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EURODISC
Mid-Term Workshop
Harris Pratsinis, Eleni Mavrogonatou, Dimitris
Kletsas
Laboratory of Cell Proliferation and
Ageing, Institute of Biology, NCSR
Demokritos, Athens, Greece
Lab 6
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How does this work relate to understanding disc
degeneration ?

• Intervertebral disc degeneration is associated
  with
• Decrease in cell density viability in the disc
• Alterations in the synthesis metabolism of
  ECM-components

These processes are governed by growth factors
cytokines
Therapeutic approaches may involve stimulation of
the disc cells using growth factors cytokines
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How does this work relate to other work in this
project ?
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How does this work relate to other work in this
project ?
AIM To study disc cell repair and the
intracellular signalling cascades involved
Task 37 To collect conditioned media from disc
cells subjected to physical stresses Task 38 To
identify autocrine and paracrine growth factor
activities in the above conditioned media
Task 39 To examine intracellular signalling
cascades triggered by growth factors, canditioned
media and physical stresses
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DO PHYSICAL MECHANICAL STRESSES AFFECT THE
SECRETION OF AUTOCRINE GROWTH FACTORS BY
INTERVERTEBRAL DISC CELLS ?
Test of media conditioned by stressed cultures
(compared to media conditioned by untreated ones)
for their ability to stimulate DNA synthesis (and
eventually proliferation) of target cultures of
the same cell type (autocrine regulation) or
neighboring cell type (paracrine regulation)
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MECHANICAL STRESS (Methodology)
Preparation of Conditioned Media (Ulm) Cell
culture in monolayer (supplemented with
serum) Plating in collagen gels (900,000
cells/gel, serum-free) After 2-3 days
stimulation Hydrostatic pressure (0.5
hour) Cyclic strain (24 hours various
stretching intensities) Collect conditioned media
dissolved gels 1 or 24 hours after the end of
the stimulation
Test of autocrine/paracrine activity
(Athens) Plating in monolayer (supplemented with
serum) After confluency, arrest for 2 days
(serum-free) Stimulation with Conditioned Media
(plus tritiated thymidine) Count incorporated
radioactivity after 2 days
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AUTOCRINE GROWTH STIMULATION OF BOVINE NUCLEUS
PULPOSUS CELLS AFTER CYCLIC STRAIN
ABSENCE OF AUTOCRINE GROWTH REGULATION AFTER A
HYDROSTATIC PRESSURE-STRESS OF BOVINE NUCLEUS
PULPOSUS CELLS
3HTHYMIDINE INCORPORATION ( of the respective
control)
Medium conditioned by NP cells (Ulm C4011N)
stimulates DNA synthesis in NP cell cultures
(Athens C 6502 T/N)
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AUTOCRINE / PARACRINE GROWTH STIMULATION OF HUMAN
ANNULUS FIBROSUS CELLS AFTER CYCLIC STRAIN
Medium conditioned by AF cells (Ulm T4007A)
stimulates DNA synthesis in AF cell cultures
(Athens H 6007 T/A)
Recovery duration
3HTHYMIDINE INCORPORATION ( of the respective
control)
Unstretched
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CONDITIONED MEDIUM GROWTH PROMOTING ACTIVITY OF
BOVINE INTERVERTEBRAL DISC CELLS AFTER CYCLIC
STRAIN IS DONOR-DEPENDENT
NS Non Significant (pgt0.05, t-test)
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PHYSICAL STRESS (Methodology)
Preparation of Conditioned Media
(Oxford) Plating in monolayer culture (200,000
cells/6-well-plate, supplemented with
serum) Culture to sub-confluence in medium with
1mg/ml glucose and in normal osmolarity (280
mOsm) Remove serum and apply stimuli Collect
conditioned media at the end of the stimulation
(48 hours)
Test of autocrine/paracrine activity
(Athens) Plating in monolayer (supplemented with
serum) After confluency, arrest for 2 days (in
serum-free medium with 1mg/ml glucose and in
normal osmolarity) Stimulation with Conditioned
Media (plus tritiated thymidine) Count
incorporated radioactivity after 2 days
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AUTOCRINE GROWTH REGULATION AFTER PHYSICAL
STRESSES OF BOVINE INTERVERTEBRAL DISC CELLS
Medium conditioned by nucleus pulposus cells
(Oxford) tested in Nucleus pulposus cells
(Athens)
Open bars Normal Osmolarity Hatched bars High
Osmolarity
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AUTOCRINE / PARACRINE GROWTH REGULATION AFTER
PHYSICAL STRESSES OF BOVINE INTERVERTEBRAL DISC
CELLS
Medium conditioned by outer annulus fibrosus
cells (Oxford) tested in Annulus fibrosus
cells Nucleus pulposus
cells
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What have we learned so far ?

