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Wei Li, Liying Zhang, Dulei Zou, Yangluowa Qu, Sanming Li, Tingting Liu, Juan Li, Zuguo Liu; An ultra-thin amniotic membrane as carrier in corneal epithelium tissue-engineering. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1674.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic membrane (AM) is widely used as a carrier for limbal epithelial stem cell ex vivo expansion. However, the thick stroma of AM affects the postoperative corneal transparency. In this study, we developed an ultra-thin AM (UAM) and evaluated its biomaterial characteristics and efficacy in corneal epithelium tissue-engineering.
Cryo-preserved AM was treated with 0.02% EDTA, after that AM epithelium was removed by scraper. Stromal side of the denuded AM (DAM) was then digested with type IV collagenase at 37℃ for different durations to generate UAM. The characteristics including optical transmittance, thickness, decellularization and microstructure were evaluated. Intact AM (IAM) and DAM were served as control. The components of UAM were analyzed by immunostraining of collagen I, III, IV, VII, perlecan and Laminin 5. Rabbit limbal epithelial cells were cultured on UAM and DAM, cell proliferation and differentiation markers such as p63, Ki67, K3, K12, K14 were detected by immunofluorescent staining or Western blot. The engineered corneal epithelium was transplanted to the ocular surface of total limbal stem cells deficiency (LSCD) rabbit model, and the cornea was observed by slit-lamp microscope postoperatively.
Compared with IAM and DAM, UAM showed favorable optical transmittance in both moist and dry form, ultra-thin, accellular, compact and regularly collagen fibrils arrangement. The rabbit corneal limbal epithelial cells seeded on UAM formed a multilayered epithelial structure and showed better cell polarity than those on the DAM. K14 and P63 were highly expressed in UAM based tissue-engineered corneal epithelium than those in DAM at protein level. Postoperatively, the UAM based tissue-engineered corneal epithelium showed higher transparence in rabbits with total LSCD. Histology of cornea after transplantation showed better stratification of UAM based grafts compared with DAM based grafts.
UAM preserves compact layer of the amniotic membrane and maybe an ideal carrier for corneal epithelium tissue-engineering.
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