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.     

Elastic modulus and collagen organization of the rabbit cornea: Epithelium to endothelium

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.     

A histological study of the outer layer of rabbit fish (Siganus javus) eye

The rabbit fish is an economically valuable teleost species which lives in shallow coastal waters amongst aquatic plants. Two species of rabbit fish, Siganus sutore and Siganus javus, have been identified in the sea south of Iran. In this study, in order to investigate the histology of the outer layer of the S. javus eye, the eyes of 12 healthy specimens of S. javus were extracted and histologic sections were prepared. The sections were studied under a light microscope after staining with hematoxylin and eosin and Masson's trichrome stains. The outer layer was composed of the cornea cranially and the sclera caudally. The sclera contained an episclera zone and two cartilaginous segments with connective tissue correlation. The cornea included dermal components (stratified cuboidal epithelium, Bowman's membrane, and dermal stroma with occasional flattened cells); the scleral part consisted of two main layers (irregular fibers of connective tissue overlaying the second scleral stroma layer). The fibers of the scleral stroma were the only component present at the posterior part of the cornea. These results reveal that the eye of S. javus does not possess Descemet's membrane or endothelium in the cornea.     

Corneal splitting in the developing lamprey Petromyzon marinus L. eye

The cornea of the adult lamprey has both dermal (spectacle) and scleral components. These are separated by a thin mucoid layer that allows free movement of the globe. This study has shown that during the larval (ammocoete) stage, the lamprey cornea develops in a manner similar to that of other lower vertebrates. Just prior to the period of transformation to the adult parasite, the outer dermal portion of the ammocoete cornea (spectacle) consists of an anterior stratified columnar epithelium with goblet cells at the surface. The stroma of the dermal cornea consists of a thick outer layer of orthogonally oriented collagen with branching fibroblasts and a thin, loosely organized inner layer with slender elongated fibroblasts. The scleral cornea is lined internally by a flattened monolayer of mesodermal cells, the corneal endothelium. Its narrow stroma is composed entirely of thin, orthogonally aranged, collagen-fiber lamellae, and is bounded externally by a thin continuous mesothelial layer of cells that abuts directly onto the loose stromal component of the dermal cornea. During the early stages of transformation, the anterior epithelium of the dermal cornea becomes stratified squamous in type. Later, the inner loose stroma of the dermal cornea (spectacle) begins to separate from the scleral cornea components, and a third complete mesothelial layer forms a distinct inner border for the dermal cornea. A mucoid layer is formed between the dermal (spectacle) and scleral corneas and remains throughout the adult life.     

The mucinous layer of corneal endothelial cells

PURPOSE: The goal of this study was to characterize the morphology of the mucinous layer on rabbit, bovine, owl, and human corneal endothelial cells. METHODS: Corneoscleral buttons were fixed using cetylpyridinium chloride to stabilize "mucus" and the tissue was prepared for transmission electron microscopy. Photomicrographs were measured to determine the thickness of the endothelial and epithelial mucinous layer in the central cornea. RESULTS: The endothelial mucinous layer was seen as a nearly uniform electrodense region on the apical aspect of the endothelium. It was found to be 0.9 microm, 0.9 microm, 0.9 microm, and 0.5 microm thick in rabbit, bovine, owl, and human, respectively. The owl endothelium had an additional less electrodense layer with a granular appearance and a thickness of about 200 microm. The mucinous layer on the epithelium was similar in appearance to that on the endothelium and across species. CONCLUSIONS: The morphologic similarity of the endothelial and epithelial mucinous layers is a serendipitous finding that should prove valuable in experimental design. Ultimately, it is hoped that studies of the posterior corneal surface will deepen our knowledge of endothelial protection.     

Hypoxia adaptation in the cornea: Current animal models and underlying mechanisms

The cornea is an avascular, transparent tissue that is essential for visual function. Any disturbance to the corneal transparency will result in a severe vision loss. Due to the avascular nature, the cornea acquires most of the oxygen supply directly or indirectly from the atmosphere. Corneal tissue hypoxia has been noticed to influence the structure and function of the cornea for decades. The etiology of hypoxia of the cornea is distinct from the rest of the body, mainly due to the separation of cornea from the atmosphere, such as prolonged contact lens wearing or closed eyes. Corneal hypoxia can also be found in corneal inflammation and injury when a higher oxygen requirement exceeds the oxygen supply. Systemic hypoxic state during lung diseases or high altitude also leads to corneal hypoxia when a second oxygen consumption route from aqueous humor gets blocked. Hypoxia affects the cornea in multiple aspects, including disturbance of the epithelium barrier function, corneal edema due to endothelial dysfunction and metabolism changes in the stroma, and thinning of corneal stroma. Cornea has also evolved mechanisms to adapt to the hypoxic state initiated by the activation of hypoxia inducible factor (HIF). The aim of this review is to introduce the pathology of cornea under hypoxia and the mechanism of hypoxia adaptation, to discuss the current animal models used in this field, and future research directions.     

