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Abstract

The effects of short-term use of oral glucocorticoid (GC) on the skeleton are not well defined. To address this gap, the influences of 7 days, 21 days of GC administration on femurs of intact rats were investigated. Forty 4-month-old female Sprague–Dawley rats were randomly divided into control group (Cont) and prednisone-treated group (Pre) and administered either distilled water or prednisone acetate at doses of 3.5 mg/kg/day for 0, 7 and 21 days, respectively. All the femurs were harvested for dual-energy X-ray absorptiometry scan, biomechanical testing and micro computed tomography scan. The whole body weight, femur bone mineral density (BMD), all three-point bending test parameters, microstructural parameters increased or improved significantly in Cont at day 21 when compared to day 0. The whole body weight, distal femur BMD, Young’s modulus, bending stiffness, density of tissue volume and trabecular thickness (Tb.Th) decreased, while structure model index and trabecular separation (Tb.Sp) increased significantly in Pre at day 21 when compared to age-matched control but had no significant differences between day 7 and day 21. Our data demonstrate that 7-day use of prednisone does not influence on rats’ femur, and 21-day use of prednisone slows in rate of whole body weight gain, decreases femur metaphysis BMD and bone stiffness which mainly due to the deteriorated bone microstructure.

Keywords

Prednisone short-term femur bone mineral density bone mechanics microstructure

Introduction

Glucocorticoids (GCs) are widely used in clinical application for their unsurpassed anti-inflammatory and immunomodulatory effects, but always limited by substantial adverse outcomes such as osteoporosis, diabetes, obesity and so on during a prolonged use (dosage ≥7.5 mg/day prednisone-equivalent for longer than 3 months).^1,2 The incidence rate of GC-induced osteoporosis is approximately 50% in patients treated for 6 months or longer.³ Compared with long-term use of GCs, the role of short-term use of GCs on the skeleton is more complex and at time considered paradoxical.^4
–6 Ogoshi et al.⁷ have reported that the whole body bone mineral density (BMD) and bone formation rate increased in 3-month-old rats treated with GC for 4 weeks. However, some experimental evidences have indicated that GCs excess induce an initial but transient loss of BMD because of an early up-regulation of bone resorption.^8,9 Fujita et al.¹⁰ also support that bone mineral content decrease in rats treated with GC for 6 weeks.

To develop a better understanding about the characteristics of short-term use of GCs on the skeleton, an experimental model of 4-month-old female rats treated with GC at 3.5 mg/kg/day for 7 and 21 days has been established. Our goals in this study are to determine the following:

Whether the short-term administration of oral GC shows a positive or negative effect on femur BMD, biomechanical properties and microstructure in vivo.

What differences of femoral BMD, biomechanical properties and microstructure between two time periods: 7 and 21 days.

Materials and methods

Animals and experimental protocols

This study was carried out in accordance with the recommendations in the guide for the Care and Use of Laboratory Animals of Guangdong Laboratory Animal Monitoring Institute. Permit Number: SYXK (GUANGDONG) A2008036. Forty 4-month-old female Sprague–Dawley rats (Animal Center of Guangdong Medical University) were given chow and tap water ad libitum, and maintained approximately at 24–28 degree under a 12-h light cycle (7:00 a.m. to 7:00 p.m.) and allowed free access to water and diets containing 1.11% calcium and 0.74% phosphorus.

Forty 4-month-old healthy female Sprague–Dawley rats were randomly divided into two groups: control group (n = 20) and prednisone-treated group (n = 20) by mean body weight standardization, all rats were administered either distilled water as vehicle control (Cont), or prednisone acetate (CAS:125-10-0, Zhejiang Xianju Pharmacy Ltd., Zhejiang, China) at doses of 3.5 mg/kg/day (Pre) for 0, 7, and 21 days respectively. The volume for the oral gavage was 5 ml/kg/day.

Sample collection and applications

Rats were weighted every week. At the endpoint, the rats were killed by cardiac puncture under anaesthesia using 1 mg/kg sodium pentobarbitone and the femurs were isolated and harvested for tests. The right femurs were scanned BMD by dual-energy X-ray absorptiometry (DXA) and then mechanical three-point bending test was performed by material testing system. The left femurs were harvested for micro-computed tomography (micro-CT) analysis.

Measurement of BMD

The right femurs rats were wrapped with saline-saturated gauze to maintain their moisture and stored at −20°C. After thawed at room temperature, the bones were moisturized by soaking them in saline solution with the residual muscle removed. The whole and distal femurs BMD were scanned with Prodigy DXA scanner (GE Lunar Prodigy, Fairfield, Connecticut, USA) to measure the BMD (g/cm²).

