Grand Challenges in Musculoskeletal Pain Research: Chronicity, Comorbidity, Immune Regulation, Sex Differences, Diagnosis, and Treatment Opportunities

Musculoskeletal pain involves bones, joints, and related muscular tissues and includes most debilitating pain conditions such as low back pain, arthritic pain, and widespread muscle pain. Chronic musculoskeletal pain is the most predominant among chronic pain conditions and presents a serious challenge to primary care. Pain arising from musculoskeletal tissues is characteristic of deep pain and has important differences from that of cutaneous pain [Table 1; (1)]. Compared to cutaneous tissue injury, the injection of the same amount of inflammatory agent into the joint induces more intense inflammation and greater and more widespread neuronal activation in the pain transmission pathways (2). Musculoskeletal nociceptive inputs appear more effective in inducing neuronal excitation and produce greater sensory disturbances (3, 4), which may explain predominant chronic pain conditions involving deep tissues. The underlying etiology and pathology of chronicmusculoskeletal pain conditions are poorly understood even after decades of research.


INTRODUCTION
Musculoskeletal pain involves bones, joints, and related muscular tissues and includes most debilitating pain conditions such as low back pain, arthritic pain, and widespread muscle pain. Chronic musculoskeletal pain is the most predominant among chronic pain conditions and presents a serious challenge to primary care. Pain arising from musculoskeletal tissues is characteristic of deep pain and has important differences from that of cutaneous pain [ Table 1; (1)]. Compared to cutaneous tissue injury, the injection of the same amount of inflammatory agent into the joint induces more intense inflammation and greater and more widespread neuronal activation in the pain transmission pathways (2). Musculoskeletal nociceptive inputs appear more effective in inducing neuronal excitation and produce greater sensory disturbances (3,4), which may explain predominant chronic pain conditions involving deep tissues. The underlying etiology and pathology of chronic musculoskeletal pain conditions are poorly understood even after decades of research.

