Zoledronate and Clodronate affecting bone repair in non-critical defects grafted with carbonated nanostructured hydroxyapatite: an in vivo study fostering clinical reasoning
DOI:
https://doi.org/10.2340/biid.v12.45131Keywords:
Bisphosphonates, clodronate, zolendronate, carbonated hydroxyapatite, femur, in vivo studyAbstract
Objective: This study aimed to compare the effect of two different generations of bisphosphonates (BPs) on bone repair assisted by an alloplastic bone graft (spheres of nanostructured carbonate apatite/calcium, CHA) in a non-critical defect in the rat femur.
Materials and Methods: Thirty-six female Wistar rats were randomly assigned into six groups: Control group (blood clot), carbonate apatite (CHA) alone, Zoledronate (Zol) with blood clot, Clodronate (Clo) with blood clot, Zol+CHA, and Clo+CHA. Drugs were administered intraperitoneally (Zol: 0.6 mg/kg; Clo: 20 mg/kg) every 30 days for 60 days before surgery. Standardized monocortical femoral defects (2 mm) were created and filled according to group assignment. After 30 days of healing, samples were harvested for histological and histomorphometrical evaluation. New bone formation and remnant biomaterial were quantified. Statistical analysis included the Kruskal–Wallis test and Dunn’s post hoc (p < 0.05), along with Pearson and Spearman correlation analyses.
Results: Histological analysis revealed enhanced new bone formation in groups treated with BPs, especially when combined with CHA. The Zol+CHA group exhibited the highest new bone formation (24.0 ± 4.0%), significantly greater than Control (2.0 ± 0.5%; p = 0.011) and CHA (5.0 ± 1.2%; p = 0.0017). The Clo and Clo+CHA groups also showed significant improvements (19%) compared to the Control (p = 0.03) and CHA (p = 0.04). Remnant biomaterial was significantly greater in Zol+CHA (15.0 ± 2.0) and Clo+CHA (15.0 ± 2.0%) than in CHA alone (8.0 ± 1.0%; p = 0.022), suggesting inhibition of bone graft resorption by BPs. Correlation analysis revealed a strong positive association between remnant biomaterial and new bone formation (Spearman ρ = 0.94, p = 0.005; Pearson r = 0.88, p = 0.021), supporting the biological synergy of CHA and BPs in bone repair.
Conclusion: Both bisphosphonates enhanced bone repair in the femoral defect model, demonstrating a synergistic effect when combined with nanostructured CHA. Zoledronate required the presence of the biomaterial to exert its osteogenic influence, while Clodronate stimulated new bone formation independently. These findings indicate that generation-specific differences among bisphosphonates may guide their future use in bone tissue engineering strategies.
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Landesberg R, Woo V, Cremers S, Cozin M, Marolt D, Vunjak-Novakovic G, et al. Potential pathophysiological mechanisms in osteonecrosis of the jaw. Ann N Y Acad Sci. 2011;1218:62–79. https://doi.org/10.1111/j.1749-6632.2010.05835.x DOI: https://doi.org/10.1111/j.1749-6632.2010.05835.x
