Morphological and biochemical characteristics of osteogenesis during drug therapy of experimental osteoporosis

Relevance. Osteoporosis is a clinical and economic problem on a global scale. A significant contribution to solving the problem of effective treatment of osteoporosis can be the creation of drugs based on unique biologically active compounds.

The aim of the study was the morphological substantiation of the experimental model of osteoporosis and a comprehensive (multifactorial) assessment of the effectiveness of its drug therapy.

Materials and methods. The study was carried out on 40 mature female Wistar rats, which comprised 6 groups. A model of osteoporosis was formed in animals of four experimental groups (6 animals in each): the first and second groups (respectively) consisted of young rats, the third and fourth — old ones. Animals of the second and fourth groups were injected with a drug tested for its ability to activate the process of osteosynthesis. Rats of the fifth and sixth groups (young and old, 8 animals each), subjected to sham surgery, served as controls. As a result of the use of the histo-morphometric method and atomic absorption spectroscopy in the diaphysis of the femur, the thickness of the layers of the compact substance, the number of bone plates and osteocytes, as well as the amount of collagen, calcium and phosphorus were determined. Using enzyme immunoassay, bone remodeling markers — osteocalcin, sclerostin, osteoprotegerin, fibroblast growth factor-23 and nuclear factor kappa-β activator ligand (RANKL) — were determined in the blood serum. Statistical processing of the data was carried out using the GraphPad PRISM (USA) program to determine the median, upper and lower quartiles. Differences were considered significant at p < 0.01.

Results. Modeling of osteoporosis induces atrophic thinning of the compact substance, a decrease in the number of osteocytes and bone plates in the diaphysis of the femur, a decrease in the content of collagen, calcium and phosphorus in them, a decrease in the concentration of osteocalcin, sclerostin, fibroblast growth factor, osteoprotegerin and an increase in the concentration of RANKL in the blood plasma, more pronounced in old animals. As a result of the use of the drug X3 for the treatment of osteoporosis, the following were revealed: a significant increase in the thickness of the compact substance, the number of osteocytes and bone plates in the diaphysis, the content of collagen, calcium and phosphorus in them, an increase in the concentration of biochemical markers of osteosynthesis, and a mild imbalance of RANKL. The increase in plasma levels of markers of bone remodeling was most pronounced when the drug X3 was combined with vitamin D3.

Conclusion. 1. The used surgical-endocrine method of modeling osteoporosis leads to pronounced degenerative changes in osteocytes and their derivatives in all parts of the compact bone substance, and also causes significant disturbances in the mineral composition and an imbalance of bone remodeling markers, more pronounced in senile rats. 2. The tested drug X3, used for the treatment of osteoporosis, has a high degree of effectiveness, since it stimulates regenerative osteogenesis, restores the damaged structure of bone tissue elements, its organic and mineral components. 3. The restorative effect of the drug is more pronounced in senile rats.

1. Bolland MJ, Grey AB, Gamble GD, Reid IR. Effect of osteoporosis treatment on mortality: a meta-analysis // J Clin Endocrinol Metab. 2010. No 95. P. 1174–1181.

2. MacLean C, Newberry S, Maglione M, et al. Systematic review: comparative effectiveness of treatments to prevent fractures in men and women with low bone density or osteoporosis // Ann Intern Med. 2007. No 148. Р. 197–213.

3. Cauley JA, Thompson DE, Ensrud KC, et al. Risk of mortality following clinical fractures // Osteoporosis Int. 2000. No 11. Р. 556–561.

4. Kanis JA. World Health Organization Scientific Group. Assessment of Osteoporosis at the Primary Health-Care Level Technical Report Sheffield. UK: WHO Collaborating Centre, Uni versity of Sheffield; 2008.

5. National Osteoporosis Foundation 2004. Disease statistics.

6. National Osteoporosis Foundation. Advocacy News & Updates. American’s Bone Health: The State of Osteoporosis and Low Bone Mass. Accessed April 18. 2011.

7. Riggz BL, Melton D. Osteoporosis. Etiology, diagnosis, treatment. CJSC Binom Publishing House. M. 2000. P. 309–313. In Russian

8. International Osteoporosis Foundation. 2010.

9. EFFO and NOF. Who are candidates for prevention and treatment for osteoporosis? // Osteoporos Int. 1997. No 7. Р. 1–32.

10. Management of Osteoporosis in Postmenopausal Women: 2010 Position Statement of The North American Menopause Society // Menopause. 2010. Vol.17. No 1. Р. 25–54.

