Support of acellular porcine corneal stroma for growth of corneal epithelium and stromal cell in vitro

AIM: To determine whether acellular porcine cornea stroma (APCS) could support the growth of the rabbit corneal cells in vitro . METHODS: APCS was prepared. The rabbit's corneal epithelium and stromal cells were cultured and seeded on APCS in vitro . The observation of phase contrast photograph and histological examination were performed. RESULTS: Histological examination showed the epithelium grew on the scaffold of APCS in 2-3 layers at 10th day. The stromal cells adhered to the surface of the scaffold after 24 hours and invaded into the interlaminar of the material at 5th day. CONCLUSION: These results indicate that APCS can support the growth and proliferation of the corneal epithelium and stromal cells in vitro .     

脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长

目的:探讨脱细胞猪角膜基质体外能否支持兔角膜细胞的生长。方法:体外培养兔角膜上皮细胞和基质细胞,并接种到制备的脱细胞猪角膜基质上,倒置相差显微镜和组织学观察细胞生长情况。结果:上皮细胞能在脱细胞猪角膜基质上贴附生长,10d时可形成2~3层的复层结构。基质细胞在脱细胞猪角膜基质上贴附生长后可向材料深层迁徙。结论:制备的脱细胞猪角膜基质体外可支持兔角膜上皮细胞和基质细胞的生长。     

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.     

种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究

目的研究种植人骨髓间充质干细胞（MSCs）的猪角膜基质治疗兔角膜损伤的可能性。方法用全骨髓贴壁法分离纯化人MSCs并传代,流式细胞仪检测免疫表型及诱导成脂、成骨分化鉴定。12只新西兰白兔随机分为2组,实验组取第3代MSCs接种于去上皮的猪角膜基质上,培养4 d后移植到广泛损伤的兔角膜上,对照组单纯移植去上皮猪角膜基质。术后2、4、8周,取各实验眼行组织学检查,观察移植的MSCs及猪角膜基质的存活、转归及移植局部的反应。免疫组织化学、免疫荧光染色检测移植后角膜上皮细胞角蛋白12的表达。结果培养获得的MSCs中CD29阳性者占95.97%,CD44阳性者占96.49%,CD90阳性者占92.79%,CD105阳性者占94.66%,CD34阳性者占0.59%,CD45阳性者占0.36%,符合MCSs的免疫表型,并可以诱导成脂及成骨分化。实验组MSCs接种到去上皮猪角膜基质后贴附、生长迅速,术后植片在植床上存活良好,无排斥反应,角膜较对照组透明,新生血管少,而对照组在移植后发生排斥反应。实验组角膜免疫组织化学及免疫荧光染色均检测出CK12阳性细胞。结论种植MSCs的猪角膜基质移植到损伤兔角膜后可以存活,MSCs可以分化为角膜上皮样细胞,具有构建组织工程角膜的潜能。     

Construction of a full-thickness human corneal substitute from anterior acellular porcine corneal matrix and human corneal cells

AIM: To construct functional human full-thickness corneal replacements. METHODS: Acellular porcine corneal matrix (APCM) was developed from porcine cornea by decellulariztion. The biomechanical properties of anterior-APCM (AAPCM) and posterior-APCM (PAPCM) were checked using uniaxial tensile testing. Human corneal cells were obtained by cell culture. Suspending ring was designed by deformation of an acupuncture needle. MTT cytotoxicity assay was used to check the cytotoxicity of suspending ring soaking solutions. A new three-dimensional organ culture system was established by combination of suspending ring, 48-well plate and medium together. A human full-thickness corneal substitute was constructed from human corneal cells with AAPCM in an organ coculture system. Biochemical marker expression of the construct was measured by immunofluorescent staining and morphological structures were observed using scanning electron microscopy. Pump function and biophysical properties were examined by penetrating keratoplasty and follow-up clinical observations. RESULTS: There were no cells in the AAPCM or PAPCM, whereas collagen fibers, Bowman’s membrane, and Descemet’s membrane were retained. The biomechanical property of AAPCM was better than PAPCM. Human corneal cells grew better on the AAPCM than on the PAPCM. There was no cytotoxicity for the suspending ring soaking solutions. For the constructed full-depth human corneal replacements keratocytes scattered uniformly throughout the AAPCM and expressed vimentin. The epithelial layer was located on the surface of Bowman’s membrane and composed of three or four layers of epithelial cells expressing cytokeratin 3. One layer of endothelial cells covered the stromal surface of AAPCM, expressed Na+/K+ATPase and formed the endothelial layer. The construct was similar to normal human corneas, with many microvilli on the epithelial cell surface, stromal cells with a long shuttle shape, and zonula occludens on the interface of endothelial cells. The construct withstood surgical procedures during penetrating keratoplasty. The corneal transparency increased gradually and was almost completely restored 7d after surgery. CONCLUSION: AAPCM is an ideal scaffold for constructing full-thickness corneal replacement, and functional human full-thickness corneal replacements are successfully constructed using AAPCM and human corneal cells.     

