Calcium Regulates Cyclic Compression-Induced Early Changes ... · Calcium Regulates Cyclic...
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Calcium Regulates Cyclic Compression-Induced Early Changes In Chondrocytes During In Vitro Tissue Formation by Igal Raizman A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department Laboratory Medicine and Pathobiology University of Toronto © Copyright by Igal Raizman 2009
Transcript of Calcium Regulates Cyclic Compression-Induced Early Changes ... · Calcium Regulates Cyclic...
Calcium Regulates Cyclic Compression-Induced Early Changes In Chondrocytes During In Vitro Tissue Formation
by
Igal Raizman
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Igal Raizman 2009
ii
Calcium Regulates Cyclic Compression-Induced Early Changes
In Chondrocytes During In Vitro Tissue Formation
Igal Raizman
Master of Science
Graduate Department of Laboratory Medicine and PathobiologyUniversity of Toronto
2009
AbstractA single application of cyclic compression to bioengineered cartilage improves tissue formation
through cell shape changes that are mediated by α5β1 integrin and membrane-type
metalloprotease (MT1-MMP). To determine if this response is controlled by calcium, we
investigated how calcium regulated cell shape changes, MT1-MMP and integrin activity in
response to stimulation. Stimulation-induced changes in cell shape and MT1-MMP expression
were abolished with chelation of extracellular calcium, and reinstated with its re-introduction.
Spreading and retraction were inhibited by blocking the stretch-activated and L-Type voltage-
gated channels, respectively; channel blocking also inhibited MT1-MMP upregulation.
Channels’ role was confirmed through treatment with calcium A23187 ionophore, which
alleviated the effects of channel blocking. Calcium regulated the integrin-mediated signalling
pathway, which was facilitated through the kinase Src. Both calcium- and integrin-mediated
pathways converged on activating ERK in response to stimulation. Understanding the molecular
mechanisms regulating chondrocyte mechanotransduction may lead to the development of
improved bioengineered cartilage.
iii
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embe
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or a
ll th
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and
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•D
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itlan
a Pr
ada
for h
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repa
ratio
n of
CPP
•M
enat
Atti
a fo
r her
ass
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with
con
foca
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Par
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for h
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(Adv
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thei
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alua
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assi
stan
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ith S
EM im
agin
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•D
r. M
arc
Gry
npas
for c
hairi
ng m
y th
esis
def
ence
iv
Tab
le o
f Con
tent
sA
BST
RA
CT
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. II
AC
KN
OW
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DG
ME
NT
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II
TA
BL
E O
F C
ON
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IV
LIS
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GU
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LIS
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AB
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1.1
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OD
UC
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1.1.
2 C
artil
age:
Rol
e &
Fun
ctio
n ...
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1.1.
3 A
rtic
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Car
tilag
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1.
1.4
Com
posi
tion
& A
rchi
tect
ure
of A
rtic
ular
Car
tilag
e ...
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. 3
1.1.
5 C
hond
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1.
1.6
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race
llula
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rix
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ctur
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1.
1.8
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cellu
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atri
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1.2
CA
RTI
LAG
E PA
THO
LOG
Y &
TISS
UE
ENG
INEE
RIN
G ..
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1.2.
2 A
rtic
ular
Car
tilag
e In
jury
& R
epai
r Obs
tacl
es ..
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1.2.
3 Ti
ssue
Eng
inee
ring
App
roac
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1.2.
4 C
alci
um P
oly-
Phos
phat
e (C
PP) a
nd B
ipha
sic
Con
stru
cts .
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.. 19
1.3
MEC
HA
NIC
AL
STIM
ULA
TIO
N ..
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1 1.
3.2
Mec
hano
tran
sduc
tion
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1.3.
3 In
tegr
ins a
s Mec
hano
sens
ors .
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1.3.
4 C
alci
um S
igna
lling
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1.3.
5 C
ell M
orph
olog
y ....
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1.4
HY
POTH
ESIS
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CH
APT
ER
TW
O: P
APE
R M
AN
USC
RIP
T ...
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2.1
INTR
OD
UC
TIO
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2.2
MET
HO
DS .
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3R
ESU
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2.4
DIS
CU
SSIO
N ...
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8 2.
5FI
GU
RES
&LE
GEN
DS .
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5
CH
APT
ER
TH
RE
E: D
ISC
USS
ION
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..... 7
6
CH
APT
ER
FO
UR
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FER
EN
CE
S ...
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. 94
CH
APT
ER
FIV
E: A
PPE
ND
IX A
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. 109
5.2
BO
VIN
E A
RTI
CU
LAR
CA
RTI
LAG
E C
HO
ND
RO
CY
TES
EXPR
ESSI
ON
OF
THE
L-TY
PE V
GC
C ..
......
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... 11
1 5.
3TI
SSU
E FO
RM
ATI
ON
NO
T A
FFEC
TED
WIT
H IN
HIB
ITO
R T
REA
TMEN
TS ..
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.... 1
12
5.4
MT1
-MM
PEX
PRES
SIO
N IN
CH
ON
DR
OC
YTE
S C
ULT
UR
ED O
N M
ON
OLA
YER
.....
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.. 113
5.
6C
HO
ND
RO
CY
TE C
ELL
AR
EAD
ISTR
IBU
TIO
NS
FOLL
OW
ING
STI
MU
LATI
ON
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5.7
CEL
L SP
REA
DIN
G A
PPEA
RS
TO B
E D
EPTH
-DEP
END
ENT
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.. 116
CH
APT
ER
SIX
: APP
EN
DIX
B ..
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. 117
v
LIS
T O
F FI
GU
RE
S
FIG
UR
E 1.
1:A
RTI
CU
LAR
CA
RTI
LAG
E TI
SSU
E IS
FO
RM
&FU
NC
TIO
N ..
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..... 2
FI
GU
RE
1.2:
THE
CO
MPO
SITI
ON
OF
AR
TIC
ULA
R C
AR
TILA
GE .
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. 6
FIG
UR
E 1.
3:TH
E PR
OTE
OG
LYC
AN
AG
GR
ECA
N ..
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.. 8
FIG
UR
E 1.
4:ZO
NA
L A
RC
HIT
ECTU
RE
OF
AR
TIC
ULA
R C
AR
TILA
GE
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..... 1
0 FI
GU
RE
1.5:
DIA
GR
AM
MA
TIC
REP
RES
ENTA
TIO
N O
F A
BIP
HA
SIC
CO
NST
RU
CT
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.... 2
0 FI
GU
RE
1.6:
APP
LIC
ATI
ON
OF
CO
NFI
NED
CO
MPR
ESSI
VE
MEC
HA
NIC
AL
FOR
CES
.....
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.. 23
FIG
UR
E 2.
1:C
HA
RA
CTE
RIZ
ATI
ON
OF
CH
ON
DR
OC
YTE
RES
PON
SE T
O C
YC
LIC
CO
MPR
ESSI
ON
.....
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. 67
FIG
UR
E 2.
2:EX
TRA
CEL
LULA
R C
ALC
IUM
REG
ULA
TES
CEL
L SP
REA
DIN
G ...
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... 68
F I
GU
RE
2.3:
REG
ULA
TIO
N O
F C
ELL
SPR
EAD
ING
&R
ETR
AC
TIO
N B
Y D
ISC
TIN
CT
CA
LCIU
M C
HA
NN
ELS
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... 69
F I
GU
RE
2.4:
MT1
-MM
PU
PREG
ULA
TIO
N IS
DEP
END
ENT
UPO
N E
XTR
AC
ELLU
LAR
CA
LCIU
M ...
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... 70
FI
GU
RE
2.5:
CA
LCIU
M C
HA
NN
ELS
REG
ULA
TE M
T1-M
MP
EXPR
ESSI
ON
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71
F IG
UR
E 2.
6:C
ALC
IUM
ION
OPH
OR
E R
EVER
SES
THE
EFFE
CTS
OF
CH
AN
NEL
INH
IBIT
ION
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... 72
FI
GU
RE
2.7:
SRC
INH
IBIT
OR
PP2
AB
OLI
SHED
CEL
L SP
REA
DIN
G &
MT1
-MM
PU
PREG
ULA
TIO
N ..
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.... 7
3 FI
GU
RE
2.8:
CO
NV
ERG
AN
CE
OF
INTE
GR
IN A
ND
CA
LCIU
MSI
GN
ALL
ING
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.. 74
FIG
UR
E 2.
9:PR
OPO
SED
MEC
HA
NIS
M ..
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.. 75
FIG
UR
E 5.
1:C
HO
ND
RO
CY
TE A
CTI
N C
YTO
SKEL
ETO
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.. 70
FIG
UR
E 5.
2:B
OV
INE
AR
TIC
ULA
R C
AR
TILA
GE
CH
ON
DR
OC
YTE
S EX
PRES
SIO
N T
HE
L-TY
PE V
GC
C ..
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. 71
FIG
UR
E 5.
3:TI
SSU
E FO
RM
ATI
ON
IS N
OT
AFF
ECTE
D W
ITH
INH
IBIT
OR
TR
EATM
ENT .
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.. 72
FIG
UR
E 5.
4:M
T1-M
MP
EXPR
ESSI
ON
IN C
HO
ND
RO
CY
TES
CU
LTU
RED
ON
MO
NO
LAY
ER ..
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.... 7
3 FI
GU
RE
5.5:
PHO
SPH
OR
YLA
TIO
N O
F ER
KFO
LLO
WIN
G M
ECH
AN
ICA
LST
IMU
LATI
ON
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..... 7
4 FI
GU
RE
5.6:
CH
ON
DR
OC
YTE
CEL
L A
REA
FOLL
OW
ING
MEC
HA
NIC
AL
STIM
ULA
TIO
N...
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.... 7
5 FI
GU
RE
5.7:
CEL
L SP
REA
DIN
G A
PPEA
RS
TO B
E D
EPTH
DEP
END
ENT
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. 76
FIG
UR
E 6.
1:G
ENE
EXPR
ESSI
ON
OF
CH
ON
DR
OC
YTE
SUB
POPU
LATI
ON
PH
ENO
TYPE
MA
RK
ERS
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.. 137
FI
GU
RE
6.2:
HIS
TOLO
GIC
AL
APP
EAR
AN
CE
OF
TISS
UES
AFT
ER C
ULT
UR
E ...
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138
FIG
UR
E 6.
3:EF
FEC
TO
F M
ECH
AN
ICA
L ST
IMU
LATI
ON
ON
MA
TRIX
AC
CU
MU
LATI
ON
.....
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139
FIG
UR
E 6.
4:M
T1-M
MP
GEN
E &
PRO
TEIN
CH
AN
GES
FO
LLO
WIN
G M
ECH
AN
ICA
L ST
IMU
LATI
ON
......
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.... 1
40
FIG
UR
E 6.
5:M
MP-
13G
ENE
&PR
OTE
IN C
HA
NG
ES F
OLL
OW
ING
MEC
HA
NIC
AL
STIM
ULA
TIO
N ...
......
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......
......
..... 1
41
FIG
UR
E 6.
6:A
CC
UM
ULA
TIO
N O
FM
ATR
IX M
OLE
CU
LES
BY
DEE
P ZO
NE
CH
ON
DR
OC
YTE
S ...
......
......
......
......
......
......
......
142
LIS
T O
F T
AB
LE
S
TAB
LE1.
1:C
OLL
AG
ENS
OF
AR
TIC
ULA
R C
AR
TILA
GE
......
......
......
......
......
......
......
......
......
......
......
......
......
......
......
......
..... 7
TA
BLE
1.2:
PRO
TEO
GLY
CA
NS
OF
AR
TIC
ULA
R C
AR
TILA
GE
......
......
......
......
......
......
......
......
......
......
......
......
......
......
......
... 9
TAB
LE1.
3:C
UR
REN
TLY
KN
OW
N IN
TEG
RIN
S ...
......
......
......
......
......
......
......
......
......
......
......
......
......
......
......
......
......
......
. 30
TAB
LE1.
4:C
LASS
IFIC
ATI
ON
OF
VO
LTA
GE-
GA
TED
CA
LCIU
M C
HA
NN
ELS
......
......
......
......
......
......
......
......
......
......
......
.. 36
TAB
LE6.
1:D
NA
CO
NTE
NT
OF
TISS
UE
FOR
MED
BY
CH
ON
DR
OC
YTE
SU
BPO
PULA
TIO
NS
......
......
......
......
......
......
......
..... 1
45
1
CH
APT
ER O
NE:
BA
CK
GR
OU
ND
1.1
Intr
oduc
tion
With
the
wor
ld p
opul
atio
n co
ntin
ually
agi
ng, c
artil
age-
rela
ted
path
olog
ies
are
beco
min
g m
ore
pron
ounc
ed a
nd th
eir
toll
on th
e w
orld
s’ h
ealth
car
e sy
stem
s in
eve
r in
crea
sing
. It i
s es
timat
ed
that
ove
r 36
mill
ion
indi
vidu
als,
in t
he U
nite
d St
ates
alo
ne,
are
affli
cted
with
som
e fo
rm o
f
carti
lage
pat
holo
gy a
risen
from
arth
ritis
or s
ports
inju
ry [B
uckw
alte
r & M
anki
n, 9
8b].
Giv
en th
e
debi
litat
ing
natu
re o
f se
vere
joi
nt p
ain,
and
the
lim
ited
abili
ty o
f ca
rtila
ge t
o se
lf-re
pair,
gre
at
effo
rt ha
s be
en d
edic
ated
to e
luci
datin
g w
ays
to re
pair
nativ
e ca
rtila
ge ti
ssue
. Unf
ortu
nate
ly, d
ue
to th
e tis
sue’
s co
mpl
ex b
iom
echa
nica
l pro
perti
es, l
ittle
pro
gres
s ha
s be
en m
ade
and
mos
t clin
ical
treat
men
ts re
sort
to p
rost
hetic
repl
acem
ents
of t
he d
amag
ed ti
ssue
. Con
sequ
ently
, the
cre
atio
n of
bioe
ngin
eere
d ar
ticul
ar c
artil
age
tissu
e ha
s be
en s
ugge
sted
as
a vi
able
pos
sibl
e al
tern
ativ
e to
curr
ent c
linic
al a
ppro
ache
s.
1.1.
2 C
artil
age:
Rol
e &
Fun
ctio
n
The
desi
gnat
ion
of c
artil
age
is g
iven
to
a sp
ecia
lized
typ
e of
den
se c
onne
ctiv
e tis
sue
that
is
com
pose
d of
cho
ndro
cyte
cel
ls e
mbe
dded
in a
rich
mat
rix c
ompo
sed
of m
ainl
y of
pro
teog
lyca
ns,
colla
gens
and
ela
stin
fibr
es. D
epen
ding
on
the
rela
tive
abun
danc
e of
thes
e co
mpo
nent
s ca
rtila
ge
is fu
rther
cla
ssifi
ed in
to th
ree
dist
inct
type
s, no
tabl
y: e
last
ic c
artil
age,
fibr
ocar
tilag
e, a
nd a
rticu
lar
(hya
line)
carti
lage
[Te
men
off
& M
ikos
, 00
b].
Brie
fly,
elas
tic c
artil
age
is c
hara
cter
ized
by
a
sign
ifica
nt p
rese
nce
of e
last
in in
the
extra
cellu
lar m
atrix
(EC
M) a
nd is
foun
d in
the
ear a
nd n
ose
of in
divi
dual
s. Fi
broc
artil
age,
whi
ch is
fou
nd a
t the
end
s of
tend
ons
and
ligam
ents
, pos
sess
es a
high
er c
onst
ituen
cy o
f col
lage
ns. H
yalin
e ca
rtila
ge, i
n tu
rn, p
osse
sses
a w
hite
, gla
ssy
appe
aran
ce
and
is m
ost o
ften
foun
d in
the
linin
g of
arti
cula
ting
surf
aces
[Mow
& L
ai, 0
9].
2
1.1.
3 A
rtic
ular
Car
tilag
e
Arti
cula
r ca
rtila
ge, a
spe
cial
ized
for
m o
f hy
alin
e ca
rtila
ge, i
s a
uniq
ue c
onne
ctiv
e tis
sue
of th
e
mus
culo
skel
etal
sys
tem
tha
t fo
rms
a 3-
4mm
thi
ck l
ayer
cov
erin
g th
e ar
ticul
atin
g en
ds o
f
diar
thro
dial
(sy
novi
al)
join
ts,
whi
ch e
nabl
es s
moo
th a
rticu
latio
n an
d co
nseq
uent
ly m
ovem
ent
and
activ
ity (
Figu
re 1
) [M
ow &
Lai
, 09]
. Due
to th
eir
role
of
prov
idin
g fle
xion
and
ext
ensi
on,
join
ts a
re r
epea
tedl
y su
bjec
t to
com
plic
ated
mot
ions
at
vary
ing
spee
ds.
Thro
ugho
ut s
uch
activ
ities
, joi
nts
are
expo
sed
to a
wid
e ra
nge
of f
orce
s, at
tim
es to
talin
g up
to s
ever
al ti
mes
the
body
wei
ght [
Mow
& L
ai, 0
9;B
row
er &
Hsu
, 69]
. The
pre
senc
e of
arti
cula
r car
tilag
e at
the
ends
of d
iarth
rodi
al j
oint
s se
rves
a d
ual
purp
ose:
1)
even
tra
nsfe
r of
for
ces
to n
eigh
bour
ing
subc
hond
ral
bone
pla
tes
and,
2)
alon
g w
ith t
he s
ynov
ial
fluid
to
prov
ide
a ne
arly
fric
tionl
ess
surf
ace
for t
he jo
int-f
orm
ing
bone
s to
mov
e fr
eely
ove
r one
ano
ther
dur
ing
mot
ion
[Hub
eret
al.,
00].
Figu
re 1
: A
rticu
lar
carti
lage
tis
sue
is a
spe
cial
ized
con
nect
ive
tissu
e lin
ing
the
ends
of
artic
ulat
ing
join
ts a
nd s
erve
s a
dual
fu
nctio
n of
: 1)
pro
vidi
ng f
or a
nea
rly f
rictio
nles
s jo
in m
ovem
ent,
and
2) f
acili
tatin
g th
e tra
nsfe
r or
mec
hani
cal
load
s du
ring
mov
emen
t. Ill
ustra
tion
adap
ted
from
Mow
& L
ai, 1
979.
Whe
n ca
rtila
ge is
load
ed (i
.e. d
urin
g w
alki
ng o
r phy
sica
l act
ivity
) an
initi
al in
crea
se in
pre
ssur
e
sque
ezes
tis
sue
fluid
out
of
the
solid
ext
race
llula
r m
atrix
, ca
usin
g th
e tis
sue’
s m
atrix
to
cons
olid
ate
and
the
cells
pre
sent
to u
nder
go c
ompr
essi
ve d
efor
mat
ion.
How
ever
, the
incr
ease
in
nega
tive
char
ge d
ensi
ty d
ue t
o pr
otei
ns e
mbe
dded
in
the
extra
cellu
lar
mat
rix,
alon
g w
ith t
he
3
rela
tivel
y lo
w fl
uid
perm
eabi
lity
of th
e m
atrix
, ass
ists
in m
inim
izin
g w
ater
esc
ape.
The
resu
lting
hydr
osta
tic p
ress
ure
(~95
% o
f re
sist
ance
), al
ong
with
the
sol
id e
xtra
cellu
lar
mat
rix (
~5%
resi
stan
ce),
is th
en a
ble
to w
ithst
and
the
com
pres
sive
for
ces
expe
rienc
ed b
y th
e jo
int [
Pear
leet
al.,
05;W
ong
& C
arte
r, 03
].
The
abili
ty o
f arti
cula
r car
tilag
e to
with
stan
d su
ch li
fe-lo
ng ta
mec
hani
cal l
oads
with
out a
ny
sign
ifica
nt e
rosi
on in
hea
lthy
indi
vidu
als,
is m
ade
poss
ible
by
the
tissu
e’s
uniq
ue m
orph
olog
ical
and
biom
echa
nica
l pro
perti
es [
Gro
dzin
sky
et a
l., 0
0;M
ow &
Lai
, 09;
Pear
le, W
arre
n &
Rod
eo,
05].
Nor
mal
and
mod
erat
e jo
int
load
ing
serv
es t
o m
aint
ain
carti
lage
hea
lth a
nd f
unct
ion
thro
ugho
ut th
e lif
etim
e of
an
indi
vidu
al. I
t is
wel
l acc
epte
d th
at m
echa
nica
l for
ces,
and
chan
ges
in l
oadi
ng,
resu
lt in
met
abol
ic a
ltera
tions
tha
t al
low
the
tis
sue
to r
espo
nd t
o th
ese
chan
ges
[Eck
stei
net
al.,
06]
. Fai
lure
to p
rope
rly re
spon
d to
suc
h ch
ange
s re
sults
in ti
ssue
pat
holo
gy a
nd
dege
nera
tion,
of w
hich
ost
eoar
thro
sis i
s a p
rime
exam
ple
[Buc
kwal
ter,
95].
1.1.
4 C
ompo
sitio
n &
Arc
hite
ctur
e of
Art
icul
ar C
artil
age
Sim
ilarly
to
othe
r ca
rtila
ges,
artic
ular
car
tilag
e is
con
side
red
to b
e a
perm
eabl
e, v
isoe
last
ic
mat
eria
l co
nsis
ting
of t
hree
prin
cipa
l ph
ases
: a
solid
pha
se m
ainl
y co
mpo
sed
of a
col
lage
n
netw
ork
and
embe
dded
pro
teog
lyca
ns;
a flu
id p
hase
of
wat
er;
and
an i
on p
hase
con
tain
ing
elec
troly
tes
and
diss
olve
d ga
ses
[Hub
er, T
rattn
ig &
Lin
tner
, 00]
. A d
istin
guis
hing
cha
ract
eris
tic
of a
rticu
lar
carti
lage
, as
com
pare
d to
mos
t ot
her
tissu
es,
is t
he l
ack
of v
ascu
lar,
neur
al a
nd
lym
phat
ic i
nner
vatio
n. A
s a
resu
lt, a
ll nu
trien
t an
d w
aste
exc
hang
es o
ccur
sol
ely
thro
ugh
diff
usio
n to
and
from
the
syno
vial
flui
d. T
his
fluid
, whi
ch a
lso
serv
es to
lubr
icat
e th
e jo
int,
is a
plas
ma
ultra
filtra
te c
onta
inin
g el
ectro
lyte
s, sm
all m
olec
ules
and
glu
cose
[O'H
ara
et a
l., 9
0].
4
1.1.
5 C
hond
rocy
tes
Cho
ndro
cyte
s, th
e so
le c
ellu
lar
com
pone
nt o
f ar
ticul
ar c
artil
age,
con
stitu
te u
p to
5%
of
the
tissu
e’s
tota
l vo
lum
e, a
nd a
re e
mbe
dded
in
an e
xtra
cellu
lar
mat
rix (
ECM
). Th
e ce
lls o
rigin
ate
from
mes
ench
ymal
ste
mce
lls (
MSC
s) w
hich
, in
mat
ure
indi
vidu
als,
are
loca
lized
to
the
bone
mar
row
. C
hond
rocy
tes
aris
e du
ring
embr
yoge
nesi
s w
hen
mes
ench
ymal
ste
m c
ells
und
ergo
diff
eren
tiatio
n an
d be
gin
secr
etin
g ca
rtila
gino
us m
atrix
[Buc
kwal
ter &
Man
kin,
98a
]. W
ith ti
me,
chon
droc
ytes
des
tined
to
beco
me
bone
acq
uire
a h
yper
troph
ic p
heno
type
and
beg
in p
rodu
cing
prot
eins
that
are
requ
ired
for m
atrix
cal
cific
atio
n. O
ther
cho
ndro
cyte
s, on
the
perip
hery
, con
tinue
to s
ecre
te c
olla
gen
and
othe
r m
atrix
mol
ecul
es, a
nd c
reat
e th
e ch
arac
teris
tic E
CM
of
carti
lage
.
The
sign
als
cont
rolli
ng i
n w
hich
dire
ctio
n th
e ch
ondr
ocyt
es m
atur
e ha
ve n
ot b
een
fully
eluc
idat
ed. M
atur
e ch
ondr
ocyt
es a
re c
ompl
etel
y en
case
d in
mat
rix, a
nd h
ave
a lim
ited
abili
ty to
prol
ifera
te in
viv
o. In
add
ition
, due
to th
eir r
ole
of m
aint
aini
ng th
e EC
M th
roug
h se
cret
ion
of it
s
cons
titue
nts,
they
pos
sess
pro
min
ent
endo
plas
mic
ret
icul
um a
nd g
olgi
app
arat
i [T
emen
off
&
Mik
os, 0
0a].
Alth
ough
cho
ndro
cyte
s w
ithin
arti
cula
r car
tilag
e ar
e sa
id to
be
of a
sin
gle
cell
type
,
min
or b
ut i
mpo
rtant
phe
noty
pic
diff
eren
ces
exis
t be
twee
n ch
ondr
ocyt
es d
epen
ding
on
thei
r
rela
tive
posi
tion
in th
e tis
sue.
For
inst
ance
, cho
ndro
cyte
s lo
cate
d at
the
artic
ular
sur
face
are
mor
e
flatte
ned
in m
orph
olog
y an
d ar
e kn
own
to e
xpre
ss p
rote
ins
requ
ired
for l
ubric
atio
n [S
chum
ache
r
et a
l., 9
4]. I
n co
ntra
st, c
hond
rocy
tes
resi
ding
clo
ser
to t
he s
ubch
ondr
al b
one
are
roun
ded
and
expr
ess p
rote
ins t
hat a
re re
quire
d fo
r mat
rix c
alci
ficat
ion
[Xu
et a
l., 9
4].
Ow
ing
to t
he l
ow c
ell
dens
ity,
artic
ular
car
tilag
e is
sai
d to
be
a m
atri
x-co
ntin
uous
stru
ctur
e,
indi
catin
g th
at e
ach
cell
is f
ully
sur
roun
ded
by e
xtra
cellu
lar
mat
rix m
olec
ules
, a
feat
ure
that
proh
ibits
bot
h ce
ll-to
-cel
l co
ntac
t an
d ce
llula
r m
igra
tion
thro
ugh
the
ECM
. D
espi
te t
heir
low
num
bers
, th
e ch
ondr
ocyt
es p
lay
a cr
ucia
l ro
le i
n re
gula
ting
tissu
e pr
oper
ties
thro
ugh
the
5
synt
hesi
s, se
cret
ion,
org
aniz
atio
n an
d de
grad
atio
n of
the
sur
roun
ding
EC
M [
Kin
ner
et a
l.,
05;M
uir,
95].
The
mec
hani
cal
char
acte
ristic
s of
the
tis
sue
depe
nd o
n th
e pr
oper
ties
and
cons
titue
nts
of
the
ECM
, pr
imar
ily:
1)
colla
gens
, w
hich
pr
ovid
e te
nsile
st
reng
th.
2)
Prot
eogl
ycan
s, w
hich
are
neg
ativ
ely
char
ged
and
trapp
ed w
ithin
the
colla
gen
netw
ork
and
attra
ct
catio
nsan
d w
ater
, th
us a
llow
ing
the
tissu
e to
with
stan
d co
mpr
essi
on. 3
) Th
e in
ters
titia
l flu
id
who
se c
ompo
sitio
n in
fluen
ces c
ellu
lar r
espo
nses
to m
echa
nica
l loa
ding
[Hal
let a
l., 9
6].
1.1.
6 E
xtra
cellu
lar M
atri
x
The
extra
cellu
lar m
atrix
of a
rticu
lar c
artil
age,
whi
ch m
ay b
e co
nsid
ered
as
a fib
er-r
einf
orce
d ge
l,
is p
rimar
ily c
ompo
sed
of w
ater
and
mac
rom
olec
ules
suc
h as
col
lage
ns,
prot
eogl
ycan
s, an
d
nonc
olla
geno
us p
rote
ins.
Hum
an a
rticu
lar
carti
lage
has
a s
ubst
antia
l flu
id c
onte
nt (
prim
arily
wat
er b
ut a
lso
diss
olve
d io
ns, g
ases
and
met
abol
ites)
, whi
ch c
orre
spon
ds to
60-
80%
of t
he to
tal
wet
wei
ght
of t
he t
issu
e. T
he r
emai
ning
20-4
0% o
f th
e to
tal
tissu
e w
eigh
t is
attr
ibut
ed t
o a
varie
ty
of
extra
cellu
lar
mat
rix
mol
ecul
es,
prim
arily
: co
llage
ns
(50-
70%
, dr
y w
eigh
t),
prot
eogl
ycan
s (1
5-30
%, d
ry w
eigh
t), a
nd o
ther
non
-col
lage
nous
pro
tein
s (1
5-20
%, d
ry w
eigh
t)
(Fig
ure
2).
The
prec
ise
com
posi
tion
of m
acro
mol
ecul
es,
alon
g w
ith t
he f
luid
pha
se,
is
resp
onsi
ble
for
the
inhe
rent
stru
ctur
e an
d m
echa
nica
l pr
oper
ties
of a
rticu
lar
carti
lage
; an
inte
ract
ion
betw
een
the
fluid
pha
se a
nd th
e va
rious
mat
rix m
acro
mol
ecul
es p
rovi
des
the
tissu
e’s
stiff
ness
and
its r
esili
ence
to m
echa
nica
l loa
ds [P
earle
, War
ren
& R
odeo
, 05]
.
6
Figu
re 2
: Th
e co
mpo
sitio
n of
arti
cula
r ca
rtila
ge t
issu
e by
wei
ght
(cho
ndro
cyte
s ex
clud
ed):
The
extra
cellu
lar
mat
rix i
s fu
rther
di
vide
d in
to it
s con
stitu
ent c
olla
gens
, pro
teog
lyca
n an
d ot
her n
on-c
olla
geno
us p
rote
ins.
Col
lage
ns, w
hich
con
stitu
te th
e m
ajor
por
tion
of d
ry w
eigh
t of t
he E
CM
, are
a fa
mily
of p
rote
ins
that
con
sist
of
thre
e po
lype
ptid
e ch
ains
, cal
led
α ch
ains
, whi
ch h
ave
tripl
e-he
lix c
onfig
urat
ion.
To d
ate,
43
dist
inct
α c
hain
s ha
ve b
een
iden
tifie
d an
d ar
rang
e to
form
at l
east
27
know
n pr
otei
ns
that
are
def
ined
as
colla
gens
[Ey
re, 0
4]. D
epen
ding
on
the
parti
cula
r ty
pe o
f co
llage
n, th
e th
ree
cons
titue
nt α
cha
ins
may
all
be id
entic
al o
r di
ffer
ent.
How
ever
, reg
ardl
ess
of th
e is
ofor
m, a
ll α
chai
ns c
onta
in a
t lea
st o
ne d
omai
n co
mpo
sed
of re
peat
ing
–Gly
cine
-X-Y
seq
uenc
es to
faci
litat
e
the
corr
ect f
orm
atio
n of
the
tripl
e he
lix. G
lyci
ne, b
eing
the
smal
lest
am
ino
acid
, is
requ
ired
as
larg
er a
min
o ac
ids
wou
ld n
ot f
it in
to th
e re
stric
ted
spac
es in
the
cent
re o
f th
e tri
ple-
helix
. Mos
t
colla
gens
for
m s
upra
mol
ecul
ar a
ssem
blie
s, su
ch a
s fib
rils
and
netw
orks
, an
d ar
e di
vide
d in
to
seve
ral s
ubgr
oups
bas
ed o
n st
ruct
ural
and
fun
ctio
nal c
hara
cter
istic
s [C
rem
eret
al.,
98]
. Whi
le
the
mos
t abu
ndan
t and
cha
ract
eris
tic c
olla
gen
of a
rticu
lar c
artil
age
is C
olla
gen
II, o
ther
type
s ar
e
also
fou
nd to
a le
sser
ext
ent:
V, V
I, IX
, X a
nd X
I. Th
e ex
tens
ive
cros
s-lin
king
of
the
vario
us
colla
gen
type
s is
vita
l to
the
tis
sue’
s m
ater
ial
stre
ngth
[Ey
re,
04;P
oole
et a
l., 0
1].
A b
rief
over
view
of
the
colla
gens
fou
nd i
n ar
ticul
ar c
artil
age
tissu
e, a
nd t
heir
resp
ectiv
e ro
les,
is
7
pres
ente
d in
the
tabl
e be
low
(Tab
le I)
. Col
lage
n ty
pe I,
whi
ch is
not
nor
mal
ly fo
und
in th
e tis
sue,
is o
ften
used
as a
mar
ker f
or c
ellu
lar d
ediff
eren
tiatio
n du
ring
cultu
re [S
chna
bele
t al.,
02]
.
Tabl
e I:
Col
lage
ns a
re a
key
com
pone
nt o
f ar
ticul
ar c
artil
age’
s ex
trace
llula
r m
atrix
, and
con
stitu
te t
he g
reat
er p
ortio
n of
the
tis
sue’
s dr
y w
eigh
t. Th
ey a
re a
fam
ily o
f pr
otei
ns th
at c
onsi
st o
f th
ree
poly
pept
ide
chai
ns b
ound
in a
trip
le-h
elix
con
figur
atio
n.
List
ed b
elow
are
the
colla
gens
, alo
ng w
ith th
eir r
espe
ctiv
e fu
nctio
ns, t
hat a
re fo
und
in a
rticu
lar c
artil
age
tissu
e.
Col
lage
nFu
nctio
nR
efer
ence
Col
lage
n I
Usu
ally
not
pre
sent
in h
ealth
y ar
ticul
ar c
artil
age,
and
is
used
as
a m
arke
r fo
r ce
llula
r de
-diff
eren
tiatio
n du
ring
cultu
re
[Sch
nabe
l, M
arlo
vits
, Ec
khof
f, Fi
chte
l, G
otze
n,
Vec
sei
& S
chle
gel,
02]
Col
lage
n II
Form
s th
e ba
ckbo
ne o
f th
e ca
rtila
ge h
eter
opoy
mer
ic
fibril
s. R
espo
nsib
le f
or t
he t
ensi
le s
treng
th o
f tis
sue.
M
ost p
rom
inen
t car
tilag
e in
hea
lthy
tissu
e.
[Cre
mer
, R
oslo
niec
&
K
ang,
98
;Eyr
e,
04]
Col
lage
n V
Parti
cipa
tes
in t
he f
orm
atio
n of
the
fib
rilla
r co
llage
n ne
twor
k.[G
else
et a
l., 0
3]
Col
lage
n V
IU
biqu
itous
mat
rix c
ompo
nent
of
mos
t tis
sues
. Fo
und
pred
omin
atel
y in
th
e pe
ricel
lula
r m
atrix
(a
roun
d ch
ondr
ocyt
es).
Inte
ract
s w
ith
a va
riety
of
EC
M
prot
eogl
ycan
s.
[Cha
nget
al
.,97
]
Col
lage
n IX
Act
s as
a m
acro
mol
ecul
ar b
ridge
bet
wee
n co
llage
n fib
rils
and
betw
een
colla
gen
fibril
s an
d ot
her
ECM
co
mpo
nent
s. R
egul
ates
fib
ril s
ize.
Fou
nd p
redo
min
atel
y in
the
peric
ellu
lar m
atrix
(aro
und
chon
droc
ytes
).
[Poo
le, 9
7]
Col
lage
n X
Secr
eted
by
chon
droc
ytes
in th
e zo
ne o
f car
tilag
e th
at is
de
stin
ed t
o be
cal
cifie
d, a
nd i
n zo
nes
of s
econ
dary
os
sific
atio
n. P
rovi
des
supp
ort
as t
he c
artil
age
mat
rix i
s de
grad
ed d
urin
g en
doch
ondr
al o
ssifi
catio
n
[Alin
iet a
l., 9
4]
Prot
eogl
ycan
s, th
e se
cond
larg
est c
onst
ituen
t of
the
ECM
, are
a s
peci
fic c
lass
of
glyc
opro
tein
s
that
con
sist
of g
lyco
amin
ogly
can
(GA
G) c
hain
s cov
alen
tly b
ound
to th
e ce
ntra
l cor
e pr
otei
n. T
he
8
mos
t pr
edom
inan
t pr
oteo
glyc
an f
ound
in
artic
ular
car
tilag
e is
Agg
reca
n, w
hich
con
sist
s of
a
prot
ein
core
and
cov
alen
tly b
ound
ker
atan
sul
phat
e an
d ch
ondr
oitin
sul
phat
e gl
ycoa
min
ogly
can
chai
ns. A
ggre
can
mon
omer
s bi
nd to
hya
luro
nan,
an
un-b
ranc
hed
poly
sacc
harid
e (F
igur
e 3)
, to
form
la
rge
mul
ti-m
olec
ular
ag
greg
ates
th
at
are
dist
ribut
ed
with
in
the
colla
gen
netw
ork
(mol
ecul
ar w
eigh
t in
the
mill
ions
). Th
e in
tera
ctio
n be
twee
n th
e A
ggre
can
mon
omer
s an
d
hyal
uron
an is
fac
ilita
ted
and
stab
ilize
d by
a p
olyp
eptid
e te
rmed
Lin
k Pr
otei
n. S
uch
dist
ribut
ion
of p
rote
ogly
can
aggr
egat
es s
erve
s se
vera
l pu
rpos
es:
the
intri
nsic
ele
ctric
al c
harg
es o
f th
e
prot
eogl
ycan
s pr
ovid
e a
high
fix
ed-c
harg
e, a
nd t
he h
ydro
phili
c na
ture
ent
raps
wat
er a
nd t
hus
allo
ws t
he ti
ssue
to w
ithst
and
com
pres
sion
.
Figu
re 3
: A d
iagr
am o
f the
pro
teog
lyca
n ag
grec
an, w
hich
con
sist
s of
cov
alen
tly b
ound
ker
atan
sul
phat
e an
d ch
ondr
oitin
sul
phat
e gl
ycoa
min
ogly
can
(GA
G)
chai
ns.
Agg
reca
n m
onom
ers
bind
to
hyal
uron
an,
via
link
prot
ein,
to
form
lar
ge m
ulti-
mol
ecul
ar
aggr
egat
es th
at a
re d
istri
bute
d w
ithin
the
colla
gen
netw
ork
of th
e ar
ticul
ar c
artil
age.
Agg
reca
n se
rves
a p
rimar
y ro
le o
f pro
vidi
ng
the
osm
otic
resi
stan
ce n
eces
sary
for c
artil
age
to re
sist
com
pres
sive
load
s. D
iagr
am ta
ken
from
(Pea
rleet
al.,
200
5)
Oth
er s
mal
ler
prot
eogl
ycan
s, be
long
ing
to a
fam
ily o
f le
ucin
e-ric
h pr
oteo
glyc
ans
(SLR
Ps),
are
also
fou
nd in
the
ECM
, alth
ough
to a
less
er e
xten
t. D
espi
te th
eir
rela
tive
low
abu
ndan
ce in
the
ECM
, kno
ckou
t stu
dies
hav
e in
dica
ted
that
they
pla
y a
cruc
ial r
ole
in p
rovi
ding
mat
rix s
tabi
lity
9
(Knu
dson
& K
nuds
on, 2
001;
Rou
ghle
y, 2
006;
Pool
eet
al.,
200
1). T
he p
rote
ogly
cans
, alo
ng w
ith
thei
r res
pect
ive
role
s are
list
ed in
the
tabl
e be
low
(Tab
le II
).
Tabl
e II:
Pro
teog
lyca
ns,
the
seco
nd l
arge
st co
nstit
uent
of
the
ECM
, ar
e a
spec
ific
clas
s of
gly
copr
otei
ns t
hat
cons
ist o
f gl
ycoa
min
ogly
can
chai
ns c
oval
ently
bou
nd to
the
cent
ral c
ore
prot
ein.
List
ed b
elow
are
they
maj
or p
rote
ogly
cans
foun
d in
arti
cula
r ca
rtila
ge ti
ssue
, alo
ng w
ith th
eir r
espe
ctiv
e fu
nctio
ns (w
here
kno
wn)
. Tab
le a
dapt
ed fr
om (P
oole
et a
l., 2
001)
Prot
eogl
ycan
Func
tion
Agg
reca
nTh
roug
h bi
ndin
g to
hya
luro
nan,
via
Lin
k Pr
otei
n, A
ggre
can
aggr
egat
es f
orm
a s
truct
ural
bac
kbon
e th
at i
s re
sista
nt t
o co
mpr
essio
n an
d pr
ovid
es s
tiffn
ess.
It is
also
the
maj
ority
pr
oteo
glyc
an b
y w
eigh
t.Pe
rleca
nLo
cate
d on
th
e ce
ll su
rface
an
d fa
cilit
ates
ce
ll-m
atrix
in
tera
ctio
ns a
nd a
dhes
ion.
Big
lyca
nB
ind
TGF-
β. F
ound
onl
y in
the
per
icel
lula
r m
atrix
. Pr
ecise
fu
nctio
n ha
s not
bee
n el
ucid
ated
.D
ecor
inB
ind
TGF-
β.
Foun
d on
ly
in
the
inte
rterri
toria
l m
atrix
. R
egul
ates
col
lage
n fib
ril fo
rmat
ion.
Equ
imol
ar to
agg
reca
n an
d is
conc
entra
ted
in p
eric
ellu
lar s
ites.
Ver
sican
Reg
ulat
es th
e fo
rmat
ion
of c
olla
gen
mac
rofib
rils.
Last
ly, o
ther
non
-col
lage
nous
pro
tein
s co
nstit
ute
the
rem
aini
ng 1
5-20
% o
f the
EC
M. B
elon
ging
to th
is g
roup
of p
rote
ins
is L
ink
Prot
ein,
whi
ch a
cts
as a
link
bet
wee
n hy
alur
onan
and
Agg
reca
n,
as w
ell
as s
ever
al p
rote
ins
invo
lved
in
cell-
mat
rix b
indi
ng:
Cho
ndro
adhe
rin a
nd C
D44
. A
lso
pres
ent
is C
artil
age
Olig
omer
ic P
rote
in t
hat
bind
s to
Col
lage
n Ty
pe I
I an
d is
bel
ieve
d to
be
invo
lved
in m
acro
fibril
ass
embl
y.
1.1.
7 Zo
nal a
rchi
tect
ure
Mat
ure
artic
ular
car
tilag
e po
sses
ses
a zo
nal a
rchi
tect
ure,
and
is o
ften
clas
sifie
d in
to fo
ur d
istin
ct
zone
s in
suc
ceed
ing
horiz
onta
l sec
tions
. Fro
m th
e ar
ticul
ar s
urfa
ce a
nd p
roce
edin
g de
eper
to th
e
subc
hond
ral
bone
, ca
rtila
ge i
s se
para
ted
into
the
sup
erfic
ial,
mid
dle
(als
o re
ferr
ed t
o as
trans
ition
al a
nd ta
ngen
tial),
dee
p (r
adia
l),an
d ca
lcifi
ed z
ones
(fig
ure
4)[P
earle
, War
ren
& R
odeo
,
10
05].
The
zone
s va
ry in
thei
r stru
ctur
e an
d co
mpo
sitio
n, a
nd it
is s
ugge
sted
that
eac
h zo
ne p
lays
a
disc
rete
ro
le
in
resp
ondi
ng
to
mec
hani
cal
load
s. A
dditi
onal
ly,
thou
gh
artic
ular
ca
rtila
ge
chon
droc
ytes
are
cat
egor
ized
as
poss
essi
ng id
entic
al p
heno
type
s, tra
nsie
nt m
etab
olic
diff
eren
ces
betw
een
chon
droc
ytes
of
diff
eren
t si
zes
and
shap
es i
n di
ffer
ent
zona
l lo
catio
ns h
ave
been
obse
rved
in-v
itro
[Dar
ling
et a
l., 0
4;H
ause
lman
net
al.,
98;
Hid
aka
et a
l., 0
6;Sc
hmid
tet a
l., 0
4].
Figu
re 4
: Mat
ure
artic
ular
car
tilag
e po
sses
ses
a zo
nal a
rchi
tect
ure,
and
can
be
clas
sifie
d in
to f
our
dist
inct
zon
es in
suc
ceed
ing
horiz
onta
l sec
tions
. Fro
m th
e su
rfac
e an
d pr
ocee
ding
dee
per t
owar
ds th
e su
bcho
ndra
l bon
e ar
e th
e: s
uper
ficia
l, m
iddl
e, d
eep
and
the
calc
ified
zon
es.
The
zone
s va
ry i
n th
eir
com
posi
tion,
fib
ril a
lignm
ent,
and
the
resi
ding
cho
ndro
cyte
s’ p
heno
type
s. It
is
sugg
este
d th
at e
ach
zone
pla
ys a
dis
cret
e ro
le in
resp
ondi
ng to
mec
hani
cal l
oads
. Illu
stra
tion
adap
ted
from
(Pea
rleet
al.,
200
5)
The
supe
rfic
ial z
one,
whi
ch c
onst
itute
s th
e up
per-
mos
t 10-
20%
of c
artil
age,
is th
e th
inne
st z
one
of a
rticu
lar
carti
lage
and
for
ms
the
glid
ing
surf
ace.
In
com
paris
on t
o th
e ot
her
zone
s, th
e
supe
rfic
ial z
one
has
the
high
est c
onte
nt o
f w
ater
(~8
4%, w
et w
eigh
t), T
ype
II c
olla
gen
(~85
%,
dry
wei
ght)
and
the
low
est
cont
ent
of p
rote
ogly
cans
(~1
5%, d
ry w
eigh
t). T
he d
ense
ly p
acke
d
colla
gen
fibre
s, co
llect
ivel
y kn
own
as la
min
a sp
lend
ens,
are
thin
in
diam
eter
and
are
orie
nted
para
llel t
o th
e ar
ticul
ar s
urfa
ce. T
he p
aral
lel a
rran
gem
ent d
eter
min
es th
e m
echa
nica
l pro
perti
es
of th
e tis
sue,
by
prov
idin
g te
nsile
stre
ngth
that
aid
s in
res
istin
g sh
ear
stre
sses
pro
duce
d du
ring
mov
emen
t. It
has
also
been
spe
cula
ted
that
suc
h an
arr
ange
men
t act
s as
a b
arrie
r, an
d pr
even
ts
the
pass
age
of l
arge
r m
olec
ules
(e.
g. a
ntib
odie
s) f
rom
the
syn
ovia
l flu
id i
nto
carti
lage
. Th
e
11
resi
dent
cho
ndro
cyte
s po
sses
s a
decr
ease
d ce
llula
r vol
ume,
and
are
flat
tene
d al
ong
the
dire
ctio
n
of s
hear
stre
ss (p
aral
lel t
o su
rfac
e) [H
uber
, Tra
ttnig
& L
intn
er, 0
0]. O
f the
pro
teog
lyca
ns fo
und
in t
his
zone
, de
corin
and
big
lyca
n co
nstit
ute
the
pred
omin
ant
ones
[Po
ole
et a
l., 9
6].
An
addi
tiona
l pro
teog
lyca
n, S
uper
ficia
l Zon
e Pr
otei
n (S
ZP),
is u
niqu
ely
expr
esse
d in
this
zon
e an
d
is i
nvol
ved
in t
he l
ubric
atio
n at
the
joi
nt-c
artil
age
inte
rfac
e [S
chum
ache
r, B
lock
, Sc
hmid
,
Ayd
elot
te &
Kue
ttner
, 94
]. R
ecen
t st
udie
s su
gges
ted
that
the
sup
erfic
ial
zone
con
tain
s a
subp
opul
atio
n of
cho
ndro
cyte
s w
ith p
roge
nito
r-lik
e ch
arac
teris
tics,
and
it ha
s be
en s
pecu
late
d
that
the
cel
ls m
ay h
ave
the
capa
bilit
y to
pro
duce
car
tilag
e w
ith h
ealth
y st
ruct
ural
and
bioc
hem
ical
pro
perti
es [D
owth
wai
teet
al.,
04]
.
Dire
ctly
bel
ow t
he s
uper
ficia
l zo
ne i
s th
e in
term
edia
te (
also
kno
wn
as t
rans
ition
al o
r m
iddl
e)
zone
, whi
ch c
orre
spon
ds to
the
larg
est t
he z
one
in te
rms
of th
ickn
ess
(40-
60%
of f
ullt
hick
ness
).
Due
to th
e la
yer’
s pu
rpos
e of
ser
ving
as
a tra
nsiti
on b
etw
een
the
supe
rfic
ial a
nd d
eep
zone
s, th
e
cont
ents
of
wat
er, c
olla
gen
and
prot
eogl
ycan
s (o
f w
hich
agg
reca
n is
the
mos
t pr
omin
ent)
are
inte
rmed
iate
and
are
in-b
etw
een
the
cont
ents
of t
he su
perf
icia
l and
dee
p zo
nes [
Pear
le, W
arre
n &
Rod
eo,
05].
The
rela
tive
abun
danc
e of
pro
teog
lyca
ns p
rovi
des
the
mid
dle
zone
with
the
appr
opria
te m
echa
nica
lpr
oper
ties
that
are
req
uire
d to
with
stan
d an
d ab
sorb
the
com
pres
sive
forc
es t
rans
mitt
ed t
o th
e jo
int.
The
chon
droc
ytes
in
this
zon
e ar
e sp
heric
al a
nd a
re r
ich
in
synt
hetic
org
anel
les
such
as
the
Endo
plas
mic
Ret
icul
um a
nd G
olgi
bod
ies.
The
colla
gen
fibril
s
are
larg
er c
ompa
red
to s
uper
ficia
l zon
e an
d ar
e ar
rang
ed r
ando
mly
with
res
pect
to th
e ar
ticul
ar
surf
ace
[Poo
le,
Koj
ima,
Yas
uda,
Mw
ale,
Kob
ayas
hi &
Lav
erty
, 01
]. A
cha
ract
eris
tic p
rote
in
rest
ricte
d to
the
mid
dle
zone
is th
e C
artil
age
Inte
rmed
iate
Lay
er P
rote
in (
CIL
P), a
nd w
hile
its
exac
t fu
nctio
n ha
s no
t be
en f
ully
elu
cida
ted
evid
ence
sug
gest
s its
am
ount
inc
reas
es w
ith a
ge
[Mor
iet a
l., 0
6].
12
The
deep
(or
rad
ial)
zone
has
the
low
est
wat
er a
nd c
olla
gen
cont
ents
, ho
wev
er t
he c
olla
gen
fibril
s are
of t
he la
rges
t dia
met
er a
nd p
rote
ogly
can
cont
ent i
s max
imal
. The
cho
ndro
cyte
s pos
sess
seve
ral
prop
ertie
s th
at a
re u
sual
ly a
ttrib
uted
to
hype
rtrop
hic-
like
chon
droc
ytes
, pr
imar
ily:
expr
essi
on o
f al
kalin
e ph
osph
atas
e an
d co
llage
n Ty
pe X
. Th
e ce
lls a
re a
ligne
d in
col
umns
perp
endi
cula
r to
the
artic
ular
surf
ace
(as w
ell a
s to
the
subc
hond
ral p
late
). Si
mila
rly, t
he c
olla
gen
fibre
s ar
e or
ient
ed p
erpe
ndic
ular
to
the
join
t su
rfac
e an
d pe
netra
te t
he u
nder
lyin
g ca
lcifi
ed
carti
lage
whi
ch st
reng
then
s the
bon
ding
of c
artil
age
to b
one.
Imm
edia
tely
bel
ow th
e de
ep z
one
is
the
tidem
ark
whi
ch s
erve
s as
an
inte
rfac
e be
twee
n th
e de
ep z
one
and
the
calc
ified
lay
er. T
he
tidem
ark
has
not
been
ful
ly c
hara
cter
ized
; ho
wev
er i
t is
kno
wn
to c
onta
in c
ryst
als
of c
alci
um
salts
and
hya
luro
nan.
The
tide
mar
k is
ofte
n co
nsid
ered
to b
e th
e bo
unda
ry b
etw
een
carti
lage
and
bone
[H
uber
, Tra
ttnig
& L
intn
er, 0
0].
The
calc
ified
laye
r fac
ilita
tes
the
trans
mis
sion
of f
orce
s ac
ross
the
join
ts a
nd s
erve
s to
anc
hor t
he
carti
lage
to
the
subc
hond
ral
bone
. A
lthou
ghdu
ring
deve
lopm
ent
the
chon
droc
ytes
of
the
calc
ified
zon
e ar
e ac
tive,
the
cel
ls b
ecom
e qu
iesc
ent
as t
he c
artil
age
ages
and
min
eral
izes
.
Con
sequ
ently
, th
e ca
lcifi
ed z
one
cont
ains
rel
ativ
ely
few
met
abol
ical
ly a
ctiv
e ce
lls t
hat
are
entra
pped
with
in a
cal
cifie
d m
atrix
. D
espi
te t
he m
iner
aliz
atio
n, h
owev
er,
the
arra
ngem
ent
of
colla
gen
fiber
s in
the
cal
cifie
d la
yer
mak
es i
t le
ss s
tiff
than
bon
e. T
he s
tiffn
ess
of t
he z
one
(ela
stic
mod
ulus
= 0
.32
GPa
) is
app
roxi
mat
ely
an o
rder
of
mag
nitu
de g
reat
er t
han
non-
min
eral
ized
car
tilag
e, b
ut a
n or
der
of m
agni
tude
les
s th
an b
one
[Men
te &
Lew
is,
94].
This
appa
rent
gra
dien
t in
stiff
ness
, fro
m n
on-m
iner
aliz
ed c
artil
age
to b
one,
is e
ssen
tial f
or th
e pr
oper
func
tiona
lity
of c
artil
age
as it
allo
ws t
he a
ppro
pria
te d
istri
butio
n of
forc
es a
long
the
join
t [H
uber
,
Trat
tnig
& L
intn
er, 0
0].
13
1.1.
8 Pe
rice
llula
r Mat
rix
In a
dditi
on to
hor
izon
tal s
ubdi
visi
ons
into
the
resp
ectiv
e zo
nes,
a ci
rcum
fere
ntia
l diff
eren
tiatio
n
of m
atrix
com
pone
nts
exis
ts a
roun
d in
divi
dual
cho
ndro
cyte
s. Ea
ch o
f th
e zo
nes
can
be f
urth
er
divi
ded
into
thr
ee d
istin
ct r
egio
ns o
r m
atric
es:
the
peric
ellu
lar,
terr
itoria
l an
d in
terte
rrito
rial
regi
ons/
mat
rices
[Poo
le, 9
7]. W
hile
eac
h of
the
regi
ons p
lays
a c
ruci
al ro
le in
the
func
tiona
lity
of
the
tissu
e (te
rrito
rial
regi
ons
allo
ws
for
chon
droc
yte
atta
chm
ent
to
the
ECM
, an
d th
e
inte
rterr
itoria
l re
gion
con
tribu
tes
to t
he m
echa
nica
l pr
oper
ties
of t
he t
issu
e),
the
peric
ellu
lar
mat
rix (P
CM
) reg
ion
is o
f par
ticul
ar in
tere
st.
With
in th
e EC
M in
divi
dual
cho
ndro
cyte
s ar
e su
rrou
nded
by
a na
rrow
reg
ion
of m
atrix
, ter
med
the
peric
ellu
lar
mat
rix, t
hat
is c
hara
cter
ized
by
a hi
gher
con
cent
ratio
n of
pro
teog
lyca
ns,
finer
arra
ngem
ent o
f col
lage
n fib
res,
and
the
pres
ence
of C
olla
gen
Type
VI [
Pool
e, 9
7].T
oget
her,
the
PCM
and
the
surr
ound
ed c
hond
rocy
te h
ave
been
gro
uped
to a
form
a b
asic
stru
ctur
al u
nit t
erm
ed
the
“cho
ndro
n”.
In
each
cho
ndro
n, t
he c
hond
rocy
te i
s lin
ked
at i
ts s
urfa
ce t
o a
filam
ento
us
coat
ing
of c
arbo
hydr
ate-
rich
mol
ecul
es a
nd e
nclo
sed
bya
fibril
lar p
eric
ellu
lar c
apsu
le [G
uila
ket
al.,
06].
Alth
ough
the
pre
cise
fun
ctio
n of
the
per
icel
lula
r m
atrix
is
not
fully
elu
cida
ted,
cons
ider
able
evi
denc
e su
gges
ts it
is in
volv
ed in
the
regu
latio
n of
bio
chem
ical
and
bio
mec
hani
cal
inte
ract
ions
bet
wee
n th
e ce
lls a
nd t
he s
urro
undi
ng E
CM
[A
lexo
poul
oset
al.,
05;
Gra
ffet
al.,
03;G
uila
ket
al.,
05]
. It
is w
ell
acce
pted
tha
t in
hea
lthy
artic
ular
car
tilag
e th
e re
sidi
ng
chon
droc
ytes
mus
t fu
nctio
n sy
nerg
istic
ally
with
the
EC
M i
n or
der
to a
bsor
b, r
edis
tribu
te a
nd
trans
mit
phys
iolo
gica
l com
pres
sive
and
she
arin
g fo
rces
to th
e su
bcho
ndra
l bon
e. A
n in
tera
ctio
n
betw
een
the
cell
surf
ace
and
ECM
com
pone
nts
sign
ifica
ntly
inf
luen
ces
met
abol
ism
, ge
ne
expr
essi
on a
nd c
ellu
lar
resp
onse
s to
gro
wth
fac
tors
. B
ecau
se c
hond
rocy
tes
are
com
plet
ely
surr
ound
ed b
y th
e PC
M a
ny s
igna
ls –
of e
ither
mec
hani
cal o
r ch
emic
al n
atur
e –
orig
inat
ing
in
14
the
ECM
mus
t pa
ss t
hrou
gh t
he p
eric
ellu
lar
envi
ronm
ent
befo
re t
hey
are
sens
ed b
y th
e
chon
droc
ytes
. As
such
it h
as b
een
hypo
thes
ized
that
the
PCM
may
fun
ctio
n as
a tr
ansd
ucer
of
biom
echa
nica
l and
bio
chem
ical
sig
nalli
ng, s
elec
tivel
y tra
ppin
g, m
odify
ing,
or
reta
inin
g so
lubl
e
med
iato
rs s
uch
as g
row
th fa
ctor
s [L
arso
net
al.,
02;
Lee
et a
l., 0
0]. T
he lo
wer
per
mea
bilit
y of
the
PCM
, rel
ativ
e to
the
ECM
, has
bee
n po
stul
ated
to in
hibi
t flu
id fl
ux n
ear t
he c
ells
by
a fa
ctor
of
30,
thus
hav
ing
a co
nsid
erab
leef
fect
on
conv
ectiv
e tra
nspo
rt an
d di
ffus
ion
from
and
to
the
chon
droc
yte
[Ale
xopo
ulos
, Set
ton
& G
uila
k, 0
5]. S
imila
rly, i
t has
bee
n pr
opos
ed th
at a
ny d
irect
inte
ract
ions
bet
wee
n ce
ll su
rfac
e re
cept
ors
and
the
ECM
mus
t be
fac
ilita
ted
by c
onst
ituen
t
mac
rom
olec
ules
pre
sent
with
in th
e PC
M. I
n fa
ct, i
t has
bee
n pr
evio
usly
sho
wn
that
rete
ntio
n of
nativ
e PC
M s
igni
fican
tly i
nflu
ence
s th
e m
etab
olic
act
ivity
of
chon
droc
ytes
in
vitr
o [L
arso
n,
Kel
ley,
Bla
ckw
ood,
Ban
es &
Lee
, 02]
.
