Fibroblast Cell Behavior on Bound and Adsorbed Fibronectin onto Hyaluronan and Sulfated Hyaluronan Substrates

Abstract

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.

Improved fibronectin-immobilized fibrinogen microthreads for the attachment and proliferation of fibroblasts

“…28 Various studies have investigated adhesive proteins on polymer carriers to regulate cellular activities. 29 A recent study revealed that natural and synthetic polymer surfaces that had been modified with FN could improve cell adhesion and accelerate cell proliferation. 30 In this study, we describe the fabrication process for Fbg microfiber scaffolds by using a simple and cost-effective micron-sized silicone tube.…”

Characterization of fibroblast morphology on bioactive surfaces using vertical scanning interferometry

“…One approach to address this dedifferentiation and tissue scarcity in the field of cartilage regeneration is focused on initiating phenotypic changes in cells from non-traditional tissues using protein-coated surfaces. Modification of surfaces is a widely employed technique for controlling cell attachment, proliferation, and differentiation (Barbucci et al, 2005;Carlsson et al, 1979;McKeehan and Ham, 1976). One method utilizes protein coatings to modify the surface and investigate the ability of Matrix Biology 25 (2006) 523 -533 www.elsevier.com/locate/matbio dermal fibroblasts to effectively serve as an alternate cell source to chondrocytes (French et al, 2004).…”

“…A common and simple method to coat surfaces with proteins is protein adsorption (Barbucci et al, 2005). Protein adsorption occurs by applying an aqueous solution of proteins to a surface and allowing the proteins to adhere to the substrate over several hours.…”

“…Protein adsorption occurs by applying an aqueous solution of proteins to a surface and allowing the proteins to adhere to the substrate over several hours. Protein adsorption for cellular studies and applications depends on many variables, including protein surface density, differences in adsorption between material surfaces, protein conformation on the surface, surface roughness, and retention of biological activity post-adsorption (Barbucci et al, 2005;Gaudet et al, 2003;Horbet, 1996;Sethuraman and Belfort, 2005;Tang and Eaton, 1995). The characteristics of the surface coating can potentially be varied by altering the method of protein application.…”

“…Previous studies have used atomic force microscopy (Barbucci et al, 2005;Han et al, 1998;Morra et al, 2003;Sousa et al, 2004;Vianello et al, 2000), X-ray photoelectron spectroscopy (Glidle et al, 2003;Morra et al, 2003;Sousa et al, 2004), attenuated total reflectance-infrared (Barbucci et al, 2005;Han et al, 1998;Morra et al, 2003), total internal reflection microscopy (Dubin-Thaler et al, 2004), and scanning electron microscopy (Barbucci et al, 2005;Glidle et al, 2003;Han et al, 1998;Tsai et al, 2004) to characterize protein-coated surfaces. These techniques have been used to gain information about surface roughness, surface density, cell adhesion, and average protein height (Gaudet et al, 2003;Kuhlmeier et al, 2003;Sousa et al, 2004).…”