New invasion assay using endothelial cells grown on native human basement membrane

A new invasion assay is introduced using endothelial cells grown on native human basement membrane (BM). The source of the BM was human amnion. The amnion is a uniform tissue composed of an epithelial layer resting on a continuous basement membrane overlying an avascular collagenous stroma. The epithelium was removed exposing the basement membrane (BM) surface. Human umbilical cord endothelium or bovine capillary endothelium were cultivated on the BM surface. Human squamous carcinoma cells were inoculated onto the BM surface in the presence or absence of the endothelial monolayer. Tumor cells attached readily to both the endothelial monolayer or the BM surface alone. Tumor cells which invaded the basement membrane and underlying collagenous connective tissue were collected on a Millipore filter applied to the opposite side of the amnion. Tumor cells invaded the devitalized amnion connective tissue in the absence of endothelium. The presence of either bovine or human endothelium significantly reduced the rate of tumor cell invasion. This system should be useful for further quantitative studies of the interaction between endothelium and tumor cells with regard to the mechanism of invasion.     

兔角膜内皮细胞载体的体外培养及移植的研究

以特定曲率的壳聚糖-硫酸软骨素共混膜为载体，构建兔角膜内皮。研究了共混膜的透光性、体外酶降解性以及兔角膜内皮细胞在载体上的细胞贴附性、细胞形态及膜强度等性质。结果表明，共混膜的透光率达90％以上，生物降解性良好。该共混膜有良好的细胞贴附性及机械强度；兔角膜内皮细胞可在共混膜上长成良好的单层，细胞形态较好，并贴附牢固，振荡后细胞无脱落，膜片保持完整。将培养好的载体植入到去除内皮层的兔角膜中，术眼在56天内基本保持透明，说明体外构建的内皮可执行角膜内皮层的部分功能。     

Localization of hyaluronic acid, chondroitin sulfate, and CD44 in rabbit cornea.

To demonstrate the localization of hyaluronic acid (HA) in rabbit cornea, the biotinylated HA-binding region, which specifically binds to the HA molecule, was applied to the tissue. Localization of chondroitin sulfate (CS) and CD44, a possible cell surface receptor for HA, were also examined by immunohistochemistry. The stainability of HA changed depending on the fixatives used. Reaction products for HA were distinctly detected in epithelial cells and stromal keratocytes, but faintly in the extracellular matrix of the stroma when unfixed cryosections were applied. No positive reaction was found in the endothelium, except that the positive deposits formed a continuous layer on the apical surface of the endothelium. Electron microscopy using samples fixed with 2% paraformaldehyde revealed gold particles indicating HA labeling the intercellular space of the epithelium and stromal extracellular matrix. No intracellular deposition was detected in epithelial cells, whereas the gold labeling was seen in vacuolar structures of stromal keratocytes. Immunodeposits for CS were intensely localized in the epithelium and stroma, and weakly in the endothelium. Immunoreactivity for CD44 was found in the epithelial, endothelial and stromal cells. In particular, immuno-deposits for CD44 were detected in basal parts of epithelial cells, while they were localized in the apical surface of endothelial cells. These results suggest that HA is synthesized in and secreted from epithelial and stromal cells of rabbit cornea, while the localization of HA in the apical surface of the endothelium is closely associated with that of CD44. Moreover, the presence of CS in corneal tissue may play a role in its transparency, as has been suggested for keratan sulfate and dermatan sulfate.     

Oxygen consumption by the component layers of the cornea

1. The structural integrity of the cornea is maintained by an active fluid transport system which depends on metabolism. Experiments were designed to establish the respiratory activity of each corneal layer.2. A rapid micropolarographic technique was used to determine the oxygen consumptions of the individual layers of the rabbit cornea.3. Inherent problems of the determinations have been made minimal by the use of both denuded and whole corneal tissue preparations. Four independent measures were obtained for each limiting layer (epithelium and endothelium).4. Results show that the endothelium, epithelium and stroma use 21, 40 and 39% respectively of the total oxygen consumption of the cornea. On the basis of volumes of oxygen per unit volume tissue, epithelial oxygen utilization is about ten times that of the stroma and approximately 0.2 that of the endothelium.5. The endothelium has a larger oxygen uptake than previously reported.6. The present results, in conjunction with other studies, indicate that the ratio of glycolytic to oxidative activity in the rabbit cornea is 0.87:0.13.     

