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138 Journal of Traditional Chinese Medicine 2006; 26(2):138-147

Basic Investigation
Acupuncture Increases Bone Strength by Improving Mass and
Structure in Established Osteoporosis after Ovariectomy in Rats
WenPing Zhang, Masayuki Kanehara, YanJun Zhang, ZhiFeng Yu, GuoXia Zhang, YouXin Yang,
Saori Tachi & Torao Ishida
Research Institute of Traditional Chinese Medicine (ITCM), Suzuka University of Medical Science (SUMS), Japan

The effects of acupuncture on bone biomechanical properties and histomorphometry in ovariectomized (OVX)
rats were studied. Twenty-four 8-week-old female Sprague-Dawley rats were randomly assigned to three
groups: sham, model and acupuncture. Rats in the model and acupuncture groups were ovariectomized, while
those in the sham group underwent a sham operation. All rats were anesthetized and fastened for 15 minutes,
and for the acupuncture group, needling on Pishu (BL20) and Shenshu (BL23) was performed. Blood and
urine were collected to measure serum osteocalcin ( OC ) and urinary calcium, phosphorus or
deoxypyridinoline (Dpd). After 16 weeks of treatment, all the rats were killed and their tibiae and femora
were removed. The tibiae were used for analyses of bone histomorphometry and the femora for a three-point
bending test. Acupuncture gave significant protection against ovariectomy-caused decline on femoral strength
in the mechanical test, increased the trabecular bone volume and thickness, lowered the trabecular separation
of tibiae and restricted the excretion of phosphorus and Dpd, while promoting the concentrations of serum
osteocalcin as compared with model rats. These results seemed to indicate that acupuncture on the points of
Pishu (BL20) and Shenshu (BL23) not only promoted the bone formation but also suppressed the bone
resorption induced by OVX in osteoporotic rats, which suggests that it would be a potentially useful and
convenient method in preventing osteoporosis.

INTRODUCTION subcutaneously or administered nasally, and there is

With the continuing demographic shift in populations also a high incidence of side effects including pain at
toward an old society, osteoporosis has become a the injection site, flushing, and nasal irritation. On the
major public health problem. Fear of fracture itself other hand, treatment of osteoporosis with
can reduce the quality of life in women who have antiresorptive agents, such as estrogen and
been diagnosed as having osteoporosis. Hormone bisphosphonates, induces a modest increase in bone
repalcement therapy1 remains the mainstay for the mass,3,4 but these agents do not seem capable of
prevention of postmenopausal osteoporosis, because restoring lost bone completely to premenopausal
the biggest culprit in the process of bone loss is levels, because of their inhibitory effect on bone
estrogen dificiency.2 However, hormone replacement formation as well as bone resorption.5,6 The
therapy is not accepted universally due to the limitations and weakness of antiresorptive therapy
contraindications in some patients, low compliance have led to a search for safe, effective alternatives.
and reluctance of many women because of the fear And acupuncture, as an important part of traditional
and dislike of possible side effects and long-term risks. Chinese medicine (TCM) and easily acceptable
Besides, calcitonin is effective in relieving pain, therapy, with advantages of convenience, safety,
improving bone density, but it has to be given efficacy with few side effects, is commonly used in
Journal of Traditional Chinese Medicine 2006; 26(2): 138-147 139

