Clinical Study of Desuzumab in the Treatment of Knee Osteoarthritis
Conditions: Knee Osteoarthritis
Sponsor: Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

Clinical research program of Union 
Hospital Affiliated to Tongji Medical 
College of Huazhong University of science 
and technology  
  
Study Protocol 
 
  
entry name  
Clinical study of denosumab in the 
treatment of knee osteoarthritis 
Scheme No  
UHTC0715  
Version number 
  
V2.0  
Version date 
2025.03.01 
Main researchers 
Hongtao, Tian 
Professional 
department  
Department of orthopedics, Union 
Hospital, Tongji Medical College, 
Huazhong University of science and 
technology 
Estimated start and end date    2021.08-2025.12 
        

Table of contents 
Program summary ..........................................................................................................4  
Information of main researchers ....................................................................................6  
List of abbreviations .......................................................................................................7  
1. introduction............................................................................................................. 8 
1.1 
Research background .......................................................................................8  
1.1.1 
Basic overview ..........................................................................................8  
1.1.2 
The previous research foundation of this study ........................................9  
1.2 
Type of study ..................................................................................................10  
1.3 
Research basis ................................................................................................10  
1.4 
Risk / benefit assessment ............................................................................... 11  
1.4.1 
Known potential risks ............................................................................. 11  
1.4.2 
Probability of injury ............................................................................... 11  
1.4.3 
Degree of injury....................................................................................... 11  
1.4.4 
Known potential benefits ........................................................................11  
1.4.5 
Potential risk / benefit assessment .......................................................... 11  
1.4.6 
discuss .................................................................................................... 12  
2. Study purpose / end point...................................................................................... 12  
3. research design ..................................................................................................... 12  
3.1 
overall design ................................................................................................ 12  
3.2 
sample size..................................................................................................... 13  
3.3 
Blinding and Unblinding procedures ............................................................ 13  
3.4 
Definition of study start, end and completion ............................................... 13  
4. Study population .................................................................................................. 13  
4.1 
diagnostic criteria .......................................................................................... 13  
4.2 
Exclusion criteria .......................................................................................... 14  
4.3 
Subject grouping method .............................................................................. 14  
4.4 
Subject withdrawal criteria ........................................................................... 14  
4.5 
Subject recruitment ....................................................................................... 14  
5. Research intervention ........................................................................................... 15  
5.1 
Study intervention description ...................................................................... 15  
5.2 
Research drugs or medical devices or diagnostic reagents or questionnaires, 
etc ................................................................................................................................ 15  
5.3 
Research intervention discussion .................................................................. 15  
5.4 
Research intervention related matters ............................................................ 15  
6. Relevant regulations on concomitant medication, permitted drugs, drugs with 
caution, banned drugs and concomitant treatment ...................................................... 16  
7. Items and times of clinical and laboratory examinations to be carried 
out ............................................................................................................................... 16  
8. price ..................................................................................................................... 16  
8.1 Primary and secondary end points/outcome evaluation..................................... 16  
8.2 safety evaluation................................................................................................. 16  
9. adverse events and serious adverse events................................................................ 16  

10. statistical analysis and statistical methods...............................................................17  
11. medical treatment and protection of subjects .........................................................17 
11.1 risk assessment and risk disposal measures of subjects in the study Plan........17 
11.2 medical treatment and protection of subjects during the study..........................19  
11.3 medical treatment and protection of subjects after the study.............................19 
12. supporting documents and precautions...................................................................19 
12.1 informed consent process .................................................................................19 
12.2 privacy protection.............................................................................................20  
12.3 specimens Collection and use of data................................................................20  
12.4 quality control and quality assurance................................................................20  
12.5 data processing and record keeping...................................................................20  
12.5.1 data collection and management................................................................21  
12.5.2 research data retention ...............................................................................21  
12.6 publication and data sharing agreement............................................................21  
12.7 statement of conflict of interest........................................................................ 21  
13. references............................................................................................................... 21 
 
 
 
 
 
 
 
 
 
 
 

Program summary 
Study name 
Clinical study of denosumab in the treatment of knee 
osteoarthritis 
Research Introduction 
As an activator of inhibiting nuclear factor kB receptor, 
denosumab affects osteoclast differentiation and development 
by inhibiting OPG/RANKL/RANK bone regulatory axis 
pathway. Therefore, denosumab is widely used in the 
treatment of bone diseases such as osteoporosis. Osteoporosis 
is closely related to knee osteoarthritis. RANKL-RANK 
pathway also plays a key role in the pathogenesis of knee 
osteoarthritis. Therefore, we propose the hypothesis that 
denosumab can effectively treat knee osteoarthritis. 
 
