Eur Spine J (2014) 23:2437–2448 DOI 10.1007/s00586-014-3463-z
ORIGINAL ARTICLE
Short-term glucocorticoid treatment causes spinal osteoporosis in ovariectomized rats W. Bo¨cker • T. El Khassawna • N. Bauer • K. Brodsky • D. Weisweiler • P. Govindarajan • G. Schlewitz • M. Kampschulte • L. Du¨rselen • U. Thormann • G. Szalay • R. Schnettler • A. C. Langheinrich • C. Heiss
Received: 5 July 2013 / Revised: 4 July 2014 / Accepted: 6 July 2014 / Published online: 31 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose In humans, glucocorticoid-induced osteoporosis is the most common cause of medication-induced osteoporosis. Recent clinical data suggest that glucocorticoid therapy increases the risk of vertebral fractures within a short treatment period. Therefore, this study aimed at investigating vertebral bone in a rat model of glucocorticoid-induced postmenopausal osteoporosis. Methods Fifty Sprague–Dawley rats were randomly assigned into three groups: 1) untreated controls, 2) Shamoperated group, and 3) ovariectomized rats treated with glucocorticoid (dexamethasone) for 3 months (3M) after recovery from bilateral ovariectomy. Osteoporotic bone status was determined by means of the gold standard dual energy X-ray absorptiometry (DEXA) scan. Vertebral bodies were examined using lCT, histological analysis, mRNA expression analysis, and biomechanical compression
Electronic supplementary material The online version of this article (doi:10.1007/s00586-014-3463-z) contains supplementary material, which is available to authorized users. W. Bo¨cker T. El Khassawna K. Brodsky D. Weisweiler P. Govindarajan R. Schnettler C. Heiss Laboratory of Experimental Trauma Surgery, Justus-Liebig University, Giessen, Germany e-mail: [email protected] W. Bo¨cker G. Schlewitz U. Thormann G. Szalay R. Schnettler C. Heiss (&) Department of Trauma Surgery, University Hospital of Giessen-Marburg, Giessen, Germany e-mail: [email protected]
testing. Further systemic effects were studied biochemically using serum marker analysis. Results Dexamethasone treatment showed at 3M a significantly lower bone mineral density in ovariectomized rats compared to Sham-operated control (p \ 0.0001) as analyzed in vivo by DEXA. Furthermore, Z scores reached levels of -5.7 in the spine indicating sever osteoporotic bone status. Biomechanical testing of compression stability indicated a lower functional competence (p \ 0.0001) in the spine of treated rats. lCT analysis showed significant reduction of bone volume density (BV/TV%; p \ 0.0001), significantly enhanced trabecular spacing (Tb.Sp; p \ 0.0001) with less trabecular number (Tb.N; p \ 0.001) and complete loss of trabecular structures in glucocorticoid-treated ovariectomized rats. Histological analysis by osteoblast and osteoclast activities reflected a higher bone catabolism reflected by osteoclast counts by TRAP (p \ 0.019) and lower bone catabolism indicated by ALPstained area (p \ 0.035).Serum analysis showed a significant increase in osteocalcin (p \ 0.0001), osteopontin (p \ 0.01) and insulin (p \ 0.001) at 3M. Expression M. Kampschulte Department of Radiology, University Hospital of Giessen-Marburg, Giessen, Germany L. Du¨rselen Institute of Orthopedic Research and Biomechanics, Centre of Musculoskeletal Research, University of Ulm, Ulm, Germany A. C. Langheinrich Department of Radiology Frankfurt/Main, BGU-Frankfurt, Giessen, Germany
N. Bauer Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig University, Giessen, Germany
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analysis of molecular markers in the vertebral body revealed lower expression in tenascin C in the OVX-steroid animals at 3M. Conclusions Short-term glucocorticoid treatment of ovariectomized rats indicates according to DEXA standards a severe osteoporotic bone status in vertebral bone. Nonetheless, dysfunctional bone anabolism and enhanced bone catabolism are observed. Alterations of bone extracellular matrix proteins that correlate to inferior mechanical stability and affected microstructure were noticed and suggest further investigation. Treatment with dexamethasone was also seen to affect insulin and osteopontin levels and thus osteoblast function and maturation. This described animal model presents a recapitulation of clinically obtained data from early phase glucocorticoid-induced osteoporosis observed in patients. Keywords Glucocorticoid Osteoporosis Postmenopausal Animal model Rat DEXA
Introduction Osteoporosis is a systemic disorder of the skeleton, which is associated with an increased risk of vertebral fractures. Although osteoporosis already causes the majority of fractures in the industrialized world, the incidence of osteoporotic fractures will double within the next 10 years with serious social-economic consequences [1, 2]. Nearly, half of these osteoporotic fractures occur within the spine [3]. One of the major functions of the spinal bone is to resist mechanical forces. Bone strength depends on the quantity and quality of bone. The bone quantity is defined by bone mineral density (BMD) and the overall geometry, hence, the lower BMD is the higher the risk of osteoporotic vertebral fractures. Furthermore, quality is dependent on the macro- and microstructure of the bone, mineralization, and collagen matrix status. These parameters correlate with the vertebral bone resistance to fractures. Osteoporosis of the vertebral bone, therefore, is characterized by low bone mass and micro-architectural deterioration of bone tissue leading to bone fragility [4–8]. Several risk factors were identified for the development of osteoporosis, such as low peak BMD, genetic predisposition, low body mass index, immobilization, estrogen deficiency, and glucocorticoid therapy [4, 5, 8]. Postmenopausal osteoporosis is the most common cause of vertebral fractures, while glucocorticoid-induced osteoporosis is the second leading cause and the most common reason for secondary osteoporosis. Long-term glucocorticoid usage is between 1 and 3 % among adults worldwide [9]. Fractures may occur in as many as 30–50 % of patients
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receiving chronic glucocorticoid therapy [10–12]. Among these patients, fractures occur more frequently in postmenopausal women at sites enriched in cancellous bone, such as the vertebrae [13, 14]. As with vertebral fractures occurring in postmenopausal osteoporosis, vertebral fractures associated with glucocorticoid therapy are often asymptomatic [12]. When assessed by X-ray-based morphometric measurements of vertebral bodies, 37 % of postmenopausal women on chronic ([6 months) oral glucocorticoid therapy sustain one or more vertebral fractures [12]. Vertebral fractures occur early after exposure to glucocorticoids, at a time when BMD declines rapidly [15]. The early rapid loss of bone predisposes to vertebral fractures. Cross-sectional and longitudinal studies reveal a greater loss in trabecular bone as compared with cortical bone [16, 17]. The vertebral bodies are predominantly composed of trabecular bone (95 % trabecular, 5 % cortical bone [18, 19]) and are, therefore, more susceptible to the effects of glucocorticoids than the cortical bone found in long bones such as the distal radius and proximal femur [20]. Although fractures can occur early in the course of glucocorticoid therapy, their incidence is also related to the dose and duration of glucocorticoid exposure. Doses as low as 2.5–7.5 mg of prednisolone equivalents (equates 0.38–1.12 mg dexamethasone) per day can be associated with a 2.5-fold increase in vertebral fractures, but the risk is greater at higher doses for prolonged periods of time [21, 22]. Following exposure to prednisone equivalents of 10 mg daily (equates 1.5 mg dexamethasone) for longer than 90 days, the risk of fractures of the spine is increased by 17-fold [22]. The effects of glucocorticoids on bone loss are multifactorial including indirect or systemic effects as well as direct effects on bone remodeling [20, 23].Glucocorticoids reduce osteoblastic bone formation. This occurs through a reduction in osteoblast number and function resulting in a decreased osteoblastogenesis [24]. But glucocorticoids also increase osteoclastic bone resorption through an increase in receptor activator of NF-jB ligand (RANKL) and a reduction in osteoprotegerin (OPG) [25]. Negative effects of glucocorticoids on bone quality and bone mass eventually lead to increased risk of vertebral fractures both directly and indirectly [23]. In human subjects, glucocorticoid-induced osteoporosis occurs in two phases: a rapid, early phase in which BMD is reduced, presumably due to excessive bone resorption, and a slower, progressive phase in which BMD declines due to impaired bone formation [26]. Here, we present an in-depth investigation of an animal model of osteoporosis that mimics a critical clinical condition of postmenopausal women who receive steroid therapy. The ovariectomized rat model utilizes short-term treatment with the glucocorticoid dexamethasone. The
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study shall emphasize on the combined effect of steroid therapy and estrogen deficiency through ovariectomy on bone quality. Nevertheless, its main aim is the thorough examination of the treatment effects on macro- and microstructures as well as cellular and molecular changes within vertebral bodies leading to the inferior bone quality and quantity. Furthermore, such examination shall enlighten future research to understand the detailed mechanisms by which glucocorticoids induce osteoporosis. This understanding could provide a new fundament for novel treatment strategies for osteoporotic vertebral fractures.
