Osteoarthritis and Cartilage 28 (2020) 356e362

Molecular and imaging biomarkers of local inflammation at 2 years

after anterior cruciate ligament injury do not associate with patient
reported outcomes at 5 years
A. Struglics y *, A. Turkiewicz z, S. Larsson y, L.S. Lohmander y, F.W. Roemer y x k,
R. Frobell y, M. Englund z
y Orthopaedics, Department of Clinical Sciences Lund, Lund University, Lund, Sweden
z Clinical Epidemiology Unit, Orthopaedics, Department of Clinical Sciences Lund, Lund University, Lund, Sweden
x Quantitative Imaging Center (QIC), Department of Radiology, Boston University School of Medicine, Boston, MA, USA
k Department of Radiology, University of Erlangen-Nuremberg, Erlangen, Germany

a r t i c l e i n f o s u m m a r y

Article history: Objective: To estimate the association between molecular or imaging inflammatory biomarkers at 2 years
Received 5 June 2019 after anterior cruciate ligament (ACL) injury and patient-reported outcomes at 5 years.
Accepted 19 December 2019 Methods: For 116 ACL-injured patients, molecular biomarkers of inflammation (synovial fluid and serum
cytokines) and Hoffa- and effusion-synovitis as visualized on magnetic resonance imaging (MRI) were
Keywords: assessed 2 years post-injury. Knee injury and Osteoarthritis Outcome Score (KOOS) and SF-36 were
Anterior cruciate ligament injury
assessed at 2 and 5 years. We used multiple imputation to handle biomarker values that were below the
Biomarkers
level of detection or missing, and linear regression for statistical analyses.
Inflammation
Synovitis
Results: None of the synovial fluid cytokines or imaging biomarkers of inflammation at 2 years were
Patient reported outcomes associated with any of the patient-reported outcomes at 5 years. With each log10 unit higher of serum
tumor necrosis factor concentration the knee-related quality of life of KOOS was increased (i.e., better
outcome) by 35 (95% confidence interval 7 to 63) points.
No other serum biomarker measured at 2 years was associated with patient-reported outcome at 5 years.
Conclusion: Local joint inflammation assessed by biomarkers in synovial fluid and Hoffa- and effusion-
synovitis on MRI at 2 years after an ACL injury did not associate with patient-reported outcomes at 5
years. Thus, chronic inflammation in the ACL-injured knee, as reflected by the biomarkers studied here,
seems not to be a key determinant for the long-term patient-reported outcomes.
© 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Introduction Cytokines related to inflammation such as tumor necrosis factor

Injury to the anterior cruciate ligament (ACL) is associated with
increased risk for the development of knee osteoarthritis (OA).
Some 10e20 years after an ACL injury between 10% and 90% of the
patients develop knee OA1,2. The mechanisms involved are thought
to be mainly due to the acute soft tissue and/or osteochondral
injury and chronic effects of altered biomechanics of the knee.
However, acute or chronic local inflammation elicited by the
trauma or by changes in biomechanics may also be important3e6.
* Address correspondence and reprint requests to: A. Struglics, Lund University,
Faculty of Medicine, Department of Clinical Sciences Lund, Orthopaedics, BMC C12,
Klinikgatan 28, SE-221 84, Lund, Sweden. Tel: 46-46-222-0762.
E-mail address: andre.struglics@med.lu.se (A. Struglics).
(TNF), interleukin (IL)-1b, IL-6, and interferon (IFN)-g in serum and
synovial fluid in OA patients have been reported to be associated
with joint pain7e10. Two studies have reported on the association
between markers of inflammation and patient-reported outcomes
(PROs; i.e., self-assessed health) in knee-injured patients11,12. Thus,
the association between synovitis or inflammation-related mole-
cules and PROs is not well understood. ACL injury is one of the most
frequently used human models to study the development and
progression of knee OA, and anti-inflammatory treatment in the
early stage is currently under evaluation as a treatment strategy to
lower the risk of post-traumatic OA13.
Our aim was to evaluate associations between local inflamma-
tion in the knee joint, indicated by molecular or imaging bio-
markers, 2 years after ACL injury with PROs at the 5-year follow-up.

https://doi.org/10.1016/j.joca.2019.12.010
1063-4584/© 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
 A. Struglics et al. / Osteoarthritis and Cartilage 28 (2020) 356e362 357

