New Technologies in Clinical Trials in Corneal Diseases and Limbal Stem Cell Deficiency: Review from the European Vision Institute Special Interest Focus Group Meeting.

To discuss and evaluate new technologies for a better diagnosis of corneal diseases and limbal stem cell deficiency, the outcomes of a consensus process within the European Vision Institute (and of a workshop at the University of Cologne) are outlined. Various technologies are presented and analyzed for their potential clinical use also in defining new end points in clinical trials. The disease areas which are discussed comprise dry eye and ocular surface inflammation, imaging, and corneal neovascularization and corneal grafting/stem cell and cell transplantation. The unmet needs in the abovementioned disease areas are discussed, and realistically achievable new technologies for better diagnosis and use in clinical trials are outlined. To sum up, it can be said that there are several new technologies that can improve current diagnostics in the field of ophthalmology in the near future and will have impact on clinical trial end point design.

Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures.

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.

Pterygium Pathology: A Prospective Case-Control Study on Tear Film Cytokine Levels.

Pterygium is a common eye disease, linked to an increased exposure to UV radiation and dry environments. The associated pathology culminates in visual impairment and, in some rare cases, blindness. However, there remains a lot of uncertainty concerning the pathogenesis of this fibrovascular lesion. As the composition of the tear film provides a reflection into the pathological changes at the ocular surface, tear analysis represents an ideal approach to gain insight in the progression of disease following pterygiectomy. This study enrolled 19 patients and age/gender-matched healthy controls. Tear film levels of interleukin- (IL-) 6, IL-8, and vascular endothelial growth factor (VEGF) were investigated over time, and preoperative concentrations were linked to corneal neovascularization and pterygium size. Diminished tear film levels were found in unilateral patients who show no clinical signs of pterygium recurrence over a period of one year. Hence, our results highlight the potential of using the course of IL-6, IL-8, and VEGF levels in tears as biomarkers for recovery. In addition, when focusing on the affected eyes (i.e., primary and recurrent pterygium), we detected fold changes in preoperative cytokine concentrations to correspond with disease severity. As our proposed biomarkers did not reveal a linear relationship with corneal neovascularization nor the invasive behaviour of pterygium, no exact role in the pterygium pathology could be established. Hence, our data point to these factors being contributors rather than decisive players in the pathological processes.

Immunocytochemical characterization of ex vivo cultured conjunctival explants; marker validation for the identification of squamous epithelial cells and goblet cells.

Tissue-engineered products are at the cutting edge of innovation considering their potential to functionally and structurally repair various tissue defects when the body's own regenerative capacity is exhausted. At the ocular surface, the wound healing response to extensive conjunctival damage results in tissue repair with structural alterations or permanent scar formation rather than regeneration of the physiological conjunctiva. Conjunctival tissue engineering therefore represents a promising therapeutic option to reconstruct the ocular surface in severe cicatrizing pathologies. During the rapid race to be a pioneer, it seems that one of the fundamental steps of tissue engineering has been neglected; a proper cellular characterization of the tissue-engineered equivalents, both morphologically and functionally. Currently, no consensus has been reached on an identification strategy and/or markers for the characterization of cultured squamous epithelial and goblet cells. This study therefore evaluated the accuracy of promising markers to identify differentiated conjunctival-derived cells in human primary explant cultures through immunocytochemistry, including keratins (i.e., K7, K13, and K19) and mucins (i.e., MUC1, MUC5AC, and PAS-positivity). Comparison of the in vivo and in vitro cellular profiles revealed that the widely used goblet cell marker K7 does not function adequately in an in vitro setting. The other investigated markers offer a powerful tool to distinguish cultured squamous epithelial cells (i.e., MUC1 and K13), goblet cells (i.e., MUC5AC and PAS-staining), and conjunctival-derived cells in general (i.e., K19). In conclusion, this study emphasizes the power alongside potential pitfalls of conjunctival markers to assess the clinical safety and efficacy of conjunctival tissue-engineered products.

Pterygium-The Good, the Bad, and the Ugly.