Belfrage O, Isaksson H, Tägil M. Local treatment of a bone graft by soaking in zoledronic acid inhibits bone resorption and bone formation. A bone chamber study in rats. BMC Musculoskelet Disord. 2012;13:240. https://doi.org/10.1186/1471-2474-13-240 DOI: https://doi.org/10.1186/1471-2474-13-240
Toker H, Ozdemir H, Ozer H, Eren K. A comparative evaluation of the systemic and local alendronate treatment in synthetic bone graft: a histologic and histomorphometric study in a rat calvarial defect model. Oral Surg Oral Med Oral Pathol Oral Ra-diol. 2012;114(5 Suppl):S146–52. https://doi.org/10.1016/j.oooo.2011.09.027 DOI: https://doi.org/10.1016/j.oooo.2011.09.027
Peng H, Ling T, Zhang Y, Xie T, Pei X, Zhou K, et al. Nanowhiskers orchestrate bone formation and bone defect repair by modulating immune cell behavior. ACS Appl Mater Interfaces. 2023;15(7):9120–34. https://doi.org/10.1021/acsami.2c21865 DOI: https://doi.org/10.1021/acsami.2c21865
Bosemark P, Isaksson H, McDonald MM, Little DG. Augmentation of autologous bone graft by a combination of bone morpho-genic protein and bisphosphonate increased both callus volume and strength. Acta Orthop. 2013;84(1):106–11. https://doi.org/10.3109/17453674.2013.773123 DOI: https://doi.org/10.3109/17453674.2013.773123
Marx RE. Pamidronate (Aredia) and zoldronate (Zometa) – induced avascular necrosis of the jaws: a growing epidemic. J Oral Maxillofac Surg. 2003;61:1115. https://doi.org/10.1016/S0278-2391(03)00720-1 DOI: https://doi.org/10.1016/S0278-2391(03)00720-1
Roelofs AJ, Thompson K, Ebetino FH, Rogers MJ, Coxon FP. Bisphosphonates: molecular mechanisms of action and effects on bone cells, monocytes and macrophages. Curr Pharm Des. 2010;16:2950–60. https://doi.org/10.2174/138161210793563635 DOI: https://doi.org/10.2174/138161210793563635
Senel FC, Duman MK, Muci E, Cankaya M, Pampu AA, Ersoz S, et al. Jaw bone changes in rats after treatment with zoledronate and pamidronate. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010;109:385–91. https://doi.org/10.1016/j.tripleo.2009.10.011 DOI: https://doi.org/10.1016/j.tripleo.2009.10.011
AlRowis R, Aldawood A, AlOtaibi M, Alnasser E, AlSaif I, Aljaber A, et al. Medication-related osteonecrosis of the jaw (MRONJ): a review of pathophysiology, risk factors, preventive measures and treatment strategies. Saudi Dent J. 2022;34(3):202–10. https://doi.org/10.1016/j.sdentj.2022.01.003 DOI: https://doi.org/10.1016/j.sdentj.2022.01.003
Im GI, Qureshi SA, Kenney J, Rubash HE, Shanbhag AS. Osteoblast proliferation and maturation by bisphosphonates. Biomaterials. 2004;25(18):4105–15. https://doi.org/10.1016/j.biomaterials.2003.11.024 DOI: https://doi.org/10.1016/j.biomaterials.2003.11.024
Knoch VF, Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, et al. Effects of bisphosphonates on proliferation and oste-oblast differentiation of human bone marrow stromal cells. Biomaterials. 2005;26:6941–9. https://doi.org/10.1016/j.biomaterials.2005.04.059 DOI: https://doi.org/10.1016/j.biomaterials.2005.04.059
Altundal H, Gursoy B. The influence of alendronate on bone formation after autogenous free bone grafting in rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005;99:285–91. https://doi.org/10.1016/j.tripleo.2004.05.022 DOI: https://doi.org/10.1016/j.tripleo.2004.05.022
