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Previous studies have shown that the nucleus could offer structural support to the lens capsule. This study investigated the biomechanical performance of porcine lens with and without nucleus for 4 mm, 4.5 mm, 5 mm, 5.5 mm and 6 mm capsulotomy and its potential impact on the stretch ratio of capsular bag when the anterior capsulotomy edge was stretched. Our simulation results showed higher strain for the capsular bag with nucleus, which is crucial for the porcine lens to tolerate more stretch without failure. This simulation could support future study on the optimization of capsulotomy based on patient specific condition, that is, the geometry of lens.

The cornea is a transparent and vitally multifaceted component of the eye, playing a pivotal role in vision and ocular health. It has primary refractive and protective functions. Typical corneal dysfunctions include opacities and deformities that result from injuries, infections, or other medical conditions. These can significantly impair vision. The conventional challenges in managing corneal ailments include the limited regenerative capacity (except corneal epithelium), immune response after donor tissue transplantation, a risk of long-term graft rejection, and the global shortage of transplantable donor materials. This review delves into the intricate composition of the cornea, the landscape of corneal regeneration, and the multifaceted repercussions of scar-related pathologies. It will elucidate the etiology and types of dysfunctions, assess current treatments and their limitations, and explore the potential of regenerative therapy that has emerged in both in vivo and clinical trials. This review will shed light on existing gaps in corneal disorder management and discuss the feasibility and challenges of advancing regenerative therapies for corneal stromal scarring.

Background: Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities., Methods: A total of 24 donor corneal rims with informed consent were used-18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC's conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring., Results: By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters-stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)-were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring., Conclusions: We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC-in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments., (© 2024. The Author(s).)

The limbus is a transition from the cornea to conjunctiva and sclera. In human eyes, this thin strip has a rich variation of tissue structures and composition, typifying a change from scleral irregularity and opacity to corneal regularity and transparency; a variation from richly vascularized conjunctiva and sclera to avascular cornea; the neural passage and drainage of aqueous humor. The limbal stroma is enriched with circular fibres running parallel to the corneal circumference, giving its unique role in absorbing small pressure changes to maintain corneal curvature and refractivity. It contains specific niches housing different types of stem cells for the corneal epithelium, stromal keratocytes, corneal endothelium, and trabecular meshwork. This truly reflects the important roles of the limbus in ocular physiology, and the limbal functionality is crucial for corneal health and the entire visual system. Since the anterior limbus containing epithelial structures and limbal epithelial stem cells has been extensively reviewed, this article is focused on the posterior limbus. We have discussed the structural organization and cellular components of the region beneath the limbal epithelium, the characteristics of stem cell types: namely corneal stromal stem cells, endothelial progenitors and trabecular meshwork stem cells, and recent advances leading to the emergence of potential cell therapy options to replenish their respective mature cell types and to correct defects causing corneal abnormalities. We have reviewed different clinical disorders associated with defects of the posterior limbus and summarized the available preclinical and clinical evidence about the developing topic of cell-based therapy for corneal disorders., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Corneal endothelial dysfunction is one of the leading causes of corneal blindness, and the current conventional treatment option is corneal transplantation using a cadaveric donor cornea. However, there is a global shortage of suitable donor graft material, necessitating the exploration of novel therapeutic approaches. A stem cell-based regenerative medicine approach using induced pluripotent stem cells (iPSCs) offers a promising solution, as they possess self-renewal capabilities, can be derived from adult somatic cells, and can be differentiated into all cell types including corneal endothelial cells (CECs). This review discusses the progress and challenges in developing protocols to induce iPSCs into CECs, focusing on the different media formulations used to differentiate iPSCs to neural crest cells (NCCs) and subsequently to CECs, as well as the characterization methods and markers that define iPSC-derived CECs. The hurdles and solutions for the clinical application of iPSC-derived cell therapy are also addressed, including the establishment of protocols that adhere to good manufacturing practice (GMP) guidelines. The potential risks of genetic mutations in iPSC-derived CECs associated with long-term in vitro culture and the danger of potential tumorigenicity following transplantation are evaluated. In all, this review provides insights into the advancement and obstacles of using iPSC in the treatment of corneal endothelial dysfunction.

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.

Introduction: Corneal blindness due to scarring is treated with corneal transplantation. However, a global problem is the donor material shortage. Preclinical and clinical studies have shown that cell-based therapy using corneal stromal stem cells (CSSCs) suppresses corneal scarring, potentially mediated by specific microRNAs transported in extracellular vesicles (EVs). However, not every CSSC batch from donors achieves similar anti-scarring effects., Objectives: To examine miRNA profiles in EVs from human CSSCs showing "healing" versus "non-healing" effects on corneal scarring and to design a tool to select CSSCs with strong healing potency for clinical applications., Methods: Small RNAs from CSSC-EVs were extracted for Nanostring nCounter Human miRNA v3 assay. MicroRNAs expressed > 20 folds in "healing" EVs (P < 0.05) were subject to enriched gene ontology (GO) term analysis. MiRNA groups with predictive regulation on inflammatory and fibrotic signalling were studied by mimic transfection to (1) mouse macrophages (RAW264.7) for M1 phenotype assay; (2) human corneal keratocytes for cytokine-induced fibrosis, and (3) human CSSCs for corneal scar prevention in vivo. The expression of miR-29a was screened in additional CSSC batches and the anti-scarring effect of cells was validated in mouse corneal wounds., Results: Twenty-one miRNAs were significantly expressed in "healing" CSSC-EVs and 9 miRNA groups were predicted to associate with inflammatory and fibrotic responses, and tissue regeneration (P <10 -6 ). Overexpression of miR-29a and 381-5p significantly prevented M1 phenotype transition in RAW264.7 cells after lipopolysaccharide treatment, suppressed transforming growth factor β1-induced fibrosis marker expression in keratocytes, and reduced scarring after corneal injury. High miR-29a expression in EV fractions distinguished human CSSCs with strong healing potency, which inhibited corneal scarring in vivo., Conclusion: We characterized the anti-inflammatory and fibrotic roles of miR-29a and 381-5p in CSSCs, contributing to scar prevention. MiR-29a expression in EVs distinguished CSSCs with anti-scarring quality, identifying good quality cells for a scarless corneal healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

A transparent cornea is paramount for vision. Corneal opacity is one of the leading causes of blindness. Although conventional corneal transplantation has been successful in recovering patients' vision, the outcomes are challenged by a global lack of donor tissue availability. Bioengineered corneal tissues are gaining momentum as a new source for corneal wound healing and scar management. Extracellular matrix (ECM)-scaffold-based engineering offers a new perspective on corneal regenerative medicine. Ultrathin stromal laminar tissues obtained from lenticule-based refractive correction procedures, such as SMall Incision Lenticule Extraction (SMILE), are an accessible and novel source of collagen-rich ECM scaffolds with high mechanical strength, biocompatibility, and transparency. After customization (including decellularization), these lenticules can serve as an acellular scaffold niche to repopulate cells, including stromal keratocytes and stem cells, with functional phenotypes. The intrastromal transplantation of these cell/tissue composites can regenerate native-like corneal stromal tissue and restore corneal transparency. This review highlights the current status of ECM-scaffold-based engineering with cells, along with the development of drug and growth factor delivery systems, and elucidates the potential uses of stromal lenticule scaffolds in regenerative therapeutics.

