Isolation of Antibacterial Nano-Hydroxyapatite Biomaterial from Waste Buffalo Bone and Its Characterization

: pp. 133–141
Central Department of Chemistry, Tribhuvan University
Central Department of Chemistry, Tribhuvan University, Research Centre for Applied Science and Technology (RECAST), Tribhuvan University
Central Department of Chemistry, Tribhuvan University
Department of Microbiology
Central Department of Chemistry, Tribhuvan University, Research Centre for Applied Science and Technology (RECAST), Tribhuvan University

Hydroxyapatite nanoparticles were isolated from a biowaste, buffalo bone, via the thermal decomposition method. The resulting white powdered material was characterized by Fourier Transformed Infrared (FTIR) spectroscopy, X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), and Energy Dispersive X-ray (EDX) analysis. The FTIR spectra confirmed that a heat treatment of the bone powder at the temperature at or above 1223 K removed the organic moieties leading to the formation of a pure inorganic biomineral. The XRD analyses showed that the obtained material was nanocrystalline HAp (nano-HAp) with an average grain diameter of 25 nm, while their rod-shaped particles with their tightly agglomerated morphology were confirmed by the SEM analysis. Besides Calcium (Ca), Phosphorous (P), and Oxygen (O), trace amounts of Aluminum (Al), Magnesium (Mg), Copper (Cu), Zirconium (Zr) and Carbon (C) were also found by EDX analysis. Antibacterial activity of nano-HAp against six standard isolates was investigated by the agar well diffusion method and found to be more susceptible to Acinetobacter baumannii while other standard strains such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus showed lesser susceptibility and no antibacterial activity was noticed against Salmonella typhi and Methicillin resistant Staphylococcus aureus (MRSA) with the analysed concentration of nano-HAp suggesting its potential application in biomedical fields.

  1. Manalu, J.L.; Soegijono, B.; Indrani, D.J. Characterization of Hydroxyapatite Derived from Bovine Bone. Asian J. Appl. Sci.2015, 3 (4), 758-765.
  2. Lim, K.T.; Kim, J.W.; Kim, J.; Chung, J.H. Development and Evaluation of Natural Hydroxyapatite Ceramics Produced by the Heat Treatment of Pig Bones. J. Biosyst. Eng.2014, 39, 227-234.
  3. Ooi, C.Y.; Hamdi, M.; Ramesh, S. Properties of Hydroxyapatite Produced by Annealing of Bovine Bone. Ceram. Int.2007, 33, 1171-1177.
  4. Kolmas, J.; Groszyk, E.; Kwiatkowska, R.D. Substituted Hydroxyapatites with Antibacterial Properties. Biomed Res. Int.2014, 2014,Article ID 178123.
  5. Rana, M.; Akhtar, N.; Rahman, S.; Hasan, Z. Extraction and Characterization of Hydroxyapatite from Bovine Cortical Bone and Effect of Radiation. Int. J. Biosci.2017, 11, 20-30.
  6. Balu, S.; Sundaradoss, M. V.; Andra, S.; Jeevanandam, J. Facile Biogenic Fabrication of Hydroxyapatite Nanorods Using Cuttlefish Bone and Their Bactericidal and Biocompatibility Study. Beilstein J. Nanotechnol.2020, 11, 285-295.
  7. Savvova, O. Biocide Apatite Glass-Ceramic Materials for Bone Endoprosthetics. Chem. Chem. Technol. 2013, 7, 109-112.
  8. Savvova, О.; Babich, O.; Fesenko, O. Investigation of Structure Formation in Calciumsilicophosphate Glass-Ceramic Coatings for Dental Implants. Chem. Chem. Technol. 2018, 12, 244-250.
  9. Nur, A.; Budiman, A. W.; Jumari, A.; Nazriati, N.; Fajaroh.; F. Electrosynthesis of Ni-Co/Hydroxyapatite As a Catalyst for Hydrogen Generation via the Hydrolysis of Aqueous Sodium Borohydride [NaBH4] Solutions. Chem. Chem. Technol. 2021, 15, 389-394.
