Safety outcomes and long-term effectiveness of ex vivo autologous cultured limbal epithelial transplantation for limbal stem cell deficiency.

PURPOSE: To evaluate the safety and effectiveness of ex vivo autologous cultured limbal stem cell transplantation (CLET). METHODS: We reviewed the clinical records of 59 consecutive patients treated with 65 CLETs. Efficacy was graded 1 year after surgery as successful, partially successful or failed. A safety analysis was performed considering side effects and complications that were recorded during the first year after CLET and those reported later than 1 year, including the events related to subsequent treatments. RESULTS: The mean post-CLET follow-up was 6.0±4.1 years. 69% of CLETs had either one or more adverse events (AEs), or adverse drug reactions (ADRs), within 1 year of surgery, with inflammation being the most common (42%), followed by corneal epithelium defects/disepithelialisation (31%), and blood coagula under the fibrin (24%). One year after surgery, 41% of the 59 primary CLET procedures were successful, 39% partially successful and 20% failed. The most common ADRs recorded for the primary unsuccessful CLETs were ulcerative keratitis, melting/perforation, and epithelial defects/disepithelialisation. Six failed CLETs required reconstructive penetrating keratoplasty (PK). Among CLETs with a favourable outcome, 13 underwent corrective PK (mean 4.8±3.4 years), and thereafter seven eyes maintained integrity of the corneal epithelium, five showed corneal surface failure, and one had recurrent epithelial defects. Corneal graft rejection episodes were reported in 71% and 58% of patients following corrective or reconstructive PK, respectively. Seven primary CLETs with a favourable outcome worsened thereafter, and the overall 3-year long-term effectiveness was 68%. CONCLUSIONS: This study addresses important issues regarding possible risks associated with disarray of the ocular surface homeostasis following autologous CLET in patients with limbal stem cell deficiency, despite the fact that the majority of patients experienced a favourable long-term benefit.

Effect of 60 kHz and 150 kHz Femtosecond Lasers on Corneal Stromal Bed Surfaces: A Comparative Study.

Purpose. To compare the effect of 60 kHz and 150 kHz femtosecond (FS) laser on the corneal stromal bed surfaces (SBS). Methods. Sixteen human donor corneal tissues unsuitable for transplantation were used. Anterior and posterior lamella was obtained using 60 kHz and 150 kHz FS laser. A standard depth of 400 µ m was set for anterior lamellar keratoplasty (ALK) and endothelial lamellar keratoplasty (ELK). The quality and smoothness of the SBS post-FS laser dissection were graded for statistics. Results. No intraoperative complications were found. The side cuts were straight, and the SBS appeared smoother in cuts obtained using 150 kHz. The average values of the SBS quality of the anterior lamellar cut were found to be 2.25 (±0.28) for 60 kHz and 3.125 (±0.25) for 150 kHz (P = 0.0039). Whereas, 2 (±0.4) for 60 kHz and 2.75 (±0.28) for 150 kHz (P = 0.0273) was the quality observed in endothelial cuts. No significant difference was found between anterior and posterior cuts performed using the same FS laser (60 kHz or 150 kHz) (P > 0.05). Conclusions. The 60 kHz and 150 kHz FS lasers are equally effective in performing lamellar dissection for ALK and ELK. 150 kHz FS laser allows a tighter spot and layer separation which creates a slightly smoother SBS.

Limbal stem cell deficiency and ocular phenotype in ectrodactyly-ectodermal dysplasia-clefting syndrome caused by p63 mutations.