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.

Valproic acid (VPA), widely used for the treatment of neurological disorders, has anti-fibrotic activity by reducing collagen production in the postoperative conjunctiva. In this study, we investigated the capacity of VPA to modulate the postoperative collagen architecture. Histochemical examination revealed that VPA treatment was associated with the formation of thinner collagen fibers in the postoperative days 7 and 14 scars. At the micrometer scale, measurements by quantitative multiphoton microscopy indicated that VPA reduced mean collagen fiber thickness by 1.25-fold. At the nanometer scale, collagen fibril thickness and diameter measured by transmission electron microscopy were decreased by 1.08- and 1.20-fold, respectively. Moreover, delicate filamentous structures in random aggregates or closely associated with collagen fibrils were frequently observed in VPA-treated tissue. At the molecular level, VPA reduced Col1a1 but induced Matn2, Matn3, and Matn4 in the postoperative day 7 conjunctival tissue. Elevation of matrilin protein expression induced by VPA was sustained till at least postoperative day 14. In primary conjunctival fibroblasts, Matn2 expression was resistant to both VPA and TGF-β2, Matn3 was sensitive to both VPA and TGF-β2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-β2. MATN2, MATN3, and MATN4 localized in close association with COL1A1 in the postoperative conjunctiva. These data indicate that VPA has the capacity to reduce collagen fiber thickness and potentially collagen assembly, in association with matrilin upregulation. These properties suggest potential VPA application for the prevention of fibrotic progression in the postoperative conjunctiva. KEY MESSAGES: VPA reduces collagen fiber and fibril thickness in the postoperative scar. VPA disrupts collagen fiber assembly in conjunctival wound healing. VPA induces MATN2, MATN3, and MATN4 in the postoperative scar., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo-SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo-SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT-PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo-SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno-free component efficiently preserved CSK characteristics, with non-inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Introduction: Refractive stromal lenticules from Small Incision Lenticule Extraction (SMILE), though usually discarded, hold a potential for various ophthalmic applications, including refractive correction, stromal volume expansion, and biomechanical strengthening of the cornea., Objectives: To investigate the effect of lenticule customization on lenticule neurite length profile and the excitatory response (calcium signaling) and the potential of reinnervation., Methods: Human and porcine stromal lenticules were treated by (1) excimer laser reshaping, (2) ultraviolet A-riboflavin crosslinking (CXL), and (3) decellularization by sodium dodecyl sulfate (SDS), respectively. The overall neurite scaffold immuno-positive to TuJ1 (neuron-specific class III β-tubulin) expression and population of active neurite fragments with calcium response revealed by L-glutamate-induced Fluo-4-acetoxymethyl ester reaction were captured by wide-field laser-scanning confocal microscopy, followed by z-stack image construction. The NeuronJ plugin was used to measure neurite lengths for TuJ1 (NL-TuJ1) and calcium signal (NL-Ca). Reinnervation of lenticules was examined by the ex vivo grafting of chick dorsal root ganglia (DRG) to the decellularized human lenticules. Differences between groups and controls were analyzed with ANOVA and Mann-Whitney U test., Results: The customization methods significantly eliminated neurites inside the lenticules. NL-TuJ1 was significantly reduced by 84% after excimer laser reshaping, 54% after CXL, and 96% after decellularization. The neurite remnants from reshaping and CXL exhibited calcium signaling, indicative of residual excitatory response. Re-innervation occurred in the decellularized lenticules upon stimulation of the grafted chick embryo DRG with nerve growth factor (NGF 2.5S)., Conclusion: All of the lenticule customization procedures reduced lenticule neurites, but the residual neurites still showed excitatory potential. Even though these neurite remnants seemed minimal, they could be advantageous to reinnervation with axon growth and guidance after lenticule reimplantation for refractive and volume restoration of the cornea., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Corneal opacification is the fourth most common cause of blindness globally behind cataracts, glaucoma, and age-related macular degeneration. The standard treatment of serious corneal scarring is corneal transplantation. Though it is effective for restoring vision, the treatment outcome is not optimal, due to limitations such as long-term graft survival, lifelong use of immunosuppressants, and a loss of corneal strength. Regulation of corneal stromal wound healing, along with inhibition or downregulation of corneal scarring is a promising approach to prevent corneal opacification. Pharmacological approaches have been suggested, however these are fraught with side effects. Tissue healing is an intricate process that involves cell death, proliferation, differentiation, and remodeling of the extracellular matrix. Current research on stromal wound healing is focused on corneal characteristics such as the immune response, angiogenesis, and cell signaling. Indeed, promising new technologies with the potential to modulate wound healing are under development. In this review, we provide an overview of cell-free strategies and some approaches under development that have the potential to control stromal fibrosis and scarring, especially in the context of early intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jhanji, Billig and Yam.)

