ORIGINAL ARTICLE

Effects of combined therapy of alendronate and low-intensitypulsed ultrasound on metaphyseal bone repair after osteotomyin the proximal tibia of aged rats

Hiroshi Aonuma • Naohisa Miyakoshi • Yuji Kasukawa •

Keiji Kamo • Hiroshi Sasaki • Hiroyuki Tsuchie •

Toyohito Segawa • Yoichi Shimada

Received: 19 June 2012 / Accepted: 17 June 2013

� The Japanese Society for Bone and Mineral Research and Springer Japan 2013

Abstract Bisphosphonates and low-intensity pulsed

ultrasound (LIPUS) are both known to maintain or promote

callus formation during diaphyseal fracture healing. How-

ever, the effect of these treatments on the repair of meta-

physeal fractures has not been elucidated. To evaluate the

effects of bisphosphonates and/or LIPUS on cancellous

bone healing, an osteotomy was performed on the proximal

tibial metaphysis of 9-month-old Sprague–Dawley rats

(n = 64). Treatment with alendronate (1 lg/kg/day), LI-

PUS (20 min/day), or a combination of both was admin-

istered for 2 or 4 weeks, after which changes in bone

mineral density (BMD), bone histomorphometric parame-

ters, and the rate of cancellous bony bonding were mea-

sured. Alendronate suppressed bone resorption parameters

at 2 weeks (p = 0.019) and increased bone volume and

BMD at 4 weeks (p = 0.034 and p = 0.008, respectively),

without affecting bony bonding. LIPUS had no significant

effect on any of the histomorphometric parameters at 2 or

4 weeks, but significantly increased in BMD at 4 weeks

(p = 0.026) as well as the percentage of bony bonding at

both 2 and 4 weeks (p \ 0.01). The combined therapy also

showed significantly increased BMD compared with the

control group at 4 weeks (p = 0.010) and showed a trend

toward increased bony bonding. In conclusion, alendronate

and LIPUS cause an additive increase in BMD at the

affected metaphysis: alendronate increases the bone vol-

ume at the osteotomy site without interrupting metaphyseal

repair, whereas LIPUS promotes metaphyseal bone repair,

without affecting bone histomorphometric parameters.

Keywords Metaphyseal bone repair � Alendronate �Low-intensity pulsed ultrasound � Combined therapy

Introduction

Fragility fractures or fractures as a result of a fall are highly

prevalent in older patients and with an aging population,

and the increased prevalence of these fractures is likely to

become a social and economic burden worldwide. The

reduced bone quality and bone mass at the fracture sites of

these elderly patients is an important issue that complicates

successful fracture repair, often resulting in delayed or non-

union fractures. In addition, a prolonged treatment period

for cancellous bone fractures that occur close to a joint may

create other complications, such as joint contracture.

Shortening the time to complete bone union at sites of

cancellous bone fracture is necessary in order to avoid

these complications, especially in elderly patients.

Bisphosphonate therapy for osteoporosis has been

shown to increase bone mineral density (BMD), reduce the

risk of osteoporotic fracture [1] and decrease the risk of

subsequent fracture after a fragility fracture [2–4]. For

instance, in patients with low trauma hip fracture, studies

suggest that bisphosphonate therapy, if administered close

to the time of fracture, can increase BMD and improve the

stability of screw fixation in the repair site [5, 6]. Conse-

quently, it is reasonable to suggest that bisphosphonates

may improve fracture repair rates by increasing BMD and

saving bone stock. However, bisphosphonates also have a

potent inhibitory action on bone resorption, and, thus ,there

is the concern that bisphosphonate treatment immediately

after fracture may delay healing by affecting normal bone

metabolism and turnover at the fracture site [7].

H. Aonuma (&) � N. Miyakoshi � Y. Kasukawa � K. Kamo �H. Sasaki � H. Tsuchie � T. Segawa � Y. Shimada

Department of Orthopedic Surgery, Akita University Graduate

School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan

e-mail: aonuma1230@yahoo.co.jp

123

J Bone Miner Metab

DOI 10.1007/s00774-013-0492-3

Low-intensity pulsed ultrasound (LIPUS) is a clinically

available modality for accelerating fracture healing in fresh

fractures and nonunions. In several clinical trials, LIPUS

reduced the time to radiological healing of fresh fractures

by 37–42 % [8–10], and encouraged healing in 73–85 % of

individuals with a nonunion [11, 12]. In particular, LIPUS

is considered to be more beneficial in patients with risk

factors for fracture healing, such as those who smoke, or

those with larger fracture gaps [13]. LIPUS may, therefore,

improve the local condition at the fragile fracture site and

have a positive effect on fracture healing.

