Corneal pannus, Herbert's pits and conjunctival inflammation in older children in Papua New Guinea.

PURPOSE: The prevalence of trachomatous inflammation-follicular (TF) in Papua New Guinea (PNG) suggests antibiotic mass drug administration (MDA) is needed to eliminate trachoma as a public health problem but the burden of trichiasis is low. As a result, WHO issued bespoke recommendations for the region. If ≥ 20% of 10-14-year-olds have both any conjunctival scarring (C1 or C2 or C3) and corneal pannus and/or Herbert's pits, MDA should be continued. Equally, if ≥ 5% of that group have both moderate/severe conjunctival scarring (C2 or C3) and corneal pannus and/or Herbert's pits, MDA should be continued. METHODS: We identified 14 villages where > 20% of 1-9-year-olds had TF during baseline mapping undertaken 4 years and 1 month previously. Every child aged 10-14 years in those villages was eligible to be examined for clinical signs of corneal pannus, Herbert's pits and conjunctival scarring. A grading system that built on existing WHO grading systems was used. RESULTS: Of 1,293 resident children, 1,181 (91%) were examined. Of 1,178 with complete examination data, only one (0.08%) individual had concurrent scarring and limbal signs. CONCLUSIONS: The WHO-predefined criteria for continuation of MDA were not met. Ongoing behavioural and environmental improvement aspects of the SAFE strategy may contribute to integrated NTD control. Surveillance methods should be strengthened to enable PNG health authorities to identify future changes in disease prevalence.

3D bioprinting as a prospective therapeutic strategy for corneal limbal epithelial stem cell deficiency.

Limbal epithelial stem cells (LESCs) are responsible for the maintenance and repair of the corneal surface. Injuries and diseases of the eye may result in a vision condition called limbal stem cell deficiency (LSCD). Without limbal stem cells, the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy as a treatment method was first described in 1997, and LESCs cultured from either patients or donors have been used to treat LSCD successfully. However, the main source of cornea for LSCD treatment is from donors, which are too few to meet the demand (less than 1:70 of cases). The global shortage of donor cornea promotes the need for studies exploring corneal limbus alternatives. Many problems still remain unresolved, such as original geometry reconstruction, corneal epithelial regeneration, and ocular optical function restoration. 3D bioprinting has garnered tremendous attention in recent years, and significant advances have been made in fabricating cell-laden scaffolds. These advancements could lead to a promising treatment for LSCD. It is possible that alternative limbus stem cells can be constructed using 3D printing, which, in corneal limbus regeneration, enables personalized corneal implants and fabrication of single- or multilayer corneal limbus equivalents with corneal limbal stem cells. In this review, the progress, applications, and limitations of the most influential works regarding current treatments of LESC deficiency are discussed. The advantages of 3D bioprinting are illustrated, and some first promising steps toward the creation of a functional cornea limbus with 3D bioprinting are discussed. Finally, insights into the prospects and technical challenges facing the future research of 3D bioprinting of corneal limbus alternatives in vivo and in vitro are provided.

Measurement of the Elastic Modulus of Cornea, Sclera and Limbus: The Importance of the Corneal-Limbus-Scleral Biomechanical Unit.

BACKGROUND: Energy storage, transmission and dissipation are important considerations of normal mechanical homeostasis. In this paper we present a new technique termed vibrational optical coherence tomography (VOCT) to study the anterior anatomic structures of the pig eye to better understand how energy applied to the cornea is dissipated without delamination occurring. METHODS: VOCT uses infrared light and an applied sinusoidal audible sound wave to image and measure the resonant frequency and modulus of individual macromolecular components of tissue non-invasively. We have measured the resonant frequencies and calculated the moduli of tissues in the anterior portion of the pig eye using VOCT. RESULTS: While both pig and human eyes have similar resonant frequencies, they do differ in the peak amplitudes near the frequencies of 80, 120, 150 and 250 Hz. It is known that the stroma of pig cornea is much thicker than that of human corneas and these differences may explain the normalized peak height differences. The similarity of the resonant frequency peaks near 80, 120, 150 and 250 Hz of cornea, sclera and limbus suggest that the anatomically described layers in these tissues are connected into a single biomechanical unit that can store external mechanical energy and then transmit it for dissipation. Since the energy stored and dissipated is proportional to the modulus and the ability of the tissue to deform under stress, energy storage in these tissues is related to the stiffness. CONCLUSIONS: It is concluded that stored energy is transmitted to the posterior segment of the eye for dissipation through the attachment with the sclera. This mechanism of energy dissipation may protect the cornea from changes in shape, curvature, and refractive power. However, ultimately, energy dissipation through thinning of the sclera may cause globe elongation observed in subjects with myopia and glaucoma.

