Experimental Gerontology 57 (2014) 114–121
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Molecular evidence of osteoblast dysfunction in elderly men with osteoporotic hip fractures☆ Ursula Föger-Samwald a,⁎, Janina M. Patsch a,b, Doris Schamall a, Afarin Alaghebandan a, Julia Deutschmann a, Sylvia Salem c, Mehdi Mousavi d, Peter Pietschmann a a
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Immunology and Infectiology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria Department of Radiodiagnostics, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria Department of Orthopaedics, St. Vincent Hospital Vienna, Stumpergasse 13, A-1060 Vienna, Austria d Department of Trauma Surgery, Danube Hospital, Langobardenstrasse 122, A-1220 Vienna, Austria b c
a r t i c l e
i n f o
Article history: Received 11 March 2014 Received in revised form 28 April 2014 Accepted 20 May 2014 Available online 24 May 2014 Section Editor: Werner Zwerschke Keywords: Osteoporosis Hip fracture Gene expression Bone formation
a b s t r a c t Osteoporosis is extremely frequent in post-menopausal women; nevertheless, osteoporosis in men is also a severe and frequently occurring but often underestimated disease. Increasing evidence links bone loss in male idiopathic osteoporosis and age related osteoporosis to osteoblast dysfunction rather than increased osteoclast activity as seen in postmenopausal osteoporosis. The aim of this study was to investigate gene expression of osteoblast related genes and of bone architecture in bone samples derived from elderly osteoporotic men with hip fractures (OP) in comparison to bone samples from age matched men with osteoarthritis of the hip (OA). Femoral heads and adjacent neck tissue were collected from 12 men with low-trauma hip fractures and consecutive surgical hip replacement. Bone samples of age matched patients undergoing hip replacement due to osteoarthritis served as controls. One half of the bone samples was subjected to RNA extraction, reverse transcription, and realtime polymerase chain reactions. The second half of the bone samples was analyzed by static histomorphometry. From each half samples from four different regions, the central and subcortical region of the femoral head and neck, were analyzed. OP patients displayed a significantly decreased RUNX2, Osterix and SOST expression compared to OA patients. Major microstructural changes in OP bone were seen in the subcortical region of the neck and were characterized by a significant decrease of bone volume, and a significant increase of trabecular separation. In conclusion, decreased local gene expression of RUNX2 and Osterix in men with hip fractures strongly supports the concept of osteoblast dysfunction in male osteoporosis. Major microstructural changes in the trabecular structure associated with osteoporotic hip fractures in men are localized in the subcortical region of the femoral neck. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Primary osteoporosis is an age related multifactorial disease characterized by impaired bone mass and bone microarchitecture leading to decreased bone strength and consequently to an increased risk of fragility fractures (Rachner et al., 2011). Bone loss associated with aging starts earlier and is more pronounced in women than in men (Clarke and Khosla, 2010). Nevertheless, 1 in 8 men older than 50 years will ☆ Funding sources: This work was supported by the Austrian Federal Bank (Grant No. 12544 to PP). ⁎ Corresponding author at: Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria, Währinger Gürtel 18-20, 1090 Wien, Austria. E-mail addresses: [email protected] (U. Föger-Samwald), [email protected] (J.M. Patsch), [email protected] (D. Schamall), afi[email protected] (A. Alaghebandan), [email protected] (J. Deutschmann), [email protected] (S. Salem), [email protected] (M. Mousavi), [email protected] (P. Pietschmann).
http://dx.doi.org/10.1016/j.exger.2014.05.014 0531-5565/© 2014 Elsevier Inc. All rights reserved.
experience an osteoporotic fracture (Melton et al., 1992) and 30% of all osteoporotic hip fractures worldwide occur in men (Johnell and Kanis, 2006). With an aging population male osteoporosis will become an even increasing socioeconomic burden. Moreover, fracture-related morbidity and mortality is higher in men than in women (Khosla, 2010). In 50% of men with osteoporosis a secondary cause such as an underlying disorder or drug-induced bone loss can be identified (Gielen et al., 2011). In the absence of secondary causes primary osteoporosis is diagnosed and termed ‘idiopathic osteoporosis’ in young men and ‘age related osteoporosis’ in men older than 70 years (Gielen et al., 2011). At the tissue level, the pathophysiology of osteoporosis is characterized by loss of bone material due to a negatively balanced bone remodeling process favoring bone resorption over bone formation. Bone resorption exceeds bone formation induced either by an increased activity of osteoclasts or a decreased activity of osteoblasts. Identifying excessive bone resorption by osteoclasts as the key mechanism driving rapid postmenopausal bone loss in women, the main focus of osteoporosis research has been on osteoclast dysfunction. Hence, standard therapies available for the
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treatment of osteoporosis are antiresorptive agents targeting osteoclastogenesis and mature osteoclasts (Rachner et al., 2011). However, several studies based on histomorphometry (Ciria-Recasens et al., 2005; Fratzl-Zelman et al., 2011; Johansson et al., 1997; Kurland et al., 1997; Zerwekh et al., 1992) and studies with cell cultures derived from bone tissues from osteoporotic patients (Hu et al., 2013; Marie et al., 1991; Pernow et al., 2006; Ruiz-Gaspa et al., 2010) provide evidence for an impact of osteoblast dysfunction on the pathophysiology of male osteoporosis. In previous work we reported that, in comparison to healthy controls, local gene expression of the major osteoblast transcription factor RUNX2, the Wnt signaling pathway members WNT10B and SOST and the osteoclast regulating gene RANKL is significantly decreased in iliac crest biopsies of men with idiopathic osteoporosis (Patsch et al., 2011). This study supports the concept of decreased bone formation as the underlying pathophysiological mechanism of male osteoporosis and was the first local gene expression study in an exclusively male idiopathic study population. Previous local gene expression analyses of osteoporotic bone tissue predominantly were performed with female study populations or included both, men and women. However, as gene expression patterns are expected to be influenced by various factors including sex and age, studies with carefully selected study cohorts will help us to get a more detailed understanding on pathophysiological mechanism underlying male and female osteoporosis. The aim of the present study was to investigate local gene expression of osteoblast related genes in femoral heads of elderly men with osteoporotic hip fractures in comparison to men with osteoarthritis. Furthermore, histomorphometric characteristics of the collected bone samples were related to the local expression of the investigated genes. Extending the concept of osteoblast dysfunction to male ‘age related osteoporosis’, we hypothesized to find a decreased local expression of osteoblast related genes in bone samples from men with osteoporotic hip fractures. 2. Patients and methods 2.1. Human bone tissue samples Femoral heads and the adjacent femoral neck were obtained from male patients undergoing total hip arthroplasty surgery due to fragility fractures of the hip (OP) and from male patients undergoing total hip arthroplasty surgery due to osteoarthritis of the hip (OA). Patients were recruited at the Department of Trauma Surgery, Danube Hospital, Vienna, Austria and the Department of Orthopaedics, St Vincent Hospital, Vienna, Austria respectively. During preoperative preparation patients were asked to participate in this study and collaborating physicians checked for predefined inclusion and exclusion criteria. Exclusion criteria for the OP group included hip fractures caused by high energy trauma (e.g. car accidents), alcohol abuse, preoperative lab findings giving evidence for severe renal or hepatic failure, or other major chronic diseases. Moreover, patients with clinical signs or established diagnosis of liver cirrhosis, hyperthyroidism, hypogonadism, any malignancy within the last five years or other severe pathologies were excluded from enrolment. Exclusion criteria for the OA group were fragility fractures, clinical diagnosis of osteoporosis, or the previous use of specific antiosteoporotic drugs other than vitamin D and calcium supplements. Inclusion criteria for both groups, OP and OA, were a minimum age of 70 years and a signed informed consent. The study was approved by the local ethic committees.