Most studies have evaluated bisphosphonate adminis-

tration and LIPUS exposure in animal models with

diaphyseal long bone fractures [14–18]. However, fractures

in elderly patients are more commonly found in cancellous

bone, such as in the proximal hip, knee, humerus and distal

forearm. Indeed, a fracture of the proximal tibia is one of

most common sites of cancellous bone fracture in the

elderly. Loss of reduction is common following fixation of

fractures at the proximal tibia, occurring in 30 to 79 % of

elderly patients [19–21]. Furthermore, flexion contracture

of the knee joint or muscle weakness of the quadriceps

muscles is often observed in patients older than 40 years of

age [22]. Thus, shortening the time to union in the treat-

ment of cancellous bone fractures in the knee joint will

help to avoid these complications.

To our knowledge, there are no studies concerning the

combined use of bisphosphonates and LIPUS for meta-

physeal bone repair. Aspenberg [23] suggested that bis-

phosphonate administration may enhance the structural

properties of cancellous bone by augmenting the fixation

stability of the fracture and screw. Additionally, LIPUS has

been shown to enhance time to healing in fracture repair by

improving the quality of the newly formed callus. Thus, we

hypothesized that, together, these two treatment strategies

could have a synergistic effect on fracture repair of can-

cellous bone, with the aim of shortening the time to bone

union at the cancellous site. This combination therapy

would yield a new strategy for the treatment of fractures in

cancellous bone to help alleviate the economic burden that

will ensue with our global aging population. Here, we

sought to investigate the effect of alendronate, a commonly

used bisphosphonate, in combination with LIPUS on met-

aphyseal bone repair in a rat proximal tibial osteotomy

model.

Materials and methods

Animals

Nine-month-old female Sprague–Dawley rats (Charles

River Laboratory Inc., Kanagawa, Japan) were housed in

a controlled environment at 22 �C with a 12 h light/dark

cycle. It has been reported that the tibial growth or body

weight gain of rats reaches a plateau at 9 months of age

[24, 25], and thus 9-month-old rats were considered to

be aged rats in this study. The rats were allowed free

access to water and pair-fed standard food (CE-2, Clea

Japan Inc., Tokyo, Japan) containing 1.14 % calcium,

1.06 % phosphorus, and 250 IU vitamin D3 per 100 g

[26, 27].

Experimental design

Sixty-four rats were randomized into four groups (n = 16

in each group): (1) control group (saline administration

with sham-LIPUS); (2) alendronate group (alendronate

administration with sham-LIPUS); (3) LIPUS group (saline

administration with LIPUS); and (4) combined group

(alendronate administration with LIPUS). A cancellous

bone osteotomy was performed on the right proximal tibia

of each rat. Briefly, a lateral parapatellar incision was made

from the knee joint of the right hind limb through the

proximal half of the tibia. Using an electrical power saw

(Yoshida Medical Inc., Japan), an incomplete mid-sagittal

osteotomy was created from the joint surface around one-

quarter of the proximal tibia, without extending to the

caudal cortex [28]. The osteotomized tibia was closed

using a non-absorbable suture. After surgery, the rats were

allowed to move freely. Rats with an abnormal gait or

impaired locomotion were not observed post-operatively.

Alendronate administration and/or LIPUS were initiated on

the third day after the osteotomy and continued until the

rats were sacrificed under anesthesia with pentobarbital at 2

or 4 weeks (n = 8 in each group). The right tibia from each

rat was harvested and fixed in 10 % neutral buffered for-

malin. All animal experiments were approved by the

‘‘Guidelines for Animal Experiment’’ of Akita University

School of Medicine.

Alendronate administration

A solution of alendronate (Teiroc Injection 10 mg, Teijin

Pharma, Tokyo, Japan) was prepared in saline at a con-

centration of 0.02 mg/ml. Rats in the alendronate and

combination groups received a daily subcutaneous injec-

tion of alendronate (1 lg/kg). This dosage of alendronate

was equivalent to the dosage used in humans (5 mg/day)

by oral administration and chosen in accordance with that

used by others in previous animal studies [17, 29, 30].

Saline was selected as a vehicle control, and 0.2 ml of

saline was administered as a subcutaneous injection to rats

in the control and LIPUS groups. Body weights were

measured weekly and injection dosages were adjusted

accordingly.

J Bone Miner Metab

123

Ultrasound intervention

LIPUS was provided by a Sonic Accelerated Fracture

Healing System (SAFHS; Teijin Pharma, Tokyo, Japan).

LIPUS signal strength and duration of treatment were

consistent with the recommended clinical conditions for

this device. This device is the same that is used in the clinic

for patients with delayed or non-union fractures, and its

efficacy has been shown previously using animal models

[16, 31, 32]. The ultrasound signal generated with a

transducer consisted of a burst width of 200 ls containing

1.5 MHz sine waves at a frequency of 1.0 kHz, and a

spatial average-temporal average (SATA) intensity of

30 mW/cm2. Rats were anesthetized with an intraperito-

neal injection of ketamine (20 mg/kg) (Sankyo, Tokyo,

Japan) and xylazine (1.5 mg/kg) (ZENOAQ, Fukushima,

Japan) before exposure to LIPUS or sham-LIPUS for

20 min per day. Sufficient gel was used during the appli-

cation of the ultrasound, and a rubber band was employed

to fix the transducer against the antero-medial side of the

osteotomized tibia (Fig. 1) such that the LIPUS could be

routinely and consistently applied at the healing site.