present clinics and is gradually reconsidered in many lateral to the lower border of the 11th thoracic
countries. Concerning the therapy for osteoporosis, as vertebra in the back and Shenshu (BL23) is located
a new field of acupuncture, it is still not well inferior and lateral to the spinous process of the
recognized or widely used in clinics so far. The aim of second lumbar spine. In order to obtain the typical
the present study is to reproduce in rats objective reaction of acupuncture, the needles were
postmenopausal clinical situation and osteoporotic manipulated further by twistling approximately 90 °
patients that are under chronic therapy with coordinately at a frequency of about 2 Hz (120
acupuncture for 16 weeks, and to investigate whether turns/minute) for a minute, separately after the
it could prevent further bone loss due to high bone insertion and before the withdrawal of needles, all the
turnover and prolong the process of osteoporosis needles were kept in place for 15 minutes. The
treatment were conducted 5 times a week and
induced by OVX and whether it would be a useful and
continued for 16 weeks. Rats were weighed weekly
beneficial therapy for osteoporosis.
throughout the whole experiment.
Experimental Animals
Twenty-four 8-week-old virgin Sprague-Dawley rats,
weighing 220 ± 7g, were purchased from Clea Co.,
Ltd. (Tokyo, Japan) and housed individually at a room
temperature of 22 ± 2℃ and humidity of 55 ± 10%
with a 12-h light/dark cycle in metallic cages.
Throughout the experimental period, rats had free
access to tap water and were fed standard CE2 rat
chow (Clea Co., Ltd., Tokyo, Japan). The animals
were randomly assigned to the next three groups:
sham control group (S), model control group (M) and
acpuncture group (Acp). The sham operation for S
group (n=8) and ovariectomy for M (n=8) and Acp
(n=8) rats were perfomed under nembutal
Fig. 1: Acupucnture was manipulated at points of BL
(I.P.,30mg/kg) anestehsia at 8 weeks of age. The 20 (thin arrow) and BL23 (thick arrow), respectively.
animals were permitted 7 days to recover from the
Blood and Urine Chemistry
surgery, then the experiment started.
A 24-h urine sample was collected from rats that were
Acupucnture Manipulation
housed individually for 24 h in metabolic cages and
All rats were fastened onto an autopsy table for 15 withheld from food at 13 and 17 weeks after operation,
minutes after ether inhalation. Stainless acupucnture to measure the urinary excretion of calcium,
needles with a diameter of 0.22mm and a length of phosphorus and deoxypyridinoline (Dpd), a specific
0.8cm were inserted perpendicularly to a depth of biomarker of bone resorption.8 Blood was collected
about 0.3 cm into the points of Pishu (BL20) and from the ophthalmic veins soon after the collection of
Shenshu (BL23) in the Acp group rats (Fig. 1). The urine, when the stomachs were empty. Serum was
principle of points selection follows the standard on stored at -80 ℃ until osteocalcin (OC), a marker of
rats recorded in the teaching material of experimental osteoblast activity, was measured.9 Serum osteocalcin
acupuncturology.7 The location of Pishu (BL20) is was measured with a commercial kit (Rat Gla-OC
140 Journal of Traditional Chinese Medicine 2006; 26(2): 138-147

Competitive EIA Kit, Takara Biomedicals). And cortical edges were excluded.13 Cancellous bone
urinary calcium, phosphorus and Dpd were volume was measured and expressed as a percentage
determined at Shionogi laboratory (Osaka, Japan) by of bone volume (BV/TV, %). Trabecular thickness
using the OCPC method for calcium, (Tb.Th, μ m), number (Tb.N, No./mm) and
phosphomolybdic acid method for phosphorus and the separation (Tb.Sp μ m ) were derived from the
CLIA method for Dpd. Dpd was normalized by the measures of bone surface and volume.14,15
urinary creatinine (Cr).
Statistical analysis
Mechanical testing
All results were expressed as mean ± SD for each
At the end of the experiment, the left femur was
group. Statistical differences among three groups were
removed and cleaned from the adjacent tissue and
evaluated by student t-test. P values less than 0.05
readied for mechanical testing. After collection, the
were considered to be significant.
left femur was immediately placed into Nacl (9g/L) at
4 ℃ and the femoral failure load was determined 24 RESULTS
hours later, using three-point bending test, with a table Uterine and body weights
top type tester EZ test series (Shimazu Co., Osaka,
Rats were weighed weekly throughout the whole
Japan). The femur was placed horizontally with the
experiment. The weight changes with age were shown
anterior aspect facing downwards on two supports
in Fig. 2. OVX induced a significant body weight gain
separated by a distance of 1 cm and equidistant from
from three weeks of postoperation, however, no
its ends. Central loading was then applied at a
significant difference was seen between the M and
constant deformation rate of 5mm/min until the bone
Acp group, even though Acp rats were slightly (but
fractured to determine its strength.10, 11
not significantly) heavier by 3-18% than the M rats.
Bone histomorphometry As an indicator of estrogenic activity, the uteri were
The excised right tibiae were stained for 7 days in a dissected and weighed on a balance scale at the killing
Villanueva bone stain solution (Wako, Tokyo, Japan), time. The uteri were markedly atrophied in the OVX
dehydrated in a vacuum desiccator with graded animals (the uterus weights were 0.79 ± 0.2, 0.09 ±
ethanol and acetone; and then infiltrated with equal 0.13, 0.18±0.14 in groups S, M and Acp respectively;
parts of acetone and methacrylate monomer for S vs. M & Acp, P<0.001), however, the megascopic
overnight, methyl methacrylate monomer for 24 hours, uteri in Acp rats were not severely atrophied as those
prepolymerized embedding medium for 24 hours in a in M group, as well as slightly heavier (but not
refrigerator and finally embedded in a vacuum oven at significantly) than M uteri weights.
35℃ with 40 cm Hg pressure.12 To characterize static
Biomechanical testing
cancellous bone, 10-μ m -thick sagittal sections were
cut with a saw microdome (SP 1600 Leica instruments As shown in Table 1, ovariectomy did not
GmbH, Nussloch), and image analysis was performed significantly affect the properties of the femur in
in the secondary spongiosa of the proximal tibia three-point bending test. However, acupuncture
metaphysis with a semiautomatic image-analysis clearly increased the max-force, deflection, strain and
system (Image plus Ver.4.0, Planetron Co. Ltd.) energy ( vs. M, P<0.05), even significantly than the S
without knowledge of group allocation. Trabecular group in some parameters. Though no statistical
bone morphometry measurements were performed in difference was seen between the M and Acp groups
a mean tissue area of 7.65mm2 beginning at 0.8 to on the parameter of max-stress, it showed a 19%
2.3mm distal to the epiphyseal growth plate and the
Journal of Traditional Chinese Medicine 2006; 26(2): 138-147 141

