Bieder, D.

Toegel, S.

Viernstein, H.

Walzer, S.M.

Windhager, R.

Elsevier - Publisher Connector 

Brazilin blocks the induction of NFKB1/P50 in human chondrocytes and synoviocytes 

Abstracts / Osteoarthritis and Cartilage 22 (2014) S57–S489S148Purpose: Monocytes play a pivotal role in controlling tissue inﬂam-
mation and repair. They inﬁltrate the tissues and develop into twomain
subsets of macrophages in response to local signals: inﬂammatory (M1-
) and tissue-repair (M2-) macrophages (M4). Here we investigated
whether M1 and M24 differentially modulate chondrogenesis of mes-
enchymal stem/stromal cells (MSC).
Methods: Monocytes isolated from buffy-coats were cultured for 5 days
with GM-CSF or MCSF to induce respectively M1- or M24-polarization.
MSC were isolated from the bone marrow of a total of 9 patients and
expanded for two passages. MSC or skin ﬁbroblasts (as control cells) and
polarized macrophages were cultured in collagen scaffolds up to 3
weeks alone or after being mixed (at the ratio 1:1). In order to deter-
mine the reached cell number, selected constructs were generated by
combining carboxyﬂuorescein succinimidyl ester (CFSE) labeled MSC
with macrophages. To investigate the role of soluble factors in modu-
lating MSC differentiation, conditioned media harvested from M14 and
M24 were supplemented during the chondrogenic culture of MSC.
Resulting tissues and/or isolate cells were assessed histologically (gly-
cosaminoglycan, GAG), biochemically (GAG contents), cytoﬂuorimetri-
cally (percentage of cells expressing or not CD45, and CFSE) and by RT-
PCR (Sox9 and collagen-II)
Results: Coculture of MSC/M24 in scaffolds resulted in statistically
signiﬁcant 1.9-fold higher GAG content than what would be expected
(deﬁned as chondro-induction). Chondro-induction was lower (1.30.4)
and less reproducible when coculture was performed withM14 and did
not occur with skin ﬁbroblasts. GAG contents of constructs generated by
solely macrophages were undetectable. Histological analyses of con-
structs conﬁrmed the biochemical results. In the coculture there was no
modulation of the chondrogenic genes. As compared to monocultures,
in co-culture MSC and M24 numbers decreased less markedly (at day 7,
MSC were 84% and 42% of the initial number, M24 were 26% of the
initial number and undetectable, respectively for co-cultured and
monocultured cells).
Conclusions: We have demonstrated that coculture MSC/M42 results
in synergistic cartilage tissue formation, which is not mediated by
soluble factors alone. Further studies are envisioned to investigate
whether M42 modulate the survival of speciﬁc MSC sub-populations.
Finally, in vivo studies are necessary to assess the clinical relevance of
our ﬁndings in the context of cartilage repair.
242
POSSIBLE CHONDROPROTECTIVE EFFECT OF CANAKINUMAB: AN IN
VITRO STUDY ON HUMAN OSTEOARTHRITIC CHONDROCYTES
S. Cheleschi y, N.A. Pascarelli y, O.M. Lucherini y, E. Crainz z, S. Tenti y,
M. Galeazzi y, G. Collodel x, A. Fioravanti y, L. Cantarini y. yDept. of Med.,
Surgery and NeuroSci., Rheumatology Unit, Siena, Italy; zOrthopaedics
and Traumatology Clinic, Univ. Hosp. of Siena, Siena, Italy; xDept. of
Molecular and Dev.al Med., Siena, Italy
Purpose: Canakinumab is a human IgGkmonoclonal antibody targeting
Interleukin (IL)-1b; its action involves the neutralization of IL-1b sig-
naling, resulting in suppression of inﬂammation in patients with
autoimmune disorders. IL-1b plays an important role also in the
destruction of cartilage and it is the most important cytokine in the
pathogenesis of osteoarthritis (OA). In response to IL-1b, chondrocytes
secrete other proinﬂammatory cytokines, neutral metalloproteinases
(MMPs), nitric oxide (NO); furthermore IL-1b inhibits chondrocytes
proliferation and induces apoptosis. The aim of our study was to eval-
uate the possible in vitro effect of canakinumab on cultures of human
OA chondrocytes cultivated in the presence or absence of tumor
necrosis factor (TNF)-a.
Methods: Human articular cartilage was obtained from the femoral
