Penetrating keratoplasty in Asian eyes: the Singapore Corneal Transplant Study.

PURPOSE: The Singapore Corneal Transplant Study (SCTS) is a 16-year prospective study of 2100 consecutive corneal transplants performed between January 1991 and November 2006 in patients from Southeast Asia at a single tertiary center. The indications, complications, long-term survival rates, and risk factors for graft failure of penetrating keratoplasty (PK) performed in Asian eyes are reported. DESIGN: Prospective cohort study. PARTICIPANTS: Of the 2100 corneal transplants, 1130 consecutive PKs were performed from January 1991 to December 2003. One graft per patient was selected, leaving 901 grafts for analysis. METHODS: Data were obtained from the Singapore Eye Bank's SCTS database. Cases were classified into optical, therapeutic, and tectonic indications and 9 corneal disease groups. Twenty-four demographic, preoperative, intraoperative, and donor risk factors were subjected to Kaplan-Meier survival analysis, univariate analysis, and multivariate analysis by Cox proportional hazards regression modeling. MAIN OUTCOME MEASURE: Graft failure, defined as the irreversible loss of optical clarity. RESULTS: Study patients were Asian, comprising Chinese (72.7%), Indian (11.54%), and Malay (11.1%) ethnicities (mean age, 56.65 years). The mean follow-up period was 36.8+/-35.5 months. Indications for surgery were optical (87.0%), therapeutic (8.1%), and tectonic (4.88%). Main diagnoses were pseudophakic/aphakic bullous keratopathy (23.4%), postinfectious scarring (12.9%), regrafts (12.4%), keratoconus (9.7%), and posttraumatic scarring (7.3%). Kaplan-Meier survival rates for optical grafts were 86.6%, 72.0%, 63.7%, and 52.0% at 1, 3, 5, and 10 years, respectively; survival rates for therapeutic grafts were 78.4%, 58.3%, and 37.3% at 1, 3, and 5 years, and those for tectonic grafts were 68.3% and 41.7% at 1 and 3 years. Endothelial rejection and late endothelial decompensation accounted for 50.51% of failures. Multivariate analysis revealed 9 predictors for graft failure: recipient gender, age, graft size, graft endothelial status, primary corneal disease, glaucoma, inflammation, perforation, and corneal vascularization. CONCLUSIONS: The long-term outcome for optical indications in Asian eyes follows a trend in endothelium-related attrition similar to that seen in the West. Tectonic and therapeutic keratoplasty for corneal infections and perforation, however, constitute a significant proportion of corneal transplantation performed in Asia and carry a graver prognosis in terms of graft survival.

The Boston keratoprosthesis in Stevens-Johnson syndrome.

PURPOSE: To evaluate the use of the Boston keratoprosthesis (KPro) in patients with Stevens-Johnson syndrome (SJS). DESIGN: Retrospective, noncomparative, interventional case series. METHODS: Sixteen eyes of 15 patients with SJS underwent KPro surgery at the Massachusetts Eye and Ear Infirmary from January 2000 through December 2005. The preoperative, operative, and postoperative findings were recorded. All patients underwent either the type I or type II Boston KPro surgery by one surgeon (C.H.D.). Retention of the prosthesis, best-corrected visual acuity, the need for surgical revision, and postoperative complications were recorded. The outcomes were compared with those of an earlier group of patients from the 1990s. RESULTS: The mean age of patients was 50+/-18 years (range, 23 to 74 years), and the mean duration of their disease was 10+/-6.6 years. The mean follow-up period was 3.6+/-1.5 years (range, 10.2 months to 5.6 years). Ten eyes underwent type II KPro surgery, whereas six eyes underwent type I KPro surgery. Twelve eyes (75%) achieved a visual acuity of 20/200 or better after surgery, with eight eyes (50%) achieving excellent vision of 20/40 or better. Visual acuity was maintained at 20/200 or better over a mean period of 2.5+/-2.0 years. Preexisting glaucoma was found to be a significant risk factor for visual loss. There were no cases of KPro extrusion or endophthalmitis. CONCLUSIONS: KPro in SJS has improved, largely because of the introduction of vancomycin prophylaxis and better glaucoma treatment. It seems to be superior to standard penetrating keratoplasty, with or without allografted stem cell transplantation, as judged from the literature. However, the outcome of the KPro in SJS is still substantially less favorable than in nonautoimmune diseases.

