Evaluation of reconstructed human corneal endothelium sheets made with porcine Descemet's membrane in vitro and in vivo

被引:11
作者
Lu, Qing [1 ]
Peng, Rong-Mei [1 ]
Feng, Na [2 ]
Wen, Ming-Da [2 ]
He, Lin-Hui [1 ]
Hong, Jing [1 ]
机构
[1] Peking Univ, Dept Ophthalmol, Beijing Key Lab Restorat Damaged Ocular Nerve, Hosp 3, 49 North Garden Rd, Beijing 100191, Peoples R China
[2] YuMing Biotechnol Co Ltd, Qingdao, Peoples R China
基金
中国国家自然科学基金;
关键词
Tissue engineering; Human corneal endothelial cells; Porcine Descemet 's membrane; Corneal endothelial sheets; AMNIOTIC MEMBRANE; CELLS; CARRIER; TRANSPLANTATION; HYDROGEL;
D O I
10.1016/j.exer.2020.108125
中图分类号
R77 [眼科学];
学科分类号
100212 ;
摘要
Purpose: To identify the feasibility of reconstructing corneal endothelial sheets by seeding non-infected monoclonal human corneal endothelial cells (HCECs) onto porcine Descemet's membrane (DM) and verifying the function in vitro and in vivo. Methods: Denuded porcine DM was decellularized for haematoxylin and eosin staining, and DNA was removed via incubation with ethylene glycol diglycidyl ether (EGDE). The physical properties of the incubated DMs were evaluated and compared to those of unincubated DMs. The non-infected monoclonal HCECs were examined by chromosome analysis and the cell proliferation was evaluated by BrdU-labelling. Then HCECs at passage 30 were then seeded on the DM and cultured for approximately 5 days. The cell growth, density and expression of the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase), the tight-junction-associated protein zonula occludens (ZO-1) and acetylated alpha tubulin were examined by electron microscopy and immunocytochemistry to compare HCECs cultivated on porcine DM and those cultured in vitro. Cells on the reconstructed HCEC sheets were labeled with DiI, and the sheets were subsequently transplanted into cat eyes via DM endothelial keratoplasty (DMEK). The corneal transparency, thickness, anterior segment, and HCEC density were monitored in vivo, and the corneal endothelial cell morphology and histological structure were examined ex vivo 98 days after surgery. Results: No significant differences were observed in the elongation at break of the DMs and the thickness of the DMs incubated with EGDE compared to those of the unincubated DMs (P > 0.05). Results of chromosome analysis shown the number of the HCEC cell line was still 46 and no abnormal chromosome structure was found. BrdU-labelling shown the HCECs stopped proliferating after 5 days and the cells formed a single layer. The cells transferred to porcine DM formed tight connections with the substrate and generated layers of hexagonal cells on day 5. Adjacent cells cultivated on DM were closely attached to each other, tightly adhered to the porcine DM and expressed the Na+/K+-ATPase, ZO-1 protein and acetylated alpha tubulin, as did HCECs cultured in vitro. In addition, the HCEC density on DMs was 3020.14 +/- 52.30 cells/mm2. After surgery, the corneas gradually became transparent, and the thickness decreased to 525.33 +/- 56.23 mu m at day 98 after the transplantation, while the control corneas showed consistent oedema during the monitoring period. The HCEC density was 2521.60 +/- 78.24 cells/mm(2) in vivo 98 days after transplantation. The histological results showed that the DiI-labeled cells were dense in the transplanted area and had a hexagonal or polygonal morphology and a normal ultrastructure; adjacent cells were closely attached to each other and tightly adhered to the porcine DM. Conclusions: Seeding non-infected monoclonal HCECs on porcine DM could reconstruct functional corneal endothelial sheets. These results may help uncover new applications for tissue-engineered endothelium in endothelial keratoplasty. Superscript/Subscript Available
引用
收藏
页数:10
相关论文
共 54 条
[1]   Microtubules and actin crosstalk in cell migration and division [J].
Akhshi, Tara Kafiyeh ;
Wernike, Denise ;
Piekny, Alisa .
CYTOSKELETON, 2014, 71 (01) :1-23
[2]  
Anshu A., DESCEMETS STRIPPING
[3]   Highly acetylated tubulin permits enhanced interactions with and trafficking of plasmids along microtubules [J].
Badding, M. A. ;
Dean, D. A. .
GENE THERAPY, 2013, 20 (06) :616-624
[4]  
Bourne W M, 1998, Trans Am Ophthalmol Soc, V96, P229
[5]  
Chamberlain W., DESCEMET ENDOTHELIAL
[6]  
Choi J. S., BIOENGINEERING ENDOT
[7]   Bioengineering endothelialized neo-corneas using donor-derived corneal endothelial cells and decellularized corneal stroma [J].
Choi, Jin San ;
Williams, James K. ;
Greven, Margaret ;
Walter, Keith A. ;
Laber, Patrick W. ;
Khang, Gilson ;
Soker, Shay .
BIOMATERIALS, 2010, 31 (26) :6738-6745
[8]  
Cooper DKC, 2014, XENOTRANSPLANTATION, V21, P97, DOI [10.1111/xen.12082, 10.1111/xen.12098]
[9]   Tensile mechanical and creep properties of Descemet's membrane and lens capsule [J].
Danielsen, CC .
EXPERIMENTAL EYE RESEARCH, 2004, 79 (03) :343-350
[10]   Feasibility and safety of porcine Descemet's membrane as a carrier for generating tissue-engineered corneal endothelium [J].
Diao, Yu-Mei ;
Hong, Jing .
MOLECULAR MEDICINE REPORTS, 2015, 12 (02) :1929-1934