As a secondary aim, we assessed the cross-sectional association
between molecular and imaging biomarkers of inflammation with
PROs at 2 years after the ACL injury. Since local inflammation
rapidly changes in the acute knee trauma phase, due to severity of
injury and the variable time between injury and aspiration5, we
chose to analyze the 2-year exposure data as our aim was to capture
patients in a more stable phase of prolonged (chronic) inflamma-
tion, which we hypothesized to be a potential risk factor for worse
long-term patient-relevant outcomes.
quantification (LLOQ)5. Accordingly, only cytokines (here termed
molecular biomarkers) with more than 20% of the values above
LLOQ were accepted for analysis: i.e., synovial fluid IL-6, IL-8, IL-10
and TNF, serum IL-6, IL-8, IL-10, IL-12p70, IFN-g and TNF (Table I).
The cytokine data, as a marker of inflammation, were used in a
previous study for the prediction of structural knee OA at 5 years16.
Magnetic resonance imaging (MRI) protocol and assessment

At all time points, knee magnetic resonance imaging was per-

Materials and methods
Subjects
We used data from the randomised controlled Knee ACL,
NONsurgical vs surgical (KANON) trial (ISRCTN 84752559)
comparing a surgical and a non-surgical treatment strategy of acute
ACL injuries including 121 young and active individuals with an
acute ACL injury to a previously uninjured knee14,15. The study was
approved by the regional (Lund) ethical review board.
Inflammatory biomarkers in synovial fluid and serum
Synovial fluid and serum levels of IL-1b, IL-6, IL-8, IL-10, IL-
12p70, IFN-g, and TNF were assessed in the KANON samples using a
multiplex Human Pro-inflammatory 7-plex immunoassay5. At the
2-year follow up, cytokine data from 119 serum and 81 synovial
fluid samples were available; a large proportion of these samples
had cytokine concentrations below the lower limits of
formed using a 1.5 T system (Gyroscan Intera, Philips, Best, The
Netherlands) with a circular polarised surface coil using identical
sequences for all subjects. The MRI sequence protocol has been
described in detail17. The 2-year follow-up MRIs were assessed
according to the ACL OsteoArthritis Score (ACLOAS) instrument by
one musculoskeletal radiologist (FWR)18. ACLOAS is based on non-
enhanced MRI and includes assessment of inflammation of whole-
joint synovitis based on signal changes in Hoffa's fat pad (Hoffa-
synovitis) and a composite measure of the amount of intraarticular
joint fluid and synovial tissue thickening on fluid sensitive images
(effusion-synovitis). Both features are graded 0e3 depending on
the amount of signal alterations in Hoffa's fat pad or in terms of the
estimated maximal distention of the synovial cavity. Reliability data
using the ACLOAS instrument have been published18,19. At the 2-
year follow up, 119 subjects (i.e., 98% of the subjects at baseline) had
available Hoffa- and effusion-synovitis scores (here termed imag-
ing biomarkers of inflammation). Of the original 121 subjects, 116
(96%) also had molecular biomarker and PRO data at 2 years. The
presence of synovitis was stratified into Hoffa grades 0, 1 and  2,

Number of subjects Number of samples Samples with values above LLOQ

Total N N N (%) Concentration

Cytokines
sfIL-6 116 81 53 (65) 2.44 (0.96, 8.56)
sfIL-8 116 81 81 (100) 15.76 (11.40, 26.29)
sfIL-10 116 81 25 (31) 3.00 (2.43, 4.07)
sfTNF 116 81 76 (94) 4.07 (3.41, 4.90)
sIL-6 116 116 25 (22) 2.23 (1.58, 4.04)
sIL-8 116 116 116 (100) 7.71 (6.19, 10.52)
sIL-10 116 116 51 (44) 5.89 (4.13, 10.84)
sIL-12p70 116 116 31 (27) 5.69 (3.23, 32.00)
sIFN-g 116 116 24 (21) 3.65 (1.96, 6.80)
sTNF 116 116 116 (100) 10.86 (9.24, 13.03)
Synovitis
Hoffa grades
0, n 1, n  2, n
Grade 0 23 e e
1 e 59 e
2 e e 31
3 e e 3
Total N (%) 23 (20) 59 (51) 34 (29)
Effusion grades
0, n  1, n
Grade 0 81 e
1 e 26
2 e 5
3 e 4
Total N (%) 81 (70) 35 (30)

LLOQ ¼ lower limit of quantification.

