OBJECTIVE: This study aimed to investigate corneal limbus changes in patients with type 2 diabetes mellitus (DM) using in vivo confocal microscopy (IVCM) and explore the correlation between their ocular manifestations and systemic status.
METHODS: Fifty-five patients with type 2 DM and 20 age-matched controls were included. The following IVCM parameters were compared between the 2 groups: palisades of Vogt (POV), corneal epithelial thickness (CET), basal cell density (BCD), subbasal nerve plexus, and dendritic cell density. All subjects underwent blood and urine sampling for laboratory analysis, including fasting blood glucose, glycated hemoglobin, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, C-reactive protein, urinary albumin-to-creatinine ratio, urine albumin, and urine creatinine. The correlations between IVCM parameters and blood biomarkers were detected. Receiver operating characteristic curve was used for selecting the cutoff value of risk factors for corneal stem cell injury in patients with DM.
RESULTS: Compared with controls, patients with DM displayed a significant reduction of POV (superior region, P = 0.033; inferior region, P = 0.003; nasal region, P < 0.001; temporal region, P < 0.001), central CET (44.8 ± 3.6 μm vs. 51.9 ± 3.6 μm, P < 0.001), central corneal BCD (7415.5 ± 563.2 cells/mm 2 vs. 9177.9 ± 977.8 cells/mm 2 , P < 0.001), and peripheral corneal BCD (6181.3 ± 416.5 cells/mm 2 vs. 8576.3 ± 933.2 cells/mm 2 , P < 0.001). Dendritic cell density (41.0 ± 33.7 cells/mm 2 vs. 24.6 ± 7.8 cells/mm 2 , P = 0.001) was significantly higher in the DM group. The following weak correlations were shown between IVCM parameters and blood biomarkers: central corneal BCD was negatively correlated with DM duration (r = -0.3, P = 0.024), TC (r = -0.36, P = 0.007), and LDL (r = -0.39, P = 0.004). The presence of POV in the superior region was negatively correlated with TC (r = -0.34, P = 0.011) and LDL (r = -0.31, P = 0.022). Cutoff values of 1.215 mmol/L for HDL, 1.59 mmol/L for TG, or 4.75 mmol/L for TC were established to distinguish patients with a high risk from a low risk for stem cell damage.
CONCLUSIONS: Patients with type 2 DM displayed a lower positive rate of typical POV and a decrease in BCD, CET, and subbasal nerve density. The most relevant indicators for stem cell phenotypes were DM duration, TC, and LDL. Lipid status in diabetic patients could be a predictor of risk for developing corneal limbal stem cell deficiency. Further studies with larger sample sizes or basic research are needed to verify the results.

OBJECTIVE: Ethanol and mitomycin C (MMC) are clinically used to treat corneal diseases such as LASEK and LASIK surgery. In this study, we investigated the effects of time-dependent alcohol and MMC in cultured rat limbal stem cells (LSCs) to determine the appropriate time for the use of this compound in the clinical setting.
METHODS: LSCs (N = 10 eyes) isolated from male Wistar rats were cultured and characterized; then, isolates were divided into three groups. One group was exposed to a 20% concentration of ethanol for 5, 10, 15, 20, 25, and 30 s, and cell viability was assessed one, three, and five days following ethanol exposure using an MTT assay. To investigate the effect of MMC, cells in the second group were treated with 0.02% MMC in various periods (i.e., 15 s, 30 s, 60 s, 90 s, and 120 s) and time-dependent responses of cultured LSCs were recorded. Cells in the third group were co-treated with ethanol and MMC; then, dose and time dependency was evaluated.
RESULTS: In comparison with the viable cells in the control group, ethanol markedly decreased the viability of cells in a time-dependent manner in days one and three. On day five, the viability of LSCs was improved significantly (p < 0.05) in comparison with day one. The number of viable progenitor cells was significantly decreased after MMC treatment in a time-dependent manner, as determined by the MTT assay (p < 0.001). The use of mitomycin, along with alcohol, decreased cell viability in all groups treated with ethanol + MMC compared to the control on days one, three, and five (p < 0.0001).
CONCLUSIONS: Our findings suggest that ethanol and MMC reduced cell viability in cultured LSCs in a time-dependent manner. In addition, when LSCs were exposed to alcohol alone, they had a better recovery process within 5 days in comparison to when exposed to mitomycin alone or mitomycin + alcohol.

To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD).
A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial.
Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes.
A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients\' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial\'s Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly.
Corneal epithelial reconstruction rate was the primary end point.
Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed.
The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named \"Nepic,\" is now approved as a cellular and tissue-based product in Japan.
Proprietary or commercial disclosure may be found after the references.
Proprietary or commercial disclosure may be found after the references.

OBJECTIVE: To investigate the effect of long-term extended soft contact lens wear on limbal and central corneal cell morphology, and limbal architecture.
METHODS: Each participant attended a study visit involving in vivo confocal microscopy of central corneal and limbal epithelium. Scans were graded by five masked graders for three features: central epithelial irregularity, limbal epithelial irregularity and the prominence of palisades of Vogt. The variability of grades between different graders and the difference of grades between extended wearers and daily soft/non-contact lens wearers were assessed.
RESULTS: Nineteen participants (9 extended soft contact lens wearers and 10 daily soft/non-contact lens wearers) aged 31-65 years were enrolled in this study. Scans from 37 eyes were included in the analysis. Agreement between graders for each feature was moderate to good with inter class correlation >0.7. While there were no significant differences in central epithelial cell irregularity (p = 0.527) and the prominence of palisade of Vogt (p = 0.182) between extended or daily soft/non-contact lens wearers, limbal epithelial cell irregularity showed a trend with increased irregularity in extended soft contact lens wearers (p = 0.091).
CONCLUSIONS: While no differences in limbal cell morphology and structureor central epithelial cell wasfound in thissubjective grading study of extended wearers compared to daily soft/non-contact lens wearers, further studies using a larger sample size or a longitudinal study design are warranted.