The location of the fluid pump in the cornea

1. Fluid transport across rabbit corneal tissue has been investigated by observing the movement of fluid interfaces under the microscope, or by mounting the tissue between two chambers and observing the displacement of menisci within capillary tubes.2. In both cases, the endothelial layer supported on a thin sheet of connective tissue is capable of pumping fluid in a direction out of the cornea, against a head of pressure. The volume of fluid moved may amount to twelve times the thickness of the endothelial cells in an hour.3. This active fluid movement accounts for the prevention of swelling of the normal corneal stroma. The hypothesis that corneal hydration is regulated by the sodium pump in the epithelial layer is not supported by these experiments.     

In vitro culture characteristics of corneal epithelial, endothelial, and keratocyte cells in a native collagen matrix

The objective of this investigation was to demonstrate the effectiveness of a tissue-engineered collagen sponge as a substrate for the culture of human corneal cells. To that end, human kerotocyte, epithelial, and endothelial cells were cultured separately on collagen sponges composed of native fibrillar collagen with a pore size of approximately 0.1 mm. Co-culture experiments were also performed (epithelial/endothelial and epithelial/keratocyte cultures). Proliferation of keratocytes and matrix production was assessed. The morphology of the epithelial and endothelial cell cultures was characterized by histology and scanning electron microscopy. Keratocytes cultured on collagen sponges exhibited increased matrix synthesis over time as well as proliferation and repopulation of the matrix. Epithelial and endothelial cells showed the ability to migrate over the collagen sponge. The thickness of the epithelial layer was influenced by soluble factors produced by endothelial cells. The morphology of the bottom layer of epithelial cells was influenced by the presence of keratocytes in the culture. These studies indicate that human corneal cells exhibit normal cell phenotype when cultured individually on an engineered collagen sponge matrix and co-culture of different cell types in the cornea can influence cell behavior.     

准分子激光联合板层角膜移植术在角膜瘢痕性混浊治疗中的应用

目的：探讨应用准分子激光联合板层角膜移植术治疗角膜瘢痕性混浊眼病的临床疗效。方法：对20例病变深度均未累及角膜后弹力层和内皮细胞层的角膜瘢痕性混浊患者（其中角膜云翳5例、斑翳3例、白斑12例）,按照角膜混浊的范围和累及层次,分别采用准分子激光新技术行PTK、LASIK板层或全厚植片深板层角膜移植术进行治疗;术后观察患者角膜植片愈合情况、角膜内皮细胞密度,并比较手术前后光学增视效果。结果：20例患者角膜植床和植片愈合良好,随访期内均未发生排斥现象,手术眼视力较手术前均有不同程度提高。结论：按照病变累及角膜组织解剖上的不同深度,合理选择激光联合板层角膜移植术的不同术式治疗角膜瘢痕性混浊,治疗效果良好,并发症少,免疫排斥发生率低,可在临床推广应用。     

Leukocyte-mediated injury to corneal endothelial cells. A model of tissue injury.

Although leukocyte-mediated cell injury has long been suggested as a major mechanism of general tissue injury in acute and chronic inflammation, only limited data have been developed to directly implicate leukocytes in ocular tissue injury. In an effort to unravel the role and mechanisms of leukocyte-mediated injury to the cornea, an isolated corneal cup model was developed. For this model, peripheral bovine leukocytes were added to isolated bovine corneal cultures (corneal cup) in vitro, after which endothelial cell damage and death were evaluated morphologically. In general, corneal endothelial cell swelling was observed within 5-10 minutes after leukocyte exposure, followed by detachment of the corneal endothelium at a 30-75-minute interval. Grossly, the detached endothelium appeared as a floating sheet. Using trypan blue dye exclusion, cells in the sheet were found to be nonviable. When examined ultrastructurally, the sheet was found to consist of leukocytes and endothelial cell debris. Light-microscopic examination of the corneal cup at 5-10 minutes after leukocyte action demonstrated extensive endothelial cell damage, as indicated by cell membrane thickening, cytoplasmic vacuolization, and changes in nuclear shape. These changes in the endothelial cells were confirmed by electron microscopy. Vacuolization and swelling of the endothelium appeared as the first changes, induced by leukocyte interactions with the endothelial cell layer. In general, the endothelial cell nucleus became convoluted, mitochondria swelled, and the endoplasmic reticulum became dilated. These changes become more pronounced as the endothelial cells become detached from the underlying Descemet's membrane. Leukocyte interaction with corneal epithelium, on the other hand, did not result in cell detachment, but only cell damage. These studies demonstrate the ability of leukocytes to denude Descemet's membrane of endothelial cells and destroy corneal endothelial cells in vitro. They also suggest that the corneal cup model system will enhance both morphologic and biochemical evaluation of the mechanisms and consequences of leukocyte-mediated injury to the cornea, and thereby provide new insights into the mechanisms of endocular inflammation and tissue injury in vivo.     