Vernal keratoconjunctivitis with a limbal mass lesion developing independently of severe papillae formation at the tarsal conjunctiva: a case report.

BACKGROUND: A hypertrophic limbal mass lesion is an uncommon finding of vernal keratoconjunctivitis; it normally occurs in eyes with severe papillae formation in the tarsal conjunctiva. We present a case with a limbal mass lesion in a patient with relatively mild allergic findings in the tarsal conjunctiva. CASE PRESENTATION: A 12-year-old Japanese boy displaying allergic conjunctivitis presented with a mass lesion at the inferior limbus in the left eye. Relatively mild papillae formation was found on the tarsal conjunctiva in both eyes. We diagnosed that the mass lesion resulted from limbal vernal keratoconjunctivitis and resected it for therapeutic purposes. Histopathological examination showed that eosinophils, lymphocytes, and fibroblasts were present in the subepithelial lesion and the substantia propria of the mass lesion. Immunohistochemical staining detected diffuse and rich infiltration of CD3-positive T lymphocytes and a relatively small number of CD20-positive B lymphocytes and CD138-positive plasma cells that tended to aggregate. The histopathologic features suggested that the limbal mass lesion had similar structures to the papillae at the tarsal conjunctiva of vernal keratoconjunctivitis. CONCLUSION: The limbal mass lesion as a finding of vernal keratoconjunctivitis can occur even if the papillae formation at the patient's tarsal conjunctiva is mild.

Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds.

Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14-21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.

Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment.

Corneal stroma-derived mesenchymal stem cells (CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells (LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.

Recent Advances in Stem Cell Therapy for Limbal Stem Cell Deficiency: A Narrative Review.

Destruction of the limbus and depletion of limbal stem cells (LSCs), the adult progenitors of the corneal epithelium, leads to limbal stem cell deficiency (LSCD). LSCD is a rare, progressive ocular surface disorder which results in conjunctivalisation and neovascularisation of the corneal surface. Many strategies have been used in the treatment of LSCD, the common goal of which is to regenerate a self-renewing, transparent, and uniform epithelium on the corneal surface. The development of these techniques has frequently resulted from collaboration between stem cell translational scientists and ophthalmologists. Direct transplantation of autologous or allogeneic limbal tissue from a healthy donor eye is regarded by many as the technique of choice. Expansion of harvested LSCs in vitro allows smaller biopsies to be taken from the donor eye and is considered safer and more acceptable to patients. This technique may be utilised in unilateral cases (autologous) or bilateral cases (living related donor). Recently developed, simple limbal epithelial transplant (SLET) can be performed with equally small biopsies but does not require in vitro cell culture facilities. In the case of bilateral LSCD, where autologous limbal tissue is not available, autologous oral mucosa epithelium can be expanded in vitro and transplanted to the diseased eye. Data on long-term outcomes (over 5 years of follow-up) for many of these procedures is needed, and it remains unclear how they produce a self-renewing epithelium without recreating the vital stem cell niche. Bioengineering techniques offer the ability to re-create the physical characteristics of the stem cell niche, while induced pluripotent stem cells offer an unlimited supply of autologous LSCs. In vivo confocal microscopy and anterior segment OCT will complement impression cytology in the diagnosis, staging, and follow-up of LSCD. In this review we analyse recent advances in the pathology, diagnosis, and treatment of LSCD.

[Panorama of limbal alterations (French translation of the article)]. / Panorama des altérations limbiques cornéennes.

The corneal limbus is a privileged region on the border between two quite different microenvironments, where corneal epithelial stem cells, numerous melanocytes, and antigen-presenting cells are all concentrated within a richly vascularized and innervated stroma. This situation within the ocular surface confers on it the key functions of barrier, epithelial renewal and defense of the cornea. As an immunological crossroads and since the corneoscleral limbus is directly exposed to external insults such as caustic agents, ultraviolet radiation, microbial agents, and allergens, it is the potential site of many tumoral, degenerative or inflammatory pathologies and may progress under certain conditions to limbal stem cell deficiency.

Strategies for reconstructing the limbal stem cell niche.

