Skip to main content
SpringerLink
Log in

Dietary Effects of Nanopowder Eggshells on Mineral Contents, Bone Turnover Biomarkers, and Regulators of Bone Resorption in Healthy Rats and Ovariectomy-Induced Osteoporosis Rat Model

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Postmenopausal osteoporosis is a critical issue for female health worldwide. This current study was designed to evaluate the role of nanopowder eggshell (NPES) in healthy and ovariectomy-induced osteoporosis rats. Fifty-six female rats were divided into healthy rats (35) and ovariectomized rats (21). The healthy rats were subdivided into five groups (G1–G5) and received one of the following treatments: saline, 20 or 40 mg/kg of calcium carbonate, and 20 or 40 mg/kg of NPES. The 21 ovariectomized rats were divided into three groups (G6–G8) and received either saline, 40 mg/kg of calcium carbonate, or 40 mg/kg of NPES. Biochemical and histopathological assessments of bone formation and resorption were performed. Biomarkers of bone formation (calcium and osteocalcin (OCN)) and calcium content in left femur ashes were significantly higher in healthy rats given 40-mg/kg NPES than in healthy control rats and healthy rats given 40-mg/kg calcium carbonate. The ovariectomized groups had significantly lower levels of vitamin D3, OCN, and osteoprotegerin (OPG) than the healthy control. Alanine transaminase (ALT), alkaline phosphatase (ALP), and receptor activator of nuclear factor-κB ligand (RANKL) were significantly increased in the ovariectomized group than in the healthy control group. Treatment with NPES and calcium carbonate reduced liver enzymes in ovariectomized rats. NPES treatment significantly increased Vit D3, OCN, OPG, and bone ash mineral content (calcium, magnesium, zinc, and phosphorus) in ovariectomized rats. NPES also increased femur cortical thickness, osteoblast number, and collagen fiber. The current study suggests that NPES can modulate bone turnover biomarkers and increase bone trace elements. Moreover, NPES alleviates bone resorption in ovariectomy-induced osteoporosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data Availability

The data will be available from the authors on reasonable request.

Abbreviations

ALT:

Alanine transaminase

ALT:

Alkaline phosphatase

AST:

Aspartate transaminase

ES:

Eggshells

NPES:

Nanopowder eggshells

OCN:

Osteocalcin

OPG:

Osteoprotegerin

RANKL:

Receptor activator of nuclear factor-κB ligand

References

  1. Lee, S. J., Graffy, P. M., Zea, R. D., Ziemlewicz, T. J., & Pickhardt, P. J. (2018). Future osteoporotic fracture risk related to lumbar vertebral trabecular attenuation measured at routine body CT. Journal of Bone and Mineral Research, 33(5), 860–867.

    CAS  PubMed  Google Scholar 

  2. Ma, Y., Wu, X., Xiao, X., Ma, Y., Feng, L., Yan, W., Chen, J., & Yang, D. (2020). Effects of teriparatide versus percutaneous vertebroplasty on pain relief, quality of life and cost-effectiveness in postmenopausal females with acute osteoporotic vertebral compression fracture: A prospective cohort study. Bone, 131, 115154.

    CAS  PubMed  Google Scholar 

  3. Johnell, O., & Kanis, J. (2006). An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporosis International, 17(12), 1726–1733.

    CAS  PubMed  Google Scholar 

  4. Paruk, F., Tsabasvi, M., & Kalla, A. (2021). Osteoporosis in Africa—Where are we now. Clinical Rheumatology, 40(9), 3419–3428.

    CAS  PubMed  Google Scholar 

  5. Migliorini, F., Maffulli, N., Spiezia, F., Peretti, G. M., Tingart, M., & Giorgino, R. (2021). Potential of biomarkers during pharmacological therapy setting for postmenopausal osteoporosis: A systematic review. Journal of Orthopaedic Surgery and Research, 16(1), 1–13.

    Google Scholar 

  6. Yun, G. W., Kang, J. H., & Lee, H. (2018). Effects of Korean herbal medicine (Cheong-A-Won) for treatment of bone mineral density in women with osteoporosis: A randomized, double blind, placebo controlled trial. European Journal of Integrative Medicine, 20, 84–89.

    Google Scholar 

  7. Zhao, S., Mo, X., Wen, Z., Liu, M., Chen, Z., Lin, W., Huang, Z., & Chen, B. (2022). Declining serum bone turnover markers are associated with the short-term positive change of lumbar spine bone mineral density in postmenopausal women. Menopause, 29(3), 335–343.