EDC/NHS cross-linked collagen foams as scaffolds for artificial corneal stroma

In this study, a highly porous collagen-based biodegradable scaffold was developed as an alternative to synthetic, non-degradable corneal implants. The developed method involved lyophilization and subsequent stabilization through N-ethyl-N'-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) cross-linking to yield longer lasting, porous scaffolds with a thickness similar to that of native cornea (500 microm). For collagen-based scaffolds, cross-linking is essential; however, it has direct effects on physical characteristics crucial for optimum cell behavior. Hence, the effect of cross-linking was studied by examining the influence of cross-linking on pore size distribution, bulk porosity and average pore size. After seeding the foam with human corneal keratocytes, cell proliferation, cell penetration into the scaffold and ECM production within the scaffold were studied. After a month of culture microscopical and immunohistochemical examinations showed that the foam structure did not undergo any significant loss of integrity, and the human corneal keratocytes populated the scaffold with cells migrating both longitudinally and laterally, and secreted some of the main constituents of the corneal ECM, namely collagen types I, V and VI. The foams had a layer of lower porosity (skin layer) both at the top and the bottom. Foams had an optimal porosity (93.6%), average pore size (67.7 microm), and chemistry for cell attachment and proliferation. They also had a sufficiently rapid degradation rate (73.6+/-1.1% in 4 weeks) and could be produced at a thickness close to that of the natural corneal stroma. Cells were seeded at the top surface of the foams and their numbers there was higher than the rest, basically due to the presence of the skin layer. This is considered to be an advantage when epithelial cells need to be seeded for the construction of hemi or full thickness cornea.     

Human corneal endothelial cell sheets for transplantation: Thermo-responsive cell culture carriers to meet cell-specific requirements

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.     

In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: direct contact studies

Synthetic biodegradable polymers have many potential therapeutic applications. In ophthalmology, biodegradable polymers have been used as viscoelastic agents and surgical implants. Other potential applications include controlled release of drugs and growth factors, gene therapy, and tissue engineering. In the present study, in vitro biocompatibility of three biodegradable polymers, 50:50 PDLGA, 85:15 PDLGA, and Inion GTR membrane was evaluated in comparison to tissue culture polystyrene by investigating cell proliferation and potential acute toxicity by the WST-1 cytotoxicity/cell proliferation test, the ATP test, and the lactate dehydrogenase (LDH) test. Evaluations were conducted with cell line cultures from various ocular tissues, human corneal epithelial cells (HCE), rabbit stromal fibroblasts (SIRC), bovine corneal endothelial cells (BCE), human conjunctival epithelial cells (IOBA-NHC), and human retinal pigment epithelial cells (ARPE-19) by direct contact studies by plating the cells on the polymer film specimens in 96-wells. The proliferation results show that cell lines from various ocular tissues attached and grew on PDLGA 50:50, PDLGA 85:15, and Inion GTR membrane. Cytotoxicity experiments with the LDH and ATP tests showed no or extremely slight toxic adverse effects. These polymers have potential to be used as scaffolds in cell transplantation devices or as surgical implants.     

