Swelling studies on the cornea and sclera: the effects of pH and ionic strength.

The biophysical properties of the cornea and sclera depend on the precise maintenance of tissue hydration. We have studied the swelling of the tissues as a function of pH and ionic strength of the bathing medium, using an equilibration technique that prevents the loss of proteoglycans during swelling. Synchrotron x-ray diffraction was used to measure the average intermolecular and interfibrillar spacings, the fibril diameters, and the collagen D-periodicity. We found that both tissues swelled least near pH 4, that higher hydrations were achieved at lower ionic strengths, and that sclera swelled about one-third as much as cornea under most conditions. In the corneal stroma, the interfibrillar spacing increased most with hydration at pH values near 7. Fibril diameters and D-periodicity were independent of tissue hydration and pH at hydrations above 1. Intermolecular spacings in both tissues decreased as the ionic strength was increased, and there was a significant difference between cornea and sclera. Finally, we observed that corneas swollen near pH 7 transmitted significantly more light than those swollen at lower pH levels. The results indicate that the isoelectric points of both tissues are close to pH 4. The effects of ionic strength can be explained in terms of chloride binding within the tissues. The higher light transmission achieved in corneas swollen at neutral pH may be related to the fact that the interfibrillar fluid is more evenly distributed under these conditions.     

Interpretation of the meridional X-ray diffraction pattern from collagen fibrils in corneal stroma

The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae.Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found.A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.     

Relations for lipid bilayers. Connection of electron density profiles to other structural quantities.

Three relations are derived that connect low angle diffraction/scattering results obtained from lipid bilayers to other structural quantities of interest. The first relates the area along the surface of the bilayer, the measured specific volume, and the zeroth order structure factor, F(0). The second relates the size of the trough in the center of the electron density profile, the volume of the terminal methyl groups, and the volume of the methylene groups in the fatty acid chains. The third relates the size of the headgroup electron density peak, the volume of the headgroup, and the volumes of water and hydrocarbon in the headgroup region. These relations, which are easily modified for neutron diffraction, are useful for obtaining structural quantities from electron density profiles obtained by fitting model profiles to measured low angle x-ray intensities.     

Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.     

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.     

The core proteins of large and small interstitial proteoglycans from various connective tissues form distinct subgroups.

Large and small proteoglycans were separately isolated from a number of connective tissues and compared to determine the extent of structural similarity. This was studied by enzyme-linked immunosorbent assays and by the peptide patterns obtained when 125I-labelled proteoglycans were digested with trypsin. All the large proteoglycans, i.e. from tendon, sclera, cartilage and aorta, appear to contain the structure typical for the hyaluronic acid-binding region, both shown by enzyme-linked immunosorbent assay and by content of peptides unique for this region. These proteoglycans also share other structural features of the protein core, as indicated by immunological cross-reactivity and similar peptide patterns. The large proteoglycans from aorta in addition show the presence of unique structures both upon immunoassay and with regard to peptide pattern. Among the small proteoglycans two groups can be identified, although amino acid composition and protein core sizes are grossly similar. One group consists of the small proteoglycans from aorta and cartilage having similar peptide maps and showing immunological cross-reactivity in enzyme-linked immunosorbent assay. The other distinctly different group consists of the small proteoglycans from bone, cornea, sclera and tendon, which among them show identity in enzyme-linked immunosorbent assay and similar peptide patterns. Proteoglycans from the two groups, however, show partial immunological cross-reactivity.     

Shared and distinct structural features of interstitial proteoglycans from different bovine tissues revealed by electron microscopy

Large and small interstitial proteoglycans were purified from different bovine tissues, i.e. cartilage, sclera, tendon, aorta, cornea, and bone. The structure of the molecules was compared using the glycerol spraying/rotary shadowing technique for electron microscopy. Large proteoglycans from sclera and tendon have a core protein with a domain structure similar to that previously reported for cartilage proteoglycans (Paulsson, M., M?rgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heineg?rd, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). It is comprised of a pair of globules at one end of the molecule, connected by a short extended segment, followed by a long extended domain which is terminated by a third globular domain. Large aorta proteoglycans show a somewhat different structure, with only one globular domain at each end of a long extended segment. Large sclera and aorta proteoglycans form aggregates with hyaluronate and cartilage link protein in a manner similar to that of large cartilage proteoglycans. The large proteoglycans show considerable tissue variability with regard to number, length, and spacing of glycosaminoglycan side chains. The small proteoglycans reveal a small globular core protein to which one or two glycosaminoglycans are attached. Although the main structural features do not differ, proteoglycans of the S1 class have an average glycosylation close to two glycosaminoglycans/molecule, while that of the S2 class is close to one. Differences in glycosaminoglycan length were observed between tissues and between the S1 and S2 class of proteoglycan derived from a single tissue.     

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.     

Ageing of the human corneal stroma: structural and biochemical changes.

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm2 to 3.46 nm2, and an increase in scleral collagen from approx. 2.65 nm2 to 3.19 nm2. These changes may be due to an increase in the extent of non-enzymic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

In Situ D-periodic Molecular Structure of Type II Collagen

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structure of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.     

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.     

Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma.

Ultrastructural data from x-ray diffraction studies of the cornea were used to estimate the refractive indices of the collagen fibrils and extrafibrillar material of human, ox, trout, and rabbit corneas. X-ray diffraction measurements of the size and spacing of the collagen fibrils and the separation between the constituent molecules of the fibrils were taken from a previous species study. The tissue volume fractions occupied by the stromal components were estimated and their refractive indices were calculated using the Gladstone-Dale law of mixtures. For the fibrils and extrafibrillar material, the refractive indices in the human cornea were 1.411 and 1.365; for the ox 1.413 and 1.357; for the rabbit 1.416 and 1.357; and for the trout 1.418 and 1.364, respectively. An alternative estimate based on the physical properties and chemical composition of bovine cornea, accounting for interfibrillar type VI collagen and cellular water, produced similar estimates of 1.416 and 1.356 for the fibrils and extrafibrillar material, respectively.     