The epithelial cell layer that covers the surface of the cornea provides a protective barrier while maintaining corneal transparency. The rapid and effective turnover of these epithelial cells depends, in part, on the limbal epithelial stem cells (LESCs) located in a specialized microenvironment known as the limbal niche. Many disorders affecting the regeneration of the corneal epithelium are related to deficiency and/or dysfunction of LESCs and the limbal niche. Current approaches for regenerating the corneal epithelium following significant injuries such as burns and inflammatory attacks are primarily aimed at repopulating the LESCs. This review summarizes and assesses the clinical feasibility and efficacy of current and emerging approaches for reconstruction of the limbal niche. In particular, the application of mesenchymal stem cells along with appropriate biological scaffolds appear to be promising strategies for long-term revitalization of the limbal niche.

Anterior segment optical coherence tomography angiography to evaluate the peripheral fitting of scleral contact lenses.

PURPOSE: The aim of this study was to show the potential applicability of optical coherence tomography angiography (OCTA) for the evaluation of the peripheral fitting of fully scleral contact lenses. METHODS: A pilot study was proposed fitting three different scleral contact lenses (Irregular Corneal Design [ICD]) with different sagittal heights (4200, 4800, and 5600 mm) in a healthy volunteer of 27 years old. We evaluated by means of optical coherence tomography (OCT, DRI Triton) the apical clearance achieved with each of the three lenses fitted. The impact over scleral flow was assessed with the OCTA module of the same device. RESULTS: The apical clearance was 310, 901, and 1680 µm with the scleral lenses of sagittal heights 4200, 4800, and 5600 µm, respectively. With OCTA, we evaluated the impact of the lens bearing on the conjunctival vascular flow, observing an area of vascular interruption of 0, 25, and 75% with the lenses of 4200, 4800, and 5600 µm of sagittal heights, respectively. The vascular interruption was induced in the perilimbar area, suggesting the need of readjusting the limbal clearance zone of the lens. CONCLUSION: Fully scleral contact lens fitting may be optimized with the use of OCTA, allowing the practitioner to perform the fitting with better control of the peripheral bearing of the lens on the conjunctival tissue, assessing the impact on vascular structures. This potential use of OCTA must be investigated further in future studies including large samples of eyes.

Demonstration of P-selectin expression and potential function in human corneal epithelial cells.

In response to an unexpected observation of apparent localisation by immunocytochemistry, we have investigated the potential expression and function of P-selectin (CD62P) in human corneal epithelial cells. The SV40 immortalised cell line, HCE-T (validated by STR profiling), along with multiple donor corneal-limbal tissue samples, were examined for P-selectin expression using a combination of immunocytochemistry, Western blotting, RT-PCR and immunohistochemistry. Potential expression of the major ligand for P-selectin (P-selectin glycoprotein ligand-1; PSGL-1; CD162) was also examined by immunocytochemistry and RT-PCR. A selective inhibitor of P-selectin-PSGL-1 binding (KF38789) was subsequently tested for effects on HCE-T cells using a cell culture gap-closure assay. HCE-T cells as well as primary epithelial cultures derived from donor corneal-limbal tissue, displayed positive immunostaining for P-selectin. Staining was particularly evident at cell-cell boundaries and at the outer edge of expanding epithelial islands. P-selectin expression was confirmed by Western blotting and RT-PCR (validated by product sequencing), as well as by immunohistochemistry performed on serial sections of corneal-limbal tissue stained for P-selectin, keratin 3 and p63. PSGL-1 was detected by RT-PCR and immunocytochemistry in both corneal epithelial cells as well as human limbal fibroblasts (HLF). KF38789 (5 µM) significantly reduced closure of a 500-µm gap between confluent sheets of HCE-T cells over an 8-hr period (by ∼40%, p < 0.01; paired two-tailed T test), but had no effect on culture gap-closure by either HLF or murine 3T3 fibroblasts. These results provide evidence of P-selectin expression in human corneal epithelial cells and suggest a potential role for this glycoprotein in facilitating the net movement of confluent sheets of human corneal epithelial cells.

Comparison of Two Rabbit Models with Deficiency of Corneal Epithelium and Limbal Stem Cells Established by Different Methods.