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submerged in 4% paraformaldehyde, subsequently in 70% ethanol and then stored at 4 °C. All analyses were performed in four different regions of the bone samples. The four regions were defined as peripheral region of the femoral head (pHd), central region of the femoral head (cHd), peripheral region of the femoral neck (pN), and central region of the femoral neck (cN) (Fig. 1). Samples from the central as well as the peripheral region included only trabecular bone. 2.3. Histomorphometry Cubes (7 mm × 7 mm × 7 mm) were cut out from the above described regions of each bone sample with a bone saw and processed for histomorphometry as described previously (Patsch et al., 2011). Briefly, all bone cubes were fixed with ethanol, dehydrated in ascending ethanol series, and embedded in polymethyl methacrylate (PMMA). Histomorphometry on Goldner stained sections was performed using the semiautomatic OsteoMeasure histomorphometry system (Osteometrics Inc.; Atlanta, GE). Within the trabecular compartment of each section nine random fields of view were imaged with an ×2 objective. Histomorphometric structure parameters including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), and bone surface density (BS/TV) were analyzed according to the standards of the American Society of Bone and Mineral Research (Dempster et al., 2013). 2.4. RNA extraction Sample preperation for RNA extraction was performed as described previously (Patsch et al., 2011). Briefly, a small cube (approximately 5 mm × 5 mm × 5 mm) was cut out from the above described regions of each bone sample using RNAse-free instruments. The bone cube, containing bone tissue and bone marrow, was flash frozen in liquid nitrogen and together with two small steel beads placed in a grinding mill (3 min, 30 Hz) for tissue homogenization. Total RNA was extracted using TRIzol™ reagent (Invitrogen, Carlsbad, CA), chloroform extraction and isopropanol precipitation according to the manufacturer's protocol. RNA quality and quantity were checked by photometry at 260 and 280 nm. 2.5. Reverse Transcription and Quantitative PCR cDNA was synthesized from 1 μg of total RNA using a cDNA synthesis kit (High Capacity cDNA Reverse Transcripton Kit; Applied Biosystems,
pHd
pN cHd cN pN
2.2. Sample preperation To facilitate histomorphometric, as well as gene expression analysis, femoral heads and the adjacent femoral necks were cut into two halves right after hip replacement surgery. One half selected for subsequent gene expression analysis was submerged in RNA-Later™ (Ambion, Warrington, UK) and stored as instructed by the manufacturer. The second half selected for subsequent histomorphometric analysis was
Fig. 1. Schematic diagram of the proximal femur and the sites from which samples were removed.
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Foster City, CA). Real Time PCR was performed with 100 ng of each sample for the genes RUNX2, RANKL (TNFSF11), OPG (TNFRSF1), Osterix (SP7), Osteocalcin (BGLAP), Sclerostin (SOST) and GAPDH using predesigned TaqMan Gene Expression Assays (Applied Biosystems; Hs00231692_m1, Hs00243519_m1, Hs00171068_m1, Hs00541729_m1, Hs00609452_g1, Hs00228830_m1, and Hs99999905_m1 respectively). Reactions were performed in triplicates and each reaction was performed with 9 μl cDNA diluted in water, 1 μl TaqMan Gene Expression Assay and 10 μl mastermix buffer (TaqMan Universal PCR Mastermix, Applied Biosystems). Cycler conditions used to perform real time PCR reactions on a thermal cycler (Abi Prism Sequence Detection System 7900HT; Applied Biosystems) were 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. All experiments were normalized to the housekeeping gene GAPDH. Results were calculated applying the ΔΔCt method and are presented as fold increases relative to GAPDH expression (mean Ct values: RUNX2: 28.9 ± 2.6, Osterix: 31.6 ± 2.9, SOST: 30.8 ± 2.8, Osteocalcin: 24.5 ± 3.8, RANKL: 32.5 ± 3.0, OPG: 30.7 ± 2.4).
3.3. Gene expression
2.6. Statistical methods
3.3.1. Differences in gene expression between OP and OA patients First we compared the mean expression of all regions analyzed in each patient between the two groups OA and OP (Fig. 3). The expression of RUNX2, Osterix and SOST was significantly decreased in bone samples from OP patients compared to OA patients. RANKL, OPG, and Osteocalcin expression did not differ between the two groups. Also the ratio of the expression of the two antagonizing molecules RANKL and OPG did not differ between the two groups. To obtain more insights into spatial patterns of differences in expression levels, we compared the expression of RUNX2, Osterix, and SOST in the four regions analyzed between OA and OP patients (Fig. 4). The expression of RUNX2 was significantly decreased in the central region of the head and the central region of the neck in OP patients compared to OA patients. Osterix expression was significantly decreased in both the peripheral and the central region of the head and SOST expression in the central region of the head and both the peripheral and central region of the neck in OP patients compared to OA patients. Spatial differences are only given for a limited set of genes as the RNA extracted from the bone samples was in some cases not sufficient to analyze the expression of the whole set of genes.
Data are reported either as median values and the range or as means ± standard deviation. Differences in gene expression levels and histomorphometric indices between patients with OP and OA were assessed using Mann–Whitney tests (SPSS Statistics 21; SPSS, Inc., Chicago, IL, USA). For each parameter either the different regions of each bone sample as described above were analyzed separately and/or the mean values of the regions were compared between OP and OA patients. For correlation analyses, Spearman coefficients were calculated. The critical value for statistical significance was set at p b 0.05.
3.3.2. Differences in gene expression between the femoral head and the femoral neck To obtain more insights into spatial differences in expression levels, we compared the expression of RUNX2, Osterix, and SOST between the femoral head and the femoral neck in both groups of patients. In bone samples from OA patients, Osterix and SOST expression were significantly higher in the femoral head compared to the femoral neck. In bone samples from OP patients, the expression of the four genes investigated did not differ between the femoral head and the femoral neck (Fig. 5).
3. Results 3.1. Patients Twelve patients undergoing total hip arthroplasty surgery due to fragility fractures of the hip (OP) and ten male patients undergoing total hip arthroplasty surgery due to osteoarthritis of the hip (OA) were included in this study. Gene expression was analyzed in bone samples from nine OP patients (mean age 81 ± 7 years) and nine OA patients (mean age 80 ± 7 years). Bone microarchitecture was analyzed in bone samples from seven OP patients (mean age 83 ± 6 years) and ten OA patients (mean age 83 ± 6 years). Some samples were excluded from gene expression studies as we failed to isolate sufficient amount of RNA. Due to technical reasons, i.e. limited perfusion or crush artifacts, some samples were excluded from microstructural analysis. Exclusions were randomly distributed compared to the original samples and therefore did not change the study population.
3.2. Histomorphometry Major microarchitectural changes as analyzed by static histomorphometry were seen in the peripheral region of the neck. BV/ TV was significantly decreased and Tb.Sp was significantly increased in the peripheral region of the neck in bone samples from OP patients compared to OA patients. Tb.N, Tb.Th, and BS/TV (data not show) did not differ between the two groups in the peripheral region of the neck. For the other regions analyzed no significant differences between the two groups were seen for all parameters. Comparing the means of all 4 regions analyzed in one patient, BV/TV and Tb.Th were significantly decreased in bone samples from OP patients compared to OA patients. Histomorphometric data are shown in Fig. 2. Cellular components were only rarely observed in our sections and therefore were not assessed.