It ha
s al
so b
een
sugg
este
d th
at th
e pr
imar
y fu
nctio
n of
the
PCM
is to
pro
vide
a p
rote
ctiv
e ef
fect
for
the
chon
droc
ytes
du
ring
load
ing
thro
ugh
an
“ada
ptiv
e w
ater
lo
ss
from
th
e PC
M
prot
eogl
ycan
s” [P
oole
, 97]
. Se
vera
linv
estig
ator
s ha
ve s
uppo
rted
the
notio
n th
at a
cho
ndro
n ac
ts
as a
com
pres
sion
res
ista
nt,
fluid
fill
ed ‘
blad
der’
whi
ch i
s ab
le t
o ab
sorb
mec
hani
cal
load
s,
defo
rm a
nd r
ecov
er c
ompl
etel
y w
hen
unlo
aded
. Rec
ent
stud
ies
have
sho
wn
that
the
obs
erve
d
mis
mat
ch b
etw
een
elas
tic m
odul
i of
the
PCM
and
EC
M r
esul
ts in
a s
igni
fican
t stre
ss s
hiel
ding
effe
ct fo
r the
cho
ndro
cyte
s [A
lexo
poul
os, S
etto
n &
Gui
lak,
05]
.
Whi
le th
e PC
M’s
com
posi
tion
is s
imila
r to
that
of t
he E
CM
, dis
tinct
diff
eren
ces
in c
olla
gen
and
prot
eogl
ycan
com
posi
tion
exis
t. O
f the
col
lage
ns fo
und
in a
rticu
lar c
artil
age,
Col
lage
n Ty
pe V
I
(see
Tab
le I)
is fo
und
excl
usiv
ely
in th
e pe
ricel
lula
r reg
ion
and
is u
sed
as a
mar
ker f
or c
hond
rons
[Poo
le, 9
7]. S
uppo
rting
the
ide
aof
the
PC
M a
s a
sign
al t
rans
duce
r is
the
abi
lity
of T
ype
VI
colla
gen
to i
nter
act
with
a w
ide
arra
y of
EC
M c
ompo
nent
s in
clud
ing
bigl
ycan
, hy
alur
onan
,
15
perle
can,
fib
rom
odul
in a
s w
ell
as T
ype
II c
olla
gen
[Bid
anse
tet
al.,
92;
Kie
ltyet
al.,
92]
; in
addi
tion,
it a
lso
serv
es a
s a
site
of c
hond
rocy
te a
ttach
men
t [A
lexo
poul
os, S
etto
n &
Gui
lak,
05]
.
Sim
ilarly
, typ
e IX
Col
lage
n is
als
o co
ncen
trate
d w
ithin
the
PC
M, h
owev
er, s
mal
ler
quan
titie
s
may
als
o be
fou
nd in
the
terr
itoria
l mat
rix -
none
is f
ound
in th
e in
terte
rrito
rial r
egio
ns [
Pool
e,
97].
In te
rms o
f pro
teog
lyca
ns, t
he re
gion
has
bee
n sh
own
to b
e ric
h in
dec
orin
.
1.2
Car
tilag
e Pa
thol
ogy
& T
issue
Eng
inee
ring
The
loss
of
artic
ular
car
tilag
e, t
ypic
ally
thr
ough
inj
ury
or d
isea
se,
ofte
n re
sults
in
limite
d
mob
ility
, pai
n, a
nd d
ebili
tatin
g ef
fect
s in
indi
vidu
als.
It is
est
imat
ed th
at c
artil
age
dege
nera
tion
due
to o
steo
arth
ritis
and
spo
rts-r
elat
ed i
njur
ies,
affli
cts
som
e 36
mill
ion
indi
vidu
als
in N
orth
Am
eric
a [T
emen
off &
Mik
os, 0
0a].
As
the
popu
latio
n ag
es, t
he n
umbe
r of i
ndiv
idua
ls w
ith jo
int
dam
age
is o
nly
expe
cted
to in
crea
se a
nd b
urde
n bo
th th
e he
alth
car
e sy
stem
and
the
popu
latio
n.
1.2.
2A
rtic
ular
Car
tilag
e In
jury
& R
epai
r Obs
tacl
es
Arti
cula
r ca
rtila
ge i
njur
ies
may
be
clas
sifie
d in
to t
hree
dis
tinct
cat
egor
ies:
mat
rix d
isru
ptio
ns,
parti
al th
ickn
ess
defe
cts,
and
full
thic
knes
s de
fect
s. In
Mat
rix d
isru
ptio
ns a
blu
nt tr
aum
a fo
rce
resu
lts in
dam
age
to th
e EC
M, b
utin
man
y ca
ses
the
resi
dent
cho
ndro
cyte
s ar
e ab
le to
repl
enis
h
ECM
mac
rom
olec
ules
thr
ough
inc
reas
ed a
nabo
lic a
ctiv
ity.
In m
ore
seve
re i
njur
ies,
such
as
parti
al t
hick
ness
def
ects
, di
srup
tion
of c
artil
age
tissu
e oc
curs
but
doe
s no
t pr
opag
ate
to t
he
subc
hond
ral
bone
. Whi
le a
cel
lula
r re
spon
se i
s in
itiat
ed t
o re
pair
the
dam
age,
for
rea
sons
yet
unkn
own,
the
cellu
lar r
espo
nse
is te
rmin
ated
bef
ore
the
tissu
e is
repa
ired.
In th
e m
ost s
ever
e ty
pe
of i
njur
y, d
amag
e tra
nsve
rse
the
entir
e th
ickn
ess
of t
he t
issu
e an
d pe
netra
tes
the
subc
hond
ral
bone
. In
such
cas
es, t
he d
efec
t is
fill
ed w
ith a
fib
rin c
lot
and
clas
sica
l w
ound
hea
ling
ensu
es.
How
ever
, th
e re
sulta
nt t
issu
e di
spla
ys a
fib
roca
rtila
ge p
heno
type
, an
d po
sses
ses
infe
rior
16
mec
hani
cal p
rope
rties
that
resu
lt in
the
even
tual
deg
rada
tion
of th
e tis
sue
over
a p
erio
d of
yea
rs
[Tem
enof
f & M
ikos
, 00a
].
Inte
rest
ingl
y en
ough
, the
200
-yea
r-ol
d ob
serv
atio
n th
at “
carti
lage
onc
e de
stro
yed
neve
r he
als”
(as
stat
ed b
y H
unte
r in
174
31 ) re
mai
ns a
ppro
pria
te e
ven
toda
y, a
s ad
ult
artic
ular
car
tilag
e
poss
esse
s a
very
lim
ited
capa
city
for
self-
repa
ir. T
he t
issu
e’s
avas
cula
r na
ture
, th
e lim
ited
prol
ifera
tive
and
met
abol
ic c
apac
ity o
f cho
ndro
cyte
s, al
ong
with
the
inab
ility
of c
hond
rocy
tes
to
mig
rate
thro
ugh
the
ECM
are
all
fact
ors
that
pre
clud
e pr
oper
tiss
ue h
ealin
g [H
unzi
ker,
99;C
outts
et a
l., 9
7].
Whi
le c
linic
al c
artil
age
rest
orat
ion
tech
niqu
es a
re a
vaila
ble,
all
poss
ess
limita
tions
and
are
of
deba
tabl
e ef
fect
iven
ess.
Brie
fly, i
n pa
tient
s w
ith s
mal
ler
lesi
ons,
atte
mpt
s ar
e m
ade
to r
esto
re
join
t-sur
face
car
tilag
e th
roug
h su
rgic
al i
mpl
anta
tion
ofau
togr
afts
or
thro
ugh
utili
zatio
ns o
f
tech
niqu
es th
at e
ncou
rage
nat
ive
tissu
e re
pair
proc
esse
s [O
'Dris
coll,
98]
. In
the
form
er te
chni
que,
carti
lage
def
ects
are
rep
lace
d w
ith s
mal
ler
plug
s fr
om a
less
wei
ght-b
earin
g re
gion
of
the
join
t.
Whi
le th
e pr
oced
ure
has
been
sho
wn
to d
ecre
ase
pain
in s
ome
patie
nts,
it su
ffer
s fr
om s
ever
al
limita
tions
: 1)
the
res
ulta
nt d
amag
e to
the
don
or s
ite, 2
) th
e am
ount
of
carti
lage
ava
ilabl
e fo
r
trans
plan
tatio
n, 3
) di
scre
panc
y be
twee
n na
tive
and
trans
plan
ted
tissu
e pr
oper
ties,
and
4) t
he
diff
icul
ty to
mat
ch th
e to
polo
gy o
f the
gra
fts w
ith th
ein
jure
d si
te. A
s suc
h, th
e lo
ng-te
rm su
cces
s
of th
is a
ppro
ach
rem
ains
to b
e ve
rifie
d [M
artin
et a
l., 0
7]. O
ther
tech
niqu
es a
imin
g to
enh
ance
nativ
e tis
sue
rege
nera
tive
prop
ertie
s th
roug
h ph
arm
acol
ogic
al a
gent
s, el
ectri
cal
stim
ulat
ion,
or
even
tra
nspl
anta
tion
of e
xtra
cho
ndro
cyte
s ha
ve n
ot b
een
com
plet
ely
succ
essf
ul a
nd t
he
effe
ctiv
enes
s de
crea
ses
with
the
pat
ient
s’ a
ge a
nd e
xten
t of
dam
age
[O'D
risco
ll, 9
8]. I
n ca
ses
whe
re
carti
lage
da
mag
e is
se
vere
, th
e op
timal
tre
atm
ent
cons
titut
es
the
repl
acem
ent
of
1 Hun
ter W
(174
3) P
hilo
s Tra
ns 4
70:5
14
17
artic
ulat
ing
join
ts w
ith sy
nthe
tic p
rost
hesi
s (i.e
. hip
repl
acem
ents
). H
owev
er, f
ailu
re ra
tes a
s hig
h
as 2
0% h
ave
been
repo
rted
afte
r see
min
gly
succ
essf
ul im
plan
tatio
ns [S
oder
man
et a
l., 0
1].
As
a re
sult
from
the
var
ious
lim
itatio
ns o
f cu
rren
tly a
vaila
ble
clin
ical
app
roac
hes,
tissu
e
engi
neer
ing
has
emer
ged
as a
nov
el t
hera
peut
ic a
ltern
ativ
e fo
r tis
sue
rege
nera
tion.
It
is h
oped
that
tis
sue
engi
neer
ing
appr
oach
es
wou
ld
allo
w
the
prop
ertie
s (s
truct
ural
, bi
olog
ical
,
biom
echa
nica
l) of
gra
fted
tissu
es t
o be
spe
cific
ally
tai
lore
d to
the
aff
ecte
d jo
ints
[B
eris
et a
l.,
05].
1.2.
3 Ti
ssue
Eng
inee
ring
App
roac
hes
Bec
ause
of
curr
ent
surg
ical
lim
itatio
ns i
n th
e re
pair
of c
ompl
ex c
artil
age
lesi
ons,
tissu
e
engi
neer
ing
hold
s gr
eat p
rom
ise
for
the
gene
ratio
n of
fun
ctio
nal t
issu
e in
vitr
ofo
r su
bseq
uent
impl
anta
tion
in v
ivo.
In g
ener
al, t
issu
e en
gine
erin
g of
arti
cula
r car
tilag
e ca
n be
ach
ieve
d th
roug
h
thre
e di
ffer
ent s
trate
gies
: util
izin
g cu
lture
dis
hes,
scaf
fold
s, or
subs
trate
s.
The
first
app
roac
h, t
erm
edau
tolo
gous
cho
ndro
cyte
im
plan
tatio
n, u
tiliz
es t
he p
atie
nt’s
ow
n
chon
droc
ytes
to
enco
urag
e ca
rtila
ge r
epai
r an
d re
gene
ratio
n. I
n th
is s
trate
gy a
sam
ple
of t
he
patie
nt’s
car
tilag
e is
har
vest
ed a
nd th
e re
side
nt c
hond
rocy
tes
are
isol
ated
and
exp
ande
d, th
roug
h
grow
th i
n m
onol
ayer
cul
ture
, in
vitr
o. F
ollo
win
g a
perio
d of
exp
ansi
on,
cells
are
col
lect
ed,
trans
plan
ted
back
into
the
defe
ct s
ite, a
nd c
onfin
ed th
ere
utili
zing
a p
erio
stea
l fla
p [G
illog
lyet
al.,
98].
Alth
ough
ther
e is
evi
denc
e th
at th
e tre
atm
ent i
s eff
ectiv
e at
alle
viat
ing
pain
, the
resu
lting
tissu
e is
at l
east
par
tially
fibr
ocar
tilag
inou
s an
d th
us is
sub
ject
to d
egra
datio
n ov
er ti
me
[Bre
inan
et a
l., 9
7].
Mor
eove
r, w
hile
it
is r
elat
ivel
y ea
sy t
o gr
ow c
ells
in
a 2D
mon
olay
er,
a lim
iting
feat
ure
to th
is te
chni
que
is th
e in
abili
ty to
sha
pe th
e ce
lls in
to a
pre
-def
ined
, thr
ee-d
imen
sion
al
shap
e th
at i
s ab
le t
o pe
rfec
tly f
it th
e de
fect
site
. Lik
ewis
e, c
ontin
ued
mon
olay
er e
xpan
sion
is
know
n to
res
ult i
n an
irre
vers
ible
loss
of
chon
droc
yte-
phen
otyp
e an
d ce
llula
r de
diff
eren
tiatio
n
18
whi
ch r
esul
ts in
infe
rior
extra
cellu
lar
mat
rix th
at is
inad
equa
te f
or a
rticu
latin
g jo
ints
[Sc
hnab
el,
Mar
lovi
ts,
Eckh
off,
Fich
tel,
Got
zen,
Vec
sei
& S
chle
gel,
02;M
artin
, M
iot,
Bar
bero
, Ja
kob
&
Wen
dt, 0
7;D
arlin
g &
Ath
anas
iou,
05]
.
The
seco
nd s
trate
gy in
volv
es th
e se
edin
g of
cel
ls in
to p
orou
s m
ater
ials
(sc
affo
lds)
that
may
be
untre
ated
or
coup
led
with
che
mic
als,
such
as
grow
th f
acto
rs, t
o pr
omot
e ce
ll an
d/or
mat
rix in
-
grow
th.
Upo
n se
edin
g w
ith c
artil
age-
form
ing
cells
the
sca
ffol
d is
gro
wn
in c
ultu
re a
nd
subs
eque
ntly
impl
ante
d di
rect
ly in
to th
e de
fect
site
[Mar
tin, M
iot,
Bar
bero
, Jak
ob &
Wen
dt, 0
7].
Bec
ause
the
chon
droc
ytes
are
cul
ture
d in
a 3
D s
truct
ure,
whi
ch m
ay b
e cr
eate
d in
a v
arie
ty o
f
geom
etrie
s, th
ey a
llow
a b
ette
r ap
proa
ch t
o fix
irr
egul
arly
-sha
ped
defe
cts.
Mor
e im
porta
ntly
,
how
ever
, gro
win
g ch
ondr
ocyt
es in
3D
has
bee
n sh
own
to b
e co
nduc
ive
to th
e m
aint
enan
ce o
f
cell
phen
otyp
e an
d ce
ll re
tent
ion
in t
he d
efec
t si
te [
Stew
art
et a
l., 0
0].
Scaf
fold
s m
ay b
e
cons
truct
ed u
tiliz
ing
eith
er b
iode
grad
able
or
non-
biod
egra
dabl
e co
mpo
nent
s to
fur
ther
tailo
r to
indi
vidu
al n
eeds
. Unf
ortu
nate
ly, s
ever
al s
tudi
es h
ave
show
n th
at th
e re
sulti
ng ti
ssue
pos
sess
es
infe
rior m
echa
nica
l pro
perti
es a
nd lo
w c
olla
gen
cont
ent,
as c
ompa
red
to n
ativ
e tis
sue.
It h
as b
een
hypo
thes
ized
that
the
abse
nce
of w
eigh
t bea
ring,
or m
echa
nica
l loa
ding
, dur
ing
tissu
e fo
rmat
ion
is r
equi
red
for
prop
er E
CM
gen
e ex
pres
sion
and
pro
tein
syn
thes
is t
o oc
cur.
Add
ition
ally
,
beca
use
subs
trate
s ca
nnot
be
anch
ored
ful
ly in
the
impl
ante
d tis
sue,
they
may
be
disl
odge
d by
shea
ring
forc
es d
urin
g lo
com
otio
n [T
emen
off &
Mik
os, 0
0a].
As
all t
he p
revi
ous
stra
tegi
es h
ave
limita
tions
, rec
ent a
ppro
ache
s to
car
tilag
e tis
sue
engi
neer
ing
have
focu
sed
on d
evel
opin
g bi
phas
ic a
rticu
lar c
artil
age
cons
truct
s th
at in
clud
e a
bone
-inte
rfac
ing
com
pone
nt.
The
biph
asic
con
stru
cts
are
com
pose
d of
car
tilag
e tis
sue
form
ed o
n th
e to
p of
a
subs
trate
that
ser
ves
as th
e bo
ne in
terf
ace
com
pone
nt. S
imila
rto
scaf
fold
s, su
bstra
tes
are
poro
us,
whi
ch a
llow
s fo
r bo
ne i
n-gr
owth
and
sub
sequ
ent
anch
orag
e in
the
und
erly
ing
bone
. Th
e
19
subs
trate
s m
ay b
e ei
ther
bio
degr
adab
le o
r no
n-bi
odeg
rada
ble
and
may
be
coup
led
to m
olec
ules
mod
ulat
ing
tissu
e gr
owth
. The
sub
stra
te a
cts
to b
oth
supp
ort c
artil
age
form
atio
n an
d fa
cilit
ates
fixat
ion
afte
r im
plan
tatio
n. M
oreo
ver,
as t
he c
artil
age
tissu
e is
alre
ady
form
ed p
rior
to
impl
anta
tion,
lat
eral
int
egra
tion
to t
he a
djac
ent
nativ
e ca
rtila
ge i
s po
ssib
le [
Gry
npas
et a
l.,
02;R
edm
anet
al.,
05;
Kuo
et a
l., 0
6].
To e
nabl
e th
e co
nstru
ctio
n of
a b
ipha
sic
cons
truct
for
tissu
e im
plan
tatio
n, a
sui
tabl
e bo
ne
subs
titut
e m
ust
be u
sed.
The
ide
al m
ater
ial
shou
ld e
xhib
it se
vera
l pr
oper
ties
in o
rder
to
be
cons
ider
ed a
dequ
ate.
Firs
tly,
the
mat
eria
l m
ust
not
be c
ytot
oxic
and
allo
w f
or t
he p
rope
r
atta
chm
ent a
nd g
row
th o
f se
eded
cho
ndro
cyte
s, as
wel
l as
the
form
atio
n of
car
tilag
e tis
sue.
In
addi
tion,
the
sub
stitu
te b
one
mat
eria
l sh
ould
be
oste
ocon
duct
ive,
as
to a
llow
rap
id i
nteg
ratio
n
with
the
host
bon
e, a
nd b
iode
grad
able
(at a
pre
dete
rmin
ed ra
te) i
n or
der t
o be
eve
ntua
lly re
plac
ed
by t
he i
n-gr
owin
g ho
st b
one.
As
impo
rtant
ly,
the
subs
trate
mus
t be
stro
ng e
noug
h to
ful
fill
requ
ired
load
-bea
ring
func
tions
bef
ore
sign
ifica
nt b
one
in-g
row
th o
ccur
s, an
d th
e de
grad
atio
n
prod
ucts
of
the
subs
trate
mus
t no
t in
duce
an
infla
mm
ator
y re
actio
n th
atm
ay j
eopa
rdiz
e th
e
impl
anta
tion
[Cou
ttset
al.,
01]
.
1.2.
4 C
alci
um P
oly-
Phos
phat
e (C
PP) a
nd B
ipha
sic
Con
stru
cts
Our
lab
has
pre
viou
sly
deve
lope
d an
d ch
arac
teriz
ed a
por
ous
calc
ium
pol
ypho
spha
te (
CPP
)
subs
trate
that
pos
sess
es m
echa
nica
l pro
perti
es a
ppro
xim
atin
g ca
ncel
lous
bon
e. U
tiliz
ing
the
CPP
we
are
able
to fo
rm b
ipha
sic
cons
truct
s in
vitr
o, c
onsi
stin
g of
car
tilag
inou
s tis
sue
grow
n on
top
of th
e po
rous
cer
amic
sub
stra
te (F
igur
e 5)
[Kan
dele
t al.,
06]
. O
win
g to
the
poro
us p
rope
rties
of
CPP
(37
vol
.% p
oros
ity),
chon
droc
ytes
are
abl
e to
ent
er i
nto
the
pore
s of
the
im
med
iate
subs
urfa
ce a
nd p
rodu
ce c
artil
agin
ous t
issu
e th
at is
anc
hore
d to
the
surf
ace
of th
e su
bstra
te [P
illia
r
et a
l., 0
1]. L
ikew
ise
the
pres
ence
of i
nter
conn
ecte
d em
pty
pore
s in
the
subs
trate
allo
ws
for b
one
20
in-g
row
th a
fter
impl
anta
tion,
and
thu
s pr
ovid
ing
a se
cure
fix
atio
n in
to t
he h
ost
bone
. Th
e
subs
trate
is
not
cyto
toxi
c to
the
cel
ls a
nd t
he r
esul
ting
tissu
e ha
s be
en p
revi
ousl
y sh
own
to
rese
mbl
e th
e na
tive
tissu
e an
d to
cont
ain
Col
lage
n Ty
pe
II
and
vario
us
char
acte
ristic
prot
eogl
ycan
s. C
PP is
bio
degr
adab
le a
nd th
e de
grad
atio
n pr
oduc
ts, c
alci
um a
nd p
hosp
hate
, do
not
inci
te a
n in
flam
mat
ory
reac
tion;
as
the
subs
trate
deg
rade
sw
ith t
ime,
it
will
ulti
mat
ely
be
repl
aced
with
nat
ural
hos
t bo
ne [
Gry
npas
, Pill
iar,
Kan
del,
Ren
lund
, Fili
aggi
& D
umitr
iu, 0
2].
Unf
ortu
nate
ly, s
imila
rto
the
carti
lage
form
ed in
sca
ffol
ds, t
he c
artil
age
tissu
e fo
rmed
con
tain
ed
less
EC
M a
nd p
osse
sses
onl
y a
frac
tion
of t
he m
echa
nica
l pr
oper
ties
of n
ativ
e tis
sue.
Suc
h
mec
hani
cal
defic
ienc
ies
may
aff
ect
the
tissu
e’s
abili
ty t
o re
surf
ace
a la
rge
join
t de
fect
and
to
prov
ide
adeq
uate
load
-bea
ring
func
tiona
lity
to th
e de
fect
site
[Wal
dman
et a
l., 0
2].
Figu
re 5
: A
dia
gram
mat
ic r
epre
sent
atio
n (le
ft) o
f a
biph
asic
con
stru
ct o
f in
-vitr
o fo
rmed
arti
cula
r ca
rtila
ge t
issu
e gr
own
on
Cal
cium
Pol
ypho
spha
te (C
PP) s
ubst
rate
. On
the
right
is th
e co
rres
pond
ing
hist
ogra
m o
f a 4
-wee
k-ol
d ar
ticul
ar c
artil
age
tissu
e as
gr
own
in o
ur la
b.
21
1.3
Mec
hani
cal s
timul
atio
n
It ha
s be
en k
now
n fo
r ye
ars
that
the
mec
hani
cal
envi
ronm
ent
of a
tis
sue
regu
late
s its
gro
wth
,
deve
lopm
ent a
nd, w
hen
requ
ired,
its
rem
odel
ling
[Gro
dzin
sky,
Lev
enst
on, J
in &
Fra
nk, 0
0;K
im
et a
l., 9
4;B
eaup
reet
al.,
00]
. On
a ce
llula
r lev
el m
echa
nica
l for
ces
have
bee
n sh
own
to in
fluen
ce
cellu
lar f
unct
ion
thro
ugh
regu
latio
n of
gen
e an
d pr
otei
n ex
pres
sion
, ind
uctio
n of
diff
eren
tiatio
n,
apop
tosi
s an
d/or
mai
nten
ance
of
cellu
lar
hom
eost
asis
[G
rodz
insk
y, L
even
ston
, Ji
n &
Fra
nk,
00;W
ang
& T
ham
patty
, 06
]. A
lthou
gh t
he u
nder
lyin
g m
echa
nism
s by
whi
ch c
ells
sen
se
mec
hani
cal f
orce
s and
con
vert
them
into
bio
logi
cal r
espo
nses
are
not
fully
und
erst
ood,
man
y ce
ll
type
s (i.
e.
Tend
on
and
AC
L fib
robl
asts
, C
ardi
ac
Fibr
obla
sts,
smoo
th
mus
cle
cells
, an
d
chon
droc
ytes
) ha
ve b
een
show
n to
res
pond
to
chan
ges
in t
heir
mec
hani
cal
envi
ronm
ent
[Gro
dzin
sky,
Lev
enst
on, J
in &
Fra
nk, 0
0;W
aldm
anet
al.,
03;
Yan
get
al.,
04;
Hsi
ehet
al.,
00;
Lee
et a
l., 9
9]. W
hile
the
unde
rlyin
g m
echa
notra
nsdu
ctio
n si
gnal
ling
path
way
s ha
ve n
ot b
een
fully
eluc
idat
ed,
seve
ral
pote
ntia
l si
gnal
ling
even
ts h
ave
been
sug
gest
ed t
o be
the
prim
ary
trigg
ers
whi
ch u
ltim
atel
y in
fluen
ce c
ell f
unct
iona
lity.
The
se e
xtra
cellu
lar e
vent
s inc
lude
cel
l def
orm
atio
n,
incr
ease
d hy
dros
tatic
pre
ssur
e, c
ompr
essi
on o
f th
e ch
arge
d ex
trace
llula
r m
atrix
, ch
ange
s in
osm
olar
ity a
nd p
H,
as w
ell
as f
luid
flo
w [
Zhan
get
al.,
06;
Kni
ght
et a
l.,06
b;K
nigh
tet
al.,
06a;
Gui
lak,
Ale
xopo
ulos
, Upt
on, Y
oun,
Cho
i, C
ao, S
etto
n &
Hai
der,
06].
As
chon
droc
ytes
are
fou
nd i
n a
load
-bea
ring
tissu
e, t
hey
are
expo
sed
to a
wid
e va
riety
of
mec
hani
cal
forc
es,
incl
udin
g te
nsio
n, c
ompr
essi
on a
nd s
hear
. It
has
been
dem
onst
rate
d th
at,
unde
r ph
ysio
logi
cal
cond
ition
s, th
e pr
esen
ce o
f m
echa
nica
l st
imul
atio
n is
req
uire
d fo
r th
e
deve
lopm
ent a
nd m
aint
enan
ce o
f hea
lthy
artic
ular
car
tilag
e tis
sue
[Eck
stei
n, H
udel
mai
er &
Put
z,
06].
In f
act,
it is
now
evi
dent
that
arti
cula
r ca
rtila
ge u
nder
goes
atro
phy
unde
r re
duce
d lo
adin
g
cond
ition
s su
ch a
s po
stop
erat
ive
imm
obili
zatio
n. O
n a
cellu
lar
leve
l, m
echa
nica
l lo
adin
g of
22
artic
ular
car
tilag
e tis
sue
resu
lts i
n ce
ll de
form
atio
n, h
ydro
stat
ic p
ress
ure
grad
ient
s, flu
id f
low
,
defo
rmat
ion
of th
e ch
arge
d ex
trace
llula
r mat
rix a
nd th
e ac
com
pany
ing
chan
ges i
n os
mol
arity
and
pH [G
rodz
insk
y, L
even
ston
, Jin
& F
rank
, 00;
Hub
er, T
rattn
ig &
Lin
tner
, 00]
. The
se e
xtra
cellu
lar
even
ts a
re b
elie
ved
to i
nflu
ence
the
bio
synt
hetic
act
ivity
of
chon
droc
ytes
thr
ough
an
arra
y of
intra
cellu
lar e
vent
s su
ch a
s m
odul
atio
n of
the
cyto
skel
eton
, nuc
leus
def
orm
atio
n an
d al
tera
tions
of i
ntra
cellu
lar
conc
entra
tions
of
ions
-
nam
ely
Ca2+
[Won
g &
Car
ter,
03].
Expe
rimen
tally
,
chon
droc
ytes
ha
ve
been
sh
own
to
be
mec
hano
sens
itive
in
-vitr
o,
and
depe
ndin
g on
th
e
mag
nitu
de, d
urat
ion
(sta
tic v
s. dy
nam
ic)
and
type
of
stra
in (
i.e.,
osm
otic
, she
ar, c
ompr
essi
on),
have
alte
red
thei
r bio
synt
hetic
act
ivity
. In
gene
ral,
high
freq
uenc
ies
and
low
-mod
erat
e st
rain
s ar
e
asso
ciat
ed w
ith in
crea
sed
bios
ynth
etic
act
ivity
, whi
le s
tatic
com
pres
sion
, low
freq
uenc
y or
hig
h
stra
ins
have
res
ulte
d in
a d
ecre
ased
syn
thes
is [
Dav
isso
net
al.,
02;
Mau
cket
al.,
00;
Wal
dman
,
Spite
ri, G
rynp
as, P
illia
r, H
ong
& K
ande
l, 03
;Wal
dman
et a
l., 0
4;W
aldm
anet
al.,
06]
.
Bec
ause
of
the
vita
l ro
le m
echa
nica
l lo
adin
g pl
ays
in t
he d
evel
opm
ent
and
mai
nten
ance
of
heal
thy
carti
lage
in
vivo
, it
has
been
sug
gest
ed t
hat
the
appl
icat
ion
of m
echa
nica
l st
imul
atio
n
may
be
bene
ficia
l to
the
in-v
itro
form
atio
n of
car
tilag
e tis
sue.
Sev
eral
stu
dies
hav
e de
mon
stra
ted
that
loa
ding
cho
ndro
cyte
s, in
a c
ontro
lled
man
ner,
resu
lts i
n im
prov
ed t
issu
e fo
rmat
ion
[De
Cro
oset
al.,
06;
Wal
dman
, Cou
to, G
rynp
as, P
illia
r & K
ande
l, 06
]. Fo
r ins
tanc
e, th
e ap
plic
atio
n of
fluid
-indu
ced
shea
r fo
rces
to
chon
droc
ytes
see
ded
in m
onol
ayer
has
bee
n sh
own
to i
ncre
ase
prot
eogl
ycan
syn
thes
is. S
imila
rly, a
pplic
atio
n of
mec
hani
cal f
orce
s to
car
tilag
e ex
plan
ts, i
sola
ted
chon
droc
ytes
gro
wn
in s
caff
olds
and
/or
the
surf
aces
of
subs
trate
s ha
s re
sulte
d in
the
stim
ulat
ed
synt
hesi
s of c
artil
agin
ous E
CM
mac
rom
olec
ules
[Shi
eh &
Ath
anas
iou,
03]
.
In o
ur la
b, w
e ha
ve p
revi
ousl
y sh
own
that
a s
ingl
e ap
plic
atio
n of
cyc
lic c
ompr
essi
on re
sults
in a
grea
ter
accu
mul
atio
n of
EC
M m
acro
mol
ecul
es, a
nd th
e co
nseq
uent
impr
ovem
ent i
n th
e tis
sue’
s
23
mec
hani
cal
prop
ertie
s (F
igur
e 6)
. Sp
ecifi
cally
, th
e ap
plic
atio
n of
uni
axia
l cy
clic
com
pres
sive
forc
es o
f ap
prox
imat
ely
9.81
mN
(eq
uiva
lent
to
1g)
for
30 m
inut
es a
t 1H
z ha
ve s
igni
fican
tly
enha
nced
EC
M p
rodu
ctio
n (1
5-30
%) a
nd im
prov
ed th
e m
echa
nica
l pro
perti
es o
f the
tiss
ue (b
oth
load
-bea
ring
capa
city
and
stif
fnes
s) b
y tw
ofol
d [W
aldm
an, C
outo
, Gry
npas
, Pill
iar
& K
ande
l,
06].
Figu
re 6
: A
dia
gram
mat
ic r
epre
sent
atio
nof
the
appl
icat
ion
of c
onfin
ed c
ompr
essi
ve m
echa
nica
l fo
rces
30
min
, 1g
m,
1Hz)
th
roug
h a
com
plia
nt a
garo
se p
lug
on in
vitr
ofo
rmed
car
tilag
e tis
sue
grow
n on
Cal
cium
Pol
ypho
spha
te (C
PP) s
ubst
rate
.
Stud
ies
aim
ing
to e
luci
date
the
und
erly
ing
mec
hani
sm(s
) re
gula
ting
the
tissu
e’s
resp
onse
to
cycl
ic c
ompr
essi
onha
ve d
emon
stra
ted
the
exis
tenc
e of
a b
ipha
sic
resp
onse
, co
mpo
sed
of
sequ
entia
l cat
abol
ic a
nd a
nabo
lic c
hang
es, i
n re
spon
se to
this
mec
hani
cal s
timul
atio
n. B
riefly
, it
was
sho
wn
that
mec
hani
cal
stim
ulat
ion
indu
ces
an E
RK
-med
iate
d up
regu
latio
n of
Mat
rix
Met
allo
prot
eias
e (M
MP)
-3 a
nd M
MP-
13 (2
hou
rs p
ost s
timul
atio
n) th
roug
h ac
tivat
ion
of th
e A
P-
1 co
nsen
sus
sequ
ence
[D
e C
roos
, D
haliw
al,
Gry
npas
, Pi
lliar
& K
ande
l, 06
]. A
P-1
cons
ensu
s
sequ
ence
s ar
e fo
und
in m
ost
MM
Ps,
and
trans
crip
tiona
ly r
egul
ate
thei
r ac
tivity
[C
raw
ford
&
Mat
risia
n, 9
6]. F
ollo
win
g th
e up
regu
latio
n of
MM
P-13
and
MM
P-3,
whi
ch re
sulte
d in
incr
ease
d
24
tissu
e de
grad
atio
n, s
igni
fican
t in
crea
ses
in t
ype
II c
olla
gen
and
aggr
ecan
gen
e ex
pres
sion
was
obse
rved
, ulti
mat
ely
resu
lting
in in
crea
sed
synt
hesi
s an
d ac
cum
ulat
ion
of th
e tis
sue’
s EC
M (
24
hour
s po
st s
timul
atio
n)[D
e C
roos
, Dha
liwal
, Gry
npas
, Pill
iar &
Kan
del,
06].
The
upre
gula
tion
of
MM
P-13
, in
turn
, was
sho
wn
to b
e de
pend
ent u
pon
the
activ
atio
n of
Mem
bran
e Ty
pe-1
MM
P
(MT1
-MM
P), w
hich
is k
now
n to
be
mec
hano
sens
itive
in s
ever
al c
ell t
ypes
[Y
amag
uchi
et a
l.,
02;B
radl
eyet
al.,
01]
and
is b
elie
ved
to b
e cr
ucia
l to
mat
rix re
mod
ellin
g [H
olm
beck
et a
l., 9
9].
The
activ
atio
n of
MT1
-MM
P, w
hich
was
fac
ilita
ted
thro
ugh
ERK
ind
uced
bin
ding
of
early
grow
th f
acto
r-1
(Egr
-1)
to t
he M
T1-M
MP
prom
oter
, is
bel
ieve
d to
cau
se d
egra
datio
n in
the
peric
ellu
lar
mat
rix a
nd th
us e
licit
the
anab
olic
pha
se o
f th
e re
mod
ellin
g re
spon
se [
De
Cro
oset
al.,
07].
Whe
n th
e M
T1-M
MP
upre
gula
tion
was
bl
ocke
d,
thro
ugh
deco
y M
T1-M
MP
olig
onuc
leot
ide
or th
roug
h M
T1-M
MP
siR
NA
, the
incr
ease
in ti
ssue
EC
M a
ccum
ulat
ion
was
not
obse
rved
follo
win
g m
echa
nica
l stim
ulat
ion
(sub
mitt
ed).
Sinc
e se
vera
l stu
dies
hav
e su
gges
ted
that
MT1
-MM
P is
impo
rtant
in re
gula
ting
cell
mor
phol
ogy
(for
ins
tanc
e, i
t re
gula
tes
the
appr
opria
te c
ell
shap
e an
d cy
toar
chite
ctur
e re
quire
d fo
r ad
ipos
e
diff
eren
tiatio
n an
d al
low
s ce
lls to
adj
ust t
heir
cellu
lar a
rchi
tect
ure
in re
spon
se to
mat
rix s
tiffn
ess
[Ben
ingo
et a
l., 0
4;C
hun
et a
l., 0
6])
and
owin
g to
the
role
cel
l mor
phol
ogy
play
s in
fac
ilita
ting
chon
droc
ytes
’ re
spon
se t
o m
echa
nica
l st
imul
i [C
ampb
ell
et a
l., 0
7;K
nigh
t, To
yoda
, Le
e &
Bad
er,
06b;
Spite
riet
al.,
08;
Smith
et a
l., 9
5],
the
rel
atio
nshi
p be
twee
n M
T1-M
MP
and
cell
shap
e ch
ange
s fol
low
ing
mec
hani
cal s
timul
atio
n w
as in
vest
igat
ed. I
t was
obs
erve
d th
at fo
llow
ing
cycl
ic c
ompr
essi
on c
hond
rocy
tes
unde
rwen
t a ra
pid,
tran
sien
t cel
l spr
eadi
ng a
nd re
turn
ed to
pre
-
stim
ulat
ed m
orph
olog
y w
ithin
six
hou
rs.
This
spr
eadi
ng a
nd r
etra
ctio
n w
as a
ssoc
iate
d w
ith
incr
ease
d ac
cum
ulat
ion
of n
ewly
syn
thes
ized
pro
teog
lyca
n an
d co
llage
n. C
hond
rocy
te s
prea
ding
was
sho
wn
to b
e de
pend
entu
pon
the
α5β1
inte
grin
, as
bloc
king
it (o
r the
β1
subu
nit)
abol
ishe
d
25
spre
adin
g. H
owev
er b
lock
ing
the
inte
grin
onl
y pa
rtial
ly i
nhib
ited
the
com
pres
sion
-indu
ced
incr
ease
in
mat
rix a
ccum
ulat
ion,
sug
gest
ing
othe
r m
echa
nose
nsiti
ve c
ompo
nent
s m
ay b
e
invo
lved
. It w
as f
urth
er s
how
n th
at c
ell r
etra
ctio
n fo
llow
ing
mec
hani
cal s
timul
atio
n is
par
tially
med
iate
d by
MT1
-MM
P, a
s si
lenc
ing
MT1
-MM
P in
hibi
ted
both
the
cel
lula
r re
tract
ion
and
mat
rix a
ccum
ulat
ion
(sub
mitt
ed).
Whi
le i
t is
app
aren
t th
at b
oth
the
mor
phol
ogic
al c
hang
es (
spre
adin
g/re
tract
ion)
and
the
upre
gula
tion
of M
T1-M
MP
are
corr
elat
ed a
nd c
ruci
al to
fac
ilita
ting
the
chon
droc
ytes
’ re
spon
se
to
cycl
ic
com
pres
sion
, th
e un
derly
ing
mec
hani
sms
of
mec
hano
trans
duct
ion
are
still
no
t
unde
rsto
od.
26
1.3.
2M
echa
notr
ansd
uctio
n
Gen
eral
ly s
peak
ing,
mec
hano
trans
duct
ion
refe
rs to
the
cellu
lar p
roce
sses
that
are
resp
onsi
ble
for
the
conv
ersi
on o
f m
echa
nica
l st
imul
i in
to b
ioch
emic
al s
igna
ls w
hich
allo
w c
ells
to
adap
t an
d
resp
ond
to s
urro
undi
ng e
nviro
nmen
tal c
ues.
Thes
e cu
es, i
n tu
rn,a
re k
now
n to
influ
ence
nea
rly
ever
y si
ngle
cel
lula
r ac
tivity
, in
clud
ing
grow
th,
diff
eren
tiatio
n, m
igra
tion,
apo
ptos
is,
gene
expr
essi
on a
nd p
rote
in sy
nthe
sis
[Ale
ngha
t & In
gber
, 02a
]. W
hile
the
type
and
the
stre
ngth
of t
he
appl
ied
mec
hani
cal s
timul
i var
y co
nsid
erab
ly in
diff
eren
t tis
sues
(i.e
. vas
cula
r en
doth
elia
l cel
ls
are
expe
rienc
e sh
ear f
orce
s to
talli
ng a
few
pas
cals
, car
tilag
e m
ay e
xper
ienc
e co
mpr
essi
ve fo
rces
up
to
20M
Pa)
all
mec
hano
trans
duct
ion
proc
esse
s ca
n be
di
vide
d in
to
four
ge
nera
l
phas
es[M
illw
ard-
Sadl
er &
Sal
ter,
04b;
Urb
an, 9
4]:
I.M
echa
noco
uplin
g –
forc
es a
t th
e tis
sue
leve
l ar
e tra
nsla
ted
into
loc
al a
ctio
ns a
t th
e
surf
aces
of t
he re
sidi
ng c
ells
.
II.
Cou
plin
g –
fo
rces
at t
he c
ell s
urfa
ce (
outs
ide)
are
tran
sduc
ed in
to b
ioch
emic
al c
ells
in
the
cyto
plas
m (i
nsid
e)
III.
Sign
al t
rans
mis
sion
–si
gnal
tra
nsdu
ctio
n ta
kes
plac
e to
the
eff
ecto
r ta
rget
(i.e
. th
e
alte
ratio
n of
cellu
lar g
ene
expr
essi
on)
IV.
ECM
cou
plin
g –
Cha
nges
in th
e ef
fect
or c
ell l
ine
resu
lt in
cha
nges
in th
e EC
M a
nd/o
r the
alte
red
ECM
env
ironm
ent r
esul
ts in
the
mod
ifica
tion
of in
trace
llula
r sig
nalli
ng.
Furth
erm
ore,
the
cellu
lar
resp
onse
s to
mec
hani
cal s
timul
i can
be
sepa
rate
d in
to r
apid
res
pons
es
(sec
onds
to m
inut
es) o
r lon
g-te
rm re
spon
ses
(hou
rs to
day
s). R
apid
resp
onse
s ar
ise
as a
resu
lt of
the
activ
atio
n of
var
ious
intra
cellu
lar s
igna
lling
pat
hway
s, w
hich
ulti
mat
ely
resu
lt in
cha
nges
in
the
ECM
com
posi
tion
or ti
ssue
func
tiona
lity
[Mill
war
d-Sa
dler
& S
alte
r, 04
b].
Des
pite
the
cruc
ial r
ole
mec
hano
trans
duct
ion
play
s in
cel
lula
r fu
nctio
ns, c
ellu
lar
surv
ival
, and
dise
ase
(i.e.
de
afne
ss,
mus
cula
r dy
stro
phie
s, ca
ncer
, an
d de
velo
pmen
tal
diso
rder
s),
the
27
unde
rlyin
g m
echa
nism
s ha
ve n
ot b
een
fully
elu
cida
ted
yet [
Jaal
ouk
& L
amm
erdi
ng, 0
9]. A
key
feat
ure,
dis
tingu
ishi
ng m
echa
notra
nsdu
ctio
n fr
om o
ther
type
s of
sig
nal t
rans
duct
ion,
is th
e la
ck
of a
lig
and-
med
iate
d ac
tivat
ion
of c
ell
surf
ace
rece
ptor
s; i
nste
ad,
activ
atio
n oc
curs
thr
ough
mec
hani
cal s
timul
i ind
uced
dis
torti
ons
in th
e ce
llula
r mem
bran
e, s
urro
undi
ng E
CM
, or t
he c
ells
’
inte
rnal
com
pone
nts
[Ale
ngha
t & In
gber
, 02b
;Orr
et a
l., 0
6]. T
hese
gen
eral
ized
phy
sica
l cha
nges
are
sens
ed, a
nd c
onve
rted
to a
bio
chem
ical
sig
nal,
by s
ever
al c
lass
es o
f ce
llula
r co
mpo
nent
s,
nam
ely
inte
grin
s (w
hich
are
loca
lized
in f
ocal
con
tact
s be
twee
n th
e ce
ll an
d th
e EC
M)
and
ion
chan
nels
(whi
ch a
re lo
cate
d th
roug
hout
the
cellu
lar m
embr
ane)
.
1.3.
3In
tegr
ins a
s Mec
hano
sens
ors
Cel
l ad
hesi
on t
o th
e su
rrou
ndin
g EC
M p
lays
mul
tiple
rol
es i
n as
sem
blin
g, m
aint
aini
ngan
d
coor
dina
ting
tissu
es i
n m
ultic
ellu
lar
orga
nism
s [H
uven
eers
& D
anen
, 09
]. H
owev
er,
besi
des
func
tioni
ng a
s st
ruct
ural
anc
hor
poin
ts,
cell-
mat
rix a
dhes
ion
site
s pl
ay a
cru
cial
rol
e in
trans
mitt
ing
mec
hani
cal
stim
uli
into
ce
lls
thro
ugh
coup
ling
to
inte
grin
s an
d th
e ac
tin
cyto
skel
eton
. The
pre
cise
com
posi
tion
of th
e ad
hesi
on c
ompl
ex (
its m
atur
ity)
and
the
natu
re o
f
the
unde
rlyin
g EC
M b
oth
regu
late
how
ext
erna
l for
ces a
re se
nsed
by
the
cells
.
Adhe
sion
Com
plex
es:
One
of
the
earli
est a
bilit
ies
of c
ells
to r
espo
nd to
mec
hani
cal f
orce
s re
quire
s th
e at
tach
men
t of
cells
to
the
unde
rlyin
g EC
M.
Rec
ently
it
has
been
sug
gest
ed t
hat
cell-
asso
ciat
ed h
yalu
rona
n
regu
late
s th
e ea
rly s
tage
s of
cel
l at
tach
men
t, pa
rticu
larly
in
chon
droc
ytes
[C
ohen
et a
l.,
03;Z
imm
erm
anet
al.,
02;
Coh
enet
al.,
06]
. St
udie
s ha
ve s
how
n th
at t
reat
men
t of
cel
ls w
ith
hyal
uron
idas
e dr
astic
ally
redu
ces c
ell a
ttach
men
t, an
d th
at lo
nger
per
iods
of t
ime
are
requ
ired
for
thes
e ce
lls t
o fir
mly
adh
ere
to t
he s
ubst
rate
; sp
ecifi
cally
, it
was
det
erm
ined
tha
t hy
alur
onan
-
28
med
iate
d at
tach
men
t oc
curs
with
in s
econ
ds a
fter
the
cell’
s in
itial
enc
ount
er w
ith a
sur
face
[Zai
del-B
aret
al.,
04;
Zim
mer
man
, Gei
ger &
Add
adi,
02].
The
hyal
uron
an-m
edic
ated
adh
esio
n, w
hich
is tr
ansi
ent,
is r
epla
ced
with
in a
few
min
utes
with
inte
grin
-con
tain
ing
cont
acts
(in
tegr
ins
are
disc
usse
d be
low
). Th
e ea
rlies
t in
tegr
in-c
onta
inin
g
adhe
sion
site
s ar
e te
rmed
foca
l com
plex
es (F
X) a
nd a
re fo
rmed
by
the
activ
atio
n of
sm
all R
ho-
fam
ily G
TPas
e R
ac [
Rot
tner
et a
l., 9
9]. T
hese
foc
al c
ompl
exes
are
sm
all,
dot-l
ike
and
prov
ide
early
cel
l atta
chm
ent i
n pr
otru
ding
are
as s
uch
as th
e le
adin
g ed
ge. T
he c
ompo
nent
ial m
akeu
p of
FXs
is d
ynam
ical
ly r
egul
ated
in r
espo
nse
to c
ell s
igna
ls a
nd th
e ex
ogen
ous
tens
ion
(i.e.
mat
rix
rigid
ity),
and
may
var
y in
diff
eren
t cel
l typ
es[B
errie
r & Y
amad
a, 0
7]. N
onet
hele
ss, i
t is
curr
ently
belie
ved
that
the
earli
estm
olec
ules
pre
sent
at t
he F
X a
re th
e α V
β3 in
tegr
in, t
alin
and
pax
ilin.
As
the
FXs
mat
ure
(for
exa
mpl
eas
a r
esul
t of
inc
reas
ed s
tress
) ad
ditio
nal
mol
ecul
ar c
ompo
nent
s
may
be
adde
d in
clud
ing,
vin
culin
, α-a
ctin
in, f
ocal
adh
esio
n ki
nase
(FA
K),
Arp
2/3
[Zai
del-B
ar,
Coh
en, A
ddad
i & G
eige
r, 04
]. Fo
cal c
ompl
exes
are
hig
hly
dyna
mic
and
last
for
a f
ew m
inut
es
befo
re b
eing
repl
aced
, dis
man
tled
or, t
hrou
gh th
e ac
tivat
ion
of R
ho, m
atur
e in
to fo
cal a
dhes
ions
(FA
)[R
ottn
er, H
all &
Sm
all,
99].
Foca
l adh
esio
ns a
re m
ore
pron
ounc
ed in
siz
e an
d in
volv
e th
e
addi
tiona
l re
crui
tmen
t of
mol
ecul
es s
uch
as z
yxin
and
the
conc
omita
nt a
ssem
bly
of a
n ac
tin-
bund
le.
This
tra
nsiti
ons
appe
ars
to b
e ac
tom
yosi
n-dr
iven
and
inv
olve
d co
ntra
ctili
ty o
f th
e
mem
bran
e th
at r
esul
ts i
n fo
rce
gene
ratio
n at
the
cel
l-mat
rix a
dhes
ion
site
[Za
idel
-Bar
, Coh
en,
Add
adi &
Gei
ger,
04;B
errie
r & Y
amad
a, 0
7]. W
hile
the
biol
ogic
al re
leva
nce
of F
A w
as in
itial
ly
unce
rtain
, in
rece
nt y
ears
it w
as s
how
n th
at F
As
are
foun
din
viv
oin
are
as o
f hi
gh f
luid
she
ar
stre
ss (i
.e. b
lood
ves
sels
) and
that
they
pla
y a
cruc
ial r
ole
durin
g de
velo
pmen
t [C
ukie
rman
et a
l.,
01;R
omer
et a
l., 0
6].
It is
bel
ieve
d th
at l
ocal
mec
hani
cal
forc
es a
ctiv
ate
the
grow
th a
nd
mat
urat
ion
of F
As [
Riv
elin
eet
al.,
01]
.
29
Des
pite
thei
r siz
e FA
rem
ain
high
ly d
ynam
ic w
ith v
ario
us c
ompo
nent
s, in
par
ticul
ar v
incu
lin a
nd
α-ac
tinin
, int
erch
angi
ng b
etw
een
thei
r FA
-ass
ocia
ted
and
cyto
plas
mic
poo
ls. I
n ad
ditio
n, F
A c
an
furth
er m
atur
e in
to f
ibril
lar
adhe
sion
s (F
Bs)
in
whi
ch t
he α
5β1
inte
grin
ass
ocia
tes
with
fibro
nect
in fi
brils
[Zam
iret
al.,
99]
. Vis
ually
, FB
s ap
pear
as
elon
gate
d fib
rils
or a
n ar
ray
of d
ots
and
are
loca
lized
to
mor
e ce
ntra
l re
gion
s in
cel
ls.
Unl
ike
FAs,
whi
ch c
onta
in h
igh
leve
ls o
f
paxi
llin
and
vinc
ulin
, FB
s po
sses
s hi
gh le
vels
of
tens
in a
nd li
ttle
phos
phot
yros
ine[
Zam
ir, K
atz,
Aot
a, Y
amad
a, G
eige
r &
Kam
, 99
]. It
has
been
sug
gest
ed t
hat
FBs
play
a r
ole
in m
atrix
reor
gani
zatio
n, a
s th
e pl
iabi
lity
of th
e EC
M w
as s
how
n to
influ
ence
FB
ass
embl
y [K
atz
et a
l.,
00].
Whi
le th
e ex
act m
echa
nism
regu
latin
g th
e tra
nsfo
rmat
ion
of F
As i
nto
FBsh
as n
ot b
een
fully
eluc
idat
ed, i
t was
dem
onst
rate
d th
at c
-Src
-nul
l cel
ls f
ail t
o fo
rm F
Bs,
indi
catin
g th
at c
-Src
may
be in
volv
ed in
this
pro
cess
[Vol
berg
et a
l., 0
1].
Inte
grin
s:
The
adhe
sion
of
the
cells
to
the
surr
ound
ing
ECM
is
med
iate
d, i
n pa
rt, b
y sp
ecia
lized
trans
mem
bran
e ad
hesi
on r
ecep
tors
ter
med
int
egrin
s. Th
is r
ecep
tor
fam
ily e
ncom
pass
es s
ever
al
non-
cova
lent
ly b
ound
het
erod
imer
ic tr
ansm
embr
ane
prot
eins
com
pose
d of
an
α (1
20-1
80 k
Da)
and
β (9
0-11
0 kD
a) s
ubun
its. W
ithin
eac
h su
buni
t is
foun
d a
larg
e ex
trace
llula
r do
mai
n, w
hich
inte
ract
s w
ith v
ario
us E
CM
com
pone
nts,
a si
ngle
tran
smem
bran
e sp
anni
ng d
omai
n, a
nd a
sho
rt
cyto
plas
mic
tai
l w
hich
int
erac
ts w
ith v
ario
us a
dapt
er m
olec
ules
in
the
cyto
plas
m [
Hum
phrie
s,
00].
Cur
rent
ly 2
4 di
ffer
ent m
amm
alia
n re
cept
ors
are
know
n, w
hich
are
com
pose
d of
18
diff
eren
t
α an
d 8
diff
eren
t β s
ubun
its; t
he s
peci
ficity
of
the
inte
grin
tow
ards
a p
artic
ular
liga
nd a
nd it
s
dow
nstre
am c
ellu
lar
effe
ct a
re r
egul
ated
by
the
resp
ectiv
e su
buni
ts (
Tabl
e II
I) [
Bra
kebu
sch
&
Fass
ler,
05;T
akad
aet
al.,
07]
. In
tegr
ins
are
able
to
reco
gniz
e a
wid
e ar
ray
of p
hysi
olog
ical
ligan
ds, i
nclu
ding
bot
h so
lubl
e an
d su
rfac
e-bo
und
prot
eins
. Bin
ding
of l
igan
ds a
ttach
ed to
a ri
gid
30
surf
ace
resu
lts in
the
gene
ratio
n of
for
ce a
t the
foc
al c
onta
cts,
and
dire
ctly
influ
ence
s th
e ce
ll’s
cont
ract
ile a
ppar
atus
. In
tegr
in b
indi
ng t
o th
eir
resp
ectiv
e lig
ands
req
uire
s th
e pr
esen
ce o
f
diva
lent
cat
ions
[Arn
aout
et a
l., 0
5].
Tabl
e 3:
The
cur
rent
ly k
now
n m
amm
alis
n in
tegr
ins a
nd th
eir l
igan
d-bi
ndin
g sp
ecifi
citie
s, ad
apte
d fr
om [4
9].
Inte
grin
Lig
and
Inte
grin
Lig
and
α1β1
Lam
inin
, col
lage
nαL
β2IC
AM
, IC
AM
-4α2
β1La
min
in, c
olla
gen,
thro
mbo
spon
din,
E-
αMβ2
ICA
M, i
C3b
, fac
tor X
, fib
rinog
en,
ICA
M-4
, hep
arin
α3β1
Lam
inin
, thr
ombo
spon
din,
PAR
αXβ2
ICA
M, i
C3b
, fib
rinog
en, I
CA
M-4
,
hi
llα4
β1Th
rom
bosp
ondi
n,
MA
dCA
M-1
, VC
AM
-1,
αDβ2
ICA
M, V
CA
M-1
, fib
rinog
en, f
ibro
nect
in,
vitro
nect
in, C
yr61
, pla
smin
ogen
α5β1
Fibr
onec
tin, o
steo
pont
in,
fibril
lin, t
hrom
bosp
ondi
n,
AD
AM
, CO
MP,
L1
αIIb
β3Fi
brin
ogen
, thr
ombo
spon
din,
,
fibro
nect
in, v
itron
ectin
, vW
F, C
yr61
,
ICA
M-4
, L1,
CD
40 li
gand
α6β1
Lam
inin
, thr
ombo
spon
din,
AD
AM
, Cyr
61
αVβ3
Fibr
inog
en, v
itron
ectin
, vW
F,
thro
mbo
spon
din,
fibr
illin
, ten
asci
n,
PEC
AM
-1, f
ibro
nect
in, o
steo
pont
in,
BSP
, MFG
-E8,
AD
AM
-15,
CO
MP,
α7β1
Lam
inin
α6β4
Lam
inin
α8β1
Tena
scin
, fib
rone
ctin
,
oste
opon
tin, v
itron
ectin
,
αVβ5
Ost
eopo
ntin
, BSP
, vitr
onec
tin, C
CN
3,
LAP-
TGF-
β
α9β1
Tena
scin
, VC
AM
-1,
oste
opon
tin, u
PAR
,
plas
min
, ang
iost
atin
,
αVβ6
LAP-
TGF-
β, fi
bron
ectin
, ost
eopo
ntin
,
AD
AM
α10β
1La
min
in, c
olla
gen
α4β7
MA
dCA
M-1
, VC
AM
-1, f
ibro
nect
in,
oste
opon
tinα1
1β1
Col
lage
nαE
β7E-
cadh
erin
αVβ1
LAP-
TGF-
β,
αVβ8
LAP-
TGF-
β
31
Man
y in
tegr
ins
are
know
n to
exi
st i
n di
ffer
ent
activ
atio
n st
ates
(ac
tive,
int
erm
edia
te,
and
inac
tive)
whi
ch c
orre
spon
d to
thei
r rel
ativ
e af
finity
sta
tes
tow
ards
ext
race
llula
r lig
ands
. Util
izin
g
thes
e di
ffer
ent
affin
ity s
tate
s in
tegr
ins
are
utili
zed
by c
ells
for
bot
h ou
tsid
e-in
and
ins
ide-
out
sign
allin
g,
whe
reby
cy
topl
asm
ic
chan
ges
influ
ence
th
e in
tegr
ins
abili
ty
to
bind
/atta
ch
extra
cellu
lar
ligan
ds (
insi
de-o
ut)
whi
ch th
en is
abl
e to
influ
ence
cel
lula
r fu
nctio
nalit
y (o
utsi
de-
in) [
Ingb
er, 0
3]. I
nteg
rins
utili
ze th
eir c
ytop
lasm
ic d
omai
ns,c
omm
only
refe
rred
to a
s th
e ta
il, to
elic
it va
rious
intra
cellu
lar s
igna
lling
resp
onse
s th
roug
h in
tera
ctio
n w
ith v
ario
us a
dapt
er p
rote
ins
and
the
actin
cyt
oske
leto
n. O
win
g to
the
intri
nsic
lack
of e
nzym
atic
act
ivity
, and
lack
of a
n ac
tin
bind
ing
dom
ain,
all
of t
he i
nteg
rins’
fun
ctio
nalit
y is
fac
ilita
ted
thro
ugh
inte
grin
ass
ocia
ted
adap
ter m
olec
ules
[Arn
aout
, Mah
alin
gam
& X
iong
, 05]
.
