Abstract
Osteoclasts and chondroclasts are necessary, during endochondral ossification, for the resorption of primary bone and calcified cartilage septa, respectively. The bisphosphonates inhibit mineralized tissue resorption by various mechanisms according to the different types of this drug, which can affect bone remodeling during skeletal growth. The objective of the present study is to analyze the way that alendronate (ALN) and etidronate (ETN) can affect osteoclastogenesis and bone formation during endochondral ossification of the long bones of growing rats. Newborn Wistar rats were treated daily with ETN, ALN, or sterile saline solution (control) for 21 days. Their femur and tibiae epiphyses were radiographed and analyzed by light, scanning and transmission electron microscopy. The expression of genes related to osteogenesis and to osteoclast differentiation and activity were analyzed by real-time quantitative polymerase chain reaction. The ETN group presented reduced body weight, disorganized growth plate and an extended area of cartilage in the ossification zone with little bone matrix; in the ALN group, this area was not altered. The ALN presented latent TRAP-positive cells, whereas in the ETN group, they were activated. The expression of NFκB1 and 2, OPG, Spp1 and Runx2 in the ossification zone was reduced by both bisphosphonates. RANKL expression was reduced by ETN, whereas ALN decreased the expression of RANK. The results also indicated that, in addition to the anti-resorptive effect of the drugs, disturbances in bone deposition occurred concomitantly with the reduced expression of osteogenesis-related genes.
Similar content being viewed by others
References
Arana-Chavez VE, Bradaschia-Correa V (2009) Clastic cells: mineralized tissue resorption in health and disease. Int J Biochem Cell Biol 41:446–450
Bachrach LK, Ward LM (2009) Clinical review 1: bisphosphonate use in childhood osteoporosis. J Clin Endocrinol Metab 94:400–409
Bianco P, Cancedda FD, Riminucci M, Cancedda R (1998) Bone formation via cartilage models: the “borderline” chondrocyte. Matrix Biol 17:185–192
Boyce BF, Yao Z, Xing L (2009) Osteoclasts have multiple roles in bone in addition to bone resorption. Crit Rev Eukaryot Gene Expr 19:171–180
Bozal CB, Martinez AB, Cabrini RL, Ubios AM (2005)Effect of ethane-1-hydroxy-1,1-bisphosphonate (EHBP) on endochondral ossification lesions induced by a lethal oral dose of uranyl nitrate. Arch Toxicol 79:475-481
Bradaschia-Correa V, Massa LF, Arana-Chavez VE (2007) Effects of alendronate on toot eruption and molar root formation of young growing rats. Cell Tissue Res 330:475–485
Bradaschia-Correa V, Barrence FA, Ferreira LB, Massa LF, Arana-Chavez VE (2012) Effect of alendronate on endochondral ossification in mandibular condyles of growing rats. Eur J Histochem 56:e24
Bradaschia-Correa V, Moreira MM, Arana-Chavez VE (2013) Reduced RANKL expression impedes osteoclast activation and tooth eruption in alendronate-treated rats. Cell Tissue Res 353:79–86
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
D’Aoust P, McCulloch CA, Tenenbaum HC, Lekic PC (2000) Etidronate (HEBP) promotes osteoblast differentiation and wound closure in rat calvaria. Cell Tissue Res 302:353–363
Ding M, Danielsen CC, Hvid I (2008) The effects of bone remodeling inhibition by alendronate on three-dimensional microarchitecture of subchondral bone tissues in guinea pig primary osteoarthrosis. Calcif Tissue Int 82:77–86
Fischer JE, Rosenberg E, Santora AC, Reszka AA (2013) In vitro and in vivo responses to high and low doses of nitrogen-containing bisphosphonates suggest engagement of different mechanisms for inhibition of osteoclastic bone resorption. Calcif Tissue Int 92:531–538
Hadjidakis DJ, Androulakis II (2006) Bone remodeling. Ann N Y Acad Sci 1092:385–396
Kamoun-Goldrat A, Ginisty D, Le Merrer M (2008) Effects of bisphosphonates on tooth eruption in children with osteogenesis imperfect. Eur J Oral Sci 116:195–198
Katoh Y, Tsuji H, Matsui H, Maruta K, Morita Y (1991) Effects of ethane-1-hydroxy-1,1-diphosphonate on cell differentiation, and proteoglycan and calcium metabolism, in the proximal tibia of young rats. Bone 12:59–65
Kim MS, Jung SY, Kang JH, Kim HJ, Ko HM, Jung JY, Koh JT, Kim WJ, Kim SM, Lee EJ, Kim SH (2009) Effects of bisphosphonate on the endochondral bone formation of the mandibular condyle. Anat Histol Embryol 38:321–326
Larsson A, Larsson SE (1978) The effects of ethylene-1-hydroxy-1,1-disphosphonate on cellular transformation. Acta Pathol Microbiol Scand A 86:211–223
