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Zinc inhibits ovariectomy induced microarchitectural changes in the bone tissue
Journal of Nutrition & Intermediary Metabolism 3 (2016) 33e40
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Zinc inhibits ovariectomy induced microarchitectural changes in the bone tissue Payal Bhardwaj a, *, Durg Vijay Rai a, b, Mohan Lal Garg a a b
Department of Biophysics, Panjab University, Chandigarh, India Faculty of Biomedical Engineering, Shobhit University, Meerut, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2015 Received in revised form 5 December 2015 Accepted 21 December 2015 Available online 23 December 2015
Purpose: The purpose of the current study was to examine the effect of zinc supplementation as a nutritional supplement in case of osteopenia induced microarchitectural changes in rat model. Methods: Forty eight animals in two batches of twenty four animals each were assigned to four groups: Control, Zinc, Ovariectomized (OVX) and OVX þ Zinc. The treatment period was continued for eight weeks. Histoarchitecture analysis was performed on both the bones i.e. femur and tibia using light as well as electron microscopy. Also, the bone calcium content was estimated using atomic absorption spectrophotometer. Results: The body weight of the animals in the OVX group was significantly higher in comparison to the control animals. The body weight was found to increase significantly upon zinc supplementation to OVX animals till the 4th week and then was almost comparable till the termination of treatment period. Calcium content in both femur and tibia were found to be significantly reduced in the ovariectomized group. The connectivity of trabeculae was lost following ovariectomy. Zinc administration restored bone calcium content as well bone tissue morphology including trabecular thickness. Conclusion: These findings suggest that changes in the trabecular bone attributed to estrogen deficiency are arrested by zinc supplementation. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Zinc Bone microarchitecture Ovariectomy Trabecular bone Electron microscopy Calcium
1. Introduction Osteoporosis is a skeletal disease characterised by low bone mass and structural weakening of the bone material that leads to reduced bone strength [1]. Postmenopausal osteoporosis is by far the commonest form of age-related bone loss. In women, bone loss is exacerbated after menopause due to a decrease in estrogen production. It is estimated that 10 million individuals have osteoporosis while another 34 million suffer from low bone density. Upon survey, it has been found that 61 million individuals will develop osteoporosis or low bone density by 2020 [2]. Beyond medical costs, there is the physical burden of living with osteoporosis and its impact on the daily life style, including restrictions in daily activities, loss of confidence (due to fear of falling and fracture) and loss of independence [3]. The development of osteoporosis is thought to be primarily
* Corresponding author. Department of Biophysics, Panjab University, Basic Medical Sciences block, Chandigarh, 160014, India. E-mail address: [email protected] (P. Bhardwaj).
related to ageing, genetic factors. Some modifiable factors, such as smoking, excess alcohol consumption, life style and deficiency or excess of some of the components of diet, are also associated with osteoporosis [4]. Therapies for osteoporosis fall into two categories: antiresorptive drugs, which slow bone resorption, and anabolic drugs, which stimulate bone formation. However, the adverse effects of these medications include malignant tumour formation with hormone therapy and gastrointestinal tolerance problems with bisphosphonates, which may exclude their long-term use [5,6]. Thus, there is a need for some alternative that can improve bone health without inducing adverse effects. Growing evidence of the benefits of natural foods for bone health presents alternatives for the prevention or treatment of osteoporosis [7,8]. Trace elements play a major role in the growth and development of skeleton, of which zinc is of particular interest to us. The fact that the organic component in the bone is mainly composed of protein and that most of the bone mineral portion is calcium implies that the essential nutrients required for bone health are protein and calcium [9]. In addition this, certain minerals, vitamins and trace metals are also required for the
http://dx.doi.org/10.1016/j.jnim.2015.12.333 2352-3859/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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maintenance of bone composition and microarchitecture [10]. Zinc is an essential trace element, is a component of 200 enzymes and is also well known to be necessary for normal mineralization and collagen synthesis in bone. Yamaguchi et al. studied the direct effects of zinc on the proliferative activity of bone cells and found that it has a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. Zinc produced remarkable increases in alkaline phosphatase activity and protein concentration in osteoblasts [11,12]. The role of zinc has been studied previous in terms of zinc deficiency or adequate zinc diet. Zn-deficient diets have impairment in growth plate chondrocyte proliferation, body length gain [13,14] and bone mass [15] in animals. However, to the best of our knowledge, little research on the association between zinc supplementation and bone micro architecture in the osteopenic condition has been conducted. To address this issue, the present study investigated the effect of zinc supplementation on bone calcium content and microarcitecture in osteopenic rat model.
2. Material and methods To carry out the present study, forty eight female Wistar rats (12 weeks old, 100e150g) in a batch of two were procured from the Central Animal House of Panjab University, Chandigarh. The animals were housed in polypropylene cages bedded with rice husk. They were given clean drinking tap water and standard animal pellet diet (Ashirwad Industries, Kharar, Punjab, India), throughout the experiment ad libitum. The animals were randomly divided into four groups comprising twelve animals in each group. They were acclimatized for one week to laboratory conditions prior to the start of the experimental procedure. Group 1 (Control) was given standard diet as well as drinking water. Group 2 (Zinc) was given zinc supplement as zinc sulphate in drinking water at a dose level of 227 mg of ZnSO4.7H2O per litre [16]. Group 3 (OVX) animals were bilaterally ovariectomized and were kept under normal feeding conditions without any zinc supplementation. Group 4 (OVX þ Zinc) animals were bilaterally ovariectomized and given zinc in drinking water (227 mg of ZnSO4.7H2O per litre). The treatment period was continued for eight weeks. The experimental design and procedures were approved by the Ethical Committee on Animal Experiments of the Central Animal House, Panjab University, Chandigarh, India. It was not possible to carry the measurements in a single batch. Therefore, measurements of all the parameters were done in two batches. These batches were segregated in different time over a period of one year. The first batch includes the ash content analysis and the second batch includes the histological analysis using light and electron microscopy.
2.1. Surgical procedure for bilateral ovariectomy Ovariectomy was performed in female Wistar rats by the method as described in our previous paper [17]. The ovariectomized Wistar rat model is the FDA recommended model and has been widely used for the rapid development of osteoporosis for purposes of investigating aspects of pathogenesis and treatment of postmenopausal bone loss [18]. At the completion of termination period, the animals were sacrificed by decapitation under anesthesia (using diethyl ether) and both the femora and tibiae were extracted. Bones of all the animals were cleared off from the muscle and cartilage and were further processed.
