Cultured bone on biomaterial substrates: A tissue engineering approach to treat bone defects

In the present thesis, a tissue engineering approach to treat bone defects was investigated. Such strategy was based on the use of patient own cultured bone marrow stromal cells (BMSCs) in association with biomaterials to produce autologous living bone equivalents. When engineering such implants, three main factors had to be taken into account: (i) the cells, (ii) the culture technology and (iii) the biomaterial scaffolds. The capacity of BMSCs to proliferate, differentiate along the osteogenic lineage and form a bone like tissue was demonstrated in various in vitro assays making use of biochemical, immunological, microscopic and gene expression techniques. The ability of the cells to produce bone in vivo was established using an ectopic (extra osseous) implantation model. Results indicated that BMSC cultures were composed of a heterogeneous population containing a subpopulation of cells with high proliferative capacity and with potential to differentiate into bone forming cells. Both the growth and the differentiation pattern of these cells could be manipulated, to a certain degree, through the use of bioactive factors during culture. After implantation, the bone forming capacity of the cultures proved to be related to the amount of early osteoprogenitors and precursors cells that could be induced into starting the osteogenic differentiation process. In bone marrow aspirates, this subpopulation appeared to decrease with donor age and to be strongly dependent on the donor, indicating that the aspiration procedure plays an important role in the obtained bone marrow cell population. In order to evaluate the in vivo bone formation capacity of BMSC cultures prior to implantation, an experimental method was developed in which the amount of early osteoprogenitors and precursors cells could be quantified. With regard to the technology design, data indicated that the culture of cells on the biomaterial scaffolds prior to implantation resulted in implants with faster in vivo bone forming ability as compared to scaffolds implanted after cell seeding. In addition, two biodegradable polymeric systems were proposed as scaffolds to be used in the described bone engineering approach after evaluating their ability to support bone marrow cell growth, differentiation and in vivo bone formation. In summary, although the complete knowledge of the factors controlling BMSC rowth and osteogenic differentiation still needs to be further expanded, the obtained results suggest that the bone tissue engineering approach described in this thesis presents a great potential for the repair of bone defects and will become an advantageous alternative to the traditional autologous bone grafting.