复合自体脂肪干细胞的脱细胞猪角膜基质移植治疗兔角膜碱烧伤

目的:研究复合脂肪干细胞的脱细胞猪角膜基质对碱烧伤角膜的治疗效果。方法:制备晾干的脱细胞猪角膜基质,取第3代体外培养的兔自体脂肪干细胞,体外种植到脱细胞猪角膜基质上,培养3d后用于板层角膜移植。结果:板层角膜移植2mo后,术眼角膜基本恢复透明,未发生排斥反应,新生血管较少,3mo后新生血管基本消退。结论:复合自体脂肪干细胞的脱细胞猪角膜基质对兔碱烧伤角膜具有良好的修复能力。     

脱细胞猪角膜基质体外支持皮肤细胞生长的实验研究

目的 探讨脱细胞猪角膜基质体外是否支持皮肤细胞的生长.方法 制备脱细胞猪角膜基质(前期实验已完成).体外培养人皮肤表皮细胞和成纤维细胞,取第3代人皮肤成纤维细胞接种在脱细胞猪角膜基质的中间基质面,培养3 d后,将材料翻转,取第3代人皮肤表皮细胞接种在材料的上皮面上,再培养10 d后,取细胞-支架复合体制作石蜡切片,HE染色后光镜下观察.结果 组织学观察显示皮肤的表皮细胞和成纤维细胞体外均可在脱细胞猪角膜基质上黏附生长.接种10 d后,皮肤表皮细胞在材料表面形成复层结构,可见角化的细胞.接种13 d可见成纤维细胞存活并在支架层间生长.结论 脱细胞猪角膜基质有良好的细胞相容性,在体外能支持皮肤细胞的生长和增生.     

Decellularization of porcine corneas and repopulation with human corneal cells for tissue‐engineered xenografts

Purpose: To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering. Methods: Corneal cells were isolated from human corneoscleral rims. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra‐acetic acid (EDTA, 0.1%), aprotinin (10 KIU/ml) and 0.3% sodium dodecyl sulphate. Haematoxylin–eosin (HE) and 4,6‐diamidino‐2‐phenylindole (DAPI) staining were performed to confirm removal of the corneal cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using DNA Purification Kit (Fermentas, St. Leon‐Rot, Germany). Alcian blue staining was carried out to analyse the structure of the extracellular matrix (ECM). Corneal stromal cells were injected into the DPCs; limbal corneal epithelial cells and corneal endothelial cells were seeded onto the anterior and posterior surfaces of the DPCs, respectively. Evaluation was undertaken at days 14 and 30. The phenotypical properties of the cultivated corneal cells were investigated using Immunolocalization of type I collagen, keratocan, lumican, cytokeratin 3 (AE5) and type VIII collagen. Results: Haematoxylin–eosin and DAPI staining showed efficient elimination of porcine corneal cells, whereas alcian blue confirmed gross preservation of the ECM. The quantitative analysis of the DNA content showed a significant reduction (mean before decellularization: 75.45 ± 13.71 ng/mg; mean after decellularization: 9.87 ± 2.04 ng/mg, p < 0.001). All three types of corneal cells were efficiently cultured and expanded on the DPCs. Conclusions: Decellularized porcine corneas might serve as a potential scaffold for tissue engineering of the cornea, possibly providing xenogenic substrate for corneal transplantation.     

