# New Insights on Mechanical Stimulation of Mesenchymal Stem Cells for Cartilage Regeneration
## 3 Influencing the Mechanical Environment  
The characteristic structure of the articular cartilage is the result of the dynamic processes that occur within the joint. Chondrocytes experience biomechanical stimuli like compression, shear stress, and hydrostatic pressure [19]. These forces are perceived as a shifting of currents, electrical fields, or changes in osmolarity and so converted into intracellular signals, influencing mechanisms like transcription, exocytosis, and activity of Na+/K+-ATPase [21]. Growth factors such as TGF-\(\beta\), IGF, and bone morphogenetic proteins (BMP)-2,-4,-7 are necessary to stimulate chondrogenic processes and require the presence of calcium ions (Ca\({}^{2+}\)) to regulate cell functions, such as the synthesis of extracellular matrix components. Physical stimuli have been associated with the regulation of Ca\({}^{2+}\) entry, primarily through voltage-operated calcium channels (VOCCs), transient receptor potential (TRP) channels, and purinergic receptors [22]. Furthermore, VOCC inhibitors have shown to reduce cartilage degradation and the progression of osteoarthritis [23], suggesting their importance in both physiological and pathological milieux. TRP channels, such as TRP vanilloid 4 (TRPV4), which have been linked to upregulation of the SOX9 pathway, or TRPC1, able to guide chondrogenesis in stem cells and regulate the activity of other voltage-dependent ion channels, are also highly involved. Inhibitors for these two receptors, 2-aminoethoxydiphenylborane (2-APB) and Ruthenium Red, have been shown to prevent MSC chondrogenesis induced by pulsed electromagnetic fields (PEMFs) [24]. The structure of the articular cartilage comprises several layers: First, a thin superficial zone, where the collagen fibers are aligned parallel to the surface and the chondrocytes are numerous and flattened. This layer is in tight contact with the synovial fluid, and it can resist shear stresses. Under the first zone, there is a transitional zone which contains especially proteoglycans (PGs) employed for compressive resistance. Here, the collagen is arranged obliquely. Just below, the deep zone can cushion the compressions and presents collagen fibers organized perpendicular to the surface, high concentrations of proteoglycans, and columns of cells. Lastly, the tide mark separates this last zone from the calcified cartilage [25] (Figure 1). Figure 1: Structure of the articular cartilage. The structure of the articular cartilage comprises several layers: First, a superficial zone, where the collagen fibers are aligned horizontally, the chondrocytes are numerous and flattened, and the proteoglycan content is low. Under the first zone, there is a transitional zone which contains high levels of proteoglycans, randomly arranged collagen, and round-shaped cells. Just below, the deep zone presents vertically aligned collagen fibers, a high concentration of proteoglycans, and columns of cells. For the sake of simplicity, this tissue could be imagined like a biphasic model: one phase is represented by the interstitial water that permeates the other phase, made up of the solid components of the ECM (collagen, PGs and other proteins). These two phases are extremely interdependent in terms of functionality and biomechanics. Compression is one of the forces experienced by cartilage, which leads to an internal increase of hydrostatic pressure of the aqueous phase. As a result, the water leaks from the ECM towards the capsule, but the structure of the cartilage will remain unaltered thanks to the PG component. More precisely, the effect of uniaxial compression will be compensated by the tensile stiffness generated by the repulsive forces between the negatively charged carboxylic or sulfonic groups of the glycosaminoglycans (GAGs). When the pressure ceases, the water is again attracted inside the interstices. Tissue compressibility under load reaches even a millimeter, but, when the spring back is not able to compensate hard and long compressions, the structure can be damaged [26].