Supporting Information
Wiley-VCH 2013
69451 Weinheim, Germany
Non-Invasive and In Situ Characterization of the Degradation of
Biomaterial Scaffolds by Volumetric Photoacoustic Microscopy**
Yu Shrike Zhang, Xin Cai, Junjie Yao, Wenxin Xing, Lihong V. Wang,* and Younan Xia*
anie_201306282_sm_miscellaneous_information.pdf
anie_201306282_sm_movie_s1.wmv
anie_201306282_sm_movie_s2.wmv
anie_201306282_sm_movie_s3.wmv
anie_201306282_sm_movie_s4.wmv
1
Experimental Section
Materials
:
Gelatin (type A, from porcine skin), sorbitan monooleate (Span
®
80), and toluene
(99.8%) were all obtained from Sigma
-
Aldrich and used as received for fabricating the gela
tin
microspheres with uniform sizes. PLGA (lactide:glycolide = 50:50 and 75:25), and 1,4
-
dioxane
(99.8%) were also obtained from Sigma
-
Aldrich and used as received for fabricating the
scaffolds. The water used in all fabrications was obtained by filtering
through a set of cartridges
(Millipore).
Preparation of inverse opal scaffolds
:
PLGA inverse opal scaffolds were fabricated
according to our previously reported protocols.
[
1b, 6c, 6d, 9
]
Briefly, uniform gelatin microspheres
(about 1.5 wt.%) were collected
as a suspension in methanol in a 50
-
mL centrifuge tube. The
tube was gently tapped to pack the microspheres into a cubic close
-
packed lattice. The lattice
was placed in an oven heated at 70
o
C for 1 h to induce necking between the adjacent
microspheres. A
fter cooling to room temperature, the lattice was carefully harvested using a
spatula, placed on a filter paper to remove methanol, and then infiltrated with a PLGA solution in
1,4
-
dioxane (30 wt.%, containing
a
mass ratio of 1:1 of the two types of PLGAs
,
50:50 and
75:25
). After removing the excess PLGA solution with filter paper, the pellet containing PLGA
solution was frozen in a refrigerator (
-
20
o
C) for 5 h, and then lyophilized in a freeze
-
dryer
(Labconco) overnight. The sample was immersed in ethanol, vacuumed briefly to eliminate air
bubbles trapped in the sample, and placed in 900 mL of water heated at 45
o
C under magnetic
stirring for 3
–
4 h to
dissolve the gelatin microspheres. To fabricate formazan
-
doped scaffolds,
0.5 wt.%
(
i.e.
, 1.67 wt.% to PLGA)
MTT formazan (Sigma
-
Aldrich) was added into the PLGA
solution in 1,4
-
dioxane and
this
solution was used to infiltrate the gelatin lattice. All the
other
procedures were
kept
the same as
for
making plain scaffolds. The scaffolds were then
stored
in
PBS (Invitrogen) prior to use.
Animals and implantation of scaffolds
:
All animal experiments were performed in
accordance with protocols approved by the W
ashington University Department of Comparative
Medicine and the Animal Studies Committee. Athymic nude mice 4
–
5 weeks old were obtained
from Harlan and housed under specific pathogen
-
free conditions in the animal facility at
Washington University. All the s
caffolds were sterilized in 70% ethanol for at least 2 h prior to
implantation. The scaffolds were then implanted subcutaneous
ly
in the ears of the mice.
[6c]
Briefly,
animals in all groups were anesthetized by administration of gaseous isoflurane (2%,
2
Butl
er Inc., Dublin) and aseptically prepared. One parasagittal incision was made on the top side
of the ear of each animal approximately 0.5 cm to the head. Subcutaneous pockets were created
lateral to the incision by using blunt dissection. One scaffold was
inserted into the subcutaneous
pocket using a pair of tweezers (one scaffold per mouse). The skin incision was then closed with
9
-
0 Ethilon suture (Ethicon) and secured using GLUture topical tissue adhesive (Abott
Laboratories).
Degradation of inverse opal
scaffolds
: For
in vitro
experiments, i
nverse opal scaffolds
immersed in 5 mL of either PBS or PBS containing 0.025 wt.% lipase (Sigma
-
Aldrich) at 37
o
C.
