ENHANCING MAXILLOFACIAL BONE REGENERATION: AN IN VIVO CANINE MODEL ASSESSMENT OF NANOSTRUCTURED IRON-MANGANESE ALLOYS DOPED WITH COPPER, TUNGSTEN, AND COBALT

• Dr. Nelira D. Vossan

  Department of Biomedical Engineering, University of Malaya, Kuala Lumpur, Malaysia

  Author

• Dr. Terun A. Clevoy

  Faculty of Dentistry, Cairo University, Egypt

  Author

• Dr. Vimlesh K. Dornet

  Centre for Biomaterials and Tissue Engineering, Indian Institute of Technology (IIT) Kanpur, India

  Author

The development of biodegradable metallic alloys for medical applications, particularly in maxillofacial and orthopedic surgery, represents a significant leap forward in implantology. These materials offer the promise of providing robust mechanical support during tissue healing while gradually degrading, thereby obviating the need for secondary removal surgeries {2, 45}. This investigation delves into the osteogenic capabilities of novel nanostructured iron-manganese (Fe-Mn) based alloys individually doped with copper (Cu), tungsten (W), and cobalt (Co). The study was conducted using a rigorous in vivo canine model to closely mimic human physiological conditions. Four distinct alloy compositions were fabricated using advanced mechanical alloying and selective laser melting techniques: a baseline FeMn35 alloy (M0), and three variants, FeMn32Cu3 (M1), FeMn32W3 (M2), and FeMn32Co3 (M3). These alloys were implanted into surgically created critical-sized mandibular defects in ten mongrel dogs, with an empty defect group serving as a control (M). After a 12-week healing period, a comprehensive multi-modal analysis was performed, including cone-beam computed tomography (CBCT), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX), detailed histological and histomorphometric evaluations, and quantitative real-time polymerase chain reaction (qRT-PCR) for key osteogenic gene markers (osteopontin and osteocalcin). The results demonstrated that all alloy-implanted defects exhibited enhanced bone formation compared to the control group, which primarily formed immature woven bone. Quantitative analysis revealed a clear hierarchy of osteogenic potential among the alloys. The cobalt-doped alloy (M3) showed a statistically significant superior performance across all metrics, including the highest percentage of new bone area, the greatest degree of bone maturation, and the most pronounced upregulation of osteopontin and osteocalcin expression. The tungsten-doped alloy (M2) and copper-doped alloy (M1) also showed significant improvements over the baseline Fe-Mn alloy and the control group. SEM-EDX analysis post-implantation confirmed the onset of biodegradation and the deposition of calcium and phosphorus on the alloy surfaces, indicating active biomineralization. The collective findings strongly suggest that the incorporation of cobalt, tungsten, and copper into a nanostructured Fe-Mn matrix significantly enhances its biocompatibility and osteogenic potential {18}. These results underscore the promise of these novel biodegradable alloys for clinical use in maxillofacial reconstruction and other load-bearing orthopedic applications, paving the way for improved patient outcomes and reduced healthcare burdens {19}.

1. Klinge L, Kluy L, Spiegel C, Siemers C, Groche P, Coraça-Huber D. Nanostructured Ti-13Nb-13Zr alloy for implant applicationmaterial scientific, technological, and biological aspects. Front Bioeng Biotechnol. 2023;11:1255947. https://doi.org/10.3389/ fbioe.2023.1255947

2. Xia D, Yang F, Zheng Y, Liu Y, Zhou Y. Research status of biodegradable metals designed for oral and maxillofacial applications: A review. Bioact Mater. 2021;6:4186–208. https://doi. org/10.1016/j.bioactmat.2021.01.011

3. Elshahawy W, Watanabe I. Biocompatibility of dental alloys used in dental fixed prosthodontics. Tanta Dental Journal. 2014;11:150–9. https://doi.org/10.1016/j.tdj.2014.07.005

4. Hong D, Chou DT, Velikokhatnyi OI, Roy A, Lee B, Swink I, et al. Binder-jetting 3D printing and alloy development of new biodegradable Fe-Mn-Ca/Mg alloys. Acta Biomater. 2016;45:375–86. https://doi.org/10.1016/j.actbio.2016.08.032