Inflammation related cytokines and Hoffa- and effusion-synovitis at 2 years after ACL injury. Con-
Table I centrations (pg/ml) in synovial fluid (sf) and serum (s) are expressed as median values with 25th and 75th Osteoarthritis
percentiles in parenthesis andCartilage
358 A. Struglics et al. / Osteoarthritis and Cartilage 28 (2020) 356e362

and into effusion grades 0 and  1 (Table I). The data from these
imaging biomarkers of inflammation were used in a previous study
for the prediction of structural knee OA at 5 years16.
Patient-reported outcome measures
PRO measures included the Knee injury and Osteoarthritis
Outcome Score (KOOS)20,21 and the Medical Outcomes Study 36-
item short-form health survey (SF-36) divided into mental (MCS)
and physical (PCS) component summaries22. The outcome scores
for KOOS (0e100 scale, worst to best) and SF-36 (0e100 scale,
worst to best) for the 2 and 5-year follow-ups of the KANON study
have been reported14,15.
variables was used as the imputation model. For the values below
LLOQ, the limit for the interval censoring was set to the LLOQ of
respective biomarker to inform the model on the censoring
mechanism. All biomarkers were included in one imputation
model, together with the values of the respective PRO at both 2 and
5 years, imaging biomarkers of inflammation and age and sex to
preserve multivariable associations. In a sensitivity analysis, we
repeated the main analysis on complete data, i.e., excluding persons
with missing biomarker values.
All statistical analyses were done using IBM SPSS Statistics
(version 24) and Stata 15, StataCorp. 2017; Stata Statistical Software
Release 15, College Station, TX; StataCorp LP.

Statistics Results

For the present study we included 116 patients who had mo-
lecular and imaging biomarkers of inflammation as well as PROs
measures at 2 years after injury, and who had PROs reported at the
5-year follow-up (Table II).
We used linear regression to evaluate the association between
molecular or imaging biomarkers of inflammation and PROs (out-
comes at 5 years) and a separate model was fit for each PRO. We
used two sets of regression models: multivariable, all biomarkers
were included in one model (mutually adjusted for each other);
univariable, each biomarker was evaluated in a separate model.
Additionally, we adjusted for age and sex, and for the 2-year value
of the respective PRO. We present the estimates with 95% confi-
dence intervals (CI).
Missing values of the molecular biomarkers: i.e., a biological
sample was obtained but the value was below lower limit of
quantification (LLOQ) or there was no biological sample available
(only the case for synovial fluid samples), were imputed using a
multiple imputation approach. For each PRO (the outcome) we
created 30 imputed datasets using the fully conditional specifica-
tion, i.e., chained equations23,24. To achieve normally distributed
residuals the molecular biomarkers were log10 transformed; in-
terval regression for a continuous partially observed (censored)
Demographics and descriptive data
At the 2-year follow-up, included subjects (N ¼ 116) had a mean
age of 28.2 years (SD 4.9) and 30 (26%) were women. Eighty-one of
these subjects had synovial fluid available for analysis; the main
reasons for not contributing with synovial fluid at the 2 year visit
was due to either “dry” knees or refusal to undergo aspiration due
to experiences of discomfort during the previous aspirations. Be-
tween 31% and 100% of the synovial fluid and between 21% and
100% of the serum samples had biomarker concentrations above
LLOQ (Table I). At 2 years post injury, 20% of the subjects had no
signs of Hoffa-synovitis (grade 0) and 29% of the subjects had signs
of inflammation based on Hoffa grades 2 or 3; 70% of the patients
had no signs of effusion-synovitis at 2 years (grade 0) and 8% (n ¼ 9)
had signs of inflammation based on effusion-synovitis grades 2 or 3
(Table I). Combining Hoffa- and effusion-synovitis, 50 subjects
(43%) had MRI defined inflammation (based on sum grades  2) at
the 2-year follow up (detailed data not shown).
At 2 and 5 years post injury the means of the five subscales of
KOOS were between 65 and 94, and between 69 and 96, respec-
tively; the means of SF-36 subscales were between 80 and 86, and
between 84 and 86, respectively (Table II). Due to low variation in
the outcomes of activity of daily living (ADL) and MCS (i.e., almost
all participants scoring maximum) we did not analyse these out-
comes any further.

