Calcified Tissue International

, Volume 75, Issue 3, pp 225–230

Effects of Doxycycline on Mechanical Properties of Bones in Rats with Ovariectomy-Induced Osteopenia

Authors

  • M. Pytlik
    • Department of PharmacologyMedical University of Silesia
    • Department of PharmacologyMedical University of Silesia
  • W. Janiec
    • Department of PharmacologyMedical University of Silesia
Article

DOI: 10.1007/s00223-004-0097-x

Cite this article as:
Pytlik, M., Folwarczna, J. & Janiec, W. Calcif Tissue Int (2004) 75: 225. doi:10.1007/s00223-004-0097-x

Abstract

Tetracyclines have been reported to inhibit bone resorption and intensify bone formation. The aim of the present study was to investigate the effects of doxycycline (20 mg/kg PO daily for 28 days) on bone mechanical properties in bilaterally ovariectomized and sham-operated rats. The experiment was carried out on 3-month-old Wistar rats. Mechanical properties of the whole femur (extrinsic stiffness, ultimate and breaking load, deformation caused by applied load) and the femoral neck (load at fracture) as well as bone mass and bone mineral content in the tibia, femur, and L4 vertebra were examined. Bilateral ovariectomy resulted in decreases in bone mineral content/bone mass ratio and worsening of mechanical properties of the femoral neck. The changes were counteracted by doxycycline. Doxycycline reversed the effect of ovariectomy on load at fracture of the femoral neck. Doxycycline did not significantly affect the mechanical properties of bones in the sham-operated rats.

Keywords

DoxycyclineOvariectomyMechanical properties of bonesRat

Tetracyclines belong to potential agents which could be applicable in the treatment of excessive bone loss [1]. Apart from their broad-spectrum antimicrobial activity, tetracyclines exert many effects on the host organism, anticollagenase activity being one of the most widely recognized. Tetracyclines incorporate into bones and influence bone remodeling. Their ability to inhibit bone resorption was observed in different experimental models [2, 3, 4, 5, 6]. There are also reports on their increasing bone formation effects [6, 7, 8, 9]. However, unfavorable effects of tetracyclines on bones were also described [10, 11, 12].

Postmenopausal osteoporosis is a heterogeneous disorder that leads to fracture within 15–20 years from the cessation of the ovarian function [13]. Estrogen deficiency is responsible for the increased bone remodeling rate (bone resorption and formation), with the balance tilted in favor of resorption causing progressive loss of bone mass and strength [14]. The model of bilaterally ovariectomized rats, used in the present study, mimics the accelerated bone loss observed in postmenopausal women due to estrogen deficiency [15].

The ultimate aim of pharmacotherapy in osteoporosis is decreasing the incidence of fracture [16]. There are only a few published reports on the effect of tetracyclines on the mechanical properties of bones. This is, as far as we know, the first study to examine the effects of a tetracycline on bone mechanical properties in bone loss induced by estrogen deficiency. The aim of the present work was to examine the effects of doxycycline on mechanical properties of bones in normal (sham-operated) and bilaterally ovariectomized rats.

Materials and Methods

Animals

The experiments were carried out on 3-month-old female Wistar rats, given distilled water to drink and fed a standard diet ad libitum. The animals were divided into 4 groups (n = 5–8): I—sham-operated control group, II—ovariectomized control group, III—sham-operated group receiving doxycycline, and IV—ovariectomized group receiving doxycycline. The procedure of the experiments on the animals was approved by the Bioethical Board, Medical University of Silesia.

Bilateral ovariectomy or a sham operation was performed under ether anesthesia. A longitudinal incision was made inferior to the rib cage on the dorsolateral body wall. The ovaries were exteriorized, ligated, and excised. Rats subjected to the sham surgical procedure only had the ovaries exteriorized and then replaced.

Treatment with doxycycline started 3–4 days after the surgical procedure. Doxycycline (Doxycyclinum; Polfa, Poland) was administered as doxycycline hydrochloride by daily oral gavage for 4 weeks at a dose equivalent to 20 mg/kg of doxycycline daily. The control groups received 0.9% saline in the same volume of 2 ml/kg PO daily. The animals were weighed every day.

The day after the end of the administration of doxycycline or saline the animals were sacrificed and the right and left tibial and femoral bones and L4 vertebra were prepared. In the isolated bones, mass and macrometric parameters were determined (length, diameter of the diaphysis in the midlength).

