as the blank control to identify the total vBMD and trabecular
anhydrous ethanol was added to assist with the removal of water.
At first the grid was dried thoroughly in air and then at 35 ꢃC for
24 h to get the sample for photography under the TEM. The
shape and size of the TEM image was obtained from counting
over 100 species in a randomly selected region. All determina-
tions were carried out in triplicate grids. The TEM was operated
at 80 kV, electron beam accelerating voltage. The nano-images
were recorded on an imaging plate (Gatan Bioscan Camera
Model 1792) with 20 eV energy windows at 6000–400 000ꢀ. The
images were digitally enlarged.
vBMD in the femur of the mice receiving 4a–c plus prednisone.
2.3.3. Bone weight and mineral content. After the mice were
sacrificed the left femur of the mice was collected immediately.
After the complete removal of the muscle, the length of the femur
was measured and then immersed in a mixed solution of chlo-
roform and methanol (2 : 1) twice (each 3 h). To remove the fat
ꢃ
the femur was heated at 120 C for 6 h, and then cooled. After
recording dry weight the femur was calcined in a furnace at
800 ꢃC for 8 h and then cooled to weigh the ash weight. The
weight ratio of the ash to dry femur (the mineral content of the
femur) was calculated. The femur ash was dissolved in 0.5 mL of
hydrochloric acid (6 N) and diluted to 5.00 mL with ultrapure
water, from which 0.05 mL of the solution was drawn and diluted
to 1.00 mL with ultrapure water before use. The calcium content
of the aqueous solution was measured with the method of
o-methyl-phenolphthalein complexing ketone.41,42 The phos-
phorous content of the aqueous solution was measured with the
use of molybdenum blue.43,44
2.4.3. The size of the nano-particle of 4a–c in normal saline
(NS). The size of the nano-particle of 4a–c in NS was measured
on a Malvern’s Zeta Sizer (Nano-ZS90) with DTS (Nano)
Program. The concentration of the solution of 4a–c in NS was
1.1 mM. The testing temperature was 25 ꢃC. The time interval was
30 s. The mean size and half-peak width of ten times were
recorded.
2.4.4. The zeta-potential of 4a–c in NS. The surface zeta-
potential of the nano-particle of 4a–c in NS was measured on a
ZetaPlus Potential Analyzer (ZetaPlus S/N 21394, Brookhaven
Instruments Coorpo-ration) with BIC Zeta Potential Analyzer.
The concentration of the solution of 4a–c in NS was 1.1 mM and
2.3.4. Bleeding ceased time. After the 4-week treatment of
estradiol, the mice from the groups 4a–c were weighed to record
the body weight. Thirty and ninety minutes after the last
administration, the mice were given an in vivo tail bleeding ceased
time assay following a standard procedure. In brief, the mouse
was placed in a tube holder with its tail protruding, and a 2 mm
cut was made on the tail. Flowing blood was gently wiped away
with a filter paper every 30 s until it stopped to yield the bleeding
ceased time.45,46
ꢃ
the testing temperature was 25 C. The zeta potential measure-
ment was repeated for 6 runs per sample, and the data was
calculated automatically using the software from the electro-
phoretic mobility based on Smoluchowski’s formula.
3. Results and discussion
2.3.5. Uterine weight. After the 4-week treatment of estradiol
the mice in groups 4a–c were sacrificed with pentobarbital
sodium (40.0 mg kgꢂ1, i.p.) anesthesia and dissected immediately
to obtain the uterine weight.
3.1. Total vBMD and trabecula vBMD of the femur of 4a–c
treated mice
The anti-osteoporosis efficacy of the mice in groups of 4a–c was
quantitatively measured by CT and pQCT to determine 3D bone
geometry and the size-independent vBMD. This is shown in
Fig. 3 and Fig. 4. In Fig. 3 we found that the total vBMD in the
femur of the mouse receiving NS plus prednisone was signifi-
cantly lower than that of the femur of the mouse receiving NS
alone. We can infer that prednisone effectively induced the
2.4. Characterizing the nano-image
2.4.1. Scanning electron microscope (SEM) image of 4a–c in
solid state. The characterization of the nano-image of the powder
from a 1.1 mM solution of 4a–c in ultrapure water was identified
on SEM (JEM-1230, JEOL, Tokyo, Japan) at 50 kV. The solu-
tion was attached on to a copper plate and lyophilized via
double-side tape (Euromedex, France). The specimens were
coated with 20 nm of gold–palladium using JEOL JFC-1600
AUTO FINE COATER. The coater was operated at 15 kV,
30 mA, 200 mTorr (argon) for 60 seconds. The shape and size
distribution of the nano-species were measured by counting more
than 100 particles in a randomly selected region on the SEM
alloy. All the measurements in triplicate grids were performed.
The images were recorded on an imaging plate (Gatan Bioscan
Camera Model 1792) with 20 eV energy windows at 100–
10 000ꢀ, and were digitally enlarged.
Fig. 3 Total vBMD and images of pQCT scanning at a distance from
the proximal femur. Growth plate corresponding to <6% of the total
length of the femur of the treated mice. (a) Total vBMDs is represented
with mean ꢁ SD mg cmꢂ3, n ¼ 12, PDN ¼ prednisone, dose of 4a–c ¼
110 nmol kgꢂ1. (b) Compared to NS + PDN, 4b + PDN and 4c + PDN
p < 0.01; (c) compared to NS + PDN and 4c + PDN p < 0.01; (d)
compared to NS + PDN p < 0.01.
2.4.2. Transmission electron microscopy (TEM) images of 4a–
c in water. The characterization of the nano-image of 4a–c in
aqueous solution was identified on TEM (JSM-6360 LV, JEOL,
Tokyo, Japan). A 1.1 mM solution of 4a–c in ultrapure water was
dripped onto a former-coated copper grid and then a drop of
This journal is ª The Royal Society of Chemistry 2012
J. Mater. Chem., 2012, 22, 21740–21748 | 21743