Eel calcitonin (elcatonin) suppressed callus remodeling but did not interfere with fracture healing in the femoral fracture model of cynomolgus monkeys.

Abstract

We investigated the effect of eel calcitonin (elcatonin) on the process of fracture repair in the osteotomized femur of cynomolgus monkeys, since they possess a Haversian remodeling system similar to that of humans. Alendronate was used for comparison. Twenty female cynomolgus monkeys (Macaca fascicularis), aged 18-22 years, were allocated into five groups: control (CNT, n = 4), low-dose elcatonin group (0.5 U/kg; ELL, n = 4), high-dose elcatonin group (5 U/kg; ELH, n = 4), low-dose alendronate group (10 microg/kg; ALL, n = 4) and high-dose alendronate group (100 microg/kg; ALH, n = 4). All animals were given subcutaneous injections twice a week for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless steel plate. After fracture, treatments were continued until sacrifice at 26 weeks after surgery. The femora were assessed by micro CT, contact microradiograph, three-point bending mechanical test and histomorphometry. Micro CT showed that callus sizes in elcatonin-treated groups were similar to CNT, whereas alendronate-treated groups had larger calluses than those in the CNT and elcatonin-treated groups. Fracture lines almost disappeared in the CNT and elcatonin-treated groups but remained clear in the alendronate-treated groups. Total area did not differ significantly between the elcatonin-treated groups and the CNT but was significantly greater in the ALH compared to the CNT and elcatonin-treated groups, due to increased callus area in the ALH group. Callus remodeling was less suppressed in the elcatonin-treated groups than in the alendronate-treated groups when compared with callus remodeling in the CNT. Although no significant differences in structural mechanical properties such as ultimate load, stiffness and work to failure were found among all groups, ultimate stress was significantly reduced in the ALH group compared with CNT and ELL groups. In conclusion, mild suppression of callus remodeling by elcatonin did not impair overall fracture healing process. In contrast, alendronate delayed structural fracture healing process by strongly suppressing callus remodeling.

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ORIGINAL ARTICLE
Eel calcitonin (elcatonin) suppressed callus remodeling
but did not interfere with fracture healing in the femoral fracture
model of cynomolgus monkeys
Takeshi Manabe Æ Æ Satoshi Mori Æ Æ Tasuku Mashiba Æ Æ Yongping Cao Æ Æ Yoshio Kaji Æ Æ
Ken Iwata Æ Æ Satoshi Komatsubara Æ Æ Tetsuji Yamamoto Æ Æ Azusa Seki Æ Æ Hiromichi Norimatsu
Received: 15 January 2008/Accepted: 24 August 2008/Published online: 3 April 2009
? The Japanese Society for Bone and Mineral Research and Springer 2009
Abstract
(elcatonin) on the process of fracture repair in the osteo-
tomized femur of cynomolgus monkeys, since they possess
a Haversian remodeling system similar to that of humans.
Alendronate was used for comparison. Twenty female
cynomolgus monkeys (Macaca fascicularis), aged 18–
22 years, were allocated into five groups: control (CNT,
n = 4), low-dose elcatonin group (0.5 U/kg; ELL, n = 4),
high-dose elcatonin group (5 U/kg; ELH, n = 4), low-dose
alendronate group (10 lg/kg; ALL, n = 4) and high-dose
alendronate group (100 lg/kg; ALH, n = 4). All animals
were given subcutaneous injections twice a week for
3 weeks. Then fracture was produced surgically by trans-
versely cutting the midshaft of the right femur and fixing
with stainless steel plate. After fracture, treatments were
We investigated the effect of eel calcitonin
continued until sacrifice at 26 weeks after surgery. The
femora were assessed by micro CT, contact microradio-
graph,three-pointbending
histomorphometry. Micro CT showed that callus sizes in
elcatonin-treated groups were similar to CNT, whereas
alendronate-treated groups had larger calluses than those in
the CNT and elcatonin-treated groups. Fracture lines
almost disappeared in the CNT and elcatonin-treated
groups but remained clear in the alendronate-treated
groups. Total area did not differ significantly between the
elcatonin-treated groups and the CNT but was significantly
greater in the ALH compared to the CNT and elcatonin-
treated groups, due to increased callus area in the ALH
group. Callus remodeling was less suppressed in the elca-
tonin-treated groups than in the alendronate-treated groups
when compared with callus remodeling in the CNT.
