Molecular Pathways Involved in Knee Pain

Knee osteoarthritis (OA) is the most common form of arthritis and the most common cause of knee pain in the world. The rate of knee arthritis is as high as that of cardiac disease and is the most common problem in individuals over the age of 65.

Central Sensitization (CS) is a marker of widespread pain sensitivity that can occur throughout the central nervous system distribution, leading to changes in the spinal cord as well as in the brain. The presence of CS increases the complexity of the clinical picture and can negatively affect treatment outcomes. CS is present in \>20% of patients suffering from knee OA indicating that in the majority of individuals suffering with painful knee OA, knee pain should be related to molecular changes in the joint. CS might be also associated with discrete synovial fluid proteomic signatures due to the generation by the joint of chemical mediators (e.g. nerve growth factor) that drive CS, or CS might moderate the relationship between synovial fluid proteomic signatures and symptoms due to alterations in pain processing.

The aim of this study is to explore the potential molecular links between pain and structure on knee pain using synovial fluid proteomics. A secondary purpose is to explore the association of knee pain with biomarkers of stress, metabolism and dietary habits.

In a single session, ultrasound-guided synovial fluid, blood urine and saliva extraction, clinical assessment, completion of a questionnaire booklet and knee x-rays will be conducted. The clinical assessment will measure three features of central sensitisation (sensitivity to blunt pressure on the most painful knee, changes in pain felt during repeated light pricking of the knee skin, and reduction in pain that accompanies inflation of a blood pressure cuff on the non-dominant arm), features of leg strength (dynamometer, time up-and-go test) and features of balance (sway). Participant involvement at each session is expected to last less than 3 hours.

Individuals over 45 having complaints of knee pain for 3-6 months are eligible to participate. The clinical assessments, questionnaire completion and subsequent statistical analysis are expected to be completed within 18 months of study commencement.

The findings can provide more insight into the traits of knee pain, allow the examination of possible correlations to each other, and highlight potential detrimental effects of them on knee joint health.

Osteoarthritis (OA) is the most common cause of disability in the elderly population and most individuals suffering from osteoarthritis are managed in the primary care setting. Knee OA is the most common form of arthritis and the most common cause of knee pain in the world. The rate of knee arthritis is as high as that of cardiac disease and is the most common problem in individuals over the age of 65. In the United Kingdom, 10% of 65 to 74-year-old individuals consult their general practitioners about OA per year. Out of the entire population, 4% attend their general practitioners as a result of knee OA, and half of them (2%) consult their general practitioner for the first time or with the acute flare of knee arthritis.

There is a considerable body of evidence showing augmented central nervous system (CNS) processing in OA. Central Sensitization (CS) is a marker of widespread and centrally augmented pain that refers to those neurophysiological processes that can occur throughout the CNS distribution, leading to changes in the spinal cord as well as in the brain. The presence of CS increases the complexity of the clinical picture and negatively affects a range of outcomes (e.g. pain, disability, negative affect, quality of life) following treatment. CS is not present within all patients with chronic pain rendering identification of those patients and decision-making for the right management approach even harder. Clinically, CS manifests as hypersensitivity to pain, that sometimes spreads beyond peripheral generators and is a marker for pain chronicity. CS of nociceptive pathways is a mechanism of clinical pain amplification in OA and is present in \>20% of patients suffering from knee OA. This means that in the majority of individuals suffering with painful knee OA, knee pain should be related to molecular changes in the joint. CS might be also associated with discrete synovial fluid proteomic signatures due to the generation by the joint of chemical mediators (e.g. nerve growth factor) that drive CS, or CS might moderate the relationship between synovial fluid proteomic signatures and symptoms due to alterations in pain processing. Radiographic knee joint changes and pain levels are associated with knee cartilage loss but whether CS indices are linked to radiographic changes has not been established. High levels of CS might increase the risk of cartilage loss by increasing the levels of pain or might be associated with already existing changes.

