脱水猪角膜基质为载体体外构建猫角膜内皮组织

目的:以脱水猪角膜基质为载体体外构建猫角膜内皮组织,并观察其形态结构,以寻找体外构建角膜组织最佳载体材料及方法,最终目的是体外构建可用于移植的角膜内皮组织。方法:以脱水处理的猪角膜基质片为载体,将猫角膜内皮细胞接种于去除猪角膜内皮细胞的后弹力膜上,在添加了表皮生长因子和层粘连蛋白的培养液中培养7 d,分别观察倒置显微镜下、组织学切片及扫描电镜下内皮组织的形态结构。结果:倒置显微镜下,组织培养的猫角膜内皮细胞形成单层内皮组织,排列较规律,细胞间连接紧密;组织学观察发现,培养的猫角膜内皮细胞形成完整的内皮组织,贴附于脱水基质的后弹力膜上,与正常的角膜内皮组织结构相似;扫描电镜下,组织培养的猫角膜内皮细胞间连接紧密,细胞大小不甚一致,胞核清晰。 结论:以脱水猪角膜基质为载体,体外成功构建出猫角膜内皮组织,其形态结构近似于正常角膜内皮组织。     

聚乳酸支架材料与角膜上皮样细胞的体外生物相容性

目的观察聚乳酸支架材料与角膜上皮样细胞的体外生物相容性。方法通过热压法与流延法分别制作聚乳酸支架材料。复苏培养前期实验获得的角膜上皮样细胞,免疫荧光化学法鉴定,将第三代角膜上皮样细胞分别种植于上述2种聚乳酸支架材料,显微镜下观察此两种支架材料上细胞的存活与生长情况;采用伊红染色观察支架材料上细胞数量与形态,噻唑蓝法检测细胞活力。结果热压法聚乳酸支架材料为纤维交织立体结构,而流延法材料为实性结构,无孔隙。细胞种植于支架材料后,细胞种植4d后镜下可见热压法聚乳酸支架材料组大量细胞存活,且紧贴着支架材料生长并向支架材料内伸展,伊红染色可见材料上大量着色的细胞;流延法组细胞存活极少,伊红染色仅见极少细胞附着在材料上。结论热压法聚乳酸支架材料具备纤维孔隙结构,支持角膜上皮样细胞的存活与生长,具备与角膜上皮样细胞有较好的生物相容性,有进一步开发角膜上皮样细胞移植支架的前景。     

新型角膜支架材料的制备及生物相容性研究

首先合成了一种新型可降解的三乙烯基交联剂,并以偶氮二异丁氰(A IBN)为引发剂与乙烯基吡咯烷酮(NVP)通过自由基聚合制备了一种新型的交联NVP角膜支架材料。对材料的吸水率、接触角和降解性能进行了测定,通过体内埋植实验和细胞培养对材料进行了生物相容性评价。结果表明材料的吸水率达到104%,接触角为41,°降解速率较为恒定;体内埋植实验研究表明,3个月后,材料已大部分降解,材料内出现胶原和角膜基质细胞,周围组织无明显的炎症反应;细胞培养实验表明,角膜上皮细胞在材料上可以较好的生长,没有明显的细胞毒性,具有良好的生物相容性,这种合成的新型交联NVP角膜支架材料将在角膜组织工程中具有潜在的应用价值。     

异种脱细胞角膜基质囊袋移植的生物相容性研究

目的探讨脱细胞猪角膜基质移植入兔角膜囊袋后的生物学反应。方法猪角膜通过不同方式去除细胞及免疫源性成分，保留角膜组织基质的弹力纤维及胶原纤维，将其切取为直径为4mm的植片，植入兔角膜囊袋内，在不同时间点观察生物材料在角膜内生物学反应。结果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人。结论新型可降解角膜基质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。     