KOOS 2 years, 5 years, Association between biomarkers at 2 years and PROs at 5 years
mean (SD) mean (SD)
(longitudinal analysis)
Pain 87.1 (15.2) 90.9 (11.9)
Other symptoms 80.4 (19.6) 84.1 (16.1)
Plotting the data suggested no apparent association between
Activities of daily living 93.9 (10.8) 95.9 (8.0)
Sport and recreation 70.8 (27.6) 77.1 (23.4)
molecular biomarkers at 2 years and PROs at 5 years (Fig. S1). The
Knee related QOL 65.0 (23.8) 69.4 (22.2) multivariable adjusted model (our main model) indicated that
KOOS4 75.8 (20.0) 80.4 (16.4) higher serum levels of log10 TNF at the 2-year follow-up were
SF-36 associated with better outcome at the 5-year follow-up for the
2 years, 5 years, KOOS subscale of knee-related quality of life (QOL) (mean effect of
mean (SD) mean (SD) 35.0, 95% CI 6.9 to 63.2; Table III). No other imaging or molecular
PCS 79.6 (19.1) 84.5 (16.1) biomarker in serum or synovial fluid was associated with PRO
MCS 85.7 (14.8) 85.8 (15.4) outcomes at 5 years. The univariable models (unadjusted or
KOOS ¼ knee injury and osteoarthritis outcome score (ranges from 0 to 100, with adjusted) yielded similar results, with the addition of associations
higher scores indicating better results). KOOS4 ¼ includes four KOOS subscales: between serum TNF and KOOS-pain and KOOS4, and between
pain, other symptoms, function in sports and recreation, and knee related quality serum IL-10 and knee-related QOL (Table IV, Table S1). In a sensi-
of life (QOL) scores. SF-36 ¼ Medical Outcomes Study 36-itmes short-form health
tivity analysis using only non-missing data, the results were
survey. MCS ¼ mental component summary; PCS ¼ physical component sum-
mary. SD ¼ standard deviation. essentially the same (Table S2).
Of the imaging biomarkers of inflammation, in the univariable
Patient reported outcomes model, those with effusion-synovitis had worse values of SF-36 PCS
Table II at 2 and 5 years after ACL Osteoarthritis (mean effect of 8.5, 95% CI -14.8 to 2.2; Table IV). None of the
injury (n ¼ 116) andCartilage other imaging biomarkers for inflammation was associated with
the PROs at 5 years (Tables III and IV, Table S1).
 A. Struglics et al. / Osteoarthritis and Cartilage 28 (2020) 356e362 359

Synovial fluid variables KOOS pain KOOS other symptoms KOOS sport/rec KOOS QOL KOOS4 SF-36 PCS

log10 sfIL-6 2.1 (5.7, 1.5) 3.3 (7.6, 1.1) 5.0 (11.6, 1.6) 0.1 (6.7, 6.9) 2.0 (6.7, 2.8) 0.2 (4.6, 4.2)
log10 sfIL-8 0.7 (11.8, 10.4) 2.9 (17.3, 11.5) 1.9 (20.6, 24.5) 7.5 (31.9, 17.0) 3.6 (19.5, 12.3) 8.7 (23.1, 5.6)
log10 sfIL-10 1.4 (12.1, 14.9) 5.3 (12.2, 22.9) 13.2 (12.7, 39.0) 0.7 (26.9, 28.3) 4.9 (13.4, 23.2) 1.3 (16.4, 18.9)
log10 sfTNF 7.1 (12.3, 26.6) 1.3 (24.8, 22.3) 0.5 (38.0, 36.9) 19.7 (19.1, 58.5) 7.8 (16.8, 32.4) 11.6 (11.4, 34.6)
Hoffa grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Hoffa grade 1 2.2 (2.5, 6.9) 0.8 (7.2, 5.7) 4.1 (5.9, 14.1) 2.1 (7.0, 11.1) 1.8 (4.6, 8.2) 1.7 (5.1, 8.5)
Hoffa grades  2 1.5 (4.0, 6.9) 3.1 (10.4, 4.2) 4.0 (7.5, 15.6) 6.0 (4.5, 16.5) 2.3 (5.1, 9.7) 3.7 (4.1, 11.5)
Effusion grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Effusion grades  1 3.7 (0.6, 8.0) 5.5 (0.6, 11.5) 3.2 (5.6, 11.9) 0.8 (7.6, 9.1) 3.0 (2.8, 8.8) 2.2 (8.3, 3.9)