The investigators will recruit 140 individuals with OA-related knee pain. They will also use standardised quantitative sensory testing (QST) such as pressure pain detection threshold (PPT), temporal summation (TS) and conditioned pain modulation (CPM). PPT specifically, has been used in past knee pain studies and is considered a valid and reliable method to establish tenderness around the knee joint. Similarly, TS has been used previously to establish whether individuals demonstrate amplified, centrally driven localised knee pain. Conditioned pain paradigms are commonly used to assess the function of endogenous pain inhibitory pathways in humans. In this technique, a painful test stimulus is evaluated in the absence and then in the presence of a second, also painful (conditioning), stimulus applied to a remote region of the body. In a typically functioning nociceptive system, the amount of pain experienced with the primary test stimulus will be reduced during the presentation of the secondary conditioning stimulus. Decreased inhibition of experimental pain is found in many patients with idiopathic pain syndromes. It predicts the tendency to develop future chronic pain. The purpose of using QST is to establish objective and quantifiable data that will allow the stratification of patients into 'sensitised' and 'non-sensitised' and permit further analysis.

Quantitative Sensory Testing is a reliable and valid method to assess for the presence of CS and demonstrates predictive capacity in relation to musculoskeletal (MSK) treatment outcomes. The testing consists of pressure pain threshold (PPT), punctate thresholds, temperature sensitivity, temporal summation (TS) and conditioned pain modulation (CPM) used to quantify noxious or innocuous stimuli within healthy individuals and patients alike. QST has been used, among others, as a screening and assessment tool for sensory abnormalities in patients with pain disorders, as well as to assist in the stratification of patients and evaluate the clinical aspects of peripheral and CS.

Regional pain and symptoms of depression and/or anxiety have been shown to facilitate the development of constant pain therefore, signs of depression or anxiety are important to explore correlation of symptoms at a single time-point. Signs of depression and anxiety can be identified with the use of the Hospital Anxiety and Depression Scale (HADS) that has been shown to be valid in multiple populations. The stress response involves activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis. Any physical or psychological threat to homeostasis triggers release of corticotrophin-releasing hormone in the hypothalamus, and ultimately raises levels of steroid hormones such as cortisol in the blood stream and saliva. In the short term, cortisol helps to meet the demands of stress by mobilizing energy stores, and assists recovery from stress by inhibiting further release of corticotrophin-releasing hormone. However, continuing stress promotes maladaptive functioning of the HPA axis, which, in turn, may compromise metabolism, impair immune function, and alter cardiovascular control. Cortisol levels in saliva have been implicated in chronic pain presentations (pain severity) and can be considered a useful biological biomarker that can be used to explore psychopathological associations, prognosis and treatment outcomes.

Nutritional information collected via a standardised food frequency questionnaire (FFQ) and metabolic evidence from body secretions can provide significant insight about the course of OA, as the presence of glucosamine and chondroitin sulfate in faecal samples has shown to improve the symptoms of the condition and delay its progression. Nutritional information and metabolic evidence from faecal samples can be used to explore associations of gut microbiota with pain sensitivity and stress levels.

The widely utilised Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) will be also used for the evaluation of knee OA. It is a self-administered questionnaire made of 24 items and consists of three subscales covering pain, stiffness and physical function. It has been used extensively and is considered a valid and reliable tool. For evaluating quality of life, stiffness, generalised well-being, sleeping difficulties and understanding of the diagnosis and treatment, the investigators will use patient-reported outcome measure 'The Versus Arthritis Musculoskeletal Health Questionnaire' (MSK-HQ). MSK-HQ covers a broad aspect of musculoskeletal conditions, and recent studies have shown it to be reliable and valid. Frailty has been also found to be a predictor of disability and a determinant of treatment outcomes and the Simple Frailty Questionnaire (FRAIL) is considered a valid and reliable tool for that purpose.

Both the 30-second sit to stand test (30CST) and the 'time up and go' (TUG) test will be used to see if patients have improved their lower limb fitness levels. 30CST has shown excellent reliability and validity. TUG has been widely used in clinical setups and is a valid tool to assess necessary functional mobility.

