Cibere, J.

Huebner, J.L.

Hunt, M.A.

Kraus, V.B.

Peters, S.

Pollock, C.L.

Saxne, T.

Sayre, E.C.

Elsevier - Publisher Connector 

Biomarkers of cartilage degradation and synthesis relevant to knee osteoarthritis: relationships with dynamic knee joint load and changes following exercise 

Abstracts / Osteoarthritis and Cartilage 20 (2012) S54–S296 S83reference subjects. Also, to perform a time dependent analysis and
investigate correlations between cartilage and bone biomarkers, and pro-
inﬂammatory cytokines.
Methods: SFwas aspirated (0 - 23 days from injury) from the acutely injured
knee of 111 individuals (mean age 27 years (13-64), 22%women) with acute
joint effusion and blood in the SF (i.e. hemarthrosis). Concentrations of sGAG
(sulphated glycosaminoglycan) were measured by Alcian blue precipitation
whereas cartilage ARGS, bone biomarker (osteocalcin [OCL], secreted protein
acidic and rich in cysteine [SPARC] and osteopontin [OPN]) and pro-inﬂam-
matory cytokine (IL-1b, IL-6, IL-8 and TNF-a) concentrations were analyzed
using electrochemiluminescence immunoassays. Biomarker concentrations
of cartilage, bone and cytokines were compared to SF concentrations from
knees of age and sexmatched healthy reference subjects and were analyzed
with regard to time between injury and aspiration (same day [n¼29], 1 day
[n¼31], 2-3 days [n ¼ 19], 4-7 days [n ¼ 20] and 8-23 days [n ¼ 12]).
Results: SF-concentrations of ARGS (p<0.001), SPARC (p<0.001), OPN
(p<0.001), and all cytokines (p<0.001), but not sGAG (p¼0.06) or OCL
(p¼0.992), were signiﬁcantly higher in injured knees compared to knees of
reference subjects. SF concentrations of cartilage markers sGAG and ARGS
were signiﬁcantly elevated in knees aspirated later than 1 day after injury,
whereas concentrations of SPARC and OPN were elevated in knees aspi-
rated the same day (day 0) as injury and at all time points thereafter (i.e.
day 1-23, ﬁg. 1). The OPN concentrations were higher with increased time
between injury and aspiration. Cytokine concentrations were elevated at
all time points of aspiration (from day 0-23). Cytokine concentrations were
lower with increased time between injury and aspiration at time-points
after day 1 (i.e. day 2-23). There were positive correlations between OPN
and the cartilage markers (sGAG, r¼0.61 and ARGS, r¼0.59), and between
SPARC and all measured cytokines (r>0.33).
Conclusions: Compared to knees of age and sex matched healthy refer-
ence subjects, acutely injured knees with hemarthrosis have elevated SF
concentrations of the cartilage biomarker ARGS, bone biomarkers SPARC
and OPN and the pro-inﬂammatory cytokines IL-1b, IL-6, IL-8 and TNF-a.
Our results suggest that there could be large variations in these concen-
trations within the ﬁrst month of injury and that there may be a relation
between OPN and cartilage markers and between SPARC and inﬂamma-
tion. Increased concentrations of SF pro-inﬂammatory cytokines directly
after knee injury may activate proteases leading to degradation of cartilage
aggrecan more than 1 day after knee injury.
Fig. 1. sGAG, ARGS, SPARC and OPN concentrations in SF aspirated from the
acutely injured knee (hemarthrosis) of individuals at different time points
after injury. The data is expressed as the mean log 10 (95% conﬁdence
interval) concentrations. The dotted lines represent the mean log10 (95%
conﬁdence interval) concentrations in the SF from knees of age and sex
matched healthy reference subjects. sGAG: sulphated glycosaminoglycan;
SPARC: secreted protein acidic and rich in cysteine; OPN: osteopontin.
Ă154
BIOMARKERS OF CARTILAGE DEGRADATION AND SYNTHESIS RELEVANT
TO KNEE OSTEOARTHRITIS: RELATIONSHIPS WITH DYNAMIC KNEE
JOINT LOAD AND CHANGES FOLLOWING EXERCISE
M.A. Hunt 1,2, C.L. Pollock 1, V.B. Kraus 3, T. Saxne 4, S. Peters 1,
J.L. Huebner 3, E.C. Sayre 2, J. Cibere 1,2. 1Univ. of British Columbia,
Vancouver, BC, CANADA; 2Arthritis Res. Ctr. of Canada, Vancouver, BC,
CANADA; 3Duke Univ., Durham, NC; 4 Lund Univ., Lund, Sweden
Purposes: 1) To examine relationships between biomarkers of artic-
ular cartilage degradation and synthesis with dynamic knee joint load
in patients with knee osteoarthritis (OA); 2) To compare changes in
these biomarkers following 10 weeks of exercise vs. no exercise.
Methods: Seventeen (8 male, 9 female; mean age 66.1, SD 11.3)
individuals with radiographically-conﬁrmed medial tibiofemoral OA
and varus malalignment participated in this pilot study. All partici-
pants underwent a baseline testing session where serum and urine
