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1 Departments of Endocrinology and
2 Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, brug 124, PO Box 7057, 1081 HV Amsterdam, The Netherlands
(Requests for offprints should be addressed to N Bravenboer; Email: n.bravenboer{at}vumc.nl)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several in vivo studies have shown that many biochemical signal molecules are involved in the translation of mechanical stimuli into bone formation including glucose-6-phosphate dehydrogenase (G6PD) (Skerry et al. 1989, Lanyon 1992), c-fos (Raab-Cullen et al. 1994, Lean et al. 1996), cAMP (Davidovitch et al. 1984), cyclooxygenase (COX-2) (Forwood 1996), nitric oxide (NO) and prostanoid (Pitsillides et al. 1995), insulin-like growth factors (IGFs) (Raab-Cullen et al. 1994, Lean et al. 1995, Bravenboer et al. 2001), transforming growth factor-ß (TGF-ß) (Raab-Cullen et al. 1994, Bravenboer et al. 2001), protein kinase B (PKB or Akt) (Skerry & Suva 2003), and glutamate transporter (GLAST) (Mason et al. 1997, Skerry 1999, Skerry & Genever 2001, Skerry & Suva 2003). These responses to in vivo mechanical loading are time- and spatially-dependent. Early strain related changes within 5 min after loading are shown in osteocytes in which the G6PD activity is increased (Skerry et al. 1989, Lanyon 1992), whereas an increase of IGF-I mRNA expression is located on trabecular surfaces and in osteocytes of the diaphysial cortex (cortical and trabecular osteocytes) of rat caudal vertebrae within 6 h after a single loading session (Lean et al. 1995).
The aim of this study was to characterize the role of IGF-I mRNA in the cortical tibia shaft during the translation of mechanical stimuli into bone formation. To this end, we developed an in situ hybridization method especially for bone tissue to detect the local osteogenic response on cellular level 6 h after a single period of dynamic loading. To induce a single period of mechanical loading the four-point bending model of Forwood and Turner has been used (Turner et al. 1991, 1994, Forwood et al. 1998) resulting in bone formation in the rat tibia 5–8 days after stimulation (Forwood et al. 1996).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study comprised two parts: (1) validation study of the four-point bending system to verify bone formation after a single mechanical loading session and (2) detection of changes in mRNA expression of IGF-I after a single mechanical loading session with non-radioactive in situ hybridization.
The animal experiments were in accordance with the governmental guidelines for care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the VU University Medical Center Amsterdam, The Netherlands.
Validation study four-point bending system
Female Wistar rats of 12-weeks old (Harlan, Zeist, The Netherlands) were randomly assigned into two groups (n = 3 per group): LOAD and SHAM. The right tibiae underwent ‘medio-lateral’ loading (LOAD) (distance between the centers of the loading pads: upper pads, 11 mm and lower pads, 23 mm) or sham-loading (SHAM) (opposed pads were placed at the inner position: 11 mm) using the four-point bending system of Forwood and Turner (Fig. 1
, Turner et al. 1991, Forwood et al. 1998). Since loading will result in bending and squeezing of the tibia and sham-loading only in squeezing of the tibia, the SHAM group was used as control for the LOAD group. The left non-loaded tibiae served as contra-lateral controls (CONTROL). The four-point bending model (Forwood et al. 1996) was used to generate a single period of dynamic loading of the right tibia in rats in vivo. The rats were subjected to a single episode of loading comprising 300 cycles (2 Hz) using a peak magnitude of 60 N, which generates a mean strain of 2664 µstrain in the loaded tibia compared with a mean strain of 350 µstrain in the sham-loaded tibia (Forwood et al. 1998). Tetracycline (25 mg/kg body weight) was administered intraperitoneally to the rats, 5 and 12 days after the single loading session. The rats were killed 15 days after loading.
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In vivo mechanical loading in situ hybridization
Fifteen female 12-week-old Wistar rats (235 ± 12 g) (Harlan) were randomly assigned to three weight-matched groups (n = 5/group): LOAD, SHAM, and CONTROL. The right tibiae underwent ‘medio-lateral’ loading (LOAD), sham-loading (SHAM), or no loading (CONTROL) using the four-point bending system of Forwood and Turner (Fig. 1
, Turner et al. 1991, Forwood et al. 1998). The left tibiae served as contra-lateral controls. The four-point bending model (Forwood et al. 1996) was used to generate a single period of dynamic loading of the right tibia in rats in vivo in order to detect acute changes of IGF-I mRNA locally in bone tissue after stimulation by mechanical stress. The rats were subjected to a single episode of loading comprising 300 cycles (2 Hz) using a peak magnitude of 60 N. The loading experiment was performed under general anesthesia (2% isoflurane in 1 l/min O2 and 2 l/min N2O). The rats were killed exactly 6 h after loading. This time point was based on literature (Lean et al. 1995) which was confirmed by a time-course pilot experiment at our laboratory using real-time reverse transcriptase (RT)-PCR analysis (H W van Essen, personal communication). The tibiae were dissected and immediately fixed in 4% (wt/vol) paraformaldehyde (buffered in PBS, pH 7.4) at 4 °C for 24 h.
Tissue
After the fixation, the tibiae were decalcified in 10% EDTA with 0.5% paraformaldehyde in PBS at 4 °C for 4
weeks. Finally, the tibiae were washed in PBS and dehydrated through a series of ethanol and xylene at room temperature and embedded in paraffin.
Control brains were dissected rostrally to the cerebellum (interaural coordinate 0 mm) and the hippocampus (interaural coordinate 4 mm) in three coronal blocks and immediately fixed in 4% (wt/vol) paraformaldehyde (buffered in PBS, pH 7.4) at 4 °C for 24 h, followed by washing in PBS, dehydration through a series of ethanol and xylene at room temperature and embedding in paraffin.
Reagents
All restriction enzymes and modifying enzymes were purchased from Roche Molecular Biochemicals, as well as digoxigenin-UTP, anti-digoxigenin Fab fragments, nitro-blue-tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate, and blocking reagent. Nylon membranes were purchased from Qiagen. Polyvinyl alcohol was obtained from Aldrich (Milwaukee, WI, USA). Euparal mounting medium was purchased from Chroma Gesellschaft (Schmid GmbH, Köngen, The Netherlands). Silane-coated glass slides were obtained from Sigma-Aldrich (St Louis, MO, USA).
rRNA and human IGF-I cDNA was kindly provided by Dr S C van Buul-Offers (Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Utrecht, The Netherlands).
Synthesis of digoxigenin-labeled complementary RNA (cRNA) probes
Standard in vitro transcription reactions were carried out using T7- and Sp6-RNA polymerase with digoxigenin-UTP as a substrate (Melton et al. 1984). cDNA encoding ribosomal 28S RNA and human IGF-I (259 bp, containing exon 2 and 3, 120–379 nt, gene ID X00173 [GenBank] ) (Jansen et al. 1983) were used as a template for the synthesis of antisense and sense digoxigenin-labeled RNA probe. The probe was specific for the mRNAs analyzed. The IGF-I probe was checked for cross-hybridization using in situ hybridization on spleen and growth plate cartilage (Smink et al. 2002).
Non-radioactive in situ hybridization
Serial, longitudinal, tibia sections (5 µm), which were cut in posterior–anterior direction, and cross-sectional control brain sections (5 µm) were mounted onto RNase-free silane-coated glass slides and dried at 56 °C for at least 3 days. In situ hybridization was performed on every 50th section with a total of five slides per tibia (i.e., sections I, II, III, IV, and V; Fig. 1). Sections I, II, III, IV, and V included the loading zone of the loaded and sham-loaded tibia and were taken to obtain regular sampling throughout the whole tibia. Corresponding sections of the right and the left tibia sections of one rat were mounted on the same glass slide. All sections were dewaxed, rehydrated, and rinsed in water. The sections were pretreated with 0.2 M HCl for 15 min at room temperature, permeabilized in proteinase K (15 µg/ml) for 30 min at 37 °C, and subjected to an acetylation treatment (Wilkinson 1992). The sections were rinsed in 2 x SSC (0.3 M sodium chloride and 0.03 M sodium citrate) and kept in this solution until the start of the hybridization.