房水中转化生长因子水平与角膜内皮损伤及修复的关联分析

目的 探讨房水中转化生长因子水平与角膜内皮损伤及修复的关联.方法 研究选取本院就诊患者102例,检测患者房水中总TGF-β2和活化TGF-β2的水平,监测分析角膜内皮细胞密度水平、角膜内皮六角形细胞的比例水平以及中央角膜厚度.分析讨论房水中TGF-β2水平与角膜内皮损伤及修复的关联.使用SPSS软件处理数据.结果 对治疗前总TGF-β2水平与表征角膜内皮细胞损伤状态的四项指标水平进行相关分析结果提示,治疗前研究样本总TGF-β2水平与角膜内皮细胞密度之间的相关系数为-0.123、于角膜内皮六角细胞水平之间的相关系数为-0.198,另外治疗前总TGF-β2水平与中央角膜厚度水平之间的相关系数为0.254,且均P＜0.05;治疗前活化TGF-β2水平与表征角膜内皮细胞损伤状态的指标水平相关分析结果也提示了类似结果,除了活化TGF-β2水平与内皮细胞密度的r为-0.145,未见统计学意义之外,活化TGF-β2水平与其他指标的相关系数均有统计学意义.治疗后总TGF-β2水平以及活化TGF-β2水平与四项指标的相关系数也发现了类似规律,且均P＜0.05.结论 总TGF-β2水平以及活化TGF-β2水平增高与角膜内皮损伤有关,与内皮细胞密度、角膜内皮六角细胞比例下降,中央角膜厚度则会增加密切相关.     

Ocular cell monolayers cultured on biodegradable substrates.

The aim of this study was to culture retinal pigment epithelial (RPE) and corneal endothelial cells on biodegradable substrates for future use in monolayer transplantation in the eye. The biodegradable polymers, poly-l-lactic (PLLA) and poly-dl-lactic-co-glycolic acid (85:15) (PLGA) (both of molecular weight 105 kd) were the biomaterials used. All materials were seeded with either pig/human retinal pigment epithelial cells or rabbit corneal endothelial cells and were maintained in tissue culture conditions. Upon confluency, the cell density was calculated and cell viability determined. All monolayers were stained with phalloidin-rhodamine for F-actin and antibodies to the tight junction (zonula occludens) protein, ZO1, to demonstrate the presence of tight junctions. The final cell density of human RPE monolayers on PLLA films was 2950 cells/mm(2) (+/-185). The final cell density of pig RPE on PLLA and PLGA film was 2350 cells/mm(2) (+/-152 and 178, respectively). Rabbit corneal endothelial cells had a final cell density of 2650 cells/mm(2) (+/-164). F-actin staining revealed a circumferential ring of actin filaments in all of the cells grown on substrates. ZO(1) immunohistochemistry demonstrated staining along the lateral cell borders of all cell types. The successful culture of retinal pigment epithelial and corneal endothelial monolayers on these substrates may have potential for transplanting cell monolayers in the eye to improve vision.     

A novel simple technique for en face endothelial observations using water-soluble media -'thinned-wall' preparations

A new, easily applicable technique providing en face preparations for light microscopy observations of the rat aorta and human thin wall arteries is described here. The major steps of the technique include attachment of the fixed and flattened vessel with the endothelium face down on a glass slide, covered with a water-soluble adhesive medium; drying and softening the vessel wall with another water-soluble medium; removal of the adventitia and most of the media; detaching the layer by placing the glass slide in water; and final attachment of the layer with the endothelium upwards. On such 'thinned-wall' preparations, 40-50 microm in thickness, the stained endothelial cells are clearly visible. Because of the preparation thickness and the use of water-soluble media during the preparation, some subendothelial lipid accumulations, characteristic of the early stages of atherosclerosis process, are well preserved.     

Electrical coupling between smooth muscle and endothelium in arterioles of the guinea-pig small intestine

Equations describing the steady-state passive electrical properties of arterioles have been derived. The arteriole was modelled as having two thin layers of cells (muscle and endothelium) with strong electrical coupling between cells within a layer and variable coupling between the layers. The model indicated that spread of membrane potential changes was highly dependent on the thickness of cells within the layers. The model was also used to identify the optimal experimental strategy for detecting coupling between the two layers, and experiments were carried out on arterioles from the guinea-pig small intestine. Thickness of the endothelial layer was measured using electron microscopy and was found to be around 0.5 microm. Electrical input resistance was measured in intact arterioles and compared to input resistance of arterioles from which the endothelium had been removed. The experiments confirmed that there was a strong electrical coupling between the muscle and endothelium in these vessels.     

Replication of dengue and junin viruses in cultured rabbit and human endothelial cells.

