改良聚羟乙基丙烯酸甲酯-聚甲基丙烯酸甲酯一体化人工角膜植入兔与猴角膜的实验研究

目的：探讨聚羟乙基丙烯酸甲酯（PHEMA）海绵支架材料与角膜组织的生物相容性,评价改良聚羟乙基丙烯酸甲酯（PHEMA）一聚甲基丙烯酸甲酯（PMMA）一体化人工角膜植入龟与猴角膜的初步临床效果。方法：通过两个阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。实验分为两个部分完成。第1部分（A组）：将PHEMA海绵边裙材料植入10只正常兔角膜板层间,术后2,4wk及2,3,4mo分别行组织病理、免疫组织化学及电镜检查,观察海绵边裙与角膜组织的生物学愈合情况。第2部分（B组）：8只兔眼和2只猴眼角膜囊袋内I期植入一体化人工角膜,术后临床观察材料与组织的愈合情况;术后3mo时行Ⅱ期手术切除术眼角膜中央前板层角膜组织,暴露人工角膜中央镜柱,术后随访时间3～6mo,初步观察临床治疗效果。结果：1）A组接受角膜板层间边裙植入术的10只兔眼,随访期间未见并发症：组织病理学显示,PHEMA海绵边裙材料植入术后2wk始有成纤维细胞长入,术后2～3mo时多量成纤维细胞长入并伴有新生血管生长;免疫组织化学显示,海绵孔隙中长入的细胞和角膜基质细胞均对波形蛋白免疫反应呈阳性;电镜下可见,成纤维细胞在海绵材料间隙中生长,细胞生长状态良好,并分泌胶原和细胞外基质。2）B组接受一体化人工角膜移植术的,6只兔眼一体化人工角膜均在位,男2只兔眼I期术后有前板层角膜基质融解。接受一体化人工角膜移植术的2只猴眼,I期和Ⅱ期术后均未见明显并发症。结论：PHEMA海绵能与角膜组织良好的生物学愈合;改良PHEMA-PMMA一体化人工角膜植入术后能获得相对稳定的治疗效果。     

改良聚羟乙基丙烯酸甲酯-聚甲基丙烯酸甲酯一体化人工角膜植入兔与猴角膜的实验研究(英文)

目的:探讨聚羟乙基丙烯酸甲酯(PHEMA)海绵支架材料与角膜组织的生物相容性,评价改良聚羟乙基丙烯酸甲酯(PHEMA)—聚甲基丙烯酸甲酯(PMMA)一体化人工角膜植入兔与猴角膜的初步临床效果。方法:通过两个阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。实验分为两个部分完成。第1部分(A组):将PHEMA海绵边裙材料植入10只正常兔角膜板层间,术后2,4wk及2,3,4mo分别行组织病理、免疫组织化学及电镜检查,观察海绵边裙与角膜组织的生物学愈合情况。第2部分(B组):8只兔眼和2只猴眼角膜囊袋内I期植入一体化人工角膜,术后临床观察材料与组织的愈合情况;术后3mo时行Ⅱ期手术切除术眼角膜中央前板层角膜组织,暴露人工角膜中央镜柱,术后随访时间3 ～6mo,初步观察临床治疗效果。结果:1)A组接受角膜板层间边裙植入术的10只兔眼,随访期间未见并发症:组织病理学显示,PHEMA海绵边裙材料植入术后2wk始有成纤维细胞长入,术后2 ～3mo时多量成纤维细胞长入并伴有新生血管生长;免疫组织化学显示,海绵孔隙中长入的细胞和角膜基质细胞均对波形蛋白免疫反应呈阳性;电镜下可见,成纤维细胞在海绵材料间隙中生长,细胞生长状态良好,并分泌胶原和细胞外基质。2)B组接受一体化人工角膜移植术的,6只兔眼一体化人工角膜均在位,另2只兔眼I期术后有前板层角膜基质融解。接受一体化人工角膜移植术的2只猴眼,I期和Ⅱ期术后均未见明显并发症。结论:PHEMA海绵能与角膜组织良好的生物学愈合;改良PHEMA-PMMA一体化人工角膜植入术后能获得相对稳定的治疗效果。     