higher tendency for Acp rats compared with the M


Body weight (g)
0 1w 3w 5w 7w 9w 11w 13w 15w 17w

T im e after surgery (w eeks)

S ham M odel A cp

Fig. 2: Body weights changes. The OVX groups had a significantly higher weights in postoperation (vs S,
P<0.05). Even though Acp group tended to be 3-18% heavier than the M group from 11 weeks in postoperation,
significance was not seen.

Table 1: Mechanical properties of the femoral midshaft. Significant difference from sham,
model rats are depicted as “*”or “+”, respectively
Groups Max-force (N) Max-stress (MPa) Max-deflection (mm) Max-strain (%) Energy (J)
S(8) 167.63±19.44 95.07±17.05 1.18±0.09 25.24±1.54 0.1±0.02
M(8) 163.45±24.86 92.59±16.87 1.12±0.16 23.95±4.05 0.09±0.03
+ +
Acp(8) 190.11±25.31 110.44±19.84 1.33±0.11* 28.18±2.62* 0.13±0.02*+

Histomorphometry Biochemical markers of bone turnover

Fig. 3 provided a summary of the trabecular bone The serum osteocalcin concentration in the OVX rats
was significantly higher than that in the S rats, and
volume and structural parameters from the static
Acp rats was further significantly higher compared
histomorphometry. Ovariectomy led to a significant
with the model rats after 13 and 17 weeks of surgery,
decrease in trabecular volume. A decrease (P<0.05) in respectively. The excretion of urinary calcium did not
the trabecular volume, number and a larger separation show any changes among the three groups at both 13
(P<0.05) in the OVX rats compared with intact S rats and 17 weeks of postsurgery, however, the
were observed. The addition of acupuncture phosphorus concentration for Acp rats was
counteracted the effect of ovariectomy. Rats in the significantly lower than the sham and model rats at 17
weeks after the operation, and at 13 and 17 weeks of
Acp group had a significantly higher bone volume and
postoperation, the urinary excretion of Dpd in the rats
thickness than the M rats, whereas no difference was of OVX groups was significantly higher than in the
seen between the two groups in trabecular number and sham rats, in addition, the Dpd excretion in the OVX
separation, in spite of the tendency of lower numbers rats acupuncted was significantly lower than that in
and separation for Acp rats (Fig. 4). the model rats (Fig. 5).
142 Journal of Traditional Chinese Medicine 2006; 26(2): 138-147

180 *+
40 *+

* 100
BV/TV (%)

20 60
10 20
Sham Model Acp
Sham M odel Acp

C 7 D
1000 *
Tb.N (#/mm)

T b.S P (   m )

* * 600

2 400

1 200

0 0
S ham M odel A cp Sham Model Acp

Fig. 3: Histomorphometric values of the various groups on proximal tibial metaphysis. (A) Bone volume
(%). (B) Trabecular thickness (μm). (C) Trabecular number (#/mm). (D) Trabecular separation (μm).
“*”and “+” show significantly different from the sham control and OVX model control rats, respectively.