Limbal epithelial stem cells are required for the maintenance and repair of the corneal epithelial surface. The difficulty in obtaining human corneal tissue for research purposes means that animal models for studying the corneal and limbal epithelium are extremely useful. Porcine corneal tissue represents an attractive experimental model, however, functional analysis of the limbal epithelial cell population is needed to validate the use of this tissue. Single cell clonal analysis revealed that holoclone-generating cells were enriched in the limbus as compared with the central cornea (38.3% vs 8.3%) and that label-retaining cells were also enriched in the limbus and compared with the central cornea (44.7 ± 6.4 vs 4.7 ± 1.5). Furthermore, it was demonstrated that in a 3D-printed organ culture system, porcine tissue was capable of maintaining and healing the corneal epithelium. Ki67 staining of corneal sections revealed that in response to central epithelial wounding, a greater proportion of progenitors in the basal limbal epithelium enter an actively dividing state. The authors present a comprehensively validated model system for studying the interactions between limbal niche factors and limbal epithelial stem cell fate.

BACKGROUND: Sutureless and glue-free conjunctival autograft as a treatment modality for primary pterygium is recently gaining popularity but conventional technique of suturing conjunctival autograft is still practised widely.
OBJECTIVE: To compare the outcome of sutureless and glue-free technique with sutures for limbal conjunctival autografting in management of primary pterygium.
METHODS: A prospective interventional study was carried out in 50 consecutive eyes with primary nasal pterygium requiring surgical excision. Simple excision under local anaesthesia was performed followed by closure of the bare sclera by sutureless and glue-free conjunctival autograft in 25 eyes of 25 patients (group 1) and by the conventional method of suturing conjunctival autograft using interrupted 10-0 nylon sutures in 25 eyes of 25 patients (group 2), followed by bandaging for 24 hours in both the groups. Surgical time was recorded for both the techniques. Postoperative discomfort was assessed using preformed questionnaires. The patients were followed up for 6 months. During follow up, graft related complications and recurrence if any were noted.
RESULTS: Mean surgical time for group 1 (23.20±1.55 minutes) was significantly less as compared to group 2 (37.76±1.89 minutes); (p=0.001). Postoperative symptoms were seen in less number of patients (20%) and were of shorter duration (2 weeks) in group 1 as compared to group 2 with 20 (80%) patients having symptoms lasting for 4 weeks; (p<0.001). Recurrence rate and conjunctival granuloma formation rate for group 1 (0%) and for group 2 (4%) were statistically insignificant.
CONCLUSIONS: Sutureless and glue-free conjunctival autograft technique is simple, easy, safe, effective and less time consuming than sutured limbal autograft technique with less postoperative discomfort and adverse events encountered with the use of suture material. Postoperative results of both techniques are comparable. Hence sutureless and glue-free conjunctival autografting is a good technique for the treatment of primary pterygium.

Stem cell-based therapy has become an attractive and promising approach for the treatment of severe injuries or thus-far incurable diseases. However, the use of stem cells is often limited by a shortage of available tissue-specific stem cells; therefore, other sources of stem cells are being investigated and tested. In this respect, mesenchymal stromal/stem cells (MSCs) have proven to be a promising stem cell type. In the present study, we prepared MSCs from bone marrow (BM-MSCs) or adipose tissue (Ad-MSCs) as well as limbal epithelial stem cells (LSCs), and their growth, differentiation, and secretory properties were compared. The cells were grown on nanofiber scaffolds and transferred onto the alkali-injured eye in a rabbit model, and their therapeutic potential was characterized. We found that BM-MSCs and tissue-specific LSCs had similar therapeutic effects. Clinical characterization of the healing process, as well as the evaluation of corneal thickness, re-epithelialization, neovascularization, and the suppression of a local inflammatory reaction, were comparable in the BM-MSC- and LSC-treated eyes, but results were significantly better than in injured, untreated eyes or in eyes treated with a nanofiber scaffold alone or with a nanofiber scaffold seeded with Ad-MSCs. Taken together, the results show that BM-MSCs\' therapeutic effect on healing of injured corneal surface is comparable to that of tissue-specific LSCs. We suggest that BM-MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain.
CONCLUSIONS: Damage of ocular surface represents one of the most common causes of impaired vision or even blindness. Cell therapy, based on transplantation of stem cells, is an optimal treatment. However, if limbal stem cells (LSCs) are not available, other sources of stem cells are tested. Mesenchymal stem cells (MSCs) are a convenient type of cell for stem cell therapy. The therapeutic potential of LSCs and MSCs was compared in an experimental model of corneal injury, and healing was observed following chemical injury. MSCs and tissue-specific LSCs had similar therapeutic effects. The results suggest that bone marrow-derived MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain.