The objective of this proposal is to develop tools for characterization of engineered tissues using the optical techniques of light scattering and fluorescence. An integrated instrument is proposed which will incorporate several optical techniques. This system will be applied to model systems to assess and characterize its capabilities and will ultimately be applied to monitor engineered bone tissues. The educational objectives include course development, student recruitment and outreach activities.
The main goal of this project was to assess the potential of optical imaging approaches as a tool for characterizing non-invasively essential components of engineered tissues. Engineered tissue development typically relies on the deposition of cells (usually stem cells) on a biomaterial scaffold, followed by the stimulation of cells via different types of stimuli in order to induce differentiation towards a certain lineage, deposition and ultimately remodeling of matrix proteins and degradation of the original scaffold. We have shown that optical imaging modalities can offer important insights on the status of each one of these components, i.e. the cells, the matrix and the scaffold, in a non-destructive manner that allows dynamic sample monitoring. For example, we have shown that we can use two-photon excited fluorescence (TPEF) imaging performed at two excitation and at two emission wavelength regions, in order to identify biochemical differences between undifferentiated mesenchymal stem cells (MSCs) and MSCs that have differentiated towards adipogenic and osteogenic pathways. Specifically, we have shown that three chromophores, namely NADH, FAD and lipofuscin, are the main contributors to the natural cell fluorescence and that the redox ratio (defined as FAD/(NADH+FAD)) decreases upon the onset of differentiation. Since TPEF is an imaging method that allows high resolution images of thick samples, the redox ratio can be used to non-invasively monitor the detailed differentiation patterns of cells within three-dimensional scaffolds. In addition, we have demonstrated that important scaffold aspects can be characterized by TPEF imaging. The scaffolds we have examined consist of silk, a very promising biomaterial because of its mechanical properties, biocompatibility and potential control for structure and degradation. We have shown that endogenous fluorescence can serve as an indicator of secondary and tertiary structure, especially in terms of b-sheet content and alignment, properties that define to a great extent the strength and mechanical stability of the scaffolds. Furthermore, we have shown that light scattering spectroscopy can be employed to characterize down to the nanoscale the deposition of mineral deposits onto silk. This process is critical in bone tissue engineering and the ability to monitor it non-invasively offers great opportunities to gain important insights in its dynamic progression. Finally, in the context of matrix characterization, we have developed automated algorithms to assess the organization of collagen fibers. Collagen is the dominant matrix protein in many tissues, and, due to its non-centrosymmetric nature, it is a strong source of endogenous second harmonic generation (SHG) signal. SHG imaging is another type of high-resolution optical imaging and it is being used extensively to image and characterize collagen fibers. In summary, we have found that a combination of high-resolution, multi-wavelength optical modalities can be used to characterize important aspects of the cell, matrix and scaffold components of engineered tissues. These techniques can be performed simultaneously or sequentially using the same instrument and can monitor dynamically the changes that occur as engineered tissues develop. These new capabilities promise to improve significantly our understanding of the factors that optimize functional tissue development for the repair or replacement of damaged tissues.