11. WHO. FRAX® WHO fracture risk assessment tool: calculation tool. Accessed February 14. 2011.

12. Bairamov AA, Maevskij EI, Shabanov PD. Correction of bone remodeling in experimental osteoporosis. Reviews on Clinical Pharmacology and Drug Therapy. 2019;17(4):43–50. doi.org/10.17816/RCF17443-50. In Russian

13. Lesnyak OM. Audit of the state of the problem of osteoporosis in the Russian Federation // Preventive medicine. 2011;(2):7–10. In Russian

14. Cattail VM, Arki RA. Pathophysiology of the endocrine system. M. Binomi. 2010. 335 p. In Russian

15. Povoroznyuk VV, Grigor’eva NV. Menopause and osteoporosis. K.: Health, 2004. 356 p. In Russian

16. Maevskij EI, Roshchenfel’d AS, Grishina EV, Kondrashova MN. Correction of metabolic acidosis by maintaining the functions of mitochondria. (monograph). Pushino, ITEB RAS, 2001. 155 p. In Russian

17. Maevskij EI, Uchitel’ ML, Bajramov AA., et al. Correction of hormonal activity with substrate compositions in men and women // Gastroenterology of St. Petersburg. 2017;(2–3):55–56. In Russian

18. Mitochondrial processes in the temporal organization of life. Edited by M. N. Kondrashovа, V. V. Dynnik, Yu. G. Kaminskij, et al. Pushchino, 1978. 182 p. In Russian

19. Therapeutic effect of succinic acid. Sb. Edited by M. N. Kondrashova. Pushchino, 1976. 255 p. In Russian

20. Maevskij EI, Grishina EV, Haustova YaV, et al. Again about preparations containing succinate // Gastroenterology of St. Petersburg. 2017;(1):91–92. In Russian

21. Vasilieva AA, Simonova MA, Bairamov AA, et al. Сorrection of the functional state of female rats after unilateral ovariectomy using a succinate containing composition. Cardiometry, 2017. No 10. P. 86–92. DOI: 0.12710/cardiometry.2017.8692.

22. Succinic acid in medicine, food industry, agriculture. Sb. Edited by M. N. Kondrashova, Yu. G. Kaminskij, E. I. Maevskij. Pushchino, 1996. 300 p. In Russian

23. Kotelnikov GP, Bulgakov SV. Osteoporosis. M. GEOTAR Mediz. 2010. 504 p. In Russian

24. Kronenberg GM, Melmed SH, Polonski KS, Larsen PR. Endocrinology according to Williamson. Reproductive endocrinology. M. Reid Elsiver. 2011. 410 p. In Russian

25. Hodzhkin S. Mayo Clinic on Osteoporosis. M.: Astrel. 2007. 237 p. In Russian

26. Levitskiy AP, Makarenko OA, Den’ga OV, et al. Eksperimental’nye metody issledovaniya stimulyatorov osteogeneza: metodicheskie rekomendatsii. Kiev: Avitsenna; 2005. P. 31–38. In Russian

27. Frol’kis V, Povoroznyuk V, Evtushenko O, Grigorieva N. Eksperimental’nyy osteoporoz. Doctor. 2003;(6):48–52. In Russian

28. Bairamov AA, Mamina NSh, Karonova TL, Shabanov PD. Possibilities of in vivo validation of a model of experimental osteoporosis. Reviews on Clinical Pharmacology and Drug Therapy. 2020;18(4):365–367. In Russian. DOI: 10.7816/RCF184365-367.

29. Zamaraeva TV. Method for determining the content of collagen proteins by hydroxyproline // Modern methods in biochemistry. M.: Medicine, 1977. C. 262–264. In Russian

30. Bertchenko GN, Lipkin SM. Features of Bone Tissue Research. In: Microscopic Techniques: A Guide for Physicians and Laboratory Technicians. Edited by D. S. Sarkisov, Yu. L. Perov. Moscow: Meditsina, 1996. P. 463. In Russian

31. Sharayev PN. Method for determining free and bound hydroxyproline in blood serum. Laboratory Work. 1981;(5):283–285. In Russian

32. Patent RUS2582973C1. Bajramov AA, Shabanov PD, Maevskij EI, et al. Antiosteoporoznoe sredstvo. In Russian

33. GOST 33044-2014 “Principles of Good Laboratory Practice”. In Russian

34. Vega D, Maalouf NM, Sakhaee K. The role of receptor activator of nuclear factor-kappaB (RANK)/ RANK ligand/osteoprotegerin: clinical implications // J Clin Endocrinol Metab. 2007. Vol. 92. No 12. Р. 4514–4521.

35. Dobronravov VA. Modern Insights into the Pathophysiology of Secondary Hyperparathyroidism: The Role of Fibroblast Growth Factor 23 and Klotho. Nephrology. 2011;(4):11–20. In Russian

36. Shutov EV. Significance of fibroblast growth factor-23 in patients with chronic kidney disease — a review of modern research // Attending physician. 2012. No. 8. P. 35–42. In Russian

37. Kneissel M. The promise of sclerostin inhibition for the treatment of osteoporosis // IBMS BoneKEy. 2009. No. 6. Р. 259–264.

38. Grebennikova TA, Belaya ZhE, Rozhinskaya LYa, Mel’nichenko GA. Canonical signaling pathway of Wnt/βcatenin: from the history of discovery to clinical application // Therapeutic archive. 2016;10(88):74–75. In Russian

39. Sims NA, Chia LY. Regulation of sclerostin expression by paracrine and endocrine factors // Clin Rev Bone Miner Metab. 2012. No.10. Р. 98–107.

40.