2.2. Elemental analysis The method of Szpunar et al. [19] was partially modified for bone digestion. Bone specimens were digested in concentrated nitric acid, and trace element analysis for the bone tissue was performed using an atomic absorption spectrophotometer (model 3100; PerkinElmer). Calcium hollow cathode lamp was operated at a slit width of 0.7 nm, which was selected to isolate 422.7 nm lines. Calcium standard (1000 ppm) was used for calibrating the system. 2.3. Micro architectural analysis Microscopy was done to analyze the microstructure of bone that includes arrangement and geometry of the trabecular region. Also, the adipocytes present in the bone marrow were analyzed using light microscopy. 2.3.1. Light microscopy 2.3.1.1. Bone sample preparation 2.3.1.1.1. Bone tissue preparation (fixation and embedding) for Hematoxylin-eosin staining. After excision, the trabecular regions (sectioned from the metaphyseal region along the growth plate) of the femora and tibia of all the groups were sectioned and fixed immediately in 4% formaldehyde prepared in 0.1M phosphate buffer (pH 7.2) overnight. After this, all the samples were decalcified in 10% Ethylene Diamine Tetra Acetic acid (EDTA) prepared in 0.05M TriseHCl buffer, pH 7.2. All the samples were removed from demineralising solution and washed in phosphate buffer saline. Then they were treated in five different gradients of alcohol (starting from 30% ethanol to absolute alcohol) for 1hr. After this they were dipped in absolute alcohol and benzene (1:1) for half an hour, only benzene for half an hour and then in wax for 6 h. Samples were then embedded in the paraffin wax (melting temperature 60 C) using plastic cassettes. Plastic cassettes were then inserted into the microtome so that the wax block faces the blade. The thin bone tissue sections (of about 4e10 mm thin) were then sliced and further processed for different staining protocols. HE staining was done to visualize the diaphyseal cortical bone thickness of both the femora and the tibia upon ovariectomy and zinc supplementation. After deparaffinising in xylene for about 15 min, the sections were immersed in the graded alcohol solutions (90% ethanol to 30% ethanol). After this the slides were dipped in Hematoxylin for 1e2 min. The slides were rinsed under running tap water in staining box until the water is no longer coloured (~5 min). Sections were again immersed in eosin stain for 1e2 min and rinsed until water became clear. Then again the slides were kept in gradient of ethanol and were shifted to xylene till they were cover slipped. Then the slides were viewed and analyzed under the light microscope (Leica). 2.3.1.2. Bone marrow preparation. Adipocytes were analyzed by extracting bone marrow cells from the femur bone. The marrow cells were then stained with h and e stain as according to the standard protocol. The stained sections were then analyzed under light microscope (Leica). 2.3.2. Scanning electron microscopy 2.3.2.1. Bone sample preparation. The bone samples were processed for the assessment of microarchitecture as per the method followed by Chang et al. [20]. Longitudinal section of all the bone samples (trabecular region sectioned from the metaphyseal growth plate) were immersed in a tissue fixative solution (4% formaldehyde in phosphate buffer saline) for at least one day and then rinsed with the Phosphate buffer saline (PBS) before characterization. For SEM, the bone was polished and etched in a hydrogen peroxide
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(35% H2O2) at 40 C for 2 h; the etched specimens were then dehydrated by 10 min immersions in five different concentrations (50%e100%) of ethanol solution. The dried bone specimens were mounted on the aluminium stubs (covered by carbon tape) and sputter-coated with gold. After this, the endosteal portion (trabecular region) of the femora and tibiae's microstructures were examined by scanning electron microscopy (Fine coat Ion sputter JSM-6100 JEOL) operating at 15 KV in the Sophisticated Analytical Instrumentation Facility, CIL, Panjab University, Chandigarh, India.
ovariectomized group when compared to the normal control. Femur (p < 0.001) and tibia (p < 0.001) calcium concentrations were enhanced in the OVX þ Zinc group and were restored to normal in the case of femur bone. However, the tibia calcium levels upon zinc supplementation to the OVX animals were not fully recovered (p < 0.05) when compared to the normal control animals. Zinc treatment alone to the control animals also showed increased calcium levels in both the bone tissues and the increase was significant (p < 0.05) in the case of tibia.
2.4. Statistical analysis
3.4. Scanning electron microscopy of the trabecular region of bone (both femur and tibia) tissue
Data are expressed as mean (SD). One-way analysis of variance was used to evaluate differences among groups. NewmaneKeuls post hoc test was used for multiple comparisons between groups. Differences were considered statistically significant at P < 0.05. The statistical analysis software SPSS (version 14) was adopted to process the data in the present study. 3. Results 3.1. Dose administered to each animal Zinc supplementation in the drinking water can be considered as an appropriate and customary method for supplementing nutrition. To calculate the dose of elemental zinc taken by an animal, each animal's daily consumption of drinking water was recorded which varied between 25 and 30 ml. Taking into consideration the daily consumption of drinking water and the average body weight of animals, we estimated zinc consumption to vary between 8 and 9 mg/kg body weight [17]. 3.2. Body weight measurement The variation in the body weights of all the animals subjected to different treatments are shown in Table 1. Body weights were measured at the beginning of treatment period and then were recorded at the 2nd, 4th and 8th week. The body weights of the animals of all the groups were found to increase progressively till the end of eighth week. At the second week, no weight change was observed in the OVX group when compared to the control group. At the 4th (p < 0.05) and 8th (p < 0.01) week, the mean body weight of the animals in the ovariectomized group was significantly higher in comparison to the control animals. Zinc supplementation to the OVX group showed increased body weight in comparison to the control at the 2nd and 4th week, and at the 8th week mean body weight of the animals was almost comparable to that of the control group. The whole data is also summarized as body weight gain (Table 2) for all the treatment groups. 3.3. Calcium content in bone tissue Mean values of calcium concentration in both femur (p < 0.001) and tibia (p < 0.001) were found to be significantly reduced in the
Figs. 1 and 2 show the scanning electron microscopic images of the trabecular portion of the femur and tibia endosteal region respectively in different groups. As seen in Figs. 1 and 2 at a magnification of 500, the femur and tibia bone of control animals consisted of dense and well organized trabecular bone tissue. No resorbed areas were identified in both the bones of the control group. Micrographs of both the bones of ovariectomized group had resorbed trabecular region. The connectivity of trabeculae is lost following ovariectomy. The connectivity is more disrupted in the case of tibia when compared to the femur group. Zinc supplementation to the control animals showed similar well arranged pattern of the trabeculae as compared to the control. Administration of the zinc to the OVX rats markedly reduces bone loss as is evident from the micrographs showing dense network of trabeculae. 3.5. Hematoxylin and eosin staining of the trabecular region H and E stained sections of the trabecular portion of the rat femur bone at the lower magnification (5), revealed normal trabecular architecture with the preservation of trabecular plate and interconnectivity between trabeculae. Bone marrow cells were seen in the spaces between the trabeculae (Fig. 3). However, as can be seen from the micrograph of the OVX group, there was deterioration and the thinning of the trabecular bone structure accompanied by the increase in the bone marrow. In addition, there was widening of the spaces between the trabeculae as a result of trabecular bone loss. Zinc supplementation to the OVX animals resulted in thickening of the trabecular network making it dense as that of the control bone (Table 4). Zinc supplementation to the normal control animals maintained the connectivity of the trabeculae, similar to that of the control bone. 3.6. Bone marrow cells (adiposity) The ovariectomized group exhibited a significant elevation in the number of adipocytes as compared to the control group. Also, it can be observed that ovariectomy leads to the formation of large sized adipocytes (Fig. 4). Zinc supplementation to the OVX animals down regulated the adipocyte number. However, the adipocyte number remains almost similar to that of the control group when zinc was supplementation alone to the control animal.
