Human Oral Mucosal Fibroblasts from Limbal Stem Cell Deficient Patients as an Autologous Feeder Layer for Epithelial Cell Culture.

PURPOSE: To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use. METHODS: HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens-Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD. RESULTS: HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups. CONCLUSIONS: HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

Descemet's membrane endothelial keratoplasty (DMEK) versus Descemet's stripping automated endothelial keratoplasty (DSAEK) for corneal endothelial failure.

BACKGROUND: Corneal endothelial transplantation has become the gold standard for the treatment of corneal endothelial dysfunctions, replacing full thickness transplantation, known as penetrating keratoplasty. Corneal endothelial transplantation has been described using two different techniques: Descemet's membrane endothelial keratoplasty (DMEK) and Descemet's stripping automated endothelial keratoplasty (DSAEK). Both are still performed worldwide. OBJECTIVES: To compare the effectiveness and safety of Descemet's membrane endothelial keratoplasty (DMEK) versus Descemet's stripping automated endothelial keratoplasty (DSAEK) for the treatment of corneal endothelial failure in people with Fuch's endothelial dystropy (FED) and pseudophakic bullous keratopathy (PBK). SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Trials Register) (2017, Issue 7); MEDLINE Ovid; Embase Ovid; LILACS BIREME; the ISRCTN registry; ClinicalTrials.gov and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP). The date of the search was 11 August 2017. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and non-randomised paired, contralateral-eye studies in any setting where DMEK was compared with DSAEK to treat people with corneal endothelial failure. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the search results, assessed trial quality and extracted data using the standard methodological procedures expected by Cochrane. Our primary outcome was best corrected visual acuity (BCVA) measured in logarithm of the Minimum Angle of Resolution (logMAR). Secondary outcomes were endothelial cell count, graft rejection, primary graft failure and graft dislocation. We graded the risk of bias of non-randomised studies (NRSs) using ROBINS-I. MAIN RESULTS: We did not identify any RCTs but found four non-randomised studies (NRSs) including 72 participants (144 eyes), who had received DSAEK in the first eye followed by DMEK in the fellow eye. All the studies included adult participants where there was evidence of FED and endothelial failure requiring a corneal transplant for the treatment of visual impairment. We did not find any studies that included PBK. The trials were published between 2011 and 2015, and we assessed them as high risk of bias due to potential unknown confounding factors since DSAEK preceded DMEK in all participants. Two studies reported results at 12 months, one at 6 months, and one between 6 and 24 months. At one year, using DMEK in cases of endothelial failure may result in better BCVA compared with DSAEK (mean difference (MD) -0.14, 95% confidence interval (CI) -0.18 to -0.10 logMAR, 4 studies, 140 eyes, low-certainty evidence). None of the participants had severe visual loss (BCVA of 1.0 logMAR or more; very low-certainty evidence). Regarding endothelial cell count data (4 studies, 134 eyes) it is hard to draw any conclusions since two studies suggested no difference and the other two reported that DMEK provides a higher cell density at one year (very low-certainty evidence). No primary graft failure and only one graft rejection were recorded over four studies (144 eyes) (very low-certainty evidence). The most common complications reported were graft dislocations, which were recorded in one or two out of 100 participants with DSAEK but were more common using DMEK, although this difference could not be precisely estimated (risk ratio (RR) 5.40, 95% CI 1.51 to 19.3; 4 studies, 144 eyes, very low-certainty evidence). AUTHORS' CONCLUSIONS: This review included studies conducted on people with corneal endothelium failure due to FED for whom both DMEK and DSAEK can be considered, and found low-certainty evidence that DMEK provides some advantage in terms of final BCVA, at the cost of more graft dislocations needing 're-bubbling' (very low-certainty of evidence).

Neurotrophic keratopathy.