Testing of bone mechanical properties

After measuring BMD, the right femurs were used to determine the bone mechanical properties through three-point bending by material testing system (858 Mini Bionix MTS Systems, Eden Prairie, Minnesota, USA). The bones were tested with a 1-mm indenter, at a speed of 0.01 mm/s with a 15-mm span for femur. Force and deflection were automatically recorded. The three-point bending test output parameters of femur included elastic load (the force required to cause bone specimens to deform, N), maximum load (the maximum force the bone can resist, N), bending load (fracture load, the force required to cause bone fracture, N) and the maximum deflection (maximum degree of the bone displacement, mm). The stiffness (load–displacement curve slope, N/mm), Young’s modulus were calculated based on the parameters mentioned earlier.

Micro-computed tomography scanning analysis

The left femurs were harvested for micro-CT analysis by a Viva CT40 (Scanco Medical, Zurich, Switzerland) under high-resolution conditions (X-ray energy 70 kVp, 114 μA, 8 W and integration time 200 ms). Briefly, the region of interest was the metaphysis extending 1.5–3.5 mm proximally from the distal growth plate cartilage of the femur. Cortical bone was excluded from the measurement. Three-dimensional (3-D) images and longitudinal sections of mineralized distal femoral metaphysis were generated using a Gaussian filter (sigma 0.8, support 1) and a threshold of 350. The 3-D image analysis was performed to determine bone volume/tissue volume (BV/TV), structure model index (SMI), BMD of tissue volume (density of TV), and microarchitecture parameters such as trabecular thickness (Tb.Th), number (Tb.N), and separation (Tb.Sp). The 2-D cross-sectional images of mid-shaft of femur were captured and measured parameters such as tissue area (T.Ar), marrow area (Ma.Ar) and cortical thickness (Ct.Th) by image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Silver Spring, Maryland, USA).

Statistical analysis

Data are presented as mean ± standard deviation. Variance analysis was performed for comparison of BMD, mechanics evaluation and Micro-CT parameters between in Cont group and Pre-treatment group, 0, 7s and 21 days time period by SPSS statistical software (Version 17.0, SPSS Inc., USA). Statistical differences in the means among the groups were analysed by LSD multiple comparison test when the variance was homogeneous and Tamhane’s T2 multiple comparison test by rank when the variance was not homogeneous. Probabilities ( p) less than 0.05 were considered significant.

Results

Body weight

The body weight was 243.45 ± 8.07 g, 265.05 ± 10.93 g, 284.04 ± 13.01 g, respectively, at days 0, 7 and 21 in the Cont groups and was 245.13 ± 7.21 g, 267.58 ± 10.05 g, 270.02 ± 12.70 g, respectively, at days 0, 7 and 21 in the Pre group. The body weight increased with time ( p = 0.000, day 21 vs. day 0 in Cont). Seven days use of prednisone had no significant effects on the body weight and 21 days use of prednisone slowed the body weight gain ( p = 0.004 vs. day 21 in Cont).

BMD in distal femur and total femur

Total femur BMD and distal femur BMD increased with time and both were significantly higher at day 21 than that at day 0 in Cont group (increased by 8.23%, p = 0.024 and 9.91%, p = 0.015, respectively). In Pre group, distal femur BMD decreased (by 12%) at day 21 and was lower than that at day 21 in the Cont group ( p = 0.002). No statistic difference was noted in total femur BMD between Pre group at day 21 and Cont group at day 21 (Figure 1).

Figure 1.

Effects of 0, 7 and 21 days prednisone treatment on femur BMD in rats. Notes: *p < .05: vs. Cont at day 0; #p < .05: vs. Cont at day 21. Cont: control group; Pre: prednisone-treatment group; BMD: bone mineral density.

Bone mechanics testing of femur

As shown in Figure 2, the three-point bending test of the femur demonstrated that Young’s modulus, bending load, maximum load and bending stiffness increased (by 15.40%, p = 0.022, 22.52%, p = 0.016, 22.60% p = 0.013 and 12.10% p = 0.016, respectively) with time in Cont group and were obviously higher at day 21 than that at day 0 in Cont group. Young’s modulus and stiffness decreased (by 7.78%, p = 0.012 and 9.25%, p = 0.015, respectively) at day 21 in Pre group and were lower than that in the Cont group at day 21, while maximum load and bending load exhibited no significant change in Pre group at day 21.

Figure 2.

Effects of 0, 7 and 21 days prednisone treatment on three-point bending testing parameters of femur in rats. *p < .05: vs. Cont at day 0; #p < .05: vs. Cont at day 21. Cont: control group; Pre: prednisone-treatment group.