Hybridization was performed in a solution containing 50% formamide, 2 x SSC, 1 x Denhardt’s solution, 250 µg/ml tRNA, 480 µg/ml herring sperm RNA, 10% dextran-sulfate and the rRNA digoxigenin-labeled cRNA probe at a concentration of 250 pg/µl and the human IGF-I (hIGF-I) digoxigenin-labeled cRNA probe at a concentration of 1500 pg/µl. Sections were hybridized overnight at 53 °C. After hybridization, sections were washed with 50% formamide in 2 x SSC at the hybridization temperature for 30 min and treated with RNase A (1 unit/ml) for 30 min at 37 °C. Subsequently, sections were rinsed in 2 x SSC, treated with 1% blocking reagent for 30 min, and incubated with sheep anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (1:1500) O/N at 4 °C.
Chromogenesis was performed in the dark with 0.38 mg/ml nitro-blue-tetrazolium chloride (NBT) and 0.19 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in the presence of 6% (wt/vol) polyvinyl alcohol (De Block & Debrouwer 1993) resulting in a blue precipitate. The sections were counterstained with nuclear fast red. Thus, positive cells will have a blue cytoplasmatic staining, whereas negative cells will be pink. Finally, the sections were dehydrated through a series of ethanol and mounted with Euparal (Waldeck Gmbh & Co Division Chroma, Münster, Germany). Sense probes were used to investigate the level of non-specific binding.
Quantification and statistics
First, a semi-quantitative screening of five different animals per group was performed to collect the in situ hybridization data. All tibiae were scored in a semi-quantitative manner which was defined as follows: no expression (0), low expression (1), average expression, (2) and high expression (3) as shown in Fig. 2.
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ANOVA (one-way ANOVA) was used for statistical analysis using SPSS version 9.0 for Windows. A P value of < 0.05 was considered to reflect statistical significance.
Photography
Brightfield photographs were made using Leica microscope (DM4000B) with a digital camera (Leica DC500) and the Leica software program IM50 (Leica Microsystems, Rijswijk, The Netherlands).
IGF-I protein assay
IGF-I protein was measured in serum by a RIA for the quantitative measurement of human somatomedin-C (Bio-Source, International, Camarillo, CA, USA). The intra-assay coefficient of variation was < 5.2% (n = 15) for levels between 925 and 2000 ng/ml and the inter-assay coefficient of variation was < 10% (n = 20) for levels between 120 and 500 ng/ml. The minimal detectable concentration was 35 ng/ml.
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Results |
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The loaded right tibiae of the rats showed lamellar bone formation at the endosteal surface (Fig. 3A–C) and woven bone formation at the periosteal surface (Fig. 3A). The MS/BS expressed as a percentage of the bone surface of the loaded tibiae was 58%, whereas the contralateral control tibiae showed a MS/BS of 40%. The MS/BS of sham-loaded tibiae and its contralateral control tibiae was for both 38% (Table 1).
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The IGF-I protein concentrations in serum, expressed as mean ± S.E.M., were 1506 ± 126 ng/ml in the control group, 1409 ± 155 ng/ml in the load group, and 1408 ± 93 ng/ml in the sham group respectively. Differences between groups were not observed (P value = 0.808).
Ribosomal 28S RNA expression in tibiae
Analysis of ribosomal 28S RNA in control tibiae using non-radioactive in situ hybridization exhibited cytoplasmatic expression within every cell type, including all osteocytes (Fig. 4A), osteoblasts (Fig. 4A), chondrocytes, and bone marrow cells (data not shown). Control hybridizations of control tibiae with the sense rRNA probe showed no signals (Fig. 4B).
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The control brain showed IGF-I mRNA expression in Purkinje cells of the cerebellum and in neurons of the medulla oblongata (data not shown). In the internal control, i.e. the growth plate, IGF-I mRNA was located in chondrocytes of the proliferative and the hypertrophic zone (Fig. 4C). In control tibiae, IGF-I mRNA was expressed in osteoblasts, which were situated against the surface of trabecular bone (Fig. 4C) and endocortical bone (Fig. 4E). IGF-I mRNA expression was also observed in osteocytes, which were lying within the first lamella at the endosteal side of the shaft (Fig. 4E) and some trabecular osteocytes (Fig. 4C). The endocortical osteocytes, which were located within the deeper lamellae (Fig. 4E), and the periosteal osteocytes (data not shown) did not express IGF-I mRNA. IGF-I mRNA was expressed in the intracortical endothelial cells of blood vessels (Fig. 4F) and in the periosteum of control tibiae (data not shown). Some cells of the bone marrow i.e. megakaryocytes, macrophages, and myeloid cells also expressed IGF-I mRNA (data not shown).