Materials and methods Experimental design This study is a part of trans-regional project concerned with the establishment of clinically relevant osteoporotic rat model as the foundation for biomaterial-aided healing of osteoporosis fractures. Recently, we have reported the osteoporotic bone status of long bone (femur and tibia) of these particular animal populations used for this experimental setup [27]. Nonetheless, osteoporotic fractures of the spine are of more relevance to the clinical challenge. 10-week-old healthy female Sprague–Dawley rats (n = 50) were purchased from Charles River (Sulzfeld, Germany). The average initial weights of animals were 250–290 g, and were maintained under standard laboratory conditions. Animals underwent an acclimatization period of 4 weeks before the experimental procedures. This study was performed in full compliance with our institutional and German protection laws and approved by the ethical commission of the local governmental institution (‘‘Regierungspra¨sidium’’ Giessen/Germany, permit number: 89/2009). The animals were divided into three groups: control (n = 10), Sham (n = 20), ovariectomy and steroid (OVXsteroid) (n = 20).At the age of 14 weeks, Sham group animals underwent laparotomy after being anesthetized with intra-peritoneal injection of 62.5 mg/kg bodyweight ketamine (HostaketÒ, Hoechst, Germany) and 7.5 mg/kg B.wt. xylazine (RompunÒ, Bayer, Germany) and were fed with standard diet. OVX-steroid rats were ovariectomized bilaterally with a dorsal approach. Thereafter, the animals received glucocorticoid injection of 0.3 mg/kg bodyweight dexamethasone-21-isonicotinate (Voren-DepotÒ, Boehringer Ingelheim, Germany), applied once every 2 weeks and also received a regular diet. The steroid therapy started post-operatively after the ovariectomy of the animals and was continued until euthanasia. Animals of the control group that reported the initial bone status were euthanized immediately (10 weeks of age).However, both
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experimental groups Sham and OVX-steroid groups were monitored at M1 and M3 post-treatment. Measurement of bone parameters by dual energy X-ray absorptiometry (DEXA) Dual energy X-ray absorptiometry is considered the standard clinical method to evaluate bone quality in vivo and its experimental implementation is a strong indicator of the treatment effects. The rats were anesthetized as described above and positioned ventrally with arms separated from the trunk to scan the whole body and scanned by DEXA (Lunar ProdigyÒ, GE Healthcare, Germany). After the scan, regions of interest (ROI) were marked with respective to spine (Fig. 1). The measured parameters included BMD (g/cm2), bone mineral content (BMC) (g) and adipose tissue (%). Z scores were calculated with the formula, Z score = [measured BMD – age-matched BMD]/agematched population SD. The animals were scanned immediately after ovariectomy and laparotomy to obtain the baseline measurements (M0) at M1 and M3 posttreatment. Analysis was performed using the small-animal mode of the enCORE software (GE Healthcare, v. 13.40) and was calibrated at each start of the experiment. Biomechanical testing of compression stability Biomechanical competence testing was performed to evaluate bone quality of the vertebrae, therefore, a compression test was carried out on both Th8 and Th9 (N = 8 of each per time point for each group). Briefly, vertebral bodies were placed between two flat-ended rods of 10 mm diameter in a materials testing machine (Z10, Zwick, Ulm, Germany) and compressed to failure at a displacement rate of 10 mm/min. The failure load in N was defined as the maximum compressive load occurring in the load deformation curves. Mean and standard deviations were calculated and a paired Wilcoxon test was used to prove the significance. Micro-computed tomography (micro-CT) To assess bone geometry, structural index and volume Th10 vertebral body bone samples (n = 6–10 per group) at time point 0, M1 and M3 (Sham; n = 6–10, OVX-steroid; n = 3) were analyzed using micro-computed tomography (lCT). Bones were transferred into 4 % phosphate buffered paraformaldehyde upon harvesting and stored in parafilm to prevent dehydration during the scan. Th10 was scanned in a micro-computer tomography (micro-CT) manufactured by SkyScan (SkyScan1072_80 kV, Kontich, Belgium) with a maximum spatial resolution of 8 lm at 10 % modulation transfer function (MTF). The X-ray system is based on a
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Fig. 1 In vivo DEXA analysis indicated lower bone parameter caused by the treatment in both rat’s spine and pelvis. a Fixed regions of interest (RIO) were scanned and measured in DEXA for spine black box (1), and for pelvis red box (2). b BMD decreased progressively throughout treatment time and was lower in the OVXsteroid when compared to Sham at M3 in both spine (upper panel)
and pelvis (lower panel). c BMC was lower in both ROIs of the experimental group only at M3 when compared to the Sham. d Percent of tissue fat was increased in both Sham and OVX-steroid groups only at the spine. e Analysis exhibited a reduction of the Z score in OVX-steroid compared to Sham at M3
microfocus tube (20–80 kV, 0–100lA) reaching a minimum spot size of 8 lm at 8 W generating X-rays in conebeam geometry. The X-ray detector consists of a 12-bit digital CCD high-resolution (1024 9 1024 pixel) camera with fiber optic 3.7:1 coupling to an X-ray scintillator and digital frame-grabber. Samples were positioned on a computer controlled rotation stage and scanned 180° around the vertical axis in rotation steps of 0.45° at 75 kV. Tibia was scanned en-bloc with an isotropic spatial resolution of (9 lm)3 voxel size. The cortical bone was excluded by means of marking the outer bonds by free hand. For standardized morphometry, the metaphyseal volume of interest (VOI) was set with an offset of 0.5 mm and a longitudinal extension of 3 mm. The gray-scale threshold was set to 75 to separate bone from the surrounding tissue using an adaptive threshold method. Raw data were reconstructed with a modified Feldkamp cone-beam reconstruction modus resulting in two-dimensional cross-sectional images with an 8-bit gray-scale resolution. The following parameters were measured: relative bone volume (BV/TV), relative bone surface (BS/TV), mean trabecular thickness (Tb.Th), mean trabecular separation (Tb.Sp), trabecular number (Tb.N), and structure model index (SMI). Thresholding, definition of ROI and VOI as well as semi-automated morphometric measurement were performed with the SkyScan-CT-analyzer software (CTAn, Sky Scan). The scanning was performed at time points of M0, M1 and M3.
and M3) fixed in paraformaldehyde (4 % PFA) were decalcified in 10 % EDTA in 0.3 M tris buffer (pH 7.4) for 2–3 weeks at room temperature. The samples were then dehydrated through standard graded alcohol solutions and embedded in paraffin. Tissues were sectioned longitudinally to obtain 5-lm-thick sections with microtome (Microm HM 355S, Thermo Scientific, Germany). Following staining procedures were then performed: (1) alkaline phosphatase (ALP) enzyme-histochemical staining to investigate osteoblasts activity considered osteoblasts were ALP positive cells residing on bone surface and were counted randomly in 15–20 microscopic fields per section. (2) Tartrate-resistant acid phosphatase (TRAP) enzymehistochemical staining to investigate osteoclasts activity considered osteoclasts were TRAP-positive multinuclear cells located on the bone surface and were counted randomly in 15–20 microscopic fields per section. Briefly, sections were deparaffinized, for TRAP treated with sodium acetate buffer and incubated in Naphthol-AS-TR phosphate (N6125-1G, Sigma, Germany) and sodium tartrate (Merck, Germany) at 37 °C for 60 min, for ALP sections samples were treated with Tris and then incubated in BCIP/NBT phosphate substrate at 37 °C for 60 min. Both were then visualized with Nova RED (Vector, SK4800, CA) and finally counter stained with Hematoxylin. (3) MMP9 antibody (ab76003, Abcam, MA); (4) tenascin C antibody (ab108930, Abcam, MA); (5) biglycan antibody (ab58562, Abcam, MA). Briefly, deparaffinized sections (N = 3/group/time point) blocked with freshly prepared 3 % H2O2 for 5 min were incubated with the primary antibody dilution as stated above in DAKO buffer, Germany). Sections were afterward incubated with secondary antibody diluted in 1 % BSA in TBS with serum of species of interest in a 1:8 ratio (for 30 min at RT). Finally, secondary antibodies were bound to be visualized by
Bone processing and histochemistry Histological analysis serves in visualizing and quantifying the various cell types affected by the treatment; as well describe matrix proteins, mineralization and adipose tissue. Vertebral bone samples (L3, n = 6 per group at each M0
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incubating sections with ELITE vectastain ABC kit (PK 6100, Vector, CA), for 30 min at RT and then incubated with nova red solution (Vector, SK-4800, CA) for 5 min at RT. The slides were counterstained with hematoxylin. Histomorphometry was performed on macroscopic pictures, the pictures were randomly taken for each paraffin section for both enzyme-histochemical and immunohistochemical analyses, with at least 15 images at 409 magnification. For ALP and TRAP total trabecular surface (mm), osteoclasts count (N), and trabecular bone surface covered by osteoclasts (mm) were measured for TRAP analysis. Trabecular surface (mm), and trabecular surface positive for ALP (mm) were determined. All analyses were performed with Image J 1.45 software (NIH, USA). Measurement of markers of bone and energy metabolism Analysis of OCN, OPN, insulin, ACTH, PTH, RANKL, and leptin serum concentrations was performed in duplicates at the Novartis Pharma Ag with a Luminex 200TM multiplexing instrument using two commercially available test kits (rat bone panel 2, catalogue number MXRABN20N02005, Millipore GmbH, Schwalbach, Germany; rat bone panel 3, catalogue number MXRABN300N02002, Millipore GmbH, Schwalbach, Germany). Statistical analysis was done with mean results. RNA isolation, cDNA synthesis and real-time PCR analysis Molecular testing of gene expression reflects the cellular changes examined by histological analysis, geometrical changes examined by lCT and biomechanical competence. Therefore, bone samples (L1 vertebral bodies) were isolated after animal euthanasia. L1 vertebral body bone samples were stored in RNA later solution at -80 °C for mRNA expression analysis. L1 vertebral bodies (n = 10–12 per group) were ground in liquid nitrogen using mortar and pestle to avoid RNA degradation. The extraction of total RNA was performed according to the TRIzolÒ manufacturer’s protocol (Invitrogen, Germany). 1 lg of total RNA was reverse transcribed to cDNA with quantitect reverse transcriptase kit (Cat No 205313, Qiagen, Germany). Real-time analysis was done with the BioRad iQ5 PCR cycler in a total reaction volume of 25 ll comprising 12.5 ll quantifast SYBR green PCR master mix (Cat No 204054, Qiagen, Germany), 1 ll cDNA, 300 nM of each primer, and 11 ll RNAse free water. Beta 2 microglobulin (b2M) was used as the housekeeping gene, which was chosen after comparing it to RPL27, RPL13A, 18srRNA, b-acting, and GAPDH (Table S1) based on GeNorm software parameters.