    PubMed  PubMed Central  Google Scholar 

  8. Boyle, W. J., Simonet, W. S., & Lacey, D. L. (2003). Osteoclast differentiation and activation. Nature, 423(6937), 337–342.

    CAS  PubMed  Google Scholar 

  9. Udagawa, N., Koide, M., Nakamura, M., Nakamichi, Y., Yamashita, T., Uehara, S., Kobayashi, Y., Furuya, Y., Yasuda, H., & Fukuda, C. (2021). Osteoclast differentiation by RANKL and OPG signaling pathways. Journal of Bone and Mineral Metabolism, 39(1), 19–26.

    CAS  PubMed  Google Scholar 

  10. Tang, H., Zhu, X., Wu, L., Mo, X., Deng, F., & Lei, S. (2019). Integrative analysis confirmed the association between osteoprotegerin and osteoporosis. Chinese Medical Sciences Journal, 34(2), 147–156.

    PubMed  Google Scholar 

  11. Gambacciani, M., & Levancini, M. (2014). Hormone replacement therapy and the prevention of postmenopausal osteoporosis. Przeglad Menopauzalny = Menopause Review, 13(4), 213.

    PubMed  PubMed Central  Google Scholar 

  12. Li, M., & Xu, D. (2020). Antiresorptive activity of osteoprotegerin requires an intact heparan sulfate-binding site. Proceedings of the National Academy of Sciences, 117(29), 17187–17194.

    CAS  Google Scholar 

  13. Migliorini, F., Colarossi, G., Baroncini, A., Eschweiler, J., Tingart, M., & Maffulli, N. (2021). Pharmacological management of postmenopausal osteoporosis: A level i evidence based-expert opinion. Expert Review of Clinical Pharmacology, 14(1), 105–119.

    CAS  PubMed  Google Scholar 

  14. Schierbeck LL, Rejnmark L, Tofteng CL, et al. (2012). Effect of hormone replacement therapy on cardiovascular events in recently postmenopausal women: Randomised trial. Bmj, 345, e6409.

  15. Fazil, M., Baboota, S., Sahni, J. K., Ameeduzzafar, & Ali, J. (2015). Bisphosphonates: Therapeutics potential and recent advances in drug delivery. Drug Delivery, 22(1), 1–9.

    CAS  PubMed  Google Scholar 

  16. An, J., Yang, H., Zhang, Q., Liu, C., Zhao, J., Zhang, L., & Chen, B. (2016). Natural products for treatment of osteoporosis: The effects and mechanisms on promoting osteoblast-mediated bone formation. Life Sciences, 147, 46–58.

    CAS  PubMed  Google Scholar 

  17. Mijan, M. A., Lee, Y.-K., & Kwak, H.-S. (2014). Effects of nanopowdered eggshell on postmenopausal osteoporosis: A rat study. Food Science and Biotechnology, 23(5), 1667–1676.

    CAS  Google Scholar 

  18. Lee, Y.-K., Jung, S. K., Chang, Y. H., & Kwak, H.-S. (2017). Highly bioavailable nanocalcium from oyster shell for preventing osteoporosis in rats. International Journal of Food Sciences and Nutrition, 68(8), 931–940.

    CAS  PubMed  Google Scholar 

  19. Yadav, M., Pareek, N., & Vivekanand, V. (2022). Eggshell and fish/shrimp wastes for synthesis of bio-nanoparticles. Agri-Waste and Microbes for Production of Sustainable Nanomaterials (pp. 259–280). Elsevier.

    Google Scholar 

  20. Brun, L. R., Lupo, M., Delorenzi, D. A., Di Loreto, V. E., & Rigalli, A. (2013). Chicken eggshell as suitable calcium source at home. International Journal of Food Sciences and Nutrition, 64(6), 740–743.

    CAS  PubMed  Google Scholar 

  21. Dri, N., Brun, L., Di Loreto, V., Lupo, M., & Rigalli, A. (2011). Study of the composition of the egg shell. A low cost calcium supplement. Bone, 6(49), 1381.

    Google Scholar 

  22. Hirasawa, T., Omi, N., & Ezawa, I. (2001). Effect of 1α-hydroxyvitamin D3 and egg-shell calcium on bone metabolism in ovariectomized osteoporotic model rats. Journal of Bone and Mineral Metabolism, 19(2), 84–88.

    CAS  PubMed  Google Scholar 

  23. Rahman, M., Netravali, A., Tiimob, B., & Rangari, V. (2014). Bioderived “green” composite from soy protein and eggshell nanopowder. ACS Sustainable Chemistry & Engineering, 2(10), 2329–2337.

