ORIGINAL ARTICLE ABSTRACT Objective: To determine the effect of zinc on salt induced bone damage in rats. Study Design: Laboratory based randomized control trial. Place and Duration of Study: The Anatomy department of Islamic International Medical College, Rawalpindi, hosted the conduction of research with the cooperation of National Institute of Health, Islamabad. The study th th commenced on 17 September 2015 and completed on 17 March 2016. Materials and Methods: Forty five female Sprague Dawley rats, 10-12 weeks old were used in the study. The animals were randomly divided into 3 groups. The rats in experimental group A fed on high salt diet (8%NaCl) whereas animals in experimental group B were given high salt diet supplemented with zinc (50mg/kg/day) for eight weeks however, the diet of control group was not tempered with. Blood samples were drawn at the start of intervention through tail vein and at the end of the experiment by intracardiac puncture for hormonal assay. All rats were dissected, left humeri and femora were removed, decalcified and five micrometer (µm) sections were obtained after tissue processing. Tissues were stained with Haematoxylin and eosin (H&E) for histological parameters. The quantitative data was analyzed by using Statistical Package for Social Sciences (SPSS) version 21 and was expressed as Mean + S.D. One Way Analysis of Variance (ANOVA) followed by Post hoc tukey test was applied for inter group comparison of parameters. T-test was applied for intragroup comparison of values. Result having p-value <0.05 was considered statistically significant. Results: Marked histological changes were identified in the experimental groups. These changes were of greater severity in high salt diet group as compared to the zinc supplemented group in which reverse beneficial effects were observed. Fall in serum calcium and alkaline phosphatase levels were deemed substantial in group A with respect to group B. Conclusion: Zinc has a Protective role against High salt exposed diet induced damage on the histomorphology of bone tissue. Key Words: Cortical Bone, Hypercalciuria, Osteoblast, Salt, Zinc. researches, precise associations of the trace elements with bone health are not clear as yet. Inverse of negative balance between bone formation and resorption has been evaluated with the help of 3 trace elements. st Low bone mass is a silent epidemic of the 21 century and figures are set to increase worldwide. Considering the elements which affect bone metabolism is of utmost importance for the prevention of osteoporosis. Although nutrition is an important determinant of bone health, but the 4 effects of the micronutrients is little understood. Bone is a systematized tissue which acclimates and changes according to certain factors and its organization varies due to diverse functional 5 requirements. The net result of unaltered healthy bone mass is sustained by a balanced bone 6 formation and resorption activity. Imbalance results in a progressive metabolic ailment called 7 8 osteoporosis, becoming a public health problem, Introduction The world is under continuous threat of increase 1 d i e t - re l a t e d n o n - c o m m u n i c a b l e a i l m e n t s . Unbalanced and excessive salt intake is often closely associated with development of hypertension and 2 other cardiovascular diseases. However, awareness regarding relationship of zinc to sodium induced osteoporosis is still in a gray area. Despite of previous Effect of Zinc on Salt Induced Impaired Remodeling in Long Bones of Rats 1 2 3 Kaukab Anjum , Rehana Rana , Sumaira Abbasi JIIMC 2016 Vol. 11, No.3 Correspondence: Dr. Kaukab Anjum Assistant Professor, Anatomy Wah Medical College, WahCantt E-mail: kanjumq@gmail.com 1 Department of Anatomy Wah Medical College, WahCantt 2 Department of Anatomy Islamic International College Riphah International University, Islamabad 3 Department of Anatomy Federal Medical & Dental College, Islamabad Funding Source: NIL ; Conflict of Interest: NIL Received: Mar 24, 2016; Revised: Apr 07, 2016 Accepted: Aug 01, 2016 Effect of Zinc on Bones of Salt Loaded Rats 113 9 u ps ett i n g 2 0 0 m i l l i o n p e o p l e wo r l d w i d e . Characterized by lessened structural integrity and proneness to fractures, it is more prevailing than 10 myocardial infarct, breast cancer and stroke It is imperative to explore and develop nutritional strategies for osteoporosis prevention as the life threatening outcomes and increase in annual cost associated with disease morbidity requires a quick 11 fix. 