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Statistical analysis Statistical analysis for BMD, BMC, fat (%), Z score, biomechanics, semi-quantitative reverse transcriptase PCR, lCT was performed by two-way ANOVA accompanied by Bonferroni’s multiple comparison test to determine the variation between groups at each particular time point. Furthermore, one-way ANOVA followed by Bonferroni’s multiple comparison tests was performed to determine variation across time points in a particular group, as well as for serological analysis of normally distributed data (ACTH). Statistical analysis for TRAP and ALP staining as well as serological analysis of OCN, OPN and insulin was performed using Kruskal–Wallis test followed by Dunns multiple comparison test. All the above statistical analysis was performed by using the statistical software Graphpad prism version 5. Unless mentioned, the asterisks indicate the significance level (*p \ 0.05, **p \ 0.01, ***p \ 0.001 and ****p \ 0.0001).
Results Effects of steroid therapy on BMD, BMC and Z scores in bilaterally ovariectomized rats Dual energy X-ray absorptiometry measurements were obtained from two regions of interest (ROIs), the spine and the pelvis (Fig. 1a).There was a significant decrease in the BMD at M3 in the spine and pelvic ROIs when comparing Sham to OVX-steroid (p \ 0.0001, for both; Fig. 1b). Across time points, BMD of Sham significantly increased with time at both M1 and M3, reaching significance at time point M3. BMD of vertebral and pelvic bone of OVX-steroid significantly declined with time (p \ 0.05 M1 versus M3, for both ROIs). Furthermore, significantly lower BMC was observed in spinal and pelvic bone of the experimental group only at M3 when compared to the Sham. Across time points, vertebral and pelvic bone of Sham rats showed significant increase at M3 compared to its baseline (Fig. 1c). Percent fat showed no significant differences between Sham and OVX-steroid in both skeletal sites at either time points. Across time points, significant increase was observed at M3 in vertebral bone of Sham and OVX-steroid rats when compared to time point M0 (Fig. 1d). Significant decrease in Z score was observed in OVXsteroid rats at time point, with Z scores reaching -5.7 M3 in spine (p \ 0.0001) and -4.4 in pelvis (p \ 0.0001; Fig. 1e). Effects of steroid therapy on compression stability of vertebral bone Estrogen deficiency and steroidal injection lead to an inferior biomechanical competence in the spin after 3M of
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treatment. The compression test utilized here to test integrity of trabecular bone in Th8 and Th9, values showed a significantly lower failure load in Th8 (p \ 0.001) and Th9 (p \ 0.0001) in vertebral bone of OVX-steroid group when compared to the Sham (Fig. 2).
Fig. 2 Inferior biomechanical competence resulting from steroid treatment in ovariectomized rats. Both Th8 and Th9 show the global effect of the treatment on the mechanical properties of the spin in the treated rats after 3M of treatment. Load at failure showed that 30–40 % lower force was needed in a compression test to collapse vertebra from treated rats compared to the Sham
Fig. 3 Detrimental effects of dexamethasone on spine microstructure of ovariectomized rat. a Image reconstruction of young rats (M0) and (M3) treated and Sham rats exhibited a loss of trabecular density and affected cortical bon in the OVX-steroid animals (lower panel). b– c Bone volume parameters show a lower BV/TV and BS/TV in OVX-steroid group compared to the Sham. d–f Properties of trabecular bone showed no change in Tb.Th; nonetheless, lower Tb.N leading to higher Tb.Sp was seen. g SMI indicates shape change in trabecular bone and reflected a rod-like trabecular bone in the OVXsteroid compared to the Sham. BV bone volume, TV trabecular volume, BS bone surface, SMI structure model index, Tb.N trabecular number, Tb.Sp trabecular separation
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Effects of steroid therapy on bone volume and structure in lCT analysis The biomechanical testing inferior properties were also conformed through investigating bone parameters using lCT. Representative reconstructed lCT scan images depicting effected bone micro-architecture reflecting effect of treatment on the trabecular and cortical bone in the spin after 3M (Fig. 3a). The Images show lower trabecular networking and thinner cortical bone. BV/TV (%) was computed to determine the volume of Th10 bone compared to total volume of the sample. High significant difference was observed in BV/TV (%) on comparison between Sham and OVX-steroid group rats with OVX-steroid group depicting lower BV/TV (%) compared to Sham rats at M3 (p \ 0.0001; Fig. 3b). Across time points, BV/TV (%) non-significantly reduced in Sham group at time points M1 and M3 compared to baseline, whereas there was a significant decrease with time significant at each time points of M3 compared to baseline and M1 in OVX-steroid rats (p \ 0.0001). Relative bone surface
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(BS/TV) (1/mm) was also significantly lower in OVX-steroid rats at M3 (p \ 0.001; Fig. 3c). Trabecular thickness (Tb.Th) showed no significant changes observed between groups and time point (Fig. 3). Trabecular number (Tb.N) is considered as the inverse of the mean distance between the mid-axes of the observed bone structure. Trabecular number was significantly lower in OVX-steroid rats compared to Sham rats at M3 compared to Sham rats (p \ 0.001, Fig. 3e). Trabecular separation (Tb.Sp) as determined by the thickness of the marrow cavity was significantly higher in OVX-steroid rats at M3 compared to Sham rats (p \ 0.0001, Fig. 3f). On comparison across time points, significant increase in trabecular separation was observed in OVX-steroid at M3 compared to baseline (p \ 0.0001) whereas in Sham rats, trabecular separation increased at M3 (Fig. 3f). Structure model index (SMI) enables to quantify the characteristic form of the 3D structure in terms of the amount of plates and rods composing the bone structure. The results show that SMI was significantly high in OVX-steroid compared to Sham rats at M3 (p \ 0.001, Fig. 3g).
the Sham rats after 3M of treatment (p = 0.019, Fig. 4a).Moreover, MMP9 was more intense in the regions of osteoclasts and osteocytes in trabecular bone of the OVX-steroid spine compared to general distribution of MMP on the surface of trabecular bone in the Sham spine (Fig. 4c). On the other hand, osteoblast-mediated bone anabolism was investigated by detecting ALP and BGN. ALP staining indicated a reduction of osteoblast activity in the OVX-steroid rats as osteoblast/Tb surface (N/mm) parameter was seen lower when compared to Sham rats after 3M (p = 0.035, Fig. 4b).Further, BGN-stained area was lower in the OVX-steroid than the Sham (p = 0.042), and it was deteriorated and located on the borders of the trabecular bone as to the BGN localization all over the trabecular surface in the Sham (Fig. 4d). Furthermore, TNC was scarcely distributed in osteoid regions at M3 OVX-steroid, while it was well distributed in the osteocyte regions and the trabecular bone borders among Sham rats (Fig. 4e). This lower expression of TNC in the treated animals was also confirmed in relative expression analysis of TNC at M3 (Fig. 4f, note graph is in DCt).
Effects of steroid therapy on bone metabolism
Effects of bone turnover markers
The role of osteoclasts on bone catabolism was detected by TRAP and MMP9 staining. TRAP-positive cells were increased on the bone surface in OVX-steroid compared to
Markers of bone turnover were tested in the serum to assess bone metabolism. OCN suggesting a limitation in bone formation (Fig. 5a) and OPN (Fig. 5b), an inductor of bone
Fig. 4 Unbalanced bone metabolism leads to lesser bone quality in OVX-steroid rats spin. a, c Higher osteoclast activity was observed in OVX-steroid compared to Sham at M3 through a higher TRAPpositive area correlated to trabecular area p = 0.019; MMP9 shows higher intensity at M3 and differences in signal localization between the two groups. b, d Lower osteoblast activity in OVX-steroid inferred by a lower ALP positive area correlated to trabecular area when compared to Sham rats at M3 (p = 0.035); BGN signal was more intense at the trabecular bone borders indicating a misallocation
in the OVX-steroid when compared to the Sham at M3 (p = 0.042). e Sham vertebra showed no visible change on their bone matrix mineralization as seen from the TNC signal intensity, nonetheless OVX-steroid vertebrae exhibited at M3 a decreased signal and localization differences influenced by the treatment where only small areas around the osteocytes responded to the staining. f Lower relative expression analysis of TNC in the OVX-steroid group confirmed the influence of the treatment on TNC expression (graph depicted DCt)
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Fig. 5 Serum markers indicated a discrepant bone turnover in the OVX-steroid rats at M3. a Undercarboxylated osteocalcin was increased in the OVX-steroid serum. b Osteopontin higher serum levels indicated a discrepant osteoblasts function and extracellular
matrix due to the treatment. c Higher insulin serum levels indicated an indirect effect on the OPG/RANKL ratio leading to promote resorption. d ACTH unchanged levels reflect the overall body health in both treated and control rats
remodeling, were increased in the OVX-steroid group. However, an increase in the insulin levels in the OVXsteroid rats serum was also detected (Fig. 5c). Furthermore, ACTH that regulates the glucocorticoid pathway and indicates the general condition of the animals showed no difference between the groups (Fig. 5d).