Ow
ing
to th
e ric
h ex
trace
llula
r mat
rix s
urro
undi
ng c
hond
rocy
tes,
and
the
know
n ro
le o
f int
egrin
s
to i
nter
act
with
EC
M c
ompo
nent
s, in
tegr
ins
have
lon
g be
en s
ugge
sted
as
one
mec
hani
sm b
y
whi
ch c
hond
rocy
tes
sens
e an
d re
spon
d to
mec
hani
cal
forc
es. P
revi
ous
stud
ies
have
sug
gest
ed
that
int
egrin
s m
edia
te m
ultip
le a
spec
ts o
f ch
ondr
ocyt
e m
echa
notra
nsdu
ctio
n in
res
pons
e to
com
pres
sive
and
she
ar f
orce
s [S
pite
ri, Y
oung
, Sim
mon
s, K
ande
l & P
illia
r, 08
;Mill
war
d-Sa
dler
& S
alte
r, 04
a;O
node
raet
al.,
05]
. In
par
ticul
ar,
chon
droc
ytes
hav
e be
en s
how
n to
sec
rete
inte
rleuk
in-4
in r
espo
nse
to m
echa
nica
l com
pres
sion
thro
ugh
a si
gnal
ling
casc
ade
activ
ated
by
the
α1β1
inte
grin
; whe
n ch
ondr
ocyt
es w
ere
stim
ulat
ed in
the
pres
ence
of
a G
RD
DSP
pep
tide,
whi
ch c
ompe
tes
for
the
inte
grin
-mat
rix L
igan
d bi
ndin
g sp
ots,
the
resp
onse
was
abo
lishe
d
[Mill
war
d-Sa
dler
& S
alte
r, 04
a]. I
n an
othe
r m
onol
ayer
stu
dy, m
echa
nica
l stim
ulat
ion
faile
d to
resu
lt in
incr
ease
d pr
oteo
glyc
an s
ynth
esis
in c
hond
rocy
tes
stim
ulat
ed in
the
pres
ence
of b
lock
ing
antib
ody
for
αVβ5
su
gges
ting
that
th
is
parti
cula
r in
tegr
in
also
pl
ays
a ro
le
in
mec
hano
trans
duct
ion
[Hol
ledg
eet
al.,
08]
. Stu
dies
con
duct
ed in
3D
cul
ture
furth
er su
ppor
ted
the
32
role
of
inte
grin
in m
edia
ting
chon
droc
ytes
’ re
spon
se to
mec
hani
cal f
orce
s w
here
, for
inst
ance
,
bloc
king
the
α5β1
inte
grin
regu
late
d ce
ll m
orph
olog
y an
d in
fluen
ced
synt
hesi
s of
col
lage
ns a
nd
prot
eogl
ycan
s in
resp
onse
to sh
ear f
orce
s [Sp
iteri,
You
ng, S
imm
ons,
Kan
del &
Pill
iar,
08].
Whi
le th
e ro
le in
tegr
ins
play
in c
hond
rocy
te m
echa
notra
nsdu
ctio
n ha
s be
en il
lust
rate
d th
roug
h
expe
rimen
ts u
tiliz
ing
bloc
king
ant
ibod
ies
or p
eptid
e-co
mpe
ting
ligan
ds, t
he p
reci
se d
owns
tream
even
ts h
ave
not b
een
fully
elu
cida
ted.
Upo
n in
tera
ctio
n be
twee
n an
inte
grin
and
the
resp
ectiv
e
mat
rix li
gand
a 2
pN
talin
-dep
ende
nt a
dhes
ion
poin
t for
ms
[Jia
nget
al.,
03]
. Thi
s ea
rly a
dhes
ion
sign
als f
or th
e ac
tivat
ion
of R
ac a
nd C
dc42
whi
ch u
ltim
atel
y re
gula
te th
e na
ture
and
ext
ent o
f the
adhe
sion
(FX
, FC
or
FB)[
Schw
artz
& S
hatti
l, 00
]. U
pon
the
form
atio
n of
the
adhe
sion
var
ious
adap
ter
mol
ecul
es m
ay i
nter
act
with
the
new
ly f
orm
ed c
ompl
ex a
nd,
depe
ndin
g on
the
com
posi
tion
of a
dapt
er m
olec
ules
, sen
d va
rious
dow
nstre
am s
igna
ls. O
ne m
olec
ule
of in
tere
st is
the
foca
l adh
esio
n ki
nase
(FA
K),
whi
ch is
one
of t
he fi
rst m
olec
ules
that
are
recr
uite
d to
the
site
[Sch
war
tz, 0
1]. U
pon
recr
uitm
ent,
FAK
aut
opho
spho
ryla
tes
to r
evea
l a S
rc-h
omol
ogy-
2 (S
H2)
bind
ing
dom
ain
whi
ch th
en e
nabl
es it
to in
tera
ct w
ith o
ther
kin
ases
such
as S
rc a
nd F
yn [S
chal
ler
et a
l., 9
4;Sc
hlae
pfer
et a
l., 9
7]. T
hese
mol
ecul
es, e
ither
in c
ohor
t or
inde
pend
ently
, are
abl
e to
phos
phor
ylat
e ot
her
com
pone
nts
of t
he c
ompl
ex s
uch
as p
axili
n an
d te
nsin
, an
d th
us e
licit
a
dive
rse
arra
y of
dow
nstre
am s
igna
ls [V
uori
et a
l., 9
6]. I
t is
inte
rest
ing
to n
ote
that
dep
endi
ng o
n
the
perc
eive
d m
echa
nica
l fo
rce,
diff
eren
t ph
osph
oryl
atio
n si
tes
on F
AK
and
Src
are
act
ivat
ed
thus
inf
luen
cing
the
dow
nstre
am c
ellu
lar
resp
onse
; fo
r in
stan
ce i
t w
as s
how
n th
at F
AK
-
tyro
sine
397
is s
peci
fical
ly p
hosp
hory
late
d up
on b
indi
ng o
f in
tegr
ins
to r
igid
sur
face
s w
here
as
othe
r su
rfac
es r
egul
ate
the
phos
phor
ylat
ion
of o
ther
site
s [R
enet
al.,
00]
. Cel
lula
r re
spon
ses
to
mec
hani
cal f
orce
s al
so d
epen
d on
the
spec
ific
ECM
com
pone
nts
to w
hich
inte
grin
s ca
n bi
nd. A
stud
y in
vest
igat
ing
the
effe
cts
of b
iaxi
al s
tretc
h on
car
diac
fib
robl
asts
not
ed t
hat
whi
le J
NK
1
33
activ
atio
n w
as s
een
in c
ells
pla
ted
on f
ibro
nect
in, v
itron
ectin
or
lam
inin
whi
le E
RK
2 w
as o
nly
activ
ated
in
cells
pla
ted
on f
ibro
nect
in [
Mac
Ken
naet
al.,
98]
. Si
mila
rly,
cycl
ic s
tretc
h of
vasc
ular
sm
ooth
mus
cle
resu
lted
in in
crea
sed
apop
tosi
s in
cel
ls g
row
n on
col
lage
n I m
atrix
, but
not o
n ot
her m
atrix
es [Y
ano
et a
l., 9
6].
Of
the
mul
tiple
inte
grin
s kn
own
to b
e ex
pres
sed
in m
amm
alia
n ce
lls, o
f pa
rticu
lar
inte
rest
are
thos
e in
tegr
ins
whi
ch a
re k
now
n to
be
expr
esse
d in
arti
cula
r car
tilag
e, n
amel
y: α
1β1,
α5β
1 (th
e
clas
sica
l “f
ibro
nect
in”
rece
ptor
), α1
0β1
and
the
αVβ5
[Lo
eser
, 00]
. Of
thes
e, t
he β
1 in
tegr
ins
have
bee
n th
e m
ost s
tudi
ed. U
pon
activ
atio
n, β
1 in
tegr
in si
gnal
ling
resu
lts in
the
activ
atio
n of
the
Ras
-MA
PK p
athw
ay [
Shak
ibae
iet
al.,
99;
Shak
ibae
i &
Mer
ker,
99].
This
sig
nalli
ng p
athw
ay
play
s a
cruc
ial
role
in
mai
ntai
ning
cel
lula
r fu
nctio
n (i.
e. g
row
th, d
iffer
entia
tion,
mig
ratio
n) i
n
man
y ce
ll ty
pes
thro
ugh
a st
ep-w
ise
activ
atio
n of
a c
asca
de o
f pr
otei
ns. U
pon
activ
atio
n, R
as,
whi
ch is
mem
bran
e an
chor
ed, p
hosp
hory
late
s an
d ac
tivat
es th
e Se
r/Thr
kin
ase
Raf
. Raf
, in
turn
,
phos
phor
ylat
es m
itoge
n ac
tivat
ed p
rote
in k
inas
e ki
nase
(M
APK
K)
also
kno
wn
as E
xtra
cellu
lar
Reg
ulat
ed K
inas
e (E
RK
). U
pon
activ
atio
n, E
RK
tran
sloc
ates
from
the
cyto
sol t
o th
e nu
cleu
s an
d
regu
late
s th
e ac
tivity
of
trans
crip
tion
fact
ors
such
as
jun,
c-fo
s,c-
myc
and
AP-
1 [M
acK
enna
,
Dol
fi, V
uori
& R
uosl
ahti,
98;
Shak
ibae
i & M
erke
r, 99
;Kar
in, 9
5].
Thes
e tra
nscr
iptio
n fa
ctor
s, in
turn
, reg
ulat
e th
e tra
nscr
iptio
n of
gen
e an
d ac
tivat
enu
mer
ous c
ellu
lar r
espo
nses
.
1.3.
4C
alci
um S
igna
lling
An
addi
tiona
l mec
hani
sm b
y w
hich
cel
ls a
re a
ble
to s
ense
to re
spon
d to
mec
hani
cal s
timul
atio
n
is th
roug
h th
e ut
iliza
tion
of c
alci
um s
igna
lling
. Cal
cium
(Ca2+
) is
a ub
iqui
tous
sec
ond
mes
seng
er
that
is
know
n to
reg
ulat
e a
dive
rse
rang
e of
cel
lula
r fu
nctio
ns i
nclu
ding
gen
e tra
nscr
iptio
n,
met
abol
ism
, m
uscl
e co
ntra
ctio
n,
prol
ifera
tion
and
mec
hano
trans
duct
ion.
Th
is
dive
rse
func
tiona
lity
of C
a2+ io
ns is
attr
ibut
ed to
the
abili
ty o
f ce
lls to
var
y th
eir
Ca2+
sign
als
in s
pace
,
34
time,
am
plitu
de a
s w
ell t
hrou
gh e
xpre
ssio
n of
sel
ectiv
e in
trace
llula
r co
mpo
nent
s th
at a
re e
ither
Ca2+
depe
nden
t or r
espo
nsiv
e [B
errid
geet
al.,
00]
.
The
intra
cellu
lar c
once
ntra
tion
of is
Ca2+
regu
late
d by
two
sim
ulta
neou
s pr
oces
ses,
one
of w
hich
one
aim
s to
intro
duce
cal
cium
into
the
cyto
plas
m (‘
on’ r
eact
ions
), w
hile
the
othe
r is
empl
oyed
to
rem
ove
cyto
plas
mic
cal
cium
thr
ough
the
use
of
buff
ers,
pum
ps,
and
ion
exch
ange
rs (
‘off
’
reac
tions
).It
is g
ener
ally
bel
ieve
d th
at i
n re
stin
g ce
lls, i
n w
hich
an
equi
libriu
m i
s es
tabl
ishe
d
betw
een
the
‘on’
and
‘of
f’ m
echa
nism
s, th
e in
trace
llula
r co
ncen
tratio
n of
cal
cium
([C
a2+] i)
is
appr
oxim
atel
y 10
0nM
. W
hen
cells
are
stim
ulat
ed,
thro
ugh
even
ts s
uch
as d
epol
aris
atio
n or
mec
hani
cal d
efor
mat
ion,
the
equi
libriu
m s
hifts
tow
ards
the
‘on’
reac
tions
, cau
sing
the
[Ca2+
] ito
incr
ease
to le
vels
that
may
surp
ass 1
μM [B
ootm
anet
al.,
01]
.
In g
ener
al,
Ca2+
utili
zed
in s
igna
lling
is
deriv
ed e
ither
fro
m i
nter
nal
stor
es o
r fr
om t
he
extra
cellu
lar
med
ium
. Int
erna
lly, c
alci
um is
pre
dom
inat
ely
stor
ed in
the
endo
plas
mic
ret
icul
um
(ER
) or
its
mus
cle
equi
vale
nt t
he s
arco
plas
mic
ret
icul
um (
SR),
alth
ough
in
som
e in
stan
ces
calc
ium
m
ay
be
sequ
este
red
in
the
mito
chon
dria
[B
ootm
an,
Col
lins,
Pepp
iatt,
Pr
othe
ro,
Mac
Ken
zie,
de,
Tra
vers
, To
vey,
Seo
, B
errid
ge,
Cic
colin
i &
Lip
p, 0
1;C
ollin
set
al.,
01]
. Th
e
rele
ase
of s
eque
ster
ed c
alci
um is
reg
ulat
ed b
oth
by c
alci
um it
self
and
by a
gro
up o
f se
cond
ary
mes
seng
ers
whi
ch i
nclu
de:
inos
itol-1
,4,5
-trip
hosp
hate
(IP
3),
cycl
ic A
DP
ribos
e (c
AD
PR)
and
nico
tinic
aci
d ad
enin
e di
nucl
eotid
e ph
osph
ate
(NA
AD
P) [
Ber
ridge
et a
l., 0
3].
Brie
fly,
the
activ
atio
n of
man
y ce
ll-su
rfac
e re
cept
ors
by th
eir l
igan
ds (i
.e. g
row
th fa
ctor
s, ho
rmon
es) r
esul
ts
in
the
activ
atio
n of
ph
osph
olip
ase
C
(PLC
) w
hich
ca
taly
zes
the
hydr
olys
is
of
phos
phat
idyl
inos
itol
4,5-
biop
hosp
hate
(PI
P 2)
to p
rodu
ce t
he I
P 3an
d di
acyl
glyc
erol
. U
pon
its
prod
uctio
n, I
P 3di
ffus
es in
to th
e cy
topl
asm
and
,thr
ough
act
ivat
ion
of s
peci
fic r
ecep
tors
on
the
ER/S
R (
IP3R
s) c
ause
s th
e re
leas
e of
sto
red
calc
ium
ions
. Sim
ilarly
, cA
DPR
indu
ces
the
rele
ase
35
of E
R-s
tore
d ca
lciu
m t
hrou
gh b
indi
ng t
o R
yano
dine
Rec
epto
rs (
RyR
s), w
hich
are
stru
ctur
ally
and
func
tiona
lly a
nalo
gous
to
IP3R
s [B
ootm
an,
Col
lins,
Pepp
iatt,
Pro
ther
o, M
acK
enzi
e, d
e,
Trav
ers,
Tove
y, S
eo,
Ber
ridge
, C
icco
lini
& L
ipp,
01;
Ber
ridge
, B
ootm
an &
Rod
eric
k, 0
3].
Rec
ently
it
was
sho
wn
that
NA
AD
P al
so p
osse
sses
the
abili
ty t
o in
duce
the
rel
ease
of
stor
ed
calc
ium
fro
m E
R/S
R s
tore
s, al
thou
gh th
e re
cept
or th
roug
h w
hich
it is
act
ing
is n
ot y
et k
now
n
[Gen
azza
ni &
Gal
ione
, 97]
.
Exte
rnal
cal
cium
is
utili
zed
by c
ells
thr
ough
sev
eral
spe
cial
ized
Ca2+
influ
x ch
anne
ls t
hat
are
grou
ped
toge
ther
bas
ed o
n th
eir
activ
atio
n m
echa
nism
s [B
errid
ge,
Lipp
& B
ootm
an,
00].
Vol
tage
-Gat
ed C
alci
um C
hann
els
(VG
CC
) ar
e ac
tivat
ed b
y th
e de
pola
rizat
ion
of t
he c
ellu
lar
mem
bran
e an
d he
lp re
gula
te v
ario
us c
ellu
lar p
roce
sses
incl
udin
g se
cret
ion,
con
tract
ion
and
gene
expr
essi
on [
Cat
tera
llet
al.,
05]
. St
ruct
ural
ly,
mam
mal
ian
VG
CC
s ar
e co
mpr
ised
of
5 di
stin
ct
subu
nits
(α1,
α 2, β
, γ a
nd δ
) whi
ch e
xist
in d
iffer
ent i
sofo
rms
and
are
diff
eren
tially
exp
ress
ed in
tissu
es th
roug
hout
the
body
. The
larg
est o
f sub
units
, the
α1
subu
nit (
190-
250
kDa)
, com
prom
ises
the
cond
uctio
n po
re (
chan
nel),
the
vol
tage
sen
sor,
gatin
g ap
para
tus,
and
cont
ains
sev
eral
regu
lato
ry si
tes a
cted
on
by se
cond
mes
seng
ers.
The
rem
aini
ng fo
ur su
buni
ts in
any
giv
en V
GC
C
serv
e to
re
gula
te
the
chan
nel’s
ga
ting
and
impa
rt di
stin
ctiv
e co
nduc
tanc
e,
curr
ent,
and
phar
mac
olog
ical
pr
oper
ties
to
vario
us
chan
nel
subt
ypes
[B
errid
ge,
Lipp
&
B
ootm
an,
00;C
atte
rall,
Per
ez-R
eyes
, Sn
utch
& S
tries
snig
, 05
]. Th
e va
rious
sub
type
s ar
e cl
assi
fied
acco
rdin
g to
ele
ctro
phys
iolo
gica
l an
d ph
arm
acol
ogic
al p
rope
rties
. B
riefly
, al
l ch
anne
ls a
re
divi
ded
eith
er i
nto
Low
-Vol
tage
Act
ivat
ed (
LVA
) or
Hig
h-V
olta
ge A
ctiv
ated
(H
VA
) gr
oups
.
LVA
cha
nnel
s re
quire
a s
trong
dep
olar
izat
ion
of t
he m
embr
ane
(-70
mV
), w
hile
the
HV
A
chan
nels
req
uire
a s
mal
ler
depo
lariz
atio
n (-
20 m
V)
[Lac
inov
a, 0
5]. S
ubse
quen
tly, c
hann
els
in
each
gro
up a
re fu
rther
div
ided
acc
ordi
ng to
thei
r res
pect
ive
curr
ent c
ondu
ctan
ce p
atte
rns
(Tab
le
36
4).
The
first
cha
ract
eriz
ed L
VA
cha
nnel
pos
sess
ed a
lar
ge,
slow
dec
ayin
g, c
urre
nt a
ndw
as
term
ed L
-Typ
e (L
for l
arge
and
long
last
ing)
. A p
harm
acol
ogic
al p
rope
rty s
hare
d am
ongs
t all
L-
type
cha
nnel
s is
the
ir se
nsiti
vity
to
1,4-
DH
Ps,
a w
ide
clas
s of
dru
gs,
with
inh
ibito
ry a
ctio
n
(nife
dipi
ne,
niso
ldip
ine,
isr
adip
ine)
. O
ther
HV
A c
hann
els,
whi
ch p
osse
ss a
sm
alle
r/sho
rter
cond
ucta
nce
and
are
inse
nsiti
ve to
DH
Ps, w
ere
disc
over
ed a
nd te
rmed
acc
ordi
ng to
sen
sitiv
ity to
othe
r sel
ectiv
e in
hibi
tors
: N-ty
pe (N
for n
euro
nal;
sens
itive
to ω
-con
otox
in G
VIA
), P/
Q-ty
pe (P
Purk
inje
cel
ls; f
or s
ensi
tive
to ω
-Aga
IVA
) and
R-ty
pe (R
for r
apid
and
resi
stan
t; re
sist
ant t
o th
e
abov
e na
med
toxi
ns).
The
sing
le s
ubty
pe th
at b
elon
gs to
the
HV
A g
roup
is th
e T-
type
cha
nnel
whi
ch is
nam
ed fo
r its
tiny
and
tran
sien
t con
duct
ance
[Ber
ridge
, Lip
p &
Boo
tman
, 00;
Cat
tera
ll,
Pere
z-R
eyes
, Snu
tch
& S
tries
snig
, 05;
Laci
nova
, 05]
.
Tabl
e 4:
Cla
ssifi
catio
n of
VG
CC
, ada
pted
from
[Lac
inov
a, 0
5]
Cha
nnel
Cla
ssTy
pe o
f Con
duct
ance
Subu
nit
HV
A
L-Ty
pe (L
ong-
, Lar
ge)
α 1S
(Ca V
1.1)
α 1C
(Ca V
1.2)
α 1D
(Ca V
1.3)
α 1F
(Ca V
1.4)
P/Q
-Typ
e (P
urki
nje
Cel
ls)
α 1A
(Ca V
2.1)
N-T
ype
(Neu
rona
l)α 1
B (C
a V2.
2)
R-T
ype
(Rap
id)
α 1E
(Ca V
2.3)
LVA
T-Ty
pe (T
iny,
Tra
nsie
nt)
α 1G
(Ca V
3.1)
α 1H
(Ca V
3.2)
α 1I
(Ca V
3.3)
37
In c
ontra
st t
o th
e V
GC
C, e
xtra
cellu
lar
calc
ium
can
ent
er c
ells
thr
ough
mec
hani
cally
act
ivat
ed
calc
ium
cha
nnel
s th
at r
espo
nd t
o ce
ll de
form
atio
n –
thei
r op
en p
roba
bilit
y in
crea
ses
with
mem
bran
e st
retc
h. O
win
g to
thei
r sen
sitiv
ity to
com
pres
sion
, she
ar s
tress
as
wel
l as
stre
tch
thes
e
chan
nels
are
ofte
n re
ferr
ed to
as
mec
hano
trans
duce
rs a
s th
ey c
onve
rt di
rect
phy
sica
l for
ces
into
biol
ogic
al s
igna
ls [
Gui
bert
et a
l., 0
8]. U
nlik
e th
e V
GC
Cs,
thes
e st
retc
h ac
tivat
ed c
hann
els
are
less
sel
ectiv
e fo
r the
pre
cise
spe
cies
of i
ons,
and
tend
to a
llow
for i
ons
of s
imila
r siz
e/ch
arge
to
pass
thro
ugh
the
cond
uctio
n po
re. O
f pa
rticu
lar
inte
rest
is th
e ca
tioni
c st
retc
h ac
tivat
ed c
hann
el
(SA
C),
whi
ch a
llow
s fo
r ca
lciu
m to
pas
s th
roug
h th
e co
nduc
tion
pore
, alb
eit n
on s
elec
tivel
y as
both
sod
ium
and
pot
assi
um h
ave
may
pas
s as
wel
l [W
u &
Dav
is,
01].
Prev
ious
res
earc
h
sugg
este
d tw
o m
ajor
rol
es t
hat
SAC
s m
ay a
ssum
e: (
1) i
nduc
tion
of s
usta
ined
mem
bran
e
depo
lariz
atio
n vi
a ca
tion
influ
x, th
us a
ctiv
atin
g V
GC
Cs
and
indu
cing
incr
ease
in [
Ca2+
] i, o
r (2
)
allo
w f
or lo
cal i
ncre
ases
in c
ytos
olic
cal
cium
and
thus
influ
ence
loca
l sig
nalli
ng p
athw
ays
[Wu
& D
avis
, 01;
Zou
et a
l., 0
2]. W
hile
the
chan
nel h
as n
ot b
een
fully
cha
ract
eriz
ed o
n a
mol
ecul
ar
leve
l, an
d its
mec
hani
sm o
f act
ivat
ion
not f
ull e
luci
date
d, th
ree
mod
els
have
bee
n pr
opos
ed: (
1)
the
“bila
yer m
odel
” pr
opos
es th
at m
echa
nica
l def
orm
atio
ns in
the
lipid
bily
aer c
ause
the
chan
nel
to a
ctiv
ate
[Mar
oto
et a
l., 0
5],
(2)
the
“tet
her
mod
el”,
in
turn
, pr
opos
es t
hat
the
chan
nel
is
dire
ctly
lin
ked
to e
xtra
cellu
lar
or i
ntra
cellu
r pr
otei
ns t
hat
upon
def
orm
atio
n ge
nera
te a
nd p
ass
tens
ion
to th
e ch
anne
l[Pia
oet
al.,
03]
, (3)
last
ly, t
he “
seco
ndar
y si
gnal
mod
el”
sugg
ests
that
a
dist
ance
mec
hani
cal-s
ensi
tive
prot
ein
gene
rate
s a
diff
usib
le s
econ
d m
esse
nger
whi
ch c
ause
s th
e
chan
nel
to o
pen
[Kal
apes
iet
al.,
05]
. U
nlik
e vo
ltage
-gat
ed c
hann
els,
very
few
sel
ectiv
e
inhi
bito
rs o
f SA
Cs
are
curr
ently
kno
wn.
The
triv
alen
t lan
than
ide,
gad
olin
ium
(Gd3+
), is
the
mos
t
wid
ely
used
inhi
bito
r, ho
wev
er it
s in
hibi
tory
–al
beit
not m
ajor
-ef
fect
on
othe
r typ
es o
f cal
cium
chan
nels
hav
e be
en d
ocum
ente
d [C
atte
rall,
Per
ez-R
eyes
, Snu
tch
& S
tries
snig
, 05;
Wu
& D
avis
,
38
01].
A s
ingl
e sp
ecifi
c in
hibi
tor
(GsM
Tx-4
) ha
s be
en i
sola
ted
from
tar
antu
la (
Gra
mm
osto
la
spat
ulat
a) v
enom
[Suc
hyna
et a
l., 0
0].
Prev
ious
st
udie
s ha
ve
impl
icat
ed
calc
ium
si
gnal
ling
in
chon
droc
yte
mec
hano
trans
duct
ion
follo
win
g flu
id s
hear
forc
es [D
'And
rea
et a
l., 0
0], h
ydro
stat
ic fo
rces
[Miz
uno,
05]
, com
pres
sive
forc
es [
Rob
erts
et a
l., 0
1;Pi
nggu
an-M
urph
yet
al.,
05b
], an
d m
icro
pipe
tte in
dent
atio
n [K
ono
et
al.,
06].
How
ever
, th
e un
derly
ing
mec
hani
sm(s
) th
roug
h w
hich
ca
lciu
m
faci
litat
es
the
chon
droc
ytes
’ res
pons
e to
mec
hani
cal s
timul
i hav
e no
t bee
n fu
lly e
luci
date
d. O
f the
mul
titud
e of
calc
ium
cha
nnel
s, ch
ondr
ocyt
es h
ave
been
sho
wn
to p
redo
min
atel
y ex
pres
s th
e L-
Type
VG
CC
[Sha
oet
al.,
05;
Zusc
iket
al.,
97]
, and
the
catio
nic
SAC
(as
show
n th
roug
h ga
dolin
ium
inhi
bito
r
stud
ies)
[Gui
lak
et a
l., 9
9;Pe
rkin
set
al.,
05]
. Rec
ent s
tudi
es h
ave
show
n th
at s
elec
tive
inhi
bito
rs
of V
GC
Cs
can
redu
ce t
he e
xpre
ssio
n of
MM
P-3
and
MM
P-13
in
oste
oarth
ritic
can
ine
chon
droc
ytes
[B
oile
auet
al.,
05]
, po
ssib
ly s
ugge
stin
g th
at t
he u
preg
ulat
ion
of t
hese
MM
Ps
follo
win
g m
echa
nica
l st
imul
atio
n m
ay b
e at
tribu
ted,
at
leas
t pa
rtial
ly,
to c
alci
um s
igna
lling
.
Sim
ilarly
, st
udie
s in
hum
an p
ulm
onar
y ar
tery
end
othe
lial
cells
hav
e sh
own
that
cal
cium
can
regu
late
the
bind
ing
activ
ity o
f AP-
1 (a
regu
lato
r of M
T1-M
MP
expr
essi
on)[
Fant
ozzi
et a
l., 0
3],
whi
le o
ther
stu
dies
hav
e sp
ecifi
cally
impl
icat
ed c
alci
um c
urre
nts
thro
ugh
the
L-Ty
pe V
GC
C in
regu
latio
n of
AP-
1 bi
ndin
g ac
tivity
[Pre
mku
mar
et a
l., 0
0]. F
urth
er e
vide
nce
that
cal
cium
may
be
invo
lved
in f
acili
tatin
g th
e im
prov
emen
t of
carti
lage
tiss
ue f
ollo
win
g m
echa
nica
l stim
ulat
ion
is
seen
in
st
udie
s th
at
indi
cate
d th
at
intra
cellu
lar
calc
ium
le
vels
ca
n in
fluen
ce
aggr
ecan
mR
NA
[Alfo
rdet
al.,
03]
and
colla
gen
type
-X e
xpre
ssio
n [B
onen
& S
chm
id,
91],
as w
ell
as
mod
ulat
e th
e ra
tio o
f co
llage
ns ty
pe I
/type
II
in a
rticu
lar
chon
droc
ytes
[D
eshm
ukh
& S
awye
r,
77].
Oth
er st
udie
s hav
e im
plic
ated
the
SAC
s in
regu
latio
n of
agg
reca
n, so
x9 a
nd c
olla
gen
type
s I,
39
II,
IX,
and
XI
gene
exp
ress
ion,
as
treat
men
t w
ith G
d3+in
fluen
ced
thei
r ex
pres
sion
[Pe
rkin
s,
Der
foul
, Ast
& H
all,
05].
Rec
ently
it w
as o
bser
ved
that
sev
eral
ion
chan
nels
, inc
ludi
ng V
GC
Cs,
are
co-lo
caliz
ed in
thei
r
expr
essi
on w
ith β
1-in
tegr
ins
in m
ouse
lim
b-bu
d ch
ondr
ocyt
es [M
obas
heri
et a
l.,02
;Sha
kiba
ei &
Mob
ashe
ri, 0
3]. O
ther
stu
dies
, alb
eit i
n va
scul
ar s
moo
th m
uscl
es c
ells
, hav
e sh
own
that
the
α5β1
inte
grin
reg
ulat
es a
tyr
osin
e ph
osph
oryl
atio
n ca
scad
e in
volv
ing
Src
and
othe
r fo
cal
adhe
sion
prot
eins
that
con
trol t
he fu
nctio
n of
the
L-ty
pe V
GC
C [W
uet
al.,
01b
]. In
fact
, it i
s no
w k
now
n
that
bot
h Sr
c an
d Py
k2 (a
n ad
ditio
nal f
ocal
adh
esio
n as
soci
ated
pro
tein
) are
abl
e to
bin
d to
the
II-
III
linke
r an
d C
-term
inal
reg
ions
of
the
α 1c
subu
nit o
n L-
type
VG
CC
and
diff
eren
tially
reg
ulat
e
the
chan
nel’s
act
ivity
[D
ubui
set
al.,
06]
. C
onse
quen
tly,
it is
qui
te p
ossi
ble
that
cal
cium
is
invo
lved
in m
echa
notra
nsdu
ctio
n pa
thw
ays,
in c
hond
rocy
tes,
both
inde
pend
ently
thro
ugh
vario
us
ion
chan
nels
as w
ell a
s thr
ough
syne
rgis
tic a
ctio
n w
ith c
o-lo
caliz
ed in
tegr
ins.
40
1.3.
5C
ell M
orph
olog
y
Reg
ulat
ion
of c
ell
mor
phol
ogy
thro
ugh
adhe
sion
and
de-
adhe
sion
bet
wee
n th
e ce
lls a
nd t
heir
surr
ound
ing
ECM
is c
ritic
al fo
r num
erou
s ce
llula
r fun
ctio
ns, i
nclu
ding
mig
ratio
n, d
evel
opm
ent,
apop
tosi
s an
d re
spon
se t
o m
echa
nica
l st
imul
i [W
oods
et a
l., 0
7;Sp
iteri
et a
l., 0
6].
Rap
id c
ell
shap
e ch
ange
s ha
ve b
een
char
acte
rized
in
num
erou
s tis
sues
and
are
now
kno
wn
to p
lay
an
esse
ntia
l rol
e in
the
body
. In
the
endo
thel
ium
, for
inst
ance
, the
reg
ulat
ion
of c
ell-c
ell a
nd c
ell-
ECM
adh
esio
ns c
ause
s ra
pid
chan
ges
in c
ell s
hape
and
vas
cula
r arc
hite
ctur
e in
resp
onse
to b
oth
norm
al a
nd d
isea
se p
roce
sses
[Dud
ek &
Gar
cia,
01]
. Sim
ilarly
, cel
l spr
eadi
ng h
as b
een
obse
rved
in m
any
othe
r ce
ll ty
pes
incl
udin
g fib
robl
asts
, pl
atel
ets,
B c
ells
, sm
ooth
mus
cle
cells
, an
d
chon
droc
ytes
[Spi
teri,
Pill
iar &
Kan
del,
06;T
haka
ret a
l., 0
9;Fl
eire
et a
l., 0
6;C
aval
cant
i-Ada
met
al.,
07].
In p
artic
ular
, in
ter
ms
of m
echa
nica
l st
imul
i, it
was
pre
viou
sly
show
n th
at t
he
appl
icat
ion
of m
echa
nica
l fo
rces
, bo
th c
ompr
essi
ve a
nd s
hear
, ca
n en
hanc
e th
e fo
rmat
ion
of
stre
ss f
ibre
s an
d th
roug
h ch
ange
s in
the
cel
lula
r ac
tin c
ytos
kele
ton
influ
ence
cel
l m
orph
olog
y
[Mill
war
d-Sa
dler
& S
alte
r, 04
a;Sp
iteri,
Pill
iar
& K
ande
l, 06
;Gui
lak,
95;
Kni
ght,
Toyo
da, L
ee &
Bad
er, 0
6b].
Thus
, it
is b
elie
ved
that
cel
l sp
read
ing
can
serv
e as
a m
echa
nism
thr
ough
whi
ch
cells
sens
e, in
tegr
ate
and
resp
onse
to e
xter
nal m
echa
nica
l for
ces.
In r
espo
nse
to e
xter
nal
stim
uli
whi
ch c
ause
s ce
ll sp
read
ing,
act
in p
olym
eriz
atio
n dr
ives
the
asse
mbl
y of
ear
ly c
onta
cts
betw
een
the
cell
and
the
ECM
, ulti
mat
ely
lead
ing
to th
e fo
rmat
ion
of
foca
l co
mpl
exes
and
/or
foca
l ad
hesi
ons
[Pol
lard
& B
oris
y, 0
3].
Thes
e ev
ents
are
tig
htly
regu
late
d by
the
smal
l GTP
ases
Rac
1 an
d R
ho w
hich
reg
ulat
e ac
tin p
olym
eriz
atio
n (s
prea
ding
)
and
acto
myo
sin
cont
ract
ility
(ret
ract
ion)
, res
pect
ivel
y [B
alle
stre
met
al.,
01;
Witt
man
net
al.,
03]
.
Thes
e G
TPas
es
are
in
may
be
re
gula
ted
by
inte
grin
-med
iate
d ad
hesi
on
whi
ch
indu
ces
auto
phos
phor
ylat
ion
of F
AK
at t
yros
ine
397
whi
ch a
ctiv
ates
FA
K a
nd c
reat
es a
dditi
onal
bin
ding
41
site
s fo
r Src
. The
act
ivat
ed F
AK
-Src
com
plex
then
stim
ulat
es R
ac1
activ
ity th
roug
h re
crui
tmen
t
and
phos
hpor
ylat
ion
of p
130C
as a
nd i
ts a
ssoc
iate
d ad
apte
r pr
otei
ns (
Doc
k190
, ELM
O1,
Crk
),
whi
ch fu
nctio
ns a
s a
Gua
nine
-Exc
hang
e Fa
ctor
(GEF
) and
act
ivat
es R
ac1
resu
lting
in m
embr
ane
prot
rusi
ons
[Bru
gner
aet
al.,
02;
Kiy
okaw
aet
al.,
98]
. Thu
s, th
e ex
act l
ocal
izat
ion
and
activ
atio
n
of in
tegr
ins
is a
ble
to r
egul
ate
loca
lized
act
ivity
of
GEF
s an
d co
ordi
nate
mem
bran
e pr
otru
sion
s
[Huv
enee
rs &
Dan
en, 0
9]. I
nter
estin
gly,
MT1
-MM
P ha
s be
en s
how
n to
pla
y a
role
in re
gula
ting
inte
grin
clu
ster
ing
whi
ch f
urth
er i
mpl
icat
es t
his
MM
P in
reg
ulat
ing
cell
mor
phol
ogy
and
cons
eque
ntly
mat
rix a
ccum
ulat
ion
[Tak
ino
et a
l., 0
6].
As
spre
adin
g pr
ogre
sses
, Rho
A a
ctiv
ity g
radu
ally
incr
ease
s in
con
cert
with
for
mat
ion
of s
tress
fibre
s an
d th
e m
atur
atio
n of
foc
al c
onta
cts.
This
inc
reas
ed R
hoA
act
ivity
pro
mot
es m
yosi
n
activ
ity a
nd th
us r
esul
ts in
the
cont
ract
ility
(re
tract
ion)
of
the
actin
cyt
oske
leto
n [H
uven
eers
&
Dan
en,
09].
Whi
le t
he e
xact
mec
hani
sm b
y w
hich
Rho
A i
s ac
tivat
ed h
as n
ot b
een
fully
eluc
idat
ed,
one
poss
ible
exp
lana
tion
is t
hat
tens
ion
caus
ed d
urin
g th
e sp
read
ing
trigg
ers
Src-
med
iate
d ac
tivat
ion
spec
ific
GEF
s (p
115R
hoG
EF,
Gef
12,
p190
Rho
GEF
) th
at a
ctiv
ate
Rho
A
[Huv
enee
rs &
Dan
en, 0
9;D
anen
et a
l., 0
2]. I
n co
ntra
st, i
t has
bee
n sh
own
that
act
ivat
ion
of F
AK
-
Src
sign
allin
g ca
n re
sult
in in
crea
sed
phos
phor
ylat
ion
of E
ndop
hilin
A2
(a p
rote
in in
volv
ed in
endo
cyto
sis)
at
Tyr3
15 p
hosp
hory
latio
n w
hich
inh
ibits
its
act
ivity
and
thu
s pr
olon
gs t
he
prot
eoly
tic a
ctiv
ity o
f MT1
-MM
P an
d th
e de
grad
atio
n of
the
surr
ound
ing
ECM
[Wu
et a
l., 0
5].
The
incr
ease
d de
grad
atio
n of
the
ECM
wou
ld th
en w
eake
n th
e fo
cal a
dhes
ion
stab
ility
and
cau
se
the
cell
to re
tract
–an
eve
nt w
hich
may
sign
al th
e ce
ll to
incr
ease
its m
etab
olic
act
ivity
.
Non
ethe
less
, de
spite
the
hea
vy i
nvol
vem
ent
of i
nteg
rins
and
thei
r as
soci
ated
pro
tein
s in
cel
l
spre
adin
g an
d re
tract
ion,
it
is n
ow a
ccep
ted
that
cal
cium
may
exe
rt a
pote
nt e
ffec
t on
the
se
proc
esse
s. St
udie
s ha
ve s
how
n th
at u
nder
cer
tain
load
ing
cond
ition
s ch
ondr
ocyt
es e
xhib
it a
rapi
d
42
(~8
min
utes
) re
orga
niza
tion
of t
heir
actio
n cy
tosk
elet
on i
n a
calc
ium
-dep
ende
nt m
anne
r
[Eric
kson
et a
l., 0
3].
In a
n ad
ditio
nal
stud
y, c
hond
rocy
tes
assu
med
a f
latte
ned
fibro
blas
t-lik
e
phen
otyp
e (m
arke
d by
the
app
eara
nce
of s
tress
fib
ers)
upo
n tre
atm
ent
with
Gd3+
; up
on t
he
rem
oval
of
the
inhi
bito
r, th
e ce
lls a
ssum
ed th
eir
norm
al m
orph
olog
y su
gges
ting
SAC
s ar
e al
so
invo
lved
in re
gula
tion
chon
droc
yte
mor
phol
ogy
[Per
kins
, Der
foul
, Ast
& H
all,
05].
1.4
Hyp
othe
sis
Ow
ing
to t
he c
lose
int
erac
tion
of c
alci
um c
hann
els,
inte
grin
s an
d th
e cy
tosk
elet
on,
we
hypo
thes
ized
the
fol
low
ing:
im
prov
ed t
issu
e fo
rmat
ion
follo
win
g th
eap
plic
atio
n of
cyc
lic
com
pres
sion
occ
urs
via
a tra
nsie
nt c
ell s
prea
ding
, act
ivat
ion
of M
T1-M
MP,
and
sub
sequ
ent c
ell
retra
ctio
n w
hich
are
all
regu
late
d by
con
verg
ing
calc
ium
and
inte
grin
sign
allin
g pa
thw
ays.
Res
earc
h G
oals
1.In
vest
igat
e th
e ro
le c
alci
um p
lays
in
chon
droc
yte
cell
spre
adin
g an
d re
tract
ion
follo
win
g m
echa
nica
l stim
ulat
ion.
1.1.
Det
erm
ine
whe
ther
the
res
pons
e is
reg
ulat
ed b
y ex
trace
llula
r ca
lciu
m a
nd,
if so
, de
term
ine
whi
ch c
alci
um c
hann
els a
re im
plic
ated
.
1.2.
Det
erm
ine
whe
ther
cal
cium
is in
volv
ed in
reg
ulat
ing
MT1
-MM
P ex
pres
sion
fol
low
ing
mec
hani
cal s
timul
atio
n.
2.In
vest
igat
e th
e in
volv
emen
t of i
nteg
rins,
and
thei
r res
pect
ive
adap
ter p
rote
ins,
in th
e re
spon
se
to c
yclic
com
pres
sion
.
3.D
eter
min
e w
heth
er
calc
ium
si
gnal
ling
path
way
s in
tera
ct
with
th
e in
tegr
in
sign
allin
g pa
thw
ays i
n m
edia
ting
the
chon
droc
ytes
’ res
pons
e to
mec
hani
cal s
timul
atio
n.
43
CH
APT
ER
TW
O: P
APE
R M
AN
USC
RIP
T
44
Cal
cium
reg
ulat
es c
yclic
com
pres
sion
-indu
ced
earl
y ch
ange
s in
chon
droc
ytes
dur
ing
in
vitr
otis
sue
form
atio
n.
Igal
Rai
zman
, MSc
; Rita
Kan
del,
MD
CIH
R-B
ioE
ngin
eeri
ng o
f Ske
leta
l Tis
sue
Tea
m, M
ount
Sin
ai H
ospi
tal,
Uni
vers
ity o
f
Tor
onto
, Tor
onto
, Can
ada
M5G
1X
5.
Run
ning
title
: Cal
cium
reg
ulat
ion
of c
hond
rocy
te m
echa
notr
ansd
uctio
n
Key
wor
ds: C
alci
um, C
hond
rocy
tes,
Tis
sue-
engi
neer
ing,
Mec
hano
tran
sduc
tion
Man
uscr
ipt s
ubm
itted
to C
ell C
alci
um.
45
Abs
trac
t:
A s
ingl
e ap
plic
atio
n of
cyc
lic c
ompr
essi
on (
30m
in)
to b
ioen
gine
ered
car
tilag
e re
sults
in
impr
oved
tiss
ue f
orm
atio
n th
roug
h se
quen
tial c
atab
olic
and
ana
bolic
cha
nges
med
iate
d vi
a ce
ll
shap
e ch
ange
s th
at a
re r
egul
ated
by
α5β1
int
egrin
and
mem
bran
e-ty
pe m
etal
lopr
otea
se (
MT1
-
MM
P). T
o de
term
ine
if ca
lciu
m e
xerts
a r
egul
ator
y ro
le o
ver
this
pro
cess
, we
inve
stig
ated
how
calc
ium
regu
late
d ce
ll sh
ape
chan
ges,
MT1
-MM
P an
d in
tegr
in a
ctiv
ity in
resp
onse
to m
echa
nica
l
stim
ulat
ion.
St
imul
atio
n-in
duce
d ch
ange
s in
ce
ll sh
ape
and
MT1
-MM
P ex
pres
sion
w
ere
abol
ishe
d w
ith c
hela
tion
of e
xtra
cellu
lar
calc
ium
, an
d re
inst
ated
with
its
re-
intro
duct
ion.
Spre
adin
g w
as i
nhib
ited
by b
lock
ing
the
stre
tch-
activ
ated
cha
nnel
(w
ith g
adol
iniu
m),
whi
le
retra
ctio
n w
as p
reve
nted
by
bloc
king
the
L-Ty
pe v
olta
ge-g
ated
cha
nnel
(w
ith n
ifedi
pine
); bo
th
com
poun
ds i
nhib
ited
MT1
-MM
P up
regu
latio
n. C
hann
els’
rol
e w
as f
urth
er c
onfir
med
with
the
use
of e
ither
inhi
bito
r in
conj
unct
ion
with
the
calc
ium
A23
187
iono
phor
e, w
hich
rest
ored
cel
lula
r
resp
onse
. C
alci
um r
egul
ated
the
int
egrin
-med
iate
d si
gnal
ling
path
way
, w
hich
was
fac
ilita
ted
thro
ugh
the
kina
se S
rc. B
oth
calc
ium
-an
d in
tegr
in-m
edia
ted
path
way
s co
nver
ged
on a
ctiv
atin
g
ERK
in
re
spon
se
to
stim
ulat
ion.
W
hile
bo
th
inte
grin
s an
d ca
lciu
m
sign
allin
g m
edia
te
chon
droc
yte
mec
hano
trans
duct
ion,
ca
lciu
m
appe
ars
to
play
th
e m
ajor
re
gula
tory
ro
le.
Und
erst
andi
ng
the
unde
rlyin
g m
olec
ular
m
echa
nism
s in
volv
ed
in
chon
droc
yte
mec
hano
trans
duct
ion
may
lea
d to
the
dev
elop
men
t of
im
prov
ed b
ioen
gine
ered
car
tilag
e an
d a
bette
r tre
atm
ent f
or o
steo
arth
ritis
.
46
2.1
Intr
oduc
tion
Arti
cula
r car
tilag
e co
vers
the
artic
ulat
ing
ends
of b
ones
in s
ynov
ial j
oint
s. D
ue to
lack
of
vasc
ular
sup
ply
the
tissu
e po
sses
ses
a lim
ited
abili
ty fo
r sel
f-re
pair,
and
with
inju
ry o
r dis
ease
it
ofte
n fa
ils t
o pr
oper
ly h
eal.
Cur
rent
atte
mpt
s to
add
ress
thi
s is
sue
have
bee
n on
ly p
artia
lly
succ
essf
ul [
Dar
ling
& A
than
asio
u, 0
3]. C
onse
quen
tly, r
epai
r of
car
tilag
e de
fect
s w
ith in
vitr
o-
form
ed c
artil
age
tissu
e is
a p
rom
isin
g al
tern
ativ
e ap
proa
ch t
hat
wou
ld c
ircum
vent
man
y
limita
tions
inhe
rent
in c
urre
nt tr
eatm
ent a
ppro
ache
s. U
nfor
tuna
tely
the
biom
echa
nica
l pro
perti
es
of b
ioen
gine
ered
car
tilag
e tis
sue
are
infe
rior
to t
he n
ativ
e tis
sue,
a f
acto
r w
hich
pre
clud
es i
ts
utili
zatio
n cl
inic
ally
. In
our
lab
, w
e ha
ve s
how
n th
at t
he a
pplic
atio
n of
30
min
utes
of
com
pres
sive
forc
es e
nhan
ces
colla
gen
and
prot
eogl
ycan
acc
umul
atio
n by
arti
cula
r cho
ndro
cyte
s
grow
n on
the
sur
face
of
a po
rous
cer
amic
sub
stra
te [
Wal
dman
, Sp
iteri,
Gry
npas
, Pi
lliar
&
Kan
del,
04;W
aldm
an, C
outo
, Gry
npas
, Pill
iar &
Kan
del,
06].
This
incr
ease
in m
atrix
app
ears
to
be d
epen
dent
on
a bi
phas
ic r
emod
ellin
g pr
oces
s, w
hich
inv
olve
s an
ini
tial
cata
bolic
pha
se
(cha
ract
eriz
ed b
y ER
K a
ctiv
atio
n an
d up
regu
latio
n of
MT1
-MM
P) f
ollo
wed
by
a su
bseq
uent
anab
olic
res
pons
e w
hich
inc
reas
es t
he a
mou
nt o
f ex
trace
llula
r m
atrix
(EC
M)
synt
hesi
s an
d
accu
mul
atio
n [D
e C
roos
, Dha
liwal
, Gry
npas
, Pill
iar &
Kan
del,
06].
Furth
erm
ore,
it w
as o
bser
ved
that
th
is
stim
ulat
ion-
indu
ced
incr
ease
in
m
atrix
sy
nthe
sis
is
depe
nden
t up
on
trans
ient
chon
droc
yte
spre
adin
g an
d su
bseq
uent
ret
ract
ion,
med
iate
d th
roug
h th
e α5
β1 in
tegr
in
and
was
depe
nden
t on
the
upre
gula
tion
of m
embr
ane-
type
1 m
etal
lopr
otei
nase
(M
T1-M
MP)
, whi
ch w
as
obse
rved
to
occu
r im
med
iate
ly f
ollo
win
g m
echa
nica
l st
imul
atio
n [D
e C
roos
, Ja
ng,
Dha
liwal
,
Gry
npas
, Pi
lliar
&
K
ande
l, 07
](s
ubm
itted
fo
r pu
blic
atio
n).
H
owev
er,
the
unde
rlyin
g
mec
hani
sm(s
) by
whi
ch c
hond
rocy
tes
sens
e m
echa
nica
l fo
rces
and
con
vert
them
to
the
prev
ious
ly d
elin
eate
d pa
thw
ays
are
poor
ly u
nder
stoo
d. F
or e
xam
ple,
it
is n
ot k
now
n w
heth
er
ERK
act
ivat
ion
occu
rs p
rior t
o ce
ll sp
read
ing.
47
As
calc
ium
flu
xes
have
bee
n sh
own
to o
ccur
in
mec
hani
cally
stim
ulat
ed c
hond
rocy
tes
[Alfo
rd, Y
ello
wle
y, J
acob
s &
Don
ahue
, 03;
D'A
ndre
a, C
alab
rese
, Cap
ozzi
, Gra
ndol
fo, T
onon
&
Vitt
ur,
00;G
uila
k, Z
ell,
Eric
kson
, G
rand
e, R
ubin
, M
cLeo
d &
Don
ahue
, 99
;Zha
ng,
Wan
g &
Che
n, 0
6], a
nd c
an o
ccur
with
in s
econ
ds o
f stim
ulat
ion
[Kon
o, N
ishi
kori,
Kat
aoka
, Uch
io, O
chi
& E
nom
oto,
06;
Ping
guan
-Mur
phy
et a
l., 0
5a],
it ha
s be
en p
ropo
sed
that
the
first
sig
nalli
ng e
vent
in r
espo
nse
to m
echa
nica
l st
imul
i m
ay i
nvol
ve c
alci
um. O
f th
e m
ultit
ude
of c
alci
um c
hann
els
desc
ribed
, ch
ondr
ocyt
es h
ave
been
sho
wn
to e
xpre
ss t
he L
-Typ
e V
olta
ge G
ated
Cal
cium
Cha
nnel
(VG
CC
) and
the
catio
nic
stre
tch
activ
ated
cat
ioni
c ch
anne
l (SA
C) t
hat a
re p
erm
issi
ve to
calc
ium
ion
flu
xes
[Per
kins
, D
erfo
ul,
Ast
& H
all,
05;S
hao,
Alic
knav
itch
& F
arac
h-C
arso
n,
05;Z
usci
k, G
unte
r, Pu
zas
& R
osie
r, 97
]. St
udie
s in
can
ine
chon
droc
ytes
hav
e sh
own
that
sele
ctiv
e in
hibi
tors
of
VG
CC
s ar
e ab
le to
red
uce
the
expr
essi
on o
f M
MP-
3 an
d -1
3 [B
oile
au,
Mar
tel-P
elle
tier,
Bru
net,
Tard
if, S
chrie
r, Fl
ory,
El-K
atta
n, B
oily
& P
elle
tier,
05],
whi
le st
udie
s in
hum
an p
ulm
onar
y ar
tery
end
othe
lial
cells
hav
e sh
own
that
cal
cium
sig
nalli
ng r
egul
ates
the
bind
ing
activ
ity o
f A
P-1
(a k
now
n re
gula
tor
of M
T1-M
MP
expr
essi
on)
[Fan
tozz
i, Zh
ang,
Plat
oshy
n, R
emill
ard,
Cow
ling
& Y
uan,
03]
. Mor
eove
r, ch
ondr
ocyt
es a
ppea
r to
reor
gani
ze th
eir
actin
cyt
oske
leto
n in
a c
alci
um-d
epen
dent
man
ner
[Eric
kson
, N
orth
rup
& G
uila
k, 0
3],
and
intra
cellu
lar
calc
ium
leve
ls h
ave
been
sho
wn
to in
fluen
ce a
ggre
can,
col
lage
n ty
pes
I, II
, and
X
expr
essi
on le
vels
[Alfo
rd, Y
ello
wle
y, J
acob
s &
Don
ahue
, 03;
Bon
en &
Sch
mid
, 91;
Des
hmuk
h &
Saw
yer,
77].
Inte
rest
ingl
y, io
n ch
anne
ls c
o-lo
caliz
e w
ith β
1 in
tegr
ins [
Mob
ashe
ri, C
arte
r, M
artin
-
Vas
allo
& S
haki
baei
, 02;
Shak
ibae
i & M
obas
heri,
03]
, and
som
e st
udie
s ha
ve s
how
n th
at c
ross
-
talk
bet
wee
n th
e io
n ch
anne
ls a
nd in
tegr
ins
exis
t whi
ch c
an r
egul
ate
cell
func
tion
[Wu,
Dav
is,
Mei
ning
er,
Wils
on &
Dav
is,
01b]
. A
s su
ch,
calc
ium
sig
nalli
ng c
ould
be
invo
lved
in
the
48
regu
latio
n of
bot
h th
e ce
ll m
orph
olog
y ch
ange
s, an
d th
e an
abol
ic/c
atab
olic
resp
onse
that
occ
urs
in o
ur sy
stem
.
The
role
of
calc
ium
in
med
iatin
g en
hanc
ed c
artil
age
tissu
e fo
rmat
ion
in r
espo
nse
to
cycl
ic c
ompr
essi
on h
as n
ot b
een
eluc
idat
ed. T
he a
im o
f th
is s
tudy
, the
refo
re, w
as to
det
erm
ine
whe
ther
the
initi
al c
hond
rocy
te re
spon
se th
at le
ads
to th
e cy
clic
com
pres
sion
-indu
ced
incr
ease
in
mat
rix a
ccum
ulat
ion,
nam
ely
cell
spre
adin
g an
d M
T1-M
MP
expr
essi
on,
are
regu
late
dby
calc
ium
. In
addi
tion,
bec
ause
of t
he p
utat
ive
role
of α
5β1
inte
grin
in th
is p
roce
ss, w
e in
vest
igat
ed
whe
ther
the
calc
ium
sig
nalli
ng p
athw
ay is
invo
lved
in r
egul
atin
g th
e in
tegr
in a
ssoc
iate
d ki
nase
Src.
Und
erst
andi
ng th
e m
echa
nism
by
whi
ch m
echa
nica
l com
pres
sion
resu
lts in
incr
ease
d m
atrix
accu
mul
atio
n m
ay fu
rther
hel
p us
to im
prov
e th
e pr
oper
ties o
f in
vitr
o-fo
rmed
car
tilag
e tis
sue.
49
2.2
Met
hods
Cel
l Cul
ture
Cho
ndro
cyte
s w
ere
isol
ated
by
sequ
entia
l enz
ymat
ic d
iges
tion
from
bov
ine
(6 to
9 m
onth
old)
met
acar
pal-p
hala
ngea
l jo
ints
as
desc
ribed
pre
viou
sly
[Boy
leet
al.,
95]
. Brie
fly, c
artil
age
harv
este
d fr
om 2
-3 j
oint
s w
as p
oole
d to
geth
er i
n or
der
to o
btai
n su
ffic
ient
cel
ls, a
nd d
iges
ted
with
0.5
% p
rote
ase
(Sig
ma
Che
mic
al C
o., M
I) f
or 1
hou
r at
37°
C, f
ollo
wed
by
dige
stio
n w
ith
0.1%
co
llage
nase
(R
oche
M
olec
ular
B
ioch
emic
als,
IN)
over
nigh
t un
der
stan
dard
cu
lture
cond
ition
s (3
7°C
, 5%
CO
2). C
hond
rocy
tes
wer
e se
eded
in H
am’s
F-1
2 su
pple
men
ted
with
5%
feta
l bov
ine
seru
m (S
igm
a C
hem
ical
Co.
) at a
den
sity
of 1
60,0
00 c
ells
/mm
2on
to th
e su
rfac
es o
f
calc
ium
pol
ypho
spha
te (C
PP) s
ubst
rate
s (2
mm
hei
ght x
4m
m d
iam
eter
). C
ultu
res
wer
e gr
own
at
37°C
und
er st
anda
rd c
ell c
ultu
re c
ondi
tions
.
Mec
hani
cal S
timul
atio
n
Follo
win
g 3
days
of
stat
ic c
ultu
re,
cells
wer
e su
bjec
ted
to u
niax
ial,
conf
ined
cyc
lic
com
pres
sion
(1k
Pa,
1 H
z, 3
0min
) us
ing
a M
AC
H-1
mec
hani
cal
stim
ulat
ion
(Bio
synt
ech,
Mon
treal
, CA
N) a
s d
escr
ibed
pre
viou
sly
[De
Cro
os, D
haliw
al, G
rynp
as, P
illia
r & K
ande
l, 06
].
To a
void
dis
lodg
ing
the
smal
l am
ount
of t
issu
e fo
rmed
by
this
tim
e, th
e ce
lls w
ere
subj
ecte
d to
com
pres
sive
forc
ed th
roug
h a
com
plia
nt 2
% a
garo
se g
el d
isk
(3.5
mm
dia
met
er x
8 m
m h
eigh
t)
that
was
pla
ced
on t
op o
f th
e ce
lls.
The
cont
rol
cons
truct
s w
ere
mai
ntai
ned
unde
r id
entic
al
cultu
re c
ondi
tions
but
did
not
rece
ive
any
mec
hani
cal s
timul
atio
n.
Cal
cium
Stu
dies
To in
vest
igat
e th
e ro
le o
f cal
cium
in m
echa
notra
nsdu
ctio
n, c
onst
ruct
s w
ere
prei
ncub
ated
in s
erum
fre
e H
ams
F-12
alo
ne (
as a
con
trol)
or s
uppl
emen
ted
with
EG
TA (
10μM
), or
EG
TA
50
(10μ
M)
com
bine
d w
ith C
aCl 2
(5μM
), fo
r 4
hour
s pr
ior
to m
echa
nica
l st
imul
atio
n.
Thes
e
sam
ples
wer
e th
en s
ubje
cted
to m
echa
nica
l stim
ulat
ion
unde
r the
sam
e co
nditi
ons.
To d
eter
min
e
whi
ch c
alci
um p
erm
issi
ve c
hann
els
are
invo
lved
, cho
ndro
cyte
s w
ere
prei
ncub
ated
for 4
hou
rs in
the
abse
nce
orpr
esen
ce o
f th
e io
n ch
anne
l inh
ibito
rs, n
ifedi
pine
and
gad
olin
ium
(Si
gma)
whi
ch
wer
e di
ssol
ved
in D
MSO
and
then
dilu
ted
with
HA
Ms
F-12
to f
orm
a 2
0μM
sol
utio
n (0
.01%
DM
SO).
