Biomaterials with Potential Use in Bone Tissue Regeneration-Collagen/Chitosan/Silk Fibroin Scaffolds Cross-Linked by EDC/NHS

Abstract

Blending of different biopolymers, e.g., collagen, chitosan, silk fibroin and cross-linking modifications of these mixtures can lead to new materials with improved physico-chemical properties, compared to single-component scaffolds. Three-dimensional scaffolds based on three-component mixtures of silk fibroin, collagen and chitosan, chemically cross-linked, were prepared and their physico-chemical and biological properties were evaluated. A mixture of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) was used as a cross-linking agent. FTIR was used to observe the position of the peaks characteristic for collagen, chitosan and silk fibroin. The following properties depending on the scaffold structure were studied: swelling behavior, liquid uptake, moisture content, porosity, density, and mechanical parameters. Scanning Electron Microscopy imaging was performed. Additionally, the biological properties of these materials were assessed, by metabolic activity assay. The results showed that the three-component mixtures, cross-linked by EDC/NHS and prepared by lyophilization method, presented porous structures. They were characterized by a high swelling degree. The composition of scaffolds has an influence on mechanical properties. All of the studied materials were cytocompatible with MG-63 osteoblast-like cells.

Keywords: EDC/NHS; chitosan; collagen; silk fibroin; three-dimensional scaffolds.

Figure 1

The graphical presentation of the possible use of the material.

Figure 2

The steps of the tissue engineering process. In the red circle, the part of the tissue engineering round to which the article relates is crossed—design, physico-chemical characterization and in vitro study on cells of materials consisting of ternary mixtures based on silk fibroin, collagen and chitosan cross-linked with EDC/NHS mixture.

Figure 3

The scheme of preparing and cross-linking biopolymeric scaffolds.

Figure 4

FTIR-ATR spectra of biopolymeric scaffolds: (A) 50/50 Coll/CTS mixtures with 10, 20, and 30% of SF; (B) 50/50 SF/Coll mixtures with 10, 20, and 30% of CTS; (C) 50/50 SF/CTS mixtures with 10, 20, and 30% of Coll.

Figure 5

Swelling behavior of the studied scaffolds, cross-linked by EDC/NHS: (A) Coll/CTS mixture with 10, 20 and 30% of SF; (B) SF/Coll mixture with 10, 20 and 30% of CTS; (C) SF/CTS with 10, 20 and 30% of Coll.

Figure 6

Result of liquid uptake in the studied scaffolds, cross-linked by EDC/NHS: (A) Coll/CTS mixture with 10, 20 and 30% of SF; (B) SF/Coll mixture with 10, 20 and 30% of CTS; (C) SF/CTS with 10, 20 and 30% of Coll.

Figure 7

The result of moisture content measurements of biopolymeric three-component scaffolds cross-linked by EDC/NHS; *—represents statistically significant differences between samples (p ≤ 0.05).

Figure 8

SEM images of Coll/CTS mixtures with 10, 20 and 30% of SF, cross-linked by EDC/NHS, for dry scaffolds and scaffolds after 168 h of immersion in PBS: (A) magnification 500×; (B) magnification 150×.

Figure 9

SEM images of SF/Coll mixtures with 10, 20 and 30% of CTS, cross-linked by EDC/NHS, for dry scaffolds and scaffolds after 168 h of immersion in PBS: (A) magnification 500×; (B) magnification 150×.

Figure 10

SEM images of SF/CTS mixtures with 10, 20 and 30% of Coll, cross-linked by EDC/NHS, for dry scaffolds and scaffolds after 168 h of immersion in PBS: (A) magnification 500×; (B) magnification 150×.

Figure 11

Results of (A) porosity and (B) density measurements of biopolymeric three-component scaffolds cross-linked by EDC/NHS; *—represents statistically significant differences between samples (p ≤ 0.05).

Figure 12

The results of mechanical properties studies of biopolymeric scaffolds, cross-linked by EDC/NHS: (A) Young’s modulus; (B) maximum force; (C) maximum deformation; *—represents statistically significant differences between samples (p ≤ 0.05); ns—represents no statistically significant differences (p ≤ 0.05).

Figure 13

Metabolic activity after 1, 4 and 7 days of MG-63 cells cultured on studied scaffolds (n = 3, mean ± SD; p ≤ 0.05; *—significantly different between sample and TCPS in 1 day; **—significantly different between sample and TCPS in 4 days; #—significantly different between sample and TCPS in 7 days).