EXPERIMENTAL MUSCULOSKELETAL PAIN -RELEVANCE TO CHRONICITY
Human muscle pain can be induced experimentally by injecting the hyperalgesic agents such as hypertonic saline (5), capsaicin (6), glutamate (7), serotonin (8), or nerve growth factor (9, 10) into the muscle. To avoid invasive procedures, a short-wave diathermy-induced human muscle pain model has been developed (11). These human muscle pain models help to advance our understanding of the mechanisms and improve the treatment of muscle pain. A limitation of these models, however, is that the induced muscle pain hypersensitivity would resolve within minutes to hours [e.g., (5,12)]. Nerve growth factor induces relatively long-lasting hyperalgesia for 4-7 days to a few weeks after injection into the muscle in humans (9, 13, 14). Chronic pain is defined as pains that persist or recur for longer than 3 months (15). While acute and chronic pain shares similar neural mechanisms, chronic pain is underlying by distinct mechanisms of central sensitization and still unsettled involvement of the immune system (16)(17)(18). Although these human experimental models mimic aspects of acute or persistent pain, they are not necessarily suitable for studying pain chronicity, a deteriorating and devastating problem for patients.
Animal persistent musculoskeletal pain models have been developed to simulate chronic pain conditions in humans. A variety of methods can be used to assess muscle/joint pain in animals,  (19). Pain hypersensitivity occurs in multiple joints after 10 days and lasts up to 3 weeks. Later studies revealed that a systemic disease was induced in this model that included skin lesions, destruction of bone and cartilage, impairment of liver function, and lymphadenopathy, which made it difficult to differentiate pain behavior from generalized malaise and debilitation, and led to ethical concerns (20). Injection of inflammatory irritants into the joints and muscles, including CFA, carrageenan, zymosan, mustard oil, formalin, capsaicin, bee venom, acidic saline, lipopolysaccharide, inflammatory cytokines, monosodium iodoacetate, and sodium urate crystals, has been used to produce tissue injury and hyperalgesia [(21), review]. These models mainly reflect early injury responses and there is still a significant challenge to develop models that can be translated into human chronic musculoskeletal pain conditions [see (22)]. Surgical interventions are employed to model chronic low back pain, the most commonly seen chronic musculoskeletal pain [reviewed in (23)]. The procedures include the compression of the dorsal root ganglion (24), disruption of the lumbar intervertebral disc (25), implantation of tissues into the lumbar epidural space to mimic disc herniation (26), injection of complete Freund's adjuvant into the intervertebral disc or nucleus pulposus (27), and surgical application of zymosan into the epidural space to induce inflammation of the dorsal root ganglion (28). These approaches lead to behavioral hyperalgesia resembling human low back pains such as discogenic and radicular back pain and low back pain related to local inflammation. The pain hypersensitivity after surgical interventions can last for up to months (24,25,27), longer than that after a simple injection of algesic mediators. To mimic soft tissue musculoskeletal pain such as tendinopathy, a constriction injury of the tendon of the rat masseter muscle produces pain hypersensitivity that lasts for months (29,30). These models may be employed to identify distinct mechanisms that contribute to the development of chronic musculoskeletal pain.
The new International Classification of Diseases-11 has updated the chronic pain classification to include the Primary chronic pain category to designate chronic pain that cannot directly be ascribed to any disease of structural injury. Fibromyalgia, a chronic condition characterized by widespread pain involving musculoskeletal tissues, is a type of primary chronic musculoskeletal pain and women appear to be affected more than men (31,32). A reserpine myalgia model has been presented to model fibromyalgia in animals (33). In this model, a daily subcutaneous injection of reserpine (1 mg/kg) was repeated for 3 consecutive days induced decreased muscle pressure threshold and allodynia. While the reserpine approach is promising, the model's relevance to fibromyalgia is still an issue. The reserpine-induced myalgia would only persist for about 1 week and the sex difference in fibromyalgia prevalence is not reproduced (33,34). Reserpine depletes monoamine neurotransmitters norepinephrine, dopamine, and serotonin by inhibiting vesicular monoamine transporters (35). Reserpine-induced pain suggests that these monoamines exert a net inhibitory effect on nociception. It is interesting to note that Catechol-O-methyltransferase (COMT) metabolizes catecholamines, but inhibition of COMT leads to enhanced catecholamine levels and pain hypersensitivity via beta2/3-adrenoceptors (36). The imbalance of monoamines in fibromyalgia and functional pain syndrome requires further investigation.
The preclinical muscle/bone/joint pain models have contributed greatly to our understanding of the biological mechanisms underlying musculoskeletal pain. However, there are still gaps in our understanding, particularly with the development of pain chronicity. Most studies have settled to use the observed persistent pain within days or a couple of weeks after the injury as a surrogate of chronic pain in humans, despite that the early persistent pain could still be a type of acute pain and may miss characteristics of chronic pain that occurs late. Recent studies have reported month-long upregulation of microglial markers in the spinal cord after nerve injury and differential cytokine profiles between the early and late phases of hyperalgesia (37). The transcription factor NF-kB is known for its immediate pro-inflammatory role, but it also contributes to the resolution of inflammation at the late phase of inflammation (38). Reevaluation of the CFA inflammatory hyperalgesia model indicates that the temporal course of mechanical hyperalgesia consists of an initial developing phase with peak hyperalgesia at 4-24 h, a subsequent attenuating phase of a few weeks, followed by a late persistent (chronic) phase that lasted for months (18). Importantly, different cellular mechanisms are involved in the early acute phase and late chronic phase of CFA-induced hyperalgesia, as suggested by a late downregulation of astroglial glutamate transporters that occurs at a time when hyperalgesia transitions into the persistent chronic phase (18). Thus, preclinical studies on chronic musculoskeletal pain need to attend to the late chronic phase of pain hypersensitivity. The central mechanisms involved in the transition of chronic muscle pain should also be studied (39,40). We are challenged to differentiate the factors relevant to the transition and maintenance of musculoskeletal pain chronicity. Myalgia is one of the major symptoms of Covid-19 (41,42). It remains to be seen whether it could develop into a chronic problem.

