Purpose

The purpose of this study was to determine the influence of mucin expression in an immortalized human corneal epithelial cell line (hTCEpi) on the surface properties of cells, such as wettability, contact angle, and surface heterogeneity.

Methods

hTCEpi cells were cultured to confluence in serum-free medium. The medium was then replaced by stratification medium to induce mucin biosynthesis. The mucin expression profile was analyzed using quantitative PCR and Western blotting. Contact angles were measured using a two-immiscible liquid method, and contact angle hysteresis was evaluated by tilting the apparatus and recording advancing and receding contact angles. The spatial distribution of mucins was evaluated with fluorescently labeled lectin.

Results

hTCEpi cells expressed the three main ocular mucins (MUC1, MUC4, and MUC16) with a maximum between days 1 and 3 of the stratification process. Upon stratification, cells caused a very significant increase in contact angle hysteresis, suggesting the development of spatially discrete and heterogeneously distributed surface features, defined by topography and/or chemical functionality. Although atomic force microscopy measurements showed no formation of appreciable topographic features on the surface of the cells, we observed a significant increase in surface chemical heterogeneity.

Conclusions

The surface chemical heterogeneity of the corneal epithelium may influence the dynamic behavior of tear film by "pinning" the contact line between the cellular surface and aqueous tear film. Engineering the surface properties of corneal epithelium could potentially lead to novel treatments in dry eye disease.

Mucins are large glycoproteins expressed by epithelial cells of both the conjunctiva and cornea, and principle components of the glycocalyx. They are thought to play an important role in determining the interactions between the cornea/conjunctiva and the overlying tear film. The purpose of this study was to characterize the membrane-associated corneal mucin expression pattern from multiple species commonly used in ophthalmic research and drug development to better define the biochemical attributes of the ocular surface. Humans, rhesus macaques and dogs were found to have a very similar pattern of mucin expression, with mucin 16 (MUC16) being the most prevalent mucin transcript. In contrast, the rabbit had a unique mucin expression pattern with all mucin transcripts expressed at relatively similar levels. To determine if there were spatial differences in expression, peripheral and central corneal epithelium were individually isolated and evaluated for mucin expression. In all species examined, MUC1, MUC4 and MUC16 had higher peripheral corneal expression when compared with central, which reached statistical significance in MUC1 (rhesus and dog). The data demonstrated variation in corneal epithelial membrane-associated mucin expression between species, with the rabbit having a distinct expression pattern. These differences may be reflective of the environment, pathogen exposure or tear film dynamics of the respective species. The species differences, as well as regional mucin expression patterns, characterized in this study further define the biochemical composition of the ocular surface and may play an important role in tear film stability.

Currently available artificial corneas can develop post-implant complications including epithelial downgrowth, infection, and stromal melting. The likelihood of developing these disastrous complications could be minimized through improved formation and maintenance of a healthy epithelium covering the implant. We hypothesize that this epithelial formation may be enhanced through the incorporation of native corneal basement membrane biomimetic chemical and physical cues onto the surface of the keratoprosthesis. We fabricated hydrogel substrates molded with topographic features containing specific bio-ligands and developed an in vitro wound healing assay. In our experiments, the rate of corneal epithelial wound healing was significantly increased by 50% in hydrogel surfaces containing topographic features, compared to flat surfaces with the same chemical attributes. We determined that this increased healing is not due to enhanced proliferation or increased spreading of the epithelial cells, but to an increased active migration of the epithelial cells. These results show the potential benefit of restructuring and improving the surface of artificial corneas to enhance epithelial coverage and more rapidly restore the formation of a functional epithelium.

Purpose

The purpose of this study was to determine the influence of mucin expression in an immortalized human corneal epithelial cell line (hTCEpi) on the surface properties of cells, such as wettability, contact angle, and surface heterogeneity.

Methods

hTCEpi cells were cultured to confluence in serum-free medium. The medium was then replaced by stratification medium to induce mucin biosynthesis. The mucin expression profile was analyzed using quantitative PCR and Western blotting. Contact angles were measured using a two-immiscible liquid method, and contact angle hysteresis was evaluated by tilting the apparatus and recording advancing and receding contact angles. The spatial distribution of mucins was evaluated with fluorescently labeled lectin.

Results

hTCEpi cells expressed the three main ocular mucins (MUC1, MUC4, and MUC16) with a maximum between days 1 and 3 of the stratification process. Upon stratification, cells caused a very significant increase in contact angle hysteresis, suggesting the development of spatially discrete and heterogeneously distributed surface features, defined by topography and/or chemical functionality. Although atomic force microscopy measurements showed no formation of appreciable topographic features on the surface of the cells, we observed a significant increase in surface chemical heterogeneity.