The medium was changed every day. One group of scaffolds (N = 4)
was fixed onto glass slides
using a s
mall amount of epoxy (one scaffold per slide), and
was subject
ed to non
-
invasive PAM
imaging. T
he scaffolds in the other group (N = 3
for each time point
) were used for mass loss
measurement. Briefly, three scaffolds were removed from the medium each week,
washed
thoroughly with water
,
and allowed to
air
-
dry. The
mass
of each dry scaffold was measured
using a microbalance (Cahn C
-
30)
,
then
, each scaffold was dissolved in 0.2 mL 1,4
-
dioxane
,
and
absorbance at 562 nm was obtained by a spectrophotometer (Tecan
Infinite 200). The amount of
MTT formazan was calculated according to a calibration curve. For
in vivo
experiments, one
group of implanted scaffolds (N = 3) was monitored non
-
invasively using PAM, and the
scaffolds in the other group (N = 3 for each time
point) w
ere
used for mass loss measurement.
Three scaffolds were explanted each week and freeze
-
dried. Each scaffold/tissue construct was
dissolved in dichloromethane (DCM), and the insoluble parts were removed by centrifugation.
The DCM solution containin
g dissolved PLGA was freeze
-
dried and the mass was measured
using a microbalance. To determine the background mass caused by soluble tissue components,
ear tissue
samples
with approximately the same volume w
ere
subjected to the same analysis.
Photoacoustic
microscopy (PAM) and signal processing
:
Two PAM systems were used for
this study: optical
-
resolution PAM (OR
-
PAM) and acoustic
-
resolution PAM (AR
-
PAM).
For
PAM, the optical and ultrasonic foci are configured coaxially and confocally
. For the OR
-
PAM,
the o
ptical focal spot size was approximately 5
μm
,
much smaller in diameter than the acoustic
focus
, so
the lateral resolution was predominantly determined by
the optical focal spot size, the
so
-
called optical resolution
.
For t
he
AR
-
PAM, the
optical focal spot
size was 2 mm in diameter
in a clear medium
,
much larger than the ultrasonic focus
, so
the lateral resolution was
predominantly determined by
the ultrasonic focus, the so
-
called acoustic resolution
.
3
OR
-
PAM was used to monitor the degradation of scaffolds
in vitro
.
[13]
Briefly,
The
nanosecond pulsed laser beam from a tunable dye laser (CBR
-
D, Sirah) pumped by a Nd:YLF
laser (INNOSLAB, Edgewave) was tightly focused into the sample by an optical objective (NA
=
0.1 in air). An optical
-
acoustic beam combiner c
omposed of a layer of silicone oil sandwiched
by two prisms was used for the coaxial and confocal alignment of the optical illumination and
acoustic detection. The resultant ultrasound waves were first focused by a concave acoustic lens
(NA
=
0.5 in water)
ground into the bottom of the combiner, and then detected by a wideband
ultrasonic transducer (V214
-
BB
-
RM, Olympus
-
NDT), which had a central frequency of 50 MHz
and a 6
-
dB nominal bandwidth of 70%. A correction lens offset the optical aberration of the
pr
ism.
The O
R
-
PAM could achieve a lateral resolution of approximately 5 μm, an axial
resolution of approximately1
5
μm, and a penetration depth of
about 1
mm.
AR
-
PAM was used to monitor the degradation of scaffolds
in vivo
.
[14]
Briefly, o
ptical
excitation
wa
s
provided by the same laser system as for OR
-
PAM, and the
photo
acoustic signals
were detected using
an
ultrasonic transducer.
The light was coupled into a multimode optical
fiber
(
M30L02, Thorlabs
)
and reshaped by a conical lens to form a ring pattern on t
he tissue
surface. The ring
-
shaped light pattern was then weakly focused into the sample by an optical
condenser,
the light focus
overlapped with the tight detection ultrasonic focus
.
