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This supplement contains the following items:

1. Original protocol, final protocol, summary of changes.

2. Original analysis plan, final statistical analysis plan, summary of

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Original Protocol


1 VIDEO protocol NHMRC project 605501

VIDEO PROTOCOL

VItamin D Effect on Osteoarthritis study

Scientific title

Does vitamin D supplementation prevent progression of knee osteoarthritis?

A randomised controlled trial

Sponsor Institution

Menzies Research Institute & Monash University

Chief Investigators

A/Prof Changhai Ding

Prof Graeme Jones

Prof Flavia Cicuttini

Dr Tania Winzenberg

Dr Anita Wluka

Dr Steve Quinn

Contact details:

A/Prof Changhai Ding

Menzies Research Institute

University of Tasmania

17 Liverpool Street, Hobart TAS 7000

Phone – (03) 62267730 Fax – (03) 62267704



1. Background

Osteoarthritis (OA) is characterized by gradual loss of articular cartilage and changes of other

joint structures (i.e., subchondral bone and meniscus) leading, eventually, to total joint

replacement. It is the most common joint disorder in the world and in Western populations is

one of the most frequent causes of pain, loss of function and disability in adults 1.

Approximately 25% of people 55 years of age or older have had knee pain on most days in a

month in the past year. Of these about half have radiographic knee OA and are considered to

have symptomatic OA 1. The recognition of arthritis as a National Health Priority Area and

the establishment of the “Bone and Joint Decade 2000 – 2010” organization highlight the

importance of arthritis and musculoskeletal disorders. There is clearly an urgent need for

research that investigates innovative and cost-effective approaches to slow the progression of

OA.

Vitamin D deficiency [defined as serum level of 25-hydroxy-vitamin D(25-(OH)D) < 50

nmol/l] is very common in older people. High rates of vitamin D deficiency have been

reported in all sectors of the community especially in Tasmania and Victoria where this study

will be conducted.

Pathophysiological processes of vitamin D in normal and OA joints

Vitamin D may have direct effects on chondrocytes in osteoarthritic cartilage. Vitamin D

receptors (VDR) have been demonstrated in human articular chondrocytes (HAC) of

osteoarthritic cartilage, especially the superficial zone 2. VDR expression by HAC is often

associated with sites where matrix metalloproteinases (MMPs) expression is prevalent, and

1α-25(OH)2D3 contributes to the regulation of MMP and PGE2 production by HAC in

osteoarthritic cartilage 2. Thus, articular cartilage, including cartilage affected by OA, seems

to be sensitive to the effects of vitamin D.

Vitamin D may also exert an effect on OA through bone. During bone growth, vitamin D

regulates the transition from growth plate cartilage to bone 3. Chronic vitamin D inadequacy

in adults has adverse effects on calcium metabolism, osteoblast activity, matrix ossification,

and bone density, resulting in increased bone turnover and enhanced bone loss 4. Thus

vitamin D deficiency could impair the ability of bone to respond optimally to

pathophysiological processes in OA, and predispose to disease progression.

Muscle weakness is another possible mediator of effects of vitamin D on OA. Some studies

have demonstrated that low 25-(OH)D (calcidiol) levels are associated with reduced muscle

function. As reviewed by Bischoff et al 5, it is postulated that the beneficial effect of vitamin

D on muscle strength is mediated by 1,25(OH)2D binding to a vitamin D–specific nuclear

receptor in muscle tissue leading to de novo protein synthesis, muscle cell growth, and

improved muscle function.

In summary, there is potential for vitamin D supplementation to have beneficial effects on

OA through its direct action on cartilage, and/or its effects on bone and muscle health.

Evidence from observational studies

Cross-sectional data

An early study 6 reported that in women aged between 27 to 79 years, low serum 25-(OH)D

levels were associated with prevalent knee osteophytes but this association became non-

significant after adjustment for BMI and age. In this study, serum 25-OHD levels were not

associated with joint space narrowing. However, recent studies reported at the 2008

American College of Rheumatology Annual Meeting suggested that serum 25-(OH)D levels

were associated with OA. Kinjo 7 reported that in the Third National Health and Nutrition



Examination Survey (NHANES III), women (but not men) with knee OA who used chronic

analgesics were more likely to have low 25-(OH)D levels than subjects with asymptomatic

OA. Similar findings were observed in back pain, suggesting vitamin D may be an important

pain modulator in some groups or that reverse causation applies whereby subjects with pain

have less sun exposure as a result. Chaganti 8 reported that in the community-dwelling

ambulatory elderly men, those with low serum 25-(OH)D levels were twice as likely to have

prevalent radiographic hip OA.

Radiographic assessment of OA is two dimensional in nature, lacks sensitivity to change and

is highly susceptible to measurement error through factors such as joint position. Magnetic

resonance imaging (MRI) has more recently been used to study OA. Standard techniques

such as fat-saturated T1-weighted spoiled gradient echo and T2-weighted proton density-

weighted fast spin echo sequences, have been utilized to directly assess knee structural

alterations such as cartilage volume, cartilage defects, subchondral bone changes and

meniscal lesions. This has increased our understanding of early joint changes. In the

Tasmania Older Adult Cohort (TASOAC) study (NHMRC, 302204), in cross-sectional

analysis, serum 25-(OH)D level was significantly and positively associated with knee

cartilage volume in older men and women. This was a continuous association with no

evidence to support a threshold 9. Furthermore, 25-(OH)D insufficiency was weakly and

positively associated with medial (yes vs no: β=29 mm2, P=0.12) and lateral tibial bone area

(yes vs no: β=21 mm2, P=0.07) in women in adjusted analysis. Because decreased cartilage

volume and increased bone area play important roles in the aetiology of knee OA 10, these

results suggest vitamin D appears insufficiency appears detrimental to joint health.

Longitudinal data

Data from the Framingham OA cohort study suggests that vitamin D insufficiency

contributes to the progression and possibly development of OA. In this study, the risk of

progression of OA in older patients with knee OA was three times higher in the middle and

lowest tertiles for both intake and serum levels of vitamin D as compared with patients in the

highest tertiles. Low serum levels of vitamin D also predicted loss of cartilage, as assessed by

loss of joint space (OR: 2.3, 95% CI: 0.9, 5.5), as well as osteophyte growth (OR: 3.1, 95%

CI: 1.3, 7.5) 11. Two studies at the 2008 American College of Rheumatology Annual

Scientific Meeting provided further evidence suggesting vitamin D levels are important for

OA. Javaid 12 reported from the Multicentre Osteoarthritis (MOST) Study, that there were no

significant associations between low serum vitamin D status and incident radiographic knee

OA; however, 25-(OH)D as measured by quartiles was predictive of incident radiographic

knee OA, with the lowest risk in the middle quartiles. This quadratic association remained

significant (P=0.007) after adjusting for vitamin D supplement use and baseline season. Lo 13

reported that in those with symptomatic knee OA, a high baseline vitamin D level was

associated with a lower odds of increase in the medial: lateral tibial BMD ratio (M:L BMD)

and higher odds of decrease in M:L BMD ratio over 1 year. High M:L BMD may be related

to increased medial tibiofemoral OA.

The only longitudinal MRI data is from our TASOAC study and this strongly supports a role

of vitamin D in OA. We reported that higher baseline serum levels of 25-(OH)D predicted

reduced loss of cartilage volume over 2 years, and increases in vitamin D levels were

associated with a further protective association 9. These suggest vitamin D is important in

maintaining knee cartilage health in older adults.

