1. No category

Knee Osteoarthritis: A Clinical Trial Examining Therapeutic Effects of HighRosmarinic Acid Spearmint Tea and Investigations into Relationships of Pain
with Modifiable Lifestyle Factors and Biomarkers of Joint Metabolism and
Inflammation
by
Adele Erin Connelly
A Thesis
Presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Doctor of Philosophy
In
Human Health and Nutritional Sciences + Toxicology
Guelph, Ontario, Canada
© Adele Erin Connelly, April, 2015
Abstract
Knee Osteoarthritis: A Clinical Trial Examining Therapeutic Effects of High-Rosmarinic
Acid Spearmint Tea and Investigations Into Relationships of Pain with Modifiable Lifestyle
Factors and Biomarkers of Joint Metabolism and Inflammation
A. Erin Connelly
University of Guelph, 2015
Advisors:
Dr.Alison M. Duncan &
Dr.Amanda J. Wright
Osteoarthritis (OA) is a chronic, progressive disease involving the degeneration of
cartilage and joint tissue, resulting in pain and disability. This thesis investigated OA knee pain
and physical function, namely associations with lifestyle factors and biomarkers as well as a
novel therapeutic product to manage symptoms of knee OA. In a survey of healthy adults with
knee OA (n=197), OA characteristics, health history and information about smoking history,
alcohol consumption, height, body weight, medication and supplement intake, and exercise
habits were collected. The Western Ontario and McMaster Universities Osteoarthritis Index
(WOMAC) was used to assess pain. OA medication use and higher body mass index (BMI)
category were associated with higher pain. Supplement use and meeting physical activity
guidelines (150 min/week) were associated with lower pain. Next, associations between OA
symptoms and biomarkers of joint metabolism and inflammation (n=54) were analyzed using
Spearman correlation coefficients, controlling for age, gender and BMI. Serum hyaluronic acid
was significantly associated with better performance in the 6-minute walk test (6MWT) and stair
climb test (SCT). Serum matrix mellatoproteinase-3 was significantly associated with higher
pain. Interleukin-6 was significantly associated with higher pain, stiffness and worse
performance in the SCT. Finally, in a randomized, double-blind clinical trial, participants (n=46)
consumed tea brewed from a high-rosmarinic acid (rosA) spearmint or a commercially available
spearmint twice daily for 16 weeks. Pain significantly decreased within the high-rosA group, but
not the control group and scores for stiffness and physical disability significantly decreased
within both groups. Increased quality of life score on the bodily pain index in the short form
(SF)-36 questionnaire was observed within the high-rosA group only. In the 6MWT, only the
high-rosA group increased distance walked at Week 16, but the increase was not statistically
significant. There were no changes in the SCT within either group. Overall, this thesis found
associations between OA symptoms and biomarkers of joint metabolism and inflammation and
characterized the relationship between OA pain and certain lifestyle factors. Also, for the first
time, a decrease in pain with consumption of a high-rosA acid spearmint tea was observed,
rationalizing further consideration in the management of knee OA pain.
Dedication
This thesis is dedicated to my grandmother, Elizabeth Connelly. Your love of knowledge and
education is inspiring and has served as one of the greatest influences in my life. You are a
model of strength, courage, and resolve.
iv
Acknowledgements
I am forever grateful for everyone who has supported, challenged, and helped me throughout this
process. I generally gravitate to strong female role models, and I really hit the jackpot with these
three; Dr. Alison Duncan, Dr. Amanda Wright, and Dr. Amy Tucker. Alison and Amanda, you
are both such well-rounded, hard working professors. You successfully balance family, research,
teaching, students, business, and I have learned so much from watching you in action. You both
want the best for your students and work hard for that to happen. Alison, it is hard to
comprehend how anyone can accomplish as much as you do in a day/week/year and I am
grateful for all you have done for me, including passing on an appreciation for statistics.
Amanda, thank you for all of your patience with me. You always challenge me to be the best
version of myself, as a scientist and as a person, and I am indebted to you for your persistence.
Thank you both for providing me with this wonderful opportunity and guiding me through this
process with kindness and support. Amy, thank you so much for being a mentor and a friend. I
don’t know how other people get through doctoral degrees without an ‘Amy’. You think about
science in such a complete, creative, and thoughtful way. I have learned so much from you and
had so many revelations in your office. Thank you for your patience, kindness, and guidance.
Dr. Laima Kott, thank you for your invaluable contributions to this thesis and for serving on my
advisory committee. Dr. Lindsay Robinson and Dr. Monica Maly, thank you for your important
roles in my examination. Thank you to both Dr. Bill Bettger and Dr. Jim Kirkland for serving on
my advisory and comprehensive exam committees. Bill, you have been an important role model
and I have learned so much for you. Jim, I am grateful for our time together and it has also been
a pleasure being your teaching assistant for the last 4 years and I will miss it.
I have had the opportunity to work with many different types of people, all of whom were
important to this process, and my growth, and I value those experiences. Natasha, you were a life
saver during a stressful time, I am so grateful that we got to work together and I know that you
are going to do great things. Thank you to all my past and present research collaborators and
lab/HNRU friends.
v
HHNS is truly a remarkable place. I have met and been inspired by so many wonderful people in
this department. Brilliant, hard-working, fun, athletic, charitable, weird, and overall amazingly
well-rounded individuals walk the halls and amaze me with their seemingly effortless
awesomeness. I have made so many wonderful friendships in this department and they have
made me a better person, scientist, and intramural athlete (excluding softball), and they are dear
to me. Jessica Ralston, your friendship, support and kindness has meant the world to me. You
have taught me so many things and I admire your intelligence, creativity and insane work ethic.
Kaitlin Roke, I look up to you in so many ways, you are so smart, so kind, and so caring. Tara
McDonald and Emily McIntosh, you are incredible scientists and humans and I am grateful for
your friendship. Thank you to my wonderful partner and friend, Andrew. You have been a major
part of this process and always know exactly what I need. Thank you for making me laugh, being
my sounding board, a shoulder to cry on, and a kick in the pants (when needed). Thank you for
your patience, love, and encouragement.
I need to thank my tiny, furry friend, Domi, who has been by my side and who always brings a
smile to my face. I am lucky to have a large, warm supporting family who have been so proud
and excited for me during this process. Mom and Dad, you have shown me what life is like when
you are passionate about your job, work hard, and have big goals. Thank you for your
unconditional support, trust, and love. You are my biggest cheerleaders, source of strength, and
best friends.
vi
Table of Contents
Abstract
Dedication……………………………………………………………………………………....iv
Acknowledgments………………………………………………………………………............v
Table of Contacts……………………………………………………………………………….vii
List of Tables……………………………………………………………………………............xi
List of Figures…………………………………………………………………………………...xiii
List of Abbreviations…………………………………………………………………………....xvi
List of Appendices……………………………………………………………………………...xix
Chapter 1: Introduction and Review of the Literature……………………………………..1
1.1. Introduction…………………………………………………………………………...........1
1.2 Review of the Literature……………………………………………………………………2
1.2.1 Osteoarthritis……………………………………………………………………...2
1.2.1.i Definition of Osteoarthritis……………………………………………...2
1.2.1.i.a Clinical Diagnosis………………………………………..........2
1.2.1.ii. Prevalence of OA………………………………………………………3
1.2.1.iii. Societal and Individual Costs…………………………………………3
1.2.2 Pathology of OA…………………………………………………………….........5
1.2.2.i. Tissues involved………………………………………………………..6
1.2.2.i.a Cartilage……………………………………………………….6
1.2.2.i.b. Bone ………………………………………………………….8
1.2.2.i.c. Synovial Membrane…………………………………………..9
1.2.2.i.d. Menisci and Ligaments………………………………………10
vii
1.2.2.ii Pain Pathology………………………………………………….10
1.2.3 Risk factors for OA……………………………………………………………….12
1.2.3.i. Non-Modifiable Risk Factors…………………………………………...13
1.2.3.i.a. Age……………………………………………………….…....13
1.2.3.i.b. Female Sex………………………………………………….....14
1.2.3.ii. Modifiable Risk Factors………………………………………………..16
1.2.3.ii.a. Obesity…………………………………………………..........16
1.2.3.ii.b. Knee Injury…………………………………………………...16
1.2.3.ii.c Physical Activity……………………………………………...18
1.2.3.ii.d. Diet………………….……………………………………….18
1.2.4 Measuring OA and the Progression of OA……………………………………….19
1.2.4.i. Imaging……………………………………………………………........19
1.2.4.i.a Radiography/X-rays…………………………………………...19
1.2.4.i.b. MRI…………………………………………………………...20
1.2.4.ii. Clinical Symptoms……………………………………………………..21
1.2.4.ii.a. Pain…………………………………………………………...22
1.2.4.ii.b. Physical Function…………………………………………….23
1.2.4.iii. Biomarkers…………………………………………………………….25
1.2.5. Non-Surgical Treatment and Management of OA……………………………….28
1.2.5.i. Management Recommendations for All Individuals with Knee OA…..29
1.2.5.i.a. Education and Self-Management…………………………......29
1.2.5.i.b.Weight Loss/Weight Maintenance………………………….....30
1.2.5.i.c. Exercise……………………………………………………….31
viii
1.2.5.ii. Management Recommendations for some with Knee OA……………………..32
1.2.5.ii.a. Pharmaceuticals………………………………………………33
1.2.5.ii.b. Biomechanical Interventions and Aids………………………34
1.2.5.ii.c. Natural Health Products/Supplements………………………..35
1.2.5.ii.d. Dietary Constituents…………………………………………..39
1.2.5.ii.d.1. Rosmarinic Acid…………………………………….39
1.2.6 Summary…………………………………………………………………………...45
Chapter 2: Aims of the Thesis…………………………………….…………………………...46
Chapter 3: Modifiable lifestyle factors are associated with lower pain in adults with knee
osteoarthritis………………………………………………………………………………........47
3.1 Abstract……………………………………………………………………………...48
3.2 Introduction……………………………………………………………………….....48
3.3 Methods……………………………………………………………………………...50
3.4 Results……………………………………………………………………………….53
3.5 Discussion…………………………………………………………………………...59
3.6 Conclusions………………………………………………………………………….65
3.7 Acknowledgments…………………………………………………………………...65
3.8 Bridge to Chapter 4………………………………………………………………….66
Chapter 4: Serum biomarkers of joint metabolism and inflammation in relation to clinical
symptoms and physical function in adults with knee osteoarthritis………………………..67
4.1 Abstract……………………………………………………………………………..68
4.2 Introduction……………………………………………………………………........69
4.3 Methods……………………………………………………………………………..70
4.4 Results…………………………………………………………………………........73
4.5 Discussion…………………………………………………………………………...77
4.6 Conclusions…………………………………………………………………….........82
ix
4.7 Acknowledgments…………………………………………………………………...82
4.8 Bridge to Chapter 5…………………………………………………………….........84
Chapter 5: High-Rosmarinic Acid Spearmint Tea in the Management of Knee
Osteoarthritis Symptoms……………………………………………………………………...85
5.1 Abstract……………………………………………………………………………..86
5.2 Introduction……………………………………………………………………........86
5.3 Methods……………………………………………………………………………..88
5.4 Results…………………………………………………………………………........92
5.5 Discussion…………………………………………………………………………..99
5.6 Conclusions………………………………………………………………………....100
5.7 Acknowledgments…………………………………………………………………..101
Chapter 6: Summary, General Discussion, and Future Directions…………………….......102
6.1 Summary of Results…………………………………………………………….......102
6.2 General Discussion………………………………………………………………....103
6.3 Future Directions…………………………………………………………………...106
6.4 Concluding Remarks……………………………………………………………….108
References……………………………………………………………………………………...109
Appendices…………………………………………………………………………………….145
x
List of Tables
Chapter 1:
Table 1.1 Standards developed by the American College of Rheumatology for the
diagnosis of knee OA based on type of examinations……………………………………3
Table 1.2 Alterations in pain processing that occur in OA..............................................11
Table 1.3 Odds Ratio for developing knee OA for different risk factors (Adapted from
Blagojevic et al 2010)……………………………………………………………………12
Table 1.4 Joint structures and their endpoint and outcomes visualized with MRI……..21
Table 1.5 Different methods of assessing pain in individuals with knee OA…………..23
Table 1.6 Performance-based and self-reported physical function assessments………..24
Table 1.7 OARSI recommended biomarkers for continued investigation………………27
Table 1.8 Results from systematic reviews and meta-analysis examining exercise
interventions in knee OA………………………………………………………………...32
Table 1.9 Results from systematic reviews and meta-analysis examining pain relieving
effects of pharmaceutical products in adults with hip or knee OA………………………34
Table 1.10 Most commonly studied NHP and supplement products for the treatment of
OA………………………………………………………………………………………..37
Chapter 3:
Table 3.1 Summary of participant characteristics (N = 197)……………………………54
xi
Table 3.2 Health history and lifestyle factors across mild, moderate and severe WOMAC
pain categories…………………………………………………………………………...55
Table 3.3 Stepwise multiple regression examining factors related to pain score in adults
with knee osteoarthritis…………………………………………………………………..59
Chapter 4:
Table 4.1 Participant characteristics, WOMAC scores and physical function tests…….74
Table 4.2 Serum biomarker levels……………………………………………………….75
Chapter 5:
Table 5.1 Participant characteristics and outcome measures at baseline………………..95
Table 5.2 SF-36 scores for Weeks 0 and 16 for the high-rosA and control groups…….97
Table 5.3 Six-minute walk test distances (m) for Weeks 0, 8, and 16 for the high-rosA
and control groups……………………………………………………………………….98
Table 5.4 Biomarker levels in the high-rosA and control groups in Week 0 and Week
16………………………………………………………………………………………...98
xii
List of Figures
Chapter 1:
Figure 1.1 The burden of OA is affected by direct, indirect and intangible costs.
(Modified from Hunter et al 2014)………………………………………………………4
Figure 1.2 Cross-sectional picture of healthy knee joint on the left and characteristic
changes to those structures in OA on the right. Taken from Hunter 2011………………5
Figure 1.3 Complex cyclic processes in cartilage, bone, and synovial membrane in knee
OA contribute to the disease pathogenesis. Chondrocytes produce matrix degrading
enzymes (MMP and ADAMTS) and inflammatory mediators that can act on the
chondrocytes or enter the synovial membrane to cause inflammation. Chondrocytes
produce type X collagen in a repair attempt, but it is not suitable for adult ECM. In the
bone, osteophytes and bone marrow lesion lead to an adverse biomechanical environment
and are potentially painful. Adapted from Lee et al
(2013a)……………………………………………………………………………………8
Figure 1.4 Aging alone does not cause OA. In aging, changes that affect joint function
and tissues increase susceptibility to OA. For the development of symptomatic OA,
additional risk factors or OA factors, need to be present. (Modified from Anderson and
Loeser 2010)……………………………………………………………………………14
Figure 1.5 Management of knee OA should follow a sequential, pyramidal approach
where options at the bottom of the pyramid are suitable for all with OA, simple, nonsurgical options are suitable for some, advanced non-surgical options are suitable for few
with OA and surgery is the last, least desirable options. Modified from Dieppe and
Lohmander 2005………………………………………………………………………..29
Figure 1.6 Chemical structure of Rosmarinic Acid…………………………………….40
xiii
Chapter 3:
Figure 3.1 Chi-square analysis revealed significant differences in the percent of
participants in the mild pain (0-125), moderate pain (126-250), and severe pain (251-500)
categories in terms of BMI category [normal weight (N), overweight (OW), or obese
(OB)] (A), self-reported health (B), use of medications for OA (C), use of prescription
medications for OA (D), use of supplements (E), and met physical activity guidelines (F).
BMI = body mass index………………………………………………………………….58
Chapter 4:
Figure 4.1 Significant associations between serum HA and physical function tests in all
participants. Serum HA was measured by ELISA in 54 participants with knee OA and
correlated with performance in the 6MWT (A) and SCT (B) as objective measures of
physical function…………………………………………………………………………76
Figure 4.2 Significant associations between serum MMP3 and IL-6 and WOMAC pain
score in all participants. Serum MMP3 was measured by ELISA in 54 participants with
knee OA and correlated with WOMAC pain score (A). Serum IL-6 was measured by a
multi-plex assay in 53 participants with knee OA and correlated with WOMAC pain
score (B)………………………………………………………………………………….76
Figure 4.3 Significant associations between serum IL-6 and physical function tests in all
participants. Serum IL-6 was measured by multi-plex in 53 participants with knee OA
and correlated with performance in the 6MWT (A) and SCT (B) as objective measures of
physical
function…………………………………………………………………………………..77
Chapter 5:
Figure 5.1 Participant flow through the trial (CONSORT diagram)……………………93
xiv
Figure 5.2 Mean scores and standard error are presented for (A) WOMAC pain scores,
(B) WOMAC stiffness scores, (C) WOMAC physical function score and (D) WOMAC
total scores for the high-rosA group (circles) and the control group (triangles). The
change within groups from baseline to week 16 was examined; significant differences
within the high-rosA group are indicated with a cross (+) and significant differences
within the control group are indicated with an asterisk (*). rosA, rosmarinic acid;
WOMAC, Western Ontario and McMaster Universities Osteoarthritis Index………..96
xv
List of Abbreviations
6MWT
6-minute walk test
AAOS
American Academy of Orthopaedic Surgeons
ACL
Anterior cruciate ligament
ACR
American College of Rheumatology
ADAMTS
A disintegrin and metalloproteinase with thrombospondin
motifs
AGEs
Advanced glycation end products
ASU
Avocado/soybean unsaponifiables
AIMS
Arthritis Impact Measurement scale
BMI
Body mass index
BMLs
Bone marrow lesions
BW
Body weight
CGRP
Calcitonin gene related peptide
CI
Confidence Interval
CIA
Collagen-induced arthritis
COMP
Cartilage oligomeric matrix protein
COX-2
Cyclooxygenase-2
CRP
C-reactive protein
CVD
Cardiovascular diseases
ECM
Extra cellular matrix
ELISA
Enzyme-linked immunosorbent assay
ES
Effect size
EULAR
European League Against Rheumatism
FDA
Food and Drug Association
GAG
Glycosaminoglycan
GI
Gastrointestinal
GSH
Glutathione
HA
Hyaluronic acid
xvi
HO-1
Heme oxygenase-1
HRT
Hormone replacement therapy
IL
Interleukin
IKK-b
Inhibitor of nuclear factor kappa-B kinase subunit beta
JSN
Joint space narrowing
KL
Kellgren-Lawrence
KOOS
Knee injury and OA outcome score
KOS-ADL
Knee outcomes survey activities of daily living scale
LIF
Leukemia inhibitory factor
LPS
Lipopolysaccharide
MMP
Matrixmetalloproteinase
MPQ
McGill pain questionnaire
MS
Millisecond
MSK
Musculoskeletal
MRI
Magnetic resonance imaging
NHP
Natural health product
NO
Nitric oxide
NOAEL
No observed adverse effect level
NRS
Numerical rating scale
NSAIDs
Nonsteroidal anti-inflammatory drugs
NF-kB
Nuclear factor-kB
Nrf2
Nuclear factor-like 2
OA
Osteoarthritis
OARSI
Osteoarthritis Research Society International
OMERACT
Outcome measures in rheumatology
OR
Odds ratio
PASE
Physical Activity Scale for the Elderly
PGE2
Prostaglandin E2
PIIANP
Procollagen type II N-terminal propeptide
QoL
Quality of life
RA
Rheumatoid arthritis
xvii
RCT
Randomized controlled trial
ROS
Reactive oxygen species
RosA
Rosmarinic acid
SCT
Stair-climb test
SMD
Standard mean difference
SOD
Superoxide dismutase
TBARS
Thiobarbituric acid reactive substances
TEAC
Trolox equivalent antioxidant capacity
TNFa
Tumor necrosis factor-alpha
VAS
Visual analog scale
WHO
World Health Organization
WOMAC
Western Ontario and McMaster Universities Osteoarthritis
Index
xviii
List of Appendices
Chapter 1:
Appendix 1. A table displaying the range and diversity of diseases for which rosA has been
examined for its potential therapeutic effects…………………………………………………140
Chapter 3:
Appendix 2. Certificate of approval from University of Guelph Human Research Ethics’
Board............................................................................................................................................123
Appendix 3. Participant informed consent form…………………………………………….....124
Appendix 4. Lifestyle Questionnaire…………………………………………………………..127
Appendix 5. Sample of 24-hour food recall sheet……………………………………………..134
Chapter 5:
Appendix 6. Certificate of approval from University of Guelph Human Research Ethics’
Board…………………………………………………………………………………………...135
Appendix 7. Participant informed consent…………………………………………………….136
Appendix 8. Recruitment poster……………………………………………………………….145
Appendix 9. Telephone participant eligibility questionnaire…………………………………..146
Appendix 10. In-person participant eligibility questionnaire……..…………………………...150
Appendix 11. Participant tea brewing instructions…………………………………………….160
Appendix 12. Example study diary sheet………………………………………………………161
Appendix 13. Anthropometric case report form……………………………………………….162
Appendix 14. Physical function test case report form…………………………………………164
Appendix 15. Biomarker results from clinical trial....................................................................167
xix
Chapter 1: Introduction and Review of the Literature
1.1 Introduction
Osteoarthritis (OA) is a progressive disease characterized by degeneration of articular cartilage
and alteration of joint tissues, resulting in pain, stiffness and disability (Martel-Pelletier and
Pelletier 2010). It is the most common form of arthritis and a leading cause of pain and disability
in older adults (Loser et al 2012). The knees, hips and hands are most commonly affected, with
knee OA having the greatest impact on disability (Jordan et al 2004b). The pain in OA is the
most significant clinical issue and impacts function, mobility and quality of life (Hawker 2012).
There is no cure for OA or method of halting joint tissue destruction. Treating OA pain is
complex as pain is a multifactorial biopsychosocial phenomenon that is not completely
understood. Additionally, pain and other symptoms of OA are not strongly correlated to
radiographic evidence of the disease (Bijlsma et al 2011). This complicates research trying to
evaluate the efficacy of different therapies. Current analgesic treatments are not effective and the
overall lack of successful treatment options remains a large and critical gap in the management
of OA. Many of the limitations in discovering effective treatments for OA stem from an
incomplete understanding of OA etiology, a poor ability to measure and define the disease or
assess disease progression, and response to new treatment options (Felson 2014). In general, this
thesis investigates the impact of lifestyle factors, including a bioactive-enriched spearmint tea, on
the management of knee OA and relationships between potential biomarkers and symptoms of
OA. Investigating novel diet-derived therapeutic products is an important area of research that
holds potential for managing symptoms of OA. The literature review provides an overview of
OA, including its pathology and risk factors, as well as methods for measuring OA and current
management strategies.
1
1.2 Review of the Literature
1.2.1 Osteoarthritis
1.2.1.i Definition of Osteoarthritis
OA is a disease of the synovial joints where degeneration of joint tissue results in pain, stiffness,
and impaired physical function (Lane et al 2011; Litwic et al 2013). Pain is the most prominent
and disabling symptom of OA, resulting in reduced participation in activities and negative effects
on mood, sleep, and overall quality of life (Dieppe and Lohmander 2005; Lane et al 2011;
Shimura et al 2013). OA can arise in any synovial joint in the body, but is most common in the
hands, hips, knees and spine (Dieppe and Lohmander 2005). Knee OA results in the most severe
disability and the knee is the joint most extensively studied in OA research (Dieppe and
Lohmander et al 2005; Hunter and Felson 2006; Hayashi et al 2014). Although there is no
standard definition for OA, it can be defined structurally or symptomatically (Nelson et al 2012;
Litwic et al 2013). Radiographic OA is used in research settings and is based on structural
characteristics of the joint, including joint space narrowing (JSN), osteophytes, bone cysts, and
sclerosis visualized on radiographs (Felson et al 1997). Symptomatic OA is used in clinical and
research settings and is based on radiographic evidence, as well as assessment of pain, stiffness,
and cracking of joints (Altman et al 1986).
1.2.1.i.a Clinical Diagnosis
Diagnosis of OA is based on an overall clinical impression of age, history, location of joint
abnormalities, and sometimes, radiographic evidence (Wu et al 2005). The American College of
Rheumatology developed standards for diagnosis of knee OA in 1986 that are still used today
(Table 1.1) (Altman et al 1986; Litwic et al 2013).
2
Table 1.1 Standards developed by the American College of Rheumatology for the diagnosis of
knee OA based on history and type of examination.
History and Physical
History, Physical
History, Physical
Examination
Examination and
Examination and
Radiographic Findings
Clinical Findings
Knee pain, and
Knee pain
Must Knee pain
osteophytes
Have
And at least 3 of:
- Over 50 years of age
- Less than 30 minutes
of morning stiffness
- Crepitus on motion
- Bony tenderness
- Bony enlargement
- No palpable warmth
of synovial membrane
And at least 1 of;
- Over 50 years of age
- Less than 30 minutes of
morning stiffness
- Crepitus on motion
And at least 5 of;
- Over 50 years of age
- Less than 30 minutes of
morning stiffness
- Crepitus on motion
- Bony tenderness
- Bony enlargement
- No palpable warmth of
synovial membrane
- ESR < 40 mm/hour
- RF < 1:40
- Synovial fluid signs of
OA
*Adapted from Altman et al 1986.
ESR = erythrocyte sedimentation rate; RF = rheumatoid factor.
1.2.1.ii. Prevalence of OA
In Canada, OA affects approximately 4.4 million people (Bombardier et al 2012). Worldwide,
OA is the most common joint disorder (Storheim and Zwart 2014), and the World Health
Organization (WHO) estimates that 10% of the world’s population have clinical problems
attributed to OA (Woolf and Pfleger 2003). Of the total OA burden, knee OA is estimated to
account for 83% (Vos et al 2013; Murray et al 2013). Due to increased longevity, reduced
physical activity levels, and increasing prevalence of obesity, the burden of OA is increasing
(Bombardier et al 2012; Hunter et al 2014). It has been predicted that by 2040, more than 10
million Canadians will be diagnosed with OA (Bombardier et al 2012).
1.2.1.iii. Societal and Individual Costs
OA is a costly disease with Canada, United States of America, United Kingdom, France, and
Australia spending approximately, 1 to 2.5% of the gross national product on care for arthritis
(Hunter et al 2014). In 2010 in Canada, direct and indirect health care costs of OA were
estimated to be $10.2 billion and $17.3 billion, respectively (Bombardier et al 2012). Direct costs
3
include drugs, side effects of treatment, non-pharmacological treatments, visits to health
professionals, home adaptations, tests, hospitalizations, surgical procedures and use of
community services (Figure 1) (Gupta et al 2005; Bombardier et al 2012; Chen et al 2012).
Indirect costs are incurred based on absence from work, productivity losses at work, leaving the
work force, caregiver time off work, and premature mortality (Gupta et al 2005; Bombardier et al
2012; Chen et al 2012).
The burden of OA must also be evaluated in terms of its impact on quality of life and other
intangible costs, as there are significant physical and psychological effects on the individual
(Figure 1.1) (Chen et al 2012; Hunter et al 2014). Pain, suffering, decreased quality of life,
depression and anxiety, and reduced participation in activities are common in adults with OA
(Chen et al 2012; Hunter et al 2014). Adults with OA report more pain, worse quality of life,
greater number of hospitalizations and reduced productivity than those without (daCosta
DiBonaventura et al 2012). The total economic burden of OA in Canada is expected to rise to
over $1,455 billion in 2040 (based on 2010 values) (Bombardier et al 2012).
Figure 1.1 The burden of OA is affected by direct, indirect and intangible costs. (Modified from
Hunter et al 2014).
4
1.2.2 Pathology of OA
OA is a heterogeneous disease, involving complex and interacting mechanical, biological,
biochemical, molecular, and enzymatic feedback loops with cartilage degeneration as the
common, final event (Martel Pelletier and Pelletier 2010; Umlauf et al 2010). Despite this
degeneration, OA is an active process and a network of mechanisms reacting to stress or injury
on the joint (Umlauf et al 2010; Loeser et al 2012). All joint features are affected in OA (Figure
1.2) (Hunter 2011). Structural changes include cartilage fibrillation, degeneration of articular
cartilage, thickening of subchondral bone, osteophyte formation, synovial inflammation,
degeneration of ligaments and meniscus, hypertrophy of joint capsule, cellular and molecular
changes in nerves, as well as changes to periarticular muscle, bursa, fat pads (Figure 1.2)
(Goldring and Goldring 2006; Martel Pelletier and Pelletier 2010; Loeser et al 2012). The loss of
cartilage and modifications to bone and synovial membrane contribute to an unfavourable
biomechanical environment which increases stress on the joint and furthers the progression of
cartilage degradation (Goldring and Goldring 2006; Heijink et al 2012).
Figure 1.2 Cross-sectional picture of healthy knee joint on the left and characteristic changes to
those structures in OA on the right. Taken from Hunter 2011.
5
1.2.2.i. Tissues involved
In OA, there is degeneration and alteration of the cartilage, bone, synovial membrane, menisci,
and ligaments, which contributes to an unfavourable biomechanical and biochemical
environment (Hunter 2011).
1.2.2.i.a Cartilage
Articular cartilage covers the ends of bones in synovial joints to provide a smooth surface with
low friction for efficient gliding, load bearing, impact absorption, and to sustain shear forces
during movement (Martel-Pelletier et al 2008; Struglics and Hansson 2012). It can accomplish
these functions because of the structure and composition of the extra cellular matrix (ECM),
which is composed of collagen, proteoglycans, and water (Martel Pelletier and Pelletier 2010). A
fibrillar collagen network, composed of type II collagen linked together by cartilage oligomeric
matrix protein (COMP) and chondroadherinin, is arranged in triple helices and gives cartilage its
stiffness and tensile strength (Martel-Pelletier et al 2008; Sofat 2009; Goldring and Goldring
2010). The other main component of cartilage is the proteoglycan aggrecan. This is a large
aggregating polymeric structure with a large protein core and 3 globular domains (Sofat 2009).
Glycosaminoglycan (GAG) side chains on aggrecan contain a large number of negatively
charged groups, which attract and retain water to form a gel that gives cartilages its compressive
ability (Sofat 2009). There are no blood vessels, lymphatic vessels, or nerves in cartilage and
chondrocytes are the only cell type present (Martel Pelletier and Pelletier 2010). As such,
chondrocytes are responsible for the synthesis, breakdown, and maintenance of the ECM and
respond to mechanical forces, osmotic stresses, growth factors, cytokines, and other
inflammatory mediators (Sofat 2009; Goldring and Goldring 2010; Loeser et al 2012; Struglics
and Hansson 2012).
The dynamic equilibrium between synthesis and degradation of ECM maintained by
chondrocytes is disrupted in OA, with degradation being favoured (Lee et al 2013a). Catabolic
activity increases as a response to cartilage loss, and cell proliferation and enhanced production
of matrix proteins occurs (Goldring and Marcu 2009; Goldring and Goldring 2010; Martel
Pelletier and Pelletier 2010). Despite the repair attempt, proteoglycans are degraded, exposing
type II collagen fibrils which are then accessible for degradation (Loeser et al 2012).
6
Chondrocytes produce and release matrix metalloproteinase (MMP) and a disintegrin and
metalloproteinase with thrombospondin motifs (ADAMTS) enzymes, responsible for cleaving
collagen fibrils and aggrecans, respectively (Anderson and Loeser 2010). In a cyclical manner,
cleaved parts of ECM can act on chondrocytes or diffuse to the synovial membrane to initiate
kinase signaling, producing more enzymes and inflammatory mediators (Figure 1.3) (Goldring
2012; Loeser et al 2012). Although the exact roles of these mediators are unknown, interleukin
1-beta (IL-1b), tumor necrosis factor-alpha (TNFa), interleukin 6 (IL-6), leukemia inhibitory
factor (LIF), interleukin 17 (IL-17), interleukin 18 (IL-18), nitric oxide (NO), prostaglandin E2
(PGE2), and leukotriens are increased in OA and involved in the disease pathogenesis (MartelPelletier et al 2008; Umlauf et al 2010). It is not known if inflammation in OA occurs as an
initiating event or secondary to cartilage breakdown (Conde et al 2011). With the loss of
cartilage and increased inflammatory and oxidative stress, apoptosis of chondrocytes occurs (Lee
et al 2013a). Chondrocytes synthesize new aggrecan and collagen in a repair attempt, but the
new aggrecan is similar to juvenile proteoglycans and the collagen produced is type X and
neither of these can maintain the same mechanical properties as healthy adult cartilage (Figure
1.3) (Lohmander et al 1999; Martel-Pelletier et al 2008). This abnormal composition contributes
to an adverse biomechanical environment, further stimulating degradation (Loeser et al 2012).
The changes to the cartilage are not directly responsible for OA pain as cartilage is aneural and
not capable of generating pain, although the other joint structures are richly innervated (Lee et al
2013a).
7
Figure 1.3 Complex cyclic processes in cartilage, bone, and synovial membrane in knee OA
contribute to the disease pathogenesis. Chondrocytes produce matrix degrading enzymes (MMP
and ADAMTS) and inflammatory mediators that can act on the chondrocytes or enter the
synovial membrane to cause inflammation. Chondrocytes produce type X collagen in a repair
attempt, but it is not suitable for adult ECM. In the bone, osteophytes and bone marrow lesions
lead to an adverse biomechanical environment and are potentially painful. Adapted from Lee et
al (2013a).