Mechanical Tests

Mechanical properties of the femur were assessed using the set constructed at the Department of Pharmacology, Medical University of Silesia, in cooperation with Hottinger Baldwin Messtechnik GmbH (HBM).

In the present study, the mechanical properties of intact left femurs were studied using a bending test with three-point loading. The load was applied perpendicularly to the long axis of the femur in the midlength of the bone supported on its epiphyses. The load increased at a rate of 100 N/minute. The load was measured by the sensor with a strain gauge (HBM) and the deformation was measured by an inductive sensor (HBM). The signals sent by the sensors were amplified and registered using the XY recorder. The bone load–deformation curves representing the relationship between load applied to the bone and deformation in response to the load were analyzed. The load–deformation curve can be divided into the elastic deformation region and the plastic deformation region. Within the elastic deformation region, the slope of the load–deformation curve, representing the extrinsic stiffness or rigidity of bone, was tested. Within the plastic deformation region, the ultimate load and the breaking load, as well as the deformation caused by the applied loads, were determined. The ultimate load is the maximum load sustained by the bone. The breaking load is the load at which the bone actually breaks.

Mechanical properties of the femoral neck were studied using a compression test. The load was applied to the head of the femur along the long axis of the femur. The bone was prepared for measurement by fixing the diaphysis, which was cut at 1.7 cm from the proximal end of the right femur, in a metacrylate plate. The applied load increased at a rate of 100 N/minute. The load was measured by the sensor with a strain gauge (HBM); the signals sent by the sensor were amplified and registered using the XY recorder. The load causing the fracture of the femoral neck was determined.

In order to determine the content of mineral substances in bones, the left tibia and femur and L4 vertebra were mineralized at 620°C for 48 hours and weighed.

Statistical Analysis

Results are presented as the mean ± SEM. Statistical estimation was performed using ANOVA followed by post-hoc Duncan’s multiple-range test.

Results

Bilateral ovariectomy resulted in faster body mass gain (by 63.5%), a substantial decrease in the mass of the uterus (by 75%), and an increase (by 77%) in the mass of the thymus in comparison with the sham-operated control rats (Table 1). Changes in the skeletal system of the ovariectomized control rats were also observed. Although bone mass, length, diameter, and mineral content did not statistically significantly differ from those of the sham-operated control rats, there were significant decreases in the bone mass/body mass ratios (by 8% in the tibia and 9.6% in the femur) and the bone mineral content/body mass ratios (by 13.7%–16.2%). The bone mineral content/bone mass ratios decreased in the tibia (by 6.5%) and in the femur (by 7.3%), indicating a decrease in bone mineral density. Mechanical properties of the femur worsened in the ovariectomized control group in comparison with the sham-operated control group (Table 2). The force causing fracture of the femoral neck was statistically significantly lower than that in the sham-operated control group (by 15.9%). The investigated parameters of the whole femur did not statistically significantly change.
Table 1

Effect of doxycycline (20 mg/kg PO daily), administered for 4 weeks, on body mass gain and bone parameters in rats

Parameter

 

I—Sham-operated control group

II—Ovariectomized control group

III—Sham-operated group treated with doxycycline

IV—Ovariectomized group treated with doxycycline

ANOVA

Initial body mass (g)

 

199.59 ± 7.47

196.16 ± 6.73

201.86 ± 3.59

212.70 ± 4.31

NS

Body mass gain after 28 days (g)

 

47.27 ± 2.89

77.28 ± 2.45***

32.56 ± 4.97**

71.57 ± 2.98***

P < 0.001

Uterus mass (mg)

 

370.41 ± 35.72

92.51 ± 6.73***

388.87 ± 48.52

86.04 ± 6.66***

P < 0.001

Thymus mass (mg)

 

364.90 ± 19.65

645.77 ± 74.29***

265.53 ± 43.85

607.14 ± 50.70**

P < 0.001

Bone mass (mg)

Tibia

506.14 ± 17.36

514.79 ± 16.15

500.36 ± 8.69

523.44 ± 12.39

NS

 

Femur

701.00 ± 21.24

700.59 ± 18.47

684.57 ± 11.40

707.72 ± 11.73

NS

 

