Chitosan-Coated Collagen Membranes Promote Chondrocyte Adhesion, Growth, and Interleukin-6 Secretion

Abstract

Designing scaffolds made from natural polymers may be highly attractive for tissue engineering strategies. We sought to produce and characterize chitosan-coated collagen membranes and to assess their efficacy in promoting chondrocyte adhesion, growth, and cytokine secretion. Porous collagen membranes were placed in chitosan solutions then crosslinked with glutaraldehyde vapor. Fourier transform infrared (FTIR) analyses showed elevated absorption at 1655 cm^-1 of the carbon-nitrogen (N=C) bonds formed by the reaction between the (NH₂) of the chitosan and the (C=O) of the glutaraldehyde. A significant peak in the amide II region revealed a significant deacetylation of the chitosan. Scanning electron microscopy (SEM) images of the chitosan-coated membranes exhibited surface variations, with pore size ranging from 20 to 50 µm. X-ray photoelectron spectroscopy (XPS) revealed a decreased C-C groups and an increased C-N/C-O groups due to the reaction between the carbon from the collagen and the NH2 from the chitosan. Increased rigidity of these membranes was also observed when comparing the chitosan-coated and uncoated membranes at dried conditions. However, under wet conditions, the chitosan coated collagen membranes showed lower rigidity as compared to dried conditions. Of great interest, the glutaraldehyde-crosslinked chitosan-coated collagen membranes promoted chondrocyte adhesion, growth, and interleukin (IL)-6 secretion. Overall results confirm the feasibility of using designed chitosan-coated collagen membranes in future applications, such as cartilage repair.

Keywords: chitosan; chondrocytes; collagen; interleukin (IL)-6; membrane.

Figure 1

Protein composition of chitosan-coated collagen membranes. Following chitosan-coated collagen designed under various conditions, the produced membranes were subjected to Fourier transform infrared (FTIR) analyses. Spectra were obtained and plotted: (a) Unmodified collagen membrane, extensively washed (W); (b) 0.1% chitosan-coated collagen membrane, extensively W and non-crosslinked; (c) 1% chitosan-coated collagen membrane, non-W and non-crosslinked; (d) 0.1% chitosan-coated collagen membrane, crosslinked with glutaraldehyde (G) vapor and W; (e) 0.1% chitosan-coated collagen membrane, crosslinked with G vapor and non-W; (f) 1% chitosan-coated collagen membrane, non-W; (g) 1% chitosan-coated collagen membrane, extensively W; (h) 1% chitosan-coated collagen membrane, crosslinked with G and W; and (i) 1% chitosan-coated collagen membrane, crosslinked with G and non-W.

Figure 2

Scanning electron microscopy (SEM) analyses of chitosan-coated collagen membranes. Following material synthesis, chitosan-coated and non-coated membranes crosslinked with glutaraldehyde vapor were processed and analyzed under SEM. Structural analyses were performed on the surface of the membrane, and on the cross-sections (n = 4).

Figure 3

X-ray photoelectron spectroscopy (XPS) spectra of chitosan-coated collagen membranes. Materials with or without chitosan at various concentrations were synthesized and subjected to XPS analyses. Chemical composition spectra of the chitosan-coated and non-coated collagen membranes were compared (n = 4).

Figure 4

Stress-strain characterization of chitosan-coated and non-coated collagen membranes crosslinked with glutaraldehyde (GTA) vapor. Analyses were performed on dried membranes (n = 4). Ch: Chitosan.

Figure 5

Stress-strain characterization of chitosan-coated and non-coated collagen membranes crosslinked with glutaraldehyde vapor. Analyses were performed on humidified (H) membranes.

Figure 6

Weight lost of chitosan-coated collagen membrane following incubation with cell culture medium. Following the design of the different membranes, these were weighted at time zero. They were then immersed in culture medium and maintained at 37 °C for 48 and 96 h. At the end of each incubation period, membrane were washed with sterile water and dried for 4 days. The weight of each membrane was registered and compared to the initial one; n = 4. (a) Membrane weights at 48 h; (b) Membrane weights at 96 h.

Figure 7

Chondrocyte adhesion on chitosan-coated collagen membranes. Cells were seeded and cultured for 24 h. The cells were then stained with Hoechst dye, observed under an ultraviolet epifluorescence microscope, and photographed. Photographs (200× magnification) are representative of six separate experiments. (a) Collagen membrane alone; (b) 0.1% chitosan coated collagen membrane; (c) 1% chitosan-coated collagen membrane.

Figure 8

Effect of chitosan-coated collagen membranes on chondrocyte growth and metabolic activity. Following material synthesis, chondrocytes were seeded onto this material and cultured for various time periods. Cell growth was assessed by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT); with results presented as means ± standard deviation (SD) (n = 5). A difference was considered statistically significant at p < 0.05.

Figure 9

Level of live chondrocytes following growth on chitosan-coated collagen membranes. Chondrocytes were seeded onto the membranes and culture for 48 and 96 h. At the end of each culture period, cells were detached and the live cells were determined using trypan bleu exclusion assay; n = 3.

Figure 10

Effect of chitosan-coated collagen membranes on chondrocyte IL-6 secretion. Cells were cultured on membranes for various time periods. The supernatants were collected to quantitate IL-6 levels by sandwich enzyme-linked immunosorbent assay. Values are means ± SD (n = 4). The difference was considered statistically significant at p < 0.05.