Controlled Release of Molecules to Enhance Cell Survival and Regeneration.

Controlled release or controlled drug delivery comprises the set of techniques and approaches to improve bioavailability through improved safety and/or efficacy using a carrier material for the molecule of interest. The predictability and tunability of these carriers make them ideal for protection, localization, and sustained presentation of a wide range of therapeutics, including growth factors implicated in cell survival and regeneration. Here we provide a method for encapsulating epidermal growth factor in a degradable polymer matrix for delivery to the cornea. Additional notes are included to demonstrate the wide-ranging capabilities of such methods for other materials, therapeutic agents, and sites of action within the eye.

A sustainable approach to derive sheep corneal scaffolds from stored slaughterhouse waste.

Aim: The escalating demand for corneal transplants significantly surpasses the available supply. To bridge this gap, we concentrated on ethical and sustainable corneal grafting sources. Our objective was to create viable corneal scaffolds from preserved slaughterhouse waste.Materials & methods: Corneas were extracted and decellularized from eyeballs that had been refrigerated for several days. These scaffolds underwent evaluation through DNA quantification, histological analysis, surface tension measurement, light propagation testing, and tensile strength assessment.Results: Both the native and acellular corneas (with ~90% DNA removed using a cost-effective and environmentally friendly surfactant) maintained essential optical and biomechanical properties for potential clinical use.Conclusion: Our method of repurposing slaughterhouse waste, stored at 4°C for several days, to develop corneal scaffolds offers a sustainable and economical alternative xenograft model.

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Bioprinting of anisotropic functional corneal stroma using mechanically robust multi-material bioink based on decellularized cornea matrix.

Corneal scarring is a common cause of blindness, affecting millions globally each year. A huge gap between the demand and supply of donor tissue currently limits corneal transplantation, the only definitive therapy for patients with corneal scarring. To overcome this challenge, researchers have harnessed the efficacy of 3D bioprinting to fabricate artificial corneal stromal constructs. With all the different bioinks available, the decellularized corneal matrix-based bioprinted construct can fulfill the required biological functionality but is limited by the lack of mechanical stiffness. Additionally, from a biophysical standpoint, it is necessary for an ideal corneal substitute to mimic the anisotropy of the cornea from the central optic zone to the surrounding periphery. In this study, we enhanced the mechanical robustness of decellularized cornea matrix (DCM) hydrogel by blending it with another natural polymer, sonicated silk fibroin solution in a defined ratio. Although hybrid hydrogel has an increased complex modulus than DCM hydrogel, it has a lower in vitro degradation rate and increased opaqueness due to the presence of crystalline beta-sheet conformation within the hydrogel. Therefore, we used this multi-material bioink-based approach to fabricate a corneal stromal equivalent where the outer peripheral corneal rim was printed with a mechanically robust polymeric blend of DCM and sonicated silk fibroin and the central optic zone was printed with only DCM. The bioprinted corneal stroma thus maintained its structural integrity and did not break when lifted with forceps. The two different bioinks were encapsulated with human limbus-derived mesenchymal stem cells (hLMSC) individually and 3D bioprinted in different patterns (concentric and parallel) to attain a native-like structure in terms of architecture and transparency. Thus, the bilayer cornea constructs maintained high cell viability and expressed keratocyte core proteins indicating optimal functionality. This approach helped to gain insight into bioprinting corneas with heterogeneous mechanical property without disturbing the structural clarity of the central optic zone.

Apical cell expansion maintained by Dusky-like establishes a scaffold for corneal lens morphogenesis.

The Drosophila corneal lens is entirely composed of chitin and other apical extracellular matrix components, and it is not known how it acquires the biconvex shape that enables it to focus light onto the retina. We show here that the zona pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells and prevents them from undergoing apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other zona pellucida domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. We find that artificially inducing apical constriction by activating myosin contraction is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape in controlling the morphogenesis of overlying apical extracellular matrix structures such as the corneal lens.