1.2.2.i.b. Bone
Bone alterations are recognized as important in the pathogenesis of OA, although less well
understood than changes in cartilage (Dieppe and Lohmander 2005). In OA, the structure and
properties of subchondral bone are altered with increases in subchondral plate thickness,
modification of the architecture of the trabecular bone, formation of new bone at joint margins,
and changes in the vascularity (Figure 1.3) (Dieppe and Lohmander 2005; Goldring and
Goldring 2010; Martel Pelletier and Pelletier 2010). Bone marrow lesions (BMLs) are also
8
common and consist of bone marrow necrosis, trabecular abnormalities, bone marrow fibrosis,
and edema (Figure 1.3) (Raynauld et al 2008; Ding et al 2010). These are hypothesized to result
from excessive repetitive loading or an acute inflammatory response (Raynauld et al 2008;
Loeser et al 2012). There is evidence that subchondral bone changes precede cartilage
degradation (Goldring and Goldring 2010; Martel Pelletier and Pelletier 2010). Changes in bone
could promote abnormal cartilage metabolism through reduced structural support, impaired
nutrient supply, and provision of catabolic factors (Ding et al 2010; Goldring and Goldring 2010;
Martel Pelletier and Pelletier 2010). Increased bone stiffness could also make the cartilage less
effective at accommodating mechanical loads (Goldring and Goldring 2010). As bone is richly
innervated, it may be a potential source of pain in OA (Cotofana et al 2013). Indeed, several
studies have correlated bone abnormalities observed by magnetic resonance imaging (MRI) with
knee pain in OA (Link et al 2003; Kornaat et al 2006; Cotofana et al 2013).
1.2.2.i.c. Synovial Membrane
The synovial membrane is a thin sheet of connective tissue that covers all structures in the joint
except the cartilage (Martel Pelletier and Pelletier 2010). It is well vascularized and innervated
and contains cells that phagocytize debris and secrete hyaluronic acid (HA) and lubricin to
facilitate gliding during movement (Martel Pelletier and Pelletier 2010; Loeser et al 2012).
Inflammation of the synovial membrane as well as synovial hypertrophy and hyperplasia occur
in OA (Martel Pelletier and Pelletier 2010). Products of cartilage degradation diffuse into the
synovial membrane where they are phagocytosed by macrophages, initiating the release of
cytokines, reactive oxygen species (ROS) and synovial inflammation (Figure 1.3) (Martel
Pelletier and Pelletier 2010). These cytokines are then released into the synovial fluid where they
can diffuse into the cartilage to act on chondrocytes, continuing the cycle of degradation and
inflammation (Martel Pelletier and Pelletier 2010). Synovial cells are also capable of producing
MMPs and ADAMTS in response to inflammatory signaling (Bondeson et al 2010). ROS
produced in synovial inflammation are also believed to play a role in OA by breaking down
ECM, up regulating matrix degrading enzymes, and inhibiting anabolic signals (Goldring and
Berenbaum 2004; Im et al 2008). As the synovial membrane is richly innervated, synovial
inflammation is potentially an important source of pain in OA and significant relationships
9
between synovitis and OA symptoms has been reported in several studies (Benito et al 2005;
Loeuille et al 2005; Martel Pelletier and Pelletier 2010; Sowers et al 2011; Ballegaard et al
2014).
1.2.2.i.d. Menisci and Ligaments
The knee menisci are two pads of fibrocartilage that lie between the femur and tibia and are
attached to the joint capsule (Englund et al 2012). The menisci are an integral part of the
biomechanical system of the knee joint and work to stabilize the joint, resist tension,
compression, and sheer stress, and absorb shock during dynamic movement (Martel Pelletier and
Pelletier 2010; Englund et al 2012). There are 3 ligaments in the knee. These consist of
collagenous fibers arranged as parallel bundles that provide anterioposterior and rotational
stability of the knee, prevent hyperextension, and provide proprioceptive information (Vincent et
al 2012; Witt and Vilensky 2014). Pathological changes in the menisci and ligaments are
common in adults with knee OA, even in those without previous injury (Ding et al 2010; Loeser
et al 2012). These pathologic changes include matrix disruption, fibrillation, cell clusters,
calcification, and cell death, which can alter knee kinematics and strain areas of cartilage that are
not accustomed to those loads, promoting cartilage degradation (Loeser et al 2012). In addition,
increases in vascular penetration and innervation have been reported in OA menisci, suggesting a
potentially important source of pain in OA (Ashraf et al 2011).
1.2.2.ii Pain Pathology
It is not well understood how the process of joint degeneration in OA causes pain (Witt and
Vilensky 2012; Heijink et al 2012). The structural origins of pain in OA also are unclear, but
pain fibers innervate the synovial membrane, ligaments, menisci, and subchondral bone (Witt
and Vilensky 2012). The central mechanisms of interpreting pain signals are also not well
understood, but there is an emotional component of chronic pain (McDougall 2006; Lee et al
2013a). Depression and anxiety are common co-morbidities in OA and have been associated
with the chronic pain experienced (Hawker et al 2011). Pain is multifactorial and in OA,
believed to arise from nociceptive and neuropathic mechanisms (Mease et al 2011). Nociceptive
pain occurs with local tissue damage, where mechanical or chemical stimulation of nociceptors
causes the transmission of a pain signal from the joint to the dorsal root ganglion in the spinal
10
cord and then up the spinothalamic tract to cortical centers for processing (Lee et al 2013a).
When a tissue is damaged, chemicals are released that cause physiological pain and which can
also sensitize nociceptors (Mease et al 2011; McDougall and Lindon 2012). Neuropathic pain is
generated from damage to the nerves (Hochman et al 2010) and has been detected in individuals
with OA using questionnaires that identify the type and quality of pain sensations (i.e. burning,
numbness, etc.) (Hochman et al 2010; Ohtori et al 2012). In OA and other chronic pain
conditions, ongoing activation of pain fibers produces continuous neurotransmitter release into
the spinal cord, causing increased neuronal activity and alterations in peripheral and central pain
processing (McDougall 2006; Mease et al 2011). Table 1.2 shows and defines a number of these
alterations in pain processing that occur in OA.
Table 1.2 Alterations in pain processing that occur in OA.
Alteration in Pain
Explanation/Definition
Processing
Allodyina
Innocuous stimuli is perceived and transmitted
as noxious stimuli (i.e. pain while walking).
Hyperalgeisa
Heightened pain intensity to a noxious insult (i.e.
increased sensitivity to painful sensations).
Peripheral and
Decrease in the activation threshold of a neuron,
central sentization from chronic nociceptor stimulation.
Enlargement of
Increase in the area that a nociceptor innervates.
receptor field
Can occur beside injured or inflamed tissue.
Spontaneous firing Nociceptors send signals in the absence of
mechanical stimulation (i.e. pain at rest).
Altered cortical
Individual variability in central processing of
processing
nociceptive stimuli. Has been hypothesized to
explain the lack of association between structural
damage and pain in OA.
Reference
Witt and
Vilensky 2012
McDougall
2006
Hochman et al
2010
Mease et al
2011
McDougall
2006
Lee et al 2013
The same inflammatory mediators involved in joint destruction play a role in the altered pain
processing in OA (Omoigui 2007; Lee et al 2013a). During inflammatory conditions, the number
of cytokine receptors on neurons is increased (Witt and Vilensky 2012). Cytokines can directly
or indirectly stimulate neurons and lower the firing threshold to initiate the alterations described
in Table 1.2 (Konttinen et al 2012). Inflammatory mediators can also activate silent nociceptors
in a joint. These are pain fibers that are not active unless the body is under stress (Witt and
Vilensky 2012). Neuropeptides are chemical mediators released from the terminals of joint
afferents that can cause neurogenic inflammation (McDougall 2006). The neuropeptides,
11
substance P, calcitonin-gene-related-peptide, and neurokinin-1, are believed to play a role in OA
pain (Konttinen et al 2012; Witt and Vilensky 2012). It has also been suggested that neurogenic
inflammation may contribute to joint damage by promoting and amplifying the inflammatory
response (Kidd et al 2003).
1.2.3 Risk factors for OA
Several large prospective cohort studies have provided a wealth of information on risk factors for
the development of OA. The main non-modifiable risk factors associated with incidence of OA
are age and female sex, while family history and developmental conditions that affect bone
growth or joint shape have also been identified (Felson et al 2000; Blagojevic et al 2010).
Modifiable risk factors for OA include body mass index (BMI), joint injury, physical activity and
diet. Table 1.3 presents odds ratios (OR) calculated from a meta-analysis examining risk factors
for the onset of knee OA (Blagojevic et al 2010).
Table 1.3 Odds Ratio for developing knee OA for different risk factors (Adapted from
Blagojevic et al 2010)
Risk
OR
Studies
Notes
Factor
(95% CI) Included
Age
Not
15
OR wasn’t calculated due to differing classification
calculated
of age groups. Age was a risk in all studies.
Female
1.84
8
Gender was often used an as adjustment factor and
Gender
(1.32-2.55)
gender specific effects were not often reported.
BMI
2.18
23
OR for overweight versus normal.
(1.86-2.55)
2.63
17
OR for obese versus normal.
(2.28-3.05)
Knee
3.86
16
Injury
(2.61–5.70)
Physical
Not
21
OR wasn’t calculated due to varied definitions of
Activity
calculated
physical activity. Higher quality studies showed a
general increased risk with intense exercise.
*BMI = body mass index; OR = odds ratio
12
1.2.3.i. Non-Modifiable Risk Factors
1.2.3.i.a. Age
Age is the primary risk factor for OA and the incidence of OA increases with age (Anderson and
Loeser 2010; Litwic et al 2013). However, everyone who ages does not get OA (Anderson and
Loeser 2010). It is generally thought that normal changes with aging increase the susceptibility
to OA, but additional risk factors are required for the development of symptomatic or clinical
OA (Figure 1.4) (Anderson and Loeser 2010). Radiographic damage in a knee also increases
with age, even in the absence of disease, demonstrating that mild joint degradation may occur
and accumulate with aging (Ding et al 2010; Anderson and Loeser 2010). It is nearly impossible
to distinguish if these radiographic changes are a part of normal aging or preclinical OA that has
not yet developed. Aging also decreases the ability of the joint to respond to stressors and
maintain homeostasis in the cartilage, as observed by the fact that older adults develop OA more
rapidly than younger adults following similar injuries (Roos et al 1995; Anderson and Loeser
2010).
Cellular changes that occur with aging can also increase the susceptibility to OA (Figure 1.4). As
there is little to no cell division or cell death in adult cartilage, chondrocytes can accumulate agerelated changes over time (Heijink et al 2012). The accumulation of advanced glycation end
products (AGEs) can increase collagen cross-linking, making cartilage more brittle (Heijink et al
2012). There is also evidence that aged chondrocytes show a senescence-associated secretory
phenotype that includes increased production of cytokines, chemokines and MMPs that may
contribute to matrix degradation (Heijink et al 2012; Loeser et al 2012).
13
Figure 1.4 Aging alone does not cause OA. In aging, changes that affect joint function and
tissues increase susceptibility to OA. For the development of symptomatic OA, additional risk
factors or OA factors, need to be present. (Modified from Anderson and Loeser 2010).
1.2.3.i.b. Female Sex
The prevalence of OA is similar in both sexes until the age of 50, after which the prevalence in
women increases significantly (Felson et al 1997; Srikanth et al 2005; Maleki-Fischbach and
Jordan 2010). Not only are women diagnosed with OA more often, they usually have more
severe pain and disability than men (Keefe et al 2000; Perrot et al 2009; Sims et al 2009; Elbaz et
al 2011; Tonelli et al 2011; Glass et al 2014).
Pre-clinical studies have shown that a complex and incompletely understood relationship exists
between cartilage metabolism and circulating gonadal steroids (Reginster et al 2003). Estrogen
receptors are present on human and animal chondrocytes and estrogen has been found to have
chondroprotective roles via its effect on growth factors, cytokines, MMPs, and ROS (Rosner et
al 1982; Tanko et al 2008). A 10-year prospective cohort study found that lower serum estradiol
14
and urinary estrogen metabolite levels were associated with increased risk of knee OA (Sowers
et al 2006). Because of this evidence, the effect of hormone replacement therapy (HRT) on the
development and progression OA has been thoroughly examined. Several cross-sectional
(Hannan et al 1990; Samanta et al 1993; Nevitt et al 1996; Spector et al 1997) and longitudinal
(Zhang et al 1998; Hart et al 1999) studies have found that HRT use is associated with a
decreased risk of knee OA, although other studies have not found an association (Sowers et al
1996: Erb et al 2000; Maheu et al 2000; Nevitt et al 2001). Some have even demonstrated an
increased risk (Oliveria et al 1996; Sandmark et al 1999). Although it is possible that changes in
estrogen levels can account for the increased risk of OA in women, it is a complicated
relationship that has yet to be fully elucidated.
Several studies have found that men have larger cartilage volume and thickness, and a larger
subchondral bone/cartilage area than women after controlling for BMI (Otterness and Eckstein
2007; Maleki-Fischbach and Jordan 2010). This relationship has also been found in children. In a
study with children aged 9-18 years (n=92), boys had greater cartilage thickness and volume than
girls, after accounting for body weight, bone size, and physical activity (Jones et al 2000). There
was a significant association between increased cartilage volume and vigorous activity for more
than 20 minutes, so it was suggested that increased physical activity in young boys may account
for cartilage differences (Jones et al 2000). These gender differences in cartilage may play a role
in the initiation of knee OA, but more research in this area is required.
Psychosomatic influences may also mediate the greater proportion of women diagnosed with
OA, as radiographic OA is common, but a diagnosis of OA relies on pain as well. Some studies
have shown that women more commonly employ emotion-focused pain coping strategies and
pain catastrophizing than men when dealing with pain or stressful events (Keefe et al 2000;
France et al 2004). Emotion-focused pain coping and pain catastrophizing have been associated
with higher pain in OA (Keefe et al 2000). However, when Keefe et al (2000) controlled for pain
catastrophizing, gender differences in OA pain were eliminated. Increased risk of OA for
females is well established, and although hypotheses exist to explain this, the reasons are still
unclear.
15
1.2.3.ii. Modifiable Risk Factors
1.2.3.ii.a. Obesity
Obesity is a well-established risk factor for the development OA (Cooper et al 2000; Hunter
2009; Elbaz et al 2011; Goulston et al 2011; Lee at el 2013b; Riddle and Stratford 2013). A
prospective cohort study revealed that losing weight (≥ 2 BMI units) was associated with a 50%
decrease in risk of OA (Felson et al 1992). Obesity can result in excessive mechanical demand
and increased loading and forces on the knee joint, which can directly damage articular cartilage
(Messier et al 1996; Heijink et al 2012). This has been demonstrated in animal studies where
abnormal loads due to obesity result in alterations in the composition, structure, metabolism and
mechanical properties of cartilage (Griffin et al 2009; Guilak 2011). In addition, slower walking
with short and wide steps, greater stance duration and other altered gait and joint biomechanics
in obese adults may contribute to OA risk; however, the specific role of such changes in terms of
OA is not fully understood (Guilak 2011; Runhaar et al 2011). Obese individuals are also at a
higher risk for hand OA, indicating that the relationship between obesity and OA is not just
mechanical (Katz et al 2010). Adipose tissue is a source of local and systemic inflammation and
a number of adipokines associated with obesity (IL-6, IL-1, TNFa) have also been shown to
influence cartilage biology (Katz et al 2010; Issa and Griffin 2012). In addition, hypertension,
hypercholesterolemia, and high blood glucose are associated with knee OA, independent of BMI,
indicating that a common set of metabolic factors are involved in those diseases (Hart et al 1995;
Sowers et al 2009, Issa and Griffin 2012). Obesity is a primary risk factor for knee OA, with
strong evidence for the role of biomechanical and metabolic factors contributing to disease
initiation and progression.
1.2.3.ii.b. Knee Injury
Knee injury is a strong risk factor for the development of knee OA due to direct damage to the
tissues, disruption of biomechanics, and a large inflammatory response (Roos et al 1995;
Blagojevic et al 2010; Litwic et al 2013). It has been reported that 50-60% of patients with
injuries to ligaments or menisci are diagnosed with OA 12-15 years later and that this
relationship is not altered by surgical intervention (Roos et al 1995; Jarvholm et al 2005;
Blagojevic et al 2010). Anterior cruciate ligament (ACL) injury plus traumatic meniscus damage
seems to be the strongest predictor of early joint cartilage changes and OA (Neuman et al 2011;
16
Englund et al 2012). Indeed, in animal models ACL transection and damage to the meniscus is
used to induce rapid and extensive OA-like damage to the joint (Goldring 2012).
Knee cartilage adapts to cyclic loading and thickens in areas of greatest loading, with the thickest
cartilage found at the lateral facet of the tibia and thinnest cartilage on the medial facet (Vincent
et al 2012). With knee injury, the meniscus or ligament loses its function and loading during
weight bearing activities can occur in areas of cartilage that are not adapted (Vincent et al 2012).
Loading in non-adapted regions has been shown to lead to cartilage fibrillation, loss of
proteoglycans, increased surface friction, increased sheer stress, trabecular bone changes,
subchondral bone lesions, joint malalignment, up regulation of catabolic factors, and cartilage
degradation (McDougall 2006; Englund et al 2012; Vincent et al 2012).
The large immune and inflammatory response that occurs with injury could initiate a cycle of
inflammation and degradation in the knee joint (McDougall 2006; Lotz 2010). Immediately
following an injury, inflammatory mediators are released into the joint from nerves,
synoviocytes, and vascular endothelium to orchestrate healing responses (McDougall 2006). The
trauma itself and inflammatory mediators cause cell necrosis, collagen rupture, GAG loss, and
rupture of blood vessels in the joint capsule, synovial membrane, menisci, or subchondral bone
that leads to intraarticular bleeding (Lotz 2010). Intraarticular bleeding promotes ECM loss and
leads to synovial cell hypertrophy and synovitis (Hooiveld et al 2003; Lotz 2010). Shortly
thereafter, there is an activation of viable cells to respond to the trauma, which also includes the
generation of ROS, matrix degrading enzymes, and more inflammatory mediators (Marks and
Donaldson 2005; Lotz 2010). In joint injury, there is also an increase of nociceptors and
activation of silent nociceptors (McDougall and Linton 2012). Injured regions in healed joints
display truncated and varicose nerves and have been reported to be full of inflammatory
neuropeptides including, substance P and calcitonin gene related peptide (CGRP), which have
been shown to increase pain responses and contribute to the inflammation (McDougall and
Linton 2012).
17
1.2.3.ii.c Physical Activity
The role of physical activity as a risk factor for knee OA is controversial (Ding et al 2010;
Barbour et al 2014). There are studies showing protective (Hart et al 1999), damaging (Felson et
al 1997; Cheng et al 2000; Cooper et al 2000), and no (Felson et al 2007; Barbour et al 2014)
effects, as well as increasing risk with different levels of physical activity (McAlindon et al
1999; Lin et al 2013). Importantly, the lack of a standard definition of physical activity or
method of measuring levels makes quantifying risk difficult. Examining the risk of OA from
sporting participation is also complicated because of the different types of sports, duration of
participation, and frequent occurrence of joint injuries (Kujala et al 1995). Repetitive, excessive
forces on the knee joint from intense exercise or sport participation may compromise structural
integrity of the cartilage and promote degradation (Lin et al 2013). The risk of OA development
does seem to be moderately increased with sporting participation, which could be mediated
through associated joint injury (Bennell et al 2011b). However, joint loading and movement is
needed for bone and joint health, and therefore walking or other low impact activities may be
protective for knee OA (Felson et al 2007; Barbour et al 2014). In older adults without knee OA
or previous knee injuries, vigorous activity was associated with increased cartilage volume and
regular walking was associated with reduced risk of BMLs (Racunica et al 2007). The risk of
knee OA with physical activity is most likely only present with high levels or risky types of
exercise and is different between different ages, types of activities and genetics.
1.2.3.ii.d. Diet
The role of diet as a risk factor for OA is also controversial. Low intake of vitamins has been
examined as potential risk for the initiation of OA. Vitamin D has been most extensively studied,
as it has biological functions in cartilage, bone and muscle (McAlindon et al 1996b; Tetlow and
Woolley 2001; Ding et al 2009). However, cross-sectional studies have not found associations
between serum levels of 25(OH)D and radiographic knee OA (Al-Jarallah et al 2011; Muraki et
al 2011). Furthermore, in 3 large, prospective cohort studies, baseline intake of vitamin D and
serum levels of 25(OH)D were not related to incident knee OA, 6.5 to 22 years later (McAlindon
et al 1996b; Bergink et al 2009; Konstari et al 2012). The role of ROS in the pathogenesis of OA
has led to the hypothesis that high intake of antioxidant micronutrients could help to protect
against OA by mediating oxidative damage (Jordan et al 2004a). An inverse association between
18
fruit intake and BMLs was found in healthy adults, suggesting that fruit constituents, including
antioxidants, could have a role in protecting against knee structural damage (Wang et al 2007).
Along with its antioxidant actions, vitamin C has a role in collagen synthesis, which may
increase its relevance to OA. Vitamin E is a potent chain-breaking antioxidant and protector
against lipid peroxidation (Jordan et al 2004a; Jeon et al 2013). However, baseline intake of
vitamin C and E was not associated with incidence of OA 6.5 years later (McAlindon et al
1996a). The role of diet as a risk factor for the initiation of OA remains controversial and the
complex interplay of bioactive components in whole foods and other lifestyle factors associated
with diet quality complicate analysis.
1.2.4 Measuring OA and the Progression of OA
Measuring OA and its progression is a significant challenge in OA research and clinical practice.
In the research setting, the disease can be defined radiographically but symptoms are necessary
for medical diagnosis. Unfortunately, there is a weak correlation between radiographic and
symptomatic evidence in OA (Dieppe and Lohmander 2005). Importantly, this limits the ability
to measure response to a novel treatment. In 1996, the Outcome Measures in Rheumatology
(OMERACT) group recommended that all clinical trials include the assessment of pain, physical
function, patient global assessment and for studies longer than 1 year, imaging (Altman et al
1996). Quality of life, physician global assessment, inflammation, biological markers, time to
surgery, and analgesic consumption were also included as potential endpoints (Altman et al
1996).
1.2.4.i. Imaging
1.2.4.i.a Radiography/X-rays
Radiography is the main method in epidemiology and research to define and measure OA
(Petersson 1996; Lawrence et al 1998). Radiographs can visualize the space between bones,
osteophytes, subchondral cysts, and bone sclerosis (Guermazi et al 2011). Cartilage cannot be
visualized, so as an indirect measure, the space between the joint and JSN is used (Hayes et al
2005). The Kellgren-Lawrence (KL) scale from 0-4 is used to define severity of OA, based on
JSN and the presence of osteophytes (Ding et al 2010). However, in some studies, KL scores are
19
based on JSN alone and a separate osteophyte score is given, making comparisons between
studies challenging (Altman et al 2007; Neogi et al 2009). Various positions can be used when
taking a knee x-ray, but in research, the gold standard is the standing (i.e. weight bearing) anteroposterior with knees fully extended (Altman et al 1996). However, only the medial and lateral
compartments can be viewed and the patello-femoral joint is missed (Hayes et al 2005; Bedson
and Croft 2008). It has also been postulated that improvements in joint pain during a clinical trial
may alter the ability of the patient to extend the knee, changing joint position and altering JSN,
without changing actual cartilage structure (Mazzuca et al 2002). Another limitation of
radiographs is that they are not sensitive to change. It has been shown that when radiographic
OA is detected, at least 10% of knee cartilage has already been lost (Jones et al 2004).
Radiographs do not correlate well with joint symptoms and, as such, cannot be used alone to
evaluate OA patients (Bedson and Croft 2008). A number of cross-sectional studies have found
weak associations between radiographic OA and pain (Hart et al 1999; Hannan et al 2000;
Barker et al 2004; Bedson and Croft et al 2008). Structural joint damage most likely predisposes
the joint to pain, but the severity of the joint damage bears little relation to the severity of the
pain experienced (Dieppe and Lohmander 2005). However, it has been argued that accounting
for factors that affect pain leads to strong associations between KL score and pain. Neogi et al
(2009) controlled for psychosocial and genetic factors by comparing knees within a participant
and found a strong correlation between pain and KL score. Similarly, Pereira et al (2013)
observed a strong correlation when controlling for depressive symptoms. Despite limitations,
radiography still remains the main method to define OA in research settings.
1.2.4.i.b. MRI
MRI is a powerful, non-invasive imaging tool that overcomes many of the limitations of the
radiograph as all joint structures are visualized. Additionally, joint abnormalities can be detected
before changes can be visualized on a radiograph allowing for the tracking of early joint
abnormalities to OA (Spector and Cooper 1993; Colwell et al 2001; Amin et al 2005; Hayes et al
2005; Ding et al 2007). Different MRI techniques allow for examination of many types joint
abnormalities and biochemical properties of cartilage (Hayes et al 2005) (Table 1.4). However,
interpreting MRI images require fully trained and experienced readers to be able to distinguish
between signal abnormalities from pathological changes (Guermazi et al 2011; Hayashi et al
20
2014). The relevance of MRI outcomes to symptomatic knee OA is not clear. In systematic
reviews of MRI outcomes and knee pain, just over half of the studies demonstrated statistically
significant associations (Yusuf et al 2010; Hunter et al 2013). BMLs and synovitis/effusion have
the strongest evidence for association with pain (Yusuf et al 2011). However, joint lesions on
MRI are common in individuals with no knee pain and KL scores of 0, and it is unknown if these
are pre-OA lesions or innocuous tissue changes that occur with aging (Hayes et al 2005; Hunter
et al 2013). Standardization of MRI outcome measurements and defined parameters for knee OA
is required in this area (Hayes et al 2005; Guermazi et al 2011).
Table 1.4 Joint structures and their endpoint and outcomes visualized with MRI
Joint Structure Abnormality
Endpoint
Cartilage defects
Graded and based on location, size, severity
Cartilage volume or surface area Measured in millimeters
Bone marrow lesions
Presence of, size, site, severity
Osteophytes
Length
Meniscal or ligament tears
Graded on severity
Joint effusion
Presence of fluid, small-large
Synovial cysts
Presence of, small-large
Synovitis
Presence of, mild to marked
*Information taken from Hayes et al 2005 and Guermazi et al 2011
Although MRI is more sensitive and can visualize cartilage, radiography is widely available, has
better defined outcome measures, takes less time and has a much lower cost (Guermazi et al
2011). Finding strong correlations between pain and either imaging method will be difficult until
potential cofounders that affect pain are understood and accounted for.
1.2.4.ii. Clinical Symptoms
Symptoms of OA can vary greatly among patients and include joint pain and stiffness, swelling,
decreased function, and cracking noise with joint movement (Dieppe and Lohmander 2005;
Alshami 2014). Pain is the main concern for those with OA, what drives heath care use, and
plays a large role in deciding treatment options (Hawker et al 2011; Bombardier et al 2012).
There are countless ways to measure clinical symptoms of OA and many different methods are
used in the literature. In addition, quality of life, sleep quality, mood, pain coping strategies,
participation in valued experiences, and others are recognized as contributing to the full OA
experience and are being measured in research settings (Hawker et al 2011; Lane et al 2011).
21
1.2.4.ii.a. Pain
Pain is a subjective experience with a neurophysiologic basis (Hunter et al 2009). It is not well
understood but it is complex and influenced by a multitude of factors (Neogi et al 2009).
Measures of pain can include the severity, intensity, frequency, in activity, and impact on
mobility, mood, sleep, and quality of life (Alshami 2014). In OA research, pain can be measured
with a simple presence or absence of pain question, pain rating scale, or questionnaire (Neogi et
al 2009; Allen et al 2010). Multiple methods are used in the literature, which can make
comparisons between studies difficult. Table 1.5 presents pain assessment methods used in OA
research. There are a number of multidimensional pain questionnaires available, which are
proposed to provide a more comprehensive assessment of pain and overall response to a
treatment in OA (Dworkin et al 2011). Dworkin et al (2011) examined responsiveness of various
outcome measures in OA clinical trials and despite heterogeneity, found non-significant
differences in favour of using the WOMAC pain scale. Overall, there is no pain measure
recommended for use above others. Clinicians and researchers should choose a questionnaire
specific to their purpose (Hawker et al 2011).
22
Table 1.5 Different Methods of Assessing Pain in Individuals with Knee OA
Pain
Intended
Number and Type of
Outcome
Assessment
Population
Questions
One-dimensional Pain Assessments
Pain in the last # General
Yes/No
Pain or not
of days, weeks,
or months
Visual analog
General
1
Score (0scale (100mm)
100)
or numerical
rating scale
Multidimensional Pain Questionnaires
SF-36 - Bodily
General
2 Likert questions about
Pain Subscale
pain intensity as a
subscale from the
medical outcomes SF36
McGill Pain
Chronic
4 subscales; 78 pain
Questionnaire
pain
descriptors are
conditions
categorized into
subclasses
Arthritis Impact Arthritis
5 questions (Likert
Measurement
scale)
Scale – pain
subscale
Lequesne
OA
3 subscales with 10
Algofunctional
questions
Index
WOMAC pain
OA
5
subscale
Examples of
Use
Neogi et al 2009
Glass et al 2014,
Allen et al 2010,
Riecke et al
2010; Tonelli et
al 2011
Score (0100), higher
score = lower
pain
Tonelli et al
2011; Terwee et
al 2006
A score is
assigned to
each pain
descriptor
Score (0-25),
usually scales
are combined
Creamer et al
2000; Hughes
and Carr 2002
Score (0-24)
Oben et al 2009;
Case et al 2003
France et al
2004, Keefe et
al 2000
Score (0-500 Barker et al
for VAS or 0- 2004, Wluka et
20 for Likert) al 2002
McAlindon et al
2013; Messier et
al 2004; Brand
et al 2001
VAS = Visual analog scale; WOMAC = Western Ontario and McMaster Universities
Osteoarthritis Index.
1.2.4.ii.b. Physical Function
Physical function can be defined as the ability to move around and perform daily activities
(Terwee et al 2006; Bennell et al 2011a; Dobson et al 2013). There is no gold standard of
assessment and debate exists about the use of self-reported or performance-based measures for
assessing physical function (Terwee et al 2006). Self-reporting is easier, less time consuming and
23
not influenced by observer bias but it can be influenced by culture, language, and education level
and may not reflect actual ability of the participant (Dobson et al 2013). Performance-based
measures examine functioning in an artificial situation, can be influenced by motivation to
participate, and may not reflect how the person functions in their own environment (Terwee et al
2006). Performance based measurements examine what a patient can do in a specific
timed/distance test, while the self-report is what the patient thinks they can do, and it has been
suggested that pain plays a larger role in that aspect (Terwee et al 2006). Table 1.6 presents
different assessment methods of physical function used in OA research. Assessment of physical
function is complex and overall self-reported and performance-based outcome measures capture
different, but complementary, aspects of physical function and should both be included when
assessing physical function (Dobson et al 2013; Stratford and Kennedy 2006). An international,
multidisciplinary expert advisory group reviewed performance-based measures and
recommended that the 30-s chair-stand test, 40-m fast-paced walk test, a stair-climb test, timed
up-and-go test and 6-min walk test be used for assessment of physical function in hip and knee
OA (Dobson et al 2013). There is no consensus on which self-reported questionnaire of physical
function is superior, as it depends on the population being studied and the purpose of the
assessment.
Table 1.6 Performance-Based and Self-Reported Physical Function Assessments
Assessment
Measures
Outcome Variable Examples of
Use
Performance Based
Chair stand test Assesses ability to rise and
Count (#/repetitions McAlindon
then sit back down in a chair, performed in 30
et al 2013
as well as lower body
seconds) with
strength and power
greater values
indicating better
performance
Self-paced walk Assesses time taken to walk
Time (seconds) with McAlindon
test
short distances (<50 meters)
lower values
et al 2013
indicating better
performance
Stair-climb test Assesses ability to ascend and Time (seconds) with Rejeski et al.
descend a flight of stairs, as
lower values
1995,
well as strength, power, and
indicating better
Messier et al
balance
performance
2004, Miller
et al 2006
Timed up and go Assesses ability to rise from a Time (seconds) with Oliveira et al
24
6-minute walk
test
Self-Reported
Knee Outcome
Survey-Activities
of Daily Living
Scale
(KOS-ADL)
chair, walk 3 meters, turn
around, walk back to chair
and sit down, as well
mobility, strength, balance
and agility
Assesses endurance and
ability to walk longer
distances
lower values
indicated better
performance
2012
Distance (meters)
with a greater
distance indicating
better performance
Rejeski et al.
1995,
Messier et al
2004, Miller
et al 2006
Examines symptoms and
functional limitations in daily
living caused by knee
pathologies with 17 questions
and 2 Likert subscales
Score (0-100)
greater score means
no knee related
symptoms or
functional
limitations
Score (0 – 10)
Collins et al
2011
Score (0-100)
Tonelli et al
2011, Riecke
et al 2010
Arthritis Impact
Measurement
scale (AIMS)
Measures different aspects of
the impact of arthritis,
including physical function.
