@article{TomaschMaleinerHeheretal., author = {Tomasch, Janine and Maleiner, Babette and Heher, Philipp and Rufin, Manuel and Andriotis, Orestis G. and Thurner, Philipp J. and Redl, Heinz and Fuchs, Christiane and Teuschl-Woller, Andreas H.}, title = {Changes in Elastic Moduli of Fibrin Hydrogels Within the Myogenic Range Alter Behavior of Murine C2C12 and Human C25 Myoblasts Differently}, series = {Froniers in Bioengineering and Biotechnology}, volume = {10}, journal = {Froniers in Bioengineering and Biotechnology}, pages = {836520}, abstract = {Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates ("2D") or embedded in fibrin hydrogels ("3D") with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels' fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7-30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.}, subject = {Tissue Engineering}, language = {en} } @article{AngelovaDaskalovaFilipovetal., author = {Angelova, Liliya and Daskalova, Albena and Filipov, Emil and Monforte Vila, Xavier and Tomasch, Janine and Avdeev, Georgi and Teuschl-Woller, Andreas Herbert and Buchvarov, Ivan}, title = {Optimizing the Surface Structural and Morphological Properties of Silk Thin Films via Ultra-Short Laser Texturing for Creation of Muscle Cell Matrix Model}, series = {Polymers}, volume = {2022}, journal = {Polymers}, number = {14(13), 2584}, abstract = {Temporary scaffolds that mimic the extracellular matrix's structure and provide a stable substratum for the natural growth of cells are an innovative trend in the field of tissue engineering. The aim of this study is to obtain and design porous 2D fibroin-based cell matrices by femtosecond laser-induced microstructuring for future applications in muscle tissue engineering. Ultra-fast laser treatment is a non-contact method, which generates controlled porosity-the creation of micro/nanostructures on the surface of the biopolymer that can strongly affect cell behavior, while the control over its surface characteristics has the potential of directing the growth of future muscle tissue in the desired direction. The laser structured 2D thin film matrices from silk were characterized by means of SEM, EDX, AFM, FTIR, Micro-Raman, XRD, and 3D-roughness analyses. A WCA evaluation and initial experiments with murine C2C12 myoblasts cells were also performed. The results show that by varying the laser parameters, a different structuring degree can be achieved through the initial lifting and ejection of the material around the area of laser interaction to generate porous channels with varying widths and depths. The proper optimization of the applied laser parameters can significantly improve the bioactive properties of the investigated 2D model of a muscle cell matrix. Keywords: biopolymers; femtosecond laser processing; muscle cell matrix 2D model; muscle tissue engineering; silk fibroin.}, subject = {Tissue Engineering}, language = {en} } @article{DeiningerWagnerHeimeletal., author = {Deininger, Christian and Wagner, Andrea and Heimel, Patrick and Salzer, Elias and Monforte Vila, Xavier and Weißenbacher, Nadja and Grillari, Johannes and Redl, Heinz and Wichlas, Florian and Freude, Thomas and Tempfer, Herbert and Teuschl-Woller, Andreas and Traweger, Andreas}, title = {Enhanced BMP-2-Mediated Bone Repair Using an Anisotropic Silk Fibroin Scaffold Coated with Bone-like Apatite}, series = {Int. J. Mol. Sci.}, volume = {23}, journal = {Int. J. Mol. Sci.}, number = {1 / 283}, abstract = {The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.}, subject = {Tissue Engineering}, language = {en} } @article{TomaschMaleinerHromadaetal., author = {Tomasch, Janine and Maleiner, Babette and Hromada, Carina and Szwarc-Hofbauer, Dorota and Teuschl-Woller, Andreas}, title = {Cyclic Tensile Stress Induces Skeletal Muscle Hypertrophy and Myonuclear Accretion in a 3D Model}, series = {Tissue Eng. Part A.}, volume = {2023}, journal = {Tissue Eng. Part A.}, number = {Mar}, pages = {257 -- 268}, abstract = {Skeletal muscle is highly adaptive to mechanical stress due to its resident stem cells and the pronounced level of myotube plasticity. Herein, we study the adaptation to mechanical stress and its underlying molecular mechanisms in a tissue-engineered skeletal muscle model. We subjected differentiated 3D skeletal muscle-like constructs to cyclic tensile stress using a custom-made bioreactor system, which resulted in immediate activation of stress-related signal transducers (Erk1/2, p38). Cell cycle re-entry, increased proliferation, and onset of myogenesis indicated subsequent myoblast activation. Furthermore, elevated focal adhesion kinase and β-catenin activity in mechanically stressed constructs suggested increased cell adhesion and migration. After 3 days of mechanical stress, gene expression of the fusogenic markers MyoMaker and MyoMixer, myotube diameter, myonuclear accretion, as well as S6 activation, were significantly increased. Our results highlight that we established a promising tool to study sustained adaptation to mechanical stress in healthy, hypertrophic, or regenerating skeletal muscle.