X-ray scattering used to map the preferred collagen orientation in the human cornea and limbus

Many properties of connective tissues are governed by the organization of the constituent collagen. For example, the organization of collagen in the cornea and the limbus, where the cornea and sclera meet, is an important determinant of corneal curvature and hence of the eye's focusing power. We have used synchrotron X-ray scattering to map the orientation of the collagen fibrils throughout the human cornea, limbus, and adjacent sclera. We demonstrate a preferred orientation of collagen in the vertical and horizontal directions that is maintained to within about 1 mm from the limbus, where a circular or tangential disposition of fibrils occurs. The data are also used to map the relative distribution of both the total and the preferentially aligned collagen in different parts of the tissue, revealing considerable anisotropy. The detailed structural information provided is an important step toward understanding the shape and the mechanical properties of the tissue.     

Material properties of the cornea and sclera: a modelling approach to test experimental analysis

Material properties of cornea and sclera are important for maintaining the shape of the eye and the requisite surface curvatures for optics. They also need to withstand the forces of external and internal musculature and fluctuations in intraocular pressure (IOP). These properties are difficult to measure and variable results have been reported. A previously published experimental procedure, from which the material properties of the eyeball coats were obtained, has been modelled in this paper using Finite Element Analysis, in order to test the accuracy of the experiment. Material parameters were calculated from the model and the resulting relationships between stress and strain for the cornea and sclera compared to their experimentally obtained counterparts. The comparison between model and experiment was close for the sclera but more varied for the cornea. The pressure vessel model can be applied for measuring the material properties of the sclera but is less accurate for the cornea.     

The integration of the corneal and limbal fibrils in the human eye.

The precise orientation of the collagen fibrils in human cornea and sclera and the method by which these two areas fuse together at the limbus have never been determined, despite the importance of this information. From a consideration of the mechanics of the system, fibril orientation in the tissue has the potential to affect the curvature of the cornea so, by inference, refractive problems such as astigmatism involving an incorrect curvature of the cornea may be related to fibril orientation. The high intensity and small beam size of a synchrotron x-ray source has enabled us to study fibril orientation in post-mortem human cornea and sclera. Previously we have revealed two preferred directions of orientation in the cornea (Meek, K. M., T. Blamires, G. F. Elliot, T. Y. Gyi, and C. J. Nave. 1987. Curr. Eye Res. 6:841-846) and a circumcorneal annulus in the limbus (Newton, R. H., and K. M. Meek. 1998. Invest. Ophthalmol. & Visual Sci. 39: 1125-1134). Here we present the results of our investigation into the relationship between these two features. Our measurements indicate that the corneal fibrils oriented in the two preferred directions bend at the limbus to run circumferentially. On the basis of these results we propose a model as to how the human cornea and sclera fuse together.     

The Expression of Tenascin-X in Developing and Adult Rat and Human Eye

Tenascin-X has been studied in developing and adult rat eye and in foetal and adult human eyes, using immunohistochemistry and frozen sections. The data were compared with the distribution of tenascin-C. The immunoreactivity for tenascin-X was seen in a basement membrane-like feature in different structures of embryonic (E) day 16–17 rat eyes. Postnatal (P) day 2 and older rat eyes showed immunoreactivity for tenascin-X in different connective tissues. In the epithelial basement membrane zone of the cornea, immunostaining was positive in P5 eyes, negative in P10 and P15 eyes and again positive in P30 and adult eyes. In the 20-week-old human foetus, immunoreactivity for the tenascin was seen in the posterior parts of the conjunctival stroma adjacent to the sclera and in a basement membrane-like fashion in anterior conjunctiva. In the adult human eye, immunoreactivity for tenascin-X was seen in the anterior one-third stroma of cornea as thin fibrils, in the stroma of the limbus and conjunctiva, and in blood vessels. Immunostaining for tenascin-C was seen in the posterior aspect of the further cornea, and in mesenchyme adjacent to cornea in E16–17 rat eyes. Corneal keratocytes and Descemet's membrane showed immunoreactivity for tenascin-C in P2–P15 rat eyes. Sclera and the junction of the cornea, and sclera expressed tenascin-C in P2 and older rat eyes. In human foetal eyes, immunostaining for tenascin-C was seen in the anterior parts of the corneal stroma, in the basement membrane zone and Bowman's membrane of the corneal epithelium, in the posterior one-fifth of the corneal stroma and the sclera starting from the junction of the cornea and sclera. In normal human adult eyes, immunostaining for tenascin-X was seen in the anterior one-third stroma of cornea, in the stroma of limbus and conjunctiva, and in blood vessels. The association of tenascin-X and basement membranes in early development evokes a question of its potential function in the development of the basement membrane. The results also suggest the association of tenascin-X with connective tissue development as well as the association of tenascin-C with the migration of keratocytes during the development of the corneal stroma.     