Jakobsen T, Baas J, Bechtold JE, Elmengard B, Søballe K. Soaking morselized allograft in bisphosphonate can impair implant fixation. Clin Orthop Relat Res. 2007;463:195–201. https://doi.org/10.1097/BLO.0b013e31813c6696 DOI: https://doi.org/10.1097/BLO.0b013e31813c6696
Seo SW, Cho SK, Storer SK, Lee FY. Zoledronate reduces unwanted bone resorption in intercalary bone allografts. Int Orthop. 2010;34:599–603. https://doi.org/10.1007/s00264-009-0748-7 DOI: https://doi.org/10.1007/s00264-009-0748-7
Kellesarian SV, Abduljabbar T, Vohra F, Gholamiazizi E, Malmstrom H, Romanos GR, et al. Does local ibandronate and/or pamidronate delivery enhance osseointegration? A systematic review. J Prosthodont. 2018;27(3):240–9. https://doi.org/10.1111/jopr.12571 DOI: https://doi.org/10.1111/jopr.12571
Fang H, Zhu D, Yang Q, Chen Y, Zhang C, Gao J, et al. Emerging zero-dimensional to four-dimensional biomaterials for bone regeneration. J Nanobiotechnol. 2022;20(1):26. https://doi.org/10.1186/s12951-021-01228-1 DOI: https://doi.org/10.1186/s12951-021-01228-1
Ruggiero SL, Mehrotra B, Rosenberg TJ, Engroff SL. Osteonecrosis of the jaws associated with the use of bisphosphonates: a review of 63 cases. J Oral Maxillofac Surg. 2004;62:527. https://doi.org/10.1016/j.joms.2004.02.004 DOI: https://doi.org/10.1016/j.joms.2004.02.004
Vasconcelos AC, Berti-Couto SA, Azambuja AA, Salum FG, Figueiredo MA, Da Silva VD, et al. Comparison of effects of clodro-nate and zoledronic acid on the repair of maxilla surgical wounds – histomorphometric, receptor activator of nuclear factor-kB ligand, osteoprotegerin, von Willebrand factor, and caspase-3 evaluation. J Oral Pathol Med. 2012;41(9):702–12. https://doi.org/10.1111/j.1600-0714. 2012.01140.x DOI: https://doi.org/10.1111/j.1600-0714.2012.01140.x
Crépin S, Laroche ML, Sarry B, Merle L. Osteonecrosis of the jaw induced by clodronate, an alkylbisphosphonate: case report and literature review. Eur J ClinPharmacol. 2010;66:547–54. https://doi.org/10.1007/s00228-010-0822-5 DOI: https://doi.org/10.1007/s00228-010-0822-5
Dong L, Jiang L, Xu Z, Zhang X. Denosumab, teriparatide and bisphosphonates for glucocorticoid-induced osteoporosis: a Bayesian network meta-analysis. Front Pharmacol. 2024;15:1336075. https://doi.org/10.3389/fphar.2024.1336075 DOI: https://doi.org/10.3389/fphar.2024.1336075
Valiense HB, Fernandes GVO, Moura BS, Calasans-Maia JA, Alves ATNN, Rossi AM, et al. Effect of Carbonate-apatite on bone repair in non-critical size defect of rat calvaria. Key Eng Mat. 2012;493–4:258–62. https://doi.org/10.4028/www.scientific.net/KEM.493-494.258 DOI: https://doi.org/10.4028/www.scientific.net/KEM.493-494.258
Mavropoulos E, Hausen M, Costa AM. Biocompatibility of carbonated hydroxyapatite nanoparticles with different crystallini-ties. Key Eng Mat. 2012;493–4:331–6. https://doi.org/10.4028/www.scientific.net/KEM.493-494.331 DOI: https://doi.org/10.4028/www.scientific.net/KEM.493-494.331
Le Geros RZ. Crystallography studies of the carbonate substitution in the apatite structure. Thesis. University of New York; 1967.