A lattice model for computing the transmissivity of the cornea and sclera.

The method of photonic band structure is used to calculate the frequencies of light that propagate in lattice models of the cornea and sclera of the mammalian eye, providing an explanation for transparency in the cornea that first properly accounts for multiple scattering of light. Each eye tissue is modeled as an ordered array of collagen rods, and photonic band structure methods are used to solve Maxwell's equations exactly for these models, a procedure that automatically effectively includes all orders of multiple scattering. These calculations show that the dispersion relation for the cornea is linear in the visible range, implying that the cornea is transparent. We show that the transmissivity is approximately 97% by using an effective medium approximation derived from the photonic band structure results and applicable in the visible region. In contrast, the dispersion relation for the model in the sclera is not linear in the visible region, and there are band gaps in this region that could play an important role in the transmission of light in the sclera.     

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.     

Modeling and experimental investigation of electromechanical properties of scleral tissue; a CEM model using an anisotropic hyperelastic constitutive relation

Biomechanics and Modeling in Mechanobiology - The sclera is a soft tissue primarily consisting of collagen fibers, elastin, and proteoglycans. The proteoglycans are composed of a core protein and...     

Experimental confirmation of calculated phases and electron density profile for wet native collagen.

An experimental procedure is developed to phase the reflections obtained in x-ray diffraction investigations of collagen in native wet tendons. Phosphotungstic acid was used for isomorphous addition in phase determination and was located by electron microscopy. Structure factors (with phases) were obtained from the electron microscopy data for the heavy metal. Structure-factor magnitudes for collagen with and without the heavy metal were obtained from the x-ray diffraction data. The first 10 orders were investigated. Standard Argand diagrams provided two solutions for each of these, except the weak sixth order. In each case, one of the two possible solutions agrees well with the phases proposed on theoretical grounds by Hulmes et al. The present results suggest that their other proposed phases are probably correct. An electron density profile along the unit cell of the fibril is presented that shows a distinct step, as expected on the basis of the hole-overlap model. The overlap region is 48% of the length of the unit cell.     

X-ray diffraction evidence for the existence of 102.0- and 230.0-nm transverse periodicities in striated muscle

Synchrotron radiation techniques have enabled us to record meridional x-ray diffraction patterns from frog sartorius muscle at resolutions ranging from approximately 2,800 to 38 nm (i.e., overlapping with the optical microscope and the region normally accessible with low angle diffraction cameras). These diffraction patterns represent the transform of the low resolution structure of muscle projected on the sarcomere axis and sampled by its repeat. Altering the sarcomere length results in the sampling of different parts of this transform, which induces changes in the positions and the integrated intensities of the diffraction maxima. This effect has been used to determine the transform of the mass projection on the muscle axis in a quasicontinuous fashion. The results reveal the existence of maxima arising from long-range periodicities in the structure. Determination of the zeroes in the transforms has been used to obtain phase information from which electron density maps have been calculated. The x-ray diffraction diagrams and the resulting electron density maps show the existence of a series of mass bands, disposed transversely to the sarcomere axis and distributed at regular intervals. A set of these transverse structures is associated with thin filaments, and their 102.0-nm repeat suggests a close structural relationship with their known molecular components. A second set, spaced by approximately 230.0 nm, is also present; from diffraction theory one has to conclude that this repeat simultaneously exists in thick and thin filament regions.     

Ageing of the human corneal stroma: structural and biochemical changes

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm² to 3.46 nm², and an increase in scleral collagen from approx. 2.65 nm² to 3.19 nm². These changes may be due to an increase in the extent of non-enzymatic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. II. Binding sites of proteoglycans on the cell surface

Exogenous proteoglycans stained for electron microscopy with colloidal gold and/or cuprolinic blue bind to the surface of cultured arterial smooth muscle cells at two different sites. (I) About 20% of the proteoglycans adsorbed to the cells from the culture medium interact as monomeric and multimeric proteoglycans with smooth or coated membrane areas. (II) The bulk of exogenous proteoglycans exhibits high affinity binding to cell membrane-associated 10 nm fibrils containing or being closely associated with fibronectin and to collagen. It is suggested that the self association of proteoglycans and their binding to the cell membrane and to cell surface-associated fibronectin and collagen are important for maintaining an appropriate micro-environment for the cultured cells.     

A collagen-binding 59-kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG-S1 and PG-S2 (decorin).

We have determined the primary structure of a 59 kd collagen binding protein which is present in many types of connective tissues, e.g. cartilage, tendon, skin, sclera and cornea. The amino acid sequence, deducted from a 2662 bp cDNA clone, predicts a 42 kd protein with a high content of leucine residues. Most of the protein consists of homologous 23 amino acid residues repeats with predominantly leucine residues in conserved positions. Similar leucine rich repeats have been identified in a number of proteins including the small interstitial proteoglycans decorin and PG-S1. The 59 kd protein and the two proteoglycans are homologous in their entire sequences suggesting that they have evolved from a common ancestral gene. The 59 kd protein and decorin are also functionally related in that both bind to collagen type I and II, and affect their fibrillogenesis. The substitution with glycosaminoglycan chains appears to be a feature shared by all three members of this family of leucine rich motif extracellular proteins, since the 59 kd protein isolated from cartilage is substituted with at least one keratan sulfate chain.