WIDESPREAD AND COMORBID PAIN CONDITIONS
Widespread pain is commonly seen in musculoskeletal pain conditions, which is characteristic of deep tissue pain that refers to the areas remote from injury. The temporomandibular joint disorders (TMJD) patients not only have pain in the TMJ and muscles of mastication but also pain in other muscles and joints (43,44). Somewhat conceptually overlapping with widespread pain, patients with chronic musculoskeletal pain frequently have other comorbid pain conditions: fibromyalgia and TMJD (45,46), migraine (47), and visceral pain (48), TMJD and headache (49), and Ehlers-Danlos syndromes (joint pain) and Chiari Malformation (headache and neck pain) (50). A causal relationship between the comorbid pain conditions is often difficult to determine regarding which condition triggers the other, but the involvement of multiple body structures suggests a central-mediated effect (51,52).
Widespread and comorbid muscle pains can be reproduced in animal models. Repeated unilateral injections of acidic saline into the gastrocnemius muscle of rats produce bilateral hyperalgesia that lasts for up to 30 days (53), which mimics persistent widespread muscle pain in humans. Unilateral injection of CFA into the masseter muscle induces bilateral behavioral hyperalgesia (54). Combined masseter muscle inflammation and stress induce visceral hypersensitivity similar to that seen in comorbid TMJD and irritable bowel syndrome patients (55). Interestingly, the inflammatory pain of the craniofacial muscle in rats can spread to the hind paw, but not vice versa (56). Since primary sensory afferents from the craniofacial region project to a wide region of the brain, in contrast to distinct somatotopy of spinal afferents (57), craniofacial musculoskeletal pain tends to induce comorbid pain conditions. The cellular mechanisms underlying the comorbidity of chronic musculoskeletal pain remain to be elucidated.

HOMEOSTATIC IMMUNE REGULATION IN PERSISTENT PAIN
The development of persistent or chronic pain largely depends on the interactions between the nervous and immune systems, which involves glia that function as immune cells in the brain (17,58). The pain-related neuroimmune interactions are reciprocal and involve neurotransmitters and their receptors and immune mediators including cytokines and their receptors. In inflammatory hyperalgesia after injection of CFA into the masseter muscle, there is reactive astrogliosis, induction of proinflammatory cytokine IL-1b, and the coupling of NMDA receptor phosphorylation through IL-1 receptor signaling (59). In ischemic myalgia, IL-1b signals through IL-1 receptor to upregulate acid-sensing ion channels to induce nociceptor sensitization (60). Activation of P2X4 receptors on muscle macrophages leads to IL-1b release and muscle hyperalgesia (61).
Despite evidence from preclinical studies, clinical trials for the treatment of chronic pain with glial modulators have been unsuccessful (62), which would suggest our incomplete understanding of the mechanisms. From imaging studies on chronic low back pain patients, it is observed that glial activity is negatively correlated with the levels of pain and IL-1b, suggesting an inhibitory role of glia related to the translocator protein, the marker used for glial activation in the study (63). This inhibitory role of glial activity has largely been overlooked. The immune system provides balanced regulation to maintain normal function. The effector and regulatory T cells (Teffs and Tregs), for example, are pro-and anti-inflammatory, respectively (64). The depletion of Tregs delays pain resolution (65) and enhances neuropathic pain (66). Tumor necrosis factor (TNF) is proinflammatory through the TNFR1 while antiinflammatory via TNFR2 (67). Deletion of TNFR2 hampers, but activation of TNFR2 promotes recovery from neuropathic pain (65). The known proinflammatory NF-kB pathway is involved in the development of pain hypersensitivity but also involved in bone marrow stromal cell-produced pain relief in a tendinopathy model (68), suggesting dual roles of NF-kB in hyperalgesia and pain relief according to circumstances. These results suggest that the perturbation of the balanced or homeostatic immune regulation, not only immune activation, leads to disease conditions including chronic pain. In search of treatment strategies targeting the immune system, it would be ideal to return to homeostasis by strengthening the antiinflammatory/protective profile, instead of simply shutting off immune activation.