The flavivirus dengue and the arenavirus Junin are both associated with a hemorrhagic shock syndrome in man. We have demonstrated the replication of these viruses in vitro in both rabbit and human endothelial cells by viral titers and immunofluorescent antibody studies. Rabbit endothelium established in continuous culture was derived from vena cava, while human cells in primary culture were derived from umbilical veins. In rabbit endothelium, dengue-2 virus passaged through monkey kidney monolayer cells (LLC-MK2) or human lymphoblastoid cells (raji) produced significantly more virus than the seed obtained from suckling mouse brain (MB). Inoculation of actively dividing, subconfluent human endothelial cells with the LLC-MK2 degue virus produced higher viral titers than inoculation of confluent cells. The appearance of Junin virus was delayed beyond that of dengue virus in rabbit endothelial cells although equivalent titers of virus were produced. In human cells, Junin virus was less productive than dengue virus and produced characteristic cycles of virus release. This is the first direct evidence for replication of human hemorrhagic fever viruses in endothelial cells.     

Establishment of a human in vitro model of the outer blood-retinal barrier

The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age-related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE-19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein-derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE-19. Both human umbilical vein-derived endothelial and ARPE-19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein-derived endothelial cells in the trilayer became fenestrated when co-cultured with the ARPE-19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE-19 cells. These cells also revealed VE-cadherin and ZO-1 at cell-cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO-1, were localized to cell-cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein-conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten-fold reduction) whilst transfer of a 4 kDa FITC-conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional-like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE-19 and human umbilical vein-derived endothelial cells can be co-cultured on the amniotic membrane and that the resultant cross-talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer-retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD.     

Diffusion of oxygen at the endothelial surface of the rabbit cornea.

1. An in vitro investigation was made to determine the oxygen tension level required at the endothelial surface of rabbit cornea to produce a net oxygen flux into the cornea across this surface when the epithelial surface was exposed to air. 2. The experimental design was based on a mathematical model which showed that the direction of oxygen flux measured in an agar layer adjacent to the endothelium was the same as the direction of oxygen flux across the endothelial surface. 3. From micro-electrode measurements of oxygen tension in the agar layer, it was found that an oxygen tension greater than 102 mmHg at the endothelial surface was required tocause a net flux of oxygen into the cornea. 4. Comparing this result to the in vivo situation,it was concluded that all layers of the rabbit cornea receive oxygen from the atmosphere under open eye conditions.     

Functional reconstruction of corneal endothelium using nanotopography for tissue-engineering applications

Dysfunction in the corneal endothelium, which controls the hydration and transparency of the cornea, is one of the common reasons for transplantation. A tissue-engineered corneal endothelium is of interest for corneal regeneration and for in vitro testing of ocular drugs. In the native environment, corneal endothelial cells interact with the nanotopography of the underlying Descemet's membrane. This study showed that nanotopography enhanced bovine corneal endothelial cell (BCEC) responses, creating a monolayer which resembled the healthy corneal endothelium. Topographies of different geometries were first tested to identify those that would elicit the most significant responses. A BCEC monolayer was then generated on both micro- and nanoscale pillars and wells. The BCEC monolayer cultured on topographies exhibited polygonal geometries with well-developed tight junction proteins. Scanning electron microscopy revealed that cells on pillars showed a higher density of microvilli, which was similar to native corneal endothelium. BCECs on nanopillars displayed a lower coefficient of variation of area (0.31) that was within the range of healthy corneal endothelium. More importantly, a BCEC monolayer cultured on nanopillars also had an enhanced Na(+)/K(+)-ATPase immunofluorescence expression, mRNA upregulation and a higher Na(+)/K(+)-ATPase activity. These results suggest that nanopillar substrate topography may provide relevant topographical cues, which could significantly enhance the formation and function of corneal endothelium.     

超声乳化术中不同灌注液对角膜内皮细胞的影响

目的:观察超声乳化术中使用不同灌注液(世可和平衡盐液)对兔眼角膜内皮细胞结构及功能的影响。方法:3.5月龄纯种日本大耳白兔,随机分为正常对照组、空白对照组、世可组和平衡盐液组,采用接触性角膜内皮显微镜进行角膜内皮细胞形态学定量测定。锥兰-茜素红活性染色,观察各组角膜内皮细胞微细结构的变化。结果:术后6h,各组较正常对照组细胞密度和六边形细胞百分率降低;细胞面积和六边形细胞百分率均高于正常对照组;光镜显示空白对照组角膜内皮细胞形态改变明显,而世可组和平衡盐液组角膜内皮细胞改变轻微,无细胞坏死。结论:在超声乳化术中,灌注液对角膜内皮的损伤为化学性损伤,在相同手术条件下,世可对角膜内皮细胞具有良好的保护作用。