新型可降解支架材料植人角膜的生物学反应研究

目的 探讨可降解交联剂交联乙烯基吡咯烷酮对兔角膜组织的生物学反应的影响。方法 将乙烯基吡咯烷酮植入兔角膜囊袋内3个月，观察生物材料在角膜内生物学反应。结果 可降解交联剂交联乙烯基吡咯烷酮合成材料植入兔角膜囊袋3个月，生物相容性良好，材料逐渐降解，材料内有胶原和角膜基质细胞长入。结论 新型可降解交联剂交联乙烯基吡咯烷酮合成材料植入后未见兔角膜有明显的炎症反应，材料的组织相容性好，可作为组织工程角膜的支架材料。     

原花青素交联的支架材料的生物相容性研究

目的 探讨原花青素作为交联剂的壳聚糖、胶原蛋白、硫酸软骨素、透明质酸钠合成的支架材料植入兔角膜的生物相容性.方法 将壳聚糖、胶原蛋白、硫酸软骨素、透明质酸钠等溶液按照一定的比例混合,加入原花青素和戊二醛溶液作为交联剂,制成2种支架材料分别植入2组新西兰大白兔角膜的囊袋内.在不同的时间点进行裂隙灯显微镜观察照相,并进行组织病理学检查,比较不同的支架材料对角膜的影响.结果 原花青素交联组术后1周有结膜睫状充血,角膜水肿;2周后充血水肿逐渐消失.兔眼角膜情况稳定,术后4个月基本透明;材料与角膜的生物相容性好,5个月后材料大部分吸收溶解,角膜透明.组织病理学检查可见兔角膜标本中材料内有角膜基质细胞长入.原花青素交联的支架材料比戊二醛交联的支架材料刺激性小.结论 原花青素交联的壳聚糖、胶原蛋白、硫酸软骨素、透明质酸钠组成的支架材料与角膜组织具有良好的生物相容性,原花青素可能比戊二醛更适合作为支架材料的交联剂.     

人工角膜支架材料在动物体内应用的生物学反应

目的 探讨3种人工角膜支架材料经氩等离子体（Argon plasma)处理后植入兔角膜对角膜组织的生物学反应的影响。方法 选用3种支架材料：由80％的聚丙烯和20％聚丁烯化合而成的多孔材料；Polyester多孔材料；Expended polytetrafluoroethylene。处理组材料经Argon plasma处理。将盘状支架材料植入兔角膜囊袋内。结果 材料经处理后角膜水肿较未处理明显减轻，8周后3种材料中角膜水肿均消失。材料1，2植入时新生血管出现延迟，范围小，炎细胞浸润明显减少。术后6周纤维细胞迁徙入材料内。当材料植入角膜后角化蛋白（KS）含量降低，Dermatan(DS)含量增高，并有大分子葡萄糖聚糖（GAG）出现。结论 Argon-plasma处理可明显减轻兔角膜炎症反应，增加材料的组织相容性。     

异种脱细胞角膜基质材料的生物相容性研究

目的：研究脱细胞猪角膜基质材料在角膜组织中的生物相容性,为组织工程角膜的构建寻找一种良好的支架材料。方法：将用去垢剂联合酶的方法脱细胞处理的猪角膜基质材料植入兔角膜基质囊袋中,进行裂隙灯观察并照相。不同时间取材HE染色,进行光镜检查。结果：脱细胞猪角膜基质材料植入兔角膜基质囊袋,观察4mo,生物相容性良好,材料逐渐降解吸收。结论：脱细胞猪角膜基质材料植入兔角膜中无炎症、无免疫排斥反应,生物相容性好,可作为组织工程角膜的支架材料。     

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、4周及2、3、4个月分别行组织病理、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3个月时行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—6个月,初步观察临床治疗效果。结果（1）A组：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2周始有成纤维细胞长入,术后2—3个月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：6只兔眼的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。2只猴眼于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。（中华眼科杂志．2007,43：602-607）     

Argon—Plasma对人工角膜支架材料组织相容性的影响

目的 探讨3种人工角膜支架材料经Argon-Plasma处理后植入兔膜对兔角膜组织的影响。方法 选用3种支架材料：材料1：聚丙烯和聚丁烯（80:20）化合而成的多孔材料;材料2：Polyester多孔材料;材料 3:Expended polytetrafluoroethglene。上述3种材料切成直径6mm的圆盘状,每种材料分为处理组和非处理组。处理组材料经Argon-Plasma处理。选用63只新西兰大白兔,共分为7组,每组9只,6组为实验组及对照组（3组为处理组,3组为非处理组）,1组为手术对照组。以显微手术的方法,将盘状支架材料植入囊袋内,手术对照组仅制备囊袋不植入材料。术后定期观察角膜水肿及新生血管的生长情况。术后28、42、84d取角膜进行角膜组织学及免疫组织化学分析。结果 当材料经处理后角膜水肿较未处理明显减轻,8周后3种材料中角膜水肿均消失。材料1、2植入时新生血管出现延迟,范围也较小,同时炎细胞浸润明显减少。术后6周纤维细胞迁徒入材料内,术后12周空腔填充大量纤维细胞。材料处理与否不影响胶原蛋白的沉积。结论 Argon-Plasma处理可明显减轻兔角膜炎症反应,增加材料的组织相容性。     