Although no significant differences in structural mechani-
cal properties such as ultimate load, stiffness and work to
failure were found among all groups, ultimate stress was
significantly reduced in the ALH group compared with
CNT and ELL groups. In conclusion, mild suppression of
callus remodeling by elcatonin did not impair overall
fracture healing process. In contrast, alendronate delayed
structural fracture healing process by strongly suppressing
callus remodeling.
mechanicaltestand
Keywords
Fracture healing ? Micro-CT ? Cynomolgus monkey
Calcitonin ? Bisphosphonate ?
Introduction
Antiresorptive agents are used widely in the treatment of
osteoporosis in postmenopausal woman [1–9]. These
agents have been shown to prevent bone loss and
T. Manabe ? T. Mashiba ? Y. Cao ? Y. Kaji ? K. Iwata ?
S. Komatsubara ? T. Yamamoto ? H. Norimatsu
Department of Orthopedic Surgery, Faculty of Medicine,
Kagawa University, Kagawa, Japan
S. Mori (&)
Department of Bone and Joint Surgery,
Seirei Hamamatsu General Hospital, 2-12-12 Sumiyoshi,
Hamamatsu, Shizuoka 430-8558, Japan
e-mail: stmori@sis.seirei.or.jp
Y. Cao
Department of Orthopedic Surgery,
First Hospital of Peking University, Peking, China
A. Seki
Hamri Corporation, Tokyo, Japan
H. Norimatsu
Shikoku Medical College, Kagawa, Japan
123
J Bone Miner Metab (2009) 27:295–302
DOI 10.1007/s00774-009-0046-x

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osteoporotic fractures by suppressing the osteoclastic
resorption of bone [1, 2]. Secondly, they are effective
because they reduce bone remodeling by suppressing bone
formation activity [1, 2, 10, 11]. Alendronate reduces
activation frequency by 90% after 2 years treatment in
postmenopausal women [11].
Since osteoporotic patients are prone to fracture, phy-
sicians are often confronted with the situation of fracture
occurring in patients under treatment. However, few stud-
ies have focused on the effect of antiresorptive agents on
the healing process after the patients sustain fractures
during treatment. We have demonstrated that bisphospho-
nates such as incadronate and alendronate treatment
resulted in strong and enlarged calluses but delayed callus
remodeling in fractured femora of rats [12–16]. Thus, it is
possible that strong suppression of osteoclastic bone
resorption may prolong the natural fracture healing
process.
Elcatonin, a synthetic derivative of eel calcitonin, con-
sists of 31 amino acids with stable ethylene linkage as a
result of substituting the intra-chain disulfide bond of cal-
citonin. Elcatonin is physicochemically and biologically
stable, and inhibits bone resorption through direct action on
the calcitonin receptors on osteoclast [17]. In Japan, this
drug has been approved in clinical treatment of osteopo-
rosis, mainly for the analgesic effect, in addition to the
effect of bone loss prevention [18–21].
Because of its analgesic effect, elcatonin is frequently
used for pain relief in patients with fractures, especially
immediately after fracture. Based on this clinical usage, it
is important to examine the effects of this agent on the
fracture healing process.
In this study, we examined the effect of elcatonin on the
fracture healing process compared with the effect of
alendronate, using the femoral fracture model in cyno-
molgus monkey that possesses intracortical remodeling
similar to humans.
Materials and methods
Animals
Twenty female cynomolgus monkeys (Macaca fascicu-
laris)aged 18–22 years
Philippines) with normal menstrual cycle were housed in a
room maintained at 22 to 30?C and a 12-h light-dark cycle,
with free access to water and restricted access to standard
monkey breeder pellets (Republic Flour Mills Corp., Lu-
guna, Philippines) of 100 g/day. All animal procedures
were conducted in accordance with the National Institutes
of Health (NIH) guidelines, following a protocol approved
by the Kagawa University Animal Study Committee.