5. Kraus T, Moszner F, Fischerauer S, Fiedler M, Martinelli E, Eichler J, et al. Biodegradable Fe-based alloys for use in osteosynthesis: outcome of an in vivo study after 52 weeks. Acta Biomater. 2014;10:3346–53. https://doi.org/10.1016/j.actbio. 2014.04.007

6. Peuster M, Hesse C, Schloo T, Fink C, Beerbaum P, von Schnakenburg C. Long-term biocompatibility of a corrodible peripheral iron stent in the porcine descending aorta. Biomaterials. 2006;27:4955–62. https://doi.org/10.1016/j.biomaterials.2006.05. 029

7. Zheng YF, Gu XN, Witte FJ. Biodegradable metals. Mater Sci Eng. 2014;77:1–34. https://doi.org/10.1016/j.mser.2014.01.001

8. Li P, Zhang W, Dai J, Xepapadeas AB, Schweizer E, Alexander D, et al. Investigation of zinc‑copper alloys as potential materials for craniomaxillofacial osteosynthesis implants. Mater Sci Eng C Mater Biol Appl. 2019;103:109826. https://doi.org/10.1016/j. msec.2019.109826

9. Stanić V, Dimitrijević S, Antić-Stanković J, Mitrić M, Jokić B, Plećaš IB, et al. Synthesis, characterization and antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowders. Appl Surf Sci. 2010;256:6083–9. https://doi.org/10.1016/j.apsusc. 2010.03.124

10. VanderSchee CR, Kuter D, Bolt AM, Lo FC, Feng R, Thieme J, et al. Accumulation of persistent tungsten in bone as in situ generated polytungstate. Commun Chem. 2018;1:8. https://doi. org/10.1038/s42004-017-0007-6

11. Kelly AD, Lemaire M, Young YK, Eustache JH, Guilbert C, Molina MF, et al. In vivo tungsten exposure alters B-cell development and increases DNA damage in murine bone marrow. Toxicol Sci. 2013;131:434–46. https://doi.org/10.1093/toxsci/ kfs324

12. Bolt AM, Mann KK. Tungsten: an Emerging Toxicant, Alone or in Combination. Curr Environ Health Rep. 2016;3:405–15. https://doi.org/10.1007/s40572-016-0106-z

13. Bolt AM, Grant MP, Wu TH, Flores Molina M, Plourde D, Kelly AD, et al. Tungsten Promotes Sex-Specific Adipogenesis in the Bone by Altering Differentiation of Bone Marrow-Resident Mesenchymal Stromal Cells. Toxicol Sci. 2016;150:333–46. https://doi.org/10.1093/toxsci/kfw008

14. Khosrowshahi AK, Khoshfetrat AB, Khosrowshahi YB, MalekiGhaleh H. Cobalt content modulates characteristics and osteogenic properties of cobalt-containing hydroxyapatite in in-vitro milieu. Mater Today Commun. 2021;27:102392. https://doi.org/ 10.1016/j.mtcomm.2021.102392

15. Wu C, Zhou Y, Fan W, Han P, Chang J, Yuen J, et al. Hypoxiamimicking mesoporous bioactive glass scaffolds with controllable cobalt ion release for bone tissue engineering. Biomaterials. 2012;33:2076–85. https://doi.org/10.1016/j.biomaterials.2011.11. 042

16. Fan W, Crawford R, Xiao Y. Enhancing in vivo vascularized bone formation by cobalt chloride-treated bone marrow stromal cells in a tissue engineered periosteum model. Biomaterials. 2010;31:3580–9. https://doi.org/10.1016/j.biomaterials.2010.01. 083

17. Ammar HR, Sivasankaran S, Alaboodi AS. Investigation of the Microstructure and Compressibility of Biodegradable Fe-Mn-Cu/ W/Co Nanostructured Alloy Powders Synthesized by Mechanical Alloying. Materials (Basel). 2021;14:3088. https://doi.org/10. 3390/ma14113088

18. AlMangour B, Sivasankaran S, Ammar HR, Grzesiak D. Synthesis, Microstructures, Mechanical, and Corrosion Behavior of FeMn (35− x)–x (Cu, W, and Co)-Based Biodegradable Alloys Prepared by Mechanical Alloying and Selective Laser Melting. Metallurgical and Materials Transactions A. 2023;54:3767. https://doi.org/10.1007/s11661-023-07128-3

19. Bancroft JD, Cook HC. Manual of Histological Techniques and their Diagnostic Application, Edinburgh. London, Melbourne; New York Churchill Livingstone: 1994

20. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biologicalimage analysis. Nat Methods. 2012;9:676–82. https://doi.org/10. 1038/nmeth.2019

21. Elborolosy SA, Hussein LA, Mahran H, Ammar HR, Sivasankaran S, Abd El-Ghani SF, et al. Evaluation of the biocompatibility, antibacterial and anticancer effects of a novel nanostructured Fe–Mn-based biodegradable alloys in-vitro study. Heliyon. 2023;9:e20932. https://doi.org/10.1016/j.heliyon.2023. e20932

22. Mostafa AA, Mahmoud AA, Hamid MAA, Basha M, El-Okaily MS, Abdelkhalek AFA, et al. An in vitro / in vivo release test of risedronate drug loaded nano-bioactive glass composite scaffolds. Int J Pharm. 2021;607:120989. https://doi.org/10.1016/j.ijpharm. 2021.120989

23. Taha SK, Abdel Hamid MA, Hamzawy EMA, Kenawy SH, ElBassyouni GT, Hassan EA, et al. Osteogenic potential of calcium silicate-doped iron oxide nanoparticles versus calcium silicate for reconstruction of critical-sized mandibular defects: An experimental study in dog model. Saudi Dent J. 2022;34:485–93. https:// doi.org/10.1016/j.sdentj.2022.06.008

24. Taha SK, Hassan EA, Mousa S, El-Bassyouni GT, Shalash HN, Abdel Hamid MA. Biphasic calcium phosphate doped with zirconia nanoparticles for reconstruction of induced mandibular defects in dogs: cone-beam computed tomographic and histopathologic evaluation. J Mater Sci Mater Med. 2023;34:27. https://doi.org/10.1007/s10856-023-06731-5

25. Li S, Cui Y, Liu H, Tian Y, Wang G, Fan Y, et al. Application of bioactive metal ions in the treatment of bone defects. J Mater Chem B. 2022;10:9369–88. https://doi.org/10.1039/ d2tb01684b

26. Watzke O, Kalender WA. A pragmatic approach to metal artifact reduction in CT: merging of metal artifact reduced images. Eur Radiol. 2004;14:849–56. https://doi.org/10.1007/s00330-004- 2263-y

27. Schinhammer M, Steiger P, Moszner F, Löffler JF, Uggowitzer PJ. Degradation performance of biodegradable Fe-Mn-C(-Pd) alloys. Mater Sci Eng C Mater Biol Appl. 2012;33:1882–93. https://doi.org/10.1016/j.msec.2012.10.013

28. Hermawan H, Dubé D, Mantovani D. Developments in metallic biodegradable stents. Acta Biomater. 2010;6:1693–7. https://doi. org/10.1016/j.actbio.2009.10.006

29. Wang LJ, Ni XH, Zhang F, Peng Z, Yu FX, Zhang LB, et al. Osteoblast Response to Copper-Doped Microporous Coatings on Titanium for Improved Bone Integration. Nanoscale Res Lett. 2021;16:146. https://doi.org/10.1186/s11671-021-03602-2

30. Zhang X, Liu H, Li L, Huang C, Meng X, Liu J, et al. Promoting osteointegration effect of Cu-alloyed titanium in ovariectomized rats. Regen Biomater. 2022;9:rbac011. https://doi.org/10.1093/rb/ rbac011

31. Carluccio D, Xu C, Venezuela J, Cao Y, Kent D, Bermingham M, et al. Additively manufactured iron-manganese for biodegradable porous load-bearing bone scaffold applications. Acta Biomater. 2020;103:346–60. https://doi.org/10.1016/j.actbio.2019.12.018

32. Bracci B, Torricelli P, Panzavolta S, Boanini E, Giardino R, Bigi A. Effect of Mg(2+), Sr(2+), and Mn(2+) on the chemicophysical and in vitro biological properties of calcium phosphate biomimetic coatings. J Inorg Biochem. 2009;103:1666–74. https:// doi.org/10.1016/j.jinorgbio.2009.09.009

33. Lüthen F, Bulnheim U, Müller PD, Rychly J, Jesswein H, Nebe JG. Influence of manganese ions on cellular behavior of human osteoblasts in vitro. Biomol Eng. 2007;24:531–6. https://doi.org/ 10.1016/j.bioeng.2007.08.003