VIDEO study



VIDEO study will be the first randomised controlled trial to explore the effects of the vitamin

D supplementation on knee structural changes (cartilage and bone) utilizing pioneering MRI

techniques and limb muscle strength assessment in OA patients. It has been supported by

National Health & Medical Research Council (NHMRC ID 605501).

Sub-studies

In this study, we will also explore the vitamin D supplementation on bone microarchitecture

and density at the distal radius and distal tibia assessed by three-dimensional high-resolution

peripheral quantitative computed tomography (HR-pQCT) 14. Although effects of vitamin D

on bone mineral density assessed by dual energy X-ray absorptiomtry (DXA) have widely

reported 15, the effects of vitamin D supplementation on bone microarchitecture have not

been reported.

Furthermore, we will determine the effect of vitamin D supplementation on blood pressure

(clinic, ambulatory, upper arm and central measures) and arterial stiffness (aortic pulse wave

velocity). Vitamin D deficiency is associated with increased ischaemic and non-ischaemic

cardiovascular disease16.While these associations are thought to occur independently from

traditional risk factors for cardiovascular disease, several lines of evidence suggest that

vitamin D deficiency may influence blood pressure via mechanisms including activation of

the renin–angiotensin–aldosterone system (RAAS) 17-19. A recent systematic review reported

that there is accumulating evidence to support the hypothesis that vitamin D deficiency

contributes to hypertension20. This study also concluded that randomised, placebo-controlled

trials are “greatly needed to clarify and definitively prove the effect of vitamin D on blood

pressure”20. VIDEO study aims to be the first to assess this. Central and ambulatory blood

pressure will be the main outcomes because central blood pressure is the actual pressure load

experienced by the heart (and other organs such as the kidneys and brain) rather than the

pressure at the upper arm21, and ambulatory blood pressure is regarded as the “gold standard”

technique because it correlates with target organ damage and provides more accurate

information on daily (including night time) blood pressure fluctuations 22.

These 2 sub-studies will be performed with collaboration of Prof Ego Seeman from

University of Melbourne and Dr James Sharman from Menzies, and are self-funded.

A third sub-study (also self-funded) is to examine the effects of vitamin D supplementation

on the function of deep lumbo-pelvic stabilising muscles. The protective deep lumbo-pelvic

stabilising muscles [including, but not exclusively, transversus abdominus (TrAb) and lumbar

multifidus (LM); also known as “core muscles”] become dysfunctional shortly after the onset

of low back pain, and that ongoing muscle dysfunction is associated with persistent low back

pain23, 24. Muscle weakness can be a sign of vitamin D deficiency25, and therefore, vitamin D

supplementation may have beneficial effects on functionally important core muscles.

2. Aims

To determine whether vitamin D supplementation reduces the loss of knee cartilage

volume (~2% per year less loss than placebo group), as assessed by MRI, over a 2-

year period in patients with symptomatic knee OA.

To determine whether vitamin D supplementation significantly prevents the

progression of knee cartilage defects and enlargement of tibial bone area over a 2-year

period in patients with knee OA.

To determine whether vitamin D supplementation significantly reduces loss of limb

muscle strength over a 2-year period in patients with knee OA.



To determine whether vitamin D supplementation significantly increase bone

microarchitecture and density at the distal radius and distal tibia in patients with knee

OA.

To determine whether vitamin D supplementation significantly reduce blood pressure

(both upper arm and central) and aortic pulse wave velocity in older people.

To determine whether vitamin D supplementation improves measures of deep lumbo-

pelvic stabilising muscle function, particularly transversus abdominus and lumbar

multifidus

3. Methods

3.1 Study design

VIDEO is a randomised, placebo-controlled double-blind clinical trial.

We will recruit 400 subjects with symptomatic knee OA, 200 patients in Southern Tasmania

and 200 in Melbourne, by using a combined strategy, including collaboration with general

practitioners, specialist rheumatologists, and orthopaedic surgeons, as well as advertising

through local media. This is the approach we have taken to successfully recruit participants

for large community-based trials across a range of musculoskeletal conditions.

3.2 Inclusion criteria:

1) Age 50-79 years old;

2) Men and women with symptomatic knee OA for at least 6 months with a pain visual

analogue scale (VAS) of at least 20 mm;

3) Meet the America College of Rheumatology (ACR) criteria for symptomatic knee OA

assessed by a rheumatologist 26;

4) Have an ACR functional class rating of I, II and III 27;

5) Have relatively good health (0-2 according to the investigator’s global assessment of

disease status on a 5-point Likert scale, range 0 [very well] to 4 [very poor]); and

6) Have serum vitamin D level of >12.5 nmol/L and <60 nmol/L.

7) Is able to read, speak and understand English, capable of understanding the study

requirements and willing to co-operate with the study instructions.

3.3 Exclusion criteria:

1) Patients with severe radiographic knee OA (grade 3 according to Altman’s atlas 28);

2) Patients with severe knee pain (on standing more than 80 mm on a 100-mm VAS);

3) Any contra-indication to having an MRI.

4) Patients with rheumatoid arthritis, psoratic arthritis, lupus, or cancer;

5) Patients with severe cardiac or renal function impairment

6) Patients with hypersensitivity to vitamin D;

7) Patients with any condition possibly affecting oral drug absorption (eg, gastrectomy or

clinically significant diabetic gastroenteropathy);

8) having significant trauma to the knees including arthroscopy or significant injury to

ligaments or menisci of the knee within 1 year preceding the study;

9) having anticipated need for knee or hip surgery in the next 2 years;

10) having taken Vitamin D supplements in last 30 days;

11) having taken an investigational drug in last 30 days.

4. Study medications and allocation



Participants in the intervention arm will receive 50,000 IU (1.25 mg) compounding vitamin

D3 (cholecalciferol, Nationwide Compounding Pharmacy, Melbourne) capsules given once

monthly 29, 30, and the control arm will receive an identical inert placebo (provided by the

same company). Allocation of participants will be based on computer generated random

numbers. Allocation concealment will be ensured by the used of identical inert placebo, and

the use of a central automated allocation procedure, with security in place to ensure allocation

data cannot be accessed or influenced by any person. Thus the randomized controlled trial

(RCT) will be double blind. Monthly treatment at this dose will achieve serum vitamin D

levels above 60 nmol/l in all compliant subjects 29, and will have less cost and better

compliance than daily treatment. Toxicity is extremely unlikely with this dose. All

participants will be provided recommended standard of care.

5. Scheduled visits and measurements

Timetable and measures to be made

Pre-

screening

0 months 3 months 6 months 12

months

24

months

MRI

Knee pain (VAS)

Knee radiograph

Serum 25-(OH)D

Serum calcium, phosphate,

Creatinine

Micro-CT (in Melbourne)

DXA (in Hobart)

Core musculature measure

Lower limb muscle strength

Hand grip strength

Weight

Height

Skin fold

Girth measurements

Upper arm pressure

Central blood pressure

Ambulatory blood pressure

Arterial stiffness

Physical activity (IPAQ)

Medications

WOMAC



Sun exposure

Low back pain

Foot pain

Depression

Quality of life

Cigarette smoking

Previous knee injury,

occupation

Pill counts

Adverse events

If the participant withdraws after a minimum of 6 months of treatment, he/she will be requested to

have a second knee MRI (HR-pQCT or DXA) scan.

5.1 MRI assessment of structural changes

This will be assessed in the right knee. Knees will be imaged in the sagittal plane on a 1.5-T

whole body magnetic resonance unit with use of a commercial transmit-receive extremity coil.

Fat-saturated T1-weighted spoiled gradient echo and T2-weighted proton density-weighted

fast spin echo sequences will be used.