Table 1 Body weight of all the four groups at specific time intervals after surgery. Groups
Initial body weight (g)
Control Zinc OVX OVX þ zinc
130 126 130 130
± ± ± ±
7 5 7 11
Body weight (g) after 2nd week 150 149 155 165
± ± ± ±
8 9 17 12a
Body weight (g) after 4th week 177 176 192 202
± ± ± ±
8 8 16a 14c
Body weight (g) after 8th week 217 215 239 245
± ± ± ±
9 7 19b 15
Each value is the mean ± SD (n ¼ 6).Values with a superscript are significantly different from the control group (a, p < 0.05; b, p < 0.01; c, p < 0.001) or from Ovx group (d, p < 0.05; e, p, <0.01; f, p < 0.001).
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Table 2 Percentage body weight gain during the treatment period of all the four groups at specific time intervals after surgery. Groups
Body weight gain after 2nd week
Body weight gain after 4th week
Body weight gain after 8th week
Control Zinc OVX OVX þ Zinc
15 18 19 27
18 18 24 22
23 22 24 21
Table 3 Effect of OVX and zinc supplementation on the calcium concentrations of femur and tibia tissue. Groups
Femur calcium (mg/g of tissue)
Tibia calcium (mg/g of tissue)
Control Zinc Ovx Ovx þ Zinc
204 ± 14 216 ± 16 157±5c 194 ± 10f
191 ± 6 201±7a 153±8c 177±7f,a
Each value is the mean ± SD (n ¼ 6).Values with a superscript are significantly different from the control group (a, p < 0.05; b, p < 0.01; c, p < 0.001) or from Ovx group (d, p < 0.05; e, p, <0.01; f, p < 0.001).
consistent with the earlier reports suggesting that ovariectomy increases food consumption and body mass [21,22]. Estradiol efficiently initiates a negative-feedback satiation signal and also thought to exert inhibitory effects on feeding by augmenting glucagon-mediated satiety signalling [23e25]. Also, the complex interaction between estrogen and leptin levels in the central nervous system and peripheral tissues also functions to control food intake, body weight, and adiposity [26,27]. Therefore, reduction in the estrogen levels as a consequence of post menopausal condition would cause inhibition of the satiety signals and thus, leads to excessive food intake, increase in the body weight and also the adiposity.
4. Discussion 4.2. Mechanism of zinc on bone resorption 4.1. Body weight gain Body weights of the animals of all the groups were found to increase progressively till the termination of the treatment period (Tables 1 and 2). However, the percentage body weight gain calculated was found to be consistently higher in the ovariectomized group in comparison to the rest of the groups. This increase in the body weight following ovariectomy could be attributed to the excessive intake of food known as hyperphagia. Our results are
Following ovariectomy, to confirm the efficacy of surgery protocol, the estradiol levels were evaluated. For this, the estradiol levels have been checked using competitive binding assay shown in our previous paper [17]. The results showed significant decrease in the estradiol levels following ovariectomy which gives the confirmation of the success of the surgery protocol. At a molecular level, the decline in the estradiol levels after ovariectomy might upregulate the production of several cytokines, differentiation and
Fig. 1. Electron micrographs of the trabecular region of the femur (endosteal surface) at a magnification (500) of the control group showing well organized trabeculae; zinc group showing densely formed trabecular arrangement; OVX group showing loss of trabecular connectivity; OVX þ Zinc group showing dense trabecular network. Arrows indicate resorbed areas.
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Fig. 2. Electron micrographs of the trabecular region of the tibia (endosteal surface) at a magnification (500) of the control group showing well organized trabecular network; Zinc group showing similar pattern of trabeculae as that of the control; OVX group showing large areas of resorption and poorly organized trabeculae network; OVX þ Zinc group showing well arranged trabeculae almost similar to that of control. Arrows indicate resorbed areas in the bone tissue. The resorbed areas in the trabecular region of ovariectomized bone.
Fig. 3. H and E stained light micrographs of the femur collagenous trabecular region of Control; Zinc; OVX and OVX þ Zinc group. Arrows indicate trabeculae.
maturation of osteoclast, and finally protection of osteoclast against apoptosis. Also, declined estogen levels will cause more secretion of RANKL from the osteoblast cells instead of osteoprotegrin protein. OPG is a decoy receptor which binds to the RANKL and prevents it from activating RANK. Thus, with estrogen loss after surgical
removal of ovaries, the osteoprotegrin levels decline, leaving RANKL to bind to RANK and allowing differentiation of osteoclasts. Osteoclasts will then develop ruffled border, get attached to the bone surface and cause bone resorption [28e31]. Zinc supplementation to the ovariectomized animals caused
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Table 4 Quantitative analysis of light micrographs of the trabecular region for all the treatment groups. Groups
Trabecular thickness (mm)
Control Zinc OVX OVX þ Zinc
207 225 124 175
± ± ± ±
21 19 14c 19f
Each value is the mean ± SD (n ¼ 6).Values with a superscript are significantly different from the control group (a, p < 0.05; b, p < 0.01; c, p < 0.001) or from Ovx group (d, p < 0.05; e, p, <0.01; f, p < 0.001).
