Knee pain will be graded by a VAS from 0 to 100 mm, with 0 indicating "No pain" and 100 indicating "Worst possible pain".

WOMAC is a self-administered, disease-specific questionnaire that assesses the severity of OA symptoms. The reliability and validity of this index have been confirmed in many clinical trials. WOMAC subscales consist of pain (5 items), stiffness (2 items), and physical function (17 items). Each item is rated from 0 to 4, totaling scores of 0-20, 0-8, and 0-68 for pain, stiffness, and function subscales, respectively.

WOMAC is a self-administered, disease-specific questionnaire that assesses the severity of OA symptoms. The reliability and validity of WOMAC have been confirmed in many clinical trials. WOMAC subscales consist of pain (5 items), stiffness (2 items), and physical function (17 items). Each item is rated from 0 to 4, totaling scores of 0-20, 0-8, and 0-68 for pain, stiffness, and function subscales, respectively.

WOMAC is a self-administered, disease-specific questionnaire that assesses the severity of OA symptoms. The reliability and validity of WOMAC have been confirmed in many clinical trials. WOMAC subscales consist of pain (5 items), stiffness (2 items), and physical function (17 items). Each item is rated from 0 to 4, totaling scores of 0-20, 0-8, and 0-68 for pain, stiffness, and function subscales, respectively.

PGA-OA score will be assessed using a 100 mm visual analogue scale (higher is worse).

The SF-12 Quality of Life questionnaire includes 8 multi-item domains (physical function, social function, role-emotional, role-physical, bodily pain, general health, mental health, and vitality). These can be combined into 2 summary measures (physical and mental component summary measures).

TUG is a functional performance measure specifically studied in persons with OA of the hip and knee, which directly evaluates an individual's ability to transfer, ambulate, and maintain balance during transitions. The TUG assesses the time it takes participants to get up from a standard-height chair, walk 3 m, turn and return to the chair, and sit down again. The TUG has good interrater and intrarater reliability and validity for functional testing in older adults.

The chair-stand test will use a standard chair with a 47-cm seat height. Participants start the test seated, with arms crossed over the chest, and are instructed to rise to a full stand and return to the initial seated position as many times as possible in 30 seconds. The total number of completed chair stands is averaged across two trials and used for analysis. A greater number of chair stand repetitions is interpreted as better performance.

Both knees will be assessed with the participant supine and the knee in 30° flexion. The suprapatellar bursa will be scanned according to the Outcome Measures in Rheumatology (OMERACT) atlas. Maximal depth of effusion and synovial thickness will be measured in millimeters. Power Doppler signal observed in the synovial membrane in both longitudinal and transverse planes will be scored using a semi-quantitative grading system, from 0 to 3 (0 = absent, 1 = mild, 2 = moderate, 3 = marked or severe).

The consumption of rescue medication will be recorded at each visit and in the daily logs.

The blood samples will be collected in the morning after an overnight fast at baseline and the following visits to measure the level of CRP in serum.

Stool and saliva samples will be collected at baseline and the following visits. Microbial diversity will be quantified via the Shannon diversity index (α diversity) and unweighted Unifrac distance (β diversity), and microbiota composition will be identified on different levels, including phylum, family, and genus.

Adverse events and serious adverse events will be measured and recorded.

The grip strength of the participants' dominant hand will be measured using a calibrated Jamar dynamometer with the participants in the sitting position (Patterson Medical, Ltd., Nottinghamshire, UK). Three grip strength measurements will be taken at 10 s intervals, and the maximum value of the three measurements will be used as the participant's final grip strength.

A trained technician will measure the BMD of all participants. The total body BMD and multiple site-specific BMD (i.e., the lumbar spine, pelvis, trunk, femoral neck, trochanteric, and ward's triangle) will be all measured. The dual-energy X-ray absorptiometry (DXA) scan results are reported as absolute values of BMD (g/cm2). The same DXA machine will be used for all participants. All technicians will receive training to ensure the reproducibility of the BMD measures.

Body composition assessment provides insights into the nutritional status and functional capacity. It helps understand nutrition in the developmental origins of health and disease and in monitoring therapeutic interventions. The whole body muscle mass will be measured using DXA.

Body composition assessment provides insights into the nutritional status and functional capacity. It helps understand nutrition in the developmental origins of health and disease and in monitoring therapeutic interventions. The whole body fat mass will be measured using DXA.

PPT measures sensitivity to pain evoked by mechanical stimulation of nociceptors. PPT will be assessed at the center of patellar in the index knee and at the distal radioulnar joint (right side unless contraindicated) by applying an algometer (1 cm2 rubber tip; Wagner, FDIX25) at a rate of 0.5 kg/s, with the calculation of averaging three trials at each site.

Mechanical temporal summation (mTS) measures an augmented response to repetitive mechanical stimulation. mTS will be assessed using a weighted 60 g von Frey monofilament at the center of patellar in the index knee and at the distal radioulnar joint (right side unless contraindicated). Subjects first provided a numerical pain rating to a trial of four stimulations. Subsequently, the monofilament was applied repeatedly over the skin of the same site at a frequency of 1 Hz for 30 s. Subjects provided a pain rating at the completion of the train of 30 simulations, and 15 s post stimulation. Temporal summation was defined as being present when, compared with the initial trial, the subject reported increased pain following the repeated mechanical stimulation at the site being tested. If there is no difference between the two trials, then the participant is not defined as temporal summation.

HADS is a self-report scale and consists of 14 items, 7 of which investigate depression and 7 anxiety symptoms, providing a score of 0 to 21 for each domain, where higher scores equal greater involvement of either anxiety or depression. The purpose of the scale is not to make a diagnosis. It is to determine the risk group by screening for anxiety and depression in a short time in patients with physical illness. In addition, the scale can be used to evaluate the change in the emotional status of patients.

The PD-Q includes three questions about knee pain intensity rated via NRSs ("no pain" (score 0) to "worst pain possible" (score 10)), pain course pattern rated via a diagram displaying four possible pain course patterns (scored -1 to 2 depending on which pain course pattern is selected), a question about pain radiation (answered yes/no, and scored 2 or 0 respectively), and seven questions about somatosensory phenomena each rated on 6-point Likert scales ("never" (score 0) to "very strongly" (score 5)). Total scores are obtained by summing scores for all items, ranging from 0 to 38. Higher scores indicate more neuropathic-like symptoms. Scores of ≤12 indicate pain is unlikely to be neuropathic and scores of ≥19 suggest pain is likely to have a neuropathic component. Scores ≥13 and ≤18 indicate uncertain results and the possibility of a neuropathic component.