28 questions in 6 domains on
a 5-point Likert scale
Knee injury and Evaluate knee problems in
Osteoarthritis
young-middle aged people
Outcome Score with OA following a knee
(KOOS)
injury with 42 Likert
questions
WOMAC
17 questions designed for
Physical Function adults with hip or knee OA
Subscale
Tonelli et al
2011
Score (0-68, Likert
or 0-1700, VAS)
Messier et al
2004, Miller
et al 2006;
Wluka et al
2002;
McAlindon
et al 2013
VAS = Visual analog scale; WOMAC = Western Ontario and McMaster Universities
Osteoarthritis Index.
1.2.4.iii. Biomarkers
A biomarker is a measurable characteristic evaluated as an indicator of normal biological
processes, pathogenic processes or pharmacological responses to therapeutic interventions (van
Spill et al 2010). Molecules that are released during metabolism of joint tissues could serve as
biomarkers of the OA process. The ‘BIPED’ framework, proposed by Bauer et al (2006) is
25
widely used and classifies the roles of biomarkers. A biomarker can belong in more than one of
the categories.

Burden of disease biomarkers measure the extent or severity of OA by correlating levels
to extent of severity of OA.

Investigative biomarkers capture markers that do not meet criteria for other categories.

Prognosis biomarkers indicate new or worsening OA by examining association between
marker and onset or progression of OA.

Efficacy biomarkers indicate a response to a treatment.

Diagnostic biomarkers are used to differentiate someone with OA from someone without.
Currently, there are no biomarkers accepted for use in any of the BIPED categories (Felson
2014). In 2011, the Osteoarthritis Research Society International (OARSI) and the United States’
Food and Drug Association (FDA) recommended further investigation of 16 urine and serum
biomarkers that reflect different tissues and processes involved in OA (Table 1.7). No
biomarkers were considered appropriate to serve as the primary outcome measure in clinical
trials, although the continued study of biomarkers was encouraged. Also, as no single biomarker
can represent all the complex biological changes occurring in the joint, it was recommended that
a panel of biomarkers should be measured in research settings. As presented in Table 1.7, the
selected biomarkers represent the main tissues affected in OA (i.e. cartilage, bone, synovial
membrane) as well as different pathological processes (i.e. collagen degradation and synthesis).
26
Table 1.7 OARSI recommended biomarkers for continued investigation
Biomarker*
Pathological Process
Proposed BIPED
Classification#
Urinary Biomarkers
CTX-II
Type II collagen degradation
B, P, E, D
Urinary or Serum Biomarkers
NTX-I
Bone resorption
P, E
CTX-I
Bone resorption
B, P, D
C1, 2C
Type I and II collagen degradation
D
C2C
Type II collagen degradation
E, D
Coll2-1
Type II collagen degradation
D, B
Serum Biomarkers
COMP
Cartilage degradation
B, P, D
HA
Synovitis
B, P, E, D
PIIANP
Type II collagen synthesis
B, P, D
CPII
Type II collagen synthesis
D
CS-846
Aggrecan synthesis/turnover
P
MMP-3
Enzyme involved in tissue breakdown
E
Modified from Kraus et al 2011
#B = burden of disease; I = investigative; P = prognosis; E = efficacy; D = diagnostic
*C1, 2C= collagenase-generated neoepitope of types I and II collagens; C2C = collagenasegenerated neoepitope of type II collagen; COMP = cartilage oligomeric matrix protein; CPII =
type II collagen carboxy-propeptide; CTX-I = carboxy-telopeptide of type I collagen; CS-846 =
aggrecan chondroitin sulfate 846 epitope; CTX-II = carboxy-telopeptide of type II collagen; HA
= hyaluronan; NTX-I = N-telopeptide of type I collagen; MMP-3 = metalloproteinases-3.
Despite the promise of biomarkers, there are limitations. Many of these molecules arise from
multiple body tissues and serum levels may not be representative of degradation in the target
joint. In addition, measuring biomarkers from synovial joint fluid is difficult as collection is
challenging and dilution can be uncontrolled (Martel-Pelletier et al 2008). Serum and urine
concentrations of biomarkers can also be affected by synovial clearance as well as metabolism,
degradation, and clearance from the circulation, which is not currently understood (MartelPelletier et al 2008). Age, gender, ethnicity, BMI, physical activity, diurnal variation, and other
physiological processes or diseases may also affect biomarker levels complicating their utility
(Martel-Pelletier et al 2008). However, biomarkers of OA deepen the understanding of disease
progression and their inclusion in research will hopefully aid in the discovery of new treatments.
27
1.2.5. Non-Surgical Treatment and Management of OA
There is no cure or disease modifying therapy for OA. Therefore treatment goals include pain
management and maintenance or improvement of physical function (Hochberg et al 2012;
McAlindon et al 2014). International, evidence-based management guidelines recommend a
combination of non-pharmacological and pharmacological options (Hunter 2009). In general,
management of knee OA should follow a sequential, pyramidal approach where effective and
less risky options are used by all individuals with knee OA and only where these options fail,
more risky options are utilized (Dieppe and Lohmander 2005; Hunter 2009), as displayed in
Figure 1.5. Invasive procedures like intraarticular injections of corticosteroids or hyaluranon are
reserved for individuals who do not respond to other treatment options (Dieppe and Lohmander
2005). Total joint replacement is an effective treatment for end-stage OA, but only suitable for
those who have exhausted available conservative interventions (Choong and Dowsey 2011;
Dowsey et al 2014).
28
Figure 1.5 Management of knee OA should follow a sequential, pyramidal approach where
options at the bottom of the pyramid are suitable for all with OA, simple, non-surgical options
are suitable for some, advanced non-surgical options are suitable for few with OA and surgery is
the last, least desirable options. Modified from Dieppe and Lohmander 2005.
1.2.5.i. Management Recommendations for All Individuals with Knee OA
OA guidelines are consistent in stating that education and self-management, weight loss (if
overweight/obese), and exercise should be included as core management practices for knee OA
(Hochberg et al 2012; AAOS, 2013; Fernandes et al 2013; McAlindon et al 2014).
1.2.5.i.a. Education and Self-Management
Patient-education programs are a method for achieving self-management. This describes an
individual’s ability to manage the symptoms, treatment, physical, and psychological
consequences of a chronic condition (Du et al 2011). Although education is widely recognized as
an important aspect of disease management and included in all management guidelines, evidence
from the literature is not overwhelmingly positive. In a meta-analysis of 14 randomized
controlled trials (RCTs) examining effects of self-management programs in OA patients, a
significant effect size (ES) for pain relief of 0.06 (95% confidence interval (CI) = 0.02 to 0.10)
was found (Chodosh et al 2005). In 2013, a meta-analysis with 29 RCTs comparing selfmanagement programs to attention control, usual care, information alone, or another intervention
29
concluded that low to moderate quality evidence exists for beneficial effects of self-management
programs in persons with OA (Kroon et al 2014). There seems to be a small benefit of selfmanagement and education for pain in adults with OA. Even with small effects, education should
be incorporated into OA management plans, as there is no risk of adverse effects.
1.2.5.i.b.Weight Loss/Weight Maintenance
Although weight loss is included in all the guidelines, weight loss as a treatment for knee OA has
not been extensively examined in the literature. A meta-analysis examined 4 weight-loss RCTs
in knee OA patients and found that 5% weight reduction was associated with insignificant
improvements pain (ES = 0.20, 95% CI = 0 to 0.39), but significant improvements in physical
function (ES=0.23, 95% CI = 0.04 to 0.42) (Christensen et al 2007). Miller et al (2006) presented
similar findings in older, obese adults with knee OA where greater weight loss following a 6months intensive weight loss program (dietary changes, nutrition and physical activity education,
and supervised exercise 3 days/week) resulted in better physical function scores, 6MWT
distances, and faster SCT times, when compared to a weight stable group. In an 18-week RCT
examining weight loss from diet, exercise, diet plus exercise, or a healthy control group in
overweight or obese adults with knee OA, only the diet plus exercise group had significant
reductions in pain and improvements in physical function (Messier et al 2004). The diet-only
intervention group lost more weight than other groups, but no significant differences in pain or
physical function between diet only and healthy lifestyle group were present (Messier et al
2004). Alternatively, in obese patients with knee OA, a calorie-restricted intensive weight loss
RCT resulted in reductions in pain and physical function scores with no difference between verylow energy diet (415 kcal/day) and low-energy diet (810 kcal/day) groups, given their respective
diet for 8 weeks, followed by 1,200 kcal/day for 8 weeks in both groups (Riecke et al 2010).
However, in a longer study in obese adults with knee OA, an intensive low energy diet group lost
significantly more weight than a minimal attention control group, but there were no significant
differences in pain at 1 year (Bliddal et al 2011). As described, education programs, exercise,
and diet interventions are used in weight loss studies, which makes it difficult to decipher the
effect of each intervention alone. To examine weight loss alone and eliminate the effect of
attention and education that comes in an intervention study, Riddle and Stratford (2013)
examined longitudinal data from 2 large cohort studies following adults with knee OA for 3030
months or 3 years. A significant dose-response relationship was found between body weight
changes and changes in pain and physical function scores for weight loss. Overall, weight loss
for those who are overweight represents an important method to controlling pain and improving
function in patients with knee OA.
1.2.5.i.c. Exercise
Although weight loss is often an outcome of exercise interventions trials, there are also benefits
of exercise without weight loss. A number of systematic reviews and meta-analyses have been
published in this area examining aerobic and strengthening exercises for the treatment of knee
OA (Table 1.8). All meta-analyses concluded that exercise has a small to moderate positive
effect on pain and physical function in adults with knee OA. Heterogeneity between populations
(age, gender, BMI, disease duration) and exercise type, duration, frequency, and intensity existed
in all meta-analyses and limited the strength of conclusions. Examining exercise ‘dose’ is
difficult as it is a factor of frequency and intensity, and the duration of intervention varies widely
between studies. In addition, blinding is not possible in exercise studies and most have been less
than 6 months, so conclusions cannot be made about long-term benefits. There was some
evidence of greater effects with one-type exercise programs over mixed (Jamtvedt et al 2008;
Juhl et al 2014); however, no differences between types of exercise (i.e. walking vs
strengthening) was found. The fact that a specific type of exercise is not beneficial over others
can be seen as a positive, as OA patients can choose an exercise program that they prefer, which
may benefit adherence, which is known to be an important factor in maintaining positive effects
of exercise (Roddy et al 2005). Exercise should be an important part of an OA management
program as improvements are observed with a variety of exercises.
31
Table 1.8 Results from Systematic Reviews and Meta-Analysis Examining Exercise
Interventions in knee OA
Reference (Number
Exercises Included
Results ES or SMD# (95% CI)
of studies included)
Roddy et al 2005 (13)
Aerobic walking and
Aerobic Walking
quadriceps strengthening
Pain: ES = 0.52 (0.34 to 0.70)
PF: ES = 0.46 (0.25 to 0.67)
Quadriceps Strengthening
Pain: ES = 0.32 (0.23 to 0.42)
PF: ES = 0.32 (0.23 to 0.41)
Devos-Comby et al
Strength, walking, aerobic, PF: ES = 0.29 (0.23 to 0.36)
2006 (16)
balance, and flexibility
Fransen and
Strength, aerobic, walking, Pain: SMD = 0.40 (0.30 to 0.50)
McConnell 2008 (32)
balance, & tai chi
PF: SMD = 0.37 (0.25 to 0.49)
Jansen et al 2011 (12)
Strength training and
Strength training;
mixed exercise (strength,
Pain: ES = 0.38 (0.23 to 0.54)
aerobic, and range of
PF: ES = 0.41 (0.17 to 0.66)
motion exercises)
Mixed exercise;
Pain: ES = 0.34 (0.19 to 0.49)
PF: ES = 0.25 (0.03 to 0.48)
Juhl et al 2014 (48)
Aerobic, resistance,
Aerobic
performance, mixed
Pain: SMD = 0.67 (0.39 to 0.94)
PF: SMD = 0.56 (0.24 to 0.87)
Resistance
Pain: SMD = 0.62 (0.45 to 0.79)
PF: SMD = 0.60 (0.37 to 0.83)
Performance
Pain: SMD = 0.48 (0.11 to 0.85)
PF: SMD = 0.56 (0.14 to 0.98)
Tanaka et al 2014 (17) Strength and aerobic
Pain: SMD = 0.57 (0.40 to 0.74)
#
Physical function is self-reported.
CI = confidence interval; ES = effect size; PF = physical function; SMD = standard mean
difference.
1.2.5.ii. Management Recommendations for some with Knee OA
The guidelines do not completely agree on their recommendations for pharmaceuticals,
biomechanical interventions and aids, and dietary constituents and natural health products
(NHPs). Dieppe and Lohmander (2005) suggest that these interventions should be used in a
supervised fashion in those where safer methods have failed (Figure 1.5).
32
1.2.5.ii.a. Pharmaceuticals
Despite management guidelines, medication is the primary intervention used by OA patients.
Medication can promote function by alleviating symptoms, but also can reduce function by
causing drug-related morbidity and mortality (Ross et al 2001). Results from RCTs have
revealed that nonsteroidal anti-inflammatory drugs (NSAIDS) are more effective at relieving
pain in OA than acetaminophen but are also associated with a greater risk of adverse effects.
Therefore, acetaminophen remains the first line pharmaceutical option for pain management
(McAlindon et al 2014). Despite the widespread use of the drug and agreement between
guidelines, there is not much data to support acetaminophen for pain relief in OA. As presented
in Table 1.9, three meta-analyses have found small effects of acetaminophen over placebo.
However, Bannuru and McAlindon (2010) reported an increase risk for gastrointestinal (GI)
adverse effects with acetaminophen consumption. When examining epidemiological data, Garcia
Rodriguez et al (2001) reported that users of acetaminophen at doses greater than 2 g had a
greater risk of upper GI complications (relative risk = 3.7, 95% CI = 2.6 to 5.1). Topical
NSAIDs should be used before oral NSAIDs. A meta-analysis in 2004 found that topical
NSAIDS were effective at relieving pain only up to 2 weeks (Lin et 2004); however, later metaanalyses found pain relief for topical NSAIDS in RCTs longer than 4 weeks (Biswal et al 2006;
Towheed 2006). There is a lower risk of GI adverse effects from topical NSAIDS when
compared to oral NSAIDS (Lin et al 2004; Chou et al 2006), but increased risk of local adverse
effects, like burning, rash and itch (Towheed 2006; Chou et al 2006). Non-selective NSAIDS
and oral COX-2 inhibitors are widely used to manage OA pain and have been shown to be
effective over placebo and acetaminophen in meta-analyses (Table 1.9). Despite these superior
effects, all studies, as well as management guidelines, highlight the increased risk for serious
gastrointestinal ulcers with bleeding, perforation, or obstruction, renal dysfunction,
cardiovascular events, and the risk of death (Zhang et al 2004; Towheed 2006; Chou et al 2006;
Argoff and Gloth 2012; Hochberg et al 2012; McAlindon et al 2014). Generally, opioids are only
recommended in situations where there are serious or absolute contraindications to NSAIDS
(Adebajo 2012). A systematic review revealed moderate pain relieving effect of codeine,
33
oxycodone, and morphine over placebo but patients were 4 times as likely to withdraw from
studies because of adverse events (Nuesch et al 2009; McAlindon et al 2014).
Despite the widespread use of pharmaceuticals for pain relief in OA, the evidence from metaanalyses reveals mild to moderate benefits and risk of adverse events, especially in older, chronic
users (Zhang et al 2004; Craig et al 2014). Only OA patients at low-risk for adverse events
should use pharmaceuticals, and short-term use and conservative dosing should be followed as
well as individual management strategies based on patient history, co-morbidities and risk for
adverse events (Hochberg et al 2012; McAlindon et al 2014).
Table 1.9 Results from Systematic Reviews and Meta-Analysis Examining Pain Relieving
Effects of Pharmaceutical Products in Adults with Hip or Knee OA
Reference
Number of
ES or SMD (95% CI) for
RCTs included
pain relief
Acetaminophen vs. Placebo
Zhang et al 2004
2
ES = 0.21 (0.02 to 0.41)
Towheed et al 2006
7
SMD = 0.13 (0.04 to 0.22)
Bannuru and McAlindon 2010
10
ES = 0.18 (0.11 to 0.25)
Topical NSAIDs vs. Placebo
Lin et 2004
13
ES = 0.40 (0.15 to 0.65)*
Biswal et al 2006
4
ES = 0.28 (0.14 to 0.42)
Towheed 2006
4
SMD = 0.33 (0.18 to 0.48)
Oral NSAIDs vs. Placebo
Bjordal et al 2004 (All oral NSAIDs)
23
ES = 0.32 (0.24 to 0.39)
Lee et al 2005 (Only COX-2 inhibitors) 15
ES = 0.44 (0.33 to 0.55)
Oral NSAIDs vs. Acetaminophen
Zhang et al 2004
8
ES = 0.20 (0.10 to 0.30)
Wegman et al 2004
5
SMD = 0.33 (0.15 to 0.51)
Towheed 2006
10
SMD = 0.25 (0.17 to 0.33)
Verkleij et al 2011
15
ES = 0.29 (0.22 to 0.35)
Opioids vs Placebo
Nuesch et al 2009
22
SMD = 0.28 (0.20 to 0.35)
* This result was at 2 weeks; no significant difference was present at 4 weeks vs. placebo
CI = confidence intervals; COX-2 = cyclooxygenase 2; ES = effect size; NSAIDs = nonsteroidal
anti-inflammatory drugs; RCTs = randomized controlled trials; SMD = standard mean
difference.
1.2.5.ii.b. Biomechanical Interventions and Aids
Biomechanical interventions for knee OA include knee braces, knee sleeves, taping, foot
orthoses and aids include canes, crutches and walkers. There is not much agreement between the
34
guidelines in this area. However, it is generally agreed upon that none of these are sufficient as
stand-alone treatment. Meta-analysis and systematic reviews are limited by poor quality of trials
and heterogeneity of interventions. There are multiple types of braces for knee OA, from a
simple sleeve to an unloader brace that aims to change the distribution of the force on the knee
(Page et al 2011). Insoles have been advocated as easy ways to decrease loading on a
compartment of the knee and decrease pain (Page et al 2011). Two systematic reviews examined
braces and insoles together and concluded that there is evidence of small beneficial effects on
pain and function in adults with knee OA (Brouwer et al 2005; Raja and Dewan 2011). It was
noted that long-term adherence is an important factors to consider and that longer high-quality
trials are required. Alternatively, Wang et al (2012) examined insoles and shoes alone in a metaanalysis and did not find any effects on function in adults with knee OA. Taping the knee at the
patella aims to realign the patella to reduce stress and unload painful soft tissues (Page et al
2011). A meta-analysis of 2 RCTs for taping did not suggest any beneficial effects on pain,
function, or gait but pooled analysis was not possible due to differences in reporting (Wang et al
2012). In general, appropriate footwear, walking aids and assistive technologies to reduce pain
and increase participation in activities for adults with knee OA is recommended and should be
based on individual situations (Fernandes et al 2013).
1.2.5.ii.c. Natural Health Products/Supplements
With the lack of strong pain relieving effects of pharmaceuticals and the challenge of chronic
pain in OA, there is a great opportunity for NHPs, oral supplement products, and dietary
constituents in the management of OA (Elder et al 2012). There is a demand for these products,
as arthritis is among the top 6 conditions for which NHPs and supplements are used (Lapane et al
2012). Evidence of the demand and potential for these products includes the fact that The
Arthritis Society provides free educational guides including “Nutrition and Arthritis” and
“Complementary and Alternative Therapies”.
A number NHPs and supplements have claimed to modify the course of OA and to control
symptoms, but there is no consensus in the guidelines on the efficacy of these products. A large
number and variety of products have been examined. The most commonly studied are
glucosamine, chondroitin, capsaicin, avocado/soybean unsaponifiables (ASU), and rosehip
35
(Table 1.10) (de Silva et al 2011; Lapane et al 2012). Glucosamine and chondroitin are
constituents of GAGs in the ECM of articular cartilage and have been used as therapeutic agents
for OA since the 1980’s (McCarty 1994; Lee et al 2010). However, most OA management
guidelines recommend against the use of glucosamine and chondroitin, based on small or nonsignificant ES in systematic reviews (Reichenbach et al 2007; Lee et al 2010; Wandel et al
2010), inconsistent results between industry and independent trials (Clegg et al 2006), and
heterogeneity among studies (Vlad et al 2007; Wandel et al 2010). The use of glucosamine
continues to rise despite a lack of evidence of its effectiveness (Block et al 2010). Capsaicin is a
compound present in chili peppers that, when applied topically, causes acute pain and sensitivity
by stimulating pain fibers and release of substance P in the skin (Mason et al 2004). Repeated
applications desensitize pain fibers and, possibly, deplete substance P (Mason et al 2004). Metaanalyses in adults with OA and other chronic pain conditions have found moderate pain relieving
effects of capsaicin over placebo (Zhang and Li Wan Po 1994; Mason et al 2004; de Silva et al
2011). However, capsaicin is associated with transient rash, itching, and burning, when
compared to placebo. ASU mixtures are made up of unsaponifiable fractions of avocado and
soybean oil and are suggested to have anti-inflammatory effects (Christensen et al 2008b). A
systematic review and meta-analysis of short-term trials in adults with hip or knee OA found
small benefit for ASU over placebo with an ES for pain relief of 0.39 (95% CI = 0.01 to 0.76)
(Christensen et al 2008b). However, a recent meta-analysis revealed ASU supplementation up to
12 months resulted in pain 8 points lower than placebo (95% CI = 1 to 16) (Cameron and
Chrubasik 2014). Powder made from the seeds and husks of the fruit of the wild-briar hedgerow
rose have shown anti-inflammatory actions in in vitro studies (Christensen et al 2008a). A metaanalysis of 3 RCTs showed a benefit of rosehip powder over placebo with an ES of 0.37 (95% CI
= 0.13 to 0.60) (Christensen et al 2008a). Data from rosehip studies is promising, but its efficacy
and safety require confirmation in larger and longer RCTs. Chinese herbal mixture SKI306X is a
purified extract from a mixture of 3 oriental herbal medicines (Clematis mandshurica,
Trichosanthes kirilowiia, and Prunella Vulagris) (Jung et al 2001). Pooling results from 2 studies
demonstrated that treatment for 4 weeks resulted in pain reduction of 17.36 (95% CI = 12.15 to
22.57) compared to placebo (Cameron and Chrubasik 2014). A proprietary extract from
Boswellia serata, 5-Loxin, has shown anti-inflammatory actions in vitro and when results from
2 clinical trials carried out by the same authors were pooled, results revealed that treatment with
36
5-Loxin for 90 days resulted in a significant decrease in pain of 16.94 (95% CI = 11.50 to
22.39) compared to placebo (Cameron and Chrubasik 2014). For the remainder of the products
described in Table 1.10, no meta-analyses were available due to low number or quality of
studies, heterogeneity between trials, and different preparation of extracts (Cameron and
Chrubasik 2014). For almost all products listed the proposed mechanism for beneficial effects in
OA stems from in vitro anti-inflammatory and anti-oxidant mechanisms.
Table 1.10 Selected references from commonly studied NHP and supplement products for the
treatment of OA
Product
MetaProposed
Comment
References
Analysis
Mechanism
Results
Glucosamine
Inconclusive AntiInconsistent
Lee et al 2010;
inflammatory and results between
Vlad et al 2007;
suggested
industry and
Wandel et al 2010
cartilage sparing
independent trials
and significant
heterogeneity
between trials
Chondroitin
Inconclusive AntiInconsistent
Lee et al 2010;
inflammatory and results between
Reichenbach et al
suggested
industry and
2007; Wandel et al
cartilage sparing
independent trials 2010
and significant
heterogeneity
between trials
Capsaicin
Moderate
Desensitization of Associated with
Zhang and Li Wan
(topical)
pain relieving pain fibers
transient rash,
Po 1994; Mason et
effects over
itching, and
al 2004; de Silva et
placebo
burning
al 2011
Avocado/soybean ES for pain
AntiMinimum of 3
Christensen et al
unsaponifiables
relief = 0.39
inflammatory,
months
2008b; Cameron
(95% CI =
anti-oxidant,
supplementation
and Chrubasik
0.01 to 0.76) anabolic and anti- suggested
2014; Cameron et
catabolic in
al 2009; Henrotin
human
et al 2010
chondrocytes
Rosehip powder
ES for pain
AntiLonger trials for
Christensen et al
relief = 0.37
inflammatory,
confirmation of
2008a; Cameron et
(95% CI =
anti-oxidant
efficacy and
al 2009
0.13 to 0.60)
safety required
Chinese herbal
Pain relief
AntiModest
Cameron and
mixture
17.36 (95%
inflammatory
improvements
Chrubasik 2014
37
(SKI306X)
CI = 12.15 to
22.57)
Boswellia serrata
extract (5Loxin)
Pain relief
16.94 (95%
CI = 11.50 to
22.39)
Not available
Antiinflammatory,
analgesic
S-adenosyl
methionine
Not available
Ginger extract
(Zingiber
officinale)
Not available
Antiinflammatory,
anabolic actions
in human
chondrocytes
Antiinflammatory,
anti-oxidant
Devils claw
(Harpagphytum
procumbens)
Not available
Antiinflammatory,
anti-oxidant
Pycnogenol
pine bark extract
Not available
Willow bark
(Salix
daphnoides)
Not available
Antiinflammatory,
anti-oxidant
Antiinflammatory
Purple passion
fruit peel extract
Not available
Methylsulfonylm
ethane
Antiinflammatory
Antiinflammatory,
anti-oxidant
observed with 4
to 6 weeks
supplementation
Longer trials for
confirmation of
efficacy and
safety
Modest
improvements in
pain, up to 12
weeks
Similar pain
reducing effects
as NSAIDs in
short and long
term trials
Evidence of pain
relief from poorly
designed studies
with various types
of extracts
Inconclusive
results from
different products
of the roots
Modest evidence
for improvements
in pain
Inconclusive
results from 2
studies
Cameron and
Chrubasik 2014
Debbi et al 2011
De Silva et al 2011
Cameron et al
2009
Cameron and
Chrubasik 2014;
Cameron et al
2009
Cameron and
Chrubasik 2014;
Farid et al 2007
De Silva et al
2011; Cameron
and Chrubasik
2014
Farid et al 2010
Modest pain
relieving effects
observed after 8
week
supplementation
Curcumin
Not available AntiModest pain
Henriotin et al
inflammatory,
relieving effects
2010; Nakagawa et
anti-oxidant,
after 8 week
al 2014
analgesic
supplementation
CI = confidence interval; ES = effect size; NSAIDS = non-steroidal anti-inflammatory drugs
38
1.2.5.ii.d. Dietary Constituents
Supplementation with vitamin E and D has also been investigated for the treatment of knee OA.
However, no significant differences from placebo was observed in pain, function or structural
joint changes between supplementation with 500 IU vitamin E/day for 6 months or 2 years in
RCTs in adults with knee OA (Brand et al 2001; Wluka et al 2002). Similarly, supplementation
of 2,000 IU of vitamin D/day for 2 years did not result significant differences from placebo in
terms of pain, function or structural joint changes (McAlindon et al 2013). Another RCT enrolled
patients with knee OA and vitamin D insufficiency (defined as serum 25(OH)D < 50 nmol/L)
and demonstrated small, but significant improvements in pain and function over placebo in
patients randomized to 60,000 IU vitamin D/day for 10 days followed by 60,000 IU vitamin
D/month for 12 months (Sanghi et al 2013). The authors concluded that the changes bordered on
being of no difference. There is not strong evidence to suggest that a specific vitamin will be able
to treat the structural or symptomatic features of knee OA. However, a balanced diet, resulting in
an adequate dietary intake of vitamins may slow progression of knee OA and protect against
worsening (Cao et al 2013).
1.2.5.ii.d.i. Rosmarinic Acid
With the significant role of inflammation and oxidative stress in joint tissue breakdown and
alterations in pain processing in OA, a product that can counter these actions holds potential to
control pain. An example of such a potential compound is rosmarinic acid (rosA). This is a
polyphenolic carboxylic acid that is an ester of caffeic acid and 3,4-dihydrophenyllactic acid
(Figure 1.6) (Peterson and Simmonds, 2003). RosA is a naturally occurring compound consisting
of 2 phenolic rings, both of them containing 2 ortho-dihydroxy groups (Furtado et al 2008). A
carbonyl group, an unsaturated double bond and a carboxylic acid group are located between the
two rings (Figure 1.6).
39
Figure 1.6 Chemical structure of rosmarinic acid
Sources of RosA
RosA was first isolated from rosemary in 1958 by two Italian chemists, Scarpati and Oriente
(Peterson and Simmonds, 2003). RosA is found in 39 plant families and it has been isolated
from many species of the Lamiaceae family, which comprise the majority of oil-rich culinary
and medicinal herbs (Petersen and Simmonds, 2003; Petersen 2013). These plants include basil,
rosemary, sage, savory, marjoram, oregano, thyme, lavender, perilla, self-heal, hyssop, lemon
balm, peppermint, fennel, painted nettle, comfrey, thyme, rosemary, oregano, peppermint, and
spearmint (Youn et al 2003; Ranjbar et al 2006; Pearson et al 2010). It has been suggested that
certain species of this family (i.e. oregano, sage, thyme, spearmint, peppermint) have antioxidant
constituents in amounts significant enough to make a contribution to the dietary intake of
phenolic antioxidants (Nurmi et al 2006). RosA is also present in the roots of the medicinal plant
saliva miltiorrhiza, also known as Dashen (Liu et al 2010).
Toxicity of RosA
Safety of a dry-spearmint extract containing 15.4% rosA was assessed in a 90-day study in
Sprague-Dawley rats given up to 1,948 mg dry spearmint extract/kg bw/day, by gavage
40
(equivalent to 300 mg rosA/kg bw/day or 21 g rosA/day for a 70 kg adult) (Lasrado et al 2015).
There were no adverse effects on body weight, food consumption, neurological parameters,
hematology, clinical chemistry, gross pathology, and histopathology and so, the no-observed
adverse effect level (NOAEL) in rats can be assumed to be 1,948 mg/kg bw/day. In 19 healthy
adults consuming perilla frutescens extracts containing 50 or 200 mg rosA for 21 days, no
adverse events were reported and there were no abnormalities detected in complete blood cell
counts, hepatic and renal function tests, total protein and proteinograms, electrolytes, lipids, uric
acid, and concentrations of creatine phosphokinase (Takano et al 2003).
Pharmacokinetics of RosA
In animal and human studies, rosA has been shown to be rapidly absorbed, metabolized,
conjugated, and excreted in the urine following oral consumption. Metabolities of rosA include
methylated rosA, caffeic acid, ferulic acid, and coumaric acid (Baba et al 2003; 2005). In male
Sprague-Dawley rats, rosA, methylated rosA and coumaric acid were detected at peak levels 0.5,
1, and 8 hours, respectively, after rosA administration (Baba et al 2003). In the urine, rosA, and
rosA metabolities methylated rosA, caffeic acid, ferulic acid, and coumaric acid were detected,
with 83% being excreted 8 to 18 hours after administration. In the plasma and urine, the majority
of components were conjugated.
Similar results were reported in 6 healthy men following consumption of perilla extract
containing 200 mg rosA (Baba et al 2005). In plasma, rosA and it’s conjugates reached peak
levels of 1.15  0.28 umol/L at 0.5 hours after adminstration, with ferulic acid and methylated
rosA also being detected. In the urine, rosA, methylated rosA, caffeic acid, ferulic acid and a
trace of courmaric acid was detected, with the majority being present in conjugated forms. The
proportion of rosA and its metabolites excreted in the urine after oral administration to humans
was 6.3% of the total dose with 75% of these appearing 6 hours after consumption (Baba et al
2005).
41
Biological Activities
In the last 5 years, research exploring the biological activities of rosA has greatly expanded,
resulting in several recent extensive reviews (Bulgakov et al 2012; Khojasteh et al 2014; Kim et
al 2015). Additionally, rosA has been examined for its potential therapeutic effects in a large
number of diseases (Appendix 1). RosA has demonstrated antimicrobial, anti-tumor, anti-viral,
anti-allergenic, antioxidant, and anti-inflammatory actions (Youn et al 2003; Petersen and
Simmonds 2003; Bulgakov et al 2012; Khojasteh et al 2014; Kim et al 2015).
