Synthesis of hydroxyapatite nanoparticle and role of its size in hydroxyapatite/chitosan–gelatin biocomposite for bone grafting

In the research of bone tissue engineering and regeneration, nano-hydroxyapatite (nHAp), chitosan (CS), and gelatin (GeL)-based scaffold shows promising result because of their potentials of tailored properties by manipulating 3D networks. In this work, nHAp was synthesized by wet chemical precipitation method using calcium nitrate tetrahydrate [Ca(NO3)2∙4H2O] and di-ammonium hydrogen phosphate (NH4)2HPO4. Glutaraldehyde was used as the cross-linking agent to prepare 3D networks through a freeze-drying technique. X-ray diffraction (XRD) analysis confirmed the formation of single-phase nHAp. Fourier Transformation Infrared Spectroscopy (FTIR) spectra revealed the presence of hydroxyl (-OH) and phosphate $$\left( {{\text{PO}}_{4}^{3- } } \right)$$ groups into the sample which confirmed the formation of hydroxyapatite. Raman Spectroscopy analysis elicited the conservancy of the nHAp and scaffold structure. Scanning Electron Microscopy (SEM) images revealed the formation of a 3D interconnected porous scaffold with a pore size in the range of 30–250 μm. Energy Dispersive X-ray Spectroscopy (EDS) of the scaffold affirmed that the prepared nHAp is calcium abundant nHAp. The cytotoxicity of the above scaffolds was studied by VERO cells, which revealed that the prepared samples were non-cytotoxic. Mechanical testing demonstrated that inclusion of higher nHAp concentration leads to increase the mechanical properties of the scaffolds.