The corneal endothelium located on the posterior corneal surface is responsible for regulating stromal hydration. This is contributed by a monolayer of corneal endothelial cells (CECs), which are metabolically active in a continuous fluid-coupled efflux of ions from the corneal stroma into the aqueous humor, preventing stromal over-hydration and preserving the orderly arrangement of stromal collagen fibrils, which is essential for corneal transparency. Mature CECs do not have regenerative capacity and cell loss due to aging and diseases results in irreversible stromal edema and a loss of corneal clarity. The current gold standard of treatment for this worldwide blindness caused by corneal endothelial failure is the corneal transplantation using cadaveric donor corneas. The top indication is Fuchs corneal endothelial dystrophy/degeneration, which represents 39% of all corneal transplants performed. However, the global shortage of transplantable donor corneas has restricted the treatment outcomes, hence instigating a need to research for alternative therapies. One such avenue is the CEC regeneration from endothelial progenitors, which have been identified in the peripheral endothelium and the adjacent transition zone. This review examines the evidence supporting the existence of endothelial progenitors in the posterior limbus and summarizes the existing knowledge on the microanatomy of the transitional zone. We give an overview of the isolation and ex vivo propagation of human endothelial progenitors in the transition zone, and their growth and differentiation capacity to the corneal endothelium. Transplanting these bioengineered constructs into in vivo models of corneal endothelial degeneration will prove the efficacy and viability, and the long-term maintenance of functional endothelium. This will develop a novel regenerative therapy for the management of corneal endothelial diseases.

(1) Objective: To study the anti-fibrotic effects of Lycium barbarum polysaccharides (LBP) on corneal stromal fibroblasts and assess LBP's effect on cell viability. (2) Methods: Primary human corneal keratocytes of passage 3 to 6 were used for all experiments. Cells are pretreated with LBP solution for 24 h and then transforming growth factor beta 1 (TGFβ1) for 48 h and collected for experiments. Fibrotic protein analysis was performed using immunofluorescence and Western blot. The effect of LBP on cell viability was assessed using the MTS assay. (3) Results: LBP significantly reduced the expression of fibrotic proteins, including α-SMA and extracellular matrix proteins (collagen type I and III). LBP significantly decreased the viability of myofibroblasts but not the fibroblasts. Conclusions: In this study, LBP was effective in the prevention of fibrosis gene expression. Further studies to assess the underlying mechanism and pharmacological properties will facilitate the formation of a topical LBP solution for in vivo studies.

Introduction: The tear proteomics and neuromediators are associated with clinical dry eye parameters following refractive surgery., Purpose: To investigate and compare the tear proteomic and neuromediator profiles following small incision lenticule extraction (SMILE) versus laser-assisted in-situ keratomileusis (LASIK)., Methods: In this randomized controlled trial with paired-eye design, 70 patients were randomized to receive SMILE in one eye and LASIK in the other eye. Tear samples were collected preoperatively, and 1 week, 1, 3, 6 and 12 months postoperatively, and were examined for protein concentration changes using sequential window acquisition of all theoretical fragment ion mass spectrometry (SWATH-MS). The data were analyzed with DAVID Bioinformatics Resources for enriched gene ontology terms and over-represented pathways. Tear neuromediators levels were correlated with clinical parameters., Results: Post-SMILE eyes had significantly better Oxford staining scores and tear break-up time (TBUT) than post-LASIK eyes at 1 and 3 months, respectively. Tear substance P and nerve growth factor levels were significantly higher in the LASIK group for 3 months and 1 year, respectively. SMILE and LASIK shared some similar biological responses postoperatively, but there was significant up-regulation in leukocyte migration and wound healing at 1 week, humoral immune response and apoptosis at 1 month, negative regulation of endopeptidase activity at 3 to 6 months, and extracellular structure organization at 1 year in the post-LASIK eyes. Tear mucin-like protein 1 and substance P levels were significantly correlated with TBUT ( r  = -0.47, r  = -0.49, respectively)., Conclusion: Significant differences in the tear neuromediators and proteomics were observed between SMILE and LASIK, even though clinical dry eye signs have subsided and became comparable between 2 procedures., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Background: Corneal stromal stem cells (CSSC) reduce corneal inflammation, prevent fibrotic scarring, and regenerate transparent stromal tissue in injured corneas. These effects rely on factors produced by CSSC to block the fibrotic gene expression. This study investigated the mechanism of the scar-free regeneration effect., Methods: Primary human CSSC (hCSSC) from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferon-γ and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor β3 (hTGFβ3) and hTGFβ1 were examined by immunofluorescence and qPCR. TGFβ3 knockdown for > 70% in hCSSC [hCSSC-TGFβ3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGFβ3(si) were transplanted in a fibrin gel to mouse corneas, respectively, after wounding by stromal ablation. Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined., Results: hTGFβ3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFβ3 at days 1 and 3 post-injury, along with the reduced expression of mouse inflammatory genes (CD80, C-X-C motif chemokine ligand 5, lipocalin 2, plasminogen activator urokinase receptor, pro-platelet basic protein, and secreted phosphoprotein 1). By day 14, hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes (fibronectin, hyaluronan synthase 2, Secreted protein acidic and cysteine rich, tenascin C, collagen 3a1 and α-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGFβ3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring., Conclusion: This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3, inducing a scar-free tissue response.

Corneal transplantation remains the ultimate treatment option for advanced stromal and endothelial disorders. Corneal tissue engineering has gained increasing interest in recent years, as it can bypass many complications of conventional corneal transplantation. The human cornea is an ideal organ for tissue engineering, as it is avascular and immune-privileged. Mimicking the complex mechanical properties, the surface curvature, and stromal cytoarchitecure of the in vivo corneal tissue remains a great challenge for tissue engineering approaches. For this reason, automated biofabrication strategies, such as bioprinting, may offer additional spatial control during the manufacturing process to generate full-thickness cell-laden 3D corneal constructs. In this review, we discuss recent advances in bioprinting and biomaterials used for in vitro and ex vivo corneal tissue engineering, corneal cell-biomaterial interactions after bioprinting, and future directions of corneal bioprinting aiming at engineering a full-thickness human cornea in the lab., Competing Interests: The authors declare no conflict of interest.

Background: Persistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). This study investigated CE reformation using human adipose mesenchymal stem cells (ADSC), which derived epithelial progenitors via mesenchymal-epithelial transition (MET)., Methods: STEMPRO human ADSC were cultured with specific inhibitors antagonizing glycogen synthase kinase-3 and transforming growth factor-β signaling, followed by culture under a defined progenitor cell targeted-epithelial differentiation condition to generate epithelial-like cells (MET-Epi), which were characterized for cell viability, mesenchymal, and epithelial phenotypes using immunofluorescence and flow cytometry. Tissue-engineered (TE) MET-Epi cells on fibrin gel were transplanted to corneal surface of the rat LSCD model caused by alkali injury. Epithelial healing, corneal edema, and haze grading, CE formation were assessed by fluorescein staining, slit lamp bio-microscopy, anterior segment optical coherence tomography, and immunohistochemistry., Results: CD73 high /CD90 high /CD105 high /CD166 high /CD14 negative /CD31 negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-β4, and cytokeratin (CK)-5, 9. Under defined epithelial differentiation culture, these progenitors generated MET-Epi cells expressing cell junction proteins ZO1 and occludin. When transplanted onto rat corneal surface with LSCD-induced PED, TE-MET-Epi achieved more efficient epithelial healing, suppressed corneal edema, and opacities, when compared to corneas without treatment or transplanted with TE-ADSC. CE markers (CK3, 12, and CDH1) were expressed on TE-MET-Epi-transplanted corneas but not in other control groups., Conclusion: Human ADSC-derived epithelial-like cells, via MET, recovered the CE from PED associated with LSCD. ADSC can be a viable adult stem cell source for potential autologous epithelial cell-based therapy for corneal surface disorders.