research objective 
To explore the clinical efficacy of denosumab in the 
treatment of knee osteoarthritis. 
research design 
Prospective research 
Total number of 
enrolled patients 
30 cases 
Number of study 
groups  
Number of control 
groups 
15 cases /15 cases 
diagnosis 
Knee osteoarthritis 

Inclusion Criteria 
⚫ 
patients with knee osteoarthritis whose inclusion criteria 
meet the diagnostic criteria for arthritis  
⚫ 
no drug contraindications  
⚫ 
aged between 45 and 75  
⚫ 
promised to follow the research procedures and cooperate 
with the implementation of the whole process of the study 
⚫ 
patients understand the relevant treatment process patients 
have the ability of informed consent patients have not 
taken drugs that affect observation recently  
⚫ 
subjects must meet all the inclusion criteria to be eligible 
to participate in the study 
 
Exclusion criteria 
 
⚫ mental illness  
⚫ patients with malignant tumors;  
⚫ patients with other infectious diseases patients with 
metabolic bone disease, diabetes, hyperthyroidism  
⚫ unable to actively cooperate with the treatment of patients 
⚫ hypocalcemia 
Research intervention 
Denosumab, 60mg single injection, has been listed. Basis of 
super indications: denosumab reduces bone resorption by 
inhibiting OPG/RANK/RANKL pathway, which is widely 
used in the treatment of osteoporosis. Osteoporosis is closely 
related to knee arthritis. RANKL-RANK pathway also plays 
an important role in the pathogenesis of knee osteoarthritis. 

Evaluation criteria: 
primary endpoint: 
secondary endpoint: 
safety evaluation: 
⚫ Primary end point / outcome: VAS pain score, OKS
function of knee joint at 1,3 and 6 months.  
⚫ secondary end point / outcome: WOMAC score, incidence 
of adverse reactions, EQ-5D quality of life score. 
⚫ For patients' benefit, some participants opted to withdraw 
from the trial post-denosumab injection and proceed with 
knee replacement, with intraoperative synovial tissue 
obtained for H&E, IHC staining, and WB analysis. 
⚫ safety evaluation: incidence of adverse reactions; physical 
examination; Important signs (such as body temperature, 
pulse, blood pressure); Radiological or other imaging 
evaluation; Laboratory evaluation 
Statistical method: 
sample size:  
analysis set:  
primary efficacy 
endpoint:  
safety endpoint: 
The required sample size is calculated by using the 
non-inferiority design scheme. The alpha value is set 
to be 5%, the efficacy power is set to be 80%, and 
the effective rates of both drugs are 90%. Under the 
condition of non-inferiority limit value of 10%, the 
number of patients required in each group is 
calculated to be 15, and the total number of patients 
planned to be enrolled is 30; Among them, there 
were 15 people in the denosumab group and 15 
people in the placebo group (the same amount of 
normal saline); Primary end point / outcome: OKS 
function of knee joint at 1,3 and 6 months, VAS pain 
score; Secondary end point / outcome: WOMAC 
score, incidence of adverse reactions, EQ-5D quality 
of life score; H&E, IHC staining and WB analysis of 
synovium from patients after knee replacement; 
Safety end point: incidence of adverse reactions, 
radiological or other imaging evaluation, laboratory 
evaluation; 
 
 
Study duration 
2021.08-2025.12 
Participants' 
participation time 
6 months 
Research unit / 
location 
Domestic single center 
Union Hospital Affiliated to Tongji Medical College of 
Huazhong University of science and technology 

 
Information of main researchers 
Name, qualification and contact information of main researchers 
Hongtao,Tian, chief physician, practicing physician certificate, physician 
qualification 
certificate, 
GCP 
certificate 
13955875563 
(see 
the 
qualification documents of main researchers for details) 
List of abbreviations 
abbreviation 
 
Chinese full text 
 
VAS  
Pain score 
OKS  
Oxford hip score 
WOMAC  
Quality of life score 
EQ-5D  
Survival treatment score 
H&E 
Hematoxylin and eosin 
IHC 
Immunohistochemical 
WB 
Western blot 
 