L4 vertebra

243.43 ± 12.62

258.81 ± 14.83

252.67 ± 7.54

242.66 ± 7.27

NS

Bone mass/body mass (mg/100 g body mass)

Tibia

204.90 ± 4.45

188.43 ± 3.69*

213.67 ± 2.94

184.32 ± 5.72**

P < 0.001

 

Femur

283.99 ± 5.56

256.68 ± 5.10**

292.30 ± 3.32

249.15 ± 5.86***

P < 0.001

 

L4 vertebra

98.36 ± 3.81

94.91 ± 5.18

107.86 ± 2.74

85.31 ± 1.82*

P < 0.01

Bone mineral content (mg)

Tibia

223.54 ± 8.37

212.52 ± 7.40

221.02 ± 5.90

223.95 ± 7.43

NS

 

Femur

302.39 ± 11.31

280.43 ± 11.10

295.05 ± 7.85

296.54 ± 5.58

NS

 

L4 vertebra

82.10 ± 4.00

78.26 ± 3.21

80.23 ± 2.65

77.52 ± 1.50

NS

Bone mineral content/body mass (mg/100 g body mass)

Tibia

90.46 ± 2.10

77.72 ± 1.32***

94.24 ± 1.05

78.89 ± 3.27***

P < 0.001

 

Femur

122.35 ± 2.69

102.48 ± 1.99***

125.83 ± 1.73

104.43 ± 2.90***

P < 0.001

 

L4 ertebra

33.17 ± 1.11

28.61 ± 0.74**

34.20 ± 0.69

27.29 ± 0.72***

P < 0.001

Bone mineral content/bone mass ratio

Tibia

0.442 ± 0.005

0.413 ± 0.005**

0.441 ± 0.007

0.428 ± 0.005

P < 0.01

 

Femur

0.431 ± 0.005

0.400 ± 0.007**

0.431 ± 0.006

0.419 ± 0.004a

P < 0.01

 

L4 vertebra

0.338 ± 0.006

0.305 ± 0.012

0.318 ± 0.007

0.321 ± 0.011

NS

Bone length (mm)

Tibia

36.33 ± 0.25

36.77 ± 0.41

36.63 ± 0.19

37.28 ± 0.05

NS

 

Femur

33.54 ± 0.29

34.04 ± 0.38

33.87 ± 0.27

34.42 ± 0.08

NS

Bone diameter (mm)

Tibia

2.63 ± 0.06

2.79 ± 0.01

2.69 ± 0.06

2.73 ± 0.06

NS

 

Femur

3.36 ± 0.08

3.30 ± 0.06

3.31 ± 0.03

3.41 ± 0.05

NS

Results are presented as means ± SEM (N = 5–8). ANOVA and then post-hoc Duncan’s multiple-range test were used for estimation of statistical significance. NS—not significant in ANOVA. *—Significantly different (Duncan’s test) from the sham-operated control group (I); *P < 0.05, **P < 0.01, ***P < 0.001

a Significant difference (Duncan’s test) between the ovariectomized group treated with doxycycline (IV) and the ovariectomized control group (II); P < 0.05.

Table 2

Effects of doxycycline (20 mg/kg PO daily), administered for 4 weeks, on the mechanical properties of the femur in rats

Parameter

 

I—Sham-operated control group

II—Ovariectomized control group

III—Sham-operated group treated with doxycycline

IV—Ovariectomized group treated with doxycycline

ANOVA

Extrinsic stiffness (N/mm)

 

262.28 ± 13.32

225.03 ± 12.03

232.77 ± 6.38

244.43 ± 10.14

NS

Load (N)

Ultimate

88.13 ± 4.49

81.67 ± 3.65

94.21 ± 5.28

87.03 ± 4.95

NS

 

Breaking

86.84 ± 4.48

73.90 ± 6.67

92.74 ± 5.63

82.66 ± 6.16

NS

Deformation (mm)

At ultimate load

0.34 ± 0.02

0.37 ± 0.02

0.43 ± 0.04

0.37 ± 0.04

NS

 

At breaking load

0.36 ± 0.03

0.43 ± 0.03

0.45 ± 0.04

0.42 ± 0.06

NS

Load at fracture of the femoral neck (N)

 

80.41 ± 4.43

67.66 ± 2.39*

71.86 ± 4.87

83.06 ± 2.72a

P < 0.05

Results are presented as means ± SEM (N = 5–8). ANOVA and then post-hoc Duncan’s multiple-range test were used for estimation of statistical significance. NS—not significant in ANOVA. *—Significantly different (Duncan’s test) from the sham-operated control group (I); *P < 0.05

a Significant difference (Duncan’s test) between the ovariectomized group treated with doxycycline (IV) and the ovariectomized control group (II); P < 0.05

Administration of doxycycline at a dose of 20 mg/kg PO daily for 28 days to the sham-operated rats caused slower body mass gain than in the sham-operated control rats. There were no significant changes in the examined parameters of the skeletal system.

