Evaluation of L929 fibroblast attachment and proliferation on Arg-Gly-Asp-Ser (RGDS)-immobilized chitosan in serum-containing/serum-free cultures

https://doi.org/10.1263/jbb.104.69Get rights and content

In this study, chitosan membranes prepared by the solvent casting method were modified with the Arg-Gly-Asp-Ser (RGDS) sequence of fibronectin using the photochemical immobilization technique. The results obtained from attenuated total reflection-Fourier transform infrared spectra and X-ray photoelectron spectroscopy studies confirmed the successful immobilization of RGDS on chitosan membranes. The immobilized peptide concentration was determined by ninhydrin analysis on the order of 10−7 mol/cm2. In vitro cell culture studies were performed with L929 mouse fibroblasts to investigate the effect of biomodification on fibroblast cell behaviour in serum-free and 10% serum-containing media. The results obtained from cell culture studies pointed out the specific interactions between biosignal RGDS molecules and fibroblast cells. A triggered cell attachment and proliferation were observed on RGDS-modified chitosan membranes that were more distinguishable in serum-free medium. In addition, the photochemical immobilization technique was realized in the presence of a photomask that was used to immobilize the RGDS molecules in a defined micropattern. L929 mouse fibroblasts attached on the RGDS-micropatterned areas indicating integrin-mediated interactions.

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Materials

Chitosan derived from crab shell (> 85% deacetylation; cat. no. C 3646) and RGDS with a FW of 433.4 were purchased from Sigma (Munich, Germany). Photochemical cross-linker sulfosuccinimidyl 6 (4′-azido-2′-nitrophenyl-amino) hexanoate (sulfo-SANPAH) with a FW of 492.4 was obtained from Pierce (Rockford, IL, USA). Phosphate-buffered saline (PBS) tablets were purchased from Sigma. Tissue culture clusters, flasks and plates were purchased from Nunc (Hannover, Germany). Dulbecco’s modified Eagle’s

Biomodification of chitosan membranes

The RGDS tetrapeptide is known to cause a distinct increase in cell attachment compared to the tripeptide sequence (RGD), confirming that peptides with higher integrin affinity cause higher cell attachment activity (6). In this study, RGDS was immobilized on the surface of chitosan membranes to construct a bioactive material for stimulated fibroblast attachment and growth. Photochemical immobilization technique which does not require specific activated groups on the surfaces (21, 22, 24) was

Acknowledgments

This study was financially supported by Grant No. 03K120570 from the Turkish Republic Prime Ministry State Planning Organization and by FIS-CNR (Rome) and FIRB RBNE01458S grants. A. Karakeçili acknowledges the grant provided by the Consorzio Interuniversitario per i Sistemi a Grande Interfaccia (CSGI, Florence, Italy) for the period spent at LAMSUN at University of Catania.

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