改良PHEMA-PMMA一体化人工角膜治疗兔角膜碱烧伤的实验研究

目的评价改良聚羟乙基丙烯酸甲酯（PHEMA）-聚甲基丙烯酸甲酯（PMMA）一体化人工角膜时兔角膜碱烧伤的治疗效果。方法通过两阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。将PHEMA海绵边裙材料植入10只正常兔角膜板层间，另10只碱烧伤兔眼角膜囊袋内1期植入一体化PHEMA—PMMA人工角膜，术后用组织病理、免疫组织化学和电镜检查观察海绵边裙与角膜组织的生物学愈合情况。术后2个月行II期手术．术后随访观察3～6个月。结果接受板层间植入术的10只兔角膜未见任何并发症。术后2周成纤维细胞长入PHEMA海绵，术后2～3个月多量细胞长入伴有新生血管；海绵孔隙中生长细胞和角膜基质细胞波形蛋白（Vim）免疫反应阳性；电镜下，细胞在海绵材料间隙中生长状态良好，并分泌胶原和细胞外基质。接受人工角膜移植术的10只碱烧伤兔眼中，1眼1期术中角膜穿孔而被排除，另9眼Ⅱ期术后，3眼2周内人工角膜镜柱偏位，6眼观察期间人工角膜在位。结论PHEMA海绵能够与角膜组织达到良好的生物学愈合；人工角膜的片型和整体强度有待进一步改良。     

壳聚糖复合人工角膜小型猪植入实验研究

目的 探讨壳聚糖纳米生物一体化人工角膜的生物相容性及植入小型猪角膜的治疗效果.方法 10只小型猪（藏香猪）均以左眼作为实验眼,建立角膜碱烧伤模型.人工角膜植入手术分两期进行：Ⅰ期手术将人工角膜植入碱烧伤猪角膜板层囊袋内;3个月后行Ⅱ期手术（角膜前板层开窗术）,钻切中央角膜前板层组织,暴露人工角膜光学区.术后观察6个月,行眼前段OCT检查、病理及免疫组化检测.结果 10只小型猪中1例在Ⅰ期术后2个月出现术眼感染性角膜溃疡,前板层溶解,最终导致人工角膜脱出.9例完成人工角膜Ⅱ期开窗手术,术后3例发生人工角膜前增生膜,4例出现后增生膜（其中2例前、后兼有）,余4例角膜中央光学区维持透明,荧光素染色无渗漏.组织学检查发现人工角膜裙边材料孔隙中有纤维组织长入;眼前段OCT显示猪眼前段解剖构基本正常;免疫组化分析发现前、后增生膜样本中血管内皮生长因子（VEGF）呈现高表达.结论 壳聚糖纳米生物一体化人工角膜的组织生物相容性良好,其裙边材料有利于角膜纤维组织及新生血管长入,该人工角膜的小型猪植入实验获有相对稳定的移植治疗效果.     

聚酯纤维网的角膜生物相容性实验研究

目的探讨聚酯纤维网植入兔角膜板层后与角膜组织的生物相容性及其与角膜的生物愈合特性。方法选择20只健康新西兰兔,体重2.0～2.5 kg,右眼均行手术作为实验组,左眼作为正常对照组。实验组中15只右眼角膜板层植入聚酯纤维网,并分为术后2周、1个月、3个月、5个月、8个月亚组,作为不同时段的取材期,每亚组3只右眼;其余5只右眼角膜仅制备囊带而不植入材料,作为手术对照亚组;术后定期观察角膜水肿和新生血管生长情况,并取角膜行光镜检查。结果术后早期实验组兔眼不同程度地出现角膜水肿和新生毛细血管的长入,经术后恢复或拆线后均呈自行性消退和闭锁。在最长为8个月的观察期中,植入物在角膜组织中稳定。与植入材料相贴的角膜板层上下界面,成纤维细胞数量明显增加,网孔部位尤其明显,并最终形成胶原纤维紧密的连接。所有植入材料的兔眼均未见角膜坏死、溃疡及植入物脱出。结论聚酯纤维网在兔角膜组织中具有良好的生物相容性。     

两种软性聚甲基丙烯酸羟乙酯一体式人工角膜植入碱烧伤兔角膜的研究

目的 评估两种软性聚甲基丙烯酸羟乙酯(PHEMA)一体式人工角膜(Ⅰ型和Ⅱ型)植入碱烧伤角膜后的临床结果.方法 实验研究.将12枚两种人工角膜植入(各6枚)碱烧伤后的兔角膜,进行裂隙灯、眼底照相、B超及超声生物显微镜(UBM)检查.结果 所有植入物均稳定存留于角膜内,实验观察最长达72周.所有的人工角膜位置无移位,无结膜瓣裂开,无漏水,无感染,指测眼压正常,B超显示后节情况正常,UBM显示大部分动物出现虹膜粘连(10只眼),眼底照相无视网膜脱离.Ⅰ型有3只眼发生后增殖物;Ⅱ型无后增殖物发生.每一例人工角膜表面可见增生的结膜覆盖光学区域.结论 新型的PHEMA一体式人工角膜没有界面问题,手术一次植入,其中Ⅰ型无需摘除晶体和玻璃体切除,Ⅱ型光学区后突出部分可能会降低后增殖膜的发生,并且采用的软性水凝胶材料使眼压测量成为可能,其长期效果需要进一步观察.     

