Influence of Charge and Heat on the Mechanical Properties of Scaffolds from Ionic Complexation of Chitosan and Carboxymethyl Cellulose

Abstract

As one of the most abundant, multifunctional biological polymers, polysaccharides are considered promising materials to prepare tissue engineering scaffolds. When properly designed, wetted porous scaffolds can have biomechanics similar to living tissue and provide suitable fluid transport, both of which are key features for in vitro and in vivo tissue growth. They can further mimic the components and function of glycosaminoglycans found in the extracellular matrix of tissues. In this study, we investigate scaffolds formed by charge complexation between anionic carboxymethyl cellulose and cationic protonated chitosan under well-controlled conditions. Freeze-drying and dehydrothermal heat treatment were then used to obtain porous materials with exceptional, unprecendent mechanical properties and dimensional long-term stability in cell growth media. We investigated how complexation conditions, charge ratio, and heat treatment significantly influence the resulting fluid uptake and biomechanics. Surprisingly, materials with high compressive strength, high elastic modulus, and significant shape recovery are obtained under certain conditions. We address this mostly to a balanced charge ratio and the formation of covalent amide bonds between the polymers without the use of additional cross-linkers. The scaffolds promoted clustered cell adhesion and showed no cytotoxic effects as assessed by cell viability assay and live/dead staining with human adipose tissue-derived mesenchymal stem cells. We suggest that similar scaffolds or biomaterials comprising other polysaccharides have a large potential for cartilage tissue engineering and that elucidating the reason for the observed peculiar biomechanics can stimulate further research.

Keywords: carboxymethyl cellulose; charge complexation; chitosan; dehydrothermal treatment; freeze-drying; mesenchymal stem cells; polyelectrolytes; porous scaffolds; tissue engineering.

Figure 1

Illustration of the design leading to multifunctional and cross-linker-free biocomposite scaffolds by charge complexation of chitosan (CS) and carboxymethyl cellulose (CMC).

Figure 2

SEM images top (A) and cross section (B). (C) Photographs, (D) porosity, and (E) density of dry and non-neutralized scaffolds of chitosan (CS100), carboxymethyl cellulose (CS0), and chitosan–carboxymethyl cellulose biocomposites (CS60, CS50, and CS40). Statistically significant differences **p < 0.05, *p < 0.05.

Figure 3

Top-view and cross-sectional images obtained by SEM (in dry state) and CLSM (in hydrated state) of heated and neutralized CS50 (A) and CS40 (B). (C) Porosity of heated and neutralized CS50 (A) and CS40 (B). (D) A scaffold with a density of 0.118 g cm^–3 supported 500 g. (E) Photographs of dry CS50/105 °C/N scaffolds after hydration with ultrapure water and biofluid for 28 days.

Figure 4

¹³C solid-state NMR spectra of chitosan (CS100), carboxymethyl cellulose (CS0), CS50/N, and CS50/105 °C/N.

Figure 5

Potentiometric charge titration isotherms as a function of pH for CS50 (A) and CS40 (B) compared to CMC and CS. (C) Overall amino and carboxylate charge per mass for CS50 and CS40 and the effect of heat treatment.

Figure 6

Swelling (A, B) and weight loss (C, D) of nonheated (CS50 and CS40) and heated (CS50/105 °C/N and CS40/105 °C/N) upon equilibration in cell growth media at 37 °C. (E) Images of “CS50/105 °C/N” taken after the weight loss test at different times. Statistically significant differences **p < 0.05, *p < 0.05

Figure 7

Stress–strain curves (A, B) and comparative mechanical properties (D, E) of neutralized scaffolds of CS–CMC composites and (C) photographs of the CS50/105 °C/N scaffold before and after compression test. Statistically significant differences **p < 0.03.

Figure 8

MTT viability assay of mesenchymal stem cells (MSCs) after 5 days of cultivation on CS50/105 °C/N and CS40/105 °C/N at low (40 000 cells per scaffold) and high (200 000 cells per scaffold) (A). Fluorescence images of a live/dead calcein-AM (green) and PI (red) staining of MSCs cultured on CS50/105 °C/N (B) and CS40/105 °C/N (C). Data are represented as mean ± SD (n = 4); * Indicates significant difference with a confidence interval of 95% and p ≤ 0.05.