samples were collected, followed by a three-dimensional motion
analysis. Motion analysis data from digital cameras and force plat-
forms were combined to calculate the external knee adduction
moment (KAM) impulse - a measure of medial compartment knee
joint load. Following baseline testing, participants were randomized
to either 10 weeks of: 1) physiotherapist-supervised lower limb
muscle strengthening exercises, or 2) no exercises (control). Those in
the exercise group performed a series of six exercises designed to
strengthen the hip abductors, quadriceps, and hamstrings muscles, as
well as minimize dynamic varus malalignment. Exercises were per-
formed at home at least four times per week and were partially
supervised through ﬁve visits (weeks 1, 2, 3, 5, 8) with the study
physiotherapist to conﬁrm proper and safe performance and
progression of resistance. Identical follow-up testing, including
collection of serum and urine samples, was conducted 11 weeks after
baseline. Biomarkers assessed included: urinary C-telopeptide of type
II collagen (uCTX-II) and urinary type II collagen cleavage neoepitope
(uC2C) both normalized for creatinine and indicative of collagen
degradation, serum cartilage oligomeric matrix protein (sCOMP) also
indicative of cartilage degradation, serum hyaluronic acid (sHA)
indicative of synovial inﬂammation and serum C-propeptide of type II
procollagen (sCPII) indicative of collagen synthesis. Ratios of degra-
dation to synthesis biomarker levels were also computed. Biomarker
values were log-transformed and linear regression analyses were
performed to evaluate the association of each biomarker with the
KAM impulse, adjusted for sex, age, and physical activity. Similar
linear regression models were computed to assess between-group
differences in the change values of each biomarker, adjusting for age
and sex.
Results: Nine participants (5 male) were randomized to the exercise
group, while the other eight participants (3 male) received no addi-
tional treatment during the study. Based on baseline data only,
signiﬁcant positive associations with KAM impulse were only seen for
uCTX-II (p¼0.02) and the ratio of uCTX-II:sCPII (p<0.01). Biomarker
concentrations (log transformed) at baseline and follow-up for both
groups are summarized in Table 1. One participant in the control
group was lost to follow-up and did not return for ﬁnal testing.
Compared with the no exercise group, participants in the exercise
group reported signiﬁcant reductions (p¼0.04) in sCOMP levels
following the intervention (non-log transformed reduction of 0.8 U/L
vs. 0.9 U/L increase in the non-exercising group). sHA (p¼0.10) and
uCTX-II (p¼0.11) were only slightly reduced in the exercise group
compared to controls.
Conclusions: This research provides initial evidence of the relationship
between biomarkers and knee joint load measures in patients with medial
tibiofemoral knee OA as well as changes in circulating biomarker
concentrations following non-pharmacological treatment. Speciﬁcally, it
appears that uCTX-II is associated with knee joint load, while sCOMP is
amenable to change following exercises speciﬁcally designed to ofﬂoad
cartilage.
Mean (sd) natural log-transformed biomarker concentrations for both groups at baseline and follow-up
Baseline Exercise
Group (n¼9)
Baseline No
Exercise
Group (n¼8)
Follow-up
Exercise
Group (n¼9)
Follow-up No
Exercise
Group (n¼7)
Adjusted Between-groups
Change Difference (95% CI)
p-value
URINARY MARKERS
uCTX-II (log ng/mmol creatinine) 5.40 (0.81) 5.97 (0.57) 5.32 (0.93) 6.25 (0.68) -0.33 (-0.71, 0.04) 0.11
uC2C (log ng/mmol creatinine) 2.45 (0.68) 2.46 (0.76) 2.57 (0.81) 2.71 (0.58) -0.13 (-0.84, 0.58) 0.73
SERUM MARKERS
sHA (log U/L) 3.47 (0.93) 3.80 (0.96) 3.26 (1.13) 4.21 (0.86) -0.79 (-1.67, 0.08) 0.10
sCOMP (log U/L) 2.20 (0.21) 2.26 (0.17) 2.11 (0.24) 2.36 (0.13) -0.16 (-0.30, -0.02) 0.04
sCPII (log U/L) 6.56 (0.19) 6.44 (0.53) 6.50 (0.36) 6.71 (0.40) -0.34 (-0.93, 0.24) 0.27
RATIOS
[uCTX-II]:sCPII -1.16 (0.74) -0.46 (0.49) -1.18 (0.97) -0.46 (0.81) 0.01 (-0.63, 0.66) 0.97
[uC2C]:sCPII -4.11 (0.69) -3.98 (1.19) -3.93 (0.98) -4.01 (0.73) 0.22 (-0.59, 1.03) 0.61
[sHA]:sCPII -3.09 (0.99) -2.64 (0.87) -3.24 (1.37) -2.50 (0.77) -0.45 (-1.43, 0.53) 0.39
Abstracts / Osteoarthritis and Cartilage 20 (2012) S54–S296S84155
SYNOVIAL FLUID PREPARATION TO IMPROVE IMMUNOASSAY
PRECISION FOR BIOMARKER RESEARCH USINGMULTIPLEX PLATFORMS
C. Jayadev, R. Rout, W. Jackson, A. Price, P. Hulley. Univ. of Oxford, Oxford,
