In this study we visualized and quantified the axial heterogeneity in corneal collagen fiber organization and its potential effects on corneal biomechanics using a combination of NLO-HRMac imaging and mechanical testing. Collagen fibers were visualized in 3-D, and fiber interconnectivity was measured as a function of stromal depth. Our results show that collagen fiber patterns are much more complex than conventionally depicted and that there is a significant axial heterogeneity in the interconnectivity of these fibers. We were also able to identify new collagen fiber types—bow spring and anchorlike fibers—which, to the best of our knowledge have not been reported. Finally, we show that the anterior corneal stroma has a significantly increased effective elastic modulus compared with that of the middle and posterior stroma. Although specific measurements of fiber branching and elasticity were not made on the same cornea, these findings strongly suggest that the collagen fiber interaction may influence the biomechanical properties of the cornea.
Different forms of corneal axial heterogeneity have been reported by multiple investigators using a variety of imaging modalities. Abahussin et al.23
describe a decrease in preferred collagen orientation in the anterior cornea using x-ray diffraction imaging. Swelling studies conducted by Müller et al.24
showed that corneal swelling is limited to the posterior and middle stroma, suggesting a structural and biomechanical heterogeneity. Transmission electron microscopy images show a larger degree of interconnectivity between fibrils in the anterior stroma,25
and that the collagen fibrils that make up the larger fibers occasionally bifurcate.5
Finally, on a cellular level, confocal microscopic studies show an axial heterogeneity in keratocyte density, which has been found to decrease with increasing stromal depth in both rabbit25
and human corneas.26
Overall, our findings are consistent with these earlier reports of heterogeneity14
and indicate that collagen organization is highly complex, showing a logarithmic decrease in collagen fiber interconnectivity with increasing stromal depth. This axial heterogeneity is the result of a marked difference in collagen fiber patterns, which change gradually with stromal depth. As a result, fewer fibers are aligned along the preferred meridians, which would cause a drop in preferred orientation on x-ray diffraction images.23
At the posterior end, near Descemet's membrane, fibers run in parallel almost entirely uninterrupted for several millimeters. These patterns are consistent with the classic model described in the literature of orthogonally arranged sheets of fibers.27,28
About halfway through the stroma, collagen fibers branch more frequently. However, most fibers follow the curvature of the cornea and run for long distances. Overall, this area appears to be a transition zone between the posterior third and the highly complex anterior third. Anteriorly, the amount of interconnectivity markedly increases. Most fibers appear to branch and fuse with several other fibers. Contrary to the classic model of interwoven bands of collagen, we observed true intertwining. Fibers split and joined other fibers, suggesting that individual fibrils contribute to multiple fibers, which may be running in parallel to the original fiber or may move off at an angle. The branching and fusing patterns on the fibrillar (nano-) scale that are visible on electron microscopy images are mirrored on a microscopic level, where they result in the intertwining of fibers seen in this study. This intertwining links fibers much more tightly than mere interweaving.
The insertions into the ALL observed in this study have been reported as early as 1849. Drawings of light microscopic sections by Bowman showed the presence of collagen fibers extending from the ALL into the stroma, where they appeared to be fusing with fibers following the corneal curvature.29
More recently, transmission electron microscopy studies have shown insertion of collagen fibers into the ALL at electron-dense plaques, with short extensions into the underlying stroma.25
In this study, we observed fibers extending upward from the densely intertwined meshwork just beneath the ALL, arcing upward and fusing with the ALL. In some cases, these fibers descended again and fused with other collagen fibers, forming a near-parabolic arc. These bow spring fibers are highly reminiscent of the load-bearing elements of girders and bridges. This population of fibers seems to exist in addition to the sutural fibers described by Morishige et al.,10–12
which insert into the ALL and terminate there, rather than arcing and fusing with the meshwork underneath the ALL. It is conceivable that bow spring fibers represent a variation of sutural fibers and that they serve a similar function.
In addition, we observed fibers that inserted from the limbus about midway between Bowman's and Descemet's layer, and rather than following the curvature of the cornea, ran in a nearly straight line for several millimeters before branching and fusing with the meshwork just underneath the bow spring fibers. These anchorlike fibers directly connected the most highly intertwined areas of the cornea with the limbus and bear a resemblance to the anchoring fibers described by Aghamohammadzadeh et al.,6
which also insert from the limbus and stretch out toward the central cornea. Their connection to the meshwork underlying the bow spring fibers suggests that they serve an anchoring function.
Our data further show that the degree of collagen fiber intertwining was linked to local stromal elastic modulus. The highly intertwined anterior third of the cornea was significantly stiffer than the much less intertwined posterior two thirds. This observation matches data published by Kohlhaas et al.30
obtained through extensometry measurements of corneal strips, which also show a stiffer anterior stroma. Interestingly, despite using a vastly different approach and measuring tensile rather than compressive modulus, they also reported a ratio of approximately 2:1 between anterior and posterior flap rigidity.
