Jae Lim Lee

Clinical efficacy of amniotic membrane transplantation in the treatment of various ocular surface diseases.

PURPOSE To investigate the efficacy of permanent amniotic membrane transplantation (AMT) for the treatment of various ocular surface diseases. METHODS The medical records of 62 eyes from 58 patients who had undergone permanent AMT were reviewed. The amniotic patches were grafted for the treatment of neurotrophic ulcers (n=15), inflammatory corneal ulcers (n=15), scleral ulcers (n=11), painful bullous keratopathy (n=8) and pterygium as an adjuvant to a conjunctival autograft (n=13). Cryo-preserved or freeze-dried amniotic membrane (AM) were used. The overall success rate, the interval to epithelialization, pain-subsiding time, and complications were evaluated. The pain relief and the full epithelialization interval in the bullous keratopathy patients given the cryo-preserved AM were compared with those given the freeze-dried AM. RESULTS The success rate in the patients with neurotrophic ulcer, inflammatory corneal ulcer, scleral ulcer and bullous keratopathy were 93.3%, 66.7%, 92.9% and 100%, respectively. A conjunctival autograft with AMT showed a 100% success rate without recurrence. The time to re-epithelialization was 24.4+/-24.2, 20.4+/-5.8, and 16.9+/-7.0 days in patients with neurotrophic, inflammatory and scleral ulcers, respectively (p=0.431). The pain relief interval in the cryo-preserved and freeze-dried AM group was 17.7 and 23.3 days, and the re-epithelialization interval was 29 and 22 days, respectively, which was insignificant. CONCLUSIONS AMT has a high success in the treatment of neurotrophic, scleral ulcer, bullous keratopathy and pterygium with a low rate of complications but presented only partial success in the treatment of inflammatory corneal ulcers. The clinical efficacy of AMT was not related to the methods used to preserve the AM.

Experience of Comamonas acidovorans keratitis with delayed onset and treatment response in immunocompromised cornea.

Purpose To report 2 cases of Comamonas Acidovorans keratitis in immunocompromised cornea. Methods A complete review of the medical records of the two cases of Comamonas acidovorans keratitis. Results We found some similarities in clinical courses of two cases. Both of them showed development of keratitis during the management with corticosteroids, delayed onset, slow response to antibiotics, and relatively less affected corneal epithelium. Conclusions Comamonas Acidovorans is known as a less virulent organism. However it can cause an indolent infection that responds slowly even to adequate antibiotics therapy in immunocompromised corneas.

Long-term outcome in ocular intractable surface disease with Seoul-type keratoprosthesis.

Purpose: To evaluate the long-term clinical efficacy of the Seoul-type keratoprosthesis (S-KPro). Methods: S-KPros were implanted into 4 unsighted and 5 sighted eyes in 9 patients: 6 patients were diagnosed with Stevens-Johnson syndrome, 2 had chemical burns, and 1 suffered from ocular pemphigoid. The preoperative visual acuity ranged from light perception to hand motion. The average follow-up period was 62.8 months. We evaluated several clinical factors, including visual acuity, visual field, number of additional grafting procedures, number of capsulotomy procedures, and the interval between retinal detachment and skirt exposure. Results: The S-KPro showed anatomic success for an average of 62.8 months in 66.7% of the eyes. The average visual acuity preservation time was 31.6 months. Localized glaucomatous visual field defect was not found in any of the sighted patients; however, diffuse visual field constriction was observed after long-term follow-up. Average time of skirt exposure and mean number of additional grafting procedures were 12.9 months and 2.44, respectively. Retinal detachments were developed in all the patients at a mean time interval of 2 months after S-KPro exchange. Conclusions: S-KPro achieved visual rehabilitation for an average of 31.6 months with long-term anatomic stability in patients with severe intractable ocular surface disease.

Biocompatibility of nanocomposites used for artificial conjunctiva: in vivo experiments.

