On the Mechanical Roles of Glycosaminoglycans in the Tensile Properties of Porcine Corneal Stroma

Abstract

Purpose: The cornea is primarily composed of collagen fibrils that are embedded in a ground substance rich in proteoglycans and other glycoproteins. It is known that glycosaminoglycan (GAG) side chains of proteoglycans form anti-parallel duplexes between collagen fibrils. The present work was done in order to investigate the mechanical function of GAGs in defining the tensile properties of porcine corneal stroma.

Methods: Porcine corneal stromal strips dissected from the nasal–temporal direction were divided into control, buffer-treated, and enzyme-treated groups. The samples in the control group were used immediately after dissection. However, the buffer-treated and enzyme-treated samples were, respectively, incubated for 18 hours at 37°C in a buffer solution made up of 100-mM sodium acetate at pH 6.0 or in an enzyme solution containing keratanase II. The Blyscan assay was used to quantify the total GAG content and assess GAG depletion in the samples treated with the enzyme and buffer solutions. Uniaxial tensile tests were also performed to determine the effect of GAG removal on mechanical properties of the cornea.

Results: The GAG content in enzyme-treated samples was significantly lower than that of the normal and buffer-treated specimens (P < 0.05). Moreover, GAG-depleted strips showed significantly softer mechanical responses in comparison with the control and buffer samples (P < 0.05).

Conclusions: Removing GAGs from the corneal extracellular matrix led to significant tensile property reduction; supporting the hypothesis that there exists a strong correlation between the GAG content and mechanical properties of the corneal stroma.

The cornea is a transparent tissue that covers the front of the eye; and serves as a protective layer for the delicate internal contents of the eye. In addition, the cornea has a central role in proper vision because of its unique optical properties. It transmits over 95% of incoming light, has a refractive power of about 43 diopters, and is responsible for most of the refractive power in the eye. These unique optical properties are primarily because of the exceptionally organized microstructure of the corneal extracellular matrix (i.e., stroma). 

The mechanical and physical properties of the cornea are primarily governed by the stroma, a mesenchymal connective tissue making up approximately 90% of the total corneal thickness. The corneal stromal collagen fibrils are embedded in parallel-to-the-surface lamellae. The fibrils are spaced apart by a ground substance rich in proteoglycans (PGs) and other glycoproteins.¹ The PGs, composed of a core protein to which glycosaminoglycans (GAGs) are attached, bind at specific sites along the collagen fibrils. GAGs are negatively charged linear complex molecules that are essential for regulating corneal collagen fibrillar assembly and organization.² The mechanical role of GAGs in defining corneal mechanical properties is not yet fully understood. 

The syntheses and sulfation of GAGs occur inside the Golgi apparatus.³ Five classes of GAGs, i.e., chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate, and hyaluronic acid, are found in mammalian tissues: Except for hyaluronic acid, which is a non-sulfated GAG, the others are attached to a core protein and form PGs. GAGs play different functions in the extracellular matrix of soft tissues. For example, they are important in a variety of cell functions such as adhesion, proliferation, motility, and differentiation.^4–8 They also play a role in growth factor signaling, the mechanical properties of a tissue, and regulating the collagen structure.^9,10 PGs and their GAG side chains, because of their regulatory effects on the diameter and specific organization of collagen fibrils, are important for the unique microstructure and transparency of the corneal extracellular matrix.^11–15 Thus, any changes in the GAG content and composition are expected to influence corneal mechanical properties. 

The major sulfated GAGs in the cornea are CS, DS, and KS, which appear as side chains of decorin, biglycan, lumican, keratocan, fibromodulin, and mimecan PGs.^16–20 KS, the predominant corneal GAG, is composed of repeating disaccharides consisting of galactose (Gal) and N-acetylglucosamine.²¹ The densities and ratios of KS, CS, and DS are important in defining the corneal microstructure and subsequently its proper optical and mechanical functions. For example, the increase in collagen interfibrillar spacing and disorganization of collagen regular packing during corneal wound healing and in scar tissue are believed to be due to raised levels of CS and DS and a reduction of KS.^12,22–24 Macular corneal dystrophy is a heritable disease condition in which the cornea becomes cloudy because individuals are unable to produce KS.^25,26 Furthermore, the deficiency of GAG-degradative enzymes in mucopolysaccharidoses, such as Hurler and Scheie syndromes, causes an accumulation of DS resulting in clouding of the cornea and loss of the visual acuity.²⁷ Finally, the loss of tensile strength and a reduction in GAG density have been reported in keratoconus.^28–31 Thus, it could be hypothesized that GAG content reduction could lead to corneal mechanical property weakening. The primary objective of the present study was to test this hypothesis by removing GAGs enzymatically and quantifying their effects on corneal tensile properties. 

