Produzione,caratterizzazione e studio dell'attività biologica di TAT-OP1 fattore osteogenetico per l'ingegneria tissutale

Osteogenic protein-1 (OP-1 or BMP-7) is a member of Bone Morfogenic Proteins’s family (BMPs) and consists of 431 amino acid. BMPs are multi-functional growth factors belonging to the transforming growth factor ? (TGF-?) superfamily. Implicated in a variety of functions as the formation of cartilage and bone, and the development of non-osteogenic tissues (heart, nerve), BMPs are secreted as a precursor approximately four times longer than the mature form, and share a C-terminal distinctive pattern ..C…CXGXC…CC…CXCX.. containing seven cysteines, the active region of the proteins. In this study we prepared a recombinant fusion protein, called TAT-OP1, including TAT sequence, an Arg rich peptide derived from HIV protein usually used to perform the cell transfection, and a portion of OP-1 sequence. The construct TAT-OP1, 162 aminoacids long, starts with an N-terminal 6His-tag followed by TAT sequence (all together 30 AA), a peptidase specific cleavage site (spanning 6 AA) and the C-terminal OP-1 domain (126 AA) containing the cysteines motive. Obtained by recombinant DNA technology, the protein TAT-OP1 has been purified by immobilized metal ion affinity chromatography (IMAC) and RP-HPLC, then characterized by SDS-PAGE, aminoacid analysis, UV, CD, and mass spectrometry. In order to demonstrate the osteogenic potential of recombinant fusion protein TAT-OP1, we treated osteoblastic cell line MC3T3-E1 by using different treatment conditions (pulse- 200 nM and in continous- 5.5, 13.5 and 27 nM treatment). After 7 and 14 days of cellular treatment performed by application of both stimulation methods, the presence of calcium salt deposits, alkaline phosphatase activity and the expression of osteogenic markers (osteopontin, osteocalcin and Cbfa1/Runx2) were detected by colorimetric and immunoflorescence assays. To investigate the possible applications of this protein in bone tissue engineering, we developed a research model using adherent fibroblastic cells isolated from umbilical cord blood (UCBMSCs) and a three-dimensional (3D) synthetic scaffold, named Puramatrix Hydrogel TM to mimic the native micro-environment. Moreover, PLGA micro-beads were employed to allow a controlled release of TAT-OP1 with the aim to maintain a suitable level of protein for prolonged times, enhancing its stimulation efficacy. The primary cultures of UCBMSCs were separated by density-gradient method and were phenotypically characterized in 2D system for the expression of CD105, CD90, CD166, nestin, c-kit, CD31, CD34 and CD38 and for their multi-differentiative potential. As detected on MC3T3-E1 cells, TAT-OP1 demostrated to stimulate nodule calcium formation and the expression of alkaline phosphatase when the cells were treated by both of stimulation methods. After encapsulation into Puramatrix Hydrogel TM, the cellular response to TAT-OP1 stimulation was evaluated by using electron microscopy analysis, to detect the production of bone like ECM. After 27 days of stimulation with TAT-OP1 (200 nM), microfibrils were observed partially aggregated around the cells. Calcification nodules and Hydroxyapatite crystals were detected only in the cultures encapsulated into Puramatrix Hydrogel TM and treated with PLGA microspheres-controlled release system. Further investigations will define the utility of this technical approach to improve the in vitro study of osteogenic differentiation and the biological activity of TAT-OP1 for clinical application in the field of bone tissue engineering