12 Salt being most ubiquitous of food flavorings and a 13 known risk factor for osteoporosis, imposes hazards on human wellbeing. High urinary excretion of calcium with increase salt intake leads to impaired 14 bone health. Human population has exceeded the daily limit of 15 2000 mg of Na /day as recommended by WHO. Different communities have different intakes (Western 2300-4300 mg Na/day , Asian 5300mg- 16 6000mg of Na/ day) Sodium in this range is adversely affecting people including osteoporosis, hypertension , increase urinary tract stones and 17 stroke. It took 75 years to realize that zinc is a crucial trace 18 element although it has been used therapeutically in Ayurveda but its nutritional significance in public 19 health was recognized recently. As it is a vital 20 element and human body contains only 2-3 grams. 21 even a small deficiency is a disaster. Zinc can be a hidden link for the prevention of osteoporosis due to 22 its regulatory role in bone metabolism. It has the ability to stimulate the differentiation and proliferation of osteoblasts and inhibiting osteoclast 22 like cells formation from bone marrow Zinc ,by stimulating apoptotic cell death of mature osteoclasts can inhibit bone resorption and have 23 direct positive effect on bone metabolism. Other than bones which act as a zinc sink zinc is stored in 24 muscles and skin. So free available quantity is negligible and only food source can be utilized when 25 required to prevent conditions like bone loss, 26 27 gastric ulcers night blindness. Therefore, this experimental study will highlight the potential benefits of Zn supplementation in reducing bone loss more accurately and eventually will give desired awareness to masses regarding positive link between zinc and bone health. Materials and Methods The study was a laboratory based randomized control trial carried out in the Anatomy department of Islamic International Medical College Rawalpindi. It was initiated after the approval of the Ethical Review Committee. The research was carried out with the collaboration of National Institute of Health (NIH) Islamabad and Army Medical College. It took six months to complete this study. Inclusion criteria were forty five, 12 weeks old, adult female Sprague Dawley rats weighing 250-300g. Pregnancy, male rats and any evident pathology were also considered as exclusion factors. Forty five rats grouped by using random number table method, selected by non-probability convenient sampling, were randomly divided in to three groups (15 animals in each group) and were allowed to adjust in well aired new environment in a temperature range of 20-26°C. The rats in group A 28 (N=15) were given diet having 8% NaCl for eight weeks. Rats in group B (N=15) were given high salt diet supplemented with zinc at a dose of 50mg/kg 29 body weight. The rats of group C (N=15) served as controls, they were given standard laboratory diet. Water was provided ad libitum. The dose of NaCl and Zinc was set based on previous studies. Dissection was done after eight weeks. Blood was drawn through intracardiac puncture for assessing serum calcium and alkaline phosphatase (ALP) level at the end of intervention. The left humeri and femora of rats were removed and immediately fixed in 10% neutral buffered formaldehyde for 2 days. Decalcification was performed using aqueous solution of 5-10% nitric acid for 24-48 hours. Transverse sections from the mid diaphysis were obtained, processed and embedded in paraffin wax 30 to form blocks. Five μm thick sections were obtained by mounting blocks on rotary microtome. Haematoxylin and eosin was used for histological study of specimen. Cortical bone thickness of diaphysis of humeri and femora was measured with the help of ocular micrometer. The thickness of cortical bone was measured by counting the number of divisions of eye piece of linear ocular micrometer, placed perpendicularly from underneath the periosteum to endosteum. Cortical bone width of opposite side was measured in a same manner per section under 4X objective and results were averaged. Parametric data was analyzed by using Statistical JIIMC 2016 Vol. 11, No.3 Effect of Zinc on Bones of Salt Loaded Rats 114 Package for Social Sciences (SPSS) version 21. Quantitative data was expressed as Mean + S.D. One Way Analysis of Variance (ANOVA) followed by Post hoc tukey test was applied for inter group comparison of parameters.t-test was applied for intra group comparison of values. Result having p- value <0.05 was considered statistically significant. Results Mean thickness of the humeral cortical bone was 5 3 . 