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。     

异种角膜片层间植入的组织相容性及形态学研究

采用层间囊袋分离法在２８只兔眼角膜上行角膜层间植入术，植片由人尸体角膜制成，最后进行观察并取得结果的２３只，术后观察３个月，角膜保持透明，形态学的主要特点是植片内角膜细胞缺如，内皮细胞形态结构未见明显影响，提示人眼角膜植片与兔眼角膜具有较好的组织相容性，为非人类角膜材料的应用提供了初步的经验与依据。     

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。     

脱细胞猪角膜基质的生物相容性与组织工程化兔角膜上皮移植的研究

目的 探讨脱细胞猪角膜基质的生物相容性,评价组织工程化角膜上皮组织作供体的可行性,观察支架材料的细胞化情况和种子细胞的存活情况.方法 实验研究.采用完全随机化设计的方法,用Dispase-Triton-X-100处理猪角膜基质,脱去角膜细胞;以角膜基质囊袋内植入的方法,观察异种角膜基质植入后的生物相容性,A组:脱细胞猪角膜基质,B组:新鲜猪角膜基质,C组:空白对照组.以组织工程化雄性角膜上皮组织为供体,同种雌性为受体,作板层角膜移植,观察角膜的混浊、水肿、新生血管等情况;组织病理学和免疫组化方法检测支架材料的细胞化情况,Y染色体性别决定基因(SRY)-聚合酶链反应(PCR)方法追踪种子细胞的存活情况.结果 猪角膜基质植入兔角膜囊袋后,角膜逐渐恢复透明,排斥反应指数＜6,组织病理学观察角膜结构完整,胶原纤维平行排列,少许细胞长入脱细胞猪角膜基质边缘,各组免疫组化检测未见CIM+、CD8+T淋巴细胞浸润.组织工程化角膜上皮作异体板层角膜移植后,3～4 d上皮光滑,10～20 d变为透明;15 d时角膜上皮、基质、内皮完整,上皮细胞约4或5层结构,少许基质细胞长入支架,1个月时可见角膜上皮细胞约7或8层细胞,基质纤维排列规则,多量细胞长入脱细胞角膜基质.上皮细胞表达CK3,支架内新生细胞表达波形蛋白.SRY-PCR结果显示种子细胞可以在受体内长期存活.结论 脱细胞猪角膜基质生物相容性良好,组织工程化角膜上皮可作为板层角膜移植的供体,脱细胞猪角膜基质细胞化良好,种子细胞可以在受体内长期存活.     

Engineering the crystalline lens with a biodegradable or non-degradable scaffold

Both a biodegradable hyaluronic acid (HA) and a nondegradable polymeric gel were evaluated as scaffolds for tissue engineering the lens in Dutch Belt pigmented and New Zealand white rabbits. Following removal of the crystalline lens through a 2 mm capsulorhexis, a collagen patch or a silicone plug was placed in the capsule bag to seal the capsulotomy. In Part I, a cross-linked HA or cohesive solution of HA was injected into the capsule bag. In Part II, a synthetic polymer (SP) was injected into the capsular bag of one eye and HA followed by SP (HA/SP) was injected into the capsule bag of the opposite eye. At 3 months focal Nd:YAG laser photocoagulation was performed in an attempt to remove some retained HA gel in one eye. Overall the regenerated lenses of the HA gel groups were spherical with excellent cortical structure and clarity and a spherical nucleus of condensed HA gel. In the one eye treated with focal photocoagulation, partial clearing of the retained HA gel was noted. In the SP and HA/SP eyes, lens regrowth around the polymer was generally clear in the anterior and peripheral capsule bag and more opacified posterior to the polymeric scaffold. In summary, naturally regenerating lens tissue was directed to grow in a more normal, regular pattern by providing a biodegradable hyaluronic acid scaffold or a nondegradable polymeric optical scaffold.     

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.     

