Cartilaginous tissue is a connective tissue composed of specialized cells (e.g., chondrocytes and fibroblasts) that produce a large amount of extracellular matrix (ECM), which is comprised mostly of collagen fibers, abundant ground substance rich in proteoglycan, and elastic fibers. It is characterized by its avascular structures within the tissue, implying that nutrition for normal tissue cells and maintaining a healthy ECM, is mainly supplied through diffusion from nearby vascularized tissues and synovial fluid. Poor nutritional supply to the cartilaginous tissue is believed to be an important factor leading to tissue degeneration. Moreover, due to the complex collagen fiber structures, the solute diffusion properties in cartilaginous tissues are mainly anisotropic (i.e., orientation dependent) in three-dimensional (3D) space. Thus, the determination of nutrient solute anisotropic diffusion properties is crucial for understanding the mechanism of nutrient transport in cartilaginous tissues. Furthermore, characterization of the solute diffusive transport properties in cartilaginous tissues will delineate the relationship between solute diffusion and tissue morphology for further understanding the pathophysiology and etiology of tissue dysfunction and degeneration.

Fluorescence recovery after photobleaching (FRAP) is a versatile and widely used tool for the determination of local diffusion properties within solutions, cells, and tissues due to its high spatial resolution offering the possibility to microscopically examine a specific region of a sample. However, there is a lack of FRAP techniques which can determine the two-dimensional (2D) and 3D anisotropic solute diffusion properties in cartilaginous tissues. Therefore, the objective of this project is to develop novel FRAP techniques for determining 2D and 3D anisotropic solute diffusion properties in cartilaginous tissues.

First, a new 2D FRAP technique solely based on the spatial Fourier analysis (SFA) was developed to determine the 2D anisotropic diffusion tensor in cartilaginous tissues. The major innovations of this study included the derivation of a close-form solution for the 2D diffusion equation by solely using Fourier transform and the complete determination of three independent components of the 2D diffusion tensor. The new theory was validated by computer simulated FRAP experiments indicating the high accuracy and robustness. The new method was applied to determine the 2D diffusion tensor of 4kDa FITC-Dextran in porcine TMJ discs. It was found that the diffusion of this solute in TMJ discs was inhomogeneous and anisotropic. This study has provided a new method to quantitatively investigate the relationship between transport properties and tissue composition and structure. The obtained transport properties are crucial for future development of numerical models studying nutritional supply within the TMJ disc.

Next, the relationship between solute diffusion properties and tissue morphology was investigated by using the new FRAP technique and scanning electron microscopy (SEM). The SEM results demonstrated that the collagen fibers in the TMJ disc aligned anteroposteriorly in the medial, intermediate and lateral regions while aligning mediolaterally in the posterior region. Interestingly, fibers aligned in both the anteroposterior and mediolateral directions were found in the anterior region of the TMJ disc. The diffusion properties were highly correlated with tissue morphology. It was found again that the solute diffusion in the TMJ disc was anisotropic and inhomogeneous, which suggested that tissue structure (i.e., the collagen fiber alignment) and composition (e.g., water content) could be key factors that affect the solute diffusion properties within TMJ discs.

Lastly, a new 3D MP-FRAP technique was fully developed for determining 3D anisotropic solute diffusion in cartilaginous tissues. A closed-form solution for the 3D anisotropic diffusion equation was derived by using SFA and all the components of the 3D diffusion tensor were obtained by averaging the diffusivities over a shell of a spherical surface in the frequency domain. The new method was well validated by analyzing computer simulated MP-FRAP data as well as measuring the diffusivities of FITC-Dextran (FD) molecules in the glycerol/PBS solutions. Quantitative analysis of 3D MP-FRAP experiments in the ligament tissues was demonstrated as an in vitro application of our new technique. The results demonstrated that the 3D diffusion properties of two types of FD solutes (FD70 and FD150) in the ligament tissue slices were anisotropic and the diffusion along the fiber orientation was always faster than the other two directions.

The advantages of the new 2D and 3D FRAP techniques includes (1) the boundary and initial conditions for these analyses are flexible, so bleaching volume could be any 2D or 3D geometries, (2) the real first recovery image frame or stack right after photobleaching is not required for the diffusion tensor calculation, and (3) the diffusion tensor can be calculated without measuring the point spread function or optical transfer function of the microscope. Due to these features, our techniques can be conveniently carried out on a commercial confocal or multiphoton laser scanning microscope for the 2D or 3D anisotropic diffusion measurements. Future work for this project involves incorporating high-speed fluorescence imaging techniques into our FRAP methods in order to enhance the capabilities and broaden the applications of our method. In addition, investigating diffusion properties in cartilaginous tissues by using other imaging modalities [e.g., magnetic resonance imaging (MRI) and computed tomography (CT)] may lead to translational applications for the FRAP techniques developed in this dissertation.