脱细胞猪角膜基质的基底膜在修复兔角膜损伤中的作用

目的 探讨脱细胞角膜基质支架材料表面保存基底膜样结构对修复角膜损伤的必要性. 方法 将采用反复冻融及酶消化法制备的脱细胞猪角膜基质,移植到兔角膜损伤模型上,通过大体及组织学分别观察材料表面基底膜结构的有无对修复兔角膜基质损伤的影响. 结果 脱细胞猪角膜基质材料表面有基底膜的实验组（n=8）兔角膜损伤全部修复,无穿孔,支架材料与受体角膜整合.材料表面无基底膜的对照组（n=8）中,有6例植入的支架材料溶解,兔角膜穿孔.两组结果比较差异有显著统计学意义. 结论 脱细胞角膜基质支架材料表面具有基底膜样结构,有利于形成正常角膜上皮屏障,从而防止了生物材料的过快降解,有利于材料与宿主基质的整合和改建.     

Porous matrix and primary-cell culture: a shared concept for skin and cornea tissue engineering

Skin and cornea both feature an epithelium firmly anchored to its underlying connective compartment: dermis for skin and stroma for cornea. A breakthrough in tissue engineering occurred in 1975 when skin stem cells were successfully amplified in culture by Rheinwald and Green. Since 1981, they are used in the clinical arena as cultured epidermal autografts for the treatment of patients with extensive burns. A similar technique has been later adapted to the amplification of limbal-epithelial cells. The basal layer of the limbal epithelium is located in a transitional zone between the cornea and the conjunctiva and contains the stem cell population of the corneal epithelium called limbal-stem cells (LSC). These cells maintain the proper renewal of the corneal epithelium by generating transit-amplifying cells that migrate from the basal layer of the limbus towards the basal layer of the cornea. Tissue-engineering protocols enable the reconstruction of three-dimensional (3D) complex tissues comprising both an epithelium and its underlying connective tissue. Our in vitro reconstruction model is based on the combined use of cells and of a natural collagen-based biodegradable polymer to produce the connective-tissue compartment. This porous substrate acts as a scaffold for fibroblasts, thereby, producing a living dermal/stromal equivalent, which once epithelialized results into a reconstructed skin/hemicornea. This paper presents the reconstruction of surface epithelia for the treatment of pathological conditions of skin and cornea and the development of 3D tissue-engineered substitutes based on a collagen-GAG-chitosan matrix for the regeneration of skin and cornea.     

LYVE-1蛋白在小鼠碱烧伤角膜的表达及意义

目的观察淋巴管内皮透明质酸受体(LYVE-1)在小鼠碱烧伤角膜内的表达情况,探讨角膜新生淋巴管形成的时间过程及角膜疾病后新生淋巴管形成的作用。方法应用NaOH溶液制作小鼠角膜碱烧伤模型,分别于角膜碱烧伤后第1d、3d、5d、7d和12d取材。采用免疫组化法,观察正常角膜和碱烧伤后不同时间段角膜内LYVE-1的表达情况。结果在正常角膜组织中,LYVE-1表达于角膜上皮细胞和内皮细胞内。在角膜碱烧伤后1d、3d和5d,LYVE-1主要表达于角膜上皮内及入侵角膜基质的炎性细胞内;碱烧伤后7d,可见大量LYVE-1呈条索样表达于角膜基质中,并可见少量LYVE-1阳性表达于开放状态的淋巴管;碱烧伤后12d,角膜基质内新生淋巴管数量增多。结论正常角膜组织储备LYVE-1生物因子,在炎性角膜中,LYVE-1可能在角膜新生淋巴管运输透明质酸(HA)的过程中发挥重要作用。     

Effects of inhibition of urokinase-type plasminogen activator (u-PA) by amiloride in the cornea and tear fluid of eyes irradiated with UVB