Coenzyme Q10 and Vitamin E Regulate the Bioactivity of Human Corneal Fibroblast Cells.

Purpose: Corneal fibroblasts are involved in the wound healing of the cornea with proliferation, migration, and differentiation processes. Coenzyme Q10 (CoQ10) and vitamin E can enhance corneal wound healing when applied after a corneal lesion as an eye drop. Thus, this study was performed to determine the potential efficiency of a CoQ10 ophthalmical solution containing a CoQ10 and vitamin E D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-derived formulation in human corneal fibroblasts (HCFs) in vitro. Methods: Primary HCFs were obtained from cadaveric corneal tissue, and cell viability was determined using MTT assay at 24 and 72 h. Cell migration was evaluated using an in vitro wound healing assay, and mRNA expressions of collagen type I (COL-I), collagen type III (COL-III), lumican, hyaluronan, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitors of MMP (TIMP)-1, TIMP-2, interleukin (IL)-1ß, IL-6, IL-8, and IL-10 were assessed using reverse transcription polymerase chain reaction at 24 and 72 h. Results: At various concentrations of CoQ10 ophthalmical solution (CoQ10-os), cell viability and wound healing rates of HCFs increased compared with the control group. The expressions of COL-I, COL-III, lumican, and hyaluronan were increased by CoQ10-os, whereas those of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were not affected by CoQ10-os at 24 and 72 h. In treating HCFs with a CoQ10-os medium, IL-1ß, IL-6, and IL-8 decreased, whereas IL-10 was significantly increased in a time- and dose-dependent manner. Conclusions: The findings indicate that CoQ10 and vitamin E-TPGS are potent regulators of the bioactivity of HCFs, thus supporting their potential application as ophthalmical solutions in therapies aimed at the fast regeneration of damaged cornea tissues.

The Role of Vimentin in Human Corneal Fibroblast Spreading and Myofibroblast Transformation.

Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, cell-matrix adhesion, and fibrotic transformation. Here, we assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibroblasts. Overall, although knockout (KO) of vimentin did not dramatically impact corneal fibroblast spreading and mechanical activity (traction force), cell elongation in response to PDGF was reduced in vimentin KO cells as compared to controls. Blocking vimentin polymerization using Withaferin had even more pronounced effects on cell spreading and also inhibited cell-induced matrix contraction. Furthermore, although absence of vimentin did not completely block TGFß-induced myofibroblast transformation, the degree of transformation and amount of αSMA protein expression was reduced. Proteomics showed that vimentin KO cells cultured in TGFß had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Interestingly, proteins associated with LRRC15 in other cell types, such as collagen, fibronectin, ß1 integrin and α11 integrin, were also upregulated. Overall, our data show that vimentin impacts both corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.

Biologicals and Biomaterials for Corneal Regeneration and Vision Restoration in Limbal Stem Cell Deficiency.

The mammalian cornea is decorated with stem cells bestowed with the life-long task of renewing the epithelium, provided they remain healthy, functional, and in sufficient numbers. If not, a debilitating disease known as limbal stem cell deficiency (LSCD) can develop causing blindness. Decades after the first stem cell (SC) therapy is devised to treat this condition, patients continue to suffer unacceptable failures. During this time, improvements to therapeutics have included identifying better markers to isolate robust SC populations and nurturing them on crudely modified biological or biomaterial scaffolds including human amniotic membrane, fibrin, and contact lenses, prior to their delivery. Researchers are now gathering information about the biomolecular and biomechanical properties of the corneal SC niche to decipher what biological and/or synthetic materials can be incorporated into these carriers. Advances in biomedical engineering including electrospinning and 3D bioprinting with surface functionalization and micropatterning, and self-assembly models, have generated a wealth of biocompatible, biodegradable, integrating scaffolds to choose from, some of which are being tested for their SC delivery capacity in the hope of improving clinical outcomes for patients with LSCD.

The Neuropeptide α-Melanocyte-Stimulating Hormone Prevents Persistent Corneal Edema following Injury.