The
role
of
calc
ium
flu
xes
was
fur
ther
con
firm
ed b
y ut
ilizi
ng th
e A
2318
7 io
noph
ore
(10μ
M)
in c
onju
nctio
n w
ith t
he i
nhib
itors
.To
inv
estig
ate
the
invo
lvem
ent
of S
rc,
cons
truct
s
wer
e pr
einc
ubat
ed in
the
pres
ence
of
PP2
(4-a
min
o-5-
(4-c
hlor
ophe
nyl)-
7-(t-
buty
l)pyr
azol
o[3,
4-
d]py
rimid
ine)
or i
ts n
egat
ive
cont
rol P
P3 (4
-Am
ino-
7-ph
enyl
pyra
zol[3
,4-d
]pyr
imid
ine)
(20
μM),
purc
hase
d fr
om C
albi
oche
m, f
or 4
hou
rs p
rior t
o m
echa
nica
l stim
ulat
ion
.
Cho
ndro
cyte
Mor
phol
ogy
and
Area
Follo
win
g m
echa
nica
l stim
ulat
ion,
tiss
ue-C
PP c
onst
ruct
s wer
e w
ashe
d th
ree
times
in c
old
phos
phat
e bu
ffer
ed s
alin
e (P
BS)
and
fix
ed i
n 2.
5% g
luta
rald
ehyd
e fo
r at
lea
st a
n ho
ur.
Subs
eque
ntly
, con
stru
cts w
ere
rinse
d th
ree
addi
tiona
l tim
es in
PB
S, d
ehyd
rate
d in
gra
ded
etha
nol
(up
to 1
00%
), cr
itica
l po
int
drie
d (B
al-T
ec, L
iech
tens
tein
; C
PD 0
30),
and
sput
ter
coat
ed w
ith
gold
(D
ento
n V
acuu
m,
NJ;
Des
k II
). T
he c
ells
at
the
surf
ace
of t
he C
PP s
ubst
rate
wer
e
visu
aliz
ed b
y se
cond
ary
elec
tron
imag
ing
unde
r sc
anni
ng e
lect
ron
mic
rosc
opy
(SEM
, FEI
, OR
;
XL3
0). R
esul
ting
imag
es w
ere
impo
rted
into
Imag
e J (
http
://rs
b.in
fo.n
ih.g
ov/ij
/) an
d ch
ondr
ocyt
e
area
was
det
erm
ined
by
traci
ng t
he c
ell
outli
ne a
nd c
alcu
latin
g th
e ar
ea u
sing
the
Im
age
J
prog
ram
, util
izin
g th
e sc
ale
bar i
n th
e im
ages
. Onl
y ce
lls th
at w
ere
in d
irect
con
tact
with
the
CPP
subs
trate
wer
e m
easu
red.
At
leas
t th
ree
cons
truct
s w
ith a
tot
al o
f at
lea
st 1
00 c
ells
wer
e
exam
ined
for e
ach
cond
ition
.
51
Wes
tern
Blo
t Ana
lysi
s
The
tissu
e w
as r
emov
ed f
rom
the
CPP
and
hom
ogen
ized
with
a m
otor
ized
pes
tle o
n ic
e
(100
μl) i
n R
IPA
Buf
fer (
50 m
M T
ris-H
CL
pH 7
.4, 1
50 m
M N
aCl,
1% T
riton
x-1
00, 1
% S
odiu
m
deox
ycho
late
, 0.
1%
SDS,
1m
M
EDTA
)
with
pr
otea
se
inhi
bito
rs
NaF
(1
mM
) an
d
phen
ylm
ethy
lsul
fony
l flu
orid
e (1
mM
) ad
ded
imm
edia
tely
prio
r to
use
. P
rote
in e
xtra
cts
wer
e
clar
ified
by
cent
rifug
atio
n fo
r 10
min
at 1
2,00
0gan
d pr
otei
n co
nten
t qua
ntifi
ed u
tiliz
ing
a B
CA
prot
ein
assa
y ki
t (Pi
erce
, Roc
kfor
d, I
L). P
rote
ins
from
eac
h sa
mpl
e (2
0μg
) w
ere
sepa
rate
d by
sodi
um-d
odec
yl-s
ulfa
te p
olya
cryl
amid
e ge
l ele
ctro
phor
esis
(SD
S-PA
GE)
(12%
acr
ylam
ide,
1.5
h
at 1
50V
), el
ectro
blot
ted
(1.5
h, 0
.3m
A) o
nto
nitro
cellu
lose
mem
bran
es (B
ioTr
ace
NT,
Pal
l Life
Scie
nces
, Pen
saco
la, F
L) th
en in
cuba
ted
with
ant
ibod
ies
reac
tive
with
tota
l ER
K1/
2 or
pho
spho
-
ERK
1/2
(1:2
000;
Cel
l Sig
nalin
g, B
ever
ly, M
A) i
n tri
s-bu
ffer
ed s
alin
e, 0
.1%
Tw
een-
20 (1
0m
M
Tris
, pH
7.5
, 150
mM
NaC
l, 0.
05%
v/v
Tw
een-
20)
cont
aini
ng 5
.0%
non
-fat
dry
milk
ove
rnig
ht
at 4
°C. I
mm
unor
eact
ivity
was
vis
ualiz
ed u
sing
sec
onda
ry a
ntib
ody
conj
ugat
ed w
ith h
orse
radi
sh
pero
xida
se (
1:20
00,
Cel
l Si
gnal
ing)
and
enh
ance
d ch
emilu
min
esce
nce
(EC
L Pl
us;
Am
ersh
am
Bio
scie
nces
).
Sem
i-qua
ntifi
catio
n of
gen
e ex
pres
sion
by
RT-P
CR
To d
eter
min
e th
e ex
pres
sion
of M
T1-M
MP
follo
win
g m
echa
nica
l stim
ulat
ion,
tota
l RN
A
was
ext
ract
ed f
rom
stim
ulat
ed a
nd u
nstim
ulat
ed ti
ssue
s us
ing
Triz
ol (
Invi
troge
n, C
arls
bad,
CA
)
acco
rdin
g to
the
man
ufac
ture
r's d
irect
ions
. Tot
al R
NA
was
reve
rse
trans
crib
ed (R
T) in
to c
DN
A
usin
g re
vers
e tra
nscr
ipta
se
(Sup
ersc
ript
II,
Invi
troge
n)
acco
rdin
g to
th
e m
anuf
actu
rer's
inst
ruct
ions
. Rel
ativ
e ge
ne e
xpre
ssio
n w
as e
xam
ined
by
sem
i-qua
ntita
tive
RT-
PCR
usi
ng T
aq
52
Poly
mer
ase
(Inv
itrog
en) a
nd p
rimer
s in
reac
tions
des
igne
d to
am
plify
the
sequ
ence
of i
nter
est a
s
outli
ned
prev
ious
ly [
De
Cro
os, J
ang,
Dha
liwal
, Gry
npas
, Pill
iar
& K
ande
l, 07
]. 18
S rR
NA
was
the
hous
ekee
ping
gen
e ag
ains
t whi
ch g
ene
expr
essi
on w
as n
orm
aliz
ed. P
CR
pro
duct
s w
ere
run
on a
1.6
%-a
garo
se g
el s
tain
ed w
ith e
thid
ium
bro
mid
e. B
and
inte
nsity
was
sem
i-qua
ntifi
ed b
y
dens
itom
etry
usi
ng L
ab W
orks
softw
are
(V4.
0, M
edia
Cyb
erne
ctic
s).
Stat
istic
al A
naly
sis
All
expe
rimen
ts w
ere
perf
orm
ed in
dup
licat
e an
d al
l exp
erim
ents
rep
eate
d at
leas
t thr
ee
times
. The
dat
a w
ere
pool
ed a
nd e
xpre
ssed
as
mea
n ±
stan
dard
err
or o
f th
e m
ean
(SEM
). Th
e
resu
lts w
ere
eval
uate
d us
ing
a on
e-w
ay a
naly
sis
of v
aria
nce
(AN
OV
A)
with
a b
onfe
rron
i pos
t-
hoc
corr
ectio
n. S
tatis
tical
sign
ifica
nce
was
ass
igne
d at
p<0
.05.
53
2.3
Res
ults
Cha
ract
eriz
atio
n of
ERK
pho
spho
ryla
tion
and
cell
spre
adin
g fo
llow
ing
cycl
ic c
ompr
essi
on
ERK
MA
P K
inas
e ac
tivat
ion
and
cell
spre
adin
g ar
e am
ongs
t the
ear
ly c
hang
es in
duce
d in
chon
droc
ytes
by
mec
hani
cal s
timul
atio
n. T
o ex
amin
e w
hich
of t
hem
occ
urre
d fir
st w
e ex
amin
ed
the
time
cour
se o
f ER
K p
hosp
hory
latio
n (F
igur
e 1A
) and
cel
l sha
pe (F
igur
e 1C
) at v
ario
us ti
mes
up to
30
min
utes
follo
win
g th
e in
itiat
ion
of m
echa
nica
l stim
ulat
ion.
Sig
nific
ant u
preg
ulat
ion
of
ERK
pho
spho
ryla
tion
was
obs
erve
d by
5 m
inut
es (5
fold
incr
ease
), w
ith th
e le
vels
pea
king
by
15
(10
fold
incr
ease
) m
inut
es o
f st
imul
atio
n. A
lbei
t stil
l sig
nific
antly
ele
vate
d co
mpa
red
to c
ontro
l
cons
truct
s, a
decl
ine
in p
hosp
hory
latio
n w
as n
oted
with
25
min
utes
of s
timul
atio
n. C
hond
rocy
te
area
pea
ked
(two-
fold
inc
reas
e) w
ith 5
min
utes
of
cycl
ic c
ompr
essi
on a
nd,
alth
ough
slig
htly
decr
easi
ng, r
emai
ned
wel
l ab
ove
cont
rol
valu
es a
t th
e su
bseq
uent
tim
e po
ints
. Thi
s su
gges
ted
that
cha
nges
in c
ell s
hape
and
act
ivat
ion
of th
e M
AP
Kin
ase
sign
allin
g w
ere
rapi
d. A
s m
axim
al
cell
spre
adin
g oc
curr
ed p
rior t
o m
axim
al E
RK
act
ivat
ion,
it su
gges
ted
that
cel
l spr
eadi
ng w
as th
e
earli
er c
hang
e so
onl
y ce
ll sh
ape
chan
ges w
ere
quan
tifie
d in
the
subs
eque
nt e
xper
imen
ts.
Extr
acel
lula
r cal
cium
regu
late
s cel
l mor
phol
ogy
Giv
en th
at th
e ch
ondr
ocyt
es re
spon
ded
with
in 5
min
utes
to c
yclic
com
pres
sion
, and
sin
ce
calc
ium
sig
nalli
ngis
kno
wn
to b
e in
volv
ed i
n m
echa
notra
nsdu
ctio
n an
d re
gula
tion
of c
ell
mor
phol
ogy,
the
role
of
extra
cellu
lar
calc
ium
in r
egul
atin
g ce
ll sp
read
ing
was
inve
stig
ated
. As
show
n in
Fig
ure
2A c
ells
that
wer
e st
imul
ated
in th
e pr
esen
ce o
f EG
TA-tr
eate
d m
edia
fai
led
to
spre
ad fo
llow
ing
mec
hani
cal s
timul
atio
n (E
GTA
trea
ted:
58.
8 ±
1.81
μm
2 ; EG
TA u
ntre
ated
: 86.
5
± 5.
44 μ
m2 ).
This
did
not
rep
rese
nt a
del
ayed
res
pons
e as
no
spre
adin
g w
as s
een
as l
ate
as 6
hour
s af
ter
stim
ulat
ion.
A
s EG
TA c
an c
hela
te i
ons
othe
r th
an c
alci
um,
we
conf
irmed
tha
t
calc
ium
was
res
pons
ible
for
thi
s ef
fect
by
addi
ng c
alci
um b
ack
into
the
med
ia a
fter
EGTA
54
treat
men
t but
prio
r to
mec
hani
cal s
timul
atio
n. T
he fi
nal a
mou
nt o
f cal
cium
cho
sen
to b
e ad
ded
to
the
med
ia w
as d
eter
min
ed th
roug
h do
se-r
espo
nse
expe
rimen
ts w
hich
ens
ured
all
catio
n bi
ndin
g
capa
city
of
EGTA
was
sat
urat
ed.
Add
ition
of
calc
ium
int
o th
e EG
TA-tr
eate
d m
edia
prio
r to
mec
hani
cal s
timul
atio
n al
low
ed f
or c
ell s
prea
ding
sim
ilar
to th
at s
een
in c
onst
ruct
s un
derg
oing
cycl
ic c
ompr
essi
on in
unt
reat
ed m
edia
as d
eter
min
ed m
orph
olog
ical
ly b
y SE
M a
nd b
y m
easu
ring
cell
size
(Fig
ure
2B).
Cel
l spr
eadi
ng a
nd re
trac
tion
are
regu
late
d by
two
dist
inct
ion
chan
nels
in c
hond
rocy
tes
As
extra
cellu
lar c
alci
um in
fluen
ced
chon
droc
yte
resp
onse
to c
yclic
com
pres
sion
, the
role
of tw
o ca
lciu
m tr
ansi
tory
cha
nnel
s, th
e vo
ltage
-gat
ed c
alci
um c
hann
el a
nd th
e ca
tioni
c st
retc
h-
activ
ated
cha
nnel
s, in
reg
ulat
ing
thes
e ce
ll sh
ape
chan
ges
wer
e in
vest
igat
ed (
Figu
re 3
).
Invo
lvem
ent o
f the
se c
hann
els
wou
ld a
lso
prov
ide
addi
tiona
lsup
port
fort
he in
volv
emen
t of i
on
(cal
cium
) flu
xes
regu
latin
g ce
ll re
spon
se t
o cy
lic c
ompr
essi
on.
Mec
hani
cal
stim
ulat
ion
in t
he
pres
ence
of
nife
dipi
ne, a
sele
ctiv
e in
hibi
tor o
f the
L-ty
pe V
GC
C, h
ad n
o ef
fect
on
cell
spre
adin
g
obse
rved
imm
edia
tely
follo
win
g m
echa
nica
l stim
ulat
ion
(0 h
ours
); ho
wev
er w
hile
unt
reat
ed c
ells
retra
cted
to th
eir o
rigin
al c
ell s
ize
by 6
hou
rs, t
hose
exp
osed
to n
ifedi
pine
rem
aine
d si
gnifi
cant
ly
mor
e sp
read
rel
ativ
e to
the
uns
timul
ated
con
trols
. In
con
trast
, co
nstru
cts
stim
ulat
ed i
n th
e
pres
ence
of g
adol
iniu
m, a
sel
ectiv
e in
hibi
tor o
f the
stre
tch
activ
ated
cat
ioni
c ch
anne
l (G
d3+),
did
not e
xhib
it cy
clic
com
pres
sion
-indu
ced
cell
spre
adin
g fo
llow
ing
the
stim
ulat
ion,
sug
gest
ing
that
the
L-Ty
pe V
GC
C is
invo
lved
in c
ell r
etra
ctio
n w
hile
the
stre
tch
activ
ated
cha
nnel
is in
volv
ed in
regu
latin
g ce
ll sp
read
ing.
To s
uppo
rt th
e ro
le o
f ca
lciu
m a
nd t
o ex
clud
e in
dire
ct e
ffec
t of
the
inh
ibito
rs i
n th
e
regu
latio
n of
ce
ll m
orph
olog
y, c
onst
ruct
s w
ere
mec
hani
cally
stim
ulat
ed i
n th
e pr
esen
ce o
f
55
calc
ium
ion
opho
re A
2318
7 an
d ei
ther
nife
dipi
ne o
r ga
dolin
ium
. Tr
eatm
ent
with
the
A23
187
iono
phor
e pa
rtial
ly
reve
rsed
th
e in
hibi
tion
of
gado
liniu
m,
and
mec
hani
cally
st
imul
ated
chon
droc
ytes
spr
ead
imm
edia
tely
fol
low
ing
mec
hani
cal s
timul
atio
n, c
ompa
red
to u
nstim
ulat
ed
chon
droc
ytes
. Si
mila
rly m
echa
nica
l st
imul
atio
n in
the
pre
senc
e of
bot
h ni
fedi
pine
and
the
A23
187
iono
phor
e re
vers
ed t
he e
ffec
ts o
f ni
fedi
pine
-inhi
bitio
n as
cho
ndro
cyte
s ha
d pa
rtial
ly
retra
cted
by
six
hour
s po
st m
echa
nica
l st
imul
atio
n as
com
pare
d to
veh
icle
tre
ated
con
stru
cts
(Fig
ure
3E).
Extr
acel
lula
r cal
cium
regu
late
d M
T1-M
MP
expr
essi
on
We
had
dete
rmin
ed p
revi
ousl
y th
at M
T1-M
MP
expr
essi
on fo
llow
ing
cycl
ic c
ompr
essi
on
play
s a
role
in
mod
ulat
ing
cell
shap
e ch
ange
s ob
serv
ed f
ollo
win
g cy
clic
loa
ding
. Th
us w
e
exam
ined
whe
ther
blo
ckin
g th
e ca
lciu
m i
nflu
x pr
even
ted
chan
ges
in M
T1-M
MP
mR
NA
expr
essi
on f
ollo
win
g st
imul
atio
n (F
igur
e 4)
. A
s ex
pect
ed,
a si
gnifi
cant
upr
egul
atio
n in
MT1
-
MM
P, a
ppro
xim
atel
y fo
ur-f
old,
gen
e ex
pres
sion
was
seen
in c
onst
ruct
s stim
ulat
ed in
the
abse
nce
of a
ny c
hem
ical
pre
-trea
tmen
t. I
n co
ntra
st,
no i
ncre
ase
in M
T1-M
MP
mR
NA
was
see
n in
cons
truct
s th
at
wer
e m
echa
nica
lly
stim
ulat
ed
in
the
pres
ence
of
EG
TA-tr
eate
d m
edia
.
Upr
egul
atio
n of
MT1
-MM
P ex
pres
sion
was
reve
rsed
by
addi
tion
of c
alci
um to
the
EGTA
-trea
ted
med
ia,
to l
evel
s si
mila
r to
tha
t se
en i
n m
echa
nica
lly s
timul
ated
unt
reat
ed c
ontro
l co
nstru
cts.
Uns
timul
ated
con
trol
cons
truct
s th
at w
ere
plac
ed i
n EG
TA-tr
eate
d m
edia
sho
wed
upr
egul
ated
MT1
-MM
P ge
ne e
xpre
ssio
n (a
ppro
xim
atel
y th
ree-
fold
) in
the
abse
nce
of m
echa
nica
l stim
ulat
ion
sugg
estin
g a
role
for c
alci
um in
regu
latio
n of
bas
al le
vels
of M
T1-M
MP
in c
hond
rocy
tes
(Fig
ure
4B).
56
The
effe
ct o
f bl
ocki
ng L
-Typ
e V
GC
C a
nd S
AC
on
the
indu
ctio
n of
MT1
-MM
P m
RN
A
expr
essi
on f
ollo
win
g cy
clic
com
pres
sion
was
ana
lyze
d as
wel
l. A
s sh
own
in F
igur
e 5,
bot
h
nife
dipi
ne a
nd G
d3+pr
even
ted
com
pres
sion
-indu
ced
upre
gula
tion
of M
T1-M
MP
mR
NA
as
com
pare
d to
the
corr
espo
ndin
g un
treat
ed c
ontro
ls (p
<0.0
5). I
nter
estin
gly,
sim
ilar t
o th
e ob
serv
ed
in E
GTA
-trea
ted
cons
truct
s, bo
th n
ifedi
pine
and
gad
olin
ium
indu
ced
upre
gula
tion
of M
T1-M
MP
in th
e ab
senc
e of
mec
hani
cal s
timul
atio
n.
Trea
tmen
t of
cons
truct
s w
ith th
e A
2318
7 io
noph
ore
reve
rsed
the
inh
ibito
ry e
ffec
ts o
f bo
th n
ifedi
pine
and
gad
olin
ium
res
ultin
g in
upr
egul
atio
n of
MT1
-MM
P ge
ne e
xpre
ssio
n fo
llow
ing
cycl
ic c
ompr
essi
on s
uppo
rting
the
rol
e of
cal
cium
in
thes
e pr
oces
ses (
Figu
re 6
B).
Invo
lvem
ent o
f int
egri
n si
gnal
ling
in c
ell s
prea
ding
and
MT1
-MM
P ex
pres
sion
The
α5β1
int
egrin
has
als
o be
en s
how
n to
reg
ulat
e ch
ondr
ocyt
e sp
read
ing
(and
MT1
-MM
P ex
pres
sion
) fol
low
ing
cycl
ic c
ompr
essi
on. I
nteg
rins
trans
late
the
sign
al b
y ac
tivat
ion
of a
dapt
er p
rote
ins
such
as
Src.
To
furth
er e
valu
ate
the
role
of
calc
ium
in
this
pro
cess
we
exam
ined
whe
ther
cal
cium
iono
phor
e w
ould
rev
erse
the
inhi
bito
ry e
ffec
ts in
duce
d by
blo
ckin
g
inte
grin
act
ivat
ion
thro
ugh
the
use
of th
e Sr
c in
hibi
tor,
PP2.
PP3
ser
ved
as a
neg
ativ
e co
ntro
l as
was
the
med
ia w
ith th
e ve
hicl
e. A
s ex
pect
ed g
iven
the
invo
lvem
ent o
f α5β
1, tr
eatm
ent w
ith P
P2
prev
ente
d ce
ll sp
read
ing
and
upre
gula
tion
of M
T1-M
MP
follo
win
g cy
clic
exp
ress
ion.
C
ell
spre
adin
g di
d oc
cur
in t
he c
ontro
ls (
vehi
cle
and
PP3)
so
the
cells
wer
e ab
le t
o re
spon
d to
mec
hani
cal s
timul
atio
n (F
igur
e 7)
. The
iono
phor
e A
2318
7 w
as a
ble
to o
verc
ome
the
inhi
bito
ry
effe
ct o
f PP
2 (p
<0.0
5) a
s ce
ll si
ze in
crea
sed
with
cyc
lic c
ompr
essi
on a
lthou
gh n
ot to
the
sam
e
leve
l obs
erve
d in
con
trol c
onst
ruct
s (v
ehic
le o
nly)
. Inc
reas
ing
the
iono
phor
e co
ncen
tratio
n di
d
not h
ave
any
furth
er e
ffec
t on
cell
shap
e su
gges
ting
that
it w
as u
nlik
ely
that
the
parti
al e
ffec
t was
due
to a
n in
suff
icie
nt a
mou
nt o
f the
dru
g.
57
Regu
latio
n of
ERK
MAP
Kin
ase
activ
atio
n
To e
xam
ine
whe
ther
cal
cium
als
o re
gula
tes
ERK
MA
PK a
ctiv
atio
n w
e ex
amin
ed t
he
effe
ct o
f ca
lciu
m c
hann
el b
lock
ers
on E
RK
pho
spho
ryla
tion
(Fig
ure
8).
As
expe
cted
cyc
lic
com
pres
sion
res
ulte
d in
inc
reas
ed E
RK
pho
spho
ryla
tion.
Inh
ibiti
on o
f ei
ther
cal
cium
cha
nnel
(SA
C o
r L-
type
VG
CC
) di
d no
t pr
even
t ER
K p
hosp
hory
latio
n fo
llow
ing
stim
ulat
ion,
and
actu
ally
inc
reas
ed t
he p
hosp
hory
latio
n in
Gd3+
-trea
ted
cons
truct
s, al
thou
gh v
aria
bilit
y in
the
resp
onsi
vene
ss o
f the
cel
ls to
thes
e dr
ugs
was
obs
erve
d. M
echa
nica
l stim
ulat
ion
in th
e pr
esen
ce
of P
P2 d
id n
ot p
reve
nt E
RK
pho
spho
ryla
tion,
how
ever
whe
n th
e ce
lls w
ere
stim
ulat
ed i
n th
e
pres
ence
of
both
the
Src
inh
ibito
r, PP
2, a
nd n
ifedi
pine
, ER
K p
hosp
hory
latio
n w
as i
nhib
ited
com
plet
ely
(p <
0.0
5).
58
2.4
Disc
ussio
n
We
have
pre
viou
sly
show
n th
at t
he m
echa
nica
l st
imul
atio
n-in
duce
d im
prov
emen
t in
tissu
e pr
oper
ties
is f
acili
tate
d th
roug
h tra
nsie
nt c
hond
rocy
tes
spre
adin
g an
d re
tract
ion
that
are
regu
late
d vi
a α5
β1 i
nteg
rin,
ERK
MA
Pkin
ase
activ
atio
n an
d up
regu
latio
n of
MT1
-MM
P
expr
essi
on (
subm
itted
for
pub
licat
ion)
. W
hile
cal
cium
has
bee
n im
plic
ated
in
othe
r st
udie
s
exam
inin
g ch
ondr
ocyt
e m
echa
notra
nsdu
ctio
n [R
ober
ts, K
nigh
t, Le
e &
Bad
er, 0
1;Ta
naka
et a
l.,
05;Z
hang
, Wan
g &
Che
n, 0
6],
its r
ole
in r
egul
atin
g th
e m
echa
nism
(s)
lead
ing
to th
e im
prov
ed
carti
lage
for
mat
ion
as a
res
ult o
f m
echa
nica
l stim
ulat
ion
have
not
bee
n fu
lly in
vest
igat
ed. T
his
stud
y de
mon
stra
ted
that
the
tra
nsie
nt c
ell
spre
adin
g an
d re
tract
ion,
as
wel
l as
the
ass
ocia
ted
upre
gula
tion
of M
T1-M
MP,
that
follo
ws
a si
ngle
app
licat
ion
of c
yclic
com
pres
sion
are
regu
late
d
via
calc
ium
as
treat
men
t of
the
med
ia w
ith E
GTA
, prio
r to
stim
ulat
ion
abol
ishe
d th
ese
chan
ges
and
re-in
trodu
ctio
n of
cal
cium
was
abl
e to
reve
rse
this
inhi
bito
ry e
ffec
t. C
ell s
prea
ding
and
cel
l
retra
ctio
n w
ere
regu
late
d th
roug
h tw
o di
stin
ct c
alci
um p
erm
issi
ve i
onic
cha
nnel
s, th
eca
tioni
c
stre
tch-
activ
ated
cha
nnel
(SA
C)
and
the
L-ty
pe v
olta
ge g
ated
cha
nnel
(V
GC
C),
resp
ectiv
ely.
Des
pite
the
diff
eren
tial
regu
latio
n of
cel
l m
orph
olog
y, i
nhib
ition
of
eith
er c
hann
el p
reve
nts
upre
gula
tion
of M
T1-M
MP
gene
exp
ress
ion
follo
win
g st
imul
atio
n. T
reat
men
t with
the
calc
ium
iono
phor
e A
2318
7 pa
rtial
ly r
ever
sed
the
inhi
bito
ry e
ffec
ts o
f ni
fedi
pine
and
gad
olin
ium
on
cell
mor
phol
ogy,
and
com
plet
ely
reve
rsed
the
eff
ects
of
stim
ulat
ion
on M
T1-M
MP
expr
essi
on.
Inhi
bitio
n of
the
activ
atio
n of
inte
grin
ass
ocia
ted
kina
se S
rc a
bolis
hed
cell
spre
adin
g an
d M
T1-
MM
P up
regu
latio
n, b
ut t
reat
men
t w
ith t
he A
2318
7 io
noph
ore
parti
ally
re-
esta
blis
hed
this
cellu
lar
resp
onse
. The
two
path
way
s, ca
lciu
m a
nd in
tegr
in a
ssoc
iate
d ki
nase
Src
con
verg
ed o
n
the
activ
atio
n of
ER
K M
AP
Kin
ase,
as i
nhib
ition
of b
oth
path
way
s are
requ
ired
to a
chie
ve d
own-
regu
latio
n of
ER
K p
hosp
hory
latio
n in
resp
onse
to m
echa
nica
l stim
ulat
ion.
59
The
role
of c
alci
um in
regu
latin
g th
ese
early
eve
nts
was
dem
onst
rate
d th
roug
h th
e us
e of
EGTA
, whi
ch a
bolis
hed
thes
e st
imul
atio
n-in
duce
d ch
ange
s, an
d th
e re
intro
duct
ion
of c
alci
um
into
the
med
ia w
hich
retu
rned
the
cellu
lar r
espo
nse
to m
echa
nica
l stim
ulat
ion.
Whi
le E
GTA
can
chel
ate
a va
riety
of
catio
ns (
nam
ely
Ca2+
, Na2+
, K+ ),
it w
as th
e re
-intro
duct
ion
of c
alci
um in
to
the
med
ia th
at re
turn
ed c
ell r
espo
nse
to n
orm
al le
vels
, sug
gest
ing
that
the
mec
hani
sm re
gula
ting
this
res
pons
e is
attr
ibut
ed to
cal
cium
and
not
oth
er m
edia
sol
uble
cat
ions
. The
rol
e of
cal
cium
influ
x w
as f
urth
er c
onfir
med
thro
ugh
the
use
of th
e A
2318
7 io
noph
ore
alon
e or
in c
onju
nctio
n
with
nife
dipi
ne o
r ga
dolin
ium
. A
2318
7 io
noph
ore
has
been
pre
viou
sly
show
n to
ele
vate
intra
cellu
lar c
alci
um le
vels
in o
ur la
bora
tory
[How
arth
et a
l., 9
3]. I
t is
inte
rest
ing
that
iono
phor
e
treat
men
t w
as o
nly
able
to
parti
ally
rev
erse
the
inh
ibito
ry e
ffec
ts o
f th
e ni
fedi
pine
and
gado
liniu
m o
n ce
ll m
orph
olog
y. T
he re
ason
s fo
r thi
s in
com
plet
e re
vers
al a
re n
ot k
now
n, b
ut o
ne
poss
ible
exp
lana
tion
may
be
that
the
loc
aliz
atio
n of
cal
cium
, an
d no
t m
erel
y its
pre
senc
e,
follo
win
g m
echa
nica
l st
imul
atio
n is
an
impo
rtant
asp
ect
of t
his
resp
onse
. L
ocal
ized
cal
cium
curr
ents
hav
e be
en p
revi
ousl
y do
cum
ente
d to
inf
luen
ce l
ocal
sig
nalli
ng c
asca
des
and
thus
activ
ate
sign
allin
g pa
thw
ays
in s
peci
fic l
ocat
ions
in
the
cell
[Jan
mey
, 98
;Zou
, Li
fshi
tz,
Tuft,
Foga
rty &
Sin
ger,
02].
Cel
l sp
read
ing
was
sho
wn
to b
e re
gula
ted
thro
ugh
the
SAC
, w
hich
is
know
n to
be
activ
ated
thro
ugh
dire
ct p
hysi
cal d
efor
mat
ion
of th
e ce
llula
r m
embr
ane
[Wu
& D
avis
, 01;
Zou,
Lifs
hitz
, Tuf
t, Fo
garty
& S
inge
r, 02
]. O
win
g to
pre
viou
s st
udie
s im
plic
atin
g SA
C a
s th
e in
itial
step
in m
echa
notra
nsdu
ctio
n [G
uila
k,95
;Kni
ght,
Bom
zon,
Kim
mel
, Sha
rma,
Lee
& B
ader
, 06a
],
it is
not
une
xpec
ted
that
we
foun
d th
at th
is c
hann
el w
as in
volv
ed in
our
sys
tem
. Whi
le it
is n
ot
poss
ible
to c
hara
cter
ize
the
expr
essi
on o
f the
SA
C in
cho
ndro
cyte
s, as
the
chan
nel h
as n
ot b
een
fully
seq
uenc
ed o
r ch
arac
teriz
ed [
Mar
oto,
Ras
o, W
ood,
Kur
osky
, Mar
tinac
& H
amill
, 05]
, its
60
pres
ence
in
chon
droc
ytes
has
bee
n su
ppor
ted
thro
ugh
the
use
of g
adol
iniu
m [
Gui
lak,
Zel
l,
Eric
kson
, Gra
nde,
Rub
in, M
cLeo
d &
Don
ahue
, 99;
Mou
wet
al.,
07;
Perk
ins,
Der
foul
, Ast
& H
all,
05].
In c
ontra
st, w
e ob
serv
ed th
at th
e L-
Type
VG
CC
regu
late
d ch
ondr
ocyt
e re
tract
ion
follo
win
g
mec
hani
cal
stim
ulat
ion.
Whi
le
the
VG
CC
ha
s be
en
show
n to
be
in
volv
ed
in
mec
hano
trans
duct
ion
in
othe
r st
udie
s [G
omez
et
al.,
02;G
uibe
rt,
Duc
ret
&
Savi
neau
,
08;L
acin
ova,
05]
, its
rel
atio
nshi
p to
cho
ndro
cyte
mor
phol
ogy
was
not
exp
lore
d by
oth
ers.
In
prel
imin
ary
stud
ies
we
wer
e ab
le t
o de
term
ine
that
arti
cula
r ca
rtila
ge c
hond
rocy
tes
expr
ess
VG
CC
mR
NA
as
dete
rmin
ed b
y R
T-PC
R (
data
not
sho
wn)
, sim
ilar
to w
hat
was
obs
erve
d by
Zusc
ik e
t al.,
in g
row
th p
late
cho
ndro
cyte
s [Z
usci
k, G
unte
r, Pu
zas
& R
osie
r, 97
]. Th
e se
quen
tial
role
of
the
SAC
and
the
L-ty
pe V
GC
C i
s in
lin
e w
ith l
itera
ture
sug
gest
ing
that
one
of
the
func
tiona
l out
com
es o
f SA
C a
ctiv
atio
n, a
t lea
st in
sm
ooth
mus
cle
cells
, is
cell
depo
lariz
atio
n an
d
subs
eque
nt a
ctiv
atio
n of
VG
CC
s [Zo
u, L
ifshi
tz, T
uft,
Foga
rty &
Sin
ger,
02].
Che
latio
n of
ext
race
llula
r ca
lciu
m a
bolis
hed
the
stim
ulat
ion-
indu
ced
upre
gula
tion
of
MT1
-MM
P su
gges
ting
that
eith
er c
alci
um d
irect
ly r
egul
ates
MT1
-MM
P ex
pres
sion
, or
tha
t
MT1
-MM
P ex
pres
sion
is d
epen
dent
upo
n ce
ll m
orph
olog
y ch
ange
s. Th
e fo
rmer
is s
uppo
rted
by
seve
ral
stud
ies
show
ing
that
cal
cium
can
reg
ulat
e th
e bi
ndin
g ac
tivity
of
AP-
1 (a
reg
ulat
or o
f
MT1
-MM
P ex
pres
sion
) in
hu
man
pu
lmon
ary
arte
ry
endo
thel
ial
cells
[F
anto
zzi,
Zhan
g,
Plat
oshy
n, R
emill
ard,
Cow
ling
& Y
uan,
03]
, an
d th
at L
-type
VG
CC
gen
erat
ed c
urre
nts
are
impl
icat
ed in
dire
ct re
gula
tion
AP-
1 bi
ndin
g ac
tivity
[Pre
mku
mar
, Mis
hra,
Ove
rhol
t, Si
mon
son,
Che
rnia
ck &
Pra
bhak
ar, 0
0]. I
t is
inte
rest
ing
to n
ote
that
in in
hibi
tor-
treat
ed, b
ut m
echa
nica
lly
unst
imul
ated
sam
ples
, an
inc
reas
e in
MT1
-MM
P ge
ne e
xpre
ssio
n w
as o
bser
ved.
Sin
ce t
his
chan
ge w
as o
bser
ved
with
out a
ny m
orph
olog
ical
cha
nges
in c
ell s
hape
it s
ugge
sts
that
cal
cium
sign
allin
g ex
erts
a ro
le re
gula
ting
cons
titut
ive
expr
essi
on o
f MT1
-MM
P. F
urth
er s
uppo
rting
this
61
notio
n is
the
rest
orat
ion
of M
T1-M
MP
gene
exp
ress
ion
with
the
iono
phor
e tre
atm
ent,
perh
aps
furth
er i
ndic
atin
g th
e ex
iste
nce
of a
sep
arat
e ca
lciu
m s
igna
lling
pat
hway
tha
t co
ntro
ls M
T1-
MM
P ex
pres
sion
. T
his
is i
n ke
epin
g w
ith p
revi
ous
wor
k by
Loh
i et
al.
who
sho
wed
tha
t
iono
phor
e tre
atm
ent c
an in
duce
MM
P ex
pres
sion
, suc
h as
MM
P-9
and
MM
P-2.
Alth
ough
they
did
not o
bser
ve a
cha
nge
in M
T1-M
MP
in th
eir s
yste
m [L
ohi &
Kes
ki-O
ja, 9
5], i
t is p
ossi
ble
that
this
is
a re
sult
of t
he d
iffer
ent
cell
type
(fib
rosa
rcom
a) u
sed,
as
the
tum
our
cells
m
ay h
ave
diff
eren
t cal
cium
sign
allin
g m
echa
nism
s [W
hitfi
eld,
92]
.
Due
to
the
prev
ious
ly d
ocum
ente
d ro
le o
f α5
β1 i
nteg
rin i
n m
edia
ting
cell
shap
e an
d
MT1
-MM
P ch
ange
s, w
e ex
amin
ed t
he r
ole
of t
he k
inas
es S
rc a
nd F
AK
, w
hich
are
kno
wn
dow
nstre
am m
odul
ator
s of
inte
grin
act
ivat
ion,
in r
egul
atin
g th
ese
chan
ges,
and
inve
stig
ated
the
role
of
calc
ium
in th
is p
roce
ss. T
he r
espo
nse
to c
yclic
com
pres
sion
was
fac
ilita
ted
thro
ugh
the
Src,
as
its in
hibi
tion
abol
ishe
d bo
th c
ell s
prea
ding
and
MT1
-MM
P up
regu
latio
n. N
o ch
ange
s in
FAK
pho
spho
ryla
tion
wer
e se
en w
ith m
echa
nica
l st
imul
atio
n (d
ata
not
show
n).
This
may
be
expl
aine
d by
not
ing
that
diff
eren
t ty
pes
of m
echa
nica
l fo
rces
can
diff
eren
tially
reg
ulat
e th
e
phos
phor
ylat
ion
of F
AK
and
Src
[R
en,
Kio
sses
, Si
eg,
Ote
y, S
chla
epfe
r &
Sch
war
tz,
00].
Alte
rnat
ivel
y, i
t is
pos
sibl
e th
at t
he c
ell-m
atrix
int
erac
tions
for
med
dur
ing
spre
adin
g ar
e
som
ewhe
re b
etw
een
foca
l ad
hesi
ons
and
fibril
lar
adhe
sion
s an
d th
us t
he c
onst
itutio
nof
asso
ciat
ed k
inas
es c
ould
pre
dom
inan
tly re
ly o
n Sr
c. F
ibril
lar a
dhes
ions
are
kno
wn
to p
lay
a ro
le
in m
atrix
reo
rgan
izat
ion
[Kat
z, Z
amir,
Ber
shad
sky,
Kam
, Y
amad
a &
Gei
ger,
00],
and
it is
belie
ved
that
the
ir fo
rmat
ion
(thro
ugh
mat
urat
ion)
is
depe
nden
t up
on S
rc a
ctiv
ity [
Vol
berg
,
Rom
er, Z
amir
& G
eige
r, 01
]. S
imila
rly, s
ince
the
α5β
1 in
tegr
in c
an b
e lo
caliz
ed w
ith M
T1-
MM
P in
inte
rcel
lula
r co
ntac
ts [
Gal
vez
et a
l., 0
2], a
nd S
rc p
hosp
hory
latio
n ha
s be
en p
revi
ousl
y
show
n to
reg
ulat
e M
T1-M
MP
activ
ity [
Wu,
Gan
, Y
oo &
Gua
n, 0
5]it
is n
ot s
urpr
isin
g th
at
62
inhi
bitio
n of
Src
can
inf
luen
ce t
he m
echa
nose
nsiti
ve u
preg
ulat
ion
of M
T1-M
MP
follo
win
g
stim
ulat
ion.
The
dual
regu
latio
n of
cel
l mor
phol
ogy
and
MT1
-MM
P is
not
une
xpec
ted
as o
ther
s ha
ve
show
n th
at b
oth
inte
grin
s an
d ca
lciu
m c
hann
els
are
com
pone
nts
of a
cel
lula
r m
echa
nose
nsin
g
com
plex
. VG
CC
s ar
e kn
own
to c
o-lo
caliz
e w
ith β
1 in
tegr
ins
in m
ouse
lim
b-bu
d ch
ondr
ocyt
es
[Mob
ashe
ri, C
arte
r, M
artin
-Vas
allo
& S
haki
baei
, 02;
Shak
ibae
i &
Mob
ashe
ri, 0
3], a
nd s
tudi
es
usin
g va
scul
ar s
moo
th m
uscl
e ce
lls s
how
ed th
at α
5β1
inte
grin
can
regu
late
the
func
tion
of th
e L-
Type
VG
CC
[Wu,
Dav
is, M
eini
nger
, Wils
on &
Dav
is, 0
1b].
It is
now
know
n th
at S
rc c
an b
ind
to
both
the
II-I
II li
nker
and
C-te
rmin
al r
egio
ns o
f th
e α 1
Csu
buni
t of
the
L-Ty
pe V
GC
C a
nd th
us
regu
late
its
activ
ity [D
ubui
s, R
ockl
iffe,
Hus
sain
, Boy
ett,
Wra
y &
Gaw
ler,
06].
Our
dat
a su
gges
ts
that
cal
cium
pla
ys a
maj
or r
egul
ator
y ro
le in
med
iatin
g th
is r
espo
nse,
pos
sibl
y to
the
exte
nt o
f
lyin
g up
stre
am to
α5β
1 an
d re
gula
ting
its a
ctiv
atio
n. T
his
idea
is s
uppo
rted
by th
e ob
serv
atio
n
that
blo
ckin
g ca
lciu
m s
igna
lling
com
plet
ely
abol
ishe
d ce
ll sp
read
ing
in r
espo
nse
to m
echa
nica
l
stim
ulat
ion,
and
tha
t io
noph
ore
treat
men
t w
as a
ble
to p
artia
lly r
ever
se t
he e
ffec
ts o
f Sr
c
inhi
bitio
n. M
oreo
ver,
the
abili
ty o
f ca
lciu
m to
influ
ence
bas
al le
vels
MT1
-MM
P ex
pres
sion
in
unst
imul
ated
sam
ples
fur
ther
im
plic
ates
cal
cium
in
exer
ting
the
maj
or r
egul
ator
yro
le i
n
med
iatin
g ch
ondr
ocyt
e re
spon
ses.
The
activ
atio
n of
ER
K,
how
ever
, m
ay b
e su
gges
tive
of a
para
llel p
athw
ay w
here
bot
h si
gnal
ling
casc
ades
con
verg
e to
regu
late
ER
K, w
hich
then
exe
rts a
regu
lato
ry ro
le o
n A
P-1
and
MT1
-MM
P ex
pres
sion
. To
fully
add
ress
this
, it w
ould
be
nece
ssar
y
to v
isua
lize
calc
ium
flu
xes
in r
espo
nse
to m
echa
nica
l st
imul
atio
n in
rea
l-tim
e an
d de
term
ine
whe
ther
inhi
bitio
n of
Src
act
ivity
influ
ence
s th
ese
curr
ents
. Unf
ortu
nate
ly, d
ue to
the
tech
nica
l
limita
tions
of m
echa
nica
lly s
timul
atin
g ou
r bip
hasi
c 3D
con
stru
cts,
it w
as n
ot p
ossi
ble
for u
s to
mon
itor c
alci
um fl
uxes
in re
al-ti
me.
63
Last
ly, i
t is
wor
th n
otin
g th
at th
e ob
serv
atio
n th
at s
igni
fican
t cha
nges
in c
ell m
orph
olog
y
occu
rred
prio
r to
the
pea
k in
pho
spho
ryla
tion
of E
RK
sug
gest
s th
at p
hysi
cal
chan
ges
in c
ell
shap
e pr
eced
e/in
itiat
e th
e do
wns
tream
sig
nalli
ng c
asca
des
such
as
ERK
MA
P K
inas
e. P
ast
stud
ies
in o
ur la
b in
dica
ted
that
MT1
-MM
P up
regu
latio
n in
res
pons
e to
cyc
lic c
ompr
essi
on is
depe
nden
t on
ERK
act
ivat
ion
[De
Cro
os, J
ang,
Dha
liwal
, Gry
npas
, Pill
iar
& K
ande
l, 07
], an
d
owin
g to
MT1
-MM
P’s
role
in
faci
litat
ing
cellu
lar
retra
ctio
n (s
ubm
itted
for
pub
licat
ion)
, it
is
poss
ible
tha
t th
e ac
tivat
ion
of E
RK
may
not
be
invo
lved
in
cell
spre
adin
g bu
t ra
ther
the
subs
eque
nt
even
ts.
This
no
tion
is
supp
orte
d by
st
udie
s w
hich
ob
serv
ed
that
ph
ysic
al
defo
rmat
ions
in th
e ce
llula
r m
embr
ane
com
pris
e th
efir
st s
tep
in m
echa
notra
nsdu
ctio
n [G
uila
k,
95;K
nigh
t, B
omzo
n, K
imm
el, S
harm
a, L
ee &
Bad
er, 0
6a].
Bas
ed o
n th
e re
sults
obt
aine
d, w
e w
ould
lik
e to
sug
gest
a p
athw
ay t
hrou
gh w
hich
mec
hani
cal
forc
es r
esul
t in
im
prov
ed f
orm
atio
n of
car
tilag
e tis
sue
in v
itro
(fig
ure
9):
cycl
ic
com
pres
sion
cau
ses
cellu
lar d
efor
mat
ions
whi
ch a
ctiv
ate
SAC
, res
ultin
g in
cal
cium
influ
x. T
his
influ
x re
sults
in c
ell s
prea
ding
and
act
ivat
ion
of th
e L-
Type
VG
CC
and
α5β
1 in
tegr
in w
ith it
s
asso
ciat
ed S
rc k
inas
e. T
his r
esul
ts in
the
phos
phor
ylat
ion
of E
RK
and
subs
eque
nt u
preg
ulat
ion
of
MT1
-MM
P.
Sim
ulta
neou
sly,
Sr
c si
gnal
ling
may
re
sult
in
incr
ease
d ph
osph
oryl
atio
n of
Endo
phili
n A
2 (a
pro
tein
invo
lved
in M
T1-M
MP
endo
cyto
sis)
, whi
ch in
hibi
ts e
ndoc
ytos
is a
nd
prol
ongs
the
pro
teol
ytic
act
ivity
of
MT1
-MM
P [W
u, G
an,
Yoo
& G
uan,
05]
.The
inc
reas
ed
degr
adat
ion
of th
e EC
M w
ould
then
wea
ken
any
cell-
mat
rix a
dhes
ions
cau
sing
the
cells
to re
tract
to th
eir o
rigin
al m
orph
olog
y, w
hile
the
degr
adat
ion
prod
ucts
of t
he E
CM
will
sig
nal t
he c
ells
to
incr
ease
syn
thes
is o
f no
vel
mat
rix m
olec
ules
(fo
r ex
ampl
e, f
ibro
nect
in f
ragm
ents
hav
e be
en
show
n pr
evio
usly
to in
crea
se p
rote
ogly
can
synt
hesi
s by
chon
droc
ytes
[Hom
andb
erg
et a
l., 9
2]).
64
In s
umm
ary,
cal
cium
cur
rent
s th
roug
h SA
C a
nd L
-Typ
e V
GC
C c
hann
els
appe
ar t
o
regu
late
the
trans
ient
cel
l spr
eadi
ng a
nd re
tract
ion,
resp
ectiv
ely,
that
are
obs
erve
d fo
llow
ing
the
appl
icat
ion
of c
yclic
com
pres
sive
for
ces.
In a
dditi
on,
calc
ium
exe
rts a
reg
ulat
ory
role
on
the
inte
grin
-med
iate
d si
gnal
ling
path
way
s, th
at i
s fa
cilit
ated
thr
ough
the
kin
ase
Src.
Fur
ther
unde
rsta
ndin
g of
th
e un
derly
ing
mol
ecul
ar
mec
hani
sms
invo
lved
in
ch
ondr
ocyt
e
mec
hano
trans
duct
ion
will
ulti
mat
ely
lead
to
the
deve
lopm
ent
of i
mpr
oved
bio
engi
neer
ed
carti
lage
and
may
lead
to a
bet
ter t
reat
men
t for
ost
eoar
thrit
is a
nd o
ther
car
tilag
e pa
thol
ogie
s.
Ack
now
ledg
emen
ts:
I.R. w
as s
uppo
rted
by f
ello
wsh
ip f
rom
CA
N-a
rthrit
is S
ocie
ty a
nd O
ntar
io S
tude
nt S
chol
arsh
ip
OG
S. T
he r
esea
rch
was
sup
porte
d by
a g
rant
fro
m th
e C
anad
ian
Inst
itute
s of
Hea
lth R
esea
rch
(CIH
R).
We
than
k M
r. H
arry
Boj
arsk
i an
d R
ydin
g-R
egen
cy M
eat
Pack
ers
for
prov
idin
g th
e
tissu
es f
or th
ese
stud
ies,
Dr
Svitl
ana
Prad
afo
r m
anuf
actu
ring
the
CPP
sub
stra
tes
and
Dr.
Jian
Wan
g fo
r tec
hnic
al a
ssis
tanc
e.
65
2.5
Figu
res &
Lege
nds
Figu
re 1
: C
hara
cter
izat
ion
of c
hond
rocy
te r
espo
nse
to c
yclic
com
pres
sion
. (A
) A
tim
e co
urse
of
a co
nfin
ed c
yclic
com
pres
sion
stim
ulat
ion
over
1, 3
, 5, 1
5 an
d 25
min
utes
sho
ws
an
incr
ease
in E
RK
pho
spho
ryla
tion
as e
arly
as
five
min
utes
dur
ing
mec
hani
cal s
timul
atio
n, w
ith
max
imal
pho
spho
ryla
tion
obse
rved
by
15 m
inut
es. (
B) R
epre
sent
ativ
e w
este
rn b
lot i
ndic
atin
g th
e ph
osph
oryl
atio
n of
ER
K (
pER
K)
and
the
resp
ectiv
e to
tal
ERK
ove
r th
e tim
e co
urse
. (C
) R
epre
sent
ativ
e SE
M
mic
rogr
aphs
of
ch
ondr
ocyt
es
as
seen
th
roug
h sc
anni
ng
elec
tron
mic
rosc
opy.
(D
) C
hond
rocy
te c
ell
spre
adin
g is
obs
erve
d as
ear
ly a
s 5
min
utes
int
o a
cycl
ic
com
pres
sion
. * d
enot
es s
tatis
tical
ly s
igni
fican
t diff
eren
ce (p
<0.0
5) a
s co
mpa
red
to u
nstim
ulat
ed
cont
rol c
onst
ruct
s at
the
resp
ectiv
e tim
e po
ints
. Res
ults
are
exp
ress
ed a
s m
eans
± S
EM (n
=3, i
n du
plic
ates
).
Figu
re 2
: C
hond
rocy
te c
ell
spre
adin
g fo
llow
ing
cycl
ic c
ompr
essi
on i
s re
gula
ted
by
extr
acel
lula
r ca
lciu
m.(
A)
Rep
rese
ntat
ive
SEM
mic
rogr
aphs
(A
1) u
nstim
ulat
ed c
hond
rocy
tes,
(A2)
stim
ulat
ed c
hond
rocy
tes
afte
r st
imul
atio
n, (
A3
and
A5)
uns
timul
ated
cho
ndro
cyte
s in
EG
TA-tr
eate
d m
edia
at0
and
6 h
ours
, (A
4, A
6) st
imul
ated
cho
ndro
cyte
s in
EGTA
-trea
ted
med
ia
at 0
and
6 h
ours
, (A
7) u
nstim
ulat
ed c
hond
rocy
tes
in E
GTA
-trea
ted
med
ia s
uppl
emen
ted
with
ca
lciu
m a
t 0 h
ours
, and
(A8)
stim
ulat
ed c
hond
rocy
tes i
n EG
TA-tr
eate
d m
edia
supp
lem
ente
d w
ith
calc
ium
at
0 ho
urs.
(B)
Qua
ntifi
catio
n of
cho
ndro
cyte
are
a in
con
trol
(C)
and
stim
ulat
ed (
S)
cons
truct
s at
0 a
nd 6
hou
rs p
ost
mec
hani
cal
stim
ulat
ion
in t
he p
rese
nce
of E
GTA
or
vehi
cle.
*d
enot
es s
tatis
tical
ly s
igni
fican
t di
ffer
ence
(p<
0.05
) as
com
pare
d to
res
pect
ive
unst
imul
ated
sa
mpl
es (n
=3, i
n du
plic
ates
).
Figu
re 3
: Cho
ndro
cyte
cel
l spr
eadi
ng a
nd c
ontr
actio
n fo
llow
ing
mec
hani
cal s
timul
atio
n ar
e co
ntro
lled
by t
wo
diff
eren
t io
n ch
anne
ls.
Cho
ndro
cyte
s w
ere
stim
ulat
ed i
n th
e pr
esen
ce o
f N
ifedi
pine
(10
μM
), G
adol
iniu
m (
10 μ
M),
or c
ontro
l ve
hicl
e (0
.01%
DM
SO i
n H
AM
’S-F
12)
Cho
ndro
cyte
s vi
sual
ized
by
SE
M
at
0 ho
urs
(A)
or
6 ho
urs
(B)
and
area
qu
antif
ied
mor
phom
etric
ally
(C, D
). Tr
eatm
ent w
ith th
e A
2318
7 io
noph
ore
parti
ally
reve
rsed
the
effe
cts
on
nife
dipi
ne a
nd g
adol
iniu
m (
E).
(A1)
uns
timul
ated
, (A
3) s
timul
ated
, (A
2) u
nstim
ulat
ed a
nd
gado
liniu
m t
reat
ed, (
A4)
stim
ulat
ed a
nd g
adol
iniu
m t
reat
ed, (
A5)
uns
timul
ated
and
nife
dipi
ne
treat
ed,
(A6)
stim
ulat
ed a
nd n
ifedi
pine
tre
ated
. (B
1) u
nstim
ulat
ed,
(B3)
stim
ulat
ed,
(B2)
un
stim
ulat
ed a
nd g
adol
iniu
m tr
eate
d, (B
4) s
timul
ated
and
gad
olin
ium
trea
ted,
(B5)
uns
timul
ated
an
d ni
fedi
pine
tre
ated
, (B
6) s
timul
ated
and
nife
dipi
ne t
reat
ed. *
deno
tes
stat
istic
ally
sig
nific
ant
diff
eren
ce (p
<0.0
5) a
s com
pare
d to
resp
ectiv
e un
stim
ulat
ed sa
mpl
es.
Figu
re
4:
U
preg
ulat
ion
of
MT
1-M
MP
in
resp
onse
to
m
echa
nica
l st
imul
atio
n in
ch
ondr
ocyt
es is
dep
ende
nt e
xtra
cellu
lar
calc
ium
. C
ells
wer
e st
imul
ated
in th
e pr
esen
ce o
f 10
μM E
GTA
, 10
μM E
GTA
with
the
addi
tion
of 5
μM
CaC
l 2, o
r con
trol v
ehic
le (P
BS
in H
AM
’S
F-12
). (A
) C
hela
tion
with
EG
TA a
bolis
hed
the
up-r
egul
atio
n of
MT1
-MM
P m
RN
A f
ollo
win
g m
echa
nica
l st
imul
atio
n. R
e-in
trodu
ctio
n of
cal
cium
int
o th
e ch
elat
ed m
edia
ret
urne
d th
e ex
pect
ed
incr
ease
M
T1-M
MP
mR
NA
ex
pres
sion
fo
llow
ing
mec
hani
cal
stim
ulat
ion.
(C
) R
epre
sent
ativ
e ge
l illu
stra
ting
the
effe
cts o
f cal
cium
che
latio
n an
d re
-intro
duct
ion
on M
T1-M
MP
gene
ex
pres
sion
. *
deno
tes
stat
istic
ally
si
gnifi
cant
di
ffer
ence
(p
<0.0
5)
as
com
pare
d to
un
stim
ulat
ed c
ontro
l co
nstru
cts
at t
he r
espe
ctiv
e tim
e po
ints
. Rsu
lts a
reex
pres
sed
as m
eans
±
SEM
(n=3
, in
dupl
icat
es).
66
Figu
re
5:
Upr
egul
atio
n of
M
T1-
MM
P in
re
spon
se
to
mec
hani
cal
stim
ulat
ion
in
chon
droc
ytes
is
depe
nden
t up
on c
alci
um. C
hond
rocy
tes
wer
e st
imul
ated
in
the
pres
ence
of
Nife
dipi
ne (
10 μ
M),
or G
adol
iniu
m (
10 μ
M),
orve
hicl
e (0
.01%
DM
SO i
n H
AM
’S-F
12).
(A)
pre-
treat
men
t w
ith N
ifedi
pine
and
Gad
olin
ium
abo
lishe
d th
e up
-reg
ulat
ion
of M
T1-M
MP
mR
NA
, as
see
n in
veh
icle
-trea
ted
cons
truct
s, fo
llow
ing
mec
hani
cal
stim
ulat
ion.
(B
) Pr
e-tre
atm
ent o
f co
nstru
cts
with
Nife
dipi
ne r
esul
ted
in a
sig
nific
antly
incr
ease
d M
T1-M
MP
mR
NA
ex
pres
sion
in
unst
imul
ated
con
stru
cts,
sugg
estin
g ca
lciu
m s
igna
lling
is
invo
lved
in
regu
latin
g co
nstit
utiv
e M
T1-M
MP
leve
ls in
cho
ndro
cyte
s. (C
) Rep
rese
ntat
ive
gel i
llust
ratin
g th
e ef
fect
s of
N
ifedi
pine
and
Gad
olin
ium
on
MT1
-MM
P ge
ne e
xpre
ssio
n. *
den
otes
sta
tistic
ally
sig
nific
ant
diff
eren
ce (p
<0.0
5) a
s co
mpa
red
to u
nstim
ulat
ed c
ontro
l con
stru
cts
at th
e re
spec
tive
time
poin
ts.
Res
ults
are
exp
ress
ed a
s mea
ns ±
SEM
(n=3
, in
dupl
icat
es)
Figu
re 6
: T
reat
men
t w
ith c
alci
um i
onop
hore
rev
erse
d th
e ef
fect
s of
nife
dipi
ne/g
olin
ium
in
hibi
tion
on M
T1-
MM
P fo
llow
ing
cycl
ic c
ompr
essi
on.
(A)
Trea
tmen
t w
ith t
he A
2318
7 io
noph
ore
also
ret
urne
d M
T1-M
MP
expr
essi
on t
o st
imul
ated
con
stru
cts
whi
ch m
imic
ked
the
chan
ges
seen
in
vehi
cle-
treat
ed c
onst
ruct
s. (B
) R
epre
sent
ativ
e ge
l ill
ustra
ting
the
effe
cts
of
A23
187
iono
phor
e tre
atm
ent
on M
T1-M
MP
gene
exp
ress
ion
in n
ifedi
pine
/gad
olin
ium
tre
ated
co
nstru
cts.
* de
note
s st
atis
tical
ly s
igni
fican
t di
ffer
ence
(p<
0.05
) as
com
pare
d to
uns
timul
ated
co
ntro
lcon
stru
cts
at th
e re
spec
tive
time
poin
ts. R
esul
ts a
re e
xpre
ssed
as
mea
ns ±
SEM
(n=3
, in
dupl
icat
es).
