Tissue Engineering and Regeneration in Dentistry by Rachel J. Waddington Alastair J. Sloan & Alastair J. Sloan

Author:Rachel J. Waddington,Alastair J. Sloan & Alastair J. Sloan
Language: eng
Format: epub
ISBN: 9781118741078
Publisher: John Wiley & Sons, Inc.
Published: 2016-11-30T00:00:00+00:00

Cell tracking

In order to address these questions, many new approaches to cell imaging and tracking are currently under development. It is now possible to commercially obtain transgenic mice that constitutively express the green fluorescent protein (GFP) under the direction of promoter sequences to, for example, ubiquitin C and β‐actin. From these GFP‐cells, subpopulations enriched for a nominated cell surface marker can be obtained, providing a better understanding of their function and dynamics obtained by study in vivo following cell transplantation, in vitro as monolayer cell cultures or in 3D scaffolds, or by using ex vivo organ culture models (Sloan et al., 2013). In such model systems, the image signal of GFP‐cells is continually present even after cell proliferation, which offers a great advantage for long‐term tracking of cells compared with other cell labelling methods, such as loading cells with Q dots. These cells are usually monitored using confocal microscopy, which can provide information regarding cell migration, particularly in response to a wound healing event. Loss of signals in cells loaded with Q dots, however, can give information regarding cellular proliferation, monitored using imaging systems such as second‐harmonic generation microscopy. Gene reporter systems, attached to promoter sequences for osteogenic markers, such as Osterix, osteopontin, osteocalcin, and type I collagen, are able to provide event‐specific information about the rate of MSC osteogenic differentiation towards osteoblasts. Alkaline phosphatase has also historically been proposed as a marker of osteoblast differentiation, but caution should be taken when using as a sole marker since high alkaline phosphatase activity is also noted in some MSCs, particularly iPS cells (Bassaneze et al., 2013; Zhang et al., 2014a). A variety of gene reporter systems have been utilised, including luciferase or red fluorescent protein (de Almeida et al., 2011).

Currently, noninvasive, real‐time, and longitudinal imaging is available to track bone marrow stromal cells in dermal wound healing models. Some of these techniques have been successful in imaging to a depth of 1 mm. Techniques that have been established with detailed methodological protocols for microscopy techniques, including optical coherence tomography, multiphoton microscopy, two‐photon excited fluorescence, second‐harmonic generation microscopy, and coherent anti‐Stokes Raman scattering (CARS). These techniques appear to possess promising applications for in vitro and ex vivo studies. Certain techniques, such as CARS, have been successfully employed in detecting nonlabelled cells and in identifying molecular vibration of chemical bonds in signature proteins or DNA (Masia et al., 2013). The limited penetration of these techniques through tissues may currently limit similar use of noninvasive techniques for monitoring bone healing in vivo. Repeated opening the wound to reveal the healing bone would affect the natural continuum of the process, and thus, analysis using these techniques may for the immediate future require tissue sample collection over specified time points to coincide with the key events of bone wound repair. Their advantage, however, is that they have the capacity to provide data for multiple biomarkers relating to cell behaviour, ECM/osteoid formation, and mineral deposition within a single tissue specimen.

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