Measurement of BMD

BMD of the entire excised tibia (including both cortical

and cancellous bones) was measured by dual-energy X-ray

absorptiometry (DEXA, Hologic QDR-4500, Hologic, MA,

USA) in the anterior plane. Bones were scanned in the

‘‘small animal’’ scan mode, with the ‘‘regional high-reso-

lution’’ scan option. The region of interest (ROI) was

20 mm in length from the proximal edge of the tibia and

total width of the tibia (Fig. 2). Triplicate analysis of five

different tibias, with new placement after each determina-

tion, showed that the coefficient of variation of measure-

ment ranged from 0.72 to 1.45 %.

Sample preparation

After BMD measurements, the right proximal half of the

tibia from each rat, including the osteotomy site, was

decalcified with neutral 10 % ethylenediaminetetraacetic

acid (EDTA) for approximately 4 weeks and embedded in

paraffin. Three micron-thick mid-frontal slices were then

sectioned and stained with Hematoxylin and Eosin (H-E)

for cancellous bone histomorphometry.

Bone histomorphometry

Bone histomorphometric analysis at a magnification of

9200 was performed with a semiautomatic graphic

system (Histometory RT CAMERA, System Supply,

Nagano, Japan). Measurements were obtained at 390 lm

caudally from the lowest point of the growth plate and

medially from the endosteal surface. The histomorpho-

metric parameters were measured, including the volume

of cancellous bone per tissue volume (BV/TV; %),

osteoid surface (OS/BS; %), and eroded surface (ES/

BS; %) [33].

Evaluation of bone union after osteotomy

H-E stained sections obtained from each osteotomized tibia

were used to evaluate cancellous bone union. The semiau-

tomatic graphic system, at 9100 magnification, was used to

measure the length of bone union, defined as bone-to-bone

Fig. 1 LIPUS exposure. LIPUS treatment was administered under

anesthesia to the right ante-medial side of the proximal tibia at the

mid-sagittal osteotomy site. The transducer was fixed by a rubber

band during treatment

Fig. 2 Schema showing the region of interest (ROI) for bone mineral

density measurements. Tibiae were scanned in an anteroposterior

view using dual-energy X-ray absorptiometry. The ROI measured

20 mm from the proximal tip of the tibia and included the entire

surface area within that region. The arrowheads indicate the fracture

site

J Bone Miner Metab

123

bonding at the osteotomy site, and the length of the total

osteotomy line; these measurements were taken within the

same area as that for the morphometry measurements.

Cartilaginous bonding was also regarded as bony union,

whereas fibrous bonding was defined as a nonunion. The

proportion of bone union in the total length of the osteotomy

line was calculated [28].

Statistical analysis

All values are presented as the mean ± SD. Statistical

differences among treatment groups were compared using

Scheffe’s post hoc test for multiple comparisons using an

analysis of variance (ANOVA). Two-factor factorial

ANOVA was performed to evaluate the effect of alendro-

nate alone or LIPUS alone and the interaction between

these interventions on bone histomorphometric parameters

and the percentage of bony union. All statistical analyses

were performed using Stat View 5.0 J for Windows (SAS

Institute, NC, USA).

Results

BMD

After 2 weeks of treatment, neither rats in the alendro-

nate group nor rats in the LIPUS group showed a sig-

nificant change in BMD compared with the control

group (Table 1). Similarly, the combined treatment of

alendronate and LIPUS also failed to significantly

increase BMD as compared with the other three groups

at 2 weeks (multiple comparisons). However, at 4 weeks

after treatment, the combined treatment showed a sig-

nificant 12 % increase in BMD of the proximal tibial

osteotomy site as compared with the control group

(p = 0.010; multiple comparisons). In addition, alendr-

onate alone and LIPUS alone were significant factors

contributing to the increase in BMD at 4 weeks (without

interaction; p = 0.008 and p = 0.026, respectively; two-

factor factorial ANOVA).

Histological findings

Fibrous tissue unions were observed at most of the

boundaries of the osteotomy site in the control and

alendronate groups at 2 weeks (Fig. 3a, b), whereas few

boundaries were observed in the LIPUS and combined

groups (Fig. 3c, d). In addition, at 2 weeks, there were two

cases of nonunion in the alendronate group and one case of

extremely poor bonding (\20 % bony bonding) in the

LIPUS group. By 4 weeks, cancellous bone unions were

observed in the control group; although some immature

bones still remained (Fig. 3e). Favorable bony bonding was

identified in the alendronate, LIPUS and combined groups

at this time point (Fig. 3f–h).