increase in the estrogen levels suggesting the role of zinc in the secretion of estrogen from glands other than ovary like suprarenal glands and adipose tissues. Further studies need to be carried out to find out the exact mechanism as to how the zinc stimulates estrogen secretion from the other glands. Our previous study showed that Zinc directly improves the antioxidant defense system of bone and indirectly decreases oxidative stress by recovering estrogen levels. It has also been shown in the study that there was zinc loss from the bone tissue in the case of ovariectomized animals as found from the lowered zinc concentrations in the bone tissue as well as serum [17] that gets improved when zinc was supplemented externally to the animals in drinking water daily. Enhanced bone resorption might cause changes in the mineral content of the bone of which the most important one is the calcium. Calcium concentrations in the femur and tibia tissue were found to be significantly reduced following ovariectomy and improved upon zinc supplementation (Table 3). The improvement in the calcium concentration directly reflects the mineral portion of the bone tissue suggesting a plausible role of zincon bone metabolism. It has also been observed from the previous data that in the case of ovariectomy, zinc loss occurs as a result of enhanced bone resorption. When zinc was given to the OVX animals, improvement in the zinc and copper levels of bone
tissue causes increased osteoblastic activity [17] which further induces mineralization and cellular protein synthesis. 4.3. Micro architecture of bone Microarchitecture of bone can be a determinant of bone fragility independent of bone density [32]. Deterioration of the bone microstructure as a result of any pathological disorder, will cause substantial decrease in the bone strength and hence bone quality. To identify the structural changes accompanying estrogen deficiency-induced bone loss and the dietary effects on bone metabolism, we have examined the changes in cancellous bone microstructure using light microscopy and scanning electron microscopy in an ovariectomized osteopenic rat model. Increase in the number of resorbed areas in the OVX group could be due to the increased osteoclastic activity as indicated by increased TRAP-5b levels [17]. Increased number of resorbed areas in the OVX group is further substantiated by the decreased estrogen levels and increased oxidative stress observed in our previous study. Bone resorption following ovariectomy leads to dissolution of mineral component present in the bone and thus causing fragmentation of collagen matrix. Trabecular bone consists of rod or plate like structures known as trabeculae. Inter trabecular spaces are occupied by the bone marrow cells. Both the cortical and trabecular bone tissue have the similar functions but they differ in the geometry and arrangement. Furthermore, following ovariectomy, resorbed trabecular (Figs. 1e3) regions were observed. There were much larger portions of the endocortical surface lacking trabecular connections in the OVX animals. This is due to the enhanced bone resorption following ovariectomy. Zinc supplementation to the control animals showed similar well arranged pattern of the trabeculae as compared to the control. Administration of the zinc to the OVX rats markedly reduces bone loss as can be evident from the micrographs, showing dense network of trabeculae.
Fig. 4. Adipocytes in the bone marrow section (shown by arrow) in the Control, Zinc, OVX and OVX þ Zinc supplemented group (20).
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4.4. Adipogenesis
References
In the present work, adipose tissue content of the bone marrow of the bones was found to increase following ovariectomy (Fig. 4). This substantiates previous observations in the postmenopausal women [33] and the OVX rats [34]. An increased adipose tissue accumulation in the bone marrow has also been reported in association with age-related bone loss [35]. Osteopenic condition leads to the differentiation of adipocytes at the expense of osteogenic cells. Increase in the adipocytes can also be linked to an increased bone resorption. It has been reported earlier by Benayahu et al. [36] that adipocytes have the potential to stimulate osteoclast differentiation and, also secrete cytokines such as tumour necrosis factor-alpha (TNFea) and interleukin (IL-6). So from this, we can say that the increased adipocytes as observed in the OVX condition might have increased the levels of various cytokines which are required for the proliferation of osteoclast cells. The increase in the osteoclast cells following ovariectomy has been observed in our study as reported in the previous paper [17]. Zinc supplementation to the OVX animals decreased the number of adipocytes cells thereby suggesting the role of zinc in the differentiation of bone marrow cells into osteogenic cells rather than the adipocytes. Zinc ion may protect bone by decreasing adipocyte formation, which may indirectly promote osteoblast proliferation, mineral and bone formation, and also inhibiting osteoclast formation, activation and bone resorption [37]. Thereby summarizing the above mentioned outcomes, we can state that concomitance between increased bone marrow adipose cells number and osteoclasts activity, with reduced bone trabeculae of the estrogen deficient rats may suggest a role for adipose cells in ovariectomy induced bone loss. Zinc supplementation following ovariectomy has been shown to regulate the number of adipocytes, enhances bone calcium concentration and thus may help in normalizing the structure deficits to a greater extent. The improvement in the micro architecture could be due to the augmentation in the collagen synthesis as well as mineralization. Zinc has been shown to cause differentiation of osteoblasts from immature osteoblasts. Zinc directly activates aminoacyl-tRNA synthetase in osteoblast cells and stimulates collagen synthesis. This will cause the deposition of extracellular matrix which would later on mineralize [38]. This shows that zinc is playing a very important role in the bone mineral metabolism and hence exerts bone protecting effect when supplemented in postmenopausal condition.
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Asarian, Estradiol increases glucagon's satiating potency in ovariectomized rats, Am. J. Physiol. Regul. Integr. Comp. Physiol. 281 (2001) 1290e1294. [26] R. Torto, S. Boghossian, M.G. Dube, P.S. Kalra, S.P. Kalra, Central leptin gene therapy blocks ovariectomy-induced adiposity, Obes. (Silver Spring) 14 (2006) 1312e1319. [27] Q. Gao, G. Mezei, Y. Nie, Y. Rao, C.S. Choi, I. Bechmann, C. Leranth, D. ToranAllerand, C.A. Priest, J.L. Roberts, X.B. Gao, C. Mobbs, G.I. Shulman, S. Diano, T.L. Horvath, Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals, Nat. Med. 13 (2007) 89e94. [28] S. Bord, S. Beavan, D. Ireland, A. Horner, J.E. Compston, Mechanisms by which high-dose estrogen therapy produces anabolic skeletal effects in postmenopausal women: role of locally produced growth factors, Bone 29 (2001) 216e222. [29] B.F. Boyce, T. Yamashita, Z. Yao, Q. Zhang, F. Li, L. Xing, Roles for NF-kappa B and c-Fos in osteoclasts, J. Bone Min. Metab. 23 (2005) 11e15. [30] L.C. Hofbauer, C.A. Kuhne, V. Viereck, The OPG/RANKL/RANK system in metabolic bone diseases, J. Musculoskelet. Neuronal Interact. 4 (2004) 268e275. [31] Paola Narducci, Renato Bareggi, Vanessa Nicolin, Receptor activator for Nuclear factor kappa b Ligand (RANKL) as an osteoimmune regulator in bone
5. Conclusion Zinc is an important constituent of bone tissue and may certainly associates with the bone micro architecture. From this study, we confirm that zinc supplementation if given at early stages of bone loss will prevent the bone tissue deterioration. Conflict of statement The authors: Dr. Payal Bhardwaj, Prof. DV Rai and Prof. ML Garg state no conflict of interest. Acknowledgements Authors would like to acknowledge the financial assistance provided by Indian Council of Medical Research (ICMR, No. 3/1/2/4/ 10-RHN, dated 9th Aug 2010) in terms of Senior Research Fellowship (SRF) and contingency for the acquisition of various chemicals required to conduct the current research work.
