Injury, Int. J.

Care Injured 47 S1 (2016) S39S42

Contents lists available at ScienceDirect

Injury
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n j u r y

Ultrasound and fragility fracture: is there a role?

Wing-Hoi Cheunga,b,*, Kwok-Sui Leunga,b, Simon Kwoon-Ho Chowa
a
Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong
b
Orthopaedic Research Laboratory of Translational Medicine Research and Development Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced
Technology, Chinese Academy of Sciences, China

KEYWORDS ABSTRACT

osteoporosis Osteoporotic fracture is known to have impaired healing capacity and therefore takes longer time to
fracture healing heal, as compared with younger one. The mechanism of impaired osteoporotic fracture healing is multi-
low intensity pulsed ultrasound factorial, where lower responsiveness to mechanical loading is generally believed to be one factor, yet
LIPUS
not absolutely confirmed. In recent years, low intensity pulsed ultrasound (LIPUS) is demonstrated
estrogen receptor
to have good efficacy in treating normal fracture healing, as proven by many randomized controlled
trials, as well as in vitro and animal evidences. The effects of LIPUS on osteoporotic fracture healing
was also validated in an animal study, which revealed that osteoporotic fractured bone of SD rats
showed radiologically and biomechanically comparable responses to LIPUS as age-matched normal
fracture healing, in terms of callus width, bridging rate, bone volume fraction, and stiffness etc. Gene
expression profiling also confirmed that osteoporotic fractured bone responded to LIPUS very well
by upregulating Col1 and BMP2 (osteogenesis) at early phase, VEGF (angiogenesis) at middle phase
and RANKL (remodeling) at late phase. These confirm that osteoporotic bones respond well to LIPUS
as good as normal bone. These findings may be associated with estrogen receptors (ERs), as estrogen
depletion is sensed and relayed by ERs and ERs also function as mechano-sensors. A previous study
observed a delayed ERs expression pattern in fracture callus of OVX rats, as compared with SHAM rats,
which correlated well with the expression pattern of BMP-2 (callus formation-related gene). Hence,
the responses of osteoporotic fractured bone to LIPUS may be related to the local ERs expression at
fracture callus that needs further experiments to validate.
2016 Elsevier Ltd. All rights reserved.

Osteoporotic fracture healing responsiveness to mechanical
stimulation?
Osteoporotic fracture is a critical medical challenge with
increasing aging population and the prevalence is high too. In
the USA, there are more than 1.5 million of such fracture cases
each year [1] and therefore the related healthcare cost is very
high. The capacity for fracture repair has been reported to
decrease with age [2]. Many reports indicate the differences
of mechano-biology between osteoporotic and normal bones
[3] and osteoporosis impairs both early phase [4] and late
phase of fracture healing with 40% reduction in callus cross-
sectional area, 23% decrease of bone mineral density (BMD) and
fivefold decrease in mechanical properties [5]. The mechanism
of impaired osteoporotic fracture healing is multi-factorial
and a number of evidences showed that poor sensitivity of
osteoblasts to mechanical signals [6,7], impaired angiogenesis
[810], and reduced mesenchymal stem cells [11,12] may play
a role in the impaired healing. Acceleration of osteoporotic
* Corresponding author at: Department of Orthopaedics and Traumatology,
5/F Lui Che Woo Clinical Sciences Building, Prince of Wales Hospital, The Chinese
University of Hong Kong, Shatin, New Territories, Hong Kong SAR.
E-mail address: louis@ort.cuhk.edu.hk (W.-H. Cheung).
fractures is always the target of orthopaedic researchers to
shorten the hospitalization and hence the economic benefits,
where mechanical stimulation, e.g. weight bearing, is a common
clinical approach. However, previous finding revealed that the
osteoblasts from osteoporotic donors were less responsive to
1% cyclic strain stretching in terms of proliferation and TGF
release, as compared with younger normal donors [6]. Hence,
there is a general belief that osteoporotic bone is less responsive
to mechanical stimulation; however, there were several
reports telling opposite findings, e.g. Leppnen et al showed
that osteoporosis was not attributable to impaired mechano-
responsiveness of aging skeleton [13]; also, male adult rats with
lower estrogen level demonstrated better mechanical responses
than females [14]. Therefore, mechanical stimulation to enhance
osteoporotic fracture healing remains controversial.
Efficacy of low intensity pulsed ultrasound on fracture healing
Low intensity pulsed ultrasound (LIPUS), a propagating
acoustic wave that transfers energy onto the treated regions,
has been well reported to accelerate fracture healing. Many
randomized controlled clinical trials confirmed the accelerated
fracture healing at different skeletal sites by LIPUS with 1742%
reduction in healing time [15,16]. Beneficial effects on complex

0020-1383/ 2016 Elsevier Ltd. All rights reserved.

