Pain alleviating effects of mesenchymal stem/stromal cells and derived extracellular vesicles in osteoarthritis

Musculoskeletal disorders pose significant burdens on both individuals and society. Osteoarthritis (OA) is a common form of musculoskeletal disease affecting around a third of the population aged over 60 years old. It is characterised by debilitating joint pain and stiffness that only partially correlates with joint structural damage. Clinically used OA treatments fail to halt disease progression and yield limited pain relief. During the search for new therapies for OA, mesenchymal stem cells (MSCs) and derived extracellular vesicles (MSC-EVs) have led to promising therapeutic outcomes such as joint function improvement and pain relief. Whether the observed pain relief effects of MSCs and MSC-EVs originate from the direct effect on pain-sensing neurones in OA joints remains unknown. This Thesis aims to uncover the mechanisms of the pain-alleviating effects of MSCs and MSC-EVs in OA joints. To study whether MSCs and MSC-EVs reduce pain through acting on pain-sensing neurones in OA joints, it is essential to examine whether the presence of MSCs and MSC-EVs influence naïve sensory neurone activity. To do this, an in vitro co-culture was established to study the excitability and ion channel activities of dorsal root ganglion (DRG) neurones, the cell bodies of knee-innervating sensory neurones located in the DRG, both in direct and indirect co-culture with MSCs. Results showed that neither the excitability of nor the tested ion channel function of naïve DRG neurone altered when co-cultured with MSCs and the MSCs conditioned medium in vitro. These results provided a proof of concept of using an in vitro co-culture model to study the impact of MSCs on DRG neurone activity. Additionally, understanding the molecular mechanisms of OA pain at different stages of the disease is also critical for unveiling the analgesic mechanisms of MSCs and MSC-EVs. Thus, a surgically induced mouse OA model, destabilisation of the medial meniscus (DMM), was used to identify molecular targets mediating OA pain. DMM operated mice were kept for 8 weeks to develop OA, during which pain-related behaviour changes were monitored by the digital ventilated cage (DVC) system and digging behaviour testing. After 8 weeks, DMM mice presented moderate OA in operated joints but not pain-related behavioural changes. Further characterisation of knee-innervating neurones showed that such neurones from DMM mice were significantly more depolarised than those from Sham mice. Additionally, DRG neurones isolated from 8-weeks DMM mice showed increased Ca2+ influx in response to P2X3R agonist α,β me-ATP, and increased P2X3R immunoreactivity was also seen in the DRG of 8-week DMM, which suggest the potential role of P2X3R in OA pain at this stage of the disease. With the establishment of in vitro and in vivo models for OA pain study, the pain alleviation effects of MSCs and MSC-EV in advanced OA were next investigated. Using the DMM induced OA model, it was found that MSCs and MSC-EVs improve pain-related behaviour changes (i.e. improved motor coordination, digging and sleep) in DMM mice, and such behavioural improvement was not the result of reduced joint damage, but rather normalised excitability of knee-innervating sensory neurones from MSCs and MSC-EVs treated DMM mice. Furthermore, in vitro study showed that MSC-EVs can normalize sensory neurone hyperexcitability induced by nerve growth factor (NGF). Together, these results suggested that MSCs and MSC-EVs improve pain in OA which may be through a direct action on peripheral sensory neurones. Mice have been the predominant species used for OA research in this Thesis. However, the genetic, biomechanical, and anatomical differences between mice and humans can hinder the clinical translation of observed outcomes. Thus, this Thesis further explored the possibility of using sheep as an animal model for OA pain research. An osteochondral defect (OD), a common clinical observation in joints leading to OA, was created in sheep, and the sensory neurone properties of these operated sheep were investigated. Increased sensory neurone excitability and increased transient receptor potential vanilloid 1 (TRPV1) ion channel function were observed in DRG neurones innervating the OD operated joint, which demonstrated the utility of sheep for the study of joint pain mechanisms. Overall, this Thesis discovered that MSCs provide pain relief in OA joints through normalising sensory neurone activity, possibly via the release of MSC-EVs. Additionally, this Thesis also established an in vitro model that can be used for further study of MSCs and sensory neurone interactions and explored the possibility of using sheep as an animal model for orthopaedic pain research.