生物角膜用于人角膜板层移植术后的临床疗效及激光扫描共焦显微镜动态观察

Clinical effect of bioengineered cornea and in vivo confocal microscopy observation after human lamellar keraplasty

背景近年来由于角膜供体材料的缺乏，异种角膜移植研究逐渐受到研究者的关注，猪角膜脱细胞基质构建的生物角膜支架具有良好的生物相容性并已制备成生物角膜材料，但其用于人眼角膜移植后的临床效果及其组织形态学变化特征尚不清楚。目的观察用生物角膜进行人板层角膜移植术后的临床效果，并利用激光扫描共焦显微镜进行活体动态观察，评估生物角膜作为新型角膜替代材料进行人眼角膜板层移植的可行性。方法采用前瞻性系列病例观察研究方法，于2014年2—8月在河南省立眼科医院纳入药物治疗无效的感染性角膜炎或陈旧性角膜白斑患者15例15眼，包括真菌性角膜炎4例，细菌性角膜炎1例，混合感染性角膜炎9例，陈旧性角膜白斑1例。采用猪角膜脱细胞基质构建的生物角膜行患眼生物角膜板层移植术，以术眼的对侧正常眼作为对照。分别于术后3d、7d、1个月、3个月、6个月、9个月和12个月对术眼进行随访，对植片愈合情况进行评分，随访时间为1年。疗效指标主要包括角膜透明度、新生血管形成、角膜植片有无溶解或感染复发、最佳矫正视力（logMAR）。分别于术后3、6、9和12个月利用激光扫描共焦显微镜对角膜进行动态观察，记录和分析移植后角膜上皮和内皮细胞的形态和密度、角膜基质的形态特征以及角膜上皮下神经纤维的恢复情况。结果术眼术后3d早期出现角膜水肿等炎症反应，术后7d炎症反应消退。术后1个月植片透明，随访期间未出现角膜溶解、感染复发现象。术后6个月时1例患者出现青光眼，术后12个月时1例患者发生排斥反应。术眼术前及术后不同时间点间logMAR、角膜透明度评分、角膜新生血管评分的总体比较差异均有统计学意义（X2=92．63、59．37、10．50，均P〈0．05），其中术后视力、角膜透明度均较术前明显改善，差异均有统计学意义（均P〈0．05）。激光扫描共焦显微镜检查结果显示，术眼术后受体角膜上皮细胞逐渐覆盖植片，完成正常的上皮化，角膜上皮细胞形态接近对侧正常眼，但角膜内皮细胞体积大于对侧眼，而植片角膜基质中无细胞生长，明显与对侧眼角膜基质不同；术后6个月时8例患者植片内有上皮下神经纤维长入，但神经纤维走形弯曲，密度明显低于对侧正常眼。术后不同时间点角膜上皮细胞密度总体比较差异均无统计学意义（F=1．48，P=0．22）。对侧正常眼、术眼术后3、6、9和12个月角膜内皮细胞密度分别为（2542±119）、（1895±129）、（1869±135）、（1854±101）和（1844±103）个／mm2，术眼术后各个时间点角膜内皮细胞密度均明显低于对侧正常眼，差异均有统计学意义（均P〈0．05）；术后6、9和12个月角膜上皮下神经纤维密度值分别为（1．26±0．13）、（3．62±0．81）和（5．98±0．44）mm／mm2，均明显高于前1个时间点的测定值，差异均有统计学意义（t′=-8．16、-7．24，均P=0．00）。结论用生物角膜行人眼角膜板层移植术后植片即可长期存活，重建眼表结构，改善患者视力，可替代人角膜供体材料。

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 vivo 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 （Xz = 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 eontralateral 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.