The cerebral mechanism of Tuina for treating knee osteoarthritis pain: study protocol for a randomized controlled parallel trial

Hui Xu Shanghai University of TCM: Shanghai University of Traditional Chinese Medicine Lianbo Xiao (  xiao_lianbo@163.com ) Shanghai University of Traditional Chinese Medicine Guanghua Hospital https://orcid.org/0000-00034226-6002 Chi Zhao Shanghai University of TCM: Shanghai University of Traditional Chinese Medicine Yuchen Xie Henan Provincial Hospital of Traditional Chinese Medicine Bingxin Kang Shanghai University of TCM: Shanghai University of Traditional Chinese Medicine Xirui Xu Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine A Xinyu Shanghai University of TCM: Shanghai University of Traditional Chinese Medicine Jun Xie Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine

obtained before and after treatment to measure clinical change. Adverse events will be documented and assessed throughout the trial.

Background
Knee osteoarthritis (KOA) is a common degenerative joint disease, which is characterized by a failure of cartilage self-repair and secondary hyperosteogeny. The main symptoms of KOA are knee joint pain and limited range of motion secondary to progressive joint deformity and instability [1][2][3]. KOA is reputedly the most common musculoskeletal disease of the lower limb and the main cause of pain and disability among elderly individuals, with 80% of individuals over the age of 65 years showing radiological symptoms of KOA [4]. In China, the overall prevalence rate of KOA is estimated at 15%, with the prevalence increasing to 50% among individual over the age of 60 years and 80% among those over the age of 75 years [5]. Pain is the most important and common symptom of KOA. Early KOA pain often occurs intermittently during exercise and weight bearing. However, with progression of joint degeneration, the pain often becomes persistent, including at rest and at night. The mechanism of KOA pain is unclear, which makes its treatment challenging.
Current management for KOA includes physical activity modi cation and pain relief using a combination of non-pharmacological and pharmacological interventions [6,7]. Among non-pharmacological treatment, massage has been used for the management of KOA pain. Massage is an ancient therapeutic art that is based on the application of pressure [8]. Tuina is a speci c massage technique that has been a component of Traditional Chinese Medicine for over 2000 years. Tuina combines traditional Chinese Medicine practice with current evidence-based parameters of intervention, such as biomechanical function, anatomy, pathology, and physiology.
Tuina is an important part of Traditional Chinese Medicine which works along the meridians and collaterals of the whole body, correcting any displacement in body structure and guiding the individual to appropriate patterns of movement based on symptomology and functional assessment. Tuina is currently rapidly gaining popularity as an alternative and complementary medicine approach owing to its proven positive therapeutic effects.
Previous randomized controlled trials (RCT) have supported the effectiveness of Tuina for the treatment of different health conditions, such as musculoskeletal disorders [9,10] and acute bronchitis [11]. With regard to KOA, two systematic reviews have shown a positive clinical effect of Tuina in improving outcomes for patients with KOA [12,13]. An RCT demonstrated the safety and effectiveness of Tuina for improving knee extension muscle strength, increasing knee joint exibility, and improving KOA-related disability [14].
Exploring the mechanism underlying the effectiveness of Tuina has been an issue of increasing research importance in the eld of alternative and complementary medicine in recent years. Currently, the central effects of Tuina largely remain to be de ned. Our randomized controlled parallel trial was designed to address this gap through a comparative evaluation of the effect of Tuina on the resting-state functional connectivity (rsFC) of the brain in patients with KOA compared to treatment using celecoxib as the control.

Objectives
The purpose of this trial will be to investigate the central mechanisms of Tuina, used as a pain management treatment for KOA, by evaluating the correlation between measured changes in the rsFC, before and after treatment, and improvement in clinical variables. This evaluation will be made against a control group, using celecoxib as the treatment.

Methods/design
Included will be patients with KOA who meet the de nition of osteoarthritis as per the 1991 American College of Rheumatology (ACR) criteria [15]. Patients will be recruited through advertisement in the orthopaedic clinic of the Shanghai Guanghua Integrated Chinese and Western Medicine Hospital, Shanghai, China. We followed the SPIRIT 2013 Statement [16] to guide the development and reporting of our trial protocol. This study has been approved by the ethics committee of Guanghua Hospital of Integrated Traditional Chinese Medicine and Western Medicine (Ethics Approval Number: 2020-K-109) and registered in the Chinese Clinical Trial Registry. All participants will be asked to provide written informed consent and will be informed that the trial will not involve the collection of biological specimens for storage.

