The approach to OA changed a lot throughout history. At first, it was thought that OA is a disease of cartilage. Later, the perception was replaced by an idea that subchondral bone is also affected, but today it is known that all the tissues in or around the joint are influenced by the disease, leading to the concept of OA as a whole joint disease.
2.1. Articular Cartilage
Articular cartilage (AC) is avascular, alymphatic, and aneural tissue with chondrocytes as the only cell type in the cartilage tissue [
5,
30,
31]. Besides chondrocytes, AC is formed by the extracellular matrix (ECM), which is composed of water (more than 70%) and organic components such as type II collagen, aggrecan, other proteoglycans (decorin, biglycan, and fibromodulin), collagens (types III, VI, IX, XI, etc., collagens), glycosaminoglycans and glycoproteins [
5,
30,
32]. Proteoglycan aggregates, built of negatively charged glycosaminoglycans (keratan sulfate and chondroitin sulfate) bound to the aggrecan core protein, that is linked with hyaluronic acid backbone, together with other matrix components, are entrapped in a network of cross-linked type II collagen fibrils, as portrayed in [
31]. Premature termination codon on aggrecan mRNA affects cartilage development, while failure in aggrecan production may have a secondary effect on type II collagen, suggesting a possible extracellular matrix/type II collagen feedback regulation [
33,
34,
35]. Although type II collagen and aggrecan are the most common proteins in the cartilage matrix, there is a distinct difference in the matrix structure around chondrocytes, where other proteins such as collagen VI, fibromodulin and matrilin 3 form the pericellular matrix [
36]. All cartilage components are synthesized by chondrocytes, which play a key role in maintaining the cartilaginous environment by balancing the production of ECM components and its degrading enzymes, providing minimal and balanced turnover between anabolic and catabolic processes [
37]. AC metabolism is stimulated by mechanical loading, detected by mechanoreceptors on the cell surface [
38]. Through the process of mechanotransduction, mechanical signals modulate the biochemical activity of chondrocytes, inducing the biosynthesis of molecules to preserve the integrity of the tissue. Surface mechanoreceptors include mechanosensitive ion channels and integrins. Integrins are transmembrane proteins that activate internal cell signaling by binding chemical molecules, such as cytokines and growth factors [
39]. The activation of these mechanoreceptors initiates intracellular signaling cascades, leading to the tissue remodeling process. Furthermore, biomechanical stimulus generated by dynamic compression during moderate exercise can reduce the synthesis of proteolytic enzymes, regulate the metabolic balance, and prevent the progression of cartilage damage [
38]. The importance of proper mechanical loading is demonstrated by the fact that insufficient biomechanical stimuli, such as immobilization, can lead to reduced thickness (>10%) and softening of AC in the knee joint, in the absence of normal joint loading [
40]. Conversely, excessive mechanical loading leads to a quantitative imbalance between anabolic and catabolic activity, resulting in the depletion of matrix components and, due to lack of AC regenerative capacity, leads to irreversible destruction, thus making it the most apparent triggering cause of OA [
38].
Figure 1. Schematic representation of cartilage extracellular matrix and its changes in osteoarthritis. (A) A healthy network of proteoglycan aggregates entangled with type II collagen fibres is seen. matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) cleavage sites are represented by red arrows; (B) Cartilage matrix changes in osteoarthritis defined by degradation of proteoglycans and cleavage of type II collagen fibres by ADAMTS and MMPs, respectively.
AC can be divided into four layers: superficial (tangential) zone, middle (transitional) zone, deep (radial) zone, and a highly mineralized zone of calcified cartilage. This calcified zone is separated from the unmineralized upper cartilage layers by a histologically defined zone called tidemark and divides the cartilage from the underlying subchondral bone [
5,
31,
32]. The layers are characterized by chondrocyte shape and positioning, as well as collagen fibrils’ orientation [
41,
42,
43,
44,
45]. In the superficial zone, chondrocytes are disk-shaped, and collagen fibrils are oriented horizontally. Collagen fibrils in the middle zone are diagonally directed and round-shaped chondrocytes are located randomly. The deep zone is defined by vertical columns of chondrocytes together with radially aligned collagen fibrils. In calcified cartilage, collagen fibrils are arranged perpendicular to the articular surface [
46]. The population of chondrocytes is scarce in this zone. They are roundly shaped, without specific positioning [
30]. A detailed representation can be seen in .