Corneal endothelial cells (CEnCs) regulate corneal hydration and maintain tissue transparency through their barrier and pump function. However, these cells exhibit limited regenerative capacity following injury. Currently, corneal transplantation is the only established therapy for restoring endothelial function, and there are no pharmacologic interventions available for restoring endothelial function. This study investigated the efficacy of the neuropeptide α-melanocyte-stimulating hormone (α-MSH) in promoting endothelial regeneration during the critical window between ocular injury and the onset of endothelial decompensation using an established murine model of injury using transcorneal freezing. Local administration of α-MSH following injury prevented corneal edema and opacity, reduced leukocyte infiltration, and limited CEnC apoptosis while promoting their proliferation. These results suggest that α-MSH has a proregenerative and cytoprotective function on CEnCs and shows promise as a therapy for the prevention and management of corneal endothelial dysfunction.

Investigating the Role of TGF-ß Signaling Pathways in Human Corneal Endothelial Cell Primary Culture.

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial-mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-ßs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-ß signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-ß1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-ß-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-ß1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-ß receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-ß1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

Anti-inflammatory and wound healing potential of medicinal maggot excretions/secretions at the ocular surface.

PURPOSE: In the skin, Lucilia sericata maggot excretions/secretions (ES) accelerate wound healing and limit inflammation. This study aimed to determine whether ES have similar beneficial effects at the ocular surface. METHODS: Human corneal epithelial cells (HCEC) were cultured with ES and cell viability was determined by the MTT assay. Additionally, mRNA expression of growth factors, antimicrobial peptides (AMPs) and cytokines was assessed by qPCR. ES ability to modulate TLR-induced IL-6 and IL-8 expression was determined by qPCR and ELISA. ES potential to promote corneal healing was evaluated in vitro by a migration assay in HCEC, and in vivo using a mouse model. RESULTS: ES did not impair HCEC viability up to 25 µg/ml. Among the factors evaluated, only hBD-2 was upregulated (2.5-fold) by 1.5 µg/ml ES after 6 hrs (P = 0.04). In HCEC, ES reduced Poly I:C-induced IL-6 and IL-8 mRNA (P ≤ 0.001) and protein (P ≤ 0.0001) expression. A similar effect was observed with Flagellin (TLR5 agonist) but it was less robust for FSL-1 (TLR2/6 agonist) and Pam3CSK4 (TLR1/2 agonist). The greatest in vitro migration effect was observed with 6.2 µg/ml ES after 44 hrs where gap area compared to vehicle was 53.3 ± 3.7% vs. 72.6 ± 5.4% (P = 0.001). In the mouse model, the maximum healing effect was present with 1.5 µg/ml ES after 12 hrs with a wound area of 19.0 ± 2.7% vs. 60.1 ± 21.6% (P = 0.003) or 77% reduction of the wound area compared to the negative control. CONCLUSIONS: ES significantly reduce in vitro TLR-induced production of inflammatory cytokines and promote corneal wound healing.

In vitro evaluation of platelet extracellular vesicles (PEVs) for corneal endothelial regeneration.

Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × g ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 1011 /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.

The NLRP1 Inflammasome Induces Pyroptosis in Human Corneal Epithelial Cells.