3.3.3. Correlation analysis Considering all bone samples, RUNX2 expression correlated significantly with the expression of Osterix (r = 0.818, p b 0.001), SOST (r = 0.702, p b 0.001), RANKL (r = 0.773, p = 0.005), and Osteocalcin (r = 0.758, p = 0.011). Moreover, Osterix expression correlated significantly with the expression of SOST (r = 0.750 p b 0.001). We also correlated gene expression levels with microstructural indices. RUNX2 expression correlated positively with Tb.Nr (r = 0.350, p = 0.046). Osterix expression correlated positively with BV/TV (r = 0.459, p = 0.007) and Tb.Th (r = 0.403, p = 0.02) and correlated negatively with Tb.Sp (r = −0.386, p = 0.027) and BS/TV (r = −0.86, p = 0.035). SOST expression was positively associated with BV/TV (r = 0467, p = 0.007) and Tb.Nr (r = 0.438, p = 0.012) and was negatively associated with Tb.Sp (r = −0.466, p = 0.007). 4. Discussion Gene expression patterns in osteoporotic bone tissues are expected to be influenced by various factors including sex and age. Therefore, studies with carefully selected study cohorts will help us to get a more detailed understanding on pathophysiological mechanism underlying male and female osteoporosis. With the aim of investigating local gene expression patterns and microstructural alterations associated with age related osteoporosis in men, we found a significantly decreased local expression of the osteoblast transcription factors RUNX2 and Osterix, and of the Wnt signaling pathway inhibitor SOST in osteoporotic bone samples compared to osteoarthritic bone samples. Microstructural changes in osteoporotic bone samples were characterized by decreased BV/TV and Tb.Th and increased Tb.Sp. Major microarchitectural differences were seen in the peripheral region of the neck. Increasing evidence links bone loss in male idiopathic osteoporosis and in age related osteoporosis to osteoblast dysfunction. Osteblasts, the cells synthesizing and mineralizing bone during bone formation and the life long process of bone remodeling, arise from mesenchymal
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OA OP Fig. 2. Histomorphometric structure parameters including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) in bone samples from the peripheral region of the femoral head (OA: n = 7, OP: n = 10), the central region of the femoral head (OA: n = 6, OP: n = 10), the peripheral region of the femoral neck (OA: n = 7, OP: n = 9), the central region of the femoral neck (OA: n = 6, OP: n = 7) and as mean value of all four regions (total) (OA: n = 7, OP: n = 10). Bone samples were taken either from men with osteoarthritis (OA) or with osteoporosis (OP). Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
stem cells (MSCs). Benisch et al. (2012) have shown that the transcriptome of hMSCs populations derived from elderly patients suffering from age related osteoporosis significantly differs from hMSCs populations derived from non osteoporotic donors (Benisch et al., 2012). Major transcription factors required for the differentiation of osteoblastic cells are RUNX2 and Osterix, which is thought to act downstream of RUNX2 (Katagiri and Takahashi, 2002; Nakashima et al., 2002). An indispensable role of both RUNX2 and Osterix for osteoblast differentiation was impressively demonstrated by the entire absence of osteoblasts after homozygous deletion of Runx2 and Osterix in mice (Komori et al., 1997; Nakashima et al., 2002). In our study, RUNX2 and Osterix expression was significantly decreased in OP patients compared to OA patients, and therefore give evidence for disturbances in osteoblast differentiation in elderly men with osteoporotic hip fractures. Decreased local expression of RUNX2 and Osterix in osteoporotic bone tissue compared to osteoarthritic and healthy control bone was also reported by others (Dragojevic et al., 2011, 2013; Giner et al., 2013) and was previously shown by our group in bone samples from men with idiopathic osteoporosis (Patsch et al., 2011). However this is, to the best of our knowledge, the first study investigating local gene expression in an exclusively male study population with age related osteoporosis. The Wnt/β-catenin signaling pathway (canonical Wnt pathway) has been established to be a master regulator of osteogenesis influencing the differentiation of mesenchymal stem cells to osteoblasts rather than chondrocytes or adipocytes (Kang et al., 2007; Rossini et al., 2013). Sclerostin, the protein encoded by SOST, antagonizes canonical WNT signaling by perturbing the interaction of Wnt ligands with their receptors. Sclerostin is expressed primarily by bone cells and specifically by osteocytes (van Bezooijen et al., 2004; Winkler et al., 2003). By antagonizing canonical WNT signaling, Sclerostin inhibits proliferation and differentiation and stimulates apoptosis in osteoblasts (Monroe et al., 2012). The clinical relevance of Sclerostin as a negative regulator of
bone mass has been impressively demonstrated by two bone sclerosing disorders, van Buechem disease and sclerostosis. These diseases are caused by specific loss of function mutations in the SOST gene and are associated with a high bone mass phenotype and increased osteoblast activity (de Vernejoul and Kornak, 2010). A possible role for Sclerostin in osteoporotic bone loss is suggested by the finding of increased serum levels in postmenopausal women (Mirza et al., 2010) and a positive association of serum Sclerostin levels with the occurrence of osteoporotic fractures (Ardawi et al., 2012). Furthermore, SOST expression was shown to be significantly higher in MSCs from donors with age related osteoporosis compared to MSCs from age matched non osteoporotic donors (Benisch et al., 2012). The authors therefore suggest an auto-inhibitory effect of Sclerostin on proliferation and self-renewal of MSCs leading to reduced bone formation in primary osteoporosis (Benisch et al., 2012). However, local expression of SOST was significantly lower in our study population of elderly men with osteoporotic hip fractures and was also significantly lower in men with idiopathic osteoporosis (Patsch et al., 2011). A decreased expression of SOST in bone tissue of elderly patients with osteoporosis is in line with our recent finding of significantly lower serum Sclerostin levels in a study cohort of geriatric patients with hip fractures compared to age matched controls (Dovjak et al., in press). As speculated by Patsch et al. (2011), the observed reduction of SOST might be due to reduced bone volume since osteocytes are the main source of sclerostin (Patsch et al., 2011) and SOST expression correlated positively with BV/TV and Tb.Nr and correlated negatively with Tb.Sp. Delgado-Calle et al. (2011) reported a reduced proportion of lacunae occupied by osteocytes in fractured bone compared to osteoarthritic and normal bone. Concomitantly the proportion of caspase-positive lacunae as a marker of apoptosis was significantly increased (Delgado-Calle et al., 2011). Moreover, the proportion of osteocyte-occupied lacunae that stained positively for Sclerostin was lower in fractured bone (Delgado-Calle et al., 2011). Taken
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OA OP Fig. 3. Relative mRNA expression (ΔΔCt) of RUNX2 (OA: n = 9, OP: n = 9), Osterix (OA: n = 9, OP: n = 9), SOST (OA: n = 8, OP: n = 9), Osteocalcin (OA: n = 5, OP: n = 6), RANKL (OA: n = 7, OP: n = 8), OPG (OA: n = 6, OP: n = 7), and the ration of RANKL/OPG (OA: n = 6, OP: n = 7) in osteoarthritic (OA) and osteoporotic (OP) bone samples. Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
together, these data may suggest that decreased local expression of SOST is a consequence of low bone mass and/or increased osteocyte apoptosis. In addition, a reduced expression of SOST in osteocytes might contribute to a reduced expression of SOST in fractured bone. Regarding the antiproliverative and proapoptotic effect of SOST expression on
osteoblast cultures, a reduction of SOST expression concomitantly with a reduction of the osteoblast transcription factors RUNX2 and Osterix seems contradictory. However, SOST is only one of several molecules regulating the Wnt signaling pathway. Thus, our data might suggest that another molecule than Sclerostin, i.e. DKK1, plays the main
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OA OP Fig. 4. Relative mRNA expression (ΔΔCt) of RUNX2, Osterix, and SOST in bone samples from the peripheral region of the femoral head, the central region of the femoral head, the peripheral region of the femoral neck and the central region of the femoral neck. Bone samples were taken either from men with osteoarthritis (OA) or with osteoporosis (OP). The corresponding number of samples (n) included in each comparison is given in the rectangles below the plots. Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