9.999K纯金的生物相容性及其与角膜生物愈合特性的研究

目的：研究9.999K纯金的生物相容性及其与角膜的生物愈合特性,寻找理想的人工角膜周边制作材料。方法：将制作有70－80个小孔、厚0.1mm、直径8mm、曲率半径7.8mm的弧形9.999K纯金薄片植入兔角膜板层中,裂隙灯观察并照相,不同时间点取材进行光镜、扫描电镜以及透射电镜检查。结果：最长观察7个月无脱出,在组织中稳定、无析出、无包裹、对周围组织无毒性。角膜成纤维细胞能长入人工制作的小孔内并合成胶原;8眼出现植入物前角膜基质层钙质沉积。术后第2、3周出现不同程度的角膜水肿与新生血管,10天左右自行消退。结论：纯金具有良好的生物相容性,通过人工制作的小孔与角膜产生生物愈合;植入物一定程度地影响了角膜的内环境;增加小孔面积后,可增加其生物相容性。     

脱细胞猪角膜基质支架的制备及其生物相容性

BACKGROUND: Studies have found that a variety of biological materials can be used for preparing corneal stroma scaffolds that have good biocompatibility, but research on preparation and biocompatibility of the acellular porcine corneal stroma scaffold is little. OBJECTIVE: To explore the preparation and biocompatibility of the acellular porcine corneal stroma scaffold. METHODS: Acellular porcine corneal stroma scaffold and its extract were prepared. Well-grown human corneal stromal cells were selected and cultured in the extract of acellular porcine corneal stroma scaffold (experimental group) or in the complete medium (control group), respectively. After 1, 2 and 3 days of culture, the proliferation ability of human corneal stromal cells was detected by MTT assay. In the meanwhile, human corneal cells were directly seeded onto the acellular porcine corneal stroma scaffold, and then the cell growth on the scaffold was detected using immunochemical method. RESULTS AND CONCLUSION: The number of human corneal stromal cells was in a rise with time in the two groups, and absorbance values had no significant difference between two groups at different time points of culture. Human corneal stromal cells grew well on the scaffold, and were positive for cell integrin β1, vimentin, aldehyde dehydrogenase 3A1, as well as CD34, CDK2 and K-Ras. These results show that the acellular porcine corneal stroma scaffold has no cytotoxicity, and has good biocompatibility.     

9.999K纯金的生物相容性及其与角膜生物愈合特性的研究

目的研究9.999K纯金的生物相容性及其与角膜的生物愈合特性,寻找理想的人工角膜周边制作材料.方法将制作有70～80个小孔、厚0.1mm、直径8mm、曲率半径7.8mm的弧形9.999K纯金薄片植入兔角膜板层中,裂隙灯观察并照相,不同时间点取材进行光镜、扫描电镜以及透射电镜检查.结果最长观察7个月无脱出,在组织中稳定、无析出、无包裹、对周围组织无毒性.角膜成纤维细胞能长入人工制作的小孔内并合成胶原;8眼出现植入物前角膜基质层钙质沉积.术后第2、3周出现不同程度的角膜水肿与新生血管,10天左右自行消退.结论纯金具有良好的生物相容性,通过人工制作的小孔与角膜产生生物愈合;植入物一定程度地影响了角膜的内环境;增加小孔面积后,可增加其生物相容性.     

三种角膜内皮细胞载体膜片的性质研究

以壳聚糖分别与硫酸软骨素、羧甲基壳聚糖、透明质酸按一定比例制备出共混膜，研究了膜片的透光性、吸水率、渗透性、力学性质、表面结构、红外图谱等物理性质。以共混膜为载体培养兔角膜内皮细胞，从而筛选出了透明度高，亲水性好，机械性能优异，最适合内皮细胞贴附生长的壳聚糖．硫酸软骨素共混膜片，并对其生物降解性、生物相容性等性质进行了研究。为了提高壳聚糖．硫酸软骨素共混膜片的通透性，在膜片制备过程中加入不同剂量的醋酸盐作为扩孔剂，结果表明当加入一定量的醋酸盐为扩孔剂时，膜片的通透性提高。     

