羟基磷灰石表面改性人工角膜支架植入兔角膜后基质金属蛋白酶-2及胶原纤维的表达

The expression level of MMP-2 and collagen of hydroxyapatite modified titanium for keratoprosthesis in the corneal stroma of rabbits

目的 探讨经羟基磷灰石（HA）表面改性的三襻式人工角膜钛支架植入兔角膜板层后基质金属蛋白酶-2（MMP-2）及胶原纤维的表达,评价其生物相容性.方法 实验研究.24只新西兰白兔采用单纯随机抽样方法随机分为3个组,HA-Ti支架组（9只）及Ti支架组（9只）分别于右眼角膜板层植入HA表面改性后的三襻式人工角膜钛支架（HA-Ti）及人工角膜钛支架（Ti）,6只仅制备囊袋不植入支架作为对照组.观察人工角膜支架的生物相容性及新生血管生长情况,并分别于术后2周、4周、16周取材,角膜组织行苏木素-伊红染色、并用免疫组织化学方法检测各组角膜基质中MMP-2的表达并进行半定量分析；用苦味酸天狼猩红苏木素染色的方法观察支架植入后对胶原的影响.多组间比较应用单因素方差分析,组间两两比较应用SNK-q检验.结果 观察期间Ti-HA支架组及Ti支架组均未见角膜感染、溶解、支架排出.早期实验组角膜有水肿及新生血管长入,HA-Ti支架组新生血管明显多于Ti支架组.组织学观察术后2周,植入HA-Ti支架组角膜基质内2周可见少量炎性细胞（中性粒细胞及噬酸细胞为主）,此后逐渐减少.支架周围2周时可看到较多成纤维细胞,16周时成纤维细胞减少,粉染的胶原纤维增多.术后2周,HA-Ti支架组、Ti支架组及对照组MMP-2阳性细胞IOD值分别为103.729±15.112、84.439±13.699、69.310±13.078,组间比较差异有统计学意义（F =6.083,P＜0.05）,但Ti支架组和对照组差异无统计学意义（F=3.209,P＞0.05）；4周时3个组MMP-2阳性细胞IOD值逐渐降低,分别为88.963±9.017、53.005±4.605、49.326±4.443,组间比较差异有统计学意义（F=47.074,P＜0.01）；16周时3个组MMP-2阳性细胞IOD值分别为72.176±12.815、50.014±8.813、46.734±11.670,组间比较差异有统计学意义（F=6.079,P＜0.05）.偏光镜下观察见角膜组织内4周有大量绿色的Ⅲ型胶原纤维及黄红色Ⅰ型胶原纤维,16周时角膜内主要为鲜红色的Ⅰ型胶原纤维,仍有少量的Ⅲ型胶原纤维.结论 兔角膜植入HA-Ti支架引起MMP-2活化,持续高表达并未造成对角膜的溶解；其影响周围角膜细胞外基质中Ⅲ型胶原纤维向Ⅰ型胶原纤维的转化,利于支架稳定,HA-Ti支架具有较好的生物相容性.

Objective To investigate the expression level of metalloproteinases-2 （MMP-2） and Collagen in a hydroxyapatite surfaced-modified of three Pan type titanium keratoprosthesis after that implanted into the corneal stroma of rabbits,further evaluate its biological compatibility.Methods Experimental study.Twenty-four New Zealand white rabbits,2.0-2.5 kg,were respectively divided into three groups.Surgery was performed in right eye of all animals.skirt of HA-Ti and Ti were respectively inserted into the corneal stroma of rabbit of experimental group A and group B ; only a sack was made without implantation in control group C.Cornea edema and corneal neovascularization were observed at scheduled times after operation; animals were sacrificed 2,4 and 16 weeks after operation and their cornea was removed and examined under light microscopy ; the surface of skirt was observed under scanning electron microscope.Results During the study period,all skirts were stable without infected,dissolved and excluded.Different degree of cornea edema and neovascularization was revealed after surgery.MMP-2 were absent in the normal corneal matrix.The expression level of MMP-2 in group A was higher than group C at all time points （F =6.083,P ＜ 0.05）,and was increased than group B at 4th （F =47.074,P ＜ 0.01）,and was increased than group C at 16th weeks too（F =6.079,P ＜0.05）.Corneal organization has a large green 4 weeks type Ⅲ collagen and yellow red type Ⅰ collagen,16 weeks corneal mainly for bright red when within the collagen type Ⅰ,still have a small amount of collagen type Ⅲ.Conclusions Rabbit cornea implanted HA-Ti skirts cause MMP-2 activation,continuous high expression didn＇t cause the cornea to dissolve; Collagen-Ⅲ turned into collagen-Ⅰ gradually in the extracellular matrix around the skirts.Hydroxyapatite modified titanium for Keratoprosthesis promoted the corneal neovasularization and improve the interfacial biointegration of skirt and host cornea.