Cartilage volume: The volumes of individual cartilage plates (medial tibial, lateral tibial and

patella) are isolated from the total volume by manually drawing disarticulation contours

around the cartilage boundaries on a section by section basis. These data are then resampled

by means of bilinear and cubic interpolation (area of 312 and 312 μm and 1.5 mm thickness,

continuous sections) for the final 3D rendering.

Cartilage defects assessment: The cartilage defects (0-4) will be graded at medial tibial and

femoral, lateral tibial and femoral, and patellar sites: grade 0, normal cartilage; grade 1, focal

blistering and intracartilaginous low-signal intensity area with an intact surface and bottom;

grade 2, irregularities on the surface or bottom and loss of thickness of less than 50%; grade

3, deep ulceration with loss of thickness of more than 50%; grade 4, full-thickness chondral

wear with exposure of subchondral bone.

Knee tibial plateau bone area: The area of medial and lateral tibial plateau bone will be

measured manually on the three reformatted images closest to tibial cartilage. An average of

these three areas will be used as an estimate of the tibial plateau bone area.

Subchondral bone marrow lesions: will be assessed on the T2-weighted MR images and

defined as discrete areas of increased signal adjacent to the subcortical bone at the lateral,

medial femur and/or tibia. Each bone marrow lesion will be scored on the basis of lesion size,

e.g., a lesion was scored as grade 1 if it was only present on 1 slice, grade 2 if present on 2

consecutive slices, or grade 3 if present on >3 consecutive slices.

Meniscal tear assessment: The menisci will be assessed in the sagittal view and be confirmed

in the coronal and axial views as previously described 31. In brief, the presence or absence of

a tear was based on the presence of a signal, which was line shaped, brighter than the dark

meniscus, and reached the surface of the meniscus at both ends within 6 defined regions

(anterior horn, body, and posterior horn at both medial and lateral tibiofemoral

compartments).

Meniscal extrusion assessment: The extent of meniscal extrusion on the medial or lateral

edges of the tibial femoral joint space for the anterior, body, and posterior horns of the



menisci will be graded (CIA19) with scores of 0 = no extrusion, 1 = partial meniscal

extrusion, and 2 = complete meniscal extrusion with no contact with the joint space.

5.2 Lower limb muscle strength

Lower limb muscle strength will be measured by dynamometry at the lower limb (involving

both legs simultaneously). The muscles measured with this technique are mainly the

quadriceps and hip flexors. The devices will be calibrated by suspending known weights at

regular intervals.

5.3 Knee pain

Knee pain, stiffness and function later will be assessed by the Western Ontario and McMaster

Universities Osteoarthritis Index (WOMAC), a self-administered questionnaire 32. Knee pain

will be also assessed using visual analogue scale (VAS).

5.4 Measurements of bone microarchitecture and density Bone macro-, micro-architecture and volumetric BMD (vBMD) were measured at the distal

radius and distal tibia using three-dimensional high-resolution peripheral quantitative

computed tomography (HR-pQCT, Xtreme CT, Scanco Medical AG, Bassersdorf,

Switzerland) 14. This system uses a two-dimensional detector array in combination with a

0.08-mm point-focus x-ray tube. This enables the simultaneous acquisition of a stack of

parallel CT slices with a resolution (voxel size) of 82 m. The following settings were used:

effective energy of 60 kVp, x-ray tube current of 95 mA, and matrix size of 1536 x 1536.

During measurement, the non-dominant arm or leg of the patient was immobilized in an

anatomically formed carbon fibre shell. The reference line was manually placed at the middle

point of the endplate of the radius and tibia on the scout view. As a default, the first CT slice

commenced at a certain distance from the reference line, i.e., 9.5 mm at radius, 22.5 mm at

tibia, respectively. One hundred and ten slices were obtained at each skeletal site over a 9.02

mm distance. The effective dose was less than 3 Sv per measurement with a measurement

time of 2.8 min.

Analyses use a threshold-based algorithm to separate the volume of interest into the cortical

and trabecular regions. Attenuation data are converted to equivalent hydroxyapatite (HA)

densities. The vBMD (mgHA/cm3) in the trabecular region (vBMDtrab), cortical region

(vBMDcort) and the combined total vBMD (vBMDtot) were computed as the average mineral

density within the trabecular, cortical and entire volume of interest respectively. Trabecular

bone volume / tissue volume (BV/TV,%) was determined by dividing the apparent trabecular

bone density by 1200 mgHA/cm3 which represents fully mineralised bone [i.e. BV/TV(%) =

100 x (vBMDtrab/1200)].

Trabecular number (TbN) was defined as the mean inverse distance between the ridges (the

centre points of trabeculae). Briefly, the three-dimensional ridges were extracted from the

original threedimensional gray-scale images. The distances of the ridges were then directly

assessed by the newly developed methods derived from distance transformation. This is not

affected by the structure of trabeculae whether it is plate- or rod-like. Trabecular thickness

(TbTh, mm) and separation (TbSp, mm) were calculated from BV/TV and TbN using

histomorphometry methods [i.e., TbTh = (BV/TV) /TbN and TbSp = (1 BV/TV)/TbN].

Cortical thickness (CTh) was calculated by dividing the mean cortical volume by the outer

bone surface.

5.5 Bone mineral density and fat mass

Bone mass was measured using dual-energy X-ray absorptiometry (DXA) at the right total

hip and spine at baseline and follow up. The instrument used was a Hologic Delphi

densitometer on array setting (Hologic, Inc., Waltham, MA). The software program was not



altered during the study timeframe. Bone mass was examined as areal BMD (g/cm2), which is

calculated by dividing the bone mineral content (BMC) by the area measured.

Fat mass and lean mass (both in kg) were also measured using DXA.

5.6 Blood pressure and arterial stiffness

Clinic upper arm blood pressure will be read twice after 5 minutes seated rest using a

validated semi-automated device (Omron HEM-907). Clinic central blood pressure will be

read twice after 5 minutes seated rest using radial applanation tonometry (SphygmoCor 8.1,

AtCor Medical). 24 hour ambulatory blood pressure monitoring (TM2430, A&D mercury)

and aortic pulse wave velocity (SphygmoCor 8.1, AtCor Medical) will be performed in a

subgroup of patients at the Hobart site. This method derives an estimate of aortic pulse wave

velocity, which is the “gold standard” clinical arterial stiffness measure 33 known to

independently correlate with mortality 34-37.

5.7 Serum vitamin D and other measurements

Serum samples were treated initially with acetronitrile to rapidly extract 25-hydroxyvitamin

D [25-(OH)D] and other hydroxylated metabolites. 25-(OH)D was then assayed utilising a

Liquid Phase radioimmunoassay (Immunodiagnostics Systems Ltd, Boldon, Tyne &

Wear, UK). Serum calcium, phosphate and renal function will be assessed using routine

biochemical methods. Serum will be stored for other measures.

5.8 Radiographic osteoarthritis

This will be assessed by a standing semiflexed AP radiograph of right knee as per the

Altman atlas 28. X-rays will also be scored for osteophytes and joint space narrowing on a

four point scale (0-3) utilising two simultaneous observers and the OARSI atlas.

5.9 Physical activity

Physical activity will primarily be assessed by pedometry at baseline and two years. An

Omron pedometer which measures vertical displacement (steps per day) will be utilised. Each

subject will be instructed prior to use and a diary will be kept. The pedometer will be worn

for seven consecutive days on two occasions. Up to seven days is required to accurately

assess habitual physical activity. We will also measure physical activity by the International

Physical Activity Questionnaire (IPAQ) 38 short version.