UNITED KINGDOM
Purpose: Synovial ﬂuid (SF) reﬂects the biological milieu of the joint and is
a potentially important source of biomarkers in osteoarthritis (OA). OA joints
usually do not offer enough SF volume for analysis of multiple markers in
a single sample by conventional enzyme-linked immunosorbent assays
(ELISA). Newer platforms i.e. Luminex and Meso Scale Discovery (MSD)
enable multiplex analysis in small (50ml) volumes. However, SF analysis is
challenging due to its complex matrix and viscous non-Newtonian nature.
The aim of the study was to optimise SF sample preparation for the best
assay precision during multiplex analysis by the Luminex and MSD
platforms.
Methods: SF was harvested from 16 patients with end-stage knee OA at
arthroplasty. All samples underwent identical processing and preparation
(Figure 1).
ĂDilutions were made with the manufacturer's assay diluent. Hyaluronic
acid (HA) was digested by treatment with 2mg/ml bovine testicular hyal-
uronidase (madeup in assaydiluent) for 1hr at RT. This included an effective
2-fold sample dilution. Experiments (Table 1) were conducted according to
the manufacturer's protocol. Samples were analysed in triplicate.Table 1
Experiment Summary
Platform Platform Info. Assay Details
Luminex Polystyrene bead system Inﬂammatory 10-plex
IL8, IL10, GMCSF, IL5,
Luminex Magnetic bead system IL6 single-plex
Meso Scale Discovery Multi-spot
Electrochemiluminescence
Inﬂammatory 4-plex (Bead events per target (for Luminex assays only) and the coefﬁcient of
variation (CV) of the calculated concentration for sample triplicates were
compared for each pre-treatment protocol. Luminex data generated from
<35 bead events per target is not acceptable. At least 50 bead events are
recommended. Intra-assay precision with a CV <25% is a widely used
acceptance criterion. Matched/repeat measure statistical tests were used
when possible as each sample was assayed after each pre-treatment.
P<0.05 was considered signiﬁcant.
Results: Luminex bead counts: In the polystyrene bead Luminex 10-plex,
the proportion of usable samples (2 replicates with 35 bead events)
was signiﬁcantly greater when samples were digested (McNemar's test,
p<0.001 for each target). In the magnetic bead Luminex IL6-plex, all
samples were usable with 50 bead events in every well for each prepa-
ration protocol.
Assay Precision:
Polystyrene bead Luminex 10-plex: Only IL6 and IL8 were above the limit
of quantiﬁcation. Comparison of the overall group mean %CV for all
usable samples was signiﬁcantly lower in digested than neat samples for
both IL-6 (p¼0.007, Mann-Whitney test) and IL-8 (p¼0.004, two-tailed t-
test).
Magnetic bead Luminex IL6-plex: Every digested samples had an intra-assay
CV <15% (group mean 6.7%). Each sample's intra-assay CV was signiﬁ-
cantly greater if it was neat or diluted (p¼0.0195, repeated measures
ANOVA; p< 0.05 for neat, 2-fold & 4-dilution by Newman-Keuls post-hoc
comparisons).
Meso Scale Discovery:For relatively abundant targets i.e. IL6 & IL8, all
but 2 samples had an intra-assay CV <25% for each pre-treatment.
There was no signiﬁcant difference in intra-assay CV for samples
under each different pre-treatment (repeat measures ANOVA). For
TNF-a, only digestion produced intra-assay CVs <25% for every
sample. However, the improved %CV of digested samples did not
reach statistical signiﬁcance (repeat measures ANOVA). ILb levels
were below the linear part of the standard curve at the lower limit of
quantiﬁcation.
Conclusions:The magnetic bead Luminex system maximises bead events
per target irrespective of sample preparation.
SF digestionwith 2mg/ml hyaluronidase (with 2-fold dilution) gives better
intra-assay precision for polystyrene and magnetic bead Luminex assays
than dilution alone. MSD assays for abundant targets show good precision
irrespective of sample pre-treatment. However, digestion may offer better
precision for less expressed targets i.e. TNF-a and is preferable to large
dilutions that may take the target below the dynamic range WeTreatment Protocols Number of Samples
(IL1b, IL6, TNFa, IL4,
IFNg & IL2)
Neat Digested 16
Neat 2xDilution
4xDilution Digested
7 (of 16)
IL1b, TNFa, IL6 & IL8) Neat 2xDilution
4xDilution Digested
7 (of 16) Same as above


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Biomarkers of cartilage degradation and synthesis relevant to knee osteoarthritis: relationships with dynamic knee joint load and changes following exercise

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