Neurotrophic Keratopathy (NK) refers to a condition where corneal epitheliopathy leading to frank epithelial defect with or without stromal ulceration (melting) is associated with reduced or absent corneal sensations. Sensory nerves serve nociceptor and trophic functions, which can be affected independently or simultaneously. Loss of trophic function and consequent epithelial breakdown exposes the stroma making it susceptible to enzymatic degradation. Nerve pathology can range from attrition to aberrant re-generation with corresponding symptoms from anaesthesia to hyperaesthesia/allodynia. Many systemic and ocular conditions, including surgery and preserved medications can lead to NK. NK can be mild (epithelium and tear film changes), moderate (non-healing epithelial defect) or severe (stromal melting and perforation). Moderate and severe NK can profoundly affect vision and adversely impact on the quality of life. Medical management with lubricating agents from artificial tears to serum/plasma drops, anti-inflammatory agents, antibiotics and anti-proteases all provide non-specific relief, which may be temporary. Contact lenses, punctal plugs, lid closure with botulinum toxin and surgical interventions like tarsorrhaphy, conjunctival flaps and amniotic membrane provide greater success but often at the cost of obscuring sight. Corneal surgery in a dry ocular surface with reduced sensation is at high risk of failure. The recent advent of biologicals such as biopolymers mimicking heparan sulfate; coenzyme Q10 and antisense oligonucleotide that suppress connexin 43 expression, all offer promise. Recombinant nerve growth factor (cenegermin), recently approved for human use targets the nerve pathology and has the potential of addressing the underlying deficit and becoming a specific therapy for NK.

Optimization of optical and mechanical properties of real architecture for 3-dimensional tissue equivalents: Towards treatment of limbal epithelial stem cell deficiency.

Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimize the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The 'optimal' RAFT TE was thin, transparent but still handleable and was produced using 0.6ml of 3mg/ml collagen. Degradation rates of the 'optimal' RAFT TE and HAM were similar. hLE achieved confluency on 'optimal' RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63α. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency.

Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in vitro.

PURPOSE: Cultured human limbal epithelial cells (HLECs) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behavior, and long-term fate after transplantation. The aim of this research was to evaluate, in vitro, quantum dot (Qdot) technology as a tool for tracking transplanted HLECs. METHODS: In vitro cultured HLECs were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony-forming efficiency assays and expression of CK3, P63alpha, and ABCG2. Sheets of cultured HLECs labeled with Qdot nanocrystals were transplanted onto decellularized human corneoscleral rims in an organ culture model and observed to investigate the behavior of transplanted cells. RESULTS: Quantum dot labeling had no detrimental effect on HLEC viability or function in vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks after transplantation in our organ culture model. In addition, it was possible to label and observe epithelial cells in intact human corneas by using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS: This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks after transplantation; however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterization in in vivo models are required.

Culture and Characterization of Oral Mucosal Epithelial Cells on a Fibrin Gel for Ocular Surface Reconstruction.

AIM OF THE STUDY: To develop a clinical grade fibrin gel for the culture of oral mucosal epithelial cells (OMEC) intended for ocular surface reconstruction in the treatment of limbal stem cell deficiency (LSCD). MATERIALS AND METHODS: Transparent fibrin gels composed of fibrinogen and thrombin were developed for the culture of epithelial cells. Oral mucosa was harvested from the buccal region of healthy volunteers and cultured as explants on fibrin gels. Tranexamic acid (TA), a clinically approved anti-fibrinolytic agent was added to prevent the fibrin gel from digesting due to cellular activity. The gels were stained for p63α (as a marker of poorly differentiated epithelial cells), CK19, CK13 and CK3 (expressed by OMEC). Epithelial cell stratification was observed using hematoxylin-eosin staining. RESULTS: Addition of TA prevented gels from dissolving during the culture period. OMEC proliferated on the fibrin gel and attained confluence over a 2-week period (±2 d) and exhibited a typical epithelial, cobblestone morphology. Basal OMEC exhibited positive staining for p63α while the superficial cells exhibited positive staining for CK3. The cells expressed a strong immunoreactivity for CK19 and CK13 suggesting that they retained a normal oral epithelial phenotype. CONCLUSION: Fibrin gels, maintained in the presence of TA, to control the rate of substrate degradation, provide a more robust yet transparent substrate for the culture and transplantation of cultured OMEC. The fibrin gels are easily standardized, the components commercially available, and produced from clinically approved materials. The resulting stratified OMEC-derived epithelium displays characteristics similar to that of a human cornea, e.g. CK3 expression. The conventional dependence on a murine feeder layer for support of epithelial cells is unnecessary with this technique and hence, provides for an attractive alternative for treatment of LSCD.