5.10 Anthropometry

Height was measured to the nearest 0.1 cm (with shoes, socks, and headgear removed) using

a stadiometer. Weight was measured to the nearest 0.1 kg (with shoes, socks, and bulky

clothing removed) using a single pair of electronic scales (Delta Model 707; Seca, Hamburg,

Germany) that were calibrated using a known weight at the beginning of each clinic. Waist

and hip circumference were measured using a tape measure. Skinfold at triceps, subscapular,

biceps, Iliac crest, supraspinale and midabdominal sits will be measured using Slim Guide

skinfold callipers.

5.11 Questionnaires

Depression (using Patients Health Questionnaire-9, PHQ-9), cigarette smoking status

(currently smoking, level of smoking, ever smoked, when gave up), previous knee injury and

occupation, low back pain, foot pain and quality of life will be assessed by questionnaires.

5.12 Real-time dynamic ultrasound of the core musculature (transversus abdominus,

internal obliques, multifidus) using a fully featured ‘big box’ diagnostic ultrasound machine

will be used to measure thickness of transversus abdominus and lumbar mulitifidus muscles

at rest and during contractions. These measures have a high degree of reliability (ICC >0.90

across a range of studies 39. We will do this a baseline, 12 and 24 months.



5.13 Safety

Adverse events will be recorded during the study period. Intensity and relationship with the

study medication will be investigated.

6. Statistics and sample size

Paired t-test and logistic regression will be used to compare changes in cartilage volume,

cartilage defects, bone marrow lesions, and meniscal abnormalities in univariable and

multivariable modelling (adjusted by confounding factors) between groups. Both intention to

treat analysis and per protocol will be utilized. Per protocol will be defined as achieving a

vitamin D level >60 nmol/l at month 3.

Based on sample size calculations, 200 patients in each arm (allowing for a 20% dropout over

the trial) will be sufficient to detect the specified differences (2.2% for medial tibial cartilage

loss, 0.9% for increase in medial tibial bone area, 15% for increase in cartilage defects)

between the placebo and vitamin D arms with at least 80% power for each outcome.

7. Termination criteria

As the aim of the study is to assess ultimate outcome following randomisation to one of the

treatment arms, participants will be strongly encouraged to attend the study. However, it is

recognised that situations will arise that will necessitate withdrawal of a participant from the

study. Such situations include a participant’s refusal to continue, moved to overseas or

interstate, deceased, institutionalised, or physical unable, etc.

In general there will be no specific withdrawal criterion relating to adverse effects of drug

therapy as the analysis will be on an intention-to-treat basis. If a participant withdraws or is

removed from the study for any reason, the reason for and date of the discontinuation and

date of the last dose of study medication should be recorded in the appropriate section of the

form.

8. Product liability

VIDEO study is not sponsored by a pharmaceutical company. We will obtain compounding

vitamin D3 (cholecalciferol) 1.25 mg capsule and placebo from Nationwide Compounding

Pharmacy in Melbourne. This is an unregistered (S4) product in Australia but it can be

obtained under the Special Access Scheme (SAS). It has been available for use through some

hospitals (including the Royal Hobart Hospital) for the treatment of vitamin D deficiency.

9. Report of the project

VIDEO study research team will provide a statement to the Tasmania Health & Medical

Human Ethics Committee and Monash University Human Research Ethics Committee

annually or as required. When the research is completed, published papers and abstracts will

inform the Ethics Committees of the outcome.

10. Ethic conduct of the trial

This study will be conducted in accordance this protocol, ICH GCP Note for Guidance on

Good Clinical Practice (CPMP/ICH/135/95) Annotated with TGA comments and NHMRC

National Statement on Ethical Conduct in Human Research 2007 and in keeping with local

regulations.

11. Informed consent

11.1 Consent Form



Before obtaining consent from each participant, he /she must be informed of the objectives,

benefits, risks and requirements of the study, as well as the nature of the test medication. An

information sheet should be given to every participant prior to screening and randomisation.

Participants will be giving their own consent after having read all content of the information

sheet and consent form. Participant and investigator each retain a copy of the signed consent

form.

11.2 Obtaining Consent

The Investigator, or a person designated by the Investigator, and under the Investigator's

responsibility, should fully inform the participant of all pertinent aspects of the VIDEO study

including the written participant information sheet. All participants should be informed to the

fullest extent possible about the study, in language and terms they are able to understand.

Participants are expected to giving their own consents by reading content in the information

sheet and consent form and giving their signatures by themselves. In those who might have

vision impairment, a research officer may read out content and ensure a thoroughly

understanding of all content before asking for signature.

Prior to a subject’s participation in the study, the written Informed Consent Form

should be signed, name filled in and personally dated by the participant or by the

participant’s legally acceptable representative, and by the person who conducted the

informed consent discussion.

A copy of the signed and dated written Informed Consent Form will be provided to

the participant. The original consent is to be stored in the participant’s individual

study file, held by the investigator. A second copy may be filed in the participant’s

file at the general practice.

The Participant information Sheet and Consent Form used for obtaining the

participant's informed consent must be the current version that has been reviewed and

approved by the appropriate Ethics Committee.

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2. Tetlow LC, Woolley DE. Expression of vitamin D receptors and matrix metalloproteinases in

osteoarthritic cartilage and human articular chondrocytes in vitro. Osteoarthritis Cartilage 2001;9:423-

31.

3. McAlindon TE. Nutraceuticals: do they work and when should we use them? Best Pract Res

Clin Rheumatol 2006;20:99-115.

4. Holick MF. High prevalence of vitamin D inadequacy and implications for health. Mayo Clin

Proc 2006;81:353-73.

5. Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. Estimation

of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin

Nutr 2006;84:18-28.

6. Hunter DJ, Hart D, Snieder H, Bettica P, Swaminathan R, Spector TD. Evidence of altered

bone turnover, vitamin D and calcium regulation with knee osteoarthritis in female twins.

Rheumatology (Oxford) 2003;42:1311-6.

7. Kinjo M. Serum 25OH Vitamin D Level and Symptomatic Knee Osteoarthritis: Analysis in a

Population-Based U.S. Sample. . Arthritis Rheum 2008;58:S239.

8. Chaganti RK, Parimi N, Dam T, Cawthon P, Nevitt M, Lane N. The Association of Serum

Vitamin D with Prevalent Radiographic Hip Osteoarthritis in Older Men. . Arthritis Rheum

2008;58:S676.

9. Ding C, Cicuttini F, Parameswaran V, Burgess J, Quinn S, Jones G. Serum levels of vitamin

D, sunlight exposure, and knee cartilage loss in older adults: the Tasmanian older adult cohort study.

Arthritis Rheum 2009;60:1381-9.

10. Ding C, Cicuttini F, Jones G. Tibial subchondral bone size and knee cartilage defects:

relevance to knee osteoarthritis. Osteoarthritis Cartilage 2007;15:479-86.



11. McAlindon TE, Felson DT, Zhang Y, et al. Relation of dietary intake and serum levels of

vitamin D to progression of osteoarthritis of the knee among participants in the Framingham Study.

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Final Protocol

(Published)


STUDY PROTOCOL Open Access

Vitamin D supplementation in the managementof knee osteoarthritis: study protocol for arandomized controlled trialYuelong Cao1,3, Graeme Jones1*, Flavia Cicuttini2, Tania Winzenberg1, Anita Wluka2, James Sharman1,Kay Nguo1 and Changhai Ding1,2*

Abstract

Background: Osteoarthritis (OA) is a common health issue worldwide in the aging population who are alsocommonly deficient in vitamin D. Our previous study suggested that higher serum 25-(OH)D levels were associatedwith reduced knee cartilage loss, implying that vitamin D supplementation may prevent the progression of kneeOA. The aim of the VItamin D Effects on OA (VIDEO) study is to compare, over a 2- year period, the effects ofvitamin D supplementation versus placebo on knee structural changes, knee pain, and lower limb muscle strengthin patients with symptomatic knee OA.