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head neck Fig. 5. Comparison of relative mRNA expression (ΔΔCt) of RUNX2, Osterix, and SOST in bone samples from the femoral head and the femoral neck. Bone samples were taken either from men with osteoarthritis (OA) or with osteoporosis (OP). The corresponding number of samples (n) included in each comparison is given in the rectangles below the plots. Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
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regulatory function of Wnt signaling in male age related osteoporosis. But, as previously speculated by Patsch et al. (2011), a decreased expression of SOST could also indicate an autoregulatory rescue mechanism. Increased bone resorption by osteoclasts is the key mechanism driving bone loss in early postmenopausal women. RANKL is a member of the tumor necrosis factor (TNF) ligand superfamily and is best known for its essential role in regulating the differentiation and activation of osteoclasts. Binding of RANKL to its signaling receptor RANK on osteoclasts triggers the activation of signaling pathways pivotal for osteoclastogenesis. Acting as a decoy receptor, osteoprotegerin (OPG) prevents binding of RANKL to RANK and thereby counteracts its osteogenic effects. In our study no significant differences for RANKL and OPG expression levels were detected in OP and OA bone. Moreover, the ratio of RANKL to OPG expression as an important determinant of osteoclastic stimulation did not differ between these two groups. This is in contrast to other studies addressing local RANKL and OPG mRNA expression levels in osteoporotic bone tissues (Abdallah et al., 2005; Dragojevic et al., 2013; Logar et al., 2007; Tsangari et al., 2004; Zupan et al., 2012). Dragojevic et al. (2013), Zupan et. al (2012), and Logar et al. (2007) found increased RANKL and RANKL/OPG expression levels in OP bone compared to OA bone (Dragojevic et al., 2013; Logar et al., 2007; Zupan et al., 2012). In the study of Dragojevic et al. (2013), OPG levels were significantly decreased and RANKL/OPG expression was significantly lower when comparing osteoporotic bone to non osteoporotic autopsy samples (Dragojevic et al., 2013). Tsangari et al. (2004) compared expression levels in osteoporotic bone to non osteoporotic autopsy samples and also found an increased RANKL/OPG expression level in osteoporotic bone (Tsangari et al., 2004). However, the aforementioned studies were done in study populations comprising both men and women, typically including more women than men. Abdallah et al. (2005) compared mRNA gene expression in iliac crest bone biopsies from patients with osteoporotic hip fractures or osteoarthritis in an exclusively female study population (Abdallah et al., 2005). They also found significantly increased RANKL/OPG expression levels in osteoporotic bone. Decreased RANKL expression and RANKL/OPG expression levels were previously reported by our group in bone samples from men with idiopathic osteoporosis (Patsch et al., 2011). In line with this, in our study cohort a trend towards a decreased expression of RANKL in osteoporotic bone samples was observed. As osteocytes are major sources of RANKL, similar to SOST, decreased expression of RANKL might be related to a reduced bone volume. However, as in our study mean Ct values for RANKL expression were relatively high, these data require further confirmation. Taken together, local RANKL/OPG expression as determinant of bone resorption was, in contrast to postmenopausal and age related osteoporosis in women, not increased in our male age related osteoporosis study cohort, suggesting pathophysiological differences between male and female age related osteoporosis. Based on the observation of the rare occurrence of OA in the femoral heads of fractured hips and of osteoporotic hip fractures in OA patients, OA of the hip and osteoporotic hip fractures have long been postulated to be inversely related (Dequeker et al., 2003). Though, when studied longitudinally and not cross-sectionally, the relationship between OA and OP is controversial (Im and Kim, 2014). OA is regarded as a whole joint disease in which articular and periarticular tissues including the bone compartment are involved (Dequeker et al., 2003). Major changes are found in the subchondral bone and are characterized by increased bone density, subchondral bone sclerosis, and osteophytes (Henrotin et al., 2012). To get a more comprehensive picture on differences between OP and OA bone, we compared structural parameters as well as gene expression of the three osteoblast related genes RUNX2, Osterix, and SOST at four different regions of the femoral head and femoral neck. Comparing the expression of the aforementioned genes between the femoral head and femoral neck, expression of Osterix and SOST was significantly higher in the femoral head of OA patients, whereas in OP patients no difference between the femoral head and the femoral
neck could be observed. Gene expression therefore seems to be spatially more homogeneous in OP bone compared to OA bone, probably reflecting major structural changes in the subchondral bone of OA patients. A significantly decreased or a trend towards a decreased expression of RUNX2, Osterix, and SOST in OP bone compared to OA bone was not restricted to the femoral head but could be observed in all four different regions. Age dependent changes in the trabecular microstructure of the proximal femur have been shown to be region and sex dependent (Cui et al., 2008; Djuric et al., 2010). Thus, it is tempting to assume the same for OP and OA associated microstructural changes. Trabecular microstructure in OP bone compared to healthy bone or OA bone was analyzed in different subregions of the proximal femur including the femoral neck, the subchondral bone, the intertrochanteric bone or iliac crest biopsies (Boutroy et al., 2011; Hordon and Peacock, 1990; Li et al., 2012; Milovanovic et al., 2012; Patsch et al., 2011; Uitewaal et al., 1987; Zupan et al., 2013). However, to the best of our knowledge, this is the first study, comparing male OP and OA bone samples regarding trabecular microstructure in the femoral head and the femoral neck concomitantly. Structural differences between OA and OP bone that we observed in our bone samples resemble these of previous studies; though in the femoral head they did not reach statistical significance. This might be due to the smaller sample size investigated in our study. In contrast to the femoral head, in the peripheral region of the femoral neck BV/TV was significantly decreased and Tb.Sp was significantly increased in OP bone compared to OA bone. We can conclude from these data that structural differences between OP and OA bone are more pronounced in the femoral neck than in the femoral head. A major strength of our study is the diligent selection of study participants including only men older than 70 years and allowing us to identify gene expression patterns specific for men with age related osteoporosis. A limitation of this study is the comparison of bone samples from men with osteoporotic hip fractures to bone samples from men with osteoarthritis of the hip but not to healthy controls; nevertheless, bone tissue from healthy aged subjects is very difficult to obtain. However, by analyzing gene expression and microstructure at four different and clearly defined regions near the subchondral bone and distal from the subchondral bone, a potential confounder by structural and molecular changes near the affected joint was minimized. Additionally, this experimental setup enabled us to minimize a potential confounder from molecular changes in close proximity to the site of fracture associated with the inflammatory response that initiates fracture healing. Another limitation of this study is that it lacks cellular analysis. Gene expression was normalized to the housekeeping gene GAPDH. GAPDH expression did not differ between the two groups investigated (comparison of Ct values: p = 0.71). We opted for the use of only one housekeeping gene as osteoporosis and osteoarthritis are slowly developing pathologies that are not thought to be associated with excessive proliferative events and the amount of RNA available for each sample was limited. We are aware of the relatively low number of study participants. Despite this, several differences in gene expression and microstructural indices were statistically significant. In conclusion, our data give evidence for osteoblast dysfunction in age related male osteoporosis that, in contrast to postmenopausal osteoporosis and age related osteoporosis in women, is not accompanied by an increased activity of osteoclasts.
Conflicts of interest PP has received research support and/or honoraria from Amgen Gmbh, Eli Lilly Gmbh, Fresenius Kabi Austria Gmbh, Merck, Sharp and Dohme Gmbh, Novartis Parma, Nycomed Pharma, Roche Austria, Servier Austria, Shire, Sinopharm and Sanofi-Aventis. JMP has received speaker honoraria from Amgen. All the other authors state that they have no conflicts of interest.
