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This investigation aims to utilise the cell-cell communications between osteoblasts and osteoclasts to create a functional tissue engineered construct that is closer to physiological remodelling than current single cell tissue engineered constructs. A ratio of osteoblasts:osteoclasts was optimised as well as a culture medium that supports both cell types. Four different materials, each with excellent properties for tissue engineering including biocompatible and biodegradable, were compared for their ability to support co-cultures. These materials are; silk fibroin, from Bombyx mori (water vapour and methanol stabilised), chitosan and Poly (1-lactin acid) (PLLA). Silk fibroin and chitosan were shown to support the growth and differentiation of both osteoblasts and osteoclasts in both mono and co-cultures, PLLA did not support osteoclast growth as determined by Tartrate resistant acid phosphatase stain and DNA concentration. The 2D films showed signs of degradation after 10 days culture according to differential scanning calorimetry, fourier transform infra red and gel permeation chromatography. Silk fibroin 3D sponges were manufactured to determine if the co-cultures adhere to, proliferate and differentiate in a 3D environment. Static and dynamic (rotary bioreactor) conditions were compared to determine if the bioreactor conditions enhanced the co-culture of osteoblasts and osteoclasts. The cells were shown to adhere to and proliferate on the sponges by scanning electron microscopy and DNA analysis. The sponges also showed early signs of degradation. The use of silk fibroin and a co-culture system appear to provide excellent potential for bone tissue engineering.

Liver cirrhosis and/or liver malignancies have been nominated as the 5th leading cause of death worldwide. The WHO reported, in 2006, that 20 million people around the globe suffer from some form or other of severe liver illness. The ultimate fate of end-stage liver disorders is hepatic dysfunction and eventually organ failure. The only curative mode of management for liver failure is liver transplantation, which is subject to many limitations. Novel alternatives, such as artificial and bio-artificial support devices only aid in temporary replacement of some liver function until an organ is available for transplantation. These newer modalities also have drawbacks or remain experimental and still demand further controlled trials to allow proof of concept and safety before transferring them to the bedside. Regenerative medicine and stem cell therapy has recently shown promise in the management of various human diseases. Recent reports of stem cell plasticity and its multipotentiality has raised hopes of stem cell therapy offering exciting therapeutic possibilities for patients with chronic liver disease. With the understanding that stem cells might not just be about making organs ex vivo, but also regenerating a patients own tissues; a concept is now developing to use stem cells to treat patients with serious disease conditions that are terminal or where conventional modes of treatment are insufficient. There exists a choice of stem cells that have been reported to be capable of self-renewal and differentiation to hepato-biliary cell lineages both in vitro and in vivo. These include: rodent and human embryonic stem cells, bone marrow haematopoietic stem cells, mesenchymal stem cells, umbilical cord blood stem cells, foetal liver progenitor cell and adult liver progenitor cells. It may, however, be argued that with a global population of 6 billion people and a global birth rate in access of 130 million per year, the products of birth ,umbilical cord and cord blood, possibly provide the most readily accessible and ethically sound alternative source of stem cells. The differentiated stem cells can be potentially exploited for gene therapy, cellular transplant, bio-artificial liver-assisted devices, drug toxicology testing and use as an in vitro model to understand the developmental biology of the liver. In this study UCB-derived nucleated cells and umbilical cord-derived Mesenchymal stem cells were exploited for liver differentiation ex vivo. These cells were cultured on extracellular matrix (ECM) protein-coated dishes and inserted into ECM incorporated scaffold 3D culture systems. Stimulation with exogenous mitogens and morphogens to induce hepatic histogenesis was experimented. Immunofluorescence analysis revealed the expression of markers specific for: hepatic stem cells (CK-19), hepatoblasts (AFP) and 4 mature hepatic and biliary epithelium markers including: albumin (ALB), and cytokeratin- 18 (CK-18) and cytokeratin-19 (CK-19) and cytokeratin-7 (CK-7) respectively. The differentiated cells displayed several features of hepatic cell kinetics and metabolic activities, including glycogen synthesis, uptake of Indocyanine green dye and cytochrome P450 activity. These cells may prove to have potential in developing cellular therapy for various liver disorders for which the current mode of therapy is inadequate and also provide an adequate in vitro model of parenchymal liver cells in toxicology and in bioartificial liver research.

The aim of the project was to test the hypothesis that substrate stiffness will affect MSC proliferation and differentiation. In order to achieve this two systems were developed; a natural fibrin substrate, whereby altering the concentration of fibrinogen changes the stiffness of the resultant gel and an artificial PDMS substrate where the stiffness is controlled by altering the degree of crosslinking. To determine the effect of various fibrin matrices, providing more physiological growth conditions, on the MSC phenotype; cells were cultured on the gels and then analysed or re-plated onto TCP before analysis. It was found that cells, that had an initial 7-day culture period on the fibrin, proliferated. and maintained their osteogenic differential potential better when compared to cells pre-cultured on TCP. Similarly, a concentration relationship between colony number and fibrin concentration was seen with a decrease in colony number as the fibrin number increased suggesting that' progenitor cell numbers are better maintained on low-stiffness gels. Furthermore, direct culture on the gels demonstrated a stiffness related increase in colony number. PDMS is easily produced with a large range of mechanical properties. Uncoated PDMS does not support MSC attachment and growth in vitro and therefore an acrylic acid coating was applied. Although XPS analysis was unable to establish that a complete coating was deposited on all of the substrates, once coated the PDMS supported MSC attachment and growth. CFU-f efficiency was not directly altered by the mechanical properties of the underlying substrate, however, the differentiation of the cells showed a trend; with an increase in osteoblastic differentiation as the stiffness increased. This trend was also seen under high-density culture conditions with no correlation to the rate of proliferation. Although the exact mechanism is unknown the data presented here supports the concept that substrate signals influence MSC growth and differentiation.