Current understanding of the treatment and outcome of acute primary angle-closure glaucoma: an Asian perspective.

INTRODUCTION: Primary angle-closure glaucoma (PACG) is a major cause of blindness among Asians. A better understanding of the disease will improve the treatment and outcome of this condition. METHODS: A literature review of all recent publications on PACG was carried out. Articles were retrieved using a key word search of MEDLINE, PubMed and Science Citation Index databases. RESULTS: Following laser peripheral iritodomy for acute angle-closure, Asians were found to have a higher tendency to develop a subsequent rise in intraocular pressure compared to Caucasians. Furthermore, the extent and severity of visual field damage was more severe in Asians than Caucasians, particularly in eyes that presented insidiously with chronic PACG. Prophylactic laser iridotomy in the contralateral eye was found to be highly effective in preventing acute angle-closure attacks. CONCLUSION: PACG is more difficult to manage and is associated with more severe long-term visual morbidity in Asians than Caucasians. Regular follow-up of patients with PACG is important for the early detection of progression of the disease and visual field deterioration.

LASIK in the presbyopic age group: safety, efficacy, and predictability in 40- to 69-year-old patients.

OBJECTIVE: To report the safety, efficacy, and predictability of LASIK in a presbyopic population and to examine possible differences between age groups. DESIGN: Retrospective, descriptive, comparative consecutive case series. PARTICIPANTS: Seven hundred ten eyes of 424 patients between 40 and 69 years of age who underwent LASIK by the same surgeon between January 1999 and September 2005. Patients had manifest refractive spherical errors ranging from -10.5 to +6 diopters (D) and cylinder of up to 2.50 D. METHODS: LASIK was performed with IntraLase femtosecond laser or Hansatome microkeratomes and VISX Star (S4 or S2; VISX Inc., Santa Clara, CA) or Baush & Lomb Technolas 217z (Zyoptix or PlanoScan; Bausch & Lomb, Claremont, CA) excimer lasers. There were 511 myopic eyes (spherical equivalent [SE], -5.03+/-2.02 D) and 199 hyperopic eyes (SE, +2.21+/-1.21 D). Patients were divided into 3 groups: group 1 (40 to 49 years old; n = 359 eyes), group 2 (50 to 59 years old; n = 293 eyes), and group 3 (60 to 69 years old; n = 58 eyes). MAIN OUTCOME MEASURES: Uncorrected visual acuity (UCVA) and best spectacle-corrected visual acuity (BSCVA), retreatment rates, safety, efficacy, and predictability. RESULTS: The mean age of patients in groups 1, 2, and 3 was 45, 53, and 63 years, respectively. With increasing age, there was a trend toward higher retreatment rates and more myopic end points. Safety, efficacy, and predictability were comparable between all groups. At the final follow-up (mean+/-standard deviation, 10.4+/-7.4 months), 80% to 100% of eyes had 20/30 or better UCVA and 81% to 90% were within +/-1.00 D. No eyes lost more than 2 lines of BSCVA, and only 9 eyes (1.3%) lost 2 lines. Subgroup analysis of eyes with a follow-up of 12 months or more was performed. The results were compared with those with shorter follow-up. Both subgroups had comparable outcomes; the duration of follow-up did not affect the visual outcomes. CONCLUSIONS: Despite a trend toward worse final BSCVA and higher retreatment rates in older patients, a greater risk of visual loss after LASIK was not observed. LASIK for myopia and hyperopia has reasonable safety, efficacy, and predictability profiles in the 40- to 69-year-old presbyopic population.

New grading system for the evaluation of chronic ocular manifestations in patients with Stevens-Johnson syndrome.

