Imaging for Non-destructive Assessment of Engineered Tissues

Destructive analysis at multiple in vitro and in vivo time points creates a costly critical bottleneck in development of engineered tissues and regenerative medicine approaches. Our laboratory research the applicability of a multimodal tissue diagnostic platform combining optical (Time-Resolved Fluorescence Spectroscopy (TRFS)) and ultrasound backscatter microscopy (UBM) technologies for label-free, real-time, non-destructive in vitro and in vivo analysis of composition, structure and function of engineered tissue constructs. A few examples of such studies conducted in collaboration with Dr. Athanasiou and Dr. Leach in Biomedical Engineering and Dr. Leigh Griffiths in the School of Veterinary Medicine are depicted below.

Chondrogenic differentiation of stem cells derived from human adipose tissue (ASC) on a 3-D matrix

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Representative FLIM images. Top: control and scaffolds cultured in chondrogenic conditions for (B) 1, (C) 3, (D) 5, and (E) 7 weeks. Bottom: Average fluorescence lifetime histogram of chondrogenic samples for each time point and control samples over 7 weeks (n=4 for each week). The increased lifetime values vs week of differentiation suggests accumulation of collagen II and formation of collagen crosslinks over 7 weeks). These findings correlate with immunostaining and collagen assay results.

TRFS and UBM of vascular patch material implanted in a porcine carotid arteriotomy model

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Pig carotid sample with CorMatrix® graft. TRFS results: (a) fluorescence spectra, (b) τ, and (c) LC-2 values from normal and grafted tissue areas. Standard deviation (SD) is shown as shaded areas. UBM image of vessel showing (d) grafted (arrow C) and (f) normal area with corresponding registered Verhoeff van Gieson (VVG)- stained histology section in (e) and (g), respectively.

 

The effects of specific treatments designed to alter the amount of collagen and collagen cross-links in engineered cartilage constructs can be detected based on intrinsic tissue fluorescence signatures.

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Parameters between the non-destructive and destructive assays (optical, biochemistry, and biomechanical methods) with high and significant correlation. (a) Significant and high correlation was observed for collagen/ww with EY (r=0.81, p<0.0001) and for collagen/ww with average lifetime over 400/40 nm (r=0.69, p=0.0015). (b) Significant and high correlation was also observed for GAG/ww with HA (r=0.70, p=0.0012). However, no significance was found between GAG/ww and the average lifetime over 400/40 nm (r=-0.26, p=0.31). This indicated that GAG had a minor contribution compared to collagen to the overall fluorescence emission of the construct. (c) Significant and high correlation was observed between the average lifetime over 400/40 nm and EY (r=0.57, p=0.01) but no significance was found between the average lifetime and HA (r=-0.07, p=0.77), suggesting the potential nondestructive optical assessment of mechanical properties of cartilage samples.

Related Publications

B.Z. Fite, M. Decaris, Y.H. Sun, Y. Sun, A. Lam, C.K.L. Ho, J.K. Leach, L. Marcu. “Noninvasive Multimodal Evaluation of Bioengineered Cartilage Constructs Combining Time-Resolved Fluorescence and Ultrasound Imaging.” Tissue Eng Part C Methods, 17(4): 495-504, 2011. (Link) (PDF)

H. Fatakdawala, L.G. Griffiths, S. Humphrey, L. Marcu, “Time-resolved fluorescence spectroscopy and ultrasound backscatter microscopy for nondestructive evaluation of vascular grafts,” J. Biomed. Opt., 19(8), 080503 (2014). (Link)

Y. Sun, D. Responte, H. Xie, J. Liu, H. Fatakdawala, J. Hu, K.A. Athanasiou, and L. Marcu. “Nondestructive Evaluation of Tissue Engineered Articular Cartilage Using Time-Resolved Fluorescence Spectroscopy and Ultrasound Backscatter Microscopy” Tissue Engineering Part C: Methods. March 2012, 18(3): 215-226. (Link)

 

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