guidelines, Thomson et al. reported that rats, which were ovariectomized at the age of 12 weeks, showed after 52 weeks of ovariectomy a BMD of 0.145 g/cm2 and 15 g BMC in DEXA. In comparison, our model showed after 12 weeks of ovariectomy and steroid treatment a BMD of 0.16 g/cm2 and 9.3 g BMC. The more pronounced alterations in bone parameters in our model reflect the effects of the additional steroid treatment. Despite being known to reduce the mechanical properties of bone in rats, OVX alone did not lead to a comparable loss of bone density as seen in postmenopausal women. Therefore, additional treatments (i.e., dietary, drug, etc.) are necessary to induce a significant loss of bone mass in rats [31]. Nonetheless, the ovariectomized rats are still the most commonly utilized animal model for postmenopausal osteoporosis. However, OVX rats show two phases of bone loss. A 100 days after ovariectomy, a rapid loss of bone mass is seen, which is followed by a relative stabilization of bone mass on an osteopenic level. Further, 270 days post-ovariectomy the rat model comes into a phase of slow loss of cancellous bone mass. This reflects an imbalanced bone metabolism with predominance of bone loss, which is in its rate lower than that of the osteoporotic patients. Therefore, additional treatments shall be needed to recapitulate osteoporotic bone status of patients [31–33]. Additional treatment with corticosteroids has been reported to alter bone metabolism. The osteoporotic bone status was mainly caused by a decrease in bone formation with only minimal changes in bone resorption. Such an anabolic decrease is more comparable to osteoporotic patients than that of ovariectomized rat models. Further, animal models with steroid treatment combined with ovariectomy showed the clearest bone loss [31]. The rats treated in this study showed a Z score of -5.7 after 3M of treatment. Biomechanical competence and bone structural properties were significantly lower than the
Discussion The steroid-induced osteoporosis is one of the most important forms of secondary osteoporosis. Only few months after steroid treatment starts bone catabolism increases, and after few years about 30–50 % of patients show osteoporosis manifestation [28]. Many rat models of osteoporosis were reported either with estrogen deficiency resulting from bilateral ovariectomy alone or combined with diet or steroid treatment. This study used dexamethasone as a glucocorticoid to induce osteoporotic status in OVX rats. Nonetheless, this study is a part of trans-regional project concerned with detailed investigation of clinically relevant osteoporotic rat model. Unfortunately and due to animal welfare and ethical committee decisions the study had to utilize the reports which meticulously investigated bone alteration of the mature rat at 3M after OVX [29, 30] as recommended in the Food and Drug Administration (FDA) guidelines of animal models for osteoporosis as controls. However, bone parameters obtained through treatment in this study in lined with the decline of bone parameters indicated in the FDA guidelines. Wornski et al. [30] reported that rats which were ovariectomized at the age of 11 weeks showed a 13.2 % loss in trabecular bone volume (TBV) 5 weeks after ovariectomy. These animals also had a 10.2 % reduction in osteoblast surface (OBS) and 12 % increase in osteoclast surface (OCS). The OVX ? steroid animals presented in this study showed 20.07 % reduction of OBS and 15.11 % in OCS compared to the Sham. In the FDA
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Sham control. Investigated bone metabolism reflected a lower bone anabolism and a higher bone catabolism. To our knowledge, this is the first study clearly demonstrating that dexamethasone treatment in postmenopausal (ovariectomized) rats results in osteoporosis of the vertebral bone. This in lines with observations in patients where fractures occur more frequently in postmenopausal women treated with glucocorticoids [16, 17]. Previous studies reported contradictory results on glucocorticoid exposure to rats and showed either increased BMD or decreased BMD [34, 35]. Such fluctuation may have resulted from different factors like age of the animal and dose of the glucocorticoid. Nevertheless, clinical and experimental data from other animal species have clearly demonstrated that glucocorticoids cause a reduction in BMD and eventually osteoporosis [36]. In this experimental setup, we could show that dexamethasone significantly reduced BMD in young mature rats to a Z score of -5.7 in a comparison to age-matched healthy controls. Histological analysis referring to osteoclasts (TRAP) and osteoblasts (ALP) was performed to evaluate the effects of combined treatment on bone metabolism. Increased osteoclast numbers defined as TRAP-positive cells residing on the bone surface were seen in OVX-steroid rats, which in accordance with the osteoclasts were reported to play an important role in mediating bone loss caused by glucocorticoids [37, 38]. Glucocorticoids are known to increase the expression of M-CSF, which causes differentiation of pre-osteoclast to osteoclast [23, 39]. Moreover, dexamethasone in particular was reported to enhance the DNA binding activities of the transcription factors leading to accelerated osteoclast formation [40]. Such osteoclast-mediated bone resorption was exhibited in this study through the higher intensity of MMP9 in IHCs at M3 and its differences in signal localization in the OVXsteroid group. Furthermore, the fact that short-term treatment with dexamethasone primarily caused an increase in osteoclast numbers concords with the clinical observation that glucocorticoid-induced osteoporosis occurs in two phases: a rapid, early phase in which BMD is reduced, presumably due to excessive bone resorption, and a slower, progressive phase in which BMD declines [26]. The data provided here could show that the short-term treatment indeed recapitulate the rapid, early phase of glucocorticoidinduced osteoporosis observed in patients. Histomorphometrical analysis of ALP- and BGNstained area normalized to trabecular area showed lower ALP and BGN at the OVX-steroid rat groups at time point 3M, suggesting that dexamethasone treatment at this early phase affects bone anabolism. This agrees with previous studies which reported a diminished osteoblast function after glucocorticoids treatment [41]. Nonetheless, investigated markers of osteoblast function [42] showed no
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difference between the control and experimental RANKL, leptin and PTH levels in serum as well as OCN relative expression were not changed (Fig. S1 and Fig. S2). Furthermore, osteopontin (OPN) is a regulator of the mineralization process, expressed by osteoblasts in advance to mineral deposition [43], however, OPN is also an important extracellular matrix protein expressed by immature osteoblasts [44]. The higher OPN levels in the OVX-steroid rats at 3M suggest the presence of dysfunctional osteoblasts. Such observations are also seen in other bone diseases that show lower bone competence and BMD such as neurofibromatosis type 1 [45]. In the light of these findings, we investigated TNC as a bone matrix marker, which is a cell adhesive and anchorage molecule that responds in correlation with integrin to bone mechanical properties [46]. Indeed, we could show that intensity and distribution are affected within the bone matrix and that its relative expression is down regulated, which could have affected osteoblast maturation and mineralization [36, 47]. In support to the histological findings, lCT and DEXA data we found in compression analysis a significantly lower strength of vertebral bone in dexamethasone-treated rats, which indicates that the bone of these animals is susceptible to fracture. This is conclusive to clinical data in humans where even short-term treatment with glucocorticoids leads to a rapid bone loss and increase in vertebral fracture risk [41]. Intriguingly, TNC expression levels and IHC observations suggest that lower mechanical competence in the vertebral body of treated rats interact with ECM proteins. As TNC binds to integrin and is known to be regulated through mechanotransduction [46].Interestingly, this accords with the clinical data that shows a higher vertebral fracture risk due to rapid bone loss after shortterm treatment with glucocorticoids [41]. Furthermore, bone extracellular matrix comprising both collagenous and non-collagenous matrix proteins plays a major role in bone structural and functional properties. There are few experimental studies in rats investigating the osteoporotic effect of glucocorticoids. In contrast to the current results, in both sham-operated and parathyroidectomized male rats a treatment with a continuous rate infusion of dexamethasone (16.25 lg/kg/day) for 19 days resulted in a net effect favoring bone formation as reflected by increased bone mass assessed by histomorphometry [35]. Overall, there are conflicting results in the literature regarding the net effect of corticosteroids on bone metabolism. Treatment of male rats with 1 mg/kg/day methylprednisolone of 90 days duration resulted in a reduced bone strength and quality, interestingly, markers of bone metabolism were not assessed [48]. One reason for the discrepant results of various studies could be the fact that moderate doses of prednisolone resulted in increased bone stability in rats, whereas high doses had the opposite effect
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[49]. Therefore, dexamethasone was implemented in this study to avoid such fluctuating effects on the musculoskeletal system of the rat. Nevertheless, glucocorticoids direct and indirect influence on bone mineralization through affecting calcium absorption and resorption, indicating that glucocorticoid-induced reduction of bone formation is associated with a decrease in the mineral apposition rate [9]. Moreover, treatment with dexamethasone was associated with a decline of all markers of bone metabolism including OCN, TRAP5b and PTH, which has been also observed in humans [50]. Here, we show that the several crucial markers of dysregulated bone metabolism were not differentially expressed or rather biochemically altered through serological tests. However, important markers stand out, while ACTH insignificant change indicates that the treatment is not harmful for systemic functions of the body an increase of insulin was noted. Insulin is a regulator of glucose homeostasis in the body that regulates osteoblasts by decreasing osteoprotegerin (OPG) expression [51]. Subsequently, the OPG/RANKL ratio is decreased and resorption is promoted, which then lead to an increase in the release of undercarboxylated osteocalcin. Our findings of elevated undercarboxylated osteocalcin and insulin levels in serum suggest that glucosteroids, which are insulin resistant, are causing the insulin serum level to increase and finally promote resorption. Furthermore, the increased osteopontin level in the serum could also suggest a dysfunctional bone anabolism through immature osteoblasts [43, 44] and that macrophages are releasing osteopontin to facilitate the attachment of osteoblasts [52] in order to balance bone metabolism. In summary, our data showed that short-term treatment of postmenopausal (ovariectomized) Sprague–Dawley rats with dexamethasone results in an increased bone catabolism and decreased bone anabolism associated with misallocation of bone matrix proteins. Altogether the obtained data suggest that this animal model is suitable to further investigate the role of glucocorticoids treatment on bone metabolism mediated by bone matrix proteins such as integrins, and the implementation of such knowledge in counter therapy against risk of osteoporotic fractures. Furthermore, the current study is encouraging to carry the described treatment for longer period to investigate whether it will recapitulate the decline in bone loss reported in patients. Further, the authors recommend the investigating of extracellular matrix proteins in comparison with an OVX control as such data are not described in the FDA guide lines. Acknowledgments This study was solely funded by DFG, German Research Foundation (SFB/TRR 79, T1). The authors sincerely thank Mrs. Stengel A, Ms. Sparer S, Mrs. Bergen I and Mrs. Hartmann S
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None.