Control hybridization of tibiae sections with the corresponding sense RNA probe did not show signals (Fig. 4D).
Effect of mechanical loading on IGF-I mRNA expression in osteocytes of the tibia shaft
Mechanical loading induced IGF-I mRNA expression in osteocytes within multiple layers at the endosteal side of the shaft of the tibia in contrast to the contra-lateral control tibia, where IGF-I mRNA expression was only seen within the superficial layer at the endosteal side of the shaft (Fig. 4E and F). Quantitative analysis of the osteocytes within the endosteal side of the shaft showed a twofold increase of IGF-I mRNA expression 6 h after mechanical loading (Figs 4F and 5). The proportion of IGF-I mRNA-positive osteocytes was 29.3 ± 12.9% (mean ± S.D.) for loaded tibiae (n = 5), 16.7 ± 4.4% (mean ± S.D.) for sham-loaded tibiae (n = 5), and 14.7 ± 4.2% (mean ± S.D.) for contra-lateral control tibiae (n = 10; Fig. 5). Mechanical loading significantly increased the number of osteocytes, which express IGF-I mRNA (P < 0.01, load versus contra-lateral control and P < 0.05, load versus sham (Fig. 5)).
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No differences in IGF-I mRNA expression between loaded, sham-loaded, and contra-lateral control tibiae were observed in the osteoblasts, chondrocytes, and bone marrow cells by semi-quantitative screening. No differences in morphology of the cells were observed between groups.
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Discussion |
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These results confirm previous findings. Lean et al.(1995) showed an increase of IGF-I mRNA in cortical and trabecular osteocytes of the eighth caudal vertebra 6 h after mechanical stimulation with the invasive vertebra compression model demonstrated by in situ hybridization and Raab-Cullen et al.(1994) showed an increase of IGF-I mRNA in the periosteum 4 h after loading with the four-point bending model demonstrated by northern blot (Raab-Cullen et al. 1994). Besides, IGF-I protein concentration in bone is also increased after stimulation by mechanical stress using additional weight-bearing (rat-with-backpack) as was previously reported (Bravenboer et al. 2001). The current study is the first study which showed a twofold increase of IGF-I mRNA in osteocytes in a non-invasive mechanical loading model (four-point bending model) at cellular level in vivo (non-radioactive in situ hybridization).
In addition, the twofold increase of IGF-I mRNA was specifically detected in the osteocytes at the endosteal side of the shaft within the deeper lamellae. It has generally been accepted that osteocytes are responsible for the predominant sensing of mechanical strains in bone (Klein-Nulend et al. 1995). It can be concluded that the osteocytes at the endosteal side of the shaft and the inner lamellae are mechanosensitive, since these osteocytes synthesize IGF-I mRNA 6 h after mechanical loading. This is also shown by Gross et al.(2002) who reported that mice overexpressing IGF-I in osteoblasts had an increased periosteal bone formation, suggesting that IGF-I increased sensitivity of the osteocytes and the osteoblasts.
Furthermore, we suggest that the mechanosensitive osteocytes, which synthesize IGF-I mRNA 6 h after mechanical loading, are important for the increased lamellar bone formation after mechanical loading. Forwood et al.(1996) reported that a single short period of loading, using the four-point bending model, resulted in an increased lamellar bone formation rate at the endosteal surface of rat tibia. Mechanical loading with an external bending load of 60 N in vivo will result in an increased woven bone formation rate at the periosteal surface and increased lamellar bone formation rate at the endosteal surface (Forwood et al. 1998). This has been confirmed in the validation study at our laboratory which showed that a single mechanical loading session was sufficient to induce lamellar bone formation at the endosteal side of the tibiae and the woven bone at the periosteal bone side. The lamellar bone formation is located at the endosteal side of the shaft, which is similar to the location of newly synthesized IGF-I mRNA after mechanical loading. This study also showed that IGF-I mRNA expression was not observed in the osteocytes at the periosteal side of the shaft, but was restricted to the sub-endocortical osteocytes and the osteocytes in multiple lamellae. The IGF-I mRNA may be less involved by the woven bone formation, which is the result of irritation of the periosteum (Forwood et al. 1998). This suggests that IGF-I mRNA in the mechanosensitive osteocytes is specifically important for lamellar bone formation.