羟基磷灰石表面修饰的人工角膜钛支架体外生物相容性研究

目的采用细胞培养法观察人工角膜纯钛支架经羟基磷灰石(HA)表面修饰后,其生物相容性是否增加。方法采用第4～6代兔角膜基质成纤维细胞直接接种于HA-Ti、Ti及盖玻片表面,培养3、24、48、72h后,用丫啶橙染色法观察材料表面细胞的黏附、伸展和增殖情况,在扫描电子显微镜下观察材料表面的细胞形态及细胞外基质产生情况。结果细胞接种3、24、48、72h后,HA-Ti表面的活细胞数多于其他材料表面(P0.05)。细胞接种3h,细胞扩展面积:HA-Ti盖玻片Ti。48h后扫描电子显微镜观察发现HA-Ti表面的细胞扩展面积最大,细胞张力丝最长。72h后,HA-Ti表面完全被胶原覆盖。结论HA表面修饰增加了人工角膜纯Ti支架的生物活性。     

一种新型生物人工肝膜材料体外激活血小板的实验

目的：体外研究一种新型生物人工肝反应器材料-丙稀酰胺接枝改性聚丙烯膜（PP-g-AAm）对血小板的激活，并评价其血液相容性。方法： 模拟体内条件将PP-g-AAm膜和对照组未改性的聚丙烯（PP）膜与富含血小板血浆（PRP）接触后，用ELISA法检测血浆中β－血小板球蛋白（β-TG）的表达， 用流式细胞术检测血小板活化标志CD62P和CD63的表达，用扫描电镜检测两种膜上血小板粘附情况。 结果： 两种膜材料接触PRP 30 min后，血浆β-TG的表达均高于空白对照组（P<0.01, P<0.05），两材料组之间差异显著（P<0.05）； PP-g-AAm膜激活血小板表达CD62P、CD63的百分率都明显少于PP膜（P<0.05， P<0.01）；两种膜材料接触PRP 1 h后，扫描电镜观察材料表面粘附的血小板都有明显变形，但PP-g-AAm膜表面粘附的血小板明显少于PP膜。结论： PP-g-AAm膜对血小板的激活明显少于PP膜，具有较好的血液相容性。     

角膜基质培养液对离体角膜上皮重建的影响

目的：观察角膜基质培养产物对 离体角膜上皮细胞存亡的影响，旨在体外构建组织工程角膜上皮中，寻找促进角膜上皮细胞 增殖以及预防离体角膜上皮细胞凋亡的途径。方法：应用流式细胞仪,对基础培养1－6代的兔角膜缘上皮细胞凋亡（亚2倍体DNA）进行检测及对比分析，并同时对 与角膜基质共育的角膜缘上皮细胞的凋亡情况进行了对比。结果：基础 培养的第六代角膜缘上皮细胞出现明显的细胞凋亡，凋亡率为19.88％±4.69％；与角膜 基质共育的角膜缘上皮细胞凋亡率则为6.84％±1.74％，明显低于无角膜基质培养下的同 代细胞（P<0.01），并与基础原代细胞凋亡率（7.01％±2.23％）比较无显著差异 （P>0.05）。结论：在体外重建角膜上皮组织中，角膜基质成分 对离体角膜缘上皮细胞培养有减少细胞凋亡的作用。     

Bioactive self-assembled peptide nanofibers for corneal stroma regeneration

Defects in the corneal stroma caused by trauma or diseases such as macular corneal dystrophy and keratoconus can be detrimental for vision. Development of therapeutic methods to enhance corneal regeneration is essential for treatment of these defects. This paper describes a bioactive peptide nanofiber scaffold system for corneal tissue regeneration. These nanofibers are formed by self-assembling peptide amphiphile molecules containing laminin and fibronectin inspired sequences. Human corneal keratocyte cells cultured on laminin-mimetic peptide nanofibers retained their characteristic morphology, and their proliferation was enhanced compared with cells cultured on fibronectin-mimetic nanofibers. When these nanofibers were used for damaged rabbit corneas, laminin-mimetic peptide nanofibers increased keratocyte migration and supported stroma regeneration. These results suggest that laminin-mimetic peptide nanofibers provide a promising injectable, synthetic scaffold system for cornea stroma regeneration.     

