Nanostructural and mechanical changes in the sclera following proteoglycan depletion

The mechanical properties of ocular tissues, such as the sclera, have a major impact on healthy eye function, and are governed by the properties and composition of the microstructural components. For example, biomechanical degradation associated with myopia occurs alongside a reduction of proteoglycans (PGs). In this study, the role of PG degradation in the nanomechanical properties of the porcine sclera is explored. In-vitro enzymatic degradation of PGs was conducted with α-amylase and chondroitinase ABC enzymes. Collagen fibril morphology and nanomechanical stiff ness were measured with atomic force microscopy (AFM). The elastic modulus of the tissue was reduced in all enzyme-treated samples relative to controls. In addition, collagen fibril organization was disrupted by PG depletion. Our data demonstrate that PGs play an important role in determining not only the mechanical properties at these length scales, but also collagen fibril arrangement.


Introduction
The sclera is the dense outer coating of the eye, which provides the structural framework that defines the shape of the eye.It is mainly composed of collagen, elastin, and interfibrillar PGs.There is substantial evidence that profound bio-Fig.1. PG depletion by using chondroitinase ABC.mechanical changes occur in the sclera with conditions such as myopia, which is characterized by scleral weakening.Alongside biomechanical changes, a reduction of collagen fibril diameter and PG content have been reported in myopic eyes.However, few studies to date have determined how PG content in the sclera affects its mechanical properties and nanostructure.In this study, in-vitro degradation of PGs in the porcine sclera was conducted in order to determine how this affects nanoscale changes in its structure and mechanical properties.

Materials and methods
Porcine eyes were obtained from a local abbatoir (n = 5).The sclerae were dissected and cryosectioned for AFM testing following the methods and experimental procedure described previously. 1 The cryosectioned samples were treated with the following solutions: 2mg/ml α-amylase in phosphate buffered saline (PBS), 2mg/ ml α-amylase in ultra-clean distilled water, chondroitinase ABC buffer, 100% ultra clean distilled water (control group), and 100% PBS (control group).The nanotopography and elastic modulus were measured before and after one-hour treatment at the same locations with AFM using the Peakforce QNM method. 1 All tests were conducted in liquid.
As shown in Figure 1, chondroitinase ABC depletes two types of major sulphated PGs in the sclera: 1 chondroitin sulfate PGss and dermatan sulfate PGs. 2 Sulphated glycosaminoglycan (sGAG, a major component of PGs) content was analyzed with the dimethylmethylene blue (DMMB) assay before and after α-amylase treatment.Although α-amylase is one of the major enzymes found in tear fluid, few studies have shown which PGs in the eye are affected by α-amylase.

Results
DMMB assays indicated that sGAG content was reduced after α-amylase treatment in all three regions (8.9% to 22.6%).Collagen fibril diameter was significantly reduced in all groups incubated with enzyme solutions, and remained unchanged in control groups.Collagen fibril D-periodicity remained unchanged in all groups after incubation.The gap zone depth increased significantly in all groups after incubation with enzymes, decreasing after 100% PBS incubation.As shown in Figure 2, the elastic modulus decreased in all groups after incubation with enzyme solutions and significantly increased after incubation with 100% PBS buffer.

Conclusion
These data demonstrate that collagen fibril mechanical properties are substantially altered by salt concentration and that PGs play an important role in determining the nanostiffness and structure of the sclera.Collagen D-periodicity remained unchanged in all groups after treatment, indicating that PG depletion does not degrade collagen fibrils.Elastic properties were reduced in samples incubated with amylase solutions, decreasing even further in those incubated with chondroitinase solution.This result indicated that PG degradation will cause a reduction in the elastic modulus of collagen fibrils.