异种角膜脱细胞基质和几丁糖载体构建角膜基质层的比较

目的探索异种角膜脱细胞基质和几丁糖为载体构建生物角膜组织的可行性，评价二者的结构、功能和生物相容性。方法l％TritonX-100及冷冻干燥处理获得猪角膜脱细胞基质，网状几丁糖材料制备成似角膜片状形态，将体外培养的兔角膜基质细胞种植于两种载体，体外培养2周，形成细胞载体复合物。对构建的角膜组织进行苏木精-伊红染色、扫描电镜观察；同时将两种生物材料移植到兔角膜基质囊袋内，观察其生物相容性。结果角膜基质细胞在两种载体上皆可黏附生长，并分泌细胞外基质。脱细胞基质载体内细胞形态不规则，位于胶原板层间，形成类似正常组织的角膜基质结构；细胞可在几丁糖网状纤维上黏附并包绕生长。两种载体移植到兔角膜囊袋后，脱细胞基质降解较慢，与宿主角膜组织相融性良好，未发生明显排斥及毒性反应；几丁糖载体降解较迅速，但降解产物诱导较严重的排斥反应。结论异种角膜脱细胞基质保留正常组织的胶原板层结构，并具角膜的韧性和良好的生物相容性，更适于作为体外构建生物角膜的载体，而几丁糖材料则需要在构成结构和性能等方面进一步改进．     

猪角膜脱细胞基质的制备及生物相容性的实验研究

目的探讨猪角膜脱细胞基质（CACM）的制备方法，评价其组织学特性和生物相容性。方法应用1％TritonX-100去垢剂振洗，经冷冻干燥处理得到猪CACM，通过苏木精-伊红染色、扫描电镜观察行组织学检测。将猪CACM植入兔角膜板层间观察10周，取材做组织切片，评价其生物组织相容性。结果组织学检测证实角膜细胞完全脱净，胶原纤维排列疏松，板层结构同正常角膜；扫描电镜CACM表面未见细胞结构，纵切面上胶原板层间出现清晰的空穴状裂隙；CACM植入兔角膜层间后，观察期内未见明显排异反应。结论TdtonX．100可以有效地脱净角膜细胞，保存胶原排列的结构特征，经过冷冻干燥可形成多孔隙且胶原排列疏松的板层结构，适合作为载体材料，猪CACM与兔角膜生物相容性好。     

脱细胞角膜材料的生物相容性研究

目的研究异种脱细胞猫角膜板层角膜移植术后的组织相容性。方法24只18周龄的健康新西兰白兔随机平均分成3组：A组为脱细胞猫角膜材料→兔板层移植组,B组为猫→兔异种新鲜角膜植片板层移植组,C组为兔→兔同种异体新鲜角膜植片板层移植组。用扫描电镜观察术后不同时期术眼角膜的组织病理学改变,抽取兔血测定CD4^＋/CD8^＋T细胞百分比。结果术后1个月3个组间角膜植片炎症反应指数组间比较差异均无统计学意义;2个月A组与B组、A组与C组间比较差异均有统计学意义（P〈0.05）。3个月A组与B组、A组与C组间比较差异均有统计学意义（P〈0.05）。3个组间术后同期7、15、30d兔外周血CD4＋T细胞和CD8＋T细胞活化率差异均无统计学意义（P〉0.05）。组织切片显示术后3个月内,B、C两组角膜植片炎症反应轻,植片内皮细胞吸收,组织结构基本正常。A组角膜植片炎症反应显著,结构较紊乱,新生血管增生。扫描电镜可见B、C2组角膜上皮结构基本正常,细胞连接较好。A组扫描电镜可见有上皮缺损。结论脱细胞处理的异种猫角膜移植后生物相容性较新鲜角膜差,免疫反应无显著差异。     