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ORIGINAL ARTICLE
Short-term glucocorticoid treatment causes spinal osteoporosis in ovariectomized rats W. Bo¨cker • T. El Khassawna • N. Bauer • K. Brodsky • D. Weisweiler • P. Govindarajan • G. Schlewitz • M. Kampschulte • L. Du¨rselen • U. Thormann • G. Szalay • R. Schnettler • A. C. Langheinrich • C. Heiss
Received: 5 July 2013 / Revised: 4 July 2014 / Accepted: 6 July 2014 / Published online: 31 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose In humans, glucocorticoid-induced osteoporosis is the most common cause of medication-induced osteoporosis. Recent clinical data suggest that glucocorticoid therapy increases the risk of vertebral fractures within a short treatment period. Therefore, this study aimed at investigating vertebral bone in a rat model of glucocorticoid-induced postmenopausal osteoporosis. Methods Fifty Sprague–Dawley rats were randomly assigned into three groups: 1) untreated controls, 2) Shamoperated group, and 3) ovariectomized rats treated with glucocorticoid (dexamethasone) for 3 months (3M) after recovery from bilateral ovariectomy. Osteoporotic bone status was determined by means of the gold standard dual energy X-ray absorptiometry (DEXA) scan. Vertebral bodies were examined using lCT, histological analysis, mRNA expression analysis, and biomechanical compression
Electronic supplementary material The online version of this article (doi:10.1007/s00586-014-3463-z) contains supplementary material, which is available to authorized users. W. Bo¨cker T. El Khassawna K. Brodsky D. Weisweiler P. Govindarajan R. Schnettler C. Heiss Laboratory of Experimental Trauma Surgery, Justus-Liebig University, Giessen, Germany e-mail: [email protected] W. Bo¨cker G. Schlewitz U. Thormann G. Szalay R. Schnettler C. Heiss (&) Department of Trauma Surgery, University Hospital of Giessen-Marburg, Giessen, Germany e-mail: [email protected]
testing. Further systemic effects were studied biochemically using serum marker analysis. Results Dexamethasone treatment showed at 3M a significantly lower bone mineral density in ovariectomized rats compared to Sham-operated control (p \ 0.0001) as analyzed in vivo by DEXA. Furthermore, Z scores reached levels of -5.7 in the spine indicating sever osteoporotic bone status. Biomechanical testing of compression stability indicated a lower functional competence (p \ 0.0001) in the spine of treated rats. lCT analysis showed significant reduction of bone volume density (BV/TV%; p \ 0.0001), significantly enhanced trabecular spacing (Tb.Sp; p \ 0.0001) with less trabecular number (Tb.N; p \ 0.001) and complete loss of trabecular structures in glucocorticoid-treated ovariectomized rats. Histological analysis by osteoblast and osteoclast activities reflected a higher bone catabolism reflected by osteoclast counts by TRAP (p \ 0.019) and lower bone catabolism indicated by ALPstained area (p \ 0.035).Serum analysis showed a significant increase in osteocalcin (p \ 0.0001), osteopontin (p \ 0.01) and insulin (p \ 0.001) at 3M. Expression M. Kampschulte Department of Radiology, University Hospital of Giessen-Marburg, Giessen, Germany L. Du¨rselen Institute of Orthopedic Research and Biomechanics, Centre of Musculoskeletal Research, University of Ulm, Ulm, Germany A. C. Langheinrich Department of Radiology Frankfurt/Main, BGU-Frankfurt, Giessen, Germany
N. Bauer Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig University, Giessen, Germany
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analysis of molecular markers in the vertebral body revealed lower expression in tenascin C in the OVX-steroid animals at 3M. Conclusions Short-term glucocorticoid treatment of ovariectomized rats indicates according to DEXA standards a severe osteoporotic bone status in vertebral bone. Nonetheless, dysfunctional bone anabolism and enhanced bone catabolism are observed. Alterations of bone extracellular matrix proteins that correlate to inferior mechanical stability and affected microstructure were noticed and suggest further investigation. Treatment with dexamethasone was also seen to affect insulin and osteopontin levels and thus osteoblast function and maturation. This described animal model presents a recapitulation of clinically obtained data from early phase glucocorticoid-induced osteoporosis observed in patients. Keywords Glucocorticoid Osteoporosis Postmenopausal Animal model Rat DEXA
Introduction Osteoporosis is a systemic disorder of the skeleton, which is associated with an increased risk of vertebral fractures. Although osteoporosis already causes the majority of fractures in the industrialized world, the incidence of osteoporotic fractures will double within the next 10 years with serious social-economic consequences [1, 2]. Nearly, half of these osteoporotic fractures occur within the spine [3]. One of the major functions of the spinal bone is to resist mechanical forces. Bone strength depends on the quantity and quality of bone. The bone quantity is defined by bone mineral density (BMD) and the overall geometry, hence, the lower BMD is the higher the risk of osteoporotic vertebral fractures. Furthermore, quality is dependent on the macro- and microstructure of the bone, mineralization, and collagen matrix status. These parameters correlate with the vertebral bone resistance to fractures. Osteoporosis of the vertebral bone, therefore, is characterized by low bone mass and micro-architectural deterioration of bone tissue leading to bone fragility [4–8]. Several risk factors were identified for the development of osteoporosis, such as low peak BMD, genetic predisposition, low body mass index, immobilization, estrogen deficiency, and glucocorticoid therapy [4, 5, 8]. Postmenopausal osteoporosis is the most common cause of vertebral fractures, while glucocorticoid-induced osteoporosis is the second leading cause and the most common reason for secondary osteoporosis. Long-term glucocorticoid usage is between 1 and 3 % among adults worldwide [9]. Fractures may occur in as many as 30–50 % of patients
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receiving chronic glucocorticoid therapy [10–12]. Among these patients, fractures occur more frequently in postmenopausal women at sites enriched in cancellous bone, such as the vertebrae [13, 14]. As with vertebral fractures occurring in postmenopausal osteoporosis, vertebral fractures associated with glucocorticoid therapy are often asymptomatic [12]. When assessed by X-ray-based morphometric measurements of vertebral bodies, 37 % of postmenopausal women on chronic ([6 months) oral glucocorticoid therapy sustain one or more vertebral fractures [12]. Vertebral fractures occur early after exposure to glucocorticoids, at a time when BMD declines rapidly [15]. The early rapid loss of bone predisposes to vertebral fractures. Cross-sectional and longitudinal studies reveal a greater loss in trabecular bone as compared with cortical bone [16, 17]. The vertebral bodies are predominantly composed of trabecular bone (95 % trabecular, 5 % cortical bone [18, 19]) and are, therefore, more susceptible to the effects of glucocorticoids than the cortical bone found in long bones such as the distal radius and proximal femur [20]. Although fractures can occur early in the course of glucocorticoid therapy, their incidence is also related to the dose and duration of glucocorticoid exposure. Doses as low as 2.5–7.5 mg of prednisolone equivalents (equates 0.38–1.12 mg dexamethasone) per day can be associated with a 2.5-fold increase in vertebral fractures, but the risk is greater at higher doses for prolonged periods of time [21, 22]. Following exposure to prednisone equivalents of 10 mg daily (equates 1.5 mg dexamethasone) for longer than 90 days, the risk of fractures of the spine is increased by 17-fold [22]. The effects of glucocorticoids on bone loss are multifactorial including indirect or systemic effects as well as direct effects on bone remodeling [20, 23].Glucocorticoids reduce osteoblastic bone formation. This occurs through a reduction in osteoblast number and function resulting in a decreased osteoblastogenesis [24]. But glucocorticoids also increase osteoclastic bone resorption through an increase in receptor activator of NF-jB ligand (RANKL) and a reduction in osteoprotegerin (OPG) [25]. Negative effects of glucocorticoids on bone quality and bone mass eventually lead to increased risk of vertebral fractures both directly and indirectly [23]. In human subjects, glucocorticoid-induced osteoporosis occurs in two phases: a rapid, early phase in which BMD is reduced, presumably due to excessive bone resorption, and a slower, progressive phase in which BMD declines due to impaired bone formation [26]. Here, we present an in-depth investigation of an animal model of osteoporosis that mimics a critical clinical condition of postmenopausal women who receive steroid therapy. The ovariectomized rat model utilizes short-term treatment with the glucocorticoid dexamethasone. The
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study shall emphasize on the combined effect of steroid therapy and estrogen deficiency through ovariectomy on bone quality. Nevertheless, its main aim is the thorough examination of the treatment effects on macro- and microstructures as well as cellular and molecular changes within vertebral bodies leading to the inferior bone quality and quantity. Furthermore, such examination shall enlighten future research to understand the detailed mechanisms by which glucocorticoids induce osteoporosis. This understanding could provide a new fundament for novel treatment strategies for osteoporotic vertebral fractures.