: The corneal endothelium regulates corneal hydration to maintain the transparency of cornea. Lacking regenerative capacity, corneal endothelial cell loss due to aging and diseases can lead to corneal edema and vision loss. There is limited information on the existence of corneal endothelial progenitors. We conducted ultrastructural examinations and expression analyses on the human transition zone (TZ) at the posterior limbus of corneal periphery, to elucidate if the TZ harbored progenitor-like cells, and to reveal their niche characteristics. Within the narrow TZ (~190 μm width), the inner TZ-adjacent to the peripheral endothelium (PE)-contained cells expressing stem/progenitor markers (Sox2, Lgr5, CD34, Pitx2, telomerase). They were located on the inner TZ surface and in its underlying stroma. Lgr5 positive cells projected as multicellular clusters into the PE. Under transmission electron microscopy and serial block face-scanning electron microscopy and three-dimensional (3D) reconstruction, the terminal margin of Descemet's membrane was inserted beneath the TZ surface, with the distance akin to the inner TZ breadth. Porcine TZ cells were isolated and proliferated into a confluent monolayer and differentiated to cells expressing corneal endothelial markers (ZO1, Na + K + ATPase) on cell surface. In conclusion, we have identified a novel inner TZ containing progenitor-like cells, which could serve the regenerative potential for corneal endothelium., Competing Interests: The authors declare no conflict of interest.

Purpose: We study the density and excitatory response of neurites, and Schwann cells (SCs) in fresh and cryopreserved stromal lenticules derived from small incision lenticule extraction (SMILE)., Methods: Human stromal lenticules (n = 23) were immunostained for β III-tubulin and imaged using spinning disk confocal laser microscopy, followed by three-dimensional reconstruction, to reveal neurite distribution. The lenticule neurite density (LND) was assessed using a validated neurite tracing and length measurement method with NeuronJ. LND was compared among groups of different lenticule thickness (71-165 μm) obtained from -3 to >-6 diopters (D) corrections. SCs were identified by marker expression and the laser effect on SC-neurite interaction was examined under transmission electron microscopy (TEM). Fresh porcine SMILE-lenticules (n = 18) were used for LND comparison among storage conditions and functional excitatory calcium response assay., Results: Using a validated neurite length measurement method, we found an inverse correlation of LND with lenticule thickness. Higher LND was found in thinner lenticules obtained from lower power of correction (r = -0.8925, P < 0.0001), whereas total lenticule neurite lengths did not alter significantly with regards to lenticule thickness. SCs were identified by GAP43 and p75NTR expression and were closely associated with lenticule neurites under TEM. In porcine lenticules, LND and excitatory calcium response were reduced after cold and cryogenic storage, when compared to fresh lenticules., Conclusions: The stromal neurites showed variations in density related to SMILE lenticule thickness and cryopreservation. With the presence of SC support and excitatory response, these neurite residues could retain minimal functionality that might serve as a potential advantage in the event of lenticule implantation.

Corneal transplantation is the treatment of choice for patients with advanced corneal diseases. However, the outcome may be affected by graft rejection, high associated costs, surgical expertise, and most importantly the worldwide donor shortage. In recent years, bioprinting has emerged as an alternative method for fabricating tissue equivalents using autologous cells with architecture resembling the native tissue. In this study, we propose a freeform and cell-friendly drop-on-demand bioprinting strategy for creating corneal stromal 3D models as suitable implants. Corneal stromal keratocytes (CSK) were bioprinted in collagen-based bioinks as 3D biomimetic models and the geometrical outcome as well as the functionality of the bioprinted specimens were evaluated after in vitro culture. We showed that our bioprinting method is feasible to fabricate translucent corneal stromal equivalents with optical properties similar to native corneal stromal tissue, as proved by optical coherence tomography. Moreover, the bioprinted CSK were viable after the bioprinting process and maintained their native keratocyte phenotypes after 7 days in in vitro culture, as shown by immunocytochemistry. The proposed bioprinted human 3D corneal models can potentially be used clinically for patients with corneal stromal diseases., (© 2019 Wiley Periodicals, Inc.)

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 (TiO 2 ) 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/TiO 2 hybrids were safe and biocompatible. In particular, the 1% reduced LCGO/TiO 2 (1rLCGO/TiO 2 ) 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/ TiO 2 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., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The conjunctiva is a clear tissue covering the white part of the eye and lines the back of the eyelids. Conjunctival diseases, such as symblepharon, cause inflammation, discharges, and photophobia. The treatment often requires excision of large parts of conjunctiva. Tissue engineering of conjunctival cells using human amniotic membrane (HAM) denuded of its epithelium as a basement membrane scaffold has been shown to be effective for covering conjunctival defects. However, most epithelial denudation protocols are time-consuming and expensive or compromise HAM's basement membrane structure and matrix components. We have previously described a method to de-epithelialize HAM using ice-cold urea (uHAM). In this report, we used this method to provide tissue-engineered constructs with cultivated conjunctival epithelial cells on uHAM in two patients, one with a giant conjunctival nevus and the other with a large symblepharon. Autologous conjunctival epithelial cells harvested from incisional biopsies of these two patients were cultured on the uHAM scaffold. The transplantation of tissue-engineered constructs to patients' ocular surface immediately after the removal of lesions showed successful reconstruction of the ocular surface. Postoperatively, there were neither recurrence of lesions nor epithelial defects throughout the follow-up (up to 7 and 19 months, respectively). This report highlights the translational potential of an efficient and inexpensive method to prepare de-epithelialized HAM as a basement membrane scaffold for cell-based tissue-engineered treatments of ocular surface disorders. Stem Cells Translational Medicine 2019;8:620&626., (© 2019 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

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.

