Autologous Corneal Limbal Stem Cell Transplantation Combining Amniotic Membrane Transplantation On The Treatment Of Limbal Stem Cells Deficiency

Objective To observe the autologous corneal limbus stem cells combining amniotic membrane transplantation of treatment effectiveness and safety on the corneal limbal stem cells deficiency.Methods Using prospective study method,observe the patients between April 2017and November 2017 in Tongji hospital treatment of corneal limbal stem cells deficiency follow-up.All patients were diagnosed stem cells of limbal of cornea deficiency by the same physician,adopt autograft limbal stem cell transplantation combining amniotic membrane transplantation surgery treatment.3 cases of monocular completeness stem cells of limbal of cornea deficiency,using the source of the contralateral eye corneal limbal stem cells;Monocular part of the stem cells of limbal of cornea deficiency,use the ipsilateral eye sources of stem cells of limbal of cornea.Use slit lamp microscope photography,in the sight of OCT,living donor corneal confocal microscope,imprint cytology and clinical pathology examination.Records the preoperative and postoperative 2 weeks,1 month and 3months of vision,new blood vessels area and its complications,3-10 months of follow-up.Results A total of 11 patients were included in this study,including 6 cases of alkali burns,2 cases of thermal burn,2 cases of blast injury,1 case of papilloma.Preoperative visual acuity range from HM/30 cm to 0.5,averaged 0.16±0.15.New blood vessels covers an area range from 11.19 to 91.89mm²,averaged 52.38±52.38mm².Section before OCT showed preoperative corneal thickness 367-634μm,averaged 474.36±77.96μm,corneal coverage before vascular film thickness is 100-286μm,averaged 180.30±76.83μm.Now done in 11 patients were more than 3 months follow-up,1 case of patients with postoperative 2 months follow-up,patients with postoperative central corneal transparency,new blood vessels area averaged 24.22±17.60mm²（P=0.01）,postoperative visual acuity range from FC/50 cm to 0.6,averaged 0.29±0.21（P=0.04）,postoperative and preoperative vision exists difference.Postoperative corneal thickness range from 402 to580μm,averaged 508.00±91.60μm（P=0.39）.2 patients postoperative 3 months the cornea vascular membrane still cover around a third size,one regular autograft limbal corneal limbal stem cells transplantation combined amniotic membrane transplantation,stem cell graft taken from the contralateral eye.1 patients accept corneal transplantation after 3months.8 underwent confocal microscopy,visible disordered eye keratocyte form,keratocyte and nerve fiber density decreased,corneal endothelial cell counts.Intraoperative resection before corneal membrane pathological HE staining,the microscopic results suggest surface coating squamous epithelium,epithelial hyperplasia of fibrous tissue and small blood vessels.Preoperative complete eyes in 7 patients living donor corneal confocal microscopy,affected eyes of shallow keratocyte range from 397.40to 709.50/mm²,averaged 519.09±101.86/mm²;Normal eyes superficial keratocyte range from 657.20 to 847.20/mm²,averaged 724.00±82.83/mm²（p﹤0.05）.Affected eyes layer keratocyte number range from 365.8 to 365.8/mm²,averaged 449.43±80.04/mm²;Normal eyes layer keratocyte number range from 503.00 to 752.40/mm²,averaged620.95±96.48/mm²（p﹤0.05）.Affected eyes deep keratocyte range from 317.70 to535.40/mm²,averaged 390.90±63.31.Normal eyes deep keratocyte number range from382.4 0to 594.20/mm²,averaged 499.50±70.34/mm²（p﹤0.05）.Affected eyes endothelial cells range from 1006.80 to 2343.20/mm²,averaged 1573.71±477.46/mm^2.Normal endothelial cell count range from 2217.80 to 3335.70/mm²,averaged 2873.34±419.07/mm²（p﹤0.01）.Three patients by reason of corneal vascular membrane area is large,failed to complete the confocal microscopy.Imprint cytology revealed preoperative corneal goblet cell populations,epithelial squamous metaplasia,postoperative corneal goblet cells and squamous epithelial cells.Resection of intraoperative corneal blood vessel membrane wax block slice,immunofluorescence examination,choose CK3,CK12,CK13and P63α,the experimental results suggest vascular membrane mainly express conjunctival epithelium of keratin,a small amount of keratin expression corneal epithelium with almost no expression of corneal limbus stem cell antibody.1 case of patients with corneal limbus stem cell deficiency recurrence during the follow-up period,the rest of the patients with no recurrence.Conclusion For monocular completeness or part of corneal limbus stem cells,autologous stem cells of limbus of cornea combined amniotic membrane transplantation can obtain certain therapeutic effect.