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

Authors: SHARMIN RAHMAN, KAZI HANIUM MARIA, MOHAMMAD SAIF ISHTIAQUE, ARIJUN NAHAR, HARINARAYAN DAS, SHEIKH MANJURA HOQUE

Abstract: 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 ($\vert $NH$_{2})$ of gelatin with the aldehyde groups ($\vert $CHO) of chitosan. The X-ray diffraction (XRD) performed on different scaffolds indicates that the incorporation of a certain amount of hydroxyapatite has no influence on the chitosan/gelatin network and at the same time, the organic matrix does not affect the crystallinity of hydroxyapatite. Transmission electron microscope (TEM) images show the needle-like crystal structure of hydroxyapatite nanoparticle. Scanning Electron Microscope (SEM) analysis shows an interconnected porous network in the scaffold where HAP nanoparticles are found to be dispersed in the biopolymer matrix. Fourier transforms infrared spectroscopy (FTIR) confirms the presence of hydroxyl group (OH$^{-})$, phosphate group (PO$_{4}^{3-})$, carbonate group (CO$_{3}^{2-})$, 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 MTT-assay. 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.

Keywords: Hydroxyapatite, chitosan, gelatin, FTIR, MTT-assay

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