Bone histomorphometry at the osteotomy site

of the proximal tibia

Two weeks after treatment, the OS/BS measurement in the

alendronate group was significantly lower than that of the

control and LIPUS groups (p = 0.011 and p = 0.007,

respectively; multiple comparisons; Table 2). In the two-

factor factorial ANOVA, alendronate alone, but not LIPUS

alone, significantly contributed to the decrease observed in

the OS/BS and ES/BS (without interaction; p \ 0.001 and

p = 0.019, respectively). After 4 weeks, no significant

differences in bone histomorphometric parameters were

observed among the four groups (multiple comparisons).

However, there was a significant link between alendronate

administration and increase in BV/TV or decrease in OS/

BS (p = 0.034 and p = 0.040, respectively; independent

of LIPUS; two-factor factorial ANOVA).

Percentage of bony bonding at the osteotomy site

of the proximal tibia

The percentage of bony bonding after 4 weeks of treatment

was significantly higher in the LIPUS group at 65 % of the

control group (p = 0.025; multiple comparisons; Table 3),

and there was a trend for an increase in the combined group

that was almost as high as the LIPUS group but did not

Table 1 Bone mineral density (BMD) at the osteotomy site of the proximal tibia

Control Alendronate LIPUS Combined Two-factor factorial ANOVA

Alendronate LIPUS Interaction

2 weeks 0.313 ± 0.016 0.323 ± 0.017 0.306 ± 0.033 0.312 ± 0.020 N.S. N.S. N.S.

4 weeks 0.309 ± 0.022 0.333 ± 0.017 0.330 ± 0.016 0.345 ± 0.022a p = 0.008 p = 0.026 N.S.

All values are mean ± SD

N.S. not significant, Control Saline + Sham LIPUS, Alendronate Alendronate + Sham LIPUS, LIPUS Saline + LIPUS, Combined

Alendronate + LIPUSa p = 0.010 vs control group by one-way analysis of variance (ANOVA) using Scheffe’s post hoc test

J Bone Miner Metab

123

reach significance. Two-factor factorial ANOVA showed

that LIPUS alone, but not alendronate alone, was a

significant contributor to increasing the percentage of bony

bonding at the cancellous osteotomy site at the proximal

Fig. 3 Histological sections of the osteotomy site stained with

Hematoxylin and Eosin (H-E). An interruption to the growth plate

(arrowhead) was observed in all sections. At 2 weeks after osteot-

omy, most of the boundaries at the osteotomy site were filled with

fibrous tissues (short arrows) in the control (a) and alendronate

(b) groups, while the boundaries included newly formed bone in the

LIPUS (c) and combined (d) groups. At 4 weeks after osteotomy, an

increase in mature trabecular bone (long arrows) was observed in the

alendronate (f), LIPUS (g) and combined (h) groups as compared

with the control (e), for which immature woven bone was observed

Table 2 Bone histomorphometric indices at the osteotomy site of the proximal tibia

Control Alendronate LIPUS Combined Two-factor factorial ANOVA

Alendronate LIPUS Interaction

2 weeks (%)

BV/TV 35.27 ± 5.54 34.18 ± 9.35 30.77 ± 7.30 32.55 ± 6.69 N.S. N.S. N.S.

OS/BS 6.67 ± 1.51 3.33 ± 1.79a 6.84 ± 2.26b 4.55 ± 1.66 p \ 0.001 N.S. N.S.

ES/BS 12.15 ± 3.93 8.67 ± 2.88 11.65 ± 2.34 9.65 ± 3.13 p = 0.019 N.S. N.S.

4 weeks (%)

BV/TV 29.90 ± 5.82 35.61 ± 7.42 31.35 ± 10.53 37.67 ± 5.83 p = 0.034 N.S. N.S.

OS/BS 7.22 ± 1.88 5.81 ± 2.74 8.51 ± 1.34 6.68 ± 2.28 p = 0.040 N.S. N.S.

ES/BS 13.09 ± 4.67 12.37 ± 3.93 14.92 ± 4.37 14.40 ± 4.51 N.S. N.S. N.S.

All values are mean ± SD. Percentage of cancellous bone volume (BV/TV), osteoid surface (OS/BS) and eroded surface (ES/BS) were measured

N.S. not significant, Control Saline + Sham LIPUS, Alendronate Alendronate + Sham LIPUS, LIPUS Saline + LIPUS, Combined

Alendronate + LIPUSa p = 0.011 vs control group at 2 weeksb p = 0.007 vs alendronate group at 2 weeks by one-way analysis of variance (ANOVA) using Scheffe’s post hoc test

J Bone Miner Metab

123

tibia at 2 and 4 weeks after treatment (p = 0.009 and

p = 0.002, respectively, independent of alendronate).

