Impact of keratocyte differentiation on corneal opacity resolution and visual function recovery in male rats.

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea's collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.

Mesenchymal Stem Cell Exosomes as Immunomodulatory Therapy for Corneal Scarring.

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-1ß, 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.

Experiment-Based Validation of Corneal Lenticule Banking in a Health Authority-Licensed Facility.

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 in vivo.

Cellular therapy of corneal epithelial defect by adipose mesenchymal stem cell-derived epithelial progenitors.

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-ß 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: CD73high/CD90high/CD105high/CD166high/CD14negative/CD31negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-ß4, 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.

A sintered graphene/titania material as a synthetic keratoprosthesis skirt for end-stage corneal disorders.

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.

Safety and Feasibility of Intrastromal Injection of Cultivated Human Corneal Stromal Keratocytes as Cell-Based Therapy for Corneal Opacities.

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.

Inhibiting glycogen synthase kinase-3 and transforming growth factor-ß signaling to promote epithelial transition of human adipose mesenchymal stem cells.

BACKGROUND: This study was aimed to investigate the epithelial differentiation of human adipose-derived mesenchymal stem cells (ADSCs) by inhibiting glycogen synthase kinase-3 (GSK3) and transforming growth factor ß (TGFß) signaling. METHODS AND RESULTS: STEMPRO human ADSCs at passage 2 were treated with CHIR99021 (GSK3 inhibitor), E-616452 (TGFß1 receptor kinase inhibitor), A-83-01 (TGFß type 1 receptor inhibitor), valproic acid (histone deacetylase inhibitor), tranylcypromine (monoamine oxidase inhibitor) and all-trans retinoic acid for 72 h. The mesenchymal-epithelial transition was shown by down-regulation of mesenchymal genes (Slug, Zinc Finger E-box Binding Homeobox 1 ZEB1, integrin α5 ITGA5 and vimentin VIM) and up-regulation of epithelial genes (E-cadherin, Epithelial Cell Adhesion Molecule EpCAM, Zonula Occludens-1 ZO-1, occludin, deltaN p63 δNp63, Transcription Factor 4 TCF4 and Twist Family bHLH Transcription Factor TWIST), compared to untreated ADSCs. Cell morphology and stress fiber pattern were examined and the treated cells became less migratory in scratch wound closure assay. The formation of cell junction complexes was observed under transmission electron microscopy. Global gene expression using GeneChip® Human Genome U133 Array (Affymetrix) showed that the treatment up-regulated 540 genes (containing genes for cell cycle, cytoskeleton reorganization, chemotaxis, epithelium development and regulation of cell migration) and down-regulated 483 genes. CONCLUSION: Human ADSCs were transited to epithelial lineage by inhibiting GSK3 and TGFß signaling. It can be an adult stem cell source for epithelial cell-based therapy.

Decellularization of human stromal refractive lenticules for corneal tissue engineering.

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.

Application of Graphene as Candidate Biomaterial for Synthetic Keratoprosthesis Skirt.

PURPOSE: Synthetic keratoprostheses are required for visual rehabilitation in patients with end-stage corneal blindness. This study aimed to assess the biocompatibility of graphene material and its potential as a novel synthetic keratoprosthesis skirt material for corneal tissue engineering. METHODS: Human corneal stromal fibroblasts were cultured on material surfaces including pristine graphene film, graphene foam, pristine titanium (Ti) discs, and tissue culture plastic surface (TCPS). Cell attachment was assayed by immunostaining of paxillin and vinculin. Cell viability and proliferation were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Click iT 5-ethynyl-2'-deoxyuridine (EdU) assays. The growth of fibroblasts on three-dimensional graphene foam was examined by scanning electron microscopy, and cytokine release was analyzed by enzyme-linked immunosorbent assay. Graphene films were implanted into rabbit corneal stromal pockets and examined by slit-lamp biomicroscopy, anterior segment optical coherence tomography, in vivo confocal microscopy, and histology. RESULTS: Pristine graphene demonstrated good biocompatibility with human stromal fibroblasts in terms of cell adhesion, viability, and proliferation. Cells on graphene films showed higher number than on TCPS control. Cells grown on graphene had 10% more proliferation than on Ti. The expression levels of IL-6 and IL-8 were reduced when cells were seeded on graphene foam as compared to Ti and graphene film. Implantation of graphene film into rabbit stroma (n = 6) did not show any signs of infection, neovascularization, or inflammation. CONCLUSIONS: Graphene displayed excellent short-term biocompatibility with corneal cells and tissue. This demonstrates that graphene can be developed as a tissue engineering material for use in cornea.

Effectiveness of antimicrobial peptide immobilization for preventing perioperative cornea implant-associated bacterial infection.

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.