Purpose: To evaluate the biocompatibility of nanocomposites used for artificial conjunctiva. Methods: Fifty New Zealand white rabbits were used for the experiments. Nanocomposites of poly ϵ-caprolactone (PCL) and of PCL coated with polyvinyl alcohol (PCL+PVA), polyvinyl pyrrolidone (PCL+PVP), or chitosan (PCL+C), and amniotic membrane (AM) as a control, were cut into small disks with a diameter of 3.5 mm. The disks were inserted underneath the conjunctiva to measure their inflammation-inducing properties. To investigate epithelial adhesion and goblet cell differentiation, the disks were transplanted after round conjunctival excision. Cultivated conjunctival epithelial cells on nanocomposite were then transplanted onto the abdomen of Balb/c athymic mice. The number of inflammatory cells and the density of goblet cells were measured using hematoxylin and eosin, periodic-acid–Schiff, and immunohistochemistry after 2 weeks. Results: The number of inflammatory cells found inside of the inserts was as follows: 21 ± 4.9 for controls, 21 ± 15.1 for PCL, 49.6 ± 26.0 for PCL+PVP, 40.2 ± 17.1 for PCL+C, and 13.8 ± 3.9 for PCL+PVA. In PCL+PVA, the accumulation of inflammatory cells was significantly lower than in the controls (p < 0.01, Mann-Whitney U). The reepithelialization of conjunctival cells was accomplished in more than 75% of all disks except for the PCL+C. In addition, we found the differentiation of goblet cells in the following order from greatest to least: amniotic membrane, PCL, and PCL+PVP. Conclusions: Nanocomposites of PCL were biocompatible in rabbit conjunctiva, suggesting that PCL may be considered as a candidate for use in the development of artificial conjunctiva.

Isolation of Putative Corneal Epithelial Stem Cells from Cultured Limbal Tissue

Purpose To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. Methods Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37℃ or 4℃ for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37℃ in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. Results The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3×104 cell/ml and 8.06×105 cell/ml at 37℃and 4℃ incubations; the number increased to 1.21×106 cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67±2.13% and 6.63±2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61±0.42% and 5.21±4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67±2.24% and 1.17±6.13%, respectively (p<0.05). Conclusions Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.

Short-term Efficacy of Topical Immunosuppressive Agents on the Survival of Cultivated Allo-Conjunctival Equivalents.

Purpose To investigate the short-term efficacy of topical immunosuppressive agents on the survival of cultivated allo-conjunctival equivalents. Methods Twenty-five eyes of New Zealand white rabbits were included. Temporal conjunctivae were trephined to a diameter of 7.5 mm, and then cultured allo-conjunctival epithelial cells on amniotic membrane were transplanted onto them. Various immunosuppressants including steroid, cyclosporine, and rapamycin were applied topically four times a day for a week. Epithelial defects and graft edema were graded daily. Numbers of inflammatory cells were measured in H&E. PKH26 and cytokeratin 4 and 7 were immunostained. Results Earlier epithelialization was observed in 1% steroid-treated eyes and defects persisted significantly in 0.5% CsA applied eyes. In histology, PKH26 positive cells considered as donor cells were only found in 1% steroid or 0.01% rapamycin applied eyes. 1% steroid- or 0.01% rapamycin-applied eyes both showed positive staining for keratin-4 and -7. Inflammatory cells were less found in 1% steroid or 0.01% rapamycin treated eyes. Conclusions Topical steroid or rapamycin can help to suppress acute inflammation and enhance the acute survival of transplanted conjunctival cells.

Efficient Cultivation Conditions for Human Limbal Epithelial Cells

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Efficient cryopreservative conditions for cultivated limbal and conjunctival epithelial cells.

