Efficacy and mechanism of rabbit corneal stromal cell-induced adipose-derived mesenchymal stem cell amnion transplantation for corneal alkali burn

Abstract

Background Corneal chemical burn is one of blinding eye diseases.Previous therapies for corneal chemical burn is limited to certain extent.However, transplantation of adipose-derived mesenchymal stem cells (ADSCs) for corneal diseases is drawing more and more attention. Objective This study was to observe the effect of rabbit ADSCs transplantation for ocular alkali burns and explore its mechanism. Methods Rabbit corneal stromal cells (CSCs) were isolated and cultured by suspended matrix method, and rabbit ADSCs were obtained and digested from inguinal fat tissue with enzyme digestion method (0.25% trypsin) and identified by flow cytometry.CSCs cocultured with ADSCs, and CSCs-induced ADSCs were identified by double-label of with immunofluorescence and reverse transcription PCR (RT-PCR). Then induced or uninduced ADSCs were inoculated on amniotic membrane to prepared ADSCs-amnion patch.Corneal alkali burn models were established in the right eyes of 60 New Zealand rabbits by placing a filter paper with 1% NaOH solution at the central cornea for 50 seconds.The models were randomized into the induced ADSCs+ amnion implanted group, the uninduced ADSCs+ amnion implanting group, amnion implanted group and model group.Corneal opacification and neovascular area were examined and corneal inflammation was graded by slit lamp microscope 1 week, 2 weeks and 1 month after surgery.The contents of CD45, interferon-γ (IFN-γ) and interleukin-10 (IL-10) in corneal homogenate as well as vascular endothelial growth factor (VEGF) in aqueous humor were detected by ELISA assay.The use and care of experiment animals followed the Statement of ARVO. Results ADSCs showed the positive responses for CD105, CD29, CD44 with the positive rate 90.23%, 88.56% and 98.88%, respectively.CSCs was positively reactive for vimentin.The double-label staining was positive after coculture of CSCs with ADSCs.Hematoxylin-eosin stain exhibited that ADSCs grew well on the amnion.Corneal porcelain opacity and a lot of new blood vessels were seen in the model group, and corneal was clear in the induced ADSCs+ amnion implanted group 1 month after surgery.The inflammatory scores were 1.65±0.18, 2.05±0.17, 2.68±0.25, 2.90±0.18, and the areas of neovasculization were (10.59±1.78), (22.58±1.63), (37.98±1.90), (45.37±1.65)mm2 respectively in the induced ADSCs+ amnion implanted group, uninduced ADSCs+ amnion implanted group, amnion implanted group and the model group.The inflammatory scores of 1 week, 2 weeks, 1 month after operation among the four groups had statistically significant differences（F=280.826, 330.172, 465.707, all at P=0.000), and the areas of neovasculization of 1 week, 2 weeks, 1 month after operation among the four groups had statistically significant differences（F=60.020, 670.811, 1 510.231, all at P=0.000), the inflammatory scores in the induced ADSCs+ amnion implanted group were remarkably lower than those of the other groups, the areas of neovasculization in the induced ADSCs+ amnion inplanted group were smaller than those of the other groups (all at P<0.01). In 1 month after surgery, the contents of CD45, IL-10, IFN-γ in cornea and VEGF in aqueous humor were statistically different among the groups（F=916.545, 1 739.358, 462.134, 129.126, all at P=0.000). Compared with the uninduced ADSCs+ amnion implanted group, amnion implanted group and the model group, CD45 and IFN-γ contents were declined, and IL-10 content was elavated in the induced ADSCs+ amnion implanted group (all at P<0.01). In addition, VEGF contents in aqueous humor were significantly lower than those in the other groups (all at P<0.01). Conclusions Rabbit CSCs-induced ADSCs amnion patch transplantation is effective for the reconstruction of ocular surface after alkali damage probably by differentiation of ADSCs into epithelial-like cell after CSCs induced.Moreover, amnion can alleviate immuno-inflammatory response and suppress neovascularization. Key words: Adipose-derived mesenchymal stem cells; Transplantation; Amniotic membrane; Alkali burn, ocular; Differentiation; Corneal stromal cells