The purpose of the present study was to test our hypothesis that amiloride, a specific u-PA inhibitor, effectively decreases u-PA activity in cornea as well as in tear fluid and favourably affects corneal healing. Therefore, comparative histochemical and biochemical studies of u-PA and the effects of amiloride were performed on rabbit corneas and tear fluid using the sensitive fluorogenic substrate Z-Gly-Gly-Arg-7-amino-4-trifluoromethylcoumarin. Rabbit eyes were repeatedly irradiated with UVB for 9 days and during the irradiation topically treated with amiloride (1 mg/ml saline) or placebo (saline) (dropwise, 5 times daily). Results show that in placebo-treated eyes, UVB evoked the appearance of u-PA activity in cornea and tear fluid in early stages of irradiation, and u-PA levels increased during irradiation. Corneal epithelium was gradually lost and remnants of the epithelium as well as keratocytes in the upper part of corneal stroma showed high u-PA activity. Finally, corneas lost their epithelium completely. In corneal stroma, numerous u-PA-containing inflammatory cells were present. Corneas were vascularized. When amiloride was dropped on the eye surface on the first day of irradiation and subsequently daily until the end of the experiment, u-PA activity in both cornea and tear fluid was strongly inhibited. Corneas were covered with a continuous epithelium until the end of the experiment. The number of inflammatory cells was significantly decreased. Corneal vascularization was reduced by 50%. In conclusion, early application of amiloride inhibited u-PA activity in UVB-irradiated corneas as well as in tear fluid and diminished the development of corneal pathology.     

血管内皮生长因子C在小鼠碱烧伤角膜内的表达及意义

目的观察血管内皮生长因子C(VEGF-C)在小鼠角膜碱烧伤后不同时间段角膜组织内的表达情况,探讨VEGF-C在小鼠角膜碱烧伤后新生淋巴管形成过程中的作用。方法制作小鼠角膜碱烧伤模型,分别于碱烧伤后1d、3d、5d、7d、12d和18d取材。采用免疫组化法(SP),观察VEGF-C在正常角膜和碱烧伤后不同时间段角膜组织内的表达情况。应用淋巴管内皮透明质酸受体(LYVE-1)标记淋巴管,观察小鼠碱烧伤角膜内新生淋巴管的形成情况。结果在正常小鼠角膜组织内,VEGF-C表达于角膜上皮层和内皮层。在碱烧伤角膜内,VEGF-C主要表达于角膜基质内入侵的炎性细胞和角膜上皮层细胞,并且表达增高,于碱烧伤后3d,VEGF-C的表达达到高峰(P<0.01)。碱烧伤后7d,VEGF-C的表达下降至正常水平,可见阳性表达LYVE-1的处于开放状态的新生淋巴管。结论VEGF-C可能参与小鼠角膜碱烧伤后角膜新生淋巴管形成过程。     

Mitochondrial DNA common deletion in the human eye: a relation with corneal aging

The most frequent mitochondrial DNA (mtDNA) mutation is a 4977 bp deletion known as the common deletion (mtDNA(CD4977)). mtDNA(CD4977) is related to skin photo-aging and to chronological aging of cells with high-energy demands such as neurons and muscle cells. The human eye contains both sun-exposed (cornea, iris) and high-energy demand structures (retina). In this study, we employed a highly sensitive quantitative PCR technique to determine mtDNA(CD4977) occurrence in different structures of the human eye. We found that the cornea, the most anterior structure of the eye, contains the highest amount of mtDNA(CD4977) (2.6%, 0.25% and 0.06% for the cornea, iris and retina, respectively). Within the cornea, mtDNA(CD4977) is almost exclusively found in the stroma, the cellular layer conferring transparency and rigidity to the human cornea (8.59%, 0.13% and 0.05% in the stroma, endothelium and epithelium, respectively). Moreover, we show that mtDNA(CD4977) accumulates with age in the corneal stroma. Taken together, our results suggest that mtDNA(CD4977) is related to photo-aging rather than chronological aging in the human eye. Similar to the involvement of mtDNA(CD4977) in skin photo-aging phenotypes, we believe that the clinical manifestations of corneal aging, including clouding and stiffening, are associated with the accumulation of mtDNA(CD4977) in the corneal stroma.     

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.     

Fas和Fas配体在角膜移植免疫排斥反应中的表达

目的 了解Fas和Fas配体(FasL)在发生免疫排斥的角膜植片中的表达情况，探讨其在角膜移植免疫排斥反应中的作用。方法 角膜移植术后排斥反应的患者42例，于再次行穿通性角膜移植术时取其排斥的移植片；正常角膜6例。进行免疫组织化学染色，观察正常角膜和移植片中上皮、基质和内皮层的Fas及：Fas配体的表达。结果 在6例正常角膜中，角膜上皮、内皮Fas和FasL．为阳性表达。42例移植片中，角膜上皮．Fas和FasL均有表达。有新生血管形成及免疫细胞浸润的角膜基质中，血管内皮细胞、基质细胞FasL为阳性表达，Fas在部分浸润的免疫细胞中有表达；移植片内皮细胞层广泛破坏。结论 Fas、FasL在角膜移植片中的表达，可能与角膜移植免疫排斥反应有关。     

Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction

To investigate the feasibility of using acellular porcine limbal stroma for limbal stem cell microenvironment reconstruction. Limbal reconstruction was performed in rabbit partial limbal defect models. Rabbits were randomly divided into four groups: acellular porcine limbal stroma, de-epithelized rabbit limbal autograft stroma, de-epithelized porcine limbal stroma and acellular porcine corneal stroma transplantation groups. In both the acellular porcine limbal stroma and de-epithelized rabbit limbal autograft stroma groups, cornea transparency and epithelium integrity were sustained and graft rejection was not observed. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1, but it returned to K3-/P63+/Ki67+(phenotype characteristic of limbal epithelium) by postoperative months 3 and 6. In the de-epithelized porcine limbal stroma group, acute and serious immune rejection occurred by postoperative days 8-10. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1. In the acellular porcine corneal stroma group, there were some new vessel invasion into the peripheral cornea and mild corneal opacity. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative months 1, 3, and 6. In conclusion, acellular porcine limbal stroma possessed very low immunogenicity, retained a good original limbal ECM microenvironment, and thus the reconstructed rabbit limbal microenvironment maintained limbal epithelial stem cell stemness and proliferation.     

Electron histochemical study on synthesis and breakdown of glycogen in the rabbit cornea.

Glycogen synthetase and phosphorylase activity was studied in the rabbit cornea electron histochemically. Glycogen synthetase and phosphorylase were located in the cytoplasmic matrices of the corneal epithelium, but glycogen synthetase was found to be mainly in the superficial half of the epithelium, and phosphorylase mainly in the deep layer. Since the cells in the deep layer of the corneal epithelium contain rich cytoplasmic organelles which have carbohydrate metabolism from glycogen or glucose through the Embden-Meyerhof pathway to the TCA cycle, glycogen synthesized in the superficial half of the corneal epithelium by glycogen synthetase may be transferred to the deep layer of the epithelium and broken down by phosphorylase. Phosphorylase in the superficial half of the corneal epithelium may break down overproduced glycogen so as to avoid the deposition of a large number of glycogen particles for maintenance of corneal transparency.     

The regulation of corneal hydration by a salt pump requiring the presence of sodium and bicarbonate ions

1. The use of polyacrylamide gel salt bridges enables trans-membrane potentials to be measured to an accuracy of 20 muV over long periods.2. The technique is applied to measure electrical potentials across corneal endothelia of rabbits.3. In de-epithelialized corneas which translocate water, a spontaneous potential of 550 muV is found across the endothelium (tissue resistance 20 Omega cm(2)).4. This electrical potential (and water translocation) is reduced to zero when sodium is absent from the Ringer, and by about 80% when bicarbonate ions are absent. Removal of chloride has no such effect.5. Under a variety of conditions, the potential correlates with the observed translocation of fluid across corneal endothelium. The translocated fluid is shown to be isotonic with sodium in the Ringer and therefore the potential correlates with ;active' sodium transport.6. The potential and water translocation are abolished in the presence of ouabain at concentrations greater than 10(-5)M.7. The potential (lens-side negative) is of the wrong polarity to explain the net sodium transport (into the lens-side) by a sodium ion ;pump'.8. The current does not equal the net sodium flux under short circuit conditions. They differ in magnitude and polarity.9. A model is proposed where the endothelium ;pumps' salt out of the corneal stroma into the aqueous humour.10. Flux equations are derived for a condition where the membrane (corneal endothelium) separates an ion exchanger (corneal stroma) from free solution (aqueous humour), where the usual relationship for free-free solutions Deltapi = c(s)Deltamu(s) does not apply.11. The model is of use only when the stroma is well stirred. It may be used in whole corneas retaining their epithelium but it may not be used in de-epithelialized corneas.12. The model predicts that the presence of an ;active' salt flux out across the endothelium would create passive water and salt fluxes. The passive water flux would also travel out of the stroma across the endothelium; the passive salt flux would travel, in the opposite direction, into the stroma across the endothelium.13. The kinetics of the passive water efflux, as a swollen cornea reverts to physiological hydration (the temperature reversal phenomenon) are predicted extremely well if the ;active' salt flux is chosen at 3.3 x 10(-7) m-mole. cm(-2) sec(-1).14. The value of the active salt flux which cannot be measured directly is extrapolated to be somewhat greater than 2.8 x 10(-7) m-moles. cm(-2) sec(-1); in good agreement with that required by the model to explain the temperature reversal phenomenon.15. The model is further used to calculate the salt concentration difference across the endothelium (which drives salt passively into the stroma) at various stromal hydrations.16. When an appropriate salt concentration is applied across the endothelium of de-epithelialized cornea, it generates a potential of the same polarity and similar magnitude to that found across the endothelium of equilibrated whole cornea. The endothelium acts like a cation exchange membrane.17. Additionally the calculated salt concentration difference across the endothelium correlates well with the measured transendothelial potentials in whole cornea as the corneal hydration varies.18. It is concluded that the model of an endothelial neutral salt ;pump' regulating corneal hydration is self consistent. The spontaneous potential found across the endothelium could be caused by the consequential passive flux of salt in the opposite direction.     