Limbal stem cell defect model is an important animal model that provides a basis for the study of ocular surface diseases. The rabbit cornea is of moderate size and is widely used in such studies as an experimental animal model. At present, the main modeling methods are alkali burns, and corneal limbus girdling and corneal epithelium doctoring. Each method has its own characteristics. In this study, we observed rabbit models with severe ocular surface defect established by the two methods and changes after amniotic membrane transplantation. In the first, second, third, and fourth week after operation, the clinical manifestations, corneal transparency, and new vessels were observed according to the standard rating scale of ocular surface, compared between the two methods, and then statistically analyzed. In the fourth week after operation, the rabbits were sacrificed and their corneas and corneal limbus were extracted from sclera, embedded by optimum cutting temperature compound, frozen, and sliced for hematoxylin and eosin staining and pathological examination. There were two groups in this study. Group 1 (alkali burns) had more severe complications, such as, conjunctiva, nubecula, new vessel hyperplasia, and so on, compared to group 2 (corneal limbus girdling and corneal epithelium doctoring). In addition, there were striking differences in corneal transparency and new vessels between the two groups (p < 0.05). Corneal transparency in group 1 was lower than in group 2. New vessels in group 1 were less in the first 2 weeks, but obviously increased compared to group 2 in the subsequent weeks. Alkaline burn could be used to study new vessel hyperplasia, while corneal limbus girdling and corneal epithelium doctoring are more suitable for studying stem cell transdifferentiation, interactive roles of stem cells and microenvironment, and so on.

Inhibition effect of curcumin on UVB-induced secretion of pro-inflammatory cytokines from corneal limbus epithelial cells.

AIM: To study the effects of curcumin on the secretion of interleukin (IL)-6 and IL-8 by corneal limbus epithelial cells. METHODS: Human corneal limbus epithelial cells were isolated and cultured from donor eyes and irradiated by UVB at different dosages with or without curcumin. MTT test was used for studying the effects of UVB and curcumin on the cell viability. The role of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways on the UVB-induced secretion of IL-6 and IL-8 were tested by addition of their inhibitors to the culture with or without UVB-radiation. Levels of various signal pathways, IL-6 and IL-8 in the cells and in the conditioned culture medium were measured by ELISA analysis. RESULTS: UVB at 20 mJ/cm2 or less and curcumin at 20 µmol/L or less did not affect the cell viability of cultured limbus epithelial cells (P>0.05). UVB irradiation at 10 and 20 mJ/cm2 induced a significant increase of secretion of IL-6 and IL-8 and upregulated NF-κB and phosphorylated MAPK pathways of cultured limbus epithelial cells (P<0.05). Various signal pathway inhibitors, including SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor) and BAY11-7082 (NF-κB inhibitor) significantly decreased the UVB-induced secretion of IL-6 and IL-8 secretion (P<0.05). Curcumin at 5-20 µmol/L significantly inhibited UVB-induced secretion of IL-6 and IL-8 by limbus epithelial cells in a dose-dependent manner; while curcumin alone did not affect the secretion of IL-6 and IL-8. The upregulation of NF-κB and MAPK pathways induced by UVB treatment was significantly inhibited by curcumin, suggesting that NF-κB and MAPK pathways are involved in the inhibitory effect of curcumin on UVB-induced production of IL-6 and IL-8. CONCLUSION: Curcumin may be a promising agent to be explored for the prevention and treatment of pterygium.

Three-dimensional structure of the mammalian limbal stem cell niche.

Although the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its three-dimensional (3D) architecture remains incomplete. The LSC niche was explored on freshly excised and organ-cultured corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full-field optical coherence microscopy (FFOCM). Limbal crypt features were detected in 90% of organ-cultured human corneoscleral rims, extending between the palisades of Vogt as radially oriented rectangular (74% of eyes) and/or rounded (23% of eyes) forms, often branching off to, or becoming interconnected by, sub-scleral radially or circumferentially oriented crypts (in 56% of eyes). Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss-crossing invaginations beneath the scleral surface. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of sampled limbal volume in humans and 9% in pigs. FFOCM combined with fluorescence, and confocal fluorescence microscopy, showed presence of p63-α+ cells and cytokeratin-3+ cells in the limbal crypts. To assess colony forming efficiency (CFE), limbal epithelial cells were cultured at low density with mitomycin-arrested 3T3 feeders. CFE increased with limbal crypt volume and was not significantly decreased in organ-cultured cornea, despite degradation of the epithelial roof, suggesting that stem cells remain protected at the base of crypts during organ culture. CFE in human samples was significantly greater than in pig, and CFE in mouse was zero. Crypt architecture in the three species appears associated with eye exposure to light. LSC density increased with percentage limbal volume occupied by crypts.

Isolation of microvascular endothelial cells from cadaveric corneal limbus.

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with foetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 µg/mL), hydrocortisone (1 µg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF(+) cells formed aligned cell-to-cell 'trains' when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32-80 years) or duration in eye bank corneal preservation medium prior to use (3-10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p < 0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. METHODS: MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. RESULTS: Human L-MSC cultures were typically CD34⁻, CD45⁻ and HLA-DR⁻ and CD73⁺, CD90⁺, CD105⁺ and HLA-ABC⁺. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. CONCLUSIONS: L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.