Characterization Of In Vitro Generated Autologous Osteoid Tissue
Bone disease and injury is expected to increase in the near future due to an ageing population, causing social and economic burden due to disability and lowered quality of life. Critical sized defects, the smallest bony injury that will not heal completely over the lifetime of an individual, are significant orthopedic and oral-maxillofacial issues, as they require bone grafts to facilitate repair. As the gold standard autograft is in short supply with additional disadvantages, bone tissue engineering seeks to alleviate this demand. It combines cells isolated from patients with biomaterials to produce bone tissue. This research looks into the feasibility of bone banking: the cryopreservation and storage of a patient’s cells that can be reanimated at the time of injury or disease and the engineering of a bone graft in vitro. Chitosan and nano-hydroxyapatite composite scaffolds were fabricated to produce appropriate pore diameters and porosities. A final concentration of 8% (w/v) chitosan and 5% (w/v) nano-hydroxyapatite were used in the final scaffold, where imaging studies (scanning electron microscopy, micro-computed tomography) showed pore diameters with a 106 µm average with 79% porosity. These scaffolds were subjected to testing, such as degradation, pH, sterilization, swelling studies, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy in order to characterize physical and chemical properties. Using an osteogenic sarcoma cell line (SAOS-2), scaffolds were then subjected to in vitro cell studies including the LIVE/DEAD® viability assay and the MTS cell proliferation assay to characterize biocompatibility. Results indicate scaffolds are biocompatible and non-cytotoxic. Further three-dimensional in vitro testing was performed using SAOS-2 in a custom-made perfusion bioreactor for 14 days. After this period isolated cells were detected on the scaffold using histological and fluorescent microscopy techniques. Further in vitro testing was investigated using mesenchymal stem cells isolated from rats. However, these cells reached senescence rapidly, therefore, the supply of cells were limited and differentiation down the osteoblastic lineage and three-dimensional culture was unable to be accomplished. This research demonstrated the development of a biocompatible scaffold with appropriate structural parameters that promote viable cells when cultured in a bioreactor. Future study should investigate the scaffolds mechanical and structural properties to determine suitability for bearing load and the production of a homogenous scaffold matrix that results in homogenous osteoid tissue. Cell culture optimization in terms of mesenchymal stem cell proliferation and extracellular matrix production also needs to be investigated in terms of modifying tissue culture factors such as shear stress and seeding density.
Advisor: Dias, George
Degree Name: Bachelor of Medical Science with Honours
Degree Discipline: Anatomy
Publisher: University of Otago
Keywords: Bone tissue engineering; Bone; regeneration; Chitosan; Scaffold; 3D CULTURE; Tissue engineering
Research Type: Thesis