 
 
 
 
 

1. Introduction  
1.1 Research background  
1.1.1 Basic overview  
At present, about 60% of the elderly over 60 years old in China suffer from 
knee osteoarthritis, and there are more than 100 million patients with knee 
osteoarthritis in China. It is estimated that the world's elderly population will 
reach 2.02 billion in 2050, and China will reach 480 million, accounting for 
almost 5% of the global elderly population, making it the country with the 
largest elderly population in the world1. At present, the treatment of knee 
osteoarthritis is only analgesic drugs to alleviate symptoms, and there is no 
effective treatment to reverse the progress of knee osteoarthritis. Therefore, 
many patients with knee osteoarthritis eventually face knee replacement 
surgery. In 2019, there were nearly 400000 knee replacement operations in 
China, and the number will increase by 30% next year, which is undoubtedly 
a huge economic expenditure for patients. Denosumab is widely used in the 
treatment of osteoporosis by inhibiting nuclear factor kB activating factor 
(RANK). RANK and RANKL are important molecular systems that regulate 
bone remodeling and the dynamic balance of bone metabolism. In 
osteoporosis, the combination of rank and RANKL will directly promote the 
differentiation and maturation of osteoblasts, enhance their bone resorption 
activity and prevent their apoptosis. The relative transcription of RANK in 
osteoclasts and their precursor cells is significantly increased, which 
promotes bone loss and leads to osteoporosis. RANKL-RANK pathway also 
plays a key role in the pathogenesis of osteoarthritis 2. The chondrocytes and 
synovial cells of patients with knee osteoarthritis will produce interleukin-1 
(IL-1β), TNF- α and prostaglandin E2 (PGE2) and other inflammatory factors 
after being stimulated by trauma, wear and tear, promoting osteoclasts to 
secrete metalloproteinases (MMPs) and matrix protease tissue inhibitors 
(TIMPs), and these inflammatory factors can directly stimulate RANKL to 

induce the production of osteoclasts, resulting in the destruction of cartilage 
matrix, the impairment of bone regeneration ability, the irreversible 
degradation of cartilage tissue, and even apoptosis 3. At the same time, it has 
been reported in the literature that osteoporosis and knee osteoarthritis are 
closely related, and the two promote each other, forming a vicious circle 4,5. 
The symptoms of knee osteoarthritis can be relieved by treating osteoporosis. 
However, there is no report about denosumab in the treatment of knee 
osteoarthritis. Therefore, this project plans to use a prospective, single center 
randomized controlled trial to study the clinical efficacy of denosumab in the 
treatment of patients with knee osteoarthritis and osteoporosis. 
1.1.2 The previous research basis of this study 
  Xiaona, Li elaborated on the OPG/RANK/RANKL signaling pathway, which exists 
in the pathogenesis of osteoporosis and knee osteoarthritis at the same time. It has been 
reported that osteoporosis is closely related to knee osteoarthritis6. Bellido et al. showed 
that when patients with knee osteoarthritis were complicated with osteoporosis, the 
subchondral bone absorption caused by periosteal degeneration caused by osteoporosis 
in the early stage would aggravate the degeneration of articular cartilage and promote 
the development of osteoarthritis7. Shen Sheng and other researchers have shown that 
early anti-osteoporosis treatment can reduce the level of inflammatory factors in the 
joint, significantly delay the occurrence of knee osteoarthritis, and play an important 
role in the early prevention of knee osteoarthritis8. Ghodasra and others believe that 
active vitamin D3, as an important carrier for human bone absorption and promoting 
calcium absorption, is of great significance for maintaining and improving bone mass. 
Increasing the intake of active vitamin D3 in the early stage of osteoporosis can reduce 
bone loss and reduce the formation of inflammatory factors9. Osteoporosis can 
aggravate the development of knee osteoarthritis. Treating osteoporosis while treating 
knee osteoarthritis can make the treatment effect of knee osteoarthritis better. In the 
early stage, we established a rat model of knee osteoarthritis. The rats in the 
experimental group were subcutaneously injected with 60 mg denosumab, and the rats 