Serum variables1 KOOS pain KOOS other symptoms KOOS sport KOOS QOL KOOS4 SF-36 PCS

log10 sIL-6 0.5 (2.6, 3.6) 1.2 (3.2, 5.7) 4.5 (3.0, 12.1) 0.5 (6.7, 5.8) 1.7 (3.5, 6.9) 2.8 (1.7, 7.4)
log10 sIL-8 0.3 (8.6, 9.2) 0.8 (13.9, 12.3) 6.2 (25.0, 12.6) 3.8 (20.8, 13.2) 2.4 (15.1, 10.3) 2.3 (15.2, 10.7)
log10 sIL-10 0.5 (2.0, 3.1) 1.5 (5.0, 1.9) 2.0 (3.7, 7.7) 4.0 (0.6, 8.6) 1.0 (2.8, 4.7) 1.9 (1.6, 5.5)
log10 sTNF 14.1 (0.2, 28.3) 6.1 (14.8, 27.0) 10.8 (18.7, 40.2) 35.0 (6.9, 63.2) 16.0 (4.5, 36.4) 0.7 (21.4, 20.0)
Hoffa grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Hoffa grade 1 0.9 (3.6, 5.5) 2.4 (8.8, 4.0) 0.4 (9.8, 9.1) 0.2 (8.2, 8.6) 0.3 (6.4, 5.9) 0.3 (6.4, 7.0)
Hoffa grades  2 0.4 (4.5, 5.4) 3.7 (10.7, 3.3) 0.6 (9.8, 11.0) 3.2 (6.0, 12.4) 0.5 (6.3, 7.3) 1.8 (5.5, 9.1)
Effusion grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Effusion grades  1 2.3 (1.7, 6.2) 3.1 (2.6, 8.8) 3.2 (5.0, 11.4) 0.7 (8.1, 6.7) 2.0 (3.5, 7.4) 1.8 (7.8, 4.1)

1) The model did not converge for serum IL-12p70 and IFN-g. PCS ¼ physical component summary; sport/rec ¼ function in sport and recreation; QOL ¼ knee related quality
of life; KOOS4 ¼ average of pain, other symptoms, sport/rec and QOL; s ¼ serum; sf ¼ synovial fluid. SF-36 ¼ Medical Outcomes Study 36-itmes short-form health survey.

Longitudinal comparison between biomarkers and PROs using multivariable adjusted linear regression
model with multiple imputation of missing values. Molecular and imaging biomarkers of inflammation at
Table III 2-year were analyzed against PROs at 5 years after injury. The results are expressed as mean effects Osteoarthritis
(95% confidence intervals). Boldface indicates statistically significant differences. Estimates are given andCartilage
per 1 unit of either log10 molecular biomarker values (in pg/ml) or synovitis grades. Sample size ¼ 116

Variables1 KOOS pain KOOS other symptoms KOOS sport/rec KOOS QOL KOOS4 SF-36 PCS

log10 sfIL-6 1.9 (4.5, 0.6) ¡4.4 (-8.0, -0.9) 3.7 (8.8, 1.4) 1.6 (7.2, 4.0) 2.8 (6.6, 1.1) 2.2 (5.9, 1.4)
log10 sfIL-8 0.3 (8.7, 8.2) 6.1 (17.3, 5.0) 0.5 (16.1, 17.2) 0.1 (17.6, 17.4) 1.7 (12.8, 9.4) 2.4 (13.4, 8.6)
log10 sfIL-10 0.2 (10.4, 10.9) 6.7 (18.7, 5.4) 1.6 (20.5, 17.4) 6.9 (26.6, 12.8) 4.6 (17.9, 8.7) 0.4 (12.1, 13.0)
log10 sfTNF 6.4 (9.1, 21.8) 3.9 (24.0, 16.1) 8.0 (24.2, 40.2) 7.2 (23.5, 37.8) 5.2 (15.0, 25.5) 0.5 (18.2, 19.3)
log10 sIL-6 1.6 (1.5, 4.6) 2.3 (1.7, 6.4) 5.5 (1.4, 12.4) 4.1 (2.3, 10.4) 3.5 (1.4, 8.4) 2.8 (1.7, 7.3)
log10 sIL-8 0.7 (10.2, 11.6) 3.8 (18.6, 10.9) 10.0 (31.5, 11.4) 1.7 (18.7, 22.1) 2.9 (17.9, 12.2) 1.9 (16.7, 12.9)
log10 sIL-10 0.8 (2.0, 3.7) 0.0 (4.1, 4.2) 4.2 (1.9, 10.4) 5.8 (0.6, 11.1) 2.5 (1.6, 6.6) 2.0 (2.0, 6.0)
log10 sTNF 19.3 (3.7, 34.9) 12.8 (8.7, 34.4) 27.5 (3.5, 58.6) 43.5 (14.8, 72.2) 25.8 (4.3, 47.3) 7.0 (14.7, 28.6)
Hoffa grade 0 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0)
Hoffa grade 1 1.0 (6.8, 4.8) 4.1 (11.9, 3.7) 2.9 (14.3, 8.6) 1.2 (12.1, 9.6) 2.3 (10.3, 5.7) 3.0 (10.8, 4.9)
Hoffa grades  2 3.1 (9.4, 3.3) 8.4 (17.0, 0.1) 6.3 (18.9, 6.3) 1.8 (10.2, 13.7) 4.0 (12.8, 4.8) 3.5 (12.2, 5.1)
Effusion 0 0.0 0.0 0.0 0.0 0.0 0.0
Effusion grades  1 2.6 (7.3, 2.2) 4.4 (10.9, 2.0) 5.1 (14.5, 4.3) 6.6 (15.5, 2.2) 4.7 (11.2, 1.8) ¡8.5 (-14.8, -2.2)

