Vitamin D as an Intervention for Improving Quadricep Muscle Strength in Patients After Anterior Cruciate Ligament Reconstruction: A Randomized Double-Blinded, Placebo-Controlled Clinical Trial
Phase: PHASE2
Conditions: Anterior Cruciate Ligament Injuries, Quadriceps Muscle Atrophy, Anterior Cruciate Ligament Rupture, Anterior Cruciate Ligament Tear
Sponsor: Chinese University of Hong Kong

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Vitamin D as an Intervention for Improving Quadriceps Muscle Strength 
in Patients after Anterior Cruciate Ligament Reconstruction: A 
Randomized Double-Blinded, Placebo-Controlled Clinical Trial 
 
Background 
 
In Hong Kong, over 3000 Anterior Cruciate Ligament reconstructions (ACLR) are performed 
each year in order to restore the knee function after an ACL injury. The goal of the ACLR 
especially for athletes is to return to sports and recondition the athlete back to the preinjury 
level of sport. Despite a successful surgery and a strenuous rehabilitation process, some athletes 
still fail to qualify the Return-To-Play (RTP) criteria, and 23% among those who returned to 
high-risk sport would suffer a second ACL injury (1). 
 
After ACLR, the patient would undergo post-operative rehabilitation to strengthen muscle 
groups associated to the knee. In the beginning of phase one (0-4 weeks post-surgery), 
controlled mobilization is required for the protection of the ACL graft and the reduction of 
swelling. Then, phase 2 (5 - 9 weeks post-surgery) consists of controlled rehabilitative training 
while phase 3 (10-16 weeks post-surgery) involves intensive muscle strengthening and training. 
During phase 4 (17-26 weeks post-surgery), patients would undergo advanced agility and 
endurance training. Finally, phase 5 (26 weeks post-surgery onwards) aims to prepare patients 
for a gradual return to sports. Most of the patients would be expected to RTP by 12 months. 
Despite the comprehensive rehabilitation program, a systematic review showed that up to 35% 
of patients would fail to return to their preinjury level of sport (2). 
 
Quadriceps muscle weakness after ACLR is a major limiting factor for functional recovery. It 
is contributed by arthrogenic muscle inhibition and muscle atrophy, which are resulted from 
muscle disuse, joint swelling, pain, inflammation and damage of neuroreceptors in the joint 
after the surgery. Muscle size is a determinant of muscle strength. Postoperative quadriceps 
muscle atrophy is inevitable, but muscle strength should be regained through rehabilitation. As 
the patient continues to exercise during rehabilitation, the atrophic response subsides and the 
strengthening training stimulates significant hypertrophy in quadriceps muscle, and thus 
increasing the quadriceps strength (3). However, in some patients, this hypertrophic response 
is insufficient and can lead to persistent quadriceps muscle atrophy after ACLR. 
 
This study would benefit patients who suffered from persistent quadriceps weakness and 
atrophy after ACLR, thus improving the post-operative outcome. This would lead to a better 
RTP rate as well as reducing the risk of reinjury, which can be a burden to the health system 
and the society due to the additional treatments and extended time off-work. 
 
Objectives 
 
1. To determine the correlation between vitamin D status and quadriceps muscle strength at 
pre-op and at 4 months after ACLR.  
2. To conduct a randomized-controlled trial to examine the therapeutic effects of vitamin D 
supplement on improving vitamin D deficiency status in patients with quadriceps muscle 
weakness after ACLR.  
3. To examine the effect of vitamin D supplements on the quadriceps muscle strength for 
patients after ACLR.  
 

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Hypothesis  
1. Vitamin D supplements will improve the vitamin D status of patients with vitamin D 
<20ng/ml and quadriceps muscle weakness after ACLR 
2. The improved level of vitamin D will lead to an improvement in quadriceps muscle 
strength and size. 
 
Patient Recruitment & Eligibility 
This is a prospective randomized, double-blinded placebo-controlled trial with all participants 
being recruited at the out-patient clinic of Prince of Wales Hospital. Participants are randomly 
assigned into either the Vitamin D intervention group or the placebo-controlled group through 
an online computer generator sequence at the time of admission to the out-patient clinic. 60 
participants will be recruited, with 30 in each group. The study will be carried out in 
compliance with Declaration of Helsinki and the ICH-GCP. Ethical approval will be obtained 
from the local IRB before the study starts. Informed consent must be obtained from all patients 
in order to participate in the study.  
 