in the control group were subcutaneously injected with placebo (the same amount of 
normal saline). During the pain test, the rats in the experimental group could tolerate 
greater force stimulation. 
1.2 research type 
⚫ 
drug intervention research 
⚫ 
exploratory research 
⚫ 
listed 
⚫ 
in line with the recommendations of the "norms for the diagnosis and treatment of 
knee osteoarthritis" 
⚫ 
super indications 
⚫ 
a large number of literatures report that osteoporosis can aggravate the 
development of knee osteoarthritis, anti-osteoporosis treatment, and improve the 
effectiveness of the treatment of knee osteoarthritis. The incidence of adverse 
reactions such as constipation and muscle pain of denosumab is less than 10%, and 
the incidence of jaw necrosis, infection and atypical femoral fracture is less than 
1% 
⚫ 
randomized controlled study 
1.3 research basis 
According to Xiaona, Li, the study elaborated the OPG/RANK/RANKL signaling 
pathway, which exists in the pathogenesis of osteoporosis and knee osteoarthritis at the 
same time. It has been reported that osteoporosis is closely related to knee 
osteoarthritis6. Bellido et al. showed that when patients with knee osteoarthritis were 
complicated with osteoporosis, the subchondral bone absorption caused by periosteal 
degeneration caused by osteoporosis in the early stage would aggravate the 
degeneration of articular cartilage and promote the development of osteoarthritis7. Shen 
Sheng and other researchers have shown that early anti osteoporosis treatment can 
reduce the level of inflammatory factors in the joint, significantly delay the occurrence 
of knee osteoarthritis, and play an important role in the early prevention of knee 

osteoarthritis8. Osteoporosis can aggravate the development of knee osteoarthritis. 
Treating osteoporosis while treating knee osteoarthritis can make the treatment effect 
of knee osteoarthritis better. 
1.4 risk / benefit assessment 
1.4.1 known potential risks 
⚫ 
hypocalcemia 
⚫ 
limb and muscle pain 
⚫ 
urinary and respiratory system infection 
⚫ 
abdominal discomfort such as constipation 
⚫ 
rash 
⚫ 
jaw necrosis 
⚫ 
atypical femoral fracture 
⚫ 
X-ray low-dose radiation 
1.4.2 probability of injury 
⚫ 
rash, constipation, urinary and respiratory infection (>1/100, <1/10), hypocalcemia, 
jaw necrosis, atypical femoral fracture are rare (<1/10000)  
1.4.3 degree of injury 
⚫ 
mild to moderate, generally does not endanger the patient's life 
1.4.4 known potential benefits 
⚫ 
patients can receive denosumab drug treatment free of charge 
⚫ 
patients enjoy outpatient registration free of charge, at the same time, you can 
contact the doctor at any time 
⚫ 
denosumab is injected subcutaneously once every 6 months to avoid the long-term 
repeated use of analgesic drugs and gastrointestinal irritation.  
⚫ 
exempt the examination and laboratory costs during the follow-up 
⚫ 
after the patient completes the follow-up, if the patient is in the placebo group, the 
patient can be provided with denosumab treatment for half a year free of charge 
⚫ 
Some patients receiving denosumab injections requested to withdraw from the trial 
to undergo knee replacement, during which synovial tissue samples were collected 
for H&E, IHC staining, and Western blot. Knee replacement inherently requires 

partial synovectomy to prevent postoperative joint crepitus and alleviate 
inflammatory pain. The resected pathological tissues during knee replacement—
including synovium and cartilage—are routinely sent for pathological examination 
to exclude neoplasms or infections, confirm diagnoses, and guide postoperative 
management. Studies suggest that denosumab may effectively reduce 
periprosthetic fracture risk. Thus, even after trial withdrawal, patients can benefit 
from its osteoporotic therapeutic effects in preventing post-arthroplasty 
complications such as prosthetic loosening and periprosthetic fractures. In addition, 
we could also check whether there are some protective effects by denosumab on 
molecular level.  
1.4.5 potential risk / benefit evaluation 
⚫ 
Denosumab has been widely used in clinic, and its potential risk incidence is low 
and controllable. Patients with knee osteoarthritis have more benefits than potential 
risks. In order to reduce potential risks, inclusion and exclusion criteria are strictly 
set in the study design. 
1.4.6 discussion 
⚫ 
This study comprehensively evaluates the risk benefits of patients, so as to 
maximize the protection of patients' interests, provide protection for patients, and 
minimize the risk. Patients' benefits outweigh potential risks. 
 