1) The model did not converge for serum IL-12p70 and IFN-g. PCS ¼ physical component summary; sport/rec ¼ function in sport and recreation; QOL ¼ knee related quality
of life; KOOS4 ¼ average of pain, other symptoms, sport/rec and QOL; s ¼ serum; sf ¼ synovial fluid; SF-36 ¼ Medical Outcomes Study 36-itmes short-form health survey.

Longitudinal comparison between biomarkers and PROs using univariable unadjusted linear regression
model with multiple imputation of missing values. Molecular and imaging biomarkers of inflammation at
Table IV 2-year were analyzed against PROs at 5 years after injury. The results are expressed as mean effects Osteoarthritis
(95% confidence intervals). Boldface indicates statistically significant differences. Estimates are given andCartilage
per 1 unit of either log10 molecular biomarker values (in pg/ml) or synovitis grades. Sample size ¼ 116

Association between biomarkers and PROs at 2 years (cross-
sectional analysis)
To provide a more comprehensive picture of our baseline data,
we also investigated the biomarker associations with PROs at 2
years after injury. In the multivariable adjusted model, synovial
fluid levels of IL-8 were associated with better outcome for SF-
36 PCS (mean effect of 22.3, 95% CI 3.1 to 41.5; Table V). In the
sensitivity analysis (data not shown) and in the univariable unad-
justed model (Table S3) there was no association between synovial
fluid levels of IL-8 and any of the PROs.
Of the imaging biomarkers of inflammation, in multivariable
adjusted model, those with effusion-synovitis (grades  1) had
worse values of KOOS subscales (pain, other symptoms, knee
360 A. Struglics et al. / Osteoarthritis and Cartilage 28 (2020) 356e362

related QOL and KOOS4) and of SF-36 PCS (Table V). The univariable
adjusted (data not shown) and unadjusted (Table S3) models
including missing values yielded similar results. None of the other
imaging biomarkers of inflammation were associated with the
PROs at 2 years (Table V, Table S3).
Discussion
In this prospective cohort of ACL-injured patients, selected
molecular biomarkers of inflammation from synovial fluid and
MRI-defined Hoffa- and effusion-synovitis assessed at 2 years were
generally not associated with any of the PROs at 5 years. Of the
inflammation biomarkers in serum that we evaluated at 2 years,
only higher TNF concentration was associated with better KOOS
outcomes (i.e., pain, QOL and KOOS4) at the 5 year follow up. Also,
in the cross-sectional analysis we found that the presence of effu-
sion synovitis at 2 years after injury may be associated with worse
outcomes of several of KOOS subscales and SF-36 PCS at the same
time point.
We hypothesized that there would be a longitudinal association
between the markers of local knee joint inflammation and the
knee-specific KOOS outcomes, in that pro-inflammatory mediators
may activate nociceptors which over time may lead to chronic
pain25. However, there were essentially no such associations. Of
further note was the poor cross-sectional concordance between the
intra-articular molecular and imaging biomarkers of inflammation
at 2 years in this cohort, making it unclear what processes these
markers are a proxy for, as previously commented16.
Several reports indicate that in OA, high levels of TNF and IL-6 in
synovial fluid and IL-6 in serum are associated with worse pain and
function8,9,26. Longitudinal studies of knee OA patients have further
suggested that high synovial fluid levels of TNF and IL-6, just prior
to knee arthroplasty, were associated with less pain-improvement
2 years later7. How molecular biomarkers, specifically pro-inflam-
matory cytokines and MRI-defined synovitis, are associated with
PROs for knee-injured patients is less clear. ACL-injured patients,
which at the time of ACL reconstruction had high synovial fluid
levels of IL-1a, failed to achieve patient accepted symptom state of
KOOS subscale QOL 2 years after surgery11. For meniscectomized
patients, we reported that higher synovial fluid concentrations of
TNF were associated with subsequent worsening (over period of in
average 7.5 years) of the KOOS subscale ADL27. On the other hand, in
the shorter perspective of a 3-month period after injury, Watt and
co-workers found that higher synovial fluid levels of IL-6 were
associated with greater improvement of KOOS4 in knee-injured
patients12. Somewhat intriguing, but in the same line as the Watt
et al. paper, we found indications that higher serum TNF levels at 2
years may be associated with better outcome for some of the KOOS
subscales at 5 years. However, the confidence intervals were wide
suggesting uncertainty of the finding, in particular as prior reports
have suggested that higher levels of systemic pro-inflammatory
cytokines such as TNF are more related to worse mood and sick-
ness28. Further, systemic inflammation (high serum TNF and IL-6)
has been reported to be a predictor of worsening knee pain over 5
years in a population-based cohort of older adults10.
Our study has important limitations. Even though 43% of the
subjects had some MRI-based inflammation, only 8% had signs of
inflammation based on moderate or severe effusion-synovitis. Sy-
novial fluid samples were not available from all subjects, and for
some of the molecular biomarkers the proportion of samples with
concentrations below lower limit of quantification was high. Even
though we used well-accepted molecular and imaging biomarkers

