Moderate mechanical loading applied laterally to the knee has been shown to enhance bone formation  and attenuate pain perception-linked signaling , but the effect of lateral loads to the knee on the maintenance of cartilage tissues remains undetermined . The major cellular subpopulation in cartilage is constituted by chondrocytes, which are critical due in part to their role in biosynthesis of extracellular matrix as well as secretion of matrix metalloproteinases (MMPs) such as MMP1, MMP3, and MMP13. Excess loads are considered to induce degenerative activities of collagenases and gelatinases in chondrocytes, while unloading due to immobilization also presents detrimental outcomes [4, 5]. While anabolic responses to bone by various mechanical loading modalities have been demonstrated, little is known about the effects of mechanical loading to the knee on the regulation of MMPs in chondrocytes of the femoral cartilage in the knee.
Knee loading applies dynamic lateral loads to the knee joint and stimulates bone formation not only in the distal femur, but along the entire length of the femur [6, 7]. The loading force required to stimulate bone formation due to knee loading is lower than that of other mechanical loading regimens, and strains in areas of bone formation are also reduced. This characteristic makes knee loading an attractive potential treatment in accelerating fracture repair . Fractures of the femoral neck, which are a serious public health concern, experience faster healing times as a result of dynamic knee loading . To explain the observed increases in bone formation due to knee loading, studies have focused on biophysical and molecular mechanisms that occur during and following a loading treatment [9–11]. Dynamic deformations of the epiphysis cause alterations in fluid pressure in the intramedullary cavity, driving oscillatory fluid flow and molecular transport in the lacunocanalicular network in the bone matrix and in the medullary cavity . Fluid flow may cause shear stress to osteocytes, leading to osteoblast differentiation and the initiation of bone formation . Although loads are directly applied to the knee, loading effects on maintenance of cartilage tissue and chondrocytes have not been examined.
Herein we addressed a question: Can knee loading, which gently applies lateral loads to the knee, reduce degenerative activity of MMP13 in non-OA and OA cartilage in the femur? If yes, what signaling pathway mediates load-driven suppression of MMP13 activity in chondrocytes? We hypothesized that the responses to knee loading are dependent on loading intensity. Mild and moderate loads (0.5–1 N) may reduce MMP13 activity, while strong loads (3 N) may elevate it. We also hypothesized that signaling pathways linked to inflammation, and cellular proliferation and differentiation are possibly involved in the regulation of MMP13 through p38 MAPK, NFκB, or small GTPases such as Rac1. The p38 MAPK pathway is responsive to mechanical stimulation and involved in the expression of MMP13 , while the NFκB pathway is known to be involved in inflammation and tissue degradation . Furthermore, Rac1 GTPase is known to regulate cytoskeletal shape and activate p38 MAPK [15, 16].
In testing the above hypothesis, we applied knee loading to the right knee of non-OA and OA mice and provided fluid flow shear to primary human chondrocytes (h-nonOA control, and h-OA) together with a C28/I2 human chondrocyte cell line. To evaluate potential dependence on loading magnitude, knee loading was applied at three levels of 0.5 N (mild), 1 N (moderate), and 3 N (strong), and fluid flow was given to induce shear intensity of 2 and 5 dyn/cm2 (mild), 10 dyn/cm2 (moderate), and 20 dyn/cm2 (strong). Activities of collagenases, gelatinases, and MMP13 were determined using fluorescent substrates specific to their activities, and the MMP13 mRNA level was determined using quantitative real-time PCR. Western blot analysis was performed to determine the phosphorylation levels of p38 MAPK and NFκB, and activity of Rac1 GTPase was monitored using a fluorescence resonance energy transfer (FRET) technique with a biosensor specific to Rac1 GTPase. To evaluate the role of Rac1 GTPase in the regulation of MMP13, silencing of Rac1 was conducted using siRNA specific to Rac1. Furthermore, two Rac1 GTPase mutants (Rac1-L61 and Rac1-N17) were transfected to C28/I2 chondrocyte cells and the effects of dominant negative and constitutively active forms of Rac1 GTPase were evaluated on MMP13 activity.