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physiology and pathology, Acta Histochem. 113 (2011) 73e81. [32] L. Dalle Carbonare, S. Giannini, Bone microarchitecture as an important determination of bone strength, J. Endocrinol. Invest. 27 (2004) 99e105. [33] M.J. McKenna, Histomorphometric study of mast cells in normal bone, osteoporosis, and mastocytosis using a new stain, Calcif. Tissue Int. 55 (1994) 257e259. [34] Ph Lesclous, J.L. Saffar, Mast cells accumulate in rat bone marrow after ovariectomy, Cells Tissues Organs 164 (1999) 23e29. [35] M.E. Nuttall, A.J. Patton, D.L. Olivera, D.P. Nadeau, M. Gowen, Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders, J. Bone Min. Res. 13 (1998)
371e382. [36] D. Benayahu, A. Peled, D. Zipori, Myeloblastic cell line expresses osteoclastic properties following coculture with marrow stromal adipocytes, J. Cell Biochem. 56 (1994) 374e384. [37] M. Yamaguchi, M. Fukagawa, Role of zinc in regulation of protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells: zinc modulation of insulin-like growth factor-I’s effect, Calcif. Tissue Int. 76 (2005) 32e38. [38] Hyun-Ju Seo, Young-Eun Cho, Taewan Kim, Hong-In Shin, In-Sook Kwun, Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells, Nutr. Res. Pract. 4 (2010) 356e361.
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Zinc inhibits ovariectomy induced microarchitectural changes in the bone tissue Payal Bhardwaj a, *, Durg Vijay Rai a, b, Mohan Lal Garg a a b
Department of Biophysics, Panjab University, Chandigarh, India Faculty of Biomedical Engineering, Shobhit University, Meerut, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2015 Received in revised form 5 December 2015 Accepted 21 December 2015 Available online 23 December 2015
Purpose: The purpose of the current study was to examine the effect of zinc supplementation as a nutritional supplement in case of osteopenia induced microarchitectural changes in rat model. Methods: Forty eight animals in two batches of twenty four animals each were assigned to four groups: Control, Zinc, Ovariectomized (OVX) and OVX þ Zinc. The treatment period was continued for eight weeks. Histoarchitecture analysis was performed on both the bones i.e. femur and tibia using light as well as electron microscopy. Also, the bone calcium content was estimated using atomic absorption spectrophotometer. Results: The body weight of the animals in the OVX group was significantly higher in comparison to the control animals. The body weight was found to increase significantly upon zinc supplementation to OVX animals till the 4th week and then was almost comparable till the termination of treatment period. Calcium content in both femur and tibia were found to be significantly reduced in the ovariectomized group. The connectivity of trabeculae was lost following ovariectomy. Zinc administration restored bone calcium content as well bone tissue morphology including trabecular thickness. Conclusion: These findings suggest that changes in the trabecular bone attributed to estrogen deficiency are arrested by zinc supplementation. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Zinc Bone microarchitecture Ovariectomy Trabecular bone Electron microscopy Calcium
1. Introduction Osteoporosis is a skeletal disease characterised by low bone mass and structural weakening of the bone material that leads to reduced bone strength [1]. Postmenopausal osteoporosis is by far the commonest form of age-related bone loss. In women, bone loss is exacerbated after menopause due to a decrease in estrogen production. It is estimated that 10 million individuals have osteoporosis while another 34 million suffer from low bone density. Upon survey, it has been found that 61 million individuals will develop osteoporosis or low bone density by 2020 [2]. Beyond medical costs, there is the physical burden of living with osteoporosis and its impact on the daily life style, including restrictions in daily activities, loss of confidence (due to fear of falling and fracture) and loss of independence [3]. The development of osteoporosis is thought to be primarily
* Corresponding author. Department of Biophysics, Panjab University, Basic Medical Sciences block, Chandigarh, 160014, India. E-mail address: [email protected] (P. Bhardwaj).
related to ageing, genetic factors. Some modifiable factors, such as smoking, excess alcohol consumption, life style and deficiency or excess of some of the components of diet, are also associated with osteoporosis [4]. Therapies for osteoporosis fall into two categories: antiresorptive drugs, which slow bone resorption, and anabolic drugs, which stimulate bone formation. However, the adverse effects of these medications include malignant tumour formation with hormone therapy and gastrointestinal tolerance problems with bisphosphonates, which may exclude their long-term use [5,6]. Thus, there is a need for some alternative that can improve bone health without inducing adverse effects. Growing evidence of the benefits of natural foods for bone health presents alternatives for the prevention or treatment of osteoporosis [7,8]. Trace elements play a major role in the growth and development of skeleton, of which zinc is of particular interest to us. The fact that the organic component in the bone is mainly composed of protein and that most of the bone mineral portion is calcium implies that the essential nutrients required for bone health are protein and calcium [9]. In addition this, certain minerals, vitamins and trace metals are also required for the
http://dx.doi.org/10.1016/j.jnim.2015.12.333 2352-3859/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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maintenance of bone composition and microarchitecture [10]. Zinc is an essential trace element, is a component of 200 enzymes and is also well known to be necessary for normal mineralization and collagen synthesis in bone. Yamaguchi et al. studied the direct effects of zinc on the proliferative activity of bone cells and found that it has a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. Zinc produced remarkable increases in alkaline phosphatase activity and protein concentration in osteoblasts [11,12]. The role of zinc has been studied previous in terms of zinc deficiency or adequate zinc diet. Zn-deficient diets have impairment in growth plate chondrocyte proliferation, body length gain [13,14] and bone mass [15] in animals. However, to the best of our knowledge, little research on the association between zinc supplementation and bone micro architecture in the osteopenic condition has been conducted. To address this issue, the present study investigated the effect of zinc supplementation on bone calcium content and microarcitecture in osteopenic rat model.