}, subject = {fibrin}, language = {en} } @article{JohannesWeihsKarneretal., author = {Johannes, Hackethal and Weihs, Anna and Karner, Lisa and Metzger, Magdalena and Dungel, Peter and Hennerbichler, Simone and Redl, Heinz and Teuschl-Woller, Andreas Herbert}, title = {Novel Human Placenta-Based Extract for Vascularization Strategies in Tissue Engineering}, series = {Tissue Eng Part C Methods}, volume = {27}, journal = {Tissue Eng Part C Methods}, number = {11}, pages = {616 -- 632}, abstract = {There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.}, subject = {Tissue Engineering}, language = {en} } @article{HromadaHartmannOesterreicheretal., author = {Hromada, Carina and Hartmann, Jaana and Oesterreicher, Johannes and Stoiber, Anton and Daerr, Anna and Sch{\"a}dl, Barbara and Priglinger, Eleni and Teuschl-Woller, Andreas H. and Holnthoner, Wolfgang and Heinzel, Johannes Christoph and Hercher, David}, title = {Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro}, series = {Biomolecules}, volume = {2022}, journal = {Biomolecules}, number = {12, 6}, pages = {820}, abstract = {Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction.}, subject = {Tissue Engineering}, language = {en} } @article{HanetsederLevstekTeuschlWolleretal., author = {Hanetseder, Dominik and Levstek, Tina and Teuschl-Woller, Andreas and Frank, Julia Katharina and Schaedl, Barbara and Redl, Heinz and Marolt Presen, Darja}, title = {Engineering of extracellular matrix from human iPSC-mesenchymal progenitors to enhance osteogenic capacity of human bone marrow stromal cells independent of their age}, series = {Front Bioeng Biotechnol}, volume = {11}, journal = {Front Bioeng Biotechnol}, doi = {https://doi.org/10.3389/fbioe.2023.1214019}, abstract = {Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.}, subject = {aging}, language = {en} } @article{BernhardMaroltPresenLietal., author = {Bernhard, Jonathan C and Marolt Presen, Darja and Li, Ming and Monforte, Xavier and Ferguson, James and Leinfellner, Gabriele and Heimel, Patrick and Betti, Susanne L and Shu, Sharon and Teuschl-Woller, Andreas H and Tangl, Stefan and Redl, Heinz and Vunjak-Novakovic, Gordana}, title = {Effects of Endochondral and Intramembranous Ossification Pathways on Bone Tissue Formation and Vascularization in Human Tissue-Engineered Grafts}, series = {Cells}, volume = {11}, journal = {Cells}, number = {19:3070}, doi = {10.3390/cells11193070}, abstract = {Bone grafts can be engineered by differentiating human mesenchymal stromal cells (MSCs) via the endochondral and intramembranous ossification pathways. We evaluated the effects of each pathway on the properties of engineered bone grafts and their capacity to drive bone regeneration. Bone-marrow-derived MSCs were differentiated on silk scaffolds into either hypertrophic chondrocytes (hyper) or osteoblasts (osteo) over 5 weeks of in vitro cultivation, and were implanted subcutaneously for 12 weeks. The pathways' constructs were evaluated over time with respect to gene expression, composition, histomorphology, microstructure, vascularization and biomechanics. Hypertrophic chondrocytes expressed higher levels of osteogenic genes and deposited significantly more bone mineral and proteins than the osteoblasts. Before implantation, the mineral in the hyper group was less mature than that in the osteo group. Following 12 weeks of implantation, the hyper group had increased mineral density but a similar overall mineral composition compared with the osteo group. The hyper group also displayed significantly more blood vessel infiltration than the osteo group. Both groups contained M2 macrophages, indicating bone regeneration. These data suggest that, similar to the body's repair processes, endochondral pathway might be more advantageous when regenerating large defects, whereas intramembranous ossification could be utilized to guide the tissue formation pattern with a scaffold architecture.}, subject = {bone tissue engineering}, language = {en} }