SEX DIFFERENCES
The phenomenon of female predominance in chronic pain has been a topic of interest in recent decades (69)(70)(71), which has led to the National Institutes of Health (NIH) mandate to include sex as a biological variable in NIH-funded Research (72). Similar to other chronic pain conditions, women appear to be affected more than men by chronic and co-morbid musculoskeletal pain (73,74). While the relative prevalence of chronic pains in females and males can be quantified, we still need to find answers for the factors that contribute to these differences. In addition to document different levels of pain, more focus should be on the underlying differences in biology between males and females. Preclinical studies have shown male-specific involvement of Tolllike receptor 4 in persistent pain [see (71)]. A recent report shows that the upregulation of genes that escape X chromosome inactivation is correlated to the development of co-morbid chronic musculoskeletal pain after a car accident in women, but not in men (74). Elucidating the underlying differential biology in sex differences in pain would be important in avoiding the biased development of pain medicine.

DIAGNOSIS AND ETIOLOGY
Most of our knowledge about mechanisms of persistent pain is based on studies of cutaneous pain in animals, experimental subjects, and patients. Although it is tempting to generalize these findings to the pain of deep tissues such as muscle and joint, there are important differences between cutaneous and deep pain ( Table 1). Deep pains are diffuse and difficult to localize and represent a challenge in diagnosis and identification of etiology. It is often difficult to identify the direct cause of chronic musculoskeletal pain conditions such as TMJD and low back pain (75,76). The diagnosis and etiology of primary chronic musculoskeletal pain, fibromyalgia in particular (77), are still under debate. Clinical studies in this area are much needed to improve the prevention and treatment of chronic musculoskeletal pain.

TREATMENT OPPORTUNITIES
Managing musculoskeletal pain, especially when it becomes chronic, is a daily challenge in primary care. Commonly used pharmacological agents are NSAIDs, opioids, and steroids. Treatment options also include physical therapy, psychotherapy, mesotherapy, whole-body cryotherapy, and alternative or complementary therapies acupuncture, prolotherapy, percutaneous electrical nerve stimulation, and neuromuscular electrical stimulation. The current treatment provides some relief of acute or short-lasting pain but is unsatisfactory for chronic musculoskeletal pain (78)(79)(80)(81). A recent report showed that transcranial magnetic stimulation of the prefrontal cortex attenuated long-term experimental muscle pain in human subjects (14).
Cell-based therapy has shown tremendous promise in the management of chronic musculoskeletal pain in recent years. Patients with disc diseases receiving intradiscal injection of bone marrow concentrate show improvement of discogenic pain through up to a 3-year follow-up (82,83). The clinical improvement is attributable to mesenchymal stromal cells (MSCs) in the bone marrow. Clinical studies have shown the pain-reducing effect of multipotent MSCs in arthritic joint pain (84)(85)(86), rotator cuff disease (87), and discogenic pain (88,89). Large scale randomized controlled trials are necessary to substantiate these exciting findings.
Preclinical studies have addressed cellular mechanisms of MSC-produced musculoskeletal pain relief. In a tendon injury model, a single intravenous injection of bone marrow-derived MSCs produces long-term attenuation of behavioral hyperalgesia (30,90). The pain-attenuation is induced through the interaction of BMSCs with immune cells and mediators, that lead to suppression of proinflammatory cytokines and upregulation of anti-inflammatory cytokines (91), inhibition of NMDA receptor phosphorylation (92), and activation of endogenous opioid receptors (93). One pitfall of systemic MSCs is that they tend to be trapped mostly in the lungs after transplantation (94,95) and there is a case of reversible pulmonary embolism after multiple infusion of adipose tissue-derived MSCs (96). However, MSCs injected locally to the spinal cord or transplanted directly into degenerated disc appeared to survive longer (97,98). The therapeutic effect of MSCs can be further improved by modifying their phenotype before transplantation (99,100). Studies on the use of therapeutic MSCs are rapidly expanding and we are looking forward to the establishment of novel treatment strategies for chronic musculoskeletal pain.

AUTHOR CONTRIBUTIONS
KR confirms being the sole contributor to this work and approved it for publication.

FUNDING
This author's work was supported by NIH grants DE025137, DE029187, and DE029946.