2. 
Research purpose / end point 
⚫ 
main purpose: To evaluate the effectiveness of denosumab in the treatment of knee 
osteoarthritis. 
⚫ 
secondary purpose: To explore the safety of denosumab in the treatment of knee 
osteoarthritis. 
⚫ 
main research end point / outcome: VAS pain score, knee OKS function at 1,3,6 
months. 
⚫ 
secondary research end point / outcome: WOMAC score, incidence of adverse 
reactions, EQ-5D quality of life score. 
⚫ 
H&E, IHC staining and WB analysis of synovium after knee replacement; 
 

3. 
Research design 
3.1 Overall design 
⚫ 
domestic single center research 
⚫ 
this research project is a randomized controlled study, and the subjects are 
randomized into groups: 1. Denosumab group; 2. Placebo group (equivalent 
normal saline). 
⚫ 
this study adopts the principles of randomized grouping and double-blind to reduce 
the selection bias, and the two people make independent statistics to reduce the 
information bias. 
⚫ 
all patients were followed up in the outpatient department, with a period of 6 
months. A total of 3 visits were required, 1,3 and 6 months after injection. The 
follow-up examination items were blood routine examination, CRP, ESR, 
electrolyte, X-ray of the knee joint in the anterior and lateral position, magnetic 
resonance of the knee joint, and bone mineral density measurement. 
⚫ 
calculate the incidence of adverse reactions of patients in the two groups, including 
the OKs function of knee joint at 1,3 and 6 months, VAS pain score, WOMAC 
score and EQ-5D quality of life score. 
⚫ 
For patients' benefit, some participants opted to withdraw from the trial post-
denosumab injection and proceed with knee replacement, with intraoperative 
synovial tissue obtained for H&E, IHC staining, and WB analysis. 
3.2 sample size 
⚫ 
calculate the required sample size using the non-inferiority design scheme, set the 
alpha value as 5%, the efficacy power as 80%, and the effective rates of both drugs 
are 90%. Under the condition of non-inferiority boundary value of 10%, calculate 
the number of patients required in each group as 15, a total of 30 patients are 
planned to be enrolled. 
3.3 blinding and Unblinding procedures 
⚫ 
no 
3.4 definition of start, end and completion of the study 
⚫ 
after the ethical review is passed, the first patient is included as the start of the 

study 
⚫ 
the end of the study is the completion of the follow-up of the last patient 
⚫ 
complete the data collation and analysis of all patients, and report to the ethics 
institution for the completion of the study 
 
4. 
Study population 
4.1 diagnostic criteria 
⚫ 
the diagnosis is based on the "guidelines for the diagnosis and treatment of knee 
osteoarthritis" 
⚫ 
patients with knee osteoarthritis whose inclusion criteria meet the "diagnostic 
criteria for arthritis" 
⚫ 
no drug contraindications 
⚫ 
aged between 45 and 75 
⚫ 
committed to follow the research procedures, and cooperate with the 
implementation of the whole process study 
⚫ 
the patient understands the relevant treatment process 
⚫ 
the patient has the ability to give informed consent  
⚫ 
the patient has not taken drugs that affect the observation recently 
⚫ 
the subjects must meet all the inclusion criteria to be eligible to participate in the 
study 
4.2 exclusion criteria 
⚫ 
mental illness 
⚫ 
patients with malignant tumors 
⚫ 
patients with other infectious diseases 
⚫ 
patients with metabolic bone disease, diabetes and hyperthyroidism  
⚫ 
patients who cannot actively cooperate in the treatment 
⚫ 
hypocalcemia 
4.3 grouping method of subjects 
⚫ 
Strictly screen the subjects according to the inclusion criteria and group them 

according to computer randomization. 
4.4 subject withdrawal criteria 
⚫ 
subjects can withdraw unconditionally at any stage of this study 
⚫ 
the subjects have other diseases that affect life treatment or life-threatening that are 
not related to the knee joint 
⚫ 
the patients die 
⚫ 
for the non-death withdrawal patients, based on the care of the patients, continue 
to track the patients and conduct telephone follow-up with the permission of the 
patients. 
4.5 subject recruitment 
⚫ 
the research object comes from outpatient patients 
⚫ 
recruitment advertisement for publicity 
 