Establishment of a corneal epithelial cell line spontaneously derived from human limbal cells

The objective of this study was to establish a spontaneously derived human corneal epithelial cell line from a normal human limbus that retains differentiation potential and proliferative properties under continuous cell culture. After 50 passages of epithelial cells obtained from human limbal tissue a cell line spontaneously emerged. The immortalized cells showed a cobblestone appearance and displayed dense microvilli on their apical cell surface membrane. Colony forming efficiency was 5-6% and population doubling time was 19.6 h. In the mRNA level, cytokeratin (CK) 3 and 12 were detected in this cell line. In the protein level, the cells expressed CK3, CK12, CK14, CK19, vimentin, and some other proteins such as F-actin and beta-tubulin and beta(1)-integrin. They lacked p63. The immortalized cells had a heteroploid karyotype, but did not exhibit tumorigenic features. When cultured on an air-liquid interface the cells could form stratified multilayer epithelia. In summary, all these results indicated that a new human corneal epithelial cell line was spontaneously established from normal limbal tissue through serial culture. This cell line would be useful for studies of corneal epithelial biology and reconstructive corneal tissue engineering.     

体外诱导人羊膜上皮细胞分化为角膜上皮样细胞的初步研究

目的探索以人永生化角膜上皮细胞( immortalized human corneal epithelial cells,ihCEC) 培养液体外模拟角膜上皮细胞微环境,诱导人羊膜上皮细胞( human corneal epithe-lial cells,hAEC) 分化为角膜上皮样细胞的可行性。方法取( 37 ± 1) 周剖宫产人羊膜组织,酶消化法提取 hAEC; 流式细胞仪测 CD29、CD90、CD105、CD34、HLA-DR 的表达。复苏培养 ihCEC,以 0 mg·L- 1、10 mg·L- 1、20 mg·L- 1、30 mg·L- 1、40 mg·L- 1丝裂霉素 37℃作用 2 h,吸去丝裂霉素,继续培养 72 h 后 CCK8 测吸光度并计算增殖抑制率。10 mg·L- 1、20 mg·L- 1丝裂霉素处理细胞后 12 h、24 h 收集细胞培养液培养 hAEC,CCK8 测吸光度绘制生长曲线; 收集 ihCEC 细胞培养液,制备条件培养基( CM) 培养 hAEC 10 d,倒置显微镜观察细胞形态,免疫荧光检测 CK12 的表达。结果 hAEC 可表达 CD29、CD90、D105,不表达 CD34、HLA-DR; 各浓度丝裂霉素组增殖抑制率分别为 10 mg·L- 1( 65. 48% ±1. 03) 、20 mg · L- 1( 77. 01% ± 0. 99) 、30 mg · L- 1( 75. 25% ± 0. 71) 、40 mg · L- 1( 76. 90% ±0. 97) ;20 mg·L- 1丝裂霉素培养 12 h 收集的细胞培养液对 hAEC 具有明显促增殖作用; 诱导分化后 hAEC 可表达 CK12。结论以 ihCEC 细胞培养液模拟的角膜上皮细胞微环境可诱导 hAEC 分化为角膜上皮样细胞。     

Induced pluripotent stem cells as a potential therapeutic source for corneal epithelial stem cells

Corneal blindness caused by limbal stem cell deficiency (LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.     