兔脂肪干细胞复合PLGA支架的生物相容性研究

背景种子细胞和支架材料是角膜组织工程研究的主要课题。脂肪干细胞具有来源广泛、增生和分化能力强的特点而成为目前组织工程种子细胞的研究热点,聚羟基乙醇（PLGA）作为高分子可降解支架材料已成功用于构建多种组织器官。目的探讨兔脂肪干细胞的生物学特性及其复合多孔支架材料聚羟基乙醇（PLGA）的生物相容性,为进一步构建脂肪干细胞组织工程化角膜基质提供实验基础。方法取雌性新西兰大白兔颈背部脂肪组织,采用消化法分离培养兔脂肪细胞,传至第4代。将传代细胞分别以3×10^4／cm^2、3×10^4／cm^2、3×10^6／cm^2的密度接种于6孔板中,分别用成骨诱导培养液、成脂诱导培养液及成软骨诱导液进行诱导培养,并分别以质量分数1％茜素红-Tris-盐酸溶液、质量分数0．6％油红染液和免疫荧光法鉴定培养细胞的多向分化能力。将第4代细胞用稀释的DiO荧光染液重悬的脂肪干细胞按1×10^7／ml的密度接种于自制的多孔PLGA支架形成细胞-生物材料复合物,Hoechst法定量检测细胞在支架上的生长情况,并分别于接种后第1、3、7天对细胞-生物材料复合物行共焦显微镜和扫描电子显微镜检测,观察细胞在该支架上的黏附生长和基质分泌情况,评价PLGA的生物相容性。结果原代培养的脂肪细胞7～8d后可达80％～90％融合,呈成纤维细胞样外观。传代第4代的细胞成骨诱导2周后茜素红染色显示矿化结节及周围细胞着深红色;成脂诱导2周后油红0染色显示细胞质内布满红色脂滴颗粒;微团培养成软骨诱导2周后,免疫荧光染色结果显示Ⅱ型胶原表达阳性。细胞接种至PLGA支架材料第7天增生达到高峰期,扫描电子显微镜和共焦显微镜检测显示细胞在支架上贴附生长良好,能够在支架表面及孔隙内壁得到充分伸展和生长,细胞外基质分泌旺盛。结论培养的兔脂肪细胞具有脂肪干细胞的多向分化功能,与多孔PLGA支架复合具有良好的生物相容性,可作为构建组织工程化角膜基质的种子细胞和支架材料。     

组织工程角膜支架材料研究现状

支架材料是组织工程角膜的土壤,土壤微环境的好坏(即支架材料的选择)直接关系到组织工程角膜的构建及其应用。选择具有更高生物活性、透明性、机械强度等特点的支架材料是组织工程角膜研究的热点,本文就支架材料的研究现状做一综述。     

Dissociation is not required for alpha-crystallin's chaperone function

Bovine alpha-crystallin was crosslinked with glutaraldehyde under conditions designed to minimise intermolecular reactions. The crosslinked protein was too large to enter SDS polyacrylamide gels but HPLC-gel permeation chromatography revealed that the Stoke's radii of the native and crosslinked proteins were very similar. These observations indicate that only intramolecular crosslinks had formed and that the crosslinked protein could not dissociate to smaller species. The crosslinked alpha-crystallin was able to inhibit the thermally-induced precipitation of beta-crystallin and appeared to be more effective than the native protein under the same conditions. It is concluded that the chaperone activity of alpha-crystallin is a surface phenomenon and dissociation into smaller species is not required.     

转谷氨酰胺酶交联胶原凝胶构建三维角膜基质

目的检测转谷氨酰胺酶交联胶原凝胶对三维培养的角膜基质细胞的影响,探讨可提高机械性能的组织工程角膜基质层新途径。方法胶原酶消化法获取原代兔角膜基质细胞,以加入转谷氨酰胺酶与胶原凝胶交联为实验组,不加酶交联为对照组。倒置显微镜下每日观察细胞生长情况、Alamar-Blue试剂检测细胞增生、免疫荧光法检测凝胶内细胞波形蛋白、检测透光度、酶消化法检测胶原凝胶抗消化能力。结果实验组细胞胶原凝胶内附着和生长优于对照组,细胞在凝胶内呈树枝状生长。2组细胞均随培养时间延长明显增生（P=0.000）。共焦显微镜下见2组细胞胞浆波形蛋白均阳性表达,实验组细胞伪足更丰富。实验组透光度稍差于对照组。实验组抵抗胶原酶消化的能力显著增强。结论酶交联的胶原凝胶对角膜基质细胞无毒性作用,重构的基质层结构更加稳定,有利于组织工程角膜基质层的构建。     

组织工程结膜支架的研究进展

结膜重建常用于各种化学热烧伤所导致的严重结膜缺损,而组织工程结膜能有效解决在结膜移植中自体结膜移植材料缺乏及异体结膜移植材料存在免疫排斥反应等难题。具有良好生物相容性及足够生物力学强度的支架材料是构建组织工程结膜的重要条件。本文对组织工程结膜支架材料的最新研究进展及目前存在的问题进行总结。     

Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds

The keratoprosthesis (KPro; artificial cornea) is a special refractive device to replace human cornea by using heterogeneous forming materials for the implantation into the damaged eyes in order to obtain a certain vision. The main problems of artificial cornea are the biocompatibility and stability of the tissue particularly in penetrating keratoplasty. The current studies of tissue-engineered scaffold materials through comprising composites of natural and synthetic biopolymers together have developed a new way to artificial cornea. Although a wide agreement that the long-term stability of these devices would be greatly improved by the presence of cornea cells, modification of keratoprosthesis to support cornea cells remains elusive. Most of the studies on corneal substrate materials and surface modification of composites have tried to improve the growth and biocompatibility of cornea cells which can not only reduce the stimulus of heterogeneous materials, but also more importantly continuous and stable cornea cells can prevent the destruction of collagenase. The necrosis of stroma and spontaneous extrusion of the device, allow for maintenance of a precorneal tear layer, and play the role of ensuring a good optical surface and resisting bacterial infection. As a result, improvement in corneal cells has been the main aim of several recent investigations; some effort has focused on biomaterial for its well biological properties such as promoting the growth of cornea cells. The purpose of this review is to summary the growth status of the corneal cells after the implantation of several artificial corneas.     

Scleral plug of biodegradable polymers containing ganciclovir for experimental cytomegalovirus retinitis

PURPOSE: To evaluate the efficacy of a biodegradable scleral plug containing ganciclovir (GCV) in a rabbit model of human cytomegalovirus (HCMV) retinitis. METHODS: To develop a rabbit model for HCMV retinitis, HCMV solution was injected once into the vitreous cavity of pigmented rabbits. The treated animals were divided into three groups: group A received no treatment, group B was treated once with GCV solution, and group C was treated with a scleral plug containing GCV. Rabbits in group B received an intravitreal injection of GCV solution 1 week after HCMV inoculation. In group C, the scleral plug containing GCV was implanted in the vitreous of the rabbits 1 week after HCMV inoculation. Ophthalmoscopically, vitreoretinal findings in each group were graded from 0+ to 4+ every week for 4 weeks after HCMV injection. RESULTS: Eyes of group A rabbits showed whitish retinal exudates and vitreous opacities 3 days after HCMV inoculation. These materials increased gradually until 3 weeks after HCMV inoculation. Scores for vitreoretinal lesions were significantly lower in eyes of group B rabbits compared with those of group A at 1 week after GCV injection (P < 0.05). However, vitreoretinal inflammation in eyes of group B rabbits increased again thereafter, and no significant difference in inflammation between groups A and B was found 2 weeks after GCV injection. In eyes of group C, scores for vitreoretinal lesions were significantly lower compared with those in both group A and group B at 3 weeks after HCMV inoculation (P < 0.01). CONCLUSIONS: The results demonstrated that sustained release of GCV into the vitreous cavity with biodegradable scleral plugs was effective for the treatment of experimentally induced HCMV retinitis in rabbits.     

MPP5 recruits MPP4 to the CRB1 complex in photoreceptors

PURPOSE: Mutations in the human Crumbs homologue 1 (CRB1) gene are a frequent cause of Leber congenital amaurosis (LCA) and various forms of retinitis pigmentosa. CRB1 is thought to organize an intracellular protein scaffold in the retina that is involved in photoreceptor polarity. This study was focused on the identification, subcellular localization, and binding characteristics of a novel member of the protein scaffold connected to CRB1. METHODS: To dissect the protein scaffold connected to CRB1, the yeast two-hybrid approach was used to screen for interacting proteins. Glutathione S-transferase (GST) pull-down analysis and immunoprecipitation were used to verify protein-protein interactions. The subcellular localization of the proteins was visualized by immunohistochemistry and confocal microscopy on human retinas and immunoelectron microscopy on mouse retinas. RESULTS: A novel member of the scaffold connected to CRB1, called membrane palmitoylated protein (MPP) subfamily member 4 (MPP4), a membrane-associated guanylate kinase (MAGUK) protein, was identified. MPP4 was found to exist in a complex with CRB1 through direct interaction with the MPP subfamily member MPP5 (PALS1). 3D homology modeling provided evidence for a mechanism that regulates the recruitment of both homo- and heterodimers of MPP4 and -5 proteins to the complex. Localization studies in the retina showed that CRB1, MPP5, and MPP4 colocalize at the outer limiting membrane (OLM). CONCLUSIONS: These data imply that MPP4 and -5 have a role in photoreceptor polarity and, by association with CRB1, pinpoint the cognate genes as functional candidate genes for inherited retinopathies.