PURPOSE: To evaluate and grade the extent and severity of chronic ocular manifestations in Stevens-Johnson syndrome (SJS). DESIGN: Prospective multicenter case series. PARTICIPANTS: We enrolled 73 patients (138 eyes) with SJS seen between April 2003 and March 2005 at 3 tertiary referral centers. METHODS: Patients with a confirmed history of SJS and chronic ocular complications that persisted for at least 1 year from the onset of SJS were included. Their detailed medical history and ophthalmic examination results were recorded on an itemized data collection form. Complications were categorized as corneal, conjunctival, and eyelid complications, and 13 components were evaluated and graded on a scale from 0 to 3 according to their severity. MAIN OUTCOME MEASURES: These were broadly classified as corneal (superficial punctate keratopathy, epithelial defect, loss of the palisades of Vogt, conjunctivalization, neovascularization, opacification, keratinization), conjunctival (hyperemia, symblepharon formation), and eyelid (trichiasis, mucocutaneous junction involvement, meibomian gland involvement, punctal damage) complications. RESULTS: The most severely affected complication components were loss of the palisades of Vogt (114 eyes; 82.6%) and meibomian gland involvement (102 eyes; 73.9%). Visual acuity in 74 of the 138 eyes (53.6%) was worse than 20/200. The severity of corneal, conjunctival, and eyelid complications was significantly correlated with visual loss. All 13 complications were correlated significantly with logarithm of the minimum angle of resolution (logMAR) visual acuity; the correlation coefficient (R) ranged from 0.359 to 0.810 (P<0.0001); for corneal epithelial defects, R was 0.169 (P = 0.0473). Eyes with a higher total score for the 3 complication categories had poorer vision (R = 0.806; P<0.0001). Multivariate regression analysis showed that corneal neovascularization, opacification, keratinization, and cataracts significantly affected logMAR visual acuity (P<0.0001, P<0.0001, P = 0.0142, P = 0.0375, respectively). CONCLUSIONS: The authors describe a new method for grading the extent and severity of ocular involvement in patients with SJS and demonstrate that the severity of ocular involvement is correlated significantly with the final visual outcome. This new grading system provides a more objective method for evaluating SJS patients and may be adapted for use in other cicatricial ocular surface diseases.

A comparison between cultivated and conventional limbal stem cell transplantation for Stevens-Johnson syndrome.

PURPOSE: To compare the resolution of inflammation and long-term results of cultivated and conventional limbal stem cell transplantation (LSCT) in a patient with Stevens-Johnson syndrome (SJS). DESIGN: Interventional case report. METHODS: A 32-year-old man with SJS and bilateral total limbal stem cell deficiency underwent cultivated LSCT in the right eye, followed by conventional LSCT in the left eye three weeks later. The postoperative medication included dexamethasone 0.1% and ofloxacin 0.3% eyedrops and a tapering dose of systemic corticosteroid, cyclosporine, and cyclophosphamide. Tear samples were collected and analyzed for interleukin (IL) 8 levels. RESULTS: Complete corneal epithelialization was achieved 48 hours after cultivated LSCT, compared with three weeks after conventional LSCT. Ocular inflammation and IL-8 levels decreased more rapidly in the eye with cultivated LSCT. Four years after surgery, more severe corneal scarring and opacification were noted in the conventional LSCT eye. CONCLUSIONS: Cultivated LSCT resulted in a better clinical result and vision, with less stromal scarring compared with conventional LSCT.

The development of a serum-free derived bioengineered conjunctival epithelial equivalent using an ultrathin poly(epsilon-caprolactone) membrane substrate.

PURPOSE: To evaluate the use of an ultrathin poly(epsilon-caprolactone) (PCL) membrane as a substrate for the development of a serum-free-derived conjunctival epithelial equivalent. METHODS: Ultrathin PCL membranes 6 microm in thickness were prepared by solvent casting and biaxial stretching and analyzed by atomic force microscopy (AFM), scanning electron microscopy (SEM), tensile testing, and water-contact angle measurement. Rabbit conjunctival epithelial cells were cultivated on sodium hydroxide (NaOH)-treated PCL membranes and untreated PCL membranes in serum-free medium. The proliferative capacity of cultivated cells was analyzed with a bromodeoxyuridine (BrdU) ELISA proliferation assay. Conjunctival equivalents were xenografted into severe combined immune-deficient (SCID) mice. Immunostaining for tissue-specific and basement membrane-related proteins was performed. RESULTS: After biaxial stretching, the tensile strength of PCL membranes increased from 21 to 42 MPa, with a Young's modulus of 225 MPa. AFM and SEM showed that biaxially stretched PCL membranes consisted of closely packed microfibrils. PCL membranes supported the attachment and proliferation of conjunctival epithelial cells to form confluent stratified epithelial sheets. Surface modification with NaOH resulted in greater hydrophilicity and cellular proliferation than that of untreated membranes. Transplanted conjunctival equivalents underwent greater proliferation and stratification in vivo. Cultivated conjunctival cells expressed K4, K19, MUC5AC, and Ki67, whereas collagen IV and integrin beta4 were detected at the basement membrane junction. CONCLUSIONS: An ultrathin PCL membrane was shown to be biocompatible, mechanically strong enough to stand up to handling, and able to support conjunctival epithelial cell proliferation. This membrane may have potential for use as a scaffold matrix for tissue-engineered conjunctival equivalents.

