Sharmin RAHMAN, Kazi Hanium MARIA, Mohammad Saif ISHTIAQUE, Arijun NAHAR, Harinarayan DAS, Sheikh Manjura HOQUE

Evaluation of a novel nanocrystalline hydroxyapatite powder and a solid hydroxyapatite/Chitosan-Gelatin bioceramic for scaffold preparation used as a bone substitute material

Artificially fabricated hydroxyapatite (HAP) shows excellent biocompatibility with various kinds of cells and tissues which makes it an ideal candidate for a bone substitute material. In this study, hydroxyapatite nanoparticles have been prepared by using the wet chemical precipitation method using calcium nitrate tetra-hydrate $[Ca(NO_3)_2 .4H_2 O]$ and di-ammonium hydrogen phosphate $[(NH_4)_2 HPO_4 ]$ as precursors. The composite scaffolds have been prepared by a freeze-drying method with hydroxyapatite, chitosan, and gelatin which form a 3D network of interconnected pores. Glutaraldehyde solution has been used in the scaffolds to crosslink the amino groups $( |NH_2)$ of gelatin with the aldehyde groups ( |CHO) of chitosan. The X-ray diffraction (XRD) performed on different scaffolds indicates that the incorporation of a certain amount of hydroxyapatite has no influence on the chitosan/gelatin network and at the same time, the organic matrix does not affect the crystallinity of hydroxyapatite. Transmission electron microscope (TEM) images show the needle-like crystal structure of hydroxyapatite nanoparticle. Scanning Electron Microscope (SEM) analysis shows an interconnected porous network in the scaffold where HAP nanoparticles are found to be dispersed in the biopolymer matrix. Fourier transforms infrared spectroscopy (FTIR) confirms the presence of hydroxyl group $(OH^{−})$, phosphate group $(PO^{3}−_{4})$, carbonate group$(CO^{3}−_{4})$ imine group (C=N), etc. TGA reveals the thermal stability of the scaffolds. The cytotoxicity of the scaffolds is examined qualitatively by VERO (animal cell) cell and quantitatively by MTTassay. The MTT-assay suggests keeping the weight percentage of glutaraldehyde solution lower than 0.2%. The result found from this study demonstrated that a proper bone replacing scaffold can be made up by controlling the amount of hydroxyapatite, gelatin, and chitosan which will be biocompatible, biodegradable, and biofriendly for any living organism.

