Abstract
Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.
Highlights
To visualize Inferior turbinate stem cells (ITSCs), the confluent cell layer was imaged by coherent anti-Stokes Raman scattering (CARS) microscopy probing the 2845 cm−1 CH2 vibration of lipids, resulting in a specific contrast provided by lipids present within the cultivated cells (Fig. 4)
Collagen type I was ubiquitously observable in the culture via second harmonic generation (SHG) microscopy, which was co-localized to calcium hydroxyapatite deposits visualized by CARS (Fig. 5, arrows)
We demonstrated the great capability of ITSCs to differentiate into osteoblasts in vitro, providing an enhanced potential for tissue regeneration during injuries or for autologous stem cell therapies treating osteodegenerative diseases in the future
Summary
The resulting blue-shifted signal is, again, spectrally extremely narrow and can readily be detected by the use of suitable filter sets Raman spectroscopy, both in its spontaneous and coherent implementations has been increasingly used to characterize stem cells and their derivatives during the last decade[8]. Time-lapse Raman spectroscopy was successfully applied to image the differentiation of murine osteoblasts[12] Extending these promising findings to the human system, Chan et al reported the successful application of Raman spectroscopy to separate human embryonic stem cells and their cardiac derivatives[13]. We used a combination of spontaneous Raman spectroscopy as well as CARS and SHG microscopy to assess and visualize the osteogenic differentiation of adult human neural crest-derived stem cells from the inferior turbinate of the human nasal cavity. Our findings indicate that these label-free analysis methods are applicable to monitoring osteogenic differentiation of adult human stem cells, suggesting their potential use for future clinical applications
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