S40 W.-H. Cheung et al. / Injury, Int. J. Care Injured 47 S1 (2016) S39S42

tibial fractures [17] and non-unions of various bones [18] were

0.004

0.003
demonstrated clinically. A few meta-analyses also verified

SD
the different extents of positive effects of LIPUS on fracture

8
healing [19,20]. In vivo, LIPUS was shown to increase blood

Mean

0.005

0.028
flow around the fracture site [21]. At cellular level, LIPUS was
found to increase cellular activities of many cell types, e.g.

0.026
0.015
increased calcium nodule formation and alkaline phosphatase

SD
BMP-2
activity in osteoblasts [22], more -catenin nuclear translocation

4
in osteocytes [23], promoted osteogenesis in mesenchymal

Mean

0.089

0.128
stem cells [24] and stimulated proliferation/differentiation
in periosteal cells [25], which are helpful to promote fracture

0.005
0.014
healing at various phases. With all these positive scientific

SD
evidences, LIPUS is well accepted to be an effective biophysical

2
modality to modulate mechanical micro-environment and blood

0.006
Mean

0.047
flow in fracture site for accelerating fracture healing. However,
all these animal or clinical evidences are on normal fracture in

22.1
adults; the effect on osteoporotic fracture was not yet elucidated.

5.4
SD
8
Effects of LIPUS on osteoporotic fracture healing animal

Mean
Gene expression levels relative to GAPDH in mechanical sensitivity related ER-, ER-, and osteogenic related Col-1 and BMP-2. (n=5 per group at each time point).

24.3
8.9
evidence

36.7
20.3
The first animal study to depict the efficacy of LIPUS on

SD
Col-1
osteoporotic fracture healing was conducted on 120 female

4
Sprague-Dawley (SD) rats divided into four groups Sham

Mean

30.9

70.2
ovariectomy with LIPUS treatment (Sham-T), Sham ovariectomy
control (Sham-C), ovariectomy with LIPUS treatment (OVX-T)

49.2
35.4
SD
and OVX control (OVX-C) [26]. Half of the 6-month-old rats
were bilaterally ovariectomized for OVX groups (FDA-verified

2
animal model of osteoporosis [27]), while another half was sham

Mean

157.4

96.9
operated for Sham groups. All the rats were housed for 3 months
to develop osteoporosis and the reduction in BMD was confirmed

0.9573
0.0198

0.000
by peripheral quantitative computed tomography (pQCT,

SD
Densiscan 2000, Scanco Medical, Bruttisellen, Switzerland),
where 9.6%, 4.6% and 2.3% of BMD were detected at 5th lumbar 8

2.5582
0.0360
Mean

0.171
vertebra, right femoral head and right femoral shaft respectively.
They were then created closed fractures at femoral mid-shaft
according to Einhorns protocol [28]. LIPUS (pulsed 1.5 MHz,

0.4936
0.0862

0.002
SD

30.0 mW/cm2 spatial-averaged temporal-averaged intensity;

ER-

Exogen 3000+, Smith & Nephew, Memphis, TN, USA) was given
4

0.2066

0.8081

20 min/day and 5 days/week for durations of 2, 4, or 8 weeks, at 0.004

Mean

which radiography, BMD and microarchitecture measurement,

histomorphometry and mechanical testing were performed.
0.5332

0.0216

0.091

Results indicated that both the treatment groups (Sham-T and

SD

OVX-T) were of significantly enhanced callus formation, faster

2

mineralization and better remodeling than their control groups

0.0226
1.0238
Mean

0.056

(Sham-C and OVX-C) [26]. Interestingly, by comparing the results

between Sham-T and OVX-T, OVX-T showed comparable healing
responses with Sham-T group in most parameters, while OVX
0.0003

0.2470

0.004
SD

groups indicated relatively more significant differences in various

assessments than Sham groups. The better healing responses in
8

0.0005

0.7091

OVX-T than Sham-T included significantly higher CW (+15.0%

Mean

0.091

at week 4), earlier appearance of callus bridging (week 4.17 vs.

week 4.75) and higher percentages of completed healing (66.7%
0.0191

0.1169

0.014

vs. 41.6% at week 4; 100% vs. 83.3% at week 8), higher ratio of
SD

increment in BV/TV value (+26% vs. +18.7% from week 2 to 4),

ER-

faster response of endochondral ossification (faster drop in CW,

0.0540

0.1344
Mean

0.003

faster decrease in cartilage area) and a higher stiffness value

(+37.4% at week 4 and 36.9% at week 8) [26]. These findings
were consistent with a previous study using low-magnitude
0.0012
0.1183