PMMA-硅胶夹持型人工角膜植入碱烧伤兔眼的实验研究

目的探讨自制的PMMA-硅胶夹持型人工角膜植入碱烧伤兔眼角膜的手术方法和疗效。方法建立兔眼碱烧伤模型：将PMMA-硅胶夹持型人工角膜植入碱烧伤后形成白斑的兔眼角膜,观察6个月。结果人工角膜手术操作顺利,术后1只实验兔眼出现了眼内炎,人工角膜脱出,另1只兔术后7周死亡,余3只兔眼在观察期内未见角膜组织溶解、房水渗漏、青光眼、感染或人工角膜前后膜增生等并发症,人工角膜稳定在位并保持透明。结论本实验自制的PMMA-硅胶夹持型人工角膜,手术操作简单,术中及术后并发症较小,似有实际临床应用价值。     

人工角膜在兔角膜内的生物学反应

目的评价3种不同直径人工角膜在动物体内应用的生物学反应.方法将35只兔随机分为Ⅰ组(11只)、Ⅱ组(12只)、Ⅲ组(12只),分别植入3种不同直径人工角膜(4.5～7.0 mm、 7.0～9.0 mm、4.5～6.0 mm),术前将人工角膜进行纤维细胞组织培养,光学中心前表面经rf-argon plasma处理后植入兔眼,观察术后人工角膜在兔角膜中的存留,观察时间＞6个月;并分别于术后2、3、4及6周摘取兔植入人工角膜行组织及病理学检查.结果术后2周成纤维细胞可沉积在人工角膜周边多孔支架内;3周时Ⅲ组角膜上皮细胞位于人工角膜光学中心,Ⅰ、Ⅱ组未见细胞生长.4周时人工角膜内可检测出胶原蛋白沉积.前房稳定,未见房水渗漏.术后6周时人工角膜的周边支架中检测出heparan sulfate(HS)及碱性成纤维细胞生长因子,后者存在于周边支架和宿主角膜的交接处.结论体外人工角膜组织培养可增加人工角膜周边支架的纤维化,稳定光学中心;人工角膜经 rf-argon plasma处理可增加材料的组织相容性;兔角膜缘上皮细胞可迁徙至人工角膜光学中心的前表面.     

AlphaCor人工角膜移植治疗兔角膜碱烧伤

目的评价AlphaCor人工角膜移植对兔角膜碱烧伤的治疗效果。方法用1mol/LNaOH烧伤兔左眼建立角膜碱烧伤模型,选择角膜白斑、角膜缘新生血管〉2个象限的5只兔眼行人工角膜移植术。AlphaCor人工角膜移植手术分2期进行：Ⅰ期手术,将AlphaCor人工角膜植入角膜板层间;Ⅱ期手术,暴露人工角膜光学区。术后观察人工角膜、受体角膜及眼前节情况。结果碱烧伤后2～6周形成带新生血管的角膜白斑。Ⅰ期术后5例人工角膜均在位,2例人工角膜出现白色沉积物,其中1例并发角膜浅层基质溶解,另3例无并发症发生。Ⅱ期手术中麻醉死亡1例,其余术后人工角膜均在位,1例术后6周发生角膜感染、穿孔,另3例术后4～6周发生人工角膜前膜增生,未见其他并发症发生。结论 AlphaCor人工角膜移植治疗兔角膜碱烧伤具有良好的生物相容性,效果较好。     

