This work examines the use of a small number of naturally-derived materials as scaffolds, specifically for bone tissue engineering. Damaged bone is typically replaced with grafts, and second only to blood, bone is the most transplanted tissue . The ability to produce autologous grafts from patients’ own cells using an engineered scaffold would therefore be extremely valuable. This thesis reports the design and use of microparticle cell carriers for bone tissue engineering applications, as potential injectable or mouldable units.The suitability of silk fibroin (SF) and gelatin (G) blends as biomaterials was tested in two-dimensional cultures, using 3T3 fibroblasts and rat MSCs (rMSCs). The blends (25:75, 50:50 and 75:25) were shown to be biocompatible and with appropriate mechanical properties for bone tissue engineering with Young’s moduli between 36 and 59 MPa. The SF/G blends supported the osteodifferentiation of rMSCs at levels equivalent to tissue culture plastic. Although SF alone did not strongly support cell attachment, the cells that did adhere showed high levels of osteodifferentiation, measured by osteopontin expression. The inclusion of gelatin significantly improved cell attachment while retaining the ability of the SF/G blends to support osteodifferentiation. Novel microparticles were created from the same SF/G blends in a reproducible, controllable manner using a flow focussing device assembled from commercially available fittings. Cell behaviour on the 3D scaffolds mimicked the results observed on 2D films: cell adhesion was significantly improved by the addition of gelatin with the seeding efficiency of 3T3 fibroblasts increasing from 25% on SF microparticles to 69 – 81% on the blended microparticles. Osteogenic differentiation of rMSCs was observed on SF/G 25:75 microparticles in both basal and osteogenic medium, and osteopontin expression was shown to be slightly higher than for rMSCs grown on commercially available Cultispher-S microparticles. Although attempts were made to mould the particles into larger 3D structure, successful preliminary results could not be repeated.Adapting the flow focussing device from liquid-liquid to a liquid-air system allowed the rapid production of oxidised alginate microparticles, blended with extracellular matrix proteins, and with encapsulated cells. These microparticle scaffolds were also shown to support cell viability (75% of rMSCs were viable after 7 days) and osteogenic differentiation. With the potential to easily encapsulate different proteins or ECM extracts, alginate-blended microparticles are potentially useful tools for culture of different cell types. Finally, the culture of microparticles within a hollow fibre bioreactor, with the hollow fibre mimicking blood capillaries, was shown to improve cell viability compared to a non-perfused control: 20 times more 3T3 fibroblasts were harvested from the bioreactor than from the control. This showed the potential for using the hollow fibre bioreactor to rapidly produce large, viable, tissue engineered constructs in vitro.
|Date of Award
|31 Mar 2016
|Paul De Bank (Supervisor) & Julian Chaudhuri (Supervisor)