Ocular Cell Types Research Articles

Single-Cell Multiomics Profiling Reveals Heterogeneity of Müller Cells in the Oxygen-Induced Retinopathy Model.

Retinal neovascularization poses heightened risks of vision loss and blindness. Despite its clinical significance, the molecular mechanisms underlying the pathogenesis of retinal neovascularization remain elusive. This study utilized single-cell multiomics profiling in an oxygen-induced retinopathy (OIR) model to comprehensively investigate the intricate molecular landscape of retinal neovascularization. Mice were exposed to hyperoxia to induce the OIR model, and retinas were isolated for nucleus isolation. The cellular landscape of the single-nucleus suspensions was extensively characterized through single-cell multiomics sequencing. Single-cell data were integrated with genome-wide association study (GWAS) data to identify correlations between ocular cell types and diabetic retinopathy. Cell communication analysis among cells was conducted to unravel crucial ligand-receptor signals. Trajectory analysis and dynamic characterization of Müller cells were performed, followed by integration with human retinal data for pathway analysis. The multiomics dataset revealed six major ocular cell classes, with Müller cells/astrocytes showing significant associations with proliferative diabetic retinopathy (PDR). Cell communication analysis highlighted pathways that are associated with vascular proliferation and neurodevelopment, such as Vegfa-Vegfr2, Igf1-Igf1r, Nrxn3-Nlgn1, and Efna5-Epha4. Trajectory analysis identified a subset of Müller cells expressing genes linked to photoreceptor degeneration. Multiomics data integration further unveiled positively regulated genes in OIR Müller cells/astrocytes associated with axon development and neurotransmitter transmission. This study significantly advances our understanding of the intricate cellular and molecular mechanisms underlying retinal neovascularization, emphasizing the pivotal role of Müller cells. The identified pathways provide valuable insights into potential therapeutic targets for PDR, offering promising directions for further research and clinical interventions.

Pax6 expressing neuroectodermal and ocular stem cells: Its role from a developmental biology perspective.

Pax-6 emerges as a critical transcription factor that guides the fate of stem cells towards neural lineages. Its expression influences the differentiation of neural progenitors into diverse neuronal subtypes, glial cells, and other neural cell types. Pax-6 operates with other regulatory factors to ensure the precise patterning and organization of the developing nervous system. The intricate interplay between Pax-6 and other signaling pathways, transcription factors, and epigenetic modifiers underpins the complicated balance between stem cell maintenance, proliferation, and differentiation in neuroectodermal and ocular contexts. Dysfunction of Pax-6 can lead to a spectrum of developmental anomalies, underscoring its importance in these processes. This review highlights the essential role of Pax-6 expression in neuroectodermal and ocular stem cells, shedding light on its significance in orchestrating the intricate journey from stem cell fate determination to the emergence of diverse neural and ocular cell types. The comprehensive understanding of Pax-6 function gained from a developmental biology perspective offers valuable insights into normal development and potential therapeutic avenues for neuroectodermal and ocular disorders.

Targeting adenosine A2A receptors for early intervention of retinopathy of prematurity.

Retinopathy of prematurity (ROP) continues to pose a significant threat to the vision of numerous children worldwide, primarily owing to the increased survival rates of premature infants. The pathologies of ROP are mainly linked to impaired vascularization as a result of hyperoxia, leading to subsequent neovascularization. Existing treatments, including anti-vascular endothelial growth factor(VEGF) therapies, have thus far been limited to addressing pathological angiogenesis at advanced ROP stages, inevitably leading to adverse side effects. Intervention to promote physiological angiogenesis during the initial stages could hold the potential to prevent ROP. Adenosine A2A receptors (A2AR) have been identified in various ocular cell types, exhibiting distinct densities and functionally intricate connections with oxygen metabolism. In this review, we discuss experimental evidence that strongly underscores the pivotal role of A2AR in ROP. In particular, A2AR blockade may represent an effective treatment strategy, mitigating retinal vascular loss by reversing hyperoxia-mediated cellular proliferation inhibition and curtailing hypoxia-mediated neovascularization in oxygen-induced retinopathy (OIR). These effects stem from the interplay of endothelium, neuronal and glial cells, and novel molecular pathways (notably promoting TGF-β signaling) at the hyperoxia phase. We propose that pharmacological targeting of A2AR signaling may confer an early intervention for ROP with distinct therapeutic benefits and mechanisms than the anti-VEGF therapy.

