Biomedical Research ; Blindness ; etiology ; prevention & control ; surgery ; Corneal Diseases ; complications ; prevention & control ; surgery ; Corneal Transplantation ; education ; methods ; Education, Medical, Graduate ; Health Promotion ; Humans ; Ophthalmology ; education ; Singapore

INTRODUCTIONTo determine the differentiation of human limbal epithelial cells in tissue culture.

MATERIALS AND METHODSEpithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR).

RESULTSThe colony-forming efficiency of primary cultured (P0) cells was about 19.35 +/- 6.46% (mean +/- SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P <0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 +/- 1.27)% (n = 7, P0) and (3.96 +/- 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse.

CONCLUSIONSHuman limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.

Cell Differentiation ; Coculture Techniques ; Connexin 43 ; metabolism ; Cornea ; cytology ; Epithelial Cells ; Humans ; Immunohistochemistry ; Keratins ; metabolism ; Limbus Corneae ; cytology ; Protein Precursors ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; Tissue Culture Techniques

Conventional corneal transplantation, in the form of penetrating keratoplasty (PK), involves full-thickness replacement of the cornea, and is a highly successful procedure. However, the cornea is anatomically a multi-layered structure. Pathology may only affect individual layers of the cornea, hence selective lamellar surgical replacement of only the diseased corneal layers whilst retaining unaffected layers represents a new paradigm shift in the field. Recent advancements in surgical techniques and instrumentation have resulted in several forms of manual, microkeratome and femto-second laser-assisted lamellar transplantation procedures. Anterior lamellar keratoplasty (ALK) aims at replacing only diseased or scarred corneal stroma, whilst retaining the unaffected corneal endothelial layer, thus obviating the risk of endothelial allograft rejection. Posterior lamellar keratoplasty/endothelial keratoplasty (PLK/EK) involves the replacement of the dysfunctional endothelial cell layer only. Whilst significant technical and surgical challenges are involved in performing lamellar micro-dissection of a tissue which is only 0.5 mm thick, the benefits of a more controlled surgical procedure and improved graft survival rates have resulted in a shift away from conventional PK. This review details the current advances in emerging lamellar corneal surgical procedures and highlights the main advantages and disadvantages of these new lamellar corneal procedures.
Contraindications ; Corneal Transplantation ; methods ; trends ; Humans

INTRODUCTIONThis study aims to identify the demographics and hygiene behaviours associated with contact lens (CL)-related microbial keratitis in Singapore.

MATERIALS AND METHODSThe hygiene and social behaviours of lens wearers presenting with infectious keratitis in Singapore were examined using an externally validated questionnaire. Fifty-eight consecutive lens wearers who presented with infectious keratitis at Singapore National Eye Centre were surveyed using a self-administered questionnaire. Patients' demographics, CL-related behaviour, attitude to aftercare visits and microbial study results were collected and analysed.

RESULTSMore than half (55%) of the patients surveyed were female, and the average age of the participants was 25.7 ± 6.4 years. Ninety-six percent of participants used soft CL, with the majority wearing monthly disposable lenses (74.1%). Myopia was the commonest reason for use of CL. CL overwear (81%), sleeping (50.9%) and swimming (33%) with their CL were the major non-compliant behaviours discovered. When encountered with eye discomfort as a result of lens-related infective keratitis, 83% of patients sought the advice of general medical practitioners prior to presentation at the hospital. Many (59%) patients have encountered media coverage but only 24% had any behavioural change as a result. Many patients relied on the recommendations of eyecare practitioners when purchasing disinfecting solutions (26%) and other hygiene practices. Pseudomonas Aeruginosa was the most common organism grown in 22.4% of patients while 47% of patients were culture-negative.

CONCLUSIONInadequate lens cleaning, poor aftercare, sleeping and swimming with CL were the major non-compliant behaviours among participants, and these are significant factors when assessing risks in acquiring CL-related infectious keratitis. General eyecare practitioners are the first-line of contact in patients presenting with keratitis symptoms and play an important role in emphasising the need for regular aftercare and good hygiene practices to CL wearers.

Contact Lenses, Hydrophilic ; Demography ; Humans ; Hygiene ; Keratitis ; Singapore

INTRODUCTIONThis study aimed to describe and compare corneal graft survival and optical outcomes following deep anterior lamellar keratoplasty (DALK) and Descemet's stripping automated endothelial keratoplasty (DSAEK) with penetrating keratoplasty (PK), and to document tectonic success of patch grafts.

MATERIALS AND METHODSThis was a retrospective, non-randomised, comparative and descriptive cohort study. A total of 139 eyes that underwent primary keratoplasty between 2000 and 2016 were included, and the following data was extracted: demographics, clinical diagnosis and primary indication, pre- and intraoperative risk factors, postkeratoplasty outcomes, and complications. Optical success was defined as good graft clarity and best corrected visual acuity (BCVA) of 6/12 or better. Graft failure was defined as irreversible corneal oedema and loss of clarity. Tectonic success in patch grafts was defined as tectonic integrity with no repeat tectonic surgical procedure required in the postoperative period.

RESULTSThe mean follow-up duration was 3.24 ± 3.47 years in the PK group (n = 16), 1.89 ± 0.86 years in the DALK group (n = 37), 2.36 ± 1.24 years in the DSAEK group (n = 53), and 2.17 ± 1.09 years in the patch graft group (n = 33). The 3-year probabilties of survival for PK, DALK, DSAEK and patch graft were 60.9%, 94.1%, 89.9%, and 67.1%, respectively. The overall percentage of complications was significantly higher for PK (81.3%), compared to DALK (48.6%), DSAEK (49.1%), and patch graft (21.2%). In the PK and DALK groups, 100% achieved DSAEK (49.1%), and patch graft (21.2%). In the PK and DALK groups, 100% achieved BCVA of 6/12 or better, while in the DSAEK group, 96.43% achieved BCVA of 6/12 or better.