Proposed Mechanisms
The mechanisms by which rosA can exert such a range of biological activities is not understood
(Bulgakov et al 2012) although numerous possibilities have been proposed. A key mechanism
that has been shown in a variety of cell lines and conditions is the inhibition of the activation
and/or translocation of the pro-inflammatory transcription factor, nuclear factor-kB (NF-kB)
with rosA treatment (Bulgakov et al 2012; Domitrovic et al 2012; Kim et al 2015; Hwang et al
2014; Fallarini et al. 2009; Lee et al 2006; Kim et al 2008; Moon et al 2010). In one study, rosA
decreased the inhibitor of nuclear factor kappa-B kinase subunit beta
(IKK-b) downstream signaling pathway and NF-kB activation in TNFa-induced human dermal
fibroblasts (Lee et al 2006). In general, treatment of rosA to chemically induced cells results in
the inhibition of ROS generation (Moon et al 2010; Vostalova et al 2010; Kim et al. 2005;
Zdarilova et al 2009; Lee et al 2008) as well as the inhibition of production of pro-inflammatory
cytokines like IL-6, IL-1B, TNFa, and PGE2 (Vostalova et al 2010; Chu et al. 2012; Hwang et al
2014; Zdarilova et al 2009). Furthermore, rosA has been demonstrated to increase intracellular
anti-oxidant defense systems, including enhanced levels and/or activation of nuclear factor-like 2
(Nrf2) (Domitrovic et al 2012; Hwang et al 2014), heme oxygenase 1 (HO-1) (Domitrovic et al
2012; Hwang et al 2014; Lee et al 2008), glutathione (GSH) (Kim et al. 2005; Zdarilova et al
2009; Fallarini et al 2009), and superoxide dismutase SOD (Kim et al 2005; Chu et al 2012).
Perez-Fons et al (2010) reported that the antioxidant abilities of rosA are from its ability to
stabilize membranes [as measured in the thiobarbituric acid reactive substances
42
(TBARS) assay] and restrict free radical movement [as measured in trolox equivalent antioxidant
capacity (TEAC) assay].
In vitro studies have reported opposing effects of rosA on apoptosis in different cell lines. In
human leukemia U937 cells, rosA treatment sensitized TNFa-induced apoptosis through
activation of caspases, and suppression of NF-kB and ROS (Moon et al 2010). However, Kim et
al (2005), Domitrovic et al (2012), Vostalova et al (2010) all found that rosA treatment resulted
in an inhibition of chemically induced apoptosis in H9c2 cardiac muscle cells, hepatic cells, and
human keratinocyte cells, via inhibition of caspase-3 activation. Clearly, the diverse biological
activities of rosA demonstrate that this compound has potential to benefit to human health.
Further investigations to elucidate the detailed molecular pathways underlying the biological
functions are needed.
Animal Models
In animal models, several effects have been demonstrated that suggest rosA may be beneficial in
the treatment of OA, specifically anti-nociceptive, anti-inflammatory effects, and anti-arthritic
effects. In male and female swiss mice, oral administration of 3 mg/kg rosA isolated from lemon
balm inhibited glutamate induced pain (Guginski et al 2009). Similarly, oral administration of
150 mg/kg rosA extracted from thunbergia laurifolia demonstrated significant anti-noceptive
effects in laboratory tests for behavioral responses to thermal and chemical induced noxious
insults in male ICR mice (Boonyarikpunchai et al 2014). In addition, rosA decreased paw edema
3-6 hours following carrageenan injection, demonstrating an anti-inflammatory effect. Finally, in
male Wistar rats with streptozotocin induced diabetic neuropathy, oral administration of 10 and
30 mg/kg rosA for 8 weeks resulted in anti-nociceptive effects in behavioural laboratory tests
(Hasanein and Zaheri 2014). Male DBA/1 mice were intraperitoneally injected with collagen for
21 days to create a rheumatoid arthritis state and then given 0 or 50 mg/kg rosA for 15 days from
Day 21 post injection (Youn et al 2003). Animals treated with rosA had significantly decreased
clinical manifestation of collagen-induced arthritis (CIA), as indicated by significant decreases in
mean arthritis index and number of affected paws. In addition, histopathological examination of
each hindpaw revealed that animals treated with rosA had normal joint composition when
43
compared to the inflamed joints of CIA mice. Finally, cyclooxygenase-2 (COX-2) levels were
significantly decreased in the joint tissue of mice treated with rosA.
Anti-inflammatory and cartilage sparing effects of a high-rosA acid spearmint plant have also
been demonstrated in animal models. Porcine cartilage explants were exposed up to 400 g/mL
of high-rosA spearmint, up to 10 mg/mL control spearmint, or 0.64 g/mL of rosA and all
explants were exposed to either 0 or 3 g/mL of lipopolysaccharide (LPS) to induce
inflammation (Pearson et al 2010). A strong inhibition of LPS-induced PGE2 and NO production
occurred in the presence of high-rosA spearmint, but not control spearmint or rosA, highlighting
the potential for complex interactions between bioactives that may impact efficacy. In addition,
high-rosA spearmint and rosA significantly inhibited to LPS-induced increased in GAG levels,
whereas control spearmint had no effect on GAG levels.
High-rosA spearmint was also examined in 8 healthy, mature standardbread horses free of
articular inflammation who were fed either 0 or 54 mg/kg of high-rosA spearmint mixed into hay
and sweet feed for 24 days (Pearson et al 2012). Following intra-articular injection of LPS in all
horses, PGE2 and GAG synovial levels were significantly increased in control horses. LPS
injection did not increase PGE2 or GAG synovial levels in horses consuming high-rosA
spearmint. LPS injection induced a small but insignificant increase in NO in all horses; however,
there were no significant differences in NO levels between groups at any point.
Humans
There have been very few investigations with rosA in humans; although it has been examined for
its effects on mild allergic rhinitis and nasal polyps. In a randomized, placebo-controlled study,
patients with mild allergic rhinitis were given either perilla frutescens extracts containing 0 mg
rosA (n=10), 50 mg rosA (n=9), or 200 mg rosA (n=10) for 21-days (Takano et al 2003). There
were no differences in symptom scores between groups. RosA consumption did decrease levels
of polymorphonuclear leuckocytes or neutophils in nasal lavage fluid on day 3, but the changes
were not statistically significant on day 21. High-rosA spearmint tea was examined for its effects
on nasal polyps, a chronic inflammatory disease of the nasal mucosa (Goronfolah et al 2009). In
44
a double-blind, placebo-controlled, crossover trial, healthy adults with history of nasal polyp
symptoms during the previous 12 months were randomized to consume 1 cup of high-rosA
spearmint tea or 1 cup regular spearmint tea for 4 weeks each, with a 4-week washout between
treatments. Twenty-two subjects completed the study and no significant differences between the
treatments in terms of nasal stuffiness or peak nasal inspiratory flow, sleep, sense of smell or
patient’s symptoms overall.
1.2.6 Summary
OA is a chronic, progressive disease resulting in degeneration of joint tissues, leading to
significant pain, stiffness, and impaired physical function. The pathology of OA is not
completely understood, but involves mechanical, biological, biochemical, molecular, and
enzymatic feedback loops with inflammatory mediators playing large role in disease pathology
and alterations in pain processing. The inability to completely characterize and measure OA
remains a significant challenge in OA research that limits understanding of the disease and
assessment of response to novel treatments. Biomarkers of joint metabolism hold promise, but
require further investigation and validation. Effective treatment strategies in OA remain an
important and critical area for research. Many management strategies exist, but none are
particularly effective and the highly utilized pharmaceuticals have a strong risk for adverse
effects. Anti-inflammatory and antioxidant dietary constituents and NHPs hold a lot of promise
for the management of knee OA and large, high-quality investigations should continue. Overall,
investigations that deepen our understanding of OA pathology and pain will enhance the ability
to manage and treat this disease and better and faster methods of measuring OA will aid the
ability to assess new treatments.
45
Chapter 2: Aims of Thesis
As reviewed above, there is no cure for knee OA and although there are many treatment options,
many are ineffective and people with OA live with considerable pain and disability. The aim of
this thesis is to examine relationships between pain and lifestyle factors and symptoms of knee
OA and biomarkers and to investigate the effects of a novel therapeutic product in adults with
knee OA.
Study 1 investigates the association between lifestyle factors and pain in individuals with knee
OA. The specific objectives are to characterize OA disease characteristics and lifestyle factors at
different levels of pain in adults with knee OA and to investigate the relationship between those
factors and OA pain. It is hypothesized that higher pain scores would be associated with higher
BMI, bilateral knee OA, increased disease duration, pain medication use, no supplement use, no
alternative therapies use, poor self-reported health, increased number of co-morbidities, low
intake of fruits and/or vegetables, and not meeting physical activity guidelines.
Study 2 examines biomarkers of joint metabolism and inflammation and their association with
pain and physical function in adults with knee OA. The specific objective is to relate a panel of
serum biomarkers of cartilage metabolism, synovial membrane, and inflammation to OA clinical
characteristics (pain, stiffness, physical function scores, 6-minute walk test performance, stair
climb task performance) in adults with knee OA. It is hypothesized that biomarkers of synovial
membrane and inflammation will be associated with pain.
Finally, study 3 evaluates a novel treatment for the management of pain and physical function in
adults with knee OA. The specific objective is to examine the effects of daily consumption of tea
brewed from a high-rosA spearmint plant on measures of pain, stiffness, disability, quality of
life, and physical function in adults with OA of the knee. It is hypothesized that participants
consuming the high-rosA spearmint tea will have improved measures of pain, stiffness,
disability, quality of life, and physical function after 4 months.
46
Chapter 3: Modifiable lifestyle factors are associated with lower pain in adults
with knee osteoarthritis
A. Erin Connelly1, Amy J. Tucker, Ph.D.1, Laima S. Kott, Ph.D.2, Amanda J. Wright, Ph.D.1,
Alison M. Duncan, Ph.D., R.D.1
1
Human Nutraceutical Research Unit, Department of Human Health and Nutritional Sciences, 2
Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
In press, accepted by Pain and Research Management on Thursday April 17th, 2015.
47
3.1 Abstract
With no cure or effective treatments for osteoarthritis (OA), the need to identify modifiable
factors to decrease pain and increase physical function is well recognized. The objective of this
study is to examine factors that characterize OA patients at different levels of pain and to
investigate the relationships between those factors and pain. Details of OA characteristics and
lifestyle factors were collected from interviews with healthy adults with knee OA (n=197). The
Western Ontario and McMaster Universities Osteoarthritis Index was used to assess pain.
Factors were summarized across 3 pain score categories and Chi-square and Kruskal-Wallis tests
examined differences. Multiple linear regression using a stepwise selection procedure examined
associations between lifestyle factors and pain. Multiple linear regression indicated that pain was
significantly higher with the use of OA medications and higher BMI category and significantly
lower with use of supplements and meeting physical activity guidelines (≥ 150 min/week).
Stiffness and physical function scores, bilateral knee OA, BMI category, and OA medication use
were significantly higher across pain categories, whereas self-reported health, servings of fruit,
supplement use, and meeting physical activity guidelines significantly lower. No significant
differences across pain categories were found for sex, age, number of diseases, duration of OA,
ever smoked, alcoholic drinks/week, over-the-counter pain medication use, OA supplement use,
physical therapy use, servings of vegetables, or minutes walked/week. Healthy weight
maintenance, exercise for at least 150 min/week and correct use of medications and supplements
represent important modifiable factors related to reduced knee OA pain.
3.2 Introduction
Knee osteoarthritis (OA) is the most common cause of knee pain and lower limb disability in
older adults (Jordan et al 2009). Approximately 4.4 million Canadians have OA and, due to
increased longevity, reduced physical activity, and increased obesity, this number is expected to
increase to 10 million within 30 years (Bombardier et al 2012). Pain is the most important OA
symptom among sufferers and contributes to disability, fatigue and decreased quality of life
(Briggs et al 1999; Hawker et al 2011). Pain is also what drives healthcare use in OA, with direct
and indirect healthcare costs of 27 billion dollars in Canada in 2010 (Bombardier et al 2012). In
general, pain is poorly understood and, in OA specifically, much of its variability cannot be
48
explained by markers of disease severity (Bedson and Croft 2008; Felson 2014; van Spill et al
2010). Many modifiable factors, including psychological and physical health, social support,
coping behaviors, and self-efficacy, have been found to influence OA pain (Hawker 2012).
With no cure available, management of OA focuses on analgesia and maintenance of physical
function (Hunter 2009). International evidence-based guidelines suggest that OA management
begin with non-pharmacological options, including weight loss or maintenance, moderate
exercise and physical activity, physical therapies and assistive devices, followed by
pharmacological interventions, starting with over-the-counter (OTC) pain medications like
acetaminophen (Fernandes et al 2013; Hochberg et al 2012; McAlindon et al 2014). However,
non-pharmacological approaches are infrequently recommended by clinicians and used by
patients (Dhawan et al 2014; Hunter 2009). Individuals with OA also tend to use pain
medications incorrectly and many live in pain and seek to manage it by limiting activities and
movement (Merkle and McDonald 2009; Sale et al 2006). With the lack of successful treatment
options, expected rise in OA cases, and the increasing burden on the healthcare system, there is
significant need to revisit OA management strategies and to focus on modifiable lifestyle
changes to decrease pain and increase or maintain physical function (Hunter 2009; Turkiewic et
al 2014). Modifiable factors that could affect pain in OA include oral management techniques
(e.g. OTC pain medications, supplements), use of physical therapies (e.g. physiotherapy,
chiropractor, etc), assistive devices (e.g. walking aids, braces, etc), home treatment methods (e.g.
heat, ice) as well as physical activity and diet.
The most important modifiable risk factor for knee OA is body weight. Excess body weight is
the strongest and most consistent risk factor for the onset and progression of knee OA
(Blagojevic et al 2010; Elbaz et al 2011; Hunter 2009; Riddle and Stratford 2013). The excessive
loads to the knee, inflammation, and associated inactivity in overweight and obese individuals
contributes to knee OA pathogenesis and pain (Hunter 2009; Lee et al 2013b).
With the exception of diet-induced weight loss, the literature examining the effect of diet on knee
OA is mixed. Epidemiological studies have shown an increased risk of disease progression with
low intake of antioxidant micronutrients (i.e. vitamin C, vitamin E, beta-carotene), while most
49
intervention studies report no significant effects of these micronutrients on the progression of
knee OA (Brand et al 2001; McAlindon et al 1996a, 1996b, 1997, 2013; Wluka et al 2002). It is
possible that antioxidant vitamins only exert an influence on knee OA when they are in their
original food matrix; however, the association of fruit and vegetable intake with OA has not been
investigated.
The important role of exercise and physical activity in the development and management of knee
OA is recognized, but it is complex and not well understood. OA clinical guidelines also
recognize regular physical activity as pivotal in OA management (Hunter 2009; McAlindon et al
2014). Modest volumes of low impact exercise can strengthen the muscular support around the
joints, help to avoid obesity, increase mood and psychological health, and can help to preserve
function and delay disability (Cheng et al 2000; Hawker et al 2011; Hawker 2012). Exercise
intervention studies consistently show that exercise decreases pain and improves function in
adults with OA (Baker et al 2001; Ettinger et al 1997; Messier et al 2004; Roddy et al 2005).
However, no association between physical activity level and knee pain was found in a large
prospective cohort study (Mansournia et al 2012).
Although OA pain is poorly understood, it is the leading complaint by persons with OA and is
influenced by many factors. The objective of this study was to examine factors that characterize
OA patients at different levels of pain and to investigate the relationships between those factors
and OA pain.
3.3 Methods
This study is based on cross-sectional data collected as part of a screening interview for a
nutrition intervention clinical trial (n=157) (Connelly et al 2014) with additional interviews
(n=40) completed to increase the sample size for a total of 200 participants, based on previous
cross-sectional analyses (Cruz-Almedia et al 2013; Goncalves et al 2011; Terwee et al 2006;
Tonelli et al 2011; Blamely et al 2009).
For the intervention and additional interviews, males and females aged ≥18 years with selfreported physician diagnosed knee OA were recruited from the Guelph area using posters and
50
newspaper advertisements. Exclusion criteria included other systemic or rheumatic arthritis,
concomitant inflammatory processes, upcoming knee replacement surgery, smoking, clinically
significant, uncontrolled cardiovascular, hepatic, or renal disorder, and any serious medical
condition within 6 months such as heart attack, stroke, cancer, or diabetes. The study was
conducted at the Human Nutraceutical Research Unit at the University of Guelph, approved by
the University of Guelph Human Research Ethics Board (REB#13OC002), and all participants
provided written consent.
Study Procedures
Trained study coordinators conducted the interviews, which included a questionnaire developed
for the study with health history and lifestyle questions about OA diagnosis, general health,
smoking history, alcohol consumption, body weight, height, medication and supplement intake,
co-morbidities, exercise habits, and use of physical therapies. Participants were asked how many
minutes per week they participated in weight training/resistance exercise, aerobic exercise,
aquatic exercise or other types of exercise (e.g. walking, sport participation, mixed fitness
classes, etc). Body mass index (BMI) was calculated from self-reported height and body weight
and then categorized into a BMI category. Participants were asked to bring all medications and
supplements to the interview, which were grouped into medications (any prescription and OTC),
medications for OA (prescription and OTC taken specifically for OA pain), prescription
medications for OA, and over the counter pain (OTC) medications. Supplements were grouped
as OA specific and non-OA specific. Participants were considered to use physical therapy if they
currently visited a practitioner. The time spent performing different types of exercises was
summed to a total number of minutes of exercise per week, and the time spent walking was
summed to a total number of walking minutes per week. A participant was considered meeting
physical activity guidelines when total number of minutes of exercise per week met or exceeded
150 minutes.
The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) has 3
subscales to evaluate pain, stiffness and physical function associated with OA (Bellamy et al
1988). The 100 mm visual analog scale (VAS) version of the questionnaire was completed in the
51
presence of a study coordinator and scored according to the WOMAC® Osteoarthritis Index User
Guide IX (Bellamy et al 2011). If a participant had OA in both knees, they were instructed to
choose the more painful knee as the signal joint. Higher scores indicate greater pain and
stiffness, and worse physical function.
A 24-hour dietary recall was conducted by a trained study coordinator and involved leading the
participant through a series of questions regarding all food and beverage intake in the previous
24 hours. Detailed questions were asked during the recall to ensure specific amounts, preparation
methods, brand names, and complete information about foods and beverages were recorded. All
were entered into ESHA Food Processor software version 9.12 (Salem, OR). Foods were
classified into one of the four main food groups (i.e. fruits and vegetables, grain products, milk
and alternatives, and meat and alternatives) according to the current Canadian Food Guide to
Healthy Eating (Health Canada 2014). Fruit and vegetable servings were also calculated and
analyzed separately. Foods not included in these groups were classified as “other”. Based on
previous nutrition research, mixed meals were broken down into their component foods
appropriate to the serving sizes, juice was classified as a fruit or vegetable depending on content
(e.g. orange versus carrot) and potatoes and french fries were classified as vegetables (Attorp et
al 2014).
Statistics
All analyses were performed using the Statistical Analysis System (version 9.3; Cary, NC, USA)
with p<0.05 considered statistically significant. Continuous variables are presented as mean ±
standard deviation. These data were not normally distributed, as determined by the Shapiro-Wilk
test. Dichotomous and categorical variables are presented as N (%).
To examine health history and lifestyle factors at different levels of pain, WOMAC pain scores
were grouped into 4 categories, mild (0 - 125), moderate (126 – 250), severe (251 – 375), and
extreme (376 – 500). Although there is no standardized method of dividing WOMAC scores,
categories were devised similar to previous work by Blamely et al (2009) and Kim et al (2013).
Only 3 participants reported a pain score over 375 (378, 398 and 442), and were included in the
52
severe category, leaving 3 pain groups (mild, moderate, and severe). Health history and lifestyle
factors were summarized for each pain category and differences were examined with Chi-square
tests for dichotomous and categorical variables and Kruskal-Wallis tests for continuous
variables. Linear regression analysis using continuous WOMAC scores was conducted using a
stepwise multiple regression was performed to determine the best predictors of pain, setting
probability to enter the model at p<0.15. Examination of residual plots revealed that the data was
homoscedastic and the relationship was linear and the distribution of the residuals was normal.
Variance inflation factors were between 1 and 2 for all values, revealing that multicollinearlity
was not an issue.
3.4 Results
Participant Characteristics
Participant characteristics are presented in Table 3.1. There were 197 adults (69% female) with a
mean age of 63 years. Mean disease duration was 98 months and 59% of individuals had OA in
both knees. Fifty-one percent of participants reported “good” health and the mean number of
diseases or conditions, including OA, was 3. The most commonly reported co-morbidities were
cardiovascular diseases (CVD), depression, hypertension, high cholesterol, hyperthyroidism, and
cancer (data not shown). Eighty-one percent of the participants reported using at least one
medication and 69% used at least one medication for OA. A high percentage of participants
(88%) took supplements, although only 37% took an OA-specific supplement. The most
commonly consumed supplements were vitamin D, multi-vitamin and minerals, calcium, omega3/fish oil, glucosamine, and vitamin C. The most commonly reported OA-specific supplement
taken was a glucosamine product (glucosamine or a combination of glucosamine and chondriotin
or methylsulfonylmethane). Only 33% of participants reported current use of physical therapy,
with physiotherapy, acupuncture and chiropractic being the most common. Forty-eight percent of
participants met the physical activity guidelines of 150 minutes of exercise/week (36), with
walking being the most frequently reported activity. Walking minutes were highly variable with
a mean of 58 and median of 40 minutes/week. Overall average WOMAC scores revealed a mild
to moderate level of pain, stiffness, and physical function in the study participants.
53
Table 3.1 Summary of Participant Characteristics (N = 197)
Characteristic
Females: N (%)
Age (years): Mean  SD
Disease duration (months): Mean  SD
Bilateral OA: N (%)
Number of diseases: Mean  SD
Self-Reported Health: N (%)
Poor
Good
Very Good
Excellent
Ever smoked: N (%)
Number of alcoholic drinks/week: Mean  SD
Medication and Supplement Use: N (%)
Medications
Medications for OA
Prescription Medications for OA
OTC Pain Medication for OA
Supplements
Supplements for OA
Participates in Physical Therapy: N (%)
Met Physical Activity guidelines: N (%)
Walking minutes: Mean  SD
WOMAC Subscale Scores: Mean  SD
Pain score (max 500)
Stiffness score (max 200)
Physical function score (max 1700)
OA = Osteoarthritis; SD = Standard deviation.
136 (69%)
63  10.9
98.1  95.6
117 (59%)
3.0  1.5
6 (3%)
100 (51%)
79 (40%)
12 (6%)
94 (48%)
3.4  4.5
159 (81%)
136 (69%)
53 (27%)
116 (59%)
173 (88%)
73 (37%)
65 (33%)
95 (48%)
58.7  71.6
159.1  92.9
100.6  50.1
578.6  347.8
Factors by Pain Categories
Table 3.2 shows the health history and lifestyle factors for participants across the 3 pain
categories. There were no significant differences across the pain categories in terms of
distributions of sex, age, number of diseases, duration of OA, ever smoked, number of alcoholic
drinks/week, use of OTC pain medication, use of OA supplements, use of physical therapy,
servings of vegetables, or minutes walked/week.
54
Table 3.2 Health history and lifestyle factors across mild, moderate and severe WOMAC pain
categories
p-value
Mild Pain
Moderate
Severe Pain
(n=83)
Pain (n=79)
(n=35)
0.14
Age (years):
64.9  11.8
63.0  9.7
60.2  10.8
mean  SD
0.86
OA duration (months):
90.1  83.9
103.3 106.0
105.2  98.3
mean  SD
0.79
# Diseases:
2.9  1.4
3.0  1.6
3.2  1.7
mean  SD
WOMAC Scores: mean  SD
<0.0001
Stiffness score
68.3  45.1
117.6  39.9
139.3  33.0
Disability score
304.4  208.0 689.2  249.0
977.8  267.0 <0.0001
Sex: N (%)
57 (69%)
53 (67%)
26 (74%)
0.74
Females
26 (31%)
26 (33%)
9 (26%)
Males
Unilateral or Bilateral OA: N (%)
44 (53%)
27 (34%)
9 (26%)
0.002
Unilateral
39 (47%)
52 (66%)
26 (74%)
Bilateral
BMI Category: N (%)*
1 (1%)
0
0
0.008
Underweight
25
(31%)
12
(16%)
5
(14%)
Normal weight
34 (41%)
29 (38%)
8 (23%)
Overweight
22 (27%)
36 (46%)
22 (63%)
Obese
Self-Reported Health: N (%)
0
1 (1%)
5 (14%)
0.001
Poor
40 (48%)
46 (58%)
14 (40%)
Good
37 (45%)
27 (34%)
15 (43%)
Very good
6
(7%)
5
(6%)
1 (3%)
Excellent
Smoking History & Alcohol Consumption
Ever smoke: N (%)
36 (44%)
37 (47%)
21 (60%)
0.25
0.86
# Alcoholic
3.1  3.8
3.8  5.3
3.3  3.9
drinks/week:
mean  SD
Medication, Supplement, and Alternative Therapy Use: N (%)
44 (53%)
59 (75%)
33 (94%)
<0.0001
OA medications
14 (17%)
25 (32%)
15 (43%)
0.009
OA prescriptions
43 (52%)
49 (62%)
25 (71%)
0.12
OTC pain
78 (94%)
69 (87%)
26 (74%)
0.01
Supplements
37 (45%)
22 (28%)
14 (40%)
0.08
OA supplements
28 (34%)
23 (29%)
14 (40%)
0.71
Physical therapy
Serving of Fruit and Vegetables: mean SD
0.03
Fruits
2.1  1.5
1.6  1.4
1.5  1.4
55
0.49
Vegetables
2.5  1.3
2.7  1.4
2.4  1.4
Physical Activity
53 (64%)
34 (43%)
8 (23%)
0.0001
Meet guidelines:
N (%)&
Walking minutes: mean
0.09
67.1  78.0
59.6  67.4
38.6  61.7
 SD
BMI = Body mass index; OA = Osteoarthritis; OTC = Over-the counter; SD = Standard
deviation; WOMAC = Western Ontario and McMaster Universities Arthritis Index
*Underweight: BMI <18.5 mg/kg2, Normal weight: BMI = 18.5 – 24.9 mg/kg2, Overweight BMI
= 25.0 – 29.9 mg/kg2, and Obese BMI > 30.0 mg/kg2) (Health Canada 2012).
&
Physical Activity Guidelines of ≥150 minutes/week of exercise (Public Health Agency of
Canada, 2012).
WOMAC stiffness and physical function scores were different across the pain categories and
increased with increasing WOMAC pain score (p<0.0001).
Having OA in only one knee was most common in the mild pain category (53%) (p=0.008) and
this proportion decreased across pain categories. Having OA in both knees was more common in
the moderate (66%) and severe pain (74%) categories than the mild pain category (47%)
(p=0.008).
The proportion of participants in each BMI category differed across pain categories (p=0.008). In
the mild pain category, the greatest percent of participants were in the normal weight (31%) and
overweight (41%) categories, and the percent of normal weight participants was lower in the
moderate (16%) and the severe (14%) pain category. The percent of participants in the obese
category was higher across increasing pain levels (Figure 3.1A).
Self-reported health categories were also different among pain categories, with fewer participants
reporting poor health in the mild (0%) and moderate (1%) versus severe (14%) pain categories
and more participants reporting very good health in the mild (45%) versus moderate (34%) pain
categories (p=0.001) (Figure 3.1B).
Servings of fruit were highest in the mild pain category (2.1), which was significantly higher
than the moderate (1.6) and severe (1.5) pain categories (p=0.03).
56
The proportion of participants who used a medication for OA increased across pain categories
(p<0.001) (Figure 3.1C), with a similar pattern observed for prescription medications (p=0.009)
(Figure 3.1D). The opposite trend was observed with supplements, with participants in the mild
pain category reporting the highest proportion of supplement use and decreasing supplement use
across pain categories (p=0.003) (Figure 3.1E).
Participating in exercise for at least 150 min/week was most common in the mild pain category
(64%), and this percentage was lower as pain category increased (p=0.0001) (Figure 3.1F).
57
Figure 3.1 Chi-square analysis revealed significant differences in the percent of participants in
the mild pain (0-125), moderate pain (126-250), and severe pain (251-500) categories in terms of
BMI category [normal weight (N), overweight (OW), or obese (OB)] (A), self-reported health
(B), use of medications for OA (C), use of prescription medications for OA (D), use of
supplements (E), and met physical activity guidelines (F). BMI = body mass index.
Regression Analysis
Multiple linear regression using a stepwise selection procedure revealed that BMI category, use
of supplements, use of OA medications, and meeting physical activity guidelines was the best
combination of factors to predict pain score, with the model accounting for 28.4% (p<0.0001)
(Table 3.3). The model revealed that pain score increased with use of OA medications and
increasing BMI category and decreased with use of supplements and meeting physical activity
58
guidelines. Factors that were not included in the model included age, sex, disease duration, ever
smoke, alcohol/week, number of diseases, use of OA supplements, use of OA prescriptions, use
of OTC pain, use of physical therapy, fruit consumption, vegetable consumption, and walking
minutes/week.
Table 3.3 Stepwise multiple regression examining factors related to pain score in adults with
knee osteoarthritis
Variable
Unstandardized
Standard Error
R2 of model
pbeta
(%)
value
Use of OA medications
60.1
13.1
14.5
<0.001
Meet Physical Activity
-33.7
12.4
20.0
0.007
Guidelines
BMI Category
19.7
7.6
24.0
0.02
Use of Supplements
-35.3
17.8
25.4
0.05
Self-Reported Health
-16.4
9.1
26.4
0.07
Use of Medications
26.4
15.0
27.5
0.08
Bilateral Knee OA
11.8
7.7
28.4
0.13
BMI = Body mass index; OA = Osteoarthritis.
* p<0.15 was used as cutoff for inclusion in the model
3.5 Discussion
This study examined modifiable lifestyle factors and their associations with OA pain in a sample
of adults with knee OA. Sixty-nine percent of participants in the present study were women, with
a mean age of 63 years and the majority of BMIs in the overweight and obese categories. This
gender distribution, age and overweight status is reflective of Canadian OA patients (Public
Health Agency of Canada, 2010). The majority of participants had mild (WOMAC pain score 0125; n=83) or moderate (WOMAC pain score 125-250; n=79), pain, providing an opportunity to
characterize modifiable lifestyle factors in adults with relatively low OA knee pain. A number of
the lifestyle factors (stiffness score, physical function score, bilateral knee OA, BMI category,
OA medication use, self-reported health, servings of fruit, supplement use, and meeting physical
activity guidelines) were significantly different among pain categories. Additionally, linear
regression analysis revealed that BMI, meeting physical activity guidelines, OA medication use,
and supplement use accounted for 28.4% of the variance in pain score. The unexplained
variability is potentially attributable to structural, psychological, and socioeconomic factors not
measured in this study.
59
Use of medications was reported by 81% of participants, which may reflect that participants also
reported an average of 3 diseases or conditions. Use of OA medications was a significant
predictor for higher pain score, and although not included in the regression model, the use of any
medication and OA prescription significantly increased as pain category increased. Use of OA
medications has previously been associated with higher levels of pain (Fisher et al 2012; Lapane
et al 2012), which could indicate that only individuals with high pain levels take medications,
current OA pain medications are not effective at relieving pain, or that adults with OA are not
using medications correctly. It has been found that individuals with OA use pain medications at
lower doses and less frequently than recommended (Davis et al 2002; Ross et al 2001; Sale et al
2006). Pharmaceutical pain management is often seen by OA sufferers as undesirable and a last
resort, due to concerns about side effects, addiction and tolerance, and general dislike for taking
pills (Ross et al 2001; Sale et al 2006). Furthermore, patient knowledge and beliefs about selfmanagement with medication is poor (Davis et al 2002; Hill and Bird 2007). OA patients may
benefit from education on the correct use of OA medications.
Use of supplements was significant in the regression model for pain and 94% of individuals in
the mild pain category reported using a supplement. Overall, supplement use was high in this
study at 88%, which may have been influenced by the fact that screening data from a nutrition
intervention study was used and interviews were conducted in a nutrition research unit. Estimates
of supplement use in adults with OA vary from 34 to 90% based on geography, population and
the definition of supplement (Lapane et al 2012). The most commonly consumed supplements in
this study were vitamin D, multi-vitamin, calcium, omega-3/fish oil, glucosamine product, and
vitamin C. As discussed previously, research into the effects of vitamin intake on OA is mixed. It
has been suggested that in individuals with vitamin deficiencies, supplements may be beneficial
in preventing OA progression (Wang et al 2004). In addition, higher dietary intakes of vitamin C
and beta-carotene were associated with lower OA pain in adults with knee OA (McAlindon et al
1996a). The same trends of lower OA pain were observed with serum levels of vitamin D
(Muraki et al 2011) and with supplementation with vitamin E (Bhattacharya et al 2012). Vitamin
D may reduce inflammatory pain through regulation of cytokines and macrophage activity,
although more studies are warranted (Cao et al 2013). Intervention studies with omega-3 rich oils
60
in adults with OA have demonstrated significantly reduced pain (Deutsch 2007; Zawadzi et al
2013), ostensibly based on anti-inflammatory mechanisms. Although not investigated in the
present study, it is possible that, individuals with higher pain may have taken more medications
to manage the pain and been less likely to take supplements for fear of interactions or general
dislike of taking multiple pills (Davis et al 2002; Ross et al 2001).
Increasing BMI category was a significant predictor of higher pain score in this study. The
association of BMI with pain is well established (Elbaz et al 2011; Felson et al 1997; Goulston et
al 2011) and reducing body weight has been related to dose-dependent decreases in pain and
improvements in physical function (Blagojevic et al 2010).
