【作者】 李力；

【导师】 黄一飞；

【作者基本信息】 中国人民解放军军医进修学院 ， 眼科学， 2009， 博士

【摘要】 目的:研究与评估改良钛支架人工角膜的生物相容性和角膜生物愈合的过程;探讨制作这种新型人工角膜的方法。体内实验观察改良后其生物相容性是否增加,观察临床表现和并发症,并取材检测MMP-2、TIMP-2在兔角膜组织的表达,探讨角膜溶解并发症的机理,为临床治疗提供依据。方法:1.设计及制备改良钛支架人工角膜;采用喷砂(SB)工艺预处理人工角膜钛支架,使其表面变粗糙,然后用酸碱两步法在人工角膜钛支架表面快速沉积羟基磷灰石(HA)涂层,改良的人工角膜钛支架(HA/SB-Ti)外型由二襻改为三襻。采用美国Lambda Research Corporation开发的光学设计软件OSLO,按照几何光学原理设计人工角膜的镜柱,用聚甲基丙烯酸甲酯(polymethyl methacrylate PMMA)为原料按设计好的图纸手工磨制。XRD和SEM分析涂层成分、表面形貌。2.将18只正常新西兰白兔及18只新西兰白兔角膜碱烧伤模型随机分为3组,其中1组、2组右眼角膜基质层内分别植入人工角膜支架HA/SB-Ti、SB-Ti,第3组仅做角膜板层切口而不植入支架为手术对照组,另取两只正常白兔作空白对照。术后裂隙灯观察角膜水肿及新生血管产生情况,同时观察支架的存留时间和并发症。术后1个月、3个月取材,石蜡包埋后,行HE染色光镜下观察支架与组织界面愈合情况;2.5%戊二醛内固定后,透射电镜观察支架植入后对角膜组织的影响;支架取出后,SEM观察材料表面组织贴附情况及HA涂层存留情况。3.二期植入改良钛支架人工角膜镜柱,观察人工角膜植入后临床反应及并发症,于1个月、3个月取材,石蜡包埋后,行免疫组织化学检测MMP-2、TIMP-2在不同组别的表达;RT-PCR检测MMP-2、TIMP-2 mRNA在不同时间点的表达;免疫印迹法(Western blot)检测MMP-2、TIMP-2mRNA产物在不同时间点的表达。结果:1.支架经喷砂工艺后表面变粗糙,浸入SCS溶液中,表面沉积了致密的羟基磷灰石涂层,涂层由疏松的片状外层及密集的内层组成。酸碱两步法处理喷砂后的样品避免了机械打磨,其形状保持良好,且其表面涂层晶体尺寸大,生长充分,外型的三襻结构及表面的涂层均增加了支架的表面积,使支架与组织的界面变大,增加了支架的牢固性。改良人工角膜镜柱的直径为3.2mm、厚度为3.8mm、前表面曲率半径为8.874mm。2.临床观察:人工角膜支架植入术后均不同程度地出现角膜水肿和新生血管。角膜水肿于术后1周内逐渐消退,有一例植入SB-Ti支架碱烧伤模型出现角膜溶解的并发症。术后1个月、3个月取材,组织学检查见HA/SB-Ti组界面处成纤维细胞数多于SB-Ti组,术后1个月时材料/组织界面处仍有炎症细胞存在。拉出试验测定HA/SB-Ti支架植入角膜3月后的剪切力较SB-Ti支架增强(P＜0.05)。SEM检查见:钛支架被细胞外基质包裹,HA/SB-Ti支架表面有一层致密物质存在,细胞外基质在HA表面伸展良好,与HA贴附紧密,而SB-Ti组支架细胞附着量明显少。碱烧伤植入支架组中支架表面沉着的细胞外基质少于正常角膜植入支架组。TEM检查见:HA/SB-Ti支架与SB-Ti支架植入术后,支架周围的胶原纤维束紊乱,角膜基质有大量成纤维细胞,HA/SB-Ti组角膜越靠近支架中心的成纤维细胞核越圆,核仁清晰,胞浆丰富,代偿增强。碱烧伤模型组角膜基质中胶原纤维出现断裂现象,较正常兔角膜组愈合延迟。3.人工角膜镜柱二期植入后,3周后结膜充血、角膜水肿症状消失,植入改良钛支架人工角膜的动物未出现角膜溶解等并发症,1月、3月各组取材,做免疫组织化学检查,碱烧伤模型HA/SB-Ti支架植入组1月、3月MMP-2表达的平均光密度较SB-Ti支架植入组有统计学差异(P＜0.01);而3月时TIMP-2表达的平均光密度HA/SB-Ti支架较SB-Ti支架无统计学差异(P＞0.05);正常动物模型HA/SB-Ti支架植入组1月、3月MMP-2、TIMP-2表达的平均光密度较SB-Ti支架植入组表达弱,有统计学差异(P＜0.05);于2周、1月、3月、5月取材后,做RT-PCR、Western blot检测,MMP-2mRNA及MMP-2mRNA表达产物从支架植入开始升高,1月达到高峰,然后开始下降,除2周与3月MMP-2mRNA表达产物比较无统计学意义(P＞0.05)外,其余各组比较有统计学意义(P＜0.05)。而TIMP-2mRNA及TIMP-2mRNA表达产物则开始有波动,然后逐渐升高,各组比较有统计学意义(P＜0.05)。结论:1.改良的人工角膜支架增加了表面的空隙率和表面积,增强了人工角膜在角膜中的固定。2.与单纯喷砂Ti支架相比,改良的支架增加了兔角膜基质成纤维细胞在其表面的粘附、伸展、增殖和分化的能力,促进了材料与组织界面的愈合,更适应角膜的微环境。3.改良人工角膜钛支架的活性鳞片状表面对抑制角膜组织中MMP-2过度表达与激活有积极作用,MMP-2、TIMP-2的表达在改良钛支架人工角膜植入后不同时间内差异显著,并与角膜溶解的临床病理过程具有同步性。MMP-2与TIMP-2的失衡是角膜溶解的可能原因之一,通过对MMP-2、TIMP-2的研究可为尽早解决人工角膜植入后角膜溶解穿孔提供新的途径。关于MMP-2与TIMP-2的激活和抑制的分子机制,以及如何为防治角膜溶解穿孔合理地调节二者之间的平衡有待进一步研究。更多还原

【Abstract】 ObjectivesThe research is engaged in developing an improved titanium skirt for keratoprosthesis,the aim of this study was to evaluate biocompatible of keratoprosthesis of novel design.And also to assess the long term clinical outcomes implanted to rabbit corneas(including compalications),to investigate the effect of MMP-2 and TIMP-2 for the skirt inserted eyes in rabbit,and the expression of MMP-2 and TIMP-2 during the course of cornea with HA/SB-Ti skirt and SB-Ti skirt,in order to explore the potential roles of MMP-2 and TIMP-2 during the course of the skirt inserted eyes in rabbit.Methods1.The pure titanium skirt for keratoprosthesis with three hands were first Sandblasted,and then bioactive hydroxyapatite coated on Sandblasted titanium for keratoprosthesis by a