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Background Xenotransplantation is arousing more attention of researchers because of the lack of corneal donors.Biological corneal scaffolds constructed by porcine corneal acellular stroma appears to have an acceptable biocompatibility.However,its clinical effects and the histomorphological features in the corneal tissue of receipts' are still unclear.Objective This study was to evaluate the viability of bioengineered corneas as a new material of human lamellar keratoplasty and observe the in viva biological features after human keratoplasty under the laser confocal microscope.Methods A prospective serial cases observational study was carried out.Fifteen eyes of 15 patients with infectious keratitis were enrolled in Henan Eye Hospital from February to August 2014 under the approval of Ethic Committee of Henan Eye Hospital and informed consent of each patient, including 4 eyes of fungal keratitis,1 eye of bacterial keratitis, 9 eyes of mixed infectious keratitis and 1 eye of leucoma.Corneal lamellar transplantation was performed on the eyes with the bioengineered corneas as grafts and the follow-up time was one year.The survival of grafts was assessed and scored, and the indices including corneal transparency,neovascularization, graft lysis or inflammatory recurrence and visual acuity (logMAR) were evaluated.The morphology and density of corneal epithelial cells and endothelial cells, corneal stroma and subepithelial neural fibers were examined by laser confocal microscope 3,6,9 and 12 months after surgery.Results Postoperative inflammatory response was seen 3 days and disappeared 7 days after surgery.The grafts were clear 1 month after surgery, and no corneal dissolution was found during the follow-up duration.Glaucoma occurred in 1 eye at 6 months and graft rejection occurred in another eye 12 months after surgery.The logMAR, corneal transparency scores and corneal neovascularization scores improved after surgery in comparison with before surgery,with a significant difference among various time points (x2 =92.63,59.37,10.50 ,all at P＜0.05).Complete epithelization of grafts was seen.Compared with the contralateral eyes,the morphology of epithelial cells was similar and endothelial cells were enlarged in the operated eyes.In addition,no stromal cell structure was seen in the corneal stroma in the operative eyes.Subepithelial nerve fibers appeared in 8 eyes at 6 months, but the fiber density was lower in the operated eyes than that in the contralateral eyes.Significant difference was found in epithelial cell density among different time points (F=1.48, P =0.22).The endothelial cell densities were (2 542 ± 119), (1 895 ± 129), (1 869 ± 135), (1 854 ± 101) and (1 844 ± 103)/mm2 before surgery and 3, 6, 9 and 12 months after surgery, showing significant differences between preoperation and postoperative time points (all at P＜0.05).The subepithelial nerve fibers densities were (1.26± 0.13),(3.62±0.81) and (5.98±0.44) mm/mm2 at 6,9 and 12 months after surgery,with significant differences between adjacent two time points (t'=-8.16 ,-7.24;both at P=0.00).Conclusions Bioengineered corneal grafts survive well in human eye after lamellar transplantation,which can reconstruct ocular surface and improve the visual acuity.Biological cornea can replace human corneal materials.
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Background Graft rejection is a primary cause of corneal transplantation failure,especially in high-risk keratoplasty.How to extend the survival time of graft is a problem to be solved.Objective This study was to investigate the influence of immune tolerance on high-risk rat keratoplasty induced by donor bone marrowderived immature dendritic cells (imDCs) transfected by chemokine receptor 7 (CCR7) recombinant adenovirus (Ad).Methods Bone marrow-derived imDCs were isolated and cultured from femur marrow of one male Wistar donor rat.The cells were transfected using recombinant Ad vector with rat CCR7 gene and resuspended in 500 μl PBS containing 1％ fetal bovine serum with the cells 1 × 107.High-risk corneal transplantaion models were established using monolateral corneal alkali-burn method in 60 SD rat recipients, and then allograft keratoplasty was performed with the 30 Wistar rats as donors.The models were randomized into the PBS group,imDCs group,imDCs with blank Ad vector group and imDCs with Ad-CCR7 group following the corresponding solution injection via caudal vein on preoperative day 7 and postoperative day 3 respectively.The survival time of graft was evaluated under the slit lamp microscope once per day.On the 14th day after operation, corneal sections were prepared from 6 eyes of each group for the pathological examination,and the relative expression levels of T helper cell 1 (Th1)-related