PDF

___

1. Mobini S, Javadpour J, Hosseinalipour M, Ghazi-Khansari M, Khavandi A et al. Synthesis and characterisation of gelatin–nano hydroxyapatite composite scaffolds for bone tissue engineering. Advances in Applied Ceramics 2008; 107 (1): 4-8. doi: 10.1179/174367608X246817
2. Bigi A, Boanini E, Panzavolta S, Roveri N, Rubini K. Bonelike apatite growth on hydroxyapatite–gelatin sponges from simulated body fluid. Journal of Biomedical Materials Research Part A 2002; 59 (4): 709-715. doi: 10.1002/jbm.10045
3. Sharma C, Dinda AK, Potdar PD, Chou CF, Mishra NC. Fabrication and characterization of novel nanobiocomposite scaffold of chitosan–gelatin–alginate–hydroxyapatite for bone tissue engineering. Materials Science and Engineering C 2016; 64: 416-427. doi: 10.1016/j.msec.2016.03.060
4. Maji K, Dasgupta S. Hydroxyapatite-chitosan and gelatin based scaffold for bone tissue engineering. Transactions of the Indian Ceramic Society 2014; 73 (2): 110-114. doi: 10.1080/0371750X.2014.922424
5. Vallet-Regi M, Gonzalez-Calbet JM. Calcium Phosphates as substitution of bone tissues. Progress in Solid State Chemistry 2004; 32 (1): 1-31. doi: 10.1016/j.progsolidstchem.2004.07.001
6. Maji K, Dasgupta S, Kundu B, Bissoyi A. Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength. Journal of Biomaterials Science, Polymer Edition 2015; 26 (16): 1190-1209. doi: 10.1080/09205063.2015.1082809
7. Mohamed KR, Beherei HH, El-Rashidy ZM. In vitro study of nano-hydroxyapatite/chitosan–gelatin composites for bio-applications. Journal of Advanced Research 2014; 5 (2): 201-208. doi: 10.1016/j.jare.2013.02.004
8. Itoh S, Kikuchi M, Koyama Y, Matumoto HN, Takakuda K et al. Development of a hydroxyapatite/collagen nanocomposite as a medical device. Cell Transplantation 2004; 13: 451-461. doi: 10.3727/000000004783983774
9. Kikuchi M. Matsumoto HN, Yamada T, Koyama Y, Takakuda K et al. Glutaraldehyde cross-linked hydroxyapatite/collagen self-organized nanocomposites. Biomaterials 2004; 25 (1): 63-69. doi: 10.1016/s0142-9612(03)00472-1
10. Yamaguchi I, Tokuchi K, Fukuzaki H, Koyama Y, Takakuda K et al. Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites. Journal of Biomedical Materials Research Part A 2001; 55 (1): 20-27. doi: 10.1002/1097-4636(200104)55:1<20::AID-JBM30>3.0.CO;2-F
11. Liao SS, Cui FZ, Zhu Y. Osteoblasts adherence and migration through three-dimensional porous mineralized collagen based composite: nHAC/PLA. Journal of Bioactive and Compatible polymers 2004; 19 (2): 117-130. doi: 10.1177/0883911504042643
12. Zhang SM, Cui FZ, Liao SS, Zhu Y, Han L. Synthesis and biocompatibility of porous nano-hydroxyapatite/collagen /alginate composite. Journal of Materials Science: Materials in Medicine 2003; 14 (7): 641-645. doi: 10.1023/a:1024083309982
13. Hae-Won K, Jonathan CK, Hyoun-Ee K. Hydroxyapatite and gelatin composite foams processed via novel freezedrying and crosslinking for use as temporary hard tissue scaffolds. Journal of Biomedical Material Research Part A 2005; 72: 136-142. doi: 10.1002/jbm.a.30168
14. Chen F, Wang ZC, Lin CJ. Preparation and characterization of nano-sized hydroxyapatite particles and hydroxyapatite/chitosan nano-composite for use in biomedical materials. Materials Letters 2002; 57 (4): 858-861. doi: 10.1016/S0167-577X(02)00885-6
15. Kim HW, Kim HE, Salih V. Stimulation of osteoblast responses to biomimetic nanocomposites of gelatin– hydroxyapatite for tissue engineering scaffolds. Biomaterials 2005; 26 (25): 5221-5230. doi: 10.1016/j.biomaterials.2005.01.047
16. Ebrahimi M, Botelho M, Lu W. Synthesis and characterization of biomimetic bioceramic nanoparticles with optimized physicochemical properties for bone tissue engineering. Journal of Biomedical Materials Research 2019; 107 (8): 1654-1666. doi: 10.1002/jbm.a.36681
17. Ji YY, Zhao F, Feng SX, De YK, Lu WW et al. Preparation and characterization of hydroxyapatite/chitosan– gelatin network composite. Journal of Applied Polymer Science 2000; 77 (13): 2929-2938. doi: 10.1002/1097- 4628(20000923)77:13<2929::AID-APP16>3.0.CO;2-Q
18. Dasgupta S, Maji K, Nandi SK. Investigating the mechanical, physiochemical and osteogenic properties in gelatinchitosan-bioactive nanoceramic composite scaffolds for bone tissue regeneration: in vitro and in vivo. Materials Science and Engineering C 2019; 94: 713-728. doi: 10.1016/j.msec.2018.10.022
19. Dan Y, Liu O, Liu Y, Zhang YY, Li S et al. Development of novel biocomposite scaffold of chitosan-gelatin /nanohydroxyapatite for potential bone tissue engineering applications. Nanoscale Research Letters 2016; 11 (1): 487. doi: 10.1186/s11671-016-1669-1
20. Nguyen VC, Pho QH. Preparation of chitosan coated magnetic hydroxyapatite nanoparticles and application for adsorption of reactive blue 19 and Ni2. The Scientific World Journal 2014; 2014: 273082. doi: 10.1155/2014/273082
21. Zhu R, Yu R, Yao J, Wang D, Ke J. Morphology control of hydroxyapatite through hydrothermal process. Journal of Alloys and Compounds 2008; 457 (1-2): 555-559. doi: 10.1016/j.jallcom.2007.03.081
22. Ruys AJ, Wei M, Sorrell CC, Dickson MR, Brandwood A et al. Sintering effects on the strength of hydroxyapatite. Biomaterials 1995; 16 (5): 409-415. doi: 10.1016/0142-9612(95)98859-c
23. Sopyan I, Mel M, Ramesh S, Khalid KA. Porous hydroxyapatite for artificial bone applications. Science and Technology of Advanced Materials 2007; 8 (1-2): 116-123. doi: 10.1016/j.stam.2006.11.017
24. Pang YX, Xujin B. Influence of temperature, ripening time and calcination on the morphology and crystallinity of hydroxyapatite nanoparticles. Journal of the European Ceramic Society 2003; 23 (10): 1697-1704. doi: 10.1016/S0955- 2219(02)00413-2
25. Elhendawi H, Felfel RM, Bothaina M. Abd El-Hady, Reicha FM. Effect of synthesis temperature on the crystallization and growth of in situ prepared nanohydroxyapatite in chitosan matrix. ISRN Biomaterials 2014; 2014: 897468. doi: 10.1155/2014/897468
26. Dasgupta S, Maji K. Characterization and in vitro evaluation of gelatin–chitosan scaffold reinforced with bioceramic nanoparticles for bone tissue engineering. Journal of Materials Research 2019; 34 (16): 1-12. doi: 10.1557/jmr.2019.170
27. Nishikawa H. Thermal behavior of hydroxyapatite in structural and spectrophotometric characteristics. Materials Letters 2001; 50 (5): 364-370. doi: 10.1016/S0167-577X(01)00318-4
28. Choi D, Prashant N K. An alternative chemical route for the synthesis and thermal stability of chemically enriched hydroxyapatite. Journal of the American Ceramic Society 2006; 89 (2): 444-449. doi: 10.1111/j.1551- 2916.2005.00738.x
29. Li J, Chen Y, Yin Y, Yao F, Yao K. Modulation of nano-hydroxyapatite size via formation on chitosan—gelatin network film in situ. Biomaterials 2007; 28 (5): 781-790. doi: 10.1016/j.biomaterials.2006.09.042
30. Chang MC, Ko CC, Douglas WH. Preparation of hydroxyapatite-gelatin nanocomposite. Biomaterials 2003; 24 (17): 2853-2862. doi: 10.1016/s0142-9612(03)00115-7
31. Zhang LJ, Feng XS, Liu HG, Qian DJ, Zhang L et al. Hydroxyapatite/collagen composite materials formation in simulated body fluid environment. Materials Letters 2004; 58 (5): 719-722. doi: 10.1016/j.matlet.2003.07.009