J Appl Physiol 95: 631–634, 2003;
10.1152/japplphysiol.01049.2002.
Cortical bone dynamics, strength, and densitometry
after induction of emphysema in hamsters
Jill E. Shea,1 Scott C. Miller,1,2 David C. Poole,3 and John P. Mattson2
1
Division of Radiobiology, Department of Radiology, University of Utah, Salt Lake City 84108; and
Department of Exercise and Sport Science, University of Utah, Salt Lake City, Utah 84112; and 3Departments
of Kinesiology, Anatomy, and Physiology, Kansas State University, Manhattan, Kansas 66506
2
Submitted 14 November 2002; accepted in final form 21 February 2003
osteopenia; chronic obstructive pulmonary disease; histomorphometry
THERE IS A GROWING BODY of evidence relating chronic
obstructive pulmonary disease (COPD) to osteopenia
and osteoporosis (4, 7, 8, 11, 15). Low bone mineral
density (BMD) has been measured at the forearm, leg,
spine, and whole body of COPD patients (4, 7, 8, 11,
15). One possible explanation for the association between COPD and low bone mass is the high level of
corticosteroid use among individuals suffering from
COPD (9). Corticosteroid use is known to decrease
BMD and is also associated with an increase in fracture risk (9, 25, 27). However, recent investigations
have demonstrated low BMD in COPD patients not
receiving corticosteroids (4, 8, 15). Therefore, the decreased BMD observed in individuals with COPD may
Address for reprint requests and other correspondence: J. P. Mattson, 1850 E. 250 S., Rm. 241, Salt Lake City, UT 84112-0920 (E-mail:
John.Mattson@health.utah.edu).
http://www.jap.org
be related to the underlying lung disease and not
necessarily the treatment regimen.
The pathophysiological pathway connecting COPD
to bone loss is unknown. However, numerous characteristics of COPD patients, in addition to the high
incidence of corticosteroid use, may contribute to the
decreased BMD observed in this patient population.
For example, reduced physical activity levels in COPD
patients may be important (11), because skeletal underloading is associated with bone loss (2, 3). Other
characteristics of individuals with COPD associated
with bone loss are cigarette smoking, hypercapnia,
respiratory acidosis, vitamin D deficiency, and hypogonadism (4, 7).
Pulmonary emphysema, characterized by air space
enlargement, is a major component of COPD. Several
animal models have been utilized to gain a better
understanding of the mechanisms leading to the development of COPD and emphysema and their sequelae
(6, 20). Elastase-induced emphysema is commonly
used as a model to investigate events downstream of
the initial development of the disease (6, 13, 14, 18, 19).
Previous research has shown that there are normally
no alterations in activity levels with this model (14).
Therefore, the model controls for corticosteroid use, as
well as activity level, two factors known to influence
skeletal metabolism. Because bone loss is associated
with COPD in the patient population, it is the goal of
the present investigation to determine whether there
are any changes to the cortical bone in the elastaseinduced emphysematous hamster model.
MATERIALS AND METHODS
The protocols were approved by the University of Utah
Institutional Animal Care and Use Committee. In all respects, they conform to the Guide for the Care and Use
of Laboratory Animals [DHHS Publication No. (NIH) 8523, revised 1985, Office of Science and Health Reports,
Bethesda, MD].
Animals. After a 1-wk acclimation period, 20 adolescent
male Syrian Golden hamsters (9 wk old, 100–118 g body wt)
were divided randomly into control (Con) and emphysema
(Emp) groups. Under deep ketamine-xylazine anesthesia
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society
631
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on September 30, 2016
Shea, Jill E., Scott C. Miller, David C. Poole, and
John P. Mattson. Cortical bone dynamics, strength, and
densitometry after induction of emphysema in hamsters. J
Appl Physiol 95: 631–634, 2003; 10.1152/japplphysiol.01049.
