Imbalance of RANK, RANKL and OPG expression during tibial fracture repair in diabetic rats

  • Fernanda Penna Lima Guedes de Amorim
  • Sócrates Souza Ornelas
  • Sandoval Felicíssimo Diniz
  • Aline Carvalho Batista
  • Tarcília Aparecida da Silva
Original Paper


To clarify the mechanisms of altered bone repair in the diabetic state, we investigated RANK, RANKL and OPG expression by immunohistochemistry and RT-PCR in the fracture sites of rats that were either healthy or made diabetic by alloxan. Histomorphometric analysis of the fracture site at 7 days after fracture revealed that diabetic rats (db) have significantly less hard tissue formation at the fracture site, compared to controls. The number of RANK, RANKL and OPG positive cells was decreased in the db group; however, the RANKL/OPG ratio was similar in controls and db at this time. At day 14, numbers of RANKL and OPG positive cells and the mRNA expression for these markers were higher in the control group than in db (P = 0.008). The RANKL/OPG ratio in the db group was greater than in controls. Our results demonstrate an imbalance of RANKL/OPG expression associated with diabetes that may contribute to the delay of fracture repair during the course of diabetes.


RANK RANKL OPG Diabetes Facture repair 



The authors are grateful to Gabriela Mariângela Farias de Oliveira, Édelyn Cristina Nunes Silva and Renata Ribeiro de Souza for their helpful technical assistance. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).