LeGeros RZ. Calcium phosphates in oral biology. Monogr Oral Sci. 1991;15:1–201. DOI: https://doi.org/10.1159/000419232
Hasegawa M, Doi Y, Uchida A. Cell-mediated bioresorption of sintered carbonate apatite in rabbits. J Bone Jt Surg Am. 2003;85:142–7. https://doi.org/10.1302/0301-620X.85B1.13414 DOI: https://doi.org/10.1302/0301-620X.85B1.13414
Calasans-Maia MD, Melo BR, Resende RFB, Louro RS, Sartoretto SC, Granjeiro JM, et al. Cytocompatibility and biocompatibil-ity of nanostructured carbonated hydroxyapatite spheres for bone repair. J Appl Oral Sci. 2015;23:599–608. https://doi.org/10.1590/1678-775720150122 DOI: https://doi.org/10.1590/1678-775720150122
Valiense H, Barreto M, Resende R, Alves AT, Rossi AM, Mavropoulos E, et al. In vitro and in vivo evaluation of strontium-containing nanostructured carbonated hydroxyapatite/sodium alginate for sinus lift in rabbits. J Biomed Mater Res B. 2016;104:274–82. https://doi.org/10.1002/jbm.b.33392 DOI: https://doi.org/10.1002/jbm.b.33392
Cezar I, Kammer G, Alves A, Calasans-Maia J, Gress MA, Rossi AM, et al. Standardized study of carbonate apatite as bone substitute in rabbit’s tibia. Key Eng Materials. 2012;493–49:242–6. https://doi.org/10.4028/www.scientific.net/KEM.493-494.242 DOI: https://doi.org/10.4028/www.scientific.net/KEM.493-494.242
Alves Cardoso D, Jansen JA, Leeuwenburgh SC. Synthesis and application of nanostructured calcium phosphate ceramics for bone regeneration. J Biomed Mater Res B Appl Biomater. 2012;100(8):2316–26. https://doi.org/10.1002/jbm.b.32794 DOI: https://doi.org/10.1002/jbm.b.32794
Lebre F, Sridharan R, Sawkins MJ, Kelly DJ, O’Brien FJ, Lavelle EC. The shape and size of hydroxyapatite particles dictate inflammatory responses following implantation. Sci Rep. 2017;7:2922. https://doi.org/10.1038/s41598-017-03086-0 DOI: https://doi.org/10.1038/s41598-017-03086-0
Kurien T, Pearson R, Scammell B. Bone graft substitutes currently available in orthopaedic practice. Bone Joint J. 2013;95-B(5):583–97. https://doi.org/10.1302/0301-620x.95b5.30286 DOI: https://doi.org/10.1302/0301-620X.95B5.30286
Ferreira GZ, Filho EVZ, Rubira-Bullen IRF, Garlet GP, Santos CF, Santos PSS. Delayed alveolar bone repair and osteonecrosis associated with Zoledronic Acid therapy in rats: macroscopic, microscopic and molecular analysis. J Appl Oral Sci. 2020;28:e20200204. https://doi.org/10.1590/1678-7757-2020-0204 DOI: https://doi.org/10.1590/1678-7757-2020-0204
Zhu E, Louis L, Brooks D, Bouxsein M, Demay M. Effect of bisphosphonates on the rapidly growing male murine skeleton. Endocrinol. 2014;155(4):1188–96. https://doi.org/10.1210/en.2013-1993 DOI: https://doi.org/10.1210/en.2013-1993
Fu K, Xu Q, Czernuszka J, Triffitt J, Xia Z. Characterization of a biodegradable coralline hydroxyapatite/calcium carbonate composite and its clinical implementation. Biomed Mat. 2013;8(6):065007. https://doi.org/10.1088/1748-6041/8/6/065007 DOI: https://doi.org/10.1088/1748-6041/8/6/065007
Ayukawa Y, Suzuki Y, Tsuru K, Koyano K, Ishikawa K. Histological comparison in rats between carbonate apatite fabricated from gypsum and sintered hydroxyapatite on bone remodeling. Biomed Res Int. 2015;2015:1–7. https://doi.org/10.1155/2015/579541 DOI: https://doi.org/10.1155/2015/579541