  10. Singh, G.; Singh, R.P.; Jolly, S.S. Customized Hydroxyapatites for Bone-Tissue Engineering and Drug Delivery Applications: A Review. J. Sol-Gel Sci. Technol.2020, 94, 505-530.
  11. Fathi, M.H.; Hanifi, A.; Mortazavi, V. Preparation and Bioactivity Evaluation of Bone-like Hydroxyapatite Nanopowder. J. Mater. Process. Technol.2008, 202(1-3), 536-542.
  12. Eshtiagh-Hosseini, H.; Housaindokht, M.R.; Chahkandi, M. Effects of Parameters of Sol-Gel Process on the Phase Evolution of Sol-Gel-Derived Hydroxyapatite. Mater. Chem. Phys.2007, 106(2-3), 310-316.
  13. Han, Y.C.; Wang, X.Y.; Li, S.P. Change of Phase Composition and Morphology of Sonochemically Synthesised Hydroxyapatite Nanoparticles with Glycosaminoglycans during Thermal Treatment. Adv. Appl. Ceram.2009, 108, 400-405.
  14. Giardina, M.A.; Fanovich, M.A. Synthesis of Nanocrystalline Hydroxyapatite from Ca[OH]2 and H3PO4 Assisted by Ultrasonic Irradiation. Ceram. Int.2010, 36, 1961-1969.
  15. Barakat, N.A.M.; Khil, M.S.; Omran, A.M.; Sheikh, F.A.; Kim, H.Y. Extraction of Pure Natural Hydroxyapatite from the Bovine Bones Bio Waste by Three Different Methods. J. Mater. Process. Technol.2009, 209(7), 3408-3415.
  16. Neira, I.S.; Guitián, F.; Taniguchi, T.; Watanabe, T.; Yoshimura, M. Hydrothermal Synthesis of Hydroxyapatite Whiskers with Sharp Faceted Hexagonal Morphology. J. Mater. Sci.2008, 43(7), 2171-2178.
  17. Iqbal, N.; Abdul Kadir, M.R.; Nik Malek, N.A.N.; Humaimi Mahmood, N.; Raman Murali, M.; Kamarul, T. Rapid Microwave Assisted Synthesis and Characterization of Nanosized Silver-Doped Hydroxyapatite with Antibacterial Properties. Mater. Lett.2012, 89, 118-122.
  18. Hassan, M.N.; Mahmoud, M.M.; El-Fattah, A.A.; Kandil, S. Microwave-Assisted Preparation of Nano-Hydroxyapatite for Bone Substitutes. Ceram. Int.2016, 42(3), 3725-3744.
  19. Ronan, K.; Kannan, M.B. Novel Sustainable Route for Synthesis of Hydroxyapatite Biomaterial from Biowastes. ACS Sustain. Chem. Eng.2017, 5(3), 2237-2245.
  20. Abdulrahman, I.; Tijani, H.I.; Mohammed, B.A.; Saidu, H.; Yusuf, H.; Ndejiko Jibrin, M.; Mohammed, S. From Garbage to Biomaterials: An Overview on Egg Shell Based Hydroxyapatite. J. Mater.2014, 2014, Article ID 802467.
  21. Rajesh, R.; Hariharasubramanian, A.; Ravichandran, Y.D. Chicken Bone as a Bioresource for the Bioceramic [Hydroxyapatite]. Phosphorus, Sulfur Silicon Relat. Elem.2012, 187(8), 914-925.
  22. Malla, K.P.; Regmi, S.; Nepal, A.; Bhattarai, S.; Yadav, R. J.; Sakurai, S.; Adhikari, R. Extraction and Characterization of Novel Natural Hydroxyapatite Bioceramic by Thermal Decomposition of Waste Ostrich Bone. Int. J. Biomater.2020, 2020,Article ID 1690178.