猪脱细胞角膜基质组织结构特点与生物相容性研究

背景构建组织工程化角膜时,载体的选择十分重要。目前有多种载体可供选用,但脱细胞角膜基质是公认的较好载体。目的观察猪脱细胞角膜基质的组织结构特点,评价其与异种角膜基质和上皮细胞的生物相容性。方法取猪角膜组织进行组织块培养,经胰蛋白酶-EDTA酶消化角膜上皮、基质、内皮细胞,支架组织于-20℃冷冻干燥后灭菌保存。猪脱细胞角膜基质石蜡切片行常规苏木精一伊红染色,光学显微镜下观察组织的形态学特征;扫描电镜下观察其组织结构特点;并对其物理特性,如抗拉性、膨胀性、含水量和透明度进行评价。将猪脱细胞角膜基质移植到兔角膜基质层内,同时与体外培养的兔角膜上皮细胞共培养4周,分析其组织和细胞生物相容性。结果经酶消化处理后猪角膜组织中未见上皮、基质和内皮细胞,其胶原纤维直径大小、排列与正常角膜组织相同,抗拉性、膨胀性、含水量和透明度等物理特性与正常猪角膜相似。猪脱细胞角膜基质行异种兔角膜基质层间移植1周时可见轻度水肿,2周后水肿基本消退,4周时透明度较好。猪脱细胞角膜基质与兔角膜基质之间贴附良好,未见炎症反应及新生血管。兔角膜上皮细胞接种于猪脱细胞角膜基质上共培养4周后CK3表达阳性。猪脱细胞角膜基质脱水前与脱水2h、4h后及正常猪角膜基质间的抗拉性、膨胀性、含水量的差异均无统计学意义（P〉0．05）,但脱水2h、4h后及正常猪角膜基质的透明度明显好于脱水前,差异均有统计学意义（P〈0．01）。结论猪脱细胞角膜基质组织结构与正常猪角膜相似,与兔角膜基质和上皮细胞具有良好的生物相容性。     

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.     

鸵鸟角膜对兔板层角膜移植的排斥反应

目的：角膜穿孔属眼科急症，角膜外伤、感染和化学伤都可引起角膜穿孔，用角膜移植治疗穿孔效果较好，但角膜供体资源在我国目前仍较为缺乏。角膜移植排斥反应是造成移植失败的主要原因。为探索异种角膜移植材料，本实验研究鸵鸟对兔板层角膜移植的植片存活时间及排斥反应特点，以便评价鸵鸟角膜的应用前景。方法：将24只≤8周龄的健康新西兰白兔随机分成两组：A组12只兔为兔一兔同种异体板层移植组；B组12只兔为鸵鸟一兔异种角膜板层移植组，治疗及观察时间均为6wk。两组术后均予结膜下注射地塞米松。术后每日在裂隙灯显微镜下观察植片存活情况和排斥反应指数；术后每周测量新生血管面积；术后1，3，5wk各组取1只兔的术眼角膜行病理检查。结果：鸵鸟板层角膜植片的平均存活时间为23d。驼鸟一兔异种板层和同种板层角膜在1，2wk排斥反应指数及角膜新生血管面积无显著差异，第3wk起差异显著。结论：鸵鸟角膜植片在移植早期能保持透明，且有较好的生物相容性。鸵鸟角膜植片后期因新生血管生长和排斥反应加重而混浊，说明单独应用地塞米松不能完全控制异种植片的排斥反应。     

自动角膜板层刀切割一个供体角膜用于两位患者

目的评估应用自动角膜板层刀切割一个供体角膜用于深板层角膜移植和角膜内皮移植的临床疗效及安全性。方法回顾性系列病例研究。应用自动角膜板层刀预先将新鲜供体角膜进行切割,将带部分基质的角膜内皮植片用于角膜内皮移植术,剩余前板层用于深板层角膜移植术。回顾性分析2010年3—8月期间在温州医学院附属眼视光医院行角膜内皮移植术的17例患者及分别来自同一供体角膜的行深板层角膜移植的17例患者。术后随访19～24个月,分别观察角膜内皮贴附情况及前板层角膜植片愈合情况。随访资料包括医学验光、前节光学相干断层扫描及角膜内皮细胞计数。手术前后视力比较采用配对t检验。结果17例角膜内皮移植患者术后角膜内皮植片均贴附良好,术后视力较术前均有提高。除1例碱烧伤患者因晶状体混浊,无法验光外,其余术后平均散光为（1．69±0．60）D。17例角膜内皮移植患者平均角膜内皮细胞计数为（2128±244）个／mm^2,平均角膜内皮植片厚度为（152±46）μm。17例前板层角膜移植患者角膜植片均透明,术后最佳矫正视力较术前提高,平均散光为（2．50±0．90）D;平均前板层角膜植片厚度为（343±39）μm。结论利用角膜板层刀将一个供体角膜用于两位患者的方法在临床实际应用中切实可行。经长期随访,角膜植片未出现明显并发症。该方法可以更充分地利用供体角膜,缓解中国供体角膜匮乏现状,意义重大。     

