The use of collagen-based bone graft substitutes for orthopaedic regenerative medicine.

Tissue engineering (TE) applies scientific and clinical techniques to restore and regenerate tissues using scaffolds, stem cells, growth factors and gene therapy, and, for orthopaedic surgery, has evolved to meet a clinical need to develop suitable alternatives to current bone grafting techniques. Collagen-based scaffolds are successfully used as skin graft substitutes but have limited mechanical properties for bone. Using established animal models, the primary objective of this thesis was to evaluate the potential of a collagen-glycosaminoglycan (CG) scaffold previously developed and optimized in our laboratory for bone repair, and to compare its healing response to three composite scaffolds also developed in our laboratory: (i) a collagencalcium phosphate (CCP) scaffold, (ii) a collagen-hydroxyapatite (CHA) scaffold and (iii) a collagen-nano hydroxyapatite (coll-nHA) scaffold. In addition, we analysed the capacity of these scaffolds to act as delivery systems for cells, growth factors and genes. In Chapter 2, a CG scaffold stiffened 7-fold over the standard skin graft collagen scaffold and a CCP scaffold with 25-fold increased stiffness over the skin graft collagen scaffold were tested in a 7mm rat calvarial model. Results demonstrated that both scaffolds, but especially the CCP scaffolds, significantly enhanced healing over untreated controls and both scaffolds elicited an appropriate tissue remodelling immune response demonstrating their potential for bone repair. In Chapter 3, contrary to expectation, the seeding and culture of mesenchymal stem cells (MSCs) onto the same CG and CCP scaffolds, prior to implantation, produced poorer results than the cell-free scaffolds used in Chapter 2. Irnmunohistochemical analysis revealed impaired healing due to the physical barrier of tissue formed during in vitro culture. This study thus improves our understanding of host response in bone tissue engineering. In Chapter 4, a novel collagen hydroxyapatite (CHA) scaffold demonstrated healing rates of a 15mm rabbit radius segmental defect equivalent to autogenous bone graft (ABG) and the CG scaffold soaked with recombinant bone morphogenetic protein-2 (rhBMP2). When the CHA scaffold was soaked in rhBMP2 at a dose exponentially lower than a commercially available rhBMP2 product, it healed the defect with complete anatomical restoration demonstrating both the off-the-shelfpotential of the product as well as its capacity to be used for growth factor delivery. In Chapter 5, as part of a gene therapy approach, nano-hydroxyapatite (nHA) particles developed in our laboratory successfully transfected MSCs with the BMP2 gene. A gene- activated matrix (GAM) was then successfully created by incorporating the transfected MSCs into a scaffold fabricated with the nHA particles and collagen, demonstrating a positive osteogenic response using MSCs in vitro, validating their potential for use in bone tissue regeneration. In conclusion, the application of novel techniques to the existing clinically-approved collagen scaffold has demonstrated considerable healing potential as off-the-shelf bone graft substitutes using established animal models. The results also demonstrate their versatility in bone tissue engineering as this study has shown the ability of these scaffolds to act as delivery devices for stem cells, growth factors and gene therapy and thus their potential to heal large defects where an off-the-shelf approach might not be sufficient.