Materials and methods Experimental design This study is a part of trans-regional project concerned with the establishment of clinically relevant osteoporotic rat model as the foundation for biomaterial-aided healing of osteoporosis fractures. Recently, we have reported the osteoporotic bone status of long bone (femur and tibia) of these particular animal populations used for this experimental setup [27]. Nonetheless, osteoporotic fractures of the spine are of more relevance to the clinical challenge. 10-week-old healthy female Sprague–Dawley rats (n = 50) were purchased from Charles River (Sulzfeld, Germany). The average initial weights of animals were 250–290 g, and were maintained under standard laboratory conditions. Animals underwent an acclimatization period of 4 weeks before the experimental procedures. This study was performed in full compliance with our institutional and German protection laws and approved by the ethical commission of the local governmental institution (‘‘Regierungspra¨sidium’’ Giessen/Germany, permit number: 89/2009). The animals were divided into three groups: control (n = 10), Sham (n = 20), ovariectomy and steroid (OVXsteroid) (n = 20).At the age of 14 weeks, Sham group animals underwent laparotomy after being anesthetized with intra-peritoneal injection of 62.5 mg/kg bodyweight ketamine (HostaketÒ, Hoechst, Germany) and 7.5 mg/kg B.wt. xylazine (RompunÒ, Bayer, Germany) and were fed with standard diet. OVX-steroid rats were ovariectomized bilaterally with a dorsal approach. Thereafter, the animals received glucocorticoid injection of 0.3 mg/kg bodyweight dexamethasone-21-isonicotinate (Voren-DepotÒ, Boehringer Ingelheim, Germany), applied once every 2 weeks and also received a regular diet. The steroid therapy started post-operatively after the ovariectomy of the animals and was continued until euthanasia. Animals of the control group that reported the initial bone status were euthanized immediately (10 weeks of age).However, both
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experimental groups Sham and OVX-steroid groups were monitored at M1 and M3 post-treatment. Measurement of bone parameters by dual energy X-ray absorptiometry (DEXA) Dual energy X-ray absorptiometry is considered the standard clinical method to evaluate bone quality in vivo and its experimental implementation is a strong indicator of the treatment effects. The rats were anesthetized as described above and positioned ventrally with arms separated from the trunk to scan the whole body and scanned by DEXA (Lunar ProdigyÒ, GE Healthcare, Germany). After the scan, regions of interest (ROI) were marked with respective to spine (Fig. 1). The measured parameters included BMD (g/cm2), bone mineral content (BMC) (g) and adipose tissue (%). Z scores were calculated with the formula, Z score = [measured BMD – age-matched BMD]/agematched population SD. The animals were scanned immediately after ovariectomy and laparotomy to obtain the baseline measurements (M0) at M1 and M3 posttreatment. Analysis was performed using the small-animal mode of the enCORE software (GE Healthcare, v. 13.40) and was calibrated at each start of the experiment. Biomechanical testing of compression stability Biomechanical competence testing was performed to evaluate bone quality of the vertebrae, therefore, a compression test was carried out on both Th8 and Th9 (N = 8 of each per time point for each group). Briefly, vertebral bodies were placed between two flat-ended rods of 10 mm diameter in a materials testing machine (Z10, Zwick, Ulm, Germany) and compressed to failure at a displacement rate of 10 mm/min. The failure load in N was defined as the maximum compressive load occurring in the load deformation curves. Mean and standard deviations were calculated and a paired Wilcoxon test was used to prove the significance. Micro-computed tomography (micro-CT) To assess bone geometry, structural index and volume Th10 vertebral body bone samples (n = 6–10 per group) at time point 0, M1 and M3 (Sham; n = 6–10, OVX-steroid; n = 3) were analyzed using micro-computed tomography (lCT). Bones were transferred into 4 % phosphate buffered paraformaldehyde upon harvesting and stored in parafilm to prevent dehydration during the scan. Th10 was scanned in a micro-computer tomography (micro-CT) manufactured by SkyScan (SkyScan1072_80 kV, Kontich, Belgium) with a maximum spatial resolution of 8 lm at 10 % modulation transfer function (MTF). The X-ray system is based on a
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Fig. 1 In vivo DEXA analysis indicated lower bone parameter caused by the treatment in both rat’s spine and pelvis. a Fixed regions of interest (RIO) were scanned and measured in DEXA for spine black box (1), and for pelvis red box (2). b BMD decreased progressively throughout treatment time and was lower in the OVXsteroid when compared to Sham at M3 in both spine (upper panel)
and pelvis (lower panel). c BMC was lower in both ROIs of the experimental group only at M3 when compared to the Sham. d Percent of tissue fat was increased in both Sham and OVX-steroid groups only at the spine. e Analysis exhibited a reduction of the Z score in OVX-steroid compared to Sham at M3
microfocus tube (20–80 kV, 0–100lA) reaching a minimum spot size of 8 lm at 8 W generating X-rays in conebeam geometry. The X-ray detector consists of a 12-bit digital CCD high-resolution (1024 9 1024 pixel) camera with fiber optic 3.7:1 coupling to an X-ray scintillator and digital frame-grabber. Samples were positioned on a computer controlled rotation stage and scanned 180° around the vertical axis in rotation steps of 0.45° at 75 kV. Tibia was scanned en-bloc with an isotropic spatial resolution of (9 lm)3 voxel size. The cortical bone was excluded by means of marking the outer bonds by free hand. For standardized morphometry, the metaphyseal volume of interest (VOI) was set with an offset of 0.5 mm and a longitudinal extension of 3 mm. The gray-scale threshold was set to 75 to separate bone from the surrounding tissue using an adaptive threshold method. Raw data were reconstructed with a modified Feldkamp cone-beam reconstruction modus resulting in two-dimensional cross-sectional images with an 8-bit gray-scale resolution. The following parameters were measured: relative bone volume (BV/TV), relative bone surface (BS/TV), mean trabecular thickness (Tb.Th), mean trabecular separation (Tb.Sp), trabecular number (Tb.N), and structure model index (SMI). Thresholding, definition of ROI and VOI as well as semi-automated morphometric measurement were performed with the SkyScan-CT-analyzer software (CTAn, Sky Scan). The scanning was performed at time points of M0, M1 and M3.
and M3) fixed in paraformaldehyde (4 % PFA) were decalcified in 10 % EDTA in 0.3 M tris buffer (pH 7.4) for 2–3 weeks at room temperature. The samples were then dehydrated through standard graded alcohol solutions and embedded in paraffin. Tissues were sectioned longitudinally to obtain 5-lm-thick sections with microtome (Microm HM 355S, Thermo Scientific, Germany). Following staining procedures were then performed: (1) alkaline phosphatase (ALP) enzyme-histochemical staining to investigate osteoblasts activity considered osteoblasts were ALP positive cells residing on bone surface and were counted randomly in 15–20 microscopic fields per section. (2) Tartrate-resistant acid phosphatase (TRAP) enzymehistochemical staining to investigate osteoclasts activity considered osteoclasts were TRAP-positive multinuclear cells located on the bone surface and were counted randomly in 15–20 microscopic fields per section. Briefly, sections were deparaffinized, for TRAP treated with sodium acetate buffer and incubated in Naphthol-AS-TR phosphate (N6125-1G, Sigma, Germany) and sodium tartrate (Merck, Germany) at 37 °C for 60 min, for ALP sections samples were treated with Tris and then incubated in BCIP/NBT phosphate substrate at 37 °C for 60 min. Both were then visualized with Nova RED (Vector, SK4800, CA) and finally counter stained with Hematoxylin. (3) MMP9 antibody (ab76003, Abcam, MA); (4) tenascin C antibody (ab108930, Abcam, MA); (5) biglycan antibody (ab58562, Abcam, MA). Briefly, deparaffinized sections (N = 3/group/time point) blocked with freshly prepared 3 % H2O2 for 5 min were incubated with the primary antibody dilution as stated above in DAKO buffer, Germany). Sections were afterward incubated with secondary antibody diluted in 1 % BSA in TBS with serum of species of interest in a 1:8 ratio (for 30 min at RT). Finally, secondary antibodies were bound to be visualized by
Bone processing and histochemistry Histological analysis serves in visualizing and quantifying the various cell types affected by the treatment; as well describe matrix proteins, mineralization and adipose tissue. Vertebral bone samples (L3, n = 6 per group at each M0
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incubating sections with ELITE vectastain ABC kit (PK 6100, Vector, CA), for 30 min at RT and then incubated with nova red solution (Vector, SK-4800, CA) for 5 min at RT. The slides were counterstained with hematoxylin. Histomorphometry was performed on macroscopic pictures, the pictures were randomly taken for each paraffin section for both enzyme-histochemical and immunohistochemical analyses, with at least 15 images at 409 magnification. For ALP and TRAP total trabecular surface (mm), osteoclasts count (N), and trabecular bone surface covered by osteoclasts (mm) were measured for TRAP analysis. Trabecular surface (mm), and trabecular surface positive for ALP (mm) were determined. All analyses were performed with Image J 1.45 software (NIH, USA). Measurement of markers of bone and energy metabolism Analysis of OCN, OPN, insulin, ACTH, PTH, RANKL, and leptin serum concentrations was performed in duplicates at the Novartis Pharma Ag with a Luminex 200TM multiplexing instrument using two commercially available test kits (rat bone panel 2, catalogue number MXRABN20N02005, Millipore GmbH, Schwalbach, Germany; rat bone panel 3, catalogue number MXRABN300N02002, Millipore GmbH, Schwalbach, Germany). Statistical analysis was done with mean results. RNA isolation, cDNA synthesis and real-time PCR analysis Molecular testing of gene expression reflects the cellular changes examined by histological analysis, geometrical changes examined by lCT and biomechanical competence. Therefore, bone samples (L1 vertebral bodies) were isolated after animal euthanasia. L1 vertebral body bone samples were stored in RNA later solution at -80 °C for mRNA expression analysis. L1 vertebral bodies (n = 10–12 per group) were ground in liquid nitrogen using mortar and pestle to avoid RNA degradation. The extraction of total RNA was performed according to the TRIzolÒ manufacturer’s protocol (Invitrogen, Germany). 1 lg of total RNA was reverse transcribed to cDNA with quantitect reverse transcriptase kit (Cat No 205313, Qiagen, Germany). Real-time analysis was done with the BioRad iQ5 PCR cycler in a total reaction volume of 25 ll comprising 12.5 ll quantifast SYBR green PCR master mix (Cat No 204054, Qiagen, Germany), 1 ll cDNA, 300 nM of each primer, and 11 ll RNAse free water. Beta 2 microglobulin (b2M) was used as the housekeeping gene, which was chosen after comparing it to RPL27, RPL13A, 18srRNA, b-acting, and GAPDH (Table S1) based on GeNorm software parameters.
