BREVETTO INTERNAZIONALE. INTERNATIONAL PATENT PCT/EP2006/060603. Engineered human osteoblast cells expressing the fluorescent eGFP

Title A new genetically engineered human cell line for the in vitro characterization of biomaterials. Description In the dentistry and orthopaedic perspectives tissue engineering is focused on the development of innovative materials whose action consists in recruiting bone progenitor cells and in stimulating their proliferation. Doing so, they favourite the development of the tissue which should be replaced. In this context, it is clear that these materials should not only allow cells adhesion and proliferation, but also ensure that the attached cells will maintain the histological features of the original tissue. Careful attention should therefore be addressed to characterize the influence of the biomaterial on bone cells behaviour and, in turn, on bone tissue formation. The ultimate approach to define the above parameters is the in vivo testing, implanting the biomaterial in artificially induced bone injury in experimental animals. This is usually carried out after biophysical and biomechanical characterization of the new material, and assessment of bio-tolerability. This is determined as lack of cytotoxic and inflammatory reaction displayed by cells growing on the biomaterial. No further characterization of biomaterial/cells interaction are conducted in vitro, due to the unavailability of adequate cellular model. The proposed cellular model is suitable for the in vitro characterization of osteoinductive and osteogenic properties of biomaterials. We genetically modified a tumor derived cell line, which has been shown to maintain the osteoblastic phenotype and to posses unlimited growing potential in vitro. By stable transfection of an expression vector, containing as reporter gene the enhanced green fluorescent protein (EGFP), we selected an immortal, genetically uniform clonal cell line, the Saos-EGFP. The engineered cell line Saos-EGFP maintains the histological features of osteoblast cells and constitutively expresses the EGFP. The presence of the heterologous protein does not perturb the physiological expression of molecular markers specific of the bone tissue. Comparative analysis carried out on parental and engineered cells demonstrated that both cell populations express molecular markers specific for bone tissue, such as osteocalcin and osteonectin, which belong to the extra cellular matrix proteins, necessary for bone tissue formation, produced by the osteoblasts. Furthermore, the distribution and cellular localization of the marker proteins were undistinguishable between parental and Saos-EGFP cells, indicating that functional properties were not altered. Another criteria of paramount importance in bone tissue histology is the cytoskeletal organization of cells. Studies have shown that the actin cytoskeletal element plays an important role in cell attachment and stabilization. Actin bundles coupled with adhesion plaques can transmit forces to the substrate and help to maintain cell shape and facilitate cell adhesion. Cytoplasmic actin fibers organization was not affected by the presence of the exogenous protein, since a prefect match in the cytoskeletal architecture between the two cell populations was observed. It was shown that the measure of EGFP fluorescence is proportional to the EGFP molecule number within each sample. This means that, having a genetically uniform clonal cell line, the fluorescence intensity is proportional to the number of cell within each sample. The high expression level of the trans-gene in the genetically modified cell line Saos-EGFP ensures a direct proportion between fluorescence intensity and cell number down to 5x103 cells per sample. This relationship is very useful to assess easily and quickly the number of cells within any sample, just determining the fluorescence intensity by a spectrophotometric reading. Thank to the immortal phenotype, the proposed cell line can be mantanined indefinitelly using standard cell culture condition.