Resumen de Tissue engineering techniques to regenerate articular cartilage using polymeric scaffolds.
Marcos Pérez Olmedilla
Articular cartilage is a connective tissue that consists of chondrocytes surrounded by a dense extracellular matrix (ECM). The ECM is mainly composed of type II collagen fibrils and proteoglycans (mainly aggrecans). The main function of articular cartilage is to provide a lubricated surface for articulation. Articular cartilage damage is common and may lead to osteoarthritis. Articular cartilage does not have blood vessels, nerves or lymphatic vessels and therefore has limited capacity for intrinsic healing and repair. Tissue engineering (TE) is a promising approach for healing degenerated cartilage. TE involves the use three-dimensional (3D) scaffolds to support cell and tissue growth. The scaffold provides a structure that facilitates chondrocyte adhesion and expansion while maintaining a chondrocytic phenotype and limiting dedifferentiation, which is a problem in two-dimensional (2D) systems. Several materials have been tested as scaffolds for the transplantation of chondrocytes into the injured cartilage. Cell attachment to the scaffolds depends on the physical and chemical characteristics of their surface. Surface morphology, rigidity, equilibrium water content, surface tension, hydrophilicity and the presence of electric charges, have an impact on cell attachment and viability. The primary aim of this thesis was to study the influence of different kinds of biomaterials (mainly scaffolds but also 2D substrates) on the response of chondrocytes to in vitro culture, including cellular adhesion, viability, proliferation, chondrocyte differentiation and ECM synthesis. 3D scaffold constructs must have an interconnected porous structure in order to allow for cell development through the network, to maintain their differentiated function, as well as to allow for the entry of nutrients and metabolic waste removal. Therefore, the effect of the hydrophilicity and pore architecture of the scaffolds was studied. A series of polymer and copolymer networks with varying hydrophilicity was synthesised and biologically tested in monolayer culture. Cell viability, proliferation and aggrecan expression were quantified. When human chondrocytes were cultured on polymer substrates in which the hydrophilic groups were homogeneously distributed, adhesion, proliferation and viability decreased monotonously with the content of hydrophilic groups in the polymer chain. Nevertheless, copolymers in which hydrophilic and hydrophobic domains alternate showed better results than the corresponding homopolymers. In order to further explore the study above, biostable and biodegradable scaffolds with different hydrophilicity and porosity were synthesised. To do so, a template of sintered microspheres (either poly(methyl methacrylate), PMMA, or poly(ethyl methacrylate), PEMA) of controlled size was used to obtain the interconnected porous structure. This technique allows the interconnectivity between pores and their size to be controlled. Highly periodic and regular pore architectures and very reproducible structures were obtained. The mechanical behaviour of the porous samples was significantly different from that of the bulk (non porous) material of the same composition. Cells fully colonised the scaffolds when the pores' size and their interconnection were sufficiently large. Another objective was to assess the chondrogenic redifferentiation in a biodegradable 3D scaffold of polycaprolactone (PCL) of human mature chondrocytes previously expanded in monolayer. This study demonstrated that chondrocytes cultured in PCL scaffolds without fetal bovine serum (FBS) - but supplemented with insulin-transferrin-selenium (ITS) and ascorbate - efficiently redifferentiated, expressing a chondrocytic phenotype characterised by their ability to synthesise cartilage-specific ECM proteins. The influence that pore connectivity and hydrophilicity of caprolactone-based scaffolds has on the chondrocyte adhesion to the pore walls, proliferation and composition of the ECM produced was also studied. Two series of caprolactone-based scaffolds were prepared, one of them of varying porosity and the other with copolymers in which the ratio of the hydrophilic/hydrophobic component varied. The number of cells inside polycaprolactone scaffolds clearly increased as porosity was increased. A minimum of around 70% porosity seems to be necessary for this scaffold architecture to allow for seeding and viability of the cells within. The results of this study suggest that some of the cells inside the scaffold adhered to the pore walls and kept the dedifferentiated phenotype characteristic of chondrocytes cultured in monolayer, while others redifferentiated. In conclusion, the findings of this thesis provide valuable insight into the field of cartilage regeneration using TE techniques. The studies carried out shed light on the right composition, porosity and hydrophilicity of the scaffolds to be used for optimal cartilage production, which could be applied for future autologous chondrocyte transplantation in patients.