2002.—Recent evidence suggests that patients suffering from
chronic obstructive pulmonary disease are also at an increased risk of developing osteoporosis. The pathophysiological mechanism(s) linking these progressive diseases is unknown. The goal of this investigation was to determine
whether there were alterations in bone mineral density and
content, cortical bone structure and strength, and indexes of
bone formation and resorption in the elastase-induced emphysematous hamster. At 3 wk after induction of emphysema, the femoral bone mineral content was 8% less (P ⫽
0.026) and the femoral fracture strength was 6% less (P ⫽
0.032) in the emphysematous hamster than in controls. The
cortical area was 8.4% less (P ⫽ 0.013) and the periosteal
mineral appositional rate was 27% less (P ⫽ 0.05) than in
controls. Additionally, the endocortical eroded surface in the
emphysematous group was about twice that in the control
group (P ⫽ 0.003). Differences in some indexes of bone formation and resorption, paralleled by differences in bone
structure and strength, were observed 3 wk after induction of
emphysema. These differences in skeletal metabolism and
strength may help explain some of the skeletal changes
associated with chronic obstructive pulmonary disease in
humans.
632
CORTICAL BONE DYNAMICS WITH EMPHYSEMA
Table 1. BMD and BMC of femurs
Femur
BMD, g/cm2
BMC, g
Middiaphysis
BMD, g/cm2
BMC, g
Emp Group
Con Group
P Value
0.119 ⫾ 0.006
0.150 ⫾ 0.012
0.123 ⫾ 0.004
0.162 ⫾ 0.008
0.162
0.026
0.108 ⫾ 0.006
0.013 ⫾ 0.001
0.113 ⫾ 0.005
0.014 ⫾ 0.001
0.406
0.045
Values are means ⫾ SD. BMD, bone mineral density; BMC, bone
mineral content; Emp, emphysema; Con, control.
J Appl Physiol • VOL
Table 2. Mechanical properties of femurs
from three-point bending test
Breaking load, N
Flexural rigidity, N/mm
Work to fracture, J/mm3
Emp Group
Con Group
P Value
41.1 ⫾ 2.8
106.5 ⫾ 15.1
0.25 ⫾ 0.07
43.8 ⫾ 3.3
118.6 ⫾ 15.1
0.20 ⫾ 0.5
0.032
0.165
0.121
Values are means ⫾ SD.
Statistics. Differences between groups were tested for significance by a two-tailed nonparametric Wilcoxon’s two-sample test (JMP, version 5.0). Values are means ⫾ SD and were
considered significant at P ⬍ 0.05.
RESULTS
Body weight and lung volume. There was no statistical difference in starting weight (110.1 ⫾ 4.7 and
113.1 ⫾ 6.3 g for Emp and Con, respectively; P ⫽ 0.517)
or final weight (122.5 ⫾ 7.5 and 129.9 ⫾ 8.6 g for Emp
and Con, respectively; P ⫽ 0.120) between the Emp
and Con groups. The saline-displacement lung volume
of the Emp group was 25% greater than that of the Con
group (1.2 ⫾ 0.4 vs. 0.9 ⫾ 0.1 g; P ⫽ 0.05). This finding
is consistent with that of Snider and Sherter (21), who
utilized the same model.
BMD and mechanical testing. The BMC of the whole
femur (P ⫽ 0.026) and the middiaphysis (P ⫽ 0.045)
were less in the Emp than in the Con group, with no
statistical differences in BMD at either site (Table 1).
The load at which fracture occurred was 6% lower in
the Emp than in the Con group (P ⫽ 0.032; Table 2).
There were no differences in the measurement of flexural rigidity (P ⫽ 0.165) or work to fracture (P ⫽ 0.121;
Table 2).
Structure and dynamics of cortical bone. The structural properties of the middiaphyseal cortical bone are
presented in Table 3. The cortical bone area of the Emp
group was smaller than that of the Con group (P ⫽
0.013). However, no difference was found in average
cortical widths (P ⫽ 0.513). The periosteal perimeter
was smaller in the Emp than in the Con group (P ⫽
0.034), although there were no differences in the endocortical perimeter (P ⫽ 0.713) or medullary area
(P ⫽ 0.967).