5. 
Research intervention 
5.1 research intervention description 
⚫ 
the intervention measures of denosumab group are subcutaneous injection of 
denosumab preparation 60mg, single injection. 
5.2 research drugs such as research drugs or medical devices or diagnostic 
reagents or questionnaires 
⚫ 
Denosumab, which has been listed. The production unit of denosumab injection is 
Amgen Manufacturing limited, 60mg (1.0ml) / piece (pre filled syringe), clear, 
colorless to light yellow solution, subcutaneously injected once every 6 months, 
stored in a refrigerator at 2-8 ℃ and protected from light. There is no indication 
for knee osteoarthritis in the drug Manual of denosumab, but it is recommended by 
the guidelines or expert consensus. Belido et al. showed that when patients with 
knee osteoarthritis were complicated with osteoporosis, subchondral bone 
absorption caused by periosteal degeneration caused by osteoporosis in the early 
stage would aggravate the degeneration of articular cartilage and promote the 
development of osteoarthritis. Shen Sheng and other researchers have shown that 

early anti osteoporosis treatment can reduce the level of inflammatory factors in 
the joint, significantly delay the occurrence of knee osteoarthritis, and play an 
important role in the early prevention of knee osteoarthritis. 
5.3 research intervention discussion 
⚫ 
summarize the relevant evidence of the safety, effectiveness and feasibility of 
research intervention or research drugs 
⚫ 
the basis for the setting of research drugs, doses and administration methods 
⚫ 
other intervention methods can improve the relevant contents of safety, 
effectiveness, feasibility and implementation details according to the 
characteristics of the intervention, Provide scientific evidence for the 
implementation of this intervention 
⚫ 
the full text of the product manual should be provided for the marketed products 
5.4 study the relevant matters of the intervention 
⚫ 
subcutaneous injection of 60 mg / 6 months 
⚫ 
clear, colorless to light yellow solution 
⚫ 
store in a refrigerator at 2-8 ℃ away from light 
 
6. 
Relevant regulations on combined medication, drugs allowed to be used, 
drugs used with caution, forbidden drugs and concomitant treatment 
⚫ 
None 
 
7. 
Items and times of clinical and laboratory examinations to be carried out 
⚫ 
blood routine examination 4 times 
⚫ 
CRP 4 times 
⚫ 
X-ray of knee joint 4 times 
⚫ 
magnetic resonance of knee joint 4 times 
⚫ 
bone mineral density measurement 4 times 
 
8. 
Evaluation 
8.1 primary and secondary endpoint / outcome evaluation 

⚫ 
primary endpoint evaluation: VAS pain score, OKs function of knee joint at 1,3,6 
months 
⚫ 
Secondary endpoint evaluation of: WOMAC score, incidence of adverse reactions, 
EQ-5D quality of life score 
⚫ 
Some participants opted to withdraw from the trial post-denosumab injection and 
proceed with knee replacement, with intraoperative synovial tissue obtained for 
H&E, IHC staining, and WB analysis. 
8.2 safety evaluation 
⚫ 
incidence of adverse reactions 
⚫ 
physical examination 
⚫ 
important signs (such as body temperature, pulse, blood pressure)  
⚫ 
radiation or other imaging evaluation 
⚫ 
laboratory evaluation 
 
9. 
Adverse events and serious adverse events 
⚫ 
events that do not endanger the patient's life and can return to normal after active 
treatment, such as limb pain, constipation, etc., are adverse times, The doctor in 
charge shall report to the attending physician within 6 hours, and the attending 
physician shall report to the chief physician within 12 hours. 
⚫ 
patients with irreversible and life-threatening events, such as jaw necrosis and 
atypical femoral fracture, are serious adverse events. The competent doctor shall 
report to the attending physician within 2 hours, and the attending physician shall 
report to the chief physician within 6 hours. The investigator will immediately 
report any SAE to the sponsor, whether or not related to the study intervention. 
 
