The process of mechanical sensing starts at the cell periphery, where the cell forms a physical connection with its environment [65]. Integrins are the main force transducer between the environment and the cell and serve as mechanical linkers between the cytoskeleton and the environment [65,66,67,68]. A family of transmembrane proteins that sit in the plasma membrane, integrins are heterodimers composed of α and β subunits. The bulk of these proteins is found in the extracellular domain with 700 aa of the α subunit and 1000 aa of β subunit protruding into the extracellular space [65,69]. By contrast, the cytoplasmic tail is 40–70 amino acids long. Integrins are maintained in a bent conformation when inactive. The activation process causes conformational changes within the cytoplasmic domain, where the protein talin binds to the β subunit and triggers the activation and conformational change of the α and β subunits [69]. Upon activation, the extracellular domains of integrins bind ECM protein such as fibronectin, collagen and others [68,70]. In the cytoplasm, the β subunit of the integrin heterodimer binds to the actin cytoskeleton through a variety of adaptor proteins. As the ECM ligand binds, the integrins activates further and clusters to initiate the assembly of the focal adhesions (FA) complex that is composed of focal adhesions kinase (FAK), vinculin, paxillin, and tensin, thus forming a linkage between the cell and the environment [68,70]. In stiff matrices there are increased number of focal adhesions and traction forces generated between FA and ECM, as compared to soft matrices that have fewer focal adhesions [68,70]. In addition, FA complexes contribute to the reorganization of the actin cytoskeleton in response to mechanical stimuli [66,67] thus translating the stimuli from the extracellular environment into a cytoskeletal change.

Immunophenotyping performed on cartilage tissue showed the expression of α1β1 (collagen type VI, II and matrilin-1), α5β1 (fibronectin) and αVβ5 (fibronectin, vitronectin and osteopontin) and lesser amounts of α3β1 (fibronectin) and αvβ3 (COMP, fibronectin, vitronectin and osteopontin) [71,72,73]. The integrin profile is dependent on ECM proteins that are present. In OA, the ECM structure is altered, which leads to an alteration in integrin profile: α2β1 (collagen type II, VI, and chondroadherin), α4β1 (fibronectin and V-CAM) and α6β1 (laminin) integrins are the predominant integrins expressed in OA [73,74]. The changes in integrin profiles highlight the adaptation of chondrocyte to the ECM environment.

When chondrocytes are harvested for ACI, they are removed from an ECM-rich environment and cultured in an ECM-free environment. In the process of adaptation to an ECM free environment, chondrocytes de-differentiate and exhibit an integrin profile change. Primary chondrocytes cultured over a period of 21 days showed a clear increase in β1 and α2 levels by immunofluorescent analysis. This increase is consistent with the increase in α2β1 complex that is found in OA, suggesting that culturing chondrocytes for prolonged periods (21 days) on plastic can lead to a disease phenotype; however, this does not rule out the formation of other integrin complexes. Furthermore, it is clear evidence that chondrocytes sense and adapt to environmental changes by altering their integrin profile. Such changes will have downstream effects on cytoskeletal arrangement and tensions [75] as well as on nuclear responses [76].

While mechanical sensing is mediated by integrins [77], the intracellular response is driven by the cytoskeleton. Rho GTPases (RhoA, Rac1 and Cdc42) are known as master regulators of actin cytoskeleton dynamics through actin nucleation (WASP/WAVE) and Diaphanous-related formins, affecting cell morphology and cell adhesion [78]. Cell morphology and adhesion are two aspects that are influenced by substrate rigidity. On stiff substrates, cells assemble stress fibers that induce high intracellular tension forces, while soft substrates do not promote stress fiber formation [79,80]. The cytoskeletal forces, mediated in part by these stress fibers, are transduced from the cytoskeleton to the nucleus through the LINC complexes (Linker of Nucleoskeleton to Cytoskeleton) [79,80].