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Acknowledgments We are grateful to Mrs Katharina Wahl for expert technical support and to Dr. Doris Moser for providing a bone saw. We further acknowledge the contribution of Philipp Becker (Orthopaedic Hospital Speising, Vienna) and of Lucia Hornek (Danube Hospital, Vienna). This research was supported by the Austrian Federal Bank (Grant No. 12544 to PP). References Abdallah, B.M., Stilgren, L.S., Nissen, N., Kassem, M., Jorgensen, H.R., Abrahamsen, B., 2005. Increased RANKL/OPG mRNA ratio in iliac bone biopsies from women with hip fractures. Calcif. Tissue Int. 76, 90–97. Ardawi, M.S., Rouzi, A.A., Al-Sibiani, S.A., Al-Senani, N.S., Qari, M.H., Mousa, S.A., 2012. High serum sclerostin predicts the occurrence of osteoporotic fractures in postmenopausal women: the Center of Excellence for Osteoporosis Research Study. J. Bone Miner. Res. 27, 2592–2602. 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Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R.T., Gao, Y.H., Inada, M., Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S., Kishimoto, T., 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755–764. Kurland, E.S., Rosen, C.J., Cosman, F., McMahon, D., Chan, F., Shane, E., Lindsay, R., Dempster, D., Bilezikian, J.P., 1997. Insulin-like growth factor-I in men with idiopathic osteoporosis. J. Clin. Endocrinol. Metab. 82, 2799–2805. Li, Z.C., Dai, L.Y., Jiang, L.S., Qiu, S., 2012. Difference in subchondral cancellous bone between postmenopausal women with hip osteoarthritis and osteoporotic fracture: implication for fatigue microdamage, bone microarchitecture, and biomechanical properties. Arthritis Rheum. 64, 3955–3962. Logar, D.B., Komadina, R., Prezelj, J., Ostanek, B., Trost, Z., Marc, J., 2007. 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Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Molecular evidence of osteoblast dysfunction in elderly men with osteoporotic hip fractures☆ Ursula Föger-Samwald a,⁎, Janina M. Patsch a,b, Doris Schamall a, Afarin Alaghebandan a, Julia Deutschmann a, Sylvia Salem c, Mehdi Mousavi d, Peter Pietschmann a a
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Immunology and Infectiology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria Department of Radiodiagnostics, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria Department of Orthopaedics, St. Vincent Hospital Vienna, Stumpergasse 13, A-1060 Vienna, Austria d Department of Trauma Surgery, Danube Hospital, Langobardenstrasse 122, A-1220 Vienna, Austria b c
a r t i c l e
i n f o
Article history: Received 11 March 2014 Received in revised form 28 April 2014 Accepted 20 May 2014 Available online 24 May 2014 Section Editor: Werner Zwerschke Keywords: Osteoporosis Hip fracture Gene expression Bone formation
a b s t r a c t Osteoporosis is extremely frequent in post-menopausal women; nevertheless, osteoporosis in men is also a severe and frequently occurring but often underestimated disease. Increasing evidence links bone loss in male idiopathic osteoporosis and age related osteoporosis to osteoblast dysfunction rather than increased osteoclast activity as seen in postmenopausal osteoporosis. The aim of this study was to investigate gene expression of osteoblast related genes and of bone architecture in bone samples derived from elderly osteoporotic men with hip fractures (OP) in comparison to bone samples from age matched men with osteoarthritis of the hip (OA). Femoral heads and adjacent neck tissue were collected from 12 men with low-trauma hip fractures and consecutive surgical hip replacement. Bone samples of age matched patients undergoing hip replacement due to osteoarthritis served as controls. One half of the bone samples was subjected to RNA extraction, reverse transcription, and realtime polymerase chain reactions. The second half of the bone samples was analyzed by static histomorphometry. From each half samples from four different regions, the central and subcortical region of the femoral head and neck, were analyzed. OP patients displayed a significantly decreased RUNX2, Osterix and SOST expression compared to OA patients. Major microstructural changes in OP bone were seen in the subcortical region of the neck and were characterized by a significant decrease of bone volume, and a significant increase of trabecular separation. In conclusion, decreased local gene expression of RUNX2 and Osterix in men with hip fractures strongly supports the concept of osteoblast dysfunction in male osteoporosis. Major microstructural changes in the trabecular structure associated with osteoporotic hip fractures in men are localized in the subcortical region of the femoral neck. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Primary osteoporosis is an age related multifactorial disease characterized by impaired bone mass and bone microarchitecture leading to decreased bone strength and consequently to an increased risk of fragility fractures (Rachner et al., 2011). Bone loss associated with aging starts earlier and is more pronounced in women than in men (Clarke and Khosla, 2010). Nevertheless, 1 in 8 men older than 50 years will ☆ Funding sources: This work was supported by the Austrian Federal Bank (Grant No. 12544 to PP). ⁎ Corresponding author at: Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria, Währinger Gürtel 18-20, 1090 Wien, Austria. E-mail addresses: [email protected] (U. Föger-Samwald), [email protected] (J.M. Patsch), [email protected] (D. Schamall), afi[email protected] (A. Alaghebandan), [email protected] (J. Deutschmann), [email protected] (S. Salem), [email protected] (M. Mousavi), [email protected] (P. Pietschmann).
http://dx.doi.org/10.1016/j.exger.2014.05.014 0531-5565/© 2014 Elsevier Inc. All rights reserved.
experience an osteoporotic fracture (Melton et al., 1992) and 30% of all osteoporotic hip fractures worldwide occur in men (Johnell and Kanis, 2006). With an aging population male osteoporosis will become an even increasing socioeconomic burden. Moreover, fracture-related morbidity and mortality is higher in men than in women (Khosla, 2010). In 50% of men with osteoporosis a secondary cause such as an underlying disorder or drug-induced bone loss can be identified (Gielen et al., 2011). In the absence of secondary causes primary osteoporosis is diagnosed and termed ‘idiopathic osteoporosis’ in young men and ‘age related osteoporosis’ in men older than 70 years (Gielen et al., 2011). At the tissue level, the pathophysiology of osteoporosis is characterized by loss of bone material due to a negatively balanced bone remodeling process favoring bone resorption over bone formation. Bone resorption exceeds bone formation induced either by an increased activity of osteoclasts or a decreased activity of osteoblasts. Identifying excessive bone resorption by osteoclasts as the key mechanism driving rapid postmenopausal bone loss in women, the main focus of osteoporosis research has been on osteoclast dysfunction. Hence, standard therapies available for the
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treatment of osteoporosis are antiresorptive agents targeting osteoclastogenesis and mature osteoclasts (Rachner et al., 2011). However, several studies based on histomorphometry (Ciria-Recasens et al., 2005; Fratzl-Zelman et al., 2011; Johansson et al., 1997; Kurland et al., 1997; Zerwekh et al., 1992) and studies with cell cultures derived from bone tissues from osteoporotic patients (Hu et al., 2013; Marie et al., 1991; Pernow et al., 2006; Ruiz-Gaspa et al., 2010) provide evidence for an impact of osteoblast dysfunction on the pathophysiology of male osteoporosis. In previous work we reported that, in comparison to healthy controls, local gene expression of the major osteoblast transcription factor RUNX2, the Wnt signaling pathway members WNT10B and SOST and the osteoclast regulating gene RANKL is significantly decreased in iliac crest biopsies of men with idiopathic osteoporosis (Patsch et al., 2011). This study supports the concept of decreased bone formation as the underlying pathophysiological mechanism of male osteoporosis and was the first local gene expression study in an exclusively male idiopathic study population. Previous local gene expression analyses of osteoporotic bone tissue predominantly were performed with female study populations or included both, men and women. However, as gene expression patterns are expected to be influenced by various factors including sex and age, studies with carefully selected study cohorts will help us to get a more detailed understanding on pathophysiological mechanism underlying male and female osteoporosis. The aim of the present study was to investigate local gene expression of osteoblast related genes in femoral heads of elderly men with osteoporotic hip fractures in comparison to men with osteoarthritis. Furthermore, histomorphometric characteristics of the collected bone samples were related to the local expression of the investigated genes. Extending the concept of osteoblast dysfunction to male ‘age related osteoporosis’, we hypothesized to find a decreased local expression of osteoblast related genes in bone samples from men with osteoporotic hip fractures. 2. Patients and methods 2.1. Human bone tissue samples Femoral heads and the adjacent femoral neck were obtained from male patients undergoing total hip arthroplasty surgery due to fragility fractures of the hip (OP) and from male patients undergoing total hip arthroplasty surgery due to osteoarthritis of the hip (OA). Patients were recruited at the Department of Trauma Surgery, Danube Hospital, Vienna, Austria and the Department of Orthopaedics, St Vincent Hospital, Vienna, Austria respectively. During preoperative preparation patients were asked to participate in this study and collaborating physicians checked for predefined inclusion and exclusion criteria. Exclusion criteria for the OP group included hip fractures caused by high energy trauma (e.g. car accidents), alcohol abuse, preoperative lab findings giving evidence for severe renal or hepatic failure, or other major chronic diseases. Moreover, patients with clinical signs or established diagnosis of liver cirrhosis, hyperthyroidism, hypogonadism, any malignancy within the last five years or other severe pathologies were excluded from enrolment. Exclusion criteria for the OA group were fragility fractures, clinical diagnosis of osteoporosis, or the previous use of specific antiosteoporotic drugs other than vitamin D and calcium supplements. Inclusion criteria for both groups, OP and OA, were a minimum age of 70 years and a signed informed consent. The study was approved by the local ethic committees.