10. Statistical analysis and statistical methods 
⚫ 
for descriptive statistics, percentage is used to describe classified data, and mean ± 
standard layer is used to describe continuous data 
⚫ 
for inferential statistics, P value and confidence interval are calculated, and it is 
pointed out that one-sided or two-sided test 

⚫ 
there is no need to preset covariates 
⚫ 
hypothetical tests (such as normal test) are required, and no transformation or 
nonparametric test is required 
⚫ 
the main observation variable is the incidence of adverse reactions of patients, 
independent sample t-test and chi square test were used to describe and analyze 
⚫ 
the primary end points / secondary end points of the study were VAS pain score, 
knee OKs function score, WOMAC score, EQ-5D quality of life score. 
Independent sample t-test was used to describe and analyze 
⚫ 
mean, non-compliance and loss of follow-up patient data, ± standard deviation, 
percentage and 95% confidence interval were used to describe the statistical 
analysis results 
⚫ 
missing values were not included in the analysis 
 
11. Medical treatment and protection of subjects 
11.1 risk assessment of subjects in the study, risk disposal measures and plans 
11.1.1 risk assessment 
⚫ 
natural risks are mainly two aspects. One is the adverse reactions of drugs used by 
patients for treatment, including gastrointestinal reactions, systemic reactions 
(alopecia, fever, rash, etc.), effects on various organs (cardiotoxicity, hepatotoxicity, 
pulmonary toxicity, neurotoxicity, urinary tract toxicity, etc.), infection, etc. 
⚫ 
There are two main aspects of human risk. One is researchers' compliance. Due to 
the tension of clinical work, many researchers are involved in clinical trials at the 
same time, so they are not familiar with the procedures or steps in the scheme, 
which increases the risk of scheme deviation and scheme violation during the 
implementation of the trial. For example, the subjects met the withdrawal criteria 
during the test but continued the test, failed to follow up or check the safety 
indicators according to the protocol requirements, and failed to record the adverse 
events during the study in time. The other is subject compliance. Different subjects 
have different purposes of participating in the clinical trial. The degree of 
cooperation of subjects also determines the success of the clinical trial. In some 

clinical studies, subjects' expectations of research intervention are too high. If the 
psychological treatment effect of patients is not achieved within a period of time, 
it may also cause poor compliance of subjects, who do not take drugs according to 
the plan or do not continue to follow-up. 
11.1.2 disposal measures and plans 
⚫ 
Improve the researchers' organization and management system and quality control 
system, and improve the system construction, including system, technical 
specifications, standard operating procedures (SOP), and strengthen the allocation 
and training of research echelons. In particular, we should improve the SOP and 
plan for emergency and critical treatment, establish a green channel for the 
transportation of emergency and intensive care units, and improve the plan for the 
transportation of patients in the hospital. First aid equipment and drugs should be 
managed in a normal and standardized way, special first aid drugs should be 
prepared according to different tests, and multi-specialty communication 
mechanism should be strengthened. Researchers participating in the trial should 
participate in the scientific and reasonable scheme design, establish targeted 
emergency plans and carry out training, strictly abide by the screening criteria of 
clinical trials, and be familiar with the clinical research scheme. 
⚫ 
Set up a medical protection and treatment team to be responsible for the prevention 
and treatment of medical risks in clinical research, regularly evaluate the risks, and 
put forward rectification measures to prevent risks. 
During the study, once a medical risk occurs, the risk disposal plan should be launched 
immediately, and the clinical researcher should immediately report to the main 
investigator and the medical protection treatment team. And immediately take measures 
to protect patients. In dealing with overweight, it should be carried out quickly, orderly 
and effectively. 
11.2 medical treatment and protection of subjects during the study 
⚫ 
subjects can withdraw unconditionally and voluntarily during any process of this 
study 
⚫ 
during the study, patients can consult doctors at any time if they have any questions 
or discomfort 

⚫ 
patients with drugs involved in this study do not need to pay their own expenses, 
and the research topic provides 
11.3 medical treatment and protection of subjects after the study 
⚫ 
for patients who have discomfort after the study, there are effective routine 
treatment measures, Targeted treatment based on pain relief, detumescence, etc. 
⚫ 
after informing the patients of the research process in detail, they can consult 
medical services through the orthopedic clinic of Wuhan Union Medical College 
Hospital 
⚫ 
this study does not require the subjects to give up the right to receive free treatment 
and compensation for the damage related to the study 
 