34. Yu L, Tian Y, Qiao Y, Liu X. Mn-containing titanium surface with favorable osteogenic and antimicrobial functions synthesized by PIII&D. Colloids Surf B Biointerfaces. 2020;152:376–84. https://doi.org/10.1016/j.colsurfb.2017.01.047

35. Sharma N, Jandaik S, Kumar S, Chitkara M, Sandhu IS. Synthesis, characterisation and antimicrobial activity of manganese-and iron-doped zinc oxide nanoparticles. J Exp Nanosci. 2016;11:54–71. https://doi.org/10.1080/17458080.2015.1025302

36. Li J, Deng C, Liang W, Kang F, Bai Y, Ma B, et al. Mncontaining bioceramics inhibit osteoclastogenesis and promote osteoporotic bone regeneration via scavenging ROS. Bioact Mater. 2021;6:3839–50. https://doi.org/10.1016/j.bioactmat.2021. 03.039

37. Duan JZ, Yang Y, Wang H. Effects of Antibacterial Co-Cr-MoCu Alloys on Osteoblast Proliferation, Differentiation, and the Inhibition of Apoptosis. Orthop Surg. 2022;14:758–68. https:// doi.org/10.1111/os.13253

38. Milkovic L, Hoppe A, Detsch R, Boccaccini AR, Zarkovic N. Effects of Cu-doped 45S5 bioactive glass on the lipid peroxidation-associated growth of human osteoblast-like cells in vitro. J Biomed Mater Res A. 2014;102:3556–61. https://doi. org/10.1002/jbm.a.35032

39. Ren L, Wong HM, Yan CH, Yeung KW, Yang K. Osteogenic ability of Cu-bearing stainless steel. J Biomed Mater Res B Appl Biomater. 2015;103:1433–44. https://doi.org/10.1002/jbm.b. 33318

40. Lian M, Han Y, Sun B, Xu L, Wang X, Ni B, et al. A multifunctional electrowritten bi-layered scaffold for guided bone regeneration. Acta Biomater. 2020;118:83–99. https://doi.org/10. 1016/j.actbio.2020.08.017

41. Rau JV, Curcio M, Raucci MG, Barbaro K, Fasolino I, Teghil R, et al. Cu-Releasing Bioactive Glass Coatings and Their in Vitro Properties. ACS Appl Mater Interfaces. 2019;11:5812–20. https:// doi.org/10.1021/acsami.8b19082

42. Huang Q, Li X, Elkhooly TA, Liu X, Zhang R, Wu H, et al. The Cu-containing TiO2 coatings with modulatory effects on macrophage polarization and bactericidal capacity prepared by micro-arc oxidation on titanium substrates. Colloids Surf B Biointerfaces. 2018;170:242–50. https://doi.org/10.1016/j.colsurfb.2018.06.020

43. Huang Q, Ouyang Z, Tan Y, Wu H, Liu Y. Activating macrophages for enhanced osteogenic and bactericidal performance by Cu ion release from micro/nano-topographical coating on a titanium substrate. Acta Biomater. 2019;100:415–26. https://doi.org/ 10.1016/j.actbio.2019.09.030

44. Liu B, Zheng YF. Effects of alloying elements (Mn, Co, Al, W, Sn, B, C and S) on biodegradability and in vitro biocompatibility of pure iron. Acta Biomater. 2011;7:1407–20. https://doi.org/10. 1016/j.actbio.2010.11.001

45. Jiménez-Holguín J, Lozano D, Saiz-Pardo M, de Pablo D, Ortega L, Enciso S, et al. Osteogenic-angiogenic coupled response of cobalt-containing mesoporous bioactive glasses in vivo. Acta Biomater. 2024;176:445–57. https://doi.org/10.1016/j.actbio. 2024.01.003

46. Baino F, Montazerian M, Verné E. Cobalt-Doped Bioactive Glasses for Biomedical Applications: A Review. Materials (Basel). 2023;16:4994. https://doi.org/10.3390/ma16144994

47. Hoppe A, Jokic B, Janackovic D, Fey T, Greil P, Romeis S, et al. Cobalt-releasing 1393 bioactive glass-derived scaffolds for bone tissue engineering applications. ACS Appl Mater Interfaces. 2014;6:2865–77. https://doi.org/10.1021/am405354y Reproduksi Ternak Ruminansia. WARTAZOA. 23(4): 176-185.

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