA expression was measured by quantitative PCR. S100 protein distribution was determined by immunofluorescent staining analysis. Results Twelve S100 mRNAs were identified in human corneal and limbal epithelial cells. S100A2, A6, A8, A9, A10, and A11 mRNA was expressed at high level, while S100A1, A3, A4, A5, A6, A7, and A12 mRNA expression was low. The intracellular localization of S100A2, A6, A8, A9, A10 and A11 protein was determined in normal corneal-limbal and SCC tissues. S100A2 and S100A10 proteins were enriched in basal limbal epithelial cells of the normal tissue. S100A8 and S100A9 were found only at the surface of peripheral corneal and limbal epithelium. S100A6 was uniformly found at the plasma membrane of corneal and limbal epithelial cells. S100A11 was found at the supralayer limbal epithelial cells adjacent to the conjunctiva. SCC tissue showed typical pathological changes with expression of cytokeartin (CK) 14 and CK4 in the epithelial cells. All SCC epithelial cells were positive of S100A2, S100A10, S100A6 and S100A11 staining. Intracellular staining of S100A8 and S100A9 was found in several layers of SCC epithelium. Expression of S100A2 and S100A10 decreased dramatically in cultured limbal epithelial cells with increased passaging, which was accompanied by a small increase of S100A9 mRNA, with no changes of S100A8 gene expression. Serum and growth hormone depletion of the culture serum caused a small reduction of S100A2 and S100A10 gene expression, which was accompanied by a small increase of S100A9 mRNA while no changes of S100A8 expression was measured. Conclusions Normal corneal and limbal epithelial cells express a broad spectrum of S100 genes and proteins. Ocular surface SCC express high levels of S100A2, S100A10, S100A8 and S100A9 proteins. The expression of S100A2 and S100A10 is associated with limbal epithelial cell proliferation and differentiation.

BACKGROUND: Pterygium is a common ocular surface disease characterized by abnormal epithelial and fibrovascular proliferation, invasion, and matrix remodeling. This lesion, which migrates from the periphery to the center of the cornea, impairs vision and causes considerable irritation. The mechanism of pterygium formation remains ambiguous, and current treatment is solely surgical excision, with a significant risk of recurrence after surgery. Here, we investigate the role of methylation in DNA sequences that regulate matrix remodeling and cell adhesion in pterygium formation. METHODOLOGY/PRINCIPAL FINDINGS: Pterygium and uninvolved conjunctiva samples were obtained from the same eye of patients undergoing surgery. The EpiTYPER Sequenom technology, based on differential base cleavage and bisulfite sequencing was used to evaluate the extent of methylation of 29 matrix and adhesion related genes. In pterygium, three CpG sites at -268, -32 and -29 bp upstream of transglutaminase 2 (TGM-2) transcription initiation were significantly hypermethylated (p

Objectives To compare the biological and ultrastructural properties of a commercially available decellularized and dehydrated human amniotic membrane (DDHAM) product with cryopreserved human amniotic membrane (CHAM) and to demonstrate the feasibility of DDHAM transplant in a case of chronic ocular surface disease. Methods Histologic examination, immunohistochemical examination for extracellular matrix molecules and growth factors, transmission and scanning electron microscopy, and atomic force microscopy were performed on both DDHAM and CHAM specimens. A DDHAM transplant was performed in a patient with chronic bullous keratopathy and ocular surface instability. Results Histologic examination and transmission electron microscopy revealed the disruption of the trilaminar structure of the basement membrane compared with CHAM, and immunohistochemical examination confirmed the loss of collagen IV and VII, laminin, and fibronectin in DDHAM. Lower levels of several growth factors were also seen in DDHAM compared with CHAM. Clinical transplant of DDHAM was, however, successful, with rapid reepithelialization. Conclusion Significant differences in composition and ultrastructure exist between DDHAM and CHAM but do not appear to compromise cell survival in vivo. Clinical Relevance The ease of storage and handling of DDHAM make it potentially valuable in ocular surface surgery, but its biological properties have not been well characterized, and there are also few data on its clinical application.

In the past 20 years, human amniotic membrane (AM) has become widely used as an ophthalmic surgical patch as well as a substrate for stem cell tissue equivalents for ocular surface reconstruction. AM reduces ocular surface scarring and inflammation, and enhances epithelialization. In addition, it shows limited immunogenicity and some anti-microbial properties. Before being applied clinically, the donor of AM is required to undergo a thorough health screening and the membrane has to undergo an accepted processing routine, which includes preservation, sterilization and de-epithelialization. There have been various articles describing methods in preserving, sterilizing and de-epithelializing AM. Each preparation technique has been reported to have differential effects on the physical and biological properties of the AM. Therefore, it is difficult to establish a standardized procedure. In this review, we discuss the present techniques and several novel, new approaches in the preparation of AM for use in ocular surface reconstruction, and their impact on AM structure and biological activity.