Synovial fluid variables KOOS pain KOOS other symptoms KOOS sport/rec KOOS QOL KOOS4 SF-36 PCS

log10 sfIL-6 2.7 (7.6, 2.3) 3.5 (9.8, 2.9) 4.0 (12.9, 4.8) 2.5 (10.1, 5.2) 3.3 (10.1, 3.6) 4.3 (10.5, 1.9)
log10 sfIL-8 11.4 (5.4, 28.3) 20.6 (1.2, 42.5) 26.6 (4.4, 57.7) 24.8 (0.3, 49.9) 20.7 (3.3, 44.6) 22.3 (3.1, 41.5)
log10 sfIL-10 5.9 (26.0, 14.1) 21.6 (47.8, 4.5) 30.7 (64.5, 3.2) 27.0 (55.1, 1.0) 24.2 (50.6, 2.3) 2.5 (26.5, 31.5)
log10 sfTNF 10.4 (16.6, 37.3) 10.7 (25.3, 46.6) 23.5 (22.7, 69.8) 13.6 (30.0, 57.2) 18.1 (18.7, 54.9) 14.6 (50.4, 21.2)
Hoffa grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Hoffa grade 1 3.5 (11.1, 4.2) 3.8 (13.4, 5.8) 7.5 (21.4, 6.3) 2.8 (14.9, 9.3) 4.0 (13.8, 5.8) 6.4 (15.9, 3.1)
Hoffa grades  2 4.8 (13.7, 4.0) 7.6 (18.6, 3.4) 14.5 (30.3, 1.2) 2.2 (16.2, 11.8) 7.0 (18.3, 4.2) 6.9 (17.8, 4.1)
Effusion grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Effusion grades  1 ¡7.9 (-14.8, -1.1) ¡10.6 (-19.4, -1.7) 8.4 (20.6, 3.7) ¡11.0 (-21.6, -0.3) ¡9.0 (-17.8, -0.1) ¡11.4 (-19.7, -3.1)

Serum variables1 KOOS pain KOOS other symptoms KOOS sport KOOS QOL KOOS4 SF-36 PCS

