• phosphonated polymer
• micro-patterned surfaces
• co-cultures
• titanium

This thesis describes cell behaviour on two kinds of substrates with different chemical and topographical surface features: micropatterned and randomly modified substrates.
Micropatterned surfaces were obtained by photoimmobilization of the polysaccharide hyaluronic acid (Hyal) on aminosilanized glass in the presence of photomasks with different geometries. The patterns obtained had different dimensions and chemistry: positive/negative spiral and squared micro-patterned surfaces of decreasing dimensions. The microstructured surfaces were characterized by AFM, SEM, ToF-SIMS, ATR/Ft-IR. SEM analysis allowed measurements of the micropattern’s dimensions: the spiral ranged from 100μm at the periphery to 1μm in the central part, the square and rectangle pattern consisted of a central square of 100x100μm and rectangles of different dimensions decreasing from the centre to the edges of the micropatterned area (2x1μm).
The behaviour of four cell types was tested on these micropatterned surfaces: human coronary artery endothelial cells (HCAEC), human dermal fibroblasts, NIH 3T3 fibroblasts and human normal osteoblasts (NHOst). Each cell type was seeded separately. The cell parameters analyzed were cell morphology, adhesion, cytoskeleton changes and distribution by using SEM, AFM and inverted optical fluorescence microscopy. Cell adhesion analysis demonstrated that HCAEC, human dermal fibroblasts and NHOst did not adhere to the immobilised Hyal but adapted their shape to the different sizes of the square and spiral patterns of silanized glass. In particular, the number of adherent HCAEC and human dermal fibroblasts depended on the dimensions of both the glass domains and the nuclei of the cells. Also, in
both geometric patterns, the reduction of the adhesive glass width induced human dermal fibroblasts to create bonds amongst themselves. NIH3T3 cells adhered inside the squares and the spiral, but reducing the adhesive glass domains width induced NIH3T3 to also adhere to immobilised Hyal probably due to the binding of cell’s specific receptor for Hyal, CD44 to photoimmobilized Hyal.
Then co-cultures of different cell types were performed on micro-structured surfaces. Cell behaviour was evaluated and monitored by inverted optical and time-lapse video microscope. A heterotypic cell-cell interaction among two or three different cell types occurred in the same chemical and topographic micro-domains. By co-culturing fibroblasts with different dimensions (human dermal fibroblasts greater than NIH3T3) with already adhered HCAEC on patterned samples with different dimensions, it was demonstrated that the success of the co-culture did not depend on cell dimensions but rather on the dimension of adhesive microdomains. The simultaneous presence of HCAEC and NIH3T3 fibroblasts did not prevent the adhesion of human normal osteoblasts on the spiral pattern. In particular, areas containing three different types of cells were visible along the glass spiral pattern mostly on the external of the spiral, where the available space to spread was wider.
Cell behaviour on randomly modified surfaces was also investigated. Randomly modified surfaces were sandblasted titanium disks, bare or coated with CMCAPh, a new phosphonate derivative of carboxymethylcellulose (CMC). The cells tested were human osteoblasts. Coating was used with the aim to increase the osteogenic activity of implant surfaces. The phosphonate polysaccharide was obtained by using a carbodiimide-like activating agent for carboxylic groups and 2-aminoethyl-phosphonic acid to create an amidic bond between the amine of the phosphonate agent and the carboxylic acids of CMC. The polymer was characterized by 31P-NMR, FT-IR and potentiometric titration. CMCAPh showed different properties from CMC and its amidated derivative polymer CMCA. Furthermore the polymer film on the titanium surface was characterized by AFM and TOF-SIMS analysis. An ATR FT-IR study was carried out to evaluate the polymer bonding mode onto the titanium surface. The effect of CMCAPh polymer in solution on normal human osteoblasts (NHOst) was studied in vitro, monitoring cell proliferation, cell differentiation and osteogenic activity and was then compared with that of the amidic derivative of carboxymethylcellulose (CMCA). Osteoblast morphology was evaluated by SEM. Adhesion analysis of normal human osteoblasts (NHOst) demonstrated a better adhesion on the titanium surface coated with CMCAPh than on bare titanium.