12. Supporting documents and precautions 
12.1 informed consent process prior to the start of the study 
⚫ 
the informed consent form obtained the consent of the ethics committee. For 
patients who meet the admission criteria, we will discuss matters related to 
informed consent with patients and sign the informed consent. In the process of 
informed consent, we will inform patients of the research process, the purpose of 
the research, possible risks and their rights, answer the questions raised by the 
research objects, and tell the research objects that they are willing to participate in 
the research, and can withdraw from the research at any time. After withdrawing 
from the research, the medical health quality of patients will not be affected.  
12.2 privacy protection 
⚫ 
this researcher actively informed patients that the protection of personal 
information involved in the research process mainly includes the following two 
aspects: 1. Information related to personal identity, such as name, gender, age, date 
of birth, work, education, home address, etc.; 2. Information related to personal 
health, such as medical records, family history and genetic history. 
⚫ 
when communicating with patients, choose a relatively private space to avoid the 
harm to patients caused by privacy leakage. When collecting patient data, the 
researcher conducted it under the condition of good confidentiality, trying to avoid 

the existence of a third party 
⚫ 
all inspection reports and imaging reports in this study are specially kept by 
Xiaoguang, Zhang 
12.3 collection and use of samples and data 
⚫ 
synovial samples obtained after knee arthroplasty for H&E, IHC staining, and WB 
analysis. 
⚫ 
this study does not involve third-party testing institutions or foreign-funded 
background research institutions. The collection of data is completed by 
Xiaoguang, Zhang and Yangyang, Shi, which is limited to the use of this research 
group.  
12.4 quality control and quality assurance 
⚫ 
data are independently collected by Xiaoguang, Zhang and Yangyang, Shi, and the 
subjective data are taken as the average value 
⚫ 
the original documents are saved in detail and summarized and reported weekly, 
Jie Jia is the person in charge of quality control 
⚫ 
participants receive relevant training before carrying out clinical trials  
12.5 data processing and record keeping 
12.5.1 data collection and management 
⚫ 
the source data to be collected includes the following parts, CRF, medical records, 
AE, OKS knee score, EQ-5D score, WOMAC score, laboratory results and H&E, 
IHC staining and WB analysis of synovium from patients after knee replacement. 
Xiaoguang, Zhang and Yangyang, Shi are responsible for this part of data 
collection. 
⚫ 
data collection is carried out by clinical researchers under the supervision of the 
person in charge, who will be responsible for the accuracy, integrity and timeliness 
of the reported data. All data shall be clear to ensure traceability. 
⚫ 
a database will be established for safekeeping. The database is password protected, 
and a logic proofreading program will be set up when the database is established.  
12.5.2 research data retention 
⚫ 
all research data and original documents shall be retained for a minimum of 2 years, 

and permission shall be obtained before destruction. 
12.6 issue agreement with data sharing 
⚫ 
participate in data sharing of all personnel 
12.7 declaration of conflict of interest 
⚫ 
declaration of consistency of all participating members that there is no conflict of 
interest 
 
13. References 
1. 
<中国中老年人群膝关节骨性关节炎患病率流行病学调查设计_陈伟.pdf>. 
2. 
Sobacchi, C., et al. (2007). Osteoclast-poor human osteopetrosis due to mutations in 
the gene encoding RANKL. Nature genetics 39, 960-962. 
3. 
He, X.F., et al. (2017). Berberine alleviates oxidative stress in rats with osteoporosis 
through 
receptor 
activator 
of 
NF-kB/receptor 
activator 
of 
NF-kB 
ligand/osteoprotegerin (RANK/RANKL/OPG) pathway. Bosnian journal of basic 
medical sciences 17, 295-301. 
4. 
Ernest, T.L. & Kondrashov, P.E. (2018). The role of excessive body weight and 
meniscal instability in the progression of osteoarthritis in a rat model. The Knee 25, 
1151-1156. 
5. 
Zhou, J., et al. (2017). Effect of intervention initiation timing of pulsed electromagnetic 
field on ovariectomy-induced osteoporosis in rats. Bioelectromagnetics 38, 456-465. 
6. 
<OPG_RANK_RANKL 信号通路研究进展_李小娜.pdf>. 
7. 
Bellido, M., et al. (2011). Improving subchondral bone integrity reduces progression 
of cartilage damage in experimental osteoarthritis preceded by osteoporosis. 
Osteoarthritis Cartilage 19, 1228-1236. 
8. 
<不同药物治疗膝骨性关节炎综合症状的效果_唐令.pdf>. 
9. 
Ghodasra, J.H., et al. (2016). Ovariectomy-Induced Osteoporosis Does Not Impact 
Fusion Rates in a Recombinant Human Bone Morphogenetic Protein-2-Dependent Rat 
Posterolateral Arthrodesis Model. Global spine journal 6, 60-68.