(SiconbrecInc.Makati,
Experimental design
After 1-month acclimatization, all monkeys were allocated
into five groups by matching body weights: control group
(CNT, n = 4), low-dose elcatonin group (ELL, n = 4),
high-doseelcatoningroup
alendronate group (ALL, n = 4) and high-dose alendronate
group (ALH, n = 4). Animals in CNT group were given
subcutaneous injection of 0.9% saline vehicle. The
remaining four groups were given subcutaneous injection
of elcatonin (Asahi Kasei Corp., Tokyo, Japan) at a dose of
0.5 U/kg per day (ELL group) or 5 U/kg per day (ELH
group), or alendronate (Teijin Pharma Limited, Tokyo,
Japan) at a dose of 10 lg/kg per day (ALL group) or
100 lg/kg per day (ALH group). Injections were given
twice a week. ELL and ALL doses are clinical doses for
human osteoporosis treatment, while ELH and ALH doses
are 10 times the corresponding clinical doses. Treatment
was initiated 3 weeks before experimental fracture, and
continued after treatment until sacrifice. Body weights
were measured weekly and doses were adjusted accord-
ingly (Fig. 1).
(ELH,
n = 4), low-dose
Fracture surgery
Fracture was produced surgically in all animals after
3 weeks of pretreatment. Under general anesthesia with a
2:1 mixture (10 mg/kg, i.m.) of ketamine hydrochloride
(Troy Laboratories Pty. Ltd., Smithfield, Australia) and
xylazine hydrochloride (Troy Laboratories Pty. Ltd.), a
transverse osteotomy was performed at the midshaft of the
Fig. 1 Experimental protocol. CNT group: vehicle (0.9% saline)
treatment, ELL group: treatment with elcatonin at 0.5 U/kg, ELH
group: treatment with elcatonin at 5 U/kg, ALL group: treatment with
alendronate at 10 lg/kg, ALH group: treatment with alendronate at
100 lg/kg, before and after fracture surgery. Subcutaneous injection
was performed twice a week
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right femur using a fine-toothed circular microsaw (Kiso
Power Co., Osaka, Japan). The fracture was repositioned
and fixed tightly with a stainless plate and screws (Osteo-
mini ACP plate, Stryker, Michigan, USA). Unrestricted
ambulation was allowed after recovery from general
anesthesia, and dosing was continued until sacrifice.
Evaluations
Anteroposterior and lateral radiographs of the fractured
femora were taken using an Acoma portable X-ray unit
(20 mA, 60 Kvp, 0.4 S; PX-20 N, Acoma X-ray Industry
Co., Ltd. Tokyo, Japan) immediately after fracture surgery
and repeated every 4 weeks until sacrifice to check fracture
alignment and fixation. The animals were sacrificed at
26 weeks after surgery. All animals were double-labeled
intravenously with calcein (8 mg/kg; Dojin Chemical
Institute, Kumamoto, Japan) at 22 and 9 days before sac-
rifice. After necropsy, the femora were excised and
dissected free of soft tissues. The stainless plate and screws
were removed carefully so as not to damage the callus.
Anteroposterior soft radiographs of the femora were taken
and the femora were frozen at -80?C wrapped in gauze
soaked in isotonic saline until micro computed tomo-
graphic (micro CT) scanning.
After thawing at room temperature, the femora were
scanned by micro CT (25 lA, 70 KV, 1.8 W; NX-LCP-
C80, Nittetsu Elex Co., Ltd. Tokyo, Japan). The bones
were placed horizontally on the table. One hundred slices
were scanned both at the proximal and distal sides of the
fracture site. Then sagittal image of the fracture were
reconstructed. After micro CT scanning, the femora were
tested for mechanical properties by a three-point bending
method using a materials testing machine (MZ500S,
Maruto, Tokyo, Japan). A femur was placed on two lower
support bars (40 mm apart) with the anterior surface
facing upward. The fracture plane was centered at the
loading point, and load was applied at a rate of
166.7 mm/min until breakage. From the load versus dis-
placement curve, the structural mechanical properties of
the fractured bone were determined as ultimate load
(maximum force that the specimen sustained), stiffness
(the slope of the linear portion of the load vs. deformation
curve before failure) and work to failure (the area under
the load vs. deformation curve before failure). These
structural parameters depend on both intrinsic material
properties and geometry.