Discussion

In elderly patients, operative treatment of metaphyseal

fractures is predisposed to numerous complications, such

as joint contracture, loss of reduction, muscle weakness,

nonunion, implant failure and the need for re-operation

[34]. As a consequence, there is a clinical need to improve

the time to union in metaphyseal fracture healing. Gian-

noudis et al. [35] proposed that manipulation of both the

local fracture environment, such as with the use of growth

factors, scaffolds and mesenchymal cells, and the systemic

environment, such as with the administration of agents that

promote bone formation and bone strength, would be a

candidate treatment option for metaphyseal bone repair.

Bisphosphonates have been reported to suppress callus

remodeling and increase the cross-sectional area at the

cortical fracture site in rats [17]. Thus, the increased cross-

sectional callus area improved the constructive potential

and strength of the repair site. Therefore, we hypothesized

that a combined therapy may offer clinicians an alternative

treatment strategy for metaphyseal bone repair in elderly

patients. To our knowledge, this is the first report to

address the effect of combining the systemic administration

of bisphosphonates with a local exposure of LIPUS to

improve metaphyseal bone healing in rats.

Here, we showed that, separately, alendronate increased

BMD and BV/TV, but had no effect on bony bonding,

whereas LIPUS promoted cancellous bone repair by

enhancing bony bonding as well as BMD. From these

separate results, we expected that the combined therapy of

alendronate and LIPUS would stimulate cancellous bone

healing after osteotomy. However, overall, the combined

therapy did not significantly progress cancellous bone

healing at 2 and 4 weeks in aged rats over the separate

administration of alendronate and LIPUS.

In terms of BMD at 4 weeks, the two-factor factorial

analysis showed an increase in BMD with LIPUS and

alendronate separately, and the multiple comparisons

showed a significant effect for the combined group as

compared with the control. From this, we concluded that

both alendronate and LIPUS have significant effects on

BMD at 4 weeks, with an additive effect of the combined

therapy on BMD.

To date, few studies have focused on bone repair at the

metaphysis using bisphosphonate treatment. Kolios et al.

[36] presented metaphyseal tibial osteotomy healing fol-

lowed by plate fixation in ovariectomized rats, where orally

supplemented alendronate had no deteriorative effect on

metaphyseal callus properties. This is similar to results

observed with a diaphyseal fracture model, where intrave-

nous administration of zoledronate did not delay endo-

chondral fracture repair within the periosteal callus of

healthy rats [18]. In the present study, alendronate increased

BV/TV at 4 weeks, with a similar increase observed in the

combined group, but not in the LIPUS group. In the mul-

tiple comparisons, neither alendronate nor combined groups

achieved significance, but in the two-factor factorial

ANOVA, alendronate significantly contributed to the

increased BV/TV. Furthermore, alendronate did not inter-

rupt cancellous bone healing as evidenced by the favorable

levels of bony bonding observed in the LIPUS and in the

combined groups. Based on these findings, as well as the

significant increase in BMD with alendronate, it appears

that bisphosphonates do not interrupt cancellous bone

healing, but induce similar effects to those observed in

cortical bone, with increases in BMD and decreases in bone

turnover at the tibial metaphysis [15, 17]. The use of bis-

phosphonate treatment after a fragility fracture in elderly

patients may therefore be beneficial for maintaining the

bone density during cancellous bone healing.

This investigation is the first to show a positive effect for

LIPUS during metaphyseal bone repair after an osteotomy

through enhanced bony bonding at the repair site. With

regard to cancellous bony union, LIPUS alone significantly

stimulated cancellous bony union at 2 and 4 weeks, as

determined by the two-factor factorial ANOVA. This

positive effect of LIPUS on the percentage of cancellous

bone union should result in a shortening of the time to

Table 3 Percentage of bony bonding at the osteotomy site of the proximal tibia

Control Alendronate LIPUS Combined Two-factor factorial ANOVA

Alendronate LIPUS Interaction

2 weeks 40.0 ± 14.5 32.0 ± 20.7 53.5 ± 22.2 55.7 ± 15.8 N.S. p = 0.009 N.S.

4 weeks 32.6 ± 11.1 39.6 ± 12.6 53.9 ± 16.4a 49.9 ± 10.8 N.S. p = 0.002 N.S.

All values are mean ± SD

N.S. not significant, Control Saline ? Sham LIPUS, Alendronate Alendronate ? Sham LIPUS, LIPUS Saline ? LIPUS, Combined

Alendronate ? LIPUSa p = 0.025 vs control group by one-way analysis of variance (ANOVA) using Scheffe’s post hoc test

J Bone Miner Metab

123

complete cancellous bone union after surgeries for can-

cellous bone fractures. We anticipated that, similar to our

findings with BMD, the combined therapy would stimulate

the cancellous bone union. However, while we observed

very similar increases in cancellous bony union between

the combined and LIPUS only groups, the increase in bony

union with the combined group was not significant in the

multiple comparisons. From this, we have concluded that

the combined therapy may offer an effective solution for

metaphyseal bone repair in elderly patients, and that these

effects may be more obvious for shortening the time to

cancellous bone union in ovariectomized rats or under

other adverse conditions for bone repair, such as presence

of diabetes mellitus, steroid use, or smoking. Previous cell-

based assays have suggested that the beneficial effects of

LIPUS on bone healing may include a positive impact on

signal transduction, gene expression, cell differentiation,

and extracellular matrix synthesis and mineralization [37–

40]. Furthermore, in a rodent study, an effect of LIPUS was

found at each sequential phase of the healing process:

during the initial inflammatory stage, soft callus formation,

hard-callus formation, and remodeling [16]. Additionally,

recent work has shown that LIPUS may accelerate fracture

healing by enhancing callus formation and maturation [41].