Pterygium is a multifaceted pathology that displays apparent conflicting characteristics: benign (e.g., self-limiting and superficial), bad (e.g., proliferative and potentially recurrent) and ugly (e.g., signs of preneoplastic transformation). The natural successive question is: why are we lacking reports showing that pterygium lesions become life-threatening through metastasis, especially since pterygium has considerable similarities with UV-related malignancies on the molecular level? In this review, we consider how our pathophysiological understanding of the benign pterygium pathology overlaps with ocular surface squamous neoplasia and skin cancer. The three UV-related disorders share the same initial insult (i.e., UV radiation) and responsive repair mechanisms to the ensuing (in)direct DNA damage. Their downstream apoptotic regulators and other cellular adaptations are remarkably alike. However, a complicating factor in understanding the fine line between the self-limiting nature of pterygium and the malignant transformation in other UV-related diseases is the prominent ambiguity in the pathological evaluation of pterygium biopsies. Features of preneoplastic transformation (i.e., dysplasia) are used to define normal cellular reactions (i.e., atypia and metaplasia) and vice versa. A uniform grading system could help in unraveling the true nature of this ancient disease and potentially help in identifying the earliest intervention point possible regarding the cellular switch that drives a cell's fate towards cancer.

Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs.

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.

LiQD Cornea: Pro-regeneration collagen mimetics as patches and alternatives to corneal transplantation.

Transplantation with donor corneas is the mainstay for treating corneal blindness, but a severe worldwide shortage necessitates the development of other treatment options. Corneal perforation from infection or inflammation is sealed with cyanoacrylate glue. However, the resulting cytotoxicity requires transplantation. LiQD Cornea is an alternative to conventional corneal transplantation and sealants. It is a cell-free, liquid hydrogel matrix for corneal regeneration, comprising short collagen-like peptides conjugated with polyethylene glycol and mixed with fibrinogen to promote adhesion within tissue defects. Gelation occurs spontaneously at body temperature within 5 min. Light exposure is not required-particularly advantageous because patients with corneal inflammation are typically photophobic. The self-assembling, fully defined, synthetic collagen analog is much less costly than human recombinant collagen and reduces the risk of immune rejection associated with xenogeneic materials. In situ gelation potentially allows for clinical application in outpatient clinics instead of operating theaters, maximizing practicality, and minimizing health care costs.

Selecting Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Studies in Isolated and Cultured Ocular Surface Epithelia.

The introduction of tissue engineering has allowed scientists to push the boundaries and treat seriously damaged ocular surface epithelia. They have managed to do this through the development of biological substitutes that restore, maintain or improve tissue function. To ensure the generation of a therapeutically safe and effective graft, knowledge on the transcriptional profile of native and cultured ocular surface epithelia is of undeniable value. Gene expression studies are, however, only as reliable as their proper selection of internal reaction controls or reference genes. In this study, we determined the expression stability of a number of reference genes: 18s rRNA, ACTB, ATP5B, CyC1, EIF4A2, GAPDH, RPL13A, SDHA, TOP1, UBC, and YWHAZ in primary isolates as well as in ex vivo cultured ocular surface epithelia explants (day 0 and/or day 14). Expression stability of the reference genes was assessed with both the geNorm and NormFinder software that use a pairwise comparison and a model-based approach, respectively. Our results extend the general recommendation of using multiple reference genes for normalization purposes to our model systems and provide an overview of several references genes that are likely to be stable in similar culture protocols.

In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds.

PURPOSE: To investigate the efficacy of recombinant human collagen type I (RHC I) and collagen-like peptide (CLP) hydrogels as alternative carrier substrates for the cultivation of limbal epithelial stem cells (LESC) under xeno-free culture conditions. METHODS: Human LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified RHC I, (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked CLP (CLP-12 EDC), (4) DMTMM- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium-) crosslinked CLP (CLP-12), (5) fibronectin-patterned CLP-12, (6) "3D limbal niche-mimicking" CLP-12, and (7) DMTMM-crosslinked CLP made from higher CLP concentration solution. Cell proliferation, cell morphology, and expression of LESC markers were analyzed. All data were compared to cultures on human amniotic membrane (HAM). RESULTS: Human LESC were successfully cultivated on six out of seven hydrogel formulations, with primary cell cultures on CLP-12 EDC being deemed unsuccessful since the area of outgrowth did not meet quality standards (i.e., inconsistence in outgrowth and confluence) after 14 days of culture. Upon confluence, primary LESC showed high expression of the stem cell marker ΔNp63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-ß4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-α1, and collagen type IV. Cells showed low expression of differentiation markers KRT3 and desmoglein 3 (DSG3). Significantly higher gene expression of KRT3 was observed for cells cultured on CLP hydrogels compared to RHC I and HAM. Surface patterning of hydrogels influenced the pattern of proliferation but had no significant effect on the phenotype or genotype of cultures. Overall, the performance of RHC I and DMTMM-crosslinked CLP hydrogels was equivalent to that of HAM. CONCLUSION: RHC I and DMTMM-crosslinked CLP hydrogels, irrespective of surface modification, support successful cultivation of primary human LESC using a xeno-free cultivation protocol. The regenerated epithelium maintained similar characteristics to HAM-based cultures.

Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants.

Short collagen-like peptides (CLPs) are being proposed as alternatives to full-length collagen for use in tissue engineering, on their own as soft hydrogels, or conjugated to synthetic polymer for mechanical strength. However, despite intended clinical use, little is known about their safety and efficacy, mechanism of action or degree of similarity to the full-length counterparts they mimic. Here, we show the functional equivalence of a CLP conjugated to polyethylene glycol (CLP-PEG) to full-length recombinant human collagen in vitro and in promoting stable regeneration of corneal tissue and nerves in a pre-clinical mini-pig model. We also show that these peptide analogs exerted their pro-regeneration effects through stimulating extracellular vesicle production by host cells. Our results support future use of CLP-PEG implants for corneal regeneration, suggesting the feasibility of these or similar peptide analogs in clinical application in the eye and other tissues. STATEMENT OF SIGNIFICANCE: Although biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable analogs that are functionally equivalent, and can be readily scaled-up are therefore very desirable for pre-clinical to clinical translation. Here, we demonstrate, using cornea regeneration as our test bed, that collagen-like-peptides conjugated to multifunctional polyethylene glycol (CLP-PEG) when grafted into mini-pigs as corneal implants were functionally equivalent to recombinant human collagen-based implants that were successfully tested in patients. We also show for the first time that these materials affected regeneration through stimulation of extracellular vesicle production by endogenous host cells that have migrated into the CLP-PEG scaffolds.

A method for quantifying limbal stem cell niches using OCT imaging.

AIMS: To evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images with histological findings. METHODS: The superior and inferior limbal region of both eyes of 50 healthy volunteers were imaged by FD-OCT. Images were processed and analysed using Matlab software. In vitro immunofluorescent staining of a cadaveric donor limbus was analysed to correlate the presence of stem cells in the visualised structures. RESULTS: FD-OCT could successfully visualise limbal crypts and the palisades of Vogt in the limbus region. Fluorescent labelling confirmed the presence of stem cells in these structures. The mean palisade ridge width (ΔPR) and the mean interpalisade epithelial rete peg width (ΔERP) were both of the order of 72 µm, leading to a palisade density (PD) of about 7.4 palisades/mm. A significant difference in ΔPR, ΔERP and PD was seen between the inferior and superior sides of the right eye and the superior sides of the left and right eye(p<0.05.). A significant influence of iris colour on parameters ΔPR, ΔERP and PD was found, and of age on PD and ΔERP (p<0.05). CONCLUSIONS: In vivo OCT imaging is a safe and effective modality to image the limbus and can be used to visualise the palisades of Vogt. Image processing using Matlab software enabled quantification and density calculation of imaged limbal palisades of Vogt. This technique may enhance targeted limbal biopsies for transplantation.

Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies.

Severe ocular surface disease can result in limbal stem cell deficiency (LSCD), a condition leading to decreased visual acuity, photophobia, and ocular pain. To restore the ocular surface in advanced stem cell deficient corneas, an autologous or allogenic limbal stem cell transplantation is performed. In recent years, the risk of secondary LSCD due to removal of large limbal grafts has been significantly reduced by the optimization of cultivated limbal epithelial transplantation (CLET). Despite the great successes of CLET, there still is room for improvement as overall success rate is 70% and visual acuity often remains suboptimal after successful transplantation. Simple limbal epithelial transplantation reports higher success rates but has not been performed in as many patients yet. This review focuses on limbal epithelial stem cells and the pathophysiology of LSCD. State-of-the-art therapeutic management of LSCD is described, and new and evolving techniques in ocular surface regeneration are being discussed, in particular, advantages and disadvantages of alternative cell scaffolds and cell sources for cell based ocular surface reconstruction.