Development of a new carbon nanotube-alginate-hydroxyapatite tricomponent composite scaffold for application in bone tissue engineering

Abstract

In recent times, tricomponent scaffolds prepared from naturally occurring polysaccharides, hydroxyapatite, and reinforcing materials have been gaining increased attention in the field of bone tissue engineering. In the current work, a tricomponent scaffold with an oxidized multiwalled carbon nanotube (fMWCNT)-alginate-hydroxyapatite with the required porosity was prepared for the first time by a freeze-drying method and characterized using analytical techniques. The hydroxyapatite for the scaffold was isolated from chicken bones by thermal calcination at 800°C. The Fourier transform infrared spectra and X-ray diffraction data confirmed ionic interactions and formation of the fMWCNT-alginate-hydroxyapatite scaffold. Interconnected porosity with a pore size of 130-170 µm was evident from field emission scanning electron microscopy. The total porosity calculated using the liquid displacement method was found to be 93.85%. In vitro biocompatibility and cell proliferation on the scaffold was checked using an MG-63 cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell attachment by Hoechst stain assay. In vitro studies showed better cell proliferation, cell differentiation, and cell attachment on the prepared scaffold. These results indicate that this scaffold could be a promising candidate for bone tissue engineering.

Keywords: alginate; chicken bone; hydroxyapatite; tissue engineering.

Figure 1

Thermogravimetric images of fMWCNT–alginate–HAP composite scaffold. Abbreviations: fMWCNT, oxidized multiwalled carbon nanotube; HAP, hydroxyapatite.

Figure 2

Fourier transform infrared spectra of alginate, fMWCNT, HAP, and fMWCNT–alginate–HAP composite scaffold. Abbreviations: fMWCNT, oxidized multiwalled carbon nanotube; HAP, hydroxyapatite.

Figure 3

X-ray diffraction patterns of alginate, fMWCNT, HAP, and fMWCNT–alginate–HAP composite scaffold. Abbreviations: fMWCNT, oxidized multiwalled carbon nanotube; HAP, hydrox yapatite.

Figure 4

Field emission scanning electron micrographs of HAP (A) and fMWCNT–alginate–HAP composite scaffold (B, C). Abbreviations: fMWCNT, oxidized multiwalled carbon nanotube; HAP, hydroxyapatite.

Figure 5

In vitro biocompatibility and cell proliferation of fMWCNT–alginate–HAP composite scaffold by MTT assay as a function of days. Notes: Values are expressed as the mean ± standard deviation (n=3). Statistical analysis was performed using two-way analysis of variance (***P<0.001). Abbreviations: fMWCNT, oxidized multiwalled carbon nanotube; HAP, hydroxyapatite; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 6

Optical microscopy images of Hoechst 3342-stained (A, B) and acridine orange-stained (C, D) cells grown on scaffold.

Figure 7

ALP assay of fMWCNT–alginate–HAP scaffolds on MG 63 cell line. Notes: Values are expressed as the mean ± standard deviation (**P<0.05, n=3). Abbreviations: ALP, alkaline phosphatase; fMWCNT, oxidized multiwalled carbon nanotube; HAP, hydroxyapatite.

Figure 8

Scaffold images showing compressive strength.