Liquid Biopsy Proteomics in Ophthalmology.

Minimally invasive liquid biopsies from the eye capture locally enriched fluids that contain thousands of proteins from highly specialized ocular cell types, presenting a promising alternative to solid tissue biopsies. The advantages of liquid biopsies include sampling the eye without causing irreversible functional damage, potentially better reflecting tissue heterogeneity, collecting samples in an outpatient setting, monitoring therapeutic response with sequential sampling, and even allowing examination of disease mechanisms at the cell level in living humans, an approach that we refer to as TEMPO (Tracing Expression of Multiple Protein Origins). Liquid biopsy proteomics has the potential to transform molecular diagnostics and prognostics and to assess disease mechanisms and personalized therapeutic strategies in individual patients. This review addresses opportunities, challenges, and future directions of high-resolution liquid biopsy proteomics in ophthalmology, with particular emphasis on the large-scale collection of high-quality samples, cutting edge proteomics technology, and artificial intelligence-supported data analysis.

Deletion of IFT20 exclusively in the RPE ablates primary cilia and leads to retinal degeneration.

Vision impairment places a serious burden on the aging society, affecting the lives of millions of people. Many retinal diseases are of genetic origin, of which over 50% are due to mutations in cilia-associated genes. Most research on retinal degeneration has focused on the ciliated photoreceptor cells of the retina. However, the contribution of primary cilia in other ocular cell types has largely been ignored. The retinal pigment epithelium (RPE) is a monolayer epithelium at the back of the eye intricately associated with photoreceptors and essential for visual function. It is already known that primary cilia in the RPE are critical for its development and maturation; however, it remains unclear whether this affects RPE function and retinal tissue homeostasis. We generated a conditional knockout mouse model, in which IFT20 is exclusively deleted in the RPE, ablating primary cilia. This leads to defective RPE function, followed by photoreceptor degeneration and, ultimately, vision impairment. Transcriptomic analysis offers insights into mechanisms underlying pathogenic changes, which include transcripts related to epithelial homeostasis, the visual cycle, and phagocytosis. Due to the loss of cilia exclusively in the RPE, this mouse model enables us to tease out the functional role of RPE cilia and their contribution to retinal degeneration, providing a powerful tool for basic and translational research in syndromic and non-syndromic retinal degeneration. Non-ciliary mechanisms of IFT20 in the RPE may also contribute to pathogenesis and cannot be excluded, especially considering the increasing evidence of non-ciliary functions of ciliary proteins.

Adeno-associated virus vectors for retinal gene therapy in basic research and clinical studies.

Retinal degenerative diseases, including glaucoma, age-related macular degeneration, diabetic retinopathy, and a broad range of inherited retinal diseases, are leading causes of irreversible vision loss and blindness. Gene therapy is a promising and fast-growing strategy to treat both monogenic and multifactorial retinal disorders. Vectors for gene delivery are crucial for efficient and specific transfer of therapeutic gene(s) into target cells. AAV vectors are ideal for retinal gene therapy due to their inherent advantages in safety, gene expression stability, and amenability for directional engineering. The eye is a highly compartmentalized organ composed of multiple disease-related cell types. To determine a suitable AAV vector for a specific cell type, the route of administration and choice of AAV variant must be considered together. Here, we provide a brief overview of AAV vectors for gene transfer into important ocular cell types, including retinal pigment epithelium cells, photoreceptors, retinal ganglion cells, Müller glial cells, ciliary epithelial cells, trabecular meshwork cells, vascular endothelial cells, and pericytes, via distinct injection methods. By listing suitable AAV vectors in basic research and (pre)clinical studies, we aim to highlight the progress and unmet needs of AAV vectors in retinal gene therapy.

Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

PLAE Web App Enables Powerful Searching and Multiple Visualizations Across One Million Unified Single-Cell Ocular Transcriptomes.