Localisation of epithelial cells capable of holoclone formation in vitro and direct interaction with stromal cells in the native human limbal crypt.

Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used in vitro clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.

Endothelial keratoplasty versus penetrating keratoplasty for Fuchs endothelial dystrophy.

BACKGROUND: Fuchs endothelial dystrophy (FED) is a condition in which there is premature degeneration of corneal endothelial cells. When the number of endothelial cells is reduced to a significant degree, fluid begins to accumulate within the cornea. As a result, the cornea loses its transparency and the individual suffers a reduction in vision. The only successful surgical treatment for this condition is replacement of part or all of the cornea with healthy tissue from a donor. The established procedure, penetrating keratoplasty (PKP), has been used for many years and its safety and efficacy are well known. Endothelial keratoplasty (EK) techniques are relatively new surgical procedures and their safety and efficacy relative to PKP are uncertain. OBJECTIVES: The objective of this review was to compare the benefits and complications related to two surgical methods (EK and PKP) of replacing the diseased endothelial layer of the cornea with a healthy layer in people with FED. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2014, Issue 1), MEDLINE (January 1950 to January 2014), EMBASE (January 1980 to January 2014), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to January 2014), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com) and ClinicalTrials.gov (www.clinicaltrials.gov). There were no date or language restrictions in the electronic searches for trials. The electronic databases were last searched on 27 January 2014. SELECTION CRITERIA: We included all randomised controlled trials (RCTs) comparing EK versus PKP for people (of any age and gender) who had been clinically diagnosed with FED. DATA COLLECTION AND ANALYSIS: Two authors independently screened the search results, assessed trial quality and extracted data using the standard methodological procedures expected by The Cochrane Collaboration. MAIN RESULTS: We included three RCTs that enrolled a total of 139 eyes of 136 participants and analysed 123 (88%) eyes. Two RCTs randomised eyes into either the endothelial keratoplasty (EK) group or penetrating keratoplasty (PKP) group and one RCT randomised eyes into either the femtosecond laser-assisted endothelial keratoplasty (FLEK) group or PKP group. The RCTs comparing EK with PKP did not show any significant differences between procedures with respect to best corrected visual acuity (BCVA) at two years (mean difference (MD) 0.14 logMAR; 95% confidence interval (CI) -0.08 to 0.36; P = 0.23) or at one year (MD 0.09 logMAR; 95% CI -0.05 to 0.23; P = 0.22), whereas the trial comparing FLEK with PKP showed significantly better BCVA after PKP (MD 0.20 logMAR; 95% CI 0.10 to 0.30; P = 0.0001). Only one RCT reported on irregular astigmatism (higher-order aberration), which was less with EK than PKP (MD -1.20 µm; 95% CI -1.53 to -0.87; P < 0.001). Only one RCT reported on endothelial cell counts (lower after FLEK than PKP: MD -969 cells/mm²; 95% CI -1161 to -777; P < 0.001), primary graft failure (higher after FLEK than PKP: RR 7.76; 95% CI 0.41 to 145.22; P = 0.10), and graft rejection (more after FLEK than PKP: RR 1.11; 95% CI 0.07 to 17.12; P = 0.94). Only one RCT reported that 27.8% of participants had graft dislocation, 2.8% had epithelial ingrowth and postoperative pupillary block, and 13.9% had intraocular pressure (IOP)-related problems in the FLEK group compared with the PKP group, in whom 10% had suture-related problems, 5% had wound dehiscence and 10% had suture revision to correct astigmatism. Overall, the adverse events in the FLEK group appeared to be more frequent than in the PKP group. No trials reported information about quality of life or economic data. The overall methodological quality of the three trials was not satisfactory as most did not perform allocation concealment or masking of participants and outcome assessors, and all trials had a small sample size. AUTHORS' CONCLUSIONS: The rapid growth of endothelial keratoplasty as the treatment of choice for FED is based upon the belief that visual recovery is more rapid, surgically induced astigmatism (regular and irregular) is less and rates of transplant rejection are lower with EK. This change in practice also assumes that the rates of long term transplant survival are equal for the two procedures. The practical differences between the surgical procedures mean that visual recovery is inherently more rapid following EK, but this review found no strong evidence from RCTs of any difference in the final visual outcome between EK and PKP for people with FED. This review also found that higher order aberrations are fewer following EK but endothelial cell loss is greater following EK. The RCTs that we included employed different EK techniques, which may have a bearing on these findings. EK procedures have evolved over the years and can be performed using different techniques, for example deep lamellar endothelial keratoplasty, Descemets stripping endothelial keratoplasty (DSEK), Descemets stripping automated endothelial keratoplasty (DSAEK), femtosecond laser-assisted endothelial keratoplasty and Descemet membrane endothelial keratoplasty (DMEK). More RCTs are needed to compare PKP with commonly performed EK procedures such as DSEK, DSAEK and DMEK in order to determine the answers to two key questions, whether there is any difference in the final visual outcome between these techniques and whether there are differences in the rates of graft survival in the long term?