Establishment of a cultivated human conjunctival epithelium as an alternative tissue source for autologous corneal epithelial transplantation.

PURPOSE: The corneal epithelium is essential for maintaining corneal transparency, and efforts have been made to develop improved techniques for corneal epithelial transplantation in patients with total limbal failure. We evaluated the suitability of transplanted cultivated human conjunctival epithelium (HCjE) as a corneal epithelium replacement in rabbits with total corneal and limbal deficiency. METHODS: HCjE cells, cultivated on human amniotic membrane (AM) to confluence and exposed to an air-liquid interface (air-lifted), were transplanted onto denuded rabbit corneas and monitored for 2 weeks. The cultivated HCjE sheet and the engrafted epithelium were analyzed by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The transplanted HCjE remained transparent, smooth, and without epithelial defects during the follow-up period. Both the cultivated HCjE cells and the engrafted epithelium manifested five to six layers of stratified squamous epithelium similar in morphology to normal corneal epithelium. The basal cells expressed the putative stem cell markers (ABCG2 and P63) and hemidesmosome and desmosome component proteins. The cytokeratins (CK4, CK13, CK3, and CK12) and MUC4 were found in the engrafted epithelium. However, MUC5AC was not expressed. The results indicate that HCjE cultivated on AM has the potential to be used as an alternative corneal epithelium. CONCLUSIONS: The transplantation of cultivated HCjE sheets is a promising technique for the treatment of eyes with limbal failure.

The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome.

PURPOSE: To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). METHODS: AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. RESULTS: BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. CONCLUSIONS: AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease.

PURPOSE: To evaluate the use of autologous serum (AS)-derived cultivated corneal epithelial transplantation for the treatment of severe ocular surface disease. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Nine eyes from 9 patients with total limbal stem cell deficiency were studied. These consisted of 2 eyes with Stevens-Johnson syndrome, 1 with chemical injury, 1 with ocular cicatricial pemphigoid, 1 with Salzmann corneal dystrophy, 1 with aniridia, 1 with graft-versus-host disease, and 2 with idiopathic ocular surface disease. METHODS: Autologous serum obtained from patients was used for cultivating corneal epithelial cells on an amniotic membrane substrate. These AS-derived corneal epithelial equivalents were compared with those derived from fetal bovine serum (FBS)-supplemented medium. At the time of surgery, complete removal of the corneal pannus and conjunctiva up to 3 mm from the limbus was performed. Allogeneic (7 cases) and autologous (2 cases) AS-derived cultivated corneal epithelial equivalents were transplanted onto the ocular surface. Postoperative follow-up included serial slit-lamp examinations with fluorescein staining, as well as photographic documentation. MAIN OUTCOME MEASURES: Ocular surface reconstruction with corneal epithelialization, graft integrity, visual acuity, and postoperative complications. RESULTS: The corneal epithelial sheets cultivated in AS- and FBS-supplemented media were morphologically similar, and demonstrated the normal expression of tissue-specific keratins and junctional specialization assembly proteins. After transplantation, complete corneal epithelialization was achieved within 2 to 5 days. All eyes demonstrated an improvement in visual acuity by > or =2 lines. During the follow-up period of 14.6+/-4.36 months, the corneal surface of all patients remained stable and transparent, without significant complications. CONCLUSIONS: Transplantation of AS-derived cultivated corneal epithelial equivalents was shown to be a feasible method of treating patients with severe ocular surface disease. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Long-term outcomes in fellow eyes after acute primary angle closure in the contralateral eye.