heads of 5 patients (range 63-71 years) with OA according to ACR cri-
teria undergoing surgery for total hip prostheses. The chondrocytes
were isolated from the articular cartilage using sequential enzymatic
digestion. The primary cultures so obtained were seeded in 6-well
plates until conﬂuence. Cells were then incubated with two concen-
trations (1mg/ml and 10mg/ml) of canakinumab alone or in combination
with TNF-a (10 ng/ml) for 48h. In these conditions we evaluated cell
viability, release of proteoglycans (PG) and NO in culture medium,
inducible nitric oxide synthase (iNOS) and MMP-1, 3, 13 expression, the
percentage of apoptosis and necrosis. After 24h we performed IL-1b
dosage (ELISA). Finally, we used a transmission electron microscope
(TEM) for morphological assessment. Data are expressed as the mean 
standard deviation of triplicate values for each experiment. Statisticalanalysis was performed using an analysis of variance followed by
Bonferroni multiple comparison tests.
Results: Canakinumab alone at the two concentrations studied hasn’t
cytotoxic effect, while TNF-a caused a signiﬁcant decrease of the per-
centage of viable cell (P < 0.001). Both concentrations of canakinumab
tested signiﬁcantly restored the cell viability in TNF-a stimulated
chondrocytes. As reported in Fig 1A, canakinumab alone at the two
concentrations studied didn’t modify signiﬁcantly IL-1b levels in the
culture medium, while TNF-a produced a signiﬁcant increase of IL-1b.
Both concentrations of canakinumab tested signiﬁcantly reduced IL-1b
production (Fig 1A). No signiﬁcant modiﬁcation of PG levels was
observed in the chondrocyte cultures treated with canakinumab alone.
The presence of TNF-a determined a signiﬁcant decrease (P < 0.001) in
PG levels and canakinumab signiﬁcantly increased the PG levels in cells
cultured with TNF-a in a dose-dependent manner. Incubation of
chondrocytes with canakinumab alone didn’t affect nitrite release. The
presence of TNF-a induced a signiﬁcant increase (P < 0.001) in NO
production. However, after co-incubation with TNF-a and canakinu-
mab, at different concentrations, the levels of NO decreased sig-
niﬁcantly (1mg/ml, P < 0.01; 10mg/ml, P < 0.01). The data of the NO
levels were conﬁrmed by the immunocytochemistry assay for iNOS.
TNF-a stimulated chondrocytes displayed a signiﬁcant increase of
MMP-1, 3, 13 gene expression; a signiﬁcant reduction was shownwhen
cells were co-treated with TNF-a and canakinumab (Fig 1B). Our
experiments conﬁrmed the pro-apoptotic effect of TNF-a and demon-
strated a protective effect of canakinumab at two concentrations
examined. The results concerning biochemical data were further con-
ﬁrmed by the morphological ﬁndings obtained by TEM.
Conclusions: It is generally accepted that IL-1b and TNF-a are the
pivotal cytokines involved in OA physiopathology. Hence, the neutral-
ization of these cytokines appears to be a logical development for OA
therapy. In the present study we showed, for the ﬁrst time, that cana-
kinumab counteracts the negative effect of TNF-a on OA chondrocyte
cultures, probably inhibiting IL-1b signaling.
Figure 1. A) IL-1b levels in the culture medium; B)MMP-1, 3, 13 gene
expression.
243
BRAZILIN BLOCKS THE INDUCTION OF NFKB1/P50 IN HUMAN
CHONDROCYTES AND SYNOVIOCYTES
D. Bieder y, S.M. Walzer y, R. Windhager y, H. Viernstein z, S. Toegel y,z.
yKarl Chiari Lab for Orthopaedic Biology, Dept. of Orthopaedics, Med.
Univ. of Vienna, Vienna, Australia; zDept. of Pharmaceutical Technology
and Biopharmaceutics, Univ. of Vienna, Vienna, Australia
Purpose: Brazilin is one major constituent of Caesalpinia sappan and is
known to possess anti-bacterial, anti-oxidative, and anti-inﬂammatory
activities. Osteoarthritis (OA) creates a disequilibrium between the
catabolic and anabolic activities of chondrocytes in remodeling the
Abstracts / Osteoarthritis and Cartilage 22 (2014) S57–S489 S149cartilage extracellular matrix (ECM). Pro-inﬂammatory signaling con-
tributes to the degradation of the cartilage matrix. Under OA conditions,
synoviocytes produce a range of inﬂammatory mediators, including IL-