Purpose: To examine the effects of cryopreservation on the viability of cultivated corneal limbal and conjunctival epithelial cells and to evaluate the optimal conditions for cryopreservation. Methods: The cultivated human limbal epithelial cells (HLECs) were stored in media including 20%, 50%, and 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) at −196°C for 1 week. The cultivated rabbit conjunctival epithelial cells were stored in 10%, 20%, and 50% FBS with 10% glycerol or DMSO as a cryoprotectant at −196°C for 1 week. After thawing, cell viability was assessed using the trypan blue vital staining and 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay. Immunofluorescent staining was performed with cytokeratin 3/12 antibody. Colony-forming efficiency (CFE) was evaluated 2 weeks after culture. Results: HLECs cryopreserved with 50% FBS showed the highest cell viability, whereas those with 20% FBS revealed the lowest survival rate (87.1% ± 0.8% and 79.8% ± 4.01%, respectively; P = 0.030). CFE of HLECs was 2.13 ± 1.35%, 2.31 ± 2.23%, and 1.94 ± 0.72% in cells with 20%, 50%, and 90% FBS, respectively (P > 0.05). For conjunctival epithelial cells, the cell viability was the highest with 50% FBS and 10% glycerol (95.0% ± 4.27%), and the lowest survival rate was observed in the condition of 10% FBS and 10% DMSO (80.0% ± 5.49%). CFE of cryopreserved conjunctival epithelial cells was 14.1% ± 1.9% in cells with 20% FBS and glycerol and 13.5% ± 2.0% in those with 20% FBS and DMSO (P > 0.05). HLECs expressed CK3/12 after cryopreservation in all conditions examined. Conclusions: The best results were yielded by 50% FBS for cell viability in HLECs. Glycerol seems to be superior to DMSO in cell viability of the rabbit conjunctival epithelium after cryopreservation.

Change of Proliferation Rate of Corneal Epithelium in the Rabbit with Orthokeratology Lens

Objective: To investigate the cell proliferation rate of normal corneal epithelium with extended orthokeratology lens (OKL) wear in comparison with extended rigid gas-permeable (RGP) lens wear. Methods: Twenty-four rabbits were fitted unilaterally with either an OKL or an RGP lens, and the other eye was used as a control. They were injected with 5-bromo-2-deoxyuridine (BrdU) 24 h prior to being sacrificed. The rabbits were sacrificed at 1, 3, 7 and 14 days after lens fitting. The cornea from the superior limbus to the center was taken at 1.0-mm intervals. The BrdU-labeled cells were counted in medium power fields (×200) in each sample using light microscopy. Results: The number of BrdU-labeled cells in the RGP lens group initially increased, but the number decreased in the corneal center and superior limbus by 32 and 8%, respectively, after 14 days. There was no statistically significant change. However, the number of BrdU-labeled cells in the OKL group decreased after 3 days, and the number of BrdU-labeled cells was reduced in the center and superior limbus by 63 and 8%, respectively, after 14 days. The change in proliferation in the corneal center in the OKL-wearing rabbits was statistically significant compared to the control (p < 0.05). Conclusions: Wearing an OKL had a greater effect on the change of the proliferation pattern in the epithelium than wearing an RGP lens, which suggests that the OKL might be less physiologic than the RGP lens is.

Adhesion complex in cultivated limbal epithelium on amniotic membrane after in vivo transplantation

Purpose: To investigate adhesion complex formation in cultivated human limbal epithelium after transplantation into the limbal deficient model. Methods: Cultivated epithelium on amniotic membrane was transplanted into limbal deficient rabbits. The transplanted rabbits and the controls were sacrificed at 1, 2, 3, and 4 weeks. The adhesion complex was examined by electron microscopy and immunohistochemistry. Results: Morphologically identifiable hemidesmosomes appeared at 1 week, and matured adhesion complex was found at 3 weeks. Collagen VII was partly stained after transplantation. The mean numbers of hemidesmosomes/2.25 μ m were 2.3 ± 0.9, 2.5 ± 0.5, 5.2 ± 1.0, and 4.0 ± 0.9 at 1, 2, 3, and 4 weeks, and all they were smaller than those in the control, respectively (p < 0.05). It reached 137.4% of the density of hemidesmosomes in human cornea at 3 weeks. The average depths of anchoring fibril were 0.10 ± 0.03, 0.27 ± 0.06, 0.45 ± 0.06, and 0.46 ± 0.12 μ m at 1, 2, 3, and 4 weeks, reaching 75.0% of that in the human cornea after 3 weeks, although they were shallower than that of the control, respectively (p < 0.05). Conclusions: Assembly of adhesion complex in cultivated epithelium transplanted in limbal deficient rabbit might recover to the level of that in the human after 3 weeks, although it was delayed compared with that in normal wound healing of the rabbit.