Eligibility criteria
The inclusion criteria for enrollment will be as follows: (1) 50-65 years of age and right-handness; (2) KOA-associated pain for a duration ≥ 6 months; (3) no other treatment pursued in the 1-month prior; (4) a score on the knee pain Numerical Rating Scale (0-10 NRS) ≥ 3 in the 1-week prior; (5) a Kellgren-Lawrence radiographic score I or II; (6) patient agreement to not pursue other therapies during the treatment period of the RCT; and (7) provision of written informed consent.

Exclusion criteria
The exclusion criteria will be as follows: (1) skin damage around the knee joint; (2) prior history of knee surgery or severe knee joint trauma; (3) secondary KOA, as a complication of rheumatoid, gouty, or infectious arthritis, or other joint disease; (4) presence of diabetes, systemic infection, severe abnormality in liver and kidney function, and malignancy; (6) allergy to celecoxib, which will be used as the 'usual care' control; (7) pregnant or lactating women; (8) presence of a mental disorder or cognitive impairment; (9) presence of active gastrointestinal disorder, including ulcers, bleeding or recurrent ulcers, and bleeding; and (10) contraindications to magnetic resonance imaging, required for rsFC, including phobias, pacemakers, de brillators, cardiac stents, and intrauterine devices.

Sample size
The focus of neuroimaging research is on determining neural mechanisms of a therapeutic effect. As such, sample size calculation for RCTs in which neuroimaging provides the primary outcome is different from that of classical RCTs. For neuroimaging studies, 12 to 15 participants per group is generally su cient to provide statistically signi cant results [17,18]. In our trial, we will include 20 patients per group, namely the Tuina and the celecoxib control group. Considering an attrition rate of 20% and the possibility of unquanti able rsFC due to head movement, our nal sample size will be set to 24 participants per group.

Recruitment strategies and enrollment
Participant registration will be conducted between June 2022 and June 2024. Written informed consent will be obtained from all participants and will include consent for the use their data in scienti c publications. Figure 1 presents the trial ow, which includes participant recruitment, eligibility screening, randomization, intervention, and outcome assessments. Figure 2 presents an overview of the trial design, conduct, review, and analysis. A completed SPIRIT 2013 checklist (Word) is included (Additional File 1).

Randomisation and blinding
An independent research staff will generate a randomly-numbered sequence, using SPSS21.0 software (SPSS Inc., Chicago, IL, USA), for complete randomization of enrolled participants to the Tuina or celecoxib treatment groups,. The number sequence will be sealed by an independent assistant in an opaque envelope containing treatment information. Eligible and consenting patients with KOA will be randomly assigned to the Tuina group and celecoxib groups using a 1: 1 allocation ratio, with 24 patients per group.
Only the therapist in charge of treatment is authorized to open the envelope and enrol participants. All outcome assessors and data statisticians will be blinded to group allocation.

Interventions
A panel of two therapists with an academic background in Tuina and complementary therapy treatments and hospital health care professionals agreed to develop a standard protocol for the Tuina treatment. The feasibility of the protocol was tested to determine whether it was realistic and workable and to identify practical issues and any adverse effects caused by the procedures. No changes were needed and no adverse events were identi ed. Participants in the pilot study were not included in the actual trial.
Clinical medicine professionals who have ≥ 3 years of working experience and expertise in Tuina were recruited. Training was provided to ensure that the agreed standard procedures are followed. Clinicians will be requested to strictly adhere to the treatment protocol, following the exact steps stated in the manual of standard procedure to minimize differences in the components of treatment provided.
Participants in the Tuina group will receive a 20-min massage session, three times a week (ideally every other day), for 6 consecutive weeks. Participants in the celecoxib group will be treated using celecoxib capsules, using a daily dosage of 200 mg orally, for 6 consecutive weeks.

Tuina treatment
Tuina is based on the meridian system theory, which considers that there are many xed channels of owing Qi energy interlaced as a network throughout the body [19]. There are mainly three kinds of Tuina techniques for the treatment of KOA: the rst is soft tissue massage to stimulate acupoints in the meridian system; the second is joint mobilization; and the third is to guide patients to practice Qi Gong energy exercises [14]. For this study we chose to stimulate acupoints in the meridian system around the knee and knee mobilization in alignment with the most common guiding ideology of Tuina to 'emphasize both bones and tendons' for the treatment of musculoskeletal diseases. Pressure will be applied to ve acupoints which are common local points used to treat knee problems and reduce knee pain [20,21]. Based on the World Health Organization standard acupuncture point location [22], the following acupoints were selected on the affected side: XUEHAI (SP10), LIANGQIU (ST37), YANGLINGQUAN (GB34), YINLINGQUAN (SP9) and XIYAN (EX-LE5). Knee joint mobilization will be performed by pressing on the inferior pole of patella and asking patients to slowly stand from sitting and return to sitting, with 3 repetitions completed.