组织工程角膜基质的体外构建及移植的实验研究

目的探讨利用组织工程技术体外构建角膜基质进行板层角膜移植的可行性和有效性。方法将猪角膜基质去细胞处理后制备成组织工程角膜基质载体;取幼兔角膜基质细胞体外培养,将其种植在载体上,体外构建成组织工程角膜基质,用PKH26荧光标记兔角膜基质细胞示踪角膜基质的构建;将16只兔的角膜基质内植入壳聚糖膜使之形成无菌性角膜溃疡,随机从16只兔中选8只,进行组织工程角膜基质移植;另外8只作为对照组,进行新鲜的同种异体兔板层角膜移植。术后对角膜进行裂隙灯、光学显微镜、透射电镜观察。结果体外构建的组织工程角膜的基质细胞具有活性,其结构与正常角膜基质相近。移植治疗无菌性角膜溃疡术后,1~2周有新生血管侵入组织工程角膜基质植片边缘,植片为灰白色半透明状;3~4周随着新生血管减退,组织工程角膜基质植片局部开始透明变薄;术后8~10周角膜溃疡完全修复,角膜恢复透明性,角膜神经可再生;观察最长达10月,角膜仍保持透明,无免疫排斥发生,与对照组疗效相同。结论体外构建的组织工程角膜基质无免疫原性、具有良好的生物相容性,可作为临床治疗角膜溃疡的移植材料。     

组织工程角膜治疗兔无菌性角膜溃疡

目的:探讨利用组织工程技术制备角膜基质进行板层角膜移植治疗无菌性角膜溃疡的可行性和疗效。方法:用去垢剂联合胰酶、DNA-RNA酶去除猪角膜基质细胞后制备成组织工程角膜基质;将16只兔的角膜基质内植入壳聚糖膜使之形成角膜溃疡,随机从16只兔中选8只行组织工程角膜基质板层移植;另外8只作为对照组,行新鲜的同种异体板层角膜移植。术后对角膜进行裂隙灯、HE染色光学显微镜检查。结果:组织工程角膜基质移植后无免疫排斥发生,术后8～10wk角膜溃疡完全修复,角膜恢复透明性,与对照组疗效相同。结论:组织工程角膜基质具有良好的生物相容性和治疗作用。     

角膜体外重建异种生物载体材料生物相容性研究

目的　研究异种 (猪 )角膜基质的生物相容性 ,评价其作为角膜体外重建载体的可行性。方法　将新鲜、脱水两种猪角膜基质分别植入新西兰白兔角膜层间 ,定期临床观察植片愈合情况 ,并于术后 2周、1、2、4、8个月取兔角膜进行组织学观察。结果　临床观察 :全部植片存活 ,12只术眼未见角膜水肿混浊、角膜新生血管、排斥反应发生 ,新鲜植片在 2个月左右已透明 ,脱水植片在 6个月后透明。组织学观察 :新鲜植片 4个月时与兔角膜基质相融愈合 ,脱水植片经角膜细胞再分布、胶原纤维改建重塑 ,于 8个月后与兔角膜基质相融愈合 ,2组植片愈合过程中未见有淋巴细胞浸润及新生血管生成。结论　异种 (猪 )角膜基质具有良好的生物相容性 ,是一种理想的角膜体外重建载体材料。     