Not meeting physical activity guidelines was a significant predictor of higher pain score and, as
pain category increased, the percent of participants meeting physical activity guidelines
decreased. Similarly, walking min/week decreased as pain category increased, although this was
highly variable and not statistically significant (p=0.09). In exercise intervention studies, pain
decreases with increased activity levels (Baker et al 2001; Ettinger et al 1997; Messier et al 2004;
Roddy et al 2005); however, the monitored exercise regimes used in interventions are not
replicated in everyday life. Indeed, physical activity levels quantified with a the Physical
Activity Scale for the Elderly (PASE) questionnaire did not correlate to WOMAC pain scores in
a large, prospective cohort study in adults with or at risk for knee OA (Mansournia et al 2012).
Participants in the present study were active, with 48% meeting the Canadian physical activity
guidelines of ≥150 min/week. Previous reports in adults with OA are highly variable, ranging
from 10% (Dunlop et al 2011), to 30-40% (Farr et al 2008; Fontaine et al 2004; Holden et al
2014) and as high as 55.9% (Barbour et al 2014). Different assessment techniques could explain
this variation, including accelerometers (Dunlop et al 2011), questionnaires (Felson et al 1997;
Fontaine et al 2004), and self-reporting of activities (Felson et al 2007; Holden et al 2014), as
well as different parameters, including distance walked/week (Felson et al 2007), years of
sporting participating (Cooper et al 2000), categories of questionnaire scores (Felson et al 2007;
Holden et al 2014), and percent of participants meeting physical activity guidelines (Barbour et
al 2014; Dunlop et al 2011). Exploring the role of exercise and physical activity levels in OA
management would benefit from standardized methods and outcome measures. It is also possible
61
that individuals with severe pain in this study did not feel that they could exercise. However,
there are reports of individuals with knee OA walking at a high cadence or velocity with low
pain levels (Messier et al 2004; White et al 2013). Further, after total knee replacement,
improvements in pain are reported, but are not associated with increases in physical activity
(Harding et al 2014), supporting the argument that pain is not the limiting factor to exercise in
knee OA. Similar to the general population, time, effort, scheduling, and commitment have been
reported as barriers to exercise in persons with OA (Ross et al 2001; White et al 2013). Exercise
is a key modifiable factor to manage pain in OA as well as to decrease risk of other chronic
diseases, which were common in this study (83%). Interestingly, all-cause mortality is higher in
adults with OA versus without OA and the increased risk for death is predicted by walking
disability, highlighting the importance of physical activity and exercise in this population
(Nuesch et al 2011).
In this study, physical function score significantly increased across pain category, indicating
worse physical functioning with higher pain. The strong correlation commonly observed between
the pain and physical function scales on the WOMAC is suggested by Terwee et al (2006) to
indicate that self-reported physical function assessments innately measure both pain and
exertion, in addition to functioning. In the SF-36 questionnaire, pain questions are placed after
the function questions, and there is lower correlation observed between scales when compared to
the WOMAC where the pain questions are asked before the physical function questions (Terwee
et al 2006). Similarly, correlations between self-reported physical function and performancebased physical function tests were strengthened when patient rated pain and exertion of the
performance-based test was combined into a composite score (Stratford and Kennedy 2006). In
addition, adults with painful knee OA report keeping still and limiting movement as a way to
manage pain. This lack of activity could contribute to de-conditioning and reduced functioning
(Davis et al 2002; Sale et al 2006). Another factor that significantly increased across pain
categories was the percentage of adults with bilateral knee OA. This has previously been
associated with higher pain scores, which may reflect the presence of additional tissue
degradation and inflammation (Adegoke et al 2012; Schiphof et al 2013).
62
Daily fruit servings reported by participants were significantly higher in the mild versus
moderate and severe pain categories. Of note, regardless of pain category, the number of daily
fruit servings (i.e. 1.5 – 2.6) was below recommended levels. Fruits are high in antioxidants and
polyphenols, and fruit consumption has been associated with lower musculoskeletal pain in
adults (Høstmark et al 2014), and berries, tart cherries, blueberries, and concord grapes,
specifically, have been found to have anti-nociceptive effects in rats (Jensen et al 2011). Healthy
eating patterns are also associated with better self-reported health, more time in exercise, and less
depressive symptoms, which have all been associated with lower pain in OA (Lee et al 2013b).
Although the 24-hour diet recall isn’t the most robust method of dietary assessment available,
increasing fruit consumption and improving diet quality are important modifiable factors that
warrant continued investigation for the potential to reduce pain in adults with knee OA.
In this study, self-reported health status was significantly different across pain categories, even
though most participants reported good or very good health. Self-reported health has been
shown to be reliable in OA populations (McAlindon et al 1999), although associations with pain
have not always been consistent. Reichmann et al (2009) found that lower income, greater
number of co-morbidities, and greater functional limitations, but not pain, were associated with
self-reported health status in adults with knee OA. In contrast, Allen et al (2010) found that
lower self-reported health was associated with higher pain scores in adults with hip or knee OA.
In this study, 91% of participants reported “good” or “very good” health, which could be related
to the fact that 82% of participants were in the mild or moderate pain categories.
Several factors were not significantly different among pain categories or significant in the
regression analysis. For example, age showed a non-significant protective directional trend with
increasing age being associated with decreased pain, consistent with previous OA research
(Schiphof et al 2013). Surprisingly, sex was not a significant factor in the regression model and
there was no difference in pain categories. Women frequently report higher pain than men in OA
(Elbaz et al 2011; Glass et al 2014; Tonelli et al 2011), but no differences in pain have been
reported between the sexes in some studies (Debi et al 2009; Dillon et al 2006). There were more
females in the severe (74%) compared to the mild and moderate (~68%) categories and more
males in the mild and moderate (~32%) than the severe (26%) categories. There were also no
63
significant differences in duration of OA across pain category. Although Rosemann et al (2007)
reported that duration of OA was a predictor of pain, several studies have shown that OA can
remain stable for years (Cooper et al 2000). Number of diseases reported did not differ across
pain category, consistent with a previous report (Leite et al 2011). However, the relationship
between co-morbidities in OA and pain is mixed and examination into specific diseases may
reveal that different co-morbidities impact OA pain in different ways. For example, strong links
between depression and pain has been found several studies in adults with OA (Hawker et al
2011; Rosemann et al 2007). OA supplement use was not significantly related to pain.
Glucosamine products were almost exclusively used in this category, and evidence for its effect
on pain is limited due to high variation among studies (McAlindon et al 2014; Vlad et al 2007).
Physical therapy was only used by 33% of adults in this study and its use did not different across
the pain categories. A systematic review and meta-analysis of physical therapies for OA
concluded that only a few physical therapy interventions were effective (Wang et al 2012), but it
is possible that effects are not seen in intervention studies due to the fact that a true placebo
group doesn’t exist for any of the physical therapies.
Limitations of this cross-sectional study include that causality of the factors studied cannot be
concluded and that the data was self-reported. In addition, a number of factors that have been
shown to affect pain were not measured in this study, including psychological variables, selfefficacy, education level, socioeconomic data, and fatigue (Blamely et al 2009; Cleveland et al
2013; Cruz-Almedia et al 2013; Hawker et al 2011). Selection bias towards adults with less
severe OA is also a possibility, as participants were required to be mobile enough to travel to the
research centre for interviews. However, this gave the opportunity to characterize individuals
with lower pain, as some studies investigating factors of OA pain examine patients waiting for
knee replacement, thereby representing a much different population and more extreme level of
pain than is representative of many people with knee OA (Cooper et al 2000). Lastly, BMI
calculations for this study were based on self-reported heights and weights, which has also been
used in previous OA research (Allen et al 2010; Fontaine et al 2004; Goulston et al 2011; Thiem
et al 2013). Given the potential for inaccuracies, BMI categories were used.
64
3.6 Conclusion
Several modifiable factors were associated with OA pain including BMI, meeting physical
activity guidelines, OA medication use, and supplement use. Having a BMI in the healthy range
is associated with lower pain levels, as well as exercising for at least 150 minutes/week. Exercise
plus a diet high in fruits and vegetables is important in weight maintenance, and increasing fruit
consumption could be important in this study population who are consuming low levels.
Supplement use was associated with lower pain and use of OA medication was associated with
higher pain. Patients should be educated on proper use of medications to manage pain in OA.
Research efforts and clinical practice should continue to focus on modifiable lifestyle changes to
decrease pain in OA.
3.7 Acknowledgments
We thank all the study participants as well as Natasha Sheikh, Svitlana Yurchenko, Kate
Faughnan and Bonnie Huang for their assistance in data collection.
65
3.8 Bridge to Chapter 4
In Study 1, several modifiable lifestyle factors were associated with OA pain, identifying
potential targets for management of OA. However, there are still no completely satisfactory
treatments and many adults with OA live in daily pain. A barrier to the development of new
therapies for OA is the low sensitivity of change in radiographs which necessitates long-term
trials with a large number of patients (Kraus et al 2011). MRI has overcome many of the
limitations of the radiograph, but is not standardized for OA outcomes, and as such, biomarkers
represent an exciting opportunity for measurement of OA. Study 2 examines serum biomarkers
of OA and their association with pain and physical function scores, as well as performance based
measures of physical function, the stair-climb test and 6-minute walk test. Study 1 examined
what modifiable lifestyle factors were associated with OA pain, and now Study 2 will examine
how serum biomarkers of joint metabolism and inflammation are associated with pain and other
symptoms of OA.
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Chapter 4: Serum biomarkers of joint metabolism and inflammation in
relation to clinical symptoms and physical function in adults with knee
osteoarthritis
A. Erin Connelly1, Amy J. Tucker1, Laima S. Kott2, Amanda J. Wright1, Alison M. Duncan1
1
Human Nutraceutical Research Unit, Department of Human Health and Nutritional Science, 2
Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
Submitted to Arthritis on April 18th, 2015, with minor revisions for thesis.
67
4.1 Abstract
The relationship between osteoarthritis (OA) symptoms and biomarkers is relatively unexplored.
In adults with knee OA, the Western Ontario and McMaster University Osteoarthritis Index
(WOMAC) assessed pain, stiffness, and physical function, and the 6-minute walk test (6MWT)
and stair climb test (SCT) assessed physical performance. Serum cartilage oligomeric matrix
protein (COMP), type-IIA collagen N-propeptide (PIIANP), hyaluronic acid (HA), matrix
metalloproteinase 3 (MMP-3), nitric oxide (NO), and interleukin 6 (IL-6) were measured by
enzyme-linked immunoassay. C-reactive protein (CRP) was measured by immunoturbidimetric
assay, and interleukin 18 (IL-18) and leukemia inhibitory factor (LIF) were measured by
multiplex immunoassay. Spearman’s coefficient examined correlations between biomarkers and
clinical variables, controlling for age, sex and body mass index (BMI). In participants (n=54;
mean age 60 ± 11.9 years, BMI 32.6 ± 7.5 kg/m2) HA was associated with better physical
performance (6MWT: R=0.33, p=0.02 and SCT: R= - 0.38, p=0.006), MMP-3 with higher pain
score (R=0.30, p=0.03) and IL-6 with higher pain (R=0.31, p=0.03) and stiffness score (R=0.30,
p=0.03) and worse physical performance (SCT: R=0.32, p=0.02). COMP, PIIANP, IL-18, CRP,
and NO were not associated with any variables and LIF was not detected. This study reports
significant associations between biomarkers and clinical characteristics in OA.
(Research supported by the Ontario Ministry of Agriculture, Food and Rural Affairs, project
#200121).
4.2 Introduction
Osteoarthritis (OA) is a major cause of disability and the number one cause of joint pain in older
adults (Dieppe and Lohmander 2005). The primary pathology in OA is joint matrix destruction
caused by an impaired balance between cartilage matrix synthesis and degradation, mechanical
stress, inflammation, and other unknown factors, leading to cartilage loss, new bone formation at
joint margins, subchondral bone changes, and synovial inflammation (Dean et al 1989; Dieppe
and Lohmander 2005). However, understanding of the mechanisms involved is incomplete due
to the disease’s slowly progressive nature, complex cartilage biochemistry, and the intricate role
of inflammation (Abramson 2008). Current treatments are ineffective and, despite the use of
pharmaceuticals, most individuals with OA suffer significant pain and impaired physical
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functioning (Messier et al 2013; Sofat et al 2011). Specifically, pain is the primary concern for
persons with OA, as it significantly affects mood, fatigue and quality of life and the symptom
that drives healthcare use (Hawker et al 2011). Investigations into OA mechanisms and diseasemodifying treatments are significantly hindered by uncertainties related to OA assessment.
Radiographs only allow for an estimation of the space between joints and large bony changes
(Bijlsma et al 2011). While magnetic resonance imaging (MRI) allows for a more specific
examination of cartilage, it is time-consuming, costly and requires complex interpretation and
improved standardization (Bijlsma et al 2011).
Biomarkers of joint metabolism have the potential to contribute to earlier OA diagnosis and to
provide a more rapid and complete picture of therapeutic responses to investigational treatments
(Felson 2014). However, no single biomarker can represent all of the complex biological
changes occurring in the joint during OA (Kraus et al 2011). To this end, in 2011, the
Osteoarthritis Research Society International and the United States’ Food and Drug Association
recommended further investigation of 16 urine and serum biomarkers that reflect different tissues
and processes involved in OA. This included the serum markers cartilage oligometric matrix
protein (COMP) as a measure of cartilage degradation, procollagen type II N-terminal propeptide
(PIIANP) as a measure of type II collagen synthesis, hyaluronic acid (HA) as an indication of
synovial membrane metabolism, and matrix metalloproteinase 3 (MMP-3) an enzyme involved
in joint tissue degradation (Kraus et al 2011). Since inflammatory pathways are known to be upregulated in OA, inflammatory cytokines also have relevance as OA biomarkers (Dieppe and
Lohmander 2005). C-reactive protein (CRP) is a marker of general inflammation that has been
investigated for its role in OA pathogenesis, with conflicting evidence (Jin et al 2013).
Interleukin (IL)-6 is a pro-inflammatory cytokine that plays a role in cartilage destruction and
has been shown to stimulate synovial proliferation, osteoclast activation, and MMP production
(Park et al 2007). IL-18 is well known for its role in rheumatoid arthritis, where it is involved in
induction of cartilage degradation through production of MMPs (Futani et al 2002). However,
serum IL-18 has not been examined in relation to OA clinical characteristics. Leukemia
inhibitory factor (LIF) plays a role in the regulation of bone metabolism and has been shown to
degrade proteoglycans ex vivo (Futani et al 2002). It has been detected in the synovial fluid of
OA patients and correlated to degree of degraded cartilage tissue (Jiang et al 2014; Westacott
and Sharif 1996).
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OA biomarker research has primarily sought to relate biomarker levels to joint structural
characteristics determined from x-rays and/or MRI (van Spill et al 2010). For example, a metaanalysis showed that serum COMP was consistently elevated in knee OA patients and was
predictive of radiographic progression over time (Hoch et al 2011). Similarly, PIIANP and
MMP-3 have been shown to predict radiographic progression in adults with knee OA (Garnero et
al 2002; Lohmander et al 2005). HA has also been positively associated with presence, severity,
and number of knees with radiographic OA (Elliott et al 2005). Despite this progress, relatively
less is known about how OA biomarkers correlate with clinical characteristics of the disease
(Bijlsma et al 2011). The objective of this study was to relate a panel of serum biomarkers of
cartilage metabolism, synovial membrane, and inflammation to OA clinical characteristics (pain,
stiffness, physical function scores, 6-minute walk test performance, stair climb task performance)
in participants with mild to moderately painful knee OA. There is a critical need to better
understand how joint and inflammatory biomarkers relate to OA pain and physical function
(Hawker et al 2011; Ishijima et al 2011; van Spill et al 2010).
4.3 Methods
Study design
A cross-sectional study using baseline data from adults with knee OA who participated in an
intervention study (Connelly et al 2014) was conducted at the Human Nutraceutical Research
Unit at the University of Guelph. The study was approved by the University of Guelph Human
Research Ethics Board (REB#11JA040) and all participants provided informed consent.
Participants
Male and female adults (>18 years old) were eligible if they had self-reported, physician
diagnosed knee OA and a screening Western Ontario and McMaster Universities Osteoarthritis
Index (WOMAC) pain score of >125. Exclusion criteria included other systemic or rheumatic
arthritis, concomitant inflammatory processes, upcoming knee replacement surgery, chemical,
radiologic, or surgical synovectomy in any large joint within the previous 3 months,
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gastrointestinal ulcers, clinically significantly or uncontrolled cardiovascular, hepatic, or renal
disorder, any serious medical condition within 6 months such as heart attack, stroke, cancer, or
diabetes, smoking, participation in a clinical trial involving an investigational or marketed drug
within the previous 6 months, and pregnancy (or intention to become pregnant), less than 6
months post-partum, lactating or less than 6 months post-lactation.
Blood collection and analysis
Participants provided a non-fasted blood sample with instructions to avoid alternative therapies
such as acupuncture, massage therapy, or strenuous physical activity or consume pain
medications for 48 hours prior. A 5 mL sample of blood was collected in a standard serum
separating tube (SST™, BD, Mississauga, Canada) by a qualified medical technician via
venipuncture. Samples were allowed to sit at room temperature for 30 minutes prior to
centrifugation (2500 rpm and 4 °C for 15 minutes) and immediately stored at -80C until
analysis.
Serum C-reactive protein (CRP) was analysed in duplicate by Lifelabs Medical Laboratory
Services (Guelph, Canada) using latex-enhanced immunoturbidimetric assay. Serum biomarker
quantification was performed in duplicate using enzyme-linked immunosorbent assay (ELISA)
for COMP, HA, MMP-3, interleukin 6 (IL-6), and nitric oxide (NO) (R&D systems, MN, USA),
and PIIANP (EMD Millipore, MO, USA) according to the manufacturers’ protocols. The assay
detection limits were 0.010 ng/mL for COMP, 0.068 ng/mL for HA, 0.009 ng/mL for MMP-3,
0.70 pg/mL for IL-6, 0.25 μmol/L for NO, and 30 ng/mL for PIIANP. Inflammatory cytokines
interleukin 18 (IL-18) and leukemia inhibitory factor (LIF) were measured in duplicate using a
custom Bio-Plex Pro cytokine kit (Bio-Rad Laboratories, CA, USA) according to the
manufacturer’s instructions. The lower limit of quantification was 1.8 pg/mL for IL–18 and 12
pg/mL for LIF.
71
Anthropometric measurements & clinical characteristics
Height was measured to the nearest millimeter using a stadiometer (Model 217, SECA,
Hanover, USA) and body weight was measured to the nearest 0.05 kg on a digital scale (SVI200F, Acculab, Barrie, Canada).
The WOMAC was used to evaluate pain, stiffness, and physical function associated with OA
with a higher score indicating greater pain and stiffness, and worse physical functioning
(Bellamy et al 1988). The visual analog scale (VAS) version of the questionnaire was completed
in the presence of a study coordinator and scored according to the WOMAC® Osteoarthritis
Index User Guide IX (Bellamy 2011).
The 6-minute walk test (6MWT) and stair climb test (SCT) are performance-based measures
administered to assess physical function. Briefly, the 6MWT was conducted by marking off a 20
m distance in a straight and flat interior hallway and asking participants to walk as quickly as
possible for 6 minutes, with standardized, verbal encouragement given at every minute, as
adapted from the American Thoracic Society (2002) and Enright (2003). The total distance
walked was determined to the nearest cm. There are no standard guidelines or current standards
for conducting a SCT and the feasibility of the test parameters are dependent on the
environmental setting (Dobson et al 2013). However, when permitted, an ascent and descent of 9
stairs test without external distractions is recommended (Dobson et al 2013). In this study, the
SCT involved participants ascending and descending a flight of 7 stairs as quickly and safely as
possible, while holding on to the handrail. Assistive devices were not used by any participants
and no verbal encouragement was provided. The time required for the total ascent and descent
was recorded to the nearest millisecond (ms).
Statistical Analysis
Since serum biomarker data were not normally distributed (determined by the Shapiro-Wilk
test), Spearman’s coefficients were used to examine correlations with clinical characteristics,
controlling for age, sex, and body mass index (BMI). There were no significant correlations
72
between serum biomarkers and disease duration (data not shown), so disease duration was not
included in the final correlation analysis. Biomarker levels were compared between females and
males using an unpaired Student’s t-test. All statistical analyses were performed using the
Statistical Analysis System (version 9.3; Cary, NC, USA) with p<0.05 considered statistically
significant. Unless otherwise indicated, data are presented as mean  standard deviation.
4.4 Results
Participant characteristics
Participant characteristics are summarized in Table 4.1. Participants had a mean age of 60  11.8
years, mean BMI of 32.6  7.5 kg/m2, and were predominately female. In this cohort of adults
with knee OA, WOMAC scores revealed a mild to moderate level of pain, and moderate levels
of stiffness and physical functioning. Physical function as assessed with the SCT and 6MWT
was worse than Canadians of a similar age group, but similar to obese and persons with knee OA
(Enright 2003; Hill et al 2011).
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Table 4.1 Participant characteristics, WOMAC scores and physical function tests (n=54)*
Participant Characteristics
Age (years)
60.0  11.8
2
BMI (kg/m )
32.6  7.5
Disease duration (months)
86.6  112.0
Males/Females (N)
16/38
WOMAC Scores
Pain score (mm)
193.4  94.4
Stiffness score (mm)
104.4  45.1
Physical function score (mm)
651.0  329.8
Physical Function Tests
SCT (seconds)
12.1  3.6
6MWT (meters)
445.6  76.3
*Mean  standard deviation.
BMI = body mass index; 6MWT = 6-minute walk test; SCT = stair climb test.
Serum Biomarkers
Mean levels of the biomarkers detected in the serum samples are presented in Table 4.2. There
were no significant differences between females and males, with the exception of MMP-3
(p=0.01). LIF was not detected (data not shown). Serum HA was significantly associated with
longer 6MWT distance (r=0.33, p=0.02) and less time taken in the SCT (r= -0.38, p=0.006) in all
participants (Figure 1). Serum MMP-3 was significantly associated with higher pain score
(r=0.30, p=0.03) in all participants (Figure 2A). Serum IL-6 was significantly associated with
higher pain score (r=0.31, p=0.03) (Figure 2B), higher stiffness score (r=0.30, p=0.03) more time
taken in the SCT (r=0.32, p=0.02) (Figure 3), and non-significantly with decreased 6MWT
distance (r= -0.24, p=0.09). Serum COMP, PIIANP, CRP, NO, and IL-18 were not significantly
associated with any clinical characteristics (data not shown).
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Table 4.2 Serum biomarker levels
Biomarker
All Participants (N=54)
Females (N=38)
Males (N=16) Mean
Mean  SD (Range)
Mean  SD
 SD
COMP (ng/mL)
309.7  123.6
301.7  130.5
329.9  105.7
(118.9 – 810.7)
PIIANP (ng/mL)
2541.6  1311.0
2594.6  1268.8
2412.3  1194.1
(1004.0 – 6979.0)
HA (ng/mL)
96.6  80.9
94.9  14.0
100.6  84.7
(14.2 – 421.8)
MMP-3 (ng/mL)
16.4  9.4
14.3  8.4a
21.1  10.2b
(4.3 – 45.7)
IL-18 (pg/mL)
62.1  31.4
57.9  28.8
72.1  35.9
(23.4 – 156.5)
IL-6 (pg/mL)
3.4  5.5
3.8  6.4
2.4  1.5
(0.7 – 11.5)
CRP (mg/L)
4.3  5.1
5.1  5.8
2.6  2.2
(0.5 – 22.0)
NO (mol/L)
24.6  12.2
25.6  14.0
22.24  11.1
(3.7 – 69.9)
a,b
Levels of MMP-3 were significantly different between males and females (p=0.01)
COMP = cartilage oligomeric matrix protein; CRP = C-reactive protein; HA = hyaluronic acid;
IL-6 = interleukin-6; IL-18 = interleukin-18; MMP-3 = matrix metalloproteinase -3; NO = nitric
oxide; PIIANP = type-IIA collagen N-propeptide; SD = standard deviation.
Figure 4.1 Significant associations between serum HA and physical function tests in all
participants. Serum HA was measured by ELISA in 54 participants with knee OA and
correlated with performance in the 6MWT (p=0.02) (A) and SCT (p=0.006) (B) as objective
measures of physical function.
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Figure 4.2 Significant associations between serum MMP3 and IL-6 and WOMAC pain
score in all participants. Serum MMP3 was measured by ELISA in 54 participants with knee
OA and correlated with WOMAC pain score (A). Serum IL-6 was measured by a ELISA in 54
participants with knee OA and correlated with WOMAC pain score (B).
Figure 4.3 Significant associations between serum IL-6 and stair climb test (SCT). Serum
IL-6 was measured by ELISA in 54 participants with knee OA and correlated with performance
in the SCT as an objective measure of physical function.
4.5 Discussion
This study examined the relationships between serum biomarkers and OA clinical characteristics
in participants with mild to moderate painful knee OA. Participants in the study represented a
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typical population of adults with knee OA, with a mean age of 60 years, a mean BMI in the
obese category, and 70% female. WOMAC scores revealed a mild to moderate level of pain and
moderate levels of stiffness and physical function. This study correlates a panel of joint and
inflammatory serum biomarkers to WOMAC scores and performance-based measures of
physical function (i.e. 6MWT and SCT) to investigate the relationship of biomarkers with OA
pain and physical function in order to advance the use of biomarkers in the diagnosis, study and
treatment of OA
Serum HA was significantly associated with longer 6MWT distance and faster SCT time in all
participants. To our knowledge, this is the first time serum HA has been correlated with
objective measures of physical function, although variations in HA levels during the day are
activity and movement related, not strictly diurnal (Criscione et al 2005). HA is a
glycosaminoglycan produced by cells in the synovial membrane and found in cartilage and
synovial fluid (Criscione et al 2005). It has a structural role in the connective tissue matrix and is
involved in joint lubrication (Elliott et al 2005; Turan et al 2007). Serum HA increases with age
and disease duration and is higher in OA patients compared to controls (Elliott et al 2005; Turan
et al 2007). Serum HA has also been positively associated with radiographic OA in several
(Elliott et al 2005; Golightly et al 2011; Sasaki et al 2013), but not all (Turan et al 2007), studies.
Joint movement pumps excess HA and water out of the joint and into the lymphatic system and
circulation (Criscione et al 2005; Goldberg et al 1991). It is possible that individuals with better
physical functioning are more active and therefore have more HA in the circulation. The
association of HA with measures of physical function in this study are reasonable as HA plays a
role in joint lubrication (Elliott et al 2005) and serum levels have been found to change with
movement (Criscione et al 2005), but more research into the relationship between serum HA and
physical functioning is required.
Serum MMP-3 was significantly associated with higher WOMAC pain scores. MMPs are
produced by chondrocytes and synovial fibroblasts and are responsible for hydrolyzing
macromolecules in the extracellular matrix (ECM) (Lohmander et al 2005). MMP-3, specifically,
plays an important role in the destruction of the ECM in OA and is higher in the serum and
synovial fluid of OA patients than healthy controls (Lohmander et al 2005; Manicourt et al
77
1994). Serum MMP-3 has not previously been found to correlate with WOMAC scores or pain
scores, but has been associated with changes in joint space narrowing (JSN) and cartilage
volume (Lohmander et al 2005; Pelletier et al 2010). In one randomized controlled trial in adults
with knee OA, serum MMP-3 decreased in the treatment group, which also experienced
increased function and decreased pain scores (Manicourt et al 2006). MMP-3 was the only
biomarker that was significantly different between males and females in the current study. This
trend for sex-related differences was also reported by Manicourt et al. (1994).
Serum IL-6 was significantly associated with higher WOMAC pain score, stiffness score and
worse performance on the SCT and non-significnaly in the 6MWT. IL-6 is a pro-inflammatory
cytokine that plays a role in cartilage destruction in OA (Shimura et al 2013; Stannus et al 2010).
It can stimulate synovial proliferation, osteoclast activation, and MMP production (Park et al
2007). In rheumatoid arthritis it has been correlated with laboratory and clinical characteristics
and blocking IL-6 can be an effective therapy (Ding et al 2006; Park et al 2007). Interestingly, it
was reported that IL-6 levels above 2.5 pg/mL increase the risk of mobility disability in
metabolic syndrome (Esposito et al 2004). This is a population that presents with relatively high
BMIs and unfavourable inflammatory profiles, similar to OA. In the current study, the mean
level of IL-6 in all participants was 3.4  5.5 pg/mL and IL-6 correlated with decreased distance
in the 6MWT and slower time in the SCT, demonstrating a significant relationship between
serum IL-6 and physical function in this sample of OA patients. Penninx et al. (2004) also found
that serum IL-6 levels were associated with poorer performance in the 6MWT and higher IL-6
levels were associated with fewer steps taken per day in individuals with OA (Stannus et al
2010). Serum IL-6 levels were not previously found to be associated with WOMAC scores (Bas
et al 2014; Brenner et al 2004; Penninx et al 2004), but significantly associated with pain
severity on a VAS scale (Shimura et al 2013). Levels have been found to decrease alongside
decreases in pain score and improvements in physical function, in randomized controlled clinical
trial (Abou-Raya et al 2014; Messier et al 2013). Similarly, baseline IL-6 levels have been
associated with change in pain while standing (Stannus et al 2013), also supporting the
connection between IL-6, pain, and physical function.
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Both MMP-3 and IL-6 were correlated with pain in this study, and although MMP-3 has not
been previously correlated with pain scores, IL-6 has been shown to up-regulate MMP-3 levels,
suggesting that the relationship between MMP-3 and pain could be mediated through IL-6.
Inflammation is a key component of OA and is associated with knee pain and progression of
cartilage degeneration (Bas et al 2014). Inflammatory mediators can activate and sensitize
afferent nociceptive sensory fibers, causing hyperalgesia and altered pain processing seen in OA
(Bellucci et al 2013).
Despite the significant associations discussed above, there were a number of biomarkers that did
not correlate with any clinical characteristics. Serum COMP, although a well-studied OA
biomarker (Verma et al 2013), was not significantly associated with any of clinical
characteristics in the current study. COMP is a non-collagenous glycoprotein that binds to
collagen to stabilize the articular cartilage collagen fiber network and is released as bound
proteins are cleaved from the cartilage matrix in OA (Wisłowska et al 2005; Clark et al 1999). It
is been shown to predict structural progression of OA in several studies (Blumenfeld et al 2013;
Clark et al 1999; Golightly et al 2011; Sharif et al 2004; Valdes et al 2014; Verma et al 2013).
However, the association of serum COMP with pain and physical function is not clearly
established. Serum COMP has been associated with non-standardized pain scores in two studies
(Sowers et al 2009; Verma et al 2013) and with WOMAC pain score in 30 adults with knee OA
(Wislowska et al 2005). However, in other reports, serum COMP was not associated with
WOMAC scores or other clinical characteristics (El-Arman et al 2010) and in a 3-year study of
OA progression, changes in serum COMP were not associated with changes in WOMAC scores
(Vilim et al 2002). COMP has also been reproducibly associated with Kellgren-Lawrence (KL)
grade and JSN from radiographs and loss of cartilage from MRI (Blumenfeld et al 2013; Clark et
al 1999: Valdes et al 2014). We hypothesize that, since COMP is a component of cartilage, it
may be a structural marker, but not a marker of pain or physical function in OA, explaining the
absence of correlations in the current study.
Serum PIIANP was also not associated with any of the clinical characteristics investigated.
Serum PIIANP is thought to indicate the rate of type II collagen synthesis as the cartilage
attempts repair to damaged cartilage (Aigner et al 1999). Type II collagen is synthesized as a
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procollagen molecule, with type IIB in normal adult cartilage and type IIA procollagen expressed
in early cartilage and at the onset of cartilage hypertrophy (Garnero et al 2002; Rousseau et al
2004). Studies have not confirmed a correlation between serum PIIANP and KL score, pain on a
VAS, or function as measured by Lequesne's index (Garnero et al 2002; Quintana et al 2008). It
was observed that patients with lower serum PIIANP had worse radiographic progression after 1
year (Garnero et al 2002). Similar to COMP, PIIANP is a marker of collagen synthesis in
cartilage, and it is reasonable that it is an indication of structural damage, but not clinical
symptoms, in adults with knee OA.
Serum CRP also did not significantly correlate with any of the clinical characteristics studied.
CRP is a marker of general inflammation and has been investigated for its role in OA
pathogenesis, with conflicting evidence (Pelletier et al 2010). A meta-analysis of 32 studies in
OA patients found that CRP levels were moderately correlated with increased pain and decreased
physical function, but not with radiographic evidence (Jin et al 2013). However, the high
correlation between CRP and obesity may limit the utility of CRP as a biomarker in OA
(Garnero et al 2001; Sowers et al 2002). For example, in this study, CRP was significantly
associated with a number of clinical characteristics only before BMI was accounted for in the
model (data not shown).
Serum IL-18 was also not significantly associated with any clinical characteristics in this study.
IL-18 is well known for its role in rheumatoid arthritis and induction of cartilage degradation
through production of MMPs (Futani et al 2002). It is reported to be higher in the plasma and
synovial fluid of OA patients relative to healthy controls and correlates with radiographic
severity (Wang et al 2013). However, this is the first study to examine serum IL-18 in relation to
OA clinical characteristics. In adults with OA, IL-18 was higher in synovial fluid than plasma
(Wang et al 2013) and, it is possible that synovial fluid levels better correlate with clinical
characteristics in OA.