Although the decrease in elastic modulus between the anterior and middle flaps matched the decrease in BPD, the posterior flap remained almost as stiff as the middle flap. In isolated cases, the effective elastic modulus even surpassed that of the anterior flap. This behavior is most likely due to the effects of postmortem swelling. As the endothelial pump shuts down after death, the cornea becomes excessively hydrated and swells. This swelling occurs primarily in the posterior portion of the stroma,31
and as a result the posterior flap contains considerably more water than the anterior and middle flaps. Swollen flaps are markedly more rigid than thinned flaps (), which explains the increase in elastic modulus for the posterior flaps. The current approach to normalizing hydration is somewhat imprecise and, due to the effect of hydration on corneal rigidity, is likely to introduce artificial variations in measured elastic modulus between individual corneas and perhaps even between different regions of the same flap. Future studies should seek to improve this method. However, it should be noted that since swelling is mostly limited to the posterior portion, the overall hydration state after thinning should have a limited effect on the anterior cornea.
Swelling studies conducted by Müller et al.24
show increased resistance to swelling, even in extreme hydration states for the anterior stroma. This behavior can be explained by the much higher degree of anterior intertwining that is visible on HRMac images. Intertwining of fibers stabilizes the cornea by mechanically linking neighboring layers, making it more difficult for interfibrillar spacing to increase. Since the tensile strength of a collagen fiber is greatest along its long axis,32
fibers that branch off and fuse with others fibers form strong links between layers, vastly increasing axial stiffness and the force required to separate adjacent layers. Conversely, in the posterior stroma the amount of intertwining is minimal, thereby allowing fibers to swell in reaction to the movement of water into the interfibrillar space, their expansion being only minimally inhibited by the presence of neighboring fibers.
With these findings, we can enhance the current model of corneal collagen organization. Using the comprehensive model described by Meek and Boote1
as a basis, we propose the following additions: (1) Bands of parallel collagen fiber bundles traversing the entire width of the cornea are limited to the posterior stroma. With decreasing distance from the ALL, the degree of fiber branching increases exponentially, resulting in a densely intertwined meshwork of fibers in the anterior stroma that exhibits less alignment along preferred directions. This structural heterogeneity matches the axial heterogeneity in elastic modulus; highly intertwined areas are more rigid than the mostly parallel fibers that make up the posterior portion of the stroma. (2) Anchorlike fibers inserting from the limbus, rather than following the curvature of the cornea at a fixed depth, traverse several layers and terminate in a network of smaller branches in the central cornea near the ALL. These fibers create mechanical links between the limbus and the central cornea and may help distribute loads. In addition, they connect spatially separate fibers across multiple layers, which prevents slipping of layers and may serve to counteract swelling forces in the middle stroma. (3) Further stabilization of corneal shape is achieved by means of interactions between the anterior stroma and the ALL in the form of sutural or bow spring fibers, which themselves are connected to the lower stromal regions and to the limbus through anchoring fibers. The lack of bow spring fibers in keratoconus corneas, which do not maintain normal curvature, strongly suggests that these fibers play an important role in maintaining corneal shape.11
These novel structural details regarding corneal collagen organization suggest that the intertwining of collagen fibers plays a role in local variations of corneal curvature and that overall corneal shape, which is a function of corneal rigidity, is ultimately controlled by collagen fiber interconnectivity. Densely intertwined regions of the cornea are more rigid and resist the outward force resulting from IOP more so than less densely intertwined areas. As such, it would be expected that stiffer regions with higher degrees of intertwining would react differently to IOP and subsequently have different local radius of curvature than the less intertwined regions. These variations may explain deviations in corneal topography and why some areas are steeper than others. Changes in corneal curvature after local stiffening have been observed in keratoconus corneas after corneal cross-linking treatment.33
observed local flattening in areas of keratoconus corneas with increased rigidity after undergoing UV cross-linking treatment compared to the untreated, structurally compromised parts of the cornea. These findings are consistent with the hypothesis that regional stiffening of the anterior cornea, through collagen intertwining, controls corneal shape, although the exact relationship between anterior corneal stiffening and corneal curvature has yet to be elucidated.
Axial heterogeneity in fiber intertwining and mechanical rigidity also has important implications for understanding the effects of refractive surgery. The two most popular methods, LASIK and PRK, reshape the cornea by removing or progressively ablating the anterior stroma. If anterior corneal stiffness influences corneal shape, then procedures extending deeper into the cornea may affect curvature to a greater extent than is accounted for by simple linear models. In addition, individual variations in anterior branching and stiffness may influence the achieved refractive results of a given procedure on an individual patient basis. Taken together, our findings on the axial heterogeneity in collagen fiber branching and mechanical stiffness may therefore help to explain, in part, some of the individual differences in achieved versus predicted outcomes after these procedures. Differences in fiber branching between eyes may also underlie the susceptibility to post-LASIK ectasia, since the minimum safe thickness of unaffected tissue may be controlled by collagen fiber interactivity.
Overall, our results show that there is a link between the arrangement of corneal collagen fibers and the compliance of the cornea. Our findings further suggest that heterogeneity of collagen fiber intertwining may regulate corneal rigidity and, by analogy, corneal shape. However, future studies are needed to identify the influence of collagen fiber branching and mechanical stiffness on corneal shape, how shape is affected by refractive surgery, and how best to assess the effect clinically, perhaps by using axial biomechanical testing.