factors,interleukin-2 (IL-2) mRNA and interferon-γ(IFN-γ) mRNA,as well as Th2-related factors, IL-4 mRNA and IL-10 mRNA, were detected by reverse transcription PCR (RT-PCR).The use and care of animals complied with the ARVO statement Results The mean survival time of grafts was (10.44±1.88) , (16.00±2.18) , (15.11±2.03) and(23.67±2.83) days in the PBS group,imDCs group,imDCs with blank Ad group and imDCs with Ad-CCR7 group, respectively, with a significant difference among the 4 groups (F =53.005, P =0.000).Compared with the PBS group, the mean survival time of grafts was considerably extended in the imDCs group,imDCs with blank Ad group and imDCs with Ad-CCR7 group (t=5.220,4.385,12.423 ,all at P=0.000) ,and a remarkble prolongation of graft survival duration was seen in the imDCs with Ad-CCR7 group in comparison with the imDCs group and the imDCs with blank Ad group (t =7.204,8.039,both at P=0.000).The relative expression levels of IFN-γ mRNA and IL-2 mRNA in the grafts were significantly lower,but the relative expression levels of IL-4 mRNA and IL-10 mRNA were significantly higher in the imDCs group,imDCs with blank Ad group and the imDCs with Ad-CCR7 group than those in the PBS group (all at P＜0.05).Compared with the imDCs group and the imDCs with blank Ad group, the relative expression levels of IFN-γ mRNA and IL-2 mRNA in the grafts were remarkably delined,but the relative expression levels of IL-4 mRNA and IL-10 mRNA were remarkably elevated in the imDCs with Ad-CCR7 group (all at P =0.000).Conclusions Application of imDCs transfected with CCR7 recombinant Ad via vena caudalis can prolong the survival time of grafts after keratoplasty of SD rats probably by affecting the balance of Th1/Th2 cytokines.
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Background Mesenchymal stem cells (MSCs) have been applied in basic and clinical researches of organ transplantation.Our previous study showed that intravenous injection of MSCs prolonged corneal allograft survival in rat.However,the effect of local administration of MSCs on corneal allograft rejection remains unclear.Objective The aim of this study was to investigate the effect of subconjunctival injection of MSCs on corneal allograft rejection in rat model of keratoplasty.Methods MSCs were isolated and cultured from femur and tibia bone marrow of clean Wistar rats,and then the cells were identified by induced differentiation of osteoblast and adipocyte.The third generation of MSCs was used in subsequent experiment.Allogenic penetrating keratoplasty was performed with the bilateral corneas of 20 Wistar rats as donor grafts and the right eyes of 40 Lewis rats as recipients.PBS 0.1 ml containing 2 × 106 MSCs and 0.1 ml PBS only was subconjunctivally injected immediately and postoperative 3 days respectively in randomized two groups,and another 6 normal Lewis rats served as the normal control group.Corneal rejection response was evaluated under the slit lamp after surgery based corneal opacity,edema and neovascularization,and the grafts were scored according to the criteria of Larkin.The corneal samples were extracted from 12 rats of the PBS control group and the MSCs group separately 10 days after surgery.The relative expressions of Th1 cytokines (interferon-γ [IFN-γ] mRNA and interleukin-2 [IL-2] mRNA) and Th2 cytokines (IL-4 mRNA and IL-10 mRNA) were detected by real-time quantitative PCR.Protein levels of IL-4 and IL-10 proteins in the corneas were assayed by ELISA.All experimental protocols involving rats were approved by the laboratory animal care and use committee of the Tianjin Medical University and treated with the ARVO statement for the use of animals in ophthalmic and vision research.Results The cells grew well with the orange stain for alizarin red in differentiated the osteoblasts and red stain for Oil red O in differentiated adipocytes.The survival time of corneal graft in the MSCs group was (11.8±1.6) days,it was significantly longer than (9.6±1.4) days in the PBS control group (P=0.004).The levels of IL-4 mRNA and IL-10 mRNA in the MSCs group were significantly higher than those in the PBS control group (both at P =0.00);while the levels of IFN-γ mRNA and IL-2 mRNA were not significantly different between the groups (both at P＞0.05).The IL-10 protein contents were (22.74 ±7.06),(68.40±12.83) and (215.41 ±44.66)pg/ml in the normal control group,PBS control group and MSCs group,showing significant difference among the three groups (F =55.06,P =0.00) and a significant increase in the MSCs group compared with the PBS control group and the normal control group (both at P ＜ 0.05).Conclusions Subconjunctival injection of MSCs prolongs the survival time of cornea allograft in penetrating keratoplasty probably by modulating the balance between Th1 and Th2 cytokines,especially by up-regulating Th2 cytokines.