The amniotic membrane in ophthalmology

Cell growth supports (i.e., scaffolds) that provide a conducive environment for normal cellular growth, differentiation, and angiogenesis are important components of tissue engineered grafts because rapid integration with the host is essential for long-term graft viability. While many of these scaffold materials are synthetic biodegradable polymers, others are naturally derived from mammalian tissue sources. Naturally occurring scaffold materials include small intestinal submucosa, acellular dermis, amniotic membrane tissue, cadaveric fascia, and the bladder acellular matrix graft. Upon implantation, these materials elicit a host-tissue response that initiates angiogenesis, encourages tissue deposition and culminates in restoration of structure and function specific to the grafted site. The sources, the methods of procurement and processing, and the effects of these naturally occurring materials on angiogenesis and tissue deposition are reviewed.

Naturally occurring scaffolds for soft tissue repair and regeneration.

Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases

The amniotic membrane (AM) is the inner layer of the fetal membranes and consist of 3 different layers: the epithelium, basement membrane and stroma which further consists of three contiguous but distinct layers: the inner compact layer, middle fibroblast layer and the outermost spongy layer. The AM has been shown to have anti-inflammatory, anti-fibrotic, anti-angiogenic as well as anti-microbial properties. Also because of its transparent structure, lack of immunogenicity and the ability to provide an excellent substrate for growth, migration and adhesion of epithelial corneal and conjunctival cells, it is being used increasingly for ocular surface reconstruction in a variety of ocular pathologies including corneal disorders associated with limbal stem cell deficiency, surgeries for conjunctival reconstruction, as a carrier for ex vivo expansion of limbal epithelial cells, glaucoma surgeries and sceral melts and perforations. However indiscriminate use of human AM needs to be discouraged as complications though infrequent can occur. These include risk of transmission of bacterial, viral or fungal infections to the recipient if the donors are not adequately screened for communicable diseases, if the membrane is not processed under sterile conditions or if storage is improper. Optimal outcomes can be achieved only with meticulous case selection. This review explores the ever expanding ophthalmological indications for the use of human AM.

Human amniotic membrane transplantation: Different modalities of its use in ophthalmology.

PURPOSE. Amniotic membrane has been applied to the ocular surface to restore corneal function. The beneficial effect of amniotic membrane transplantation may be due to the immunosuppressive effects of amniotic epithelial cells. The purpose of this study was to determine whether amniotic epithelial cells (AECs) secrete anti-inflammatory and antiproliferative factors that affect the chemotaxis of neutrophils and macrophages and suppress both T- and B-cell proliferation in vitro. METHODS. Human amniotic cells were isolated from human amniotic membrane and cultured in vitro. The supernatants from AEC cultures were collected after 48 hours of incubation. Neutrophil and macrophage chemotactic activity was tested in the presence of AEC supernatant, using 24-well migration assay chambers. Lymphocyte proliferation was tested by H 3 -thymidine incorporation. Apoptosis was examined by caspase-3 and annexin V assays, and expression of cytokines was assessed by RT-PCR. RESULTS. AEC supernatant significantly inhibited the chemotactic activity of neutrophils and macrophages toward macrophage inflammatory protein (MIP)-2 (P 0.05). The supernatant significantly reduced the proliferation of both T and B cells after mitogenic stimulation (P 0.05). Caspase-3 assays revealed that the supernatant induced apoptosis of T and B cells, but not of corneal epithelial cells and liver cells. In contrast to lymphocytes, macrophages and neutrophils were resistant to apoptosis induced by AEC supernatant. The AECs expressed message for TNF, Fas ligand (FasL), TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), TGF, and macrophage migration-inhibitory factor (MIF). However, AEC induction of apoptosis was inhibited (50%) by anti-FasL antibody but not by anti-TRAIL or anti-TNF antibodies. Moreover, AEC supernatant inhibited macrophage migration in vitro. CONCLUSIONS. AECs secrete soluble factors that inhibit cells in both the innate and adaptive immune systems. (Invest Ophthalmol Vis Sci. 2005;46:900‐907) DOI:10.1167/iovs.04-0495

Immunosuppressive factors secreted by human amniotic epithelial cells.