acid-alkali chemical treatment.The improved artificial cornea consists of a skirt and a transparent center of polymethyl methacrylate PMMA.The composition,the surface morphology and the cross-section were analyzed by means of X-ray diffraction(XRD) and scanning electron microscopy (SEM).2.A total of 18 New Zealand white rabbits and 18 alkali burned rabbit corneas were respectively divided into three groups.Skirt of HA/SB-Ti and SB-Ti were inserted into the corneal stroma of rabbits for a 1 and 3 month period.The third group did not insert skirt as surgery control.Corneal oedema and neovascularisation were evaluated.The interfacial biointegration of skirt/cornea were examined under light microscopy by HE and TEM.The extracellular matrix deposited on the surface of skirt was examined by SEM.3.The transparent center was implanted to rabbit corneas after 3 months,and then assesses the long term clinical outcomes and compalications implanted to rabbit comeas.To investigate the effect of MMP-2 and TIMP-2 for the skirt inserted eyes in rabbit,the corneas were immunohistochemically analyzed with antibodies directed against MMP-2 and TIMP-2,and the expression of MMP-2 and TIMP-2 during the course of cornea with HA/SB-Ti skirt and SB-Ti skirt,and pathohistology and computerized corneal analysis were studied on month 1 and 3. The expression of MMP-2 and TIMP-2mRNAlevel was determined by real time-polymerase chainreaction,and its protein level was determined by western blot.Results1.Dense hydroxyapatite coating was deposited on the Sandblasted specimens by an acid-alkali chemical treatment,and was composed of two sublayers,i.e.an outside loose crystal HA sublayer and an inside dense HA sublayer.The thickness of the HA coating was about 30μm.The shape of titanium skirt was well maintained by the two-step acid-alkali chemical treatment.And compared to group SB-Ti,the HA crystals of group HA/SB-Ti growed larger and more sufficient.The skirt surfaces expanded and keratoprosthesis became firm when keratoprosthesis of novel design consist of three hands.The radius of transparent center of novel design is 8.874 mm;thickness of transparent center is 3.8 mm.2.Corneal oedema and neovascularisation were found in all of the skirt inserted eyes in healthy animals,but the corneal oedema subsided within 1 week.The skirt have been retained,now up to a period of 5 months,with no severe complications such as cataract,retrocorneal membrane formation and retinal detachment,but there was one case of corneal dissolving being found on the 1 month.The number of corneal fibroblasts increased significantly in HA-Ti skirt inserted eyes compared with Ti skirt inserted eyes.The extracellular matrix deposited on the surface of HA-Ti skirt was more dense and tight than that of Ti. Compared with healthy host tissue,skirt/cornea healing after alkali burn was impaired;there were more inflammatory cells and corneal fibroblasts in alkali burn cornea/skirt interface than that of heathy cornea.The investigation of the effects of skirt surface on the interaction of group HA/SB-Ti and SB-Ti,The results showed:HA/SB-Ti