壳聚糖胶原复合膜在兔角膜基质层间的组织相容性

目的探讨壳聚糖胶原复合膜植入兔角膜基质层的组织相容性,以评价其作为人组织工程角膜修复支架材料的可行性。方法选用8只新西兰白兔,根据角膜基质层间植入膜片材料的不同平均分为A、B两组,A组植入壳聚糖胶原复合膜（实验组）、B组植入壳聚糖膜（对照组）。术后观察并记录术眼球结膜充血、角膜水肿、前房闪辉、角膜新生血管、材料的变化等情况,术后第8周取术眼角膜组织制作病理学切片进行HE染色观察,比较两组材料对角膜组织的不同影响。结果术后8周内,实验组炎症反应轻于对照组,且无角膜新生血管发生,对照组有2例发生角膜新生血管,两组膜片均于术后第2周开始降解,但在相同观察时间点,实验组膜片降解程度小于对照组。HE染色显示实验组膜片周围炎性细胞浸润轻于对照组,膜片降解程度小于对照组,降解物与角膜基质纤维融合。结论壳聚糖胶原复合膜与活体兔角膜基质组织的相容性比壳聚糖膜更优,其作为角膜损伤修复材料具有广阔前景。     

Implantation of modified poly 2 -hydroxyethy methacrylate -Polymethyl methacrylate Keratopro - stheses in rabbit and monkey corneas

AIM: To investigate the bio-colonization of poly 2-hydroxyethy methacrylate (PHEMA) sponge with cornea tissue and evaluate the therapeutic effects of modified porous poly 2-hydroxyethy methacrylate-Polymethyl methacrylate (PHEMA-PMMA) Keratoprostheses (KPro) on rabbit and monkey corneas. METHODS: The KPro were made using two-stage polymerization combined with mechanical cutting. The experiments were divided into two groups. In the control group, ten normal rabbit eyes received lamellar implantation of PHEMA sponges. The sponges were obtained 2 weeks, 1, 2, 3 and 4 months after operation. The cell proliferation and neovascularization inside the sponges were observed using light and transmission electron microscopy (TEM) and immunohistochemistry. In the experimental group, the porous PHEMA-PMMA KPros were inserted into the lamellar pockets of eight rabbit corneas and two monkey corneas (stage I operation). The healing process was investigated by slit-lamp microscopy. The anterior lamellar cornea tissues were removed 3 months after surgery, exposing the underneath transparent core (stage II operation). The operated eyes were then followed up for 3-6 months. RESULTS: No complications were observed in the control group. Under the light microscope, fibroblasts started to grow into the cornea 2 weeks after operation; lots of cells, accompanied with new blood vessels, invaded into the cornea 2-3 months after surgery. Invading cells of sponge, as well as keratocytes, were positive for vimentin. Under the electron microscope, the invading cells looked healthy and were surrounded by extracellular matrix and collagen. In eight rabbit eyes which received KPro implantation, anterior lamellar cornea melting happened in two eyes after the stage II operation. The remaining six corneas retained their central cores during observation after the stage II operation. Two operated monkey eyes were found no complication throughout the whole follow-up. CONCLUSION: The PHEMA sponge can obtain a tight fusion with the host cornea. The modified PHEMA-PMMA KPros have obtained relatively stable results after implantation into animal corneas. "     

Clinical results of implantation of the Chirila keratoprosthesis in rabbits

AIMS/BACKGROUND—An ideal keratoprosthesis (KPro) would closely resemble a donor corneal button in terms of its surgical handling, optics, and capacity to heal with host tissue in order to avoid many of the complications associated with the KPros which are currently in clinical use. This study was carried out to assess the long term clinical outcomes on implantation of the core and skirt poly(2-hydroxyethyl methacrylate) KPro in animals.
METHODS—20 KPros were made and implanted as full thickness corneal replacements into rabbits and followed for up to 21 months to date.
RESULTS—80% of the prostheses have been retained, with a low incidence of complications such as cataract, glaucoma, and retroprosthetic membrane formation which are frequently associated with KPro surgery.
CONCLUSIONS—KPros of this type may offer promise in the treatment of patients for whom penetrating keratoplasty with donor material carries a poor prognosis. Refinement of the KPro and further animal trials, including implantation into abnormal corneas, are however mandatory before human implantation could be planned.

