The Importance of Biophysical and Biochemical Stimuli in Dynamic Skeletal Muscle Models

Classical approaches to engineer skeletal muscle tissue based on current regenerative and surgical procedures still do not meet the desired outcome for patient applications. Besides the evident need to create functional skeletal muscle tissue for the repair of volumetric muscle defects, there is also growing demand for platforms to study muscle-related diseases, such as muscular dystrophies or sarcopenia. Currently, numerous studies exist that have employed a variety of biomaterials, cell types and strategies for maturation of skeletal muscle tissue in 2D and 3D environments. However, researchers are just at the beginning of understanding the impact of different culture settings and their biochemical (growth factors and chemical changes) and biophysical cues (mechanical properties) on myogenesis. With this review we intend to emphasize the need for new in vitro skeletal muscle (disease) models to better recapitulate important structural and functional aspects of muscle development. We highlight the importance of choosing appropriate system components, e.g., cell and biomaterial type, structural and mechanical matrix properties or culture format, and how understanding their interplay will enable researchers to create optimized platforms to investigate myogenesis in healthy and diseased tissue. Thus, we aim to deliver guidelines for experimental designs to allow estimation of the potential influence of the selected skeletal muscle tissue engineering setup on the myogenic outcome prior to their implementation. Moreover, we offer a workflow to facilitate identifying and selecting different analytical tools to demonstrate the successful creation of functional skeletal muscle tissue. Ultimately, a refinement of existing strategies will lead to further progression in understanding important aspects of muscle diseases, muscle aging and muscle regeneration to improve quality of life of patients and enable the establishment of new treatment options

Einführung und Optimierung eines praxisorientierten Problem-based-Learning-Moduls im Life-Science-Bereich

Der vorliegende Beitrag soll zur Diskussion rund um effektive Lehrkonzepte unter Anwendung des Problem-based-Learning-Ansatzes beitragen. Anhand eines konkreten Beispiels aus dem Life-Science-Bereich Tissue Engineering werden zunächst Herausforderungen in der Einführungsphase eines PBL-Moduls beleuchtet. Anschließend werden das aktuelle Konzept des Moduls, die vorgenommenen Maßnahmen zur Optimierung während dessen kontinuierlicher Weiterentwicklung sowie deren Wirksamkeit aus Sicht der Studierenden dargestellt. 13.05.2016 | Christine Leeb, Rita Leitner, Verena Pichler, Carina Huber-Gries, Dominik Rünzler & Veronika Jesenberger (Wien

A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a Rat Sciatic Nerve Defect Model

Over the past decade, silk fibroin (SF) has been emergently used in peripheral nerve tissue engineering. Current approaches aiming at producing SF-based nerve guidance conduits (SF-NGCs) used dissolved silk based on either aqueous solutions or organic solvents. In this study, we describe a novel procedure to produce SF-NGCs: A braided tubular structure of raw Bombyx mori silk is subsequently processed with the ternary solvent CaCl2/H2O/ethanol, formic acid, and methanol to improve its mechanical and topographical characteristics. Topographically, the combination of the treatments results in a fusion of the outer single silk fibers to a closed layer with a thickness ranging from about 40 to 75 mum. In contrast to the outer wall, the inner lumen (not treated with processing solvents) still represents the braided structure of single fibers. Mechanical stability, elasticity, and kink characteristics were evaluated with a custom-made test system. The modification procedure described here drastically improved the elastic properties of our tubular raw scaffold, favoring its use as a NGC. A cell migration assay with NIH/3T3-fibroblasts revealed the impermeability of the SF-NGC wall for possible invading and scar-forming cells. Moreover, the potential of the SF-NGC to serve as a substratum for Schwann cells has been demonstrated by cytotoxicity tests and live-dead stainings of Schwann cells grown on the inner surface of the SF-NGC. In vivo, the SF-NGC was tested in a rat sciatic nerve injury model. In short-term in vivo studies, it was proved that SF-NGCs are not triggering host inflammatory reactions. After 12 weeks, we could demonstrate morphological and functional reinnervation of the distal targets. Filled with collagen, a higher number of axons could be found in the distal to the graft (1678+/-303), compared with the empty SF-NGC (1274+/-146). The novel SF-NGC presented here shows promising results for the treatment of peripheral nerve injuries. The modification of braided structures to adapt their mechanical and topographical characteristics may support the translation of SF-based scaffolds into the clinical setting. However, further improvements and the use of extracellular matrix molecules and Schwann cells are suggested to enable silk tube based conduits to bridge long-distance nerve gaps

Enhanced cell adhesion on silk fibroin via lectin surface modification

Various tissue engineering (TE) approaches are based on silk fibroin (SF) as scaffold material because of its superior mechanical and biological properties compared to other materials. The translation of one-step TE approaches to clinical application has generally failed so far due to the requirement of a prolonged cell seeding step before implantation. Here, we propose that the plant lectin WGA (wheat germ agglutinin), covalently bound to SF, will mediate cell adhesion in a time frame acceptable to be part of a one-step surgical intervention. After the establishment of a modification protocol utilizing carbodiimide chemistry, we examined the attachment of cells, with a special focus on adipose-derived stromal cells (ASC), on WGA-SF compared to pure native SF. After a limited time frame of 20min the attachment of ASCs to WGA-SF showed an increase of about 17-fold, as compared to pure native SF. The lectin-mediated cell adhesion further showed an enhanced resistance to trypsin (as a protease model) and to applied fluid shear stress (mechanical stability). Moreover, we could demonstrate that the adhesion of ASCs on the WGA-SF does not negatively influence proliferation or differentiation potential into the osteogenic lineage. To test for in vitro immune response, the proliferation of peripheral blood mononuclear cells in contact with the WGA-SF was determined, showing no alterations compared to plain SF. All these findings suggest that the WGA modification of SF offers important benefits for translation of SF scaffolds into clinical applications

Molecular and Cellular Effects of <i>In Vitro</i> Shockwave Treatment on Lymphatic Endothelial Cells

<div><p>Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis <i>in vivo</i>. The aim of this study was to investigate <i>in vitro</i> shockwave treatment (IVSWT) effects on lymphatic endothelial cell (LEC) behavior and lymphangiogenesis. We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs. Finally, transcriptome- and miRNA analyses were conducted to gain deeper insight into the IVSWT-induced molecular mechanisms in LECs. The results indicate that IVSWT-mediated proliferation changes of LECs are highly energy flux density-dependent and LEC 2D as well as 3D migration was enhanced through IVSWT. IVSWT suppressed HUVEC 3D migration but enhanced vasculogenesis. Furthermore, we identified podoplanin^high and podoplanin^low cell subpopulations, whose ratios changed upon IVSWT treatment. Transcriptome- and miRNA analyses on these populations showed differences in genes specific for signaling and vascular tissue. Our findings help to understand the cellular and molecular mechanisms underlying shockwave-induced lymphangiogenesis <i>in vivo</i>.</p></div

Gene expression profile of sorted subpopulations.

<p>(A) Transcripts with more than two-fold stronger expression in one of the respective populations (B) Heatmap visualization of 3 replicates showing log₂-transformed gene expression of podoplanin high and podoplanin low LECs. Affymetrix.CEL files were mas5 normalized. Log2-transformed expression values were normalized centred to the median of the 25 plotted transcripts.</p