Chemical synthesis, characterization, and biocompatibility study of hydroxyapatite/chitosan phosphate nanocomposite for bone tissue engineering applications

Abstract

A novel bioanalogue hydroxyapatite (HAp)/chitosan phosphate (CSP) nanocomposite has been synthesized by a solution-based chemical methodology with varying HAp contents from 10 to 60% (w/w). The interfacial bonding interaction between HAp and CSP has been investigated through Fourier transform infrared absorption spectra (FTIR) and x-ray diffraction (XRD). The surface morphology of the composite and the homogeneous dispersion of nanoparticles in the polymer matrix have been investigated through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. The mechanical properties of the composite are found to be improved significantly with increase in nanoparticle contents. Cytotoxicity test using murine L929 fibroblast confirms that the nanocomposite is cytocompatible. Primary murine osteoblast cell culture study proves that the nanocomposite is osteocompatible and highly in vitro osteogenic. The use of CSP promotes the homogeneous distribution of particles in the polymer matrix through its pendant phosphate groups along with particle-polymer interfacial interactions. The prepared HAp/CSP nanocomposite with uniform microstructure may be used in bone tissue engineering applications.

Figure 1

X-ray diffraction patters of (a) CSP, (b) HAp, and (c) HAp/CSP nanocomposite.

Figure 2

FTIR spectra of (a) chitosan, (b) chitosan phosphate, (c) HAp powder, and (d) HAp/CSP nanocomposite.

Figure 3

TEM micrographs of (a) HAp powders and (b) HAp/CSP nanocomposite (40% w/w).

Figure 4

SEM micrographs of HAp/CSP nanocomposite samples with various contents of n-HAp particles (a) 20% (w/w), (b) 30% (w/w), (c) 40% (w/w), and (d) 50% (w/w).

Figure 5

Cell viability test using murine fibroblast cell line L929 on CSP and HAp/CSP films, CS was taken as control (n = 3).

Figure 6

Osteoblast cell proliferation on CSP and HAp/CSP films, CS was taken as control. Significant increase in cell number (P < .001) was found at day 7 in all the variations followed by retardation of growth phase.

Figure 7

AP activity of osteoblast cells grown on CSP and HAp/CSP films, CS was taken as control. Both in case of CSP and HAp/CSP enzyme activity increased up to day 14 (P < .001). HAp/CSP nanocomposite is proven a better support for osteoblast differentiation than CSP alone (P < .01).

Figure 8

SEM images of osteoblasts grown on different matrices at day 14 on (a) chitosan (CS), (b) chitosan phosphate (CSP), (c) HAp/CSP nanocomposite. Scale bar of each image is 10 μm. Arrow “1” shows a nodular cell aggregate, arrow “2” shows the extracellular matrix secreted by the cells in around themselves, and arrow “3” shows a visible mineral nodule.