Methods/design: Randomised, placebo-controlled, and double-blind clinical trial aiming to recruit 400 subjects(200 from Tasmania and 200 from Victoria) with both symptomatic knee OA and vitamin D deficiency(serum [25-(OH)D] level of >12.5 nmol/liter and <60 nmol/liter). Participants will be randomly allocated to vitamin Dsupplementation (50,000 IU compounded vitamin D3 capsule monthly) or identical inert placebo group for 2 years.The primary endpoint is loss of knee cartilage volume measured by magnetic resonance imaging (MRI) andWestern Ontario and McMaster Universities Index of OA (WOMAC) knee pain score. The secondary endpoints willbe other knee structural changes, and lower limb muscle strength. Several other outcome measures including coremuscle images and central blood pressure will be recorded. Linear and logistic regression will be used to comparechanges between groups using univariable and multivariable modeling analyses. Both intention to treat and perprotocol analyses will be utilized.

Discussion: The trial is designed to test if vitamin D supplementation will reduce loss of knee cartilage volume,prevent the progression of other knee structural abnormalities, reduce knee pain and strengthen lower limb musclestrength, thus modify disease progression in knee OA.

Trial registration: ClinicalTrials.gov identifier: NCT01176344; Australian New Zealand Clinical Trials Registry:ACTRN12610000495022

Keywords: Vitamin D, Osteoarthritis, Magnetic resonance imaging

* Correspondence: [email protected]; [email protected] Research Institute Tasmania, University of Tasmania, Private Bag 23,Hobart, TAS 7000, Australia2Department of Epidemiology and Preventive Medicine, Monash University,Melbourne, 3004, AustraliaFull list of author information is available at the end of the article

TRIALS

© 2012 Cao et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Cao et al. Trials 2012, 13:131http://www.trialsjournal.com/content/13/1/131


mailto:[email protected]

mailto:[email protected]

http://creativecommons.org/licenses/by/2.0

BackgroundOsteoarthritis (OA) is the most common form of arth-ritis in the world and one of the most common chronicconditions managed in Australian general practice [1,2].It is characterized by the gradual loss of articular cartil-age and changes to other joint structures eventuallyleading to total joint replacement. Currently there is nocure for OA, and the development of innovative andcost-effective approaches to prevent the developmentand progression of OA is urgent and important.Vitamin D comprises a group of fat-soluble secoster-

oids emcompassing two major molecules, vitamin D2

and vitamin D3. Vitamin D is circulated to the liverwhere it is converted to the prohormone calcidiol, or25-hydroxy-vitamin D (25-(OH)D), which is the best in-dicator of vitamin D status [3-5]. Vitamin D deficiency isvery common in older people. It is estimated that 20 to100% of elderly men and women in North America andEurope are vitamin D deficient (mostly defined as aserum level of 25-(OH)D< 50 nmol/liter)[6,7]. High ratesof vitamin D deficiency have also been reported in allsectors of the community of Australia, especially inTasmania and Victoria where this study will be con-ducted [8-10].It has been widely recognized that OA is a disease

affecting the whole joint, including cartilage, bone andmuscle. Through targeting these joint tissues vitamin Dsupplementation may modify disease progression in OA.Vitamin D receptors (VDRs) are found in human articu-lar chondrocytes [11], and 1α-25(OH)2D3 regulates theexpression of metalloproteinase (MMP) and prostaglan-din E2 (PGE2) in chondrocytes via VDRs [11]. Vitamin Dcould enhance the ability of bone to respond optimallyto pathophysiological processes in OA, thus prevent dis-ease progression [12,13]. Furthermore, 1,25(OH)2D leadsto de novo protein synthesis, muscle cell growth, andimproved muscle function, and thus has a beneficialeffect on muscle strength [14].Epidemiological studies have provided preliminary

evidence supporting the potential use of vitamin D forthe treatment of OA. Lower serum levels of 25-(OH)Dwere associated with greater knee pain [15] and higherprevalence of radiographic OA [16], and predicted inci-dence of knee pain [17], progression/incidence of radio-graphic OA [18,19], and loss of joint space, as well asosteophyte growth [18]. Magnetic resonance imaging(MRI) has been utilized to directly assess knee struc-tural alterations such as cartilage volume, cartilagedefects, subchondral bone changes and meniscal lesions.Using MRI, we reported that, in cross-sectional analysis,serum 25-(OH)D level was significantly and positivelyassociated with knee cartilage volume in older men andwomen, and vitamin D insufficiency was positively asso-ciated with medial and lateral tibial bone area in

women. Longitudinally, higher baseline serum levels of25-(OH)D predicted reduced loss of cartilage volumeover 2 years, and increases in vitamin D levels wereassociated with further protective association [15,16].Furthermore, serum levels of 25-(OH)D were also asso-ciated with increased leg muscle strength and quality,and thus may be important for the maintenance ofmuscle function [20].Based on this experimental and epidemiological evi-

dence, we have initiated a randomized, placebo-controlled trial (Vitamin D Effect on Osteoarthritis,VIDEO study) to determine if vitamin D supplementa-tion can reduce loss of knee cartilage volume, preventthe progression of other knee structural abnormalitiesand strengthen lower limb muscle strength, and thusmodify disease progression in knee OA. The effects ofvitamin D supplementation on the progression of kneepain will also be determined.In a sub-study, we will examine the effects of vitamin

D supplementation on the function of deep lumbo-pelvicstabilising muscles. The protective deep lumbo-pelvicstabilising muscles, which include (but not exclusively)the transversus abdominus (TrAb) and lumbar multifidusand which are known as core muscles, become dysfunc-tional shortly after the onset of low back pain, and thatongoing muscle dysfunction is associated with persistentlow back pain [18,21]. Muscle weakness can be a sign ofvitamin D deficiency [19] and therefore, vitamin D sup-plementation may have beneficial effects on functionallyimportant core muscles.Furthermore, we will determine the effect of vitamin

D supplementation on blood pressure (clinical, ambula-tory, upper arm and central measures) and arterial stiff-ness (aortic pulse wave velocity). Several lines ofevidence suggest that vitamin D deficiency may influ-ence blood pressure via mechanisms including activationof the renin-angiotensin-aldosterone system (RAAS)[22-24]. Central and ambulatory blood pressure will bethe main outcomes because central blood pressure is theactual pressure load experienced by the heart (and otherorgans such as the kidneys and brain) rather than thepressure at the upper arm [25], and ambulatory bloodpressure is regarded as the gold standard technique be-cause it correlates with target organ damage and pro-vides more accurate information on daily (includingnight time) blood pressure fluctuations [26].

Methods/designStudy designVIDEO is a randomized, placebo-controlled double-blindclinical trial. Four hundred subjects (200 from Tasmaniaand 200 from Victoria) with symptomatic knee OA andserum 25-(OH)D> 12.5 nmol/liter and < 60 nmol/literwill be recruited and randomly allocated to either the

Cao et al. Trials 2012, 13:131 Page 2 of 8http://www.trialsjournal.com/content/13/1/131


treatment or placebo control group. Recruitment meth-ods will include advertisements through the local mediaand community groups as well as liaisons with generalpractitioners, specialist rheumatologists, and orthopedicsurgeons. Ethics approval has been received from TheTasmania Health and Human Medical Research EthicsCommittee (reference number H1040) and Monash Uni-versity Human Research Ethics Committee (referencenumber CF10/1182 - 2010000616). Informed writtenconsent will be obtained from all participants.

