Abstract
The main obstacle for tissue engineering is the difficulty in producing structurally and functionally well-organized tissues from in vitro cultured cells. Thus, on one hand the research is focusing towards bioactive three-dimensional materials (scaffolds) able to stimulate specific cellular processes. In fact the problem exists that cells cultured in scaffolds have great difficulty to adhere and proliferate if they don’t recognize bioactive molecules. In this respect biological polymers are used in the preparation of synthetic matrices to be used as tissue engineering scaffolds. On the other hand biological research is focusing on morphological and functional properties of cells seeded onto bioactive materials to evaluate their viability, adherence and proliferation, fundamental steps for successful tissue engineering. Surgical specimens were treated with type Ia collagenase and cultured in FCS/EGF supplemented DMEM. Cellular characterization was carried out on 3rd passage cells. Fibroblasts were seeded on Matri-cell, a substrate rich in basal lamina constituents, or PVA-gelatin sponges. Pulmonar ovine fibroblasts were also employed to set up the experimental procedures of cell seeding on scaffolds and histological methods. Immunocytochemistry was carried out to evaluate the presence of cytokeratin, fibroblast antigen, S-100 protein, TGF-beta1, fibronectin, type I collagen. Cytochemistry allowed to examine the synthesis of glysosoaminoglycans (Alcian blu method) and glycoproteins (PAS reaction). A fibroblast-like morphology and phenotype were found in the human cells isolated and selected from yellow ligaments. An high expression of fibroblast Ag, fibronectin and type I collagen but low TGF-beta1 and no cytokeratin immunoreaction were observed. A different localization of the detected antigens was found in the isolated fibroblasts depending on the type of substrate: a strong immunoreactive network of collagen I fibres was observed around cells grown on Matri-cell compared to the granular immunoprecipitates observed in the cytoplasm of fibroblasts grown on non coated plastic. Fibronectin was detected mainly at the extracellular level in Matri-cell cultured fibroblasts. Fibroblasts seeded on PVA-gelatin are viable and adherents to the substrates. Alcian blue reaction demonstrated the active production of glysosoaminoglycans both in Matri-cell or PVA-gelatin cultured cells, suggesting that these substrates allow extracellular matrix molecule production
Theses abstracts were prepared by Professor Roger Lemaire. Correspondence should be addressed to EFORT Central Office, Freihofstrasse 22, CH-8700 Küsnacht, Switzerland.
