Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate

Abstract

Chitosan microparticles were mixed with chitosan and carboxymethyl cellulose solution to achieve a good binding between the microparticles. Three different compositions of scaffolds were made by varying the calcium phosphate (CaP) amount: 0%, 10%, and 20%. Potassium chloride was used as salt, to make pores inside the scaffolds after leaching out when immersed in phosphate buffer saline (PBS). Compressive strength and compressive modulus of both non-porous (before leaching out), and porous (after leaching out) scaffolds were measured according to the ASTM standards. The highest compressive strength of 27 MPa was reported on 10% CaP scaffolds while 20% CaP scaffolds showed the lowest. The increasing CaP content reduces the compressive strength of the scaffolds. The highest wet state compressive strength was reported on 0% CaP scaffolds with 0.36 MPs and 0.40 MPa at day 1 and day 3 respectively. In vitro cell culture studies showed good cell adhesion and cell proliferation on 10% CaP scaffolds.

Keywords: Carboxymethyl cellulose; Cell proliferation; Chitosan; Compressive strength; Scaffold.

Fig. 1.

Schematic representation of preparation of scaffolds (A) 0% CaP, (B) 10% CaP, (C) 20% CaP.

Fig. 2.

Dry state mechanical properties of non-porous and porous scaffolds, (A) Compressive strength, (B) Compressive modulus, * significance p < 0.05.

Fig. 3.

Wet state mechanical properties at day 1 and day 3, (A) Compressive strength, (B) Compressive modulus.

Fig. 4.

Swelling ratio of the scaffolds at different time points, * indicates the significant difference with respect to day1 of 10% CaP scaffolds.

Fig. 5.

XRD spectrums of scaffolds with different CaP content and different conditions, * indicates the characteristic peaks of CaP, + indicates the characteristics peaks of KCl, and # indicates the characteristics peaks of chitosan/CMC polymer.

Fig. 6.

A- SEM micrograph of chitosan MPs; A- Magnification (X) −50; B- X250; C- X2000; B- SEM micrograph of three types of scaffolds at magnification of 65; BI- before leaching; BII- after leaching.

Fig. 7.

Element mapping images and EDX Spectrum; A- SEM Image; B- Ca; C–P; D–K; magnification – 65; E- EDX spectrum.

Fig. 8.

Live/dead cell assay fluorescence images of three type of scaffolds as indicated in the image, green-live cells, red-dead cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 9.

WST-1assay results at day1, day 4, day 7, and day 14, *significance of p < 0.05.

Fig. 10.

SEM micrographs of cell attachment at day 14 of cell culture, rows- magnification (X), columns- scaffold type.