Three-year outcomes of cultured limbal epithelial allografts in aniridia and Stevens-Johnson syndrome evaluated using the Clinical Outcome Assessment in Surgical Trials assessment tool.

Limbal stem cell deficiency (LSCD) is an eye disorder in which the stem cells responsible for forming the surface skin of the cornea are destroyed by disease. This results in pain, loss of vision, and a cosmetically unpleasant appearance. Many new treatments, including stem cell therapies, are emerging for the treatment of this condition, but assessment of these new technologies is severely hampered by the lack of biomarkers for this disease or validated tools for assessing its severity. The aims of this study were to design and test the reliability of a tool for grading LSCD, to define a set of core outcome measures for use in evaluating treatments for this condition, and to demonstrate their utility. This was achieved by using our defined outcome set (which included the Clinical Outcome Assessment in Surgical Trials of Limbal stem cell deficiency [COASTL] tool) to evaluate the 3-year outcomes for allogeneic ex vivo cultivated limbal epithelial transplantation (allo-CLET) in patients who had bilateral total LSCD secondary to aniridia or Stevens-Johnson syndrome. The results demonstrate that our new grading tool for LSCD, the COASTL tool, is reliable and repeatable, and that improvements in the biomarkers used in this tool correlate positively with improvements in visual acuity. The COASTL tool showed that following allo-CLET there was a decrease in LSCD severity and an increase in visual acuity up to 12 months post-treatment, but thereafter LSCD severity and visual acuity progressively deteriorated.

Laser-assisted in-situ keratomileusis (LASIK) versus photorefractive keratectomy (PRK) for myopia.

BACKGROUND: Myopia (also known as short-sightedness or near-sightedness) is an ocular condition in which the refractive power of the eye is greater than is required, resulting in light from distant objects being focused in front of the retina instead of directly on it. The two most commonly used surgical techniques to permanently correct myopia are photorefractive keratectomy (PRK) and laser-assisted in-situ keratomileusis (LASIK). OBJECTIVES: To compare the effectiveness and safety of LASIK and PRK for correction of myopia by examining post-treatment uncorrected visual acuity, refractive outcome, loss of best spectacle-corrected visual acuity, pain scores, flap complications in LASIK, subepithelial haze, adverse events, quality of life indices and higher order aberrations. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2012, Issue 11), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to November 2012), EMBASE (January 1980 to November 2012), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to November 2012), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 15 November 2012. We also searched the reference lists of the studies and the Science Citation Index. SELECTION CRITERIA: We included randomised controlled trials comparing LASIK and PRK for the correction of any degree of myopia. DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial quality and extracted data. We summarised data using the odds ratio and mean difference. We combined odds ratios using a random-effects model after testing for heterogeneity. MAIN RESULTS: We included 13 trials (1135 participants, 1923 eyes) in this review. Nine of these trials randomised eyes to treatment, two trials randomised people to treatment and treated both eyes, and two trials randomised people to treatment and treated one eye. None of the paired trials reported an appropriate paired analysis. We considered the overall quality of evidence to be low for most outcomes because of the risk of bias in the included trials. There was evidence that LASIK gives a faster visual recovery than PRK and is a less painful technique. Results at one year after surgery were comparable: most analyses favoured LASIK but they were not statistically significant. AUTHORS' CONCLUSIONS: LASIK gives a faster visual recovery and is a less painful technique than PRK. The two techniques appear to give similar outcomes one year after surgery. Further trials using contemporary techniques are required to determine whether LASIK and PRK as currently practised are equally safe. Randomising eyes to treatment is an efficient design, but only if analysed properly. In future trials, more efforts could be made to mask the assessment of outcome.

Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

Stability of eyelid height after graded anterior-approach lid lowering for dysthyroid upper lid retraction.

OBJECTIVE: To investigate the outcome of a modified anterior approach surgical procedure for the correction of primary upper eyelid retraction in thyroid eye disease. METHODS: A retrospective review of 52 consecutive cases (in 32 patients) of anterior-approach graded upper lid lowering for the treatment of primary eyelid retraction, carried out at Moorfields Eye Hospital between 2006-2009 was conducted. Measurements of upper margin-reflex distance (MRD), upper lid skin crease height and skin fold height were taken from clinical records and photographs. A comparison between pre-operative and both early and late post-operative measurements was conducted, with a maximal follow-up of 12 months. Surgery was considered successful when all of the following criteria were met; an upper lid margin covering 0.5-1.5 mm of the superior cornea in the 12 o'clock position, smooth eyelid contour, skin crease height within 6-10 mm or upper lid skin fold within 2-5 mm of the lid margin, symmetry of lid position (difference in MRD of < 1 mm between both eyes) and patient satisfaction. RESULTS: A successful outcome was achieved in 86.5% (45/52) of lids with a single procedure. For the whole group, the mean MRD was 7.0 mm pre-operatively and 3.6 mm at 1 month after surgery. The corresponding values from photographic estimates were 6.5 mm and 3.6 mm, respectively. These values remained stable over the maximum follow-up period of 12 months. Under-correction occurred in 6/52 (11.5%) lids, one of which had persistent lateral flare, whereas over-correction occurred in 1/52 (2%). CONCLUSIONS: The described surgical approach produces reasonably predictable and stable outcome for upper eyelid lowering in patients with thyroid eye disease.

Corneal stem cells in the eye clinic.

INTRODUCTION OR BACKGROUND: Corneal opacity is a common cause of blindness. The majority of cases result from ulceration and scarring following infection or trauma, but in a proportion corneal epithelial stem cell (SC) deficiency leads to an inability to maintain a healthy corneal surface. SOURCES OF DATA: This review includes systematic reviews and individual case series of treatments for corneal epithelial SC deficiency. AREAS OF AGREEMENT: Two techniques such as transplantation of large segments of cornea from a healthy eye and ex vivo expansion of corneal SCs in the laboratory were compared. Both have merits and their clinical outcomes are similar. The smaller biopsy in the cell expansion approach has less risk for the donor eye, which is a significant advantage. AREAS OF CONTROVERSY: Treatment algorithms for different aetiologies of SC failure are evolving. The proportion of true corneal epithelial SCs in ex vivo culture is unclear and it is unknown whether these cells survive long term. GROWING POINTS: In this study, the optimum method of cell culture and transplantation is being intensively investigated. AREAS TIMELY FOR DEVELOPING RESEARCH: Development of tissues using multiple cell types, genetic modification to treat hereditary corneal disorders and development of cell therapy for other eye diseases are future possibilities.

Endothelial keratoplasty versus penetrating keratoplasty for Fuchs endothelial dystrophy.