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Statistical analysis Statistical analysis for BMD, BMC, fat (%), Z score, biomechanics, semi-quantitative reverse transcriptase PCR, lCT was performed by two-way ANOVA accompanied by Bonferroni’s multiple comparison test to determine the variation between groups at each particular time point. Furthermore, one-way ANOVA followed by Bonferroni’s multiple comparison tests was performed to determine variation across time points in a particular group, as well as for serological analysis of normally distributed data (ACTH). Statistical analysis for TRAP and ALP staining as well as serological analysis of OCN, OPN and insulin was performed using Kruskal–Wallis test followed by Dunns multiple comparison test. All the above statistical analysis was performed by using the statistical software Graphpad prism version 5. Unless mentioned, the asterisks indicate the significance level (*p \ 0.05, **p \ 0.01, ***p \ 0.001 and ****p \ 0.0001).
Results Effects of steroid therapy on BMD, BMC and Z scores in bilaterally ovariectomized rats Dual energy X-ray absorptiometry measurements were obtained from two regions of interest (ROIs), the spine and the pelvis (Fig. 1a).There was a significant decrease in the BMD at M3 in the spine and pelvic ROIs when comparing Sham to OVX-steroid (p \ 0.0001, for both; Fig. 1b). Across time points, BMD of Sham significantly increased with time at both M1 and M3, reaching significance at time point M3. BMD of vertebral and pelvic bone of OVX-steroid significantly declined with time (p \ 0.05 M1 versus M3, for both ROIs). Furthermore, significantly lower BMC was observed in spinal and pelvic bone of the experimental group only at M3 when compared to the Sham. Across time points, vertebral and pelvic bone of Sham rats showed significant increase at M3 compared to its baseline (Fig. 1c). Percent fat showed no significant differences between Sham and OVX-steroid in both skeletal sites at either time points. Across time points, significant increase was observed at M3 in vertebral bone of Sham and OVX-steroid rats when compared to time point M0 (Fig. 1d). Significant decrease in Z score was observed in OVXsteroid rats at time point, with Z scores reaching -5.7 M3 in spine (p \ 0.0001) and -4.4 in pelvis (p \ 0.0001; Fig. 1e). Effects of steroid therapy on compression stability of vertebral bone Estrogen deficiency and steroidal injection lead to an inferior biomechanical competence in the spin after 3M of
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treatment. The compression test utilized here to test integrity of trabecular bone in Th8 and Th9, values showed a significantly lower failure load in Th8 (p \ 0.001) and Th9 (p \ 0.0001) in vertebral bone of OVX-steroid group when compared to the Sham (Fig. 2).
Fig. 2 Inferior biomechanical competence resulting from steroid treatment in ovariectomized rats. Both Th8 and Th9 show the global effect of the treatment on the mechanical properties of the spin in the treated rats after 3M of treatment. Load at failure showed that 30–40 % lower force was needed in a compression test to collapse vertebra from treated rats compared to the Sham
Fig. 3 Detrimental effects of dexamethasone on spine microstructure of ovariectomized rat. a Image reconstruction of young rats (M0) and (M3) treated and Sham rats exhibited a loss of trabecular density and affected cortical bon in the OVX-steroid animals (lower panel). b– c Bone volume parameters show a lower BV/TV and BS/TV in OVX-steroid group compared to the Sham. d–f Properties of trabecular bone showed no change in Tb.Th; nonetheless, lower Tb.N leading to higher Tb.Sp was seen. g SMI indicates shape change in trabecular bone and reflected a rod-like trabecular bone in the OVXsteroid compared to the Sham. BV bone volume, TV trabecular volume, BS bone surface, SMI structure model index, Tb.N trabecular number, Tb.Sp trabecular separation
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Effects of steroid therapy on bone volume and structure in lCT analysis The biomechanical testing inferior properties were also conformed through investigating bone parameters using lCT. Representative reconstructed lCT scan images depicting effected bone micro-architecture reflecting effect of treatment on the trabecular and cortical bone in the spin after 3M (Fig. 3a). The Images show lower trabecular networking and thinner cortical bone. BV/TV (%) was computed to determine the volume of Th10 bone compared to total volume of the sample. High significant difference was observed in BV/TV (%) on comparison between Sham and OVX-steroid group rats with OVX-steroid group depicting lower BV/TV (%) compared to Sham rats at M3 (p \ 0.0001; Fig. 3b). Across time points, BV/TV (%) non-significantly reduced in Sham group at time points M1 and M3 compared to baseline, whereas there was a significant decrease with time significant at each time points of M3 compared to baseline and M1 in OVX-steroid rats (p \ 0.0001). Relative bone surface
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(BS/TV) (1/mm) was also significantly lower in OVX-steroid rats at M3 (p \ 0.001; Fig. 3c). Trabecular thickness (Tb.Th) showed no significant changes observed between groups and time point (Fig. 3). Trabecular number (Tb.N) is considered as the inverse of the mean distance between the mid-axes of the observed bone structure. Trabecular number was significantly lower in OVX-steroid rats compared to Sham rats at M3 compared to Sham rats (p \ 0.001, Fig. 3e). Trabecular separation (Tb.Sp) as determined by the thickness of the marrow cavity was significantly higher in OVX-steroid rats at M3 compared to Sham rats (p \ 0.0001, Fig. 3f). On comparison across time points, significant increase in trabecular separation was observed in OVX-steroid at M3 compared to baseline (p \ 0.0001) whereas in Sham rats, trabecular separation increased at M3 (Fig. 3f). Structure model index (SMI) enables to quantify the characteristic form of the 3D structure in terms of the amount of plates and rods composing the bone structure. The results show that SMI was significantly high in OVX-steroid compared to Sham rats at M3 (p \ 0.001, Fig. 3g).
the Sham rats after 3M of treatment (p = 0.019, Fig. 4a).Moreover, MMP9 was more intense in the regions of osteoclasts and osteocytes in trabecular bone of the OVX-steroid spine compared to general distribution of MMP on the surface of trabecular bone in the Sham spine (Fig. 4c). On the other hand, osteoblast-mediated bone anabolism was investigated by detecting ALP and BGN. ALP staining indicated a reduction of osteoblast activity in the OVX-steroid rats as osteoblast/Tb surface (N/mm) parameter was seen lower when compared to Sham rats after 3M (p = 0.035, Fig. 4b).Further, BGN-stained area was lower in the OVX-steroid than the Sham (p = 0.042), and it was deteriorated and located on the borders of the trabecular bone as to the BGN localization all over the trabecular surface in the Sham (Fig. 4d). Furthermore, TNC was scarcely distributed in osteoid regions at M3 OVX-steroid, while it was well distributed in the osteocyte regions and the trabecular bone borders among Sham rats (Fig. 4e). This lower expression of TNC in the treated animals was also confirmed in relative expression analysis of TNC at M3 (Fig. 4f, note graph is in DCt).
Effects of steroid therapy on bone metabolism
Effects of bone turnover markers
The role of osteoclasts on bone catabolism was detected by TRAP and MMP9 staining. TRAP-positive cells were increased on the bone surface in OVX-steroid compared to
Markers of bone turnover were tested in the serum to assess bone metabolism. OCN suggesting a limitation in bone formation (Fig. 5a) and OPN (Fig. 5b), an inductor of bone
Fig. 4 Unbalanced bone metabolism leads to lesser bone quality in OVX-steroid rats spin. a, c Higher osteoclast activity was observed in OVX-steroid compared to Sham at M3 through a higher TRAPpositive area correlated to trabecular area p = 0.019; MMP9 shows higher intensity at M3 and differences in signal localization between the two groups. b, d Lower osteoblast activity in OVX-steroid inferred by a lower ALP positive area correlated to trabecular area when compared to Sham rats at M3 (p = 0.035); BGN signal was more intense at the trabecular bone borders indicating a misallocation
in the OVX-steroid when compared to the Sham at M3 (p = 0.042). e Sham vertebra showed no visible change on their bone matrix mineralization as seen from the TNC signal intensity, nonetheless OVX-steroid vertebrae exhibited at M3 a decreased signal and localization differences influenced by the treatment where only small areas around the osteocytes responded to the staining. f Lower relative expression analysis of TNC in the OVX-steroid group confirmed the influence of the treatment on TNC expression (graph depicted DCt)
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Fig. 5 Serum markers indicated a discrepant bone turnover in the OVX-steroid rats at M3. a Undercarboxylated osteocalcin was increased in the OVX-steroid serum. b Osteopontin higher serum levels indicated a discrepant osteoblasts function and extracellular
matrix due to the treatment. c Higher insulin serum levels indicated an indirect effect on the OPG/RANKL ratio leading to promote resorption. d ACTH unchanged levels reflect the overall body health in both treated and control rats
remodeling, were increased in the OVX-steroid group. However, an increase in the insulin levels in the OVXsteroid rats serum was also detected (Fig. 5c). Furthermore, ACTH that regulates the glucocorticoid pathway and indicates the general condition of the animals showed no difference between the groups (Fig. 5d).