Figure 2. Microarchitectural and histologic changes of articular cartilage, subchondral bone and synovium in osteoarthritis. (A) Representation of normal joint structure and normal histologic state of the main tissue involved in osteoarthritis; (B) Early-stage osteoarthritis changes in articular cartilage, subchondral bone and synovium. Shallow cartilage erosions of the superficial tangential zone and middle transitional zone causing disruption of the extracellular matrix can be seen. Secondary chondrocyte hypertrophy and clustering is present as well. Changes to other joint tissues, mainly the reduction in subchondral bone mass, synovial thickening and inflammatory cells (lymphocytes) migration, together with changes in cellular activity and count, are depicted. (C) Late-stage osteoarthritis changes of involved tissues. Full-thickness cartilage erosions reaching the subchondral bone, chondrocyte apoptosis, subchondral bone sclerosis, osteophyte and subchondral cysts formation alongside vascular infiltration are shown. Further synovial thickening with immune cells infiltration and increased vascularization is also seen.
Of the changes observed in OA, the cartilage matrix appears to be altered early and undergoes several pathological changes. As mechanoreceptors sense physical forces, chondrocytes answer with an adaptation of their metabolic activity [
47]. One of the first changes, that results from excessive mechanical loading, is an increase of water content in the superficial zone of articular cartilage, with the loss of glycosaminoglycans and proteoglycan degradation. As OA advances, a process known as matrix swelling expands to the deep zone [
48].
In early-stage OA, when macroscopic joint changes are not yet seen, the cartilage matrix goes through changes with the help of degrading enzymes. During this stage, aggrecanases of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), particularly ADAMTS-4 and ADAMTS-5, cleave the aggrecan core protein from the hyaluronic acid backbone seen in [
49]. The cleavage site is located beneath the glycosaminoglycan (GAG)-rich part of the aggrecan, which leads to their detachment from the proteoglycan aggregate. Since GAG molecules are responsible for osmotic properties of the cartilage matrix, their function is disrupted. In early-stage, chondrocytes are trying to restore lost aggrecans by increasing their synthetic activity. Aggrecan degradation is first seen in the superficial layer, presenting as superficial cartilage fibrillation [
50]. Another family of matrix-degrading enzymes, matrix metalloproteinases (MMP), participate in matrix degradation and OA pathogenesis [
51]. MMP-13 is the most expressed proteinase in OA [
52]. Its function, but also of other MMPs such as MMP-1 and MMP-3, is a disruption of the collagen network. Although MMP-13 can degrade a number of collagens and even aggrecan and other proteins, the primary target of MMP-13 is type II collagen, which is the most common type of collagen in the cartilage structure [
52]. Perturbed equilibrium between anabolic and catabolic processes leads to the progression of OA and further structural changes [
4]. The main mediators of cartilage metabolism are cytokines, discussed below.
Following the changes in the ECM, the pericellular matrix also undergoes degradation by increased proteinase activation. During the repair of cartilage matrix damage induced by excessive mechanical loading, chondrocytes gather themselves in clusters to further increase their synthetic activity [
31,
53]. To maintain high metabolic activity, chondrocytes experience a proliferative response, but they also adjust by undergoing hypertrophic differentiation [
30]. The generation of cartilage degradation products, together with the secretion of damage-associated molecular patterns (DAMPs), further increases the release of proinflammatory mediators. They also influence the adjacent synovium to proliferate and induce inflammatory responses, ultimately leading to larger decomposition of the cartilage [
8].
