Experimental Study On Transplantation Of HUVECs For Corneal Endothelial Decompensation

Objective: Corneal endothelial diseases such as bullous keratopathy, Fuchs endothelial dystrophy, and decompensation of the endothelium after intraocular operation are mainly reasons which result in corneal opacity and vision loss. There are over one million people lose their vision all over the world each year owing to endothelial cell dysfunction and the exclusive treatment is penetrating keratoplasty. Although penetrating keratoplasty has a high success rate, it is restricted by the less of donor, rejection after transplantation and aging of donor. Therefore tissue engineering cornea become more important and the hot point in the ophthalmological area. One of the key points needs to be solved urgently is how to construct the corneal endothelial tissue. Various methods have been tried to culture corneal endothelial cells, but cannot be applied clinically because of their dependence on the more donor cornea, instability of the carrier or frangibility to operation. To explore an efficient way that not rely on the donor cornea, we culture human umbilical vein endothelial cells (HUVECs) for the first time in vitro instead of finite cornea endothelial cells in this study, then transplant it to the rabbit eyes whose endothelial tissue was wounded mechanically through the procedure of posterior lamellar endothelial keratoplasty with the allogenetic acellular cornea stroma being the carrier. In this study we will investigate the approach of operation and the physiological function of cultured human umbilical vein endothelial cells compared with the result of transplantation of simple acellular cornea strom without cells and simple removal of corneal endothelium.Methods: 1. Animal model: Thirty New-Zealand rabbits (2.5～3 kg)were used for this study. After anaesthesia, a limbal incision of 270 degree was made in the left eye for each rabbit, and the whole endothelial tissue was removed under the operating microscope. Then, 30 rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma transplantation group and 10 for control group. Posterior lamellar endothelial keratoplasty was performed on the rabbits of experimental group and stroma transplantation group after 4 weeks. 2. Preparation of the carrier: the carrier was isolated from the whole porcine cornea, 7.5 mm in diameter, 0.1 mm in thickness, and decellularized by serial digestion of combination of Triton and trypsin. The acellular corneal stroma was dyed by hematoxylin and eosin to confirm that the cells were removed thoroughly. 3. HUVECs culture: HUVECs were acquired by filling 0.1% collagenase I solution into the lumen of umbilical veins and then cultured in DMEM/F12 (1:1) with 20% fetal bovine serum and 10 ng/ml vascular endothelia growth factor. The cells were cultured for 5~7 days when the cells reach to 80%~90% confluence, the cells were seeded onto the carrier and cultured in vitro for about one week, and used for transplantation. 4. Operation: A centered, hinged flap of 9 mm in diameter, and three fourths in thickness of cornea was made. The central posterior lamellar was excised using a 7. 25 mm trephine. The donor button was placed in the recipient bed and sutured using 8-0 absorbable suture, the flap was fixed using 10-0 nylon suture. Postoperative observation was taken for 3 months.Results: Cells both on the surface of culture-dishes and on the acellular corneal stroma became adherence in 24 hours. The cells formed a monolayer after 7 days. The acellular corneal stroma was a semitransparent thin slice, which was detected histologically to be no cells but with good fibrous framework. Corneas in experimental group was relieved in edema obviously compared with that in stroma group and the control group, and showed increased translucency. The average density of endothelial cells was (2026.4±129.3) cells/mm~2 , average central corneal thickness was (505.2±25.4)μm in experimental group,(1535.6±114.5)μm in stroma group and (1493.5±70.2)μm in control group 3 months after operation.Conclusion: We succeeded initially in our study that culturing human umbilical vein endothelial cells on acellular corneal stroma and performingthe posterior lamellar endothelial keratoplasty for corneal endothelial decompensation. Human umbilical vein endothelial cells transplanted to the recipient have normal biological function and keep the cornea transparent. Thus, this study provided a new way for treating corneal opacity clinically results from corneal endothelial diseases.