2. Material and methods To carry out the present study, forty eight female Wistar rats (12 weeks old, 100e150g) in a batch of two were procured from the Central Animal House of Panjab University, Chandigarh. The animals were housed in polypropylene cages bedded with rice husk. They were given clean drinking tap water and standard animal pellet diet (Ashirwad Industries, Kharar, Punjab, India), throughout the experiment ad libitum. The animals were randomly divided into four groups comprising twelve animals in each group. They were acclimatized for one week to laboratory conditions prior to the start of the experimental procedure. Group 1 (Control) was given standard diet as well as drinking water. Group 2 (Zinc) was given zinc supplement as zinc sulphate in drinking water at a dose level of 227 mg of ZnSO4.7H2O per litre [16]. Group 3 (OVX) animals were bilaterally ovariectomized and were kept under normal feeding conditions without any zinc supplementation. Group 4 (OVX þ Zinc) animals were bilaterally ovariectomized and given zinc in drinking water (227 mg of ZnSO4.7H2O per litre). The treatment period was continued for eight weeks. The experimental design and procedures were approved by the Ethical Committee on Animal Experiments of the Central Animal House, Panjab University, Chandigarh, India. It was not possible to carry the measurements in a single batch. Therefore, measurements of all the parameters were done in two batches. These batches were segregated in different time over a period of one year. The first batch includes the ash content analysis and the second batch includes the histological analysis using light and electron microscopy.
2.1. Surgical procedure for bilateral ovariectomy Ovariectomy was performed in female Wistar rats by the method as described in our previous paper [17]. The ovariectomized Wistar rat model is the FDA recommended model and has been widely used for the rapid development of osteoporosis for purposes of investigating aspects of pathogenesis and treatment of postmenopausal bone loss [18]. At the completion of termination period, the animals were sacrificed by decapitation under anesthesia (using diethyl ether) and both the femora and tibiae were extracted. Bones of all the animals were cleared off from the muscle and cartilage and were further processed.
2.2. Elemental analysis The method of Szpunar et al. [19] was partially modified for bone digestion. Bone specimens were digested in concentrated nitric acid, and trace element analysis for the bone tissue was performed using an atomic absorption spectrophotometer (model 3100; PerkinElmer). Calcium hollow cathode lamp was operated at a slit width of 0.7 nm, which was selected to isolate 422.7 nm lines. Calcium standard (1000 ppm) was used for calibrating the system. 2.3. Micro architectural analysis Microscopy was done to analyze the microstructure of bone that includes arrangement and geometry of the trabecular region. Also, the adipocytes present in the bone marrow were analyzed using light microscopy. 2.3.1. Light microscopy 2.3.1.1. Bone sample preparation 2.3.1.1.1. Bone tissue preparation (fixation and embedding) for Hematoxylin-eosin staining. After excision, the trabecular regions (sectioned from the metaphyseal region along the growth plate) of the femora and tibia of all the groups were sectioned and fixed immediately in 4% formaldehyde prepared in 0.1M phosphate buffer (pH 7.2) overnight. After this, all the samples were decalcified in 10% Ethylene Diamine Tetra Acetic acid (EDTA) prepared in 0.05M TriseHCl buffer, pH 7.2. All the samples were removed from demineralising solution and washed in phosphate buffer saline. Then they were treated in five different gradients of alcohol (starting from 30% ethanol to absolute alcohol) for 1hr. After this they were dipped in absolute alcohol and benzene (1:1) for half an hour, only benzene for half an hour and then in wax for 6 h. Samples were then embedded in the paraffin wax (melting temperature 60 C) using plastic cassettes. Plastic cassettes were then inserted into the microtome so that the wax block faces the blade. The thin bone tissue sections (of about 4e10 mm thin) were then sliced and further processed for different staining protocols. HE staining was done to visualize the diaphyseal cortical bone thickness of both the femora and the tibia upon ovariectomy and zinc supplementation. After deparaffinising in xylene for about 15 min, the sections were immersed in the graded alcohol solutions (90% ethanol to 30% ethanol). After this the slides were dipped in Hematoxylin for 1e2 min. The slides were rinsed under running tap water in staining box until the water is no longer coloured (~5 min). Sections were again immersed in eosin stain for 1e2 min and rinsed until water became clear. Then again the slides were kept in gradient of ethanol and were shifted to xylene till they were cover slipped. Then the slides were viewed and analyzed under the light microscope (Leica). 2.3.1.2. Bone marrow preparation. Adipocytes were analyzed by extracting bone marrow cells from the femur bone. The marrow cells were then stained with h and e stain as according to the standard protocol. The stained sections were then analyzed under light microscope (Leica). 2.3.2. Scanning electron microscopy 2.3.2.1. Bone sample preparation. The bone samples were processed for the assessment of microarchitecture as per the method followed by Chang et al. [20]. Longitudinal section of all the bone samples (trabecular region sectioned from the metaphyseal growth plate) were immersed in a tissue fixative solution (4% formaldehyde in phosphate buffer saline) for at least one day and then rinsed with the Phosphate buffer saline (PBS) before characterization. For SEM, the bone was polished and etched in a hydrogen peroxide
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(35% H2O2) at 40 C for 2 h; the etched specimens were then dehydrated by 10 min immersions in five different concentrations (50%e100%) of ethanol solution. The dried bone specimens were mounted on the aluminium stubs (covered by carbon tape) and sputter-coated with gold. After this, the endosteal portion (trabecular region) of the femora and tibiae's microstructures were examined by scanning electron microscopy (Fine coat Ion sputter JSM-6100 JEOL) operating at 15 KV in the Sophisticated Analytical Instrumentation Facility, CIL, Panjab University, Chandigarh, India.
ovariectomized group when compared to the normal control. Femur (p < 0.001) and tibia (p < 0.001) calcium concentrations were enhanced in the OVX þ Zinc group and were restored to normal in the case of femur bone. However, the tibia calcium levels upon zinc supplementation to the OVX animals were not fully recovered (p < 0.05) when compared to the normal control animals. Zinc treatment alone to the control animals also showed increased calcium levels in both the bone tissues and the increase was significant (p < 0.05) in the case of tibia.