• Hydrostatic pressure does not affect autocrine
  growth factor secretion in both human bovine
  cultures
• Cyclic strain induces secretion of stimulatory
  autocrine paracrine activity (-ies) in both
  human bovine cultures, however this effect is
  donor-specific
• Preliminary results (only in bovine cultures)
  indicate that low O2 and low pH do not affect
  conditioned medium activity, while low glucose
  and high osmolarity seem to reduce it

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WHICH GROWTH FACTORS ?
Platelet-Derived Growth Factor PDGF (isoform
BB) Transforming Growth Factor-ß TGF-ß
(isoform ß1) Insulin-like Growth Factor-I
IGF-I basic Fibroblast Growth Factor bFGF
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RESPONSE OF BOVINE HUMAN INTERVERTEBRAL DISC
CELLS TO GROWTH FACTORS
3HTHYMIDINE INCORPORATION ( of control)
ng/ml
10
5
5
50
BOVINE
HUMAN
4 Nucleus Pulposus 3 Annulus Fibrosus
2 Nucleus Pulposus 4 Annulus Fibrosus
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TGF-ß SIGNALLING PATHWAY
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Kinetics of SMAD2 phosphorylation after TGF-ß
treatment in bovine intervertebral disc cells
Donor 1, Annulus
Donor 1, Nucleus
Donor 2, Annulus
Donor 2, Nucleus
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Kinetics of SMAD2 phosphorylation after TGF-ß
treatment in a human annulus fibrosus cell strain
Bovine 1, Annulus
Bovine 2, Annulus
Human, Annulus
0 10 30 60 3 6 12 24
MINUTES
HOURS
TIME AFTER TGF-ß ADDITION
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STUDY OF SIGNALLING PATHWAYS

• Measuring expression kinetics (Western
  analysis, kinase assays)
• Dominant negative mutants
• Pharmacological inhibitors

PD98059 MEK/ERK
SB203580 p38 MAPK
LY294002 PI-3-K
H-89 PKA
Wortmannin PI-3-K
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PDGF-BB and IGF-I stimulate DNA synthesis of
bovine nucleus pulposus cells by up-regulating
the ERK and the PI-3-K/Akt pathways
pERK
3HTHYMIDINE INCORPORATION (CPM)
pAkt
Actin
Ctrl
PDGF
IGF-I
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What have we learned from this part ?
PDGF, TGF-ß, IGF-I and b-FGF stimulate DNA
synthesis in bovine disc cells. On human disc
cells, however, TGF-ß has no effect or acts as
inhibitor TGF-ß stimulates the SMAD pathway in
both bovine and human disc cells PDGF and IGF-I
stimulate DNA synthesis by up-regulating the ERK
and the PI-3-K/Akt pathways
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To be continued
Identification of the growth factors and the
pathways involved in the mitogenic activity of
media conditioned by mechanically stressed disc
cells Study the role of extracellular matrix
components on the response of disc cells to
growth factors Examine the effect of growth
factors on major parameters of the
repair/regeneration process (MMPs and
TIMPs) Correlation of the results to the ageing
level
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Thanks !