纤维蛋白粘合剂粘贴双层基质透镜行兔板层角膜移植术

目的：研究纤维蛋白粘合剂(FS)粘贴的双层角膜基质透镜体内生物相容性,探讨使用该种材料行角膜移植的可行性。

方法：选取健康清洁级新西兰白兔15只,采用自身对照,以兔右眼为实验眼,左眼为对照眼。实验眼使用FS粘贴的双层角膜基质透镜作为移植材料行板层角膜移植手术,对照眼不进行人工干预。分别于术后7、14、28d使用手持裂隙灯观察双眼角膜情况,并进行生物相容性评分,同时取双眼角膜行HE染色进行组织病理学检测,观察角膜恢复情况。

结果：裂隙灯观察结果显示,至角膜移植术后28d,实验眼角膜上皮生长情况良好,角膜透明度基本恢复,水肿程度减轻,新生血管生长至角膜缘后未加重,未见上皮、内皮排斥线等排斥反应; 对照眼角膜透明,角膜上皮光滑。生物相容性评分结果显示,角膜移植术后实验眼角膜植片水肿程度逐渐减轻,透明度逐渐恢复,排斥反应较小,角膜植片的生物相容性较好; 至术后28d,实验眼和对照眼角膜透明度、水肿程度及新生血管生长程度均无差异(P>0.01)。组织病理学检测结果显示,至角膜移植术后28d,实验眼植片表面有4～5层角膜上皮细胞覆盖,角膜胶原排列整齐、规则,植片内未见明显炎性细胞浸润,双片透镜间分界消失,层间FS被机体完全吸收,植片与植床融合,未见明显分界。

结论：使用FS粘贴的双层角膜基质透镜作为植片行板层角膜移植术后恢复较好,排斥反应较小,生物相容性较好,可用于板层角膜移植。     

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.     

脱细胞猪角膜基质作为植片的深板层角膜移植治疗感染性角膜溃疡的临床观察

目的 比较脱细胞猪角膜基质与新鲜角膜组织作为植片的深板层角膜移植手术治疗感染性角膜溃疡的临床疗效。设计 回顾性病例系列。研究对象 2015年11月~2016年9月在北京佑安医院就诊的10例病变深度未累及后弹力层的感染性角膜溃疡患者。方法 回顾性分析气泡辅助下的深板层角膜移植手术资料,其中使用脱细胞猪角膜基质材料和新鲜角膜组织材料治疗的患者各5例。比较两组患者术后1年最佳矫正视力（LogMAR）、眼压、球镜度、角膜平均曲率、散光度、角膜内皮细胞数、角膜厚度、眼轴长度、角膜共聚焦显微镜表现、角膜透明度以及植片的生存情况。主要指标 术后1年患者的最佳矫正视力（LogMAR）、眼压、球镜度、角膜平均曲率、散光度、角膜内皮细胞数、角膜厚度、眼轴长度、角膜共聚焦显微镜表现、角膜透明度以及植片的生存情况。结果 在随访期间,两组所有植片均保持透明,植片生存率为100%。术后1年,脱细胞角膜基质组和新鲜角膜组的视力分别为0.53±0.21和0.33±0.06（P=0.184）,眼压分别为（8.00±2.00）mmHg和（10.33±0.58）mmHg（P=0.124）,球镜度分别为（-4.50±4.21）D和（-1.25±0.75）D（P=0.258）,角膜平均曲率分别为（47.59±5.40）D和（44.51±1.87）D（P=0.403）,散光度分别为（-5.52±1.97）D和（-5.14±1.66）D（P=0.812）,内皮细胞数分别为（1272.67±387.63）个/mm2和（1550.33±232.69）个/mm2（P=0.347）,角膜厚度分别为（439.33±67.86）μm和（534.00±14.42）μm（P=0.077）,眼轴长度分别为（23.53±0.91）mm和（23.55±1.56）mm（P=0.981）。角膜共聚焦显微镜显示,脱细胞猪角膜基质组的角膜上皮细胞可以完全覆盖植片,基底细胞和翼状细胞形态和密度与新鲜角膜组织接近,但在基底细胞下方可见高反光的沉着物,基质层细胞的密度明显低于新鲜角膜组织,并且在内皮细胞与深基质层之间仍可见脱细胞纤维排列。结论 脱细胞猪角膜基质具有良好的生物相容性,当缺乏新鲜角膜材料时,用于替代治疗感染性角膜溃疡可以取得满意的效果。     