The load vs. displacement data were normalized to
obtain intrinsic material properties such as ultimate stress,
elastic modulus, and toughness, which are independent of
cross-sectional area and shape.
Ultimate stress was calculated from the ultimate load by
the following formula:
Ultimatestress ¼ 0:125 ? Ultimateload ? L ? b=I
where L is the length between supports, b is the width of
the femur in anteroposterior direction, and I is the cross-
sectional moment of inertia.
Elastic modulus was calculated as:
Elasticmodulus ¼ Stiffness*L3=48I
Toughness was calculated from energy absorption as:
Toughness ¼ 0:75 ? work to failure*b2=LI
Cross-sectional moment of inertia (I) was calculated
withthe assumptionthat
elliptically shaped:
?
where a is the width in mediolateral direction, and t is the
average cortical thickness.
Average cortical thickness was calculated from histo-
morphometric thickness measurements obtained from each
of four quadrants of the femora section using a digitizing
system.
After mechanical testing, the segment proximal to the
fracture was fixed in 10% cold neutral buffered formalin
for 3 days, decalcified in 10% EDTA at 4?C for 4 weeks,
and then embedded in paraffin. Within 500 lm from the
original fracture line, 5-lm cross-sections were cut for
tartrate-resistant acid phosphatase (TRAP) staining. The
presence of osteoclasts was determined by TRAP activity
using a leukocyte acid phosphatase kit (Sigma Chemical
Co., St. Louis, MO, USA).
The segment distal to the fracture was fixed in 70%
ethanol, stained in Villanueva bone stain (Polysciences Inc.,
Warrington, PA, USA), dehydrated in graded ethanol,
defatted in acetone, and embedded in methyl methacrylate.
Within 500 lm from the original fracture line, 100-lm
undecalcified transverse sections were cut using a diamond
microtome saw (RM2065, Leica Instruments GmbH,
Nussloch,Germany) for
(100 Kvp, 2 lA, 5 min; lFX-1000; Fujifilm, Tokyo,
Japan). Then specimens were ground to 30 lm in thickness
for histomorphometric measurement.
Histomorphometric analysis was performed using a
semi-automated digitizing image analyzer. The system
consisted of a light or epifluorescent microscope and a
digitizing pad coupled with computer and histomorpho-
metric software (System Supply CO., Nagano, Japan)
Total cross-sectional area (T.Ar; mm2), medullary area
(Me.Ar; mm2), callus area (Cl.Ar; mm2), and thickness
(Cl.Th; lm) were measured at 409 magnification, and
bone area (B.Ar = T.Ar - Me.Ar) was calculated. Percent
bone area (%B.Ar = B.Ar 9 100/T.Ar; %) and percentage
callus area (%Cl.Ar = Cl.Ar 9 100/T.Ar; %) were also
femoralcross-section is
I ¼ ðp=64Þ ? ab3? ða ? 2tÞðb ? 2tÞ3
?
;
contact microradiographs
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calculated. Single-labeled surface, double-labeled surface,
and the interlabeling width were measured at the callus.
Mineral apposition rate (MAR; lm/day), mineralizing
surface (MS/BS; %) and bone formation rate (BFR/BS;
mm3/mm2per year) were calculated. Osteoclast measure-
ments including osteoclast number (N.Oc; #) and derived
parameters (N.Oc/BS; #/lm) were performed at 1009
magnification at the callus.
Statistical analysis
Statistical computation of data was performed using the
statistical package StatView 5.0. Differences among
treatment groups were tested by one-way analysis of
variance (ANOVA). If a significant difference was
obtained, differences between the means of two groups
were tested by Fisher’s protected least significant differ-
ence (PLSD). A P-value less than 0.05 were considered
significant.