Figu
re 7
: T
reat
men
t w
ith t
he S
rc in
hibi
tor
PP2
inhi
bite
d ce
ll sp
read
ing
and
upre
gula
tion
of M
T1-
MM
P fo
llow
ing
cycl
ic c
ompr
essi
on. (
A) T
reat
men
t with
PP2
abo
lishe
d ce
ll sp
read
ing
follo
win
g m
echa
nica
l stim
ulat
ion,
whi
ch w
as o
bser
ved
in v
ehic
le a
nd P
P3 (
nega
tive
cont
rol o
f PP
2) tr
eate
d ce
lls. (
B) T
reat
men
t of c
ells
with
PP2
prio
r to
mec
hani
cal s
timul
atio
n al
so a
bolis
hed
the
expe
cted
inc
reas
e in
MT1
-MM
P m
RN
A w
hich
was
see
n in
veh
icle
and
PP3
tre
ated
co
nstru
cts.
(C)
Rep
rese
ntat
ive
gel i
llust
ratin
g th
e ef
fect
s of
PP2
trea
tmen
t on
MT1
-MM
P ge
ne
expr
essi
on i
n st
imul
ated
con
stru
cts.
* de
note
s st
atis
tical
ly s
igni
fican
t di
ffer
ence
(p<
0.05
) as
co
mpa
red
to u
nstim
ulat
ed c
ontro
l con
stru
cts
at th
e re
spec
tive
time
poin
ts. R
esul
ts a
re e
xpre
ssed
as
mea
ns ±
SEM
(n=3
, in
dupl
icat
es).
Figu
re 8
: Cal
cium
sig
nalli
ng a
nd in
tegr
in s
igna
lling
reg
ulat
e ce
ll re
spon
se th
roug
h m
utua
l re
gula
tion
of E
RK
pho
spho
ryla
tion
follo
win
g m
echa
nica
l stim
ulat
ion.
(A) T
reat
men
t of c
ells
w
ith b
oth
the
Src
inhi
bito
r PP2
and
the
calc
ium
iono
phor
e pa
rtial
ly re
vers
ed th
e ef
fect
s of P
P2 in
te
rms
of c
ell a
rea;
the
parti
al e
ffec
t was
not
attr
ibut
ed to
insu
ffic
ient
intra
cellu
lar c
alci
um (d
ark
bar)
. (B
) Inh
ibiti
on w
ith g
adol
iniu
m d
id n
ot a
bolis
h ER
K p
hosp
hory
latio
n, n
eith
er d
id tr
eatm
ent
with
nife
dipi
ne a
nd P
P2 a
lthou
gh v
aria
bilit
y w
as o
bser
ved.
Co-
treat
men
t w
ith P
P2 a
nd
nife
dipi
ne c
ompl
etel
y ab
olis
hed
ERK
pho
spho
ryla
tion
follo
win
g m
echa
nica
l st
imul
atio
n. (
C)
Rep
rese
ntat
ive
blot
s ill
ustra
ting
the
effe
cts
of P
P2, n
ifedi
pine
and
gad
olin
ium
trea
tmen
t on
ERK
ph
osph
oryl
atio
n in
stim
ulat
ed c
onst
ruct
s. *
deno
tes s
tatis
tical
ly si
gnifi
cant
diff
eren
ce (p
<0.0
5) a
s co
mpa
red
to u
nstim
ulat
ed c
ontro
l con
stru
cts
at th
e re
spec
tive
time
poin
ts. R
esul
ts a
re e
xpre
ssed
as
mea
ns ±
SEM
(n=3
, in
dupl
icat
e).
Figu
re 9
: Pro
pose
d m
echa
nism
by
whi
ch c
hond
rocy
tes
resp
ond
to m
echa
nica
l stim
ulat
ion
with
incr
ease
d sy
nthe
sis a
nd a
ccum
ulat
ion
of e
xtra
cellu
lar
mat
rix.
67
68
69
70
71
72
73
74
75
76
CH
APT
ER
TH
RE
E: D
ISC
USS
ION
Prev
ious
ly w
e ha
d sh
own
that
a s
ingl
e ap
plic
atio
n of
cyc
lic c
ompr
essi
on o
f 30
min
utes
dura
tion
resu
lted
in e
nhan
ced
carti
lage
tiss
ue f
orm
atio
n in
vitr
o. T
his
chan
ge w
as s
how
n to
be
depe
nden
t on
tra
nsie
nt c
hang
es i
n ce
ll sh
ape
regu
late
d vi
a α5
β1 i
nteg
rin,
ERK
MA
Pkin
ase
activ
atio
n an
d up
regu
latio
n of
MT1
-MM
P ex
pres
sion
. The
cur
rent
stu
dy d
emon
stra
ted
that
the
trans
ient
cel
l spr
eadi
ng a
nd re
tract
ion,
as
wel
l as
the
asso
ciat
ed u
preg
ulat
ion
of M
T1-M
MP,
that
follo
ws
a si
ngle
app
licat
ion
of c
yclic
com
pres
sion
are
regu
late
d vi
a ca
lciu
m a
s tre
atm
ent
of th
e
med
ia w
ith E
GTA
, a
catio
n ch
elat
or,
prio
r to
stim
ulat
ion
abol
ishe
d th
ese
chan
ges
and
re-
intro
duct
ion
of c
alci
um w
as a
ble
to r
ever
se th
is in
hibi
tory
eff
ect.
Cel
l spr
eadi
ng a
nd r
etra
ctio
n
wer
e re
gula
ted
thro
ugh
two
dist
inct
cal
cium
-per
mis
sive
ion
ic c
hann
els,
the
catio
nic
stre
tch-
activ
ated
cha
nnel
and
the
L-Ty
pe v
olta
ge g
ated
cha
nnel
,res
pect
ivel
y, w
hich
als
o su
ppor
ts th
e
invo
lvem
ent o
f cal
cium
flux
es. B
oth
chan
nels
wer
e al
so s
how
n to
exe
rt a
regu
lato
ry ro
le o
n th
e
expr
essi
on o
f M
T1-M
MP
both
in
unst
imul
ated
and
stim
ulat
ed c
onst
ruct
s. C
alci
um a
lso
appe
ared
to
be i
nvol
ved
in r
egul
atin
g in
tegr
in a
ctiv
atio
n as
mec
hani
cal
stim
ulat
ion
in t
he
pres
ence
of
A23
187
iono
phor
e an
d PP
2, a
n in
hibi
tor
of in
tegr
in-a
ssoc
iate
d ki
nase
Src
, ret
urne
d
cell
spre
adin
g an
d M
T1-M
MP
upre
gula
tion
alm
ost
back
to
cont
rol
leve
ls.
The
two
path
way
s
appe
ar to
con
verg
e on
the
phos
phor
ylat
ion
of E
RK
, as
inhi
bitio
n of
bot
h pa
thw
ays
was
requ
ired
to a
chie
ve a
com
plet
e do
wn
regu
latio
n of
ER
K p
hosp
hory
latio
n in
res
pons
e to
mec
hani
cal
stim
ulat
ion.
Inte
rest
ingl
y it
was
sho
wn
that
sig
nific
ant
chan
ges
in c
ell
mor
phol
ogy
(5 m
inut
es i
nto
stim
ulat
ion)
occ
ur p
rior
to t
he p
eak
in p
hosp
hory
latio
n of
ER
K (
15 m
inut
es i
nto
stim
ulat
ion)
poss
ibly
su
gges
ting
that
ph
ysic
al
chan
ges
in
cell
shap
e pr
eced
e/in
itiat
e th
e do
wns
tream
sign
allin
g ca
scad
es s
uch
as E
RK
MA
P K
inas
e. P
ast s
tudi
es in
our
lab
show
ed th
at w
hen
ERK
77
activ
atio
n is
inhi
bite
d M
T1-M
MP
upre
gula
tion
in re
spon
se to
cyc
lic c
ompr
essi
on d
oes n
ot o
ccur
[De
Cro
os, J
ang,
Dha
liwal
, Gry
npas
, Pill
iar &
Kan
del,
07].
Sinc
e it
is p
ostu
late
d th
at M
T1-M
MP
faci
litat
es c
ellu
lar
retra
ctio
n, t
hrou
gh d
estru
ctio
n of
cel
l-mat
rix c
onta
cts,
it is
pos
sibl
e to
conc
lude
that
the
activ
atio
n of
ER
K m
ay n
ot b
e in
volv
ed in
cel
l spr
eadi
ng b
ut ra
ther
sub
sequ
ent
even
ts.
This
not
ion
is s
uppo
rted
by s
tudi
es w
hich
obs
erve
d th
at p
hysi
cal
defo
rmat
ions
in
the
cellu
lar m
embr
ane
com
pris
e th
e fir
st s
tep
in m
echa
notra
nsdu
ctio
n [G
uila
k, 9
5;K
nigh
t, B
omzo
n,
Kim
mel
, Sha
rma,
Lee
& B
ader
, 06a
]. H
owev
er, t
his
is c
ontro
vers
ial a
s se
vera
l stu
dies
in o
ther
cell
type
s, su
ch a
s C
hine
se h
amst
er o
vary
cel
ls a
nd s
moo
th m
uscl
e ce
lls, h
ave
sugg
este
d th
at
ERK
upr
egul
atio
n oc
curs
prio
r, an
d in
fac
t dr
ives
, an
y ce
ll sh
ape
chan
ges
asso
ciat
ed w
ith
phys
ical
mem
bran
e pe
rturb
atio
n or
inte
rgrin
-act
ivat
ion
[Lev
yet
al.,
03;
Sam
arak
oon
& H
iggi
ns,
02].
To
fully
del
inea
te t
he s
eque
nce
of e
vent
s in
our
sys
tem
, it
wou
ld b
e ne
cess
ary
to f
ully
inhi
bit
or k
nock
out
ERK
exp
ress
ion
prio
r to
mec
hani
cal
stim
ulat
ion
and
obse
rve
whe
ther
chan
ges
in c
ell m
orph
olog
y oc
cur.
How
ever
, ow
ing
to th
e ex
tens
ive
role
ER
K p
lays
in m
ultit
ude
of c
ellu
lar
proc
esse
s it
may
be h
ard
to c
oncl
usiv
ely
prov
e a
dire
ct i
nflu
ence
of
ERK
on
cell
shap
e in
res
pons
e to
cyc
lic c
ompr
essi
on.
Thus
, ba
sed
on t
he d
ata
obta
ined
in
our
stud
y, w
e
cann
ot d
eter
min
e de
finiti
vely
whe
ther
cel
l spr
eadi
ng d
rives
ER
K p
hosp
hory
latio
n or
that
the
two
occu
r sim
ulta
neou
sly.
The
invo
lvem
ent o
f cal
cium
in re
gula
ting
cell
resp
onse
in o
ur sy
stem
was
not
une
xpec
ted
as o
ther
s ha
ve p
revi
ousl
y do
cum
ente
d th
e ro
le o
f ca
lciu
m in
cho
ndro
cyte
mec
hano
trans
duct
ion
[Rob
erts
, K
nigh
t, Le
e &
Bad
er,
01;T
anak
a, O
hno,
Hon
da,
Tani
mot
o, D
oi,
Ohn
o-N
akah
ara,
Tafo
lla, K
apila
& T
anne
, 05;
Zhan
g, W
ang
& C
hen,
06]
. The
rol
e of
cal
cium
sig
nalli
ng in
our
syst
em w
as d
emon
stra
ted
thro
ugh
the
use
of E
GTA
whi
ch w
as s
how
n to
abo
lish
the
stim
ulat
ion-
indu
ced
incr
ease
in
cell
spre
adin
g, a
nd t
he r
eint
rodu
ctio
n of
cal
cium
int
o th
e m
edia
whi
ch
78
retu
rned
the
cellu
lar
resp
onse
to m
echa
nica
l stim
ulat
ion
(Fig
ure
2). I
t is
wor
thw
hile
not
ing
that
EGTA
is a
ble
to c
hela
te a
var
iety
of c
atio
ns (n
amel
y C
a2+, N
a2+, K
+ ), bu
t as
the
re-in
trodu
ctio
n
of c
alci
um a
lone
into
the
med
ia w
as a
ble
to re
turn
cel
l res
pons
e to
nor
mal
leve
ls, t
he m
echa
nism
regu
latin
g th
is r
espo
nse
was
attr
ibut
ed t
o ca
lciu
m a
nd n
ot o
ther
med
ia s
olub
le c
atio
ns.
The
prec
ise
amou
nt o
f ca
lciu
m t
hat
was
re-
intro
duce
d in
to t
hem
edia
(in
the
for
m o
f C
aCl 2)
was
dete
rmin
ed p
revi
ousl
y in
the
lab
thro
ugh
dose
-res
pons
e ex
perim
ents
whi
ch re
intro
duce
d ca
lciu
m
into
EG
TA-c
hela
ted
med
ia a
t var
ious
con
cent
ratio
ns u
ntil
cellu
lar r
espo
nse
was
iden
tical
to th
at
obse
rved
in E
GTA
-unt
reat
ed s
ampl
es.
It w
as im
porta
nt to
ful
ly s
atur
ate
any
EGTA
pre
sent
in
the
med
ia to
ens
ure
suff
icie
nt fr
ee c
alci
um w
as p
rese
nt. T
he la
ck o
f cel
lula
r sha
pe c
hang
es in
the
abse
nce
of c
alci
um m
ay b
e a
resu
lt of
eith
er p
reve
ntio
n of
cal
cium
-dep
ende
nt re
orga
niza
tion
of
the
actin
cyt
oske
leto
n or
act
ivat
ion
of o
ther
cal
cium
-ass
ocia
ted
dow
nstre
am s
igna
lling
eve
nts.
Cal
cium
is re
quire
d fo
r sub
stan
tial r
emod
ellin
g of
the
actin
cyt
oske
leto
n, w
hich
is n
eces
sary
for
cell
spre
adin
g, a
nd is
con
trolle
d th
roug
h ac
tivat
ion
of c
alm
odul
in a
nd o
ther
cal
cium
-dep
ende
nt
actin
bin
ding
pro
tein
s [T
oyod
aet
al.,
03]
. It
is k
now
n th
at u
nder
cer
tain
loa
ding
con
ditio
ns
chon
droc
ytes
exh
ibit
a ra
pid
(~8
min
utes
) reo
rgan
izat
ion
of th
eir a
ctin
cyt
oske
leto
n in
a c
alci
um-
depe
nden
t man
ner [
Eric
kson
, Nor
thru
p &
Gui
lak,
03]
, and
that
cal
cium
flux
es th
roug
h th
e SA
C
are
invo
lved
in
regu
latin
g ch
ondr
ocyt
e m
orph
olog
y [P
erki
ns,
Der
foul
, A
st &
Hal
l, 05
].
Inte
rest
ingl
y, h
owev
er, a
stu
dy b
y Lu
rran
t et a
l.¸su
gges
ted
that
cho
ndro
cyte
s in
fact
do
not a
lter
thei
r act
in c
ytos
kele
ton
in re
spon
se to
mec
hani
cal s
timul
atio
n, a
nd in
stea
d m
odify
thei
r vim
entin
cyto
skel
eton
[D
urra
ntet
al.,
99]
. Pa
st s
tudi
es h
ave
impl
icat
ed P
KC
, a
calc
ium
dep
ende
nt
enzy
me,
in
regu
latin
g v
imen
tin c
ytos
kele
ton
reor
gani
zatio
n in
cel
l ty
pes
such
as
astro
cyte
s
[Man
gour
aet
al.,
95]
, and
thus
it is
pos
sibl
e th
at a
sim
ilar m
echa
nism
may
exi
st in
cho
ndro
cyte
s.
PKC
, whi
ch e
ncom
pass
es a
fam
ily o
f 11
clos
ely
rela
ted
isoz
ymes
(of w
hich
the
PKC
,-1,
-2,
79
and
-fo
rms
are
calc
ium
-dep
ende
nt)
has
also
be
en
prev
ious
ly
show
n to
re
gula
te
the
Ras
/Raf
/MA
P K
inas
e pa
thw
ay i
n a
varie
ty o
f sy
stem
s [K
olch
, 00;
Mar
shal
l, 95
]an
d th
us i
s a
plau
sibl
e re
gula
tor
of t
his
syst
em a
s w
ell.
We
atte
mpt
ed t
o vi
sual
ize
the
actin
cyt
oske
leto
n
follo
win
g m
echa
nica
l stim
ulat
ion
thro
ugh
the
use
of c
onfo
cal m
icro
scop
y, a
nd a
lthou
gh it
was
just
a pr
elim
inar
y st
udy
(App
endi
x A
, Fi
gure
1)
we
obse
rved
tha
t th
ere
was
no
sign
ifica
nt
alte
ratio
n in
the
act
in c
ytos
kele
ton
with
stim
ulat
ion.
Thi
s ob
serv
atio
n su
gges
ts t
hat
the
mor
phol
ogic
al c
hang
es o
bser
ved
in o
ur s
yste
m m
ay b
e m
odul
ated
by
the
vim
entin
cyt
oske
leto
n
as w
ell.
To f
urth
er i
nves
tigat
e th
is,
the
vim
entin
cyt
oske
leto
n sh
ould
be
obse
rved
thr
ough
conf
ocal
mic
rosc
opy
as w
ell.
Inte
rest
ingl
y it
was
als
o ob
serv
ed t
hat
EGTA
tre
atm
ent
of t
he m
ediu
m r
esul
ted
in a
slig
htly
(n
ot
sign
ifica
nt)
incr
ease
in
ch
ondr
ocyt
e ce
ll ar
ea
inde
pend
ent
of
mec
hani
cal
stim
ulat
ion.
Thi
s ob
serv
atio
n ca
n be
pos
sibl
y ex
plai
ned
by e
ither
EG
TA-in
duce
d ch
ange
s in
med
ia o
smol
ality
or
to c
alci
um’s
reg
ulat
ory
func
tion
in m
aint
aini
ng c
ell
shap
e an
d vo
lum
e.
Whi
le o
smol
ality
has
bee
n sh
own
to b
e a
sign
ifica
nt r
egul
ator
of
chon
droc
yte
func
tion
[Hop
ewel
l &
Urb
an, 0
3], t
he a
utho
rs u
tiliz
ed s
olut
ions
with
dra
stic
ally
low
er o
smol
ality
(38
0
mO
smol
) th
an th
at w
hich
cou
ld b
e in
duce
d by
the
EGTA
che
latio
n in
our
sys
tem
, sug
gest
ing
that
the
min
or c
hang
es th
at m
ight
hav
e oc
curr
ed i
n ou
r sys
tem
wer
eun
likel
y to
be
resp
onsi
ble
for
chan
ges
in c
ell
shap
e. H
owev
er m
easu
rem
ents
of
the
med
ia o
smol
ality
wou
ld h
ave
to b
e
done
to d
eter
min
e th
e ac
tual
leve
ls in
our
sys
tem
. Alte
rnat
ivel
y, it
cou
ld b
e po
ssib
le to
incr
ease
the
osm
olal
ity in
our
syst
em th
roug
h th
e ad
ditio
n of
ions
such
as N
a+ , how
ever
this
may
inte
rfer
e
with
som
e ce
llula
r pr
oces
ses
whi
ch a
re a
ctiv
ated
by
Na+
chan
nels
. The
re is
som
e ev
iden
ce th
at
sodi
um m
echa
nose
nsiti
ve c
hann
els
are
expr
esse
d in
cho
ndro
cyte
s an
d m
ay p
lay
a ro
le i
n
regu
latin
g ce
ll re
spon
se a
s w
ell [
Shak
ibae
i & M
obas
heri,
03]
. Thu
s it
is m
ore
plau
sibl
e th
at th
e
80
chan
ges
in c
ell a
rea
are
attri
bute
d to
the
regu
lato
ry r
ole
calc
ium
exe
rts in
reg
ulat
ing
cell
shap
e
and
volu
me.
For
ins
tanc
e, [
Ca2+
] iha
s be
en s
how
n to
con
trol
Reg
ulat
ory
Vol
ume
Dec
reas
es
(RV
D)
and
Incr
ease
s (R
VI)
in
vario
us c
ell
type
s in
clud
ing
oste
ocyt
es a
nd c
hond
rocy
tes
[Miy
auch
iet a
l., 0
0;Y
ello
wle
yet
al.,
02]
. In
a s
tudy
by
Ker
rigan
et a
l., th
e au
thor
s sh
owed
that
chan
ges
in [
Ca2+
] i in
fluen
ces
RV
D
in o
nly
a su
bpop
ulat
ion
of f
resh
ly i
sola
ted
bovi
ne
chon
droc
ytes
[Ker
rigan
& H
all,
08],
thus
sug
gest
ing
that
the
slig
ht in
crea
se o
bser
ved
in c
ell a
rea
in o
ur sy
stem
can
be
attri
bute
d to
inte
rfer
ence
with
cal
cium
regu
latio
n in
the
cells
.
Hav
ing
conf
irmed
the
role
of
extra
cellu
lar
calc
ium
, we
furth
er c
hara
cter
ized
its
role
in
med
iatin
g th
e re
spon
se t
hrou
gh t
he u
se o
f th
e in
hibi
tors
nife
dipi
ne a
nd g
adol
iniu
m (
Gd3+
) to
sele
ctiv
ely
bloc
k ei
ther
the
vol
tage
-gat
ed L
-Typ
e ca
lciu
m c
hann
el (
VG
CC
) or
the
stre
tch-
activ
ated
cha
nnel
(SA
C),
resp
ectiv
ely
(Fig
ure
4).
In t
he p
rese
nce
of G
d3+ce
ll sp
read
ing
was
entir
ely
abol
ishe
d in
res
pons
e to
mec
hani
cal s
timul
atio
n, s
ugge
stin
g th
at c
alci
um f
low
thro
ugh
SAC
s is
dire
ctly
invo
lved
in in
fluen
cing
cyt
oske
leta
l rem
odel
ling
lead
ing
to c
ell s
prea
ding
. Thi
s
parti
cula
r ch
anne
l ha
s be
en p
revi
ousl
y sh
own
to b
e m
echa
nose
nsiti
ve [
Wu
et a
l., 0
1a;M
ouw
,
Imle
r & L
even
ston
, 07]
and
it is
pos
sibl
e th
at th
ose
resp
onse
s w
ere,
at l
east
in p
art,
attri
bute
d to
cyto
skel
etal
rea
rran
gem
ents
that
hav
e be
en f
acili
tate
d th
roug
h ca
lciu
m c
urre
nts
pass
ing
thro
ugh
the
chan
nel.
The
cha
nnel
is k
now
n to
be
activ
ated
thro
ugh
dire
ct p
hysi
cal d
efor
mat
ion
of th
e
cellu
lar
mem
bran
e [W
u &
Dav
is,
01;Z
ou,
Lifs
hitz
, Tu
ft, F
ogar
ty &
Sin
ger,
02],
whi
ch s
ome
argu
e is
the
initi
al s
tep
in m
echa
notra
nsdu
ctio
n [G
uila
k, 9
5;K
nigh
t, B
omzo
n, K
imm
el, S
harm
a,
Lee
& B
ader
, 06
a],
and
thus
it
mig
ht e
xpec
ted
that
any
sub
sequ
ent
proc
esse
s (s
prea
ding
,
retra
ctio
n, e
tc.)
be r
egul
ated
thr
ough
thi
s ch
anne
l. U
nfor
tuna
tely
it
is c
urre
ntly
im
poss
ible
to
char
acte
rize
the
expr
essi
on o
f th
e SA
C i
n ch
ondr
ocyt
es a
s th
e ch
anne
l ha
s no
t be
en f
ully
desc
ribed
[M
arot
o, R
aso,
Woo
d, K
uros
ky,
Mar
tinac
& H
amill
, 05
], bu
t its
pre
senc
e in
81
chon
droc
ytes
has
bee
n su
ppor
ted
thro
ugh
othe
r st
udie
s ut
ilizi
ng t
he i
nhib
itor
gado
liniu
m
[Gui
lak,
Zel
l, Er
icks
on,
Gra
nde,
Rub
in,
McL
eod
& D
onah
ue,
99;M
ouw
, Im
ler
& L
even
ston
,
07;P
erki
ns, D
erfo
ul, A
st &
Hal
l, 05
].
In c
ontra
st, c
ells
stim
ulat
ed in
the
pres
ence
of n
ifedi
pine
exh
ibite
d a
spre
ad m
orph
olog
y
indi
catin
g th
at e
ither
cal
cium
cur
rent
s th
roug
h th
is p
artic
ular
cha
nnel
are
not
inv
olve
d in
the
early
sta
ges
of c
ytos
kele
tal
rem
odel
ling,
or
that
the
cha
nnel
rem
ains
ina
ctiv
e th
roug
hout
the
stim
ulat
ion
itsel
f an
d th
at it
bec
omes
act
ive
sequ
entia
lly a
fter
the
SAC
s. Th
e la
tter
expl
anat
ion
is, h
owev
er, l
ess
likel
yas
we
have
obs
erve
d an
eff
ect a
nd o
ther
hav
e pr
evio
usly
rep
orte
d th
at
VG
CC
in
fluen
ces
som
e ce
llula
r re
spon
ses
to m
echa
nica
l stim
ulat
ion
in c
hond
rocy
tes
, suc
h as
the
secr
etio
n of
PTH
rP in
resp
onse
to c
yclic
stra
in[T
anak
a, O
hno,
Hon
da, T
anim
oto,
Doi
,Ohn
o-
Nak
ahar
a, T
afol
la,
Kap
ila &
Tan
ne,
05].
Non
ethe
less
, w
hile
the
cho
ndro
cyte
s sp
read
in
the
pres
ence
of n
ifedi
pine
it w
as o
bser
ved
that
thei
r sub
sequ
ent r
etra
ctio
n, w
hich
nor
mal
ly o
ccur
s by
six
hour
s po
st-s
timul
atio
n, w
as a
bolis
hed.
We
have
pre
viou
sly
show
n th
at th
e re
tract
ion
phas
e of
the
resp
onse
is
med
iate
d vi
a th
e ac
tivity
of
the
mem
bran
e-bo
und
MT1
-MM
P (s
ubm
itted
for
publ
icat
ion)
, whi
ch it
self
is re
gula
ted
thro
ugh
ERK
pho
spho
ryla
tion
[De
Cro
os, J
ang,
Dha
liwal
,
Gry
npas
, Pill
iar
& K
ande
l, 07
]. C
onse
quen
tly it
is p
ossi
ble
that
cal
cium
cur
rent
s th
roug
h th
e
VG
CC
are
not
dire
ctly
inv
olve
d in
the
rem
odel
ling
of t
he c
ytos
kele
ton,
but
rat
her
thro
ugh
activ
atio
n of
ER
K w
hich
reg
ulat
es t
he a
ctiv
ity o
f M
T1-M
MP
and
thus
ind
irect
ly i
nflu
ence
retra
ctio
n.
This
pos
sibi
lity
is s
uppo
rted
by p
revi
ous
liter
atur
e in
dica
ting
that
cal
cium
inf
lux
thro
ugh
VG
CC
is
abl
e in
fluen
ce E
RK
pho
spho
ryla
tion
in s
ever
al c
ell t
ypes
[M
ulva
ney
et a
l.,
99;G
omez
, Prit
char
d &
Her
bert,
02]
. O
vera
ll, h
owev
er, t
he s
eque
ntia
l rol
e of
the
SAC
and
the
L-ty
pe V
GC
C i
s in
kee
ping
with
lite
ratu
re s
ugge
stin
g th
at o
ne o
f th
e fu
nctio
nal
outc
omes
of
SAC
act
ivat
ion
is c
ell d
epol
ariz
atio
n an
d su
bseq
uent
activ
atio
n of
VG
CC
s [Z
ou, L
ifshi
tz, T
uft,
82
Foga
rty &
Sin
ger,
02].
The
expr
essi
on o
f th
e ch
anne
l by
the
cho
ndro
cyte
s in
our
sys
tem
was
conf
irmed
thro
ugh
RT-
PCR
(App
endi
x A
, Fig
ure
2).
To f
urth
er s
tudy
the
rol
e of
cal
cium
in
mec
hano
trans
duct
ion,
as
wel
l as
con
firm
the
effe
cts
of S
AC
and
L-T
ype
VG
CC
inhi
bitio
n w
ere
due
to c
atio
n flu
xes,
we
utili
zed
the
A23
187
iono
phor
e to
mod
ulat
e ca
lciu
m l
evel
s. Th
e ca
lciu
m i
onop
hore
A23
187
is a
mob
ile i
on-c
arrie
r
that
form
s sta
ble
com
plex
es w
ith d
ival
ent c
atio
ns (s
uch
as C
a2+) a
nd a
llow
s the
ir tra
nspo
rt ac
ross
the
cellu
lar
mem
bran
e.
Unl
ike
othe
r io
noph
ores
whi
ch f
orm
hyd
roph
ilic
pore
s in
the
cel
lula
r
mem
bran
e an
d th
us a
llow
ing
ions
to
pass
, th
e A
2318
7 io
noph
ore
bind
s di
vale
nt c
atio
ns a
nd
thro
ugh
shie
ldin
g of
thei
r cha
rge
from
the
surr
ound
ing
envi
ronm
ent f
acili
tate
s th
eir c
ross
ing
into
the
cell.
The
add
ition
of
A23
187
iono
phor
e is
util
ized
to
incr
ease
int
race
llula
r ca
lciu
m l
evel
s,
and
has
been
pre
viou
sly
show
n to
ele
vate
intra
cellu
lar c
alci
um le
vels
in c
hond
rocy
tes
[Sch
war
tz
et a
l., 9
1]. T
reat
men
t of c
ells
with
the
A23
187
iono
phor
e du
ring
cycl
ic c
ompr
essi
on w
as a
ble
to
parti
ally
rev
erse
the
eff
ects
of
nife
dipi
ne a
nd g
adol
iniu
m o
n ce
ll m
orph
olog
y. T
he r
easo
n fo
r
only
the
parti
al re
stor
atio
n of
cha
nges
in c
ell a
rea
with
the
iono
phor
e tre
atm
ent i
s no
t kno
wn,
but
one
poss
ible
exp
lana
tion
may
be
that
the
loca
lizat
ion
of c
alci
um, a
nd n
ot m
erel
y its
pre
senc
e,
follo
win
g m
echa
nica
l st
imul
atio
n is
an
impo
rtant
asp
ect
of t
his
resp
onse
. L
ocal
ized
cal
cium
curr
ents
hav
e be
en p
revi
ousl
y do
cum
ente
d to
influ
ence
loca
l sig
nalli
ng c
asca
des
and
thus
aff
ect
loca
lized
si
gnal
ling
path
way
s [J
anm
ey,
98;Z
ou,
Lifs
hitz
, Tu
ft,
Foga
rty
&
Sing
er,
02].
Alte
rnat
ivel
y, it
is p
ossi
ble
that
exp
osur
e of
chon
droc
ytes
to th
e io
noph
ore
activ
ates
add
ition
al,
mec
hani
cal s
timul
atio
n-un
rela
ted,
sig
nalli
ng p
athw
ays
that
influ
ence
pro
cess
es w
hich
indi
rect
ly
may
reg
ulat
e ce
ll ar
ea.
For
inst
ance
, in
our
lab
we
have
pre
viou
sly
show
n th
at t
reat
men
t of
bovi
ne s
ynov
ial
fibro
blas
ts w
ith t
he i
onop
hore
has
res
ulte
d in
the
pro
duct
ion
of a
gel
atin
ase
[How
arth
, Pr
itzke
r, C
ruz
& K
ande
l, 93
]w
ithou
t t
he a
pplic
atio
n of
any
mec
hani
cal
forc
es.
83
Con
sequ
ently
, tre
atm
ent
of c
hond
rocy
tes
with
A23
187
iono
phor
e m
ay e
licit
num
erou
s ot
her
cellu
lar e
ffec
ts w
hich
cou
ld p
reve
nt a
com
plet
e re
stor
atio
n of
the
resp
onse
.
Ow
ing
to
the
prev
ious
ly
esta
blis
hed
role
of
M
T1-M
MP
in
mod
ulat
ing
cell
and
chon
droc
yte
mor
phol
ogy
(sub
mitt
ed f
or p
ublic
atio
n)[C
hun,
Hot
ary,
Sab
eh,
Salti
el,
Alle
n &
Wei
ss,
06],
we
inve
stig
ated
whe
ther
the
stim
ulat
ion-
indu
ced
upre
gula
tion
MT1
-MM
P is
als
o
regu
late
d vi
a ca
lciu
m.
Che
latio
n of
ext
race
llula
r ca
lciu
m a
bolis
hed
the
stim
ulat
ion-
indu
ced
upre
gula
tion
of M
T1-M
MP
sugg
estin
g ei
ther
cal
cium
dire
ctly
exe
rts a
con
trol o
ver
MT1
-MM
P
expr
essi
on o
r tha
t MT1
-MM
P ex
pres
sion
is d
epen
dent
upo
n ce
ll m
orph
olog
y ch
ange
s (Fi
gure
3).
The
form
er is
sup
porte
d th
roug
h st
udie
s th
at h
ave
show
n th
at c
alci
um c
an r
egul
ate
the
bind
ing
activ
ity o
f A
P-1
(a r
egul
ator
of
MT1
-MM
P ex
pres
sion
) in
hum
an p
ulm
unar
y ar
tery
end
othe
lial
cells
[Fa
ntoz
zi,
Zhan
g, P
lato
shyn
, R
emill
ard,
Cow
ling
& Y
uan,
03]
, an
d th
roug
h st
udie
s th
at
impl
icat
ed L
-type
VG
CC
cur
rent
s in
dire
ct r
egul
atio
n of
AP-
1 bi
ndin
g ac
tivity
[Pr
emku
mar
,
Mis
hra,
Ove
rhol
t, Si
mon
son,
Che
rnia
ck &
Pra
bhak
ar,
00].
Furth
er s
uppo
rting
the
ide
a th
at
calc
ium
dire
ctly
regu
late
s M
T1-M
MP
expr
essi
on w
as s
een
in ti
ssue
-con
stru
cts
that
wer
e tre
ated
with
EG
TA r
esul
ting
in a
sig
nific
ant
incr
ease
in
MT1
-MM
P m
RN
A l
evel
s in
the
abs
ence
of
mec
hani
cal s
timul
atio
n. It
is in
tere
stin
g to
not
e th
at w
hile
this
incr
ease
in M
T1-M
MP
prod
uced
a
tem
pora
ry in
crea
se in
the
synt
hesi
s of
col
lage
n an
d pr
oteo
glyc
ans,
it di
d no
t res
ult i
n im
prov
ed
tissu
e fo
rmat
ion
in th
e lo
ng te
rm (
App
endi
x A
, Fig
ure
A3)
. One
may
hyp
othe
size
that
with
out
the
sync
hron
ized
cha
nges
in
cell
mor
phol
ogy,
whi
ch p
erha
ps r
esul
t in
the
act
ual
activ
atio
n of
MT1
-MM
P an
d th
e su
bseq
uent
deg
rada
tion
of t
he p
eric
ellu
lar
mat
rix,
the
new
ly s
ynth
esiz
ed
mac
rom
olec
ules
are
not
inc
orpo
rate
d in
to t
he t
issu
e’s
mat
rix. T
he o
bser
ved
chan
ges
in M
T1-
MM
P m
RN
A c
ould
not
be
repl
icat
ed w
hen
chon
droc
ytes
wer
e gr
own
on m
onol
ayer
, sug
gest
ing
that
the
shap
e of
cel
ls, w
hich
is v
ery
diff
eren
t in
mon
olay
er c
ompa
red
to 3
D c
ultu
re, i
s im
porta
nt
84
in r
egul
atin
g M
T1-M
MP
as w
ell (
App
endi
x A
, Fig
ure
A4)
. Th
e co
mpl
ete
rest
orat
ion
of M
T1-
MM
P ge
ne e
xpre
ssio
n in
the
abs
ence
of
com
plet
e re
stor
atio
n of
cel
l sh
ape
chan
ges
with
iono
phor
e tre
atm
ent m
ay a
lso
be in
dica
tive
of a
sep
arat
e ca
lciu
m s
igna
lling
pat
hway
that
exe
rts
cont
rol
over
MT1
-MM
P ex
pres
sion
or,
in c
ontra
st,
that
par
tial
spre
adin
g an
d re
tract
ion
are
suff
icie
nt to
elic
it M
T1-M
MP
gene
exp
ress
ion.
A s
tudy
by
Lohi
et a
l.sh
owed
tha
t io
noph
ore
treat
men
t can
indu
ce M
MP-
9 an
d M
MP-
2. T
he a
bsen
ce if
an
effe
ct o
n M
T1-M
MP
expr
essi
on in
thei
r sy
stem
[Lo
hi &
Kes
ki-O
ja, 9
5]m
ay r
efle
ct th
e di
ffer
ent c
ell t
ype,
fib
rosa
rcom
a, u
tiliz
ed.
Fibr
osar
com
a ce
lls w
hich
, be
ing
canc
erou
s, ar
e kn
own
to o
ften
have
abn
orm
al c
alci
um
sign
allin
g m
echa
nism
s (i.
e. c
alci
um s
igna
ls t
o di
ffer
entia
te, u
nder
go a
popt
osis
, pro
lifer
ate
are
igno
red)
[Whi
tfiel
d, 9
2].
Prev
ious
stu
dies
in
our
lab
sugg
este
d th
at c
ell
mor
phol
ogy
chan
ges,
MT1
-MM
P
expr
essi
on a
nd th
e su
bseq
uent
impr
ovem
ent i
n tis
sue
mat
rix s
ynth
esis
are
regu
late
d vi
a th
e α5
β1
inte
grin
(su
bmitt
ed f
or p
ublic
atio
n), a
nd o
win
g to
the
clos
ely
docu
men
ted
rela
tions
hip
betw
een
calc
ium
cha
nnel
s an
d in
tegr
ins,
it w
as i
mpo
rtant
to
inve
stig
ate
the
role
cal
cium
pla
ys i
n
regu
latin
g in
tegr
in a
ctiv
ity. I
n lig
ht o
f the
abo
ve re
sults
, it w
as n
ot c
lear
how
the
calc
ium
-rel
ated
and
inte
grin
-ass
ocia
ted
sign
allin
g pa
thw
ays
inte
ract
to
regu
late
cel
l sh
ape
chan
ges
and
MT1
-
MM
P ex
pres
sion
. Si
nce
inte
grin
s la
ck a
ny i
nher
ent
enzy
mat
ic a
ctiv
ity,
sign
al t
rans
duct
ion
is
acco
mpl
ishe
d vi
a ac
tivat
ion
of v
ario
us a
dapt
er p
rote
ins
such
as
FAK
and
Src
. Con
sequ
ently
, to
dete
rmin
e th
e ro
le c
alci
um p
lays
with
res
pect
to
α5β1
int
egrin
act
ivity
, th
e ad
apte
r pr
otei
ns
invo
lved
had
to
be e
luci
date
d. A
tim
elin
e ex
amin
ing
the
phos
phor
ylat
ion
of F
ocal
Adh
esio
n
Kin
ase
(FA
K) c
ondu
cted
ove
r 3, 5
, 15
and
25 m
inut
es o
f mec
hani
cal s
timul
atio
n (A
ppen
dix
A,
Figu
re 5
) in
dica
ted
that
no
sign
ifica
nt c
hang
es i
n th
e pr
otei
n’s
phos
phor
ylat
ion
stat
e oc
curs
,
sugg
estin
g FA
K is
not
invo
lved
in m
edia
ting
this
res
pons
e. I
t sho
uld
be p
oint
ed o
ut, h
owev
er,
85
that
the
antib
ody
utili
zed
to d
eter
min
e th
e ph
osph
oryl
atio
n of
FA
K b
y w
este
rn b
lot a
naly
sis
was
a ge
neric
pho
spho
-tyro
sine
ant
ibod
y th
at t
arge
ted
all
cellu
lar
prot
eins
with
pho
spho
ryla
ted
tyro
sine
res
idue
s, w
ith th
e pr
otei
n of
inte
rest
(FA
K)
iden
tifie
d th
roug
h in
cuba
tion
with
an
anti-
FAK
ant
ibod
y. C
onse
quen
tly th
e la
ck o
f di
ffer
ence
s in
FA
K p
hosp
hory
latio
n m
ay b
e po
ssib
ly
attri
bute
d to
non
-sel
ectiv
e bi
ndin
g of
the
ant
i-pTy
r an
tibod
y to
oth
er p
rote
ins
of s
imila
r
mol
ecul
ar w
eigh
t. Si
mila
rly,
FAK
con
tain
s m
ultip
le t
yros
ine
resi
dues
tha
t ar
e di
ffer
entia
lly
phos
phor
ylat
ed
and
diff
eren
tially
re
gula
te
cellu
lar
func
tion;
fo
r in
stan
ce
the
maj
or
phos
phor
ylat
ion
site
is ty
rosi
ne-3
97 w
hich
for
ms
a Sr
c H
omol
ogy
2 (S
H2)
dom
ain
capa
ble
of
inte
ract
ing
with
Src
-fam
ily k
inas
es (
i.e. S
rc,F
yn),
whi
le o
ther
site
s in
clud
e ty
rosi
ne-5
76 a
nd -
577
and
-925
whi
ch m
ay a
lso
inte
ract
with
the
kina
se G
rb2.
Rec
ently
it w
as a
lso
dem
onst
rate
d
that
pho
spho
ryla
tion
at t
yros
ine-
861
form
s an
add
ition
al S
H2
bind
ing
dom
ain
[Cal
alb
et a
l.,
96;S
chal
ler,
Hild
ebra
nd,
Shan
non,
Fox
, V
ines
& P
arso
ns,
94;S
chla
epfe
r, B
room
e &
Hun
ter,
97;Y
ano,
Gei
bel
& S
umpi
o, 9
6].
As
such
, ch
ange
s in
pho
spho
ryla
tion
in i
ndiv
idua
l ty
rosi
ne
resi
dues
may
be
mas
ked
with
ant
ibod
ies t
arge
ting
tota
l pho
spho
-tyro
sine
.
A s
econ
d ki
nase
that
is k
now
n to
be h
eavi
ly in
volv
ed in
med
iatin
g in
tegr
in s
igna
lling
,
Src,
was
like
wis
e in
vest
igat
ed in
our
sys
tem
. How
ever
, due
to a
lack
of
a re
liabl
e an
tibod
y, it
s
role
was
inve
stig
ated
thro
ugh
the
use
of th
e ch
emic
al in
hibi
tor P
P2 w
hich
sel
ectiv
ely
inhi
bits
its
activ
ity. B
ased
on
the
resu
lts (
Figu
re 7
) it
appe
ars
that
the
res
pons
e to
cyc
lic c
ompr
essi
on i
s
mai
nly
faci
litat
ed th
roug
h th
e ki
nase
Src
, as i
ts in
hibi
tion
abol
ishe
d th
e su
bseq
uent
cel
l spr
eadi
ng
and
MT1
-MM
P up
regu
latio
n. C
onst
ruct
s inc
ubat
ed w
ith th
e ch
emic
alPP
3, a
neg
ativ
e co
ntro
l for
PP2,
exh
ibite
d a
resp
onse
tha
t w
as s
imila
r to
tha
t ob
serv
ed i
n ve
hicl
e tre
ated
sam
ples
. It
is
impo
rtant
to m
entio
n th
at w
hile
PP2
is u
tiliz
ed e
xten
sive
ly in
lite
ratu
re a
s a
sele
ctiv
e in
hibi
tor o
f
Src,
it is
abl
e to
inhi
bit s
ever
al o
ther
tyro
sine
kin
ases
, alb
eit n
ot a
s ef
fect
ivel
y as
indi
cate
d by
the
86
follo
win
g IC
50va
lues
: Lc
k 0.
004
μM, F
yn 0
.005
μM
, ZA
P-70
>10
0 μM
, JA
K2
>50
μM a
nd
EGF-
R 0
.49
μM (n
ote:
Lck
and
Fyn
are
mem
bers
of t
he S
rc fa
mily
)[H
anke
et a
l., 9
6]. S
imila
rly,
som
e au
thor
s ha
ve s
how
n th
at t
he a
bilit
y of
PP2
to
inhi
bit
Src
kina
ses
may
int
erfe
re w
ith
inte
grin
-indu
ced
FAK
pho
spho
ryla
tion
[Sal
azar
& R
ozen
gurt,
01]
. A
stu
dy b
y X
ia e
t al
.,
dem
onst
rate
d th
at P
P2 c
an e
ffec
tivel
y ab
olis
h th
e ph
osph
oryl
atio
n of
Tyr
-397
of
FAK
, the
p85
subu
nit o
f PI3
k, a
nd S
er-4
73 o
f Akt
whi
ch o
ccur
upo
n th
e ac
tivat
ion
of β
1 in
getri
ns [X
iaet
al.,
04].
The
sam
e st
udy
indi
cate
d th
at P
P3 h
ad n
o ef
fect
on
any
of t
he i
nves
tigat
ed k
inas
es.
Con
sequ
ently
, whi
le P
P2 c
an b
e us
ed to
indi
cate
the
invo
lvem
ent o
f Src
kin
ase
in th
e si
gnal
ling
path
way
, it i
s no
t, in
itse
lf, s
uffic
ient
to re
ject
the
invo
lvem
ent o
f oth
er k
inas
es. T
he o
bser
vatio
n
that
no
chan
ges
in F
AK
pho
spho
ryla
tion
wer
e se
en w
ith m
echa
nica
l stim
ulat
ion,
in p
artic
ular
as
cell
spre
adin
g oc
curr
ed, c
ould
be
expl
aine
d by
not
ing
that
diff
eren
t typ
es o
f m
echa
nica
l for
ces
diff
eren
tially
regu
late
the
phos
phor
ylat
ion
of F
AK
and
Src
[Ren
, Kio
sses
, Sie
g, O
tey,
Sch
laep
fer
& S
chw
artz
, 00]
. Alte
rnat
ivel
y, it
is p
ossi
ble
that
the
cell-
mat
rix in
tera
ctio
ns fo
rmed
dur
ing
this
spre
adin
g ar
e so
mew
here
bet
wee
n Fo
cal
Adh
esio
ns a
nd F
ibril
lar
adhe
sion
s an
d th
us t
he
cons
titut
ion
of a
ssoc
iate
d ki
nase
s cou
ld p
redo
min
antly
rely
on
Src.
Fib
rilla
r adh
esio
ns a
re k
now
n
to p
lay
a ro
le in
mat
rix re
orga
niza
tion
[Kat
z, Z
amir,
Ber
shad
sky,
Kam
, Yam
ada
& G
eige
r, 00
],
and
it is
bel
ieve
d th
at t
heir
form
atio
n (th
roug
h m
atur
atio
n) i
s de
pend
ent
upon
Src
act
ivity
[Vol
berg
, Rom
er, Z
amir
& G
eige
r, 01
]. S
imila
rly, s
ince
the
α5β1
inte
grin
can
be
loca
lized
with
MT1
-MM
P in
int
erce
llula
r co
ntac
ts [
Gal
vez,
Mat
ias-
Rom
an,
Yan
ez-M
o, S
anch
ez-M
adrid
&
Arr
oyo,
02]
, and
Src
pho
spho
ryla
tion
has
been
pre
viou
sly
show
n to
regu
late
MT1
-MM
P ac
tivity
[Wu,
Gan
, Y
oo &
Gua
n, 0
5]it
is n
ot s
urpr
isin
g th
at i
nhib
ition
of
Src
can
influ
ence
the
mec
hano
sens
itive
upr
egul
atio
n of
MT1
-MM
P fo
llow
ing
stim
ulat
ion.
87
Ana
lysi
s of
the
resu
lts in
dica
ted
that
bot
h th
e α5
β1 in
tegr
in a
nd c
alci
um c
hann
els
exer
t a
sign
ifica
nt in
fluen
ce o
ver c
hond
rocy
tes’
resp
onse
to c
yclic
com
pres
sion
. It i
s pr
obab
le th
at b
oth
inte
grin
s an
d ca
lciu
m
chan
nels
ar
e co
mpo
nent
s of
a
cellu
lar
mec
hano
sens
ing
com
plex
.
Supp
ortin
g th
is n
otio
n is
the
obse
rvat
ion
that
VG
CC
s ar
e kn
own
to c
o-lo
caliz
e w
ith β
1 in
tegr
ins
in m
ouse
lim
b-bu
d ch
ondr
ocyt
es [M
obas
heri,
Car
ter,
Mar
tin-V
asal
lo &
Sha
kiba
ei, 0
2;Sh
akib
aei
& M
obas
heri,
03]
.Ano
ther
stu
dy, a
lbei
t in
vas
cula
r sm
ooth
mus
cle
cells
, has
sho
wn
that
the
α5β1
int
egrin
has
the
abi
lity
to r
egul
ate
the
func
tion
of t
he L
-Typ
e V
GC
C [
Wu,
Dav
is,
Mei
ning
er,
Wils
on &
Dav
is,
01b]
, an
d it
is n
ow k
now
n th
at b
oth
Src
and
Pyk2
(in
tegr
in-
asso
ciat
ed p
rote
ins)
are
abl
e to
bin
d to
the
II-I
II li
nker
and
C-te
rmin
al re
gion
s of
the
α 1C
subu
nit
of th
e L-
Type
VG
CC
and
this
regu
late
its
activ
ity [D
ubui
s, R
ockl
iffe,
Hus
sain
, Boy
ett,
Wra
y &
Gaw
ler,
06].
Furth
erm
ore,
in
our
syst
em,
treat
men
t w
ith t
he A
2318
7 io
noph
ore
man
aged
to
parti
ally
off
set t
he e
ffec
ts o
f Src
inhi
bitio
n by
PP2
furth
er im
plic
atin
g th
e co
nver
genc
e of
thes
e
two
path
way
s. T
hese
dat
a su
gges
t tha
t cal
cium
pla
ys a
maj
or r
egul
ator
y ro
le in
med
iatin
g th
is
resp
onse
, pos
sibl
y to
the
exte
nt o
f lyi
ng u
pstre
am to
α5β
1 an
d re
gula
ting
its a
ctiv
atio
n. T
his
idea
is s
uppo
rted
by t
he o
bser
vatio
n th
at b
lock
ing
calc
ium
sig
nalli
ng c
ompl
etel
y ab
olis
hed
cell
spre
adin
g in
res
pons
e to
mec
hani
cal
stim
ulat
ion,
and
tha
t io
noph
ore
treat
men
t w
as a
ble
to
parti
ally
reve
rse
the
effe
cts
of S
rc in
hibi
tion.
Mor
eove
r, th
e ab
ility
of c
alci
um to
influ
ence
bas
al
leve
ls M
T1-M
MP
expr
essi
on in
uns
timul
ated
sam
ples
furth
er im
plic
ates
cal
cium
in e
xerti
ng th
e
maj
or re
gula
tory
role
in m
edia
ting
chon
droc
ytes
’ res
pons
e. N
onet
hele
ss, t
o fu
lly c
onfir
m th
is, i
t
wou
ld b
e w
orth
whi
le t
o bl
ock
calc
ium
sig
nalli
ng (
i.e.
with
nife
dipi
ne)
and
activ
ate
the
α5β1
com
pone
nt th
roug
h sp
ecifi
c ac
tivat
ing
antib
odie
s. Th
is e
xper
imen
t will
allo
w u
s to
fully
con
firm
whe
ther
cal
cium
pla
ys a
maj
or r
egul
ator
y ro
le e
xerti
ng c
ontro
l ov
er i
nteg
rin a
ctiv
atio
n.
Alte
rnat
ivel
y, a
lthou
gh u
nlik
ely
base
d on
our
res
ults
, the
res
ults
may
indi
cate
that
the
calc
ium
88
activ
atio
n lie
s do
wns
tream
of i
nteg
rin a
ctiv
atio
n. S
uch
sign
allin
g pa
thw
ays
have
bee
n pr
evio
usly
show
n to
exi
st i
n sy
naps
es,
whe
re a
ctiv
atio
n of
Src
by
inte
grin
s ul
timat
ely
regu
late
d th
e
phos
phor
ylat
ion
of t
he N
MD
A c
hann
el [
Lin
et a
l., 0
3].
To f
ully
det
erm
ine
whi
ch o
f th
e
path
way
s is
act
ivat
ed f
irst i
t wou
ld b
e im
pera
tive
to in
vest
igat
e an
d m
easu
re r
eal-t
ime
calc
ium
curr
ents
in re
spon
se to
mec
hani
cal s
timul
atio
n. If
the
calc
ium
cur
rent
wer
e to
be
char
acte
rized
in
this
sys
tem
, it
wou
ld a
llow
us
to i
nhib
it or
kno
ckou
t va
rious
cel
lula
r co
mpo
nent
s an
d to
dete
rmin
e th
eir
rela
tive
orde
r in
the
pat
hway
. Fo
r in
stan
ce,
if in
hibi
ting
α5β1
int
egrin
wou
ld
abol
ish
calc
ium
cur
rent
s in
res
pons
e to
com
pres
sion
tha
t th
at w
ould
be
a de
finiti
ve i
ndic
atio
n
that
inte
grin
act
ivat
ion
trans
pire
s up
stre
am o
f cal
cium
sig
nalli
ng.
The
phos
phor
ylat
ion
of E
RK
,
how
ever
, is
sugg
estiv
e of
a p
aral
lel p
athw
ayw
here
bot
h si
gnal
ling
casc
ades
con
verg
e to
regu
late
ERK
, whi
ch th
en e
xerts
a re
gula
tory
role
on
AP-
1 an
d M
T1-M
MP
expr
essi
on.
In a
dditi
on,
asid
e fr
om p
oten
tially
est
ablis
hing
the
mec
hani
sm b
y w
hich
cho
ndro
cyte
s
sens
e an
d re
spon
d to
the
mec
hani
cal
stim
ulat
ion,
thi
s st
udy
furth
er s
heds
som
e lig
ht o
n th
e
man
ner b
y w
hich
mec
hani
cal s
timul
atio
n re
sults
in in
crea
sed
mat
rix p
rodu
ctio
n. P
revi
ous s
tudi
es
in o
ur l
ab h
ave
indi
cate
d th
at t
he m
inim
um d
urat
ion
of m
echa
nica
l st
imul
atio
n th
at i
s ab
le t
o
prod
uce
an in
crea
se in
mat
rix m
acro
mol
ecul
es w
as 3
0 m
inut
es o
f cyc
lic c
ompr
essi
on [W
aldm
an,
Spite
ri, G
rynp
as, P
illia
r, H
ong
& K
ande
l, 03
;Wal
dman
, Cou
to, G
rynp
as, P
illia
r &
Kan
del,
06].
How
ever
, th
e re
sults
obt
aine
d in
thi
s st
udy
indi
cate
tha
t al
l of
the
pre
viou
sly
char
acte
rized
cruc
ial
even
ts,
nam
ely
cell
spre
adin
g an
d ER
K p
hosp
hory
latio
n, o
ccur
muc
h ea
rlier
int
o th
e
stim
ulat
ion
cycl
e. T
his
obse
rvat
ion
rais
es th
e po
ssib
ility
that
add
ition
al c
ruci
al e
vent
s, un
know
n
as o
f ye
t, th
at m
ust
trans
pire
to
indu
ce i
ncre
ased
acc
umul
atio
n of
mat
rix m
olec
ules
. F
or
inst
ance
, th
e se
cret
ion
of v
ario
us s
igna
lling
che
mic
als,
such
as
ATP
, PT
HrP
, an
d IL
-1 i
n
resp
onse
to
mec
hani
cal
stim
ulat
ion
have
bee
n pr
evio
usly
obs
erve
d in
cho
ndro
cyte
s, an
d
89
inte
rest
ingl
y th
eir
rele
ase
have
bee
n sh
own
to i
nflu
ence
or
be i
nflu
ence
d by
eith
er c
alci
um o
r
inte
grin
rel
ated
sig
nalli
ng [
Mill
war
d-Sa
dler
et a
l., 9
9;M
illw
ard-
Sadl
eret
al.,
04;
Tana
ka, O
hno,
Hon
da, T
anim
oto,
Doi
, Ohn
o-N
akah
ara,
Taf
olla
, Kap
ila &
Tan
ne, 0
5;El
ferv
iget
al.,
01]
. It i
s
poss
ible
that
thes
e m
olec
ules
requ
ire a
long
er s
timul
atio
n pe
riod
befo
re th
ey c
an b
e re
leas
ed a
nd
accu
mul
ate
in a
suf
ficie
nt c
once
ntra
tion
to a
ct i
n an
aut
ocrin
e m
anne
r to
reg
ulat
e th
e ce
ll
resp
onse
. A
st
udy
by
Ione
scu
et
al.,
reve
aled
th
at
PTH
rP
can
mod
ulat
e ch
ondr
ocyt
e
diff
eren
tiatio
n th
roug
h re
gula
tion
of A
P-1
sign
allin
g [I
ones
cuet
al.,
01]
. Si
nce
AP-
1 al
so
regu
late
d M
T1-M
MP
expr
essi
on i
t is
pla
usib
le t
hat
PTH
rP s
igna
lling
is
also
inv
olve
d in
our
syst
em a
nd f
urth
er s
tudi
es s
houl
d be
und
erta
ken
to d
eter
min
e if
and
whe
n th
is s
igna
lling
mol
ecul
eis
sec
rete
d in
resp
onse
to c
yclic
com
pres
sion
. Alte
rnat
ivel
y, s
ince
it is
kno
wn
that
the
stim
ulat
ion-
indu
ced
incr
ease
in m
atrix
acc
umul
atio
n in
volv
es a
cat
abol
ic p
hase
cha
ract
eriz
ed b
y
degr
adat
ion
of th
e su
rrou
ndin
g m
atrix
[De
Cro
os, D
haliw
al, G
rynp
as, P
illia
r & K
ande
l, 06
], it
is
poss
ible
tha
t on
ly 3
0 m
inut
es o
f co
mpr
essi
on p
rodu
ces
a su
ffic
ient
am
ount
of
degr
adat
ion
to
elic
it th
is r
espo
nse.
To
this
end
, st
udie
s de
scrib
ing
the
kine
tics
of M
T1-M
MP
shou
ld b
e
unde
rtake
n in
our
sys
tem
to d
eter
min
e ho
w q
uick
ly a
nd e
ffic
ient
ly is
MT1
-MM
P ac
tivat
ed o
n
the
cell
surf
ace
in re
spon
se to
mec
hani
cal s
timul
atio
n.
Sim
ilarly
, pa
st s
tudi
es i
n ou
r la
b de
term
ined
th
at m
echa
nica
l s
timul
atio
n-in
duce
d
incr
ease
in
mat
rix s
ynth
esis
of
appr
oxim
atel
y 30
% i
n te
rms
of a
ccum
ulat
ion
of c
olla
gens
and
prot
eogl
ycan
s [W
aldm
an, C
outo
, Gry
npas
, Pill
iar &
Kan
del,
06].I
t was
not
cle
ar, h
owev
er, w
hy
the
obse
rved
incr
ease
was
lim
ited
to 3
0%, a
nd it
was
sus
pect
ed th
at th
is c
an b
e at
tribu
ted
to th
e
diff
eren
tial c
ontri
butio
n of
the
zone
s co
mpo
sing
arti
cula
r ca
rtila
ge. W
hen
the
indi
vidu
al z
onal
resp
onse
to c
yclic
com
pres
sion
was
inve
stig
ated
it w
as f
ound
that
thei
r re
spon
se s
till c
ould
not
expl
ain
why
the
ove
rall
incr
ease
in
the
tissu
e is
lim
ited
to t
hat
valu
e [R
aizm
anet
al.,
09]
,
90
sugg
estin
g ot
her f
acto
rs a
re in
volv
ed. T
his
stud
y pr
ovid
ed a
noth
er p
laus
ible
exp
lana
tion
for t
his
phen
omen
on, a
s ob
serv
ed i
n Fi
gure
A6
(App
endi
x A
, Fig
ure
6) w
here
the
dis
tribu
tion
of c
ell
area
s fo
llow
ing
mec
hani
cal
stim
ulat
ion
is s
how
n. A
s se
en i
n th
e fig
ure,
fol
low
ing
mec
hani
cal
stim
ulat
ion
only
a c
erta
in f
ract
ion
of t
he t
otal
cel
ls u
nder
go c
ell
spre
adin
g in
res
pons
e to
the
appl
ied
com
pres
sive
for
ces.