Methacrylated gelatin hydrogels as corneal stroma substitutes: in vivo study

Abstract

Methacrylated gelatin (GelMA) hydrogels were prepared to serve as corneal stroma equivalents. They were highly transparent (ca. 95% at 700?nm), mechanically strong and withstood handling and had high human corneal keratocyte viability (98%) after 21?days of culture period. In order to test the in vivo performance of the cell free GelMA hydrogels a pilot in vivo study was carried out using eyes of two white New Zealand rabbits. Hydrogel was implanted in a mid-stromal pocket created and without suture fixation, and observed for 8?weeks under a slit lamp. No edema, ulcer formation, inflammation or infection was observed in both the control (sham) and hydrogel implanted corneas. Corneal vascularization on week 3 was treated with one dose of anti-VEGF application. Hematoxylin and Eosin staining showed that the hydrogel was integrated with the host tissue with only a minimal foreign body reaction. Results demonstrated some degradation in the construct within 8?weeks as evidenced by the decrease of the diameter of the hydrogel from 4?mm to 2.6?mm. High transparency, adequate mechanical strength, biocompatibility and well integration with the host tissue, indicates that this hydrogel is a viable alternative to the current methods for the treatment of corneal blindness and deserves testing on larger number of rabbits and more extensively using microscopy, histology and immune histochemistry.     

脱蛋白联合冷冻干燥法制备异种骨的实验研究

目的通过脱蛋白联合冷冻干燥的方法制备异种骨,探讨其能否在保持其生物力学特性的同时良好地消除异种骨的抗原性,以满足骨移植的需求。
　　方法取牛股骨上端去除表面筋膜、结缔组织和皮质骨部分,制备成骨粒和圆柱形骨棒。将十二烷基苯磺酸钠（ABS）和脂肪醇聚氧乙烯醚硫酸钠（AES）以及蒸馏水以重量比13：7：80的比例配制复合表面活性剂。将牛骨粒或者骨棒与复合表面活性剂以重量比1：10的比例置于烧瓶中,超声振荡清洗,去除其抗原性,电镜下观察其结构。骨粒和骨棒经乙醇和乙醚脱水、脱脂,冷冻干燥。骨粒用作溶血试验和细胞毒性检测,骨棒进行新西兰大白兔的长期骨植入实验（4、12、26、52周）检测其生物相容性。乙醇抽吸法检测脱蛋白-冻干骨与未脱蛋白的单纯冻干骨的孔隙率。并且比较牛松质骨骨粒与脱蛋白-冻干松质骨骨粒的生物力学特性差异。
　　结果电镜下观察,制备的异种骨材料为天然多孔结构,保留了骨组织的三维结构,骨小梁间有200~650μm 骨髓腔。溶血率检测未超过5%,判定溶血率合格。细胞毒性检测为1级,极低细胞毒性,材料合格。长期骨植入实验表明,植入4周即有植入的骨棒与兔自身骨出现融合,未见严重炎症反应;52周时,植入骨已完全吸收。孔隙率检测表明,脱蛋白-冻干骨与未脱蛋白的单纯冻干骨相比无显著性差异,均可达到60%以上,符合骨移植材料的要求。脱蛋白-冻干骨以及未脱蛋白的单纯冻干骨的力学特性与新鲜松质骨块相比均有显著降低,但两者之间无显著性差异。结论通过脱蛋白联合冷冻干燥的方法制备的异种骨,能够在保持其生物力学特性的同时良好地消除抗原性;满足骨移植的需求。     