Purpose To compare flap adhesion strength, stromal bed quality, and tissue responses after flap preparation using nJ- and μJ-energy level femtosecond lasers. Methods All corneal flaps were created by either VisuMax laser (μJ-energy level) or femto-LDV (nJ-energy level). Flap adhesion strength in the rabbits was measured with a tension meter 1 and 2 months postoperatively. To investigate tissue responses to laser delivery, immunofluorescence staining and TUNEL assay were performed 4 and 24 hours postoperatively. To assess flap bed smoothness, human donor corneas were used. Surface irregularities were graded based on scanning electron microscopy results. Results The flap adhesion strength in the VisuMax group at month 1 and 2 was 16.95 ± 1.45 kPa and 18.33 ± 1.81 kPa, respectively; and 12.31 ± 4.15 kPa and 13.85 ± 4.78 kPa in the LDV group, respectively. No significant difference was found between the groups. Fibronectin and apoptotic cells were largely absent at the central incision site in the LDV group, but were present in the VisuMax group. The smoothness of flap beds appeared similar for both groups. An observer scored the VisuMax group 8.00 ± 1.00 and the LDV group 7.33 ± 0.58 (P = 0.387). Conclusions The flap adhesion strength increased over time after treatment with both lasers. The nJ-energy pulses produced minimal wound healing reaction and apoptotic cells along the incision plane. The application of an nJ-energy laser, which can incise the cornea without inducing significant damage to cells and wound healing reaction, offers great potential at reducing scarring following incisional laser stromal surgery.

Presbyopia remains a major visual impairment for patients, who have previously undergone laser refractive correction and enjoyed unaided distance vision prior to the onset of presbyopia. Corneal stromal volume restoration through small incision lenticule extraction (SMILE) lenticule re-implantation presents an opportunity for restoring the patients' non-dominant eye to previous low myopia to achieve a monovision. In this study, we investigated the feasibility of performing LASIK after lenticule re-implantation as a method to create presbyopic monovision. A -6.00D SMILE correction was performed in 9 rabbit eyes. The lenticules were cryopreserved for 14 days and re-implanted. Five weeks later, 3 of these eyes underwent LASIK for -5.00D correction (RL group); 3 underwent LASIK flap creation, which was not lifted (RN); and no further procedures were performed on the remaining 3 eyes. These groups were compared with 3 eyes that underwent standard LASIK for a -5.00D correction (LO); 3 that underwent creation of non-lifted flap (LN); and 3 non-operated eyes. Rabbits were euthanized 1 day post-surgery. Tissue responses were analyzed by immunohistochemistry, slit lamp and in vivo confocal microscopy (IVCM). Intrastromal irregularities and elevated reflectivity levels of the excimer-ablated plane were observed on slit lamp and IVCM, respectively in the RL group. The results were comparable (P = 0.310) to IVCM findings in the LO group. RL and LO groups showed similar fibronectin expression levels, number of CD11b-positive cells (P = 0.304) and apoptotic cells (P = 0.198). There was no difference between the RN and LN groups in reflectivity levels (P = 0.627), fibronectin expression levels, CD11b-positive cells (P = 0.135) and apoptotic cells (P = 0.128). LASIK can be performed following lenticule re-implantation to create presbyopic monovision. The tissue responses elicited after performing LASIK on corneas that have undergone SMILE and subsequent lenticule re-implantation are similar to primary procedure.

LASIK (laser-assisted in situ keratomileusis) is a common laser refractive procedure for myopia and astigmatism, involving permanent removal of anterior corneal stromal tissue by excimer ablation beneath a hinged flap. Correction of refractive error is achieved by the resulting change in the curvature of the cornea and is limited by central corneal thickness, as a thin residual stromal bed may result in biomechanical instability of the cornea. A recently developed alternative to LASIK called Refractive Lenticule Extraction (ReLEx) utilizes solely a femtosecond laser (FSL) to incise an intrastromal refractive lenticule (RL), which results in reshaping the corneal curvature and correcting the myopia and/or astigmatism. As the RL is extracted intact in the ReLEx, we hypothesized that it could be cryopreserved and re-implanted at a later date to restore corneal stromal volume, in the event of keratectasia, making ReLEx a potentially reversible procedure, unlike LASIK. In this study, we re-implanted cryopreserved RLs in a non-human primate model of ReLEx. Mild intrastromal haze, noted during the first 2 weeks after re-implantation, subsided after 8 weeks. Refractive parameters including corneal thickness, anterior curvature and refractive error indices were restored to near pre-operative values after the re-implantation. Immunohistochemistry revealed no myofibroblast formation or abnormal collagen type I expression after 8 weeks, and a significant attenuation of fibronectin and tenascin expression from week 8 to 16 after re-implantation. In addition, keratocyte re-population could be found along the implanted RL interfaces. Our findings suggest that RL cryopreservation and re-implantation after ReLEx appears feasible, suggesting the possibility of potential reversibility of the procedure, and possible future uses of RLs in treating other corneal disorders and refractive errors.