Mamun S, Haider I, Rahman T, Rubel A, Diba F. Post-operative outcomes of radiological and histopathological findings after hydroxyapatite and allogenic bone graft among jaw cyst patients: a randomized clinical trial. J Nat Inst Neurosci Bangl. 2020;6(1):48–53. https://doi.org/10.3329/jninb.v6i1.48030 DOI: https://doi.org/10.3329/jninb.v6i1.48030
Opperman KS, Vandyke K, Clark KC, Coulter EA, Hewett DR, Mrozik KM, et al. Clodronate-liposome mediated macrophage depletion abrogates multiple myeloma tumor establishment in vivo. Neoplasia. 2019;21(8):777–87. https://doi.org/10.1016/j.neo.2019.05.006 DOI: https://doi.org/10.1016/j.neo.2019.05.006
Sakti Y, Magetsari R. Structural evaluation and animal implantation of porous eggshell waste-derived hydroxyapatite graft as bone substitution. J Med Sci. 2013;45(4):176–86. https://doi.org/10.19106/jmedscie004504201304 DOI: https://doi.org/10.19106/JMedScie004504201304
Välimäki VV, Moritz N, Yrjans JJ, Vuorio E, Aro HT. Effect of zoledronic acid on incorporation of a bioceramic bone graft substi-tute. Bone. 2006;38:432–43. https://doi.org/10.1016/j.bone.2005.09.016 DOI: https://doi.org/10.1016/j.bone.2005.09.016
Dupoirieux L, Pourquier D, Picot MC, Neves M. Comparative study of three different membranes for guided bone regenera-tion of rat cranial defects. Int J Oral Maxillofac Surg. 2001;30:58–62. https://doi.org/10.1054/ijom.2000.0011 DOI: https://doi.org/10.1054/ijom.2000.0011
Zambuzzi WF, Coelho PG, Alves GG, Granjeiro JM. Intracellular signal transduction as a factor in the development of ‘smart’ biomaterials for bone tissue engineering. Biotechnol Bioeng. 2011;108(6):1246–50. https://doi.org/10.1002/bit.23117 DOI: https://doi.org/10.1002/bit.23117
Gil‐Peña H, Fernández-Iglesias Á, Fuente R, Alonso‐Durán L, Santos F, López J. Effect of clodronate administration on the structure of the primary spongiosa derived from the growth cartilage in growing rats. Int J Biochem Pharmacol. 2019;2(1):27–35. https://doi.org/10.18689/ijbp-1000106 DOI: https://doi.org/10.18689/ijbp-1000106
Nurlidar F, Kobayashi M. Succinylated bacterial cellulose induce carbonated hydroxyapatite deposition in a solution mimick-ing body fluid. Indon J Chem. 2019;19(4):858. https://doi.org/10.22146/ijc.35048 DOI: https://doi.org/10.22146/ijc.35048
Kim SW, Kim JS, Papadopoulos J, Choi HJ, He J, Maya M, et al. Consistent interactions between tumor cell IL-6 and macro-phage TNF-α enhance the growth of human prostate cancer cells in the bone of nude mouse. Int Immunopharmacol. 2011;11(7):862–72. https://doi.org/10.1016/j.intimp.2011.01.004 DOI: https://doi.org/10.1016/j.intimp.2011.01.004
Oliveira AL, Pedro AJ, Arroyo CS, Mano JF, Rodriguez G, San Roman J, et al. Biomimetic Ca-P coatings incorporating bisphos-phonates produced on starch-based degradable biomaterials. J Biomed Mater Res Appl Biomater. 2010;92B:55–67. https://doi.org/10.1002/jbm.b.31489 DOI: https://doi.org/10.1002/jbm.b.31489
Rosenqvist K, Airaksinen S, Fraser SJ, Gordon KC, Juppo AM. Interaction of bioactive glass with clodronate. Int J Pharm. 2013;452(1–2):102–7. https://doi.org/10.1016/j.ijpharm.2013.04.059 DOI: https://doi.org/10.1016/j.ijpharm.2013.04.059
Ratnayake J, Mucalo M, Dias G. Substituted hydroxyapatites for bone regeneration: a review of current trends. J Biomed Mat Res Part B Appl Biomat. 2016;105(5):1285–99. https://doi.org/10.1002/jbm.b.33651 DOI: https://doi.org/10.1002/jbm.b.33651
