Abstract
To investigate GAG-ECM (glycosaminoglycan-extracellular matrix) interactions in different cartilage types. To achieve this, we first aimed to determine protocols for consistent calculation of GAG content between cartilage types. Auricular cartilage containing both collagen and elastin was used to determine the effect of lyophilization on GAG depletion activity. Bovine articular, auricular, meniscal, and nasal cartilage plugs were treated using different reagents to selectively remove GAGs. Sulfated glycosaminoglycan (sGAG) remaining in the sample after treatment were measured, and sGAG loss was compared between cartilage types. The results indicate that dry weight of cartilage should be measured prior to cartilage treatment in order to provide a more accurate reference for normalization. Articular, meniscal, and nasal cartilage lost significant amounts of sGAG for all reagents used. However, only hyaluronidase was able to remove significant amount of sGAG from auricular cartilage. Furthermore, hyaluronidase was able to remove over 99% of sGAG from all cartilage types except auricular cartilage where it only removed around 76% of sGAG. The results indicate GAG-specific ECM binding for different cartilage types and locations. In conclusion, lyophilization can be performed to determine native dry weight for normalization without affecting the degree of GAG treatment. To our knowledge, this is the first study to compare GAG-ECM interactions of different cartilage types using different GAG extraction methods. Degree of GAG depletion not only varied with cartilage type but also the same type from different anatomic locations. This suggests specific structure-function roles for GAG populations found in the tissues.
Highlights
Proteoglycans (PGs) are soluble macromolecules contributing to the integrity and homeostasis of cartilage
Hyaluronidase was able to remove over 99% of Sulfated glycosaminoglycan (sGAG) from all cartilage types except auricular cartilage where it only removed around 76% of sGAG
The results indicate GAG-specific extracellular matrix (ECM) binding for different cartilage types and locations
Summary
Proteoglycans (PGs) are soluble macromolecules contributing to the integrity and homeostasis of cartilage They are composed of negatively charged sulfated glycosaminoglycan (sGAG) chains covalently attached to a protein core.[1] PGs in cartilage can be divided into 2 main groups, aggregating PGs and nonaggregating PGs, based on their ability to aggregate with the unsulfated GAG, hyaluronan (HA).[1] It is known that GAGs in articular cartilage contribute to viscoelastic properties.[2] To investigate the effect of GAG content on mechanical properties of cartilage, GAG can be selectively removed from the extracellular matrix (ECM) and the depleted tissue compared to native cartilage tissue.[2] Several protocols in literature are used to selectively remove GAGs in cartilage, namely, chondroitinase ABC, guanidine hydrochloride, and hyaluronidase (usually Hyal-4). Chondroitinase ABC is an enzyme that degrades chondroitin sulfate by cleaving the GAG at disaccharide linkages, while digesting HA slowly at pH 8.3 Guanidine hydrochloride depletes both aggregating and Supplementary material for this article is available on the Cartilage website at https://journals.sagepub.com/home/car
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