Fig. 4: Undecalcified proximal tibial metaphysis from a non-acupuncture treated sham rat (A), a
non-acupuncture treated control model rat (B), and an OVX rat treated with acupuncture for 16 weeks (C). Note
the reduced mass of whitely-stained bone indicative of cancellous osteopenia in the non-acupuncture treated
OVX rat. Treatment of acupuncture significantly increased cancellous mass at the end of treatment period to a
level between the sham and the model. Villanueva stain; Scale bar, 1 mm.
Journal of Traditional Chinese Medicine 2006; 26(2): 138-147 143

A Serum osteocalcin B U rin ary C alc iu m

2.5 *+ 15
* *+
* 10
   g/m l

0 0
13w 17w 13w 17w
Time after surgery (weeks) T im e after surgery (w eeks)

Sham Model Acp

Sham M odel Acp

C U rinary P hosphorus D U rin ary D e o xypyridin o lin e

300 180 *
160 *+ *
140 *+
nmol/mmol Cr

200 100
*+ 80
150 60

100 20
50 0
0 13w 17w
13w 17w T im e after surgery (w eeks)
T im e after surgery (w eeks)

Sham M odel Acp Sham M odel Acp

Fig. 5: Effects of acupuncture on serum osteocalcin, urinary calcium, phosphorus and deoxypyridinoline (Dpd)
over time. (A) Serum osteocalcin (μg/ml), (B) Urinary calcium (mg/dl), (C) Urinary phosphorus (mg/dl),
(D) Urinary Dpd (nmol/mmol Creatinine). Values were expressed as mean±SD. “*” and “+” show significantly
different from the sham and model control rats, respectively.

DISCUSSION osteoporosis rats induced by OVX. Our results of

Ovariectomized rats have been classically used as an body weight have showed a significant difference
animal with postmenopausal bone loss.16 Various between the OVX and sham rats, which is consistent
studies have demonstrated the preventive and with the expected OVX-induced weight gain. No
therapeutic effects of acupuncture and moxibustion in difference between the two OVX groups was detected,
OVX rats,17,18 other clinicians also reported that however, the trend that the Acp rats are heavier on
acupuncture may increase the bone mineral density in body weights than the model rats, seems to be
postmenopausal women.19 In order to approach the correlated to the stronger bone strength or the bone
effects and reasons for acupuncture to osteoporosis, mass of Acp rats, in which body weight-bearing is
together with simplifying and choosing more useful partially responsible for these actions, on account of
acupoints, which may provide an expremental basis weight-bearing activities being able to slow the loss of
for future clinic, we have performed the present study bone with aging.20,21,22 The purpose of treating
on needling the points of BL20 and BL23 in osteoporosis mainly consists in restoring the
144 Journal of Traditional Chinese Medicine 2006; 26(2): 138-147