log10 sIL-6 0.1 (5.4, 5.1) 1.4 (4.6, 7.5) 0.4 (10.2, 9.4) 2.9 (4.6, 10.4) 1.2 (5.1, 7.4) 1.5 (7.7, 4.6)
log10 sIL-8 2.1 (17.0, 12.7) 2.8 (22.3, 16.7) 0.6 (27.0, 28.2) 7.4 (16.4, 31.2) 1.0 (18.6, 20.7) 1.5 (17.2, 20.2)
log10 sIL-10 9.5 (15.0, 33.9) 5.2 (25.8, 36.1) 11.8 (33.4, 57.1) 9.4 (28.5, 47.4) 8.6 (22.8, 40.1) 9.1 (21.0, 39.2)
log10 sTNF 0.3 (4.5, 3.9) 1.4 (4.3, 7.1) 1.0 (7.5, 9.5) 1.4 (5.4, 8.2) 0.4 (5.7, 6.5) 1.6 (6.8, 3.7)
Hoffa grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Hoffa grade 1 4.4 (11.9, 3.2) 4.4 (14.1, 5.2) 6.6 (20.4, 7.2) 2.4 (14.3, 9.6) 4.3 (14.2, 5.6) 6.3 (15.8, 3.3)
Hoffa grades  2 4.9 (13.2, 3.4) 6.1 (16.8, 4.6) 11.6 (26.8, 3.7) 0.7 (12.4, 13.7) 5.1 (16.2, 5.9) 5.3 (15.7, 5.2)
Effusion grade 0 0.0 0.0 0.0 0.0 0.0 0.0
Effusion grades  1 ¡8.5 (-15.0, -2.1) ¡11.0 (-19.3, -2.6) 11.0 (22.8, 0.9) ¡11.5 (-21.9, -1.2) ¡10.6 (-19.1, -2.1) ¡11.6 (-19.8, -3.3)

1) The model did not converge for serum IL-12p70 and IFN-g. PCS ¼ physical component summary; sport/rec ¼ function in sport and recreation; QOL ¼ knee related quality
of life; KOOS4 ¼ average of pain, other symptoms, sport/rec and QOL; s ¼ serum; sf ¼ synovial fluid.
SF-36 ¼ Medical Outcomes Study 36-itmes short-form health survey.

Cross-sectional comparison between biomarkers and PROs using multivariable adjusted linear
regression model with multiple imputation of missing values. Molecular and imaging biomarkers of

Table V
inflammation at 2-year were analyzed against PROs at 2 years after injury. The results are expressed as Osteoarthritis
mean effects (95% confidence intervals). Boldface indicates statistically significant differences. Esti- andCartilage
mates are given per 1 unit of either log10 molecular biomarker values (in pg/ml) or synovitis grades.
Sample size ¼ 116
 A. Struglics et al. / Osteoarthritis and Cartilage 28 (2020) 356e362
of inflammation, it is possible that other biomarkers may associate
with PROs. Other timeframes of assessment of the biomarkers and
PROs might also influence the associations. Although there is no
existing evidence in this group of patients, it is possible that a
change in biomarker concentration between two time points is
better for predicting an outcome than a single measurement point
as used in the present study. We did estimate multiple associations
and thus some of the confidence intervals may exclude zero due to
this multiple testing.
In conclusion, in this prospective cohort of ACL-injured patients,
neither molecular nor MRI-assessed inflammation within the knee
joint at 2 years was associated with patient reported outcomes at 5
years post injury. The potential relationship between molecular,
structural (imaging) and patient reported outcomes remains
obscure. This is further stressed by our previous OA-prediction
study16, where the local inflammation assessed by these molecular
and imaging biomarkers of inflammation did not associate with
structural evidence of development of knee OA.
Author contributions
All authors were involved in the conception and design of the
study, or acquisition of data, or analysis and interpretation of data.
All authors contributed to drafting the article or revising it critically
for important intellectual content, and all authors gave their final
approval of the manuscript to be submitted. Additional contribu-
tions: obtaining of funding for the KANON study (LSL, RF, AS);
statistical expertise (AT, ME); collection and assembly of data (FWR,
RF, SL, AS); provision of study materials or patients (FWR, LSL, RF).
Responsibility for the integrity of the work as a whole, from
inception to finished article, is taken by A. Struglics.
Conflict of interest
Dr. Roemer is Chief Medical Officer and shareholder of BICL, LLC.
None of the other co-authors declared potential competing
interests.
Role of the funding source
The KANON study received funding for from The Swedish
Research Council, the Medical Faculty of Lund University, Region
Skåne, Governmental funding of clinical research within the na-
tional health services (ALF), The Swedish Rheumatism Association,
Thelma Zoegas Fund, The Stig & Ragna Gorthon Research Founda-
tion, Swedish National Centre for Research in Sports, Crafoord
Foundation, Tore Nilsson Research Fund, and Pfizer Global
Research.
Funding sources had no role in the design, collection, and
interpretation of the data or the decision to submit for publication.
Acknowledgements
We thank the participants of the KANON study without who this
work would not have been possible.
Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.joca.2019.12.010.
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