2.4. Statistical analysis
3.4. Scanning electron microscopy of the trabecular region of bone (both femur and tibia) tissue
Data are expressed as mean (SD). One-way analysis of variance was used to evaluate differences among groups. NewmaneKeuls post hoc test was used for multiple comparisons between groups. Differences were considered statistically significant at P < 0.05. The statistical analysis software SPSS (version 14) was adopted to process the data in the present study. 3. Results 3.1. Dose administered to each animal Zinc supplementation in the drinking water can be considered as an appropriate and customary method for supplementing nutrition. To calculate the dose of elemental zinc taken by an animal, each animal's daily consumption of drinking water was recorded which varied between 25 and 30 ml. Taking into consideration the daily consumption of drinking water and the average body weight of animals, we estimated zinc consumption to vary between 8 and 9 mg/kg body weight [17]. 3.2. Body weight measurement The variation in the body weights of all the animals subjected to different treatments are shown in Table 1. Body weights were measured at the beginning of treatment period and then were recorded at the 2nd, 4th and 8th week. The body weights of the animals of all the groups were found to increase progressively till the end of eighth week. At the second week, no weight change was observed in the OVX group when compared to the control group. At the 4th (p < 0.05) and 8th (p < 0.01) week, the mean body weight of the animals in the ovariectomized group was significantly higher in comparison to the control animals. Zinc supplementation to the OVX group showed increased body weight in comparison to the control at the 2nd and 4th week, and at the 8th week mean body weight of the animals was almost comparable to that of the control group. The whole data is also summarized as body weight gain (Table 2) for all the treatment groups. 3.3. Calcium content in bone tissue Mean values of calcium concentration in both femur (p < 0.001) and tibia (p < 0.001) were found to be significantly reduced in the
Figs. 1 and 2 show the scanning electron microscopic images of the trabecular portion of the femur and tibia endosteal region respectively in different groups. As seen in Figs. 1 and 2 at a magnification of 500, the femur and tibia bone of control animals consisted of dense and well organized trabecular bone tissue. No resorbed areas were identified in both the bones of the control group. Micrographs of both the bones of ovariectomized group had resorbed trabecular region. The connectivity of trabeculae is lost following ovariectomy. The connectivity is more disrupted in the case of tibia when compared to the femur group. Zinc supplementation to the control animals showed similar well arranged pattern of the trabeculae as compared to the control. Administration of the zinc to the OVX rats markedly reduces bone loss as is evident from the micrographs showing dense network of trabeculae. 3.5. Hematoxylin and eosin staining of the trabecular region H and E stained sections of the trabecular portion of the rat femur bone at the lower magnification (5), revealed normal trabecular architecture with the preservation of trabecular plate and interconnectivity between trabeculae. Bone marrow cells were seen in the spaces between the trabeculae (Fig. 3). However, as can be seen from the micrograph of the OVX group, there was deterioration and the thinning of the trabecular bone structure accompanied by the increase in the bone marrow. In addition, there was widening of the spaces between the trabeculae as a result of trabecular bone loss. Zinc supplementation to the OVX animals resulted in thickening of the trabecular network making it dense as that of the control bone (Table 4). Zinc supplementation to the normal control animals maintained the connectivity of the trabeculae, similar to that of the control bone. 3.6. Bone marrow cells (adiposity) The ovariectomized group exhibited a significant elevation in the number of adipocytes as compared to the control group. Also, it can be observed that ovariectomy leads to the formation of large sized adipocytes (Fig. 4). Zinc supplementation to the OVX animals down regulated the adipocyte number. However, the adipocyte number remains almost similar to that of the control group when zinc was supplementation alone to the control animal.
Table 1 Body weight of all the four groups at specific time intervals after surgery. Groups
Initial body weight (g)
Control Zinc OVX OVX þ zinc
130 126 130 130
± ± ± ±
7 5 7 11
Body weight (g) after 2nd week 150 149 155 165
± ± ± ±
8 9 17 12a
Body weight (g) after 4th week 177 176 192 202
± ± ± ±
8 8 16a 14c
Body weight (g) after 8th week 217 215 239 245
± ± ± ±
9 7 19b 15
Each value is the mean ± SD (n ¼ 6).Values with a superscript are significantly different from the control group (a, p < 0.05; b, p < 0.01; c, p < 0.001) or from Ovx group (d, p < 0.05; e, p, <0.01; f, p < 0.001).
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Table 2 Percentage body weight gain during the treatment period of all the four groups at specific time intervals after surgery. Groups
Body weight gain after 2nd week
Body weight gain after 4th week
Body weight gain after 8th week
Control Zinc OVX OVX þ Zinc
15 18 19 27
18 18 24 22
23 22 24 21
Table 3 Effect of OVX and zinc supplementation on the calcium concentrations of femur and tibia tissue. Groups
Femur calcium (mg/g of tissue)
Tibia calcium (mg/g of tissue)
Control Zinc Ovx Ovx þ Zinc
204 ± 14 216 ± 16 157±5c 194 ± 10f
191 ± 6 201±7a 153±8c 177±7f,a
Each value is the mean ± SD (n ¼ 6).Values with a superscript are significantly different from the control group (a, p < 0.05; b, p < 0.01; c, p < 0.001) or from Ovx group (d, p < 0.05; e, p, <0.01; f, p < 0.001).
consistent with the earlier reports suggesting that ovariectomy increases food consumption and body mass [21,22]. Estradiol efficiently initiates a negative-feedback satiation signal and also thought to exert inhibitory effects on feeding by augmenting glucagon-mediated satiety signalling [23e25]. Also, the complex interaction between estrogen and leptin levels in the central nervous system and peripheral tissues also functions to control food intake, body weight, and adiposity [26,27]. Therefore, reduction in the estrogen levels as a consequence of post menopausal condition would cause inhibition of the satiety signals and thus, leads to excessive food intake, increase in the body weight and also the adiposity.
4. Discussion 4.2. Mechanism of zinc on bone resorption 4.1. Body weight gain Body weights of the animals of all the groups were found to increase progressively till the termination of the treatment period (Tables 1 and 2). However, the percentage body weight gain calculated was found to be consistently higher in the ovariectomized group in comparison to the rest of the groups. This increase in the body weight following ovariectomy could be attributed to the excessive intake of food known as hyperphagia. Our results are
Following ovariectomy, to confirm the efficacy of surgery protocol, the estradiol levels were evaluated. For this, the estradiol levels have been checked using competitive binding assay shown in our previous paper [17]. The results showed significant decrease in the estradiol levels following ovariectomy which gives the confirmation of the success of the surgery protocol. At a molecular level, the decline in the estradiol levels after ovariectomy might upregulate the production of several cytokines, differentiation and
Fig. 1. Electron micrographs of the trabecular region of the femur (endosteal surface) at a magnification (500) of the control group showing well organized trabeculae; zinc group showing densely formed trabecular arrangement; OVX group showing loss of trabecular connectivity; OVX þ Zinc group showing dense trabecular network. Arrows indicate resorbed areas.
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Fig. 2. Electron micrographs of the trabecular region of the tibia (endosteal surface) at a magnification (500) of the control group showing well organized trabecular network; Zinc group showing similar pattern of trabeculae as that of the control; OVX group showing large areas of resorption and poorly organized trabeculae network; OVX þ Zinc group showing well arranged trabeculae almost similar to that of control. Arrows indicate resorbed areas in the bone tissue. The resorbed areas in the trabecular region of ovariectomized bone.
