BIOMIGAG · Biomimetic surface modification of tissue engineering scaffolds with glycosaminoglycans

Final Activity Report Summary - BIOMIGAG (Biomimetic surface modification of tissue engineering scaffolds with glycosaminoglycans)

This two-year project focused on a biomimetic surface modification of biodegradable polymer (Poly-L-lactide, PLLA) for tissue engineering applications. According to the work plan, primary surface modification of PLLA was firstly conducted via physical adsorption or covalent binding of a positively-charged polymer, namely polyethyleneimine (PEI). Contact angle, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and atomic force microscopy (AFM) revealed that the covalent immobilisation in two-step activation way was most efficient to modify the surface. Testing with an osteoblast cell line revealed an improved biocompatibility of PLLA, which was dependent on the molecular weight and way of immobilisation of PEI. Based on the acquired positive surface charge, layer-by-layer (LBL) assembly of polyelectrolytes was carried out. Components of the extracellular matrix like heparin (HEP), hyaluronic acid (HA) or its sulphated derivative (sHA) as well as gelatin (GEL) were used as polyanions. Chitosan (CHI) was chosen as polycation, having in addition anti-inflammatory and antibacterial properties. Contact angle (CA), quartz microbalance (QCM), surface plasmon (SPR) and zeta-potential measurements were carried out to monitor the LBL processes. The results revealed that for the polyelectrolyte pairs of HEP and CHI or sHA and CHI there was an continuous increase of layer mass and hydrophilicity on the resulting surface during multilayer formation, which was not observed for the polyelectrolytes pairs of HA and CHI or GEL and CHI. The pH value of polyelectrolyte solution had also great impact on the multilayer formation. For example, for the polyelectrolyte pair of sHA and CHI, the slope for the linear fit curve of angle-shift versus layer number at pH 4.0 was much higher than that obtained under pH 7.0, which confirmed the quicker assembly of polyelectrolytes at lower pH value where the sHA was supposed to be more protonated than at pH 7.0. Based on those observations the incorporation of growth factors like TGF ß1 or BMP-2 in the multilayers was carried out. Cell culture experiments were further conducted, after successful modification of PLLA with bio-active macromolecules, to evaluate surface biocompatibility. Human mesenchymal stem cells (hMSC) and primary osteoblast (hPOB) were selected for this purpose. Cell adhesion under serum-free condition was tested with all cell types on both PEI-modified and LBL surfaces. From the quantitative data it occurred that growth and differentiation assays’ incorporation of adhesive proteins to all types of LBL surface prior to cell adhesion helped the cytoskeleton organisation, which was confirmed by immunofluorescence staining results. The morphology of adhering hMSC on LBL surface differed remarkable, indicating different later growth and differentiation. Accordingly, the differentiation of hMSC into the direction of osteoblasts was most obvious on HEP and CHI surface, while HA and CHI surface supported the differentiation into chondrocytes. These results further confirmed that substrate chemical composition as well as surface topography and charge influenced cell behavior. In summary, we successfully constructed a biocompatible surface using PLLA as the substrate.

Data: CORDIS, © European Union