    CAS  Google Scholar 

  24. Hanzlik, R. P., Fowler, S. C., & Fisher, D. H. (2005). Relative bioavailability of calcium from calcium formate, calcium citrate, and calcium carbonate. Journal of Pharmacology and Experimental Therapeutics, 313(3), 1217–1222.

    CAS  PubMed  Google Scholar 

  25. Zhu, F., Liu, Z., & Ren, Y. (2018). Mechanism of melatonin combined with calcium carbonate on improving osteoporosis in aged rats. Experimental and Therapeutic Medicine, 16(1), 192–196.

    PubMed  PubMed Central  Google Scholar 

  26. Lelovas, P. P., Xanthos, T. T., Thoma, S. E., Lyritis, G. P., & Dontas, I. A. (2008). The laboratory rat as an animal model for osteoporosis research. Comparative Medicine, 58(5), 424–430.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Yang, L.-C., Wu, J.-B., Ho, G.-H., Yang, S.-C., Huang, Y.-P., & Lin, W.-C. (2008). Effects of poly-γ-glutamic acid on calcium absorption in rats. Bioscience Biotechnology and Biochemistry, 72(12), 3084–3090.

    CAS  PubMed  Google Scholar 

  28. Bancroft, J. D., & Gamble, M. (2008). Theory and practice of histological techniques. Elsevier health sciences.

  29. Lems, W. F., & Raterman, H. G. (2017). Critical issues and current challenges in osteoporosis and fracture prevention. An overview of unmet needs. Therapeutic Advances in Musculoskeletal Disease, 9(12), 299–316.

    PubMed  PubMed Central  Google Scholar 

  30. Jehle-Kunz, S., Häuselmann, H.-J., Keschawarzi, M., Lamy, O., Luzuy, F., Marcoli, N., Meier, C., Uebelhart, B., & Wiedersheim, P. (2022). Risk factors, manifestation, and awareness of osteoporosis among patients of various specialists in Switzerland: Results of a national survey. In Healthcare. MDPI.

  31. Pokorski, P., & Hoffmann, M. (2022). Valorization of bio‐waste eggshell as a viable source of dietary calcium for confectionery products. Journal of the Science of Food and Agriculture, 102(8), pp. 3193–3203.

  32. El-Shibiny, S., El-Gawad, M.A.E.-K.M.A., Assem, F. M., & El-Sayed, S. M. (2018). The use of nano-sized eggshell powder for calcium fortification of cow? s and buffalo? s milk yogurts. Acta Scientiarum Polonorum Technologia Alimentaria, 17(1), 37–49.

    CAS  PubMed  Google Scholar 

  33. Xu, H., Liu, T., Hu, L., Li, J., Gan, C., Xu, J., Chen, F., Xiang, Z., Wang, X., & Sheng, J. (2019). Effect of caffeine on ovariectomy-induced osteoporosis in rats. Biomedicine & Pharmacotherapy, 112, 108650.

    CAS  Google Scholar 

  34. Mijan, M. A., Kim, D. H., & Kwak, H. S. (2014). Physicochemical properties of nanopowdered eggshell. International Journal of Food Science & Technology, 49(7), 1751–1757.

    Google Scholar 

  35. Xu, J.-W., Gong, J., Chang, X.-M., Luo, J.-Y., Dong, L., Hao, Z.-M., Jia, A., & Xu, G.-P. (2002). Estrogen reduces CCL4-induced liver fibrosis in rats. World Journal of Gastroenterology, 8(5), 883.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Saleh, N. K., & Saleh, H. A. (2011). Olive oil effectively mitigates ovariectomy-induced osteoporosis in rats. BMC Complementary and Alternative Medicine, 11(1), 1–11.

    Google Scholar 

  37. El-Zeftawy, M., Ali, S. A. E. M., Salah, S., & Hafez, H. S. (2020). The functional nutritional and regulatory activities of calcium supplementation from eggshell for obesity disorders management. Journal of Food Biochemistry, 44(8), e13313.

    CAS  PubMed  Google Scholar 

  38. Li, H., Hao, Z., Wang, L., Yang, J., Zhao, Y., Cheng, X., Yuan, H., & Wang, J. (2022). Dietary calcium alleviates fluorine-induced liver injury in rats by mitochondrial apoptosis pathway. Biological Trace Element Research, 200(1), 271–280.

    CAS  PubMed  Google Scholar 

  39. Mohamed, N. (2016). The role of calcium in ameliorating the oxidative stress of fluoride in rats. Biological Trace Element Research, 170(1), 128–144.