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submerged in 4% paraformaldehyde, subsequently in 70% ethanol and then stored at 4 °C. All analyses were performed in four different regions of the bone samples. The four regions were defined as peripheral region of the femoral head (pHd), central region of the femoral head (cHd), peripheral region of the femoral neck (pN), and central region of the femoral neck (cN) (Fig. 1). Samples from the central as well as the peripheral region included only trabecular bone. 2.3. Histomorphometry Cubes (7 mm × 7 mm × 7 mm) were cut out from the above described regions of each bone sample with a bone saw and processed for histomorphometry as described previously (Patsch et al., 2011). Briefly, all bone cubes were fixed with ethanol, dehydrated in ascending ethanol series, and embedded in polymethyl methacrylate (PMMA). Histomorphometry on Goldner stained sections was performed using the semiautomatic OsteoMeasure histomorphometry system (Osteometrics Inc.; Atlanta, GE). Within the trabecular compartment of each section nine random fields of view were imaged with an ×2 objective. Histomorphometric structure parameters including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), and bone surface density (BS/TV) were analyzed according to the standards of the American Society of Bone and Mineral Research (Dempster et al., 2013). 2.4. RNA extraction Sample preperation for RNA extraction was performed as described previously (Patsch et al., 2011). Briefly, a small cube (approximately 5 mm × 5 mm × 5 mm) was cut out from the above described regions of each bone sample using RNAse-free instruments. The bone cube, containing bone tissue and bone marrow, was flash frozen in liquid nitrogen and together with two small steel beads placed in a grinding mill (3 min, 30 Hz) for tissue homogenization. Total RNA was extracted using TRIzol™ reagent (Invitrogen, Carlsbad, CA), chloroform extraction and isopropanol precipitation according to the manufacturer's protocol. RNA quality and quantity were checked by photometry at 260 and 280 nm. 2.5. Reverse Transcription and Quantitative PCR cDNA was synthesized from 1 μg of total RNA using a cDNA synthesis kit (High Capacity cDNA Reverse Transcripton Kit; Applied Biosystems,
pHd
pN cHd cN pN
2.2. Sample preperation To facilitate histomorphometric, as well as gene expression analysis, femoral heads and the adjacent femoral necks were cut into two halves right after hip replacement surgery. One half selected for subsequent gene expression analysis was submerged in RNA-Later™ (Ambion, Warrington, UK) and stored as instructed by the manufacturer. The second half selected for subsequent histomorphometric analysis was
Fig. 1. Schematic diagram of the proximal femur and the sites from which samples were removed.
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Foster City, CA). Real Time PCR was performed with 100 ng of each sample for the genes RUNX2, RANKL (TNFSF11), OPG (TNFRSF1), Osterix (SP7), Osteocalcin (BGLAP), Sclerostin (SOST) and GAPDH using predesigned TaqMan Gene Expression Assays (Applied Biosystems; Hs00231692_m1, Hs00243519_m1, Hs00171068_m1, Hs00541729_m1, Hs00609452_g1, Hs00228830_m1, and Hs99999905_m1 respectively). Reactions were performed in triplicates and each reaction was performed with 9 μl cDNA diluted in water, 1 μl TaqMan Gene Expression Assay and 10 μl mastermix buffer (TaqMan Universal PCR Mastermix, Applied Biosystems). Cycler conditions used to perform real time PCR reactions on a thermal cycler (Abi Prism Sequence Detection System 7900HT; Applied Biosystems) were 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. All experiments were normalized to the housekeeping gene GAPDH. Results were calculated applying the ΔΔCt method and are presented as fold increases relative to GAPDH expression (mean Ct values: RUNX2: 28.9 ± 2.6, Osterix: 31.6 ± 2.9, SOST: 30.8 ± 2.8, Osteocalcin: 24.5 ± 3.8, RANKL: 32.5 ± 3.0, OPG: 30.7 ± 2.4).
3.3. Gene expression
2.6. Statistical methods
3.3.1. Differences in gene expression between OP and OA patients First we compared the mean expression of all regions analyzed in each patient between the two groups OA and OP (Fig. 3). The expression of RUNX2, Osterix and SOST was significantly decreased in bone samples from OP patients compared to OA patients. RANKL, OPG, and Osteocalcin expression did not differ between the two groups. Also the ratio of the expression of the two antagonizing molecules RANKL and OPG did not differ between the two groups. To obtain more insights into spatial patterns of differences in expression levels, we compared the expression of RUNX2, Osterix, and SOST in the four regions analyzed between OA and OP patients (Fig. 4). The expression of RUNX2 was significantly decreased in the central region of the head and the central region of the neck in OP patients compared to OA patients. Osterix expression was significantly decreased in both the peripheral and the central region of the head and SOST expression in the central region of the head and both the peripheral and central region of the neck in OP patients compared to OA patients. Spatial differences are only given for a limited set of genes as the RNA extracted from the bone samples was in some cases not sufficient to analyze the expression of the whole set of genes.
Data are reported either as median values and the range or as means ± standard deviation. Differences in gene expression levels and histomorphometric indices between patients with OP and OA were assessed using Mann–Whitney tests (SPSS Statistics 21; SPSS, Inc., Chicago, IL, USA). For each parameter either the different regions of each bone sample as described above were analyzed separately and/or the mean values of the regions were compared between OP and OA patients. For correlation analyses, Spearman coefficients were calculated. The critical value for statistical significance was set at p b 0.05.
3.3.2. Differences in gene expression between the femoral head and the femoral neck To obtain more insights into spatial differences in expression levels, we compared the expression of RUNX2, Osterix, and SOST between the femoral head and the femoral neck in both groups of patients. In bone samples from OA patients, Osterix and SOST expression were significantly higher in the femoral head compared to the femoral neck. In bone samples from OP patients, the expression of the four genes investigated did not differ between the femoral head and the femoral neck (Fig. 5).
3. Results 3.1. Patients Twelve patients undergoing total hip arthroplasty surgery due to fragility fractures of the hip (OP) and ten male patients undergoing total hip arthroplasty surgery due to osteoarthritis of the hip (OA) were included in this study. Gene expression was analyzed in bone samples from nine OP patients (mean age 81 ± 7 years) and nine OA patients (mean age 80 ± 7 years). Bone microarchitecture was analyzed in bone samples from seven OP patients (mean age 83 ± 6 years) and ten OA patients (mean age 83 ± 6 years). Some samples were excluded from gene expression studies as we failed to isolate sufficient amount of RNA. Due to technical reasons, i.e. limited perfusion or crush artifacts, some samples were excluded from microstructural analysis. Exclusions were randomly distributed compared to the original samples and therefore did not change the study population.
3.2. Histomorphometry Major microarchitectural changes as analyzed by static histomorphometry were seen in the peripheral region of the neck. BV/ TV was significantly decreased and Tb.Sp was significantly increased in the peripheral region of the neck in bone samples from OP patients compared to OA patients. Tb.N, Tb.Th, and BS/TV (data not show) did not differ between the two groups in the peripheral region of the neck. For the other regions analyzed no significant differences between the two groups were seen for all parameters. Comparing the means of all 4 regions analyzed in one patient, BV/TV and Tb.Th were significantly decreased in bone samples from OP patients compared to OA patients. Histomorphometric data are shown in Fig. 2. Cellular components were only rarely observed in our sections and therefore were not assessed.
