Polyester scaffold: Material design and cell-protein-material interaction

Sammanfattning: Tissue engineering has emerged as a valid approach for the regeneration and restoration of bone defects. The concept of bone tissue engineering includes degradable scaffolds, osteogenic cells and osteoinductive growth factors either alone or in any combination of these three. The scaffold bulk material and its design, in particular, are essential for reaching clinically relevant treatments. It is essential that the scaffold is biocompatible and acts as a temporary extra-cellular matrix with a porous 3-dimensional structure, supporting adhesion, proliferation and differentiation of osteogenic cells. Yet another criterion of the scaffold is that is must have sufficient mechanical stability to maintain structural integrity and protect the cells with a gradual transfer of mechanical load to the developing tissue. At the same time, the scaffolds needs to be bioresorbable with a controllable degradation rate depending on its application and the rate of tissue regrowth.In this thesis, aliphatic polyester scaffolds have been modified and shown to be suitable for bone tissue engineering applications. In addition, a new microfluidic device for live imaging of cell behavior within porous 3-dimensional scaffolds has been developed.          Highly porous and degradable aliphatic polyester scaffolds with varying pore sizes and interconnected pores were fabricated. The polyesters assayed were random co-polyesters poly(L-lactide-co-ε-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO] and the homopolymer poly(L-lactide) [poly(LLA)]. The inherently different polymers yielded scaffolds with a wide range of properties with respect to surface chemistry, thermal properties, mechanical stability and degradation rate.The polyester scaffolds were shown to support the increased proliferation of bone marrow-derived stromal cells (BMSC) as well as enhanced osteogenic differentiation, with increased levels of osteocalcin gene expression, which emphasized their potential to act as cells carriers in bone tissue engineering. The potential of poly(LLA-co-CL) scaffolds and common biomedical polyesters in bone tissue engineering was further enhanced by surface functionalization. This involved two different methods of immobilization of bone morphogenetic protein-2 (BMP-2), a potent bone-growth-inducing factor, to the assayed polyesters. The first method used BMP-2 immobilized to heparin functionalized polyesters, while the second method covalently bonded BMP-2 to grafted linker groups on polyesters. Both immobilization techniques retain the bioactivity of BMP-2, and growth-factor-modified polyesters showed an increasing expression of osteogenic genes and production of osteocalcin in osteoblasts-like cells as well as increased proliferation in the mouse cell line, C3H10T1/2.The rate of degradation of electron-beam-sterilized polyester scaffolds and the subsequent loss of mechanical stability were strongly dependent on the chemical, physical and macroscopic architecture of the samples. The degradation rate and loss of mechanical integrity were much greater in porous scaffolds with hydrophilic co-monomers. By incorporating hydrophobic co-monomers with a limited ability to crystalize instead of hydrophilic co-monomers, the mechanical stability was retained for a longer time during the degradation process.The polyester supported spreading and flattened the morphology of both BMSC and osteoblast-like cells. The early cell adhesion to synthetic surfaces is mainly governed by the proteins adsorbed from its surrounding fluids. Early adhesion of BMSC to blood-plasma-coated polyesters was limited, despite the ability of the polyesters to adsorb adhesive proteins and expression of appropriate integrins on BMSC. However, adhesion to a purified adhesive matrix protein on the polyesters did occur, suggesting that pretreatment of polyester scaffolds with adhesive proteins or peptides is a feasible way to enhance the efficiency of cell loading into polyester scaffolds.                        Polyester scaffolds were combined with microfluidics and soft lithography to develop a new method for high-resolution imaging of live cells within porous scaffolds. The microfluidic device was used to frequently follow live cell proliferation and differentiation on the same spatial location within 3-dimansional porous scaffolds over a period of more than four weeks. This device is attractive for the evaluation of cells and materials intended for tissue engineering.We conclude that degradable aliphatic co-polyester scaffolds carefully designed with respect to macroscopic structure, bulk material and surface chemistry are able to meet the specific requirements of various bone tissue engineering applications. In addition, microfluidic devices permit reoccurring high resolution imaging of live cells within porous scaffolds and have a potential as a method of evaluating tissue engineering constructs.