**2. Osteoarthritis**

#### *2.1. Pain in Clinical OA*

Radiographic changes in OA are poorly correlated with pain and physical function, and the risk factors for radiographic OA are not the same as those for OA pain [9–11]. As pain is strongly correlated with person-level psychological, social, and cultural factors aside from joint damage, studies involving human OA subjects have a high risk of being influenced by confounding effects between individual subjects. Studies using a within-person, knee-matched, case-control design minimize such risks by including patients with knees discordant for the presence of pain or pain severity, and have shown that the severity of radiographic knee OA is indeed strongly associated with both the presence of frequent knee pain and severity of pain in diverse ethnic groups [12,13]. On the other hand, a community study showed that a significant number of subjects with Kellgren and Lawrence (KL) grade 4 OA, which is characterized by a marked loss of articular cartilage and profound subchondral bone changes, did not report pain [14]. As hyaline cartilage, the main focus of interest in both clinical and laboratory research on OA, is an aneural tissue, it is unable to generate nociceptive input and is an unlikely source of pain. In an arthroscopic study that probed the human knee without intra-articular anesthesia, a conscious subject reported remarkable sensitivity to the mechanical loading stimulus in anterior synovial tissues, fat pad, and capsule, but not the cartilage [15]. These findings were corroborated by the results of magnetic resonance imaging (MRI) studies correlating pain with synovitis/effusion in OA subjects [16]. It is notable that the sensation experienced with similar probing of the cruciate ligaments and menisci results in variable degrees of pain depending on the location of the probe, while among OA subjects, meniscus degeneration detected in knee MRI is not correlated with pain [17,18]. This discrepancy may be related to differences between study subjects, such as healthy volunteers vs. OA patients. Bone marrow lesions (BMLs), which in MRI scans appear as regions of hyperintense marrow signals, are associated with knee pain as well as the progression of cartilage damage [19,20]. They often occur before joint degeneration is established, and resolution of knee pain is associated with reduced BML size, suggesting that BMLs may serve as a biomarker for OA structural damage as well as pain [16,21]. In a study of two groups of subjects matched by macroscopic cartilage damage score, the expression of nerve growth factor (NGF) in osteochondral channels and osteoclast densities in subchondral bone were higher in a symptomatic than in an asymptomatic group [22]. These observations suggest that subchondral pathology is associated with symptomatic knee OA, independent of cartilage lesions.

In addition to nociceptive mechanisms arising from structural changes, recent evidence indicates that neurophysiological pathways contribute to OA pain, such that persistent tissue damage and inflammation in the joint induce mechanisms of peripheral and central sensitization [23]. In a study involving 2126 subjects with or at risk of knee OA, mechanical temporal summation (an augmented response to repetitive mechanical stimulation and a measure of central sensitization) and pressure pain thresholds (a measure of sensitivity to pain evoked by mechanical stimulation of nociceptors and

a reflection of activity-dependent peripheral sensitization) were associated with pain severity but not radiographic OA [24]. Interestingly, the duration of knee OA was not associated with these markers of pain sensitivity, suggesting that individual predisposition to sensitization rather than induction by peripheral nociceptive input from OA pathology may play a role in pain [24]. A study of patients with hand OA revealed that central sensitization was common, and lower local and widespread pressure pain thresholds and the presence of temporal summation were associated with higher hand pain intensity [25]. These results suggest that the sensitization-associated pain phenotype is common to OA in different locations. In a study of 216 patients with different degrees of knee pain, although all subjects showed high physical impairment, low quality of life, and high pain catastrophizing compared to controls, four distinct knee pain profiles with unique combinations of biochemical markers, pain biomarkers, physical impairments, and psychological factors were apparent [26]. These observations suggest that mechanism-based diagnosis and individualized treatment may be possible for the treatment of knee OA pain. In the early stages of OA, pain occurs intermittently in response to specific activities and movement, which is characteristic of nociceptive pain [27]. As OA progresses, however, pain increases, such that it becomes constant and occurs even at rest [28]. There is debate regarding whether the aggravation of pain is indicative of the development of neuropathy arising from damage to the neurons innervating the joint. In two histological studies that used samples of tissue from patients with end-stage OA, relevant changes in joint innervation for the generation of pain, such as tidemark breaching by vascular channels containing sympathetic and sensory nerves, the presence of free nerve fibers within the subchondral bone marrow, and decreased nerve fibers in synovium with inflammation, were demonstrated [29,30]. Clinical trials examining the efficacy of neuromodulating agents, such as serotonin-norepinephrine reuptake inhibitors (SNRIs) and anticonvulsants, showed inconsistent results in pain reduction, however, and none of these agents reached a high level of recommendation in recent OA treatment guidelines [31,32]. NGF induces sensitization of peripheral nociceptive terminals as well as the development of the nervous system, and plays a key role in acute and chronic pain [33]. Biologic agents that specifically block NGF revealed dramatic pain relief among OA patients, and despite their unique side effect profile, such as alterations in peripheral sensation and development of arthropathies, they are in active clinical development [34,35].

#### *2.2. Animal Models for Studying the Pathogenesis of OA Pain*