PURPOSE: Inflammasomes are multiprotein complexes that detect danger-associated signals and trigger an immunostimulatory form of cell death called pyroptosis. NLRP1 is an innate immune receptor that assembles into an inflammasome, but the primary cell types in which NLRP1 is functional have not yet been fully established. Mutations in NLRP1 are associated with diseases of barrier epithelial tissues, including skin lesions and corneal intraepithelial dyskeratosis, suggesting that NLRP1 functions within the eye. Here, we investigated the expression and activity of the NLRP1 inflammasome in primary human corneal epithelial (pHCE) cells. METHODS: The small molecule Val-boroPro (VbP) activates the NLRP1 inflammasome. Proteasome (bortezomib, MG132) and caspase-1 (VX-765, Z-VAD-FMK) inhibitors block NLRP1 activation and downstream pyroptosis, respectively. Here, we treated pHCE cells with VbP alone or in combination proteasome inhibitors and caspase-1 inhibitors. We assessed NLRP1 expression and hallmarks of pyroptosis, including lytic cell rupture, cytokine processing and release, and gasdermin D (GSDMD) processing. RESULTS: VbP triggered pyroptosis in pHCE cells, as determined by cytokine secretion, GSDMD processing, and lactate dehydrogenase (LDH) release. Proteasome and caspase-1 inhibitors completely blocked this pyroptotic cell death. In contrast, other primary ocular epithelial cells did not undergo NLRP1-dependent pyroptosis. CONCLUSIONS: Our findings demonstrate that NLRP1 forms a functional inflammasome in pHCE cells. Importantly, these data reveal that NLRP1 is a key innate immune sensor of the corneal epithelium, and moreover indicate how aberrant inflammasome activation causes corneal damage. Blockade of NLRP1 signaling may benefit patients with hyperactive NLRP1 mutations and warrants further investigation.

Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia.

BACKGROUND: Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. METHODS: Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. RESULTS: Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. CONCLUSIONS: Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

Voriconazole-Cyclodextrin Supramolecular Ternary Complex-Loaded Ocular Films for Management of Fungal Keratitis.

Fungal keratitis is one of the leading causes of ophthalmic mycosis affecting the vision due to corneal scarring. Voriconazole (VRC) is the most preferred azole antifungal agent for treating ocular mycotic infections. Ocular drug delivery is challenging due to the shorter corneal residence time of the formulation requiring frequent administration, leading to poor patient compliance. The present study aimed at improving the solubility, transcorneal permeation, and efficacy of voriconazole via the formation of cyclodextrin-based ternary complexes and incorporation of the complex into mucoadhesive films. A phase solubility study suggested a ∼14-fold improvement in VRC solubility, whereas physicochemical characterization confirmed the inclusion of VRC in the cyclodextrin inner cavity. In silico docking studies were performed to predict the docking conformation and stability of the inclusion complex. Complex-loaded films showed sustained release of voriconazole from the films and improved transcorneal permeation by ∼4-fold with an improved flux of 8.36 µg/(cm2 h) for ternary complex-loaded films compared to 1.86 µg/(cm2 h) for the pure VRC film. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and hen's egg-chorioallantoic membrane test (HET-CAM) assays confirmed that the complexes and ocular films were nonirritant and safe for ocular administration. The antifungal study performed using Aspergillus fumigatus and Fusarium oxysporum suggested improved antifungal activity compared to the pure drug film. In conclusion, the supramolecular cyclodextrin ternary complex proved to be a promising strategy for enhancing the solubility and permeability and augmenting the antifungal activity of voriconazole in the management of fungal keratitis.

An ex vivo model of human corneal rim perfusion organ culture.

The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 µl/min, the outflow facility was 0.359 ± 0.216 µl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.

Corneal Densitometry Assessed With Scheimpflug Camera in Healthy Corneas and Correlation With Specular Microscopy Values and Age.

PURPOSE: To investigate correlations between specular microscopy endothelial parameters and age with corneal densitometry values, as they are presented from a Scheimpflug device, in different levels of the cornea. METHODS: Two hundred eighty-four eyes of 142 healthy subjects were included in this observational, prospective study. Corneal densitometry was evaluated with Scheimpflug imaging system in the central 0- to 2-mm annular zone of the cornea, whereas the endothelial cell properties were assessed with the use of a noncontact specular microscope. RESULTS: Corneal densitometry values of all corneal layers were statistically significant and positively correlated with age. In univariate linear regression analysis among corneal densitometry values and the endothelial parameters, only endothelial cell density (CD) was statistically significant and inversely correlated with densitometry values in all corneal layers. In stepwise multivariate linear regression analysis, after adjustment for age, hexagonality was statistically significant and inversely correlated with posterior densitometry values, whereas coefficient of variation was positively and significantly correlated with the anterior densitometry values. When repeating stepwise multivariate linear regression analysis without adjusting for age, CD was negatively and significantly correlated with corneal densitometry values of all layers, whereas coefficient of variation was positively and significantly correlated with anterior and total corneal densitometry values. CONCLUSIONS: Corneal densitometry increases with age. It is also inversely correlated with CD, and this might be used as an indirect way to assess the status of the corneal endothelium.