While LIPUS did not show any significant changes in bone

histomorphometric parameters in the present study, it is

possible that these mechanisms may have contributed to

the favorable effects observed on cancellous bone healing.

There are several limitations in the present study. First,

we have examined the effects of combined therapy of

alendronate and LIPUS on cancellous bone healing only in

aged, not ovariectomized, rats. Many elderly patients have

osteoporosis, and thus the effects of this combined therapy

could be better evaluated under osteoporotic conditions.

However, the present study was conducted as a first step

toward determining the benefits of this combined therapy

in aged normal rats. Several previous studies have shown

that femoral or tibial mid-shaft fracture healing is delayed

and callus strength decreased in areas with low BMD in

ovariectomized rats [42, 43]. Based on these findings, we

expected that the combined therapy could exert an additive

effect on cancellous bone healing. However, for aged rats

vs. ovariectomized rats, the latter have substantially less

bone than aged rats, and thus the increases were not

obviously different in our model. It is possible that if this

study were repeated with ovariectomized rats, we may

observe a significant additive effect of alendronate and

LIPUS over each of these treatment regimens alone. Fur-

ther investigations will be needed to elucidate the signifi-

cant effects of this combined therapy on cancellous bone

healing in osteoporosis. Second, the osteotomy at the

proximal tibia may be considerably different from can-

cellous bone fractures such as those in the hip, knee, or

shoulder in clinical human cases. Animal models that

represent cancellous bone fractures are limited as com-

pared with animal models of cortical bone repair. We have

created this osteotomy model to evaluate cancellous bone

healing as a model of periarticular fractures.

In conclusion, alendronate had no adverse effects on

bony bonding, and LIPUS accelerated bone repair at the

tibial metaphysis during cancellous bone repair. In com-

bination, these treatments caused an additive increase in

BMD at the repair site. We surmise that alendronate acts by

increasing bone volume around the osteotomy site, while

LIPUS facilitates maturation of the regenerated bone. This

combined therapy may offer an effective solution for

metaphyseal fracture repair in elderly patients.

Acknowledgments We thank Teijin Pharma, Tokyo, Japan, for

kindly supplying a solution of alendronate and SAFHS. We also thank

Ms. K. Sakamoto and R. Kamoya for their technical assistance.

Conflict of interest All authors have no conflicts of interest.

References

1. Cranney A, Guyatt G, Griffith L, Wells G, Tugwell P, Rosen C,

Osteoporosis Methodology Group, The Osteoporosis Research

Advisory Group (2002) Meta-analyses of therapies for postmen-

opausal osteoporosis. IX: summary of meta-analyses of therapies

for postmenopausal osteoporosis. Endocr Rev 23:570–578

2. Black DM, Cummings SR, Karpf DB, Cauley JA, Thompson DE,

Nevitt MC, Bauer DC, Genant HK, Haskell WL, Marcus R, Ott

SM, Torner JC, Quandt SA, Reiss TF, Ensrud KE (1996) Ran-

domised trial of effect of alendronate on risk of fracture in

women with existing vertebral fractures. Fracture Intervention

Trial Research Group. Lancet 348:1535–1541

3. Reginster J, Minne HW, Sorensen OH, Hooper M, Roux C,

Brandi ML, Lund B, Ethgen D, Pack S, Roumagnac I, Eastell R

(2000) Randomized trial of the effects of risedronate on vertebral

fractures in women with established postmenopausal osteoporo-

sis. Vertebral Efficacy with Risedronate Therapy (VERT) Study

Group. Osteoporos Int 11:83–91

4. Lyles KW, Colon-Emeric CS, Magaziner JS, Adachi JD, Pieper

CF, Mautalen C, Hyldstrup L, Recknor C, Nordsletten L, Moore

KA, Lavecchia C, Zhang J, Mesenbrink P, Hodgson PK, Abrams

K, Orloff JJ, Horowitz Z, Eriksen EF, Boonen S, For the

HORIZON Recurrent Fracture Trial (2007) Zoledronic acid in

reducing clinical fracture and mortality after hip fracture. N Engl

J Med 357:1799–1809

5. Moroni A, Faldini C, Hoang-Kim A, Pegreffi F, Giannini S

(2007) Alendronate improves screw fixation in osteoporotic bone.