Keywords: complications; keratoprosthesis; optics; PHEMA     

自体角膜为载体Boston人工角膜治疗复杂性角膜盲

目的评价复杂性角膜混浊患者使用自体角膜做载体的BostonI型人工角膜植入的临床效果。方法回顾性病例研究。10例角膜盲患者（10眼）,术眼病变严重,经至少2名以上国内著名角膜病专家会诊。无法通过角膜移植复明。其中严重碱烧伤7眼,爆炸伤1眼,角膜内皮失代偿多次角膜移植失败1眼．双眼病毒性角膜炎角膜严重血管化1眼。其中9例为双眼盲。术前视力光感或手动。所有病例均一期完成手术,术中使用8．0mm或8．5mm环钻钻取患者角膜,用患者混浊病变的角膜为载体。安装Boston人工角膜。形成患者角膜．人工角膜复合体,复合体作为植片如传统角膜移植实行角膜植床和植片缝合固定,术中常规行晶状体摘除术。结果术后观察1-12个月,平均（5．7±3．8）个月,除1眼视力光感外,其余裸眼视力为0．1～0．8。手术并发症包括角膜植床出血人玻璃体腔2例,继发性青光眼2例,人工角膜后膜4例。所有术眼均无术后漏水并发症发生。结论人工角膜特别适合于穿透性角膜移植难于成功的角膜盲患者,而且是目前对严重角膜瘢痕血管化、眼睑或泪液功能不良患者有效的复明手段。我国角膜供体严重匮乏,用自体角膜为载体可作为部分BostonI型人工角膜植入手术的可行方案。     

Keratoprosthesis results in animals: An update

Background: The report presented is an update on continuing development work on modified PHEMA core-and-shirt KPros in animals. Methods: Two variations (improved wet-eye, and dry-eye) of a prototype core-and-skirt Chirila KPro are described. The clinical success rate on implantation of these versions of the Chirila KPro was assessed. Results: It was found that a significant improvement in retention rate was shown in the improved model but that the dry-eye model failed early in two of the three implanted. Conclusions: The significance of the improved strength and the reasons for disappointing results with the early dry-eye KPros are discussed. Ongoing work is briefly outlined.     

国产化人工角膜植入兔眼的稳定性评估

目的 评价国产化人工角膜在兔眼角膜中的中长期稳定性.方法 实验研究.采用长耳大白兔10只,均选右眼作为国产化Moroz改良型永久性人工角膜植入眼,左眼为对照眼.手术分两期完成,一期手术用第三眼睑(软骨组织与人耳软骨有类似效果)加固角膜前层,联合自体结膜遮盖植入人工角膜支架;3个月后进行二期手术,植人人工角膜镜柱.全部手术完成后观察6个月以上.术后抗生素滴眼预防感染,定期观察,照相记录.最终行眼组织病理学检查.并与左眼进行对照.结果 除1只兔在二期术中因麻醉意外死亡外,9只兔均顺利完成手术和术后观察,术后各观察时间点人工角膜均在位,眼表血管化良好,未发生角膜组织融解、房水渗漏、高眼压、感染等严重并发症.组织病理学检查显示:加固的软骨组织均在位,人工角膜支架与周围组织紧密结合,有较活跃的纤维组织增生包绕,角膜炎症反应很轻.对侧眼无异常.结论 国产化人工角膜具有良好的生物相容性和中长期在位稳定性,用耳软骨加固和自体结膜遮盖有助于提高人工角膜稳定性.     

Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea

AIM: To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS: A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy (TEM) and hematoxylin and eosin (H&E) staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype. RESULTS: The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process. In vitro, the methyl thiazolyl tetrazolium results revealed that extracts of the acellular ostrich corneas (AOCs) had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes. The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea (APC) transplantation. The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer. CONCLUSION: We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.     

去端肽猪皮Ⅰ型胶原在兔角膜组织中的生物相容性

目的 评价去端肽猪皮Ⅰ型胶原在兔角膜组织中的生物学反应,以探讨其作为角膜支架材料的可行性.方法 大耳白兔40只,分为5组,每组8只,一眼为实验眼,另一眼作为正常对照眼.将去端肽猪皮Ⅰ型胶原植入兔角膜前板层,术后1个月内每周2次、1个月后每月1次行肉眼、裂隙灯观察,并于每次观察后记录角膜是否透明、有无新生血管的评分情况;术后3 d、14 d、1个月、3个月及6个月,取兔角膜行组织病理学观察以及上皮细胞标志蛋白K3的免疫组化检测.评分结果采用Friedman秩和检验行组间比较,两两之间比较行Wilcoxon符号秩和检验.结果 术后角膜透明度逐渐恢复,新生血管化程度逐渐升高,至术后1个月最高,后逐渐减轻并退化;术后6个月,角膜透明度和新生血管化程度与正常眼比较,差异无统计学意义（P＞0.05）.整个观察期间未见角膜坏死、溃疡及植片脱出.结论 去端肽猪皮Ⅰ型胶原具有较好的生物相容性,进一步改进后有望成为一种新型的角膜移植支架材料.