Conclusions

The surface chemical heterogeneity of the corneal epithelium may influence the dynamic behavior of tear film by "pinning" the contact line between the cellular surface and aqueous tear film. Engineering the surface properties of corneal epithelium could potentially lead to novel treatments in dry eye disease.

Mucins are large glycoproteins expressed by epithelial cells of both the conjunctiva and cornea, and principle components of the glycocalyx. They are thought to play an important role in determining the interactions between the cornea/conjunctiva and the overlying tear film. The purpose of this study was to characterize the membrane-associated corneal mucin expression pattern from multiple species commonly used in ophthalmic research and drug development to better define the biochemical attributes of the ocular surface. Humans, rhesus macaques and dogs were found to have a very similar pattern of mucin expression, with mucin 16 (MUC16) being the most prevalent mucin transcript. In contrast, the rabbit had a unique mucin expression pattern with all mucin transcripts expressed at relatively similar levels. To determine if there were spatial differences in expression, peripheral and central corneal epithelium were individually isolated and evaluated for mucin expression. In all species examined, MUC1, MUC4 and MUC16 had higher peripheral corneal expression when compared with central, which reached statistical significance in MUC1 (rhesus and dog). The data demonstrated variation in corneal epithelial membrane-associated mucin expression between species, with the rabbit having a distinct expression pattern. These differences may be reflective of the environment, pathogen exposure or tear film dynamics of the respective species. The species differences, as well as regional mucin expression patterns, characterized in this study further define the biochemical composition of the ocular surface and may play an important role in tear film stability.

Corneal wound healing is an enormously complex process that requires the simultaneous cellular integration of multiple soluble biochemical cues, as well as cellular responses to the intrinsic chemistry and biophysical attributes associated with the matrix of the wound space. Here, we document how the biomechanics of the corneal stroma are altered through the course of wound repair following keratoablative procedures in rabbits. Further we documented the influence that substrate stiffness has on stromal cell mechanics. Following corneal epithelial debridement, New Zealand white rabbits underwent phototherapeutic keratectomy (PTK) on the right eye (OD). Wound healing was monitored using advanced imaging modalities. Rabbits were euthanized and corneas were harvested at various time points following PTK. Tissues were characterized for biomechanics with atomic force microscopy and with histology to assess inflammation and fibrosis. Factor analysis was performed to determine any discernable patterns in wound healing parameters. The matrix associated with the wound space was stiffest at 7days post PTK. The greatest number of inflammatory cells were observed 3days after wounding. The highest number of myofibroblasts and the greatest degree of fibrosis occurred 21days after wounding. While all clinical parameters returned to normal values 400days after wounding, the elastic modulus remained greater than pre-surgical values. Factor analysis demonstrated dynamic remodeling of stroma occurs between days 10 and 42 during corneal stromal wound repair. Elastic modulus of the anterior corneal stroma is dramatically altered following PTK and its changes coincide initially with the development of edema and inflammation, and later with formation of stromal haze and population of the wound space with myofibroblasts. Factor analysis demonstrates strongest correlation between elastic modulus, myofibroblasts, fibrosis and stromal haze thickness, and between edema and central corneal thickness.

Statement of significance

Tissue biomechanics during the course of corneal wound healing is documented for the first time through atomic force microscopy, and is correlated with advanced clinical imaging and immunohistochemistry. Parameters obtained from the study are applied in a multivariate statistical model to cluster the data for better classification and monitor the wound repair process. Elastic modulus of the anterior corneal stroma is dramatically altered following wounding and correlates initially with the development of edema and inflammation, and later with formation of stromal haze and population of the wound space with myofibroblasts. Importantly, the occurrence of myofibroblasts is preceded by changes in tissue mechanics, which is important to consider in light of crosslinking procedures applied to treat corneal diseases.

Ocular surface disorders, such as dry eye disease, ocular rosacea, and allergic conjunctivitis, are a heterogeneous group of diseases that require an interdisciplinary approach to establish underlying causes and develop effective therapeutic strategies. These diverse disorders share a common thread in that they involve direct changes in ocular surface chemistry as well as the rheological properties of the tear film and topographical attributes of the cellular elements of the ocular surface. Knowledge of these properties is crucial to understand the formation and stability of the preocular tear film. The study of interfacial phenomena of the ocular surface flourished during the 1970s and 1980s, but after a series of lively debates in the literature concerning distinctions between the epithelial and the glandular origin of ocular surface disorders during the 1990s, research into this important topic has declined. In the meantime, new tools and techniques for the characterization and functionalization of biological surfaces have been developed. This review summarizes the available literature regarding the physicochemical attributes of the ocular surface, analyzes the role of interfacial phenomena in the pathobiology of ocular surface disease, identifies critical knowledge gaps concerning interfacial phenomena of the ocular surface, and discusses the opportunities for the exploitation of these phenomena to develop improved therapeutics for the treatment of ocular surface disorders.