Inclusion criteriaThe inclusion criteria were as follows: age 50 to 79 years;symptomatic knee OA for at least 6 months with a painat least 20 mm on a 100 mm visual analogue scale(VAS); American College of Rheumatology (ACR) cri-teria for symptomatic knee OA assessed by a rheuma-tologist [27]; ACR functional class rating of I, II and III[28]; relatively good health, with a score of 0 to 2 on a 5-point Likert scale (with a range of 0 indicating very goodhealth to 4 indicating very poor health), according to theinvestigators global assessment of disease status; serum25-(OHD)D> 12.5 nmol/liter and < 60 nmol/liter; able toread, speak and understand English, capable of under-standing the study requirements and willing to cooper-ate with the study instructions.

Exclusion criteriaExclusion criteria were as follows: severe radiographicknee OA, grade 3 according to Altman’s atlas [29]; severeknee pain on standing (more than 80 mm on 100-mmVAS); any contraindication to having MRI; rheumatoid orpsoriatic arthritis, lupus or cancer; severe cardiac or renalimpairment; hypersensitivity to vitamin D; any conditionpossibly affecting oral drug absorption (for example, gas-trectomy or malabsorption syndromes); significant traumato knees, including arthroscopy or significant injury toligaments or menisci of the knee within one preceding thestudy; anticipated need for knee or hip surgery within thenext 2 years; history of taking vitamin D supplementswithin the previous 30 days; history of taking an investiga-tional drug within the previous 30 days.

RandomizationParticipants in each site will be randomly assigned to theintervention arm or placebo arm in a ratio of 1:1 andthe randomization will be double-blind. Allocation ofparticipants will be based on computer-generated ran-dom numbers. Allocation concealment will be ensuredby the use of an identical inert placebo, and a centralautomated allocation procedure, with security in placeto ensure allocation data cannot be accessed or influ-enced by any person.

InterventionParticipants in the intervention arm will take one cap-sule per month of 50,000 IU (1.25 mg) of a vitamin D3compound (cholecalciferol), purchased from NationwideCompounding Pharmacy, Melbourne, Australia[30], andpatients in the control arm will receive an identical inertplacebo provided by the same company. Patients arerequired to record their medication information in per-sonal diaries and a reminder will be given each month.All participants will receive the recommended standardof care. The duration of the study is 2 years.

Quality assuranceTo ensure that this trial will be of a high standard anddelivered in accordance with the trial protocol, all re-search staff will be provided with a standard protocoland case report form, and will be trained to competentlyadminister items as per protocol. The investigators, re-search assistants and outcome assessors are differentpeople. Protocols will not be altered during the studytimeframe.

Outcome measuresThe co-primary efficacy endpoints of the study will beMRI assessment of volume changes in knee cartilagefrom baseline to month 24, as well as the Western On-tario and McMaster Universities Index of OA(WOMAC) score [31] (Table 1). The secondary end-points will be other knee structural changes (cartilagedefects, tibial plateau bone area, and bone marrowlesions, meniscal tear and extrusion) from baseline tomonth 24, and lower limb muscle strength at months 3,6, 12 and 24 (Table 1).

MRI assessment of knee structural changesKnees will be imaged in the sagittal plane on a 1.5-Twhole body MRI unit using a commercial transmit-receive extremity coil. Fat-saturated T1-weighted spoiledgradient echo (GRE) and T2-weighted/proton density-weighted fast spin echo (FSE) sequences will be used.The images will be assessed by two readers blinded tothe treatment according to the methods described in ourprevious publications [15,32].Cartilage volume: the volumes of individual cartilage

plates (medial tibial, lateral tibial and patella) are isolatedfrom the total volume by manually drawing disarticula-tion contours around the cartilage boundaries on asection-by-section basis. Sagittal images will be obtainedat a partition thickness of 1.5 mm and an in-plane reso-lution of 0.31 × 0.31 mm (512 × 512 pixels), thenresampled by means of bilinear and cubic interpolation(area of 312 μm×312 μm and multiplied by 1.5 mmthickness, continuous sections) for the final 3D render-ing. Particular cartilage volume was then determined by



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summing all the pertinent voxels within the resultantbinary volume.Cartilage defects assessment: the cartilage defects (0 to

4) will be graded at medial tibial and femoral, lateral tib-ial and femoral, and patellar sites: grade 0, normal cartil-age; grade 1, focal blistering and intracartilaginous low-signal intensity area with an intact surface and bottom;grade 2, irregularities on the surface or bottom and lossof thickness of less than 50%; grade 3, deep ulcerationwith loss of thickness of more than 50%; grade 4, full-thickness chondral wear with exposure of subchondralbone.

Knee tibial plateau bone area: the area of the medialand lateral tibial plateau bone will be measured manuallyon the three reformatted images closest to the tibial car-tilage. An average of these three areas will be used as anestimate of the tibial plateau bone area.Subchondral bone marrow lesions: This will be

assessed on the T2-weighted MRI and defined as discreteareas of increased signal adjacent to the subcortical boneat the lateral, medial femur and/or tibia. Each bone mar-row lesion will be scored on the basis of lesion size, forexample, a lesion is scored as grade 1 if it occupies < 25%of the region; grade 2 if it occupies 25% to 50% of the re-gion; or grade 3 if it occupies > 50% of the region.Meniscal tear assessment: the menisci will be assessed

in the sagittal view and confirmed in the coronal andaxial views as previously described [33]. In brief, thepresence or absence of a tear is based on the presence ofa signal, which is line shaped, brighter than the dark me-niscus, and reaches the surface of the meniscus at bothends within six defined regions (anterior horn, body, andposterior horn at both medial and lateral tibiofemoralcompartments).Mensical extrusion assessment: the extent of meniscal

extrusion on the medial or lateral edges of the tibial fem-oral joint space for the anterior, body, and posteriorhorns of the menisci will be graded, where a score of0 = no extrusion, 1 = partial meniscal extrusion, and2 = complete meniscal extrusion with no contact with thejoint space.

Lower limb muscle strengthThis will be measured by dynamometry (TTM MuscleMeter, Tokyo, Japan) at the lower limb (involving bothlegs simultaneously). The muscles measured with thistechnique are mainly the quadriceps and hip flexors.The device will be calibrated by suspending knownweights at regular intervals.

WOMACKnee pain will be assessed by both WOMAC pain sub-scale (walking on a flat surface, going up/down stairs, atnight in the bed , sitting/lying and standing upright) anda 100 mm VAS.

Other measurementsCore musculature measure: Core muscle images will betaken at baseline and 12 months. Images of the coremuscles (TrAb, internal oblique muscles and LM) aretaken with real-time dynamic ultrasound using a fullyfeatured big box diagnostic ultrasound machine (PhillipsHDI 5000, Bothell, WA, US) with a hand held 7.5 mHzlinear array transducer. Images are taken of right and leftsides, both at rest and during contraction (drawing in ofabdomen) using previously published protocols [34,35].