Bone formation indexes for the periosteal and endocortical middiaphyseal surfaces are presented in Table 4. The mineral apposition rate was 27% less in the
Emp than in the Con group at the periosteal surface
(P ⫽ 0.045), with no differences in mineral apposition
Table 3. Cortical bone structural indexes measured
at midfemoral diaphysis
2
Cortical area, mm
Average cortical width, mm
Periosteal perimeter, mm
Medullary area, mm2
Endocortical perimeter, mm
Values are means ⫾ SD.
95 • AUGUST 2003 •
www.jap.org
Emp Group
Con Group
P Value
3.27 ⫾ 0.24
1.39 ⫾ 0.03
9.69 ⫾ 0.25
2.95 ⫾ 0.19
6.59 ⫾ 0.31
3.57 ⫾ 0.19
1.40 ⫾ 0.04
10.02 ⫾ 0.29
2.90 ⫾ 0.24
6.59 ⫾ 0.31
0.013
0.513
0.034
0.967
0.713
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on September 30, 2016
(150 and 7.5 mg/kg im, respectively), saline (Con) or porcine
elastase [Emp; 25 IU/100 g body wt (Sigma Chemical, St.
Louis, MO) in 0.3 ml of normal saline] was instilled intratracheally using a 27-gauge hypodermic needle, as previously
described (14, 19). Calcein (10 mg/kg body wt ip; Sigma
Chemical), a fluorochrome used for histomorphometric measurement of bone formation, was administered at 11 and 4
days before necropsy.
Tissue preparation. At 3 wk after elastase injection, the
hamsters were euthanized, and the lungs and femurs were
removed. A saline-displacement technique was used to measure excised lung volume at 0 cmH2O airway pressure (14,
19). The right femurs were wrapped in saline-soaked gauze
and placed in a ⫺20°F freezer for subsequent densitometry
and mechanical testing. The left femurs were fixed in 10%
neutral buffered formalin and used for structural and dynamic histomorphometric measurements.
Densitometry and mechanical testing. The right femurs
were completely thawed at room temperature and then
scanned using a peripheral dual-energy X-ray absorptiometer (Norland, Medical Systems, Fort Atkinson, WI; coefficient
of variation ⫽ 0.6%) adapted for small-animal research to
assess bone mineral content (BMC) and BMD of the whole
femur and the middiaphyseal shaft.
After densitometry measurement, the thawed femurs were
placed in a materials testing machine (MTS, Eden Prairie,
MN) equipped with a 5-kN load cell and then loaded to failure
in three-point bending at a rate of 10 mm/min (1, 10, 24).
Breaking load, flexural rigidity, and work to fracture were
measured from the load-deformation curve (10).
Histomorphometry of cortical bone. The left femurs were
dehydrated in graded ethanols and embedded undecalcified
in methyl methacrylate (Fisher, Los Angeles, CA). Cross
sections of the middiaphysis were cut on a low-speed bone
saw (Isomet, Buehler, Lake Bluff, IL), mounted on plastic
slides, and ground to ⬃30-␮m thickness.
A digitizing tablet, a microcomputer (Apple SE, Apple
Computer, Cupertino, CA), a fluorescence microscope (Nikon,
Tokyo, Japan), and histomorphometry software (KSS Scientific Consultants, Magna, UT) were used to measure cortical
bone formation and resorption indexes. Measurements included total surface perimeter, perimeter of single- and double-labeled surface, and interlabel width on the periosteal
and endocortical surfaces. The perimeter of eroded surface
was measured on the endocortical surface. From these measurements, percentage of single-labeled, double-labeled, and
mineralizing surface, and mineral apposition rate, surfacereferent bone formation rates, and eroded surface were calculated and expressed by use of standardized procedures and
nomenclature (17). Additionally, cortical cross-sectional area
and average and minimum cortical widths were measured.
All measurements were made by one investigator and reviewed by a second. Both investigators were blinded.