To create a high-performance reactive web application to query single-cell gene expression data across cell type, species, study, and other factors. We updated the content and structure of the underlying data (single cell Eye in a Disk [scEiaD]) and wrote the web application PLAE (https://plae.nei.nih.gov) to visualize and explore the data. The new portal provides quick visualization of over a million individual cells from vertebrate eye and body transcriptomes encompassing four species, 60 cell types, six ocular tissues, and 23 body tissues across 35 publications. To demonstrate the value of this unified pan-eye dataset, we replicated known neurogenic and cone macula markers in addition to proposing six new cone human region markers. The PLAE web application offers the eye community a powerful and quick means to test hypotheses related to gene expression across a highly diverse, community-derived database. The PLAE resource enables any researcher or clinician to study and research gene expression patterning across a wide variety of curated ocular cell types with a responsive web app.

Influenza A Virus Multicycle Replication Yields Comparable Viral Population Emergence in Human Respiratory and Ocular Cell Types.

While primarily considered a respiratory pathogen, influenza A virus (IAV) is nonetheless capable of spreading to, and replicating in, numerous extrapulmonary tissues in humans. However, within-host assessments of genetic diversity during multicycle replication have been largely limited to respiratory tract tissues and specimens. As selective pressures can vary greatly between anatomical sites, there is a need to examine how measures of viral diversity may vary between influenza viruses exhibiting different tropisms in humans, as well as following influenza virus infection of cells derived from different organ systems. Here, we employed human primary tissue constructs emulative of the human airway or corneal surface, and we infected both with a panel of human- and avian-origin IAV, inclusive of H1 and H3 subtype human viruses and highly pathogenic H5 and H7 subtype viruses, which are associated with both respiratory disease and conjunctivitis following human infection. While both cell types supported productive replication of all viruses, airway-derived tissue constructs elicited greater induction of genes associated with antiviral responses than did corneal-derived constructs. We used next-generation sequencing to examine viral mutations and population diversity, utilizing several metrics. With few exceptions, generally comparable measures of viral diversity and mutational frequency were detected following homologous virus infection of both respiratory-origin and ocular-origin tissue constructs. Expansion of within-host assessments of genetic diversity to include IAV with atypical clinical presentations in humans or in extrapulmonary cell types can provide greater insight into understanding those features most prone to modulation in the context of viral tropism. IMPORTANCE Influenza A virus (IAV) can infect tissues both within and beyond the respiratory tract, leading to extrapulmonary complications, such as conjunctivitis or gastrointestinal disease. Selective pressures governing virus replication and induction of host responses can vary based on the anatomical site of infection, yet studies examining within-host assessments of genetic diversity are typically only conducted in cells derived from the respiratory tract. We examined the contribution of influenza virus tropism on these properties two different ways: by using IAV associated with different tropisms in humans, and by infecting human cell types from two different organ systems susceptible to IAV infection. Despite the diversity of cell types and viruses employed, we observed generally similar measures of viral diversity postinfection across all conditions tested; these findings nonetheless contribute to a greater understanding of the role tissue type contributes to the dynamics of virus evolution within a human host.

Oxidative Stress and Antioxidants in Age-Related Macular Degeneration.

Oxidative stress plays a crucial role in aging-related eye diseases, including age-related macular degeneration (AMD), cataracts, and glaucoma. With age, antioxidant reparative capacity decreases, and excess levels of reactive oxygen species produce oxidative damage in many ocular cell types underling age-related pathologies. In AMD, loss of central vision in the elderly is caused primarily by retinal pigment epithelium (RPE) dysfunction and degeneration and/or choroidal neovascularization that trigger malfunction and loss of photo-sensing photoreceptor cells. Along with various genetic and environmental factors that contribute to AMD, aging and age-related oxidative damage have critical involvement in AMD pathogenesis. To this end, dietary intake of antioxidants is a proven way to scavenge free radicals and to prevent or slow AMD progression. This review focuses on AMD and highlights the pathogenic role of oxidative stress in AMD from both clinical and experimental studies. The beneficial roles of antioxidants and dietary micronutrients in AMD are also summarized.

Characterization of Porcine Ocular Surface Epithelial Microenvironment.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (β-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Biphasic expression of thyroid hormone receptor TRβ1 in mammalian retina and anterior ocular tissues.

