Chondroadherin (CHAD), a class IV small leucine rich proteoglycan/protein (SLRP), was hypothesized to play important roles in regulating chondrocyte signaling and cartilage homeostasis. However, its roles in cartilage development and function are not well understood, and no major osteoarthritis-like phenotype was found in the murine model with CHAD genetically deleted (CHAD(-/-)). In this study, we used atomic force microscopy (AFM)-based nanoindentation to quantify the effects of CHAD deletion on changes in the biomechanical function of murine cartilage. In comparison to wild-type (WT) mice, CHAD-deletion resulted in a significant ≈70-80% reduction in the indentation modulus, Eind, of the superficial zone knee cartilage of 11 weeks, 4 months and 1 year old animals. This mechanical phenotype correlates well with observed increases in the heterogeneity collagen fibril diameters in the surface zone. The results suggest that CHAD mainly plays a major role in regulating the formation of the collagen fibrillar network during the early skeletal development. In contrast, CHAD-deletion had no appreciable effects on the indentation mechanics of middle/deep zone cartilage, likely due to the dominating role of aggrecan in the middle/deep zone. The presence of significant rate dependence of the indentation stiffness in both WT and CHAD(-/-) knee cartilage suggested the importance of both fluid flow induced poroelasticity and intrinsic viscoelasticity in murine cartilage biomechanical properties. Furthermore, the marked differences in the nanomechanical behavior of WT versus CHAD(-/-) cartilage contrasted sharply with the relative absence of overt differences in histological appearance. These observations highlight the sensitivity of nanomechanical tools in evaluating structural and mechanical phenotypes in transgenic mice.

In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 μm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan–collagen adhesion. Increasing both ionic strength and [Ca2+] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca2+-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan–collagen interactions. Interestingly, we found a significant increase in aggrecan–collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan–collagen adhesion, together with aggrecan–aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.

While small leucine rich proteins/proteoglycans (SLRPs) are present in very low concentrations in the extracellular matrix (ECM), they have been shown to be critical determinants of the proper ECM assembly and function in connective tissues [1] including bone [2], cornea [3], and cartilage [4]. However, their direct and indirect roles in matrix biomechanics and the potential for osteoarthritis-related dysfunction of cartilage remain unclear. With the advent of new high resolution nanotechnological tools, the direct quantification of cartilage biomechanical properties using murine models can provide important insights into how secondary ECM molecules, such as SLRPs, affect the function and pathology of cartilage [5]. Previous nanoindentation studies of murine cartilage have assessed the effects of maturation and osteoarthritis-like degradation of cartilage on its biomechanical properties [6, 7]. Recently, murine models have received increased attention because of the availability of specific gene-knockout and gene alteration technologies [8]. For example, chondroadherin (CHAD) is a non-collagenous small leucine-rich proteoglycan (SLRP) with α-helix and β-sheet secondary structure, spatially localized in the territorial matrix (MW = 38 kDa) [9]. In articular cartilage, CHAD is distributed non-uniformly with depth [10], and binds to type II collagen and the α2β1 integrin and is hypothesized to function in the communication between chondrocytes and their surrounding matrix, as well as in the regulation of collagen fibril assembly [11, 12] (Fig. 1). The objective of the present study is to explore the role of CHAD and its depletion on the structure and nanomechanical properties of both superficial and middle/deep zone cartilage. The current methods thereby enabled depth-dependent analysis of cartilage nanostructure and dynamic energy-dissipative mechanisms.