CORTICAL BONE DYNAMICS WITH EMPHYSEMA
Table 4. Bone formation and resorption indexes
measured at midfemoral diaphysis
Emp Group
P Value
26.1 ⫾ 10.0
12.4 ⫾ 18.8
25.4 ⫾ 17.4
21.8 ⫾ 10.3
28.4 ⫾ 23.7
39.2 ⫾ 19.6
0.395
0.095
0.106
2.4 ⫾ 0.8
3.3 ⫾ 0.9
0.045
0.49 ⫾ 0.56
0.77 ⫾ 0.62
0.212
15.6 ⫾ 10.8
11.7 ⫾ 17.7
19.5 ⫾ 17.6
13.0 ⫾ 5.6
26.6 ⫾ 22.0
33.1 ⫾ 19.7
0.957
0.093
0.116
1.6 ⫾ 1.1
1.8 ⫾ 0.5
0.786
0.40 ⫾ 0.43
13.9 ⫾ 6.2
0.69 ⫾ 0.56
7.2 ⫾ 3.3
0.212
0.015
Periosteal surface
Single-labeled surface, %
Double-labeled surface, %
Mineralizing surface, %
Mineral apposition rate,
␮m/day
Bone formation rate,
␮m2 䡠 ␮m⫺1 䡠 day⫺1
Endocortical surface
Single-labeled surface, %
Double-labeled surface, %
Mineralizing surface, %
Mineral apposition rate,
␮m/day
Bone formation rate,
␮m2 䡠 ␮m⫺1 䡠 day⫺1
Resorption surface, %
Values are means ⫾ SD.
rate on the endosteal surface (P ⫽ 0.786). There were
no statistical differences in the percentage of singlelabeled, double-labeled, and mineralizing surface or
bone formation rates between the two groups on the
periosteal and endocortical surfaces. The endocortical
eroded surface was 93% greater in the Emp than in the
Con group (P ⫽ 0.015).
DISCUSSION
The present investigation demonstrated that, by 3
wk after induction of emphysema in a hamster model,
the femoral and middiaphysis BMC and middiaphyseal
cortical bone area were lower in the Emp than in the
Con group. Differences in BMC and cortical bone area
between the two groups were also reflected in the
mechanical properties of the femur, with the Emp
group having a lower femoral strength than the Con
group. The lower cortical bone areas observed in the
Emp animals may be attributed to smaller periosteal
mineral appositional rates and larger endocortical
bone eroded surfaces in the Emp than in the Con
animals. It is not possible in the present investigation
to determine when the eroded surfaces occurred, because this was a cross-sectional study. However, the
lack of significant differences in endocortical bone formation rates between the two groups suggests that the
difference may be the result of an increase in bone
resorption in the Emp group. This is not the only
inflammation model that results in rapid alterations to
bone structure and dynamics. For example, thermal
injury in mice also results in rapid and dramatic
suppression of bone formation with concomitant increases in resorption of the cortical bone by 10 days
after injury (16).
Lower femoral BMD and whole body BMD have also
been observed in patients with COPD, independent of
treatment (8). The present investigation measured a
lower femoral and middiaphyseal BMC in the Emp
J Appl Physiol • VOL
than in the Con group. The lower femoral BMC in the
Emp group supports the hypothesis that the decreased
BMD in COPD patients is due in part to the underlying
disease and not the treatment regimen per se.
Physical inactivity in COPD patients may contribute
to the reduced BMD observed in this patient population, because skeletal underloading is associated with
a reduction in BMD (2, 3). However, in a previous
study, essentially no differences in activity levels were
reported between Emp and Con groups in the elastaseinduced emphysematous hamster model (14). The
hamsters in the present investigation were mobile after the procedure, and there were no obvious differences in their mobility during the study. Additionally,
there were no statistical differences in the initial and
final weights of the two experimental groups, suggesting that differences in body weights and, thus, the
skeletal load could not account for the marked differences in skeletal structure and strength observed in
the Emp group compared with the Con group. There
was a 5.7% difference in the final mean weights between the two experimental groups. The difference in
the final weights was not statistically significant, but,
given a larger sample size, there may be weight differences that account for some of the measured differences. However, because the final weights were not
statistically different, it is unlikely that body weight
and, thus, skeletal loading account for all the observed
differences. Thus the lower BMC, cortical area, femoral
strength, and periosteal mineral apposition rate and
higher endosteal eroded surface observed in the Emp
group are likely the direct result of the elastase-induced emphysema.