  23. Fara, A.N.K.A.; Abdullah, H.Z. Characterization of Derived Natural Hydroxyapatite [HAp] Obtained from Different Types of Tilapia Fish Bones and Scales. AIP Conf. Proc.2015, 1669, 020071-0200776.
  24. Venkatesan, J.; Lowe, B.; Manivasagan, P.; Kang, K.H.; Chalisserry, E.P.; Anil, S.; Kim, D.G.; Kim, S.K. Isolation and Characterization of Nano-Hydroxyapatite from Salmon Fish Bone. Materials2015, 8(8), 5426-5439.
  25. Pandey, G.; Dhakal, K.N.; Singh, A.K.; Dhungel, S.K.; Adhikari, R. Facile Methods of Preparing Pure Hydroxyapatite Nanoparticles in Ordinary Laboratories. Bibechana2021, 18(1), 83-90.
  26. Laonapakul, T. Synthesis of Hydroxyapatite from Biogenic Wastes. KKU Eng. J.2015, 42(3), 269-275.
  27. Odusote, J.K.; Danyuo, Y.; Baruwa, A.D.; Azeez, A.A. Synthesis and Characterization of Hydroxyapatite from Bovine Bone for Production of Dental Implants. J. Appl. Biomater. Funct. Mater.2019, 17, 1-7
  28. Mohd Pu'ad, N.A.S.; Koshy, P.; Abdullah, H.Z.; Idris, M.I.; Lee, T.C. Syntheses of Hydroxyapatite from Natural Sources. Heliyon2019, 5, 1-14.
  29. Ramesh, S.; Loo, Z.Z.; Tan, C.Y.; Chew, W.J.K.; Ching, Y.C.; Tarlochan, F.; Chandran, H.; Krishnasamy, S.; Bang, L.T.; Sarhan, A.A.D. Characterization of Biogenic Hydroxyapatite Derived from Animal Bones for Biomedical Applications. Ceram. Int.2018, 44(9), 10525-10530.
  30. Uskoković, V.; Iyer, M.A.; Wu, V.M. One Ion to Rule Them All: The Combined Antibacterial, Osteoinductive and Anticancer Properties of Selenite-Incorporated Hydroxyapatite. J. Mater. Chem. B.2017, 5(7), 1430-1445.
  31. Zhang, X.; Chaimayo, W.; Yang, C.; Yao, J.; Miller, B.L.; Yates, M.Z. Silver-Hydroxyapatite Composite Coatings with Enhanced Antimicrobial Activities through Heat Treatment. Surf. Coatings Technol.2017, 325, 39-45.
  32. Ajduković, Z R.; Mihajilov-Krstev, T.M.; Ignjatović, N.L.; Stojanović, Z.; Mladenović-Antić, S.B.; Kocić, B.D.; Najman, S.; Petrović, N.D.; Uskoković, D.P. In Vitro Evaluation of Nanoscale Hydroxyapatite-Based Bone Reconstructive Materials with Antimicrobial Properties. J. Nanosci. Nanotechnol.2016, 16, 1420-1428.
  33. Padmanabhan, V.P.; Kulandaivelu, R.; Nellaiappan, S.N.T.S.; Lakshmipathy, M.; Sagadevan, S.; Johan, M.R. Facile Fabrication of Phase Transformed Cerium[IV] Doped Hydroxyapatite for Biomedical Applications - A Health Care Approach. Ceram. Int.2020, 46, 2510-2522.
  34. Jenifer, A.; Sakthivel, P.; Senthilarasan, K.; Sivaprakash, P.; Arumugam, S. In Vitro Analysis of Nickel Doped Hydroxyapatite for Biomedical Applications. Int. J. Sci. Technol. Res.2019, 8(11), 781-787.
  35. Resmim, C.M.; Dalpasquale, M.; Vielmo, N.I.C.; Mariani, F.Q.; Villalba, J.C.; Anaissi, F.J.; Caetano, M.M.; Tusi, M.M. Study of Physico-Chemical Properties and in Vitro Antimicrobial Activity of Hydroxyapatites Obtained from Bone Calcination. Prog. Biomater.2019, 8, 1-9.