guidelines, Thomson et al. reported that rats, which were ovariectomized at the age of 12 weeks, showed after 52 weeks of ovariectomy a BMD of 0.145 g/cm2 and 15 g BMC in DEXA. In comparison, our model showed after 12 weeks of ovariectomy and steroid treatment a BMD of 0.16 g/cm2 and 9.3 g BMC. The more pronounced alterations in bone parameters in our model reflect the effects of the additional steroid treatment. Despite being known to reduce the mechanical properties of bone in rats, OVX alone did not lead to a comparable loss of bone density as seen in postmenopausal women. Therefore, additional treatments (i.e., dietary, drug, etc.) are necessary to induce a significant loss of bone mass in rats [31]. Nonetheless, the ovariectomized rats are still the most commonly utilized animal model for postmenopausal osteoporosis. However, OVX rats show two phases of bone loss. A 100 days after ovariectomy, a rapid loss of bone mass is seen, which is followed by a relative stabilization of bone mass on an osteopenic level. Further, 270 days post-ovariectomy the rat model comes into a phase of slow loss of cancellous bone mass. This reflects an imbalanced bone metabolism with predominance of bone loss, which is in its rate lower than that of the osteoporotic patients. Therefore, additional treatments shall be needed to recapitulate osteoporotic bone status of patients [31–33]. Additional treatment with corticosteroids has been reported to alter bone metabolism. The osteoporotic bone status was mainly caused by a decrease in bone formation with only minimal changes in bone resorption. Such an anabolic decrease is more comparable to osteoporotic patients than that of ovariectomized rat models. Further, animal models with steroid treatment combined with ovariectomy showed the clearest bone loss [31]. The rats treated in this study showed a Z score of -5.7 after 3M of treatment. Biomechanical competence and bone structural properties were significantly lower than the
Discussion The steroid-induced osteoporosis is one of the most important forms of secondary osteoporosis. Only few months after steroid treatment starts bone catabolism increases, and after few years about 30–50 % of patients show osteoporosis manifestation [28]. Many rat models of osteoporosis were reported either with estrogen deficiency resulting from bilateral ovariectomy alone or combined with diet or steroid treatment. This study used dexamethasone as a glucocorticoid to induce osteoporotic status in OVX rats. Nonetheless, this study is a part of trans-regional project concerned with detailed investigation of clinically relevant osteoporotic rat model. Unfortunately and due to animal welfare and ethical committee decisions the study had to utilize the reports which meticulously investigated bone alteration of the mature rat at 3M after OVX [29, 30] as recommended in the Food and Drug Administration (FDA) guidelines of animal models for osteoporosis as controls. However, bone parameters obtained through treatment in this study in lined with the decline of bone parameters indicated in the FDA guidelines. Wornski et al. [30] reported that rats which were ovariectomized at the age of 11 weeks showed a 13.2 % loss in trabecular bone volume (TBV) 5 weeks after ovariectomy. These animals also had a 10.2 % reduction in osteoblast surface (OBS) and 12 % increase in osteoclast surface (OCS). The OVX ? steroid animals presented in this study showed 20.07 % reduction of OBS and 15.11 % in OCS compared to the Sham. In the FDA
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Sham control. Investigated bone metabolism reflected a lower bone anabolism and a higher bone catabolism. To our knowledge, this is the first study clearly demonstrating that dexamethasone treatment in postmenopausal (ovariectomized) rats results in osteoporosis of the vertebral bone. This in lines with observations in patients where fractures occur more frequently in postmenopausal women treated with glucocorticoids [16, 17]. Previous studies reported contradictory results on glucocorticoid exposure to rats and showed either increased BMD or decreased BMD [34, 35]. Such fluctuation may have resulted from different factors like age of the animal and dose of the glucocorticoid. Nevertheless, clinical and experimental data from other animal species have clearly demonstrated that glucocorticoids cause a reduction in BMD and eventually osteoporosis [36]. In this experimental setup, we could show that dexamethasone significantly reduced BMD in young mature rats to a Z score of -5.7 in a comparison to age-matched healthy controls. Histological analysis referring to osteoclasts (TRAP) and osteoblasts (ALP) was performed to evaluate the effects of combined treatment on bone metabolism. Increased osteoclast numbers defined as TRAP-positive cells residing on the bone surface were seen in OVX-steroid rats, which in accordance with the osteoclasts were reported to play an important role in mediating bone loss caused by glucocorticoids [37, 38]. Glucocorticoids are known to increase the expression of M-CSF, which causes differentiation of pre-osteoclast to osteoclast [23, 39]. Moreover, dexamethasone in particular was reported to enhance the DNA binding activities of the transcription factors leading to accelerated osteoclast formation [40]. Such osteoclast-mediated bone resorption was exhibited in this study through the higher intensity of MMP9 in IHCs at M3 and its differences in signal localization in the OVXsteroid group. Furthermore, the fact that short-term treatment with dexamethasone primarily caused an increase in osteoclast numbers concords with the clinical observation that glucocorticoid-induced osteoporosis occurs in two phases: a rapid, early phase in which BMD is reduced, presumably due to excessive bone resorption, and a slower, progressive phase in which BMD declines [26]. The data provided here could show that the short-term treatment indeed recapitulate the rapid, early phase of glucocorticoidinduced osteoporosis observed in patients. Histomorphometrical analysis of ALP- and BGNstained area normalized to trabecular area showed lower ALP and BGN at the OVX-steroid rat groups at time point 3M, suggesting that dexamethasone treatment at this early phase affects bone anabolism. This agrees with previous studies which reported a diminished osteoblast function after glucocorticoids treatment [41]. Nonetheless, investigated markers of osteoblast function [42] showed no
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difference between the control and experimental RANKL, leptin and PTH levels in serum as well as OCN relative expression were not changed (Fig. S1 and Fig. S2). Furthermore, osteopontin (OPN) is a regulator of the mineralization process, expressed by osteoblasts in advance to mineral deposition [43], however, OPN is also an important extracellular matrix protein expressed by immature osteoblasts [44]. The higher OPN levels in the OVX-steroid rats at 3M suggest the presence of dysfunctional osteoblasts. Such observations are also seen in other bone diseases that show lower bone competence and BMD such as neurofibromatosis type 1 [45]. In the light of these findings, we investigated TNC as a bone matrix marker, which is a cell adhesive and anchorage molecule that responds in correlation with integrin to bone mechanical properties [46]. Indeed, we could show that intensity and distribution are affected within the bone matrix and that its relative expression is down regulated, which could have affected osteoblast maturation and mineralization [36, 47]. In support to the histological findings, lCT and DEXA data we found in compression analysis a significantly lower strength of vertebral bone in dexamethasone-treated rats, which indicates that the bone of these animals is susceptible to fracture. This is conclusive to clinical data in humans where even short-term treatment with glucocorticoids leads to a rapid bone loss and increase in vertebral fracture risk [41]. Intriguingly, TNC expression levels and IHC observations suggest that lower mechanical competence in the vertebral body of treated rats interact with ECM proteins. As TNC binds to integrin and is known to be regulated through mechanotransduction [46].Interestingly, this accords with the clinical data that shows a higher vertebral fracture risk due to rapid bone loss after shortterm treatment with glucocorticoids [41]. Furthermore, bone extracellular matrix comprising both collagenous and non-collagenous matrix proteins plays a major role in bone structural and functional properties. There are few experimental studies in rats investigating the osteoporotic effect of glucocorticoids. In contrast to the current results, in both sham-operated and parathyroidectomized male rats a treatment with a continuous rate infusion of dexamethasone (16.25 lg/kg/day) for 19 days resulted in a net effect favoring bone formation as reflected by increased bone mass assessed by histomorphometry [35]. Overall, there are conflicting results in the literature regarding the net effect of corticosteroids on bone metabolism. Treatment of male rats with 1 mg/kg/day methylprednisolone of 90 days duration resulted in a reduced bone strength and quality, interestingly, markers of bone metabolism were not assessed [48]. One reason for the discrepant results of various studies could be the fact that moderate doses of prednisolone resulted in increased bone stability in rats, whereas high doses had the opposite effect
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[49]. Therefore, dexamethasone was implemented in this study to avoid such fluctuating effects on the musculoskeletal system of the rat. Nevertheless, glucocorticoids direct and indirect influence on bone mineralization through affecting calcium absorption and resorption, indicating that glucocorticoid-induced reduction of bone formation is associated with a decrease in the mineral apposition rate [9]. Moreover, treatment with dexamethasone was associated with a decline of all markers of bone metabolism including OCN, TRAP5b and PTH, which has been also observed in humans [50]. Here, we show that the several crucial markers of dysregulated bone metabolism were not differentially expressed or rather biochemically altered through serological tests. However, important markers stand out, while ACTH insignificant change indicates that the treatment is not harmful for systemic functions of the body an increase of insulin was noted. Insulin is a regulator of glucose homeostasis in the body that regulates osteoblasts by decreasing osteoprotegerin (OPG) expression [51]. Subsequently, the OPG/RANKL ratio is decreased and resorption is promoted, which then lead to an increase in the release of undercarboxylated osteocalcin. Our findings of elevated undercarboxylated osteocalcin and insulin levels in serum suggest that glucosteroids, which are insulin resistant, are causing the insulin serum level to increase and finally promote resorption. Furthermore, the increased osteopontin level in the serum could also suggest a dysfunctional bone anabolism through immature osteoblasts [43, 44] and that macrophages are releasing osteopontin to facilitate the attachment of osteoblasts [52] in order to balance bone metabolism. In summary, our data showed that short-term treatment of postmenopausal (ovariectomized) Sprague–Dawley rats with dexamethasone results in an increased bone catabolism and decreased bone anabolism associated with misallocation of bone matrix proteins. Altogether the obtained data suggest that this animal model is suitable to further investigate the role of glucocorticoids treatment on bone metabolism mediated by bone matrix proteins such as integrins, and the implementation of such knowledge in counter therapy against risk of osteoporotic fractures. Furthermore, the current study is encouraging to carry the described treatment for longer period to investigate whether it will recapitulate the decline in bone loss reported in patients. Further, the authors recommend the investigating of extracellular matrix proteins in comparison with an OVX control as such data are not described in the FDA guide lines. Acknowledgments This study was solely funded by DFG, German Research Foundation (SFB/TRR 79, T1). The authors sincerely thank Mrs. Stengel A, Ms. Sparer S, Mrs. Bergen I and Mrs. Hartmann S
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None.
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