Monday  |  Salon 8  |  01:50 PM–02:10 PM

#16046, Measuring Spatial Variation of Alignment in Engineered Tissue Constructs Using Raman Spectroscopy

Measurement of alignment and realignment of structural proteins within tissues with time is crucial to understanding their mechanical behavior and functionality. In previous studies, we developed a method to measure alignment within biological tissues using polarized Raman spectroscopy (PRS). We used PRS to develop a weighting function from highly aligned muscle tissue, then this master loading function was used with PRS spectra to calculate principal component (PC1) scores. An amplitude alignment metric was then determined by fitting a sine function to PC1 scores with respect to polarized angle. This PRS-based amplitude alignment metric provided a label-free and non-destructive method to compare alignment of proteins in soft tissues of varying composition. In this study, we show that the same PRS-based amplitude alignment metric can be used to compare relative alignment of proteins in different spatial regions within engineered tissue constructs. Engineered tissue constructs were generated using 4 mg/ml collagen type-I solutions seeded with mouse myoblast (C2C12) cells. Samples were initially fabricated to be rectangular in shape, then allowed to deform over 12 days as cells within the collagen contracted and introduced protein alignment. Protein alignment was maintained by keeping two ends of the engineered construct clamped. PRS was used to compare the variation of alignment within different regions, including the center and corner edges. In addition, we developed a computational model of the tissue construct as a hyperelastic material undergoing cell-induced contraction (uniform contraction strain). An alignment factor was defined based on principal stretch axes to predict the spatial variation of alignment in the model. PRS, immunofluorescence imaging of collagen fibers, and computational model measures of alignment all showed greater alignment at the corner edges and less alignment at the center of deformed tissue constructs. These results show the high potential of PRS to measure the spatial variation of engineered tissue alignment non-invasively.

Maedeh Lotfi   University of Florida
Hui Zhou   University of Florida
Janny Piñeiro Llanes   University of Florida
Ghatu Subhash   University of Florida
Chelsey Simmons   University of Florida
Malisa Sarntinoranont   University of Florida