T
he
AR
-
PAM
could achieve a lateral resolution of approximately 45 μm, an
axial resolution of approximately
15 μm, and a penetration depth of about 3 mm, when an ultrasonic transducer with a central
frequency of 50 MHz was used. Alternatively, when using an ultrasonic transducer with a central
frequency of 20 MHz, the AR
-
PAM ha
d a lateral resolution of approximately 80 μm, an axial
resolution of approximately 30 μm, and a penetration depth of up to 5
–
10 mm.
All v
olumetric data were processed using custom MATLAB (Mathworks) programs.
Scanning electron microscopy (SEM)
:
SEM (Nova
NanoSEM 2300, FEI) was used to
characterize the gelatin lattices and the PLGA inverse opal scaffolds. Prior to imaging, the
samples were sputtered with gold for 60 s. Images were taken at an accelerating voltage of 5 kV.
Statistics
:
All the results were pr
esented as mean ± standard error for each experimental
group. Four scaffolds were monitored for
in vitro
PAM imaging, and the other three scaffolds
were used for mass measurement at each time point.
Three
mice were monitored for
in vivo
PAM
imaging, and th
e other three
mice were sacrificed
for histology
analyses
at each time point.
4
Figure S1.
Degradation of pristine and MTT formazan
-
doped PLGA inverse opal scaffolds in
PBS and lipase solution, as measured by the mass loss assay. The addition of MTT for
mazan did
not alter the degradation profiles of the scaffolds.
5
Figure S2.
a) The change in the mass of MTT formazan as a function of degradation time for the
scaffolds in PBS, in the absence and presence of lipase, respectively. b) The change in the mass
percentage of MTT formazan relative to the scaffolds as a function of degr
adation time of the
scaffolds in PBS, in the absence and presence of lipase, respectively. The amount of MTT
formazan remained almost unchanged during the period of 6 weeks, indicating that
essentially
no
dye leaked out from the scaffold during incubation.
6
Figure S3.
An OR
-
PAM MAP image of a PLGA inverse opal scaffold doped with MTT
formazan
, showing the uniform pores and interconnecting windows
. The image
was
color
-
coded
by depth
of maximum
. MAP stands for “maximum amplitude projection”.
7
Figure
S4.
A
coronal
AR
-
PAM MAP image of a PLGA inverse opal scaffold doped with MTT
formazan
,
showing the
porous structure. MAP stands for “maximum amplitude projection”.
8
Figure S5.
Coronal
AR
-
PAM MAP images separat
ely
showing the
implanted
PLGA inverse
opa
l scaffold and the blood vessels in the ear of a nude mouse at weeks 0, 1, 3, and 6 post
implantation. MAP stands for “maximum amplitude projection”.
9
Figure S6.
a
–
c)
Coronal (top row) and sagittal (bottom row)
PAM MAP images showing the
PLGA inverse op
al scaffold subcutaneously
implanted
at the dorsal site of a nude mouse, at
week 2 post implantation, with (a) 0, (b) 1, and (c) 2 layers of chicken breast overlaid on top of
the skin. MAP stands for “maximum amplitude projection”.
1
0
Figure S7.
Coronal (
top row) and sagittal (bottom row)
PAM MAP images showing the PLGA
inverse opal scaffold subcutaneously
implanted
at the dorsal site of a nude mouse, at (a) week 2
and (b) week 4 post implantation. MAP stands for “maximum amplitude projection”.
11
Movie S1.
A 3D co
-
registered dual
-
wavelength
A
R
-
PAM movie showing
the scaffold and the
vasculature simultaneously at week 0 post implantation
,
in the ear of a nude mouse
.
Movie S2.
A 3D co
-
registered dual
-
wavelength
A
R
-
PAM movie showing
the scaffold and the
vasculature simultaneously at week 1 post implantation
,
in the ear of a nude mouse
.
Movie S3.
A 3D co
-
registered dual
-
wavelength
A
R
-
PAM movie showing
the scaffold and the
vasculature simultaneously at week 3 post implantation
,
in the ear of a nude mouse
.
Movie S4.
A 3D co
-
registered dual
-
wavelength
A
R
-
PAM movie showing
the scaffold and the
vasculature simultaneously at week 6 post implantation
,
in the ear of a nude mouse
.