    CAS  PubMed  Google Scholar 

  40. Szeleszczuk, Ł, Pisklak, D. M., Kuras, M., & Wawer, I. (2015). In vitro dissolution of calcium carbonate from the chicken eggshell: A study of calcium bioavailability. International Journal of Food Properties, 18(12), 2791–2799.

    CAS  Google Scholar 

  41. Yang, M., Wu, D., Cheng, S., Dong, Y., Wu, C., Wang, Z., & Du, M. (2022). Inhibitory effects of Atlantic cod (Gadus morhua) peptides on RANKL-induced osteoclastogenesis in vitro and osteoporosis in ovariectomized mice. Food & Function, 13(4), pp. 1975–1988

  42. Khajuria, D. K., Razdan, R., & Mahapatra, D. R. (2015). Additive effects of zoledronic acid and propranolol on bone density and biochemical markers of bone turnover in osteopenic ovariectomized rats. Revista Brasileira de Reumatologia, 55, 103–112.

    PubMed  Google Scholar 

  43. Wolf, R. L., Cauley, J. A., Baker, C. E., Ferrell, R. E., Charron, M., Caggiula, A. W., Salamone, L. M., Heaney, R. P., & Kuller, L. H. (2000). Factors associated with calcium absorption efficiency in pre-and perimenopausal women. The American Journal of Clinical Nutrition, 72(2), 466–471.

    CAS  PubMed  Google Scholar 

  44. Song, J., Liu, G., Song, Y., Jiao, K., Wang, S., Cao, T., Yu, J., & Wei, Y. (2021). Positive effect of compound amino acid chelated calcium from the shell and skirt of scallop in an ovariectomized rat model of postmenopausal osteoporosis. Journal of the Science of Food and Agriculture, 102(4), pp.1363–1371.

  45. Nagamma, T., Konuri, A., Bhat, K. M., Maheshwari, R., Udupa, P., & Nayak, Y. (2021). Modulation of inflammatory markers by petroleum ether fraction of Trigonella foenum-graecum L. seed extract in ovariectomized rats. Journal of Food Biochemistry, 45(4), e13690.

    CAS  PubMed  Google Scholar 

  46. Omelka, R., Martiniakova, M., Babikova, M., Svik, K., Slovak, L., Kovacova, V., Vozar, J., & Soltesova-Prnova, M. (2021). Chicken eggshell powder more effectively alleviates bone loss comparted to inorganic calcium carbonate: An animal study performed on ovariectomized rats. Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society, 72(6).

  47. Fang, B., Wan, Y.-Z., Tang, T.-T., Gao, C., & Dai, K.-R. (2009). Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial cellulose nanocomposite scaffolds. Tissue Engineering Part A, 15(5), 1091–1098.

    CAS  PubMed  Google Scholar 

  48. Ltaif, M., Gargouri, M., & Soussi, A. (2021). Protective effects of A. sativa against oxidative stress-induced liver damage in ovariectomized mice. BioMed Research International, 2021, Article ID 5577498, pp. 15.

  49. Feng, P., Shu, S., & Zhao, F. (2022). Anti-osteoporosis effect of fisetin against ovariectomy induced osteoporosis in rats: In silico, in vitro and in vivo activity. Journal of Oleo Science, 71(1), 105–118.

    CAS  PubMed  Google Scholar 

  50. Reddi, S., Mada, S. B., Kumar, N., Kumar, R., Ahmad, N., Karvande, A., Kapila, S., Kapila, R., & Trivedi, R. (2019). Antiosteopenic effect of buffalo milk casein-derived peptide (NAVPITPTL) in ovariectomized rats. International Journal of Peptide Research and Therapeutics, 25(3), 1147–1158.

    CAS  Google Scholar 

  51. Mansour, H. M., Fawzy, H. M., El-Khatib, A. S., & Khattab, M. M. (2021). Lapatinib ditosylate rescues memory impairment in D-galactose/ovariectomized rats: Potential repositioning of an anti-cancer drug for the treatment of Alzheimer’s disease. Experimental Neurology, 341, 113697.

    CAS  PubMed  Google Scholar 

  52. Borciani, G., Ciapetti, G., Vitale-Brovarone, C., & Baldini, N. (2022). Strontium functionalization of biomaterials for bone tissue engineering purposes: A biological point of view. Materials, 15(5), 1724.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. O’Connor, J. P., Kanjilal, D., Teitelbaum, M., Lin, S. S., & Cottrell, J. A. (2020). Zinc as a therapeutic agent in bone regeneration. Materials, 13(10), 2211.