J Bone Joint Surg Am 89:96–101

6. Cecilia D, Jodar E, Fernandez C, Resines C, Hawkins F (2009)

Effect of alendronate in elderly patients after low trauma hip

fracture repair. Osteoporos Int 20:903–910

7. Rizzoli R, Reginster JY, Boonen S, Breart G, Diez-Perez A, Fel-

senberg D, Kaufman JM, Kanis JA, Cooper C (2011) Adverse

reactions and drug–drug interactions in the management of women

with postmenopausal osteoporosis. Calcif Tissue Int 89:91–104

8. Heckman JD, Ryaby JP, McCabe J, Frey JJ, Kilcoyne RF (1994)

Acceleration of tibial fracture-healing by non-invasive, low-

intensity pulsed ultrasound. J Bone Joint Surg Am 76:26–34

J Bone Miner Metab

123

9. Kristiansen TK, Ryaby JP, McCabe J, Frey JJ, Roe LR (1997)

Accelerated healing of distal radial fractures with the use of

specific, low-intensity ultrasound. A multicenter, prospective,

randomized, double-blind, placebo-controlled study. J Bone Joint

Surg Am 79:961–973

10. Leung KS, Lee WS, Tsui HF, Liu PP, Cheung WH (2004)

Complex tibial fracture outcomes following treatment with low-

intensity pulsed ultrasound. Ultrasound Med Biol 30:389–395

11. Gebauer D, Mayr E, Orthner E, Ryaby JP (2005) Low-intensity

pulsed ultrasound: effects on nonunions. Ultrasound Med Biol

31:1391–1402

12. Rutten S, Nolte PA, Guit GL, Bouman DE, Albers GH (2007)

Use of low-intensity pulsed ultrasound for posttraumatic non-

unions of the tibia: a review of patients treated in the Netherlands.

J Trauma 62:902–908

13. Watanabe Y, Matsushita T, Bhandari M, Zdero R, Schemitsch

EH (2010) Ultrasound for fracture healing: current evidence.

J Orthop Trauma 24:S56–S61

14. Wang SJ, Lewallen DG, Bolander ME, Chao EY, Ilstrup DM,

Greenleaf JF (1994) Low intensity ultrasound treatment increases

strength in a rat femoral fracture model. J Orthop Res 12:40–47

15. Peter CP, Cook WO, Nunamaker DM, Provost MT, Seedor JG,

Rodan GA (1996) Effect of alendronate on fracture healing and

bone remodeling in dogs. J Orthop Res 14:74–79

16. Azuma Y, Ito M, Harada Y, Takagi H, Ohta T, Jingushi S (2001)

Low-intensity pulsed ultrasound accelerates rat femoral fracture

healing by acting on the various cellular reactions in the fracture

callus. J Bone Miner Res 16:671–680

17. Cao Y, Mori S, Mashiba T, Westmore MS, Ma L, Sato M, Ak-

iyama T, Shi L, Komatsubara S, Miyamoto K, Norimatsu H

(2002) Raloxifene, estrogen, and alendronate affect the processes

of fracture repair differently in ovariectomized rats. J Bone Miner

Res 17:2237–2246

18. McDonald MM, Dulai S, Godfrey C, Amanat N, Sztynda T, Little

DG (2008) Bolus or weekly zoledronic acid administration does

not delay endochondral fracture repair but weekly dosing

enhances delays in hard callus remodeling. Bone 43:653–662

19. Ali AM, El-Shafie M, Willett KM (2002) Failure of fixation of

tibial plateau fractures. J Orthop Trauma 16:323–329

20. Frattini M, Vaienti E, Soncini G, Pogliacomi F (2009) Tibial

plateau fractures in elderly patients. Chir Organi Mov

93:109–114

21. Roerdink WH, Oskam J, Vierhout PA (2001) Arthroscopically

assisted osteosynthesis of tibial plateau fractures in patients older

than 55 years. Arthroscopy 17:826–831

22. Gaston P, Will EM, Keating JF (2005) Recovery of knee function

following fracture of the tibial plateau. J Bone Joint Surg Br

87:1233–1236

23. Aspenberg P (2009) Bisphosphonates and implants: an overview.

Acta Orthop 80:119–123

24. Wronski TJ, Dann LM, Scott KS, Cintron M (1989) Long-term

effects of ovariectomy and aging on the rat skeleton. Calcif

Tissue Int 45:360–366

25. Aonuma H, Miyakoshi N, Kasukawa Y, Kamo K, Sasaki H,

Tsuchie H, Segawa T, Shimada Y (2011) Combined treatment of

alendronate and low-intensity pulsed ultrasound (LIPUS)