Purpose: We investigate whether small incision lenticule extraction (SMILE) is associated with less ectasia than laser-assisted in-situ keratomileusis (LASIK) and whether concomitant collagen cross-linking (CXL) is protective in SMILE Xtra and LASIK Xtra., Methods: Using an established LASIK rabbit ectasia model, we performed -5 diopter (D) LASIK on six eyes and -5 D SMILE on six eyes; five eyes had -5 D LASIK Xtra, five eyes -5 D SMILE Xtra. Anterior segment optical coherence tomography and corneal topography were performed preoperatively and 2, 4, and 6 weeks postoperatively. Mean (standard deviation [SD]) values of postoperative keratometry (K), maximum posterior elevation (MPE) and minimum corneal thickness (CT) were compared to preoperatively and among the surgical groups (paired t -test, analysis of variance)., Results: Mean (SD) K values decreased significantly following SMILE, SMILE Xtra, LASIK, and LASIK Xtra. The MPE increased significantly ( P < 0.05) following LASIK, SMILE, and SMILE Xtra, but not following LASIK Xtra ( P = 0.12). The MPE was less following SMILE than LASIK, but not statistically significant (week 2, 17.73 [5.77] vs. 22.75 [5.05] μm; P = 0.13); post-LASIK Xtra MPE was less than that following LASIK (week 2. 13.39 [3.05] vs. 22.75 [5.05] μm; P < 0.001). CT decreased significantly in all surgical groups; no differences were detected among the groups., Conclusions: SMILE may have less potential than LASIK to induce ectasia. LASIK Xtra and SMILE Xtra showed the smallest increase in MPE., Translational Relevance: Concomitant CXL may be protective following keratorefractive surgery and may reduce further the risk of ectasia.

Laser refractive surgery is one of the most performed surgical procedures in the world. Although regarded safe and efficient, it has side effects. All of the laser based refractive surgical procedures invoke corneal nerve injury to some degree. The impact of this denervation can range from mild discomfort to neurotrophic corneas. Currently, three techniques are widely used for laser vision correction: small incision lenticule extraction, laser-assisted keratomileusis in situ and photorefractive keratotomy. Each of these techniques affects corneal innervation differently and has a different pattern of nerve regeneration. The purpose of this review is to summarize the different underlying mechanisms for corneal nerve injury and compare the different patterns of corneal reinnervation., Competing Interests: None

Keratoconus (KC) is an ectatic corneal disease characterized by progressive thinning and irregular astigmatism, and a leading indication for corneal transplantation. KC-associated changes have been demonstrated for the entire cornea, but the pathological thinning and mechanical weakening is usually localized. We performed quantitative proteomics using Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectrometry (SWATH-MS) to analyze epithelial and stromal changes between the topographically-abnormal cone and topographically-normal non-cone regions of advanced KC corneas, compared to age-matched normal corneas. Expression of 20 epithelial and 14 stromal proteins was significantly altered (≥2 or ≤0.5-fold) between cone and non-cone in all 4 KC samples. Ingenuity pathway analysis illustrated developmental and metabolic disorders for the altered epithelial proteome with mitochondrion as the significant gene ontology (GO) term. The differential stromal proteome was related to cellular assembly, tissue organization and connective tissue disorders with endoplasmic reticulum protein folding as the significant GO term. Validation of selected protein expression was performed on archived KC, non-KC and normal corneal specimens by immunohistochemistry. This is the first time to show that KC-associated proteome changes were not limited to the topographically-thinner and mechanically-weakened cone but also non-cone region with normal topography, indicating a peripheral involvement in KC development.

Purpose: We define optical coherence tomography (OCT) measurement parameters of the corneal endothelium/Descemet's membrane (DM) complex and peripheral transition zone (TZ) and describe these measurements in an ethnically Chinese population., Methods: OCT images of the anterior segment and iridocorneal angle were obtained from 129 healthy Chinese subjects (129 eyes), aged 40 to 81 years. The scleral spur (SS) and Schwalbe's line (SL) were identified in each image. Endothelium/DM diameter, referred to as endothelial arc length (EAL), is the SL-to-SL distance. The SS-to-SL distance encompasses the TZ and trabecular meshwork (TM). Since the TZ cannot be visualized by OCT, a ratio of TZ-to-TZ+TM width was calculated from scanning electron microscopy (SEM) images obtained from 5 cadaveric corneas. The SS-to-SL distance was multiplied by this ratio to approximate in vivo TZ width., Results: From SEM measurements, the relationship TZ = 0.20*(TZ+TM) was determined. From OCT measurements, mean EAL was 12.15 ± 0.58 mm and mean TZ width was 156 ± 20 μm. For eyes with horizontal and vertical images, vertical EAL was significantly greater than horizontal EAL ( P = 0.03)., Conclusions: Corneal endothelium/DM diameter and TZ width can be obtained from OCT images. Although only combined TZ+TM is visualized on OCT, TZ width can be reasonably approximated., Translational Relevance: Emerging procedures, like endothelial cell injection and DM transplantation (DMT), require accurate measurements of endothelium/DM size for preoperative planning. Size of the TZ, which may contain progenitor cells, also could contribute to endothelial regeneration in these procedures.

Corneal neovascularization (CoNV) could be treated by novel anti-angiogenic therapies, though reliable and objective imaging tools to evaluate corneal vasculature and treatment efficacy is still lacking. Optical coherence tomography angiography (OCTA) -currently designed as a retinal vascular imaging system- has been recently adapted for anterior-segment and showed good potential for successful imaging of CoNV. However, further development requires an animal model where parameters can be studied more carefully with histological comparison. Our study evaluated the OCTA in suture-induced CoNV in a rabbit model compared to indocyanine green angiography (ICGA) and slit-lamp photography (SLP). Overall vessel density measurements from OCTA showed good correlation with ICGA (0.957) and SLP (0.992). Vessels density by OCTA was higher than ICGA and SLP (mean = 20.77 ± 9.8%, 15.71 ± 6.28% and 17.55 ± 8.36%, respectively, P < 0.05). OCTA was able to depict CoNV similarly to SLP and ICGA, though it could better detect small vessels. Moreover, the depth and growth of vessels could be assessed using en-face and serial-scans. This study validated the OCTA in a rabbit model as a useful imaging tool for translational studies on CoNV. This may contribute to further studies on OCTA for anterior-segment including serial evaluation of emerging anti-angiogenic therapies.