Administration of doxycycline at a dose of 20 mg/kg PO daily for 28 days to the ovariectomized rats did not statistically significantly affect body mass gain, mass of the uterus, and mass of the thymus in comparison with the ovariectomized control rats. There also were no significant differences between the two groups regarding the examined macrometric parameters of bones. Administration of doxycycline did not affect bone mass/body mass ratios and bone mineral content/body mass ratios. However, contrary to the ovariectomized control group, in the doxycycline-treated group there were no significant decreases in the bone mineral content/bone mass ratios in the examined bones in comparison with the sham-operated control group. In the femur, the ratio was statistically significantly higher than in the ovariectomized control group (by 4.8%), indicating better mineralization. An improvement in bone mechanical properties was observed in the doxycycline-treated ovariectomized rats. The load causing fracture of the femoral neck statistically significantly increased in comparison with the ovariectomized control group of rats (to the level of the sham-operated control group).

Discussion

In the present study, mechanical properties of the femur were studied using a three-point bending test (the whole femur) and a compressive test (the femoral neck). The extrinsic biomechanical parameters of bones were analyzed (load, deformation, stiffness), which, contrary to the intrinsic properties of material (stress, strain, Young’s modulus), are not independent of size and shape [17]. Nevertheless, it was possible to compare the values obtained in different experimental groups because there were no significant differences in the macrometric parameters of bones among them.

Numerous previous reports indicated the damaging effect of estrogen loss on bones [18, 19, 20, 21]. Estrogen deficiency leads to the increased rate of bone remodeling (both resorption and formation), and the imbalance between bone resorption and formation in favor of the former [14]. In the present study, bilateral ovariectomy caused accelerated bone loss and worsening of the mechanical properties of the femoral neck in comparison with the sham-operated control group.

Administration of doxycycline at a dose of 20 mg/kg PO daily for 4 weeks to the ovariectomized rats resulted in an improvement in bone status in comparison with the ovariectomized control rats. Doxycycline did not counteract the accelerated bone loss due to the ovariectomy, but it normalized the ratio of bone mineral content/bone mass in the investigated bones and improved the mechanical properties of the femur. Doxycycline completely reversed the effect of ovariectomy on load at fracture of the femoral neck. The favorable effect of doxycycline on the strength of the femoral neck is consistent with the improvement in biomechanical properties of the femoral neck caused by chemically modified tetracyclines CMT-3 and CMT-8 in rats with adjuvant arthritis [22].

The observed improvement in bone status induced by administration of doxycycline to the ovariectomized rats may be the effect of the intensification of bone formation and/or inhibition of bone resorption. The intensified bone formation effect of tetracyclines was observed in numerous experimental models, especially in experimentally induced disorders of bone remodeling, for example, in osteopenia caused by streptozotocin-induced diabetes [8], prednisolone-induced osteopenia [12], and osteopenia in ovariectomized aged rats [18]. The favorable effects of tetracyclines in diabetes-induced osteopenia were connected with improvement of the impaired structure and function of osteoblasts [7, 23]. Minocycline increased the expression of type I collagen mRNA in bones of ovariectomized aged rats after marrow ablation [9]. Histomorphometric measurements performed in the rats examined in the present study also indicated that both intensification of bone formation and inhibition of bone resorption played roles in the beneficial effect of doxycycline in ovariectomy-induced osteopenia [24].