Participants will be recruited according to the following criteria: 
 
Inclusion Criteria 
Exclusion Criteria 
1) Aged 18-40 with unilateral ACL 
injury 
2) Sporting injury with a Tegner 
score of 7 
3) Pre-op serum vitamin D level 
<20 ng/ml 
4) 4 months post-ACLR with 
serum Vitamin D level remained 
<20ng/ml 
5) LSI for quadriceps strength 
<70% of contralateral leg at 4-
month isokinetic assessment 
6) Both knees without history of 
injury/prior surgery  
1) Concomitant 
bone 
fracture, 
major 
meniscus injury or full-thickness chondral 
injuries requiring altered rehabilitation 
program post-operatively 
2) Pre-operative 
radiographic 
signs 
of 
arthritis  
3) Metal 
implants 
that 
would 
cause 
interference on MRI 
4) Non-HS graft for ACLR 
5) Patient non-compliant to the rehabilitation 
program 
6) Regular sunbed users  
 
 
Method 
Intervention 
For the proposed study, we will use 2000IU/day as advised by the Endocrine Society (4). 
Previous study has shown 2000 IU/day for 4 months showed significant improvement of 
muscle strength (5). Therefore, for the proposed study, we will use 2000 IU/day (a standard 
formulary used in Hospital Authority in our cluster), for a duration of 16 weeks. 
Subjects will be randomized into 2 study groups: 
Group 1: Placebo group 
Group 2: Intervention group - patients receive daily dose of 2000 IU of vitamin D3 supplements  
 

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All study tablets including the placebo will be manufactured according to Good Manufacturing 
Practice (GMP) guidelines for quality assurance. They will be taken with water at breakfast, 2 
tablets at a time and once daily. The treatment will last for 16 weeks after which the 
supplementation will be stopped. Subjects will continue with their usual lifestyles in diet and 
physical activity without receiving any other treatment for muscle health. The procurement of 
study tablets will be covered by departmental funding. 
 
Outcome Measurements 
 
1. Anthropometric Measurement  
Body Mass Index (BMI) would be calculated by the measured height and weight at 4 
months post ACLR before vitamin D supplements, during the eighth week of vitamin D 
supplements, after the completion of 16 weeks vitamin D supplements, and 12-months 
post-operation. We would measure the waist circumference as well. Subcutaneous fat 
would be measured using skinfold techniques for the triceps brachii and biceps brachii. 
2. Biochemical Assays 
Blood samples will be taken under non-fasting conditions. Serum / plasma obtained will be 
immediately stored at -80°C until analysis. Serum 25(OH) Vit-D assay: Serum 25(OH)Vit-
D levels will be measured by commercial 25(OH) Vitamin D ELISA kit (Abcam ab213966) 
according to the manufacturer’s instruction, providing the quantitative determination of 
25(OH) Vitamin D3 and 25(OH) Vitamin D2. Sensitivity: 1.98 ng/ml (Range: 0.5 ng/ml - 
1010 ng/ml). 
3. Radiological Assessment  
(a) Magnetic Resonance Imaging (MRI)  
Muscle volumes of quadricep muscle are measured using a 1.5 or 3.0 Tesla MRI 
Scanner. Axial (3mm thick cut) T1W images are obtained from the anterior superior 
iliac supine (ASIS) to patella. Quadriceps muscles were manually outlined in each axial 
slice. Muscle volume was calculated by summing all of the slice-multiplied by slice 
thickness. The quality of the muscle is assessed by analyzing the fat content of the 
muscle mass using technique that has been reported by Reeder et al (6). Bilateral legs 
will be performed before the start of the vitamin supplementation (4 months post-op) 
and with the injured side repeated after the completion of the 16-weeks supplementation 
(8 months postop).The uninjured side will be used as reference for ‘normal volume’. 
4. Self-reported activity level and muscle function  
(a) Lysholm Score (7) 
Lysholm Knee Score is a questionnaire that examines knee-specific symptoms and 
function of daily living. It consists of eight items, with a total score ranging from 0 to 
100 and a higher score indicates a better outcome with fewer symptoms of disability.  
(b) International Knee Documentation Committee Subjective Knee Form (IKDC) (8) 
IKDC is a self-reported questionnaire that measures symptoms, knee function and 
activity of daily living. The questionnaire consists of 10 questions, with a total score 
ranging from 0 to 100 and a higher score indicates greater knee function.  
(c) Tegner Score (9) 
The Tegner Activity Scale was used to assess activity level that related to sports on a 
scale of 0 to 10. Zero represents a low activity level and 10 represents the highest 
activity level.  
(d) Physical Activity Questionnaire (10) 
Level of physical activities during the past year will be evaluated with a validated 
Chinese version of the quantitative physical activity questionnaire adapted from Baecke 
et al. 