Purpose: To evaluate the safety and feasibility of intrastromal injection of human corneal stromal keratocytes (CSKs) and its therapeutic effect on a rodent early corneal opacity model., Methods: Twelve research-grade donor corneas were used in primary culture to generate quiescent CSKs and activated stromal fibroblasts (SFs). Single and repeated intrastromal injections of 2 to 4 × 104 cells to rat normal corneas (n = 52) or corneas with early opacities induced by irregular phototherapeutic keratectomy (n = 16) were performed, followed by weekly examination of corneal response under slit-lamp biomicroscopy and in vivo confocal microscopy with evaluation of haze level and stromal reflectivity, and corneal thickness using anterior segment optical coherence tomography (AS-OCT). Time-lapse tracing of Molday ION-labelled cells was conducted using Spectralis OCT and label intensity was measured. Corneas were collected at time intervals for marker expression by immunofluorescence, cell viability, and apoptosis assays., Results: Injected CSKs showed proper marker expression with negligible SF-related features and inflammation, hence maintaining corneal clarity and stability. The time-dependent loss of injected cells was recovered by repeated injection, achieving an extended expression of human proteoglycans inside rat stroma. In the early corneal opacity model, intrastromal CSK injection reduced stromal reflectivity and thickness, resulting in recovery of corneal clarity, whereas noninjected corneas were thicker and had haze progression., Conclusions: We demonstrated the safety, feasibility, and therapeutic efficacy of intrastromal CSK injection. The cultivated CSKs can be a reliable cell source for potential cell-based therapy for corneal opacities.

Optic neuropathies are the leading cause of irreversible blindness and visual impairment in the developed countries, affecting more than 80 million people worldwide. While most optic neuropathies have no effective treatment, there is intensive research on retinal ganglion cell (RGC) protection and axon regeneration. We previously demonstrated potential of human periodontal ligament-derived stem cells (PDLSCs) for retinal cell replacement. Here, we report the neuroprotective effect of human PDLSCs to ameliorate RGC degeneration and promote axonal regeneration after optic nerve crush (ONC) injury. Human PDLSCs were intravitreally injected into the vitreous chamber of adult Fischer rats after ONC in vivo as well as cocultured with retinal explants in vitro. Human PDLSCs survived in the vitreous chamber and were maintained on the RGC layer even at 3 weeks after ONC. Immunofluorescence analysis of βIII-tubulin and Gap43 showed that the numbers of surviving RGCs and regenerating axons were significantly increased in the rats with human PDLSC transplantation. In vitro coculture experiments confirmed that PDLSCs enhanced RGC survival and neurite regeneration in retinal explants without inducing inflammatory responses. Direct cell-cell interaction and elevated brain-derived neurotrophic factor secretion, but not promoting endogenous progenitor cell regeneration, were the RGC protective mechanisms of human PDLSCs. In summary, our results revealed the neuroprotective role of human PDLSCs by strongly promoting RGC survival and axonal regeneration both in vivo and in vitro, indicating a therapeutic potential for RGC protection against optic neuropathies. Stem Cells 2018;36:844-855., (© AlphaMed Press 2018.)

Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non-functional or orthodontic reason and differentiated them towards CSK phenotype using a two-step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL-differentiated CSK-like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14-day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

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.

Purpose: The pathogenesis of pterygium has been linked to limbal stem cell damage, abnormal apoptosis and cellular proliferation. In this study, we investigated the epigenetic regulation through microRNA expression in the pathogenesis of pterygium., Methods: Human full-length primary pterygia were microdissected into head and body regions. Specific microRNA and mRNA expression was assayed by TaqMan ® real-time quantitative polymerase chain reaction (qPCR). Tissue localization of target microRNAs was performed by LNA-based in situ hybridization. MicroRNA-145 (miR-145) mimics were transfected to primary culture of human pterygial cells, followed by analyses of cell cycle changes, apoptosis, p53 and MDM2 expression using flow cytometry and qPCR., Results: The expression of miR-145 was markedly higher in primary human pterygium than in limbus and conjunctiva. Both miR-143 and miR-145 were predominantly expressed in the basal pterygial epithelium. Oncogene MDM2 expression was abundant in pterygial epithelium and stroma, while the expression pattern was opposite to that of miR-145. Ectopic expression of miR-145 in pterygial cells induced G1 arrest, down-regulated MDM2 and elevated p53 expression., Conclusions: Our study showed that miR-145 suppressed MDM2 expression, which subsequently influenced the p53-related cell growth pattern in pterygial epithelium. The regulatory miR-145/MDM2-p53 loop can serve as a potential target for treatment of pterygium., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Corneal tissue is the most transplanted of all body tissues. Currently, cadaveric donor tissues are used for transplantation. However, a global shortage of transplant grade material has prompted development of alternative, cell-based therapies for corneal diseases. Pluripotent stem cells are attractive sources of cells for regenerative medicine, because large numbers of therapeutically useful cells can be generated. However, a detailed understanding of how to differentiate clinically relevant cell types from stem cells is fundamentally required. Periocular mesenchyme (POM), a subtype of cranial neural crest, is vital for development of multiple cell types in the cornea, including clinically relevant cells such as corneal endothelium and stromal keratocytes. Herein, we describe protocols for differentiation of POM from pluripotent stem cells. Using defined media containing inhibitors of TGFβ and WNT signalling, we generated neural crest cells that express high levels of the POM transcription factors PITX2 and FOXC1. Furthermore, we identified cells resembling POM in the adult cornea, located in a niche between the trabecular meshwork and peripheral endothelium. The generation and expansion of POM is an important step in the generation of a number of cells types that could prove to be clinically useful for a number of diseases of the cornea., (Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The manufacture of mechanically strong and biocompatible titania (TiO 2 ) materials is of vital importance for their application as corneal implant skirts. This study was aimed at optimizing the selection of raw powder and sintering conditions for TiO 2 ceramics. TiO 2 compacts were synthesized from five raw powders, denoted as Altair, Inframat, Alfa, Materion, and Amperit, respectively, by spark plasma sintering using different sintering parameters. The XRD and Raman results confirmed that the anatase TiO 2 phase in the Inframat powder had converted completely to rutile TiO 2 phase after sintering at 900°C and above. The nanoindentation results indicated that among the five types of TiO 2 samples sintered at 1100°C, the Inframat pellets possessed the highest Young's modulus and hardness. Additionally, when Materion samples were employed to study the effects of SPS parameters, a higher sintering temperature in the range of 1100-1300°C decreased the mechanical properties of sintered pellets probably due to the generation of more structural defects. Culture of human corneal stromal fibroblasts on the sintered sample surfaces showed that comparably high cell viability and proliferation were observed on all TiO 2 samples except Amperit compared to positive control. Furthermore, cells cultured on Inframat TiO 2 sintered in the temperature range of 900-1300°C exhibited viability and formation of focal adhesion complex similar to those on control, and those prepared at 1100°C had significantly higher cell proliferation indices than control. In conclusion, Inframat TiO 2 consolidated at 1100°C by SPS was the best formulation for the preparation of mechanically strong and biocompatible Keratoprosthesis skirt. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3502-3513, 2017., (© 2017 Wiley Periodicals, Inc.)