  36. Wang, L.; Hu, C.; Shao, L. The Antimicrobial Activity of Nanoparticles: Present Situation and Prospects for the Future. Int. J. Nanomedicine2017, 12, 1227-1249.
  37. Shrestha, P.; Cooper, B.S.; Coast, J.; Oppong, R.; Do Thi Thuy, N.; Phodha, T.; Celhay, O.; Guerin, P.J.; Wertheim, H.; Lubell, Y. Enumerating the Economic Cost of Antimicrobial Resistance per Antibiotic Consumed to Inform the Evaluation of Interventions Affecting Their Use. Antimicrob. Resist. Infect. Control2018, 7, Article number 98.
  38. Ciobanu, C.S.; Iconaru, S.L.; Chifiriuc, M.C.; Costescu, A.; Le Coustumer, P.; Predoi, D. Synthesis and Antimicrobial Activity of Silver-Doped Hydroxyapatite Nanoparticles. Biomed Res. Int.2013, 2013, Article ID 916218.
  39. Swetha, M.; Sahithi, K.; Moorthi, A.; Saranya, N.; Saravanan, S.; Ramasamy, K.; Srinivasan, N.; Selvamurugan, N. Synthesis, Characterization, and Antimicrobial Activity of Nano-Hydroxyapatite-Zinc for Bone Tissue Engineering Applications. J. Nanosci. Nanotechnol.2012, 12, 167-172.
  40. Venkatesan, J.; Kim, S.K. Effect of Temperature on Isolation and Characterization of Hydroxyapatite from Tuna [Thunnus Obesus] Bone. Materials 2010, 3, 4761-4772.
  41. Magaldi, S.; Mata-Essayag, S.; Hartung De Capriles, C.; Perez, C.; Colella, M. T.; Olaizola, C.; Ontiveros, Y. Well Diffusion for Antifungal Susceptibility Testing. Int. J. Infect. Dis.2004, 8, 39-45.
  42. Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for in Vitro Evaluating Antimicrobial Activity: A Review. J. Pharm. Anal.2016, 6(2), 71-79.
  43. Poralan, G.M.; Gambe, J.E.; Alcantara, E.M.; Vequizo, R.M. X-Ray Diffraction and Infrared Spectroscopy Analyses on the Crystallinity of Engineered Biological Hydroxyapatite for Medical Application. IOP Conf. Ser. Mater. Sci. Eng.2015, 79, 1-7.
  44. Sobczak-Kupiec, A.; Wzorek, Z. The Influence of Calcination Parameters on Free Calcium Oxide Content in Natural Hydroxyapatite. Ceram. Int.2012, 38, 641-647.
  45. Fleet, M.E. Infrared Spectra of Carbonate Apatites: Evidence for a Connection between Bone Mineral and Body Fluids. Am. Mineral.2017, 102, 149-157.
  46. Khoo, W.; Nor, F.M.; Ardhyananta, H.; Kurniawan, D. Preparation of Natural Hydroxyapatite from Bovine Femur Bones Using Calcination at Various Temperatures. Procedia Manuf.2015, 2, 196-201.
  47. Bahrololoom, M.E.; Javidi, M.; Javadpour, S.; Ma, J. Characterisation of Natural Hydroxyapatite Extracted from Bovine Cortical Bone Ash. J. Ceram. Process. Res.2009, 10(2), 129-138.
  48. Ofudje, E.A.; Rajendran, A.; Adeogun, A.I.; Idowu, M.A.; Kareem, S.O.; Pattanayak, D.K. Synthesis of Organic Derived Hydroxyapatite Scaffold from Pig Bone Waste for Tissue Engineering Applications. Adv. Powder Technol.2018, 29, 1-8.