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5. Isokinetic muscle strength assessment 
The dynamometer (Biodex System 4, Biodex Medical Systems Inc., New York, USA) will 
be used. Subjects will perform a standardized warm-up exercise (5 min cycling) followed 
by the test. Concentric/concentric contractions of knee extension/flexion will be tested at 
60°/s and 180°/s (11). Subjects will be seated on the dynamometer chair with their hips 
flexed to 85°. 
6. Knee laxity 
(a) Passive knee laxity 
To measure the anterior-posterior knee laxity, the KT-1000 knee ligament arthrometer 
(MEDmetric Corp, San Diego, CA, USA) will be used. Manual force test will be 
applied until 30lb sound signal is activated. Three trials will be performed. A side 
difference of 3 mm or above is considered clinically relevant (12). 
7. Biomechanics - Motion Analysis 
The kinematics will be assessed by the skin marker-based motion analysis system (Vicon 
MX, Oxford, UK) with the lower-body marker setup, following the OSTRC standard 
protocol using 16-camera and 16 reflective skin markers. 
(a) Single leg hop (SLH) task 
The SLH test will be performed as reported in the previous studies reported (13). Three 
trials will be performed on each leg followed by familiarization. The SLH will be 
deemed valid if the patients can achieve a maximal hop distance while maintaining their 
balance for at least 2s after landing. 
(b) Single leg squat (SLS) task  
The subject will start at the upright standing position with their toes facing forward and 
squat down at a self-determined speed. Once the pre-assigned flexion angle is reached, 
subjects will be instructed to hold the position for 10 second. A trial will be invalid if 
the subject cannot maintain the proper balance. All participants will practice 
sufficiently to achieve the prescribed knee angle (40°-50° is acceptable). 
 
Sample Size Estimation and Power Calculation  
For objective 1, when the sample size is 92, a bivariate correlation test will have 90% power 
to detect at the 0.050 level with a correlation coefficient of 0.3. The proposed sample size (150) 
will be sufficient to detect the difference and enables the fulfillment of objective 2 and 3 in this 
study (with an estimated 40% quadriceps strength deficit at 4-month post-op). For objective 2, 
when the sample size in each group is 30, a repeated-measure ANOVA (with three repeated 
measures) will have 90% power to detect at the 0.050 level, with an effect size of 0.35. 
(G*Power 3.1.9.4). 
 
Data Processing and Analysis 
Data will be reported as the mean ± standard error of the mean, except for non-normally 
distributed data which are reported as the median, minimum and maximum. All Statistical 
analysis will be performed using the Statistical Package for Social Sciences (SPSS) 25. 
Statistical significance will be set at p < 0.05. For objective 1, a bivariate Pearson’s correlation 
will be used to detect correlation between serum vitamin D levels and muscle strength and size 
at 4-month post-op, controlled for anthropometric variables. For objective 2, data will be 
analyzed by a repeated-measure ANCOVA (Control vs vitD supplementation; with repeated 
measures at 4-, 8-, 12-month post-op; other anthropometric variables as covariance).Primary 
outcome would be quadriceps muscle strength. These statistical tests will be performed after 
checking the normal distribution with Kolmogorov-Smirnov test, otherwise, a non-parametric 

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equivalent will be used. The “Intention-to-Treat” principle will be followed for data analysis 
purposes on those who have completed 16-week treatment. “Protocol Evaluable Analysis” will 
also be conducted using the 80% compliance as the cut-off. This trial will be registered at a 
clinical trial registry (ClinicalTrials.gov) before the study is implemented. 
 
Facilities and Major Equipment  
 
The proposed study will be conducted in the Prince of Wales Hospital (PWH). Subject 
inclusions, follow-up visits, clinical examinations, and questionnaire fillings will be conducted 
in the Out-patient Clinic of the Department of Orthopaedics and Traumatology, which is 
located at the 1/F of Li Ka Shing Specialist Clinic (North Wing) in the hospital. Biodex 
isokinetic dynamometer (Biodex System 4, Biodex Medical Systems Inc.), owned by our 
department, is available in Physiotherapy Department at G/F of PWH. Processing of blood 
samples for serum Vitamin D levels will be conducted in the Li Ka Shing Institute of Health 
Sciences (LiHS) in PWH, in which a full gear of centralized research facilities is available, 
including standard equipment for biochemical assays and molecular biology, such as large 
capacity refrigerated centrifuges in Centrifuge Laboratory (room 1009). 25(OH) Vitamin D 
Elisa Kit (ab213966) will be used for the analysis of the Vitamin D levels. Body composition 
including, lean body mass and body fat, as measured by bioelectrical impedance analysis (BIA, 
InBody 
Technology, 
US), 
which 
is 
owned 
by 
our 
department. 
(http://www.lihs.cuhk.edu.hk/enus/home.aspx). MRI scans will be performed at iRad Medical 
Diagnostic Centre with Philips Ingenia 1.5T MRI machine. 
 
 
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