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.     

An inverse finite element method for determining the anisotropic properties of the cornea

An inverse finite element method was developed to determine the anisotropic properties of bovine cornea from an in vitro inflation experiment. The experiment used digital image correlation (DIC) to measure the three-dimensional surface geometry and displacement field of the cornea at multiple pressures. A finite element model of a bovine cornea was developed using the DIC measured surface geometry of the undeformed specimen. The model was applied to determine five parameters of an anisotropic hyperelastic model that minimized the error between the measured and computed surface displacement field and to investigate the sensitivity of the measured bovine inflation response to variations in the anisotropic properties of the cornea. The results of the parameter optimization revealed that the collagen structure of bovine cornea exhibited a high degree of anisotropy in the limbus region, which agreed with recent histological findings, and a transversely isotropic central region. The parameter study showed that the bovine corneal response to the inflation experiment was sensitive to the shear modulus of the matrix at pressures below the intraocular pressure, the properties of the collagen lamella at higher pressures, and the degree of anisotropy in the limbus region. It was not sensitive to a weak collagen anisotropy in the central region.     

Patient-specific modeling of corneal refractive surgery outcomes and inverse estimation of elastic property changes

The purpose of this study is to develop a 3D patient-specific finite element model (FEM) of the cornea and sclera to compare predicted and in vivo refractive outcomes and to estimate the corneal elastic property changes associated with each procedure. Both eyes of a patient who underwent laser-assisted in situ keratomileusis (LASIK) for myopic astigmatism were modeled. Pre- and postoperative Scheimpflug anterior and posterior corneal elevation maps were imported into a 3D corneo-scleral FEM with an unrestrained limbus. Preoperative corneal hyperelastic properties were chosen to account for meridional anisotropy. Inverse FEM was used to determine the undeformed corneal state that produced <0.1% error in anterior elevation between simulated and in vivo preoperative geometries. Case-specific 3D aspheric ablation profiles were simulated, and corneal topography and spherical aberration were compared at clinical intraocular pressure. The magnitude of elastic weakening of the residual corneal bed required to maximize the agreement with clinical axial power was calculated and compared with the changes in ocular response analyzer (ORA) measurements. The models produced curvature maps and spherical aberrations equivalent to in vivo measurements. For the preoperative property values used in this study, predicted elastic weakening with LASIK was as high as 55% for a radially uniform model of residual corneal weakening and 65% at the point of maximum ablation in a spatially varying model of weakening. Reductions in ORA variables were also observed. A patient-specific FEM of corneal refractive surgery is presented, which allows the estimation of surgically induced changes in corneal elastic properties. Significant elastic weakening after LASIK was required to replicate clinical topographic outcomes in this two-eye pilot study.     

