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.
Achieving adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autografts or allografts has many drawbacks. A bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicate-based ceramic) is developed, which when 3D-printed combines high strength with outstanding bone regeneration ability. In this study, the performance of purely synthetic, 3D-printed Sr-HT-Gahnite scaffolds is assessed in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with bone autografts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, and histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy shows scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D-printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects and overcome the limitations of bone graft transplantation.
Biomimetic scaffolds are of great interest to tissue engineering (TE) and tissue repair as they support important cell functions. Scaffold coating with soluble collagen-I has been used to achieve better tissue integration in orthopaedy, however, as collagen persistence was only temporary such efforts were limited. Adequate coverage with cell-derived ECM collagen-I would promise great success, in particular for TE of mechanically challenged tissues. Here, we have used label-free, non-invasive multiphoton microscopy (MPM) to characterise bacterial nanocellulose (BNC) - a promising biomaterial for bone TE - and their potency to stimulate collagen-I formation by mesenchymal stem cells (MSCs). BNC fleeces were investigated by Second Harmonic Generation (SHG) imaging and by their characteristic autofluorescence (AF) pattern, here described for the first time. Seeded MSCs adhered fast, tight and very stable, grew to multilayers and formed characteristic, wide-spread and long-lasting collagen-I. MSCs used micron-sized lacunae and cracks on the BNC surface as cell niches. Detailed analysis using a collagen-I specific binding protein revealed a highly ordered collagen network structure at the cell-material interface. In addition, we have evidence that BNC is able to stimulate MSCs towards osteogenic differentiation. These findings offer new options for the development of engineered tissue constructs based on BNC.
Extensive bone loss due to trauma or disease leads to impaired healing. Current bone grafts and substitutes have major drawbacks that limit their effectiveness for treating large bone defects. A number of bone substitutes in development are undergoing preclinical testing, but few studies specifically investigate the in vivo material-tissue interactions that provide an important indicator to long-term implant safety and efficacy. This study is the first of its kind to specifically investigate in vivo material-tissue interactions at the bone-implant interface. Baghdadite scaffolds implanted in critical-sized segmental defects in sheep tibia for 26 weeks are analyzed by focused ion beam scanning electron microscopy, multiphoton microscopy, and histology. The scaffolds are seen to induce extensive bone formation that directly abut the implant surfaces with no evidence of chronic inflammation or fibrous capsule formation. Bone remodeling is influenced by slow in vivo degradation around and within the implant, causing portions of the implant to be incorporated into the newly formed bone. These findings have important implications for predicting the long-term effects of baghdadite ceramics in promoting defect healing, and support the translation of baghdadite scaffolds as a new generation of bone graft substitutes with improved properties for the repair of large bone defects.
Collagen I is the major fibrous extracellular component of bone responsible for its ultimate tensile strength. In tissue engineering, one of the most important challenges for tissue formation is to get cells interconnected via a strong and functional extracellular matrix (ECM), mimicking as closely as possible the natural ECM geometry. Still missing in tissue engineering are: (a) a versatile, high-resolution and non-invasive approach to evaluate and quantify different aspects of ECM development within engineered biomimetic scaffolds online; and (b) deeper insights into the mechanism whereby cellular matrix production is enhanced in 3D cell-scaffold composites, putatively via enhanced focal adhesion linkage, over the 2D setting. In this study, we developed sensitive morphometric detection methods for collagen I-producing and bone-forming mesenchymal stem cells (MSCs), based on multiphoton second harmonic generation (SHG) microscopy, and used those techniques to compare collagen I production capabilities in 2D- and 3D-arranged cells. We found that stimulating cells with 1% serum in the presence of ascorbic acid is superior to other medium conditions tested, including classical osteogenic medium. In contrast to conventional 2D culture, having MSCs packed closely in a 3D environment presumably stimulates cells to produce strong and complex collagen I networks with defined network structures (visible in SHG images) and improves collagen production. Copyright © 2015 John Wiley & Sons, Ltd.
Integrin-bound Src tyrosine kinase mediates ␣ IIb  3 outside-in signaling to the cytoskeleton required for platelet adhesion and thrombus formation. Src activation (signal initiation) by phosphorylation of Tyr-418 occurs at lamellipodia leading edges. However, little is known about Src inactivation mediated by C-terminal Src kinase (Csk) Tyr-529 phosphorylation. In an established platelet model cell line (A5-Chinese hamster ovary), we studied the inactivation of Src during ␣ IIb  3 -mediated adhesion to fibrinogen with live cell fluorescence resonance energy transfer (FRET) microscopy. Imaging revealed highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. The Src-Csk interaction followed a highly dynamic pattern. Every 2-3 min, Src-Csk complexes moved inward in the cell, reorganized, and formed stable focal adhesions. These accumulations were primarily seen during retraction of lamellipodia, whereas no interaction was observed during protrusions. Western blot analysis during the run time of FRET signaling revealed an increase in Cskmediated SrcTyr-529 phosphorylation with a parallel decline of tyrosine 418 phosphorylation. Mutation analysis provided additional insights into the role of Src. Although inactivation of Csk (CskK222R) had no effect on cell adhesion and spreading efficiency, cells with constitutively active expressed Src (SrcY529F) exhibited hardly any adhesion and no spreading. The few adherent cells showed weak focal adhesions that were disorganized and oversized. The data clearly demonstrate the important role of tight Src control by Csk for functional cell adhesion and spreading. The novel experimental FRET approach reported here for the inactivation of Src can be readily applied to other integrin and signaling pathways, including closely related Src family kinase members.Integrins are heterodimeric ␣/-transmembrane adhesion receptors that bind with their extracellular domains to proteins of the extracellular matrix or to counter-receptors on other cells and signal with their cytoplasmic part to the actin cytoskeleton (1-3). The signaling pathway from integrins to the actin cytoskeleton (outside-in signaling) is mediated by a network of tyrosine kinases (e.g. Src and Syk), phosphatases, and adaptor proteins. In platelets, fibrinogen binding to the ␣ IIb  3 receptor activates Src tyrosine kinases initiating downstream signaling (4). Src thereby regulates the interaction between integrin and the cytoskeleton (5).From extensive studies with platelets and platelet model cells (A5-CHO), 3 the role of Src in ␣ IIb  3 adhesion signaling is well described (6). Membrane-anchored Src is constitutively bound to the  3 cytoplasmic tail of the fibrinogen receptor. Fibrinogen binding at lamellipodia leading edges causes microclustering of integrins thus leading to trans-autophosphorylation at Tyr-418, conformational opening, and full activation of Src.Many proteins are known to influence Src activation in platelets. However, inactivation is controlled only by Csk (7, 8). With it...
To achieve adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autologous or allogeneic bone grafts has many drawbacks. We have developed a bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicatebased ceramic), which when 3D printed combines high strength with outstanding bone regeneration ability. In this study, we assess the performance of purely synthetic, 3D printed Sr-HT-Gahnite scaffolds in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with autologous bone grafts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy show evidence of scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects, and overcome the limitations of bone graft transplantation.
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