7 6 6 ± 9 . 0 6 6 μ m i n c o n t r o l g r o u p C , 53.666±7.596μm in experimental group B and lowest of all, 41.8000±15.254 μm in experimental group A. The results were significant (p˂0.05) amongst different groups (Table I) (Fig 2, 3). The difference between group C and A was 11.966 μm, being highly significant (p=0.014).The result between group C and B was insignificant (p=1.000) with difference of 0.100μm. The mean cortical thickness of group B was greater than group A with difference of -11.866μm (p˂0.05) (Table II) (Fig 1). Mean thickness of the femoral cortical bone was 4 4 . 6 0 0 ± 8 . 4 3 7 μ m i n c o n t r o l g r o u p C , 39.366±10.677μm in experimental group B and lowest of all, 30.433±9.350μm in experimental group A. The results of difference in cortical bone thickness were significant between groups (p˂0.05). The difference between group C and A was 14.166 μm, the result was highly significant (p=0.001).The insignificant difference of 5.233μm (p=0.300) was recorded between group C and B. The mean of thickness was greater in group B than group A difference being -8.933μm (p=0.036). Mean random initial and final serum calcium was 8.680±0.90333 mg/dl and 8.5333±0.9559mg/dl in control group C, 7.826±0.6123 mg /dl and 7.153±1.364mg/dl in experimental group A and 8.666±0.952 mg/dl and 8.816±0.9635mg/dl in experimental group B Initial calcium levels revealed p-value of 0.010 whereas the final levels were different in all groups (p=0.004).The mean difference between initial and final value in Control group C was 1.3800, 0.3466 in experimental group A and -1.0333 in experimental group B. Decrease in calcium level was highly significant between experimental group C and A (p=0.004), insignificant (p=0.672) between group C and B and there is significant result between group A and group B (p=0.038) (Table IV). Initial and final mean serum alkaline phosphatase level was 487.800±51.669 U/L and 478.066±53.620 U/L in control group C, 466.200±45.874U/L and 349.9333±56.0484 U/L in experimental group A and 486.066±47.373 U/L and 416.666±62.009 U/L in experimental group B (Table III) (Fig 5). Initial Serum alkaline phosphatase showed inconsequential value in all the groups (p=0.405) whereas the final levels were significant (p=0.000) The mean of difference between initial and final value in Control group C was 9.7333 U/L,116.2666 U/L in experimental group A and 69.4000 U/L in experimental group B. Comparison among groups demonstrated the highest decrease in alkaline phosphatase level between group C and A being 109.2000 U/L with significant value (p=0.000).The mean of decrease between group C and group B was 42.4666 U/L (p=0.021) which was less group A and B -66.7333 U/L (p=0.038 Table II: Mean Cor�cal Bone thickness in Humerus and femur (µm) of all groups Table I: Mul�ple comparison of cor�cal bone thickness among all groups of Humerus and Femur by Post Hoc Tukey test Discussion Bone acclimates and changes under the influence of certain elements and its organization varies due to 5 diverse functional requirements. The healthy bone mass is sustained by a balanced between bone JIIMC 2016 Vol. 11, No.3 Effect of Zinc on Bones of Salt Loaded Rats *p<0.05 115 6 formation and resorption activity. Life style, genetic and dietary factors have impact on its prevalence. Although dietary factors have limited influence but are nonetheless crucial because they modulate the achievement of maximum peak bone mass and subsequent better bone health. By developing nutritional strategies for osteoporosis prevention, the annual cost and debilitation associated with its morbidity can be lessened. The present study focused on determining the beneficial effects of zinc on high salt diet induced bone damage in long bones of rats by observing m i c ro s c o p i c q u a n t i tat i ve a n d b i o c h e m i c a l parameters. The results suggested that zinc supplementation can prevent the high salt induced deleterious effects on bones. 