  1. An YH, Friedman RJ (1999) Animal models in orthopedic research. CRC Press, Boca RatonGoogle Scholar
  2. Boyle WJ, Simonet WS, Lacey DL (2003) Osteoclast differentiation and activation. Nature 423:337–342. doi: 10.1038/nature01658 PubMedCrossRefGoogle Scholar
  3. Dib SA, Russo EMK, Chacra AR (1992) Tratado de endocrinologia clínica. Ed. Rocca, São PauloGoogle Scholar
  4. Diniz SF, Amorim FP, Cavalcante-Neto FF et al (2008) Alloxan-induced diabetes delays repair in a rat model of closed tibial fracture. Braz J Med Biol Res 41:373–379PubMedCrossRefGoogle Scholar
  5. Duarte PM, Neto JB, Casati MZ et al (2007) Diabetes modulates gene expression in the gingival tissues of patients with chronic periodontitis. Oral Dis 13:594–599. doi: 10.1111/j.1601-0825.2006.01348.x PubMedCrossRefGoogle Scholar
  6. Follak N, Kloting I, Ganzer D et al (2003) Scanning electron microscopic examinations on retarded bone defect healing in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats. Histol Histopathol 18:111–120PubMedGoogle Scholar
  7. Follak N, Kloting I, Wolf E et al (2004a) Histomorphometric evaluation of the influence of the diabetic metabolic state on bone defect healing depending on the defect size in spontaneously diabetic BB/OK rats. Bone 35:144–152. doi: 10.1016/j.bone.2004.03.011 PubMedCrossRefGoogle Scholar
  8. Follak N, Kloting L, Wolf E et al (2004b) Delayed remodeling in the early period of fracture healing in spontaneously diabetic BB/OK rats depending on the diabetic metabolic state. Histol Histopathol 19:473–486PubMedGoogle Scholar
  9. Follak N, Kloting I, Merk H (2005) Influence of diabetic metabolic state on fracture healing in spontaneously diabetic rats. Diabetes Metab Res Rev 21:288–296. doi: 10.1002/dmrr.537 PubMedCrossRefGoogle Scholar
  10. Funk JR, Hale JE, Carmines D et al (2000) Biomechanical evaluation of early fracture healing in normal and diabetic rats. J Orthop Res 18:126–132. doi: 10.1002/jor.1100180118 PubMedCrossRefGoogle Scholar
  11. Galluzzi F, Stagi S, Salti R et al (2005) Osteoprotegerin serum levels in children with type 1 diabetes: a potential modulating role in bone status. Eur J Endocrinol 153:879–885. doi: 10.1530/eje.1.02052 PubMedCrossRefGoogle Scholar
  12. Gooch HL, Hale JE, Fujioka H et al (2000) Alterations of cartilage and collagen expression during fracture healing in experimental diabetes. Connect Tissue Res 41:81–91. doi: 10.3109/03008200009067660 PubMedCrossRefGoogle Scholar
  13. Guarneri MP, Weber G, Gallia P et al (1993) Effect of insulin treatment on osteocalcin levels in diabetic children and adolescents. J Endocrinol Invest 16:505–509PubMedGoogle Scholar
  14. He H, Liu R, Desta T, Leone C et al (2004) Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss. Endocrinology 145:447–452. doi: 10.1210/en.2003-1239 PubMedCrossRefGoogle Scholar
  15. Herrero S, Calvo OM, García-Moreno C et al (1998) Low bone density with normal bone turnover in ovariectomized and streptozotocin-induced diabetic rats. Calcif Tissue Int 62:260–265. doi: 10.1007/s002239900427 PubMedCrossRefGoogle Scholar
  16. Hie M, Shimono M, Fujii K et al (2007) Increased cathepsin K and tartrate-resistant acid phosphatase expression in bone of streptozotocin-induced diabetic rats. Bone 41:1045–1050PubMedCrossRefGoogle Scholar
  17. Horcajada-Molteni MN, Chanteranne B, Lebecque P et al (2001) Amylin and bone metabolism in streptozotocin-induced diabetic rats. J Bone Miner Res 16:958–965. doi: 10.1359/jbmr.2001.16.5.958 PubMedCrossRefGoogle Scholar
  18. Katsumata K, Katsumata K Jr, Katsumata Y (1992) Protective effect of diltiazem hydrochloride on the occurrence of alloxan- or streptozotocin-induced diabetes in rats. Horm Metab Res 24:508–510PubMedGoogle Scholar
  19. Kayal RA, Tsatsas D, Bauer MA et al (2007) Diminished bone formation during diabetic fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity. J Bone Miner Res 22:560–568. doi: 10.1359/jbmr.070115 PubMedCrossRefGoogle Scholar
  20. Kobayashi-Sakamoto M, Hirose K, Nishikata M et al (2006) Osteoprotegerin protects endothelial cells against apoptotic cell death induced by Porphyromonas gingivalis cysteine proteinases. FEMS Microbiol Lett 264:238–245. doi: 10.1111/j.1574-6968.2006.00458.x PubMedCrossRefGoogle Scholar
  21. Krakauer JC, McKenna MJ, Buderer NF et al (1995) Bone loss and bone turnover in diabetes. Diabetes 44:775–782. doi: 10.2337/diabetes.44.7.775 PubMedCrossRefGoogle Scholar
  22. Lacey DL, Timms E, Tan HL et al (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176. doi: 10.1016/S0092-8674(00)81569-X PubMedCrossRefGoogle Scholar
  23. Levin ME, Boisseau VC, Avioli LV (1976) Effects of diabetes mellitus on bone mass in juvenile and adult-onset diabetes. N Engl J Med 294:241–245PubMedGoogle Scholar
  24. Macey LR, Kana SM, Jingushi S et al (1989) Defects of early fracture-healing in experimental diabetes. J Bone Joint Surg Am 71:722–733PubMedGoogle Scholar
  25. Miazgowski T, Czekalski S (1998) A 2-year follow-up study on bone mineral density and markers of bone turnover in patients with long-standing insulin-dependent diabetes mellitus. Osteoporos Int 8:399–403. doi: 10.1007/s001980050082 PubMedCrossRefGoogle Scholar
  26. Olmos JM, Perez-Castrillon JL, Garcia MT et al (1994) Bone densitometry and biochemical bone remodeling markers in type 1 diabetes mellitus. Bone Miner 26:1–8PubMedCrossRefGoogle Scholar
  27. Silvestrini G, Ballanti P, Patacchioli F et al (2005) Detection of osteoprotegerin (OPG) and its ligand (RANKL) mRNA and protein in femur and tibia of the rat. J Mol Histol 36:59–67. doi: 10.1007/s10735-004-3839-1 PubMedCrossRefGoogle Scholar
  28. So T, Lee SW, Croft M (2006) Tumour necrosis factor/tumour necrosis factor receptor family members that positively regulate immunity. Int J Hematol 83:1–11. doi: 10.1532/IJH97.05120 PubMedCrossRefGoogle Scholar
  29. Suzuki K, Kurose T, Takizawa M et al (2005) Osteoclastic function is accelerated in male patients with type 2 diabetes mellitus: the preventive role of osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) on the decrease of bone mineral density. Diabetes Res Clin Pract 68:117–125. doi: 10.1016/j.diabres.2004.08.006 PubMedCrossRefGoogle Scholar
  30. Szkudelski T (2001) The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 50:537–546PubMedGoogle Scholar
  31. Tuominen JT, Impivaara O, Puukka P et al (1999) Bone mineral density in patients with type 1 and type 2 diabetes. Diabetes Care 22:1196–1200. doi: 10.2337/diacare.22.7.1196 PubMedCrossRefGoogle Scholar
  32. Wetzler C, Kampfer H, Stallmeyer B et al (2000) Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J Invest Dermatol 115:245–253. doi: 10.1046/j.1523-1747.2000.00029.x PubMedCrossRefGoogle Scholar
  33. Yasuda H, Shima N, Nakagawa N et al (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602. doi: 10.1073/pnas.95.7.3597 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Fernanda Penna Lima Guedes de Amorim
    • 1
  • Sócrates Souza Ornelas
    • 2
  • Sandoval Felicíssimo Diniz
    • 1
  • Aline Carvalho Batista
    • 3
  • Tarcília Aparecida da Silva
    • 4
    • 5
  1. 1.Department of Pathology, Faculty of MedicineUniversity of BrasíliaBrasilia, Distrito FederalBrazil
  2. 2.Department of Molecular Pathology, Faculty of MedicineUniversity of BrasíliaBrasilia, Distrito FederalBrazil
  3. 3.Department of Stomatology (Oral Pathology), Dental SchoolUniversidade Federal de GoiásGoianiaBrazil
  4. 4.Department of Oral Pathology and Surgery, Faculty of DentistryUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  5. 5.Departamento de Clínica, Patologia e Cirurgia Odontológicas, Faculdade de OdontologiaUniversidade Federal de Minas GeraisBelo HorizonteBrazil

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