In recent years, the cultivation and expansion of primary corneal cells has made significant progress. The transplantation of cultured limbal epithelial cells represents a successful and established treatment of the ocular surface. Cultivated corneal endothelial cells are undergoing a clinical trial in Japan. Stromal keratocytes can now be expanded in vitro. A wide range of stem cell sources is being tested in vitro and animal models for their possible application in corneal cell therapy. This article gives an overview of recent advancements and prevailing limitations for the use of different cell sources in the therapy of corneal disease.

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<0.001 compared to other procedures). The corneas appeared normal on day 28 after S+P, but had a lower number of keratocytes near the laser ablated plane compared to other procedures. S+SE and S+C did not induce corneal haze and resulted similar level of fibronectin. However, S+C resulted in more inflammatory (10±2 cells; p = 0.001) and apoptotic cells (25±2 cells; p<0.001) compared to S+SE (7±1 inflammatory cells and 21±3 apoptotic cells) early post-operatively. In conclusion, each SMILE retreatment method resulted in unique tissue responses. S+SE offers advantages, such as minimal inflammation and cell death, as well as maintaining a 'flap-less' surgery, over other procedures. However, depending on the degree of enhancement, the lenticule may become too thin to be extracted and the procedure becomes more difficult to perform than S+C and S+P. S+P can maintain corneal integrity by avoiding flap creation and is technically more simple to perform than the others, but the surgery needs to be supplemented with mitomycin-C in order to reduce inflammation and modulate better wound healing.

Purpose: Pterygium is a common ocular surface disorder. Conjunctival autografting (CAG) following pterygium resection is the gold standard treatment. CAGs without Tenon's tissue provide better results but are more technically difficult to achieve. In this study, we evaluated the feasibility and reproducibility of femtosecond laser (FSL)-assisted CAG preparation., Methods: Fifteen porcine globes were fixed in a suction holder and CAGs of different diameters were created by 1) an experienced consultant and 2) a less experienced fellow using the Ziemer LDV Z8. The CAG's dimension was measured and thickness analyzed by optical coherence tomography (OCT) and histology (HE). Statistical analysis was performed by Mann-Whitney-U, Wilcoxon and Spearman-test., Results: FSL-assisted CAGs prepared at 100 μm (146.4 ± 45.7 μm) showed a significantly higher deviation from desired depth (p = 0.04) and a higher variability (p = 0.03) in thickness than those prepared at 60 μm (71.4 ± 12.7 μm). The experienced (68.3 ± 14.3 μm) and inexperienced surgeon (73.9 ± 11.9 μm) produced 60 μm grafts of comparable thickness (p = 0.6) and variability (p = 0.7). The CAG area measured after dissection (37.5 ± 12.1 mm 2 ) did not differ significantly from the FSL settings (40.6 ± 12.7 mm 2 , p = 0.3). FSL cutting time at 60 μm took 18.1 ± 2.2 s, at 100 μm 20.7 ± 2.4 s. Graft separation time was not significantly influenced by depth or surgeon. No buttonholes or CAG tags occurred during surgery., Conclusions: The FSL allowed the accurate and reliable preparation of very thin CAGs, independent of surgeon experience and may represent a valuable tool in pterygium surgery., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density.

Background: Presbyopic inlays have mostly been implanted under a corneal flap. Implantation in a pocket has advantages including less postoperative dry eye and neurotrophic effect, and better biomechanical corneal stability. This study investigated the effect of different pocket and flocket dimensions on corneal stability and refractive power after Raindrop™ implantation, and the associated wound healing response., Methodology: Ten New Zealand White rabbits had bilateral pocket Raindrop™ implantation. Eyes were allocated to 4 groups: pockets with 4mm, 6mm, and 8mm diameters, and 8mm flocket. They were examined pre-operatively, at day 1, weeks 1, 2, 3 and 4 post-surgery with anterior segment optical coherence tomography, corneal topography and in-vivo confocal microscopy. After euthanasia (week 4), CD11b, heat shock protein (HSP) 47 and fibronectin corneal immunohistochemistry was performed., Results: Corneal thickness (mean±SD) increased from 360.0±16.2μm pre-operatively to 383.9±32.5, 409.4±79.3, 393.6±35.2, 396.4±50.7 and 405±20.3μm on day 1, weeks 1,2,3 and 4 respectively (p<0.008, all time-points). Corneal refractive power increased by 11.1±5.5, 7.5±2.5, 7.5±3.1, 7.0±3.6 and 6.3±2.9D (p<0.001). Corneal astigmatism increased from 1.1±0.3D to 2.3±1.6, 1.7±0.7, 1.8±1.0, 1.6±0.9 and 1.6±0.9D respectively (p = 0.033). CT, refractive power change and astigmatism were not different between groups. The 8mm pocket and 8mm flocket groups had the least stromal keratocyte reflectivity. CD11b, fibronectin or HSP47 weren't detected., Conclusions: Anatomical and refractive stability was achieved by 1 week; the outcomes were not affected by pocket or flocket configuration. No scarring or inflammation was identified. The 8mm pocket and flocket showed the least keratocyte activation, suggesting they might be the preferred configuration.

