Multiphoton Imaging and Quantitative Analysis of Collagen Production by Chondrogenic Human Mesenchymal Stem Cells Cultured in Chitosan Scaffold

Abstract

We used the combined imaging modality of multiphoton autofluorescence and second-harmonic generation microscopy to investigate the chondrogenic process of human mesenchymal stem cells cultured in chitosan scaffold. Isolated human mesenchymal stem cells seeded onto chitosan scaffold were induced to undergo chondrogenesis by addition of the transforming growth factor-β3. After continuous culturing, the engineered tissues at the same scaffold location were imaged at different time points for up to 49 days. Using the acquired images of the chondrogenic process, we quantify tissue morphogenesis by monitoring the changes in multiphoton autofluorescence and second-harmonic generation signals from the engineered tissues. We found that the extracellular matrix generation can be modeled by an exponential function during the initial growth stage and that saturation occurs between days 11 and 14. Further, the growth rate of the extracellular matrix was found to increase toward the surface of the chitosan scaffold. Our work demonstrates the use of multiphoton microscopy for performing long-term monitoring and quantification of the tissue engineering process.

Imaging Strategies for Tissue Engineering Applications

“…7A). 83,84 Also, mesenchymal stem cells embedded in various environments such as silk, 85 chitosan, 86 acellular scaffolds, 87 and rat skin 88 were monitored using two-or multiphoton fluorescence microscopy.…”

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

“…In a study by Chen et al [109], the selected chitosan scaffold material showed strong AF that could be applied for the study of cell-biomaterial rsif.royalsocietypublishing.org J R Soc Interface 10: 20130263 interactions (described in §3.3.1). Rice et al [119] characterized silk fibroin (in the form of fibres, scaffolds, hydrogels and films) and studied the effect of hydration/dehydration, gelation time and biomechanical treatment (compression and stretching) on the autofluorescence (and SHG) signals.…”

“…a 420/20 filter when excitation occurs at 840 nm). Figure 8a shows an interesting approach for long-term monitoring and quantifying tissue morphogenesis by a combined 2-PF/SHG approach [109]. Scaffold and cells were imaged by two-photon AF and collagen I with SHG (both excited at 760 nm).…”

Chitosan facilitates structure formation of the salivary gland by regulating the basement membrane components

“…Type I, III, IV collagen are produced at high levels in wounds that are exposed to chitin-based biomaterials [40]. Similar effects were reported when chitosan scaffolds were used to culture connective tissue [41], ligaments [42], or stem cells [43]. In addition to collagen, the synthesis of other ECM components, such as glycosaminoglycans, are also increased by chitosan [44].…”