No differences in serum IGF-I concentrations were observed between loaded, sham-loaded, and control groups. This implies that the acute effect of mechanical loading is restricted to the bone region, which is deformed, because the systemic serum IGF-I concentrations are unaffected. This supports the hypothesis that the osteogenic response to mechanical loading occurs locally (Bravenboer et al. 2001). However, the level of IGF-I mRNA, which has to be produced at a single skeletal site to observe the effect on IGF-I protein in the serum, is unknown. A twofold up-regulation of IGF-I mRNA in one tibia might be too low.
The other bone cells in the tibiae and the bone marrow cells showed no difference in IGF-I mRNA expression before and 6 h after mechanical loading. Our results showed that osteoblasts and osteocytes, within the first lamella at the endosteal side of the shaft and the trabecular bone, synthesize IGF-I mRNA. These bone cells play a role during bone remodeling. Although the osteoblasts are responsible for new bone formation after loading, their IGF-I mRNA expression was not increased after mechanical stimulation; this is probably due to the fact that the endogenous IGF-I mRNA expression level in osteoblasts was very high. Several investigators have also demonstrated endogenous IGF-I mRNA expression within the osteocytes of rodents (Inaoka et al. 1995, Mason et al. 1996, Zhao et al. 2000). In contrast, a number of other studies did not observe endogenous IGF-I mRNA expression within the osteocytes of rodents and humans (Yeh et al. 1993, Lean et al. 1995, Middleton et al. 1995). These contradictory results could be explained by the fact that different bones, including tibiae, distal femurs, ulnae, and vertebrae, were examined. The daily loading of these bones varies considerably, which results in differences in gene expression. A second explanation could be the use of various molecular techniques and the preparation of the bone tissue, either undecalcified bone or decalcified bone. Finally, there is a difference in age between the studied species.
For this study, we used the four-point bending model of Forwood, because this four-point bending apparatus produces a controlled mechanical strain in the tibia of living rats. An advantage of this approach is that it does not require surgical intervention and allows normal physical activity after the loading session (Turner et al. 1991, Forwood et al. 1998). The osteogenic response will occur locally in bone. Therefore, we have used the in situ hybridization technique in order to detect the local osteogenic response at the cellular level. The non-radioactive in situ hybridization method is a powerful and sensitive technique method to localize gene expression within decalcified rat tibiae. In this study, a ribosomal 28S RNA probe was used to verify the RNA integrity of the decalcified tibiae. All bone cells, including osteocytes and osteoblasts, and chondrocytes and bone marrow cells showed ribosomal 28S RNA expression within the cytoplasm. Therefore, we conclude that the RNA integrity was maintained during the entire decalcification and embedding procedure. It is demonstrated that the IGF-I cRNA probe is specific, because the brain showed IGF-I mRNA expression in the Purkinje cells of the cerebellum and in neurons of the medulla oblongata as described earlier (Bondy et al. 1992, D’Ercole et al. 1996, Reijnders et al. 2004) and IGF-I mRNA was expressed in the chondrocytes of the proliferative and the hypertrophic zone of the growth plate as shown earlier by Reinecke and Nilsson (Nilsson et al. 1990, Reinecke et al. 2000).
Nevertheless, this study has some limitations. The applied load of 60 N is supra-physiological and the insulin-like growth factor-binding proteins (IGFBPs) have not been studied. IGF-I is one component of the IGF system. IGFBPs can influence the biological activity of IGF-I (Firth & Baxter 2002) by regulating the bioavailability of IGFs (Collett-Solberg & Cohen 1996). Therefore, it is necessary to study the effects of mechanical loading on local IGFBP expression level as well.
In conclusion, this study shows that IGF-I mRNA is twofold up-regulated within the endocortical osteocytes of the shaft and multiple layers extending into the cortical bone 6 h after mechanical loading in vivo. We conclude that these osteocytes are mechanosensitive as shown by newly synthesized IGF-I mRNA after a single short period of loading. This supports the hypothesis that these osteocytes translate mechanical stimuli into bone formation through IGF-I. The process occurs rather early in a series of cellular events, which take place after mechanical loading.
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Acknowledgements |
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References |
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Received in final form 10 September 2006
Accepted 19 September 2006
Made available online as an Accepted Preprint 27 October 2006
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P < 0.01, load versus contra-lateral control; *P < 0.05, load versus sham).