Another possible explanation for the different bone
measures observed between the Emp and Con groups
is a direct effect of the injected elastase on bone. However, in previous studies using radiolabeled porcine
elastase, a majority of the elastase was isolated in the
lung within alveolar macrophages or excreted in an
inactive state in the urine after being processed in the
liver (22, 23). Other investigators reported an increase
in the number of macrophages in the lung after intratracheal injections of elastase, supporting the role of
macrophages in isolating the elastase (12, 26). Additionally, the enzymatic activity of the elastase in the
lungs is diminished to very small amounts by 4 days
(23). However, small amounts of enzymatically active
elastase remained in the lungs for up to 4 mo (22).
Therefore, it appears from these investigations that
minimal amounts of active elastase remain in the hamster after a few days with this model; consequently,
elastase is unlikely to account for the structural and
mechanical skeletal differences observed in the present
investigation.
Systemic inflammatory cytokines are another possible contributing factor to the deleterious changes in
skeletal tissue in COPD and emphysema. Interleukin-1, interleukin-6, and tumor necrosis factor-␣, all of
which are elevated in the serum of COPD patients,
play important roles in skeletal metabolism (7). Respiratory acidosis, a decrease in blood pH due to an
95 • AUGUST 2003 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on September 30, 2016
Con Group
633
634
CORTICAL BONE DYNAMICS WITH EMPHYSEMA
This project was inspired by the work of Dr. Janet K. Bailey. We
thank Beth Bowman and K. Sue Hagemann for technical support.
DISCLOSURES
This study was supported by National Institutes of Health Grants
AR-44806 and HL-50306 and American Lung Association Grant
RG-013-N.
REFERENCES
1. Anonymous. Shear and three-point bending test of animal
bone. ASAE standards. ASAE S459: 417–419, 1992.
2. Bagi CM, Mecham M, Weiss J, and Miller SC. Comparative
morphometric changes in rat cortical bone following ovariectomy
and/or immobilization. Bone 14: 877–883, 1993.
3. Bagi CM and Miller SC. Comparison of osteopenic changes in
cancellous bone induced by ovariectomy and/or immobilization in
adult rats. Anat Rec 239: 243–252, 1994.
4. Biskobing DM. COPD and osteoporosis. Chest 121: 609–620,
2002.
5. Canzanello VJ, Kraut JA, Holick MF, Johns C, Liu CC, and
Madias NE. Effect of chronic respiratory acidosis on calcium
metabolism in the rat. J Lab Clin Med 126: 81–87, 1995.
6. Dawkins PA and Stockley RA. Animal models of chronic
obstructive pulmonary disease. Thorax 56: 972–977, 2001.
7. Dimai HP, Domej W, Leb G, and Lau KH. Bone loss in
patients with untreated chronic obstructive pulmonary disease
is mediated by an increase in bone resorption associated with
hypercapnia. J Bone Miner Res 16: 2132–2141, 2001.
8. Engelen M, Schols A, Heidendal G, and Wouters E. Dualenergy X-ray absorptiometry in the clinical evaluation of body
composition and bone mineral density in patients with chronic
obstructive pulmonary disease. Am J Clin Nutr 68: 1298–1303,
1998.
9. Goldstein MF, Fallon JJ, and Harning R. Chronic glucocorticoid therapy-induced osteoporosis in patients with obstructive
lung disease. Chest 116: 1733–1749, 1999.
J Appl Physiol • VOL
10. Hart KJ, Shaw JM, Vajda E, Hegsted M, and Miller SC.
Swim-trained rats have greater bone mass, density, strength,
and dynamics. J Appl Physiol 91: 1663–1668, 2001.
11. Iqbal F, Michaelson J, Thaler L, Rubin J, Roman J, and
Nanes M. Declining bone mass in men with chronic pulmonary
disease: contribution of glucocorticoid treatment, body mass index, and gonadal function. Chest 116: 1616–1624, 1999.
12. Kuhn C and Tavassoli F. The scanning electron microscopy of
elastase-induced emphysema. Lab Invest 34: 2–9, 1976.