Micro-CT analysis

Micro-CT data demonstrated that distal femur BV/TV, density of TV, Tb.Th, Tb.N increased with time (by 62.73%, p = 0.000, 39.38%, p = 0.003, 29.58%, p = 0.012 and 26.82%, p = 0.004, respectively) and were significantly higher in Cont group at day 21 than that in Cont at day 0, while SMI and Tb.Sp decreased (by 79.10%, p = 0.000 and 32.14%, p = 0.002, respectively) and were significantly lower in Cont group at day 21 than that in Cont at day 0. No significant differences were found between prednisone group at day 7 and Cont group at day 7. BV/TV did not exhibit significantly change, but density of TV, Tb.Th decreased by 15.12% ( p = 0.000) and 20.16% ( p = 0.005), respectively, SMI and Tb.Sp increased by 20.24% ( p = 0.002) and 22.24% ( p = 0.000), respectively, in Pre group at day 21 when compared to age-matched control (see Table 1 and Figure 3). Ma.Ar/T.Ar of femoral mid-shaft were significantly higher in Pre group at day 21 (by 8.35%, p = 0.031) than that in Cont at day 21. Ct.Th decreased in Pre at day 21, but had no significant differences between Cont and Pre groups (see Table 2 and Figure 4).

Table 1.

Effects of 0, 7 and 21 days prednisone treatment on micro-CT parameters of distal femur in rats.^a

Days	Group	BV/TV (%)	Density of TV (mg HA/ccm)	SMI	Tb.N (num.)	Tb.Th (μm)	Tb.Sp (μm)
0 day	Cont	28.72 ± 5.56	351.49 ± 21.35	1.20 ± 0.21	5.07 ± 0.37	78.10 ± 10.50	187.01 ± 17.90
0 day	Pre	29.70 ± 4.47	354.51 ± 18.73	1.07 ± 0.13	5.31 ± 0.59	72.90 ± 8.97	180.20 ± 21.81
7 days	Cont	35.21 ± 7.80	411.56 ± 23.20	0.94 ± 0.08	4.92 ± 0.34	97.60 ± 8.72	170.90 ± 17.51
7 days	Pre	37.39 ± 6.50	425.69 ± 19.87	0.96 ± 0.15	5.42 ± 0.72	101.20 ± 10.57	165.80 ± 15.25
21 days	Cont	46.45 ± 5.60^b	489.90 ± 25.30^b	0.67 ± 0.09^b	6.43 ± 0.68^b	101.20 ± 9.89^c	126.90 ± 11.07^c
21 days	Pre	43.07 ± 7.84	415.80 ± 33.73^d	0.84 ± 0.11^e	6.30 ± 0.54	80.80 ± 11.20^d	155.12 ± 12.51^d

Cont: control group; Pre: prednisone-treatment group; BV/TV: bone volume/tissue volume; density of TV: bone mineral density of tissue volume; SMI: structure model index; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation.

^aData are expressed as the mean ± standard deviation.

^bp < .01: vs. Cont at day 0.

^cp < .05: vs. Cont at day 0.

^dp < 0.01: vs. Cont at day 21.

^ep < .05: vs. Cont at day 21.

Figure 3.

Three-dimensional reconstruction images by micro-CT from distal femur in control and prednisone-treated rats. For data, see Table 1. Cont: control group; Pre: prednisone-treatment group.

Table 2.

Effects of 0, 7, and 21 days prednisone treatment on cortical bone of the femoral mid-shaft in rats.^a

Days	Group	Ma.Ar (mm)	Ma.Ar/T.Ar (%)	Ct.Th (μm)
0 day	Cont	3.64 ± 0.23	41.76 ± 7.56	426.49 ± 20.31
0 day	Pre	3.79 ± 0.50	42.05 ± 9.50	435.51 ± 17.71
7 days	Cont	3.59 ± 0.19	39.85 ± 8.05	421.56 ± 20.21
7 days	Pre	3.72 ± 0.80	40.53 ± 10.20	425.60 ± 9.07
21 days	Cont	3.85 ± 0.60	42.39 ± 7.01	429.90 ± 24.23
21 days	Pre	4.07 ± 0.54	45.93 ± 10.24^b	418.15 ± 23.64

Cont: control group, Pre: prednisone-treatment group; Ma.Ar: marrow area; Ma.Ar/T.Ar: marrow area/tissue area; Ct.Th: cortical thickness.

^aData are expressed as the mean ± standard deviation.

^bp < .05: vs. Cont at day 21.

Figure 4.

Two-dimensional images by micro-CT from femoral mid-shaft in control and prednisone-treated rats. For data, see Table 2. Cont: control group; Pre: prednisone-treatment group.