The Effect of Spatial Inhomogeneity of the Cornea on the Deformation Properties of the Eyeball and the Results of Maklakoff Applanation Tonometry

A two-component model of the eyeball that represents the cornea as a momentless, linearly elastic deformable surface and the scleral region, as an elastic element that responds to intraocular pressure changes by volume changes, has been used to analyze the effect of spatial inhomogeneity in the distribution of effective corneal stiffness on the mechanical properties of the eye. The effective stiffness of the cornea characterized both the elastic properties and the thickness of the cornea within the framework of the model. Various axisymmetric forms of the effective stiffness distribution characterized by monotonic increase along the arc between a point on the corneal surface and the apex of the cornea were studied. The considered distributions simulated both natural inhomogeneity and apical region weakening due to surgical interventions. Numerical simulation yielded the dependences of deformation parameters on intraocular pressure changes. These parameters characterized the deformation properties of both the cornea (apex displacement) and the eyeball as a whole (intraocular volume change). In the case of moderate inhomogeneity, the dependences were only slightly different from those for a homogeneous cornea with an effective stiffness equal to the mean value for the corresponding inhomogeneous distribution. A noticeable increase in the integral response of the cornea and the eyeball as a whole to changes in pressure was observed if the effective stiffness amplitude was very high (two or more times higher than the mean value). The effect of inhomogeneity on the results of tonometric measurements with a Maklakoff tonometer (flat stamp) was studied. The tonometric difference, that is, the difference between the tonometric pressure (in the loaded eye) and the true pressure (before loading), mainly depended on the average stiffness of the cornea in this case as well, with a substantial increase observed at very high stiffness amplitudes only. Apical weakening of the cornea led to an increase (although not very pronounced) of the tonometric difference.     

角膜新生血管的治疗新进展

角膜本身无血管,毛细血管网围绕角膜缘,如果血管超越角膜缘进入透明区即为病理性。角膜新生血管不是一种独立的角膜病,而是一种病理改变。由于维持角膜无血管的平衡因素被破坏,角膜缘的毛细血管侵入角膜周边部1-2 mm,即可视为角膜新生血管(CNV)形成1。随着它对感染的消除、创伤愈合、抑制免疫介导的角膜溶解有一定作用,但其结构和功能不完善,容易发生血浆渗漏,造成角膜水肿、脂质沉着以及激发的角膜瘢痕化,严重影响视力;也使角膜移植排斥反应的发生率大大增加。CNV的生长有利于病原微生物的清除和组织的修复,但严重影响了角膜的透明性,导致一系列的并发症,破坏眼球的完整性,目前国内外对治疗角膜新生血管的药物进行了大量的研究,希望通过不同的药物、不同的治疗方法解决这个大难题。     

Analytical model of corneal surgery

We present a model of the human cornea in order to study the changes in its shape resulting from surgical operations (e.g., radial keratotomy). A simple closed-form solution is given for a thin linearly elastic spherical shell model of the cornea. We assume axisymmetry and isotropy in the shell surface. The surgery is modeled by permitting Young's modulus and shell thickness to depend on position. The analytical nature of the solution permits principal shell curvatures to be explicitly calculated. The model is used to investigate the effect of surgery on corneal flattening and the associated sensitivity to intraocular pressure changes.     

扬子鳄视觉器官组织学研究

本文用光镜和电镜研究了扬子鳄(Alligator sinensis)的组织学,同时测量了其眼球的一些光学参数.扬子鳄眼球呈扁圆球形,角膜径与球径的比值为1:1.44;晶状体与角膜的比值为1:1.40。角膜内具鲍氏膜;虹膜内的括约肌、睫状体内的睫状肌均属横纹肌,视细胞椭圆体内线粒体嵴突与线粒体长轴相平行,这与报道的其它鳄类不同。虹膜内未见扩瞳肌纤维,角膜缘缺巩膜小骨片,晶状体环垫薄,因而其视觉调节能力仍然很弱。视网膜中视细胞由视杆细胞、单锥细胞、双锥细胞组成,其中以视杆细胞占多数。视细胞与神经节细胞核比值平均为2.5:1,表明扬子鳄的组织结构与其弱光视觉相适应。     

A microstructurally-based finite element model of the incised human cornea.