BACKGROUND: Fuchs endothelial dystrophy (FED), first described by Ernst Fuchsis in 1910, is a condition in which there is premature degeneration of corneal endothelial cells. When the number of endothelial cells is reduced to a significant degree fluid begins to accumulate within the cornea. As a result the cornea loses its transparency and the individual suffers a reduction in vision. The only successful surgical treatment for this condition is replacement of part or all of the cornea with healthy tissue from a cadaveric donor. The established procedure, penetrating keratoplasty (PKP), has been used for many years and its safety and efficacy are well known. Endothelial keratoplasty (EK) is a relatively new surgical procedure and the safety and efficacy relative to PKP are unknown. OBJECTIVES: The objective of this review is to collate information on the benefits and complications related to two surgical methods (PKP and EK) of replacing the diseased endothelial layer of the cornea with a healthy layer in people with FED. SEARCH STRATEGY: We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2011, Issue 2), MEDLINE (January 1950 to February 2011), EMBASE (January 1980 to February 2011), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to February 2011), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com) and ClinicalTrials.gov (www.clinicaltrials.gov). There were no date or language restrictions in the electronic searches for trials. The electronic databases were last searched on 6 February 2011. SELECTION CRITERIA: We included all randomised controlled trials (RCTs) comparing EK versus PKP for people (of any age and gender) who had been clinically diagnosed with FED. When assessing the primary and secondary outcome measures, only RCTs were included in the analysis. As per our protocol a description of data from non-randomised comparative studies is also reported. As RCTs may not detect differences in frequency of adverse events, when assessing these we included data from cohort studies with more than 50 participants and a follow-up of up to five years. DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial quality and extracted data. MAIN RESULTS: The electronic searches identified one RCT, five non-randomised comparative studies and 34 cohort studies with a sample size of over 50 participants for inclusion. The RCT was conducted in the USA and included 28 eyes of 25 participants with FED. Although this is a very good RCT, there may be potential for bias due to lack of masking of assessors and possible selective reporting not being clearly reported. In this RCT, comparing EK versus PKP, best corrected visual acuity (BCVA) was similar at 24 months (PKP 0.20 ± 0.20 logMAR; EK 0.34 ± 0.35 logMAR; P = 0.23) and higher order aberrations (HOAs) (deviations of the performance of an optical system from the predictions of paraxial optics) were lower with EK. No other data were reported by the trial. Based on data obtained from both non-randomised comparative studies and individual cohort studies the endothelial rejection rate was between 5.3% and 23.2% for PKP and 2% and 4% for EK. However the mean follow-up duration in the EK cohort studies was noticeably shorter. The rate of EK graft dislocation requiring repositioning ranged from 3% to 63%, with the majority of studies having a rate of < 10%. AUTHORS' CONCLUSIONS: There is no high quality evidence that EK is superior to PKP in the treatment of FED considering the studies that satisfied our primary and secondary outcome measures. One RCT demonstrated that HOAs are lower following EK and some lower quality evidence suggests that endothelial rejection episodes may be less with EK. These findings should be interpreted with caution as they are based on data with risk of biases. Further RCTs of visual and refractive outcomes needs to be performed in this field, comparing EK to PKP, with a larger sample size and at least five years of follow up. To avoid bias due to a surgeon's learning curve, procedures should be performed by experienced surgeons only. Quality of life and vision should also be evaluated. The risk of endothelial rejection will be difficult to address in the context of a RCT because of power considerations but large non-randomised comparative case series and corneal graft registry outcome data will be useful in this regard.

The protective effect of ophthalmic viscoelastic devices on endothelial cell loss during cataract surgery: a meta-analysis using mixed treatment comparisons.

BACKGROUND/AIMS: To compare ophthalmic viscoelastic devices (OVDs) in protecting the cornea from endothelial cell loss during cataract surgery. METHODS: A systematic review yielded 21 randomised controlled trials including 1769 patients. OVDs were classified according to the Arshinoff classification. Traditional pairwise meta-analyses were performed for each direct comparison. Mixed treatments comparisons (MTC) analysis was also performed to combine all direct and indirect comparisons. The outcome measure was loss in endothelial cell density 3 months after surgery. RESULTS: Direct comparison meta-analysis showed that viscoadaptives lead to a lower loss in cell density compared with very low viscosity dispersives, and compared with super viscous cohesives. The soft shell technique, a combination of viscous cohesives and medium viscosity dispersives, showed a lower loss compared with viscous cohesives, but was not compared with the other treatments. The MTC analysis shows that comparing all treatment options together, all mean differences were ≤ 100 cells/mm(2). The probability of being the best treatment option is 80% for viscoadaptives and 18% for the soft shell technique. CONCLUSION: Viscoadaptives may be superior to the other OVDs, but absolute differences in loss in endothelial cell density are <100 cells/mm(2).

