Nondestructive Evaluation of Tissue Engineered Articular Cartilage Using Time-Resolved Fluorescence Spectroscopy and Ultrasound Backscatter Microscopy

Abstract

The goal of this study is to evaluate the ability of a bimodal technique integrating time-resolved fluorescence spectroscopy (TRFS) and ultrasound backscatter microscopy (UBM) for nondestructive detection of changes in the biochemical, structural, and mechanical properties of self-assembled engineered articular cartilage constructs. The cartilage constructs were treated with three chemical agents (collagenase, chondroitinase-ABC, and ribose) to induce changes in biochemical content (collagen and glycosaminoglycan [GAG]) of matured constructs (4 weeks); and to subsequently alter the mechanical properties of the construct. The biochemical changes were evaluated using TRFS. The microstructure and the thickness of the engineered cartilage samples were characterized by UBM. The optical and ultrasound results were validated against those acquired via conventional techniques including collagen and GAG quantification and measurement of construct stiffness. Current results demonstrated that a set of optical parameters (e.g., average fluorescence lifetime and decay constants) showed significant correlation (p<0.05) with biochemical and mechanical data. The high-resolution ultrasound images provided complementary cross-section information of the cartilage samples morphology. Therefore, the technique was capable of nondestructively evaluating the composition of extracellular matrix and the microstructure of engineered tissue, demonstrating great potential as an alternative to traditional destructive assays.

Non-invasive and Non-destructive Characterization of Tissue Engineered Constructs Using Ultrasound Imaging Technologies: A Review

“…The collagen content in tissue-engineered constructs based on fibrin 34 and ECM deposition 19,73 have been quantitatively analyzed with gray-scale ultrasound scans in which the change of ultrasound signal intensity reflects the gradual increase in density of cells or extracellular proteins. Using an ultrasound microscopy system with a high frequency (50 MHz) single element transducer, Winterroth et al imaged the surface irregularities of an ex vivo produced oral mucosal equivalent developed on a cellular cadaveric dermis, as seeded cells adhered and grew and a keratinized protective outermost layer formed.…”

“…73 Non-invasive, non-destructive monitoring that couples structural degradation and mechanical strength changes in engineered tissue constructs would provide a pivotal tool for tissue engineers to evaluate and better design candidate scaffolds. Concurrent in vivo monitoring of both mechanical stiffness and structural changes were reported using photoacoustic imaging and shear wave elasticity imaging.…”

Quantitative Ultrasound for Nondestructive Characterization of Engineered Tissues and Biomaterials

“…22 While high-frequency (40 MHz) ultrasound images provided information on the structure and integration of the vascular graft, fluorescence spectroscopy provided complementary information on collagen and elastin content. 22 In other studies, high-frequency ultrasound imaging and time-resolved fluorescence spectroscopy have been combined to evaluate articular cartilage constructs, 23 or to monitor changes in extracellular matrix content during chondrogenic differentiation of stem cells in hydrogels. 24 Photoacoustic imaging combined with B-scan imaging can also provide enhanced and unique capabilities for visualizing biomaterials and engineered tissues.…”

Laser-induced fluorescence: Progress and prospective for in vivo cancer diagnosis

“…Fluorescence spectroscopy has been successfully applied at research and clinical levels to detect the pathologic and physiological transformations of biological tissues such as neoplasia, dermal lesions, and atherosclerosis plaques with high sensitivity and specificity [35][36][37][38][39]. The main advances in the use of fluorescence as a diagnostic and analytical tool are the fact that high sensitivity fluorescence can be detected versus a dark background.…”

“…Tissue fluorescence originates from several endogenous fluorophores including structural proteins (elastin, collagen), amino acids (tryptophan, tyrosine and phenylala-nine), porphyrins, enzyme cofactors (NADH, FAD), and flavins. The emission of these fluorophores has been investigated both in vitro and in vivo [32,35,[43][44][45]. Chromophores including NADH, collagen, elastin, porphyrins, and carotenoids, which are produced in excess from pathologies in tissues or presented in a normal state, contribute to the emission spectrum using for interpretation of diagnostic results along with other parameters.…”