异种角膜基质异位移植对大鼠外周血T细胞亚群的影响

目的: 比较3种异种角膜基质的免疫原性。方法: 将SD大鼠36只随机分为4组,每组9只。1组为对照组,2-4组为实验组。分别获取新鲜猪、兔、鸡角膜基质,等量湿重25 mg异位植入大鼠背部皮下,术后观察伤口愈合情况,并于术后7 d、14 d、28 d获取大鼠外周血,免疫荧光标记及流式细胞仪分析比较3种异种角膜基质对大鼠CD4⁺、CD8⁺、CD25⁺、CD71⁺表型的动态影响。结果: 各组植入处皮肤无红肿、无渗出及其它改变,伤口愈合良好;各组与对照组比较:猪角膜基质组各时相表达的CD4⁺、CD8⁺、CD25⁺、CD71⁺T细胞比例无显著差异(P＞0.05);兔角膜基质组术后7 d,CD4⁺T细胞升高(P＜0.05),鸡角膜基质组术后7 d,CD4⁺、CD4⁺CD71⁺升高(P＜0.01)。结论: 3种异种角膜基质对大鼠细胞免疫原性比较:鸡>兔>猪,猪角膜基质的免疫原性最低。     

单侧腮腺放射性损伤动物模型的建立

目的建立猪单侧腮腺放射性损伤动物模型。方法年满4月大梅杂交猪8头,4头双侧唾液腺造瘘,15MV-X线单侧腮腺照射,5Gy/次,3次/W,总量40Gy/8次/17d,为实验组;对侧未照射腮腺为自身对照组;2头单纯造瘘为阴性对照组;余下2头不做任何处理,为病理对照组。观察照射动物临床征象变化;每日记录唾液总量,采用SPSS11.0最小显著性差异(LSD)检验比较实验组、自身对照组、阴性对照组分泌量差异;2月后取腮腺病理学检查,比较4组腮腺显微结构变化。结果照射猪饮食下降、活动减少,进食期间频繁饮水;照射组于照射开始13±2d起分泌明显减少,LSD处理显示试验组分泌量减少、自身对照组分泌量增多、与阴性对照组间两两比较差异均有显著意义。病理学检查显示,照射腮腺总体结构尚存,细胞排列轻度紊乱,细胞数目无明显减少,部分胞核增大,胞浆相对增加,凋亡细胞少见,有区域分布趋势,阴性对照组细胞无明显变化。结论成功建立了腮腺放射性损伤模型。     

The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model

PURPOSE:

To evaluate the use of subconjunctival bevacizumab on corneal neovascularization in an experimental rabbit model for its effect on vessel extension, inflammation, and corneal epithelialization.

METHODS:

In this prospective, randomized, blinded, experimental study, 20 rabbits were submitted to a chemical trauma with sodium hydroxide and subsequently divided into two groups. The experimental group received a subconjunctival injection of bevacizumab (0.15 m; 3.75 mg), and the control group received an injection of 0.15 ml saline solution. After 14 days, two blinded digital photograph analyses were conducted to evaluate the inflammation/diameter of the vessels according to pre-established criteria. A histopathological analysis of the cornea evaluated the state of the epithelium and the number of polymorphonuclear cells.

RESULTS:

A concordance analysis using Kappa''s statistic showed a satisfactory level of agreement between the two blinded digital photography analyses. The neovascular vessel length was greater in the control group (p<0.01) than in the study group. However, the histopathological examination revealed no statistically significant differences between the groups in terms of the state of the epithelium and the number of polymorphonuclear cells.

CONCLUSIONS:

Subconjunctival bevacizumab inhibited neovascularization in the rabbit cornea. However, this drug was not effective at reducing inflammation. The drug did not induce persistent corneal epithelial defects.