Human Adenovirus Species D Interactions with Corneal Stromal Cells.

Notable among the many communicable agents known to infect the human cornea is the human adenovirus, with less than ten adenoviruses having corneal tropism out of more than 100 known types. The syndrome of epidemic keratoconjunctivitis (EKC), caused principally by human adenovirus, presents acutely with epithelial keratitis, and later with stromal keratitis that can be chronic and recurrent. In this review, we discuss the current state of knowledge regarding the molecular biology of adenovirus infection of corneal stromal cells, among which the fibroblast-like keratocyte is the most predominant, in order to elucidate basic pathophysiologic mechanisms of stromal keratitis in the human patient with EKC.

Membranes Prepared from Recombinant RGD-Silk Fibroin as Substrates for Human Corneal Cells.

A recombinant formulation of silk fibroin containing the arginine-glycine-aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal-epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.

[The in vitro effect of antiseptics on epithelial cells of human cornea and conjunctiva]. / Vozdeistvie antiseptikov na epitelial'nye kletki rogovitsy i kon"yunktivy cheloveka in vitro.

Effective and safe antiseptic eye preparations are necessary for prevention and treatment of infectious and inflammatory eye diseases. PURPOSE: in vitro evaluation of the effect of antiseptic eye drops on corneal and conjunctival epithelial cells. MATERIAL AND METHODS: Antiseptic eye drops «Bactavit¼, «Vitabact¼ and «Ocomistin¼ were the object of the study. Immortalized human corneal epithelial cell lines (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) were used as the test systems. The viability of the cells was assessed by their metabolic activity and morphology using the MTT test and phase-contrast microscopy. RESULTS: Antiseptic eye drops belonging to different groups of chemical compounds induced cytotoxic effects on the cells of corneal epithelium (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) of varying degrees, leading to morphological and functional changes in those cells. CONCLUSION: The study confirms the possibility of using cultured cells for the in vitro comparative assessment of the cytotoxic effect of antiseptic ophthalmic agents.

Olive Pomace Phenolic Compounds Stability and Safety Evaluation: From Raw Material to Future Ophthalmic Applications.

Nowadays, increasing interest in olive pomace (OP) valorization aims to improve olive's industry sustainability. Interestingly, several studies propose a high-value application for OP extracts containing its main phenolic compounds, hydroxytyrosol and oleuropein, as therapy for ocular surface diseases. In this work, the stability and accessibility of OP total phenolic and flavonoid content, main representative compounds, and antioxidant activity were assessed under different pretreatment conditions. Among them, lyophilization and supercritical CO2 extraction were found to increase significantly most responses measured in the produced extracts. Two selected extracts (CONV and OPT3) were obtained by different techniques (conventional and pressurized liquid extraction); Their aqueous solutions were characterized by HPLC-DAD-MS/MS. Additionally, their safety and stability were evaluated according to EMA requirements towards their approval as ophthalmic products: their genotoxic effect on ocular surface cells and their 6-months storage stability at 4 different temperature/moisture conditions (CPMP/ICH/2736/99), together with pure hydroxytyrosol and oleuropein solutions. The concentration of hydroxytyrosol and oleuropein in pure or extract solutions was tracked, and possible degradation products were putatively identified by HPLC-DAD-MS/MS. Hydroxytyrosol and oleuropein had different stability as standard or extract solutions, with oleuropein also showing different degradation profile. All compounds/extracts were safe for ophthalmic use at the concentrations tested.