OBJECTIVE: To describe the ocular phenotype in patients with ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome (MIM#604292) and to determine the pathogenic basis of visual morbidity. DESIGN: Retrospective case series. PARTICIPANTS: Nineteen families (23 patients) affected by EEC syndrome from the United Kingdom, Ireland, and Italy. METHODS: General medical examination to fulfill the diagnostic criteria for EEC syndrome and determine the phenotypic severity. Mutational analysis of p63 was performed by polymerase chain reaction-based bidirectional Sanger sequencing. All patients with EEC syndrome underwent a complete ophthalmic examination and ocular surface assessment. Limbal stem cell deficiency (LSCD) was diagnosed clinically on the basis of corneal conjunctivalization and anatomy of the limbal palisades of Vogt. Impression cytology using immunofluorescent antibodies was performed in 1 individual. Histologic and immunohistochemical analyses were performed on a corneal button and corneal pannus from 2 EEC patients. MAIN OUTCOME MEASURES: The EEC syndrome phenotypic severity (EEC score), best-corrected Snellen visual acuity (decimal fraction), slit-lamp biomicroscopy, tear function index, tear breakup time, LSCD, p63 DNA sequence variants, impression cytology, and corneal histopathology. RESULTS: Eleven heterozygous missense mutations in the DNA binding domain of p63 were identified in all patients with EEC syndrome. All patients had ocular involvement and the commonest was an anomaly of the meibomian glands and lacrimal drainage system defects. The major cause of visual morbidity was progressive LSCD, which was detected in 61% (14/23). Limbal stem cell deficiency was related to advancing age and caused a progressive keratopathy, resulting in a dense vascularized corneal pannus, and eventually leading to visual impairment. Histologic analysis and impression cytology confirmed LSCD. CONCLUSIONS: Heterozygous p63 mutations cause the EEC syndrome and result in visual impairment owing to progressive LSCD. There was no relationship of limbal stem cell failure with the severity of EEC syndrome, as classified by the EEC score, or the underlying molecular defect in p63. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Sixty-kilohertz femtosecond laser-assisted endothelial keratoplasty: clinical results and stromal bed quality evaluation.

PURPOSE: To evaluate preliminary clinical results and lamellar and side cut obtained with a 60-kHz femtosecond laser (IntraLase) using a new software designed to create penetrating cuts for keratoplasty to perform the donor and recipient dissection in femtosecond laser-assisted endothelial keratoplasty. DESIGN: Prospective interventional case series. METHODS: A femtosecond laser set in a raster spot pattern was used to create the posterior lamellar dissection and posterior side cut in 4 donor corneas and in 4 recipient human eyes of 4 patients with endothelial decompensation. The laser cut was performed to obtain a 150-µm thick and 9-mm wide posterior lamellar disk from the donor and the recipient eyes. Postoperatively, patients underwent slit-lamp examination and Snellen visual acuity evaluation, Visante optical coherence tomography, optical pachymetry, and endothelial microscopy analysis. Three residual donor corneas were analyzed by scanning electron microscopy and transmission electron microscopy to observe the stromal bed surface, the side cut, and the effects of the laser dissection on the cornea ultrastructure. MAIN OUTCOME MEASURES: Thickness of the implanted donor lamella, adhesion and clarity of the transplanted cornea in the postoperative follow-up, and smoothness of the interface. RESULTS: All the patients showed good adhesion and fit of the donor disk to the recipient and a circle side cut observed at the slit-lamp examination. Visante optical coherence tomography evaluation confirmed the adhesion and the precalibrated thickness of the implanted lamella. A good corneal transparency was achieved within the first 2-3 weeks. The scanning electron microscopy analysis showed a smooth stromal residual surface and a precise side cut. The transmission electron microscopy images showed that the laser randomly modified the structural design of the collagen fibers for approximately 10-µm depth, although below a regular stromal structure was maintained. CONCLUSIONS: The 60-kHz IntraLase femtosecond laser creates a smooth stromal bed and precise side cut for endothelial keratoplasty. The posterior lamellar cut performed at the same depth in both the donor and the recipient makes it possible to create a corneal disk from the donor that fits exactly with the cut in the recipient.

Techniques for culture and assessment of limbal stem cell grafts.

