Bitar, M.; (2006) In vitro human cell transplantation for engineering the hard-soft tissue interface: a soluble phosphate based glass fibre scaffold system. Doctoral thesis , University of London.

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Abstract

This work investigated the biocompatibility of soluble phosphate based glasses as scaffolds for supporting the in vitro morphogenesis the hard-soft tissue interface (enthesis) as an approach dealing with ligaments and tendons clinical problems. The short term response of human oral osteoblasts (HOB), oral fibroblasts (HOF) and flexor tendon fibroblasts (HTF) was assessed on glass discs of various compositions, and different dissolution rates, of the generic ternary form (CaO)o.ox-(Na2O)o.oy-(P20s)o5 through evaluating the maintenance of the seeded cell attachment, survival, proliferation and phenotype by using SEM, immunocytochemistry and the CyQUANT cell density kit. Subsequently, the most biocompatible ternary glass compositions were utilised for fibre production. The effect of fibre diameter of cell adhesion and survival was determined and quaternary glass fibres, of the generic composition (CaO)o.46-(Na20)o.ox-(Fe203)o.oy-(P2C>5)o.5o, where characterised in terms of diameter and solubility profiles. Three-dimensional scaffolds were produced from these fibres and the long term viability, morphology and population growth of the seeded HOB and HOF cells were determined using immunocytochemistry and direct cell count. This was coupled with application of qPCR experiments to evaluate the maintenance of differentiation of the seeded cell population. The role of extrinsic factor inclusion in enhancing in vitro, scaffold associated, tissue morphogenesis was also investigated by stimulating osteogenic differentiation in the seeded HOB cell population. An open lamellar flow bioreactor providing nutrients, oxygen and waste perfusion to the cell-scaffold culture was deigned and assessed. The feasibility of simulating the anatomical architecture of the enthesis has also been addressed as cells were seeded in co-culture on a continuous fibre arrangement. In this study, quaternary phosphate based glass fibre scaffolds containing 3 mol% iron oxide (Fe203), of approximately 30 microm in diameter, and of the composition (CaO)o.46-(Na20)o.oi-(Fe203)o.o3-(P205)o.5o have been shown to support HOB and HOF attachment, well spread morphology, survival and proliferation with no negative impact of cell differentiation. Induction of osteogenesis in the scaffold culture has resulted in up-regulating HOB related gene transcription and the flow culture system, at certain flow rates, has been verified for future use. The Co-culture system design has been successfully implemented as HOB and HOF cells were seeded with an acellular separation zone across the fibre scaffold arrangement.

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