Methods

Tissue Preparation and GAG Removal Protocol

The eyes of 6- to 8-month-old pigs were obtained from a slaughterhouse shortly after the animals were slaughtered. All samples were used immediately after they were brought to the laboratory. The epithelium was scraped off with the blunt side of a blade, and corneal scleral rings were prepared from the eyes. Then, a custom-made cutting device was used to dissect rectangular corneal strips of 5-mm width from the center of the samples in the nasal–temporal direction. The strips were dissected from the same direction so that corneal anisotropic properties would not affect the measurements.^32,33 The samples were used for both mechanical tests and GAG content studies. A digital pachymeter (DGH Technology, Inc., Exton, PA, USA) and a digital caliper (Mitutoyo Corporation, Kawasaki, Japan) were used to measure the thickness and width of the specimens, respectively. The specimens were divided into (1) a control group, (2) a buffer-treated group, and (3) an enzyme-treated group. The buffer solution was made up of 100-mM sodium acetate at pH 6.0, and the enzyme solution contained Keratanase II truncated sequence from Bacillus circulans (GlycoFineChem, Wellington, New Zealand) at 0.1 U/mL. The samples were incubated in the buffer solution (buffer group) or in the enzyme solution (enzyme group) for 18 hours at 37°C. After incubation, the samples were rinsed in PBS solution. The buffer and enzyme group samples were completely dried for 24 hours in a desiccator before incubation. The samples in the control group were used immediately after dissection. 

GAG Quantification

The GAG quantification was performed using the Blyscan assay (Biocolor Ltd., Carrickfergus, Northern Ireland). For this purpose, the specimens were first dried for 24 hours in a desiccator to obtain their dry weight. Then, they were incubated in buffer solution or enzyme solution for 18 hours. The samples in the control group were immersed in PBS until they reached approximately their after-dissection thickness. Furthermore, the samples in the buffer and enzyme groups were washed in PBS after treatment. The Blyscan assay is a quantitative dye-binding method for the analysis of GAGs in which test materials are assayed following papain extraction from the biological tissue. Thus, the corneal specimens in the control group (n = 8), buffer group (n = 8), and enzyme group (n = 8) were immersed in papain solution (P3125; Sigma-Aldrich, St. Louis, MO, USA) for 18 hours at 60°C. The protocol described in the literature was then followed to obtain the GAG content as µg/mg dry tissue.^34,35 

Mechanical Tests

An RSA-G2 machine (TA Instruments, New Castle, DE, USA) was used to measure the mechanical tensile response of the control (n = 10), buffer-treated (n = 10), and enzyme-treated (n = 10) groups. The samples in the buffer and enzyme groups were initially dried for 24 hours, immersed in the buffer or enzyme solution for 18 hours, and finally washed in PBS. The strips were allowed to air dry until their thickness reached 700 µm (i.e., the in vivo thickness of porcine cornea)^36,37 before they were mounted to the testing device. This was done in order to minimize the hydration effects on the mechanical measurements.^32,38 In other words, all mechanical experiments were performed when the thickness of samples was 700 µm and their average hydration was about 3.85 mg water/mg dry weight.³⁹ The strips were secured between grips of the RSA-G2 machine at a loading gap of about 7 mm. Sandpaper was used at the grips to increase friction and prevent any slippage. A tare load of about 20 to 30 mN was then applied in order to remove slack and determine the initial length (L₀) of the samples.³⁷ The samples were stretched to a strain of ε = 20% with a displacement rate of 2 mm/min. The engineering strain (ε) was defined as the change in the length between the grips divided by L₀. The engineering tensile stress (σ) was calculated by dividing the measured force by the initial cross-sectional area of the strips. The experiments took about a minute to complete; thus, they were done in air.³⁷ The thickness of a number of strips was measured after the experiments to confirm that no significant dehydration occurred. 

The tensile stress–strain curves were plotted and the maximum tensile stress and tangent modulus were computed to compare the mechanical response of different groups. The tangent modulus was taken as the slope of the tangent line to the stress–strain curves. The experimental data were reported as mean ± standard deviation. Furthermore, any statistically significant difference between groups was determined by performing an ANOVA with P = 0.05. Finally, the following equation was used to numerically represent the experimental measurements:³² 

\begin{equation}\sigma = \alpha {\varepsilon ^\beta } + {\sigma _0}\end{equation}

(1)

where σ₀ is the initial tensile stress, and α and β are the unknown parameters. The Levenberg–Marquardt algorithm was used to calculate the unknown constants by fitting the above numerical relation to the experimental stress–strain data. 