In e
ffec
t ro
ughl
y 30
% o
f th
e ex
amin
ed c
ells
rem
aine
d in
the
ir
sphe
rical
mor
phol
ogy
desp
ite c
yclic
com
pres
sion
. Si
nce
only
cel
ls a
t th
e C
PP i
nter
face
was
anal
yzed
, and
sin
ce c
ell s
prea
ding
app
ears
to in
crea
se w
ith th
e de
pth
of th
e tis
sue
(App
endi
x A
,
Figu
re 7
), it
is q
uite
pos
sibl
e th
at th
e to
tal p
erce
ntag
e of
non
-res
pons
ive
cells
in th
e en
tire
3D
tissu
e is
muc
h hi
gher
(pos
sibl
y as
hig
h as
70%
). A
s su
ch, i
t is
poss
ible
that
the
30%
incr
ease
in
mat
rix s
ynth
esis
is
attri
bute
d to
the
fac
t th
at o
nly
30%
of
the
cells
in
the
entir
e 3D
con
stru
ct
unde
rgo
cell
spre
adin
g an
d re
tract
ion.
Alte
rnat
ivel
y, o
win
g to
the
inh
eren
t ph
enot
ypic
al
diff
eren
ces
that
bet
wee
n ch
ondr
ocyt
es o
f di
ffer
ent
popu
latio
ns, i
t is
fea
sibl
e th
at t
he o
bser
ved
resp
onse
can
be
attri
bute
d to
onl
y a
subs
et o
f th
e ce
ll po
pula
tion
resp
ondi
ng t
o m
echa
nica
l
stim
ulat
ion.
A s
tudy
by
Shie
h et
al.,
indi
cate
d th
at c
hond
rocy
tes
from
the
supe
rfic
ial z
one
are
stiff
er th
an th
e co
rres
pond
ing
mid
dle/
deep
zon
e ch
ondr
ocyt
es, r
aisi
ng th
e po
ssib
ility
thes
e tw
o
subp
opul
atio
ns m
ay n
ot u
nder
go id
entic
al c
hang
es in
cel
l mor
phol
ogy
in re
spon
se to
mec
hani
cal
stim
ulat
ion
[Shi
eh &
Ath
anas
iou,
06]
.
Alth
ough
the
stud
y pr
ovid
ed s
igni
fican
t ins
ight
into
cho
ndro
cyte
mec
hano
trans
duct
ion,
it
poss
esse
d se
vera
l lim
itatio
ns. O
ne l
imita
tion
inhe
rent
in
the
utili
zed
syst
em i
s th
e in
abili
ty t
o
inve
stig
ate
indi
vidu
al c
ell a
rea
thro
ugho
ut th
e en
tire
thic
knes
s of
the
tissu
e. F
or th
e pu
rpos
es o
f
cons
iste
ncy
in m
easu
ring
chon
droc
yte
area
onl
y ce
lls a
t the
CPP
-tiss
ue in
terf
ace
wer
e an
alyz
ed
afte
r pe
elin
g th
e tis
sue
off.
Exam
inat
ion
of t
he e
ntire
tis
sue
by S
EM r
evea
led
a hi
ghly
den
se
tissu
e pa
cked
with
cho
ndro
cyte
s an
d EC
M m
olec
ules
whi
ch d
id n
ot a
llow
for
acc
urat
e
91
mea
sure
men
t of
cell
area
. To
ensu
re th
at th
ere
was
no
sele
ctio
n bi
as in
trodu
ced,
we
exam
ined
the
unde
r-su
rfac
e of
the
deta
ched
tiss
ue. S
prea
d ce
lls, s
imila
r to
that
atta
ched
to th
e su
bstra
te,
wer
e se
en.
As
such
it
is d
iffic
ult
to e
xtra
pola
te w
heth
er t
he r
esul
ts o
btai
ned
appl
y to
all
chon
droc
ytes
or s
impl
y to
asu
bset
of c
hond
rocy
tes
whi
ch a
re lo
cate
d at
the
inte
rfac
e. S
imila
rly,
owin
g to
a v
arie
ty o
f fa
ctor
s bo
th te
chni
cal (
such
as
poro
sity
of
the
CPP
pro
duce
d at
diff
eren
t
times
,) an
d bi
olog
ical
var
iabi
lity
in c
ells
obt
aine
d fr
om d
iffer
ent
calfs
it
is p
ossi
ble
that
a
diff
eren
t am
ount
of m
atrix
was
dep
osite
d by
the
cells
prio
r to
mec
hani
cal s
timul
atio
n. W
hile
the
diff
eren
ce c
ould
not
be
clea
rly s
een
by a
vis
ual
insp
ectio
n, a
ny m
inor
diff
eren
ces
in E
CM
-
amou
nt a
nd c
ompo
sitio
n m
ay i
nflu
ence
the
man
ner
by w
hich
the
cel
ls r
espo
nd t
o m
echa
nica
l
stim
ulat
ion.
For
ins
tanc
e, w
e ha
ve p
revi
ousl
y su
gges
ted
that
the
diff
eren
tial
resp
onse
of
supe
rfic
ial-z
one
and
deep
-zon
e ch
ondr
ocyt
es to
mec
hani
cal s
timul
atio
n ca
n be
attr
ibut
ed to
the
amou
nt o
f EC
M p
rese
nt a
roun
d th
e ce
lls a
t the
tim
e of
the
stim
ulat
ion
[Rai
zman
, De
Cro
os, S
t-
Pier
re,
Pilli
ar &
Kan
del,
09].
In t
hat
stud
y, w
e ob
serv
ed t
hat
the
resp
onse
of
deep
zon
e
chon
droc
ytes
to c
yclic
com
pres
sion
, in
term
s of
mat
rix s
ynth
esis
, var
ied
dras
tical
ly in
con
stru
cts
stim
ulat
ed 4
8 ho
ur a
part.
The
diff
eren
ce in
the
resp
onse
was
attr
ibut
ed to
a s
igni
fican
tly g
reat
er
accu
mul
atio
n of
EC
M m
olec
ules
in c
onst
ruct
s th
at w
ere
stim
ulat
ed a
t the
late
r tim
e (f
or fu
rther
info
rmat
ion
refe
r to
pape
r atta
ched
in A
ppen
dix
B).
This
obs
erva
tion
is in
kee
ping
with
pre
viou
s
rese
arch
that
indi
cate
d th
at th
e co
mpo
sitio
n of
the
ECM
, and
in p
artic
ular
of t
he P
CM
, inf
luen
ces
the
resp
onsi
vene
ss
of
chon
droc
ytes
[G
uila
k,
Ale
xopo
ulos
, H
aide
r, Ti
ng-B
eall
&
Setto
n,
05;G
uila
k, A
lexo
poul
os, U
pton
, You
n, C
hoi,
Cao
, Set
ton
& H
aide
r, 06
]. M
oreo
ver,
owin
g to
the
diff
eren
ces i
n th
e re
lativ
e ab
unda
nce
of c
hond
rocy
tes f
rom
diff
eren
t zon
e, w
hich
is a
resu
lt of
the
chon
droc
yte
extra
ctio
n pr
oces
s, th
e co
mpo
sitio
n of
the
ECM
may
diff
er sl
ight
ly fr
om e
xper
imen
t
to e
xper
imen
t. A
s su
ch, t
he p
artic
ular
resp
onse
we
are
seei
ng c
ould
be
depe
nden
t on
the
amou
nt
92
of m
atrix
, and
that
mec
hani
cal s
timul
atio
n co
nduc
ted
unde
r di
ffer
ent c
ondi
tions
(i.e
. lat
er ti
me
poin
t) m
ay re
sult
in th
e ac
tivat
ion
of o
ther
com
pone
nts o
f the
sign
allin
g pa
thw
ay.
In a
dditi
on, i
t wou
ld b
e us
eful
for
the
purp
oses
of
eluc
idat
ing
the
mec
hani
sm r
egul
atin
g
chon
droc
yte
resp
onse
to
cycl
ic c
ompr
essi
on,
to v
isua
lize
real
-tim
e ca
lciu
m f
luxe
s. N
umer
ous
tech
nolo
gies
and
dye
s ex
ist f
or th
is p
urpo
se a
nd a
re w
idel
y us
ed in
lite
ratu
re in
sin
gle-
cell
and
mon
olay
er c
ultu
res.
How
ever
, the
se m
etho
dsar
e no
t app
licab
le to
our
sys
tem
, as
the
tech
nica
l
hurd
les
of m
echa
nica
lly s
timul
atin
g a
3D ti
ssue
con
stru
ct in
side
a m
echa
nica
l stim
ulat
or p
reve
nt
us f
rom
vis
ualiz
ing
the
cells
in
real
-tim
e.
Non
ethe
less
, as
men
tione
d ab
ove,
des
igni
ng a
n
appa
ratu
s th
at w
ill a
llow
the
visu
aliz
atio
n of
cal
cium
flux
es in
real
-tim
e w
ould
be
trem
endo
usly
usef
ul in
atte
mpt
ing
to d
eter
min
e th
e re
lativ
e or
der o
f int
egrin
and
cal
cium
-cha
nnel
act
ivat
ion
in
resp
onse
to c
yclic
com
pres
sion
.
Som
e of
the
lim
itatio
ns m
entio
ned
abov
e ar
e a
dire
ct c
onse
quen
ce o
f th
e 3D
cul
ture
syst
em u
tiliz
ed i
n ou
r la
b. O
ther
stu
dies
whi
ch a
re c
ondu
cted
util
izin
g sy
stem
s su
ch a
s
mon
olay
er, p
elle
t cu
lture
s, ca
rtila
ge e
xpla
nts
or e
ven
3D c
ultu
res
utili
zing
aga
rose
or
algi
nate
allo
w f
or a
gre
ater
ana
lysi
s w
ith a
wid
er a
rray
of
tool
s du
e to
the
eas
ier
tech
nica
l as
pect
s
asso
ciat
ed w
ith t
he c
ultu
res
(i.e.
vis
ualiz
atio
n of
rea
l-tim
e ca
lciu
m c
urre
nts
in m
onol
ayer
).
How
ever
, non
e of
thes
e sy
stem
s ar
e ab
le to
gen
erat
e co
ntin
uous
laye
r of
tiss
ue to
mor
e cl
osel
y
mim
ic w
hat i
s oc
curr
ing
in a
nat
ural
in v
ivo
envi
ronm
ent w
here
cho
ndro
cyte
s ar
e su
rrou
nded
by
a sp
ecia
lized
EC
M m
atrix
and
are
situ
ated
on
top
of a
stif
f mat
eria
l (bo
ne).
As
such
, des
pite
the
limite
d ty
pe o
f ana
lysi
s w
hich
can
be
unde
rtake
n in
our
sys
tem
, thi
s sy
stem
pro
vide
s a
far m
ore
real
istic
mod
el,
and
mor
e a
phys
iolo
gica
lly a
ccur
ate
envi
ronm
ent,
to s
tudy
and
inv
estig
ate
chon
droc
yte
resp
onse
to c
ompr
essi
ve fo
rces
that
lead
to e
nhan
ced
tissu
e fo
rmat
ion.
93
Ow
ing
to th
ese
limita
tions
, oth
er e
xper
imen
ts sh
ould
be
cond
ucte
d in
the
futu
re to
furth
er
stud
y th
e in
volv
ed m
echa
nism
s. Fi
rstly
, it
wou
ld b
e im
porta
nt t
o se
lect
ivel
y kn
ock-
out
the
invo
lved
cal
cium
cha
nnel
s an
d in
tegr
ins
(i.e.
thro
ugh
the
use
of s
iRN
A) t
o fu
rther
con
firm
thei
r
invo
lvem
ent
in t
his
proc
ess.
Sim
ilarly
, it
wou
ld b
e in
tere
stin
g to
det
erm
ine
if ot
her
inte
grin
-
asso
ciat
ed p
rote
ins m
ay b
e in
volv
ed in
this
resp
onse
as w
ell a
nd, i
f so,
wha
t is t
heir
func
tion;
it is
entir
ely
poss
ible
tha
t di
ffer
ent
adap
ter
prot
eins
may
be
calle
d up
on d
epen
ding
on
the
type
of
forc
e ch
ondr
ocyt
es se
nse.
Add
ition
al s
tudi
es s
houl
d be
con
duct
ed t
o in
vest
igat
e w
hich
of
the
calc
ium
-dep
ende
nt
path
way
s ar
e ac
tivat
ed b
y th
e ca
lciu
m c
hann
els
in re
spon
se to
mec
hani
cal s
timul
atio
n. T
his
will
indi
cate
whe
ther
cal
cium
is in
volv
ed in
sim
ply
med
iatin
g th
e cy
tosk
elet
al re
arra
ngem
ent (
whi
ch
is c
alci
um d
epen
dent
) or
dire
ctly
inf
luen
ces
gene
tra
nsla
tion
thro
ugh
prot
eins
suc
h as
PK
C.
Mor
eove
r, it
wou
ld b
e im
porta
nt to
inve
stig
ate
the
poss
ible
invo
lvem
ent o
f oth
er io
nic
chan
nels
such
as
the
sodi
um a
nd p
otas
sium
cha
nnel
s, as
thes
e ch
anne
ls a
re a
lso
know
n to
be
invo
lved
in
mec
hano
trans
duct
ion
in o
ther
cel
l typ
es a
nd c
an c
o-lo
caliz
ed w
ith in
tegr
ins a
s wel
l.
Ove
rall,
an
in-d
epth
und
erst
andi
ng o
f the
mol
ecul
ar m
echa
nism
s inv
olve
d in
cho
ndro
cyte
mec
hano
trans
duct
ion,
in p
artic
ular
thos
e pa
thw
ays
whi
ch a
re r
espo
nsib
le f
or th
e sy
nthe
sis
and
mai
nten
ance
of
the
tissu
e’s
ECM
, w
ill c
ontri
bute
to
our
unde
rsta
ndin
g of
var
ious
car
tilag
e-
rela
ted
path
olog
ies
such
as
oste
oarth
ritis
. Mor
eove
r, kn
owin
g th
e un
derly
ing
mec
hani
sms
may
lead
to th
e de
velo
pmen
t of
new
stra
tegi
es f
or tr
eatm
ent o
f da
mag
ed c
artil
age
tissu
e th
roug
h th
e
use
of b
ioen
gine
erin
g ap
proa
ches
.
94
CH
APT
ER
FO
UR
: RE
FER
EN
CE
SR
efer
ence
s
Ale
ngha
t FJ,
Ingb
er D
E. M
echa
notra
nsdu
ctio
n: a
ll si
gnal
s poi
nt to
cyt
oske
leto
n, m
atrix
, and
in
tegr
ins.
Sci.S
TKE.
2002
a; 2
002:
E6.
Ale
ngha
t FJ,
Ingb
er D
E. M
echa
notra
nsdu
ctio
n: a
ll si
gnal
s poi
nt to
cyt
oske
leto
n, m
atrix
, and
in
tegr
ins.
Sci.S
TKE.
2002
b; 2
002:
E6.
Ale
xopo
ulos
LG
, Set
ton
LA, G
uila
k F.
The
bio
mec
hani
cal r
ole
of th
e ch
ondr
ocyt
e pe
ricel
lula
r m
atrix
in a
rticu
lar c
artil
age.
Act
a Bi
omat
er.2
005;
1:3
17-3
25.
Alfo
rd A
I, Y
ello
wle
y C
E, Ja
cobs
CR
, Don
ahue
HJ.
Incr
ease
s in
cyto
solic
cal
cium
, but
not
flui
d flo
w,a
ffec
t agg
reca
n m
RN
A le
vels
in a
rticu
lar c
hond
rocy
tes.
J.C
ell B
ioch
em.2
003;
90:
938-
944.
Alin
i M, C
arey
D, H
irata
S, G
rynp
as M
D, P
idou
x I,
Pool
e A
R. C
ellu
lar a
nd m
atrix
cha
nges
be
fore
and
at t
he ti
me
of c
alci
ficat
ion
in th
e gr
owth
pla
te st
udie
d in
vitr
o: a
rres
t of t
ype
X
colla
gen
synt
hesi
s and
net
loss
of c
olla
gen
whe
n ca
lcifi
catio
n is
initi
ated
. J.B
one
Min
er.R
es.
1994
; 9:1
077-
1087
.
Arn
aout
MA
, Mah
alin
gam
B, X
iong
JP. I
nteg
rin st
ruct
ure,
allo
ster
y, a
nd b
idire
ctio
nal s
igna
ling.
An
nu.R
ev.C
ell D
ev.B
iol.
2005
;21:
381-
410.
Bal
lest
rem
C, H
inz
B, I
mho
f BA
, Weh
rle-H
alle
r B. M
arch
ing
at th
e fr
ont a
nd d
ragg
ing
behi
nd:
diff
eren
tial a
lpha
Vbe
ta3-
inte
grin
turn
over
regu
late
s foc
al a
dhes
ion
beha
vior
. J.C
ell B
iol.
2001
; 15
5:13
19-1
332.
Bea
upre
GS,
Ste
vens
SS,
Car
ter D
R. M
echa
nobi
olog
y in
the
deve
lopm
ent,
mai
nten
ance
, and
de
gene
ratio
n of
arti
cula
r car
tilag
e. J
.Reh
abil.
Res.D
ev.2
000;
37:
145-
151.
Ben
ingo
KA
, Dem
bo M
, Wan
g Y
L. R
espo
nses
of f
ibro
blas
ts to
anc
hora
ge o
f dor
sal e
xtra
cellu
lar
mat
rix re
cept
ors.
Proc
.Nat
l.Aca
d.Sc
i.U.S
.A20
04; 1
01:1
8024
-180
29.
Ber
is A
E, L
ykis
sas M
G, P
apag
eorg
iou
CD
, Geo
rgou
lis A
D. A
dvan
ces i
n ar
ticul
ar c
artil
age
repa
ir. In
jury
2005
; 36
Supp
l 4:S
14-S
23.
Ber
ridge
MJ,
Boo
tman
MD
, Rod
eric
k H
L. C
alci
um si
gnal
ling:
dyn
amic
s, ho
meo
stas
is a
nd
rem
odel
ling.
Nat
.Rev
.Mol
.Cel
l Bio
l.20
03; 4
:517
-529
.
Ber
ridge
MJ,
Lipp
P, B
ootm
an M
D. T
he v
ersa
tility
and
uni
vers
ality
of c
alci
um si
gnal
ling.
N
at.R
ev.M
ol.C
ell B
iol.
2000
; 1:1
1-21
.
Ber
rier A
L, Y
amad
a K
M. C
ell-m
atrix
adh
esio
n. J
.Cel
l Phy
siol
2007
; 213
:565
-573
.
95
Bid
anse
t DJ,
Gui
dry
C, R
osen
berg
LC
, Cho
i HU
, Tim
pl R
, Hoo
k M
. Bin
ding
of t
he
prot
eogl
ycan
dec
orin
to c
olla
gen
type
VI.
J.Bi
ol.C
hem
.199
2; 2
67:5
250-
5256
.
Boi
leau
C, M
arte
l-Pel
letie
r J, B
rune
t J e
t al.
Ora
l tre
atm
ent w
ith P
D-0
2003
47, a
n al
pha2
delta
lig
and,
redu
ces t
he d
evel
opm
ent o
f exp
erim
enta
l ost
eoar
thrit
is b
y in
hibi
ting
met
allo
prot
eina
ses
and
indu
cibl
e ni
tric
oxid
e sy
ntha
se g
ene
expr
essi
on a
nd sy
nthe
sis i
n ca
rtila
ge c
hond
rocy
tes.
Arth
ritis
Rhe
um.2
005;
52:
488-
500.
Bon
en D
K, S
chm
id T
M. E
leva
ted
extra
cellu
lar c
alci
um c
once
ntra
tions
indu
ce ty
pe X
col
lage
n sy
nthe
sis i
n ch
ondr
ocyt
e cu
lture
s. J.
Cel
l Bio
l.19
91; 1
15:1
171-
1178
.
Boo
tman
MD
, Col
lins T
J, Pe
ppia
tt C
M e
t al.
Cal
cium
sign
allin
g--a
n ov
ervi
ew. S
emin
.Cel
l D
ev.B
iol.
2001
; 12:
3-10
.
Boy
le J,
Lua
n B
, Cru
z TF
, Kan
del R
A. C
hara
cter
izat
ion
of p
rote
ogly
can
accu
mul
atio
n du
ring
form
atio
n of
car
tilag
enou
s tis
sue
in v
itro.
Ost
eoar
thri
tis.C
artil
age.
1995
; 3:1
17-1
25.
Bra
dley
JM, K
elle
y M
J, Zh
u X
, And
erss
ohn
AM
, Ale
xand
er JP
, Aco
tt TS
. Eff
ects
of m
echa
nica
l st
retc
hing
on
trabe
cula
r mat
rix m
etal
lopr
otei
nase
s. In
vest
Oph
thal
mol
.Vis
.Sci
.200
1; 4
2:15
05-
1513
.
Bra
kebu
sch
C, F
assl
er R
. bet
a 1
inte
grin
func
tion
in v
ivo:
adh
esio
n, m
igra
tion
and
mor
e. C
ance
r M
etas
tasi
s Rev
.200
5; 2
4:40
3-41
1.
Bre
inan
HA
, Min
as T
, Hsu
HP,
Neh
rer S
, Sle
dge
CB
, Spe
ctor
M. E
ffec
t of c
ultu
red
auto
logo
us
chon
droc
ytes
on
repa
ir of
cho
ndra
l def
ects
in a
can
ine
mod
el. J
.Bon
e Jo
int S
urg.
Am.1
997;
79
:143
9-14
51.
Bro
wer
TD
, Hsu
WY
. Nor
mal
arti
cula
r car
tilag
e. C
lin.O
rtho
p.Re
lat R
es.1
969;
64:
9-17
.
Bru
gner
a E,
Han
ey L
, Grim
sley
C e
t al.
Unc
onve
ntio
nal R
ac-G
EF a
ctiv
ity is
med
iate
d th
roug
h th
e D
ock1
80-E
LMO
com
plex
. Nat
.Cel
l Bio
l.20
02; 4
:574
-582
.
Buc
kwal
ter J
A. O
steo
arth
ritis
and
arti
cula
r car
tilag
e us
e, d
isus
e, a
nd a
buse
: exp
erim
enta
l stu
dies
. J.
Rheu
mat
ol.S
uppl
1995
; 43:
13-1
5.
Buc
kwal
ter J
A, M
anki
n H
J. A
rticu
lar c
artil
age:
tiss
ue d
esig
n an
d ch
ondr
ocyt
e-m
atrix
in
tera
ctio
ns. I
nstr
.Cou
rse
Lect
.199
8a; 4
7:47
7-48
6.
Buc
kwal
ter J
A, M
anki
n H
J. A
rticu
lar c
artil
age:
deg
ener
atio
n an
d os
teoa
rthrit
is, r
epai
r, re
gene
ratio
n, a
nd tr
ansp
lant
atio
n. In
str.C
ours
e Le
ct.1
998b
; 47:
487-
504.
Cal
alb
MB
, Zha
ng X
, Pol
te T
R, H
anks
SK
. Foc
al a
dhes
ion
kina
se ty
rosi
ne-8
61 is
a m
ajor
site
of
phos
phor
ylat
ion
by S
rc. B
ioch
em.B
ioph
ys.R
es.C
omm
un.1
996;
228
:662
-668
.
Cam
pbel
l JJ,
Bla
in E
J, C
how
dhur
y TT
, Kni
ght M
M. L
oadi
ng a
lters
act
in d
ynam
ics a
nd u
p-re
gula
tes c
ofili
n ge
ne e
xpre
ssio
n in
cho
ndro
cyte
s. Bi
oche
m.B
ioph
ys.R
es.C
omm
un.2
007;
36
1:32
9-33
4.
96
Cat
tera
ll W
A, P
erez
-Rey
es E
, Snu
tch
TP, S
tries
snig
J. In
tern
atio
nal U
nion
of P
harm
acol
ogy.
X
LVII
I. N
omen
clat
ure
and
stru
ctur
e-fu
nctio
n re
latio
nshi
ps o
f vol
tage
-gat
ed c
alci
um c
hann
els.
Phar
mac
ol.R
ev.2
005;
57:
411-
425.
Cav
alca
nti-A
dam
EA
, Vol
berg
T, M
icou
let A
, Kes
sler
H, G
eige
r B, S
patz
JP. C
ell s
prea
ding
and
fo
cal a
dhes
ion
dyna
mic
s are
regu
late
d by
spac
ing
of in
tegr
in li
gand
s. Bi
ophy
s.J.2
007;
92:
2964
-29
74.
Cha
ng J,
Nak
ajim
a H
, Poo
le C
A. S
truct
ural
col
ocal
isat
ion
of ty
pe V
I col
lage
n an
d fib
rone
ctin
in
agar
ose
cultu
red
chon
droc
ytes
and
isol
ated
cho
ndro
ns e
xtra
cted
from
adu
lt ca
nine
tibi
al
carti
lage
. J.A
nat.
1997
; 190
( Pt 4
):523
-532
.
Chu
n TH
, Hot
ary
KB
, Sab
eh F
, Sal
tiel A
R, A
llen
ED, W
eiss
SJ.
A p
eric
ellu
lar c
olla
gena
se
dire
cts t
he 3
-dim
ensi
onal
dev
elop
men
t of w
hite
adi
pose
tiss
ue. C
ell2
006;
125
:577
-591
.
Coh
en M
, Kam
Z, A
ddad
i L, G
eige
r B. D
ynam
ic st
udy
of th
e tra
nsiti
on fr
om h
yalu
rona
n-to
in
tegr
in-m
edia
ted
adhe
sion
in c
hond
rocy
tes.
EMBO
J.2
006;
25:
302-
311.
Coh
en M
, Kle
in E
, Gei
ger B
, Add
adi L
. Org
aniz
atio
n an
d ad
hesi
ve p
rope
rties
of t
he h
yalu
rona
n pe
ricel
lula
r coa
t of c
hond
rocy
tes a
nd e
pith
elia
l cel
ls. B
ioph
ys.J
.200
3; 8
5:19
96-2
005.
Col
lins T
J, Li
pp P
, Ber
ridge
MJ,
Boo
tman
MD
. Mito
chon
dria
l Ca(
2+) u
ptak
e de
pend
s on
the
spat
ial a
nd te
mpo
ral p
rofil
e of
cyt
osol
ic C
a(2+
) sig
nals
. J.B
iol.C
hem
.200
1; 2
76:2
6411
-264
20.
Cou
tts R
D, H
eale
y R
M, O
stra
nder
R, S
ah R
L, G
oom
er R
, Am
iel D
. Mat
rices
for c
artil
age
repa
ir.
Clin
.Ort
hop.
Rela
t Res
.200
1;S2
71-S
279.
Cou
tts R
D, S
ah R
L, A
mie
l D. E
ffec
ts o
f gro
wth
fact
ors o
n ca
rtila
ge re
pair.
Inst
r.Cou
rse
Lect
.19
97; 4
6:48
7-49
4.
Cra
wfo
rd H
C, M
atris
ian
LM. M
echa
nism
s con
trolli
ng th
e tra
nscr
iptio
n of
mat
rix
met
allo
prot
eina
se g
enes
in n
orm
al a
nd n
eopl
astic
cel
ls. E
nzym
e Pr
otei
n19
96; 4
9:20
-37.
Cre
mer
MA
, Ros
loni
ec E
F, K
ang
AH
. The
car
tilag
e co
llage
ns: a
revi
ew o
f the
ir st
ruct
ure,
or
gani
zatio
n, a
nd ro
le in
the
path
ogen
esis
of e
xper
imen
tal a
rthrit
is in
anim
als a
nd in
hum
an
rheu
mat
ic d
isea
se. J
.Mol
.Med
.199
8; 7
6:27
5-28
8.
Cuk
ierm
an E
, Pan
kov
R, S
teve
ns D
R, Y
amad
a K
M. T
akin
g ce
ll-m
atrix
adh
esio
ns to
the
third
di
men
sion
. Sci
ence
2001
; 294
:170
8-17
12.
D'A
ndre
a P,
Cal
abre
se A
, Cap
ozzi
I, G
rand
olfo
M, T
onon
R, V
ittur
F. I
nter
cellu
lar C
a2+
wav
es
in m
echa
nica
lly st
imul
ated
arti
cula
r cho
ndro
cyte
s. Bi
orhe
olog
y20
00; 3
7:75
-83.
Dan
en E
H, S
onne
veld
P, B
rake
busc
h C
, Fas
sler
R, S
onne
nber
g A
. The
fibr
onec
tin-b
indi
ng
inte
grin
s alp
ha5b
eta1
and
alp
havb
eta3
diff
eren
tially
mod
ulat
e R
hoA
-GTP
load
ing,
org
aniz
atio
n of
cel
l mat
rix a
dhes
ions
, and
fibr
onec
tin fi
brill
ogen
esis
. J.C
ell B
iol.
2002
; 159
:107
1-10
86.
97
Dar
ling
EM, A
than
asio
u K
A. R
apid
phe
noty
pic
chan
ges i
n pa
ssag
ed a
rticu
lar c
hond
rocy
te
subp
opul
atio
ns. J
.Ort
hop.
Res.
2005
; 23:
425-
432.
Dar
ling
EM, A
than
asio
u K
A. B
iom
echa
nica
l stra
tegi
es fo
r arti
cula
r car
tilag
e re
gene
ratio
n.
Ann.
Biom
ed.E
ng20
03; 3
1:11
14-1
124.
Dar
ling
EM, H
u JC
, Ath
anas
iou
KA
. Zon
al a
nd to
pogr
aphi
cal d
iffer
ence
s in
artic
ular
car
tilag
e ge
ne e
xpre
ssio
n. J
.Ort
hop.
Res.
2004
; 22:
1182
-118
7.
Dav
isso
n T,
Kun
ig S
, Che
n A
, Sah
R, R
atcl
iffe
A. S
tatic
and
dyn
amic
com
pres
sion
mod
ulat
e m
atrix
met
abol
ism
in ti
ssue
eng
inee
red
carti
lage
. J.O
rtho
p.Re
s.20
02; 2
0:84
2-84
8.
De
Cro
os JN
, Dha
liwal
SS,
Gry
npas
MD
, Pill
iar R
M, K
ande
l RA
. Cyc
lic c
ompr
essi
ve
mec
hani
cal s
timul
atio
n in
duce
s seq
uent
ial c
atab
olic
and
ana
bolic
gen
e ch
ange
s in
chon
droc
ytes
re
sulti
ng in
incr
ease
d ex
trace
llula
r mat
rix a
ccum
ulat
ion.
Mat
rix
Biol
.200
6; 2
5:32
3-33
1.
De
Cro
os JN
, Jan
g B
, Dha
liwal
SS,
Gry
npas
MD
, Pill
iar R
M,K
ande
l RA
. Mem
bran
e ty
pe-1
m
atrix
met
allo
prot
eina
se is
indu
ced
follo
win
g cy
clic
com
pres
sion
of i
n vi
tro g
row
n bo
vine
ch
ondr
ocyt
es. O
steo
arth
ritis
.Car
tilag
e.20
07; 1
5:13
01-1
310.
Des
hmuk
h K
, Saw
yer B
D. S
ynth
esis
of c
olla
gen
by c
hond
rocy
tes i
n su
spen
sion
cul
ture
: m
odul
atio
n by
cal
cium
, 3':5
'-cyc
lic A
MP,
and
pro
stag
land
ins.
Proc
.Nat
l.Aca
d.Sc
i.U.S
.A19
77;
74:3
864-
3868
.
Dow
thw
aite
GP,
Bis
hop
JC, R
edm
an S
N e
t al.
The
surf
ace
of a
rticu
lar c
artil
age
cont
ains
a
prog
enito
r cel
l pop
ulat
ion.
J.C
ell S
ci.2
004;
117
:889
-897
.
Dub
uis E
, Roc
kliff
e N
, Hus
sain
M, B
oyet
t M, W
ray
D, G
awle
r D. E
vide
nce
for m
ultip
le S
rc
bind
ing
site
s on
the
alph
a1c
L-ty
pe C
a2+
chan
nel a
nd th
eir r
oles
in a
ctiv
ity re
gula
tion.
C
ardi
ovas
c.Re
s.20
06; 6
9:39
1-40
1.
Dud
ek S
M, G
arci
a JG
. Cyt
oske
leta
l reg
ulat
ion
of p
ulm
onar
y va
scul
ar p
erm
eabi
lity.
J.
Appl
.Phy
siol
2001
; 91:
1487
-150
0.
Dur
rant
LA
, Arc
her C
W, B
enja
min
M, R
alph
s JR
. Org
anis
atio
n of
the
chon
droc
yte
cyto
skel
eton
an
d its
resp
onse
to c
hang
ing
mec
hani
cal c
ondi
tions
in o
rgan
cul
ture
. J.A
nat.
1999
; 194
( Pt
3)
:343
-353
.
Ecks
tein
F, H
udel
mai
er M
, Put
z R
. The
eff
ects
of e
xerc
ise
on h
uman
arti
cula
r car
tilag
e. J
.Ana
t.20
06; 2
08:4
91-5
12.
Elfe
rvig
MK
, Gra
ff R
D, L
ee G
M, K
elle
y SS
, Soo
d A
, Ban
es A
J. A
TP in
duce
s Ca(
2+) s
igna
ling
in h
uman
cho
ndro
ns c
ultu
red
in th
ree-
dim
ensi
onal
aga
rose
film
s. O
steo
arth
ritis
.Car
tilag
e.20
01;
9:51
8-52
6.
Eric
kson
GR
, Nor
thru
p D
L, G
uila
k F.
Hyp
o-os
mot
ic st
ress
indu
ces c
alci
um-d
epen
dent
act
in
reor
gani
zatio
n in
arti
cula
r cho
ndro
cyte
s. O
steo
arth
ritis
.Car
tilag
e.20
03; 1
1:18
7-19
7.
98
Eyre
DR
. Col
lage
ns a
nd c
artil
age
mat
rix h
omeo
stas
is. C
lin.O
rtho
p.Re
lat R
es.2
004;
S118
-S12
2.
Fant
ozzi
I, Z
hang
S, P
lato
shyn
O, R
emill
ard
CV
, Cow
ling
RT,
Yua
n JX
. Hyp
oxia
incr
ease
s AP-
1 bi
ndin
g ac
tivity
by
enha
ncin
g ca
paci
tativ
e C
a2+
entry
in h
uman
pul
mon
ary
arte
ry e
ndot
helia
l ce
lls. A
m.J
.Phy
siol
Lun
g C
ell M
ol.P
hysi
ol20
03; 2
85:L
1233
-L12
45.
Flei
re S
J, G
oldm
an JP
, Car
rasc
o Y
R, W
eber
M, B
ray
D, B
atis
ta F
D. B
cel
l lig
and
disc
rimin
atio
n th
roug
h a
spre
adin
g an
d co
ntra
ctio
n re
spon
se. S
cien
ce20
06; 3
12:7
38-7
41.
Gal
vez
BG
, Mat
ias-
Rom
an S
, Yan
ez-M
o M
, San
chez
-Mad
rid F
, Arr
oyo
AG
. EC
M re
gula
tes
MT1
-MM
P lo
caliz
atio
n w
ith b
eta1
or a
lpha
vbet
a3 in
tegr
ins a
t dis
tinct
cel
l com
partm
ents
m
odul
atin
g its
inte
rnal
izat
ion
and
activ
ity o
n hu
man
end
othe
lial c
ells
. J.C
ell B
iol.
2002
; 15
9:50
9-52
1.
Gel
se K
, Pos
chl E
, Aig
ner T
. Col
lage
ns--
stru
ctur
e, fu
nctio
n, a
nd b
iosy
nthe
sis.
Adv.
Dru
g D
eliv
.Rev
.200
3; 5
5:15
31-1
546.
Gen
azza
ni A
A, G
alio
ne A
. A C
a2+
rele
ase
mec
hani
sm g
ated
by
the
nove
l pyr
idin
e nu
cleo
tide,
N
AA
DP.
Tre
nds P
harm
acol
.Sci
.199
7; 1
8:10
8-11
0.
Gill
ogly
SD
, Voi
ght M
, Bla
ckbu
rn T
. Tre
atm
ent o
f arti
cula
r car
tilag
e de
fect
s of t
he k
nee
with
au
tolo
gous
cho
ndro
cyte
impl
anta
tion.
J.O
rtho
p.Sp
orts
Phy
s.The
r.19
98; 2
8:24
1-25
1.
Gom
ez E
, Prit
char
d C
, Her
bert
TP. c
AM
P-de
pend
ent p
rote
in k
inas
e an
d C
a2+
influ
x th
roug
h L-
type
vol
tage
-gat
ed c
alci
um c
hann
els m
edia
te R
af-in
depe
nden
t act
ivat
ion
of e
xtra
cellu
lar
regu
late
d ki
nase
in re
spon
se to
glu
cago
n-lik
e pe
ptid
e-1
in p
ancr
eatic
bet
a-ce
lls. J
.Bio
l.Che
m.
2002
; 277
:481
46-4
8151
.
Gra
ff R
D, K
elle
y SS
, Lee
GM
. Rol
e of
per
icel
lula
r mat
rix in
dev
elop
men
t of a
mec
hani
cally
fu
nctio
nal n
eoca
rtila
ge. B
iote
chno
l.Bio
eng.
2003
; 82:
457-
464.
Gro
dzin
sky
AJ,
Leve
nsto
n M
E, Ji
n M
, Fra
nk E
H. C
artil
age
tissu
e re
mod
elin
g in
resp
onse
to
mec
hani
cal f
orce
s. An
nu.R
ev.B
iom
ed.E
ng20
00; 2
:691
-713
.
Gry
npas
MD
, Pill
iar R
M, K
ande
l RA
, Ren
lund
R, F
iliag
gi M
, Dum
itriu
M. P
orou
s cal
cium
po
lyph
osph
ate
scaf
fold
s for
bon
e su
bstit
ute
appl
icat
ions
in v
ivo
stud
ies.
Biom
ater
ials
2002
; 23
:206
3-20
70.
Gui
bert
C, D
ucre
t T, S
avin
eau
JP. V
olta
ge-in
depe
nden
t cal
cium
influ
x in
smoo
th m
uscl
e.
Prog
.Bio
phys
.Mol
.Bio
l.20
08; 9
8:10
-23.
Gui
lak
F. C
ompr
essi
on-in
duce
d ch
ange
s in
the
shap
e an
d vo
lum
e of
the
chon
droc
yte
nucl
eus.
J.Bi
omec
h.19
95; 2
8:15
29-1
541.
Gui
lak
F, A
lexo
poul
os L
G, H
aide
r MA
, Tin
g-B
eall
HP,
Set
ton
LA. Z
onal
uni
form
ity in
m
echa
nica
l pro
perti
es o
f the
cho
ndro
cyte
per
icel
lula
r mat
rix: m
icro
pipe
tte a
spira
tion
of c
anin
e ch
ondr
ons i
sola
ted
by c
artil
age
hom
ogen
izat
ion.
Ann
.Bio
med
.Eng
2005
; 33:
1312
-131
8.
99
Gui
lak
F, A
lexo
poul
os L
G, U
pton
ML
et a
l. Th
e pe
ricel
lula
r mat
rix a
s a tr
ansd
ucer
of
biom
echa
nica
l and
bio
chem
ical
sign
als i
n ar
ticul
ar c
artil
age.
Ann
.N.Y
.Aca
d.Sc
i.20
06; 1
068:
498-
512.
Gui
lak
F, Z
ell R
A, E
ricks
on G
R e
t al.
Mec
hani
cally
indu
ced
calc
ium
wav
es in
arti
cula
r ch
ondr
ocyt
es a
re in
hibi
ted
by g
adol
iniu
m a
nd a
milo
ride.
J.O
rtho
p.Re
s.19
99; 1
7:42
1-42
9.
Hal
l AC
, Hor
witz
ER
, Wilk
ins R
J. Th
e ce
llula
r phy
siol
ogy
of a
rticu
lar c
artil
age.
Exp
.Phy
siol
1996
; 81:
535-
545.
Han
ke JH
, Gar
dner
JP, D
ow R
L et
al.
Dis
cove
ry o
f a n
ovel
, pot
ent,
and
Src
fam
ily-s
elec
tive
tyro
sine
kin
ase
inhi
bito
r. St
udy
of L
ck-a
nd F
ynT-
depe
nden
t T c
ell a
ctiv
atio
n. J
.Bio
l.Che
m.
1996
; 271
:695
-701
.
Hau
selm
ann
HJ,
Stef
anov
ic-R
acic
M, M
iche
l BA
, Eva
ns C
H. D
iffer
ence
s in
nitri
c ox
ide
prod
uctio
n by
supe
rfic
ial a
nd d
eep
hum
an a
rticu
lar c
hond
rocy
tes:
impl
icat
ions
for p
rote
ogly
can
turn
over
in in
flam
mat
ory
join
t dis
ease
s. J.
Imm
unol
.199
8; 1
60:1
444-
1448
.
Hid
aka
C, C
heng
C, A
lexa
ndre
D, B
harg
ava
M, T
orzi
lli P
A. M
atur
atio
nal d
iffer
ence
s in
supe
rfic
ial a
nd d
eep
zone
arti
cula
r cho
ndro
cyte
s. C
ell T
issu
e Re
s.20
06; 3
23:1
27-1
35.
Hol
ledg
e M
M, M
illw
ard-
Sadl
er S
J, N
uki G
, Sal
ter D
M. M
echa
nica
l reg
ulat
ion
of p
rote
ogly
can
synt
hesi
s in
norm
al a
nd o
steo
arth
ritic
hum
an a
rticu
lar c
hond
rocy
tes-
-rol
es fo
r alp
ha5
and
alph
aVbe
ta5
inte
grin
s. Bi
orhe
olog
y20
08; 4
5:27
5-28
8.
Hol
mbe
ck K
, Bia
nco
P, C
ater
ina
J et a
l. M
T1-M
MP-
defic
ient
mic
e de
velo
p dw
arfis
m,
oste
open
ia, a
rthrit
is, a
nd c
onne
ctiv
e tis
sue
dise
ase
due
to in
adeq
uate
col
lage
n tu
rnov
er. C
ell
1999
; 99:
81-9
2.
Hom
andb
erg
GA
, Mey
ers R
, Xie
DL.
Fib
rone
ctin
frag
men
ts c
ause
cho
ndro
lysi
s of b
ovin
e ar
ticul
ar c
artil
age
slic
es in
cul
ture
. J.B
iol.C
hem
.199
2; 2
67:3
597-
3604
.
Hop
ewel
l B, U
rban
JP. A
dapt
atio
n of
arti
cula
r cho
ndro
cyte
s to
chan
ges i
n os
mol
ality
. Bi
orhe
olog
y20
03; 4
0:73
-77.
How
arth
D, P
ritzk
er K
P, C
ruz
TF, K
ande
l RA
. Cal
cium
iono
phor
e A
2318
7 st
imul
ates
pr
oduc
tion
of a
144
kD
a ge
latin
ase.
J.R
heum
atol
.199
3; 2
0:97
-101
.
Hsi
eh A
H, T
sai C
M, M
a Q
J et a
l. Ti
me-
depe
nden
t inc
reas
es in
type
-III
col
lage
n ge
ne e
xpre
ssio
n in
med
ical
col
late
ral l
igam
ent f
ibro
blas
ts u
nder
cyc
lic st
rain
s. J.
Ort
hop.
Res.
2000
; 18:
220-
227.
Hub
er M
, Tra
ttnig
S, L
intn
er F
. Ana
tom
y, b
ioch
emis
try, a
nd p
hysi
olog
y of
arti
cula
r car
tilag
e.
Inve
st R
adio
l.20
00; 3
5:57
3-58
0.
Hum
phrie
s MJ.
Inte
grin
stru
ctur
e. B
ioch
em.S
oc.T
rans
.200
0; 2
8:31
1-33
9.
Hun
zike
r EB
. Arti
cula
r car
tilag
e re
pair:
are
the
intri
nsic
bio
logi
cal c
onst
rain
ts u
nder
min
ing
this
pr
oces
s ins
uper
able
? O
steo
arth
ritis
.Car
tilag
e.19
99; 7
:15-
28.
100
Huv
enee
rs S
, Dan
en E
H. A
dhes
ion
sign
alin
g -c
ross
talk
bet
wee
n in
tegr
ins,
Src
and
Rho
. J.C
ell
Sci.
2009
; 122
:105
9-10
69.
Ingb
er D
E. M
echa
nose
nsat
ion
thro
ugh
inte
grin
s: c
ells
act
loca
lly b
ut th
ink
glob
ally
. Pr
oc.N
atl.A
cad.
Sci.U
.S.A
2003
; 100
:147
2-14
74.
Ione
scu
AM
, Sch
war
z EM
, Vin
son
C e
t al.
PTH
rP m
odul
ates
cho
ndro
cyte
diff
eren
tiatio
n th
roug
h A
P-1
and
CR
EB si
gnal
ing.
J.B
iol.C
hem
.200
1; 2
76:1
1639
-116
47.
Jaal
ouk
DE,
Lam
mer
ding
J. M
echa
notra
nsdu
ctio
n go
ne a
wry
. Nat
.Rev
.Mol
.Cel
l Bio
l.20
09;
10:6
3-73
.
Janm
ey P
A. T
he c
ytos
kele
ton
and
cell
sign
alin
g: c
ompo
nent
loca
lizat
ion
and
mec
hani
cal
coup
ling.
Phy
siol
Rev
.199
8;78
:763
-781
.
Jian
g G
, Gia
nnon
e G
, Crit
chle
y D
R, F
ukum
oto
E, S
heet
z M
P. T
wo-
pico
new
ton
slip
bon
d be
twee
n fib
rone
ctin
and
the
cyto
skel
eton
dep
ends
on
talin
. Nat
ure
2003
; 424
:334
-337
.
Kal
apes
i FB
, Tan
JC, C
oron
eo M
T. S
tretc
h-ac
tivat
ed c
hann
els:
a m
ini-r
evie
w.A
re st
retc
h-ac
tivat
ed c
hann
els a
n oc
ular
bar
omet
er?
Clin
.Exp
erim
ent.O
phth
alm
ol.2
005;
33:
210-
217.
Kan
del R
A, G
rynp
as M
, Pill
iar R
et a
l. R
epai
r of o
steo
chon
dral
def
ects
with
bip
hasi
c ca
rtila
ge-
calc
ium
pol
ypho
spha
te c
onst
ruct
s in
a sh
eep
mod
el. B
iom
ater
ials
2006
; 27:
4120
-413
1.
Kar
in M
. The
regu
latio
n of
AP-
1 ac
tivity
by
mito
gen-
activ
ated
pro
tein
kin
ases
. J.B
iol.C
hem
.19
95; 2
70:1
6483
-164
86.
Kat
z B
Z, Z
amir
E, B
ersh
adsk
y A
, Kam
Z, Y
amad
a K
M, G
eige
r B. P
hysi
cal s
tate
of t
he
extra
cellu
lar m
atrix
regu
late
s the
stru
ctur
e an
d m
olec
ular
com
posi
tion
of c
ell-m
atrix
adh
esio
ns.
Mol
.Bio
l.Cel
l200
0; 1
1:10
47-1
060.
Ker
rigan
MJ,
Hal
l AC
. Con
trol o
f cho
ndro
cyte
regu
lato
ry v
olum
e de
crea
se (R
VD
) by
[Ca2
+]i
and
cell
shap
e. O
steo
arth
ritis
.Car
tilag
e.20
08; 1
6:31
2-32
2.
Kie
lty C
M, W
hitta
ker S
P, G
rant
ME,
Shu
ttlew
orth
CA
. Typ
e V
I col
lage
n m
icro
fibril
s: e
vide
nce
for a
stru
ctur
al a
ssoc
iatio
n w
ith h
yalu
rona
n. J
.Cel
l Bio
l.19
92; 1
18:9
79-9
90.
Kim
YJ,
Sah
RL,
Gro
dzin
sky
AJ,
Plaa
s AH
, San
dy JD
. Mec
hani
cal r
egul
atio
n of
car
tilag
e bi
osyn
thet
ic b
ehav
ior:
phys
ical
stim
uli.
Arch
.Bio
chem
.Bio
phys
.199
4; 3
11:1
-12.
Kin
ner B
, Cap
ito R
M, S
pect
or M
. Reg
ener
atio
n of
arti
cula
r car
tilag
e. A
dv.B
ioch
em.E
ng
Biot
echn
ol.2
005;
94:
91-1
23.
Kiy
okaw
a E,
Has
him
oto
Y, K
obay
ashi
S, S
ugim
ura
H, K
urat
a T,
Mat
suda
M. A
ctiv
atio
n of
R
ac1
by a
Crk
SH
3-bi
ndin
g pr
otei
n, D
OC
K18
0. G
enes
Dev
.199
8; 1
2:33
31-3
336.
101
Kni
ght M
M, B
omzo
n Z,
Kim
mel
E, S
harm
a A
M, L
ee D
A, B
ader
DL.
Cho
ndro
cyte
def
orm
atio
n in
duce
s mito
chon
dria
l dis
torti
on a
nd h
eter
ogen
eous
intra
cellu
lar s
train
fiel
ds.
Biom
ech.
Mod
el.M
echa
nobi
ol.2
006a
; 5:1
80-1
91.
Kni
ght M
M, T
oyod
a T,
Lee
DA
, Bad
er D
L. M
echa
nica
l com
pres
sion
and
hyd
rost
atic
pre
ssur
e in
duce
reve
rsib
le c
hang
es in
act
in c
ytos
kele
tal o
rgan
isat
ion
in c
hond
rocy
tes i
n ag
aros
e.
J.Bi
omec
h.20
06b;
39:
1547
-155
1.
Kol
ch W
. Mea
ning
ful r
elat
ions
hips
: the
regu
latio
n of
the
Ras
/Raf
/MEK
/ER
K p
athw
ay b
y pr
otei
n in
tera
ctio
ns. B
ioch
em.J
.200
0; 3
51 P
t 2:2
89-3
05.
Kon
o T,
Nis
hiko
ri T,
Kat
aoka
H, U
chio
Y, O
chi M
, Eno
mot
o K
. Spo
ntan
eous
osc
illat
ion
and
mec
hani
cally
indu
ced
calc
ium
wav
es in
cho
ndro
cyte
s. C
ell B
ioch
em.F
unct
.200
6; 2
4:10
3-11
1.
Kuo
CK
, Li W
J, M
auck
RL,
Tua
n R
S. C
artil
age
tissu
e en
gine
erin
g: it
s pot
entia
l and
use
s. C
urr.O
pin.
Rheu
mat
ol.2
006;
18:
64-7
3.
Laci
nova
L. V
olta
ge-d
epen
dent
cal
cium
cha
nnel
s. G
en.P
hysi
ol B
ioph
ys.2
005;
24
Supp
l 1:1
-78.
Lars
on C
M, K
elle
y SS
, Bla
ckw
ood
AD
, Ban
es A
J, Le
e G
M. R
eten
tion
of th
e na
tive
chon
droc
yte
peric
ellu
lar m
atrix
resu
lts in
sign
ifica
ntly
impr
oved
mat
rix p
rodu
ctio
n. M
atri
x Bi
ol.2
002;
21
:349
-359
.
Lee
AA
, Del
haas
T, M
cCul
loch
AD
, Vill
arre
al F
J. D
iffer
entia
l res
pons
es o
f adu
lt ca
rdia
c fib
robl
asts
to in
vitr
o bi
axia
l stra
in p
atte
rns.
J.M
ol.C
ell C
ardi
ol.1
999;
31:
1833
-184
3.
Lee
V, C
ao L
, Zha
ng Y
, Kia
ni C
, Ada
ms M
E, Y
ang
BB
. The
role
s of m
atrix
mol
ecul
es in
m
edia
ting
chon
droc
yte
aggr
egat
ion,
atta
chm
ent,
and
spre
adin
g. J
.Cel
l Bio
chem
.200
0; 7
9:32
2-33
3.
Levy
Y, R
onen
D, B
ersh
adsk
y A
D, Z
ick
Y. S
usta
ined
indu
ctio
n of
ER
K, p
rote
in k
inas
e B
, and
p7
0 S6
kin
ase
regu
late
s cel
l spr
eadi
ng a
nd fo
rmat
ion
of F
-act
in m
icro
spik
es u
pon
ligat
ion
of
inte
grin
s by
gale
ctin
-8, a
mam
mal
ian
lect
in. J
.Bio
l.Che
m.2
003;
278
:145
33-1
4542
.
Lin
B, A
rai A
C, L
ynch
G, G
all C
M. I
nteg
rins r
egul
ate
NM
DA
rece
ptor
-med
iate
d sy
napt
ic
curr
ents
. J.N
euro
phys
iol.
2003
; 89:
2874
-287
8.
Loes
er R
F. C
hond
rocy
te in
tegr
in e
xpre
ssio
n an
d fu
nctio
n. B
iorh
eolo
gy20
00; 3
7:10
9-11
6.
Lohi
J, K
eski
-Oja
J. C
alci
um io
noph
ores
dec
reas
e pe
ricel
lula
r gel
atin
olyt
ic a
ctiv
ity v
ia in
hibi
tion
of 9
2-kD
a ge
latin
ase
expr
essi
on a
nd d
ecre
ase
of 7
2-kD
a ge
latin
ase
activ
atio
n. J
.Bio
l.Che
m.
1995
; 270
:176
02-1
7609
.
Mac
Ken
na D
A, D
olfi
F, V
uori
K, R
uosl
ahti
E. E
xtra
cellu
lar s
igna
l-reg
ulat
ed k
inas
e an
d c-
Jun
NH
2-te
rmin
al k
inas
e ac
tivat
ion
by m
echa
nica
l stre
tch
is in
tegr
in-d
epen
dent
and
mat
rix-s
peci
fic
in ra
t car
diac
fibr
obla
sts.
J.C
lin.In
vest
1998
; 101
:301
-310
.
102
Man
gour
a D
,Sog
os V
, Daw
son
G. P
horb
ol e
ster
s and
PK
C si
gnal
ing
regu
late
pro
lifer
atio
n,
vim
entin
cyt
oske
leto
n as
sem
bly
and
glut
amin
e sy
nthe
tase
act
ivity
of c
hick
em
bryo
cer
ebru
m
astro
cyte
s in
cultu
re. B
rain
Res
.Dev
.Bra
in R
es.1
995;
87:
1-11
.
Mar
oto
R, R
aso
A, W
ood
TG,K
uros
ky A
, Mar
tinac
B, H
amill
OP.
TR
PC1
form
s the
stre
tch-
activ
ated
cat
ion
chan
nel i
n ve
rtebr
ate
cells
. Nat
.Cel
l Bio
l.20
05; 7
:179
-185
.
Mar
shal
l MS.
Ras
targ
et p
rote
ins i
n eu
kary
otic
cel
ls. F
ASEB
J.1
995;
9:1
311-
1318
.
Mar
tin I,
Mio
t S, B
arbe
ro A
, Jak
ob M
,Wen
dt D
. Ost
eoch
ondr
al ti
ssue
eng
inee
ring.
J.B
iom
ech.
2007
; 40:
750-
765.
Mau
ck R
L, S
oltz
MA
, Wan
g C
C e
t al.
Func
tiona
l tis
sue
engi
neer
ing
of a
rticu
lar c
artil
age
thro
ugh
dyna
mic
load
ing
of c
hond
rocy
te-s
eede
d ag
aros
e ge
ls. J
.Bio
mec
h.En
g20
00; 1
22:2
52-
260.
Men
te P
L, L
ewis
JL. E
last
ic m
odul
us o
f cal
cifie
d ca
rtila
ge is
an
orde
r of m
agni
tude
less
than
th
at o
f sub
chon
dral
bon
e. J
.Ort
hop.
Res.
1994
; 12:
637-
647.
Mill
war
d-Sa
dler
SJ,
Salte
r DM
. Int
egrin
-dep
ende
nt si
gnal
cas
cade
s in
chon
droc
yte
mec
hano
trans
duct
ion.
Ann
.Bio
med
.Eng
2004
b; 3
2:43
5-44
6.
Mill
war
d-Sa
dler
SJ,
Salte
r DM
. Int
egrin
-dep
ende
nt si
gnal
cas
cade
s in
chon
droc
yte
mec
hano
trans
duct
ion.
Ann
.Bio
med
.Eng
2004
a; 3
2:43
5-44
6.
Mill
war
d-Sa
dler
SJ,
Wrig
ht M
O, F
latm
an P
W, S
alte
r DM
. ATP
in th
e m
echa
notra
nsdu
ctio
n pa
thw
ay o
f nor
mal
hum
an c
hond
rocy
tes.
Bior
heol
ogy
2004
; 41:
567-
575.
Mill
war
d-Sa
dler
SJ,
Wrig
ht M
O, L
ee H
et a
l. In
tegr
in-r
egul
ated
secr
etio
n of
inte
rleuk
in 4
: A
nove
l pat
hway
of m
echa
notra
nsdu
ctio
n in
hum
an a
rticu
lar c
hond
rocy
tes.
J.C
ell B
iol.
1999
; 14
5:18
3-18
9.
Miy
auch
i A, N
otoy
a K
, Mik
uni-T
akag
aki Y
et a
l. Pa
rath
yroi
d ho
rmon
e-ac
tivat
ed v
olum
e-se
nsiti
ve c
alci
um in
flux
path
way
s in
mec
hani
cally
load
ed o
steo
cyte
s. J.
Biol
.Che
m.2
000;
27
5:33
35-3
342.
Miz
uno
S. A
nov
el m
etho
d fo
r ass
essi
ng e
ffec
ts o
f hyd
rost
atic
flui
dpr
essu
re o
n in
trace
llula
r ca
lciu
m: a
stud
y w
ith b
ovin
e ar
ticul
ar c
hond
rocy
tes.
Am.J
.Phy
siol
Cel
l Phy
siol
2005
; 288
:C32
9-C
337.
Mob
ashe
ri A
, Car
ter S
D, M
artin
-Vas
allo
P, S
haki
baei
M. I
nteg
rins a
nd st
retc
h ac
tivat
ed io
n ch
anne
ls; p
utat
ive
com
pone
nts o
f fun
ctio
nal c
ell s
urfa
ce m
echa
nore
cept
ors i
n ar
ticul
ar
chon
droc
ytes
. Cel
l Bio
l.Int
.200
2; 2
6:1-
18.
Mor
i M, N
akaj
ima
M, M
ikam
i Y e
t al.
Tran
scrip
tiona
l reg
ulat
ion
of th
e ca
rtila
ge in
term
edia
te
laye
r pro
tein
(CIL
P) g
ene.
Bio
chem
.Bio
phys
.Res
.Com
mun
.200
6; 3
41:1
21-1
27.
103
Mou
w JK
, Im
ler S
M, L
even
ston
ME.
Ion-
chan
nel r
egul
atio
n of
cho
ndro
cyte
mat
rix sy
nthe
sis i
n 3D
cul
ture
und
er st
atic
and
dyn
amic
com
pres
sion
. Bio
mec
h.M
odel
.Mec
hano
biol
.200
7; 6
:33-
41.
Mow
, V. C
. and
Lai
, M. F
. Mec
hani
cs o
f Ani
mal
Join
ts. A
nnua
l Rev
iew
ofF
luid
Mec
hani
cs 1
1,
247-
288.