IL6 and the human limbal stem cell niche: a mediator of epithelial-stromal interaction.

The corneal epithelium is maintained by the limbal epithelial stem cells (LESCs). In this study, an in vitro model is proposed for the investigation of cell-cell interactions involving LESC maintenance. Imaging of the limbal niche demonstrated close spatial arrangement between basal limbal epithelial cells within putative LESC niche structures and fibroblasts in the stroma. Interactions of the human limbal epithelial (HLE) cells and mitotically active human limbal fibroblasts (HLF) were studied for the first time in a serum-free in vitro model that simulated aspects of the limbal niche microenvironment. HLE cocultured in a ratio 3:1 with HLF exhibited enhanced expression of the putative stem cell markers ABCG2 and p63α and holoclones were preserved as shown by colony-forming efficiency assays, clonal analysis, and colony characterisation. Interleukin 6 (IL6) was found to be up-regulated in the 3.1SF system when compared to the HLE culture with growth-arrested fibroblasts and serum (gold standard system, GS). IL6 caused a time-dependent phosphorylation of STAT3 in HLE cells. STAT3 and IL6 inhibition in 3.1SF cultures significantly reduced HLE colony-forming efficiency, suggesting a previously undetected STAT3-mediated involvement of IL6 in the maintenance of HLE cells in a progenitor-like state.

Ex vivo cultured limbal epithelial transplantation. A clinical perspective.

The term ex vivo cultured limbal epithelial transplantation (CLET) refers to the process of culturing a sheet of human limbal epithelium in the laboratory and transplanting this sheet back onto the limbal stem cell-deficient cornea of the same patient or another recipient. This emerging technology represents one of the earliest successes in regenerative medicine. CLET is, at present, best suited to patients who have unilateral total limbal stem cell deficiency arising from chemical injury and who are suitable for autologous cell culture and transplantation. Although the results of allogeneic cell transplantation are encouraging and superior to conventional stem cell transplantation techniques, insufficient follow-up precludes conclusions regarding the long-term outcomes. Other tissues, such as oral mucosal epithelium, are emerging as viable alternative sources of cells, especially for patients with bilateral disease.

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.

Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.

Ex vivo expansion and transplantation of limbal epithelial stem cells.

OBJECTIVE: To determine, using objective measures, the outcome of ex vivo cultured limbal epithelial stem cell (LESC) transplantation performed in compliance with good manufacturing practice using a novel culture system without 3T3 feeder cells. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reiger's anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye). METHODS: Allogeneic (7 eyes) or autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane. Tissue was transplanted to the recipient eye after superficial keratectomy. Impression cytology and confocal microscopy were performed 6 months after surgery with clinical follow-up to 13 months. Success was defined as an improvement in the defined clinical parameters of LESC deficiency, an improvement in visual acuity, the restoration of a more normal corneal phenotype on impression cytology, and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. MAIN OUTCOME MEASURES: Clinical parameters of LESC deficiency (loss of epithelial transparency, superficial corneal vascularization, epithelial irregularity, and epithelial breakdown), visual acuity, impression cytology and cytokeratin expression profiles, and in vivo confocal corneal confocal microscopy. RESULTS: The success rate using this technique was 60% (autografts 33%, allografts 71%). All patients with a successful outcome experienced an improvement in visual acuity of >/=2 lines Snellen acuity. Preoperatively, CK3+ and CK19+ cells accounted for 12+/-2.4% (mean +/- standard error of the mean) and 80+/-2.15% of cells, respectively, whereas postoperatively these accounted for 69+/-6.43% (P<0.0001) and 30+/-6.34% (P<0.0001) of cells, respectively. Goblet cells accounted for 8+/-1.19% of cells preoperatively and 1+/-0.35% of cells postoperatively (P<0.0001). CONCLUSIONS: These data demonstrate that it is possible to culture LESCs ex vivo in compliance with good manufacturing practice regulations. A set of objective outcome measures that confirm the efficiency of this technique in treating LESC deficiency is described. The widespread use of such standardized and objective outcome measures would facilitate a comparison between the different culture methods in use.