Results

The GAG contents of the control, buffer-treated, and enzyme-treated samples are given in Table 1. No significant difference was found between the GAG content of the buffer-treated and control samples (P = 0.9); however, the GAG content significantly decreased, about 80%, after treatment with the keratanase II enzyme. Figure 1 shows the tensile stress–strain response of GAG-depleted samples in comparison with the behavior of the buffer and control group strips. The solid lines in this figure depict the numerical curve fits. The means and standard deviations of the coefficients in Equation 1 are given in Table 2, and Figure 2 presents the maximum tensile stress and tangent modulus of the corneal strips. No significant difference was observed in the tensile properties of the control and buffer-treated samples; however, the maximum tensile stress and tangent modulus of corneal strips decreased significantly after incubation in the enzyme solution (P < 0.05). 

Table 1.

 

GAG Content of Control, Buffer-Treated, and Enzyme-Treated Corneal Samples Obtained From Blyscan Analysis

 

Figure 1.

 

A comparison of the stress–strain behavior of porcine corneal strips in the control, buffer-treated, and enzyme-treated groups. It can be seen that the buffer treatment had an insignificant effect on tensile response. Furthermore, the GAG-depleted strips had significantly softer responses compared to those of the buffer-treated and control samples. The solid lines represent the numerical fits of the average data, and error bars indicate ±1 SD.

Table 2.

 

Means and SDs of Coefficients in Equation 1

 

Figure 2.

 

Maximum tensile stress and tangent modulus of the control, buffer-treated, and enzyme-treated strips. The tensile properties of the strips after buffer treatment were similar to those of the control specimens. However, a significant amount of softening was found in the enzyme-treated specimens from which GAGs have been removed enzymatically (P < 0.05).

Discussion

The objective of the present work was to determine possible effects of GAGs on mechanical properties of the corneal stroma. For this purpose, we used keratanase II to remove GAGs from porcine corneal strips and performed uniaxial tensile experiments to quantify the mechanical response of GAG-depleted and normal corneal strips. GAGs are responsible for the maintenance of regular collagen interfibrillar spacing in corneal extracellular matrix and thus transparency of the cornea.^40–42 The three-dimensional electron microscopic reconstructions of the cornea showed that GAG chains from separate PGs joined together, forming anti-parallel duplexed associations between collagen fibrils.⁴² The absence of KS in a strain of mice with a targeted gene depletion caused significant alterations in the corneal collagen fibrillar architecture.⁴³ Considering these previous observations, we hypothesized that GAGs could affect corneal mechanical properties. 

In the present study, keratanase II was used to remove KS from porcine corneal strips. The Blyscan analysis estimated an average GAG content of 50 µg/mg dry tissue for the porcine cornea, which is in agreement with a recent study using porcine lenticules.⁴⁴ A similar amount of sulfated GAGs was also reported in bovine cornea.⁴⁵ The amount of GAGs in human cornea was reported to be 34 to 65 µg/mg dry tissue.^46,47 It is interesting to note here that the total sulfated polyanion content of the corneal stroma was proposed to be approximately constant in different species.⁴⁸ 

Previous studies have shown that KS comprises the majority of GAGs in human, bovine, and porcine corneal stroma. For example, KS is estimated to constitute about 65% of human corneal GAGs.^48–50 KS GAG chains are shorter, connecting neighboring collagen fibrils; CS and DS GAG chains are longer, interacting with several collagen fibrils.⁴² The present study showed that keratanase II enzyme removed about 80% of GAGs from samples. Although the overall density of sulfated GAGs (i.e., KS and CS/DS) is constant across the cornea, the ratio of KS GAGs and CS and DS GAGs is variable.⁵¹ For example, in both human and bovine corneas, KS is mainly present in the central region, and its density decreases toward the peripheral regions, where CS is predominantly found.⁵¹ Furthermore, DS is present throughout the bovine corneal stroma, but it is mainly found in the corneal limbus in humans.⁴⁵ The dissimilarities in the location of specific GAGs are due to species differences and characterization methods used for their detection. 

Although factors such as age and breed of animals influence corneal tensile properties reported in different studies, our tensile measurements were in agreement with previous reports.^52,53 Wollensak et al.⁵² found an average Young's modulus of 2.2 MPa at 8% strain for porcine corneas using a similar testing protocol. Furthermore, Hatami-Marbini and Jayaram⁵³ found a tensile modulus of 2.57 ± 0.68 MPa at 8% strain when the average thickness of samples was 760 µm. In the present study, the tensile modulus of the control samples at 8% strain was 2.50 ± 1.01 MPa, which agrees very well with these previous reports. 