The therapeutic use of limbal cultures for the permanent regeneration of corneal epithelium in patients with limbal stem cell deficiency (LSCD) has been reported in many studies. According to the guidelines for good manufacturing practice (GMP), strictly regulated procedures and stringent quality control tests are now required to manipulate stem cells as "medicinal products" and make engraftment safer and eventually more successful. This paper describes techniques for optimal preparation of limbal stem cell grafts, including 1) a reliable impression cytology assay for the grading of LSCD, 2) culture methods that maintain high percentages of limbal stem cells, 3) the use of specific markers for the detection of corneal, conjunctival, and limbal stem cells, namely keratin 12, mucin 1, and DeltaNp63alpha, and 4) assays to assess the presence of contaminants, such as murine fibroblasts, endotoxins, mycoplasmae, and viral particles, in the cultured graft. The use of some of these assays allowed us to obtain a regenerated normal corneal epithelium in approximately 80% of 166 LSCD patients who received transplants from 2004 to 2008.

The use of the femtosecond laser in penetrating keratoplasty.

PURPOSE: To evaluate a new technique for penetrating keratoplasty (PK) with the use of a new software algorithm for a femtosecond laser that is designed to create penetrating cuts for PK in the treatment of a number of corneal diseases. DESIGN: Prospective, interventional case series. METHODS: All eyes were treated at the Ospedale Civile di Mestre, Umberto, Italy. Seven eyes of seven patients underwent surgery for PK using a 15-kHz femtosecond laser (IntraLase, Irvine, California, USA) and a new software specifically developed for corneal surgery. Of the seven patients, five were keratoconus patients and two had bullous keratopathy. New software was used to create penetrating cuts in a top hat or mushroom configuration. After surgery, all patients were evaluated with pachymetry, corneal topography, refraction, intraocular pressure measurement, and corneal optical coherence tomography (Visante; Carl Zeiss, Oberkochen, Germany). RESULTS: On postoperative day one, all seven eyes had nearly clear corneas with a good graft of the donor corneas to the patients' corneas. At three months, all eyes had clear corneas with good endothelial cell counts and quiet anterior chambers. Normal corneal thickness was achieved in each case. At the three-month visit, suture removal was performed in five eyes to adjust for astigmatism. CONCLUSIONS: Although this is a small number of eyes, early indications are that the use of the new IntraLase software for corneal surgery creates a more favorable environment for PK as a result of a better fit of the donor cornea and a quicker visual recovery for patients.

The CORTES study: corneal transplant indications and graft survival in an Italian cohort of patients.

PURPOSE: To describe the corneal transplantation activity in Italy, to assess the long-term graft survival, and to begin to outline the potential risk factors for graft outcome. METHODS: We followed a consecutive series of penetrating (PK) and lamellar (LK) keratoplasties performed with corneas procured and distributed by the Veneto Eye Bank Foundation, which provides about one third of the corneas grafted in Italy each year. RESULTS: Data on 4415 PKs and 489 LKs performed in 174 clinical centers are reported. Keratoconus was the major transplant indication (47% and 66%, respectively, for the 2 groups), followed by regraft (14%) and bullous keratopathy (14%) in the PK group and keratitis (8%) and refractive reasons (4%) for the LKs. In the 2 groups, graft survival, after 1 year, was estimated to be 95% and 93%, respectively, showing a decrease of the survival rate during the second and third years of study. Graft survival in patients with keratoconus indication was 98% in the PK group and 95% in the LK group for the whole period of observation, whereas the patients with other indications reported a survival rate ranging from 92% after 1 year to 52% after 3 years (PK) and from 89% to 85% (LK). CONCLUSIONS: CORTES is the most extensive survey on corneal transplantation in Italy that involves a large cohort of patients and a significant number of surgeons with corneal tissues processed and distributed by a single eye bank. In the first 3 years, a picture of the epidemiology of the corneal transplant has been defined. The graft survival rates were comparable to those reported by other studies for the same follow-up period. However, the follow-up of a sample of this cohort for a further 3 years will allow us to precisely estimate the long-term graft survival and to better evaluate the risk factors related to graft failure.