A mechanical model of the human cornea is proposed and employed in a finite element formulation for simulating the effects of surgical procedures, such as radial keratotomy, on the cornea. The model assumes that the structural behavior of the cornea is governed by the properties of the stroma. Arguments based on the microstructural organization and properties of the stroma lead to the conclusion that the human cornea exhibits flexural and shear rigidities which are negligible compared to its membrane rigidity. Accordingly, it is proposed that to a first approximation, the structural behavior of the cornea is that of a thick membrane shell. The tensile forces in the cornea are resisted by very fine collagen fibrils embedded in the ground substance of the stromal lamellae. When the collagen fibrils are cut, as in radial keratotomy, it is argued that they become relaxed since there is negligible transfer of load between adjacent fibrils due to the low shear modulus of the ground substance. The forces in the cornea are then resisted only by the remaining uncut fibrils. The cutting of fibrils induces an anisotropy and inhomogeneity in the membrane rigidity. By assuming a uniform angular distribution of stromal lamellae through the corneal thickness, geometric arguments lead to a quantitative representation for the anisotropy and inhomogeneity. All material behavior is assumed to be in the linear elastic regime and with no time-dependency. The resulting constitutive model for the incised cornea has been employed in a geometrically non-linear finite element membrane shell formulation for small strains with moderate rotations. A number of numerical examples are presented to illustrate the effectiveness of the proposed constitutive model and finite element formulation. The dependence of the outcome of radial keratotomy, measured in terms of the immediate postoperative shift in corneal power, on a number of important factors is investigated. These factors include the value of the elastic moduli of the stromal lamellae (dependent on the patient's age), the incision depth, the optic zone size, the number of incisions and their positions, and the intraocular pressure. Results have also been compared with expected surgical corrections predicted by three expert surgeons and show an excellent correspondence.     

Large deformation mechanics of the enucleated eyeball

Large deformation of enucleated pig eyeballs under rigid cylindrical indenters was studied analytically and experimentally. The analytic model for the eyeball consists of a fluid-filled spherical membrane composed of an incompressible, elastic material with an exponential strain energy function. The Rayleigh-Ritz technique provided an approximate solution via a potential energy formulation. Comparison with results from tests on eyeballs and a water-filled rubber (Mooney-Rivlin) shell shows good agreement at large deflection, where membrane action dominates. Due to the highly nonlinear stress-strain relations for the sclera, the load remains relatively small until the indenter displacement approaches 40-60 percent of the eyeball radius, and then the load increases rapidly. Depending on the indenter size, either a perforation or a rupture type of failure occurs.     

Spatial mapping of collagen fibril organisation in primate cornea-an X-ray diffraction investigation

New insights are presented into the collagenous structure of the primate cornea. Wide-angle X-ray diffraction was used to map the fibrillar arrangement and distribution of collagen over three common marmoset corneas. The maps provide a point of reference to help interpret data from pathological corneas or primate models of refractive surgery. The results herein disclose a circum-corneal annulus of highly aligned collagen, 0.5-1.5 mm wide, where the cornea and sclera fuse at the limbus; a feature similar to that observed in human tissue. As in humans, the annulus is not uniform, varying in width, fibril angular spread, and collagen density around its circumference. However, more centrally the marmoset cornea exhibits a preferred lamella orientation in which proportionally more fibrils are oriented along the superior-inferior corneal meridian. This observation is in striking contrast with the situation in human cornea, where there is an orthogonal arrangement of preferentially aligned fibrils. Investigation of a further 16 corneas confirmed that approximately 33% (+/-1%) (n = 76) of fibrils in the central marmoset cornea lie within a 45 degrees sector of the superior-inferior meridian. Implications for the mechanical and optical properties of the cornea are discussed.     

