In vitro characterization of chitosan–gelatin scaffolds for tissue engineering

Abstract

Recently, chitosan–gelatin scaffolds have gained much attention in various tissue engineering applications. However, the underlying cell–matrix interactions remain unclear in addition to the scaffold degradation and mechanical characteristics. In this study, we evaluated (i) the degradation kinetics of chitosan and chitosan–gelatin scaffolds in the presence of 10 mg/L of lysozyme for dimensional stability, weight loss, and pH changes for a period of 2 months, (ii) tensile and compressive properties of films and scaffolds in wet state at 37 °C, (iii) viability of fibroblasts and human umbilical vein endothelial cells (HUVECs) on scaffolds, and (iv) the alteration in cell spreading characteristics, cytoskeletal actin distribution, focal adhesion kinase (FAK) distribution and PECAM-1 expression of HUVECs under static and 4.5, 8.5, 13 and 18 dyn/cm² shear stress conditions. Degradation results showed that gelatin-containing chitosan scaffolds had faster degradation rate and significant loss of material than chitosan. Mechanical properties of chitosan are affected by the addition of gelatin although there was no clear trend. Three-dimensional chitosan and chitosan–gelatin scaffolds supported fibroblast viability equally. However, chitosan membranes decreased cell-spreading area, disrupted F-actin and localized FAK in the nucleus of HUVECs. Importantly, the lowest shear stress tested (4.5 dyn/cm²) for 3 h washed away cells on chitosan suggesting weak cell adhesion. In the blends, effect of gelatin was dominant; actin and FAK distribution were comparable to gelatin in static culture. However, at higher shear stresses, presence of chitosan inhibited shear-induced increase in cell spreading and weakened cell adhesive strength. No significant differences were observed in PECAM-1 expression. In summary, these results showed significant influence of blending gelatin with chitosan on scaffold properties and cellular behavior.

Introduction

Chitosan is a naturally derived polysaccharide. It has gained much attention as a biomaterial in diverse tissue engineering applications due to its low cost, large-scale availability, anti-microbial activity, and biocompatibility [1]. Chitosan scaffolds with various geometries, pore sizes, and pore orientation can be obtained using controlled-rate freezing and lypohilization [2]. Chitosan marginally supports biological activity of diverse cell types and chitosan films are highly brittle with a strain at break of 40–50% in the wet state [3]. Furthermore, lysozyme-dependent chitosan degradation depends on the degree of deacetylation (DD), local pH [4], [5] and homogeneity of the source; lysozymal hydrolysis is high in acidic conditions (pH=4.5–5.5) [6] and decreases with increase in DD. Although the DD and the homogeneity of source can be controlled during polymer processing to regulate biomechanical properties, the range is marginal.

For improving the mechanical or biological properties of chitosan over a broad range, blending with other polymers is widely investigated. Gelatin is blended with chitosan to improve the biological activity since (i) gelatin contains Arg–Gly–Asp (RGD)-like sequence that promotes cell adhesion and migration, and (ii) forms a polyelectrolyte complex. Gelatin–chitosan scaffolds have been formed without or with cross-linkers such as glutaraldehyde [7] or enzymes [8] and tested in regenerating various tissues including skin [9], cartilage [10], and bone [11].

Despite these advances, the effect of various modifications on tissue remodeling processes is not clearly understood. For example, the presence of other polymers could decrease the degradation rate of chitosan by limiting lysozymal transport. In addition, cell adhesive interactions are not completely explored despite chitosan lacking a specific binding domain for integrin-mediated cell adhesion through which majority of the transmembrane signaling takes place [12]. Intracellular tension is modulated via focal adhesion (FA) complex, integrins, and the ECM [13], [14], [15]. Level of FA complex, comprised of many molecules including FA kinase (FAK), vinculin, and talin, correlate with cell spreading [16], [17]. Actin reorganizes with the redistribution and levels of FA-complex and alters cell shape and characteristics. When gelatin and chitosan are blended together, the structure formed can affect the spatial distribution of integrin ligands and polycationic chitosan interaction with the anionic cell surface. These effects influence cell adhesion, cellular bioactivity [18], [19], tissue remodeling process and ultimately the quality of the regenerated tissue.

This study focused on understanding the effect of blending gelatin with chitosan on degradation properties, mechanical properties and cell–matrix interactions. The 3-D scaffolds of various blend ratios were formed using controlled-rate freezing and lyophilization technique. Further, human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) were tested to understand the interactions of cells of two different origins. HUVECs that make up the luminal lining were tested in static culture and shear stresses. MEF found in the connective tissue that synthesize collagen [20] were tested in 3-D and 2-D cultures. Actin and FAK distribution and cell–cell junction adhesion molecule–platelet EC adhesion molecule-1 (PECAM-1, CD31) expression were evaluated [21]. These results show significant influence of blending gelatin with chitosan on the tissue remodeling process.