The retina is increasingly recognized as a target of thyroid hormone. We previously reported critical functions for thyroid hormone receptor TRβ2, encoded by Thrb, in cones, the photoreceptors that mediate color vision. TRβ1, another Thrb receptor isoform, is widely expressed in other tissues but little studied in the retina. Here, we investigate these N-terminal isoforms by RNA-sequencing analysis and reveal a striking biphasic profile for TRβ1 in mouse and human retina. In contrast to the early TRβ2 peak, TRβ1 peaks later during retinal maturation or later differentiation of human retinal organoids. This switch in receptor expression profiles was confirmed using lacZ reporter mice. TRβ1 localized in cones, amacrine cells and ganglion cells in contrast to the restricted expression of TRβ2 in cones. Intriguingly, TRβ1 was also detected in the retinal pigmented epithelium and in anterior structures in the ciliary margin zone, ciliary body and iris, suggesting novel functions in non-retinal eye tissues. Although TRβ1 was detected in cones, TRβ1-knockout mice displayed only minor changes in opsin photopigment expression and normal electroretinogram responses. Our results suggest that strikingly different temporal and cell-specific controls over TRβ1 and TRβ2 expression may underlie thyroid hormone actions in a range of ocular cell types. The TRβ1 expression pattern suggests novel functions in retinal and non-neural ocular tissues.

Cytochrome P450 1B1: A Key Regulator of Ocular Iron Homeostasis and Oxidative Stress.

Cytochrome P450 (CYP) 1B1 belongs to the superfamily of heme-containing monooxygenases. Unlike other CYP enzymes, which are highly expressed in the liver, CYP1B1 is predominantly found in extrahepatic tissues, such as the brain, and ocular tissues including retina and trabecular meshwork. CYP1B1 metabolizes exogenous chemicals such as polycyclic aromatic hydrocarbons. CYP1B1 also metabolizes endogenous bioactive compounds including estradiol and arachidonic acid. These metabolites impact various cellular and physiological processes during development and pathological processes. We previously showed that CYP1B1 deficiency mitigates ischemia-mediated retinal neovascularization and drives the trabecular meshwork dysgenesis through increased levels of oxidative stress. However, the underlying mechanisms responsible for CYP1B1-deficiency-mediated increased oxidative stress remain largely unresolved. Iron is an essential element and utilized as a cofactor in a variety of enzymes. However, excess iron promotes the production of hydroxyl radicals, lipid peroxidation, increased oxidative stress, and cell damage. The retinal endothelium is recognized as a major component of the blood–retinal barrier, which controls ocular iron levels through the modulation of proteins involved in iron regulation present in retinal endothelial cells, as well as other ocular cell types including trabecular meshwork cells. We previously showed increased levels of reactive oxygen species and lipid peroxidation in the absence of CYP1B1, and in the retinal vasculature and trabecular meshwork, which was reversed by administration of antioxidant N-acetylcysteine. Here, we review the important role CYP1B1 expression and activity play in maintaining retinal redox homeostasis through the modulation of iron levels by retinal endothelial cells. The relationship between CYP1B1 expression and activity and iron levels has not been previously delineated. We review the potential significance of CYP1B1 expression, estrogen metabolism, and hepcidin–ferroportin regulatory axis in the local regulation of ocular iron levels.

Multi-species single-cell transcriptomic analysis of ocular compartment regulons

The retina is a widely profiled tissue in multiple species by single-cell RNA sequencing studies. However, integrative research of the retina across species is lacking. Here, we construct the first single-cell atlas of the human and porcine ocular compartments and study inter-species differences in the retina. In addition to that, we identify putative adult stem cells present in the iris tissue. We also create a disease map of genes involved in eye disorders across compartments of the eye. Furthermore, we probe the regulons of different cell populations, which include transcription factors and receptor-ligand interactions and reveal unique directional signalling between ocular cell types. In addition, we study conservation of regulons across vertebrates and zebrafish to identify common core factors. Here, we show perturbation of KLF7 gene expression during retinal ganglion cells differentiation and conclude that it plays a significant role in the maturation of retinal ganglion cells.

Vaccinia Virus Protein B18R: Influence on mRNA Immunogenicity and Translation upon Non-Viral Delivery in Different Ocular Cell Types.