Finite element simulation of arcuates for astigmatism correction

In order to simulate the corneal incisions used to correct astigmatism, a three-dimensional finite element model was generated from a simplified geometry of the anterior half of the ocular globe. A hyperelastic constitutive behavior was assumed for cornea, limbus and sclera, which are collagenous materials with a fiber structure. Due to the preferred orientations of the collagen fibrils, corneal and limbal tissues were considered anisotropic, whereas the sclera was simplified to an isotropic one assuming that fibrils are randomly disposed. The reference configuration, which includes the initial strain distribution that balances the intraocular pressure, is obtained by an iterative process. Then the incisions are simulated. The final positions of the nodes belonging to the incised meridian and to the perpendicular one are fitted by both radii of curvature, which are used to calculate the optical power. The simulated incisions were those specified by Lindstrom's nomogram [Chu, Y., Hardten, D., Lindquist, T., Lindstrom, R., 2005. Astigmatic keratotomy. Duane's Ophthalmology. Lippincott Williams and Wilkins, Philadelphia] to achieve 1.5, 2.25, 3.0, 4.5 and 6.0D of astigmatic change, using the next values for the parameters: length of 45 degrees , 60 degrees and 90 degrees , an optical zone of 6mm, single or paired incisions. The model gives results similar to those in Lindstrom's nomogram [Chu et al., 2005] and can be considered a useful tool to plan and simulate refractive surgery by predicting the outcomes of different sorts of incisions and to optimize the values for the parameters involved: depth, length, position.     

兔角膜缘干细胞的研究进展

目前,角膜移植是临床上治疗角膜疾患的最有效途径,但供体角膜非常有限。新近兴起的干细胞技术,为组织工程角膜的研制和应用提供了契机。对于以角膜缘干细胞缺乏或功能障碍为特征的疾病也有治疗效果。采取角膜缘干细胞移植应是一种合理有效的治疗手段。本文主要介绍了兔角膜缘干细胞的体外分离、培养、鉴定及一些生长因子对其增殖的影响。     

Quantification of collagen organization in the peripheral human cornea at micron-scale resolution

The collagen microstructure of the peripheral cornea is important in stabilizing corneal curvature and refractive status. However, the manner in which the predominantly orthogonal collagen fibrils of the central cornea integrate with the circumferential limbal collagen is unknown. We used microfocus wide-angle x-ray scattering to quantify the relative proportion and orientation of collagen fibrils over the human corneolimbal interface at intervals of 50 μm. Orthogonal fibrils changed direction 1–1.5 mm before the limbus to integrate with the circumferential limbal fibrils. Outside the central 6 mm, additional preferentially aligned collagen was found to reinforce the cornea and limbus. The manner of integration and degree of reinforcement varied significantly depending on the direction along which the limbus was approached. We also employed small-angle x-ray scattering to measure the average collagen fibril diameter from central cornea to limbus at 0.5 mm intervals. Fibril diameter was constant across the central 6 mm. More peripherally, fibril diameter increased, indicative of a merging of corneal and scleral collagen. The point of increase varied with direction, consistent with a scheme in which the oblique corneal periphery is reinforced by chords of scleral collagen. The results have implications for the cornea's biomechanical response to ocular surgeries involving peripheral incision.     

Role of a regulatory protein from the bovine cornea in activation of cell sources of corneal regeneration in vitro

A highly purified regulatory protein isolated from the bovine cornea (RPC) was tested for the effect on the rat and newt corneas in vitro under different culture conditions. In the newt cornea, RPC stimulated limbus epithelial cells in roller cultures and cells in the basal layer of corneal epithelium in both roller and stationary cultures. In roller cultures of the rat cornea, RPC had no effect on weakly differentiated limbus cells but stimulated progenitor cells of the basal cornea layer to differentiation into definitive epithelial cells. In stationary cultures, RPC did not activate the involvement of cells in regeneration of the rat corneal tissue.     

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background

Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker.

Methodology/Principal Findings

K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker.

Conclusions/Significance

K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.     

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.