31 ALP is a marker enzyme of osteoblast activity 32 reevaluated by Ahmed who documented the decrease in calcium, ALP levels and subsequent impaired bone integrity after salt loaded diet. Decrease in the osteoblast activity due to salt overload can be the reason of low ALP levels. Furthermore, decline in the ALP activity has been demonstrated in animal models of experimental 33 induced osteoporosis. As ˃ 99% of Na and 95% of the calcium are reabsorbed in the kidneys, it is speculated that impaired renal function may be responsible for Na induced calciuria and temporarily 14 depress calcium levels. Substandard kidney function also causes hypophosphatemia and fall in 1, 25 (OH) 2 D3. All these events lead to less intestinal absorption of calcium as well as decrease availability 16 to bones. Reduction in the biomarkers of bone formation (ALP) and significant increase in the biomarkers of bone resorption has been observed due to high PTH secretion secondary to low calcium levels and consequently increase in bone 16 remodeling. In line with other publications, 34 Creedon also observed the decrease in calcium levels due to sodium induced increase urinary excretion of calcium. As a compensatory mechanism, the PTH secretion increases which causes calcium mobilization from bones at the expense of bone 15 loss. 22 As Zn is a cofactor of ALP which is an enzyme expressed by osteoblasts close to the blood vessels and is a valuable index for bone tissue development. Administration of zinc results in increase of enzyme Fig 1 : Cross-sec�on of Humerus diaphysis of A13 showing decreased cor�cal bone thickness (CBT). H&E, X4. Fig 2: Cross-sec�on of Humerus diaphysis of B7 showing increased cor�cal bone thickness (CBT). H&E, X4. Table III: Ini�al-final serum calcium (mg/dl) and Alkaline Phosphatase (U/L) level of all groups *p ˂ 0.05 Table IV: Mul�ple comparison of final calcium (mg/dl) and Alkaline Phosphatase (U/L) level *p ˂ 0.05 JIIMC 2016 Vol. 11, No.3 Effect of Zinc on Bones of Salt Loaded Rats 116 35 activity indicating enhance osteoblastic activity. Increase in levels of Calcium and ALP with significant difference (p˂0.05) in the present study is also 36 validated by Otsuka who observed increase in levels after measured zinc discharge on bone mineral density from injectable Zn-containing B-Tricalcium Phosphate. It could be attributed to intensified differentiation of osteoblastic cells to raise ALP 37 activity. Zinc plays an important role in preventing osteoporosis by stimulating bone formation, 31 reported by Ma by demonstrating increase in calcium and ALP in the femoral-diaphyseal and metaphyseal tissues. Decrease in calcium content by bone resorbing factors can be prevented by zinc 19 supplementation. Our outcome is in agreement with above results, further firming up my research. Cross section of long bones reveals four different bone types: periosteum, cortical bone, endosteum and cancellous bone. Femur diaphysis is mainly 38 composed of compact bone and cancellous bone forms a very thin layer on the inner aspect of 5 diaphysis of long bones. The cortical bone thickness is an important parameter to evaluate bone quality 39 and strength so in the present study the bone damage is assessed by measuring the cortical bone thickness in cross sections of mid diaphysis. It is revealed that humerus and femur of control group has maximum thickness of 50.7um and 44.6um respectively followed by experimental group B who took salt and zinc supplementation whereas the lowest dimensions are found in experimental group A fed on high salt diet. 32 My results are in harmony with the work of Ahmed who observed decline in the thickness of cortical bone of rats. He anticipated that high salt intake can be related with increased plasma levels of creatinine, urea, phosphate and potassium due to deranged kidney function which finally led to bone changes. Furthermore increased serum phosphate inhibits 1αhydroxylase and produced fall in 1, 25(OH) 2 D3. As a result intestinal absorption of calcium is decreased with subsequent increase in PTH secretion leading to increase osteoclastic activity. Degenerative changes in osteoblasts, osteocytes and hyperactivity of osteoclasts results in inaccurate bone remodeling with decrease in cortical bone 40 thickness. Changes in bone remodeling which is mediated by bone cells, increased osteoclastic activity and multiple resorption cavities can be the 41 reason of decrease in the thickness of cortical bone. My result is in conformity with the results of all above periodicals sharing a common point that salt intake results in osteoporosis with decrease in cortical bone thickness. Increase in cortical bone thickness after zinc supplementation in experimental group B is documented in the present study. As many published studies has confirmed that zinc has positive role in improving bone health , it is further strengthened by 22 Brzoska who reported the shielding effect of zinc diet on bone homeostasis. He postulated that increase in the bone alkaline phosphatase activity may be due to zinc