3.3.3. Correlation analysis Considering all bone samples, RUNX2 expression correlated significantly with the expression of Osterix (r = 0.818, p b 0.001), SOST (r = 0.702, p b 0.001), RANKL (r = 0.773, p = 0.005), and Osteocalcin (r = 0.758, p = 0.011). Moreover, Osterix expression correlated significantly with the expression of SOST (r = 0.750 p b 0.001). We also correlated gene expression levels with microstructural indices. RUNX2 expression correlated positively with Tb.Nr (r = 0.350, p = 0.046). Osterix expression correlated positively with BV/TV (r = 0.459, p = 0.007) and Tb.Th (r = 0.403, p = 0.02) and correlated negatively with Tb.Sp (r = −0.386, p = 0.027) and BS/TV (r = −0.86, p = 0.035). SOST expression was positively associated with BV/TV (r = 0467, p = 0.007) and Tb.Nr (r = 0.438, p = 0.012) and was negatively associated with Tb.Sp (r = −0.466, p = 0.007). 4. Discussion Gene expression patterns in osteoporotic bone tissues are expected to be influenced by various factors including sex and age. Therefore, studies with carefully selected study cohorts will help us to get a more detailed understanding on pathophysiological mechanism underlying male and female osteoporosis. With the aim of investigating local gene expression patterns and microstructural alterations associated with age related osteoporosis in men, we found a significantly decreased local expression of the osteoblast transcription factors RUNX2 and Osterix, and of the Wnt signaling pathway inhibitor SOST in osteoporotic bone samples compared to osteoarthritic bone samples. Microstructural changes in osteoporotic bone samples were characterized by decreased BV/TV and Tb.Th and increased Tb.Sp. Major microarchitectural differences were seen in the peripheral region of the neck. Increasing evidence links bone loss in male idiopathic osteoporosis and in age related osteoporosis to osteoblast dysfunction. Osteblasts, the cells synthesizing and mineralizing bone during bone formation and the life long process of bone remodeling, arise from mesenchymal
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OA OP Fig. 2. Histomorphometric structure parameters including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) in bone samples from the peripheral region of the femoral head (OA: n = 7, OP: n = 10), the central region of the femoral head (OA: n = 6, OP: n = 10), the peripheral region of the femoral neck (OA: n = 7, OP: n = 9), the central region of the femoral neck (OA: n = 6, OP: n = 7) and as mean value of all four regions (total) (OA: n = 7, OP: n = 10). Bone samples were taken either from men with osteoarthritis (OA) or with osteoporosis (OP). Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
stem cells (MSCs). Benisch et al. (2012) have shown that the transcriptome of hMSCs populations derived from elderly patients suffering from age related osteoporosis significantly differs from hMSCs populations derived from non osteoporotic donors (Benisch et al., 2012). Major transcription factors required for the differentiation of osteoblastic cells are RUNX2 and Osterix, which is thought to act downstream of RUNX2 (Katagiri and Takahashi, 2002; Nakashima et al., 2002). An indispensable role of both RUNX2 and Osterix for osteoblast differentiation was impressively demonstrated by the entire absence of osteoblasts after homozygous deletion of Runx2 and Osterix in mice (Komori et al., 1997; Nakashima et al., 2002). In our study, RUNX2 and Osterix expression was significantly decreased in OP patients compared to OA patients, and therefore give evidence for disturbances in osteoblast differentiation in elderly men with osteoporotic hip fractures. Decreased local expression of RUNX2 and Osterix in osteoporotic bone tissue compared to osteoarthritic and healthy control bone was also reported by others (Dragojevic et al., 2011, 2013; Giner et al., 2013) and was previously shown by our group in bone samples from men with idiopathic osteoporosis (Patsch et al., 2011). However this is, to the best of our knowledge, the first study investigating local gene expression in an exclusively male study population with age related osteoporosis. The Wnt/β-catenin signaling pathway (canonical Wnt pathway) has been established to be a master regulator of osteogenesis influencing the differentiation of mesenchymal stem cells to osteoblasts rather than chondrocytes or adipocytes (Kang et al., 2007; Rossini et al., 2013). Sclerostin, the protein encoded by SOST, antagonizes canonical WNT signaling by perturbing the interaction of Wnt ligands with their receptors. Sclerostin is expressed primarily by bone cells and specifically by osteocytes (van Bezooijen et al., 2004; Winkler et al., 2003). By antagonizing canonical WNT signaling, Sclerostin inhibits proliferation and differentiation and stimulates apoptosis in osteoblasts (Monroe et al., 2012). The clinical relevance of Sclerostin as a negative regulator of
bone mass has been impressively demonstrated by two bone sclerosing disorders, van Buechem disease and sclerostosis. These diseases are caused by specific loss of function mutations in the SOST gene and are associated with a high bone mass phenotype and increased osteoblast activity (de Vernejoul and Kornak, 2010). A possible role for Sclerostin in osteoporotic bone loss is suggested by the finding of increased serum levels in postmenopausal women (Mirza et al., 2010) and a positive association of serum Sclerostin levels with the occurrence of osteoporotic fractures (Ardawi et al., 2012). Furthermore, SOST expression was shown to be significantly higher in MSCs from donors with age related osteoporosis compared to MSCs from age matched non osteoporotic donors (Benisch et al., 2012). The authors therefore suggest an auto-inhibitory effect of Sclerostin on proliferation and self-renewal of MSCs leading to reduced bone formation in primary osteoporosis (Benisch et al., 2012). However, local expression of SOST was significantly lower in our study population of elderly men with osteoporotic hip fractures and was also significantly lower in men with idiopathic osteoporosis (Patsch et al., 2011). A decreased expression of SOST in bone tissue of elderly patients with osteoporosis is in line with our recent finding of significantly lower serum Sclerostin levels in a study cohort of geriatric patients with hip fractures compared to age matched controls (Dovjak et al., in press). As speculated by Patsch et al. (2011), the observed reduction of SOST might be due to reduced bone volume since osteocytes are the main source of sclerostin (Patsch et al., 2011) and SOST expression correlated positively with BV/TV and Tb.Nr and correlated negatively with Tb.Sp. Delgado-Calle et al. (2011) reported a reduced proportion of lacunae occupied by osteocytes in fractured bone compared to osteoarthritic and normal bone. Concomitantly the proportion of caspase-positive lacunae as a marker of apoptosis was significantly increased (Delgado-Calle et al., 2011). Moreover, the proportion of osteocyte-occupied lacunae that stained positively for Sclerostin was lower in fractured bone (Delgado-Calle et al., 2011). Taken
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OA OP Fig. 3. Relative mRNA expression (ΔΔCt) of RUNX2 (OA: n = 9, OP: n = 9), Osterix (OA: n = 9, OP: n = 9), SOST (OA: n = 8, OP: n = 9), Osteocalcin (OA: n = 5, OP: n = 6), RANKL (OA: n = 7, OP: n = 8), OPG (OA: n = 6, OP: n = 7), and the ration of RANKL/OPG (OA: n = 6, OP: n = 7) in osteoarthritic (OA) and osteoporotic (OP) bone samples. Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
together, these data may suggest that decreased local expression of SOST is a consequence of low bone mass and/or increased osteocyte apoptosis. In addition, a reduced expression of SOST in osteocytes might contribute to a reduced expression of SOST in fractured bone. Regarding the antiproliverative and proapoptotic effect of SOST expression on
osteoblast cultures, a reduction of SOST expression concomitantly with a reduction of the osteoblast transcription factors RUNX2 and Osterix seems contradictory. However, SOST is only one of several molecules regulating the Wnt signaling pathway. Thus, our data might suggest that another molecule than Sclerostin, i.e. DKK1, plays the main
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n: 6-8
n: 6-6
n: 5-3
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n: 7-4
n: 8-5
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n: 6-7
n: 7-4
n: 7-5
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OA OP Fig. 4. Relative mRNA expression (ΔΔCt) of RUNX2, Osterix, and SOST in bone samples from the peripheral region of the femoral head, the central region of the femoral head, the peripheral region of the femoral neck and the central region of the femoral neck. Bone samples were taken either from men with osteoarthritis (OA) or with osteoporosis (OP). The corresponding number of samples (n) included in each comparison is given in the rectangles below the plots. Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
n: 7-8
n: 7-8
n: 6-8
n: 9-6
n: 8-6
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head neck Fig. 5. Comparison of relative mRNA expression (ΔΔCt) of RUNX2, Osterix, and SOST in bone samples from the femoral head and the femoral neck. Bone samples were taken either from men with osteoarthritis (OA) or with osteoporosis (OP). The corresponding number of samples (n) included in each comparison is given in the rectangles below the plots. Mann–Whitney test was used to assess between-group differences (*p b 0.05). Data are shown as box-and-whisker plots.