1b. Upon binding to chondrocytes, these mediators activate speciﬁc
signal transduction cascades, such as the nuclear factor-kappa-B (NF-
kB) pathway, which acts as one central regulator of the catabolic pro-
cesses that suppresses the synthesis of ECM components, and induces
the expression of matrix metalloproteinases and enzymes of the
ADAMTS family, leading to perpetuated cartilage breakdown.
This study aimed to evaluate the effects of brazilin in osteoarthritic
chondrocytes and synoviocytes with particular focus on the NF-kB
pathway.
Methods: Brazilin was isolated from Caesalpinia sappan extract (CSE)
and identiﬁed using HPLC and NMR methods. Chondrocytes and syn-
oviocytes were isolated from OA patients undergoing total knee
replacement surgery. Cells were either left untreated for control sam-
ples, were treated with 10 ng/ml IL-1b, or were pre-treated with 10 mg/
ml brazilin prior to the addition of 10 ng/ml IL-1b. Genes associated
with NF-kB-mediated signal transduction were examined in primary
human chondrocytes using an NF-kB signaling array and in silico
pathway analysis. The induction and inhibition of NFKB1, its gene
product p50, and the precursor p105 were investigated in primary
human chondrocytes and synoviocytes using RT-qPCR and Western
blotting.
Results: Nine NF-kB pathway-related genes (BIRC3, CCL2, CSF1, CSF3,
IL1B, IL8, NFKB1, NFKB2 and TNFAIP3) were found to be signiﬁcantly up-
regulated by IL-1b-stimulation and signiﬁcantly down-regulated after
pre-incubation with brazilin. Pathway analysis revealed NFKB1 as one
major gene regulating the anti-inﬂammatory activities of brazilin. RT-
qPCR assays conﬁrmed that the up-regulation of NFKB1 mRNA in IL-1b-
stimulated primary chondrocytes and synoviocytes was signiﬁcantly
reduced by pre-treatment with brazilin. Western blotting showed that
IL-1b treatment increased p105 protein in chondrocytes with a peak
after 1 h followed by a steady decline, while increasing constantly the
amount of p50 over 24 h. Brazilin suppressed the IL-1b-mediated
induction of NFKB1/p50 in chondrocytes and synoviocytes.
Conclusions: The present study suggests that brazilin effectively blocks
the induction of NFKB1/p50 in cytokine-stimulated primary human
chondrocytes and synoviocytes, pointing towards a chondroprotective
potential of brazilin, which may be beneﬁcial for reducing cartilage
breakdown in OA.
244
EFFECT OF BONE MARROW SURGICAL APPROACH AND RAPIDLY
DEGRADING PRESOLIDIFIED SUBCHONDRAL CHITOSAN/BLOOD
IMPLANT ON RESURFACING OF CHONDRAL DEFECTS IN A SHEEP
MODEL
A. Bell y, M. Hurtig y, G.-E. Rivard z, C.D. Hoemann x. yUniv. of Guelph,
Guelph, ON, Canada; zHosp. Sainte-Justine, Montreal, QC, Canada;
x Ecole Polytechnique, Montreal, QC, Canada
Purpose: Bone marrow stimulation for cartilage repair is partly limited
by inadequate stem cell recruitment from the trabecular bone marrow
to the cartilage lesion. We tested the hypothesis that the cartilage repair
elicited by marrow stimulation can be improved by subchondral
delivery of rapidly degrading pre-solidiﬁed chitosan/blood implants to
bone defects with free communication with the trabecular bone mar-
row. Jamshidi needles and a drill burr were used to create 2mm
diameter bone defects with cleanly removed bone to maximize cell
recruitment.
Methods: Full-thickness 10x10mm cartilage defects were created in the
medial femoral condyle in both knees of 11 mature sheep. Based on a
pre-planned template, three 2-mm diameter bone holes were gen-
erated systematically at the corner of each defect with a Jamshidi biopsy
needle and a drill burr, and the area between the holes was perforated
with six smaller microfracture holes (Fig. 1A-B). 10 kDa chitosan/
autologous whole blood implant was presolidiﬁed ex vivo with coag-
ulation factor (recombinant human Factor VIIa, rhFVIIa, or Tissue Factor,
TF) and inserted into each of the three 2-mm diameter holes. Con-
tralateral control defects were treated with whole blood presolidiﬁed
with rhFVIIa or TF. Day 1 (N ¼ 1) and 6.5 month repair (N ¼ 10) was
evaluated bymacroscopic scoring, andmicro-CT, histomorphometry for