By initiating inflammatory processes and inducing a catabolic state of the cartilage, early surface changes seen as fibrillations extend distally, forming deep fissures, leading to cartilage delamination uncovering the calcified cartilage and the subchondral bone [
5,
31,
48]. The thinning of hyaline articular cartilage is accompanied by the expansion of underlying calcified cartilage, which additionally contributes to increased mechanical stress and the further production of catabolic factors [
8]. Furthermore, an enlarged layer of calcified cartilage advances into the overlying AC together with a duplication of the tidemark as another finding in osteoarthritic knee joints. These changes are caused by the penetration of vascular channels from the bone marrow, through the subchondral bone, calcified cartilage and tidemark to the articular cartilage, accompanied by sympathetic and sensory nerves [
54].
2.2. Subchondral Bone
Even though progressive cartilage damage and its eventual loss were the most mentioned features of the OA in the past, it is now well accepted that subchondral bone alterations and synovial inflammation also influence all other structures in the joint. Their structural changes are important disease characteristics, confirming that OA is a whole joint disease. Moreover, numerous studies suggest that interactions between cartilage and subchondral bone are fundamental for joint homeostasis, but also disease progression [
30,
31,
48,
55,
56,
57]. Relative to the slower turnover rate of the AC, subchondral bone undergoes more rapid modeling and remodeling in response to changes in the mechanical environment [
55]. Subchondral bone consists of two layers: the plate-like layer of cortical bone beneath the calcified cartilage, also known as a subchondral bone plate, and the deeper layer of subchondral trabecular or cancellous bone [
56,
57]. The structure of subchondral bone is mostly dependent on two types of cells that synthesize new bone and resorb the old one in response to the local environment—osteoblasts and osteoclasts [
57]. A study by Sanchez et al. reported that osteoblasts reply to mechanical stimulation, just like chondrocytes, by adapting their metabolic activity and secret pro-inflammatory cytokines and degradative enzymes [
58].
In established OA, the subchondral bone plate increases in volume and thickness. These changes are accompanied by alterations in subchondral trabecular bone in terms of its deterioration in the early stage and sclerosis in the late stage of OA [
59]. Furthermore, modifications of osteoblastic and osteoclastic activity result in bone turnover, causing subchondral bone lesions, cysts and osteophyte formation () [
60].
Bone marrow lesions (BML) represent micro-damage to the bone and are characterized by localized fibrosis, fat necrosis and a local increase in bone remodeling that results in microfractures of the trabecular bone [
61]. These features are commonly associated with overlying cartilage erosion and fissures, particularly with exposed subchondral bone [
62]. It has also been found that the appearance of BMLs and denuded areas of subchondral bone are related to clinical symptoms, particularly pain [
57,
63]. With the initiated increased bone turnover and neovascularization of subchondral bone, newly formed vessels together with nerves infiltrate the bone, invade the overlying cartilage tissue, making a communication channel for the exchange of biologic factors [
57]. This process is another mechanism that causes pain in OA. Subchondral bone cysts development, a hallmark of the advanced OA, is dependent on osteoclast-mediated bone resorption, a process initiated by bone damage and necrosis at sites of former BMLs [
64]. As an additional adaptive mechanism, osteophytes form at the joint margins via endochondral ossification [
4]. A recent experimental rat model found that subchondral bone changes occurred before cartilage degeneration in ACL transection, while cartilage changes occurred before the subchondral bone changes when collagenases were injected into the joint, suggesting a different pathophysiological response in primary and secondary OA models [
65]. Although the aforementioned subchondral bone structural changes are associated with subsequent cartilage loss, there is no correlation with subchondral sclerosis detected by MRI, signifying sclerosis is a late-stage characteristic of OA [
66].