200
9.
Ref
Typ
e: G
ener
ic
Mui
r H. T
he c
hond
rocy
te, a
rchi
tect
of c
artil
age.
Bio
mec
hani
cs, s
truct
ure,
func
tion
and
mol
ecul
ar
biol
ogy
of c
artil
age
mat
rix m
acro
mol
ecul
es. B
ioes
says
1995
; 17:
1039
-104
8.
Mul
vane
y JM
, Zha
ng T
, Few
trell
C, R
ober
son
MS.
Cal
cium
influ
x th
roug
h L-
type
cha
nnel
s is
requ
ired
for s
elec
tive
activ
atio
n of
ext
race
llula
r sig
nal-r
egul
ated
kin
ase
by g
onad
otro
pin-
rele
asin
g ho
rmon
e. J
.Bio
l.Che
m.1
999;
274
:297
96-2
9804
.
O'D
risco
ll SW
. The
hea
ling
and
rege
nera
tion
of a
rticu
lar c
artil
age.
J.B
one
Join
t Sur
g.Am
.199
8;
80:1
795-
1812
.
O'H
ara
BP,
Urb
an JP
, Mar
ouda
s A. I
nflu
ence
of c
yclic
load
ing
on th
e nu
tritio
n of
arti
cula
r ca
rtila
ge. A
nn.R
heum
.Dis
.199
0; 4
9:53
6-53
9.
Ono
dera
K, T
akah
ashi
I, S
asan
o Y
et a
l. St
epw
ise
mec
hani
cal s
tretc
hing
inhi
bits
cho
ndro
gene
sis
thro
ugh
cell-
mat
rix a
dhes
ion
med
iate
d by
inte
grin
s in
embr
yoni
c ra
t lim
b-bu
d m
esen
chym
al
cells
. Eur
.J.C
ell B
iol.
2005
; 84:
45-5
8.
Orr
AW
, Hel
mke
BP,
Bla
ckm
an B
R, S
chw
artz
MA
. Mec
hani
sms o
f mec
hano
trans
duct
ion.
D
ev.C
ell2
006;
10:1
1-20
.
Pear
le A
D, W
arre
n R
F, R
odeo
SA
. Bas
ic sc
ienc
e of
arti
cula
r car
tilag
e an
d os
teoa
rthrit
is.
Clin
.Spo
rts M
ed.2
005;
24:
1-12
.
Perk
ins G
L, D
erfo
ul A
, Ast
A, H
all D
J. A
n in
hibi
tor o
f the
stre
tch-
activ
ated
cat
ion
rece
ptor
ex
erts
a p
oten
t eff
ect o
n ch
ondr
ocyt
e ph
enot
ype.
Diff
eren
tiatio
n20
05; 7
3:19
9-21
1.
Piao
L, H
o W
K, E
arm
YE.
Act
in fi
lam
ents
regu
late
the
stre
tch
sens
itivi
ty o
f lar
ge-c
ondu
ctan
ce,
Ca2
+-ac
tivat
ed K
+ ch
anne
ls in
cor
onar
y ar
tery
smoo
th m
uscl
e ce
lls. P
fluge
rs A
rch.
2003
; 44
6:52
3-52
8.
Pilli
ar R
M, F
iliag
gi M
J, W
ells
JD, G
rynp
as M
D, K
ande
l RA
. Por
ous c
alci
um p
olyp
hosp
hate
sc
affo
lds f
or b
one
subs
titut
e ap
plic
atio
ns --
in v
itro
char
acte
rizat
ion.
Bio
mat
eria
ls20
01; 2
2:96
3-97
2.
Ping
guan
-Mur
phy
B, L
ee D
A, B
ader
DL,
Kni
ght M
M. A
ctiv
atio
n of
cho
ndro
cyte
s cal
cium
si
gnal
ling
by d
ynam
ic c
ompr
essi
on is
inde
pend
ent o
f num
ber o
f cyc
les.
Arch
.Bio
chem
.Bio
phys
.20
05b;
444
:45-
51.
Ping
guan
-Mur
phy
B, L
ee D
A, B
ader
DL,
Kni
ght M
M. A
ctiv
atio
n of
cho
ndro
cyte
s cal
cium
si
gnal
ling
by d
ynam
ic c
ompr
essi
on is
inde
pend
ent o
f num
ber o
f cyc
les.
Arch
.Bio
chem
.Bio
phys
.20
05a;
444
:45-
51.
104
Polla
rd T
D, B
oris
y G
G. C
ellu
lar m
otili
ty d
riven
by
asse
mbl
y an
d di
sass
embl
y of
act
in fi
lam
ents
. C
ell2
003;
112
:453
-465
.
Pool
e A
R, K
ojim
a T,
Yas
uda
T, M
wal
e F,
Kob
ayas
hi M
, Lav
erty
S. C
ompo
sitio
n an
d st
ruct
ure
of a
rticu
lar c
artil
age:
a te
mpl
ate
for t
issu
e re
pair.
Clin
.Ort
hop.
Rela
t Res
.200
1;S2
6-S3
3.
Pool
e A
R, R
osen
berg
LC
, Rei
ner A
, Ion
escu
M, B
ogoc
h E,
Rou
ghle
y PJ
. Con
tent
s and
di
strib
utio
ns o
f the
pro
teog
lyca
ns d
ecor
in a
nd b
igly
can
in n
orm
al a
nd o
steo
arth
ritic
hum
an
artic
ular
car
tilag
e. J
.Ort
hop.
Res.
1996
; 14:
681-
689.
Pool
e C
A. A
rticu
lar c
artil
age
chon
dron
s: fo
rm, f
unct
ion
and
failu
re. J
.Ana
t.19
97; 1
91 (
Pt 1
):1-
13.
Prem
kum
ar D
R, M
ishr
a R
R, O
verh
olt J
L, S
imon
son
MS,
Che
rnia
ck N
S, P
rabh
akar
NR
. L-ty
pe
Ca(
2+) c
hann
el a
ctiv
atio
n re
gula
tes i
nduc
tion
of c
-fos
tran
scrip
tion
by h
ypox
ia. J
.App
l.Phy
siol
2000
; 88:
1898
-190
6.
Rai
zman
I, D
e C
roos
JN, S
t-Pie
rre
JP, P
illia
r RM
, Kan
del R
. Arti
cula
r Car
tilag
e Su
bpop
ulat
ions
R
espo
nd D
iffer
ently
to C
yclic
Com
pres
sion
in v
itro.
Tis
sue
Eng
Part
A20
09.
Red
man
SN
, Old
field
SF,
Arc
her C
W. C
urre
nt st
rate
gies
for a
rticu
lar c
artil
age
repa
ir. E
ur.C
ell
Mat
er.2
005;
9:2
3-32
.
Ren
XD
, Kio
sses
WB
, Sie
g D
J, O
tey
CA
, Sch
laep
fer D
D, S
chw
artz
MA
. Foc
al a
dhes
ion
kina
se
supp
ress
es R
ho a
ctiv
ity to
pro
mot
e fo
cal a
dhes
ion
turn
over
. J.C
ell S
ci.2
000;
113
( Pt
20)
:367
3-36
78.
Riv
elin
e D
, Zam
ir E,
Bal
aban
NQ
et a
l. Fo
cal c
onta
cts a
s mec
hano
sens
ors:
ext
erna
lly a
pplie
d lo
cal m
echa
nica
l for
ce in
duce
s gro
wth
of f
ocal
con
tact
s by
an m
Dia
1-de
pend
ent a
nd R
OC
K-
inde
pend
ent m
echa
nism
. J.C
ell B
iol.
2001
; 153
:117
5-11
86.
Rob
erts
SR
, Kni
ght M
M, L
ee D
A, B
ader
DL.
Mec
hani
cal c
ompr
essi
on in
fluen
ces i
ntra
cellu
lar
Ca2
+ si
gnal
ing
in c
hond
rocy
tes s
eede
d in
aga
rose
con
stru
cts.
J.Ap
pl.P
hysi
ol20
01; 9
0:13
85-
1391
.
Rom
er L
H, B
iruko
v K
G, G
arci
a JG
. Foc
al a
dhes
ions
: par
adig
m fo
r a si
gnal
ing
nexu
s. C
irc.
Res.
2006
; 98:
606-
616.
Rot
tner
K, H
all A
, Sm
all J
V. I
nter
play
bet
wee
n R
ac a
nd R
ho in
the
cont
rol o
f sub
stra
te c
onta
ct
dyna
mic
s. C
urr.B
iol.
1999
; 9:6
40-6
48.
Sala
zar E
P, R
ozen
gurt
E. S
rc fa
mily
kin
ases
are
requ
ired
for i
nteg
rin-m
edia
ted
but n
ot fo
r G
prot
ein-
coup
led
rece
ptor
stim
ulat
ion
of fo
cal a
dhes
ion
kina
se a
utop
hosp
hory
latio
n at
Tyr
-397
. J.
Biol
.Che
m.2
001;
276
:177
88-1
7795
.
Sam
arak
oon
R, H
iggi
ns P
J. M
EK/E
RK
pat
hway
med
iate
s cel
l-sha
pe-d
epen
dent
pla
smin
ogen
ac
tivat
or in
hibi
tor t
ype
1 ge
ne e
xpre
ssio
n up
on d
rug-
indu
ced
disr
uptio
n of
the
mic
rofil
amen
t and
m
icro
tubu
le n
etw
orks
. J.C
ell S
ci.2
002;
115
:309
3-31
03.
105
Scha
ller M
D, H
ildeb
rand
JD, S
hann
on JD
, Fox
JW, V
ines
RR
, Par
sons
JT. A
utop
hosp
hory
latio
n of
the
foca
l adh
esio
n ki
nase
, pp1
25FA
K, d
irect
s SH
2-de
pend
ent b
indi
ng o
f pp6
0src
. Mol
.Cel
l Bi
ol.1
994;
14:
1680
-168
8.
Schl
aepf
er D
D, B
room
e M
A, H
unte
r T. F
ibro
nect
in-s
timul
ated
sign
alin
g fr
om a
foca
l adh
esio
n ki
nase
-c-S
rc c
ompl
ex: i
nvol
vem
ent o
f the
Grb
2, p
130c
as, a
nd N
ck a
dapt
or p
rote
ins.
Mol
.Cel
l Bi
ol.1
997;
17:
1702
-171
3.
Schm
idt T
A, S
chum
ache
r BL,
Kle
in T
J, V
oegt
line
MS,
Sah
RL.
Syn
thes
is o
f pro
teog
lyca
n 4
by
chon
droc
yte
subp
opul
atio
ns in
car
tilag
e ex
plan
ts, m
onol
ayer
cul
ture
s, an
dre
surf
aced
car
tilag
e cu
lture
s. Ar
thri
tis R
heum
.200
4; 5
0:28
49-2
857.
Schn
abel
M, M
arlo
vits
S, E
ckho
ff G
et a
l. D
ediff
eren
tiatio
n-as
soci
ated
cha
nges
in m
orph
olog
y an
d ge
ne e
xpre
ssio
n in
prim
ary
hum
an a
rticu
lar c
hond
rocy
tes i
n ce
ll cu
lture
. O
steo
arth
ritis
.Car
tilag
e.20
02; 1
0:62
-70.
Schu
mac
her B
L, B
lock
JA, S
chm
id T
M, A
ydel
otte
MB
, Kue
ttner
KE.
A n
ovel
pro
teog
lyca
n sy
nthe
size
d an
d se
cret
ed b
y ch
ondr
ocyt
es o
f the
supe
rfic
ial z
one
of a
rticu
lar c
artil
age.
Ar
ch.B
ioch
em.B
ioph
ys.1
994;
311
:144
-152
.
Schw
artz
MA
. Int
egrin
sign
alin
g re
visi
ted.
Tre
nds C
ell B
iol.
2001
; 11:
466-
470.
Schw
artz
MA
, Sha
ttil S
J. Si
gnal
ing
netw
orks
link
ing
inte
grin
s and
rho
fam
ily G
TPas
es. T
rend
s Bi
oche
m.S
ci.2
000;
25:
388-
391.
Schw
artz
Z, L
angs
ton
GG
, Sw
ain
LD, B
oyan
BD
. Inh
ibiti
on o
f 1,2
5-(O
H)2
D3-
and
24,2
5-(O
H)2
D3-
depe
nden
t stim
ulat
ion
of a
lkal
ine
phos
phat
ase
activ
ity b
y A
2318
7 su
gges
ts a
role
for
calc
ium
in th
e m
echa
nism
of v
itam
in D
regu
latio
n of
cho
ndro
cyte
cul
ture
s. J.
Bone
Min
er.R
es.
1991
; 6:7
09-7
18.
Shak
ibae
i M, J
ohn
T, D
e SP
, Rah
man
zade
h R
, Mer
ker H
J. Si
gnal
tran
sduc
tion
by b
eta1
inte
grin
re
cept
ors i
n hu
man
cho
ndro
cyte
s in
vitro
: col
labo
ratio
n w
ith th
e in
sulin
-like
gro
wth
fact
or-I
re
cept
or. B
ioch
em.J
.199
9; 3
42 P
t 3:6
15-6
23.
Shak
ibae
i M, M
erke
r HJ.
Bet
a1-in
tegr
ins i
n th
e ca
rtila
ge m
atrix
. Cel
l Tis
sue
Res.
1999
; 296
:565
-57
3.
Shak
ibae
i M, M
obas
heri
A. B
eta1
-inte
grin
s co-
loca
lize
with
Na,
K-A
TPas
e, e
pith
elia
l sod
ium
ch
anne
ls (E
NaC
) and
vol
tage
act
ivat
ed c
alci
um c
hann
els (
VA
CC
) in
mec
hano
rece
ptor
co
mpl
exes
of m
ouse
lim
b-bu
d ch
ondr
ocyt
es. H
isto
l.His
topa
thol
.200
3; 1
8:34
3-35
1.
Shao
Y, A
lickn
avitc
h M
, Far
ach-
Car
son
MC
. Exp
ress
ion
of v
olta
ge se
nsiti
ve c
alci
um c
hann
el
(VSC
C) L
-type
Cav
1.2
(alp
ha1C
) and
T-ty
pe C
av3.
2 (a
lpha
1H) s
ubun
its d
urin
g m
ouse
bon
e de
velo
pmen
t. D
ev.D
yn.2
005;
234
:54-
62.
Shie
h A
C, A
than
asio
u K
A. B
iom
echa
nics
of s
ingl
e zo
nal c
hond
rocy
tes.
J.Bi
omec
h.20
06;
39:1
595-
1602
.
106
Shie
h A
C, A
than
asio
u K
A. P
rinci
ples
of c
ell m
echa
nics
for c
artil
age
tissu
e en
gine
erin
g.
Ann.
Biom
ed.E
ng20
03; 3
1:1-
11.
Smith
RL,
Don
lon
BS,
Gup
ta M
K e
t al.
Effe
cts o
f flu
id-in
duce
d sh
ear o
n ar
ticul
ar c
hond
rocy
te
mor
phol
ogy
and
met
abol
ism
in v
itro.
J.O
rtho
p.Re
s.19
95; 1
3:82
4-83
1.
Sode
rman
P, M
alch
au H
, Her
berts
P. O
utco
me
of to
tal h
ip re
plac
emen
t: a
com
paris
on o
f di
ffer
ent m
easu
rem
ent m
etho
ds.C
lin.O
rtho
p.Re
lat R
es.2
001;
163-
172.
Spite
ri C
G, P
illia
r RM
, Kan
del R
A. S
ubst
rate
por
osity
enh
ance
s cho
ndro
cyte
atta
chm
ent,
spre
adin
g, a
nd c
artil
age
tissu
e fo
rmat
ion
in v
itro.
J.B
iom
ed.M
ater
.Res
.A20
06; 7
8:67
6-68
3.
Spite
ri C
G, Y
oung
EW
, Sim
mon
s CA
, Kan
del R
A, P
illia
r RM
. Sub
stra
te a
rchi
tect
ure
and
fluid
-in
duce
d sh
ear s
tress
dur
ing
chon
droc
yte
seed
ing:
role
of a
lpha
5bet
a1 in
tegr
in. B
iom
ater
ials
2008
; 29:
2477
-248
9.
Stew
art M
C, S
aund
ers K
M, B
urto
n-W
urst
er N
, Mac
leod
JN. P
heno
typi
c st
abili
ty o
f arti
cula
r ch
ondr
ocyt
es in
vitr
o: th
e ef
fect
s of c
ultu
re m
odel
s, bo
ne m
orph
ogen
etic
pro
tein
2, a
nd se
rum
su
pple
men
tatio
n. J
.Bon
e M
iner
.Res
.200
0; 1
5:16
6-17
4.
Such
yna
TM, J
ohns
on JH
, Ham
er K
et a
l. Id
entif
icat
ion
of a
pep
tide
toxi
n fr
om G
ram
mos
tola
sp
atul
ata
spid
er v
enom
that
blo
cks c
atio
n-se
lect
ive
stre
tch-
activ
ated
cha
nnel
s. J.
Gen
.Phy
siol
2000
; 115
:583
-598
.
Taka
da Y
, Ye
X, S
imon
S. T
he in
tegr
ins.
Gen
ome
Biol
.200
7; 8
:215
.
Taki
no T
, Wat
anab
e Y
, Mat
sui M
et a
l. M
embr
ane-
type
1 m
atrix
met
allo
prot
eina
se m
odul
ates
fo
cal a
dhes
ion
stab
ility
and
cel
l mig
ratio
n. E
xp.C
ell R
es.2
006;
312
:138
1-13
89.
Tana
ka N
, Ohn
o S,
Hon
da K
et a
l. C
yclic
mec
hani
cal s
train
regu
late
s the
PTH
rP e
xpre
ssio
n in
cu
lture
d ch
ondr
ocyt
es v
ia a
ctiv
atio
n of
the
Ca2
+ ch
anne
l. J.
Den
t.Res
.200
5; 8
4:64
-68.
Tem
enof
f JS,
Mik
os A
G. R
evie
w: t
issu
e en
gine
erin
g fo
r reg
ener
atio
n of
arti
cula
r car
tilag
e.
Biom
ater
ials
2000
b; 2
1:43
1-44
0.
Tem
enof
f JS,
Mik
os A
G. R
evie
w: t
issu
e en
gine
erin
g fo
r reg
ener
atio
n of
arti
cula
r car
tilag
e.
Biom
ater
ials
2000
a; 2
1:43
1-44
0.
Thak
ar R
G, C
heng
Q,P
atel
S e
t al.
Cel
l-sha
pe re
gula
tion
of sm
ooth
mus
cle
cell
prol
ifera
tion.
Bi
ophy
s.J.2
009;
96:
3423
-343
2.
Toyo
da T
, See
dhom
BB
, Yao
JQ, K
irkha
m J,
Bro
okes
S, B
onas
s WA
. Hyd
rost
atic
pre
ssur
e m
odul
ates
pro
teog
lyca
n m
etab
olis
m in
cho
ndro
cyte
s see
ded
in a
garo
se. A
rthr
itis R
heum
.200
3;
48:2
865-
2872
.
Urb
an JP
. The
cho
ndro
cyte
: a c
ell u
nder
pre
ssur
e. B
r.J.R
heum
atol
.199
4; 3
3:90
1-90
8.
107
Vol
berg
T, R
omer
L, Z
amir
E, G
eige
r B. p
p60(
c-sr
c) a
nd re
late
d ty
rosi
ne k
inas
es: a
role
in th
e as
sem
bly
and
reor
gani
zatio
n of
mat
rixad
hesi
ons.
J.C
ell S
ci.2
001;
114
:227
9-22
89.
Vuo
ri K
, Hira
i H, A
izaw
a S,
Ruo
slah
ti E.
Intro
duct
ion
of p
130c
as si
gnal
ing
com
plex
form
atio
n up
on in
tegr
in-m
edia
ted
cell
adhe
sion
: a ro
le fo
r Src
fam
ily k
inas
es. M
ol.C
ell B
iol.
1996
; 16
:260
6-26
13.
Wal
dman
SD
, Cou
to D
C, G
rynp
as M
D, P
illia
r RM
, Kan
del R
A. A
sing
le a
pplic
atio
n of
cyc
lic
load
ing
can
acce
lera
te m
atrix
dep
ositi
on a
nd e
nhan
ce th
e pr
oper
ties o
f tis
sue-
engi
neer
ed
carti
lage
. Ost
eoar
thri
tis.C
artil
age.
2006
; 14:
323-
330.
Wal
dman
SD
, Gry
npas
MD
, Pill
iar R
M, K
ande
l RA
. Cha
ract
eriz
atio
n of
car
tilag
enou
s tis
sue
form
ed o
n ca
lciu
m p
olyp
hosp
hate
subs
trate
s in
vitro
. J.B
iom
ed.M
ater
.Res
.200
2; 6
2:32
3-33
0.
Wal
dman
SD
, Spi
teri
CG
, Gry
npas
MD
, Pill
iar R
M, H
ong
J, K
ande
l RA
. Eff
ect o
f bi
omec
hani
cal c
ondi
tioni
ng o
n ca
rtila
gino
us ti
ssue
form
atio
n in
vitr
o. J
.Bon
e Jo
int S
urg.
Am.
2003
; 85-
A S
uppl
2:1
01-1
05.
Wal
dman
SD
, Spi
teri
CG
, Gry
npas
MD
, Pill
iar R
M, K
ande
l RA
. Lon
g-te
rm in
term
itten
t co
mpr
essi
ve st
imul
atio
n im
prov
es th
e co
mpo
sitio
n an
d m
echa
nica
l pro
perti
es o
f tis
sue-
engi
neer
ed c
artil
age.
Tis
sue
Eng
2004
; 10:
1323
-133
1.
Wan
g JH
, Tha
mpa
tty B
P. A
n in
trodu
ctor
y re
view
of c
ell m
echa
nobi
olog
y.
Biom
ech.
Mod
el.M
echa
nobi
ol.2
006;
5:1
-16.
Whi
tfiel
d JF
. Cal
cium
sign
als a
nd c
ance
r. C
rit R
ev.O
ncog
.199
2; 3
:55-
90.
Witt
man
n T,
Bok
och
GM
, Wat
erm
an-S
tore
r CM
. Reg
ulat
ion
of le
adin
g ed
ge m
icro
tubu
le a
nd
actin
dyn
amic
s dow
nstre
am o
f Rac
1. J
.Cel
l Bio
l.20
03; 1
61:8
45-8
51.
Won
g M
, Car
ter D
R. A
rticu
lar c
artil
age
func
tiona
l his
tom
orph
olog
y an
d m
echa
nobi
olog
y: a
re
sear
ch p
ersp
ectiv
e. B
one
2003
; 33:
1-13
.
Woo
ds A
, Wan
g G
, Bei
er F
. Reg
ulat
ion
of c
hond
rocy
te d
iffer
entia
tion
by th
e ac
tin c
ytos
kele
ton
and
adhe
sive
inte
ract
ions
. J.C
ell P
hysi
ol20
07; 2
13:1
-8.
Wu
Q, Z
hang
Y, C
hen
Q. I
ndia
n he
dgeh
og is
an
esse
ntia
l com
pone
nt o
f mec
hano
trans
duct
ion
com
plex
to st
imul
ate
chon
droc
yte
prol
ifera
tion.
J.B
iol.C
hem
.200
1a; 2
76:3
5290
-352
96.
Wu
X, D
avis
GE,
Mei
ning
er G
A, W
ilson
E, D
avis
MJ.
Reg
ulat
ion
of th
e L-
type
cal
cium
cha
nnel
by
alp
ha 5
beta
1 in
tegr
in re
quire
s sig
nalin
g be
twee
n fo
cal a
dhes
ion
prot
eins
.J.B
iol.C
hem
.20
01b;
276
:302
85-3
0292
.
Wu
X, D
avis
MJ.
Cha
ract
eriz
atio
n of
stre
tch-
activ
ated
cat
ion
curr
ent i
n co
rona
ry sm
ooth
mus
cle
cells
. Am
.J.P
hysi
ol H
eart
Cir
c.Ph
ysio
l200
1; 2
80:H
1751
-H17
61.
Wu
X, G
an B
, Yoo
Y, G
uan
JL. F
AK
-med
iate
d sr
c ph
osph
oryl
atio
nof
end
ophi
lin A
2 in
hibi
ts
endo
cyto
sis o
f MT1
-MM
P an
d pr
omot
es E
CM
deg
rada
tion.
Dev
.Cel
l200
5; 9
:185
-196
.
108
Xia
H, N
ho R
S, K
ahm
J, K
leid
on J,
Hen
ke C
A. F
ocal
adh
esio
n ki
nase
is u
pstre
am o
f ph
osph
atid
ylin
osito
l 3-k
inas
e/A
kt in
regu
latin
g fib
robl
ast s
urvi
val i
n re
spon
se to
con
tract
ion
of
type
I co
llage
n m
atric
es v
ia a
bet
a 1
inte
grin
via
bilit
y si
gnal
ing
path
way
. J.B
iol.C
hem
.200
4;
279:
3302
4-33
034.
Xu
Y, P
ritzk
er K
P, C
ruz
TF. C
hara
cter
izat
ion
of c
hond
rocy
te a
lkal
ine
phos
phat
ase
as a
pot
entia
l m
edia
tor i
n th
e di
ssol
utio
n of
cal
cium
pyr
opho
spha
te d
ihyd
rate
cry
stal
s. J.
Rheu
mat
ol.1
994;
21
:912
-919
.
Yam
aguc
hi S
, Yam
aguc
hi M
, Yat
suya
nagi
E e
t al.
Cyc
lic st
rain
stim
ulat
es e
arly
gro
wth
resp
onse
ge
ne p
rodu
ct 1
-med
iate
d ex
pres
sion
of m
embr
ane
type
1 m
atrix
met
allo
prot
eina
se in
en
doth
eliu
m. L
ab In
vest
2002
; 82:
949-
956.
Yan
g G
, Cra
wfo
rd R
C, W
ang
JH. P
rolif
erat
ion
and
colla
gen
prod
uctio
n of
hum
an p
atel
lar
tend
on fi
brob
last
s in
resp
onse
to c
yclic
uni
axia
l stre
tchi
ng in
seru
m-f
ree
cond
ition
s. J.
Biom
ech.
2004
; 37:
1543
-155
0.
Yan
o Y
, Gei
bel J
, Sum
pio
BE.
Tyr
osin
e ph
osph
oryl
atio
n of
pp1
25FA
K a
nd p
axill
in in
aor
tic
endo
thel
ial c
ells
indu
ced
by m
echa
nica
l stra
in. A
m.J
.Phy
siol
1996
; 271
:C63
5-C
649.
Yel
low
ley
CE,
Han
cox
JC, D
onah
ue H
J. Ef
fect
s of c
ell s
wel
ling
on in
trace
llula
r cal
cium
and
mem
bran
e cu
rren
ts in
bov
ine
artic
ular
cho
ndro
cyte
s. J.
Cel
l Bio
chem
.200
2; 8
6:29
0-30
1.
Zaid
el-B
ar R
, Coh
en M
, Add
adi L
, Gei
ger B
. Hie
rarc
hica
l ass
embl
y of
cel
l-mat
rix a
dhes
ion
com
plex
es. B
ioch
em.S
oc.T
rans
.200
4; 3
2:41
6-42
0.
Zam
ir E,
Kat
z B
Z, A
ota
S, Y
amad
aK
M, G
eige
r B, K
am Z
. Mol
ecul
ar d
iver
sity
of c
ell-m
atrix
ad
hesi
ons.
J.C
ell S
ci.1
999;
112
( Pt
11)
:165
5-16
69.
Zhan
g M
, Wan
g JJ
, Che
n Y
J. Ef
fect
s of m
echa
nica
l pre
ssur
e on
intra
cellu
lar c
alci
um re
leas
e ch
anne
l and
cyt
oske
leta
l stru
ctur
e in
rabb
it m
andi
bula
rcon
dyla
r cho
ndro
cyte
s. Li
fe S
ci.2
006;
78
:248
0-24
87.
Zim
mer
man
E, G
eige
r B, A
ddad
i L. I
nitia
l sta
ges o
f cel
l-mat
rix a
dhes
ion
can
be m
edia
ted
and
mod
ulat
ed b
y ce
ll-su
rfac
e hy
alur
onan
. Bio
phys
.J.2
002;
82:
1848
-185
7.
Zou
H, L
ifshi
tz L
M, T
uft R
A, F
ogar
ty K
E, S
inge
r JJ.
Vis
ualiz
atio
n of
Ca2
+ en
try th
roug
h si
ngle
st
retc
h-ac
tivat
ed c
atio
n ch
anne
ls. P
roc.
Nat
l.Aca
d.Sc
i.U.S
.A20
02; 9
9:64
04-6
409.
Zusc
ik M
J, G
unte
r TE,
Puz
as JE
, Ros
ier R
N. C
hara
cter
izat
ion
of v
olta
ge-s
ensi
tive
calc
ium
ch
anne
ls in
gro
wth
pla
te c
hond
rocy
tes.
Bioc
hem
.Bio
phys
.Res
.Com
mun
.199
7; 2
34:4
32-4
38.
109
CH
APT
ER F
IVE:
APP
END
IX A
110
Figu
re A
1: C
hond
rocy
te A
ctin
cyt
oske
leto
n as
vis
ualiz
ed b
y co
nfoc
al m
icro
scop
y.C
ontro
l and
st
imul
ated
tiss
ue c
onst
ruct
s w
ere
was
hed
in P
BS
(3x)
and
fix
ed w
ith 4
% w
/vpa
rafo
rmad
ehyd
e ov
erni
ght a
t 4°C
.Sam
ples
wer
e th
en w
ashe
d ag
ain
(3x)
with
PB
S fo
llow
ed b
y ce
ll m
embr
ane
perm
eabi
lizat
ion
usin
g 1%
Trit
on X
for
15
min
utes
at
room
tem
pera
ture
with
gen
tle s
haki
ng.
The
actin
cyt
oske
leto
n w
as s
tain
ed u
sing
pha
lloid
in-r
hoda
min
e (A
lexa
-flu
or 5
86,
Invi
troge
n)
dilu
ted
1:20
in
1% T
riton
X s
olut
ion.
Sam
ples
wer
e m
ount
ed o
nto
slid
es w
ith a
dro
p of
Pe
rmaf
luor
Mou
ntan
t so
lutio
n (a
nti-f
ade
Imm
unon
, Pi
ttsbu
rgh)
, se
aled
usi
ng a
cov
ersl
ip a
nd
stor
ed in
the
dark
at 4
°C u
ntil
anal
ysis
with
a c
onfo
cal m
icro
scop
e. I
mag
es w
ere
colle
cted
at a
re
solu
tion
of 5
12 x
512
uni
ts u
sing
a 4
0x o
bjec
tive
(Lei
ca T
CS
SP2,
Lei
ca M
icro
syst
ems,
Man
nhei
m, G
erm
any)
.Pre
limin
ary
stud
ies
sugg
este
d th
at m
echa
nica
l stim
ulat
ion
did
not a
ppea
r to
alte
r the
dis
tribu
tion
of th
e ac
tin c
ytos
kele
ton
as c
ompa
red
to c
ontro
l uns
timul
ated
con
stru
cts. 1
C
hond
rocy
te A
ctin
cyt
oske
leto
n
111
Gen
ePr
imer
Seq
uenc
ePr
oduc
t Si
zeC
av 1
.1F:
3’-
cagt
gcca
ccgc
attg
tcaa
tgac
a -5
’R
: 3’-
ctttc
ccttg
aaga
gctg
gacc
cc -5
’49
1 bp
Cav
1.2
F: 3
’-cg
aagc
ttcttc
atga
tgaa
catc
t -5’
R: 3
’-gc
ggat
ccat
gtag
aagc
tgat
gaa
-5’
928
bp
Figu
re A
2: B
ovin
e ar
ticul
ar c
artil
age
chon
droc
ytes
exp
ress
the
L-T
ype
VG
CC
. (
A)
The
expr
essi
on o
f th
e L-
Type
VG
CC
was
con
firm
ed i
n ou
r sy
stem
by
RT-
PCR
, as
the
cells
wer
e sh
own
to e
xpre
ss th
e m
RN
A o
f the
α s
ubun
it of
the
chan
nel
(B) T
he u
tiliz
ed p
rimer
seq
uenc
es,
alon
g w
ith t
he e
xpec
ted
prod
uct
size
, for
the
rec
epto
r su
buni
ts. P
rimer
seq
uenc
es w
ere
take
n fr
om A
. Ben
avid
es e
t. al
.
5.2
Bov
ine
artic
ular
car
tilag
e ch
ondr
ocyt
es e
xpre
ssio
n of
the
L-Ty
pe V
GC
C
Gen
ePr
imer
Seq
uenc
ePr
oduc
tSi
zeC
av 1
.1F:
3’-
cagt
gcca
ccgc
attg
tcaa
tgac
a -5
’R
: 3’-
ctttc
ccttg
aaga
gctg
gacc
cc-5
’49
1 bp
Cav
1.2
F: 3
’-cg
aagc
ttcttc
atga
tgaa
catc
t-5’
R: 3
’-gc
ggat
ccat
gtag
aagc
tgat
gaa
-5’
928
bp
A.
B.
112
Figu
re A
3: T
issu
e fo
rmat
ion
not a
ffec
ted
with
inhi
bito
r tre
atm
ents
.Tre
atm
ent w
ith N
ifedi
pine
al
one,
w
ithou
t m
echa
nica
l st
imul
atio
n,
indu
ces
incr
ease
d sy
nthe
sis
of
colla
gens
an
d pr
oteo
glyc
ans
with
in 2
4 ho
urs
of t
reat
men
t, ho
wev
er n
o im
prov
emen
t in
tis
sue
form
atio
n is
ob
serv
ed b
y 3
wee
ks. I
n ad
ditio
n, N
ifedi
pine
trea
tmen
t inc
reas
es c
ell p
rolif
erat
ion.
Follo
win
g th
e ap
plic
atio
n m
echa
nica
l stim
ulat
ion,
con
stru
cts
wer
e in
cuba
ted
with
[35
S]-S
O4
(2 μ
Ci/c
onst
ruct
) an
d [3 H
]-pr
olin
e (2
μC
i/con
stru
ct)
for
24 h
ours
und
er s
tand
ard
tissu
e cu
lture
con
ditio
ns t
o m
easu
re p
rote
ogly
can
and
colla
gen
synt
hesi
s re
spec
tivel
y. C
ultu
res
wer
e th
en w
ashe
d in
PB
S an
d di
gest
ed b
y pa
pain
(40
μg/
ml;
Sigm
a) i
n pa
pain
dig
estio
n bu
ffer
(40
mM
am
mon
ium
ac
etat
e, 1
mM
ED
TA,
2mM
DTT
) at
65º
C f
or 4
8 ho
urs.
The
amou
nt o
f ne
wly
syn
thes
ized
pr
oteo
glyc
an a
nd c
olla
gen
reta
ined
in
the
tissu
e w
as q
uant
ified
by
mea
surin
g ra
dioi
soto
pe
inco
rpor
atio
n us
ing
a β-
scin
tilla
tion
coun
ter
and
norm
aliz
ed t
o D
NA
con
tent
. Th
e pa
pain
di
gest
ed sa
mpl
es w
ere
asse
ssed
for D
NA
con
tent
usi
ng th
e H
oech
st 3
3258
dye
bin
ding
ass
ay a
nd
fluor
omet
ry.(
A) A
4-h
our t
reat
men
t with
Nife
dipi
ne in
duce
s an
app
roxi
mat
ely
25%
incr
ease
inth
e sy
nthe
sis
of n
ew m
atrix
mol
ecul
es i
n ch
ondr
ocyt
e co
nstru
cts.
(B)
It w
as o
bser
ved
that
N
ifedi
pine
tre
atm
ent
also
res
ults
in
incr
ease
d ch
ondr
ocyt
e pr
olife
ratio
n as
com
pare
d to
the
ve
hicl
e-an
d G
adol
inum
-trea
ted
cond
ition
s. (C
) A
one
-tim
e 4-
hour
tre
atm
ent
by N
ifedi
pine
or
Gad
olin
um d
id n
ot re
sult
in im
prov
ed ti
ssue
form
atio
n fo
llow
ing
a 3
wee
k cu
lture
per
iod
unde
r st
anda
rd c
ondi
tions
. (D
) O
win
g to
the
inc
reas
e in
DN
A c
onte
nt i
n th
e N
ifedi
pine
-trea
ted
cons
truct
s the
re is
an
appa
rent
dec
reas
e in
pro
teog
lyca
n an
d co
llage
n co
nten
t.5.
3 Ti
ssue
fo
rmat
ion
not
affe
cted
w
ith
inhi
bito
r tre
atm
ents
113
Figu
re A
4: M
T1-M
MP
expr
essi
on i
n ch
ondr
ocyt
es c
ultu
red
on m
onol
ayer
. M
T1-M
MP
gene
ex
pres
sion
is
depe
nden
t up
on t
he c
hond
rocy
tes’
env
ironm
ent
and
the
thre
e di
men
sion
al
arch
itect
ure
of th
e tis
sue.
Lik
ewis
e, th
e ro
le o
f cal
cium
in re
gula
ting
MT1
-MM
P ex
pres
sion
may
be
dep
ende
nt o
n th
e cu
lture
sys
tem
. R
epre
sent
ativ
e ge
l of
MT1
-MM
P ge
ne e
xpre
ssio
n in
ch
ondr
ocyt
es g
row
n in
mon
olay
er c
ultu
res.
The
chon
droc
ytes
exh
ibit
indi
scrim
inat
e ex
pres
sion
of
the
mR
NA
in
untre
ated
, Nife
dipi
ne a
nd G
adol
iniu
m t
reat
men
ts. T
his
sugg
ests
tha
t ca
lciu
m
regu
latio
n of
MT1
-MM
P ge
ne e
xpre
ssio
n m
ay b
e de
pend
ent
upon
the
env
ironm
ent
of t
he
chon
droc
yte
and
is d
iffer
ent
in c
hond
rocy
tes
cultu
red
in 3
D.
Cho
ndro
cyte
s w
ere
cultu
red
on
mon
olay
er a
t 80
-90%
con
fluen
ce p
rior
to t
reat
men
t w
ith e
ither
inh
ibito
rs o
r ve
hicl
e m
edia
.Tr
eatm
ent t
imes
and
con
cent
ratio
ns w
ere
othe
rwis
e id
entic
al to
thos
e ut
ilize
d pr
evio
usly
.
5.4
MT1
-MM
P ex
pres
sion
in c
hond
rocy
tes c
ultu
red
on m
onol
ayer
114
Figu
re A
5: P
hosp
hory
latio
n of
Foc
al A
dhes
ion
Kin
ase
(FA
K) i
n re
spon
se to
mec
hani
cal
stim
ulat
ion.
Cel
ls w
ere
mec
hani
cally
load
ed fo
r var
ious
per
iods
of t
ime
rang
ing
from
1 to
25
min
utes
. Tot
al c
ell p
rote
in w
as e
xtra
cted
and
ana
lyze
d by
wes
tern
blo
t for
pTy
r and
Tot
al F
AK
, as
per
the
prev
ious
ly o
utlin
ed w
este
rn b
lotti
ng m
etho
ds. N
o si
gnifi
cant
cha
nges
in F
AK
ph
osph
oryl
atio
n w
ere
obse
rved
. Thr
ee in
depe
nden
t exp
erim
ents
wer
e pe
rfor
med
in d
uplic
ate
(n=6
), an
d th
e re
sults
are
poo
led
and
expr
esse
d as
mea
n ±
SEM
.
5.5
Phos
phor
ylat
ion
of F
ocal
Adh
esio
n K
inas
e (F
AK
)
115
Figu
re A
6: C
ell A
rea
Dis
tribu
tions
Fol
low
ing
Stim
ulat
ion.
Dis
tribu
tion
of c
hond
rocy
te c
ell a
rea,
ex
pres
sed
as p
erce
ntag
e of
tota
l, in
the
vario
us c
ondi
tions
. Tot
al c
ell a
reas
in e
ach
cond
ition
w
ere
pool
ed to
geth
er a
nd se
para
ted,
util
izin
g Ex
cels
freq
uenc
y fu
nctio
n, in
to b
ins i
n th
e ra
nge
of
20um
. The
val
ue o
f eac
h bi
n w
as th
en q
uant
ified
and
exp
ress
ed a
s a p
erce
ntag
e of
the
tota
l cel
l nu
mbe
r. C
ell a
rea
was
qua
ntifi
ed im
med
iate
ly fo
llow
ing
stim
ulat
ion
(A) a
nd a
t six
hou
rs la
ter
(B) a
nd th
e di
strib
utio
n w
as d
eter
min
ed. T
hree
inde
pend
ent e
xper
imen
ts w
ere
perf
orm
ed in
du
plic
ate
(n=6
), an
d th
e re
sults
are
poo
led
and
expr
esse
d as
mea
n ±
SEM
.
5.6
Cho
ndro
cyte
cel
l are
a di
strib
utio
ns fo
llow
ing
stim
ulat
ion.
116
Figu
re A
7: C
ell s
prea
ding
app
ears
to b
e de
pth-
depe
nden
t. St
imul
ated
tiss
ue c
onst
ruct
s wer
e cu
t at
an
angl
e pr
ior t
o Sc
anni
ng E
lect
ron
Mic
rosc
opy
visu
aliz
atio
n, to
atte
mpt
to v
isua
lize
the
cell
mor
phol
ogy
grad
ient
, fol
low
ing
mec
hani
cal s
timul
atio
n. It
app
ears
that
cel
ls si
tuat
ed d
eepe
r w
ithin
the
tissu
e, c
lose
r to
the
CPP
surf
ace,
und
ergo
a m
ore
sign
ifica
nt sp
read
ing
as a
resu
lt of
m
echa
nica
l stim
ulat
ion.
5.7
Cel
l spr
eadi
ng a
ppea
rs to
be
dept
h-de
pend
ent
117
CH
APT
ER S
IX: A
PPEN
DIX
B
118
Arti
cula
r Car
tilag
e Su
bpop
ulat
ions
Res
pond
Diff
eren
tly to
Cyc
lic C
ompr
essi
on in
vitr
o
*1 I. R
aizm
an, *
1 J.N. A
mrit
ha D
e C
roos
, 1,2 J-
P. S
t-Pie
rre,
1,3 R
. M. P
illia
r and
1,2,
4 R. A
. Kan
del
*bot
h au
thor
s con
tribu
ted
equa
lly to
this
wor
k.
1 CIH
R B
ioEn
gine
erin
g of
Ske
leta
l Tis
sues
Tea
m, M
ount
Sin
ai H
ospi
tal,
and
Uni
vers
ity o
f
Toro
nto.
2 Inst
itute
of B
iom
ater
ials
and
Bio
med
ical
Eng
inee
ring,
Uni
vers
ity o
f Tor
onto
,
3 Facu
lty o
f Den
tistry
, Uni
vers
ity o
f Tor
onto
, Tor
onto
, Can
ada.
4 Add
ress
cor
resp
onde
nce
to:
Rita
Kan
del,
MD
Dep
artm
ent o
f Pat
holo
gy a
nd L
abor
ator
y M
edic
ine,
Mou
nt S
inai
Hos
pita
l 600
Uni
vers
ity A
venu
e, S
uite
6-5
00,
Toro
nto,
Ont
ario
, Can
ada
M5G
1X
5.
Tele
phon
e: 4
16-5
86-8
516
Fax:
416
-586
-862
8
E-m
ail:
rkan
del@
mts
inai
.on.
ca
Run
ning
title
: Car
tilag
e su
bpop
ulat
ion
resp
onse
Key
wor
ds: c
yclic
com
pres
sion
, sub
popu
latio
n, c
hond
rocy
tes,
deep
zon
e, su
perf
icia
l-mid
zon
e.
Man
uscr
ipt a
ccep
ted:
Jour
nal o
f Tis
sue
Engi
neer
ing
A
119
Intr
oduc
tion
Arti
cula
r car
tilag
e, a
spec
ializ
ed ti
ssue
pre
sent
at th
e en
ds o
f bon
es, a
llow
s for
smoo
th
artic
ulat
ion
and
trans
mis
sion
ofa
pplie
d fo
rces
to th
e su
bcho
ndra
l bon
e. W
hen
dam
aged
by
dise
ase
or tr
aum
a, it
has a
lim
ited
self-
repa
ir po
tent
ial d
ue, i
n pa
rt, to
its a
vasc
ular
ity. T
issu
e
engi
neer
ing
artic
ular
car
tilag
e ex
viv
ow
hich
is th
en tr
ansp
lant
ed in
to a
join
t def
ecti
s a p
rom
isin
g
ther
apeu
tic a
ppro
ach.
In p
revi
ous s
tudi
es w
e de
velo
ped
the
met
hodo
logy
to fo
rmbi
phas
ic
cons
truct
s con
sist
ing
of a
n ar
ticul
ar c
artil
age-
like
com
pone
nt a
nd a
bon
e-in
terf
acin
g co
mpo
nent
1 .Whe
n su
bmitt
ed to
mec
hani
cal s
timul
atio
n, e
ither
cyc
lic c
ompr
essi
on o
r she
ar, t
he a
mou
nt o
f
extra
cellu
lar m
atrix
(EC
M) p
rodu
ced
bych
ondr
ocyt
es in
the
in v
itro
form
ed c
artil
age
sign
ifica
ntly
incr
ease
das
did
the
mec
hani
cal p
rope
rties
2, 3.W
e ha
ve p
revi
ousl
y sh
own
that
the
incr
ease
d EC
M sy
nthe
sis i
nduc
ed b
y a
sing
le e
piso
de o
f cyc
lic c
ompr
essi
on o
f cho
ndro
cyte
s
isol
ated
from
the
full-
thic
knes
s (FT
) of b
ovin
e ar
ticul
ar c
artil
age
is a
resu
lt of
a re
mod
ellin
g
proc
ess i
nvol
ving
a c
atab
olic
resp
onse
follo
wed
by
an a
nabo
lic re
spon
se 4 .T
his s
ingl
e
appl
icat
ion
of c
yclic
com
pres
sion
incr
ease
d bo
th th
e sy
nthe
sis o
fcol
lage
n an
d pr
oteo
glyc
an
mea
sure
d 24
hou
rs p
ost-s
timul
atio
n an
d at
four
wee
ks p
ost-s
timul
atio
n3,
4 .The
initi
al re
spon
se
incl
uded
upr
egul
atio
n of
bot
h ty
pe 1
mem
bran
e-bo
und
(MT1
) mat
rix m
etal
lopr
otei
nase
(MM
P)
MT1
-MM
P as
wel
l as M
MP-
13. I
ncre
ased
MT1
-MM
P ge
ne e
xpre
ssio
n w
asde
tect
ed w
ithin
30
min
utes
of i
nitia
ting
cycl
ic c
ompr
essi
on a
nd 2
hou
rsla
ter M
MP-
13 w
as e
leva
ted.
Cha
nges
in
prot
ein
leve
ls o
ccur
red
afte
r the
gen
e ch
ange
s. B
y 24
hou
rs b
oth
MT1
-MM
P an
d M
MP-
13 h
ad
retu
rned
to c
onst
itutiv
e le
vels
of e
xpre
ssio
n4,
5.T
hese
tran
sien
t cha
nges
app
ear t
o be
crit
ical
to
the
incr
ease
d m
atrix
acc
umul
atio
n th
at o
ccur
s und
er th
ese
stim
ulat
ory
cond
ition
sas s
how
n
thro
ugh
the
use
of p
harm
acol
ogic
al in
hibi
tors
and
deco
y ol
igod
eoxy
nucl
eotid
esto
supp
ress
cata
bolic
cha
nges
of M
MP-
13 a
nd M
T1-M
MP
4, 5.A
biph
asic
con
stru
ct in
whi
ch th
ere
is m
ore
120
carti
lage
tiss
ue w
ith g
reat
er m
echa
nica
l pro
perti
es h
as th
e ad
vant
age
in th
at it
mor
e cl
osel
y
mim
ics n
ativ
e tis
sue
and
coul
d po
tent
ially
exp
erie
nce
less
pro
blem
s fol
low
ing
trans
plan
tatio
n
into
the
join
t def
ect.
Arti
cula
r car
tilag
e, in
viv
o, c
an b
e di
vide
d in
to fo
ur d
istin
ct re
gion
s: A
) sup
erfic
ial z
one
B) m
iddl
e zo
ne C
) dee
p zo
ne, a
nd D
) cal
cifie
d la
yerw
hich
toge
ther
are
ess
entia
l for
pro
per
carti
lage
func
tiona
lity.
Each
zon
e is
cha
ract
eriz
ed b
y a
spec
ific
mat
rix c
ompo
sitio
nan
d
orga
niza
tion.
In k
eepi
ng w
ith th
is,b
ioen
gine
ered
tiss
ue fr
om d
iffer
ent c
artil
age
subp
opul
atio
ns
yiel
dstis
sue
with
diff
eren
t pro
perti
es6-
10.G
iven
the
phen
otyp
ic d
iffer
ence
s obs
erve
d, it
is li
kely
that
cyc
lic c
ompr
essi
on m
ay a
ffec
t one
zon
e of
car
tilag
e pr
efer
entia
lly. I
f onl
y a
sele
ct
subp
opul
atio
n of
cho
ndro
cyte
s can
resp
ond
to m
echa
nica
l loa
ding
und
er o
ur c
ondi
tions
this
may
expl
ain
why
we
obse
rved
an
appr
oxim
atel
y 30
% in
crea
se in
tiss
ue fo
rmat
ion.
Thi
s pos
sibi
lity
is
supp
orte
d by
wor
k sh
owin
g th
at th
e pe
ricel
lula
r mat
rix is
impo
rtant
in in
fluen
cing
cho
ndro
cyte
resp
onse
to m
echa
nica
l loa
ding
11.I
ndee
d,on
e st
udy
hass
how
n th
at su
bpop
ulat
ions
of
chon
droc
ytes
gro
wn
on a
garo
se b
eads
hav
e di
ffer
entia
l res
pons
esto
com
pres
sive
mec
hani
cal
stra
in12
.Fur
ther
mor
e,th
e ce
lls in
eac
h of
the
zone
s exp
erie
nce
diff
eren
t typ
es o
f for
ces w
hen
load
edin
viv
o13
. For
inst
ance
, as a
resu
lt of
thei
r loc
atio
n,de
ep z
one
(DZ)
cho
ndro
cyte
s are
subj
ecte
d to
shea
ring
stre
sses
upo
n lo
adin
g 13
. In
cont
rast
, cho
ndro
cyte
s fro
m th
e su
perf
icia
l and
mid
zon
es (S
MZ)
enc
ount
er m
ore
tens
ile a
nd c
ompr
essi
ve fo
rces
resp
ectiv
ely
13.S
o th
ece
ll
subp
opul
atio
n us
ed to
eng
inee
r car
tilag
e m
ight
be
very
crit
ical
to th
e de
velo
pmen
t ofh
yalin
e
carti
lage
with
the
abili
ty to
surv
ive
long
term
.
In th
e cu
rren
t stu
dy, t
he e
ffec
t of c
yclic
com
pres
sion
on
mat
rix sy
nthe
sis a
nd th
e cr
itica
l
early
gen
e an
d pr
otei
n ch
ange
s tha
t occ
ur fo
llow
ing
mec
hani
cal s
timul
atio
n w
ere
exam
ined
in
DZ
and
SMZ
chon
droc
yte
subp
opul
atio
ns d
urin
g tis
sue
form
atio
n. T
his w
ill a
llow
us t
o
121
dete
rmin
e w
heth
er a
par
ticul
ar su
bpop
ulat
ion
is re
spon
sibl
e fo
r the
incr
ease
d EC
M sy
nthe
sisa
nd
prov
ide
furth
er in
sigh
t int
o th
e co
ntrib
utio
n of
MM
P-13
and
MT1
-MM
P to
mat
rix re
mod
ellin
g.
Und
erst
andi
ng th
e re
spon
se o
f diff
eren
t cho
ndro
cyte
subp
opul
atio
ns to
mec
hani
cal s
timul
atio
n
durin
g tis
sue
form
atio
n w
ill fa
cilit
ate
our u
nder
stan
ding
of t
issu
e gr
owth
and
as w
ell a
llow
for
furth
er o
ptim
izat
ion
ofca
rtila
ge ti
ssue
dev
elop
men
t in
vitr
o.
Mat
eria
ls a
nd M
etho
ds
Cel
l Iso
latio
n
Cho
ndro
cyte
s wer
e se
lect
ivel
y ha
rves
ted
from
the
uppe
r 70%
(sup
erfic
ial-m
id z
one)
and
the
botto
m 3
0% (d
eep
zone
) of c
artil
age.
from
bovi
ne m
etac
arpa
l-pha
lang
eal j
oint
s (6
to 9
mon
th
old
calv
es).
Two
diff
eren
t sub
popu
latio
ns o
f cho
ndro
cyte
s rep
rese
ntin
g th
esu
perf
icia
l-mid
zon
e
(SM
Z)an
dth
e de
ep-z
one
(DZ)
as w
ell a
sful
l-thi
ckne
ss (F
T) c
hond
rocy
tes w
ere
isol
ated
as
prev
ious
ly d
escr
ibed
by se
quen
tial d
iges
tion
with
0.5
% p
rote
inas
e (S
igm
a-A
ldric
h C
hem
ical
Co.
, St.
Loui
s, M
O) a
nd 0
.1%
Col
lage
nase
A (R
oche
App
lied
Scie
nce,
Indi
anap
olis
, IN
) at 3
7o C,
5% C
O2
1, 1
4 .Cel
ls w
ere
re-s
uspe
nded
in H
am’s
F-1
2 su
pple
men
ted
with
5%
feta
l bov
ine
seru
m
(Sig
ma-
Ald
rich)
and
seed
ed a
t a d
ensi
ty o
f 160
,000
cel
ls/m
m2
onto
the
top
surf
ace
of c
ylin
dric
al
disc
s of c
alci
um p
olyp
hosp
hate
(CPP
) sur
roun
ded
by T
ygon
tubi
ng (4
.3 m
m d
iam
eter
x6
mm
heig
ht;T
herm
opla
stic
s Pro
cess
or In
c., S
an Jo
se, C
A).
CPP
subs
trate
s (4
mm
dia
met
er x
2 m
m
heig
ht) w
ere
prep
ared
asp
revi
ousl
y de
scrib
ed 15
.Cel
ls w
ere
obta
ined
from
two
to th
ree
anim
als
and
pool
ed to
geth
er to
obt
ain
suff
icie
nt n
umbe
rs to
do
an e
xper
imen
t.
Mec
hani
cal S
timul
atio
n
Follo
win
g th
ree
days
of s
tatic
cul
ture
, the
cho
ndro
cyte
s for
med
a th
in la
yer o
f tis
sue
on to
p of
122
and
inte
grat
ed w
ith th
e su
rfac
eof
the
CPP
subs
trate
.The
se b
ipha
sic
cons
truct
s wer
e th
en
cultu
red
in se
rum
-fre
e D
MEM
ove
rnig
ht b
efor
e un
derg
oing
unia
xial
, con
fined
cyc
lic
com
pres
sion
of 1
kPa
(cor
resp
ondi
ng to
a st
rain
of 1
.4%
) usi
ng a
mec
hani
cal s
timul
ator
(MA
CH
-1, B
iosy
ntec
h, M
ontre
al, C
AN
) for
30
min
utes
at a
freq
uenc
y of
1H
z.Se
rum
-fre
e
cond
ition
s wer
e us
ed to
elim
inat
e th
e ef
fect
s of s
erum
fact
ors.
The
resu
ltant
tiss
ue fo
rmat
ion
afte
r mec
hani
cal s
timul
atio
n in
seru
m-f
ree
cond
ition
s use
d in
De
Cro
os e
t al.,
(200
6) w
as si
mila
r
to th
at se
en in
Wal
dman
et a
l., (2
005)
perf
orm
ed in
seru
m.C
yclic
com
pres
sion
of t
he in
vitr
o
form
ed c
artil
age
was
app
lied
thro
ugh
a co
mpl
iant
2%
aga
rose
gel
cyl
inde
r (3.
5 m
m d
iam
eter
x 8
mm
hei
ght;
appr
oxim
atel
y 10
±1 k
Pa c
ompr
essi
ve st
iffne
ss, n
=6) (
Sigm
a-A
ldric
h). T
he a
garo
se
plug
-tiss
ue la
yer c
onst
ruct
was
def
orm
ed u
nder
dis
plac
emen
t con
trolw
ith th
e am
plitu
de o
f
cycl
icde
form
atio
n ad
just
ed m
anua
lly to
app
ly si
nuso
idal
load
ing
to th
e co
nstru
ct3 . A
garo
se
cylin
ders
wer
e re
mov
ed im
med
iate
ly a
fter m
echa
nica
l stim
ulat
ion.
Con
trol s
ampl
es w
ere
treat
ed
iden
tical
ly to
stim
ulat
ed sa
mpl
es, b
ut d
id n
ot re
ceiv
e an
y m
echa
nica
l stim
ulat
ion.
Con
stru
cts
rem
aine
d in
seru
m-f
ree
DM
EM u
ntil
the
appr
opria
te h
arve
st ti
me-
poin
t. Pr
evio
us st
udie
s hav
e
show
n th
at c
hond
rocy
tesd
o no
t adh
ere
toth
e ag
aros
e pl
ug d
urin
g lo
adin
g 3 .
His
tolo
gica
l ass
essm
ent o
f the
diff
eren
t cho
ndro
cyte
subp
opul
atio
ns
Afte
r 3 d
ays o
f cul
ture
, the
tiss
ues f
orm
ed o
n th
e to
p su
rfac
e of
the
CPP
wer
e re
mov
ed
usin
g a
scal
pel b
lade
(no.
11)
and
em
bedd
ed in
2%
aga
r (D
ifco,
Det
roit,
MI)
. Aga
r was
use
d to
faci
litat
e ha
ndlin
g of
the
tissu
e du
ring
subs
eque
nt p
roce
ssin
g.Sa
mpl
es w
ere
fixed
in 1
0%
neut
ral-b
uffe
red
form
alin
and
em
bedd
ed in
par
affin
. Sec
tions
wer
e cu
t, st
aine
d w
ith h
emat
oxyl
in
and
eosi
n (H
&E)
or t
olui
dine
blu
e, a
nd e
xam
ined
by
light
mic
rosc
opy.
123
Anal
ysis
of g
ene
expr
essi
on
Tota
l RN
A w
as e
xtra
cted
from
the
diff
eren
tcho
ndro
cyte
subp
opul
atio
ns b
efor
e an
d
durin
g va
rious
per
iods
of c
ultu
reus
ing
TRIz
ol R
eage
nt (I
nvitr
ogen
, Car
lsba
d, C
A).
Tota
l RN
A
was
then
reve
rse-
trans
crib
ed (R
T) in
to c
DN
A u
sing
Sup
ersc
ript I
I (In
vitro
gen)
. The
resu
lting
cDN
A w
as su
bjec
ted
to P
CR
in a
n Ep
pend
orf M
aste
rcyc
ler P
CR
usi
ng p
rimer
s and
con
ditio
ns
prev
ious
ly d
escr
ibed
4, 5.
PCR
pro
duct
s wer
e ru
n on
a 1
.5%
aga
rose
gel
and
vis
ualiz
ed b
y
stai
ning
with
eth
idiu
m b
rom
ide.
Ban
d in
tens
ity w
as se
mi-q
uant
ified
by
dens
itom
etry
usi
ng L
ab
Wor
ks so
ftwar
e (V
4.0,
Med
ia C
yber
nect
ics)
.Rel
ativ
e ge
ne e
xpre
ssio
n w
as n
orm
aliz
ed to
18S
rRN
A.