collagen type I and II, and histological scoring (ICRS-2). Four intact age-
matched condyles were micro-CT scanned for baseline bone parame-
ters. 6.5 month cartilage repair tissues were also analyzed bybiochemistry for glycosaminoglycan (GAG) and collagen (% weight per
wet weight). The General Linear Model (Statistica, Statsoft, V6.2, NB,
USA) was used to analyze differences due to surgical approach (drill
hole vs 2 averaged Jamshidi holes per defect, N ¼ 20, vs intact condyles
N ¼ 4) and differences due to implant condition (N ¼ 10). p < 0.05 was
considered signiﬁcant.
Results: At day 1 post-operative, the marrow stimulation approach led
to a 65% decrease in the subchondral bone volume fraction (BVF, %)
compared to intact condyles, along with extensive subchondral bleed-
ing (Fig. 1A-D). Rapidly degrading chitosan implant was retained near
the surface of the bone defects at day 1, with no signiﬁcant effects on
bone or cartilage repair features at 6.5 months. At 6.5 months post-
operative, most medial femoral condyles (but not lateral femoral con-
dyles) developed large osteophytes. Only modest subchondral bone
repair was observed in the defect area (13% increase in BVF compared to
acute defects), and in 3 out of 20 defects, net bone resorption around
the Jamshidi biopsy hole was observed (Fig. 1G). The larger 2mm bone
holes were consistently resurfaced with collagen type II repair with
higher collagen content (18% vs 15% w/w) and lower GAG (2.4% vs 4.4%
w/w) than tissue outside the defect, while shallow microfracture holes
were poorly resurfaced (Fig. 1F). Compared to Jamshidi biopsy holes,
drill burr elicited a higher overall cartilage repair histological quality (33
 28 vs 49  25, p ¼ 0.041, N ¼ 20 marrow stimulation holes, Fig. 1H-I).
Conclusions: Blood vessels sheared by Jamshidi surgical marrow
stimulation can devitalize the subchondral bone plate and provoke
sporadic bone necrosis. In large animal condyles, bone cleanly removed
by Jamshidi biopsy or drilling is slow to repair with mineralized tissue.
2-mm diameter drill holes are resurfaced with soft repair tissue
whereas shallow microfracture holes have inadequate marrow com-
munication and are poorly resurfaced. Marrow stimulation surgical
approaches need to ﬁnd a balance between bone damage/blood vessel
rupture and subchondral implants that create optimal inﬂammatory
responses that elicit cell inﬂux and osteochondral repair.
245
A TISSUE ENGINEERED OSTEOCHONDRAL COMPOSITE FOR
CARTILAGE REPAIR: AN IN VIVO STUDY
C. Sosio y, A. Di Giancamillo y, D. Deponti y, M. Agnoletto y, F. Gervaso z,
M. Domenicucci y, A. Addis x, M. Campagnol x, C. Domeneghini k,
A. Sannino z, G. Peretti{,y. y IRCCS Istituto Ortopedico Galeazzi, Milan,
Italy; zDept. of Engineering for Innovation, Univ. of Salento, Lecce, Italy;
x Istituto Sperimentale Italiano Lazzaro Spallanzani, Rivolta d’Adda (CR),
Italy; kDept. of Hlth., Animal Sci. and Food Safety, Univ. of Milan, Milan,
Italy; {Dept. of BioMed. Sci. for Hlth., Univ. of Milan, Milan, Italy
Purpose: The aim of this work is to validate the efﬁcacy of a tissue
engineered osteochondral composite for the treatment of cartilage
lesion produced in adult pigs. The osteochondral composite was man-
ufactured by combining an osteo-compatible cylinder and a neo-
cartilagineous tissue obtained by seeding swine articular chondrocytes
into a collagen scaffold.
Methods: Articular cartilage was harvested from the trochlea of six
adult pigs and chondrocytes were isolated; after the in vitro expansion,
chondrocytes were seeded onto a collagen scaffold that was pre-inte-
grated in vitro to an osteo-compatible cylinder. The seeded osteo-
chondral scaffolds were cultured in chondrogenic medium for 3 weeks,
then they were surgically implanted in osteochondral lesions per-
formed in the trochlea of the same pigs from which the cartilage was


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Brazilin blocks the induction of NFKB1/P50 in human chondrocytes and synoviocytes

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