2.3. Synovium
The synovial membrane, together with synovial fluid, makes the synovium [
67]. Synovial fluid plays a crucial role in cartilage nutrition. The avascular cartilage uses the synovial fluid as a source of nutrients, but also as a reservoir for its degrading products [
68]. The synovial membrane consists of intima (a lining layer) and subintima (a sublining layer) thick up to 5 mm in healthy individuals [
69,
70]. Two to three layers of metabolically highly active cells (synoviocytes) lie over vascularized loose connective tissue to form the subintima, with plenty of collagen secreting fibroblasts [
69,
70,
71]. Two types of macrophages have been found in synovia: classic and inflammatory-like macrophages [
72]. In knee OA, inflammatory macrophages are important mediators as they produce VEGF, which may be a possible mechanism causing synovitis and inflammation [
69,
73]. Macrophage-like synoviocytes are CD163- and CD68-positive, but also stain positively for nonspecific esterase, an enzyme histochemical stain that differentiates macrophages and monocytes from other cell types. They form an integral part of the intima, together with fibroblast-like synoviocytes, which are CD55-positive and express class II major histocompatibility molecules [
69,
70]. Macrophage-like synoviocytes proliferate during inflammation, whereas fibroblast-like synoviocytes mainly produce hyaluronic acid and are found further from the intima [
70,
71]. The synovium produces hyaluronan and a plasminogen activator, preserving synovial fluid volume during exercise. It also secretes lubricin and hyaluronic acid, important synovial fluid components [
73].
Synovitis is recognized as an important feature in patients with OA and has been associated both with symptoms and with structural progression. The inflammation in OA causes synovia to proliferate and induces the infiltration of T and B lymphocytes as well as mast cells [
74,
75]. Synovial hypertrophy is defined as a synovial thickening of ≤4mm and effusion depth of fluid ≤4 mm or ≤4 mm in the suprapatellar recess [
76]. Stimulated by mediators of inflammation, particularly Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF), MMPs and other proteinases induce chondrocytes, synovial cells and lymphocytes to produce IL-6, IL-8, IL-15, IL-17, leukemia inhibitory factor and prostaglandin E2 (PGE2) [
70,
77,
78]. Studies demonstrated that MRI confirmed synovitis strongly correlates with pain severity expressed by the WOMAC pain score [
79]. In addition, synovitis causes the extensive production of proteolytic enzymes, causing cartilage damage, while cartilage matrix catabolism produces molecules that propagate synovial inflammation [
71].
Synovial thickening is associated with radiographic and clinical progression of knee OA, pain and dysfunction that predominantly occurs posterior to the ACL and in the suprapatellar region [
80]. It is commonly identified during arthroscopy in approximately 50% of patients with OA [
81]. Moreover, areas of inflamed synovium tend to relate to locations of cartilage degradation [
76]. However, in advanced OA, synovitis is diffuse, especially after total knee arthroplasty [
74]. Because of the increased peripheral nociceptive neuron responsiveness in synovitis, OA symptoms and pain sensitivity are progressive [
82,
83]. A large study by The European League Against Rheumatism (EULAR), which included 600 people with knee OA, demonstrated synovial hypertrophy or the effusion in 46% of patients [
84]. A study that evaluated the frequency of synovitis in OA knees using non-contrast MRI to assess synovial thickening showed that synovitis (defined as synovial thickening) was observed in 73% of OA knees compared with 0% of the control group. Synovitis was noted to be more likely to be present with increasing Kellgren–Lawrence (K/L) grade [
85]. The synovial thickening observed on MRI has been confirmed as histological synovitis, with the grade of thickening correlating with the degree of macroscopic synovitis seen at arthroscopy and microscopically. The authors also noted that the distribution of synovitis was diffuse, with no statistical difference seen between those patients with marked chondral changes and those with few chondral changes, suggesting that synovitis is present from the earliest stages of OA and is diffuse and not related only to areas of cartilage [
85,
86]. Contrast-enhanced MRI may better differentiate inflamed synovium from joint effusion, but it is still not known whether contrast-enhanced MRI assessment of synovitis can predict disease progression in OA [
76].
