TY - JOUR A1 - Hackethal, Johannes A1 - Dungel, Peter A1 - Teuschl, Andreas Herbert T1 - Frequently Used Strategies to Isolate Extracellular Matrix Proteins from Human Placenta and Adipose Tissue JF - Tissue Engineering Part C: Methods N2 - The natural extracellular matrix (ECM) provides the optimal environment for cells. Many enzymatic or non-enzymatic based strategies to extract ECM proteins from tissues were published over the past years. However, every single isolation strategy reported so far is associated with specific bottlenecks. In this study, frequently used strategies to isolate ECM from human placenta or adipose tissue using Tris-, serum-, or pepsin-based buffers were compared. The resulting ECM proteins were biochemically characterized by analysis of cellular remnants using Hoechst DNA staining, glycosaminoglycan (GAG) content by dimethylmethylene blue, visualization of protein bands using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis combined with amino acid quantification, and assessment of the proangiogenic profile using an angiogenesis array. Tris-NaCl-extracted ECM proteins showed a high heterogenic degree of extracted proteins, bioactive growth factors, and GAGs, but no collagen-I. Active serum-extracted ECM showed significant lower DNA remnants when compared with the Tris-NaCl isolation strategy. Pepsin-extracted ECM was rich in collagen-I and low amounts of remaining bioactive growth factors. This strategy was most effective to reduce DNA amounts when compared with the other isolation strategies. Pepsin-extracted ECM from both tissues easily gelled at 37°C, whereas the other extracted ECM strategies did not gel at 37°C (Tris-NaCl: liquid; serum: sponge). All relevant characteristics (DNA residues, ECM diversity and bioactivity, shape) of the extracted ECM proteins highly depend on its isolation strategy and could still be optimized. Impact statement The natural human extracellular matrix (ECM) is the ideal cell niche. Various strategies were reported to isolate human ECM components from various sources. In this article, we compared frequently used methods and compared their characteristics (DNA remnants, glycosaminoglycan content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, amino acid quantification, angiogenesis array, and gel formation). We conclude that more research is still necessary to optimize current isolation approaches for in vitro or in vivo applications of human ECM. KW - Tissue Engineering KW - Biomaterials KW - Adipose Tissue KW - extracellular matrix KW - human placenta Y1 - VL - 27 IS - 12 SP - 649 EP - 660 ER - TY - JOUR A1 - Hanetseder, Dominik A1 - Levstek, Tina A1 - Teuschl-Woller, Andreas A1 - Frank, Julia Katharina A1 - Schaedl, Barbara A1 - Redl, Heinz A1 - Marolt Presen, Darja T1 - Engineering of extracellular matrix from human iPSC-mesenchymal progenitors to enhance osteogenic capacity of human bone marrow stromal cells independent of their age JF - Front Bioeng Biotechnol N2 - 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. KW - aging KW - iPSCs KW - osteogenic differentiation KW - bone marrow stromal cells KW - extracellular matrix Y1 - U6 - http://dx.doi.org/https://doi.org/10.3389/fbioe.2023.1214019 VL - 11 ER - TY - JOUR A1 - Gollmann-Tepeköylü, Can A1 - Graber, Michael A1 - Hirsch, Jakob A1 - Mair, Sophia A1 - Naschberger, Andreas A1 - Pölzl, Leo A1 - Nägele, Felix A1 - Kirchmair, Elke A1 - Degenhart, Gerald A1 - Demetz, Egon A1 - Hilbe, Richard A1 - Chen, Hao-Yu A1 - Engert, James C A1 - Böhm, Anna A1 - Franz, Nadja A1 - Lobenwein, Daniela A1 - Lener, Daniela A1 - Fuchs, Christiane A1 - Weihs, Anna A1 - Töchterle, Sonja A1 - Vogel, Georg F A1 - Schweiger, Victor A1 - Eder, Jonas A1 - Pietschmann, Peter A1 - Seifert, Markus A1 - Kronenberg, Florian A1 - Coassin, Stefan A1 - Blumer, Michael A1 - Hackl, Hubert A1 - Meyer, Dirk A1 - Feuchtner, Gudrun A1 - Kirchmair, Rudolf A1 - Troppmair, Jakob A1 - Krane, Markus A1 - Weiss, Günther A1 - Tsimikas, Sotirios A1 - Thanassoulis, George A1 - Grimm, Michael A1 - Rupp, Bernhard A1 - Huber, Lukas A A1 - Zhang, Shen-Ying A1 - Casanova, Jean-Laurent A1 - Tancevski, Ivan A1 - Holfeld, Johannes T1 - Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification JF - Circulation KW - Toll-like receptor 3 KW - aortic valve disease KW - biglycan KW - extracellular matrix KW - osteogenesis Y1 - U6 - http://dx.doi.org/10.1161/CIRCULATIONAHA.122.063481 VL - 147 IS - 20 SP - 1518 EP - 1533 ER -