Results
General condition
The animals resumed normal activity within a few days
after fracture surgery and no health problems were
pointed out during the study period. Body weight of the
animal was not significant among the groups during the
study period.
Micro-CT
Micro-CT at the sagittal plane revealed that alendronate-
treated groups had larger callus at both periosteal and
endosteal surfaces than CNT or elcatonin-treated groups.
Especially, ALH had the largest callus. In contrast, callus
sizes were similar in elcatonin-treated groups and CNT.
Fracture lines were clearly visible in the alendronate-trea-
ted groups, but were obscure in CNT and elcatonin-treated
groups (Fig. 2).
Contact microradiographs
Contact microradiographs also confirmed that callus size
was larger in the alendronate-treated groups compared with
callus sizes in the CNT and the elcatonin-treated groups,
and was the largest in ALH (Fig. 3).
Mechanical test
The ultimate load, stiffness, and work to failure in the
femur did not differ significantly among the five groups.
When these data were normalized by cross-sectional
moment of inertia, the intrinsic material properties such as
ultimate stress, elastic modulus, and toughness tended to be
smaller in ELH and alendronate-treated groups. Ultimate
stress was significantly (P\0.05) lower only in ALH
compared with CNT (Table 1).
Histomorphometry
Total area (T.Ar) of the femur was larger in the alendro-
nate-treated groups compared with CNT but was similar in
elcatonin-treated groups and CNT. Especially, T.Ar was
significantly larger in ALH than in ELL or CNT (P\0.05
in both). Percent bone area (%B.Ar) was also significantly
larger in ALH than in ELL or CNT (P\0.05 in both).
Percent callus area (%Cl.Ar) tended to be larger in
alendronate-treated groups than in CNT, and was the
largest in ALH among the five groups. Similarly, ALH had
the greatest callus thickness (Cl.Th).
Remodeling parameters in callus were suppressed in a
dose-dependent manner following both elcatonin and
alendronate treatment, but the extent of remodeling sup-
pression tended to be less in elcatonin-treated groups.
Mineral apposition rate (MAR), bone formation rate (BFR/
BS) and mineralizing surface (MS/BS; %) were signifi-
cantly lower in elcatonin- and alendronate-treated groups
than in CNT. Especially, ALH showed the lowest values
for all dynamic parameters among the five groups.
Number of osteoclast (N.Oc/Cl.Ar) was significantly
less in both elcatonin- and alendronate-treated groups than
in CNT (Table 2).
Fig. 2 Micro CT images. The
alendronate-treated groups show
larger callus in both periosteal
and endosteal surfaces than the
CNT or elcatonin-treated
groups. Fracture lines are
clearly visible in alendronate-
treated groups but were obscure
in CNT and elcatonin-treated
groups
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Discussion
Intracortical remodeling plays an important role in restor-
ing structural and mechanical properties of fractured bone
[22–26]. Therefore, a simian fracture model that possesses
the Haversian remodeling system is expected to provide
more precise information regarding human fracture healing
process than animal models without such a system. To the
best of our knowledge, this is the first study to evaluate the
effect of calcitonin on fracture healing using nonhuman
primate. Although the statistical power of this study may
not be strong because of a limited number of animals, we
believe that the results of this study provide important
information regarding the effects of osteoporotic drug
treatment on patients who sustained fractures during
treatment.
