Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse

Abstract

Aggrecan loss in human and animal cartilage precedes clinical symptoms of osteoarthritis, suggesting that aggrecan loss is an initiating step in cartilage pathology. Characterizing early stages of cartilage degeneration caused by aging and overuse is important in the search for therapeutics. In this study, atomic force microscopy (AFM)-based force-displacement micromechanics, AFM-based wide bandwidth nanomechanics (nanodynamic), and histologic assessments were used to study changes in distal femur cartilage of wildtype mice and mice in which the aggrecan interglobular domain was mutated to make the cartilage aggrecanase-resistant. Half the animals were subjected to voluntary running-wheel exercise of varying durations. Wildtype mice at three selected age groups were compared. While histological assessment was not sensitive enough to capture any statistically significant changes in these relatively young populations of mice, micromechanical assessment captured changes in the quasi-equilibrium structural-elastic behavior of the cartilage matrix. Additionally, nanodynamic assessment captured changes in the fluid-solid poroelastic behavior and the high frequency stiffness of the tissue, which proved to be the most sensitive assessment of changes in cartilage associated with aging and joint-overuse. In wildtype mice, aging caused softening of the cartilage tissue at the microscale and at the nanoscale. Softening with increased animal age was found at high loading rates (frequencies), suggesting an increase in hydraulic permeability, with implications for loss of function pertinent to running and impact-injury. Running caused substantial changes in fluid-solid interactions in aggrecanase-resistant mice, suggestive of tissue degradation. However, higher nanodynamic stiffness magnitude and lower hydraulic permeability was observed in running aggrecanase-resistant mice compared to running wildtype controls at the same age, thereby suggesting protection from joint-overuse.

Tendon exhibits complex poroelastic behavior at the nanoscale as revealed by high-frequency AFM-based rheology

“…To measure the complex modulus of samples over a wide frequency range (1 Hz to 10 kHz), we used our custom high-frequency rheology system coupled to a commercial atomic force microscope (AFM) (MFP-3D, Asylum Research, Santa Barbara, CA) (Azadi et al, 2016; Nia et al, 2013). We used polystyrene colloidal probe tips with varying diameters of ~4.5–50 μm (Polysciences, Warrington, PA) attached to tipless cantilevers with nominal spring constant k ~ 7.4 N/m (Budget Sensors, Sofia, Bulgaria).…”

“…As tendons are primarily composed of water, about 75% of the wet weight, regulation of fluid flow is crucial to dynamic mechanical behavior. In articular cartilage, for example, the abundance of the large proteoglycan aggrecan is responsible for the tissue’s complex poroelastic properties (Azadi et al, 2016; Nia et al, 2015b, 2013, 2011). It has recently been shown that proteoglycan concentration and type can vary dramatically along and between tendons, and specifically that rotator cuff tendons contain aggrecan in addition to decorin and biglycan (Berenson et al, 1996; Matuszewski et al, 2012).…”

Using mouse models to investigate the pathophysiology, treatment, and prevention of post‐traumatic osteoarthritis

“…The magnitudes of joint loading, instability and initial structural damage required to classify models as “post‐traumatic” OA is currently ill defined, and requires clarification. However, it seems prudent to include this Type III classification, as it likely shares some of the pathophysiological mechanisms (e.g., biomechanical loading) of Type I and II, and may serve to shed light on risk factors for specific aspects of PTOA pathology and susceptibility, for example, exercise modifying OA in genetically modified mice . The boundary between physiological, protective, and sub‐critical damaging joint loading levels is not clear, and better defining the factors that might shift this balance in either direction in a given individual, would greatly benefit our understanding of what constitutes a joint “injury,” and its management to prevent the initiation and progression of OA.…”

CITED2 mediates the cross‐talk between mechanical loading and IL‐4 to promote chondroprotection

“…It has been long established that moderate mechanical loading by exercise benefits cartilage health and function by increasing cartilage thickness, proteoglycan content, and mechanical stiffness . However, nonphysiological loading such as overuse can lead to cartilage degradation . Proinflammatory cytokines and other mediators produced in joint tissues in response to nonphysiological loading play important roles in cartilage degradation and osteoarthritis (OA) pathogenesis .…”

“…4 However, nonphysiological loading such as overuse can lead to cartilage degradation. [5][6][7] Proinflammatory cytokines and other mediators produced in joint tissues in response to nonphysiological loading play important roles in cartilage degradation and osteoarthritis (OA) pathogenesis. [8][9][10][11] In particular, interleukin (IL)-1 and tumor necrosis factor-␣ can induce cartilage degradation 12 by upregulating matrix metalloproteinase (MMP) 13 gene expression in articular chondrocytes.…”