adequacy. Increase in the osteocalcin level produced by osteoblasts after zinc supplemented diet might have resulted in increase in 42 cortical bone thickness. Zinc is required for growth of osteoblasts and zinc showed decreased was bone 43 resorption. Conclusion This research indicates that zinc supplementation can be considered an appropriate dietary strategy to reduce risk of osteoporosis. Cortical bone thickness, alkaline phosphatase activity and calcium levels w e r e c o n s i d e r a b l y i n c r e a s e d a f t e r z i n c administration showing that zinc has protective role against high salt induced impaired remodeling in long bones of rats. Recommendations Effects of high salt diet can be studied for longer period of time to assess significant gross changes in long bones of rats. Effects of highs salt and zinc can be observed on the osteocytes apoptosis to evaluate their role in development and prevention of osteoporosis. Comparison of high salt diet induced effects can be studied between male and female rats to assess the difference in the degree of damage. REFERENCES 1. Boutayeb A, Boutayeb S. The burden of non communicable diseases in developing countries. International journal for equity in health. 2005; 4: 1. 2. Strazzullo P, D Elia L, Kandala NB, Cappuccio FP. Salt intake, stroke, and cardiovascular disease: meta-analysis of prospective studies. Bmj. 2009; 339. 3. Aaseth J, Boivin G, Andersen O. Osteoporosis and trace elements–an overview. Journal of Trace Elements in Medicine and Biology. 2012; 26: 149-52. JIIMC 2016 Vol. 11, No.3 Effect of Zinc on Bones of Salt Loaded Rats 117 4. Jugdaohsingh R. Silicon and bone health. The journal of nutrition, health & aging. 2007;11: 99. 5. Cvetkovic V, Najman S, Rajkovic J, Zabar AL, Vasiljevic P, Djordjevic LB, et al. A comparison of the microarchitecture of lower limb long bones between some animal models and humans: a review. Veterinarni Medicina. 2013; 58: 339-51. 6. Feng X, McDonald JM. Disorders of bone remodeling. Annual review of pathology. 2011;6: 121. 7. Watanabe K, Ikeda K. Osteocytes in normal physiology and osteoporosis. Clinical Reviews in Bone and Mineral Metabolism. 2010; 8: 224-32. 8. Ovesen J, Moller Madsen B, Thomsen JS, Danscher G, Mosekilde L. The positive effects of zinc on skeletal strength in growing rats. Bone. 2001; 29: 565-70. 9. Frings Meuthen P, Buehlmeier J, Baecker N, Stehle P, Fimmers R, May F, et al. High sodium chloride intake exacerbates immobilization-induced bone resorption and protein losses. Journal of Applied Physiology. 2011; 111: 537-42. 10. Haddad PT, Salazar M, Hernandes L. Histomorphometry of the organic matrix of the femur in ovariectomized rats treated with sodium alendronate. Revista Brasileira de Ortopedia (English Edition). 2015; 50: 100-4. 11. Hazenberg JG, Taylor D, Lee TC. The role of osteocytes and bone microstructure in preventing osteoporotic fractures. Osteoporosis international. 2007; 18: 1-8. 12. Feldman SR. Sodium chloride. Kirk-Othmer encyclopedia of chemical technology. 2005. 13. Lu L, Cheng Q, Chen J, Yang G, Wan C, Zhang Y, et al. The influence of dietary sodium on bone development in growing rats. Archives of animal nutrition. 2011; 65: 486-96. 14. Teucher B, Fairweather-Tait S. Dietary sodium as a risk factor for osteoporosis: where is the evidence? Proceedings of the Nutrition Society. 2003; 62: 859-66. 15. He F, MacGregor G. A comprehensive review on salt and health and current experience of worldwide salt reduction programmes. Journal of human hypertension. 2009; 23: 363-84. 16. Heaney RP. Role of dietary sodium in osteoporosis. Journal of the American College of Nutrition. 2006; 25: 271S-6S. 17. Teucher B, Dainty JR, Spinks CA, Majsak Newman G, Berry DJ, Hoogewerff JA, et al. Sodium and bone health: impact of moderately high and low salt intakes on calcium metabolism in postmenopausal women. Journal of Bone and Mineral Research. 2008; 23: 1477-85. 18. Kaur K, Gupta R, Saraf SA, Saraf SK. Zinc: The metal of life. Comprehensive Reviews in Food Science and Food Safety. 2014; 13: 358-76. 19. Molokwu CO, Li YV. Zinc homeostasis and bone mineral density. Obio Research and Clinical Review, Fall. 2006; 15: 7- 15. 20. Khadeer MA, Sahu SN, Bai G, Abdulla S, Gupta A. Expression of the zinc transporter ZIP1 in osteoclasts. Bone. 2005; 37: 296-304. 21. Bhowmik D, Chiranjib K. A potential medicinal importance of zinc in human health and chronic. Int J Pharm. 2010; 1: 5- 11. 