Multilayered amniotic membrane transplantation for severe ulceration of the cornea and sclera

PURPOSE To investigate tolomerase activity and p53 expression in pterygial tissue. METHODS Pterygia tissue was obtained during excisional surgery fr om 35 eyes of 35 patients, and superior bulbar conjunctival tissue from the same eye was also sampled as control when possible. Fluorescence telomeric repeat amplification protocol was used to measure telomerase activity in whole pterygium samples from 9 cases and in the epithelium and stroma of pterygium from another 10 cases. p53 protein content was measured by enzyme-linked immunosorbent assay (ELISA) in tissues obtained from 7 eyes, as well as in epithelial cell suspensions collected by brush cytology in 8 eyes. Six samples were also analyzed for UV-specific mutations in the p53 gene by the single-strand conformation polymorphism technique and DNA sequencing. A conjunctival epithelial cell line was irradiated with sublethal levels of UV-B to investigate whether telomerase activity can be induced in vitro. RESULTS In all, 63% of pterygia samples demonstrated telomerase activity, whereas all 10 paired conjunctival control samples were negative (P = 0.05, chi-square test). Of the 10 samples in which telomerase activity was measured separately in the epithelium and stroma of pterygia, 5 samples were positive in the epithelium, only 1 of which had activity in the stroma. Average telomerase activity in positive samples was 18.44 +/- 8.77 U/microg protein, compared with telomerase activity measured in a carcinoma in situ patient (33.73 U/microg), and in an immortalized conjunctival epithelial cell line (50.72 +/- 15.55 U/microg). Telomerase activity was not upregulated in this cell line by UV-B exposure. All 6 pterygia samples tested for p53 mutations did not reveal the UV-specific mutations in exons 5, 6, 7, or 8. No statistical significance was observed in the pterygium or conjunctiva p53 protein levels in epithelial cells collected by brush cytology, while p53 protein level was lower in pterygia when measured in whole tissue samples. CONCLUSIONS Telomerase activity was detected in some pterygia, mostly in the epithelium. Pterygia was not associated with an increase in epithelial p53 protein content measured by ELISA.

Telomerase activity and p53 expression in pterygia.

AIM—To study changes induced in ocular surface epithelia and the tear film by antiglaucomatous eyedrops. A β blocker (0.5% timolol) and a novel prostaglandin F2α metabolite related drug (0.12% unoprostone) were examined in a prospective, randomised fashion.
METHODS—40 patients were randomly assigned to use either 0.5% timolol (timolol group) or 0.12% unoprostone eyedrops (unoprostone group) twice a day for 24 weeks. In addition to routine ocular examinations, corneal epithelial integrity (vital staining tests, tear film break up time (BUT), anterior fluorometry, specular microscopy) and tear function (Schirmer's test, cotton thread test, tear clearance test (TCT)) were examined before and after the treatment.
RESULTS—Both eyedrops caused significant reduction in intraocular pressure from the baseline levels. No significant changes were noted in corneal integrity in both groups, except a decrease in BUT at 20 weeks in the timolol group. The timolol group demonstrated significant decreases in Schirmer's test, tear clearance test, and tear function index (Schirmer's test value multiplied by clearance test); however, no such changes were noted in the unoprostone group.
CONCLUSION—While unoprostone eyedrops caused no adverse effects on the corneal epithelial integrity and tear function, timolol caused significant impairments in tear production and turnover.

https://bjo.bmj.com/content/bjophthalmol/84/11/1250.full.pdf

Kazuomi Hanada

Papers

Multilayered amniotic membrane transplantation for severe ulceration of the cornea and sclera

Telomerase activity and p53 expression in pterygia.

Changes in ocular surface caused by antiglaucomatous eyedrops: prospective, randomised study for the comparison of 0.5% timolol v 0. 12% unoprostone.