Anal
ysis
of p
rote
in e
xpre
ssio
n
Afte
r TR
izol
ext
ract
ion
and
rem
oval
of t
he a
queo
us p
hase
and
RN
A, p
rote
insw
ere
obta
ined
from
the
rem
aini
ng in
terp
hase
and
org
anic
pha
sesr
espe
ctiv
ely.
Pro
tein
swer
e qu
antif
ied
utili
zing
a b
icin
chon
inic
aci
d (B
CA
) pro
tein
ass
ay k
it (P
ierc
e, R
ockf
ord,
IL) a
nd
spec
troph
otom
etry
at 5
62 n
m a
bsor
banc
e (T
herm
o El
ectro
n M
ultiS
kan
Ex).
Bov
ine
seru
m
albu
min
was
use
d to
gen
erat
e a
stan
dard
cur
ve.T
otal
pro
tein
s fro
m e
ach
sam
ple
(20 μg
) wer
e
mix
ed (1
:1 v
/v) w
ith sa
mpl
e bu
ffer
con
tain
ing
100
mM
Tris
-HC
L (p
H 6
.8),
4% (w
/v) S
DS,
20%
(v/v
) gly
cero
l, 0.
2% (w
/v) b
rom
ophe
nol b
lue,
and
5%
β-m
erca
ptoe
than
ol (S
igm
a-A
ldric
h) a
nd
sepa
rate
d by
a 1
2% so
dium
-dod
ecyl
-sul
fate
pol
yacr
ylam
ide
gel (
SDS-
PAG
E)(2
hou
rs a
t 150
V)
and
elec
trobl
otte
d on
to n
itroc
ellu
lose
mem
bran
es (B
ioTr
ace
NT,
Pal
l Life
Sci
ence
s, Pe
nsac
ola,
FL) f
or 1
.5 h
ours
at 0
.3 m
A. T
he m
embr
anes
wer
e w
ashe
d in
TB
S-T
(10m
M T
ris, p
H 7
.5, 1
50
mM
NaC
l, an
d 0.
05%
v/v
Tw
een-
20) a
nd su
bseq
uent
ly b
lock
ed fo
r 1 h
our a
t roo
m te
mpe
ratu
re
in 5
% (w
/v) s
olut
ion
of n
on-f
at d
ry m
ilk in
TB
S-T.
Mem
bran
es w
ere
then
pro
bed
with
a p
rimar
y
124
antib
ody
reac
tive
with
eith
er M
MP-
13 (C
albi
oche
m, 1
:150
0) o
r MT1
-MM
P (S
anta
-Cru
z
Bio
tech
nolo
gies
; 1:2
000)
ove
rnig
ht a
t 4o C
. Mem
bran
es w
ere
then
incu
bate
d w
ith se
cond
ary
antib
odie
s for
det
ectio
n of
MM
P-13
(ant
i-mou
se, 1
:500
0; S
anta
Cru
z B
iote
chno
logi
es) o
r MT1
-
MM
P (a
nti-r
abbi
t, 1:
2000
; Cel
l Sig
nalin
g) fo
r 1 h
our a
t roo
m te
mpe
ratu
re. I
mm
unor
eact
ivity
was
det
ecte
d us
ing
enha
nced
che
milu
min
esce
nce
(EC
L Pl
us) (
GE
Hea
lthca
re, B
ucki
ngha
msh
ire,
UK
) and
ban
d in
tens
ity w
as se
mi-q
uant
ified
by
dens
itom
etry
usi
ng L
ab W
orks
softw
are
(V4.
0,
Med
ia C
yber
nect
ics)
. To
com
pens
ate
for l
oadi
ngva
riatio
ns, a
ll de
nsito
met
ric v
alue
s wer
e
corr
ecte
d by
nor
mal
izin
g to
β-a
ctin
(Sig
ma;
1:5
000)
.
Extr
acel
lula
r mat
rix
accu
mul
atio
n by
cho
ndro
cyte
s fro
m th
e di
ffere
nt su
bpop
ulat
ions
Follo
win
g th
e ap
plic
atio
n of
cyc
lic c
ompr
essi
ve lo
adin
g, c
hond
rocy
te-s
eede
d co
nstru
cts
(stim
ulat
ed a
nd u
nstim
ulat
ed c
ontro
ls) w
ere
incu
bate
d w
ith [35
S]-S
O4
(2 μ
Ci/c
onst
ruct
) and
[3 H]-
prol
ine
(2μC
i/con
stru
ct) f
or 2
4 ho
urs (
37o C
, 5%
CO
2) to
mea
sure
pro
teog
lyca
n an
d co
llage
n
synt
hesi
s res
pect
ivel
y. C
ultu
res w
ere
then
was
hed
in P
BS
and
dige
sted
by
papa
in (4
0 μg
/ml;
Sigm
a-A
ldric
h) in
pap
ain
dige
stio
n bu
ffer
(40
mM
am
mon
ium
ace
tate
, 1m
M E
DTA
, 2m
M D
TT)
at 6
5ºC
for 4
8 ho
urs.
The
amou
nt o
f new
ly sy
nthe
size
d pr
oteo
glyc
an a
nd c
olla
gen
reta
ined
in th
e
tissu
e w
as q
uant
ified
by
mea
surin
g ra
dioi
soto
pe in
corp
orat
ion
usin
g a
β-sc
intil
latio
n co
unte
r and
norm
aliz
ed to
DN
A c
onte
nt. T
he p
apai
n di
gest
ed sa
mpl
es w
ere
asse
ssed
for D
NA
con
tent
usi
ng
the
Hoe
chst
332
58 d
ye b
indi
ng a
ssay
and
fluo
rom
etry
as d
escr
ibed
pre
viou
sly
(Wal
dman
et a
l.
2002
).
Extr
acel
lula
r mat
rix
accu
mul
atio
n by
dee
p zo
ne c
hond
rocy
tes a
t diff
eren
t tim
e po
ints
125
Acc
umul
atio
n of
col
lage
n an
d pr
oteo
glyc
an w
as q
uant
ified
in D
Z ch
ondr
ocyt
es a
t 1, 3
,
and
5 da
ys a
fter i
nitia
l see
ding
. For
thes
e st
udie
s,cu
lture
s wer
e in
cuba
ted
in se
rum
-fre
e D
MEM
for f
our h
ours
prio
r to
mec
hani
cal s
timul
atio
nas
it w
asno
t pos
sibl
e to
do
a 24
hr se
rum
-fre
e pr
e-
incu
batio
n fo
r the
day
1 c
ultu
res.
Cul
ture
s wer
e th
en in
cuba
ted
with
radi
oiso
tope
s im
med
iate
ly
follo
win
g m
echa
nica
l stim
ulat
ion
as d
escr
ibed
abo
ve a
nd th
e am
ount
of n
ewly
synt
hesi
zed
colla
gen
and
prot
eogl
ycan
qua
ntifi
ed.
Stat
istic
al A
naly
sis
Each
con
ditio
n w
as p
erfo
rmed
in tr
iplic
ate
and
all e
xper
imen
ts w
ere
repe
ated
at l
east
3
times
. The
dat
a w
as p
oole
d an
d ex
pres
sed
as m
ean
± SE
M. R
esul
ts w
ere
eval
uate
d us
ing
a
stud
ent’s
t-te
st o
r an
AN
OV
Afo
llow
ed b
y Fi
sher
’s p
rote
cted
leas
t sig
nific
ant d
iffer
ence
test
(pos
t-hoc
).St
atis
tical
sign
ifica
nce
was
ass
igne
d at
p<0
.05.
Res
ults
Cho
ndro
cyte
pop
ulat
ion
enri
chm
ent
To d
eter
min
e th
e ef
ficac
y of
the
harv
estin
g of
the
chon
droc
yte
subp
opul
atio
ns, i
sola
ted
cells
wer
e an
alyz
ed fo
r the
exp
ress
ion
of su
bpop
ulat
ion-
spec
ific
mar
kers
prio
r to
seed
ing
on C
PP
subs
trate
s. C
ells
isol
ated
from
DZ
show
ed g
reat
er e
xpre
ssio
n of
MM
P-13
and
Col
lage
n ty
pe X
whe
reas
exp
ress
ion
of su
perf
icia
l zon
e pr
otei
n (S
ZP)a
nd c
artil
age
inte
rmed
iate
laye
r pro
tein
(CIL
P) w
ere
pref
eren
tially
exp
ress
ed in
the
SMZ
cells
(Fig
. 1).
Expr
essi
on o
f agg
reca
n an
d ty
pe
II c
olla
gen
(col
2a1)
was
evi
dent
in a
ll th
ree
subp
opul
atio
ns w
ith a
slig
htly
gre
ater
exp
ress
ion
in
DZ
cells
(Fig
. 1).
Extra
cellu
lar m
atrix
(EC
M) a
ccum
ulat
ion
in u
nstim
ulat
ed c
onst
ruct
s als
o
diff
ered
am
ongs
t the
diff
eren
t sub
popu
latio
n(F
ig. 3
).D
Z ce
lls a
ccum
ulat
ed fo
ur-f
old
mor
e
126
colla
gen
per c
ell t
han
SMZ
cells
(SM
Z=40
9±66
cpm
/μg
of D
NA
, DZ=
233
9±57
6cp
m/u
g D
NA
;
p<0.
01).
Sim
ilarly
DZ
chon
droc
ytes
acc
umul
ated
sign
ifica
ntly
mor
e pr
oteo
glyc
ans t
han
SMZ
cells
(SM
Z=57
0±82
cpm
/μg
of D
NA
, DZ
=285
6±52
8cp
m/μ
gof
DN
A) (
p<0.
05).
This
con
firm
s
that
the
met
hod
to o
btai
n th
ese
subp
opul
atio
ns w
as a
ppro
pria
tefo
r use
in th
e su
bseq
uent
stud
ies.
His
tolo
gica
l app
eara
nce
of th
e tis
sue
subj
ecte
d to
mec
hani
cal s
timul
atio
n
To c
onfir
m th
at th
e di
ffer
ent s
ubpo
pula
tions
form
ed ti
ssue
,the
cul
ture
s afte
r 3 d
ays (
at
the
time
whe
n th
ey w
ould
be
mec
hani
cally
stim
ulat
ed) w
ere
harv
este
d, a
nd th
e tis
sue
rem
oved
from
the
top
of th
e su
bstra
te, a
nd e
xam
ined
his
tolo
gica
lly. F
igur
e 2
show
s tha
t the
diff
eren
t
chon
droc
yte
subp
opul
atio
nsca
nfo
rm ti
ssue
. Int
eres
tingl
y ce
lls fr
om th
e D
Zap
pear
ed to
form
slig
htly
thic
ker t
issu
e th
an th
e ce
lls fr
om S
MZ
and
som
e ce
lls re
sem
bled
hyp
ertro
phic
chon
droc
ytes
with
per
icel
lula
r cle
arin
g on
the
tolu
idin
e bl
ue st
aine
d tis
sues
.
Effe
ct o
f mec
hani
cal s
timul
atio
n on
diff
eren
t cho
ndro
cyte
subp
opul
atio
ns
The
effe
ct o
f cyc
lic c
ompr
essi
on o
n ac
cum
ulat
ion
of n
ewly
synt
hesi
zed
colla
gen
and
prot
eogl
ycan
sby
the
diff
eren
t sub
popu
latio
ns w
as e
xam
ined
.The
re w
as a
sign
ifica
nt in
crea
se in
accu
mul
atio
n of
pro
teog
lyca
ns a
fter c
yclic
com
pres
sion
by
SMZ
cells
(incr
ease
of 7
5%).
In
tissu
es fo
rmed
by
DZ
chon
droc
ytes
, how
ever
, pro
teog
lyca
n ac
cum
ulat
ion
decr
ease
d by
40%
(Fig
. 3A
).M
echa
nica
l stim
ulat
ion
incr
ease
d th
e ac
cum
ulat
ion
of c
olla
gen
in th
e tis
sue
form
ed b
y
SMZ
chon
droc
ytes
by
appr
oxim
atel
y 65
% c
ompa
red
to u
nstim
ulat
ed c
ontro
ltis
sue
whi
le
decr
easi
ng th
e ac
cum
ulat
ion
of c
olla
gen
in th
e tis
sue
form
ed b
y D
Z ch
ondr
ocyt
es b
y
appr
oxim
atel
y 40
% (p
<0.0
1, F
ig. 3
B).
The
appl
icat
ion
of c
yclic
com
pres
sion
did
not
sign
ifica
ntly
alte
rthe
DN
A c
onte
nt o
f the
in v
itro-
form
ed ti
ssue
s(Ta
ble
1).
127
MT1
-MM
P ge
ne a
nd p
rote
in e
xpre
ssio
n
As d
iffer
ence
s wer
eob
serv
ed in
mat
rix a
ccum
ulat
ion
by th
e di
ffer
ent c
hond
rocy
te
popu
latio
ns, t
he c
ells
wer
e ex
amin
ed fo
r cha
nges
in M
T1-M
MP
over
tim
e as
this
pro
teas
e sh
ows
very
ear
ly c
hang
es fo
llow
ing
cycl
ic c
ompr
essi
on in
full
thic
knes
s cho
ndro
cyte
s and
has
bee
n
impl
icat
ed in
the
mec
hani
sm le
adin
g to
the
rem
odel
ling
of th
e ex
trace
llula
r mat
rixre
sulti
ngin
impr
oved
mat
rix a
ccum
ulat
ion.
The
pro
file
of M
T1-M
MP
gene
exp
ress
ion
diff
ered
bet
wee
n th
e
two
diff
eren
t cho
ndro
cyte
subp
opul
atio
ns (F
ig.4
). A
sign
ifica
nt in
crea
se in
MT1
-MM
P m
RN
A
was
obs
erve
d im
med
iate
ly fo
llow
ing
mec
hani
cal s
timul
atio
n in
SM
Z ce
lls (2
.5 fo
ld in
crea
se
com
pare
d to
con
trols
, dat
a no
t sho
wn,
p<0
.05)
. The
resp
onse
was
tran
sien
t and
dec
reas
ed to
cons
titut
ive
leve
ls b
y 2
hour
s.Th
e ch
ange
s in
MT1
-MM
P ex
pres
sion
wer
e si
mila
r to
that
obse
rved
in fu
ll th
ickn
ess c
ells
as d
escr
ibed
pre
viou
sly
4 .In
cont
rast
, DZ
chon
droc
yte
MT1
-
MM
P ge
ne e
xpre
ssio
n di
d no
t inc
reas
e un
til 2
4 ho
urs f
ollo
win
g m
echa
nica
l stim
ulat
ion
(2.5
fold
incr
ease
com
pare
d to
con
trols
,p<0
.05)
. The
leve
ls o
f exp
ress
ion
at th
is ti
me
wer
esi
gnifi
cant
ly
diff
eren
t tha
n th
eex
pres
sion
inSM
Zce
lls(p
<0.0
5).T
he re
spon
ses o
f the
SM
Z an
d D
Z ce
lls
wer
eop
posi
te to
eac
h ot
her.
MT1
-MM
P pr
otei
n ch
ange
s wer
e al
so d
etec
ted
(Fig
.4C
).W
este
rn b
lot a
naly
sis s
how
ed a
sign
ifica
nt in
crea
se (2
.5 fo
ld) i
n M
T1-M
MP
prot
ein
expr
essi
on in
SMZ
chon
droc
ytes
att
wo
hour
s (p<
0.05
) whi
ch w
as d
iffer
ent f
rom
the
DZ
cells
(p<0
.05)
. The
incr
ease
was
tran
sien
t and
by 1
2 ho
urs h
ad re
turn
edto
con
stitu
tive
leve
lsin
kee
ping
with
the
gene
cha
nges
.In
cont
rast
,
sign
ifica
ntly
incr
ease
d pr
otei
n le
vels
of M
T1-M
MP
in th
e tis
sues
form
ed b
y D
Z ce
lls w
ere
only
dete
cted
at 2
4 ho
urs,
whi
ch w
as in
kee
ping
with
the
gene
cha
nges
.
128
MM
P-13
gen
e an
d pr
otei
n ex
pres
sion
The
gene
exp
ress
ion
prof
ile o
f MM
P-13
, ano
ther
mat
rix p
rote
ase
know
n to
be
invo
lved
in th
e ea
rly re
spon
se to
mec
hani
cal s
timul
atio
n, w
as a
lso
exam
ined
(Fig
.5A
). Th
ere
wer
e
sign
ifica
nt d
iffer
ence
s obs
erve
d be
twee
n th
e SM
Zan
d D
Z ch
ondr
ocyt
es im
med
iate
ly fo
llow
ing
stim
ulat
ion
(p<0
.05)
. The
exp
ress
ion
leve
ls in
SM
Z w
ere
sign
ifica
ntly
ele
vate
d (s
timul
ated
:
cont
rolr
atio
of 1
.5,p
<0.0
5) im
med
iate
ly fo
llow
ing
stim
ulat
ion
and
rem
aine
d in
crea
sed
thro
ugho
ut th
e 24
hou
r per
iod.
In c
ontra
st, i
n D
Z ch
ondr
ocyt
es th
ere
was
a d
ecre
ase
in
expr
essi
on im
med
iate
ly fo
llow
ing
stim
ulat
ion
but t
he g
ene
leve
ls in
crea
sed
until
12
hour
s (5-
fold
incr
ease
com
pare
d to
0 h
our,
p<0.
05).
The
incr
ease
was
tran
sien
t and
by
24 h
ours
MM
P-13
had
retu
rned
to c
onst
itutiv
e le
vels
of e
xpre
ssio
n.
Follo
win
g m
echa
nica
l stim
ulat
ion
ther
e w
as a
sign
ifica
nt d
ecre
ase
in M
MP-
13 p
rote
in
expr
essi
onin
the
SMZ
chon
droc
ytes
(Fig
. 5B
)but
pro
tein
exp
ress
ion
retu
rned
to c
onst
itutiv
e
leve
ls b
y 2
hour
s and
show
ed n
o fu
rther
cha
nges
.In
cont
rast
, no
sign
ifica
nt c
hang
es in
the
expr
essi
on o
f MM
P-13
wer
e ob
serv
ed in
DZ
chon
droc
ytes
at th
e tim
es e
xam
ined
.No
sign
ifica
nt
chan
ges w
ere
obse
rved
in a
ggre
can
and
colla
gen
gene
exp
ress
ion
at th
e tim
e-po
ints
inve
stig
ated
(dat
a no
t sho
wn)
.
Mat
rix
accu
mul
atio
n by
deep
zone
cho
ndro
cyte
sinf
luen
ces c
ell r
espo
nse
to m
echa
nica
l loa
ding
To in
vest
igat
e w
hy D
Z ch
ondr
ocyt
es re
spon
ded
diff
eren
tially
to m
echa
nica
l stim
ulat
ion,
the
role
of t
he E
CM
in m
echa
notra
nsdu
ctio
n w
as in
vest
igat
ed o
win
g to
the
diff
eren
ces i
n
thic
knes
s bet
wee
n SM
Z an
d D
Z tis
sue
by D
ay 3
. Thi
s was
don
e by
subj
ectin
g D
Z cu
lture
s to
cycl
ic c
ompr
essi
on a
t Day
s 1, 3
, and
5 to
test
how
EC
M a
ccum
ulat
ion
wou
ld in
fluen
ce th
e
129
resp
onse
of m
echa
nica
l stim
ulat
ion
(Fig
.6).
DZ
cells
stim
ulat
ed a
t day
1 h
ad a
sign
ifica
nt
incr
ease
in th
e ac
cum
ulat
ion
of b
oth
new
ly sy
nthe
size
d pr
oteo
glyc
ans a
nd c
olla
gens
whe
n
com
pare
d to
cor
resp
ondi
ng u
nstim
ulat
ed c
ontro
l tis
sues
(p<0
.05)
. Asi
gnifi
cant
dec
reas
e in
accu
mul
atio
n w
as d
etec
ted
incu
lture
s stim
ulat
ed a
t day
s 3an
d 5
(p<0
.05)
.D
Z cu
lture
s at D
ay 3
had
sign
ifica
ntly
less
acc
umul
ated
pro
teog
lyca
ns c
ompa
red
to D
ay 5
DZ
cultu
res (
data
not
show
n).
Dis
cuss
ion
In th
is st
udy
the
resp
onsi
vene
ss o
f sup
erfic
ial-m
id z
one
(SM
Z)or
dee
p zo
ne (D
Z)
chon
droc
ytes
to o
ne a
pplic
atio
n of
cyc
lic c
ompr
essi
ondu
ring
invi
tro
tissu
e fo
rmat
ion
was
exam
ined
. The
enr
ichm
ent o
f the
cho
ndro
cyte
subp
opul
atio
ns w
as c
onfir
med
thro
ugh
RT-
PCR
,
quan
tific
atio
n of
mat
rix sy
nthe
sis,
and
by h
isto
logy
.Cel
ls is
olat
ed fr
om th
e D
Z an
d se
eded
ont
o
CPP
subs
trate
syie
lded
a th
icke
r tis
sue
com
pare
d to
the
tissu
e fo
rmed
by
FT o
rSM
Z ce
llsw
ithin
3 da
ys o
f cul
ture
.The
FT
and
SMZ
tissu
es a
ppea
red
to b
e of
sim
ilar t
hick
ness
es.T
his w
as n
ot
unex
pect
ed a
s SM
Z m
akes
up a
bout
70%
of t
he F
T ca
rtila
ge. A
s wel
l as p
rodu
cing
mor
phol
ogic
ally
dis
tinct
tiss
ues,
thes
e ph
enot
ypic
cha
nges
tran
slat
ed in
to si
gnifi
cant
diff
eren
ces
in m
atrix
acc
umul
atio
nfo
llow
ing
mec
hani
cal s
timul
atio
n. T
here
wer
esi
gnifi
cant
incr
ease
s in
prot
eogl
ycan
and
col
lage
n ac
cum
ulat
ion
with
in 2
4 ho
urs(
p<0.
05)i
n tis
sues
form
ed b
y SM
Z
cells
,whi
le ti
ssue
s for
med
by
DZ
cells
show
ed d
ecre
ased
accu
mul
atio
n of
thes
e
mac
rom
olec
ules
. Asw
ould
be
expe
cted
bas
ed o
n ou
r pre
viou
s stu
dies
, MT1
-MM
P sh
owed
an
incr
ease
in g
ene
and
prot
ein
expr
essi
on in
the
early
tim
e po
ints
follo
win
g m
echa
nica
l stim
ulat
ion
4, 5. T
he D
Z ce
lls,w
hich
did
not
resp
ond
posi
tivel
yto
stim
ulat
ion,
did
not d
evel
opth
ese
early
chan
ges.
How
ever
the
DZ
cells
wer
e re
spon
sive
to st
imul
atio
n as
gen
e an
d pr
otei
n ch
ange
s did
130
occu
r but
at a
late
r tim
e po
int.
The
inhi
bito
ry re
spon
seof
DZ
cells
to c
yclic
com
pres
sion
seem
sto
be re
late
d to
the
amou
ntan
d/or
com
posi
tion
of th
e EC
M th
at is
gen
erat
ed b
ecau
se st
imul
atio
n of
DZ
cells
whe
n
ther
e w
as li
ttle
peric
ellu
lar m
atrix
, 24
hour
s afte
r see
ding
, had
a p
ositi
ve re
spon
se to
cyc
lic
com
pres
sion
.Tak
en to
geth
er th
ese
resu
lts in
dica
te th
at th
e SM
Z ce
lls a
re re
spon
sibl
e fo
r the
impr
ovem
ent i
n EC
M a
ccum
ulat
ion
that
occ
urs i
n de
velo
ping
car
tilag
e af
ter c
yclic
com
pres
sion
.
The
degr
adat
ive
resp
onse
by
DZ
cells
may
exp
lain
why
we
obse
rved
onl
y a
smal
l, ap
prox
imat
ely
30%
, inc
reas
e in
mat
rix a
ccum
ulat
ion
in o
ur p
revi
ous s
tudi
es. A
lthou
gh o
ur d
ata
sugg
ests
that
DZ
cells
deg
rade
s the
EC
M w
hen
expo
sed
to th
is ty
pe o
f mec
hani
cal l
oadi
ng,i
t is n
ot p
ossi
ble
to
pred
ict h
owth
ese
cells
will
beh
ave
in th
e pr
esen
ce o
f SM
Z ce
lls a
s occ
urs w
hen
they
are
mix
ed
toge
ther
to b
ioen
gine
er ti
ssue
as o
ccur
s fol
low
ing
cell
isol
atio
n fr
om c
artil
age.
It m
ay b
eth
at th
e
diff
eren
t sub
popu
latio
ns o
f arti
cula
r car
tilag
e co
mm
unic
ate
with
eac
h ot
her,
perh
aps b
y
synt
hesi
zing
for e
xam
ple,
solu
ble
med
iato
rs a
nd/o
r oth
er E
CM
com
pone
nts,
resu
lting
in n
on-
addi
tive
effe
cts.
We
have
show
n pr
evio
usly
that
cho
ndro
cyte
s are
cap
able
of c
ross
-talk
in
co-
cultu
re 16
and
alth
ough
the
mec
hani
sm(s
) has
not b
een
dete
rmin
ed, i
t lik
ely
occu
rs v
iaa
solu
ble
fact
or. A
TP17
or P
THrP
18ar
e po
ssib
le fa
ctor
s tha
t hav
e be
en sh
own
to m
edia
te in
tera
ctio
ns
betw
een
chon
droc
ytes
.
Our
dat
a al
so in
dica
tes t
hat t
he p
eric
ellu
lar m
atrix
(PC
M) p
lays
an
impo
rtant
role
in
regu
latin
g th
e ch
ondr
ocyt
es’r
espo
nse
to c
yclic
com
pres
sion
and
may
expl
ain
why
DZ
and
SMZ
cells
resp
ond
diff
eren
tly to
mec
hani
cal s
timul
atio
n. W
e ob
serv
ed th
at w
ith in
crea
sing
mat
rix
accu
mul
atio
n th
at m
echa
nica
l stim
ulat
ion
wen
t fro
m h
avin
g a
posi
tive
effe
ct o
n th
e fir
st d
ay to
a
nega
tive
effe
ct b
y th
e th
ird d
ay o
f cul
ture
. Alth
ough
the
prec
ise
func
tion
of th
e PC
Mha
s not
been
fully
elu
cida
ted,
ther
e is
con
side
rabl
e ev
iden
ce th
at su
gges
ts it
is in
volv
ed in
the
regu
latio
n
131
of b
ioch
emic
al a
nd b
iom
echa
nica
l int
erac
tions
bet
wee
n th
e ce
lls a
nd th
e su
rrou
ndin
g EC
M 11
, 19,
20. I
t is w
ell a
ccep
ted
that
in a
rticu
lar c
artil
age
the
resi
ding
cho
ndro
cyte
s mus
t fun
ctio
n
syne
rgis
tical
ly w
ith th
e EC
M in
ord
er to
abs
orb,
redi
strib
ute
and
trans
mit
phys
iolo
gica
l
com
pres
sive
and
shea
ring
forc
es to
the
subc
hond
ral b
one.
As c
hond
rocy
tes a
re c
ompl
etel
y
surr
ound
ed b
y th
e PC
M a
ny si
gnal
s fro
m th
e EC
M m
ust p
ass t
hrou
gh th
e PC
Mbe
fore
they
are
sens
ed b
y th
e ch
ondr
ocyt
es. A
s suc
h it
has b
een
hypo
thes
ized
that
the
PCM
may
func
tion
as a
trans
duce
r of b
iom
echa
nica
l for
cesa
nd it
can
do
soin
a v
arie
ty o
f way
s. Th
e PC
M in
fluen
ces t
he
fixed
cha
rge
dens
ity e
xper
ienc
ed d
urin
g ca
rtila
ge lo
adin
g, w
hich
can
affe
ct c
hond
rocy
te a
ctiv
ity
21, 2
2 .Alte
rnat
ivel
y ch
ondr
ocyt
es a
re k
now
n to
pro
duce
and
resp
ond
to fa
ctor
s suc
h as
ATP
23,
FGF
24, a
nd th
e B
MP
fam
ily 25
.As c
ompo
nent
s of t
he E
CM
,suc
h as
dec
orin
and
big
lyca
n, a
re
diff
eren
tially
pro
duce
d by
DZ
and
SMZ
cells
, the
se c
ould
regu
late
cel
ls d
irect
ly o
r ind
irect
ly b
y
sequ
este
ring
diff
eren
t gro
wth
fact
ors26
.Thi
s int
erpl
ay b
etw
een
ECM
mol
ecul
es a
nd so
lubl
e
med
iato
rs a
t the
intra
cellu
lar l
evel
add
s fur
ther
to th
e co
mpl
exity
und
erly
ing
the
chon
droc
ytes
’
resp
onse
to m
echa
nica
l loa
ding
. .A
noth
er m
echa
nism
cou
ld in
volv
e bi
ndin
g of
cho
ndro
cyte
inte
grin
s to
ECM
mol
ecul
es. T
his i
nter
actio
nre
sults
in th
e fo
rmat
ion
of a
dhes
ion
plaq
ues,
whi
ch
in tu
rn, i
s a p
rere
quis
ite fo
r cho
ndro
cyte
resp
onsi
vene
ss to
var
ious
gro
wth
fact
ors 27
. Rec
ent
wor
k ha
s sho
wn
that
the
PCM
func
tions
to p
rovi
de a
hom
ogen
ous c
ell-l
evel
stra
in a
mon
gst t
he
carti
lage
subp
opul
atio
ns w
hen
expo
sed
tova
riabl
e lo
cal E
CM
stra
ins 28
.Fur
ther
stud
y is
requ
ired
to d
elin
eate
the
exac
t mec
hani
sm(s
) reg
ulat
ing
chon
droc
yte
func
tion.
The
timin
g of
the
MM
P-13
gen
e an
d pr
otei
n ch
ange
s diff
er fr
om th
ose
obse
rved
in o
ur
prev
ious
stud
ies 4,
5.T
here
are
thre
epo
ssib
le re
ason
s for
this
. Firs
tly, t
he p
rese
nt st
udy
was
don
e
usin
g en
riche
d zo
nal p
opul
atio
ns a
nd if
ther
e is
cro
ss-ta
lk b
etw
een
the
diff
eren
t zon
es th
is c
ould
noto
ccur
thus
resu
lting
in d
iffer
ent r
espo
nses
. Sec
ondl
y th
e st
udie
s wer
epe
rfor
med
in a
132
diff
eren
t cul
ture
med
ia, D
MEM
as o
ppos
ed to
Ham
’s F
12, t
he fo
rmer
is a
cul
ture
con
ditio
n
favo
ured
by
DZ
cells
29.H
am’s
F-1
2 ha
s low
er su
lfate
com
pare
d to
DM
EM w
hich
alo
ng w
ith
othe
r diff
eren
ces i
n ch
emic
al c
ompo
sitio
n m
ay a
lso
play
a ro
le in
cel
lula
r res
pons
e.A
s wel
l,
DM
EM h
as a
hig
herC
a2+co
ncen
tratio
nth
an H
am’s
F12
(200
mg/
L an
d 33
.20
mg/
L re
spec
tivel
y)
and
prev
ious
stud
ies h
ave
show
n th
at C
a2+si
gnal
ling
is in
volv
ed in
the
mec
hano
trans
duct
ion
even
ts in
resp
onse
to fl
uid
flow
30, 3
1an
d hy
dros
tatic
pre
ssur
e 32
in c
hond
rocy
tes.
The
appl
icat
ion
of p
hysi
olog
ical
leve
ls o
f com
pres
sive
stra
in o
n ch
ondr
ocyt
es se
eded
with
in a
thre
e-di
men
sion
al
agar
ose
cons
truct
resu
lts in
intra
cellu
lar C
a2+si
gnal
ling
that
can
be
bloc
ked
with
EG
TA, a
chel
ator
of e
xtra
cellu
lar C
a2+an
d si
gnifi
cant
ly re
duce
d w
ith G
d3+, w
hich
is k
now
n to
blo
ck
volta
ge-o
pera
ted
Ca2+
chan
nels
33. A
noth
er st
udy
repo
rted
that
ext
race
llula
r Ca2+
is c
ruci
al fo
r
the
prop
agat
ion
of C
a2+w
aves
in c
ultu
red
chon
droc
ytes
follo
win
g m
echa
nica
l stim
ulat
ion
23.O
ur
resu
lts su
gges
t tha
t cho
ndro
cyte
s may
resp
ond
diff
eren
tially
dep
endi
ng o
n th
e ex
trace
llula
r Ca2+
conc
entra
tion
whi
ch w
ould
be
in k
eepi
ng w
ith th
e st
udie
s. H
owev
er,a
s the
se m
edia
diff
er in
othe
r com
pone
nts i
t is p
ossi
ble
that
one
of t
hese
oth
er, a
s yet
uni
dent
ified
, mol
ecul
es m
ay b
e
resp
onsi
ble.
Thi
rdly
, in
the
prev
ious
stud
y M
MP-
13 p
rote
in le
vels
in th
e m
edia
wer
e ev
alua
ted;
whe
reas
the
curr
ent s
tudy
mea
sure
tiss
ue le
vels
of M
MP-
13.
Nev
erth
eles
s,ev
en th
ough
ther
e ar
e
diff
eren
ces i
n th
e tim
ing
of th
e M
MP-
13 a
ltera
tions
, MT1
-MM
P ch
ange
s occ
ur w
ithin
the
first
two
hour
s in
SMZ
tissu
es a
nd th
ese
corr
elat
edw
ith in
crea
sed
mat
rix a
ccum
ulat
ion
sim
ilar t
o th
e
chan
ges w
e de
scrib
ed p
revi
ousl
y in
the
tissu
es fo
rmed
by
cells
obt
aine
d fr
om fu
ll th
ickn
ess
carti
lage
. Thi
s als
o ra
ises
the
poss
ibili
tyth
at M
T1-M
MP
mig
ht b
e m
ore
impo
rtant
than
MM
P13
in re
gula
ting
tissu
e gr
owth
in v
itro.
In su
mm
ary,
cyc
lic c
ompr
essi
on h
as a
diff
eren
tial e
ffec
t on
the
indi
vidu
al c
artil
age
subp
opul
atio
ns. S
MZ
resp
onde
d po
sitiv
ely
to a
sing
le a
pplic
atio
n of
cyc
lic c
ompr
essi
on,
133
whe
reas
DZ
resp
onde
d ne
gativ
ely.
The
DZ
resp
onse
can
be
attri
bute
d to
the
amou
nt a
nd/o
r typ
e
of m
atrix
that
acc
umul
ates
as c
yclic
com
pres
sion
appl
ied
at a
n ea
rlier
tim
epo
int i
n gr
owth
did
incr
ease
mat
rix a
ccum
ulat
ion
by D
Z ce
lls. O
ur re
sults
sugg
est t
hat S
MZ
cells
may
be
resp
onsi
ble
for t
he im
prov
ed ti
ssue
form
atio
n th
at re
sults
follo
win
g cy
clic
com
pres
sion
. For
min
g ca
rtila
ge
with
the
appr
opria
te a
mou
nt o
f SM
Z ce
lls m
ay b
e cr
itica
l to
succ
essf
ully
bio
engi
neer
arti
cula
r
carti
lage
, a p
artic
ular
pro
blem
if u
sing
ost
eoar
thrit
ic c
artil
age,
whi
ch m
ay la
ck a
supe
rfic
ial z
one
and
porti
ons o
f the
mid
zon
e, a
s the
sour
ce o
f cel
ls.
Ack
now
ledg
men
ts
This
wor
k w
as su
ppor
ted
byC
IHR
and
the
Arth
ritis
Soc
iety
. We
than
k M
r. H
arry
Boj
arsk
i and
Ryd
ing-
Reg
ency
Mea
t Pac
kers
for p
rovi
ding
the
tissu
es fo
r the
se st
udie
s.
134
Figu
re L
egen
ds
Figu
re 1
: Gen
e ex
pres
sion
of c
hond
rocy
te su
bpop
ulat
ion
phen
otyp
e m
arke
rs.T
otal
RN
A
was
ext
ract
ed fr
om su
perf
icia
l-mid
zon
e (S
MZ)
, dee
p zo
ne (D
Z) a
nd fu
ll th
ickn
ess (
FT)
chon
droc
ytes
imm
edia
tely
afte
r iso
latio
n fr
om c
artil
age.
RT-
PCR
usi
ng p
rimer
s spe
cific
for
gene
s ind
icat
ive
of p
heno
type
wer
e us
ed to
ass
ess t
he p
urity
of e
ach
subp
opul
atio
n. A
naly
ses
wer
e pe
rfor
med
in tr
iplic
ate
from
thre
e se
para
te e
xper
imen
ts (n
=9).
Rep
rese
ntat
ive
gels
from
a
sing
le e
xper
imen
t are
show
n.A
vera
ge d
ensi
tom
etric
val
ues n
orm
aliz
ed to
18S
rRN
A a
re sh
own
belo
w e
ach
gene
.
Figu
re 2
: His
tolo
gica
l app
eara
nce
of ti
ssue
s aft
er th
ree
days
ofc
ultu
re.
Cho
ndro
cyte
s wer
e is
olat
ed fr
om A
) sup
erfic
ial-m
id z
one,
B) d
eep
zone
or C
) ful
l thi
ckne
ss c
artil
age,
gro
wn
for
thre
e da
ys, r
emov
ed fr
om th
e su
rfac
e of
the
CPP
subs
trate
s, an
d pr
oces
sed
for h
isto
logi
cal
exam
inat
ion.
Sec
tions
wer
e st
aine
d w
ith e
ither
hem
atox
ylin
and
eos
in o
r tol
uidi
ne b
lue.
*i
ndic
ates
whe
re su
bstra
te w
as lo
cate
d.
Figu
re 3
: Eff
ect o
f a si
ngle
app
licat
ion
of m
echa
nica
l stim
ulat
ion
on a
ccum
ulat
ion
of n
ewly
sy
nthe
size
d co
llage
n an
d pr
oteo
glyc
an.C
hond
rocy
tes f
rom
eith
er su
perf
icia
l-mid
or d
eep
zone
w
ere
mec
hani
cally
stim
ulat
ed a
nd th
en in
cuba
ted
with
3 H-p
rolin
e an
d 35
S-SO
4to
labe
l col
lage
ns
and
prot
eogl
ycan
s res
pect
ivel
y; c
ontro
l con
stru
cts d
id n
ot re
ceiv
e an
y m
echa
nica
l stim
ulat
ion.
Tw
enty
-fou
r hou
rs la
ter,
the
amou
nt o
f new
ly sy
nthe
size
d pr
oteo
glyc
an (A
) and
col
lage
n (B
) ac
cum
ulat
ed w
as d
eter
min
ed b
y qu
antif
ying
radi
oact
ivity
in th
e tis
sue
as d
escr
ibed
und
er th
e m
etho
ds. R
esul
ts w
ere
pool
ed a
nd e
xpre
ssed
as m
ean
± st
anda
rd e
rror
of t
he m
ean
(n=9
). A
s in
dica
ted
ther
e is
a si
gnifi
cant
incr
ease
from
resp
ectiv
e un
stim
ulat
ed c
onst
ruct
s, *p
<0.0
2,
#p<0
.05.
Figu
re 4
: MT
1-M
MP
gene
and
pro
tein
exp
ress
ion
follo
win
g a
sing
le a
pplic
atio
n of
cyc
lic
com
pres
sion
.A) R
epre
sent
ativ
e ge
l sho
win
g PC
R p
rodu
ctso
ver t
ime
in b
oth
supe
rfic
ial-m
id
zone
or d
eep
zone
tiss
ues.
The
leve
ls o
f exp
ress
ion
wer
e se
mi-q
uant
ified
by
dens
itom
etry
and
th
e fo
ld c
hang
es (B
) bet
wee
n m
echa
nica
lly st
imul
ated
and
con
trol t
issu
es fo
rmed
by
diff
eren
t ch
ondr
ocyt
e po
pula
tions
ove
r a 2
4 ho
ur ti
me
perio
d fo
llow
ing
mec
hani
cal s
timul
atio
n w
ere
dete
rmin
ed. S
igni
fican
t diff
eren
ces w
ithin
eac
h zo
ne a
nd o
ver t
ime,
are
indi
cate
d, w
here
*p
=0.0
16, *
*p=0
.006
, and
#p<
0.05
with
resp
ect t
o ze
ro ti
me-
poin
t. C
) Wes
tern
blo
t ana
lysi
s and
de
nsito
met
ry w
ere
used
to d
eter
min
efo
ld c
hang
es in
MT1
-MM
P pr
otei
ns le
vels
bet
wee
n st
imul
ated
and
con
trol u
nstim
ulat
ed ti
ssue
s for
med
by
diff
eren
t cho
ndro
cyte
subp
opul
atio
ns o
ver
time.
Res
ults
wer
e po
oled
and
exp
ress
ed a
s mea
n ±
stan
dard
err
or o
f the
mea
n (n
=6).*
, #de
note
s si
gnifi
cant
diff
eren
ces w
here
*p<
0.05
with
resp
ect t
o th
e 2
hour
tim
e-po
int;
#p<0
.05
with
resp
ect
to th
e 24
-hou
r tim
e po
int.
Figu
re 5
: MM
P-13
gen
e an
d pr
otei
n ex
pres
sion
follo
win
g a
sing
le a
pplic
atio
n of
cyc
lic
com
pres
sion
in c
hond
rocy
tes f
rom
supe
rfic
ial-m
id a
nd d
eep
zone
s.A
) Rep
rese
ntat
ive
gel
show
ing
PCR
pro
duct
s. Th
e le
vels
of e
xpre
ssio
n w
ere
sem
i-qua
ntifi
ed b
y de
nsito
met
ry a
nd th
e fo
ld c
hang
es (B
) bet
wee
n m
echa
nica
lly st
imul
ated
and
con
trol t
issu
es fo
rmed
by
diff
eren
t ch
ondr
ocyt
e po
pula
tions
ove
r a 2
4 ho
ur ti
me
perio
d fo
llow
ing
mec
hani
cal s
timul
atio
n w
ere
dete
rmin
ed. S
igni
fican
t diff
eren
ces w
ithin
eac
h zo
ne a
nd o
ver t
ime,
are
indi
cate
d, w
here
*p=
0.02
with
resp
ect t
o ze
ro ti
me-
poin
t. #p
<0.0
5 to
cor
resp
ondi
ng u
nstim
ulat
ed c
ontro
l. C
) Wes
tern
blo
t
135
anal
ysis
and
den
sito
met
ry w
ere
used
to d
eter
min
efo
ld c
hang
es in
MM
P-13
prot
eins
leve
ls
betw
een
stim
ulat
ed a
nd c
ontro
l uns
timul
ated
tiss
ues f
orm
ed b
y di
ffer
ent c
hond
rocy
te
subp
opul
atio
ns o
verti
me.
Res
ults
wer
e po
oled
and
exp
ress
ed a
s mea
n ±
stan
dard
err
or o
f the
m
ean
(n=6
) Res
ults
wer
e po
oled
and
exp
ress
ed a
s mea
n ±
stan
dard
err
or o
f the
mea
n (n
=6).*
p=0.
008
with
resp
ect t
o ze
ro ti
me-
poin
t.
Figu
re 6
: Acc
umul
atio
n of
new
ly sy
nthe
size
d co
llage
n an
d pr
oteo
glyc
ans b
y de
ep z
one
chon
droc
ytes
. Cho
ndro
cyte
s fro
m th
e de
ep z
one
at e
ither
1, 3
or 5
day
s fol
low
ing
initi
atio
n of
th
e cu
lture
s wer
e m
echa
nica
lly st
imul
ated
and
then
incu
bate
d w
ith 3 H
-pro
line
and
35S-
SO4
to
labe
l col
lage
ns a
nd p
rote
ogly
cans
resp
ectiv
ely;
con
trol c
onst
ruct
s did
not
rece
ive
any
mec
hani
cal
stim
ulat
ion.
Tw
enty
-fou
r hou
rs la
ter,
the
amou
nt o
f new
ly sy
nthe
size
d pr
oteo
glyc
an (o
pen
bar)
an
d co
llage
n (f
illed
bar
s) a
ccum
ulat
ed w
ere
dete
rmin
ed b
y qu
antif
ying
radi
oact
ivity
in th
e tis
sue
as d
escr
ibed
und
er th
e m
etho
ds a
nd n
orm
aliz
ed fo
r DN
A. E
xper
imen
ts w
ere
perf
orm
ed in
du
plic
ates
and
repe
ated
thre
e tim
es (n
=6).
Res
ults
wer
e po
oled
and
exp
ress
ed a
s mea
n ±
stan
dard
er
ror o
f the
mea
n. *
deno
tes s
igni
fican
t inc
reas
e fr
om c
orre
spon
ding
uns
timul
ated
con
trol,
p<0.
05. a de
note
s sig
nific
ant d
ecre
ase
from
cor
resp
ondi
ng u
nstim
ulat
ed c
ontro
l, p<
0.05
.
136
137
138
139
140
141
142
143
144
Tabl
e 1.
DN
A c
onte
nt o
f tis
sue
form
ed b
y th
e ch
ondr
ocyt
e su
bpop
ulat
ions
.Cho
ndro
cyte
s
wer
e is
olat
ed fr
om su
perf
icia
l-mid
zon
e (S
MZ)
, dee
p zo
ne (D
Z) o
r ful
l thi
ckne
ss (F
T) o
f
carti
lage
and
gro
wn
in c
ultu
re fo
r 3 d
ays.
The
cells
wer
e m
echa
nica
lly st
imul
ated
as d
escr
ibed
unde
r the
Met
hods
. DN
A c
onte
nt w
as d
eter
min
ed 2
4 ho
urs a
fter s
timul
atio
n. T
he re
sults
from
3
expe
rimen
ts w
ere
pool
ed a
nd e
xpre
ssed
as m
ean
± st
anda
rd e
rror
of t
he m
ean
(n=
9). N
o
sign
ifica
nt d
iffer
ence
in D
NA
con
tent
was
obs
erve
d be
twee
n un
stim
ulat
ed (c
ontro
l) an
d
stim
ulat
ed ti
ssue
s for
med
by
the
diff
eren
t cho
ndro
cyte
pop
ulat
ions
.
Cho
ndro
cyte
Popu
latio
n
Con
trol t
issu
e
(μg
of D
NA
/con
stru
ct)
Stim
ulat
ed ti
ssue
s
(μg
of D
NA
/con
stru
ct)
FT9.
04 ±
0.8
8.55
± 1
.4
SMZ
9.74
± 2
.07.
35 ±
0.5
DZ
6.84
± 1
.48.
48 ±
1.2
145
Ref
eren
ce L
ist
1.W
aldm
an S
D, G
rynp
as M
D, P
illia
r RM
, Kan
del R
A. C
hara
cter
izat
ion
of c
artil
agen
ous
tissu
e fo
rmed
on
calc
ium
pol
ypho
spha
te su
bstra
tes i
n vi
tro. J
Bio
med
Mat
er R
es.
2002
;62:
323-
330.
2.W
aldm
an S
D, S
pite
ri C
G, G
rynp
as M
D, P
illia
r RM
, Kan
del R
A. L
ong-
term
inte
rmitt
ent
shea
r def
orm
atio
n im
prov
es th
e qu
ality
of c
artil
agin
ous t
issu
e fo
rmed
in v
itro.
J O
rthop
Res
. 200
3;21
:590
-596
.
3.W
aldm
an S
D, C
outo
DC
, Gry
npas
MD
, Pill
iar R
M, K
ande
l RA
. A si
ngle
app
licat
ion
of
cycl
ic lo
adin
g ca
n ac
cele
rate
mat
rix d
epos
ition
and
enh
ance
the
prop
ertie
s of t
issu
e-
engi
neer
ed c
artil
age.
Ost
eoar
thrit
is C
artil
age.
200
5.
4.D
e C
roos
JN, D
haliw
al S
S, G
rynp
as M
D, P
illia
r RM
, Kan
del R
A. C
yclic
com
pres
sive
mec
hani
cal s
timul
atio
n in
duce
s seq
uent
ial c
atab
olic
and
ana
bolic
gen
e ch
ange
s in
chon
droc
ytes
resu
lting
in in
crea
sed
extra
cellu
lar m
atrix
acc
umul
atio
n. M
atrix
Bio
l.
2006
;25:
323-
331.
5.D
e C
roos
JN, J
ang
B, D
haliw
al S
S, G
rynp
as M
D, P
illia
r RM
, Kan
del R
A. M
embr
ane
type
-
1 m
atrix
met
allo
prot
eina
se is
indu
ced
follo
win
g cy
clic
com
pres
sion
of i
n vi
tro g
row
n
bovi
ne c
hond
rocy
tes.
Ost
eoar
thrit
is C
artil
age.
200
7.
146
6.A
ydel
otte
MB
, Gre
enhi
ll R
R, K
uettn
er K
E. D
iffer
ence
s bet
wee
n su
b-po
pula
tions
of
cultu
red
bovi
ne a
rticu
lar c
hond
rocy
tes.
II. P
rote
ogly
can
met
abol
ism
. Con
nect
Tis
sue
Res
.
1988
;18:
223-
234.
7.A
ydel
otte
MB
, Kue
ttner
KE.
Diff
eren
ces b
etw
een
sub-
popu
latio
ns o
f cul
ture
d bo
vine
artic
ular
cho
ndro
cyte
s. I.
Mor
phol
ogy
and
carti
lage
mat
rix p
rodu
ctio
n. C
onne
ct T
issu
e
Res
. 198
8;18
:205
-222
.
8.K
lein
TJ,
Schu
mac
her B
L, S
chm
idt T
A, L
i KW
, Voe
gtlin
e M
S, M
asud
a K
et a
l.Ti
ssue
engi
neer
ing
of st
ratif
ied
artic
ular
carti
lage
from
cho
ndro
cyte
subp
opul
atio
ns. O
steo
arth
ritis
Car
tilag
e. 2
003;
11:5
95-6
02.
9.Si
czko
wsk
i M, W
att F
M. S
ubpo
pula
tions
of c
hond
rocy
tes f
rom
diff
eren
t zon
es o
f pig
artic
ular
car
tilag
e. Is
olat
ion,
gro
wth
and
pro
teog
lyca
n sy
nthe
sis i
n cu
lture
. J C
ell S
ci.
1990
;97
( Pt 2
):349
-360
.
10.
Wal
dman
SD
, Gry
npas
MD
, Pill
iar R
M, K
ande
l RA
. The
use
of s
peci
fic c
hond
rocy
te
popu
latio
ns to
mod
ulat
e th
e pr
oper
ties o
f tis
sue-
engi
neer
ed c
artil
age.
J O
rthop
Res
.
2003
;21:
132-
138.
11.
Gui
lak
F, A
lexo
poul
os L
G, H
aide
r MA
, Tin
g-B
eall
HP,
Set
ton
LA. Z
onal
uni
form
ity in
mec
hani
cal p
rope
rties
of t
he c
hond
rocy
te p
eric
ellu
lar m
atrix
: mic
ropi
pette
asp
iratio
n of
cani
ne c
hond
rons
isol
ated
by
carti
lage
hom
ogen
izat
ion.
Ann
Bio
med
Eng
. 200
5;33
:131
2-
1318
.
147
12.
Lee
DA
, Nog
uchi
T,K
nigh
t MM
, O'D
onne
ll L,
Ben
tley
G, B
ader
DL.
Res
pons
e of
chon
droc
yte
subp
opul
atio
ns c
ultu
red
with
in u
nloa
ded
and
load
ed a
garo
se. J
Orth
op R
es.
1998
;16:
726-
733.
13.
Pool
e A
R, K
ojim
a T,
Yas
uda
T, M
wal
e F,
Kob
ayas
hi M
, Lav
erty
S. C
ompo
sitio
n an
d
stru
ctur
e of
arti
cula
r car
tilag
e: a
tem
plat
e fo
r tis
sue
repa
ir. C
lin O
rthop
Rel
at R
es.
2001
;S26
-S33
.
14.
Kan
del R
A, B
oyle
J, G
ibso
n G
, Cru
z T,
Spe
agle
M. I
n vi
tro fo
rmat
ion
of m
iner
aliz
ed
carti
lage
nous
tiss
ue b
y ar
ticul
ar c
hond
rocy
tes.
In V
itro
Cel
l Dev
Bio
l Ani
m.1
997;
33:1
74-
181.
15.
Pilli
ar R
M, F
iliag
gi M
J, W
ells
JD, G
rynp
as M
D, K
ande
l RA
. Por
ous c
alci
um
poly
phos
phat
e sc
affo
lds f
or b
one
subs
titut
e ap
plic
atio
ns --
in v
itro
char
acte
rizat
ion.
Bio
mat
eria
ls. 2
001;
22:9
63-9
72.
16.
Gan
L, K
ande
l RA
. In
vitro
car
tilag
e tis
sue
form
atio
n by
Co-
cultu
re o
f prim
ary
and
pass
aged
cho
ndro
cyte
s. Ti
ssue
Eng
. 200
7;13
:831
-842
.
17.
Kud
irka
JC, P
anup
inth
u N
, Tes
seym
an M
A, D
ixon
SJ,
Ber
nier
SM
. P2Y
nuc
leot
ide
rece
ptor
sign
alin
g th
roug
h M
APK
/ER
K is
regu
late
d by
ext
race
llula
r mat
rix:i
nvol
vem
ent
of b
eta3
inte
grin
s. J C
ell P
hysi
ol. 2
007;
213:
54-6
4.
148
18.
Jian
g J,
Leon
g N
L, M
ung
JC, H
idak
a C
, Lu
HH
. Int
erac
tion
betw
een
zona
l pop
ulat
ions
of
artic
ular
cho
ndro
cyte
s sup
pres
ses c
hond
rocy
te m
iner
aliz
atio
n an
d th
is p
roce
ss is
med
iate
d
by P
THrP
.Ost
eoar
thrit
is C
artil
age.
200
8;16
:70-
82.
19.
Ale
xopo
ulos
LG
, Set
ton
LA, G
uila
k F.
The
bio
mec
hani
cal r
ole
of th
e ch
ondr
ocyt
e
peric
ellu
lar m
atrix
in a
rticu
lar c
artil
age.
Act
a B
iom
ater
. 200
5;1:
317-
325.
20.
Gra
ff R
D, K
elle
y SS
, Lee
GM
. Rol
e of
per
icel
lula
r mat
rix in
dev
elop
men
t of a
mec
hani
cally
func
tiona
l neo
carti
lage
. Bio
tech
nol B
ioen
g. 2
003;
82:4
57-4
64.
21.
Lai W
M, S
un D
D, A
tesh
ian
GA
, Guo
XE,
Mow
VC
. Ele
ctric
al si
gnal
s for
cho
ndro
cyte
s in
carti
lage
. Bio
rheo
logy
. 200
2;39
:39-
45.
22.
Sanc
hez
JC, P
owel
lT, S
tain
es H
M, W
ilkin
s RJ.
Elec
troph
ysio
logi
cal d
emon
stra
tion
of
Na+
/Ca2
+ ex
chan
ge in
bov
ine
artic
ular
cho
ndro
cyte
s. B
iorh
eolo
gy. 2
006;
43:8
3-94
.
23.
D'A
ndre
a P,
Vitt
ur F
. Pro
paga
tion
of in
terc
ellu
lar C
a2+
wav
es in
mec
hani
cally
stim
ulat
ed
artic
ular
cho
ndro
cyte
s. FE
BS
Lett.
199
7;40
0:58
-64.
24.
Pow
ers C
J, M
cLes
key
SW, W
ells
tein
A. F
ibro
blas
t gro
wth
fact
ors,
thei
r rec
epto
rs a
nd
sign
alin
g. E
ndoc
r Rel
at C
ance
r. 20
00;7
:165
-197
.
149
25.
Zhu
Y, O
gane
sian
A, K
eene
DR
, San
dell
LJ. T
ype
IIA
pro
colla
gen
cont
aini
ng th
e cy
stei
ne-
rich
amin
o pr
opep
tide
is d
epos
ited
in th
e ex
trace
llula
r mat
rix o
f pre
chon
drog
enic
tiss
ue a
nd
bind
s to
TGF-
beta
1 an
d B
MP-
2. J
Cel
l Bio
l. 19
99;1
44:1
069-
1080
.
26.
Hild
ebra
nd A
, Rom
aris
M, R
asm
usse
n LM
, Hei
nega
rd D
, Tw
ardz
ik D
R, B
orde
r WA
et a
l.
Inte
ract
ion
of th
e sm
all i
nter
stiti
al p
rote
ogly
cans
big
lyca
n, d
ecor
in a
nd fi
brom
odul
in w
ith
trans
form
ing
grow
th fa
ctor
bet
a. B
ioch
em J.
199
4;30
2 ( P
t 2):5
27-5
34.
27.
Cla
ncy
RM
, Red
iske
J, T
ang
X, N
ijher
N, F
renk
el S
, Phi
lips M
et a
l.O
utsi
de-in
sign
alin
g
in th
e ch
ondr
ocyt
e. N
itric
oxi
de d
isru
pts f
ibro
nect
in-in
duce
d as
sem
bly
of a
subp
lasm
alem
mal
act
in/rh
o A
/foca
l adh
esio
n ki
nase
sign
alin
g co
mpl
ex. J
Clin
Inve
st.
1997
;100
:178
9-17
96.
28.
Cho
i JB
, You
n I,
Cao
L, L
eddy
HA
, Gilc
hris
t CL,
Set
ton
LAet
al.
Zona
l cha
nges
in th
e
thre
e-di
men
sion
al m
orph
olog
y of
the
chon
dron
und
er c
ompr
essi
on: t
he re
latio
nshi
p am
ong
cellu
lar,
peric
ellu
lar,
and
extra
cellu
lar d
efor
mat
ion
in a
rticu
lar c
artil
age.
J Bio
mec
h.
2007
;40:
2596
-260
3.
29.
Sun
Y, K
ande
l R. D
eep
zone
arti
cula
r cho
ndro
cyte
s in
vitro
exp
ress
gen
es th
at sh
ow
spec
ific
chan
ges w
ith m
iner
aliz
atio
n. J
Bon
e M
iner
Res
. 199
9;14
:191
6-19
25.
30.
Yel
low
ley
CE,
Jaco
bs C
R, D
onah
ue H
J. M
echa
nism
s con
tribu
ting
to fl
uid-
flow
-indu
ced
Ca2
+ m
obili
zatio
n in
arti
cula
r cho
ndro
cyte
s. J C
ell P
hysi
ol. 1
999;
180:
402-
408.
150
31.
Yel
low
ley
CE,
Han
cox
JC, D
onah
ue H
J. Ef
fect
s of c
ell s
wel
ling
on in
trace
llula
r cal
cium
and
mem
bran
e cu
rren
ts in
bov
ine
artic
ular
cho
ndro
cyte
s. J C
ell B
ioch
em. 2
002;
86:2
90-
301.
32.
Hal
l AC
. Diff
eren
tial e
ffec
ts o
f hyd
rost
atic
pre
ssur
e on
cat
ion
trans
port
path
way
s of
isol
ated
arti
cula
r cho
ndro
cyte
s. J C
ell P
hysi
ol. 1
999;
178:
197-
204.
33.
Rob
erts
SR
, Kni
ght M
M, L
ee D
A, B
ader
DL.
Mec
hani
cal c
ompr
essi
on in
fluen
ces
intra
cellu
lar C
a2+
sign
alin
g in
cho
ndro
cyte
s see
ded
in a
garo
se c
onst
ruct
s. J A
ppl P
hysi
ol.
2001
;90:
1385
-139
1.