角膜上皮干细胞定位及功能的研究进展

角膜上皮干细胞定位于角膜缘已被广泛认可,并被命名为角膜缘干细胞,且认为角膜缘干细胞在角膜上皮的自我更新和损伤修复中起重要作用.但近年来的一些研究却提出了与之相悖的观点:整个眼表层均含有寡能干细胞,且角膜缘干细胞在维持自我更新及稳态方面并未起到至关重要的作用.这些发现提示可能还有其他干细胞存在并维持着角膜上皮的自我更新,但也有人对这些新观点提出质疑.本文通过对最新研究的不同观点及证据的阐述,对近年来角膜上皮干细胞的定位及其功能等方面的进展作一综述.     

角膜上皮干细胞的体外生长特征

目的　了解角膜上皮干细胞的生长特性。方法　用体外细胞培养的方法 ,从生长曲线、细胞倍增时间、细胞 DNA合成的活跃程度等几个方面 ,观察人角膜缘部、周边部及中央部角膜上皮细胞的生长特征。结果　角膜缘部细胞群体倍增时间最短 (4 9.79h±1.2 6 h) ,周边部次之 (6 4.89h± 2 .18h) ,中央部最长 (78.86 h± 1.38h) ,3组两两比较 P<0 .0 1;角膜缘部角膜上皮细胞 DNA合成最活跃 (CPM值 92 7.75± 47.94) ,周边部次之(CPM值 711.75± 2 9.47) ,中央部最弱 (CPM值 5 14.0 0± 72 .82 ) ,3组两两比较 P <0 .0 1。结论　结果进一部证实了角膜上皮干细胞存在于角膜缘的观点 ;也反映了角膜缘在角膜上皮创伤愈合 ,角膜上皮完整与稳定性维持过程中起着重要的作用     

使用猪角膜脱细胞基质的人板层角膜移植治疗真菌性角膜炎的临床观察

目的:观察猪角膜脱细胞基质构建的生物角膜支架用于人角膜板层移植术治疗药物难以控制的浅层真菌性角膜炎的效果.方法:对2015-06/2016-03我院收治的16例16眼真菌性角膜炎患者的临床资料进行回顾性分析.16例真菌性角膜炎进行猪角膜脱细胞基质移植,术后随访6mo.对患者术后视力、角膜植片情况、并发症及复发情况进行分析.结果:术后7~10d植片角膜上皮化.16例病例术后1mo角膜水肿,1mo后角膜水肿消失,角膜逐渐透明.术后1mo有2例出现术眼角膜上皮缺损,药物治疗均恢复.术后出现眼压高3例,给予降眼压治疗后眼压控制.随访期间未出现角膜溶解、感染复发、排斥现象.术后1、3、6mo视力分别为1.27±0.22,1.11±0.13,0.79±0.22,术后视力均较术前明显提高,术后1mo视力与术前相比无统计学差异(P=0.06),术后3、6mo视力与术前相比具有明显统计学差异(P=0.01、0.001);其中术后3mo与术后1mo视力相比无明显提高,结果无统计学差异(P=0.11),而术后6mo视力较术后1、3mo均有明显提高,结果具有显著统计学差异(P<0.001).结论:猪角膜脱细胞基质移植治疗真菌性角膜炎是安全、有效的方法.     

单纯去细胞猪角膜基质穿透性角膜移植治疗兔角膜溃疡

目的:探讨单纯去细胞猪角膜基质材料诱导兔自体角膜细胞再生及修复兔角膜溃疡的可行性.方法:新西兰白兔10只制作成角膜溃疡模型,接受单纯去细胞猪角膜材料的穿透性角膜移植术,排除发生手术并发症眼(2只),剩余8只术后进行大体观察(裂隙灯检查),观察植片透明度、新生血管、上皮修复情况.分别于3,6,8,16和24wk取材进行组织学观察,并取16wk兔行超声生物显微镜(UBM)检查、32wk兔行透射电镜检查.结果:角膜溃疡修复,眼表重建8只.初期角膜上皮反复糜烂,荧光素纳染色有荧光堆积,HE 组织学检查提示4wk时上皮细胞复层化.角膜基质细胞沿材料板层生长,排列规律;3～7d角膜缘新生血管发生,位于浅基质层,1～20d达高峰,深入植片或沿缝线环形生长,拆线后逐渐消退,32wk后闭锁仅余3～4支微血管;UBM提示角膜恢复自然曲率,植床与植片尚未修复完善.TEM 提示成纤维细胞分泌胶原原纤维,参与材料改建.结论:CACM支持细胞3D生长,促进自体细胞长入,参与角膜修复改建.     