Several studies have shown the impact of substrate stiffness on chondrocyte de-differentiation [81,82,83,84]. Q. Zhang et al. [81] investigated the effects of growing chondrocytes on a range of polydimethylsiloxane (PDMS) membranes stiffnesses: soft to stiff (stiff being similar to commercial petri dish). They showed that 78% of the cells grown on soft substrates exhibited and maintained a round chondrocyte morphology, while on stiffer substrates only 41% of the cells presented with a spherical morphology, with 59% having a stretched fibroblastic morphology [81]. E. Schuh et al. found that stiffer substrates lead to higher proliferation rates but that stiff substrates also led to phenotypic changes associated with low collagen type II and aggrecan expression, and high collagen type I expression [82]. By contrast, softer substrates promoted the maintenance of the chondrogenic phenotype with high collagen type II and aggrecan expression, and lower collagen type I expression [82]. Chondrocytes grown on different substrate rigidities also showed apparent differences in F-actin distributions [83] and actin depolarization has been shown to enhance the chondrogenic potential [85,86], while the loss of chondrocyte phenotype correlates with increased RhoA signaling and the presence of stress fibers [83,84]. On 54–135 kPa substrates, chondrocytes presented highly organized parallel stress fibers, with a wide spread polygonal morphology [83]. By contrast, on 1.4–6 kPa substrates chondrocytes had a much smaller and rounded morphology with actin filament extensions found only in few cells [83]. Because actin filaments are able to transmit force to the nucleus, substrate stiffness has been shown to contribute to lineage determination, and affect expression of NE proteins, including Lamin A/C [87,88].

Lamins are intermediate filament proteins that reside primarily within the internal periphery of the nucleus. Lamins are encoded by three genes: lamins A and C are alternative splice products of the LMNA gene, lamin B1 and lamin B2 are encoded by the LMNB1 and LMNB2 genes respectively [89]. Lamins are necessary to maintain nuclear structure and mechanical properties [90,91]. Lamins A/C primarily contribute to nuclear rigidity, while B-type lamins provide the nucleus with elastic properties [89,92]. Lamins have been shown to protect nuclear DNA against mechanical forces [93].

DNA in cells is generally found in one of two states. Heterochromatin is densely packed chromatin located at the periphery of the nucleus and is typically transcriptionally inactive [94]. Euchromatin on the other hand is gene rich with higher transcriptional activity and is located centrally with open structures [94]. The organization of chromatin is key to gene regulation and cell-fate determination [95]. Advances in microscopic imaging and molecular approaches have provided important insights into DNA localization and folding in normal versus disease states. The genome organization is an important player in regulating gene activity [96,97,98]. Lamins play an important role in chromatin organization [99], interacting with chromatin via lamina-associated domains (LADs) found mostly in heterochromatin. LADs are found in chromatin regions that contain mostly silent or weakly-expressed genes [100] and is enriched with repressive histone modifiers: H3K9me2, H3K9me3, and H3K27me3 [63]. Thus, the nuclear lamina helps to establish a repressive nuclear compartment at the nuclear periphery.

In the process of chondrocytes expansion for ACI, passage 0 (P0) chondrocytes have a rounded nucleus that is located in the center of the cell and expresses chondrocyte markers COL2A and SOX9. Using high resolution strain analysis to map mechanical strain on these chondrocytes, strain localization was distributed equally to heterochromatin and euchromatin at P0. At later passages, chondrocytes had a much flatter nucleus that was no longer centrally located in the cell. These later passage chondrocytes also had a higher strain in the heterochromatin and a higher expression of COL1A1 [101]. Interestingly, late passage chondrocytes maintained LMNB1 and LMNB2 gene levels but had a significantly lower expression of lamin A/C, suggesting that a loss of nuclear structural integrity contributes to the expression of repressed genes. It is important to note, the loss of lamin A/C is also indicative of loss of resistive force around the nucleus periphery [92]. Similarly, Nava et al. showed the application of stain on progenitor cells leads to a decrease in nuclear envelope tension to prevent DNA damage. The reduction in tension is mediated by the reduction in H3K9me3 lamina-associated heterochromatin [63].