Purpose We characterized the de-epithelialized human amniotic membrane (HAM), and compared cell attachment and proliferation efficiencies. Methods HAM was de-epithelialized by 20% ethanol (AHAM), 1.2 U/mL Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM), and 5 M urea (UHAM), respectively, followed by gentle scraping with a #15 blade. Surface topology, extracellular matrix (ECM), and growth factor content were characterized and compared to intact HAM by electron microscopies (EM), atomic force microscopy (AFM), immunohistochemistry, and Western blotting. Primary human limbal epithelial cells (LEC) attachment and proliferation efficiencies were assayed. Statistical significance was calculated by SPSS and Fisher's least significant difference test. Results EHAM, THAM, and UHAM had intact basal lamina and smooth basement membrane surface shown under transmission and scanning EM, and AFM. Cell remnants stayed on AHAM. Disrupted basement membrane and stroma was found in DHAM. Immunostaining intensity quantification and hierarchical clustering revealed that ECM composition of EHAM and UHAM resembled intact HAM. In contrast, DHAM and THAM had drastic loss of ECM and growth factor content. LEC attachment efficiency at 24 hours after seeding was the highest in EHAM (51% as on conventional culture surface), followed by UHAM and AHAM. However, cell proliferation indices at day 10 of culture were similar among different HAM substrates, suggesting repair of ECM and basement membrane by growing epithelial cells. Conclusions Urea denudation preserved the basement membrane integrity, ECM, and growth factor composition, and had higher cell attachment and proliferation efficiencies. With its short processing time, urea treatment offers a novel alternative for HAM de-epithelialization.

Purpose To investigate the potential of refractive lenticule (RL) storage and re-implantation in vivo as a method for reversing RL extraction (ReLEx) and restoring corneal stromal volume. Methods ReLEx [-6.00 diopter (D) correction] was performed on six New Zealand White rabbits in one eye. Each extracted RL was tagged and orientated before storage at -80°C for 28 days. Each RL was then re-implanted autologously in the correct orientation after flap relifting. All animals were monitored for 28 days before being euthanized for immunohistochemical analysis. Unoperated fellow eyes were used as controls. All animals had regular pre- and postoperative slit lamp photography, in vivo confocal microscopy, anterior segment optical coherence tomography (AS-OCT), keratometry, and topography. Results No intra-operative complications occurred and RL re-implantation was performed without complication. A mild intrastromal haziness was noted on day 3 after re-implantation (corneal haze grade: 2.20 ± 0.45), but corneas were clear on day 28 (0.20 ± 0.27). RL re-implantation restored central corneal thickness, and keratometric and topographic indices to near pre-operative values. Wound healing processes, marked by fibronectin and tenascin, and a few inflammatory cells were present along the re-implanted lenticular interfaces. No myofibroblasts formation, and Ki67- and TUNEL-positive cells were observed in the corneal stroma on postoperative day 28. Conclusions RL storage and re-implantation is a feasible technique for restoring stromal volume after myopic ReLEx, and may provide a method for restoring tissue in ectatic corneas, or provide an opportunity for further refractive surgery and presbyopic treatment.

Migration of cells in the ocular surface underpins physiological wound healing as well as many human diseases. Transglutaminase (TG)-2 is a multifunctional cross-linking enzyme involved in the migration of skin fibroblasts and wound healing, however, its functional role in epithelial migration has not been evaluated. This study investigated the importance of TG-2 in a murine corneal wound healing model as well as the mechanistic role of TG-2 in the regulation of related biological processes such as cell adhesion and migration of cultured human corneal epithelial (HCE-T) cells. Corneal wound closure was delayed in homozygous TG-2 deleted mice compared to wild type mice. HCE-T cells that were knocked-down for TG-2 expression through stable expression of a short-hairpin (sh) RNA targeting TG-2, were delayed in closure of scratch wounds (48 compared to 12h in control cells expressing scrambled shRNA). TG-2 knockdown did not influence epithelial cell cycle progression or proliferation, rather, it led to reduced epithelial cell adhesion, spreading and velocity of migration. At the molecular level, TG-2 knockdown reduced phosphorylation of β-3 integrin at Tyr747, paxillin at Ser178, vinculin at Tyr822 and focal adhesion kinase at Tyr925 simultaneous with reduced activation of Rac and CDC42. Phosphorylation of paxillin at Ser178A has been shown to be indispensable for the migration of corneal epithelial cells (Kimura et al., 2008) [18]. TG-2 dependent β-3 integrin activation, serine-phosphorylation of paxillin, and Rac and CDC42 activation may thus play a key functional role in enhancing corneal epithelial cell adhesion and migration during wound healing.