Serum NO was also not significantly associated with any clinical characteristics measured. NO is
a marker of oxidative stress and, along with its redox derivatives, appears to have a number of
different functions in both normal and diseased joints (Abramson 2008). Nitrate and nitrite levels
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have been found to be higher in plasma and synovial fluid of OA patients compared to healthy
adults (Karan et al 2003). However, similar to the present findings with serum, synovial fluid NO
was not previously related to WOMAC score in adults with knee OA (Brenner et al 2004).
This exploratory investigation has some noteworthy strengths and limitations. One limitation is
that sufficient synovial fluid samples were not available from this group of adults. Since several
studies have reported that inflammatory cytokines are higher in synovial fluid than serum (Wang
et al 2013), examination of both would have provided a more complete picture of the synovialserum concentration relationship. In addition, the joint and inflammatory biomarkers examined
in this study are not specific to the knee and their levels may not represent changes occurring
only in the knee joint, but rather reflect whole body changes in cartilage or inflammation. OA in
other joints was not examined or accounted for in this study, and this could also account for the
lack of association between some biomarker levels and knee specific symptoms (Bijlsma et al
2011; Felson et al 2014; Kraus et al 2011). However, whole body joint deterioration is difficult
to account for and remains an important limitation in OA biomarker research (Bijlsma et al 2011;
Felson et al 2014; Kraus et al 2011). The inclusion of participants in this study was made on the
basis of self-reported physician diagnosed knee OA, which has shown good agreement with
medical records (March et al 1998; Cheng et al 2000; Ho-Pham et al 2014). A similar approach
has been taken in previous OA studies (Parazzini et al 2003; Hootman et al 2003; Miller et al
2008). In addition, the timing of blood sampling was not controlled. However, diurnal variation
of biomarkers is not completely understood or explored. Examinations into HA, PIIANP, and
COMP revealed that differences in serum levels only occurr between before arising from bed and
1 hour following usual morning activities, with levels staying consistent for the reminder of the
day (Criscione et al 2005; Quintana et al 2008; Kong et al 2006). Strengths of this study were the
use of common and validated measures of pain and other symptoms of OA with the WOMAC
and objective measures of physical function in the 6MWT and SCT. In addition, a panel of
biomarkers was measured, representing different joint tissues and inflammation. A sample size
of 54 was sufficient to detect significant differences and is comparable to similar cross-sectional
analyses of pain and biomarkers in OA (Ishijima et al 2011; Orita et al 2011). However, future
OA biomarker investigations should consider including larger sample sizes with repeated
measurements.
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4.6 Conclusion
This study found significant correlations between serum biomarkers and clinical characteristics
in a population with mild to moderate OA knee pain. Specifically, serum HA was correlated with
physical function, serum MMP-3 was correlated with pain, and serum IL-6 was correlated with
pain and physical function. The markers of collagen metabolism, COMP and PIIANP, were not
associated with any clinical characteristics. Just as the diagnosis of OA is based on a
combination of structural and symptomatic features (Zhang et al 2010), a combination of
structural and symptomatic biomarkers may be required to fully characterize OA status.
4.7 Acknowledgments
The research was sponsored by the Ontario Ministry of Agriculture, Food and Rural Affairs
(OMAFRA #200121). We thank all the study participants as well as Natasha Sheikh and Svitlana
Yurchenko, for their assistance in data collection. Additionally, we acknowledge Premila
Sathasivam, and Mehrnoosh Kashani for their assistance with sampling, Anna Deboer, Laelie
Snook, and Jessica Ralston for their technical support, and the many undergraduate research
students and volunteers whom helped with the study.
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4.8 Bridge to Chapter 5
OA pain was examined in relation to lifestyle factors and serum biomarkers in Studies 1 and 2,
with the goal of gaining a deeper understanding of OA pain in order to treat and manage it.
Reducing OA pain could improve physical function and quality of life for adults suffering with
OA. Study 3 will now investigate a novel treatment in a randomized, double-blind clinical trial
with the primary outcome of pain reduction. Secondary outcome measures include the same
clinical characteristics examined in Study 2; physical function and stiffness scores, 6-minute
walk test performance, and stair climb task performance. In addition, evaluation of the treatment
with change in biomarker levels is presented in Appendix 15.
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Chapter 5: High-Rosmarinic Acid Spearmint Tea in the Management of Knee
Osteoarthritis Symptoms
As published with revisions for thesis:
Connelly AE, Tucker AJ, Tulk H, Catapang M, Chapman L, Sheikh N, Yurcehnko S, Flectcher
R, Kott LS, Duncan AM, Wright AJ. High-Rosmarinic Acid Spearmint Tea in the Management
of Knee Osteoarthritis Symptoms. J Med Food. 17(12):1361-1367, 2014.
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5.1 Abstract
Individuals with medically diagnosed knee osteoarthritis (OA) participated in a randomized,
double-blind study to investigate the effects of a high-rosmarinic acid (rosA) spearmint tea.
Sixty-two participants were randomized by gender and screening pain score to consume tea
brewed from a high-rosA spearmint variety or a commercially available spearmint twice daily
for 16 weeks. Pain, quality of life, and physical function at baseline and Week 16 were assessed
using the Western Ontario and McMaster Osteoarthritis Index (WOMAC), Short-form 36-item
Health Survey (SF-36), 6-Minute Walk Test (6MWT), and Stair Climb Test (SCT). Data from 46
participants (mean age=60.7; BMI=32.9 kg/m2) were analyzed. Pain score significantly
decreased from Week 0 to 16 for the high-rosA group but not for the control group and scores for
stiffness and physical disability significantly decreased from Week 0 to 16 for both groups.
Increased quality of life score on the bodily pain index in the SF-36 was observed at Week 16
within the high-rosA group only, although no significant differences were observed between the
groups. A non-significant improvement was observed in the 6MWT at Week 16 in the high-rosA
group only. There were no changes in the SCT for either group. Therefore, 16-week daily
consumption of the high-rosA and commercial spearmint teas significantly improved stiffness
and physical disability scores in adults with knee OA, but only the high-rosA tea significantly
decreased pain. Consumption of high-rosA tea warrants further consideration as a potential
complementary therapy to reduce pain in OA. Clinical Trial Registration Number:
NCT01380015.
5.2 Introduction
Osteoarthritis (OA) is a disease characterized by the degeneration of articular cartilage,
manifesting as joint pain, stiffness, and impaired function leading to physical disability and
decreased quality of life (Martel-Pelletier and Pelletier 2010). Knee OA is one of the most
common chronic diseases with an estimated prevalence of 24% among North America adults
(Bijlsma et al 2011). The pathogenesis of OA is complex and not fully understood, but is
associated with an imbalance of anabolic and catabolic activities in the cartilage in which
degenerative processes prevail, leading to the loss of cartilage (Loeser 2009). There is no
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treatment that slows cartilage breakdown, so treatment is focused on decreasing pain and
increasing function with non-pharmacological interventions like exercise and assistive devices,
as well as non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen (Zhang et al
2008). While these pharmaceuticals offer acute pain management for some individuals, serious
gastrointestinal and cardiac side effects limit their long-term use (Pincus et al 2001). It is
reported that 30-45% of individuals with OA also use supplements to manage the disease
(Lapane et al 2012; Lawson et al 2004; Singh et al 2006), with glucosamine being the most
common. However, even for glucosamine, controversy remains surrounding efficacy, after a
large National Institute of Health, randomized, double-blind glucosamine trial failed to show any
significant improvements in OA patients (Clegg et al 2006). Therefore, further research into safe,
complementary therapies for OA is required.
Rosmarinic acid (rosA) is a polyphenolic compound naturally present in spearmint, peppermint,
fennel, and other species of the Lamiaceae family and which has been hypothesized to be of
potential benefit for OA (Fletcher et al 2005; Pearson et al 2010; Petersen et al 2003; Youn et al
2003). In vitro, rosA has been shown to have anti-inflammatory, anti-oxidant,
immunosuppressant, and anti-bacterial activities (Youn et al 2003). Through selective breeding
techniques, a spearmint plant containing approximately 20 times more rosA than native
spearmint was developed (high-rosA spearmint plant clone 700B) (Fletcher et al 2005). In
porcine cartilage explants, a high-rosA spearmint extract reduced the expression of
lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) and nitric oxide release and also
inhibited glycosaminoglycan (GAG) release, suggesting a potential chondroprotective effect
(Pearson et al 2010). Also, healthy, mature standardbred horses were fed hay and sweet feeds
containing either no (n=4) or 54 mg/kg body weight of the high-rosA spearmint (n=4) for 24
days (Pearson et al 2012). Following intercarpal LPS injections to induce inflammation, the
horses consuming high-rosA spearmint had reduced synovial fluid PGE2 and GAG levels
compared to the control horses. These results demonstrate the anti-inflammatory and potential
chondroprotective actions of the high-rosA spearmint and support its use as a complementary
therapy for the management of knee OA. Therefore, a study was conducted to examine the
effects of daily consumption of tea brewed from the high-rosA spearmint plant on measures of
pain, stiffness, quality of life, and physical function in adults with OA of the knee.
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5.3 Methods
Study Design
This randomized, parallel-arm, double-blind study was conducted at the Human Nutraceutical
Research Unit of the University of Guelph. The University of Guelph Human Research Ethics
Board approved the study protocol (REB#11JA040), which was registered in the NIH clinical
trial registry (Protocol No. NCT0138001) and all participants provided written consent.
Recruitment and Screening
Adult (>18 years old) males and females were recruited from Guelph, ON and the surrounding
communities from June 2011 to June 2012 and were eligible for study inclusion if they were
non-smokers, had medically diagnosed OA of the knee and a screening Western Ontario and
McMaster University Osteoarthritis Index (WOMAC) pain score of >125. Exclusion criteria
included other systemic or rheumatic arthritis, concomitant inflammatory processes, upcoming
knee replacement surgery, chemical, radiologic, or surgical synovectomy in any large joint
within the previous 3 months, gastrointestinal ulcers, clinically significant, uncontrolled
cardiovascular, hepatic, or renal disorder, any serious medical condition within 6 months such as
heart attack, stroke, cancer, or diabetes, known allergy or hypersensitivity to mint or other food
allergies, smoking, alcohol consumption >14 drinks per week, recreational drug use,
participation in a clinical trial within the previous 6 months, and pregnancy (or intention to
become pregnant), less than 6 months post-partum, lactating or less than 6 months post-lactation.
Study Treatment Tea and Tea Protocol and Blinding
The study treatment tea was brewed from the high-rosA spearmint plant (Clone 700B) (Fletcher
et al 2005) and the control tea was brewed from commercially available spearmint tea (Distinctly
Teas Inc., Stratford, ON). Dried spearmint leaves were blended on low in a food-grade blender
for 30 seconds. Three grams of plant material were transferred into individual Teeliflip tea
bags (Riensch & Held, Germany), which were stapled. The identical control and high-rosA tea
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bags were placed in vacuumed sealed bags, coded by a person external to the study team, and
stored at room temperature until use. All participants and study team members were blinded
throughout data collection and analysis.
Participants were instructed to consume 2 cups of tea per day for a 16-week period in a provided
300 mL study mug. Brewing instructions detailed that one tea bag was to be steeped in boiling
water for 5 minutes with occasional stirring and the addition of milk, cream, sugar or sweetener
was not allowed. Three grams of high-rosA spearmint in 300 mL of water for 5 minutes was
verified by high-performance liquid chromatography to provide 130-150 mg of rosA per cup
compared with ~13 mg rosA from the control tea (data not shown). Therefore, participants in the
control and high-rosA groups consumed 26 versus 280 mg rosA per day, respectively. Analysis
of brewed tea samples by Maxxam Analytics (Mississauga, ON) ensured pesticide,
microbiological and mycotoxin levels were below the NHP Directorate Accepted Tolerance
Limits (NHPD 2012). Participants were asked to not consume any herbal teas for the duration of
the study. Consumption of coffee as well as black and green tea was allowed. Participants
recorded the times at which they consumed the study tea in a daily study diary as a measure of
compliance.
Treatment Randomization
Participants were block randomized into treatment groups based on sex and screening WOMAC
pain score. Covariate adaptive randomization (Kang et al 2008) was performed by an individual
not associated with the study. Sequentially numbered sheets of paper were used to implement the
allocation sequence.
Outcome Measures
Outcome measures were assessed at baseline, Week 8, and 16. At baseline and after completion
of the 16-week protocol, anthropometric measurements were taken. Phone check-in
conversations occurred at weeks 2, 6, 10 and 14 to discuss compliance, medications, physical
activity, and any other issues. Safety of tea consumption was assessed by recording adverse
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events. Participants were asked to maintain their regular medication and supplement use
throughout the study period but were also advised that they could increase or decrease their use
of pain medication as they felt necessary. They were asked to track all medications and
supplements in their daily study diary. Changes in pain medication consumption were quantified
by calculating total number of tablets taken per week (Constant et al 1997).
Height was measured to the nearest millimeter using a stadiometer (Model 217, SECA,
Hanover, USA) and body weight was measured to the nearest 0.05 kg on a digital scale (SVI200F, Acculab, Barrie, Canada). Two measurements were averaged for each of waist and hip
circumference determined over clothing with a tape measure (Model 201, SECA, Hanover,
USA). Blood pressure and body composition was determined with an Omron digital blood
pressure monitor and bioelectrical impedance analysis machine (Bodystat1500, Bodystat,
Isle of Man, UK), respectively. All anthropometric measurements were completed according to
standard operating procedures and completed by the same trained study coordinator (AEC).
The WOMAC is a validated, standardized 24-item questionnaire that assesses pain, disability and
joint stiffness associated with OA (Bellamy et al 1988). In the presence of a study coordinator,
the 100 mm visual analog scale (VAS) version of the WOMAC was administered and scored
according to the WOMAC® Osteoarthritis Index User Guide IX (Bellamy 2009).
The Medical Outcomes Study 36-item Short-Form General Health Survey (SF-36) is a selfadministered questionnaire that assesses 8 components of health-related quality of life (QoL) (i.e.
physical function, physical role, bodily pain, vitality, social functioning, role emotional, mental
health, and general health) and 2 composite scores (i.e. physical component score and mental
composite score) (Maruish et al 2009). In the presence of a study coordinator, participants
completed the questionnaire which was scored using Medical Outcomes software.
The 6-minute walk test (6MWT) and 7-stair climb test (SCT) are performance based measures
used to assess physical function. Briefly, the walk test was conducted by marking off a 20 m
distance in a straight and flat interior hallway and asking participants to walk as quickly as
possible for 6 minutes, with standardized, verbal encouragement given at every minute, as
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adapted from the American Thoracic Society (2002) and Enright (2003). Participants were
permitted to rest, as necessary, and to use any mobility aids, as per their normal use. The total
distance walked was determined to the nearest cm. The SCT involved participants ascending and
descending a flight of 7 stairs as quickly as was safely possible. The time required for the total
ascent/descent was recorded to the nearest millisecond (ms). The 6MWT and SCT were
administered by the same trained study coordinator (NS).
A non-fasted 5 mL blood sample was collected in a standard serum separating tube (SST™, BD,
Mississauga, Canada) tube by a qualified medical technician via venipuncture at baseline and
Week 16. Samples were allowed to sit at room temperature for 30 minutes prior to centrifugation
(2500 rpm and 4 °C for 15 minutes). For 48 hours before the blood samples were being taken,
participants were instructed to not participate in alternative therapies such as acupuncture,
massage therapy, or strenuous physical activity. Serum was sent to Lifelabs Medical
Laboratory Services (Guelph, Canada) for analysis of C-reactive protein (CRP) by latexenhanced immunoturbidimetric assay. Serum biomarker quantification was performed in
duplicate using enzyme-linked immunosorbent assay (ELISA) on duplicate samples for COMP,
HA, MMP-3, and nitric oxide (NO) (R&D systems, MN, USA), and PIIANP (EMD Millipore,
MO, USA) according to the manufacturers’ protocols. The assay detection limits were 0.010
ng/mL for COMP, 0.068 ng/mL for HA, 0.009 ng/mL for MMP-3, 0.25 μmol/L for NO, and 30
ng/mL for PIIANP. Inflammatory cytokines interleukin 18 (IL-18) and leukemia inhibitory
factor (LIF) were measured in duplicate using a custom Bio-Plex Pro cytokine kit (Bio-Rad
Laboratories, CA, USA) according to the manufacturer’s instructions. The lower limit of
quantification was 1.8 pg/mL for IL–18 and 12 pg/mL for LIF.
Statistical Analysis
All statistical analyses were performed using the Statistical Analysis System (version 9.3; Cary
NC, USA) with P<0.05 considered statistically significant. A sample size calculation based on
the outcome of total WOMAC score, using a significance level of 0.05 and power of 80%,
indicated that 25 participants per group were required. Individual WOMAC pain, stiffness and
function subscales were converted to normalized units (NU, 0 - 100) (Bellamy 2009). Data was
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examined for normality using stem leaf diagrams and box plots. Baseline anthropometric data
and outcome scores were compared between the high-rosA and control groups using unpaired ttests. Analysis for efficacy was done using the per protocol population. Baseline data for all
outcome measures were compared against Weeks 8 and 16 within the high-rosA and control
groups using unpaired Student’s t-tests. Between high-rosA and control group differences
(Week 0 to Week 8 and Week 0 to Week 16) were analyzed for all outcome measures using
repeated measures analysis of variance (ANOVA). Differences in pain medication intake
between groups was assessed using repeated measures ANOVA. As biomarker data was not
normal, the log of values was taken and differences between groups over time was examined
with repeated measures ANOVA.
5.4 Results
Participant Flow, Withdrawals and Exclusions
A total of 411 individuals completed the phone screening questionnaire, 191 individuals
completed the in-person screening questionnaire, and 62 individuals were randomized to
treatment (Figure 5.1). Twenty-eight participants in each group received the intervention, and 2
participants in the high-rosA group withdrew due to time commitment issues and 1 due to a
previously unidentified mint sensitivity. One participant in the control group withdrew due to a
diagnosis of gout, 1 due to knee replacement surgery, 1 from prolonged non-related food
poisoning illness, and 1 participant was excluded because they began smoking. In the high-rosA
group, data from 2 participants was removed from analysis due to arthroscopic surgery after 12
weeks participation in the study and 1 for non-compliance with study protocols. Complete data
was collected and analyzed for the remaining 46 participants.
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Figure 5.1 Participant flow through the trial (CONSORT diagram)
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Participant Characteristics
Participant characteristics and baseline outcome measures were not significantly different
between the high-rosA (n=22) and control (n=24) groups at baseline (Table 5.1). Participant
characteristics and anthropometric measurements were not significantly different between groups
at any time during the study (data not shown). Tea consumption compliance was 96.8 and 94.8
% for the high-rosA and control tea groups, respectively, as self-reported in daily study diaries.
This was consistent with study coordinator tracking of dispensed and returned tea bags. There
were no significant differences in pain medication tablets consumed per week between the
groups or over time (data not shown).
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Table 5.1 Participant Characteristics and Outcome Measures at Baseline
High-rosA (n=22)a
Control (n=24)a
Participant characteristics
60.5 ± 11.1
60.8 ±12.1
Age (years)
8/14
6/18
Sex (n) (males/females)
167.8 ± 9.4
164.3 ± 8.8
Height (cm)
96.8 ± 28.3
85.2 ± 19.7
Weight (kg)
34.3 ± 8.6
31.5 ± 6.9
BMI (kg/m2)
65.6 ± 18.2
58.4 ± 13.0
Waist circumference (cm)
49.9 ± 13.2
45.0 ± 9.8
Hip circumference (cm)
135.4/83.8
132.3/80.7
Blood pressure SBP/DBP (mmHg)
73.9 ± 11.5
73.8 ± 12.2
Pulse (bpm)
37.8 ± 12.0
38.0 ± 9.6
Body fat (%)
57.0 ± 16.1
51.8 ± 10.9
Lean body weight (kg)
WOMAC Scores
39.6 ± 13.9
36.1 ± 22.2
Pain
54.9 ± 19.6
52.5 ± 23.2
Stiffness
36.8 ± 17.7
39.0 ± 22.1
Physical disability
131.3 ± 42.4
127.5 ± 60.5
Total score
SF-36 Scores
56.4 ± 9.0
52.0 ±10.4
Mental component summary score
38.6 ± 6.6
39.3 ± 7.3
Physical component summary score
Physical Function Tests
448.4 ± 73.8
487.1 ± 87.2
6MWT (m)
11.4 ± 3.0
12.9 ± 4.2
SCT (seconds)
a
Values are mean ± standard deviation.
DBP = diastolic blood pressure; SBP = systolic blood pressure; 6MWT = six-minute walk test;
SCT = stair climb task.
Adverse Events
There were no serious adverse events reported during the study. Participants in the high-rosA
group reported headache (n=2), constipation (n=3), and loose bowel movements (n=1).
Participants in the control group reported dry mouth (n=1), itchy skin (n=1), and staining of
dentures (n=1). All reported adverse events were transient and short-term.
Outcome measures
WOMAC pain score significantly decreased from baseline within the high-rosA group at Week
16 (p=0.002) (Figure 5.2A). In the control group, WOMAC pain score also decreased from
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baseline; however, it was significant at Week 8 (p=0.04) but not at Week 16 (p=0.07). There
were no significant differences in WOMAC pain score between the groups at any time points in
the study. WOMAC stiffness significantly decreased from baseline to Week 16 within the highrosA (p=0.004) and control (p=0.04) groups, although there was no difference between groups
(p=0.37) (Figure 5.2B). WOMAC physical disability score significantly decreased from baseline
to Week 16 within the high-rosA (p=0.02) and the control (p=0.03) groups but no significant
differences were observed between the groups at any time point (Figure 5.2C). Generally,
WOMAC scores on all scales did not significantly differ from Week 8 to Week 16 within or
between the treatment groups.
Figure 5.2 Mean scores and standard error are presented for (A) WOMAC pain scores, (B)
WOMAC stiffness scores, (C) WOMAC physical function score and (D) WOMAC total scores
for the high-rosA group (circles) and the control group (triangles). The change within groups
from baseline to week 16 was examined; significant differences within the high-rosA group are
indicated with a cross (+) and significant differences within the control group are indicated with
an asterisk (*). rosA, rosmarinic acid; WOMAC, Western Ontario and McMaster Universities
Osteoarthritis Index.
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In the SF-36, the only significant change observed was in the QoL score for bodily pain, with an
increase observed from baseline to Week 16 within the high-rosA group (Table 5.2). There were
no other significant changes observed for SF-36 scores either within or between the rosA and
control groups (Table 5.2).
Table 5.2 SF-36 Scores for Weeks 0 and 16 for the High-rosA and Control Groups
High-rosAa
Controla
Canadian
Normb
Week 0
Week 16
Week 0
Week 16
55.7 ± 19.8
59.8 ± 23.5
55.4 ± 20.9
63.6 ± 19.9
82.3 ± 19.3
63.1 ± 19.9
76.1 ± 24.1
61.2±27.7
70.1 ± 21.4
81.3 ± 33.1
46.6 ± 7.6
70.7 ± 12.7
58.1 ± 22.1*
70.8 ± 18.4
49.7±17.8
69.3±19.0
55.7 ± 14.6
74.0 ± 14.1
74.9 ± 23.7
74.8 ± 19.4
60.5 ± 15.6
63.1 ± 19.9
51.9±19.6
58.6 ± 17.7
68.3 ± 17.7
86.9 ± 21.0
89.2 ± 22.9
76.0±25.3
81.3 ± 20.9
Social
Functioning
86.4 ± 19.0
87.9 ± 23.5
78.8±24.8
83.3 ± 20.0
Role
Emotional
80.0 ± 13.4
80.2 ± 23.4
76.8±19.3
79.0 ± 13.7
Mental
Health
38.6 ± 6.6
42.5 ± 9.0
39.3±7.3
42.9 ± 7.2
Physical
Component
score
56.4 ± 8.9
55.8 ± 13.0
51.4±10.9
53.1 ± 10.2
Mental
Component
score
a
Values are mean ± standard deviation
b
For healthy Canadians aged 55-64 years (Hopman et al 2000)
*
Change from baseline to Week 16, p<0.05
88.1 ± 18.8
Physical
Function
Physical
Role
Bodily Pain
General
Health
Vitality
87.8 ± 28.3
79.5 ± 14.7
49.0 ± 9.2
53.7 ± 8.2
The 6MWT total distance travelled did not significantly differ from baseline to Week 16 either
within or between (p=0.12) the high-rosA (p=0.94) or control groups (p=0.96) (Table 5.3). The
total time taken to complete the SCT was not significantly different from baseline to Week 16
either within or between (p=0.8) the high-rosA (p=0.43) or the control group (p=0.44).
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Table 5.3 Six-Minute Walk Test distances (m) for Weeks 0, 8, and 16 for the high-rosA and
control groups
High-RosAa
Change from
Controla (m)
Change from
b
baseline (m)
baselineb (m)
(m)
451.5 ± 73.6
439.4 ± 87.2
Week 0
451.7 ± 92.9
+ 0.2
438.3 ± 84.5
- 1.1
Week 8
473.8 ±72.3
+22.3
439.5 ± 88.2
+0.1
Week 16
a
Values are mean ± standard deviation
b
Difference from Week 0
There were no significant differences in the levels of any biomarkers between groups at any
point in the study (Table 5.4).
Table 5.4 Biomarker levels in the high-rosA and control groups in Week 0 and Week 16*
Biomarker
Treatment (mean ± SD)
Control (mean ± SD)
p-value
Week 0
Week 16
Week 0
Week 16
HA
1.85 ± 0.30
1.85 ± 0.28
1.86 ± 0.32
1.94 ± 0.34
0.64
(ng/mL)
MMP-3
1.11± 0.21
1.13 ± 0.22
1.16 ± 0.22
1.20 ± 0.19
0.95
(ng/mL)
PIIANP
3.35 ± 0.14
3.37 ± 0.14
3.33 ± 0.23
3.29 ± 0.22
0.74
(ng/mL)
COMP
1.49 ± 0.12
1.50 ± 0.15
1.45 ± 0.20
1.48 ± 0.19
0.65
(ng/mL)
IL-18
1.70 ± 0.20
1.73 ± 0.20
1.79 ± 0.20
1.78 ± 0.22
0.34
(pg/mL)
NO
1.34 ± 0.27
1.31 ± 0.19
1.30 ± 0.21
1.34 ± 0.20
0.22
(mol/L)
CRP (mg/L)
0.44 ± 0.43
0.36 ± 0.47
0.38 ± 0.50
0.44 ± 0.61
0.76
*Values are log transformed.
COMP = cartilage oligomeric matrix protein; CRP = C-reactive protein; HA = hyaluronic acid;
IL-6 = interleukin-6; IL-18 = interleukin-18; ln – log; MMP-3 = matrix metalloproteinase -3; NO
= nitric oxide; PIIANP = type-IIA collagen N-propeptide; SD = standard deviation.
5.5 Discussion
This was the first human intervention study to examine the effects of daily consumption of tea
brewed from a novel high-rosA spearmint plant on markers of pain, stiffness, and physical
function in adults with knee OA. The study was also unique in that it utilized a pragmatic design
by allowing participants to maintain their normal pain medication routine. This was intended to
explore the potential of the high-rosA tea as a complementary OA therapy.
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Anthropometric measurements did not change over the treatment period, which is important, as
weight loss is known to improve OA symptoms (Gudbergsen et al 2012; Riddle and Stratford
2013). Although the pain scores were never significantly different between groups, the
significant decrease during the treatment period within the high-rosA group is an important
finding. In the control group, pain scores only significantly decreased from Week 0 to Week 8
with no further significant decreases, suggesting a placebo effect. Placebo analgesia is a wellknown phenomenon in OA and pain research (Doherty and Dieppe 2009), which possibly
contributed to the lack of significant difference between the groups. Because of this known
placebo effect, we prioritized running a blinded study. Therefore, a commercial spearmint tea,
identical in appearance and flavor to the high-rosA tea but which contained a small amount of
rosA (approximately 15 mg per 300 mL), was used as a control. As the decreases in WOMAC
scores ceased at Week 8 in the control group, but continued to decline in the high-rosA group,
lengthening the treatment period may result in significant difference between the groups.
The SF-36 questionnaire was created to address the importance of patient perspective in the
assessment of health care outcomes (Maruish et al 2009). The significant increase in QoL bodily
pain score in high-rosA group suggests that reduced pain led to an increase in QoL in this group.
This is in agreement with the decrease in WOMAC pain score observed for the high-rosA group.
As expected, the SF-36 scores for physical function, physical role, bodily pain, and physical
component score were all below the Canadian norms at all time points studied and in both groups
(Table 5.2) (Hopman et al 2000). Individuals with musculoskeletal disorders tend to report lower
physical functioning scores compared to the general population and of those patients, OA
patients generally have worst pain and physical functioning scores (Dawson et al 2004; Kosinski
et al 1999; Picavet and Hoeymans 2004).
The study participants demonstrated impaired physical function compared with average healthy
Canadians of comparable age in the 6MWT. In the current study (mean age = 61 years), the
average distance walked in the 6MWT at baseline was 445.5 m (range; 312.0 – 611.9 m), which
falls below the average distance of 672 m for males and 611 m for females (range; 416 to 888 m)
for healthy Canadians with an average age of 65 (Hill et al 2011). This is not surprising, as
individuals with arthritis, as well as overweight/obese individuals have been documented to walk
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shorter distances in the 6MWT (Enright 2003). Although no significant differences were
observed in the 6MWT, the high-rosA group walked 22 meters further at Week 16 than at
baseline. The control group only walked 0.1 meters further at Week 16 compared with baseline.
We postulate that, at Week 16 the high-rosA group was starting to experience improvements in
physical function related to their decreased pain. While not statistically significant, the
improvement in walk distance could be meaningful as even small increases in walking distance
are important to individuals with OA (Frestedt et al 2009). The SCT is useful as it measures the
ability to negotiate stairs and this is a common challenge for individuals with OA (Bennell et al
2011a). Heiberg et al. (2012) reported that individuals with OA completed an 8-stair climb test
within the range of 10-14 seconds. This is similar to the range of 10-12 seconds recorded in the
present study where 7 stairs were used.
There were no significant changes in any of the biomarkers measured in either treatment group.
The data presented in Table 5.4 is included for reference and design of future studies.
In the current study, compliance to drinking the tea was high, which reflects our participants’
willingness to consume spearmint tea as a treatment for OA. This is important, given the lack of
effective OA treatment options (Zhang et al 2008) and reliance on pain medications to which
adherence rates are low (Sale et al 2006). Failure to adequately manage OA pain and symptoms
can negatively impact social interactions and quality of life and lead to increased personal,
healthcare, and economic costs (Davis et al 2002).
5.6 Conclusion
In this study, individuals with OA of the knee who consumed 600 mL of high-rosA spearmint tea
daily for 4 months showed significant improvements in pain scores and physical function.
Therefore, adults with knee OA may benefit from including high-rosA spearmint tea in their
daily routine. Larger and longer-term studies are required to validate the high-rosA tea as a
complementary OA treatment.
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5.7 Acknowledgments
The research was sponsored by the Ontario Ministry of Agriculture, Food and Rural Affairs
(OMAFRA #200121). We would like to thank all the study participants. Additionally, we thank
Dr. Andrew Chow for his insights during the study design and recruitment stages, Dr. Forrest
Caldwell, Premila Sathasivam, and Mehrnoosh Kashani for their assistance with sampling, and
the many undergraduate research students and volunteers whom helped with the study.
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Chapter 6. Summary, General Discussion, and Future Directions
6.1 Summary of Results
The overall aims of this thesis were to investigate associations in pain and physical function and
explore the effect of a novel dietary treatment for knee OA. These aims were addressed by
conducting two cross-sectional investigations (Study 1 and 2) and one human clinical trial (Study
3). Together, these studies explore associations in OA symptoms, and managing these symptoms
with a novel product.
Pain was significantly higher with the use of OA medications and higher BMI category and
significantly lower with use of supplements and meeting physical activity guidelines in 197
adults with knee OA. Examining differences between 3 pain categories revealed that stiffness
and physical function scores, bilateral knee OA, BMI category, and OA medication use were
significantly higher in the higher pain categories, whereas self-reported health, servings of fruit,
supplement use, and meeting physical activity guidelines was significantly lower with higher
pain category. These results provide additional information on the complex associations of OA
pain and reinforced that modifiable factors should be considered in the management of knee OA.
In 54 adults with knee OA, HA, a marker of synovial membrane metabolism, was significantly
associated with performance-based measures of physical function. MMP-3, an enzyme involved
in tissue degradation in OA, was significantly associated with higher pain score. The
inflammatory marker, IL-6, was significantly associated with higher pain score and worse
physical function. Cartilage biomarkers COMP and PIIANP and inflammatory markers IL-18,
CRP, and NO were not associated with any clinical variables. The use of serum biomarkers to
characterize OA status is important for understanding OA and testing response to novel
treatments in OA.