Transplantation of corneal endothelial cells

Though conventional corneal transplantation has achieved great success, it still has several drawbacks including limited availability of donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases. Reconstructing clinically usable corneas by applying the technology of regenerative medicine can offer a solution to these problems, as well as making corneal transplantation a non-emergency surgery and enabling the usage of banked corneal cells. In the present study, we focused on corneal endothelium that is critical for corneal transparency and investigated the reconstruction of cornea utilizing cultured human corneal endothelial cells (HCECs). We succeeded in steadily culturing HCECs by using culture dishes pre-coated with extracellular matrix produced by calf corneal endothelial cells and culture media that contained basic fibroblast growth factor and fetal bovine serum. We performed the following analysis utilizing these cultured HCECs. The older the donor was, the more frequently large senescent cells appeared in the passaged HCECs. The telomeres of HCECs were measured as terminal restriction fragments (TRF) by Southern blotting. HCECs, in vivo from donors in their seventies had a long TRFs of over 12 kilobases. Passaging shortened the TRFs but there was no difference in TRFs among donors of various ages. These results indicated that shortening of telomere length is not related to senescence of HCECs. We investigated the role of advanced glycation end products (AGEs) in the senescence of in vivo HCECs. The results indicated that AGE-protein in the aqueous humor is endocytosed into HCECs via AGE receptors expressed on the surface of HCECs and damages HCECs by producing reactive oxygen species and inducing apoptosis, suggesting that AGEs, at least partly, cause the senescence of HECEs. HCECs were cultured using adult human serum instead of bovine serum to get rid of bovine material that can be infected with prions. Primary and passage culture of HCECs was possible using adult human serum. We reconstructed the cornea using cultured HCECs and human corneal stroma. The corneal stroma, on which the cell suspension of HCECs was poured, was mildly centrifuged to enhance the HCECs attachment to the stroma. The cell density of HCECs on the reconstructed cornea reached 2,500 cells/mm2. The pump function of the reconstructed cornea was measured with an Ussing chamber. The potential difference in the reconstructed cornea and normal cornea was 0.30 mV and 0.40 mV, respectively; indicating that the pump function of the reconstructed cornea is 75% of that of the normal cornea. The reconstructed cornea was transplanted to a rabbit eye and stayed transparent for 6 months after the operation. Fluorescein labeled cultured HCECs remained on the graft 1 month after the transplantation, indicating that transplanted HCECs contributed to the transparency of the graft. The possibility of using artificial stroma or porcine corneal stroma as a carrier of cultured HCECs was investigated. The artificial stroma made of alkaline-treated collagen could not be sutured but showed good transparency, biocompatibility, and cell-attachability. Porcine corneal stroma, expressing little xeno-sugar antigen alpha-gal epitope, induced no super acute rejection but mild cellular rejection when transplanted in the cornea of animals possessing natural antibody to alpha-gal epitope. The cornea reconstructed with porcine corneal stroma and HCECs had an average cell density of 1721/mm2 and had approximately 60% of the pump function of a normal cornea. As new technologies in corneal transplantation, the application of self immature cells and the direct delivery of cultured HCECs into the anterior chamber were investigated. Part of rat mononuclear cells that were obtained from the bone marrow and injected into the rat anterior chamber transformed into corneal endothelium-like cells, suggesting that self immature cells can transform into corneal endothelial cells. Cultured rabbit corneal endothelial cells that endocytosed iron were injected into the anterior chamber of rabbits whose corneal endothelium was cryo-injured, and were pulled to Descemet's membrane by putting a magnet on the eyelid. In these rabbits, corneal edema decreased more quickly than in the control group and no intraocular pressure rise was observed during 8 weeks after the operation, suggesting that the direct delivery of cultured HCECs into the anterior chamber can be an alternative method of choice. The following obstacles should be addressed to make the transplantation of cultured corneal endothelial cells clinically applicable. 1. To reconstruct a cornea that is the same as or superior to the normal cornea, more innovation is necessary in the method of culturing and seeding HCECs. We should consider utilizing HCECs obtained from fetuses after clearing ethical issues. Moreover, we need to develop a method to enhance the cell density and the cell functions. 2. Porcine corneal stroma is promising as a carrier of HCECs instead of human corneal stroma, which is in very limited supply. The usefulness of porcine corneal stroma acellularized to prevent retrovirus infection should be evaluated. 3. To make the self immature cells applicable to corneal transplantation, we should elucidate the corneal endothelial cell specific markers and the factors that are necessary to induce self immature cells to become corneal endothelial cells. 4. The direct delivery of cultured HCECs into the anterior chamber can be an alternative method of choice when its long-term safety is confirmed.     

