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Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera.

ABSTRACT: The concept of scleral stiffening therapies has emerged as a novel theoretical approach for treating the ocular disorders glaucoma and myopia. Deformation of specific regions of the posterior eye is innately involved in the pathophysiology of these diseases, and thus targeted scleral stiffening could resist these changes and slow or prevent progression of these diseases. Here, we present the first systematic screen and direct comparison of the stiffening effect of small molecule collagen cross-linking agents in the posterior globe, namely using glyceraldehyde, genipin and methylglyoxal (also called pyruvaldehyde). To establish a dose-response relationship, we used inflation testing to simulate the effects of increasing intraocular pressure in freshly harvested rat eyes stiffened with multiple concentrations of each agent. We used digital image correlation to compute the mechanical strain in the tissue as a metric of stiffness, using a novel treatment paradigm for screening relative stiffening by incubating half of each eye in cross-linker and using the opposite half as an internal control. We identified the doses necessary to increase stiffness by approximately 100%, namely 30 mM for glyceraldehyde, 1 mM for genipin and 7 mM for methylglyoxal, and we also identified the range of stiffening it was possible to achieve with such agents. Such findings will inform development of in vivo studies of scleral stiffening to treat glaucoma and myopia.

SUBMITTER: Campbell IC

PROVIDER: S-EPMC5414911 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera.

Campbell Ian C IC Hannon Bailey G BG Read A Thomas AT Sherwood Joseph M JM Schwaner Stephen A SA Ethier C Ross CR

Journal of the Royal Society, Interface 20170401 129

The concept of scleral stiffening therapies has emerged as a novel theoretical approach for treating the ocular disorders glaucoma and myopia. Deformation of specific regions of the posterior eye is innately involved in the pathophysiology of these diseases, and thus targeted scleral stiffening could resist these changes and slow or prevent progression of these diseases. Here, we present the first systematic screen and direct comparison of the stiffening effect of small molecule collagen cross-l ...[more]

PMID: 28381643

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Project description:Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.

Project description:The mechanical properties of the microstructural components of sclera are central to eye physiology and pathology. Because these parameters are extremely difficult to measure directly, they are often estimated using inverse-modeling matching deformations of macroscopic samples measured experimentally. Although studies of sclera microstructure show collagen fiber interweaving, current models do not account for this interweaving or the resulting fiber-fiber interactions, which might affect parameter estimates. Our goal was to test the hypothesis that constitutive parameters estimated using inverse modeling differ if models account for fiber interweaving and interactions. We developed models with non-interweaving or interweaving fibers over a wide range of volume fractions (36-91%). For each model, we estimated fiber stiffness using inverse modeling matching biaxial experimental data of human sclera. We found that interweaving increased the estimated fiber stiffness. When the collagen volume fraction was 64% or less, the stiffness of interweaving fibers was about 1.25 times that of non-interweaving fibers. For higher volume fractions, the ratio increased substantially, reaching 1.88 for a collagen volume fraction of 91%. Simulating a model (interweaving/non-interweaving) using the fiber stiffness estimated from the other model produced substantially different behavior, far from that observed experimentally. These results show that estimating microstructural component mechanical properties is highly sensitive to the assumed interwoven/non-interwoven architecture. Moreover, the results suggest that interweaving plays an important role in determining the structural stiffness of sclera, and potentially of other soft tissues in which the collagen fibers interweave. STATEMENT OF SIGNIFICANCE: The collagen fibers of sclera are interwoven, but numerical models do not account for this interweaving or the resulting fiber-fiber interactions. To determine if interweaving matters, we examined the differences in the constitutive model parameters estimated using inverse modeling between models with interweaving and non-interweaving fibers. We found that the estimated stiffness of the interweaving fibers was up to 1.88 times that of non-interweaving fibers, and that the estimate increased with collagen volume fraction. Our results suggest that fiber interweaving is a fundamental characteristic of connective tissues, additional to anisotropy, density and orientation. Better characterization of interweaving, and of its mechanical effects is likely central to understanding microstructure and biomechanics of sclera and other soft tissues.

Dataset Information

Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera.

Publications

Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera.

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