22. Brzóska MM, Rogalska J, Galażyn Sidorczuk M, Jurczuk M, Roszczenko A, Kulikowska Karpińska E, et al. Effect of zinc supplementation on bone metabolism in male rats chronically exposed to cadmium. Toxicology. 2007; 237: 89- 103. 23. Yamaguchi M. Osteoporosis Treatment with New Osteogenic Factors. Journal of Molecular and Genetic Medicine. 2013; 7: 1. 24. Yamaguchi M. Role of nutritional zinc in the prevention of osteoporosis. Molecular and cellular biochemistry. 2010; 338: 241-54. 25. Maki K, Nishioka T, Nishida I, Ushijima S, Kimura M. Effect of zinc on rat mandibles during growth. American journal of orthodontics and dentofacial orthopedics. 2002;122: 410- 3. 26. Hernández Urbiola M, Giraldo Betancur A, Jimenez Mendoza D, Pérez Torrero E, Rojas Molina I, Aguilera Barreiro M, et al. Mineral content and physicochemical properties infemale rats bone during growing stage. Atomic Absorption Spectroscopy. 2011; 97: 201–6. 27. Christian P, Khatry SK, Yamini S, Stallings R, LeClerq SC, Shrestha SR, et al. Zinc supplementation might potentiate the effect of vitamin A in restoring night vision in pregnant Nepalese women. The American journal of clinical nutrition. 2001; 73: 1045-51. 28. Yatabe MS, Yatabe J, Takano K, Murakami Y, Sakuta R, Abe S, et al. Effects of a highsodium diet on renal tubule Ca2+ transporter and claudin expression in Wistar-Kyoto rats. BMC Nephrology. 2012; 13: 160. 29. Adeniyi O, Fasanmade A. Effect of dietary zinc supplementation on salt induced hypertension in rats. International Journal of Pharmacology. 2006; 2: 485-91. 30. Shady AM, Nooh HZ. Effect of black seed (Nigella sativa) on compact bone of streptozotocin induced diabetic rats. Egyptian Journal of Histology. 2010; 33: 168-77. 31. Ma ZJ, Igarashi A, Yamakawa K, Yamaguchi M. Enhancing Effect of Zinc and Vitamin K2 (Menaquinone-7) on Bone Components in the Femoral Tissue of Female Elderly Rats. Journal of Health Science. 2001; 47: 40-5. 32. Ahmed MA, Samad AAAE. Benefits of omega-3 fatty acid against bone changes in saltloaded rats: possible role of kidney. Physiological reports. 2013; 1: 106. 33. Omara EA , Shaffie NM, Et-Toumy SA , Aal WA . Histomorphometric Evaluation of Bone Tissue Exposed to E x p e r i m e n t a l O s t e o p o r o s i s a n d Tr e a t e d w i t h RetamaRaetam Extract. J App Sci Res. 2009; 5: 706-16. 34. Creedon A, Cashman KD. The effect of high salt and high protein intake on calcium metabolism, bone composition and bone resorption in the rat. British Journal of Nutrition. 2000; 84: 49-56. 35. Bortolin RH, Abreu BJdGA, Ururahy MAG, de Souza KSC, Bezerra JF, Loureiro MB, et al. Protection against T1DM- I n d u c e d B o n e L o s s b y Z i n c S u p p l e m e n t a t i o n : Biomechanical, Histomorphometric, and Molecular Analyses in STZ-Induced Diabetic Rats. 2015. 36. Otsuka M, Ohshita Y, Marunaka S, Matsuda Y, Ito A, Ichinose N, et al. Effect of controlled zinc release on bone mineral density from injectable Zn-containing β-tricalcium phosphate suspension in zinc-deficient diseased rats. Journal of Biomedical Materials Research Part A. 2004; 69: 552-60. JIIMC 2016 Vol. 11, No.3 Effect of Zinc on Bones of Salt Loaded Rats 118 37. Seo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutrition research and practice. 2010; 4: 356-61. 38. Ammann P, Shen V, Robin B, Mauras Y, Bonjour JP, Rizzoli R. Strontium ranelate improves bone resistance by increasing bone mass and improving architecture in intact female rats. Journal of bone and mineral research. 2004; 19: 2012-20. 39. Duranova H, Martiniakova M, Omelka R, Grosskopf B, Bobonova I, Toman R. Changes in compact bone microstructure of rats subchronically exposed to cadmium. Acta Veterinaria Scandinavica. 2014; 56: 64. 40. Zidan RA, Elnegris HM. Effect of homocysteine on the histological structure of femur in young male albino rats and the possible protective role of folic acid. Journal of Histology & Histopathology. 2015; 2: 16. 41. Martiniakova M, Bobonova I, Omelka R, Grosskopf B, Stawarz R, Toman R. Structural changes in femoral bone tissue of rats after subchronic peroral exposure to selenium. Acta Vet Scand. 2013; 55: 8. 42. Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung Ui, Kubota N, et al. PPAR γ insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. The Journal of clinical investigation. 2004; 113: 846-55. 43. Baltaci AK, Sunar F, Mogulkoc R, Acar M, Toy H. The effect of zinc deficiency and zinc supplementation on element levels in the bone tissue of ovariectomized rats: Histopathologic changes. Archives of physiology and biochemistry. 2014; 120: 80-5. 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