Mice
anesthetized
and
subjected
to
bilateral
full-thickness
articular
cartilage
defects
in
knee
joints
at
8
wks
of
age
-
4
Mice
euthanized
at
indicated
times
and
knee
joints
harvested
for
microscopy
Articular
cartilage
wound
healing
Equipment
and
supplies
Surgical
kit
27-gauge
needle
6-0
absorbable
polypropylene
suture
VetClose
skin
glue
CO2
chamber
Paraffin
Microtome
Polylysine-coated
slides
(Fisher
Scientific)
Brightfield
microscope
(Eclipse
E800,
Nikon)
with
mounted
camera
(2000R
Fast1394,
Retiga)
QCapturePro
software
(QImaging)
Metal
ear
punch
(Fisher
Scientific)
Grid-etched
reticle
Reagents
and
solutions
Ketamine
(100
mg/kg
body
weight)/Xylazine
(20
mg/mL)/Acepromazine
(10
mg/mL)
10%
neutral
buffered
formalin
10%
formic
acid
in
5%
formaldehyde
solution
0.1
M
EDTA
in
phosphate
buffered
saline
(PBS)
Ethanol
Toluidine
blue
(0.04%
solution
prepared
in
0.1
M
sodium
acetate
(pH
4.0))
Xylene
Procedure:
Ear
wound
punch
and
analysis
A
through-and-through
hole
(2mm
diameter)
is
created
bilaterally
in
the
center
of
the
cartilaginous
part
of
the
external
ear
using
a
metal
ear
punch.
Holes
are
measured
30
days
after
wounding
using
a
grid-etched
reticle.
Healing
area
is
calculated
by
deducting
the
diameter
of
the
residual
hole
from
2
mm.
Procedure:
Full-thickness
cartilage
injury
and
analysis
Mice
are
anesthetized
i.p.
with
rodent
cocktail
(ketamine/xylazine/acepromazine).
A
small
medial
parapatellar
skin
incision
is
made,
the
joint
capsule
opened,
and
the
patella
luxated
laterally
to
expose
the
trochlear
groove
articulating
surface.
A
full-thickness
lesion
is
made
by
creating
a
circular
defect
in
the
cartilage
with
a
sterile
needle,
using
a
circular
motion
until
the
subchondral
bone
is
reached
(confirmed
by
appearance
of
a
blood
droplet
following
removal
of
the
needle).
The
joint
capsule
is
closed
with
absorbable
sutures;
subcutaneous
tissue
is
apposed
with
a
single
mattress
suture,
and
VetClose
skin
glue
is
applied
on
the
skin
to
bridge
over
the
closed
edges.
Mice
are
euthanized
at
indicated
time
points
post-wounding
and
knee
joints
are
dissected,
fixed
(formalin),
and
decalcified
(formic
acid/formaldehyde)
for
48
h.
Mouse
joints
are
washed
twice
with
water
and
incubated
in
EDTA
for
4-6
h
at
room
temperature.
Samples
are
washed
4
times
with
water
(15
min
each
time).
Samples
are
paraffin
embedded,
mounted
in
paraffin
blocks,
and
sagittally
sectioned
at
5µ
intervals
extending
through
the
entire
trochlear
groove.
Sections
are
mounted
on
microscope
slides
and
dewaxed.
Every
third
slide
is
subjected
to
toluidine
blue
staining
(5
min),
washed
3
times
with
water,
twice
with
ethanol,
and
twice
with
xylene
before
being
coverslipped.
Stained
sections
are
viewed
using
4X
and
10X
objectives
and
images
captured
by
a
mounted
camera
and
software.
Three
observers,
who
were
blinded
with
regard
to
sample
identity,
each
scored
3
selected
sections.
Specimens
were
assigned
a
score
ranging
from
0
(no
healing)
to
14
(full
healing).