Table 1 Timetable and measures to be made

Screening Month(s)

0 3 6 12 24

Co-primary outcome measure

MRI (cartilage volume changes) ✓ ✓

WOMAC ✓ ✓ ✓ ✓ ✓

Secondary outcome measure

MRI (other structural changes) ✓ ✓

Lower limb muscle strength ✓ ✓ ✓ ✓ ✓

Other measures

Core musculature measure ✓ ✓ ✓

Hand grip strength ✓ ✓ ✓ ✓ ✓

Central and upper arm blood pressure ✓ ✓ ✓ ✓

Aortic stiffness ✓ ✓ ✓ ✓

Physical activity (IPAQ) ✓ ✓

Body fat ✓ ✓ ✓

Low foot pain ✓ ✓ ✓ ✓ ✓

Low back pain ✓ ✓ ✓ ✓

Depression ✓ ✓ ✓ ✓ ✓

Quality of life ✓ ✓ ✓ ✓

Previous knee injury and occupation ✓ ✓

Weight ✓ ✓ ✓ ✓ ✓

Height ✓ ✓

Girth measurements ✓ ✓ ✓

Knee radiograph ✓

Serum 25-(OH)D ✓ ✓ ✓

Serum calcium, phosphate, creatinine ✓ ✓

Sun exposure ✓ ✓ ✓ ✓

Cigarette smoking ✓ ✓

Diet (FFQ) and pedometer ✓ ✓

Medications ✓ ✓ ✓ ✓ ✓ ✓

Pill counts and adverse events ✓ ✓ ✓ ✓ ✓

Participants who withdraw within one year will be asked to have MRI atmonth 12; patients who withdraw after one year will be asked to have MRIstraight away. MRI: magnetic resonance imaging; WOMAC: Western OntarioMcMaster Universities Osteoarthritis Index; 25-(OHD)D: 25-hydroxy-vitamin D;FFQ: food frequency questionnaire.



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These measures have a high degree of reliability with aninterclass correlation coefficient (ICC) > 0.90 across arange of studies [36].Upper arm blood pressure, central blood pressure and

aortic stiffness: Clinical upper arm blood pressure will bemeasured twice after 5 minutes seated rest using a vali-dated device (Omron HEM-907, Kyoto, Japan). Seatedclinical central blood pressure will be recorded (immedi-ately after upper arm blood pressure) using radial appla-nation tonometry (SphygmoCor 8.1, AtCor Medical,Sydney, Australia). Aortic stiffness will be measured byelectrocardiogram-gated, sequential carotid to femoralpulse wave velocity as per expert consensus [37].Physical activity: Physical activity will primarily be

assessed using a pedometer (SW 200 Digi-Walker,Yamax Corporation, Tokyo, Japan), which measures ver-tical displacement (steps per day). The pedometer willbe worn for seven consecutive days on two occasions(baseline and 2 years) as up to seven days is required toaccurately assess habitual physical activity [38]. Wewill also measure physical activity using the Inter-national Physical Activity Questionnaire (IPAQ) shortversion [39].Body fat: Body fat will be assessed using bioelectrical

impedance analysis (BIA) (BIA analyser, Quantum II,RJL Systems, Michigan, USA). Fat-free mass, % fat-freemass, fat mass and % fat mass will be assessed [40].Hand grip strength: Hand grip strength will be

assessed to the nearest kg in both the right and left handusing a hydraulic hand dynamometer (Saehan Corpor-ation, Masan, Korea). Both hands will be alternatelymeasured in triplicate.Radiographic OA: this will be assessed at baseline by a

standing semiflexed anterior-posterior (AP) radiographas per the Altman atlas [29]. Radiographs will also beassessed simultaneously by two observers using theOsteoarthritis Research Society International (OARSI)atlas to score osteophytes and joint space narrowing ona four-point scale (0 to 3).Laboratory measurements: serum 25-(OH)D will be

assayed at month 0, 3 and 24, utilizing a Liquid Phaseradioimmunoassay (Immunodiagnostics Systems Ltd,Boldon, Tyne & Wear, UK). Serum calcium, phosphateand renal function will be assessed at month 0 and 3using routine biochemical methods.Anthropometrics and other questionnaires: Height will

be measured to the nearest 0.1 cm (with shoes removed)using a stadiometer (Leicester Height Measure, InvictaPlastics Ltd, Leicester, UK). Weight will be measured tothe nearest 0.1 kg (with shoes and bulky clothingremoved) using electronic scales (Heine S-7307, Heine,New Hampshire, USA). Waist and hip measurementswill be assessed using a tape measure to the nearest0.1 cm (Figure Finder Tape Measure, Novel Products

Inc, Illinois, USA). Sun exposure, employment statusand occupation, depression, smoking status, previousknee injury, dietary intake, low back and foot pain andquality of life will be assessed by questionnaires.

Safety assessmentsSpontaneously reported adverse events will be recordedthroughout the study. Intensity and relationship with thestudy medication will be ascribed.

Sample sizeAll sample size calculations assume α= 0.05 and β= 0.20and are performed based upon formulae provided byCohen [41]. Table 2 describes the sample size (each arm)needed to detect the specified differences between theplacebo and vitamin D arms with at least 80% power foreach outcome.Previous studies, including our own, suggest that OA

patients have a loss of cartilage volume of 4 to 5% peryear at different joint sites, respectively [42]. Vitamin Dsupplementation in doses ranging from 400 to 800 IU/dincreased the serum level of 25-(OH)D by 27 nmol/literper year in 7,964 men and women from five studies [43].We estimate from our published data [15] that thischange will lead to absolute reduction in loss of cartilagevolume by 2.2% at the medial tibial site after vitamin Dsupplementation. The sample size that is needed to de-tect this difference is calculated (Table 2).We have shown that male OA patients have an in-

crease in medial tibial bone area of 1.6 ± 2.8% per year[44], and an incidence of knee cartilage defects of 80%over 2 years [45]. There are no data known to the inves-tigators about the associations between change in vita-min D and change in bone area or cartilage defects.However, healthy subjects have been shown to have anincrease in tibial bone area of 0.7% per year (CD et al.,unpublished), and an incidence of knee cartilage defectsof 65% over 2 years in older people [46]. Assuming thatchanges in cartilage defects and bone area in OApatients will be suppressed by vitamin D supplementa-tion to the levels in the healthy subjects, the sample sizeneeded to detect these differences is given in Table 2.

Table 2 Sample size calculation

Mean(SD)

Detectabledifference

Calculatedsample size(per arm)

Loss of volume of medialtibial cartilage

4.5%± 6.5% 2.16% 143

Increase in medial tibialbone area

1.6%± 2.8% 0.9% 153

Incidence of kneecartilage defects

80% 15% 136



Therefore, 200 patients in each arm (allowing for a20% dropout over the trial) will be sufficient to detectthe differences between treatment groups.

Analysis planStatistical primary comparisons for total and subscaleWOMAC scores will be made using a repeated measuresmixed model with terms for treatment, month, centerand the corresponding baseline values as the covariates.The independent t-tests will be used to compare changesbetween groups in quantitative data from baseline to theend of follow-up. Linear regression (annual changes incartilage volume, cartilage defects, bone area and musclestrength as the dependent variables, and treatment asthe independent variable) and logistic regression (devel-opment/progression of bone marrow lesions and menis-cal abnormalities as the dependent variables, treatmentas the independent variable) analyses will be applied inunivariate and multivariate modeling adjusted for age,sex, body mass index, baseline 25-(OH)D and other dis-ease status.In secondary analysis of loss in cartilage volume, the

minimal clinically important differences (MCID) in cartil-age volume will be calculated [47] and logistic regressionwill be used to determine the association between cartilageloss (>=MCID vs. <MCID) and treatment before andafter adjustment for the covariates described above.Both intention to treat and per protocol analyses will

be utilized. Per protocol will be defined as achieving a25-(OH)D level > 60 nmol/liter at month 3. The last ob-servation carried forward method will be used in the ana-lysis of all outcomes among patients who made at leastone follow-up visit but did not complete the whole study.