Based on promising pre-clinical antioxidant and anti-inflammatory effects of a high-rosA
spearmint plant, study 3 examined this novel therapeutic product for the first time in adults with
knee OA. Participants (n=46) were randomized to consume tea brewed from the high-rosA
spearmint plant or a commercially available spearmint tea twice daily for 16 weeks in a
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randomized, double-blind study. In both groups, stiffness and physical disability scores
significantly decreased from Week 0 to Week 16. Pain score significantly decreased from Week
0 to 16 for the high-rosA group only. A non-significant increase in distance walked was observed
in the 6MWT at Week 16 in the high-rosA group only. The results from this study warrant
further exploration of this novel high-rosA spearmint tea and regular consumption of a high-rosA
spearmint tea represents a potential complementary therapy to reduce pain in OA patients.
6.2 General Discussion
OA pain is multifactorial and treating it is complicated. Previous work and findings in this thesis
have demonstrated that OA pain is modifiable and, importantly, through lifestyle and dietary
strategies (Chapter 5). Unfortunately, many older adults with OA believe that pain is a normal
part of aging and that adaptation and/or avoidance of painful activities are the only available
options (Ross et al 2001; Davis et al 2002; Sale et al 2006; Hawker 2009). Limitation of
activities can lead to social isolation and decreased movement and deconditioning, potentially
leading to more pain (Davis et al 2002). In study 1, a number of modifiable factors were
significantly associated with OA pain. Similar to other studies, BMI was associated with lower
pain (Elbaz et al 2011; Riddle and Stratford 2013; Lee et al 2013b). Also as previously shown
(Roddy et al 2005), higher levels of physical activity were associated with decreased pain.
Interestingly, in an 18-month RCT in overweight and obese adults with knee OA, weight loss
from diet plus exercise resulted in better outcomes in pain and physical function, as well as
greater decreases in IL-6 levels, than weight loss from diet or exercise alone (Messier et al 2004;
Messier et al 2013). These results demonstrate the importance of exercise and diet in weight loss
and the management of knee OA. Continuing to identify modifiable factors of OA pain and
investigating their relationship is important for managing the disease.
In study 3, pain decreased with consumption of a bioactive enriched spearmint tea. Dietary
constituents, especially ones with anti-inflammatory and antioxidant activities, have potential for
modifying pain in OA (Khanna et al 2007). Pain has a complex relationship with inflammation
(Omoigui et al 2007), and inflammatory mediators play an important role in OA disease
pathology (Loeser et al 2012; Lee et al 2013a). In study 3, both intervention groups had
decreased pain, but the decrease was only significant in the high-rosA spearmint group at the end
102
of the study. Arguably, the higher level of the rosA in the tea contributed to the significance in
pain relief. In animal models, oral administration of rosA has shown anti-nociceptive behavioural
effects in thermal and chemical induced noxious laboratory tests, as well as reductions in
chemically induced paw edema (Guginski et al 2009; Boonyarikpunchai et al 2014; Hasanein
and Zaheri 2014). In addition, rosA exhibited anti-nociceptive effects in tests examining
peripheral (i.e. glutamate injection, acetic acid-induced writhing test) and central pain
mechanisms (i.e. hot-plate test, formalin test) (Boonyarikpunchai et al 2014). Significant
reductions in OA pain have been reported from RCTs with consumption of anti-inflammatory
products and NHPs derived from fruit extracts (Farid et al 2010), plants (Cameron and Chrubasik
2014), spices (Nakagawa et al 2014) and propriety mixtures of fruit, plant, and spice extracts
(Nieman et al 2013). Further, NHP use was predictive of a lower pain score in study 1, providing
further support for their role in management of OA pain. In addition, participants in the mild OA
pain category consumed more servings of fruit than the moderate or severe pain categories. It has
been suggested that NHPs and supplements may be of most benefit for the management of OA
pain in adults with deficiencies (Cao et al 2013). Study 1 and study 3 were samples from the
same population (healthy adults with knee OA living in Guelph and surrounding communities),
and fruit and vegetable consumption was low in study 1, suggesting that additional antioxidants
or supplements could be beneficial in this group.
In study 3, participants maintained their normal routines, including pain medications.
Consumption of the high-rosA spearmint tea is meant to be a complementary to other OA
management techniques. As OA is complex and influenced by many factors, there may never be
one method that completely treats OA. Even total joint replacement must be followed by
pharmaceuticals and physical therapy (Greene and Harwin 2011). Personalized, integrated
management approaches should be used in OA treatment (Fernandes et al 2013; McAlindon et al
2014). Indeed, data from study 1 and 3 support previous research that persons with OA use a
combination of medications, supplements, physical therapies, and other strategies to manage OA
(Lapane et al 2012; Kingsbury 2014).
In the same vein, there may never be one method to completely measure and describe all aspects
of OA. The complex and multi-faceted nature of the disease suggests that a combination of pain
103
measurements, imaging measurements, and biomarker measurements will be necessary to fully
characterize OA status. In research settings, OA can be defined by radiographic or symptomatic
definitions, or by self-reported physician diagnosed. In a review examining the effect of OA
definition on prevalence and incidence estimates, Pereira et al (2011) concluded that estimates of
incidence of OA are highest and probably overestimated when radiographic definitions are used
and similar when symptomatic or self-report definitions are used. In this thesis, knee OA was
self-reported physician diagnosed. Self-reported physician diagnosed knee OA has previously
been used in various types of OA research including larger epidemiological studies (Cheng et al
2000; Parazzini 2002; Rogers et al 2002; Hootman et al 2003; da Costa DiBoniventura et al
2012) as well as smaller intervention (Callahan et al 2008; Miller et al 2008) and cross-sectional
(Keefe et al 2000). In a validation study, March et al (1998) reported that 81% of OA selfreported physician diagnosed was confirmed when using the ACR clinical criteria strictly. When
the pain question was used more loosely, 100% of the diagnoses were confirmed. Good
agreement between self-reported physician diagnosis and medical records has been reported for
other chronic diseases as well (Katz et al 1996). Self-reporting of chronic diseases also has been
shown to be fairly accurate in a Canadian population (Robinson et al 1997). There is no gold
standard for identifying cases of OA, with 25 different methods described in the literature using
radiographic endpoints and ACR criteria (Busija et al 2010). Self-reported physician diagnosed
OA is noted as an acceptable method of identifying cases, and the sensitivity of the method is
increased when a symptom question (pain or stiffness) was included in the definition (Busija et
al 2010).
People cope with OA pain in different ways. In OA, passive coping has been associated with
higher pain and lower physical functioning (Benyon et al 2010). Passive coping strategies like
worrying, resting, and retreating generally involve giving up control and allowing external
factors to influence outcomes, while active coping involves adapting to situations to try to
control the outcome (Hampson et al 1996). Another psychological variable implicated in OA
pain is self-efficacy (Marks 2012). With regards to OA, self-efficacy is the belief in one’s
capability to manage the symptoms and consequences of the disease (Marks 2012). It has been
shown to be a strong predictor of health-related behaviors and play a role in OA pain and
function (Maly et al 2007; Marks 2012). In a review, interventions that improve self-efficacy
104
also improved OA pain and physical function (Marks 2012). Although these variables were not
measured or investigated in this thesis, they can be indirectly explored in study 1 and 3. Meeting
physical activity guidelines was predictive of a lower pain score in study 1. Exercise has
psychological benefits and mood boosting effects that may play a role in pain reduction (Bennell
et al 2011b). In addition, being physically active could give individuals a sense of control and
stronger self-efficacy that could improve OA symptoms. Similarly, it is possible that in study 3,
the action of joining an intervention study gave participants a feeling of control and management
over their OA and increased their confidence in managing OA. In addition, the Hawthorne
effect, which is the observation that simply participating in a study improves disease condition,
could have played a role in disease improvement (Braunholtz et al 2001) and in addition to the
benefits of the spearmint tea, each participant had to take 10-20 minutes/day to make and drink
the 2 cups of spearmint tea. In a follow-up questionnaire to the study, many participants reported
that they enjoyed taking time to themselves each day, and although not measured, the additional
relaxation could have had an effect on pain reduction. Measuring psychological variables in OA
clinical trials will add an interesting and important component to aid in management of the
disease.
6.3 Future Directions
Many research themes have been identified in this thesis, all of which hold exciting potential for
future studies. Promising results from pre-clinical work and study 3 indicate that a larger, longer
clinical trial with the high-rosA spearmint tea should be undertaken as it has anti-inflammatory
and antioxidant actions, which has implications in joint tissue deterioration and alterations in
pain processing. OA is a slowly progressing disease, so a trial lasting at least 12 months would
be ideal. In addition, in study 3, the control group did not consume a true placebo since there was
rosA in the commercial tea, as a blinded trial was prioritized. In the future, the use of a blinded
spearmint tasting tea that does not contain rosA would allow for comparison to a placebo group.
In future RCTs, joint and inflammatory biomarkers should be in included in the outcome
measures. The primary outcome should be pain reduction, but assessment of knee structure,
stiffness, physical function, self-efficacy and psychological aspects of pain should be included.
105
The modifiable lifestyle factors identified in study 1 are also relevant to the risk of other chronic
progressive diseases, especially a healthy BMI and being physically active. It is generally known
and appreciated that a healthy weight, balanced diet and regular exercise are important for all
areas of health. Investigations into how to teach that information in a way that leads to changes
in behavior of OA patients and those at risk for OA should follow. It would be beneficial to
discover the key messages, information, and interventions that will get people with OA to
exercise, eat a healthy diet, keep a healthy BMI and maintain those practices for a lifetime.
The continued study of OA biomarkers is also crucial, as it will provide information on the
disease and well as help to improve methods to measure and monitor OA. Using an integrated
approach where biomarkers are examined in relation to structural outcomes as well as
symptomatic outcomes will help provide a more complete picture. The kinetics of biomarkers is
a specific area of research that should be examined in order to further this work. Examining the
movement of fragments of joint tissues from synovial fluid to circulation to removal from
circulation would greatly improve our understanding of serum, plasma, and urine measurements.
The kinetics of potential treatments is also important to examine, especially in regards to NHP
and supplement products. The original rationale for the use of glucosamine as a treatment for OA
was flawed in that researchers believed that the cartilage could take exogenous glucosamine
from the circulation and use it to synthesize new GAG side chains. That has not been
demonstrated and it is unknown, and probably unlikely, that any products consumed orally can
be used by the cartilage as building blocks for ECM in adults with OA. However, antiinflammatory and antioxidant products can be used by body tissues and potentially synovial
cells. Measuring levels of the product in the circulation, urine, body tissues, and synovial fluid
could help to provide an understanding the movement of an oral product that could give
powerful insight into treatment options.
Finally, as results from this thesis have demonstrated, OA is a complex disease that is affected by
many factors and that management of the disease requires a multi-component, integrated
approach. Clinical trials involving comprehensive education and interventions for diet, exercise,
106
BMI, and self-efficacy with inclusion of education on medication and supplements would be
important to systematically examine effect of integrated treatment on OA outcomes.
6.4 Concluding Remarks
There is much to be learned in the OA field. The studies in this thesis highlight the importance of
modifiable lifestyle factors in OA management, including high-rosA spearmint tea, BMI,
exercise, medications, and supplements. Consumption of a high-rosA spearmint tea represents a
potential complementary therapy to manage OA pain. Furthermore, serum biomarkers of joint
metabolism and inflammation were associated with symptoms of OA. Taken together, the results
from this research have identified modifiable lifestyle factors for managing pain in adults with
knee OA, while also demonstrating the complexity of clinical symptoms in OA and the need for
further research in this field.
107
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143
Appendices
Appendix 1. A table displaying the range and diversity of diseases for which rosA has been
examined for its potential therapeutic effects
Table 14.1 A sample of diseases explored with rosA treatment
Disease
Experimental Model
Proposed Mechanism
Cancer
C6 and HeLa cell lines
Anti-oxidant, antiproliferative
COX-2 inhibition
Erenler et al
2015
Tao et al 2014
1,2-dimethylhydrazine
induced colon cancer in
rats
Dextran sulfate sodiuminduced colitis in rats
A25-35 induced mice
Anti-inflammatory,
anti-proliferative
Karthikkumar
et al 2014
Anti-inflammatory
Neuronal SH-SY5Y
cells
Mouse
Mitochondria
protection
Decrease levels of
COX-2 and free
radicals
Inhibition of
acetylcholinesterase in
the brain
Anti-inflammatory
Urushima et al
2014
Alkam et al
2007
Camilleri et al
2013
Shimojo et al.,
2010
A549 cell line
Colitis
Alzheimer’s
Disease
Amylotropic
Lateral Sclerosis
Dementia
Scopolamine-induced
rats
Encephalitis
Mouse
Epilepsy
Pentylenotetrazole
induced seizures in
mice
Streptozotocin induced
rats
Neuro-diabetic
complications
Ischemic diabetic
stroke
Diabetic-induced
neuropathy
Diabetes-induced
vascular
dysfunction
Diabetic-induced
Reference
Cerebral artery
occlusion to diabetic
rats
Streptozotocin induced
rats
Streptozotocin induced
rats
Streptozotocin induced
Anti-oxidant
Anti-oxidant
Ozarowski et
al 2013
Swarup et al.,
2007
Coelho et al
2015
Prevention of lipid
peroxidation and
increased
acetylcholinesterase
activity in the brain
Anti-inflammatory
Mushtaq et al
2014
Anti-inflammatory,
anti-oxidant, analgesic
Anti-oxidant, antiinflammatory
Hasanein and
Zaheri 2014
Sotnikova et al
2013
Reduced connective
Jiang et al
144
Luan et al
2013
nephropathy
rats
Nephrotoxicity
Gentamicin sulphateinduced renal oxidative
damage in rats
Carbon tetrachloride
induced liver damage in
mice
Induced
ischemia/reperfusion
injury in rats
tert-butyl
hydroperoxide induced
oxidative liver damage
DEP-induced lung
injury in mice
T-cells from RA
patients
Collagen-induced
arthritis in mice
Trabeculectomy in
rabbits
OVA-sensitized mice
Bloma tropicalis mite
induced in mice
MT-4 cells
Liver injury
Lung injury
Rheumatoid
arthritis
Glaucoma
Allergy
Anti-viral
Anti-microbial
tissue growth factor
levels
Anti-oxidant
2012
Anti-oxidant, antiinflammatory
Tavafi and
Ahmadvand
2011
Domitrovic et
al 2012
Anti-oxidant, antiinflammatory
Ramalho et al
2014
Increased glutathione
and decreased lipid
peroxidation
Anti-inflammatory
Yang et al
2013
Apoptosis of T-cells
Decreased COX-2
levels
Short-term antiangiogenic effect
Anti-inflammatory
Decrease in leukocytes
and eosinophils
Anti-viral
Herpes simplex virus
type 1 (HSV-1)
acyclovir-sensitive and
clinical isolates of
acyclovir-resistant
strains
Inhibition of HSV-1
attachment to host cells
Cells exposed to
Staphylococcus aureus
Bacillus licheniformis,
Pseudomonas vulgaris,
Shigella boydii,
Salmonella typhi,
Staphylococcus aureus,
Listeria monocytogenes
and Escherichia coli
Anti-microbial
Anti-microbial
145
Sanbongi et al
2003
Hur et al 2006
Youn et al
2003
Ferreira et al
2014
Oh et al 2011
Costa et al
2012
Dubois et al
2008
Astani et al
2014
Slobodnikova
et al 2013
Hakkim et al
2012
Appendix 2. Certificate of approval from University of Guelph Human Research Ethics’ Board
146
Appendix 3. Participantinformed consent form
Date:
Protocol & Version Number:
1
Participant Initials:
Factors affecting self-reported pain, stiffness, and physical function in adults with knee
osteoarthritis.
You are being asked to participate in a participant questionnaire at the Human Nutraceutical
Research Unit (HNRU) in the Department of Human Health & Nutritional Science (HHNS) at
the University of Guelph. This research is being conducted under the supervision of Amanda
Wright, Ph.D. (Director of the HNRU and Associate Professor in HHNS), Alison Duncan, Ph.D.
(Associate Director of the HRNU and Associate Professor in HHNS) and Amy Tucker, Ph.D.
(Manager at HNRU). The Ontario Ministry of Agriculture, Food and Rural Affairs is funding
this project.
Contact Information
If you have any questions or concerns, please contact:
A. Erin Connelly, B.Sc.
(Ph.D. candidate, HHNS)
phone: (519) 824-4120 x56314 or email: [email protected]
Amy Tucker, Ph.D. (Manager, HNRU)
phone: (519) 824-4120 x53749 or email: [email protected]
Amanda Wright, Ph.D. (Co-Principal Investigator, HNRU Director, Associate Professor,
HHNS)
phone: (519) 824-4120 x54697 or email: [email protected]
Alison Duncan, Ph.D., R.D. (Co-Principal Investigator, HNRU Associate Director, Associate
Professor, HHNS)
phone: (519) 824-4120 x53416 or email: [email protected]
147
PURPOSE OF THE STUDY
The purpose of this study is to examine the relationships between osteoarthritis symptoms and a
number of lifestyle factors in adults with osteoarthritis of the knee. For this visit, you will arrive
at the HNRU and complete the Informed Consent process. Should you choose to participate, you
will be asked to complete detailed questionnaires regarding your health history and medication
use and to assess your level of disease activity using the Western Ontario and McMaster
Universities Arthritis Index (WOMAC) questionnaire. The WOMAC is a standardized
questionnaire used in the assessment and monitoring of osteoarthritis symptoms.
POTENTIAL RISKS AND DISCOMFORTS
There are minimal risks associated with participation in this visit.
POTENTIAL BENEFITS TO PARTICIPANTS AND/OR TO SOCIETY
You will benefit from participating in this visit by gaining the experience as a study participant.
PAYMENT FOR SCREENING VISIT PARTICIPATION
You will be financially compensated for your time and effort for this screening process with at
$10 gift certificate to a local coffee retailer.
COSTS FOR SCREENING VISIT PARTICIPATION
There is no direct cost for participating in this screening visit. You will only be responsible for
covering any costs related to attending this screening visit (i.e. gas money, public transportation
fees, child care fees, etc.).
CONFIDENTIALITY
Every effort will be made to ensure confidentiality of any identifying information that is obtained
in connection with this study. All participants will be assigned a number and a study code will be
used. Your name will never be used in communicating results of the study. Records will be kept
on a password-protected computer and/or in a locked file cabinet in a locked office. All data will
be kept indefinitely. In following these guidelines, participants’ confidentiality will be
maintained to the best of our ability. Results from the study may be published, but will be
presented as group data.
If requested, direct access to your research records for this study will be granted to study
monitors, auditors, the University of Guelph Research Ethics Board, and regulatory authorities
for verification of study procedures and/or data. Your confidentiality as a study participant will
not be violated during this process, to the extent permitted by applicable laws and regulations.
By signing this written informed consent form, you are agreeing to authorize such access.
PARTICIPATION AND WITHDRAWAL
You can choose whether to participate in the visit or not. If you agree to participate, you may
withdraw at any time without consequences of any kind. You may exercise the option of
removing your data from the study. You may also refuse to answer any questions you don’t
want to answer and still remain in the study. The investigator may withdraw you from this
research if circumstances arise that warrant doing so.
148
RIGHTS OF RESEARCH PARTICIPANTS
You may withdraw your consent at any time and discontinue participation without penalty. You
are not waiving any legal claims, rights or remedies because of your participation in this research
study. This study has been reviewed and received ethics clearance through the University of
Guelph Research Ethics Board. If you have questions regarding your rights as a research
participant, contact:
Sandy Auld
Research Ethics Coordinator
Room 437, University Centre
University of Guelph
Telephone: (519) 824-4120, ext. 56606
E-email: [email protected]
SIGNATURE OF RESEARCH PARTICIPANT/LEGAL REPRESENTATIVE
I have read the information provided for the study “Investigating health history and
osteoarthritis symptoms in individuals with osteoarthritis of the knee” as described herein.
My questions have been answered to my satisfaction, and I agree to participate in the screening
visit. I have been given a copy of this form.
NAME OF PARTICIPANT
(PLEASE PRINT)
SIGNATURE OF PARTICIPANT
DATE
NAME OF WITNESS
(PLEASE PRINT)
SIGNATURE OF WITNESS
DATE
149
Appendix 4. Lifestyle Questionnaire
IN-PERSON HEALTH HISTORY QUESTIONNAIRE
STUDY TITLE:
Factors affecting self-reported pain, stiffness, and physical function in adults with knee
osteoarthritis.
HNRU Coordinator:
Date:
Time:
Participant ID:
Thank you very much for your interest in this study. The purpose of this questionnaire is to
gather more information about you and your knee osteoarthritis.
Please feel free to NOT answer any questions that you are uncomfortable with
answering.Please feel free to ask the study coordinator any questions you might have.
All information provided in this questionnaire will be kept strictly confidential.
150
HEALTH HISTORY QUESTIONNAIRE
1. When were you first diagnosed with osteoarthritis of the knee?
2. Who first diagnosed you with osteoarthritis of the knee?
(i.e. family doctor, rheumatologist)
3. (a) Which knee do you have osteoarthritis in?
BOTH
RIGHT
LEFT
RIGHT
LEFT
4. Have you ever had knee replacement surgery?
YES
NO
5. Are you planning on having knee replacement surgery between
now and May 2014?
YES
NO
6. Have you ever had a synovectomy before? (i.e. surgical removal
of inflamed joint tissue)
YES
NO
7. Do you currently smoke?
YES
NO
YES
NO
YES
NO
(b) Which knee is most problematic?
(a) If NO, have you ever smoked?
(b) If YES to (a), how long since you have quit?
8. Approximately how many alcoholic drinks do you consume per
week? (1 drink = 12 oz beer, 5 oz wine, or 1.5 oz hard liquor)
9. Are you currently participating in any other research studies?
(a) If YES, please describe:
10. How would you describe your general health?
POOR
GOOD
VERY GOOD
151
EXCELLENT
11. Do you have any of the following diagnosed medical conditions:
(a) Rheumatoid arthritis?
YES
NO
(b) Cancer?
YES
NO
(c) Pre-diabetes?
YES
NO
(d) Diabetes?
YES
NO
(e) High cholesterol?
YES
NO
(f) High blood pressure?
YES
NO
(g) Impaired liver function?
YES
NO
(h) Impaired kidney function?
YES
NO
(i) Gastrointestinal ulcer?
YES
NO
(j) Autoimmune disease?
YES
NO
(k) Chronic infection?
YES
NO
(l) Depression?
YES
NO
(m) Are there any other medical conditions that might affect
your participation in the study?
YES
NO
YES
NO
(i) If YES, please describe:
12. Are you currently taking any medication and/or over-the-counter
drugs? (i.e. Tylenol, Advil, Claritin, Sudafed, etc.)
(a) If YES, please provide the study coordinator with your current medication.
The study coordinator will help you complete the following table:
152
PRODUCT
NAME
REASON
FOR USE
MEDICINAL
INGREDIENTS
DOSE
13. Are you currently taking any dietary supplements?
(i.e. multivitamin, omega-3, glucosamine)
HOW HOW
OFTEN LONG
YES
NO
(a) If YES, please provide the study coordinator with your dietary supplements.
The study coordinator will help you complete the following table:
PRODUCT
NAME
REASON
FOR USE
ACTIVE
INGREDIENTS
DOSE
14. Are you currently taking any medication for your osteoarthritis?
HOW HOW
OFTEN LONG
YES
NO
(a) If YES, please provide the study coordinator with your current osteoarthritis
medication. The study coordinator will help you complete the following table:
153
PRODUCT
NAME
REASON
FOR USE
MEDICINAL
INGREDIENTS
DOSE
HOW HOW
OFTEN LONG
15. Do you see the following health professionals for your osteoarthritis:
(make sure it is clear that these are all related to osteoarthritis)
(a) Family doctor or
rheumatologist?
YES
NO
If YES, how often? ______________
(b) Physiotherapy?
YES
NO
If YES, how often? ______________
(c) Acupuncture?
YES
NO
If YES, how often? ______________
(d) Massage therapy?
YES
NO
If YES, how often? ______________
(e) Nerve stimulation?
YES
NO
If YES, how often? ______________
(f) Other? (please list)
YES
NO
How often? ____________________
How often? ____________________
How often? ____________________
16. Do you participate in any of the following types of exercises:
(a) Weight training?
YES
NO
If YES, how often? ______________
(b) Stretching?
YES
NO
If YES, how often? ______________
154
(c) Aerobics?
YES
NO
If YES, how often? ______________
(d) Underwater?
YES
NO
If YES, how often? ______________
(e) Other? (please list)
YES
NO
How often? ____________________
How often? ____________________
How often? ____________________
17. Do you have ANY allergies? (i.e. food, medications, rag weed or
pollen)
YES
NO
(a) If YES, please list:
(b) Please describe your type of allergic reaction: (i.e. anaphylaxis, difficulty
breathing, swelling, etc.)
18 (a). What is your current height? ___________________
18 (b). What is your current body weight? _________________
Question #19 to be completed by females only
Hormone levels can affect pain sensation and perception. Since pain is a large part of
osteoarthritis, we wish to gather some information about your hormone levels.
19. Are you PRE- or POST-menopausal?
PRE
155
POST
(a) If POST-menopausal, do you use hormone replacement
therapy?
YES
NO
YES
NO
(b) If YES to using hormone replacement therapy, please specify:
(c) If PRE-menopausal, do you have regular menstrual cycles?
(d) If NO to having regular menstrual cycles, please describe the irregularities:
(e) Do you currently use oral contraceptives (or other hormonal
birth control)?
YES
NO
(f) If YES to using oral contraceptives or hormonal birth control, please specify:
________________________________
HNRU Coordinator Signature
NB: Provide the WOMAC to the participant and have them complete the form.
Complete the 24-hr food recall with the participant.
Provide the participant with a coffee voucher.
Fill out the In-person Screening Tracking Log.
156
Appendix 5. Sample of 24-hour food recall sheet
157
Appendix 6. Certificate of approval from University of Guelph Human Research Ethics’ Board
158
Appendix 7. Participant informed consent
Date:
Initials:
Protocol & Version Number:
4
Participant
CONSENT TO PARTICIPATE IN RESEARCH
Human clinical trial to investigate the effects of high rosmarinic acid spearmint tea on markers of
pain, physical function and disease activity in osteoarthritis of the knee
You are being asked to participate in a clinical research study at the Human Nutraceutical
Research Unit (HNRU) in the Department of Human Health & Nutritional Science (HHNS) at
the University of Guelph. This research is being conducted under the supervision of Amanda
Wright, Ph.D. (Director of the HNRU and Associate Professor in HHNS), Alison Duncan, Ph.D.
(Associate Director of Research at the HNRU and Associate Professor in HHNS) and Hilary
Tulk, M.Sc. (Clinical Trials Manager at HNRU). This project is funded by the Ontario Ministry
of Agriculture, Food and Rural Affairs Food Research Program.
Contact Information
If you have any questions or concerns, please contact:
A. Erin Connelly, B.Sc.
M.Sc. graduate student, HHNS
phone: (519) 824-4120 x56314 or email: [email protected]
Hilary Tulk, M.Sc.
Clinical Trials Manager, HNRU
phone: (519) 824-4120 x53749 or email: [email protected]
Amanda Wright, Ph.D.
Co-Principal Investigator, HNRU Director, Associate Professor, HHNS
phone: (519) 824-4120 x54697 or email: [email protected]
Alison Duncan, Ph.D., R.D.
Co-Principal Investigator, HNRU Associate Director, Associate Professor, HHNS
phone: (519) 824-4120 x53416 or email: [email protected]
PURPOSE OF THE STUDY
The purpose of the study is to test if daily consumption of a spearmint tea, that is high in
rosmarinic acid, improves disease activity, functional status, and/or cartilage degradation in
adults with osteoarthritis of the knee. The spearmint tea was developed at the University of
Guelph using selective breeding methods and has 15 – 20 times more rosmarinic acid than
159
regular spearmint. Rosmarinic acid has demonstrated antioxidant and anti-inflammatory
potential. This study will allow us to investigate if the high rosmarinic acid spearmint tea can be
used as a complement to traditional osteoarthritis therapies. Results of this study may also help
the commercialization and marketing of the high rosmarinic acid spearmint.
*Drs. Kott and Fletcher in the Department of Plant Agriculture at the University of Guelph have
a pending patent application for the high rosmarinic acid spearmint line and are collaborators on
the project*
PROCEDURES
This research study involves consuming two 300 mL cups of spearmint tea per day for a 4-month
period. Approximately 50 individuals will be invited to participate in this study. Half (50%) of
the participants will be randomly assigned to consume either the high rosmarinic acid spearmint
tea or a commercial spearmint tea. Participants will be asked to keep a daily study record of the
time of tea consumption, any medication or natural health products used, any physical activity or
therapies, and other significant dietary or lifestyle changes. There will be 9 visits to the HNRU,
located in room 144 of the Food Science-Guelph Food Technology Centre Building, 88
McGilvray St. at the University of Guelph. In addition, there will be 4 check-in phone calls made
to each participant. The following describes the procedures prior to and during each study visit.
STUDY TEST PRODUCTS
Throughout the treatment period you will be asked to consume two cups of spearmint tea per day
(300 mL in the morning and 300 mL in the evening). You will be consuming either a
commercially available spearmint tea or the spearmint tea with a high rosmarinic acid content
that was developed at the University of Guelph through selective breeding techniques. Both teas
are naturally caffeine free. The teas will be packaged under food grade conditions and will
undergo microbiological and toxicological testing through a certified, independent laboratory
prior to being provided to you.
STUDY DIARY
 You will be provided with a 4-week supply of tea along with detailed written instructions for
how to prepare the tea.
 You will be asked to record the date and time of consumption for each cup of tea.
 You will be asked to record any medication and natural health product usage.
STUDY VISITS
In total, you will be asked to attend 9 study visits and to participate in 4 check-in telephone calls.
Table 1 summarizes the activities you will be asked to participate in as part of each study visit or
phone call. Each activity is explained following Table 1.
.
Table 1: Activities associated with each study visit and check-in phone calls.
TYPE OF
APPOINTMENT
STUDY VISIT
ACTIVITIES
APPOINTMENT
LENGTH
Study
HNRU
1 ½ hours
 Meet all the study personnel
Orientation
 Review the study design, timeline,
and protocols
160
Week 0
HNRU
2 hours
Week 1
HNRU
30 minutes
Week 2
Phone Call
10 minutes
Week 4
HNRU
30 minutes
Week 6
Phone Call
10 minutes
Week 8
HNRU
1 ½ hours
Week 10
Phone Call
10 minutes
Week 12
HNRU
30 minutes
Week 14
Phone Call
10 minutes
Week 16
HNRU
2 hours
Week 20
HNRU
1 ½ hours
161
 Learn about and taste the high
rosmarinic acid mint tea
 Ask questions about any study
details
 Sign the Study Consent Form
 Tea distribution
 Physical measurements
 WOMAC questionnaire
 SF-36 questionnaire
 6-min walk
 Stair climb
 Blood sample
 SF sample (consenting participants
only)
 On-site check-in with study
coordinator
 Check-in call from study
coordinator
 Tea distribution
 On-site check-in with study
coordinator
 Check-in call from study
coordinator
 Tea distribution
 WOMAC questionnaire
 SF-36 questionnaire
 6-min walk
 Stair climb
 Check-in call from study
coordinator
 Tea distribution
 On-site check-in with study
coordinator
 Check-in call from study
coordinator
 Physical measurements
 WOMAC questionnaire
 SF-36 questionnaire
 6-min walk
 Stair climb
 Blood sample
 SF sample (consenting participants
only)
 WOMAC questionnaire





SF-36 questionnaire
6-min walk
Stair climb
Blood sample
Final review of study records with
study coordinator
Physical Measurements
In privacy, a trained study coordinator will measure your body weight, height, waist and hip
circumferences, and blood pressure.
 All physical measurements will be made in privacy by a trained study coordinator.
 Body weight will be measured on a digital scale.
 Height will be measured using a standard stadiometer.
 Waist and hip circumference will be measured over light clothing using a standard inelastic
tape measure.
 Blood pressure will be measured using a portable digital blood pressure monitor.
Body Composition measured by Bioelectrical Impedance Analysis (BIA):
In privacy, a trained study coordinator will measure your body composition.
BIA will not be performed on any participant with an artificial pacemaker, an implantable
cardioverter-defibrillator (ICD), or similar. Please inform the study coordinator if you have any
such device.
 BIA is averycommon technique used to determine body composition.
 You will be asked to lie comfortably on a medical bed in a private sampling area.
 The study coordinator will enter your age, sex, height and weight, and waist and hip
circumference into a handheld BIA device.
 Two electrodes will be placed on your right foot and two electrodes will be placed on your
right hand. These electrodes will be connected to the handheld BIA device.
 The measurement then begins and lasts for approximately 5 seconds.
 During the measurement, you will remain lying down with the researcher standing alongside
of you.
 Your body’s opposition to a weak electric current is measured and body water content, lean
mass and body fat are estimated.
 You will not feel the very weak electric current during the measurement and the process is
completely painless.
Western Ontario & McMaster Arthritis Index (WOMAC):The WOMAC is a standardized 24item questionnaire that assesses pain, disability and joint stiffness due to osteoarthritis.