Celecoxib treatment
Participants in the celecoxib group orally taken a celecoxib capsules (approval number J20140072, manufactured by P zer Pharmaceutical Co. Ltd.) at a daily dosage of 200 mg for 6 weeks.
MRI data acquisition MRI will be performed at the MRI Center at Guanghua Hospital of Integrated Traditional Chinese Medicine and Western Medicine. Resting-state functional magnetic resonance imaging (fMRI) will be used to quantify rsFC at baseline, before treatment, and at the end of the 6 weeks of treatment. MRI data will be acquired using a 3.0-T magnetic resonance scanner (General Electric, Wauwatosa, WI, USA), with a 32channel phase-array head coil, Participants will be asked to remain awake and keep motionless, with eyes closed, during the full scanning period.
To ensure compliance with treatment, participants will be required to register for treatment.

Outcomes evaluations
The clinical outcomes will including pain sensation, pain emotion, and pain cognition.
All evaluations will be performed twice, at baseline and after the 6-week intervention. All evaluations will be evaluated by two licensed physicians who have received training in the use of the outcome measures selected. These are the pressure pain threshold, the Numerical Rating Scale, the Pain Catastrophizing Scale, the Hamilton Anxiety Scale score, and the Hamilton Depression Scale.

Demographic / medical variables
The following demographic and medical variables will be collected for analysis: sex, age, marital status, occupation, ethnicity, education level, blood pressure, temperature, respiration, pulse, height, weight, body mass index, the combination of disease and medication, the Kellgren-Lawrence classi cation, the KOA course, and history of major surgeries.

Adverse events of treatment
The safety of patients to participate will be evaluated before and after enrollment and allocation to group, and will be based on liver and renal function. The following events will be treated immediately by the researchers until resolved: syncope, ecchymosis, pain caused by massage techniques, and symptoms, such as nausea, abdominal pain, indigestion, or increased pain and swelling with the use of celecoxib. All adverse events that occur during the trial will be recorded, including the time of occurrence, symptoms of discomfort, speci c signs, severity, speci c treatment provided, time course of improvement, time to resolution, and date of termination of the treatment and trial.

Data management and monitoring
Clinical data will be carefully saved using printed and electronic case report forms (CRFs). To guarantee data quality, only outcome assessors will access the CRFs for data entry. CRFs will be veri ed for doubleentry and accuracy. During the trial, the Guanghua Hospital of Integrated Traditional Chinese Medicine and Western Medicine will be responsible for making regular visits (once a week) to review the trial conduct. The ethics committee will monitor for protocol violations on a weekly basis and ensure that there are no con ict of interest with the sponsors or researchers. Only statisticians will access the nal trial dataset, which will only contain coded data. The safety, progress, study integrity, and design aspects will be monitored at several meetings of the research team.

Statistical analysis
Before analysis, researchers will provide a statistical scheme to the statisticians. The scheme will include the required data and processing methods. The data will be processed and analyzed by statisticians in accordance with the agreed-upon scheme.
Behavioral data analysis Measurement data: A Shapiro-Wilk test will rst be used to verify the normality of distribution of continuous variables. For data with normal distribution, independent sample t-tests will be used to compare baseline characteristics among the two groups. For non-normally distributed data and categorical variables, a Mann-Whitney U test will be used to compare between-group differences.
Count and grade data: Count data will include the ratio of men and women (sex variable) and the knee joint constituent ratio of the affected side and will be compared between the two groups using a Fourfold Table Chi-Squared Test or row * list Fisher's exact probability Test. The body mass index (BMI) classi cation, Kellgren-Lawrence classi cation, and NRSR score are grade data, with the Mann-Whitney U test used for between-group comparison and the Wilcoxon symbolic test for intra-group comparison. Statistical signi cance will be set at a p-value < 0.05 (two-sided) for all tests.

