Analysis of osteogenesis on a novel collagen glycosaminoglycan scaffold- in vitro application for bone tissue engineering

Currently, there exists a need to develop new bone graft substitutes as an alternative to conventional autografting and allografting treatments due to disadvantages such as cost, scarcity of tissue, multiple surgical procedures and the risk of infection. Tissue engineering provides an alternative solution and relies extensively on the use of porous scaffolds to provide the appropriate environment for the regeneration of tissues and organs. These scaffolds are typically seeded with cells and occasionally growth factors or subjected to biophysical stimuli in the form of a bioreactor and are either cultured in vitro to synthesise tissues which can then be implanted into an injured site or are implanted directly into the injured site and through the body\u27s own systems, regeneration of tissues or organs is induced in vivo. In our laboratory, we use a type 1 collagen glycosaminoglycan (CG) scaffold for tissue engineering applications. These scaffolds have been successfully used to clinically treat burn patients and received FDA approval in the mid-1990\u27s. This thesis examines the ability of the CG scaffold towards bone repair and regeneration. Chapter 2 of this thesis examined the ability of the CG scaffold to support attachment, growth and differentiation of human pre-osteoblastic cells hFOB 1.19. Following optimisation of hFOB cells on CG scaffold, an alamar blue viability assay was adapted for use with the hFOB cell line on CG scaffolds. However, it was shown to be a more useful as a tool to identify general cell viability rather than to accurately calculate cell numbers. Long term culture of hFOB cells on CG scaffold demonstrated that cells migrated to the centre of the scaffold by 14 days, resulting in a homogenous, confluent construct by 35 days which displayed both osteoconductive and osteoinductive qualities. Chapter 3 examined the potential of the CG scaffold to support osteogenesis of human cells under long term culture conditions up to 49 days. The effect of TGF-β¹ was examined and found to enhance osteogenesis with optimal results occurring when using an initially high exposure of 10ng/ml TGF for 7 days and reducing this to 0.2ng/ml thereafter. Cell-seeded CG constructs remained viable with fully infiltrated homogenous cell distribution; high levels of cell-mediated contraction and increased in compressive modulus reported over time. A cell capsule along the scaffold periphery and core degradation developed at 49 days; however, mineralisation was shown to be uninhibited with highest levels late stage osteogenic gene expression and mineralisation detected in the highly confluent cellular outer region of the scaffold. The effects of scaffold mechanical properties on cell behaviour were assessed in Chapter 4 which demonstrated that different crosslinking techniques can be used to produce scaffolds with varying stiffness. Results showed that CG scaffold stiffness and its ability to contract displayed an opposite effect on cell proliferation in comparison to differentiation; where the less stiff and contractible DHT-crosslinked CG constructs displayed greater osteogenic maturation while the stiffer, non contractible EDAC and GLUT-crosslinked scaffolds resulted in increased proliferation but reduced osteogenic differentiation. Chapter 5 examined the effects of exposing cell-seeded CG scaffolds to fluid flow using a flow perfusion bioreactor. This study showed that bioreactor culture improved cell distribution and osteogenic priming of hFOB pre-osteoblasts. No difference was observed in levels of cell number or metabolic activity between bioreactor and static culture. However, cell distribution improved following bioreactor culture becoming more homogenous throughout the construct and avoiding the formation of a peripheral cell capsule along the scaffold edges which is a notable problem with long term static culture (as found in Chapter 2). While bioreactor cultured scaffolds displayed lower levels of mineralisation than static cultures, the mineral was more homogeneously distributed and gene expression analysis showed that bioreactor-cultured constructs gave higher cellular expression levels of bone formation markers than static culture alone and that there was no difference in mechanical stiffness between groups. This suggests that the bioreactor can be beneficial for improving cell distribution and osteogenic priming of cells seeded onto CG scaffolds. In conclusion this thesis shows that the CG scaffold can support attachment, growth, viability and osteogenesis of human cells during Iong term culture; that CG scaffold stiffness can influence osteoblast maturation and that a flow perfusion bioreactor can be used as a tool in bone tissue engineering to improve cell distribution as well as providing osteogenic stimulation of human cells on CG scaffold. This thesis thus demonstrates excellent capabilities of the CG scaffold as a bone graft substitute