36-Item Short-Form Health Survey (SF-36):The SF-36 is a standardized 36-item health
questionnaire that assesses overall health including: (1) physical functioning; (2) limitations due
162
to physical health; (3) bodily pain; (4) general health; (5) vitality, energy, and fatigue; (6) social
functioning; (7) limitations due to emotional problems; and (8) mental health.
6-Minute Walk Test (6-min walk):The 6-min walk is a timed walking test that measures the total
distance traveled in 6 minutes. You will be asked to walk as quickly as you can over a flat
surface for 6 minutes. You may rest when necessary and can use any mobility aids, such as
canes or walkers which you normally would use.
Stair Climbing Task (stair climb):The stair climb is a timed stair climbing task that measures the
total time required to climb up a flight of 7steps, turn around and then climb down 7 steps. You
may use a handrail for help.
Blood Sample:In the privacy of the HNRU sampling area, a qualified medical technician will
collect a blood sample (two 5 mL tubes) from your arm using standard clinical methods. Blood
samples will be taken once at the beginning and once at the end of the 4-month tea consumption
period, as well as at the 1-month follow-up visit.
Synovial Fluid:You will be invited to provide synovial fluid from your knee that is affected by
osteoarthritis. Providing a synovial fluid sample is an optional procedure in this study. Synovial
fluid samples will be taken once at the beginning and once at the end of the 4-month tea
consumption period. If you agree, you will be asked to sign a separate informed consent form
specific to the synovial fluid sampling procedure. The study physician will examine the knees of
participants who agree to give a synovial fluid sample to ensure that the joint is fit for this
procedure. Please remember that you may withdraw your consent at any time.
Collection of synovial fluid is a common diagnostic and therapeutic procedure in arthritis and
will be performed by a qualified physician.
 You will be asked to lie comfortably on a medical bed in a private sampling area.
 A physician will examine your knee and determine if it is suitable for synovial fluid sampling.
 The skin on your knee will be washed with an alcohol swab.
 A sterile needle will then be inserted through the skin into the joint space and the fluid then
drawn through the needle into the syringe and the needle removed from the knee.
 The physician will apply gentle pressure to the joint.
 The skin will then be cleansed and a bandage will be applied to the needle insertion site.
 Synovial fluid samples will be analyzed for: cartilage degradation with sulphated
glycosaminoglycan and cartilage oligomeric matrix protein; oxidative stress with nitric oxide;
and inflammation with prostaglandin E2. These are markers used in assessing disease activity
in osteoarthritis and have been used in previous studies of rosmarinic acid.
Study Sample Laboratory Analysis
One tube of blood (5 mL) will be processed at the HNRU and then sent to an independent
medical laboratory (LifeLabs® Medical Laboratory Services) for same-day analysis of Creactive protein, a marker of inflammation.
The second tube of blood (5 mL) will be processed at the HNRU and a small amount of serum (2
mL) will be frozen at -80C and stored in the HNRU Wet laboratory (room 143, building #88).
Upon completion of the clinical trial, the serum samples will be analyzed at the University of
163
Guelph for biomarkers of inflammation and osteoarthritis including markers of cartilage
degradation (cartilage oligomeric matrix protein), oxidative stress (nitric oxide), and
inflammation (prostaglandin E2). These are the same biomarkers of inflammation and
osteoarthritis that will be analyzed in the synovial fluid.
Synovial fluid samples will be stored at -80C and, upon completion of the clinical trial, the
synovial fluid samples will be analyzed at the University of Guelph for markers of cartilage
degradation (glycosaminoglycan, cartilage oligomeric matrix protein), oxidative stress (nitric
oxide), and inflammation (prostaglandin E2).
All samples (i.e. serum and synovial fluid) will be stored using unique identifiers (i.e. a
participant number and study code). In this way, your confidentiality will be maintained.
Study Results Publication
Results from this study may be published, but will always be presented as group data and with no
ability to link data back to an individual (i.e. data will always remain confidential). Your
decision to be a participant in this study is voluntary and you are free to withdraw from the study
at any time. Following completion of the study analyses, a summary of the research results (both
group data and your individual data) will be mailed to you.
POTENTIAL RISKS AND DISCOMFORTS
Every effort to ensure your comfort and safety will be made during the course of this study. In
the unlikely event of a study-related injury, study staff will engage appropriate emergency
response services to assist in your care. A study coordinator certified in Standard First Aid and
CPR will always be present.
There are minimal risks associated with participation in this study. The following summarizes
the potential risks:
 If you are currently unaware of any allergies or hypersensitivies to mints, you might
experience an allergic reaction after consuming either the high rosmarinic acid or the
commercial spearmint tea.
 At 4 study visits (study visits weeks 0, 8, 16, and 20), a qualified and trained medical
technician will draw a blood sample from your forearm using a needle.
There is a chance that this process could cause you some slight discomfort as the needle is
inserted and, as with any venipuncture, there may be some minimal bruising afterwards.
These risks and potential discomforts from the blood draws will be managed by having a
qualified and experienced medical technician taking your blood. In addition, consuming
plenty of water the night before and the morning of can facilitate blood sampling. Also,
applying compression to the blood draw site will help to minimize bruising.
The following describes the potential risks and discomforts related to the synovial fluid
sampling. Providing a synovial fluid sample is optional and this procedure will only be
performed on participants who provide additional consent and whose joints are deemed fit by the
study physician.
164
 In those individuals deemed suitable by a physician and who provide consent, synovial fluid
samples will be taken from the knee joint using a syringe at 2 study visits (weeks 0 and 16).
This is a common procedure and a trained physician will perform the synovial fluid sample
collection. However, as with any needle puncture, there is a minimal chance of an infection or
bleeding into the joint. Ice or cold packs will be placed on the joint after the procedure.
 If unusual pain or swelling occurs in the knee after the synovial fluid sampling procedure and
persists for more than 5 days, participants should contact the study investigators. If a
participant experiences any unusual pain or swelling of the knee from which synovial fluid
was sampled AND a fever develops within 5 days of the synovial fluid sampling, they should
proceed to the nearest emergency room.
 If a serious adverse event occurs during a study visit at the HNRU, an ambulance will be
called. The student investigator will stay with the participant through to the hospital, if
necessary. All costs arising from a research related injury will be born by the University of
Guelph and the study researchers.
POTENTIAL BENEFITS TO PARTICIPANTS AND/OR TO SOCIETY
If you participate in this research, you will have benefit of gaining experience and skills in selftracking and self-monitoring of your osteoarthritis symptoms. In addition, you will receive a
written summary of your individual study data. Some of this data (i.e. WOMAC Index scores)
can provide insight into your arthritis symptoms and, when interpreted by a medical professional,
may be useful in guiding clinical decisions related to your disease management.
In this study, 50% of the participants will be assigned to the control commercial spearmint tea
group. Although we do not expect immediate clinical benefits in individuals who are consuming
the control commercial spearmint tea, your participation will help us to answer our research
question and you will still receive all of your individual data.
Your involvement in the study will contribute valuable insights into the health benefits of using a
spearmint tea as a complement to traditional drug-based treatment for osteoarthritis. The
knowledge generated from this study will be useful to individuals suffering from osteoarthritis
and health care professionals seeking to make recommendations of evidence-based
complementary therapies. The results may also help the University of Guelph commercialize the
high rosmarinic acid spearmint tea plant.
PAYMENT FOR PARTICIPATION
You will be financially compensated for your time and effort at an amount of $200, plus $100
extra for participation in synovial fluid sampling ($50 per sample provided). An additional $50
for completion of the study will be given (for a total of $250 without synovial fluid sampling and
$350 with synovial fluid sampling). If you withdraw from the study before completion, your
compensation will be pro-rated for your involvement.
COSTS FOR PARTICIPATION
There are no direct costs for participating in this study. You will only be responsible for covering
any costs related to ensuring you are able to attend your scheduled study visits (i.e. gas money,
165
public transportation fees, child care fees, etc.). It is our intention that, through the financial
compensation we provide for your time and effort participating in this study, we partially
reimburse you for some of the costs you may incur.
CONFIDENTIALITY
Every effort will be made to ensure confidentiality of any identifying information that is obtained
in connection with this study. All participants will be assigned a number, and a study code will
be used. Your name will never be used in communicating results of the study. Records will be
kept on a password-protected computer and/or in a locked file cabinet in a locked office. All data
will be kept for up to 25 years, as required by applicable regulations. In following these
guidelines, participants’ confidentiality will be maintained to the best of our ability. Results from
the study may be published, but will be presented as group data.
If requested, direct access to your research records for this study will be granted to study
monitors, auditors, the University of Guelph Research Ethics Board, and regulatory authorities
for verification of study procedures and/or data. Your confidentiality as a study participant will
not be violated during this process, to the extent permitted by applicable laws and regulations.
By signing this written informed consent form you are agreeing to authorize such access.
PARTICIPATION AND WITHDRAWAL
You can choose whether to be in this study or not. If you volunteer to be in this study, you may
withdraw at any time without consequences of any kind. You may exercise the option of
removing your data from the study. You may also refuse to answer any questions you don’t
want to answer and still remain in the study.
If, at any point during the study, new study-related information becomes available that might
change your willingness to continue participating in the study (i.e. new safety information, a
significant change in the study protocol, etc.), you will be provided with this information in a
timely manner. You can then choose whether to continue participating in this study or not.
The investigator may withdraw you from this research if circumstances arise that warrant doing
so. Some examples of reasons why your participation may be withdrawn include:
 There is reasonable concern about your continuation in the study.
 You miss an excessive number of study visits or doses of tea and/or study diary entries;
 You no longer meet the required study eligibility criteria;
RIGHTS OF RESEARCH PARTICIPANTS
You may withdraw your consent at any time and discontinue participation without penalty. You
are not waiving any legal claims, rights or remedies because of your participation in this research
study. This study has been reviewed and received ethics clearance through the University of
Guelph Research Ethics Board. If you have questions regarding your rights as a research
participant, contact:
Sandy Auld
Research Ethics Coordinator
Room 437, University Centre
University of Guelph
166
Telephone: (519) 824-4120, ext. 56606
E-email: [email protected]
SIGNATURE OF RESEARCH PARTICIPANT/LEGAL REPRESENTATIVE
I have read the information provided for the study “Human clinical trial to investigate the
effects of high rosmarinic acid spearmint tea on markers of pain, physical function and
disease activity in osteoarthritis of the knee” as described herein. My questions have been
answered to my satisfaction, and I agree to participate in this study. I have been given a copy of
this form.
NAME OF PARTICIPANT
(PLEASE PRINT)
SIGNATURE OF PARTICIPANT
DATE
NAME OF WITNESS
(PLEASE PRINT)
SIGNATURE OF WITNESS
DATE
167
Appendix 8. Recruitment poster
Healthy, non-smoking males and females (18 years and older) who have osteoarthritis of the
knee are needed to participate in a human nutrition study at the University of Guelph. This
research will study the health effects of spearmint tea on osteoarthritis.
 Interested individuals will be asked to:
 Complete a screening questionnaire by telephone or email.
 Visit the University of Guelph for one 1 ½ hour screening appointment to assess
eligibility for study.
If eligible to participate, individuals will be asked to:
 Visit the University of Guelph on 9 occasions over four months for:
 One 1 ½ hour study orientation visit.
 Two 2-hour study visits and two 1 ½ hour study visits
 Four of these study visits will involve a blood sample and assessments of
disease severity, pain and physical function.
 Participants may also choose to allow a medical doctor to draw a synovial
fluid sample from a knee joint at 2 of the study visits.
 Three 30-minute check-up visits and one 1 ½ hour follow-up visit.
 Consume 2 cups of mint tea per day for 4 months.
 Keep regular records of medication and dietary supplement use.
This study has been reviewed and has received clearance through the University of Guelph
Human Research Ethics Board (REB#11JA040) and will be conducted at the Human
Nutraceutical Research Unit in the Department of Human Health and Nutritional Sciences
*Financial Compensation Provided*
5
Tea Study, U of Guelph
Contact Erin at:
[email protected]
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
168
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Appendix 8. Telephone participant eligibility questionnaire
Appendix 8. Telephone participant eligibility questionnaire
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
Tea Study, U of Guelph
Contact Erin at:
[email protected]
519-824-4120 x56314
To find out more about the study and your suitability as a participant please contact Erin at 519824-4120 x56314 or [email protected]
Appendix 8. Telephone participant eligibility questionnaire
Appendix 9: Phone Screening Questionnaire
PHONE PRE-SCREENING PARTICIPANT ELIGIBILITY QUESTIONNAIRE
STUDY TITLE:
Human clinical trial to investigate the effects of high rosmarinic acid spearmint tea on
markers of pain, physical function and disease activity in osteoarthritis of the knee.
HNRU Coordinator:
Date:
Time:
Thank you for your interest in the mint tea study. I am going to provide you with some brief
details about the study to ensure your interest, and then ask you some questions to confirm that
you are eligible for the study. This will take approximately 10 minutes. Please feel free to ask me
questions at any time.
The purpose of the study is to test if daily consumption of a new spearmint tea for 4 months can
improve symptoms in adults with osteoarthritis of the knee. The new spearmint tea was
developed at the University of Guelph and has 15-20 times more rosmarinic acid than regular
spearmint. Rosmarinic acid is a plant extract that has been shown to have antioxidant and antiinflammatory effects. Approximately 50 individuals will be invited to participate in this study.
Half of the participants will be randomly assigned to consume the high rosmarinic acid
spearmint tea and the other half will be randomly assigned to consume a commercial spearmint
tea. This study will allow us to investigate if the high rosmarinic acid spearmint tea is an
effective complement to traditional osteoarthritis therapies.
If the participants asks: Study participants will be paid $200, plus $100 extra for participation
in synovial fluid sampling ($50 per sample provided). An additional $50 for completion of the
study will be given. The total reimbursement is therefore $250 WITHOUT synovial fluid
sampling and $350 WTIH synovial fluid sampling.
1. How did you hear about this study?
169
2. Do you have osteoarthritis in your knee?
YES
NO
LEFT
BOTH
6. Do you smoke?
YES
NO
7. Do you have ANY other types of arthritis or inflammatory
disease?
YES
NO
YES
NO
YES
NO
YES
NO
YES
NO
3. Which knee do you have osteoarthritis in?
RIGHT
4. When were you first diagnosed with osteoarthritis? (mm/yyyy)
5. Who diagnosed your osteoarthritis? (i.e. family doctor,
rheumatologist)
(a) If YES, please specify:
8. Do you have any other medical conditions?
(a) If YES, please specify:
9. Have you had a synovectomy in the past 3 months? (i.e. surgical
removal of inflamed joint tissue)
(a) If YES, when? (dd/mm/yyyy)
10. Are you currently taking any medications for osteoarthritis?
(a) If YES, please specify:
11. Are you currently taking any other medications including
prescription or any over the counter medications?
(a) If YES, please specify:
170
12. Are you currently taking any vitamins, minerals, herbal
medicines or dietary supplements?
YES
NO
14. Are you currently pregnant or planning on becoming pregnant?
YES
NO
15. Have you recently given birth?
YES
NO
16. Are you currently breastfeeding?
YES
NO
17. Do you have any specific allergies/hypersensitivity to mint?
YES
NO
18. Do you have any food or anaphylactic allergies?
YES
NO
(a) If YES, please specify:
13. What are you doing to manage your osteoarthritis?
Questions #14, #15 and #16 for females only
(a) If YES, please specify:
(b) If YES, please describe what type of allergic reactions you have experienced
(i.e. hive, rash, swelling, anaphylaxis):
19. Are you comfortable providing blood samples?
YES
NO
20. Have you ever provided a synovial fluid sample? (also called
arthrocentesis or joint aspiration, where a needle removes fluid
from the joint)
YES
NO
21. Participants will be required to visit the University of Guelph for
one 1 ½ hr screening appointment, one 1 ½ hr study orientation
visit, and if eligible to participate in the clinical trial, you will be
asked to attend 9 separate study visits over a 4 month period.
171
Study visits will range from 30 minutes to 2 hours and be
separated by at least 2 weeks, with flexibility in scheduling these
visits. Would your schedule accommodate these visits?
YES
NO
NB: If the individual meets the pre-screening eligibility requirements:
 Set up an in-person screening visit
 Enter the scheduled date and time of the screening visit into the Google calendar
 Fill out the Phone Screening Tracking Log
 Arrange to send the potential participant a map to the HNRU and provide parking
information
 Be sure to ask the potential participant to bring with them to the screening visit any
current medication and dietary supplements in their original packaging
If you are unsure whether the individual meets the pre-screening requirements, thank them for
their time and tell them that you will discuss their eligibility for the study with the other study
investigators and call them back by the end of the next business day.
Participant Name:
Screening ID:
Telephone #: Home -
Cell -
Email:
Preferred method of contact?
Date of Birth (dd/mm/yyyy):
Age:
In-person screening visit scheduled for:
DATE:
TIME:
COORDINATOR SIGNATURE:
172
Appendix 10. In-person participant eligibility questionnaire
IN-PERSON SCREENING PARTICIPANT ELIGIBILITY QUESTIONNAIRE
STUDY TITLE:
Human clinical trial to investigate the effects of high rosmarinic acid spearmint tea on
markers of pain, physical function and disease activity in osteoarthritis of the knee.
HNRU Coordinator:
Date:
Time:
Participant Name:
Eligibility ID:
Telephone #: Home -
Cell -
Email:
Preferred method of contact?
Date of Birth (dd/mm/yyyy):
Age:
Thank you very much for your interest in this study. The purpose of this questionnaire is to
gather more information about you and to ensure your safety as a participant in this study.
Please feel free to NOT answer any questions that you are uncomfortable with
answering.Please feel free to ask the study coordinator any questions you might have.
All information provided in this questionnaire will be kept strictly confidential.
173
HEALTH HISTORY QUESTIONNAIRE
1. When were you first diagnosed with osteoarthritis of the knee?
2. Who first diagnosed you with osteoarthritis of the knee?
(i.e. family doctor, rheumatologist)
3. (a) Which knee do you have osteoarthritis in?
BOTH
RIGHT
LEFT
RIGHT
LEFT
4. Have you ever had knee replacement surgery?
YES
NO
5. Are you planning on having knee replacement surgery between
now and February 2012?
YES
NO
6. Have you ever had a synovectomy before? (i.e. surgical removal
of inflamed joint tissue)
YES
NO
7. Do you currently smoke?
YES
NO
YES
NO
YES
NO
(b) Which knee is most problematic?
(a) If NO, have you ever smoked?
(b) If YES to (a), how long since you have quit?
8. Approximately how many alcoholic drinks do you consume per
week? (1 drink = 12 oz beer, 5 oz wine, or 1.5 oz hard liquor)
9. Are you currently participating in any other research studies?
(a) If YES, please describe:
10. How would you describe your general health?
POOR
GOOD
VERY GOOD
174
EXCELLENT
11. Do you have any of the following diagnosed medical conditions:
(a) Rheumatoid arthritis?
YES
NO
(b) Cancer?
YES
NO
(c) Pre-diabetes?
YES
NO
(d) Diabetes?
YES
NO
(e) High cholesterol?
YES
NO
(f) High blood pressure?
YES
NO
(g) Impaired liver function?
YES
NO
(h) Impaired kidney function?
YES
NO
(i) Gastrointestinal ulcer?
YES
NO
(j) Autoimmune disease?
YES
NO
(k) Chronic infection?
YES
NO
(l) Depression?
YES
NO
(m) Are there any other medical conditions that might affect
your participation in the study?
YES
NO
(i) If YES, please describe:
175
12. Are you currently taking any medication and/or over-the-counter
drugs? (i.e. Tylenol, Advil, Claritin, Sudafed, etc.)
YES
NO
(a) If YES, please provide the study coordinator with your current medication.
The study coordinator will help you complete the following table:
PRODUCT
NAME
REASON
FOR USE
MEDICINAL
INGREDIENTS
DOSE
13. Are you currently taking any dietary supplements?
(i.e. multivitamin, omega-3, glucosamine)
HOW HOW
OFTEN LONG
YES
NO
(a) If YES, please provide the study coordinator with your dietary supplements.
The study coordinator will help you complete the following table:
PRODUCT
NAME
REASON
FOR USE
ACTIVE
INGREDIENTS
176
DOSE
HOW HOW
OFTEN LONG
14. Are you currently taking any medication for your osteoarthritis?
YES
NO
(a) If YES, please provide the study coordinator with your current osteoarthritis
medication. The study coordinator will help you complete the following table:
PRODUCT
NAME
REASON
FOR USE
MEDICINAL
INGREDIENTS
DOSE
HOW HOW
OFTEN LONG
15. Do you see the following health professionals for your osteoarthritis:
(make sure it is clear that these are all related to osteoarthritis)
(a) Family doctor or
rheumatologist?
YES
NO
If YES, how often? ______________
(b) Physiotherapy?
YES
NO
If YES, how often? ______________
(c) Acupuncture?
YES
NO
If YES, how often? ______________
(d) Massage therapy?
YES
NO
If YES, how often? ______________
(e) Nerve stimulation?
YES
NO
If YES, how often? ______________
(f) Other? (please list)
YES
NO
How often? ____________________
How often? ____________________
How often? ____________________
177
16. Do you participate in any of the following types of exercises:
(a) Weight training?
YES
NO
If YES, how often? ______________
(b) Stretching?
YES
NO
If YES, how often? ______________
(c) Aerobics?
YES
NO
If YES, how often? ______________
(d) Underwater?
YES
NO
If YES, how often? ______________
(e) Other? (please list)
YES
NO
How often? ____________________
How often? ____________________
How often? ____________________
17. Are you satisfied with your current body weight?
YES
NO
18. Has you body weight changed in the past:
(a) 2 months?
YES
NO
If YES, how much? _____________
(b) 1 year?
YES
NO
If YES, how much? _____________
19. This study requires that participants maintain their body weight
for the 4-month duration of the study. Would you be
comfortable with this?
YES
NO
20. Do you have ANY allergies? (i.e. food, medications, rag weed or
pollen)
YES
NO
(a) If YES, please list:
178
(b) Please describe your type of allergic reaction: (i.e. anaphylaxis, difficulty
breathing, swelling, etc.)
21. Do you regularly consume fresh or dried mint, or mint products?
YES
NO
(a) If YES, how often?
(b) Please provide specific examples of mint products that you consume:
22. Have you ever had a reaction or hypersensitivity to mint or mint
products?
YES
NO
23. Do you consume fresh or dried herbs at an amount above typical flavoring levels for
the specific purpose of improving your general health:
(a) Basil?
YES
NO
(b) Rosemary?
YES
NO
(c) Sage?
YES
NO
(d) Marjoram?
YES
NO
(e) Oregano?
YES
NO
(f) Thyme?
YES
NO
(g) Lavender?
YES
NO
(h) Lemon balm?
YES
NO
(i) Fennel?
YES
NO
179
Question #24 to be completed by females only
Hormone levels can affect pain sensation and perception. Since pain is a large part of
osteoarthritis, we wish to gather some information about your hormone levels.
24. Are you PRE- or POST-menopausal?
(a) If POST-menopausal, do you use hormone replacement
therapy?
PRE
POST
YES
NO
YES
NO
(b) If YES to using hormone replacement therapy, please specify:
(c) If PRE-menopausal, do you have regular menstrual cycles?
(d) If NO to having regular menstrual cycles, please describe the irregularities:
(e) Do you currently use oral contraceptives (or other hormonal
birth control)?
YES
NO
(f) If YES to using oral contraceptives or hormonal birth control, please specify:
25. This study involves blood draws. Are you comfortable having
your blood taken for this study?
YES
NO
26. Are you a regular blood donor or have you donated blood in the
last two months?
YES
NO
27. Have you ever fainted from having your blood taken?
YES
NO
180
28. Have you ever provided a synovial fluid sample before? (Also
called arthrocentesis or joint aspiration, where a needle removes
fluid from the joint)
YES
NO
29. In this study, participants will be asked to consider providing a
synovial fluid sample. Participants may still continue to
participate in the study even if they choose not to do so. Would
you be comfortable having a doctor take a synovial fluid
sample for this study?
YES
NO
30. This study involves a 3-day diet history. Would you be
comfortable completing a detailed record of all the foods you
have eaten over a 3-day period?
YES
NO
31. This study requires that participants DO NOT consume any
herbal teas (other than provided by the study team) for the 4month duration of the study. Would you be comfortable with
this?
YES
NO
32. This study requires that participants continue their usual dietary
supplement routine (i.e. dosage and frequency) for the 4-month
duration of the study. Would you be comfortable with this?
YES
NO
33. This study requires that participants keep a daily record of ALL
medications and dietary supplements used (i.e. dosage and
frequency) for the 4-month duration of the study and for a 1month follow-up period. Would you be comfortable with this?
YES
NO
34. This study requires that before four of the study visits (once a
month), participants DO NOT participate in any complementary
therapies (i.e. physiotherapy, massage therapy, acupuncture,
nerve stimulation) or take any anti-inflammatory medications for
2 days (48 hours). Would you be comfortable with this?
YES
NO
35. This study requires 9 study visits to the HNRU at the University
of Guelph as follows:

Three ½ hour visits

Three 1 ½ hour visits

Two 2 hour visits

Possibly 2 additional visits, both less than ½ hour
Study visits will be separated by at least 2 weeks and there is
flexibility in their scheduling.
181
(a) Can your schedule accommodate these visits?
YES
NO
(b) Are there any particular days of the week that you would
prefer the study visits?
YES
NO
MONDAY
TUESDAY
WEDNESDAY
THURSDAY
FRIDAY
(c) Is there a particular time of day that you would prefer?
MORNING
AFTERNOON
NO PREFERENCE
________________________________
HNRU Coordinator Signature
NB: Provide the WOMAC to the participant and have them complete the form.
Complete the 24-hr food recall with the participant.
Provide the participant with a coffee voucher.
Fill out the In-person Screening Tracking Log.
182
Appendix 11. Participant tea brewing instructions
183
Appendix 12. Example study diary sheet
FEBRUARY 17, 2012 - STUDY REMINDERS
Week: 3 ◊ Thank
you for your time & participation in our study!
Study Day: 21◊ Questions about the
study? Contact our Team!
Mint Tea
Cup1 Cup2
Comments (e.g. missed tea, changes in preparation, etc.)
AM
PM
Time
Over-the-Counter, Medications/Dietary Supplements/Natural Health Products
Taken Comments (e.g. missed supplements, extra
Product Name
Quantiy Yes No over-the-counter pain medication, etc.)
Prescribed Medications
Product Name
Taken Comments (e.g. missed prescribed
Quantiy Yes No medication, started new prescription, etc.)
Other Comments (e.g. unusual circumstances, injuries or illness)
184
Appendix 13. Anthropometric case report form
HNRU CASE REPORT FORM
STUDY VISIT #1: BASELINE
Date of Visit: ________________
Date of Birth: _____________
Age: ______
ANTHROPOMETRIC MEASUREMENTS
(A) Body Height
Did not complete height
Reason:
(1) Body height: _______________ cm
(B) Body Weight
Did not complete weight
Reason:
(2) Body weight: ________________kg
C) Body Mass Index (BMI)
____________________
Did not complete BMI
Reason:
(3) Body mass index (BMI): [Weight (kg)] = _________________ kg/m2
[Height (m)]2
(D) Waist Circumference
____________________
Did not complete waist
Trial #1: ________________ cm
Reason:
Trial #2: __________________ cm
(4) Waist circumference: _____________________ cm
(E) Hip Circumference
Did not complete hip
Reason: _
Trial #1: _______________ cm
Trial #2: ________________ cm
(5) Hip circumference: ____________________
cm
HNRU Study Coordinator Initials: _____
185
HNRU CASE REPORT FORM
STUDY VISIT #1: BASELINE
(F) Blood Pressure (BP)
____________________
Did not complete BP
Reason:
Trial #1 (Diastolic / Systolic): ________________mm/Hg Trial #1 (Pulse): _______ bpm
Trial #2 (Diastolic / Systolic): _________________mm/Hg Trial #2 (Pulse): _______ bpm
(6) Blood pressure (Diastolic / Systolic): ___________________mm/Hg
(7) Pulse: ______ bpm
(G) Body Composition (BIA)
___________________
BIA Test Number: _____________
Did not complete BIA
Reason:
Did participant void bladder? _______________
(8) BIA Body fat: ________________ %
(9) BIA Lean mass: ______________ %
BIOLOGICAL SAMPLE ANALYSES
(L) C-Reactive Protein (CRP) Analysis
Did not complete CRP
Reason:
Time of collection: ______________ am / pm
(10) C-reactive protein (CRP): ______________ g/ml
HNRU Study Coordinator Initials: _____
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Appendix 14. Physical function test case report form
HNRU CASE REPORT FORM
STUDY VISIT #1: BASELINE
Date of Visit: _____________
PHYSICAL FUNCTION TESTS
(A) Stair Climbing Task (SCT)
Did not complete STR
Reason:
1. Ensure participant is wearing comfortable clothes and shoes.
2. Use of walking aids (i.e. cane, walker) is permitted during task administration.
3. Give the following instructions:
The object of this task is to climb up 7 stairs as quickly and as safely as you can. Please
begin by standing at the bottom of the stairs with your left hand on the handrail and your
toes on the line on the floor. At the word ‘go’, please climb the stairs to the top so that both
feet are on the top platform. Without any hesitation, turn around and climb down using the
same handrail. You must use the handrail and climb only one step at a time. Now I’m going
to show you. Please watch the way I turn without hesitation. Demonstrate by climbing one
flight. Are you ready to do that? Remember that the object is to climb AS QUICKLY AS
POSSIBLE, but don’t skip any steps.
4. Allow the participant to practice one flight.
5. Position the participant at the start line. Say GO and begin the timer as soon as they go.
Stop the timer once both feet are back on the floor at the bottom of the stairs.
6. Record the use of aids and the stepping pattern.
7. Record the time to complete the task.
(5) Did the participant use a walking aid (i.e. walker, cane, etc.)?
Yes
No
(6) Did the participant use a foot-over-foot stepping pattern?
Yes
No
(7) Did the participant use a foot-beside-foot stepping pattern?
Yes
No
(8) Time to complete: ____________ s
HNRU Study Coordinator Initials: ____
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(A) 6-Minute Walk Test (6MWT) Did not complete 6MWT
Reason:
(1) Measured track distance: ___________ m
Do not administer task if SBP >180 and DBP > 100 mmHg.
Use of walking aids (i.e. cane, walker) is permitted during task administration.
Give the following instructions:
The object of this task is to walk as far as possible for 6 minutes. You will walk back
and forth in this hallway. 6 minutes is a long time to walk, so you will be exerting yourself.
You may get out of breath or become exhausted. You are permitted to slow down, to stop,
and to rest as necessary. You may lean against the wall while resting, but resume walking
as soon as you are able. You will be walking back and forth around the cones. You should
pivot briskly around the cones and continue back the other way without hesitation. Now
I’m going to show you. Please watch the way I turn without hesitation. Demonstrate by
walking one lap. Are you ready to do that? Remember that the object is to walk AS FAR AS
POSSIBLE for 6 minutes, but don’t run or jog.
4. Allow the participant to practice one lap.
5. Position the participant at the start line. Say GO and begin the countdown timer as soon
as they go.
6. Do not walk with the participant. You may choose to follow at a distance behind them.
7. Provide standard reinforcement (no other words or body language) at 1 minute intervals
as follows:
 5 min left: You are doing well. You have 5 minutes to go.
 4 min left: Keep up the good work. You have 4 minutes to go.
 3 min left: You are doing well. You are halfway done.
 2 min left: Keep up the good work. You have only 2 minutes to go.
 1 min left: You are doing well. You have only 1 minute to go.
 15 sec left: In a moment I’m going to tell you to stop. When I do, just stop
right where you are and I will come to you.
8. If the participant stops; You can lean against the wall if you would like, then continue
walking whenever you feel able. If necessary, provide a wheelchair for them to sit in.
1.
2.
3.
(2) Did the participant use a walking aid (i.e. walker, cane, etc.)Yes
(3) Did the participant stop to rest?
Yes
(3a) If yes, how many times did the participant stop?
(4) Check off the number of laps completed
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_________
No
No
(5) Total distance: # of laps _________(x ________m) +________ partial = _______m
HNRU Study Coordinator Initials: _____
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Appendix 15. Biomarker results from clinical trial
Table 15.2 Pooled Biomarker results in Week 0 and Week 16 analyzed with student’s t-test
(SAS 9.2)
Biomarker
Week 0
Week 16
p-value
HA (ng/mL)
1.86
1.90
0.56
MMP-3 (ng/mL)
1.13
1.17
0.47
PIIANP (ng/mL)
3.34
3.33
0.79
COMP (ng/mL)
1.47
1.49
0.64
IL-18 (pg/mL)
1.75
1.75
0.90
1.32
1.32
0.92
NO (mol/L)
CRP (mg/L)
0.41
0.40
0.96
COMP = cartilage oligomeric matrix protein; CRP = C-reactive protein; HA = hyaluronic acid;
IL-6 = interleukin-6; IL-18 = interleukin-18; MMP-3 = matrix metalloproteinase -3; NO = nitric
oxide; PIIANP = type-IIA collagen N-propeptide.
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