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regulatory function of Wnt signaling in male age related osteoporosis. But, as previously speculated by Patsch et al. (2011), a decreased expression of SOST could also indicate an autoregulatory rescue mechanism. Increased bone resorption by osteoclasts is the key mechanism driving bone loss in early postmenopausal women. RANKL is a member of the tumor necrosis factor (TNF) ligand superfamily and is best known for its essential role in regulating the differentiation and activation of osteoclasts. Binding of RANKL to its signaling receptor RANK on osteoclasts triggers the activation of signaling pathways pivotal for osteoclastogenesis. Acting as a decoy receptor, osteoprotegerin (OPG) prevents binding of RANKL to RANK and thereby counteracts its osteogenic effects. In our study no significant differences for RANKL and OPG expression levels were detected in OP and OA bone. Moreover, the ratio of RANKL to OPG expression as an important determinant of osteoclastic stimulation did not differ between these two groups. This is in contrast to other studies addressing local RANKL and OPG mRNA expression levels in osteoporotic bone tissues (Abdallah et al., 2005; Dragojevic et al., 2013; Logar et al., 2007; Tsangari et al., 2004; Zupan et al., 2012). Dragojevic et al. (2013), Zupan et. al (2012), and Logar et al. (2007) found increased RANKL and RANKL/OPG expression levels in OP bone compared to OA bone (Dragojevic et al., 2013; Logar et al., 2007; Zupan et al., 2012). In the study of Dragojevic et al. (2013), OPG levels were significantly decreased and RANKL/OPG expression was significantly lower when comparing osteoporotic bone to non osteoporotic autopsy samples (Dragojevic et al., 2013). Tsangari et al. (2004) compared expression levels in osteoporotic bone to non osteoporotic autopsy samples and also found an increased RANKL/OPG expression level in osteoporotic bone (Tsangari et al., 2004). However, the aforementioned studies were done in study populations comprising both men and women, typically including more women than men. Abdallah et al. (2005) compared mRNA gene expression in iliac crest bone biopsies from patients with osteoporotic hip fractures or osteoarthritis in an exclusively female study population (Abdallah et al., 2005). They also found significantly increased RANKL/OPG expression levels in osteoporotic bone. Decreased RANKL expression and RANKL/OPG expression levels were previously reported by our group in bone samples from men with idiopathic osteoporosis (Patsch et al., 2011). In line with this, in our study cohort a trend towards a decreased expression of RANKL in osteoporotic bone samples was observed. As osteocytes are major sources of RANKL, similar to SOST, decreased expression of RANKL might be related to a reduced bone volume. However, as in our study mean Ct values for RANKL expression were relatively high, these data require further confirmation. Taken together, local RANKL/OPG expression as determinant of bone resorption was, in contrast to postmenopausal and age related osteoporosis in women, not increased in our male age related osteoporosis study cohort, suggesting pathophysiological differences between male and female age related osteoporosis. Based on the observation of the rare occurrence of OA in the femoral heads of fractured hips and of osteoporotic hip fractures in OA patients, OA of the hip and osteoporotic hip fractures have long been postulated to be inversely related (Dequeker et al., 2003). Though, when studied longitudinally and not cross-sectionally, the relationship between OA and OP is controversial (Im and Kim, 2014). OA is regarded as a whole joint disease in which articular and periarticular tissues including the bone compartment are involved (Dequeker et al., 2003). Major changes are found in the subchondral bone and are characterized by increased bone density, subchondral bone sclerosis, and osteophytes (Henrotin et al., 2012). To get a more comprehensive picture on differences between OP and OA bone, we compared structural parameters as well as gene expression of the three osteoblast related genes RUNX2, Osterix, and SOST at four different regions of the femoral head and femoral neck. Comparing the expression of the aforementioned genes between the femoral head and femoral neck, expression of Osterix and SOST was significantly higher in the femoral head of OA patients, whereas in OP patients no difference between the femoral head and the femoral
neck could be observed. Gene expression therefore seems to be spatially more homogeneous in OP bone compared to OA bone, probably reflecting major structural changes in the subchondral bone of OA patients. A significantly decreased or a trend towards a decreased expression of RUNX2, Osterix, and SOST in OP bone compared to OA bone was not restricted to the femoral head but could be observed in all four different regions. Age dependent changes in the trabecular microstructure of the proximal femur have been shown to be region and sex dependent (Cui et al., 2008; Djuric et al., 2010). Thus, it is tempting to assume the same for OP and OA associated microstructural changes. Trabecular microstructure in OP bone compared to healthy bone or OA bone was analyzed in different subregions of the proximal femur including the femoral neck, the subchondral bone, the intertrochanteric bone or iliac crest biopsies (Boutroy et al., 2011; Hordon and Peacock, 1990; Li et al., 2012; Milovanovic et al., 2012; Patsch et al., 2011; Uitewaal et al., 1987; Zupan et al., 2013). However, to the best of our knowledge, this is the first study, comparing male OP and OA bone samples regarding trabecular microstructure in the femoral head and the femoral neck concomitantly. Structural differences between OA and OP bone that we observed in our bone samples resemble these of previous studies; though in the femoral head they did not reach statistical significance. This might be due to the smaller sample size investigated in our study. In contrast to the femoral head, in the peripheral region of the femoral neck BV/TV was significantly decreased and Tb.Sp was significantly increased in OP bone compared to OA bone. We can conclude from these data that structural differences between OP and OA bone are more pronounced in the femoral neck than in the femoral head. A major strength of our study is the diligent selection of study participants including only men older than 70 years and allowing us to identify gene expression patterns specific for men with age related osteoporosis. A limitation of this study is the comparison of bone samples from men with osteoporotic hip fractures to bone samples from men with osteoarthritis of the hip but not to healthy controls; nevertheless, bone tissue from healthy aged subjects is very difficult to obtain. However, by analyzing gene expression and microstructure at four different and clearly defined regions near the subchondral bone and distal from the subchondral bone, a potential confounder by structural and molecular changes near the affected joint was minimized. Additionally, this experimental setup enabled us to minimize a potential confounder from molecular changes in close proximity to the site of fracture associated with the inflammatory response that initiates fracture healing. Another limitation of this study is that it lacks cellular analysis. Gene expression was normalized to the housekeeping gene GAPDH. GAPDH expression did not differ between the two groups investigated (comparison of Ct values: p = 0.71). We opted for the use of only one housekeeping gene as osteoporosis and osteoarthritis are slowly developing pathologies that are not thought to be associated with excessive proliferative events and the amount of RNA available for each sample was limited. We are aware of the relatively low number of study participants. Despite this, several differences in gene expression and microstructural indices were statistically significant. In conclusion, our data give evidence for osteoblast dysfunction in age related male osteoporosis that, in contrast to postmenopausal osteoporosis and age related osteoporosis in women, is not accompanied by an increased activity of osteoclasts.
Conflicts of interest PP has received research support and/or honoraria from Amgen Gmbh, Eli Lilly Gmbh, Fresenius Kabi Austria Gmbh, Merck, Sharp and Dohme Gmbh, Novartis Parma, Nycomed Pharma, Roche Austria, Servier Austria, Shire, Sinopharm and Sanofi-Aventis. JMP has received speaker honoraria from Amgen. All the other authors state that they have no conflicts of interest.
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Acknowledgments We are grateful to Mrs Katharina Wahl for expert technical support and to Dr. Doris Moser for providing a bone saw. We further acknowledge the contribution of Philipp Becker (Orthopaedic Hospital Speising, Vienna) and of Lucia Hornek (Danube Hospital, Vienna). This research was supported by the Austrian Federal Bank (Grant No. 12544 to PP). References Abdallah, B.M., Stilgren, L.S., Nissen, N., Kassem, M., Jorgensen, H.R., Abrahamsen, B., 2005. Increased RANKL/OPG mRNA ratio in iliac bone biopsies from women with hip fractures. Calcif. Tissue Int. 76, 90–97. Ardawi, M.S., Rouzi, A.A., Al-Sibiani, S.A., Al-Senani, N.S., Qari, M.H., Mousa, S.A., 2012. High serum sclerostin predicts the occurrence of osteoporotic fractures in postmenopausal women: the Center of Excellence for Osteoporosis Research Study. J. Bone Miner. Res. 27, 2592–2602. 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