Lerner UH (2000) Osteoclast formation and resorption. Matrix Biol 19:107–120
Li Z, Kong K, Qi W (2006) Osteoclast and its roles in calcium metabolism and bone development and remodeling. Biochem Biophys Res Commun 343:345–350
Li Y, Nakayama H, Notani T, Ahmad M, Tabata MJ, Takano Y (2008) Phosphatase actions at the site of appositional mineralization in bisphosphonate-affected bones of the rat. J Med Dent Sci 55:255–265
Maasalu K, Haviko T, Martson A (2003) Treatment of children with osteogenesis imperfect in Estonia. Acta Paediatr 92:452–455
Mackie EJ, Tatarczuch L, Mirams M (2011) The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification. J Endocrinol 211:109–121
Marshall OJ (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20:2471–2472
Massa LF, Arana-Chavez VE (2000) Ultrastructural preservation of rat embryonic dental tissues after rapid fixation and dehydration under microwave irradiation. Eur J Oral Sci 108:74–77
Massa LF, Bradaschia-Correa V, Arana-Chavez VE (2006) Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats. J Histochem Cytochem 54:713–725
Miller SC, Jee WS (1975) Ethane-1-hydroxy-1,1-diphosphonate (EHDP) effects on growth and modeling of the rat tibia. Calcif Tissue Res 18:215–231
Miller SC, Jee WS, Kimmel DB, Woodbury L (1977) Ethane-1-hydroxy-1,1-diphosphonate (EHDP) effects on incorporation and accumulation of osteoclast nuclei. Calcif Tissue Res 22:243–252
Oyhanart SR, Escudero ND, Mandalunis PM (2015) Effect of alendronate on the mandible and long bones: an experimental study in vivo. Pediatr Res 26:618–625
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36
Riminucci M, Bradbeer JN, Corsi A, Gentili C, Descalzi F, Cancedda R, Bianco P (1998)Vis-à-vis cells and the priming of bone formation.J Bone Miner Res 13:1852–1861
Rodan GA, Fleisch HA (1996) Bisphosphonates: mechanisms of action. J Clin Invest 97:2692–2696
Rossell RG (2011) Bisphosphonates: the first 40 years. Bone 49:2–19
Russell RG (2006) Bisphosphonates: from bench to bedside. Ann N Y Acad Sci 1068:367–401
Russell RG, Watts NB, Ebetino FH, Rogers MJ (2008) Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int 19:733–759
Schenk R, Merz WA, Muhlbauer R, Russell RG, Fleisch H (1973) Effect of ethane-1-hydroxy-1,1-diphosphonate (HEDP) and dichloromethylene diphosphonate (Cl 2 MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res 11:196–214
Shaw NJ, Bishop NJ (2005) Bisphosphonate treatment of bone disease. Arch Dis Child 90:206–213
Shimizu E, Tamasi J, Partridge NC (2012) Alendronate affects osteoblast functions by crosstalk through EphrinB1-EphB. J Dent Res 91:268–274
Shirai T, Kobayashi M, Nishitani K, Satake T, Kuroki H, Nakagawa Y, Nakamura T (2011) Chondroprotective effect of alendronate in a rabbit model of osteoarthritis. J Orthop Res 29:1572–1577
Sparidans RW, Twiss IM, Talbot S (1998) Bisphosphonates in bone diseases. Pharm World Sci 20:206–213
Takano Y, Sakai H, Baba O, Sakamoto Y, Terashima T, Ohya K, Kurosaki N (1998) Demonstration of putative Ca-binding domains in dentin matrix of rat incisors after daily injections of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP). Eur J Oral Sci 106:274–281
Turek J, Ebetino FH, Lundy MW, Sun S, Kashemirov BA, McKenna CE, Gallant MA, Plotkin LI, Bellido T, Duan X, Triffitt JT, Russell RG, Burr DB, Allen MR (2012) Bisphosphonate binding affinity affects drug distribution in both intracortical and trabecular bone of rabbits. Calcif Tissue Int 90:202–210
Vaananen HK, Zhao H, Mulari M, Halleen JM (2000) The cell biology of osteoclast function. J Cell Sci 113:377–381
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034.1–research0034.11
Yamamoto-Silva FP, Bradaschia-Correa V, Lima LA, Arana-Chavez VE (2013) Ultrastructural and immunohistochemical study of early repair of alveolar sockets after the extraction of molars from alendronate-treated rats.Microsc Res Tech 76:633–640
Acknowledgements
The authors thank Mr. Gaspar Lima for ultrathin sectioning. This work was supported by grants from FAPESP (2010/07509-8 and 2013/02240-9) and CNPq (Brazil).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Rights and permissions
About this article
Cite this article
Rezende, E., Bradaschia-Correa, V., Siviero, F. et al. Effects of bisphosphonates on osteogenesis and osteoclastogenesis signaling during the endochondral ossification of growing rats. Cell Tissue Res 368, 287–300 (2017). https://doi.org/10.1007/s00441-017-2574-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-017-2574-3