In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma

AIM:To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.METHODS: Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.RESULTS:HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.CONCLUSION: Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.     

以干燥脱水法保存的异种角膜基质为载体构建人工生物角膜上皮组织

目的:观察以干燥脱水法保存的鸵鸟角膜基质为载体构建人工生物角膜上皮组织的生物学特性。方法:采用组织块培养法获得新西兰大白兔角膜缘干细胞,经胰蛋白酶消化法获得细胞,种植于干燥脱水法保存的鸵鸟角膜板层基质上,采用气液界面培养法进行培养,通过倒置显微镜、透射电子显微镜、荧光显微镜观察其形态学、生长特点,超微结构及免疫学特征。结果:在干燥脱水法保存的鸵鸟角膜基质上种植兔角膜缘干细胞,接种72h后,细胞形成单层,移置气液交界面后继续培养7～10d,逐渐形成复层。经光镜、透射电镜、及免疫学检测显示其具有角膜上皮组织的生物学特性。结论:兔角膜缘干细胞能够在干燥脱水法保存的鸵鸟角膜基质载体上生长,并可形成复层,基本具有正常角膜上皮细胞的形态、超微结构和生物学特性。     

以羊膜为底物培养鸡角膜缘上皮细胞的实验研究

目的为眼表面重建术提供良好的移植材料。方法用胰酶及EDTA消化法去除羊膜上皮将消化、离心获得的鸡角膜缘上皮细胞调成1×107/mlDMEM悬液,接种在6孔培养板底的羊膜基底膜面,放人37℃,5％CO2孵箱培养。结果48h羊膜上培养细胞形成单层,较培养板上培养的同种细胞小,细胞表面的微绒毛及侧面足状突丰富,立体感明显。结论以羊膜基底膜为底物培养的鸡角膜缘上皮细胞可以着床生长并形成完整的单层上皮,为眼表重建使用羊膜及基底膜面培养的角膜上皮细胞提供了实验材料。同时表明羊膜在体外也可以促进鸡角膜缘上皮细胞生长。     

A novel biodegradable polymer scaffold for in vitro growth of corneal epithelial cells

The shortage of donor corneal tissue worldwide has led to extensive research for alternate corneal equivalents utilizing tissue engineering methods. We conducted experiments using Poly D, L lactic acid polymer along with a copolymer (Eudragit) in varying concentrations to create a biodegradable scaffold suitable for in vitro growth of corneal epithelial stem cells. It was found that stable, spherical, and porous microparticles can be prepared by combining PDLLA and Eudragit RL100 polymers in the ratio of 90:10 and 70:30. The microparticles can then be fused to form scaffold membranes with porous architecture and good water retention capacity at room temperature using methanol, which can withstand handling during transplantation procedures. The scaffolds made using a 70:30 ratio were found to be suitable for the promotion of growth of laboratory corneal epithelial stem cell lines (SIRC cell lines). This innovation can pave way for further developments in corneal stem cell research and growth, thus providing for viable laboratory-derived corneal substitutes.     

组织工程角膜上皮支架材料研究进展

体外培养角膜缘干细胞构建组织工程角膜上皮后再行角膜移植是治疗角膜缘干细胞缺乏导致眼表疾病的重要方法,选择理想的支架材料是构建组织工程角膜上皮过程中的一个关键环节。但支架材料的组织相容性、透光度,支架材料的降解与角膜修复的同步化,手术的长期有效性等问题仍有待进一步研究。细胞生物学、分子生物学、组织工程学和材料学的发展为构建组织工程角膜上皮的研究开辟了更广阔的前景。从近年来新发现或研制的支架材料的来源、优缺点及应用等方面,就构建组织工程角膜上皮支架材料的研究进展进行综述。