PURPOSE: To determine the long-term outcome of the contralateral eye in Asian persons with a unilateral attack of acute primary angle closure (APAC). DESIGN: Cross-sectional observational case series. PARTICIPANTS: Seventy-nine individuals who were examined from 4 to 10 years after a unilateral episode of APAC at 2 Singapore hospitals. METHODS: All subjects underwent a complete eye examination. The optic discs were graded clinically and photographically for the presence of glaucomatous optic neuropathy, and automated visual field tests were assessed for damage. All visual fields and optic nerve photographs (when available) underwent a second evaluation by a masked glaucoma specialist, who assessed whether the changes were compatible with glaucoma. MAIN OUTCOME MEASURES: Glaucomatous optic neuropathy, intraocular pressure (IOP), and visual impairment (defined as best-corrected visual acuity of worse than 6/12). RESULTS: Ninety of 138 eligible patients (65.2%) with APAC were examined, 79 with unilateral attacks. Subjects were predominantly Chinese (n = 68; 86%). There were 54 females (68%), and mean age was 68.5+/-8.9 years (standard deviation) at the time of APAC, with a mean duration of 6.3+/-1.5 years from the time of the APAC episode to the study examination. The contralateral eyes of 7 patients (8.9%) had definite or probable glaucoma, 2 of whom were felt to have glaucoma in that eye at the time of the attack. Three eyes had markedly cupped optic discs (cup-to-disc ratio > or = 0.9). Thirteen eyes (16.9%) had best-corrected vision worse than 6/12, due to cataract in almost half the cases. Only 1 eye had vision < 6/60, the cause of which was corneal decompensation. One other patient had only a central island remaining with vision of 6/12. Mean IOP of the study participants was 15.7+/-4.7 mmHg, with 6 subjects (7.6%) having undergone trabeculectomy. CONCLUSIONS: Definite or probable glaucoma was present at the time of diagnosis in 2 (2.5%) fellow eyes and developed in an additional 5 (6.5%) with a mean follow-up of 6 years. More than 80% of this cohort retained good vision in the contralateral eye, in contrast to the eye that underwent APAC. Unoperated cataract accounted for most of the visual impairment in this group.

Autologous serum-derived cultivated oral epithelial transplants for severe ocular surface disease.

OBJECTIVE: To evaluate the use of autologous serum (AS)-derived cultivated oral epithelial transplants for the treatment of severe ocular surface disease. METHODS: We used AS from 10 patients with severe ocular surface disease and total limbal stem cell deficiency to develop autologous cultivated oral epithelial equivalents. These were compared with epithelial equivalents derived from conventional fetal bovine serum-supplemented medium. Surgery involved removal of the corneal pannus and surrounding diseased tissue and transplantation of the AS-derived epithelial equivalents. The oral equivalents were analyzed by review of histologic and immunohistochemical findings. RESULTS: Oral epithelial sheets cultivated in AS- and fetal bovine serum-supplemented media were similar in morphology, and both formed basement membrane assembly proteins important for maintaining graft integrity. Complete corneal epithelialization was achieved within 2 to 5 days postoperatively. The ocular surface remained stable without major complications in all eyes during a mean +/- SD follow-up of 12.6 +/- 3.9 months. The visual acuity improved by more than 2 lines in 9 of 10 eyes, with transplanted oral epithelium surviving up to 19 months. CONCLUSION: The successful use of an AS-derived oral epithelial equivalent to treat severe ocular surface disease represents an important advance in the pursuit of completely autologous xenobiotic-free bioengineered ocular equivalents for clinical transplantation.

Inferior corneal decompensation following laser peripheral iridotomy in the superior iris.