Seed-to-voxel resting state functional connectivity analyses
Seed-to-voxel functional connectivity analyses will be calculated using the MATLAB R2013b platform with the CONN v17.C software functional connectivity toolbox (http://www.nitrc.org/projects/ conn). Preprocessing will be as follows: (1) removing data from the rst 10 time points to reduce machine instability and the impact of the environment; (2) spatial calibration to estimate and correct head motion; (3) time alignment to unify collection time at all levels; (4) detection of time points with large head movements as covariates of subsequent regression models to remove their effects; (5) registration of the T1 structural image to the functional space and segmented to obtain gray matter, white matter, cerebrospinal uid and related information matrix, and standardization of the functional image to Montreal Neurological Institute (MNI) space, according to the correlation matrix information; (6) Gaussian smoothing of the standardized functional image, using a 6 mm kernel; (7) removal of the smoothed functional image, which includes the in uence of 12 head movement parameters, time point signal of large head movement, white matter, cerebrospinal uid, and other covariables; and (8) a nal ltering, using a 0.01Hz ~ 0.1Hz bandwidth.

Seed-based functional connectivity analysis
Given that the thalamus and periaqueductal gray (PAG) play important roles in the ascending and descending pain pathways respectively, they will be de ned as a priori regions of interest (ROIs), using Automated Anatomical Labeling (AAL), for the functional connectivity analyses of resting-state fMRI data. A threshold of voxel-wise p < 0.005, uncorrected, and cluster-level p < 0.05 false discovery rate (FDR), corrected at cluster, will be applied in the data analysis.

Discussion
Our proposed resting-state fMRI trial will be the rst to evaluate the central mechanisms of Tuina treatment for KOA. Our rsFC analysis will enhance our understanding of the mechanisms underlying the pain relief effect of Tuina in patients with KOA.
Pain is the most important and common symptom of KOA. Early KOA pain often occurs intermittently during exercise and weight bearing; however, with knee joint disease progression, the pain often becomes persistent, at rest and at night. KOA pain can lead to joint dysfunction, reduce patients' physical tness and ability to cope with activities of daily life, as well as cause anxiety and depression [6, 23,24]. With aging of the general population, the prevalence of KOA is gradually increasing, which will cause a signi cant economic and social burden. Modern medicine is still unclear regarding the mechanism of KOA pain. Previous studies have proposed that peripheral factors, such as articular cartilage destruction and synovial in ammation, may play a key role. However, many studies have found that the severity of radiographic KOA and of the in ammatory response of the synovial membrane does not positively correlated with the pain of KOA [25][26][27]. Therefore, peripheral mechanism cannot fully explain the pain of KOA. At the same time, patients with KOA have increased pain sensitivity in non-affected areas. Researchers speculate that an "abnormal central processing" of long-term pain afferent signals may, thus, be a key factor in KOA pain [28,29].
Tuina can safely and effectively relieve the pain, stiffness, movement limitation, and other clinical symptoms of discomfort associated with KOA [30]. Tuina was included as a component of physiotherapy intervention for KOA in the 2018 Expert Consensus on Step Treatment of Knee Osteoarthritis statement in China [31]. With the recent advances in MRI technology, resting-state fMRI can now play an important role in exploring the central mechanism of Tuina. As a non-invasive, in vivo, technique, resting-state fMRI provides a high spatial resolution technique to objectively and reliably quantify the central effect of Tuina which can be correlated to clinical measures to understand the mechanisms by which Tuina relieves pain in KOA.
In summary, this resting-state fMRI trial is designed to investigate the central mechanism of Tuina in the treatment of KOA-associated pain, against treatment using celecoxib as the control. We expect that our ndings can provide a neuroimaging-based reference for the clinical application of Tuina.

Study limitations
Patients cannot be blinded to the treatment received. However, group allocation will be concealed from the researchers, therapists, and outcome assessors. This trial adheres to the principles of the Helsinki Declaration (2013) and was approved by the ethics committee of the participating hospital (Ethics approval numbers: 2020-K-109). All the participants will provide written informed consent before the trial.

Consent for publication
Consent for publication will be obtained from participants.

Availability of data and materials
The datasets and the informed consent form can be obtained from the corresponding authors upon reasonable request.

Competing interests
There are no con ict of interests to declare.

Funding
This trial will be supported by the Shanghai Municipal Key Clinical Specialty Construction Project (No.shslczdzk04801) and the Changning District Science and Technology Committee Project (No.CNKW2020Y23). The funding organization had no role in the design of the trial nor in its conduct, including data collection, management, analysis, and interpretation; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication.
6. Authors' contributions LBX will be the trial sponsor. HX and CZ will be responsible for designing and supervising the trial. HX and CZ drafted and revised the protocol manuscript, with equal contribution. YCX designed the statistical analysis scheme. BXK was responsible for clinical trial registration and ethics approval. XYA and XRX will be the principal evaluators. JX will be responsible for data collection. All the named authors meet the authorship guidelines of Trials. All authors have agreed to publication of the protocol.

Acknowledgements
Not applicable. Flowchart of the trial.