2.4. Infrapatellar Fat Pad, Menisci, Periarticular Muscles, Ligaments and Tendons
As the largest intraarticular adipose structure, Hoffa’s infrapatellar fat pad (IPFP) is located in the anterior knee compartment, between the synovium and the joint capsule [
87,
88]. As a shock absorber, Hoffa’s fat pad acts to reduce the impact of loading forces, protecting the knee from mechanical damage [
88,
89]. IPFP is a very sensitive tissue containing adipocytes, fibroblasts, leukocytes, macrophages and other immune cells [
88]. It is innervated by C-fibers that release substance P. Increased concentration of substance P is present in patients suffering from OA. Vasodilatation and migration of immune cells result in swelling, local ischemia and partial necrosis due to an increased concentration of substance P, causing structural changes in IPFP [
88]. Macrophages found in the IPFP of patients suffering from OA are increased in number and similar to macrophages of the synovium and subcutaneous fat tissue with CD11c+ and CD206+ markers [
90]. Hoffa’s fat pad is a potent producer of adipokines and cytokines such as adiponectin, leptin, IL-6 and TNF, which makes IPFP an active endocrine organ [
89,
91]. In OA patients, it is reported that IPFP and synovial fluid contain significant amounts of FGF-2, VEGF, TNF α, and IL-6 [
92]. Moreover, the severity of knee OA seems to be associated with the concentration of leptin in the synovial fluid [
93]. Collectively, alterations to Hoffa’s fat pad may be a possible cause of chronic knee pain in OA [
88].
One of the strongest risk factors for developing knee OA is meniscal lesions and injuries, due to decreases in resistance to mechanical forces, such as tension, compression and shear stress [
94]. Loss of intact meniscus function leads to OA due to joint instability and abnormal mechanical loading [
95,
96]. Moreover, in patients with radiographically confirmed OA, meniscus damage is almost always present, causing loss of load-bearing, shock absorption, lubrication and instability, which can lead to static changes in the femorotibial joint, leading to damage in the AC, as well as in the subchondral bone, contributing to the progression of knee OA [
94,
95,
97]. In healthy individuals, the collagen matrix in loaded meniscus resists biomechanical stress, allowing load transmission to other knee structures, making the knee stable during movements [
98]. If the meniscus is injured or damaged, it often results in damage to other knee structures, such as cartilage loss, changes in subchondral bone, lesions of bone marrow and synovitis [
99]. It has been demonstrated that meniscal subluxation and extrusion are an independent predictor of tibiofemoral cartilage loss and degenerative subchondral bone marrow lesions [
100]. Patients with mild and moderate knee OA showed significantly increased meniscal tear and extrusion [
101]. The history of partial or complete meniscectomy is also a risk factor associated with the incidence of OA of the knee [
102]. In summary, loss of meniscal function leads to OA and vice versa [
94].
One of the main functional limitations in knee OA is the dysfunction of the quadriceps muscle, hamstrings and hip muscles [
103,
104]. The weakness of periarticular muscles has a significant impact on knee biomechanics. However, it is not clear whether muscle weakness is associated with OA onset or OA progression [
105,
106]. Increased intramuscular fat content in the quadriceps was found to be a strong predictor of knee cartilage loss [
107,
108]. It was also noted that periarticular muscles in patients with OA are inflamed and are producing myokines [
105,
109]. Observing the molecular aspect, myokines interact with synovia, Hoffa’s fat pad, cartilage and bone, making skeletal muscles involved in OA pathogenesis [
109]. Tendon instabilities and ligament injuries have also been recognized as predisposing factors of OA, with around 50% of patients developing OA after ligament injury in a period of 10-20 years [
91]. Studies demonstrated that neuromuscular training can decrease the possibility of developing knee OA after previous knee injury [
106]. In a 10-year follow-up period, anatomic ACL reconstruction was found to reduce the risk of post-traumatic OA development [
110]. The importance of knee muscles and ligaments in everyday functional actions, such as standing up from a chair, going up and down the stairs and level surface walking is evident [
105]. In patients with symptomatic knee OA, muscle size maintenance is associated with beneficial structural changes and a reduced risk of knee joint replacement [
111].