Discussion

Despite over 50 years of GC therapy, there is still not very clearly for the best knowledge of GCs influence on skeleton and need more studies for better understanding of the skeletal effects. GC-induced osteoporosis is linked to long-term administration of GCs and has been widely and deeply studied, but the role of short-term use of GCs on the skeleton, especially on femur is less reported.^11,12

Our results indicate that 7-day treatment of prednisone does not affect the normal growth of body weight and 21 days of prednisone has prevented weight gain from continuing to increase. This is consistent with the report of Ogoshi et al. In Ogoshi’s experiment,⁸ the whole body weight of 4-week treatment of dexamethasone groups has already decreased while BMD still increase. Our previous study also showed that body weight and BMD both decreased in rats with longer treatment (12 weeks) of prednisone and the body weight loss and the dosages of prednisone showed a dose-dependent manner.^13,14 We infer that body weight may be a more sensitive indicator of response to the use of GC, lower body weight suggests that BMD (or bone mass) has been reduced or is about to be reduced. Furthermore, body weight and BMD have common related factors such as leptin level which determine that they have a close relationship and a common response to the use of GC.^15,16

In our study, femoral distal BMD do not change much at day 7, but have decreased at day 21 in prednisone group when compared to age-matched control. This suggests that an initial and transient loss of BMD is not observed in distal femur. However, there are experimental evidences indicating that GCs excess induce an initial but transient loss of BMD because of an early up-regulation of bone resorption.^8,9 But these studies only focused on the detection of biochemical markers of bone formation and bone resorption and they did not reveal the femur BMD and microstructural results at day 7 with GC treatment. Though biochemical markers are not detected in our experiment, we tended to infer that short-term (7-day) use of prednisone on rats do not initiate a transient loss of BMD based on the data of DXA and micro-CT scan. During longer treatment of prednisone (21 days), the inhibited effects of GCs on both bone formation and resorption appear to be of greater relevance and cause the decrease of BMD.^17,18 The decrease of distal femur BMD instead of total femur BMD illustrates that the effects of prednisone on cancellous bone are greater than or prior to the effects on cortical bone since distal femur is mainly composed of cancellous bone.^19
–21

BMD reflects the bone quantity, while biomechanical changes mainly reflect the bone quality.^22,23 Our data demonstrate that significant weakening of the bone quality, as shown by a correlative impairment of Young’s modulus and bending stiffness, is observed in 21 days prednisone-treated rats’ femur (after longer than 7 days of treatment). Moreover, the data obtained by micro-CT scan suggest that bone volume (bone mass) does not reduce obviously, whereas trabeculae thickness decrease, the number of rod-like trabuculae and trabeculae separation increase. Some literatures have attributed GC effects on bone biomechanics to changes in bone mass and geometry rather than to an action on bone material properties.^24,25 Some other studies also support that thinning trabeculae and unchanged trabeculae number are characteristics of GC-treated bone tissue.^26,27 When analysing this information together, we conclude that the decrease of bone quality is mainly due to the decrease of the bone stiffness which is mainly due to the adaptive change of microstructure. However, mechanical three-point bending test of femur mainly reflect the mechanical properties of diaphysis rather than metaphysis, which indicates that the geometric structure of the femoral shaft has been affected also. Usage of systemic GCs leads to enhanced osteoclastogenesis and thus endosteal resorption and cortical porosity,²⁸ and bone formation in these conditions is inhibited, resulting in a decreased periosteal bone apposition that insufficiently compensates for the endosteal resorption.²⁹ Impaired cortical bone re-modelling together with increased cortical porosity bears the potential for increased fracture risk at appendicular bone sites such as the femur and tibia.^30,31 During longer therapy of GCs, these inhibited effects of GCs on cortical will appear to be of greater relevance.

Conclusion

In conclusion, age and the usage of prednisone are two factors that exert a positive or a negative action respectively on femur BMD, biomechanics and microstructure in the rats of sexual maturity. Short-term of 7 days of prednisone treatment is no longer enough to reflect the negative changes in the rat femur in response to GC. Twenty-one day use of prednisone slows body weight gaining, decreases femur stiffness and distal femur BMD and deteriorates microstructure. These findings may help the clinician to pay more attention to the short-term adverse effects of GCs on the skeleton.

Footnotes

Human and Animal Rights and Informed Consent

All animal experiments were approved by the Academic Committee on the Ethics of Animal Experiments of the Guangdong Medical University, Zhanjiang, China. Permit Number: SYXK (GUANGDONG) A2008036

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The all costs were funded by the authors.

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Effects of short-term glucocorticoid administration on bone mineral density,biomechanics and microstructure in rats’ femur