Tetracyclines have been reported to inhibit bone resorption in vitro [2, 3, 25] and in vivo [5, 6]. Tetracyclines are inhibitors of matrix metalloproteinases, among other collagenases [1]. Collagenase has been postulated to be involved in bone resorption [18]. The activity of collagenase may limit the rate of bone resorption [26]. Several chemically modified tetracyclines that did not inhibit collagenase were poor inhibitors of bone resorption [27]. Doxycycline has the most potent anticollagenase activity among the commercially available tetracyclines [28]. Tetracyclines also affect the structure and activity of osteoclasts. The influence on osteoclasts includes decreasing the ruffled border area of osteoclasts, inducing retraction of osteoclasts, diminishing acid production, diminishing the secretion of lysosomal cysteine proteinases (cathepsins), and inhibiting gelatinase activity [4, 29]. Tetracyclines were reported to reduce in vitro the formation of mature osteoclasts and induce apoptosis of osteoclasts [27].

Administration of doxycycline at a dose of 20 mg/kg PO daily for 4 weeks to the sham-operated rats did not result in statistically significant changes in the investigated parameters of the skeletal system. Histomorphometric measurements performed in the rats examined in the present study revealed a decrease in the width of trabeculae in the femoral epiphysis after administration of doxycycline to the sham-operated rats [24]. There are also other reports indicating unfavorable effects of tetracyclines on bone remodeling. For a long time it has been known that tetracyclines exert disadvantageous side effects on the skeletal system, especially of children, leading to tooth discoloration and slowing of the longitudinal growth of bones [10, 11]. In our previous study, doxycycline at a dose of 100 mg/kg PO daily, administered to normal control rats, intensified bone resorption [12].

Summarizing, doxycycline improved the mechanical properties of the femoral neck in the ovariectomized rats, whereas it did not favorably affect the mechanical properties of bones in the sham-operated rats. The mechanism by which doxycycline exerts its favorable effects on bones of the ovariectomized rats may be speculated.

As mentioned above, the improvement in bone mechanical properties in ovariectomized rats caused by doxycycline could be the effect of inhibited bone resorption. The dose of 20 mg/kg PO daily used in the present study was an effective dose that inhibited matrix metalloproteinase activity in several rat models, including adjuvant arthritis and periodontal disease [30]. However, from two major groups of proteolytic enzymes secreted by osteoclasts suggested to participate in organic matrix degradation (matrix metalloproteinases and cysteine proteinases), only the activity of cysteine proteinases is regulated by estrogen [31]. As doxycycline in the present study seemed not to inhibit bone resorption in the sham-operated rats, it is possible that the improvement of bone status observed in the ovariectomized rats was caused by inhibition of secretion of cysteine proteinases, whose activity might have been increased by estrogen deficiency.

The favorable effect of doxycycline could be also connected with its possible influence on cytokine production. After menopause, estrogen deficiency leads to increased production of cytokines regulated by estrogen (IL-1, IL-6, and TNFα) in bone marrow and bone cells, and, consequently, an expansion of the osteoclastic pool due to increased formation of osteoclasts and elongation of their lifespan. Enhanced cytokine production also leads to increased activity of mature osteoclasts and increased osteoblastic activity [13]. Tetracyclines were reported to affect production of TNFα, IL-1 [1], and IL-6 [9].

Tetracyclines are postulated to have potential use in therapeutic management of different types of osteoporosis, various arthritides (rheumatoid arthritis, osteoarthritis, reactive arthritis), adult periodontitis, complications of diabetes, bullous and ulcerative skin lesions, and others [1]. The goal of the treatment of diseases with excessive bone loss is decreasing the incidence of fractures [16]. Doxycycline in the present study proved to be effective in improving the mechanical properties of the femoral neck, worsened due to estrogen deficiency. Still, there are many doubts about the possibilities of wider therapeutic use of tetracyclines. Tetracyclines may exert unfavorable effects on bones with normal remodeling, which may be dose dependent. The nonantimicrobial actions of tetracyclines may be responsible for complex effects, not always favorable. For example, doxycycline and minocycline were reported to augment PGE2 production in human osteoarthritis-affected cartilage in ex vivo conditions, which may underscore their therapeutic potential in the treatment of inflammatory diseases [32]. Only administration of low doses of tetracyclines may be considered in the long-term treatment of osteoporosis. The US Food and Drug Administration approved the use of doxycycline in a subantimicrobial dose as an adjunct therapy in adult periodontitis [1]. Observations of the effect of the treatment on the osseous system (bone density, fracture rate) of patients with adult periodontitis may enable assessment of its safety in relation to the whole skeleton.

Copyright information

© Springer-Verlag 2004