Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

BACKGROUND/AIM: Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. METHODS: The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. RESULTS: Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. CONCLUSIONS: These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.

Amniotic membrane transplantation for partial limbal stem cell deficiency.

AIM: To examine the efficacy, safety, and long term outcomes of amniotic membrane transplantation for corneal surface reconstruction in cases of partial limbal stem cell deficiency. METHODS: 17 eyes of 15 patients with partial limbal stem cell deficiency underwent superficial keratectomy of the conjunctivalised corneal surface followed by amniotic membrane transplantation. Cases were followed up for at least a year. RESULTS: All eyes exhibited a stable, intact corneal epithelial surface after a mean follow up period of 25.8 months with no eyes developing recurrent erosion or persistent epithelial defect. The mean time to re-epithelialisation was 22.8 days. Overall improvement in visual acuity was observed in 92.9% of 14 eyes with visual potential. Of those, five eyes gained six or more lines, two eyes gained between four and five lines, six eyes gained between one and three lines, and one eye lost three lines of Snellen acuity. Pain and photophobia were abolished in 86% of cases and substantially reduced in 14%, with all eyes exhibiting decreased vascularisation and inflammation at final follow up. CONCLUSIONS: Amniotic membrane transplantation appears to be a safe and effective method of restoring a stable corneal epithelium for cases of partial limbal stem cell deficiency and can be considered as an alternative to limbal autograft or allograft.

Amniotic membrane transplantation after extensive removal of primary and recurrent pterygia.

OBJECTIVE: To evaluate the postoperative outcome and the recurrence rate after extensive removal of primary and recurrent pterygia combined with amniotic membrane transplantation. DESIGN: A noncomparative interventional case series. PARTICIPANTS: Fifty-four eyes in 54 subjects with either primary (n = 33) or recurrent (n = 21) pterygia operated on by one surgeon (SCGT). INTERVENTION: All subjects were operated on for pterygia with an extensive excision of the lesion followed by amniotic membrane transplantation and intraoperative injection of a depot corticosteroid. MAIN OUTCOME MEASURES: Cumulative rates of conjunctival (grade 3) and corneal (grade 4) recurrence and incidence of complications. RESULTS: The mean follow-up was 12.8 +/- 4.3 months for primary and 14.3 +/- 4.9 months for recurrent pterygia. The true recurrence rate (grade 4) was 3.0%, 9.5%, and 5.6% for primary, recurrent, and all pterygia, respectively. The cumulative proportion of recurrence-free eyes at 12 months was 0.90 +/- 0.06 for primary and 0.69 +/- 0.11 for recurrent pterygia (P = 0.047, log-rank test). Removal of the semilunar fold was associated with longer survival times (P = 0.063) and decreased failure rate (P = 0.046). A similar success rate was achieved in double-head pterygia (1 recurrence in 11 eyes). CONCLUSIONS: Amniotic membrane transplantation is an effective and safe procedure for pterygium surgery, with a relatively low recurrence rate for both primary and recurrent pterygia. It can be a useful alternative to conjunctival autograft when a large conjunctival defect has to be covered, such as in primary double-head pterygia and in large recurrent pterygia.

Amniotic membrane transplantation for symptomatic conjunctivochalasis refractory to medical treatments.

UNLABELLED: PURPOSE. To determine whether preserved human amniotic membrane can restore the large conjunctival defect created during surgical removal of conjunctivochalasis. METHODS: Amniotic membrane transplantation was performed at two facilities in 40 consecutive patients (47 eyes) with symptomatic conjunctivochalasis refractory to conventional treatments. RESULTS: The majority of patients were elderly (73.1 +/- 9.7 years) and women (75%). Over a follow-up period of 6.9 +/- 4.3 months, 46 (97.8%) eyes recovered smooth, quiet, and stable conjunctival surfaces. Epithelial defects healed in 16.5 +/- 7.3 days. Episodic epiphora was resolved in 24 of 30 (83.3%) eyes and improved in five other eyes. Notable relief was also noted for such symptoms as fullness or heaviness (19/19, 100%), sharp pain (6/6, 100%), redness (14/17, 88.2%), tiredness (17/20, 80.9%), itching (11/13, 78.6%), blurry or decreased vision (6/8, 75%), burning (8/13, 61.5%), foreign body sensation (8/13, 61.5%), and crust formation (1/2, 50%). Complications included focal inflammation of the host conjunctiva adjacent to the graft (six eyes), scar formation (five eyes), and suture-induced granuloma (one eye). CONCLUSION: Amniotic membrane transplantation can be considered as an effective means for conjunctival surface reconstruction during removal of conjunctivochalasis.

Amniotic membrane transplantation for severe neurotrophic corneal ulcers.

AIMS: To evaluate whether amniotic membrane transplantation can be an effective alternative treatment for neurotrophic corneal ulcers. METHODS: Amniotic membrane transplantation was performed in 16 eyes of 15 patients with neurotrophic corneal ulcers and vision equal to or worse than 20/200. The neurotrophic state was developed following keratoplasty (four eyes), herpes zoster ophthalmicus (four eyes), diabetes mellitus (four eyes), radiation (two eyes), removal of acoustic neuroma with neuroparalysis (one eye), and herpes simplex keratitis (one eye). RESULTS: During a mean follow up period of 18.8 (SD 13.0) months, one to three layers of amniotic membrane with or without additional membrane as a patch were used for 17 procedures in 16 eyes for persistent neurotrophic corneal ulcers. All but four (76.4%) instances of amniotic membrane transplantation achieved rapid epithelialisation in 16.6 (9.0) days. Of the four eyes showing delayed healing, three eyes healed by tarsorrhaphy, and the remaining one eye with corneal perforation required penetrating keratoplasty and tarsorrhaphy. Two eyes gained vision better than 20/200. The healed corneal surface was accompanied by reduced inflammation. CONCLUSION: Amniotic membrane transplantation can be considered an effective alternative for treating severe neurotrophic corneal ulcers.