Mamoud R, Hashem A, Gouda I. Evaluation of hydroxyapatite and hydroxyapatite combined with platelet-rich plasma graft in treatment of intrabony periodontal defect – a comparative study. Dent Sci Updates. 2022;3(2):247–54. https://doi.org/10.21608/dsu.2022.122582.1112 DOI: https://doi.org/10.21608/dsu.2022.122582.1112
Yassuda DH, Costa NF, Fernandes GVO, Alves GG, Granjeiro JM, Soares GA. Magnesium incorporation into β-TCP reduced its in vivo resorption by decreasing parathormone production. J Biomed Mater Res A. 2013;101(7):1986–93. https://doi.org/10.1002/jbm.a.34502 DOI: https://doi.org/10.1002/jbm.a.34502
Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res. 1986;205: 299–308. https://doi.org/10.1097/00003086-198604 000-00036 DOI: https://doi.org/10.1097/00003086-198604000-00036
Calasans-Maia MD, Fernandes GVO, Rossi AM, Dias EP, Almeida GDS, Mitri FF, et al. Effect of hydroxyapatite and zinc-containing hydroxyapatite on osseous repair of critical size defect in the rat calvaria. Key Eng Mat. 2008;361–3:1273–6. https://doi.org/10.4028/www.scientific.net/KEM.361-363.1273 DOI: https://doi.org/10.4028/www.scientific.net/KEM.361-363.1273
Fernandes GVO, Calasans-Maia MD, Mitri FF, Bernardo VG, Rossi AM, Almeida GDS, et al. Histomorphometric analysis of bone repair in critical size defect in rats calvaria treated with hydroxyapatite and zinc-containing hydroxyapatite 5%. Key Eng Mat. 2009;396–8:15–18. https://doi.org/10.4028/www.scientific.net/KEM.396-398.15 DOI: https://doi.org/10.4028/www.scientific.net/KEM.396-398.15
Spicer PP, Kretlow JD, Young S, Jansen JA, Kasper FK, Mikos AG. Evaluation of bone regeneration using the rat critical size calvarial defect. Nat Protoc. 2012;7(10):1918–29. https://doi.org/10.1038/nprot.2012.113 DOI: https://doi.org/10.1038/nprot.2012.113
Zambuzzi WF, Fernandes GVO, Iano FG, Fernandes MS, Granjeiro JM, Oliveira RC. Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects. Braz Dent J. 2012;23:315–21. https://doi.org/10.1590/S0103-64402012000400002 DOI: https://doi.org/10.1590/S0103-64402012000400002
de Oliveira AM, Castro-Silva II, Fernandes GVO, Melo BR, Alves ATNN, Silva Júnior A, et al. Effectiveness and acceleration of bone repair in critical-sized rat calvarial defects using low-level laser therapy. Lasers Surg Med. 2014;46:61–7. https://doi.org/10.1002/lsm.2219 DOI: https://doi.org/10.1002/lsm.22198
Costa NMF, Yassuda DH, Sader MS, Fernandes GVO, Almeida GDS, Granjeiro JM. Osteogenic effect of tricalcium phosphate substituted by magnesium associated with Genderm® membrane in rat calvarial defect model. Mater Sci Eng C Mater Biol Appl. 2016;61:63–71. https://doi.org/10.1016/j.msec.2015.12.00 DOI: https://doi.org/10.1016/j.msec.2015.12.003
Soares IMV, Fernandes GVO, Cavalcante LC, Carvalho YKP, Bezerra DO, Carvalho MAM, et al. The influence of Aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats. J Appl Oral Sci. 2019;27:1–11. https://doi.org/10.1590/1678-7757-2018-010 DOI: https://doi.org/10.1590/1678-7757-2018-0103
Schemitsch EH. Size matters: defining critical in bone defect size! J Orthop Trauma. 2017;31(Suppl 5):S20–2. https://doi.org/10.1097/BOT.0000000000000978 DOI: https://doi.org/10.1097/BOT.0000000000000978
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Copyright (c) 2025 Gustavo Vicentis Oliveira Fernandes, Carlos Henrique Sardenberg Pereira, Adriana Terezinha Neves Novellino Alves, Alexandre Malta Rossi, José Mauro Granjeiro, Mônica Diuana Calasans-Maia, Gutemberg Gomes Alves
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