In the last few years, interest has grown in the use of nucleic acids as an ocular therapy for retinal genetic diseases. Recently, our research group has demonstrated that mRNA delivery could result in effective protein expression in ocular cells following subretinal injection. Yet, although mRNA therapy comes with many advantages, its immunogenicity resulting in hampered mRNA translation delays development to the clinic. Therefore, several research groups investigate possible strategies to reduce this innate immunity. In this study, we focus on B18R, an immune inhibitor to suppress the mRNA-induced innate immune responses in two ocular cell types. We made use of retinal pigment epithelial (RPE) cells and Müller cells both as immortalized cell lines and primary bovine cells. When cells were co-incubated with both B18R and mRNA-MessengerMAX lipoplexes we observed an increase in transfection efficiency accompanied by a decrease in interferon-β production, except for the Müller cells. Moreover, uptake efficiency and cell viability were not hampered. Taken together, we showed that the effect of B18R is cell type-dependent but remains a possible strategy to improve mRNA translation in RPE cells.

Angiotensin Converting Enzyme 2 (ACE2) Expression in the Aged Brain and Visual System

Multiple lines of evidence currently indicate that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gains entry into human host cells via a high-affinity interaction with the angiotensin-converting enzyme 2 (ACE2) transmembrane receptor. Research has further shown the widespread expression of the ACE2 receptor on the surface of many different immune, non-immune and neural host cell types, and that SARS-CoV-2 has the remarkable capability to attack many different types of human-host cells simultaneously. One principal neuroanatomical region for high ACE2 expression patterns occurs in the brainstem, an area of the brain containing regulatory centers for respiration, and this may in part explain the predisposition of many COVID-19 patients to respiratory distress. Early studies also indicated extensive ACE2 expression in the whole eye and the brain’s visual circuitry in aged humans. In this study we analyzed ACE2 receptor expression at the mRNA and protein level in multiple cell types involved in human vision, including cell types of the external eye and several deep brain regions known to be involved in the processing of visual signals. Here we provide evidence: (i) that many different optical and neural cell types of the human visual system provide receptors essential for SARS-CoV-2 invasion; (ii) of the remarkable ubiquity of ACE2 presence in cells of the eye and anatomical regions of the brain involved in visual signal processing; (iii) that ACE2 receptor expression in different ocular cell types and visual processing centers of the brain provide multiple compartments for SARS-CoV-2 infiltration; and (iv) of a gradient of increasing ACE2 expression from the anterior surface of the eye to the visual signal processing areas of the occipital lobe and the primary visual neocortex. A gradient of ACE2 expression from the eye surface to the occipital lobe may provide the SARS-CoV-2 virus a novel pathway from the outer eye into deeper anatomical regions of the brain involved in vision. These findings may explain, in part, the many recently reported neuro-ophthalmic manifestations of SARS-CoV-2 infection in COVID-19 affected patients.

Development and optimization of a personalized fibrin membrane derived from the plasma rich in growth factors technology

PurposeTo develop and characterize a new type of plasma rich in growth factors (PRGF) membrane for patients in which immune system is involved in the disease etiology. MethodsBlood from three healthy donors was collected to obtain the different fibrin membranes by PRGF technology. PRGF obtained volumes were activated and divided into two groups: PRGF membrane (mPRGF) obtained after incubation at 37 °C for 30 min (control); and is-mPRGF: mPRGF obtained after incubation for 30 min at 56 °C. The concentration of several growth factors, proteins, immunoglobulin E and the complement activity was determined in the different mPRGF. The proliferative potential of heat-inactivated mPRGF were assayed on keratocytes (HK) and conjunctival fibroblasts (HConF). In addition, morphological and physical features of the inactivated mPRGF were evaluated in contrast to the control mPRGF. ResultsHeat-inactivation of the mPRGF preserves the content of most of the growth factors involved in the ocular wound healing while reducing drastically the content of IgE and the complement activity. The heat-inactivated mPRGF conserve the morphological and physical characteristics of the fibrin meshwork in comparison with the control mPRGF. Furthermore, no significant differences were found in the biological activity of the control mPRGF regarding the heat-inactivated mPRGF (is-mPRGF) in any of both ocular cell types evaluated. ConclusionsThe heat-inactivation of the PRGF membranes (is-mPRGF) reduces drastically the content of IgE and complement activity while preserving the content of most of the proteins and morphogens involved in ocular wound healing. Furthermore, the morphological and physical features of the immunosafe mPRGF were also preserved after heat-inactivation.