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.     

人脐静脉内皮细胞移植治疗角膜内皮功能衰竭的动物实验研究

目的：探讨异种脱细胞角膜基质为载体培养人脐静脉内皮细胞（HUVEC）进行后板层角膜移植（PLEK）治疗角膜内皮衰竭的可行性。方法：新西兰白兔30只,随机分为3组,实验组、基质组和对照组,每组10只,术中去除角膜内皮细胞,建立角膜内皮衰竭动物模型,实验组和基质组行后板层角膜移植术,对照组仅去除角膜后板层组织,不进行移植。术后观察3mo,对三组角膜的水肿混浊程度和中央角膜厚度进行统计学分析。结果：术后7d,实验组角膜水肿程度较基质组和对照组明显减轻,透明度增加。术后3mo时,实验组内皮细胞密度为2 026.4±129.3个/mm2,中央角膜厚度平均为505.2±25.4μm,基质组中央角膜厚度平均为1 535.6±114.5μm,而对照组为1 493.5±70.2μm。结论：实验以异种脱细胞角膜基质为载体培养人脐静脉内皮细胞,行后板层角膜移植术,治疗角膜内皮衰竭取得了初步成功。移植的人脐静脉内皮细胞能够在活体上成活,并具有一定的角膜内皮细胞的生物学功能,维持角膜透明,为临床上治疗角膜内皮疾病提供了新的思路和方法。     

Decellularized porcine cornea-derived hydrogels for the regeneration of epithelium and stroma in focal corneal defects

PurposeHydrogels derived from decellularized tissues provide superior biocompatibility, tenability and tissue-specific extracellular matrix (ECM) components. Based on the preparation of decellularized porcine cornea (DPC), here we developed an injectable and transparent hydrogel for the regeneration of epithelium and stroma in focal corneal defects.MethodsThe DPC-derived hydrogel was prepared with N-cyclohexyl-N′-(2-morpholinethyl) carbodiimide metho-p-toluenesulfonate/N-hydroxysuccinimide (CMC/NHS) as cross-linkers. The characteristics of the hydrogel were analyzed and its cytocompatibility was assessed by Live/Dead and Cell Counting Kit (CCK)-8 assays. Immunofluorescence staining, quantitative PCR and Western blot analyses were performed to assess the relative protein and gene expression in corneal fibroblasts on hydrogel. The safety and efficiency of the hydrogel for repairing focal corneal defects in rabbit were measured by slit-lamp, anterior segment optical coherence tomography (AS-OCT), confocal microscopy and histological analyses.ResultsThe DPC-derived hydrogel cross-linked with CMC/NHS assumed favorable transparency, exhibited distinct mechanical properties and preserved the ECM components of native porcine cornea (NPC). In vitro experiments showed that the hydrogel maintained the phenotype, supported the proliferation and promoted the ECM synthesis of corneal fibroblasts. When injected onto rabbit corneas, the hydrogel rapidly covered, solidified and formed a smooth surface on the focal defect. Corneal epithelium was fully regenerated within 3 days. The thickness of the corneal epithelium and stroma was restored at 12 weeks after surgery without significant inflammation or scar formation. Notably, the hydrogel showed no harmful effects on the resident stroma and endothelium.ConclusionsThe DPC-derived hydrogel may represent a promising biomaterial for corneal epithelial and stromal regeneration.