Fig. 3. H and E stained light micrographs of the femur collagenous trabecular region of Control; Zinc; OVX and OVX þ Zinc group. Arrows indicate trabeculae.
maturation of osteoclast, and finally protection of osteoclast against apoptosis. Also, declined estogen levels will cause more secretion of RANKL from the osteoblast cells instead of osteoprotegrin protein. OPG is a decoy receptor which binds to the RANKL and prevents it from activating RANK. Thus, with estrogen loss after surgical
removal of ovaries, the osteoprotegrin levels decline, leaving RANKL to bind to RANK and allowing differentiation of osteoclasts. Osteoclasts will then develop ruffled border, get attached to the bone surface and cause bone resorption [28e31]. Zinc supplementation to the ovariectomized animals caused
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Table 4 Quantitative analysis of light micrographs of the trabecular region for all the treatment groups. Groups
Trabecular thickness (mm)
Control Zinc OVX OVX þ Zinc
207 225 124 175
± ± ± ±
21 19 14c 19f
Each value is the mean ± SD (n ¼ 6).Values with a superscript are significantly different from the control group (a, p < 0.05; b, p < 0.01; c, p < 0.001) or from Ovx group (d, p < 0.05; e, p, <0.01; f, p < 0.001).
increase in the estrogen levels suggesting the role of zinc in the secretion of estrogen from glands other than ovary like suprarenal glands and adipose tissues. Further studies need to be carried out to find out the exact mechanism as to how the zinc stimulates estrogen secretion from the other glands. Our previous study showed that Zinc directly improves the antioxidant defense system of bone and indirectly decreases oxidative stress by recovering estrogen levels. It has also been shown in the study that there was zinc loss from the bone tissue in the case of ovariectomized animals as found from the lowered zinc concentrations in the bone tissue as well as serum [17] that gets improved when zinc was supplemented externally to the animals in drinking water daily. Enhanced bone resorption might cause changes in the mineral content of the bone of which the most important one is the calcium. Calcium concentrations in the femur and tibia tissue were found to be significantly reduced following ovariectomy and improved upon zinc supplementation (Table 3). The improvement in the calcium concentration directly reflects the mineral portion of the bone tissue suggesting a plausible role of zincon bone metabolism. It has also been observed from the previous data that in the case of ovariectomy, zinc loss occurs as a result of enhanced bone resorption. When zinc was given to the OVX animals, improvement in the zinc and copper levels of bone
tissue causes increased osteoblastic activity [17] which further induces mineralization and cellular protein synthesis. 4.3. Micro architecture of bone Microarchitecture of bone can be a determinant of bone fragility independent of bone density [32]. Deterioration of the bone microstructure as a result of any pathological disorder, will cause substantial decrease in the bone strength and hence bone quality. To identify the structural changes accompanying estrogen deficiency-induced bone loss and the dietary effects on bone metabolism, we have examined the changes in cancellous bone microstructure using light microscopy and scanning electron microscopy in an ovariectomized osteopenic rat model. Increase in the number of resorbed areas in the OVX group could be due to the increased osteoclastic activity as indicated by increased TRAP-5b levels [17]. Increased number of resorbed areas in the OVX group is further substantiated by the decreased estrogen levels and increased oxidative stress observed in our previous study. Bone resorption following ovariectomy leads to dissolution of mineral component present in the bone and thus causing fragmentation of collagen matrix. Trabecular bone consists of rod or plate like structures known as trabeculae. Inter trabecular spaces are occupied by the bone marrow cells. Both the cortical and trabecular bone tissue have the similar functions but they differ in the geometry and arrangement. Furthermore, following ovariectomy, resorbed trabecular (Figs. 1e3) regions were observed. There were much larger portions of the endocortical surface lacking trabecular connections in the OVX animals. This is due to the enhanced bone resorption following ovariectomy. Zinc supplementation to the control animals showed similar well arranged pattern of the trabeculae as compared to the control. Administration of the zinc to the OVX rats markedly reduces bone loss as can be evident from the micrographs, showing dense network of trabeculae.
Fig. 4. Adipocytes in the bone marrow section (shown by arrow) in the Control, Zinc, OVX and OVX þ Zinc supplemented group (20).
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4.4. Adipogenesis
References
In the present work, adipose tissue content of the bone marrow of the bones was found to increase following ovariectomy (Fig. 4). This substantiates previous observations in the postmenopausal women [33] and the OVX rats [34]. An increased adipose tissue accumulation in the bone marrow has also been reported in association with age-related bone loss [35]. Osteopenic condition leads to the differentiation of adipocytes at the expense of osteogenic cells. Increase in the adipocytes can also be linked to an increased bone resorption. It has been reported earlier by Benayahu et al. [36] that adipocytes have the potential to stimulate osteoclast differentiation and, also secrete cytokines such as tumour necrosis factor-alpha (TNFea) and interleukin (IL-6). So from this, we can say that the increased adipocytes as observed in the OVX condition might have increased the levels of various cytokines which are required for the proliferation of osteoclast cells. The increase in the osteoclast cells following ovariectomy has been observed in our study as reported in the previous paper [17]. Zinc supplementation to the OVX animals decreased the number of adipocytes cells thereby suggesting the role of zinc in the differentiation of bone marrow cells into osteogenic cells rather than the adipocytes. Zinc ion may protect bone by decreasing adipocyte formation, which may indirectly promote osteoblast proliferation, mineral and bone formation, and also inhibiting osteoclast formation, activation and bone resorption [37]. Thereby summarizing the above mentioned outcomes, we can state that concomitance between increased bone marrow adipose cells number and osteoclasts activity, with reduced bone trabeculae of the estrogen deficient rats may suggest a role for adipose cells in ovariectomy induced bone loss. Zinc supplementation following ovariectomy has been shown to regulate the number of adipocytes, enhances bone calcium concentration and thus may help in normalizing the structure deficits to a greater extent. The improvement in the micro architecture could be due to the augmentation in the collagen synthesis as well as mineralization. Zinc has been shown to cause differentiation of osteoblasts from immature osteoblasts. Zinc directly activates aminoacyl-tRNA synthetase in osteoblast cells and stimulates collagen synthesis. This will cause the deposition of extracellular matrix which would later on mineralize [38]. This shows that zinc is playing a very important role in the bone mineral metabolism and hence exerts bone protecting effect when supplemented in postmenopausal condition.
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5. Conclusion Zinc is an important constituent of bone tissue and may certainly associates with the bone micro architecture. From this study, we confirm that zinc supplementation if given at early stages of bone loss will prevent the bone tissue deterioration. Conflict of statement The authors: Dr. Payal Bhardwaj, Prof. DV Rai and Prof. ML Garg state no conflict of interest. Acknowledgements Authors would like to acknowledge the financial assistance provided by Indian Council of Medical Research (ICMR, No. 3/1/2/4/ 10-RHN, dated 9th Aug 2010) in terms of Senior Research Fellowship (SRF) and contingency for the acquisition of various chemicals required to conduct the current research work.
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