    PubMed  PubMed Central  Google Scholar 

  54. Landi, E., Logroscino, G., Proietti, L., Tampieri, A., Sandri, M., & Sprio, S. (2008). Biomimetic Mg-substituted hydroxyapatite: From synthesis to in vivo behaviour. Journal of Materials Science: Materials in Medicine, 19(1), 239–247.

    CAS  PubMed  Google Scholar 

  55. Sakai, S., Hien, V. T. T., Tuyen, L. D., Duc, H. A., Masuda, Y., & Yamamoto, S. (2017). Effects of eggshell calcium supplementation on bone mass in postmenopausal Vietnamese women. Journal of Nutritional Science and Vitaminology, 63(2), 120–124.

    CAS  PubMed  Google Scholar 

  56. Kharode, Y. P., Sharp, M. C., & Bodine, P. V. (2008). Utility of the ovariectomized rat as a model for human osteoporosis in drug discovery. Osteoporosis (pp. 111–124). Springer.

    Google Scholar 

  57. Ye, M., Zhang, C., Jia, W., Shen, Q., Qin, X., Zhang, H., & Zhu, L. (2020). Metabolomics strategy reveals the osteogenic mechanism of yak (Bos grunniens) bone collagen peptides on ovariectomy-induced osteoporosis in rats. Food & Function, 11(2), 1498–1512.

    CAS  Google Scholar 

  58. Schaafsma, A., van Doormaal, J. J., Muskiet, F. A., Hofstede, G. J., Pakan, I., & van der Veer, E. (2002). Positive effects of a chicken eggshell powder-enriched vitamin–mineral supplement on femoral neck bone mineral density in healthy late post-menopausal Dutch women. British Journal of Nutrition, 87(3), 267–275.

    CAS  PubMed  Google Scholar 

  59. Hu, Y., Mu, P., Ma, X., Shi, J., Zhong, Z., & Huang, L. (2021). Rhizoma drynariae total flavonoids combined with calcium carbonate ameliorates bone loss in experimentally induced Osteoporosis in rats via the regulation of Wnt3a/β-catenin pathway. Journal of Orthopaedic Surgery and Research, 16(1), 1–9.

    Google Scholar 

Download references

Funding

The Proposal received a fund by Dr. Ragaa H. Salama from the Grant Office of Faculty of Medicine, Assiut University, Assiut, Egypt, in 2019.

Author information

Authors and Affiliations

  1. Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology, Assiut University, Assiut, 71515, Egypt

    Ragaa H. M. Salama & Ghada M. Ezzat

  2. Faculty of Medicine, Department of Histology, Assiut University, Assiut, Egypt

    Safaa S. Ali

  3. Agriculture Research Institute, Assiut, Egypt

    Tarek Hamdy M. Salama & Mohamed Abu Almged

  4. Faculty of Pharmacy, Department of Biochemistry, Assiut University, Assiut, Egypt

    Tasneem A. Alsanory

  5. Department of Radiotherapy and Nuclear Medicine, South Egypt Cancer Institute, Assiut University, Assiut, Egypt

    Aya A. Alsanory

  6. Faculty of Medicine, Department of Obstetrics and Gynecology, Assiut University Hospital, Assiut, Egypt

    Hesham Aboutaleb

Authors
  1. Ragaa H. M. Salama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Safaa S. Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tarek Hamdy M. Salama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mohamed Abu Almged
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tasneem A. Alsanory
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Aya A. Alsanory
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hesham Aboutaleb
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ghada M. Ezzat
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RS participate in study deign and supervised the research work. GM did the biochemical work, wrote the manuscript, and performed the statistical analysis. SS participate in study design, did histopathological examination, and wrote the histological results. TH participated in study design, collected the literatures, and did the experiment work. MA participated in study design. TA participated in data collection. AA participated in data collection. HT did the ovariectomy surgical procedures. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Ragaa H. M. Salama or Ghada M. Ezzat.

Ethics declarations

Ethical Approval

The study was approved by the ethical guidelines of Faculty of Medicine, Assiut University. The ethical approval number is 17300219.

Consent to Participate

The study was an animal study and was done with no need to consent to participate.

Consent to Publish

All authors read and approved the version to be published.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Salama, R.H.M., Ali, S.S., Salama, T.H.M. et al. Dietary Effects of Nanopowder Eggshells on Mineral Contents, Bone Turnover Biomarkers, and Regulators of Bone Resorption in Healthy Rats and Ovariectomy-Induced Osteoporosis Rat Model. Appl Biochem Biotechnol 195, 5034–5052 (2023). https://doi.org/10.1007/s12010-022-04038-9

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-022-04038-9

Keywords

Search

Navigation