Abstract
The unique properties of skeletal stem cells have attracted significant attention in the development of strategies for skeletal regeneration. However, there remains a crucial unmet need to develop quantitative tools to elucidate skeletal cell development and monitor the formation of regenerated tissues using non-destructive techniques in 3D. Label-free methods such as coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon excited auto-fluorescence (TPEAF) microscopy are minimally invasive, non-destructive, and present new powerful alternatives to conventional imaging techniques. Here we report a combination of these techniques in a single multimodal system for the temporal assessment of cartilage formation by human skeletal cells. The evaluation of bioengineered cartilage, with a new parameter measuring the amount of collagen per cell, collagen fibre structure and chondrocyte distribution, was performed using the 3D non-destructive platform. Such 3D label-free temporal quantification paves the way for tracking skeletal cell development in real-time and offers a paradigm shift in tissue engineering and regenerative medicine applications.
Highlights
Techniques which rely on the measurement of vibrational information such as spontaneous Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy are inherently non-invasive, non-destructive, and chemically selective
In order to validate chondrogenic development, we initially imaged unstained histological sections of the pellets for collagen distribution and cellular autofluorescence
A quantitative 3D label-free imaging system based on 2 ps lasers for optimal multimodal performance was devised, offering a non-invasive and non-destructive platform for the analysis of chondrogenic differentiation of human fetal skeletal cell populations, and enabling elucidation of temporal changes in cartilage development
Summary
Techniques which rely on the measurement of vibrational information such as spontaneous Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy are inherently non-invasive, non-destructive, and chemically selective (reviewed in Moura et al 8). Spontaneous Raman spectroscopy has been used to perform quantitative volumetric analysis of 3D stem cell cultures,[6] the required long acquisition times preclude rapid (≈10 μs per pixel) or videorate (>5 frames per second) imaging. Coherent Raman imaging techniques excite vibrational coherences in molecules which can enhance signals by more than 105 times.[9]. Research by Downes et al and our group has demonstrated that CARS microscopy can be applied to image the differentiation of adipose-derived stem cells into osteoblasts,[10] and skeletal stem cells into adipocytes,[11] respectively. 3D imaging using CARS or its multimodal combination with other label-free
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