Nanomechanical phenotype of chondroadherin-null murine articular cartilage

Abstract

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.

Keywords: Cartilage; Chondroadherin; Collagen; Murine model; Nanoindentation.

Fig. 1.

Schematic of the roles of chondroadherin in mediating chondrocyte signaling through bindings to the α2βt integrin (Camper et al., 1997; Haglund et al., 2011) and surface proteoglycan syndecans (Haglund et al., 2013).

Fig. 2.

(a) Typical indentation force versus depth loading curves (symbols) and corresponding Hertz model fit via least squares linear regression (solid lines, R² > 0.96). Data were obtained on the superficial layer of one right knee joint for 1 year old WT and CHAD^−/− specimens. The density of experimental data was reduced to increase clarity. (b) Effective indentation modulus, E_ind, for 1 year old murine cartilage superficial layer from four animals in each mouse type (mean ± SEM of ≥8 locations on the same joint surface of each animal, *: p < 0.05 between WT and CHAD^−/− via Mann–Whitney U test, #: p < 0.05 between different mice within each cohort of WT and CHAD^−/− via Kruskal-Wallis test). Data shown were taken at 1 μm/s z-piezo displacement rate; the trends are the same for 0.1 and 10 μm/s rates.

Fig. 3.

Effective indentation modulus, E_ind, for WT and CHAD^−/− murine cartilage superficial layer as a function of z-piezo displacement rate and mouse age (mean ± SEM of the average from each mouse, n = 4 animals for each model and age, except that n = 5 for WT at 4 months age).

Fig. 4.

Scanning electron microscope (SEM) images of WT and CHAD^−/− murine cartilage surface prepared via the Ohtani's procedure (Ohtani, 1987) at 11 weeks and 4 months age, and corresponding histogram of collagen diameter distribution estimated on images from n = 3 animals (≥300 fibrils in total) for each model and age.

Fig. 5.

Toluidine blue histology images of left knee joints from WT and CHAD^−/− mice showing the absence of gross-level phenotype in CHAD^−/− mice at 11 weeks and 4 months age.