The other possibility is that the goblet cells that were present at 7 days died before 14 days. Goblet cells are difficult to culture, and for this reason it is plausible that they were not able to grow or be maintained in the three-dimensional culture for a long time. In fact, the large majority of stratified conjunctival cultures do not contain goblet cells [ 39 , 40 ]. Goblet cell loss is a hallmark of squamous metaplasia, a pathological transition to a non-secretory, keratinized epithelium.
Tan et al. They observed a reduced number of goblet cells at eight days, and a complete loss after 12 days in culture. Those results are in agreement with the findings observed in our construct.
Thus, our three-dimensional model used at longer periods of time could be a useful tool to study squamous metaplasia in vitro. Further research using different culture media and supplements and analyzing the fate of goblet cells between seven and 14 days would help to elucidate the observed reduction in goblet cell numbers. The morphological study performed by SEM revealed that fibroblasts maintained an elongated shape and interacted with the fibrin, while epithelial cells completely covered the matrix surface.
Well-formed microvilli were clearly identified on the epithelial cells, which means the cells were polarized similar to the native tissue. Cell polarity is essential for the function of epithelial tissues, including the conjunctiva [ 44 ]. In addition, changes in the number of microvilli detected by SEM have been proposed as a method to identify alterations in conjunctival epithelium [ 47 ].
Finally, we observed cells having a morphology compatible with that of goblet cells. In addition, we found mucus on the surface of those cells. Epithelial cells growing on the surface of the fibrin scaffold showed higher stratification when cultured in the air-lifted condition. The ability of epithelial cells to stratify when cultured in those conditions is in agreement with previous publications.
Chung et al. Thus, these results corroborated that epithelial cells in our three-dimensional model are able to respond as they were expected to do. Conjunctival mucin secretion becomes altered in ocular surface inflammatory diseases, and we found similar changes in our experimental conditions. Goblet cells were present in all of the conditions, and the number increased in the air-lifted cultures.
Again, this response is in agreement with previous publications by several authors in other mucosal epithelium models [ 49 , 50 ]. Therefore, our results confirm the presence of goblet cells in the three-dimensional model. Moreover, there was an increase in MUC5AC secretion in air-lifted, partially desiccated, and ILtreated conditions compared to control constructs.
Similar results were observed when the air-lifted culture was additionally exposed to IL Our results are consistent with those of De Paiva et al. In dry eye disease many molecules are overexpressed. One of those is IL-6, a cytokine that has been used in several studies as a marker of inflammation. Overexpression of IL-6 in dry eye patients correlates with the symptomatic severity of disease [ 53 ]. In our research, we measured secreted levels of IL-6 and found that in the air-lifted and in the partially desiccating conditions IL-6 levels were significantly increased, which is in accordance with Zhang et al.
Overexpression of IL-6 is indicative of an inflammatory status of the cells in the constructs, and it supports the idea of using this model to study ocular surface inflammatory disease. Specifically, IL-6 is found elevated in conjunctival cytology samples from patients with dry eye disease [ 53 ], and the highest levels of IL-6 in our model were found in the partially desiccating condition, which mimicked dry eye inflammation. In summary, we have developed a structurally and physiologically relevant conjunctival model to study inflammatory diseases. We have shown that by exposing the constructs to partial desiccation for just two hours it is possible to reproduce some of the features found in dry eye disease.
Furthermore, treating the conjunctival constructs with IL mimics some of the responses found in allergic diseases. Bearing all this in mind, we conclude that this three-dimensional model is functional, responds to several stimuli, and therefore it can be used to study ocular inflammatory diseases. Formal analysis: LGP. Funding acquisition: YD. Project administration: YD. Resources: YD. Supervision: YD. Validation: LGP. Writing — original draft: LGP. Browse Subject Areas?
Click through the PLOS taxonomy to find articles in your field. Abstract The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Introduction Inflammatory ocular surface diseases are very prevalent among the global population. Materials and methods Human conjunctival tissues Human bulbar conjunctival tissues were obtained from the Barraquer Eye Bank of Barcelona Spain.
Isolation and culture of conjunctival cells Conjunctival cells were cultured as previously described [ 21 ]. Fibrin scaffold preparation Fibrin-based matrices were used as scaffolds to engineer the conjunctival-like tissue. Cell seeding of fibrin scaffolds Fibroblasts were grown inside the fibrin-based scaffolds. Experimental conditions We used four different experimental conditions in this study Fig 1. Download: PPT. Immunofluorescence and lectin-binding assays Slides were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol.
Results Fibrin-based matrices supported conjunctival cell growth Fibrin matrices supported human conjunctival cell growth.
Fig 2. Conjunctival cells grown in and on fibrin scaffold constructs. Epithelial cells maintained phenotype when cultured on fibrin scaffolds A problem that often occurs when culturing epithelial cells is epithelial-mesenchymal transition EMT [ 22 ]. Fig 3. Conjunctival cells proliferated at different rates when seeded in plasma or cryoprecipitate scaffolds. Epithelial cells and fibroblasts maintained cell morphology in the fibrin scaffolds The scaffold structure and the morphology of fibroblasts and epithelial cells incorporated into both plasma Fig 4a—4c and cryoprecipitate matrices Fig 4d—4f were studied by SEM.
Fig 5. Constructs exposed to different experimental conditions showed different features. Inflammatory responses The levels of secreted inflammatory cytokine IL-6 were measured by ELISA to determine if the different experimental conditions produced an inflammatory response in the cells contained in the three-dimensional model Fig 5f.
Effect of experimental conditions on mucous secretion The presence of mucus was evaluated in the constructs by two different techniques. Discussion While there is a large amount of ongoing conjunctival research, there is a need to develop new methodologies that can reduce the reliance on animal use and accurately simulate the in vivo physiology of this tissue. References 1. Bielory L, Syed BA.
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Decreased visual acuity may be caused by extension of scleritis to the adjacent structures, leading to inflammation of the cornea keratitis or the colored portion of the front of the eye uveitis , glaucoma , cataract , and abnormalities of the retina. Fraunfelder et al. We used four different experimental conditions in this study Fig 1. Epithelial cells cultured on the surface of fibrin scaffolds produced mucins green at days 3 left and 7 middle , but they did not produce them at day 14 right. This can cause double vision. Int Rev Immunol.
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