Biomimetic bone-like composites as osteo-odonto-keratoprosthesis skirt substitutes.

Osteo-odonto-keratoprostheses, incorporating dental laminate material as an anchoring skirt around a central poly(methyl methacrylate) (PMMA) optic, have been used to replace the cornea for many years. However, there are many intricacies associated with the use of autologous dental laminate material, surgical complexity and skirt erosion. Tissue engineering approaches to bone replacement may offer suitable alternatives in osteo-odonto-keratoprosthesis (OOKP) surgery. In this study, a hydrogel polymer composite was investigated as a synthetic substitute for the OOKP skirt. A novel high strength interpenetrating network (IPN) hydrogel composite with nano-crystalline hydroxyapatite (nHAp) coated poly (lactic-co-glycolic acid) PLGA microspheres was created to mimic the alveo-dental lamina by employing agarose and poly(ethylene glycol) diacrylate (PEGDA) polymers. The incorporation of nHAp coated PLGA microspheres into the hybrid IPN network provide a micro-environment similar to that of skeletal tissues and improve cellular response. Agarose was used as a first network to encapsulate keratocytes/3T3 fibroblasts and PEGDA (6000 Da) was used as a second network with varying concentrations (20 and 40 wt %) to produce a strong and biocompatible scaffold. An increased concentration of either agarose or PEG-DA and incorporation of nHAp coated PLGA microspheres led to an increase in the elastic modulus. The IPN hydrogel combinations supported the adhesion and proliferation of both fibroblast and ocular human keratocyte cell types during in in-vitro testing. The cells endured the encapsulation process into the IPN and remained viable at 1 week post-encapsulation in the presence of nHAp coated microspheres. The material did not induce significant production of inflammatory cytokine IL-6 in comparison to a positive control (p < 0.05) indicating non-inflammatory potential. The nHAp encapsulated composite IPN hydrogels are mechanically strong, cell supportive, non-inflammatory materials supporting their development as OOKP skirt substitutes using a new approach to dental laminate biomimicry in the OOKP skirt material.

Development of In Vitro Corneal Models: Opportunity for Pharmacological Testing.

The human eye is a specialized organ with a complex anatomy and physiology, because it is characterized by different cell types with specific physiological functions. Given the complexity of the eye, ocular tissues are finely organized and orchestrated. In the last few years, many in vitro models have been developed in order to meet the 3Rs principle (Replacement, Reduction and Refinement) for eye toxicity testing. This procedure is highly necessary to ensure that the risks associated with ophthalmic products meet appropriate safety criteria. In vitro preclinical testing is now a well-established practice of significant importance for evaluating the efficacy and safety of cosmetic, pharmaceutical, and nutraceutical products. Along with in vitro testing, also computational procedures, herein described, for evaluating the pharmacological profile of potential ocular drug candidates including their toxicity, are in rapid expansion. In this review, the ocular cell types and functionality are described, providing an overview about the scientific challenge for the development of three-dimensional (3D) in vitro models.

Reduced graphene oxide membranes in ocular regenerative medicine.

Biological membranes are currently used in Ophthalmology in order to treat different ocular disorders. These membranes have different properties such as cellular biocompatibility and promoting wound healing. Moreover, intrinsic antimicrobial properties could also be desirable because it would allow their use reducing the risk of infections. Graphene and its derivatives are promising biomaterials that already proved their bactericidal effect. However, their clinical use is limited due to the controversial results regarding their toxicity. In this work, we have developed and characterized a reduced graphene oxide membrane (rGOM) for its use in ocular Regenerative Medicine, and studied its in vitro and in vivo biocompatibility and genotoxicity with different types of human ocular cells. We proved that rGOM allowed the growth of different ocular cells without inducing in vitro or in vivo cytotoxicity or genotoxicity in the short-term. These results indicate that rGOM may be a promising candidate in Regenerative Medicine for the treatment of different ocular pathologies.