Data integrity and managementAll data obtained will be kept strictly confidential andwill be stored electronically on a database with securedand restricted access. Data transfer will be encrypted andany information capable of identifying individuals will beremoved.

WithdrawalIf a participant withdraws or is removed from the study,the reason and date of discontinuation will be recorded.Any participant who withdraws within year 1 will beasked to have MRI at the end of year 1; participants with-drawing after year 1 will be asked to have MRI on leavingthe study.

MonitoringThe trial will be overseen and monitored by a projectmanager. The project manager will visit each site toexamine trial procedures to ensure data quality andcompliance with the trial protocol.

DiscussionWe have proposed this protocol to determine if vitaminD supplementation can slow disease progression inpatients with knee OA. Vitamin D may have beneficialeffects for the treatment of OA, although there are cur-rently no recommended guidelines for this approach[48]. Hence, well-designed randomized controlled trialsare required to test if vitamin D has disease-modifyingand pain-relieving effects. Such studies also need an ap-propriate follow-up period to capture joint structuralchanges using objective measurements over the courseof OA, and this has been incorporated into the design ofthe VIDEO study.Assessing disease-modifying effects on OA requires an

accurate measurement tool that is able to evaluateimprovements in cartilage and joint health. Radiographicassessment of OA is two-dimensional, lacks sensitivityfor changes over a short period and is highly susceptibleto measurement error through factors such as variationin joint positioning [49]. MRI allows direct, accurate andreliable assessment of joint structural changes over time.These structural changes include cartilage loss, cartilagedefects, increased tibial bone area, subchondral bonemarrow lesions, meniscal tears and meniscal extrusion.Almost all these structural changes are predictive oftotal knee replacement [45,50,51], suggesting they areclinically relevant. Simultaneously, we will assess changein knee pain over time using WOMAC as a co-primaryendpoint. Thus, the findings from this study will showwhether vitamin D supplementation has both disease-modifying and symptom-relieving effects.As suggested by the 2011 Endocrine Society Clinical

Practice Guideline, all adults aged 50 to 70 years, andthose over 70 years old require at least 600 and 800 IU/dof vitamin D respectively, to maximize bone health andmuscle function. To raise blood levels of 25-(OH)Dabove 75 nmol/liter (the lowest sufficient threshold)requires at least 1500 to 2000 IU/d of supplemental vita-min D [52]. Thus, our study design provides a dose of50,000 IU monthly to achieve serum 25-(OH)D levelsabove 60 nmol/liter in all compliant subjects [53]. Thismethod will be less costly and will be more convenientthan daily treatment. Toxicity is extremely unlikely withthis dose.Two sub-studies will simultaneously be included in

this trial. Firstly, we will examine the effects of vitaminD on the function of the deep lumbo-pelvic stabilizingmuscles. Besides implications for low back pain, inhealthy people core muscles have also been implicatedin varying aspects of physical function. The lateral ab-dominal muscles are theorized to control movement andprovide stability to the trunk for functional activities andthis is supported in a number of studies [54]. Vitamin Dsupplementation may have beneficial effects on



functionally important core muscles. Secondly, we willdetermine the effect of vitamin D supplementation onblood pressure and aortic stiffness. A recent systematicreview suggested that there is accumulating evidence tosupport the hypothesis that vitamin D deficiency contri-butes to hypertension, and randomized controlled trials(RCTs) are greatly needed to clarify and to definitivelyprove the effect of vitamin D on blood pressure [55].The VIDEO study aims to be the first to assess this.In summary, knee OA is a major, but poorly under-

stood, public health problem. Vitamin D deficiency mayplay a role in the progression of OA, and based on ournovel preliminary data, the VIDEO study has beendesigned to determine whether intervening with vitaminD supplementation can in fact slow the progression ofthis disease and relieve knee pain. If correcting vitaminD deficiency can reduce rates of cartilage loss to lowerlevels as seen in older people without OA, it will signifi-cantly prolong the time it takes to reach end-stage OAeventually requiring joint replacement. This suggestsgreat potential for substantial cost savings throughreductions in joint replacement surgery, as well as po-tential for great improvements in the quality of life forpeople with OA. The success of this study will providescientific evidence for using a cost-effective and innova-tive approach to addressing this clinically significantproblem and will lend itself to an easy public healthintervention.

Trial statusUpon submission, VIDEO study is in the process ofpatient recruitment.

Abbreviations25-(OH)D: 25-hydroxy-vitamin D; ACR: American College of Rheumatology;AQOL: assessment of quality of life; BIA: bioelectrical impedance analysis;CRF: case report form; FSEL: fast spin echo; FFQ: food frequencyquestionnaire; GRE: gradient echo; ICC: interclass correlation coefficient;IPAQ: International Physical Activity Questionnaire; LM: lumbar multifidus;MCID: minimal clinically important differences; MMP: metalloproteinase;MRI: magnetic resonance imaging; OA: osteoarthritis; OARSI: OsteoarthritisResearch Society International; PHQ-9: Personal Health QuestionnaireDepression Scale-9; PGE2: prostaglandin E2; RAAS: renin-angiotensin-aldosterone system; RCT: randomized controlled trial; SOP: standardoperating procedure; TASOAC: Tasmania Older Adult Cohort;TrAb: transversus abdominus; VAS: visual analogue scales; VIDEO: Vitamin DEffects on Osteoarthritis; VDR: vitamin D receptors; WOMAC: Western OntarioMcMaster Universities Osteoarthritis Index.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsCD and GJ conceived the study, CD, GJ, FC, TW, AW, JS, KN and YCparticipated in its design and coordination, and performed the research. YC,GJ, KN and CD drafted the manuscript. All authors revised the manuscriptand gave final approval of the version to be submitted.

FundingThe VIDEO study is supported by the National Health & Medical ResearchCouncil (NHMRC ID 605501).

AcknowledgementsJodi Barling, Judy Hankin and Alice Noone have been involved in thecoordination of this study.

Author details1Menzies Research Institute Tasmania, University of Tasmania, Private Bag 23,Hobart, TAS 7000, Australia. 2Department of Epidemiology and PreventiveMedicine, Monash University, Melbourne, 3004, Australia. 3Research Instituteof Orthopaedics, Shuguang Hospital Affiliated to Shanghai University ofTraditional Chinese Medicine, Shanghai 201203, China.

Received: 4 April 2012 Accepted: 18 July 2012Published: 6 August 2012

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doi:10.1186/1745-6215-13-131Cite this article as: Cao et al.: Vitamin D supplementation in themanagement of knee osteoarthritis: study protocol for a randomizedcontrolled trial. Trials 2012 13:131.



Summary of changes

Primary outcomes

Original: Loss of knee cartilage volume from baseline to month 24.

Final: Addition of change in knee pain assessed using the Western Ontario and McMaster

Universities Index of OA (WOMAC) score.

MRI assessment of subchondral bone marrow lesions (BMLs)

Original: BMLs were scored as following:

Grade 1: BMLs were present on 1 slice.

Grade 2: BMLs were present on 2 consecutive slices.

Grade 3: BMLs were present on >3 consecutive slices.

Final: BMLs were scored using a Modified Whole Organ MR Scoring (WORMS) system:

Grade 1: BMLs occupy <25% of the region.

Grade 2: BMLs occupy 25% to 50% of the region.

Grade 3: BMLs occupy >50% of the region.

Statistical analysis plan

No change.


effect of vitamin d supplementation on tibial cartilage ...€¦ · joint structures (i.e.,...

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