Age-related macular degeneration (AMD) is a leading cause of visual impairment and irreversible blindness in most developed countries, affecting about 50 million elderly people worldwide. Retinal pigment epithelial (RPE) cell degeneration is the pathophysiological cause of AMD, leading to geographic atrophy and choroidal neovascularization. We and others have previously identified several polymorphisms on chromosome 10q26 (HTRA1 rs11200638 as well as LOC387715 rs10490924 and c.372&#95;815del443ins54) associated with AMD. In this study, we confirmed the association of our previously identified HTRA1 insertion-deletion (indel) variant (c.34delCinsTCCT) in 195 exudative AMD patients and 390 controls from the Hong Kong Chinese cohort with additional 168 patients and 210 controls from the Chengdu Chinese cohort and followed by studying its biological functions in RPE cells. Genetic analysis verified the higher prevalence of c.34delCinsTCCT allele in control subjects (8.0%) than in AMD patients (1.9%; P=7.87 × 10 -5 , odds ratio=0.229). This protective effect was validated as the haplotype of the c.34delCinsTCCT allele existed independent of the risk haplotype (P=1.17 × 10 -5 ). In vitro studies showed that recombinant HTRA1 c.34delCinsTCCT variant protein was more localized in the endoplasmic reticulum of RPE cells compared with the wild-type protein, and its secretion was delayed. Moreover, ARPE-19 cells expressing HTRA1 c.34delCinsTCCT variant had higher cell viability, lower cell apoptosis and were less responsive to anoikis, supporting its protective role. We revealed a protective AMD-associated HTRA1 variant in Chinese populations and the biological role of HTRA1 in RPE cell degeneration, indicating its involvement in AMD pathogenesis.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) and Hevin are structurally related matricellular proteins involved in extracellular matrix assembly. In this study, we compared the anterior chamber biometric parameters and iris collagen properties in SPARC-, Hevin- and SPARC-/Hevin-null with wild-type (WT) mice., Methods: The right eyes of 53 WT, 35 SPARC-, 56 Hevin-, and 63 SPARC-/Hevin-null mice were imaged using the RTVue-100 Fourier-domain optical coherence tomography system. The parameters measured were anterior chamber depth (ACD), trabecular-iris space area (TISA), angle opening distance (AOD), and pupil diameter. Biometric data were analyzed using analysis of covariance and adjusted for age, sex, and pupil diameter. Expression of Col1a1, Col8a1, and Col8a2 transcripts in the irises was measured by quantitative polymerase chain reaction. Collagen fibril thickness was evaluated by transmission electron microscopy., Results: Mice that were SPARC- and SPARC-/Hevin-null had 1.28- and 1.25-fold deeper ACD, 1.45- and 1.53-fold larger TISA, as well as 1.42- and 1.51-fold wider AOD than WT, respectively. These measurements were not significantly different between SPARC- and SPARC-/Hevin-null mice. The SPARC-null iris expressed lower Col1a1, but higher Col8a1 and Col8a2 transcripts compared with WT. Collagen fibrils in the SPARC- and SPARC-/Hevin-null irises were 1.5- and 1.7-fold thinner than WT, respectively. The Hevin-null iris did not differ from WT in these collagen properties., Conclusions: SPARC-null mice have deeper anterior chamber as well as wider drainage angles compared with WT. Therefore, SPARC plays a key role in influencing the spatial organization of the anterior segment, potentially via modulation of collagen properties, while Hevin is not likely to be involved.

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 TiO 2 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.

Corneal integrity is essential for visual function. Transplantation remains the most common treatment option for advanced corneal diseases. A global donor material shortage requires a search for alternative treatments. Different stem cell populations have been induced to express corneal cell characteristics in vitro and in animal models. Yet before their application to humans, scientific and ethical issues need to be solved. The in vitro propagation and implantation of primary corneal cells has been rapidly evolving with clinical practices of limbal epithelium transplantation and a clinical trial for endothelial cells in progress, implying cultivated ocular cells as a promising option for the future. This review reports on the latest developments in primary ocular cell and stem cell research for corneal therapy.

Refractive surgical treatment of hyperopia still remains a challenge for refractive surgeons. A new nomogram of small incision lenticule extraction (SMILE) procedure has recently been developed for the treatment of hyperopia. In the present study, we aimed to evaluate the wound healing and inflammatory responses of this new nomogram (hyperopic-SMILE), and compared them to those of hyperopic-laser-assisted in situ keratomileusis (LASIK), using a rabbit model. A total of 26 rabbits were used, and slit lamp biomicroscopy, autorefractor/keratometer, intraocular pressure measurement, anterior segment optical coherence tomography, corneal topography, and in vivo confocal microscopy examinations were performed during the study period of 4 weeks. The corneas were then harvested and subject to immunofluorescence of markers for inflammation (CD11b), wound healing (fibronectin) and keratocyte response (HSP47). The lenticule ultrastructual changes were also analyzed by transmission electron microscopy. Out results showed that hyperopic-SMILE effectively steepened the cornea. Compared to hyperopic-LASIK, hyperopic-SMILE had less postoperative wound healing response and stromal interface reaction, especially in higher refractive correction. However, compared to myopic-SMILE, hyperopic-SMILE resulted in more central deranged collagen fibrils. These results provide more perspective into this new treatment option for hyperopia, and evidence for future laser nomogram modification.

Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry.

Purpose: To investigate the early corneal wound healing and inflammatory responses after small incision lenticule extraction (SMILE) with different power of corrections and surgical experiences using a rabbit model., Methods: Twenty-four rabbits underwent SMILE with -2.00, -4.00, and -8.00 diopters (D) correction. One eye of each rabbit was operated on by surgeon 1 (experienced) and the contralateral eye was operated on by surgeon 2 (novice). Slit-lamp examination, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy were performed at 1 day and 1 week postoperatively. The corneas were harvested for immunofluorescence of markers for inflammation (CD11b), wound healing (fibronectin), and keratocyte response (HSP47)., Results: All corneas appeared clear throughout the follow-up period. In vivo confocal microscopy showed a greater reflectivity after -8.00 D than -2.00 D correction at day 1 at the lenticule anterior, posterior, and extracted lenticule planes (surgeon 1: P = .004, .041, and .038; surgeon 2: P = .012, .045, and .031). Different refractive corrections did not significantly affect the expression of CD11b, fibronectin, and HSP47. In the -2.00 D group, eyes operated on by surgeon 2 had thicker central corneal thickness evaluated by AS-OCT (P = .049) and exhibited more CD11b- and HSP47-positive cells at day 1 at the small vertical incision (P = .039 and .042)., Conclusions: The early inflammatory and wound healing responses after SMILE were minimal. In the early postoperative period, less surgical experience resulted in an increased inflammatory response in low myopic corrections. Greater keratocyte response was seen in high myopic corrections irrespective of surgeon experience. [J Refract Surg. 2016;32(5):346-353.]., (Copyright 2016, SLACK Incorporated.)