Tissue engineering has the potential to address many of the shortcomings inherent to organ transplantation and aid in the study of diseases through the creation of functional laboratory grown tissues. The design and growth of engineered tissues is a highly complex process in which it is critical to be able to control culture conditions and thus direct tissue maturation. It is therefore necessary to monitor the effects of these conditions and the progress of the tissue construct. Current analysis technologies provide a wealth of information, however they are unsuitable for repeated or prolonged measurements of the same specimen due to their individual processing requirements. Here, we present non-invasive optical methods for monitoring engineered tissues based on two photon excitation of fluorescence (TPEF) and second harmonic generation (SHG). First, we determined the nonlinear optical properties of silk fibroin based biomaterials and found that they are highly fluorescent when excited with two near infrared photons. This two photon excited fluorescence allowed us to acquire high quality images and emission spectra of the fibroin based materials. We observed spectral shifts in the TPEF emission with increases in beta sheet content and a SHG signal that was related to fibroin beta sheet alignment. Secondly, we conducted studies using endogenous sources of optical contrast to monitor human mesenchymal stem cell cultures undergoing differentiation. Using a simple TPEF based fluorescence ratio, we were were able to observe cellular biochemical changes that could be associated with differentiation. We also quantified changes in cellular morphology and found the combination of fluorescence based biochemical and morphological information revealed that not all cells are undergoing differentiation at the same rate. To increase the sensitivity of our non invasive monitoring technique, we developed a method for isolating and tracking changes in the concentrations of the intrinsic cellular fluorophores: NAD(P)H, flavoproteins and lipofuscin. We then applied this technique to monitor changes in human mesenchymal stem cells maintained under different culture (5% or 20% oxygen) conditions, and differentiation pressures. Using a redox ratio computed from the concentrations of NAD(P)H and flavoproteins we were able to observe changes in metabolic activity related to differentiation. We were also able to confirm differentiation from the changes in cell morphology detectable in the TPEF images and the presence of a SHG signal generated from fibrous collagens deposited by the cells differentiating along an osteogenic path. Quantification of the TPEF images revealed an increase in accumulation of lipofuscin, a pigment associated with oxidative stress, in the cultures maintained in 20% oxygen versus those at 5% oxygen. Overall we found that multiphoton microscopy based methods were well suited for monitoring the components of tissued engineered from human stem cells and silk fibroin based biomaterials.
|Commitee:||Garlick, Jonathan, Kaplan, David L., Vunjak-Novakovic, Gordana|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 71/03, Dissertation Abstracts International|
|Keywords:||Biomaterials, Differentiation, Nonlinear microscopy, Stem cells|
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