PURPOSE: To describe a previously unreported clinical entity of inferior corneal decompensation occurring as a long-term complication of superior laser peripheral iridotomy. DESIGN: Retrospective, non-comparative observational case series. METHODS: Chart review of 14 subjects (16 eyes) with inferior corneal decompensation. RESULTS: All patients were Chinese, most were females (71.4%), and the mean age was 69.5 +/- 8.1 year. The mean interval from iridotomy to the onset of inferior corneal decompensation was 66.1 +/- 34.0 months. The most common indication for laser iridotomy was for prophylaxis, either in the fellow eye of acute angle closure (43.8%) or for occludable angles (12.5%). CONCLUSIONS: Corneal decompensation in the inferior part of the cornea at a site remote from the iridotomy is a rare but important complication of laser iridotomy. Because many of the laser iridotomies were performed prophylactically in eyes without glaucoma, patients may need to be warned of this risk.

Modified automated lamellar therapeutic keratoplasty for keratoconus: a new technique.

PURPOSE: To describe a modified automated lamellar therapeutic keratoplasty (ALTK) technique for the treatment of keratoconus. METHODS: The ALTK procedure was modified to include the use of a Hanna trephine system to control donor and recipient diameters and was performed in 2 stages. The recipient corneal lamellar bed was first prepared by creating a laser in situ keratomileusis (LASIK) flap at a preset depth that was left to heal over. The second stage involved the use of a Hanna trephination system to perform central trephination within the lamellar flap and transplantation of a donor lamellar graft that was fashioned to precisely match the recipient corneal bed, again using the Hanna donor punch to create a central trephination within the lamellar button obtained with the ALTK unit. RESULTS: The modified ALTK procedure effectively treated a patient with moderate keratoconus with a best-corrected visual acuity of 20/80, despite rigid gas-permeable contact lenses. Postoperatively, there was a significant reduction in the irregular astigmatism, and visual acuity improved to 20/25. There were no complications, and the ALTK interface remained clear throughout the 20-month follow-up period. CONCLUSIONS: ALTK combined with Hanna trephination enables precise control of both depth and diameter of lamellar dissection during surgery, removes the need for any form of manual dissection, and may therefore improve optical and visual outcomes in lamellar keratoplasty.

Characterisation of human tear proteins using high-resolution mass spectrometry.

INTRODUCTION: The 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 METHODS: Tear 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. RESULTS: In 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. CONCLUSIONS: The results showed that the proteomic strategy used in this study was successfully applied to analyse tear proteome.

Enhancement of the mechanical and biological properties of a biomembrane for tissue engineering the ocular surface.

INTRODUCTION: In 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 METHODS: The 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. RESULTS: We 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. CONCLUSION: By 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.

Autologous cultivated conjunctival transplantation for recurrent viral papillomata.

PURPOSE: To describe the use of an autologous serum-free derived cultivated conjunctival epithelial equivalent in the treatment of extensive recurrent viral papillomata. DESIGN: Interventional case report. METHODS: A 10-year-old child with extensive recurrent viral papillomata involving the superior and inferior tarsal, forniceal, and bulbar conjunctiva underwent surgical excision of all diseased areas and double freeze-thaw cryotherapy. Autologous serum-free cultivated conjunctival equivalents were used to reconstruct the ocular surface and conjunctival fornices. RESULTS: Almost complete epithelialization was achieved within 5 days postoperatively. A good cosmetic and functional result was obtained, and no recurrences or cicatricial complications developed during 12-month follow-up. CONCLUSION: Transplantation of autologous cultivated conjunctiva was effectively used in the reconstruction of the ocular surface after extensive excision of recurrent viral papillomata. This modality of treatment may be useful in the treatment of ocular surface disorders in which extensive conjunctival and fornix reconstruction is required.

Autologous cultivated conjunctival transplantation for pterygium surgery.