CONCLUSIONFrom a similar study cohort of Asian eyes, graft survival was superior and complications were reduced for DALK and DSAEK compared to PK, but optical outcomes were comparable. Graft survival for patch graft was expectedly lower, but the incidence of complications was low.

Corneal Diseases ; surgery ; Corneal Transplantation ; Female ; Follow-Up Studies ; Graft Survival ; Humans ; Male ; Outcome Assessment (Health Care) ; Registries ; Retrospective Studies ; Singapore ; Tertiary Care Centers

INTRODUCTIONThe proteins found in tears play an important role in maintaining the ocular surface and changes in tear protein components may reflect changes in the health of the ocular surface. Proteomics provides a comprehensive approach for cataloguing all the proteins of the tear proteome, which will help to elucidate disease pathogenesis, make clinical diagnoses and evaluate the influence of medications on the structure, composition and secretion of tear proteins. In this study, an alternative proteomic strategy was investigated to explore the human tear proteome.

MATERIALS AND METHODSTear samples were obtained from patients who had pterygium and were collected on the first day and third day after pterygium surgery. Tears pooled from 6 patients were used in the analysis. Reverse-phase high-pressure liquid chromatograph (RPHPLC) was used as the first step to separate intact proteins into 21 peaks. Each fraction was then tryptic-digested and analysed by nanoLC-nano-ESI-MS/MS to characterise the protein components in each fraction.

RESULTSIn total, 60 tear proteins were identified with high confidence, including well-known abundant tear proteins, and tear-specific proteins such as lacritin and proline-rich proteins. Among them, proline-rich protein 5 was found for the first time in tear fluid. A large number of plasma proteins were also observed in tear fluid.

CONCLUSIONSThe results showed that the proteomic strategy used in this study was successfully applied to analyse tear proteome.

Eye Proteins ; analysis ; Humans ; Mass Spectrometry ; methods ; Proteome ; Tears ; chemistry

INTRODUCTIONIn this study, we have developed and optimised a novel gelatin-chitosan (GC) substrate for use as a cellular carrier for tissue-engineered conjunctival epithelium.

MATERIALS AND METHODSThe substrate was fabricated by casting and the mechanical properties of the substrate, including tensile strength and elongation, were measured. Using the MTT, cell proliferation assay with rabbit conjunctival fibroblasts, we optimised the G:C ratio to enhance cytocompatibility. Rabbit conjunctival epithelial cells were immunostained using monoclonal antibodies for keratin 4 and pancytokeratin to investigate the biological effects of the GC substrate on the proliferation and differentiation of epithelial cells.

RESULTSWe found that increasing the amount of gelatin resulted in an increase in elasticity (from 1:9 to 1:1 ratio), reaching a maximum (101.89% +/- 7.13%) at a ratio of 1:1. The MTT assay showed that the proliferation of conjunctival fibroblasts significantly increased from 0.068 +/- 0.017 to 0.177 +/- 0.011 (P = 0.014) as the gelatin was increased from 20% (1:4) to 50% (1:1). Additional studies using tissue-cultured conjunctiva explants showed that these explants grew well on the substrate, forming a multilayered epithelium. Cell morphology on this substrate was similar to that of cells grown on culture dishes alone. Positive staining of keratin 4 and pancytokeratin indicated that the substrate supported normal differentiation of conjunctival epithelial cells.

CONCLUSIONBy enhancing the proportion of gelatin, both the mechanical and biological properties of the chitosan substrate were improved. The results also suggest that this GC biomembrane may be a useful candidate for reconstructive tissue engineering of the conjunctiva.

Animals ; Biocompatible Materials ; Cell Proliferation ; Cells, Cultured ; Chitosan ; Conjunctiva ; cytology ; metabolism ; physiology ; Elasticity ; Epithelium ; physiology ; Gelatin ; Keratins ; metabolism ; Rabbits ; Tensile Strength ; Tissue Engineering

Cochlear implants (CIs) have demonstrated a clear functional benefit in children with severe-to-profound sensorineural hearing loss (SNHL) and thus have gained wide acceptance for treating deafness in the pediatric population. When evaluating young children for cochlear implantation, there are unique considerations beyond the standard issues addressed during surgery in adults. Because of advances in genetic testing, imaging resolution, CI technology, post-implant rehabilitation, and other factors, issues related to CI surgery in children continue to evolve. Such factors have led to changes in candidacy guidelines, vaccine requirements, and lowering of age requirement for surgery. In addition, differences in the anatomy and physiology of infants require special attention to ensure safety when operating on young children. This review summarizes these issues and provides guidance for surgeons treating children with SNHL.

Cochlear implants (CIs) have demonstrated a clear functional benefit in children with severe-to-profound sensorineural hearing loss (SNHL) and thus have gained wide acceptance for treating deafness in the pediatric population. When evaluating young children for cochlear implantation, there are unique considerations beyond the standard issues addressed during surgery in adults. Because of advances in genetic testing, imaging resolution, CI technology, post-implant rehabilitation, and other factors, issues related to CI surgery in children continue to evolve. Such factors have led to changes in candidacy guidelines, vaccine requirements, and lowering of age requirement for surgery. In addition, differences in the anatomy and physiology of infants require special attention to ensure safety when operating on young children. This review summarizes these issues and provides guidance for surgeons treating children with SNHL.