The primary aim of this study was to examine the
effect of elcatonin on the fracture healing process using a
femoral osteotomy model in cynomolgus monkey that
possess a Haversian intracortical remodeling system
similar to humans. Our results clearly indicated that
treatment with both elcatonin and alendronate at clinical
doses for treating human osteoporosis in Japan sup-
pressed callusremodeling but did not impair the
mechanical or structural fracture healing process. How-
ever, our data also showed that alendronate at a dose ten
Table 1 Biomechanical properties of fractured femur
CNT ELLELHALL ALH
Structural mechanical properties
Ultimate load (N)1213 ± 243 1006 ± 136 1074 ± 1701195 ± 1631229 ± 101
Stiffness (N/mm) 11027 ± 805 11301 ± 332 11163 ± 4549 11774 ± 330 12086 ± 277
Work to failure N-m702 ± 214 520 ± 191 504 ± 285523 ± 90596 ± 128
Intrinsic material properties
Ultimate stress (Mpa)40.2 ± 2.640.6 ± 6.233.3 ± 4.7 28.6 ± 2.422.1 ± 4.1a, b
Elastic modulus (Mpa)
Toughness (MJ/m3)
10510 ± 175711283 ± 144110771 ± 30448030 ± 10586240 ± 1733
833.6 ± 150.5 739.9 ± 209.0454.6 ± 230.0 451.4 ± 45.5361.4 ± 41.2
CNT group: vehicle control treatment; ELL and ELH groups: treatment with elcatonin 0.5 and 5 U/kg per day, respectively, before and after
fracture surgery
ALL, ALH groups: treatment with alendronate 10 and 100 lg/kg per day, respectively, before and after fracture surgery Data are expressed as
means ± SEM
aP\0.05 versus CNT,bP\0.05 versus ELL
Fig. 3 Contact microradio-
graphs at the fracture plane.
Callus size is larger in the
alendronate-treated groups
compared with callus sizes in
the CNT and the elcatonin-
treated groups. Especially the
largest callus size is observed at
high-dose alendronate (ALH)
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times the clinical dose (ALH) decreased some intrinsic
material properties of fractured femur through more
severe suppression of callus remodeling, although highly
increased callus volume was observed only in alendro-
nate-treated groups. In contrast, elcatonin at a dose ten
times the clinical dose (ELH) did not decrease any bio-
mechanical properties in spite of histological suppression
of callus remodeling.
Our previous study using rat femoral fracture model also
showed that alendronate increased the size and mineral
content of callus, while delaying remodeling of the callus
woven bone into lamellar bone at both 6 and 16 weeks
after the fracture surgery [15, 16].
The enlarged callus in alendronate-treated animals sta-
bilizes the fracture, despite the increased woven bone
content and delayed callus remodeling. Therefore, the
larger volume and cross-sectional area (moment of inertia)
observed in alendronate treatment appear to be an adap-
tation that compensates for the alendronate-induced delay
of woven bone remodeling into lamellar bone, which is
structurally and mechanically superior to woven bone [15].
Essentially, the same results were also obtained in a pre-
vious study using another bisphosphonate incadronate [12],
indicating that increased size, greater mineral content, and
strong inhibition of callus remodeling are the characteristic
class effects of bisphosphonates as a class of antiresorptive.
In contrast, other antiresorptive agents such as estrogen,
raloxifene and vitamin D3 analogue mildly suppress callus
remodeling and do not greatly alter the structural and
mechanical fracture healing process when compared with
control groups [15, 16].
Data on the efficacy of injectable calcitonin on fracture
are limited. Only one open randomized controlled study
showed that elcatonin reduced vertebral fracture risk by
59% with a small increase in BMD compared to placebo
after 2 years treatment [19]. Although calcitonin had a
mild effect on bone mass, the antiresorptive effect of cal-
citonin has been validated in many basic and clinical
studies [17–21, 27, 28].
In the present study, mineral apposition rate, bone for-
mation rate and mineralizing surface were significantly
lower in elcatonin- and alendronate-treated animals than in
controls. Especially, the high-dose alendronate-treated
group showed the lowest values for all dynamic parameters
among the five groups. These results showed that alendr-
onate delayed callus remodeling by strongly suppressing
remodeling of woven bone into lamellar bone. In contrast,
elcatonin mildly suppressed callus remodeling and did not
impair the overall fracture healing process. Generally the
anti-resorptive action of calcitonin is weaker than those of
bisphosphonates as also demonstrated in the current study.
