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

Abstract

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging.

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

“…Rapid developing additional nonlinear optical microscopy techniques may further broaden the promising first results for the application of engineered skin grafts. 27,28 Our findings support the use of TPLSM as a platform technology as described by Vielreicher et al 25 Based our statistical analysis on data acquired by TPLSM, we can show that hdF-supported angiogenesis in our experimental setup is generally not altered by keratinocyte coverage in a significant manner. In contrast to Liu et al, 18 we could not report an increase of vessel length or total capillary-like structures.…”

“…While this is feasible, the maximum penetration depth of this method is only about 100 mm depth from the surface of the graft, and is strongly dependent on the properties of the used scaffold, staining and characteristics of the cultivated tissue. 25 In brief, optical thickness of the vascularized dermis may limit the imaging depth of this procedure to only a few capillary diameters due to technical restrictions. Comparing our employed method, we have been able to show promising results applying TPLSM and digital image computation toward the challenge of analyzing prevascularized skin grafts.…”

Measurement of Friction-induced Changes in Pig Aorta Fibre Organization by Non-invasive Imaging as a Model for Detecting the Tissue Response to Endovascular Catheters

“…A recent review was published on the development and burgeoning use of SHG imaging in the clinic to investigate collagen remodelling in cancer in ex vivo and in vivo human tissue [28]; changes in collagen structure during fibrosis are also being monitored; ex vivo in humans, in vivo in animals, by SHG imaging [29][30][31]. In addition SHG imaging is now playing an important role in non-invasive imaging of collagen structure, formation and remodelling in tissue engineering and regenerative medicine, as reviewed recently by Vielreciher et al [32].…”

Accumulating advantages, reducing limitations: Multimodal nonlinear imaging in biomedical sciences – The synergy of multiple contrast mechanisms

“…A combination of drug delivery and tissue engineering, i.e., the dermal penetration of gold nanoparticles, has been studied in human and reconstructed skin [70]. Also in other investigations multimodal nonlinear microscopy has been used as a versatile tool to investigate artificial tissues, i.e., tissue engineered materials [109][110][111][112][113]. Cellulose scaffolds seeded with smooth muscle cells are investigated as precursor toward the development of fully functional blood vessels.…”