0.223
SD

high-frequency vibration (35 Hz, 0.3 g where g = gravitational

acceleration) with the same study design and animal model,
2

0.3069

which also demonstrated relatively better effects on osteoporotic

0.0016

0.004
Mean

fracture healing than on the age-matched non-osteoporotic one

[29]. Similar results were also found in Rubinaccis study which
Table 1

SHAM

OVX non-fractured rats treated with vibration treatment (30 Hz,

t-test
OVX

3 g) showed significant increase in cortical and medullary areas,

 W.-H. Cheung et al. / Injury, Int. J. Care Injured 47 S1 (2016) S39S42 S41

Fig. 1. Representative immunohistochemistry of ER- protein expression in ovariectomized Sprague Dawley closed femoral rat fracture model at 2 weeks post-fracture,
counter-stained with Haematoxylin. (A) Overview of the callus around the fracture site showed positive signals at various cells and tissue types including the bony callus
(BC), cartilaginous tissue (CG), progenitor cells (PGC) at 50. (B) Stronger positive signals observed in PGC and BC, and slightly weaker signals observed in hypertrophic
chondrocytes, at 100. (C) Positive signals in the BC was mostly detected at the osteoblast-like-cells (OB) and osteoclasts-like-cells (OC), at 200. (D) PGC demonstrating
positive ER- signals gradually differentiating into chondrocyte-like-cells at the soft callus and showed changes in morphology. (E) ER- positive OB, active synthesis of
newly formed bone at bony callus at 1000. (F) PGC immediately adjacent to newly formed bony callus at 1000.

periosteal and endosteal perimeters while Sham animals did not
have any effect, which illustrated that ovariectomy may sensitize
the cortical bone to mechanical stimulation [30]. All these
confirm the efficacies of LIPUS on both normal and osteoporotic
fracture healing, as well as osteoporotic bone can well respond
to mechanical stimulation as good as age-matched normal bone.
Next step is to conduct clinical trials to validate the clinical
efficacy of LIPUS on fragility fracture patients.
Molecular gene profiling of osteoporotic fracture healing
augmented by LIPUS
The detailed gene profile of osteoporotic fracture healing
augmented by LIPUS was further investigated [31]. The results
showed that collagen type 1 (Col-1) and bone morphogenetic
protein-2 (BMP-2) were significantly upregulated at week 2
post-fracture in LIPUS group than control group (3.11 and 1.95
respectively) in osteoporotic rats; vascular endothelial growth
factor (VEGF) was significantly upregulated at week 4 (3.51);
osteoporotegrin (OPG) was upregulated at week 2 post-fracture
(1.88), followed by the significant surge of RANKL expression
(1.99). This gene expression data further confirm the above fracture
measurements, indicating the process of callus formation was
increased by LIPUS during the early phase; the remodeling phase
was made sooner to occur, and that angiogenesis was increased by
LIPUS during the osteoporotic fracture repair process [31].
Impaired osteoporotic fracture healing is associated with
delayed expression of estrogen receptors
It has been known that the immediate effects of estrogen
depletion is sensed and relayed by estrogen receptors (ERs);
S42 W.-H. Cheung et al. / Injury, Int. J. Care Injured 47 S1 (2016) S39S42
also, ERs can function as mechanical signal transduction through
its ligand-independent function [32]. In addition, ERs have been
reported to localize in fracture callus [33] that indicates the
potential roles of ERs in fracture healing. Hence, there is evidence
to support that the quantity of ERs may play a role in determining
bone formation and fracture healing. When comparing the gene
expression of ERs between Sham and OVX groups, it was found
that ERs expressions were significantly higher in Sham group
at week 2 and later significantly lower at week 8 than OVX
group; in other words, OVX group demonstrated an opposite
trend (Table 1) [34]. Meanwhile, ER- correlated well with
BMP-2 significantly (r=0.545, p=0.003), where BMP-2 is known
to be important for callus formation. ERs protein expressions
were further confirmed using immunohistochemistry, which
appeared mostly in progenitor cells, osteoblasts and osteoclasts
(Fig. 1). These findings revealed that the impaired healing of
OVX-induced osteoporotic fracture may be associated with the
delayed expression of ERs. The enhancement effects of LIPUS on
osteoporotic fracture healing may be partially contributed by the
local increase of ERs expression in fracture callus, which becomes
more sensitive to mechanical signals leading to promoted
fracture healing but this however needs further experiments to
verify.
Acknowledgement
Part of the work was supported by the National Natural
Science Foundation of China (NSFC) (Reference: A.03.15.02401)
and the Asian Association for Dynamic Osteosynthesis Research
Fund (AADO-RF2010-001-2Y).
Conflict of interest
The authors have no conflict of interest.
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