mechanical function and improving the strength and experimental conditions, including the cutting
against the external force.18 The results of mechanical directions of samples with a microtome. Our results
test on the midshaft of femur, directed at the seem to make clear that acupuncture improving
middiaphysis, where rich in cortical bone, which cancellous bone and preventing bone loss may be due
fulfill essentially mechanical and preventive to changing the trabecular structure through
functions,23 show that parameters of max-force, shortening trabecular separation and increasing
max-deflection and max-strain in the Acp group are trabecular thickness rather than increasing the
significantly higher than those in the model group, but trabecular number.
the max-stress in Acp rats shows no significance in Moreover, to investigate the effects of acupuncture on
spite of 18% increased than that in the model rats. No bone metabolism in OVX rats, we have measured the
difference between the S and M groups are seen, biochemical markers (urinary excretion of calcium,
which is consistent with the relations that ovariectomy phosphorus, Dpd and serum osteocalcin level) of bone
has a more profound influence on cancellous bone turnover. Some researchers have reported that
than on cortical bone.24,25 From these data, we may ovariectomy decreases calcium absorption but others
conclude that under our experimental conditions, have reported that no significant decrease in calcium
OVX does not affect the cortical bone, whereas the absorption is observed following ovariectomy,
needling on BL20 and BL23 can strengthen the although the reasons are unclear.28,29 In the present
cortical bone on femoral diaphysis and then withstand study, the apparent calcium absorption does not differ
the extrinsic press strongly, probably by means of between the sham and model rats, which is consistent
attenuating losses of the cortical bone or adjusting the with the previous reports.29 With a significantly lower
bone structure resulted from acupuncture reaction. level of phosphorus for acupuncture rats compared
with OVX model rats, it seems to indicate that
On the other hand, bone histomorphometry is
acupuncture may depress the excretion of phosphorus.
performed on the proximal tibial metaphysis, where
Urinary Dpd is a biochemical marker of bone
is rich in cancellous bone that is mainly involved in
resorption, with a significantly decreased Dpd level in
metabolic functions. The present study shows a Acp rats than the model rats, it makes clear that
significantly increased bone volume and trabecular acupuncture may inhibit the bone absorption of
thickness for Acp rats (vs. M rats), along with the osteoporosis rats. On the other hand, that a marker
lower trabecular separation (but not significant) reflecting bone formation, serum osteocalcin in Acp
compared with the model rats on this site, which remains above the levels in model control rats seems
further strengthens the case for acupuncture on to give the reasons that acupuncture improves bone
cancellous bone. Some researchers have reported an strength and bone structure not only by depressing
obviously higher bone volume in sham control rats bone absorption but also by promoting bone formation,
(p<0.05 vs. OVX ), but not different from the OVX simultaneously.
control rats in trabecular thickness.26 Our present BL20 and BL23 are very commonly used points in
study has indicated significant larger trabecular clinic, they are usually used to treat diseases
thickness and small number for OVX rats, compared concerning kidney, stomach and intestine. Modern
with the sham rats, which are in line with other studies research has also proved their improving and
that shows significantly higher trabecular thickness adjusting functions to the kidney and gastrointestine.30
and lower number in OVX group than those in sham Osteoporosis is always involved in these organs, less
group.20,27 The divergent reported findings of intestinal absorption and more excretion are probably
histomorphometric structure seem to depend on age the main and direct reasons. As OVX and subsequent
Journal of Traditional Chinese Medicine 2006; 26(2): 138-147 145

estrogen deficiency normally lead to a decreased In conclusion, our study shows that acupuncture
intake of calcium, gastrointestinal (GI) tract improves the mechanical properties of the femoral
deterioration and the decrease in motility and cortical bone, the bone mass of the tibial cancellous
secretion of gastric juices, which are direct reasons bone in rats with established osteoporosis due to
causing a lower calcic absorption from the intestine, ovariectomy. In addition, the decreased bone
and then the poverty the poverty in calcium always resorption metabolic marker and the increased bone
results in the hypersecretion of parathyroid hormone formation marker levels for Acp OVX rats, seems to
(PTH), which may bring about the hyperfunction of indicate that it treats osteoporosis by suppressing the
bone resorption and finally cause osteopenia or bone resorption and promoting the bone formation.
osteoporosis.31 Our needling treatment with the BL20 The mechanism probably, at least in part, is due to the
and BL23 remedies deficiencies in mobility and increase in intestinal calcium intake, or the estrogenic
secretion of the gastrointestinal tract, and this should activities by means of influencing the
be a main mechanism of acupuncture for suppressing hypothalamic-pituitary-adrenal axis and subsequently
bone loss in OVX rats by promoting the absorption modulating the estrogen receptor. These findings
functions of the gastrointestine. suggest that acupuncture should be a potentially
useful, preventive and therapeutic method for
Also, it has been demonstrated that OVX may be osteoporosis, which occurs in women as a result of
associated with the hypofunction of the estrogen deficiency.
hypothalmus-pituitary-adrenal axis, and acupuncture
has been proved to be capable of enhancing the serum ACKNOWLEDGMENTS
estrogen level of ovariectomized rats, increasing the We would like to thank Mr. Masayuki Hori and Mr.
bone mineral density afterwards.33 Other studies have Kazumi Shiraiwa for consulting in the analysis of
also observed the relations between acupoints and the bone morphometry.
hypothalamic-pituitary-adrenal axis and indicated that
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