Possible explanation for this may be the difference in the
duration of pharmacological action of these agents. Calci-
tonin inhibits osteoclastic bone resorption through its
receptor abundantly expressed on the plasma membrane of
osteoclast [29–31]. Therefore, this agent dose not accu-
mulate in the bone and the duration of calcitonin action
should be limited. In contrast, bisphosphonates usually
accumulate in the bone due to the high affinity for
hydroxyapatite, and they have much longer durations of
pharmacological action. The high binding affinities of
bisphosphonates for bone may affect many important bio-
logical properties of these compounds including uptake and
retention on the skeleton, diffusion of the drug within bone,
release of absorbed drug back onto bone surfaces, and
recycling of the desorbed drug back onto bone surfaces,
and effects on mineral dynamics and cellular function
within bone [32].
Table 2 Bone histomorphometry in fracture callus
GroupCNT ELL ELH ALLALH
T.Ar (mm2)99.5 ± 12.7 95.9 ± 6.6 103.3 ± 13.7114.8 ± 17.4139.8 ± 19.3e, j
84.8 ± 3.2e, j
43.4 ± 6.2j
2034.9 ± 503.5e, j
0.1 ± 0.1b, j
0.001 ± 0.0004a, h, k
0.010 ± 0.004b, i, k
0.2 ± 0.04a, f
%B.Ar (%)77.7 ± 1.6 76.3 ± 2.7 78.1 ± 1.9 80.9 ± 1.9
%Cl.Ar (%)
Cl.Th (lm)
MAR (lm/day)
BFR/BS (mm3/mm2per year
27.9 ± 4.922.0 ± 5.227.9 ± 6.934.2 ± 6.1
1030.8 ± 230.5862.2 ± 141.3
0.8 ± 0.1e
0.024 ± 0.004a
0.130 ± 0.034e
1.0 ± 0.2d
1138.2 ± 239.2
0.6 ± 0.2d
0.015 ± 0.005a
0.097 ± 0.028e
0.2 ± 0.05a, f
1342.2 ± 283.3
0.5 ± 0.1c
0.004 ± 0.001a, j
0.035 ± 0.010b, j
0.5 ± 0.05a, j
1.4 ± 0.3
0.074 ± 0.009
MS/BS (%) 0.236 ± 0.041
N.Oc/BS (#/mm)1.5 ± 0.2
CNT group: vehicle control treatment; ELL and ELH groups: treatment with elcatonin 0.5 and 5 U/kg per day, respectively, before and after
fracture surgery
ALL, ALH groups: treatment with alendronate 10 and 100 lg/kg per day, respectively, before and after fracture surgery
Data are expressed as means ± SEM
aP\0.0001 versus CNT;bP\0.001 versus CNT;cP\0.005 versus CNT;dP\0.01 versus CNT;eP\0.05 versus CNT;fP\0.0005 versus
ELL;gP\0.001 versus ELL;hP\0.05 versus ELL;iP\0.01 versus ELL;jP\0.05 versus ELL;kP\0.05 versus ELH
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There is a weakness in the current study. Because
antiresorptive agents are administrated for osteoporosis
patients in a clinical situation, osteoporotic animal models,
such as ovariectomized, would provide more clinically
related information than normal animal model regarding
the effect of antiresorptive agents on fracture healing in the
osteoporotic patient. However, we used old enough mon-
key (18–22 years) with naturally reduced ovarian function
although it was not evaluated exactly.
Based on radiological, histomorphometric and biome-
chanical evaluation of fractured femur in cynomolgus
monkeys treated with elcatonin, we conclude that elcatonin
mildly suppresses callus remodeling and has no effect on
the natural fracture healing process and intrinsic material
properties. In contrast, high-dose alendronate induces the
formation of large, strong callus but delays the structural
and material restoration of the callus. Regarding clinical
relevance, these animal fracture data suggest that physi-
cians need not consider discontinuation of elcatonin
treatment when a patient undergoing elcatonin therapy for
osteoporosis sustains osteoporotic fractures. Moreover, the
fact that calcitonin has a potent analgesic effect may be an
additional benefit for the continuous use of this agent after
fracture.
Acknowledgments
Fukuda for histological preparation and Asahi Kasei Co. for kindly
supplying the elcatonin.
The authors thank Mika Kawada and Yoshiko
Conflict of interest statement
interest associated with this study and publication.
All authors have no conflict of
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