Disruption of anterior segment development by TGF-beta1 overexpression in the eyes of transgenic mice.

Previous experiments showed that transgenic mice expressing a secreted self-activating transforming growth factor (TGF) -beta1 did not show a phenotype in the lens and cornea until postnatal day 21, when anterior subcapsular cataracts, sporadic thickening of the corneal stroma, and thinning of the corneal epithelium were noted (Srinivasan et al., 1998). To examine the effects of higher concentrations of TGF-beta1 on the lens and cornea, we constructed transgenic mice harboring the strong, lens-specific chicken betaB1-crystallin promoter driving an activated porcine TGF-beta1 gene. In contrast to the earlier study, the transgenic mice had microphthalmic eyes with closed eyelids. Already at embryonic day (E) 13.5, the future cornea of the transgenic mice was threefold thicker than that of wild-type littermates due to increased proliferation of corneal stromal mesenchyme cells. Staining of fibronectin and thrombospondin-1 was increased in periocular mesenchyme. At E17.5, the thickened transgenic corneal stroma was vascularized and densely populated by abundant star-shaped, neural cell adhesion molecule-positive cells of mesenchymal appearance surrounded by irregular swirls of collagen and extracellular matrix. The corneal endothelium, anterior chamber, and stroma of iris/ciliary body did not develop, and the transgenic cornea was opaque. Fibronectin, perlecan, and thrombospondin-1 were elevated, whereas type VI collagen decreased in the transgenic corneal stroma. Stromal mesenchyme cells expressed alpha-smooth muscle actin as did lens epithelial cells and cells of the retinal pigmented epithelium. By E17.5, lens fiber cells underwent apoptotic cell death that was followed by apoptosis of the entire anterior lens epithelium between E18.5 and birth. Posteriorly, the vitreous humor was essentially absent; however, the retina appeared relatively normal. Thus, excess TGF-beta1, a mitogen for embryonic corneal mesenchyme, severely disrupts corneal and lens differentiation. Our findings profoundly contrast with the mild eye phenotype observed with presumably lower levels of ectopic TGF-beta and illustrate the complexity of TGF-beta utilization and the importance of dose when assessing the effects of this growth factor.     

Autoradiographic study of corneal neovascularization induced by chemical cautery

The corneas of adult rats were cauterized chemically, and the responses of the pericorneal blood vessels and the cellular constituents of the cornea were followed by light microscopic autoradiography after labeling with 3H-thymidine. As in previous experiments, this injury elicited a neovascularization as capillaries sprouted and extended centripetally from the corneoscleral limbus to the cautery site. Chemical cautery induced a response in the epithelium, endothelium, and fibroblasts of the cornea as well as in the vascular cells. Elevated labeling indices for the corneal epithelium and endothelium began at 18 and 21 hours after injury, respectively. In all of these corneal cell types, the labeling index returned to control values by 75 hours. The onset and decline of DNA synthesis in corneal fibroblasts paralleled that of the corneal epithelium and endothelium. Labeling indices of vascular cells (endothelial cells and pericytes) increased 21 hours after injury, reached a maximal level at 45 hours, and returned to control values by 1 month after cautery. The first mitoses in vascular endothelial cells and pericytes were noted 36 hours after injury, and the initial capillary sprouts appeared at 39 hours. This study demonstrates that the thymidine incorporation by cells in the pericorneal blood vessels occurs early within the postcauterization period, at least 15 hours before the first mitotic figures are detected in these same vascular cells. The significance of the temporally related elevations in labeling indices of the vascular cells and the cellular constituents of the corneal cells is uncertain, but there are many potential interrelationships between the controls of cell division and migration for cells of the vessels, epithelium, endothelium, and corneal stroma.     

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.