目的 研究微创核黄素-紫外线A行巩膜胶原交联后巩膜生物力学特性的改变及其安全性.方法 采用计算机取随机数法将56只清洁级新西兰大白兔随机分为非交联对照组和交联后1d、7d、15 d、1个月、2个月和3个月组,每组8只,其中6只用于巩膜生物力学性能检测,2只分别用于组织病理学检查和细胞凋亡检测.各组兔均取右眼为实验眼,左眼为正常对照眼.兔实验眼以质量分数0.1％核黄素溶液(不含右旋糖酐)作为光敏感剂点眼,1次/2 min,共10次,然后制作微创结膜切口,微创紫外线巩膜交联仪发光二极管(LED)端插入并贴近眼球后部巩膜照射30 min,紫外线波长为370 nm,辐照度为3 mW/cm2.照射期间用医用体温计测量巩膜温度;按照分组时间点获取实验兔球壁组织,采用千分尺测定巩膜厚度;采用微材料力学性能测试系统对巩膜条带行预拉伸、拉伸破坏试验,测定巩膜的极限应力、极限应变和8％弹性模量;制备兔眼球组织切片并行苏木精-伊红染色,评估眼球组织病理学变化;采用TUNEL法检测视网膜细胞凋亡情况. 结果 非交联对照组和交联后1d、7d、15d、1个月、2个月和3个月组实验兔及其左右眼间巩膜厚度的总体比较差异均无统计学意义(F眼别=0.02,P＞0.05;F组别 =1.71,P＞0.05).与非交联对照组比较,兔右眼交联后1d、7d、15 d、1个月、2个月和3个月组巩膜极限应力和8％弹性模量均明显增加,极限应变均明显下降,差异均有统计学意义(均P＜0.05),与交联后1d、7d、15d、1个月和2个月组比较,交联后3个月组实验眼极限应力、8％弹性模量值均下降,极限应变均增加,差异均有统计学意义(均P＜0.05);与非交联的左眼比较,交联后各时间点右眼极限应力、8％弹性模量值均增加,极限应变均下降,差异均有统计学意义(均P＜0.05).组织病理学检查显示,交联后实验兔眼角膜、巩膜、虹膜、睫状体和脉络膜组织均无明显病理学改变.细胞凋亡检测结果显示,非交联对照组和交联后1d、7d、15d、1个月、2个月和3个月组实验眼视网膜细胞凋亡率分别为(11.00±0.33)％、(12.33±1.58)％、(12.02±0.45)％、(11.81±0.85)％、(12.15±0.61)％、(12.14±0.25)％和(11.74±0.63)％,组间总体比较差异无统计学意义(F=1.78,P=0.14).结论 微创核黄素-紫外线A巩膜胶原交联术可有效增强兔巩膜的生物力学强度,且对眼球各组织无明显病理性损害.%Objective To evaluate the enhancing effects of scleral biomechanics and safety of collagen crosslinking by using minimally invasive riboflavin and ultraviolet A.Methods Fifty-six healthy New Zealand white rabbits were randomized into non-cosslinking group,post-crosslinking 1-day group,post-crosslinking 7-day group,postcrosslinking 15-day group,post-crosslinking 1-month group,post-crosslinking 2-month group and post-crosslinking 3-month group,eight for each group.Riboflavin solution at the concentration of 0.1％ was dropped once per 2 minutes for 20 minutes,and then minimally invasive riboflavin and ultraviolet A was carried out in the right eyes by putting the lighting emitting diode (LED) probe end of microinvasive ultraviolet scleral crosslinking device to irradiate the posterior sclera for 30 minutes with the wavelength of 370 nm and radiation energy of 3 mW/cm2,and the left eyes served as the normal controls.The scleral temperature was measured using clinical thermometer during the crosslinking period.The rabbits were sacrificed according to the grouping and the eyeballs were obtained.The scleral thickness was measured by callipers,the pretension and tensile failure tests of sclera strips were tested by micromaterial mechanics performance test system to measure the extreme stress,extreme strain and 8％ elastic modulus.Hematoxylin-eosin staining was employed to examine the morphology of eye tissues.Retinal cell apoptosis was detected by TUNEL assay.Results The scleral thickness was insignificantly different among the non-crosslinking group,post-crosslinking 1-day group,post-crosslinking 7-day group,post-crosslinking 15-day group,post-crosslinking 1-month group,post-crosslinking 2-month group and post-crosslinking 3-month group and between right eyes and left controls (F,es =0.02,P＞0.05;Fgroup =1.71,P＞0.05).Compared with the non-crossliking group,the extreme stress and 8％ elastic modulus of the scleras were significantly increased,and the extreme strain of the scleras was significantly decreased in post-crosslinking 1-day group,post-crosslinking 7-day group,post-crosslinking 15-day group,post-crosslinking 1-month group,postcrosslinking 2-month group and post-crosslinking 3-month group (all at P＜0.05).Not any morphological abnormalities were found in corneas,scleras,irises,ciliary bodies and choroids in various groups.The apoptosis rates of retinal cells were (11.00±0.33)％,(12.33±1.58)％,(12.02±0.45)％,(11.81±0.85)％,(12.15± 0.61)％,(12.14±0.25)％and (11.74±0.63) ％ in the non-crosslinking group,post-crosslinking 1-day group,post-crosslinking 7-day group,postcrosslinking 15-day group,post-crosslinking 1-month group,post-crosslinking 2-month group and post-crosslinking 3-month group,respectively,with no significant difference among the three groups (F =1.78,P =0.14).Conclusions Rabbit sclera collagen crosslinking by using the minimally invasive riboflavin and ultraviolet A can effectively enhance the biomechanical strength of the sclera,and this procedure is safe.
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