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Development of 3D-Printed Cartilage Constructs and Their Non-Invasive Assessment by Synchrotron-Based Inline-Phase Contrast Imaging Computed Tomography



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One goal of cartilage tissue engineering (CTE) is to create constructs for regeneration of hyaline cartilage. Three-dimensional (3D)-printed cartilage constructs fabricated from polycaprolactone (PCL) and chondrocyte-impregnated alginate mimic the biphasic nature of articular cartilage and offers great promise for CTE applications. However, ensuring that these constructs provide biologically conducive environment and mechanical support for cellular activities and articular cartilage regeneration is still a challenge. That said, the regulatory pathway for medical device development requires validation of implants such as these through in vitro bench test and in vivo preclinical examination prior to their premarket approval. Furthermore, mechano-transduction and secretion of cartilage-specific ECM are influenced by mechanical stimuli directed at chondrocytes. Thus, ensuring that these cartilage constructs have mechanical properties similar to that of human articular cartilage is crucial to their success. Non-invasive imaging techniques are required for effective evaluation of progression of these cartilage constructs. However, current non-invasive techniques cannot decipher components of the cartilage constructs, nor their time-dependent structural changes, because they contain hydrophobic and hydrophilic biomaterials with different X-ray refractive indices. The aims of this thesis were to develop 3D-printed cartilage constructs that biologically and mechanically mimic human articular cartilage and to investigate synchrotron radiation inline phase contrast computed tomography (SR-inline-PCI-CT) as a non-invasive imaging technique to characterize components of these constructs and associated time-dependent structural changes. The first objective was to determine in vitro biological functionality of the cartilage constructs over a 42-day period with regards to cell viability and secretion of extracellular matrix by traditional invasive assays. In parallel, performance of SR-inline-PCI-CT for non-invasive visualization of components and associated structural changes within the constructs in vitro over a 42-day was examined. To achieve this objective, three sample-to-detector distances (SDDs): 0.25 m, 1 m and 3 m were investigated. Then, the optimal SDD with better phase contrast and edge enhancement fringes for characterization of the multiple refractive indices within the constructs was utilized to visualize their structural changes over a 42-day culture period. Like the first objective, the second objective was to examine in vivo biological functionality of the cartilage constructs by traditional invasive assays and utilize SR-inline-PCI-CT to non-invasively visualize components of the hybrid cartilage constructs over a 21-day period post-implantation in mice. The third objective was to modulate mechanical properties of PCL framework of the 3D-printed PCL-based cartilage constructs to mimic mechanical properties of human articular cartilage. To achieve this, effect of modulation of PCL's molecular weight (MW) and scaffold's pore geometric configurations: strand size (SZ), strand spacing (SS), and strand orientation (SO), on mechanical properties of 3D-printed PCL scaffolds were studied. Then, regression models showing the effect of SZ, SS, and SO on porosity, tensile moduli and compressive moduli of scaffold were developed. Compressive and tensile properties of these scaffolds were compared with those of human articular cartilage. Then, “modulated PCL scaffolds” with mechanical and biomimetic properties that better mimic human articular cartilage was identified and recommended for fabrication of PCL-based cartilage constructs. This thesis demonstrated effective in vitro and in vivo biological performance of the 3D-printed hybrid cartilage constructs studied and presented a significant advancement in CTE applications. To be precise, cell viability was at a minimum of 77 % and secretion of sulfated GAGs and Col2 increased progressively within cartilage constructs over a 42-day in vitro. Similarly, cell viability was consistently above 70 %, and secretion of sulfated GAGs and Col2 increased post-implantation of constructs in mice over a 21-day period. Furthermore, SR-inline-PCI-CT demonstrated phase contrast and edge-enhancement fringes effective for visualization of the different components and subtle variations within the biphasic cartilage constructs, and thus, offers great potential for their non-invasive and three-dimensional visualization. Lastly, this thesis presented a significant advancement towards development of PCL constructs with mechanical behavior that mimic that of human articular cartilage. The statistical regression models developed showed the effect of SZ, SS, and SO on porosity, tensile moduli and compressive moduli of scaffolds and recommended sets of parameters for fabrication of “modulated PCL scaffolds” with mechanical properties that better mimic mechanical behavior of human articular cartilage. These “modulated PCL scaffolds” could serve as a better framework and could guide more effective secretion of cartilage-specific ECM within PCL-based constructs for CTE applications.



3D-printing, cartilage tissue engineering, synchrotron, inline phase contrast computed tomography



Doctor of Philosophy (Ph.D.)


Biomedical Engineering


Biomedical Engineering



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