Amniotic membrane transplantation or conjunctival limbal autograft for limbal stem cell deficiency induced by 5-fluorouracil in glaucoma surgeries.

PURPOSE: To determine if human amniotic membrane transplantation or limbal stem cell transplantation is effective to restore the corneal surface with partial or total limbal stem cell deficiency, respectively, caused by 5-fluorouracil (5-FU) toxicity after glaucoma surgeries. METHODS: Partial and total limbal stem cell deficiency was confirmed by impression cytology as the cause of reduced vision and corneal surface breakdown in a 69-year-old man and a 67-year-old man, respectively, who both had received a total of 105 mg 5-FU injections. Amniotic membrane transplantation or conjunctival limbal autograft was performed for corneal surface reconstruction, respectively. RESULTS: For a period of 15 months of follow-up, the visual acuity improved, and their corneal surfaces remained avascular, smooth, and without recurrence of limbal stem cell deficiency. CONCLUSION: Limbal stem cell deficiency can occur as a late complication for patients receiving 5-FU after glaucoma filtering surgeries. Partial limbal stem cell deficiency can be treated with amniotic membrane transplantation alone, whereas limbal transplantation must be considered as an alternative for total limbal stem cell deficiency to restore the corneal surface integrity and vision.

Amniotic membrane transplantation for acute chemical or thermal burns.

PURPOSE: To determine whether preserved human amniotic membrane (AM) can be used to treat ocular burns in the acute stage. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Thirteen eyes from 11 patients with acute burns, 10 eyes with chemical burns and 3 with thermal burns of grades II-III (7 eyes) and grade IV (6 eyes), treated at 7 different facilities. METHODS: Patients received amniotic membrane transplantation (AMT) within 2 weeks after the injury. MAIN OUTCOME MEASURES: Integrity of ocular surface epithelium and visual acuity during 9 months of follow-up. RESULTS: Ten patients were male and one patient was female; most were young (38.2 +/- 10.6 years). For a follow-up of 8.8 + 4.7 months, 11 of 13 eyes (84.63%) showed epithelialization within 2 to 5 weeks (23.7 +/- 9.8 days), and final visual acuity improved > or = 6 lines (6 eyes), 4 to 5 lines (2 eyes), and 1 to 3 lines (2 eyes); only one eye experienced a symblepharon. Eyes with burns of grade II to III showed more visual improvement (7.3 +/- 3 lines) than those with burns of grade IV (2.3 +/- 3.0 lines; P < 0.05, unpaired t test). In the group with grade II or III burns, none had limbal stem cell deficiency. All eyes in the group with grade IV burns did experience limbal stem cell deficiency. CONCLUSIONS: Amniotic membrane transplantation is effective in promoting re-epithelialization and reducing inflammation, thus preventing scarring sequelae in the late stage. In mild to moderate burns, AMT alone rapidly restores both corneal and conjunctival surfaces. In severe burns, however, it restores the conjunctival ocular surface without debilitating symblepharon and reduces limbal stromal inflammation, but does not prevent limbal stem cell deficiency, which requires further limbal stem cell transplantation. These results underscore the importance of immediate intervention in the acute stage of eyes with severely damaged ocular surface. Further prospective randomized studies including a control group are required to determine the effectiveness of AMT in acute chemical and thermal burns of the eye.

Amniotic membrane transplantation for symptomatic bullous keratopathy.

OBJECTIVE: To determine whether amniotic membrane transplantation can be used to treat symptomatic bullous keratopathy displaying poor visual potential. METHODS: Amniotic membrane transplantation was performed at 5 centers on 50 consecutive eyes (50 patients) with symptomatic bullous keratopathy and poor visual potential. The underlying causes of bullous keratopathy included aphakia (9 eyes), pseudophakia (19 eyes), failed grafts (9 eyes), and others (13 eyes). RESULTS: During the follow-up period of 33.8 weeks (3-96 weeks) after amniotic membrane transplantation, 43 (90%) of 48 eyes with intolerable pain preoperatively became pain free postoperatively. Among the 5 eyes with residual pain, 3 received repeated amniotic membrane transplantation, 1 required a conjunctival flap for pain relief, and 1 had reduced pain. Epithelial defects in 45 (90%) of 50 eyes created and covered by amniotic membrane healed rapidly within 3 weeks. Only 4 eyes (8%) showed recurrent surface breakdown. Epithelial edema or bullae recurred in a smaller area in 5 eyes (10%) and pseudopterygium developed in 1 eye. CONCLUSION: Amniotic membrane transplantation can be considered as an alternative to conjunctival flaps in alleviating pain, promoting epithelial healing, and preserving cosmetic appearance in patients with symptomatic bullous keratopathy and poor visual potential.