13. Lucey EC, Goldstein RH, Stone PJ, and Snider GL. Remodeling of alveolar walls after elastase treatment of hamsters.
Am J Respir Crit Care Med 158: 555–564, 1998.
14. Mattson JP and Poole DC. Pulmonary emphysema decreases
hamster skeletal muscle oxidative enzyme capacity. J Appl
Physiol 85: 210–214, 1998.
15. McEvoy CE, Ensrud KE, Bender E, Genant HK, Yu W,
Griffith JM, and Niewoehner DE. Association between corticosteroid use and vertebral fractures in older men with chronic
obstructive pulmonary disease. Am J Respir Crit Care Med 157:
704–709, 1998.
16. Miller SC, Bowman BM, Siska CC, and Shelby J. Effects of
thermal injury on skeletal metabolism in two strains of mice.
Calcif Tissue Int 71: 429–436, 2002.
17. Parfitt AM, Drenzner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, and Recker RR. Bone histomorphometry: standardization of nomenclature, symbols, and
units. J Bone Miner Res 2: 595–610, 1987.
18. Scuri M, Abraham WM, Botvinnikova Y, and Forteza R.
Hyaluronic acid blocks porcine pancreatic elastase (PPE)-induced bronchoconstriction in sheep. Am J Respir Crit Care Med
164: 1855–1859, 2001.
19. Sexton WL and Poole DC. Effects of emphysema on diaphragm blood flow during exercise. J Appl Physiol 83: 971–979,
1998.
20. Shapiro SD. Animal models of chronic obstructive pulmonary
disease: age of Klotho and Marlboro mice. Am J Respir Cell Mol
Biol 22: 4–7, 2000.
21. Snider GL and Sherter C. A one-year study of the evolution of
elastase-induced emphysema in hamsters. J Appl Physiol 43:
721–729, 1977.
22. Stone PJ, Calore JD, Snider GL, and Fanzblau C. The
dose-dependent fate of enzymatically active and inactivated tritiated methylated pancreatic elastase administered intratracheally in the hamster. Am Rev Respir Dis 120: 577–587, 1979.
23. Stone PJ, Pereira W, Biles D, Snider GL, Kagan HM, and
Franzblau C. Studies on the fate of pancreatic elastase in the
hamster lung: 14C-guanidinated elastase. Am Rev Respir Dis
116: 49–56, 1977.
24. Turner CH and Burr DB. Basic biomechanical measurements
of bone: a tutorial. Bone 14: 595–608, 1993.
25. Van Staa TP, Leufkens HG, Abenhaim L, Zhang B, and
Cooper C. Use of oral corticosteroids and risk of fracture. J Bone
Miner Res 15: 993–1000, 2000.
26. Verghese A, Franzus B, and Stout R. Characterization of
bronchoalveolar lavage cells and macrophage-derived chemoattractant activity in pancreatic elastase-induced emphysema in
hamsters. Exp Lung Res 14: 797–810, 1988.
27. Walsh LJ, Lewis SA, Wong CA, Cooper S, Oborne J, Cawte
SA, Harrison T, Green DJ, Pringle M, Hubbard R, and
Tattersfield AE. The impact of oral corticosteroid use on bone
mineral density and vertebral fracture. Am J Respir Crit Care
Med 166: 691–695, 2002.
95 • AUGUST 2003 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on September 30, 2016
increase in PCO2, may also be a contributing factor,
because there is a positive correlation between arterial
pH and BMD in COPD patients (7). Chronic respiratory acidosis in the rat is also associated with a decrease in normalized cortical bone area and an increase
in the number of osteoclasts (5). Thus numerous factors, including inflammatory cytokines, respiratory acidosis, cigarette smoking, and skeletal loading, may
contribute to the low BMD observed in COPD patients.
In conclusion, the elastase-induced emphysematous
hamster exhibited lower femoral BMC, cortical bone
area, and femoral strength within 3 wk after elastase
instillation. These differences in structural and mechanical properties in the Emp compared with the Con
group occurred without the use of corticosteroids. Finally, the depressed indexes of bone formation with
larger eroded surface observed in the present investigation may help explain why patients with COPD and
other inflammatory conditions are prone to osteopenia.