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Figure Legends
383
Fig. 1A. LIPUS exposure device in vitro. Left images show an array of ultrasound transducer and right
384
images show 6-well culture plate on the array.
385
Fig. 1B. A schematic of LIPUS device. Experimental setup with the key parameters are shown.
386
387
Fig. 2A. RIA graft-derived cells. They are fibroblast-like, spindle, and adhesive.
388
Fig. 2B. Proliferation of RIA graft-derived cells relative to day 0 level on days 2, 4, and 7 (NS, not significant;
389
*p≤ 0.05).
390
391
Fig. 3. Relative ALP activity of RIA graft-derived cells on days 7 and 14. The ALP activity in each sample
392
was expressed relative to the level in the control group on day 7 (*p≤ 0.05).
393
394
Fig. 4. Quantitative real-time polymerase chain reaction analysis of RIA graft-derived cells. The mRNA
395
expression levels of ALP (A), Runx2 (B), OSX (C), OC (D), and ATF4 (E) were evaluated and normalized
396
by the mRNA expression level of glyceraldehyde-3-phosphate dehydrogenase. The mRNA levels are
397
expressed relative to those for the controls on day 0 (value set at 1 for each marker, *p≤ 0.05).
398
399
Fig. 5. Alizarin Red S staining of the LIPUS and control groups at day 21. Photographs show that the control
400
cells (A) had less Alizarin Red S staining than the LIPUS-treated cells (B). The intensity of Alizarin Red S
401
staining in the LIPUS group was expressed relative to the level in the control group (C; *p≤ 0.05).
402
403
20
404
Table 1. Clinical data of the seven patients
Patient Gender
Age
Disease
Comorbidity
Smoking
(cigarettes/day,
years)
Other factors
affecting
osteogenic activity
55
Non-infected nonunion/Tibia
Hypertension
20, 35
51
Chronic osteomyelitis/Femur
30, 5
42
Acute osteomyelitis/Tibia
54
Acute osteomyelitis/Femur
Alcoholic liver disease
20, 35
40
Non-infected nonunion/Tibia
10, 20
60
Infected nonunion/Tibia
20, 20
44
Infected nonunion/Tibia
20, 25
405
21
406
22
407
408
Table 2. Acoustic impedance value of each material
Material
Acoustic impedance
Z, kg/(m2s)
Air
428.6
Water
1.50×106
Polystyrene
2.37×106
Silicone
1.50×106
409
23
410
Table 3. Quantitative data on acoustic output measurement
Manufacturer's
Characteristics
Measured
Characteristics
ISATA, mW/cm2
30.0
28.0
Frequency, MHz
1.5
1.5
200
200
Parameter
Pulse repetition rate, kHz
Pulse duration, μS
411
ISATA : spatial-average temporal-average intensity
24
412
Table 4. Details of the primers used for amplification
Gene
Primer sequences (5' to 3') (forward/reverse)
ALP
CTCGTTGACACCTGGAAGAGCTTCAAACCG
GGTCCGTCACGTTGTTCCTGTTCAGC
Runx2
ATGCTTCATTCGCCTCACAAAC
CCAAAAGAAGTTTTGCTGACATGG
OSX
CGGGACTCAACAACTCT
CCATAGGGGTGTGTCAT
OC
CATGAGAGCCCTCACA
AGAGCGACACCCTAGAC
ATF4
CTGACCACGTTGGATGACAC
GGGCTCATACAGATGCCTCT
GAPDH
CGTCTTCACCACCATGGAGA
CGGCCATCACGCCACAGTTT
413
*ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; OSX, osterix; OC, osteocalcin;
414
ATF4, activating transcription factor 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase
415
25
Fig. 1A
Fig. 1B
Fig. 2A
Fig. 2B
10
11
12
13
14
Fig. 3
15
16
17
Fig. 4A (ALP)
Fig. 4B (Runx2)
Fig. 4C (OSX)
Fig. 4D (OC)
18
19
20
21
22
Fig. 4E (ATF4)
23
24
25
Fig. 5A
Fig. 5B
26
27
28
Fig. 5C
29
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