surface markedly influenced the corneal fibroblasts attachment pattern and orientation in relation to the rim of the skirt,cells attaching to it perpendicularly or at an angle.The observation of the tissue sections of HA/SB-Ti skirt inserted eyes revealed that there were cells and collagen-like fibres perpendicular to or at an angle to the rough interface and that dramatic changes occured in the cellular morphology within the interface,from spindle shape on the surface to round or ovoid shape with a large cellular body and abundant cellular plasm,which suggested that the cellular function was extremely active,in accordance with the results of TEM.3.The keratoprosthesis of novel design have been retained during the experiment without severe complications such as cataract,retinal detachment and extrusion.Compared with HA/SB-Ti skirt inserted eyes from epithelium healing, corneal inflammation,neovascularization and scarring were all markedly improved in group E after injure,MMP-2 and TIMP-2 immunoreactivities were expressed in the normal corneas,predominantly in the corneal epithelium.After injury,immunoreactivities of both MMP-2 and TIMP-2 were increased notably in the healing corneal epithelium,infiltrating inflammatory cells,stromal fibroblasts and ingrowing vascular endothelial cells.The expression of MMP-2 was lower in the corneal tissue sections of HA/SB-Ti skirt inserted eyes than that in the tissue sections of SB-Ti skirt inserted eyes(P＜0.01).The Western bolt revealed no significant differences of MMP-2mRNA between group 3 month and 2 week(t test of Ct,P＞0.05);MMP-2 immunoreactivities were absent or lowly expressed predominantly in the corneal epithelium of normal corneas.The expression of MMP-2,TIMP-2 mRNA level was paralled that of protein level.Conclusions1.The titanium rough surface produced by sandblast(SB),then by a two-step acid-alkali chemical treatment and a proper solution,a hydroxyapatite coating can be quickly deposited on Sandblasted titanium skirt of keratoprosthesis, which is exempt from the procedure of polishing.The skirt surfaces expanded and keratoprosthesis became firm when keratoprosthesis of novel design consist of three hands. 2.Hydroxyapatite modified Sandblasted titanium skirt for keratoprosthesis promoted superior rabbit corneal fibroblast adhension and proliferation in comparison with Sandblasted titanium,and it could promote the interfacial biointegration of skirt and host cornea,no matter in healthy comea or diseased cornea(alkali burn cornea).Hydroxyapatite coationg improved the bioactivity of titanium.3.By Immunohistochemitry and analysis system of pathologicpicture,the relative studies showed the different expressions of MMP-2 and TIMP-2 in different period and they have simultaneous incidence with corneal dissolving. The studies of MMP-2,TIMP-2 can provide a new way to prevent the incidence of corneal dissolving after surgery for keratoprosthesis,.The exciting and inhibiting mechanism of MMP-2 and TIMP-2 as well as how to balance those in order to prevent corneal dissolving perforation are still needed to be studied.更多还原