In this study, the sleeping pattern of individuals suffering from degenerative changes of the knee will be assessed with the aim to establish whether sleep disturbance is implicated in the development of CS. Disturbed sleep is a frequent complaint of people experiencing chronic pain such as those with knee osteoarthritis (OA). Changes in sleep architecture can affect health even in the presence of apparently adequate sleep duration. Insufficient amounts of slow wave sleep are associated with hypertension, type 2 diabetes mellitus, poor cognition, and obesity. Sleep disturbances are present in 67-88% of people with chronic pain and ≥50% individuals with insomnia have chronic pain. The investigators will also use the Pittsburgh sleep quality index (PSQI) which has been used in multiple studies and validated to measure sleep disturbances. Poor cognition, as well as sleep, has been found to be a marker of persistent pain and a trait of centrally driven pain in knee pain populations. The Cognitive Failures Questionnaire (CFQ) is a valid and reliable tool to measure self-reported failures in perception, memory, and motor function.

Musculoskeletal Ultrasound scan (MSK-USS) will be also conducted on the knees of participants to establish if they have inflammation of the synovial membrane. There is enough evidence that inflammation is present in all stages of OA. Synovitis or inflammation of synovial fluid is associated with pain, disease severity and, OA progression. Synovitis manifests as synovial membrane thickening, increased vascularity, and/or joint effusion. Synovial hypertrophy, synovitis and knee effusion are linked with arthritis in the knee and associated with knee pain in osteoarthritis. The synovial fluid will be aspirated (subject to participant consent), in order to establish a phenotype which is strongly associated with OA. Studying synovial fluid biomarkers alongside clinical, radiographic and ultra-sonographic characteristics is one strategy to improve resolution and stratification into targetable OA phenotypes. Synovial fluid aspiration will be ultrasound guided as it increases the accuracy of needle placement compared to blind needling (95.8% versus 77.8%, p \< 0.001) reduces procedural pain by 43%, improves effusion detection by 200%, and volume of synovial fluid aspirated by 337% compared with blind synovial fluid aspiration. Ultrasound guidance also reduces procedural pain (43% reduction) in knees with no palpable effusion and increases the responder rate and therapeutic duration by 107% and 36% respectively.

Isometric quadriceps strength will be also assessed to establish current strength levels of vastus medialis muscle and see whether muscle strength associates with centrally driven pain or proteomic synovial concentrations. Quadriceps muscles strength deficits are associated with knee osteoarthritis. Isometric testing will be done at 30 and 60 degrees of flexion as per the protocol of a previous study.

Blood samples will be also extracted to assess the biomarkers (including serum levels and gene expression levels of various molecules) and to establish insulin resistance. Urine samples will be collected to identify the existence of collagen degradation markers (e.g. UTXII) and inflammatory regulator markers (e.g. Maresins).

Collection and study of these parameters can provide more insight into the traits of knee pain, allow the examination of possible correlations to each other, and highlight potential detrimental effects of them on knee joint health.

Eligibility Criteria:
Inclusion Criteria:

* have the ability to give informed consent
* be 45 years or over
* have complaints of knee pain for 3-6 months with or without radiographically established OA (K/L scale score ≥ 1)
* have complaints of knee pain for 3-6 months with or without satisfying the non-radiographic American College of Rheumatology criteria for knee OA
* are willing to undertake knee synovial fluid aspiration
* be able to speak, read, and write in English as all instructions and questionnaires are designed in the English language

Exclusion Criteria:

* Inability to give informed consent due to cognitive impairment or otherwise - (capacity levels are already established under General Practitioner care)
* Inability to understand key aspects of the study due to cognitive impairment or otherwise
* Giving history of additional co-morbidities such as cancer, neurological conditions, inflammatory joint diseases including rheumatoid arthritis, diabetic neuropathies, fractures or other conditions causing greater disability than their knee pain
* Acute soft tissue injury to the knee within last 3 months before potential recruitment