In vitro characterization of chitosan–gelatin scaffolds for tissue engineering
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.
Section snippets
Materials and Methods
Chitosan of >310 kD MW (DD≈85%), type A porcine skin gelatin, 5 kD MW dextran sulfate, Hen Egg White Lysozyme (46,400 U/mg) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Sigma Aldrich Chemical Co.(St. Louis, MO). Alexa Fluor 546 goat anti-mouse IgG1 and Alexa Fluor 488 phalloidin were obtained from Molecular Probes (Eugene, OR). HUVECs and endothelial growth medium-2 (EGM-2) BulletKit were from Cambrex Biosciences (Walkersville, MD). MEFs were from American Tissue Culture
Morphology of chitosan and chitosan–gelatin cylindrical scaffolds
Chitosan–gelatin cylindrical scaffolds of 14 mm diameter and 20 mm high were formed by controlled-rate freeze–drying. Initial experiments were performed by freezing blend solutions at room temperature. These results showed two phases with increased gelatin content in the bottom. To minimize separation of two components, solutions were refrigerated to form a gel prior to freeze–drying and the formed scaffolds showed uniform distribution of the two components. SEM analysis showed (Fig. 1) no
Discussions
The focus of this study was to understand the effect of blending chitosan and gelatin on various parameters important in tissue engineering. The characterization of mechanical properties of the membranes showed that gelatin compositions greatly affect the membrane stiffness of chitosan despite gelatin possessing very low stiffness relative to chitosan. The overall trend is similar to the published reports [30], although the tensile properties cannot be directly correlated due to difference in
Conclusion
The effects of blending gelatin with chitosan on scaffold stiffness properties and degradation kinetics were characterized in this study. Addition of gelatin greatly affected the stiffness of 2D and 3D scaffolds, facilitated the degradation rate and maintained the dimension in the presence of lysozyme. Evaluation of cell adhesive interactions showed decreased cell spreading area on chitosan membranes, accumulated actin and localized FAK inside HUVECs in static culture. Exposure to shear stress
Acknowledgements
Support for this research was provided by Oklahoma Center for Advancement of Science and Technology (#HR02-087R). We also thank Phoebe Doss for help on using confocal microscopy.
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