increases bone mineral density at the cancellous bone osteotomy

site in aged rats: a preliminary study. JNMA J Nepal Med Assoc

51:171–175

26. Tamura Y, Miyakoshi N, Itoi E, Abe T, Kudo T, Tsuchida T,

Kasukawa Y, Sato K (2001) Long-term effects of withdrawal of

bisphosphonate incadronate disodium (YM175) on bone mineral

density, mass, structure, and turnover in the lumbar vertebrae of

ovariectomized rats. J Bone Miner Res 16:541–549

27. Suzuki K, Miyakoshi N, Tsuchida T, Kasukawa Y, Sato K, Itoi E

(2003) Effects of combined treatment of insulin and human

parathyroid hormone (1-34) on cancellous bone mass and struc-

ture in streptozotocin-induced diabetic rats. Bone 33:108–114

28. Nozaka K, Miyakoshi N, Kasukawa Y, Maekawa S, Noguchi H,

Shimada Y (2008) Intermittent administration of human para-

thyroid hormone enhances bone formation and union at the site of

cancellous bone osteotomy in normal and ovariectomized rats.

Bone 42:90–97

29. Huang RC, Khan SN, Sandhu HS, Metzl JA, Cammisa FP Jr,

Zheng F, Sama AA, Lane JM (2005) Alendronate inhibits spine

fusion in a rat model. Spine 30:2516–2522

30. Omi H, Kusumi T, Kijima H, Toh S (2007) Locally administered

low-dose alendronate increases bone mineral density during

distraction osteogenesis in a rabbit model. J Bone Joint Surg Br

89:984–988

31. Gebauer GP, Lin SS, Beam HA, Vieira P, Parsons JR (2002)

Low-intensity pulsed ultrasound increases the fracture callus

strength in diabetic BB Wistar rats but does not affect cellular

proliferation. J Orthop Res 20:587–592

32. Coords M, Breitbart E, Paglia D, Kappy N, Gandhi A, Cottrell J,

Cedeno N, Pounder N, O’Connor JP, Lin SS (2011) The effects of

low-intensity pulsed ultrasound upon diabetic fracture healing.

J Orthop Res 29:181–188

33. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H,

Meunier PJ, Ott SM, Recker RR (1987) Bone histomorphometry:

standardization of nomenclature, symbols, and units. Report of

the ASBMR Histomorphometry Nomenclature Committee.

J Bone Miner Res 2:595–610

34. Giannoudis P, Tzioupis C, Almalki T, Buckley R (2007) Fracture

healing in osteoporotic fractures: is it really different? A basic

science perspective. Injury 38:S90–S99

35. Giannoudis PV, Einhorn TA, Schmidmaier G, Marsh D (2008)

The diamond concept–open questions. Injury 39:S5–S8

36. Kolios L, Hoerster AK, Sehmisch S, Malcherek MC, Rack T,

Tezval M, Seidlova-Wuttke D, Wuttke W, Stuermer KM,

Stuermer EK (2010) Do estrogen and alendronate improve met-

aphyseal fracture healing when applied as osteoporosis prophy-

laxis? Calcif Tissue Int 86:23–32

37. Hasegawa T, Miwa M, Sakai Y, Niikura T, Kurosaka M, Komori

T (2009) Osteogenic activity of human fracture haematoma-

derived progenitor cells is stimulated by low-intensity pulsed

ultrasound in vitro. J Bone Joint Surg Br 91:264–270

38. Naruse K, Mikuni-Takagaki Y, Urabe K, Uchida K, Itoman M

(2009) Therapeutic ultrasound induces periosteal ossification

without apparent changes in cartilage. Connect Tissue Res

50:55–63

39. Olkku A, Leskinen JJ, Lammi MJ, Hynynen K, Mahonen A

(2010) Ultrasound-induced activation of Wnt signaling in human

MG-63 osteoblastic cells. Bone 47:320–330

40. Favaro-Pıpi E, Bossini P, de Oliveira P, Ribeiro JU, Tim C,

Parizotto NA, Alves JM, Ribeiro DA, Selistre de Araujo HS,

Renno AC (2010) Low-intensity pulsed ultrasound produced an

increase of osteogenic genes expression during the process of

bone healing in rats. Ultrasound Med Biol 36:2057–2064

41. Cheung WH, Chow SK, Sun MH, Qin L, Leung KS (2011) Low-

intensity pulsed ultrasound accelerated callus formation, angio-

genesis and callus remodeling in osteoporotic fracture healing.

Ultrasound Med Biol 37:231–238

42. Namkung-Matthai H, Appleyard R, Jansen J, Hao Lin J, Maas-

tricht S, Swain M, Mason RS, Murrell GA, Diwan AD, Diamond

T (2001) Osteoporosis influences the early period of fracture

healing in a rat osteoporotic model. Bone 28:80–86

43. Wang JW, Li W, Xu SW, Yang DS, Wang Y, Lin M, Zhao GF

(2005) Osteoporosis influences the middle and late periods of

fracture healing in a rat osteoporotic model. Chin J Traumatol

8:111–116

J Bone Miner Metab

123