The effect of mean pore size in collagen-glycosaminoglycan scaffolds on cell migration and differentiation for bone tissue engineering applications
Tissue engineering was born from the belief that primary cells could be isolated from a patient, expanded in vitro and seeded onto a substrate that could be grafted back into the patient (Yang et a/, 2001) providing a biological alternative to transplantations and prosthesis. There are three main components in tissue engineering, scaffolds, cells and signalling mechanisms that make up the tissue engineering triad. This thesis focuses on the interplay between cell behaviour and scaffold structural properties. Recent studies from our laboratory have developed novel freeze-drying techniques to vary the structure of collagen-glycosaminoglycan (GAG) scaffolds producing scaffolds with mean pores ranging from 85 pm - 325 pm (Haugh et a/, 2010). The general aim of this thesis was to investigate, using this range of scaffolds, the effect of mean pore size on cell behaviour in the scaffolds and see how this behaviour varies with different cell types. A non-linear effect was seen on initial cell attachment but ultimately scaffolds with the largest pores size of 325 pm facilitated optimal osteoblast attachment, proliferation and migration. A comparison of stem cell (MSC) behaviour to semi-differentiated osteoblasts in the scaffolds demonstrated a similar non-linear effect on cell attachment but poorer MSC migration was observed highlighting the difference in cell type behaviour. Further analysis shed some light on this effect whereby MSCs were physically bigger cells and less motile. Longer term studies were carried out to determine the effect of mean pore size on osteoblast differentiation, matrix mineralisation and cell-mediated contraction. The largest scaffold mean pore size of 325 pm facilitated improved cell distribution, an earlier onset of osteogenic differentiation and a higher level of mineralisation. The final study of this thesis investigated the potential of human amniotic fluid derived stem cells as a readily accessible source of pluripotent stem cells (AFSCs). These cells demonstrated osteogenic differentiation and an ability to attach to the collagen-GAG scaffold. In conclusion, mean pore size was shown to have an effect on cell behaviour in collagen-GAG scaffolds. Ultimately the larger pores of 325 pm facilitate optimal cell attachment, migration, osteogenic differentiation, matrix mineralisation and cellmediated contraction. In addition, the osteogenic potential of a readily accessible source of pluripotent stem cells has demonstrated their future potential in bone tissue engineering.