Figure 1 shows that GAG-depleted strips had significantly softer tensile properties compared to the control and buffer-treated specimens. The maximum tensile stress (0.55 ± 0.08 MPa) and tangent modulus (0.55 ± 0.08 MPa) of enzyme-treated strips were significantly lower than those of the control and buffer-treated strips. There are different reports in the literature about the effects of GAGs on the mechanical properties of soft tissues, suggesting that the mechanical role of GAGs is tissue dependent. In general, the mechanical response of tissues with low GAG contents, such as tendons and ligaments, was found to be independent of GAGs.^54,55 However, GAGs were found to have a significant influence on the mechanical response of tissues with high GAG content, such as cartilage, aorta, and atrioventricular heart valve leaflets.^56–58 Despite this general trend, contrary results also exist. For example, Lujan et al.⁵⁴ reported no change in the viscoelasticity of ligaments after GAG removal, but Hoffman et al.⁵⁹ found a stiffness increase. Furthermore, the tensile response of the sclera remained unchanged or changed after GAG depletion depending on the testing protocol that was used.^60,61 The present study showed that GAG digestion decreased the tensile stiffness and strength of porcine corneal stroma. This finding is consistent with the reduction in tensile properties of the cornea because of keratoconus, which incorporates a significant reduction in GAG concentration.^28–31 

PGs inside the corneal extracellular matrix are attached to collagen fibrils by their core proteins, and their GAG side chains form interfibrillar duplexes acting as spacers between adjacent fibrils.^41,42,62 This means that the anti-parallel multiplexes formed by GAG chains behave as bridges and yardsticks between collagen fibrils holding them in place. Thus, GAGs are expected to modulate force transmission between collagen fibrils. The corneal stroma can be modeled as a composite material composed of reinforcing collagen fibrils in a PG matrix domain.⁶³ In this model, GAG duplexes contribute to the stress transfer between the soft, compliant matrix and stiff collagen fibrils (Fig. 3). GAG–GAG bridges between collagen fibrils are prevalent in the corneal extracellular matrix because of the large concentration of PGs. However, GAG–GAG interactions are not covalent and are subject to constant formation and dissociation. When the density of GAGs is reduced because of enzymatic digestion (or a diseased condition), the probability of formation of GAG bridges is reduced (Fig. 3), which is why a significant reduction was found in the tensile properties of the enzyme-treated samples in comparison to the control and buffer-treated strips (Figs. 1, 2). In other words, GAG removal reduced the strength of the matrix domain, which led to a reduction in the overall mechanical response of the corneal stroma. 

Figure 3.

 

A simplistic schematic plot for explaining the possible structural role of GAGs in corneal tensile properties. The corneal stroma can be considered as a composite material composed of reinforcing collagen fibrils that are placed in a PG matrix domain. In this model, GAG duplexes contribute to the stress transfer between the soft, compliant matrix and stiff collagen fibrils. The number of GAG bridges was significantly lower in samples of the enzyme-treated group, which is why they showed significantly softer tensile properties.

This work had a number of limitations. For example, the uniaxial tensile method, which does not represent natural loading conditions of the cornea, was used to characterize the mechanical response of the samples. Strip extensometry does not provide any information regarding corneal out-of-plane properties, which have been shown to be significantly different than the tensile properties.⁶⁴ The use of porcine samples could be another drawback of the present work. Future studies are needed to determine whether GAGs have the same effects on the mechanical properties of human cornea, in which the amount of GAGs and the collagen fibril orientation differ. A simple mathematical formulation was used here to numerically represent the experimental measurements. Three-dimensional network models are expected to provide a better numerical explanation for the structural roles of GAGs.^65–67 A complete digestion of GAGs was not done in this work, so the remaining GAGs might have contributed to the reported tensile properties. Finally, the mechanical measurements and KS quantification studies were not performed on the same specimens. Despite these limitations, the present work successfully showed that depleting GAGs from the corneal strips led to significant softening of their tensile behavior. This is an important finding; although it is well established that GAGs form side-by-side bridges between collagen fibrils in the corneal stroma, to the best of the author's knowledge there has not been any previous experimental study characterizing their role in defining the mechanical properties of the cornea. The experimental findings of this research may assist researchers to explain the changes in mechanical properties of the keratoconic cornea in terms of the significant GAG density reduction observed in this disease. 

Acknowledgments

The author thanks members of the Computational Biomechanics Research Laboratory at the University of Illinois at Chicago for assisting in conducting this research. 

Supported in part by a grant from the National Institutes of Health (NIH-R21EY030264). 

Disclosure: H. Hatami-Marbini, None