PURPOSE: To investigate the safety and efficacy of autologous cultivated conjunctival transplantation for the treatment of primary pterygium. DESIGN: Prospective nonrandomized clinical trial. METHODS: Forty patients with primary pterygium were recruited. Excision of the pterygium was followed by reconstruction using a serum-free derived autologous cultivated conjunctival equivalent in 22 patients (group A) and conventional denuded amniotic membrane transplantation in 18 patients (group B). In group A patients, conjunctival epithelial equivalents were constructed by the ex vivo expansion of conjunctival epithelial cells on human amniotic membranes (HAM). The main outcome measures were conjunctival epithelialization, recurrence, survival analysis, and incidence of complications. RESULTS: The mean follow-up was 14.1 +/- 3.9 months (range, 12 to 25 months). Complete epithelialization was achieved within five days after surgery in group A patients compared with approximately three weeks for group B patients. The proportion of patients that had true recurrences was 22.7% in group A and 25.0% in group B. The mean time to recurrence was 4.8 +/- 1.6 months in group A and 5.0 +/- 2.9 months in group B. No ocular complications were noted in group A, while one eye (6.0%) in group B developed scleral necrosis associated with a persistent epithelial defect. CONCLUSIONS: Transplantation of an autologous cultivated conjunctival epithelial sheet facilitated early postoperative epithelialization and recovery, and may aid in preventing serious complications associated with simple denuded HAM transplantation, such as scleral necrosis and secondary infection. This may provide a novel method for conjunctival epithelial replacement in the treatment of ocular surface disorders.

Reconstruction of the ocular surface by transplantation of a serum-free derived cultivated conjunctival epithelial equivalent.

PURPOSE: The purpose of this study was to investigate the use of a serum-free derived cultivated conjunctival epithelial sheet for ocular surface transplantation and reconstruction. METHODS: Seven subjects with various ocular surface disorders were selected for the procedure: one patient had an extensive conjunctival nevus, three patients had pterygium, two patients had persistent leaking trabeculectomy blebs, and one patient had bilateral superior limbic keratoconjunctivitis. Conjunctival epithelial cells were harvested from the forniceal conjunctiva of patients 2 weeks before the definitive surgery. Cultivation of conjunctival epithelial cells on human amniotic membrane (HAM) was carried out under serum-free conditions. At the time of transplantation, the area of diseased conjunctiva was excised and the cultured conjunctival epithelium-HAM composite was transplanted onto the surgical defect. Patients were followed up with serial slit-lamp examinations, fluorescein staining, and photographic documentation. RESULTS: A confluent stratified conjunctival epithelial sheet was formed on the HAM within 12 to 14 days. Transplanted grafts remained well-epithelialized after surgery. A successful outcome, defined as resolution of the disease, maintenance of conjunctival epithelialization, maintenance of graft integrity, and absence of significant complications, was obtained in all seven patients. A good functional and cosmetic result was achieved in all eyes. The mean follow-up period was 11.6 months (range, 6-18 months). CONCLUSIONS: Transplantation of a serum-free derived autologous cultivated conjunctival epithelial sheet on HAM was successfully performed in seven patients with ocular surface disorders. This may provide a novel method for conjunctival replacement in conditions where the normal conjunctiva is damaged or deficient.

Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system.

PURPOSE: To investigate the use of a multistep serum-free culture system in developing a conjunctival epithelial equivalent with improved in vitro and in vivo proliferative properties and to evaluate the effect of serum supplementation and culture conditions on the proliferative capacity of these cells. METHODS: Conjunctival epithelial cells were cultivated on human amniotic membrane (HAM) in a multistep serum-free culture system, under submerged and air-lifted conditions. The bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and number of cell generations were compared with those in serum-containing medium. The in vivo proliferative capability of the tissue-constructs were evaluated by xenotransplantation to SCID mice. Cultured cells were evaluated for the expression of keratin-4, -19, and -3, as well as MUC5AC goblet cell mucin. RESULTS: The epithelial cells cultivated in serum-free medium (BrdU absorbance, 1.91 +/- 0.08; cell generations, 25.6 +/- 4.5) were more proliferative than those cultivated in serum-containing medium (BrdU absorbance, 1.06 +/- 0.08; cell generations, 12.1 +/- 3.0). The serum-free-derived epithelial equivalents demonstrated a significant increase in proliferation and stratification after transplantation. Cells that were air lifted for 6 and 12 days had a reduced proliferative capacity in vitro and in vivo compared with submerged cultures. Cultured cells expressed keratin-4 and -19, and MUC5AC mRNA was detected by RT-PCR. Electron microscopy demonstrated a basal lamina with numerous hemidesmosomes. CONCLUSIONS: This is a multistep serum-free culture system for developing a conjunctival epithelial equivalent with improved proliferative and structural properties, which are crucial for enhancing graft survival and regeneration of the conjunctival surface after clinical transplantation.