  49. Liu, P.; Li, Z.; Yuan, L.; Sun, X.; Zhou, Y. Pourbaix-Guided Mineralization and Site-Selective Photoluminescence Properties of Rare Earth Substituted B-Type Carbonated Hydroxyapatite Nanocrystals. Molecules2021, 26 (3), 540.
  50. Sofronia, A.M.; Baies, R.; Anghel, E.M.; Marinescu, C.A.; Tanasescu, S. Thermal and Structural Characterization of Synthetic and Natural Nanocrystalline Hydroxyapatite. Mater. Sci. Eng. C.2014, 43, 153-163.
  51. Markovic, M. Preparation and Comprehensive Characterization of a Calcium Hydroxyapatite Reference Material Volume. J. Res. Natl. Inst. Stand. Technol. 2004, 109,253-568.
  52. Walters, M.A.; Leung, Y.C.; Blumenthal, N.C.; Konsker, K.A.; LeGeros, R.Z.A Raman and Infrared Spectroscopic Investigation of Biological Hydroxyapatite. J. Inorg. Biochem.1990, 39(3), 193-200.[90]84002-7
  53. Jaber, H.L.; Hammood, A.S.; Parvin, N. Synthesis and Characterization of Hydroxyapatite Powder from Natural Camelus Bone. J. Aust. Ceram. Soc.2018, 54, 1-10.
  54. Esmaeilkhanian, A.; Sharifianjazi, F.; Abouchenari, A.; Rouhani, A.; Parvin, N.; Irani, M. Synthesis and Characterization of Natural Nano-Hydroxyapatite Derived from Turkey Femur-Bone Waste. Appl. Biochem. Biotechnol.2019, 189, 919-932.
  55. Hariani, P.L.; Muryati, M.; Said, M.; Salni, S. Synthesis of Nano-Hydroxyapatite from Snakehead [Channa Striata] Fish Bone and Its Antibacterial Properties. Key Eng. Mater.2020, 840, 293-299.
  56. Bano, N.; Jikan, S.S.; Basri, H.; Adzila, S.; Zago, D.M. XRD and FTIR Study of A&B Type Carbonated Hydroxyapatite Extracted from Bovine Bone. AIP Conf. Proc.2019, 2068, 0201001-0201006.
  57. Shavandi, A.; Bekhit, A.E.D.A.; Ali, A.; Sun, Z. Synthesis of Nano-Hydroxyapatite [NHA] from Waste Mussel Shells Using a Rapid Microwave Method. Mater. Chem. Phys.2015, 149, 607-616.
  58. Gayathri, B.; Muthukumarasamy, N.; Velauthapillai, D.; Santhosh, S.B. Magnesium Incorporated Hydroxyapatite Nanoparticles : Preparation, Characterization, Antibacterial and Larvicidal Activity. Arab. J. Chem.2018, 11, 645-654.
  59. Ruksudjarit, A.; Pengpat, K.; Rujijanagul, G.; Tunkasiri, T. Synthesis and Characterization of Nanocrystalline Hydroxyapatite from Natural Bovine Bone. Curr. Appl. Phys.2008, 8(2-3), 270-272.
  60. Akhavan, A.; Sheikh, N.; Khoylou, F.; Naimian, F.; Ataeivarjovi, E. Synthesis of Antimicrobial Silver/Hydroxyapatite Nanocomposite by Gamma Irradiation. Radiat. Phys. Chem.2014, 98, 46-50.
  61. Feng, Q.L.; Wu, J.; Chen, G.Q.; Cui, F.Z.; Kim, T.N.; Kim, J.O. A Mechanistic Study of the Antibacterial Effect of Silver Ions on Escherichia Coli and Staphylococcus Aureus. J. Biomed. Mater. Res.2000, 52, 662-668.[20001215]52:4<662::AID-JBM10>3.0.CO;2-3
  62. Varkey, A.J. Antibacterial Properties of Some Metals and Alloys in Combating Coliforms in Contaminated Water. Sci. Res. Essays.2010, 5 (24), 3834-3839.