Characterization of tissue engineered endothelial cell networks in composite collagen-agarose hydrogels

Scaffolds constitute an important element in vascularized tissues and are therefore investigated for providing the desired mechanical stability and enabling vasculogenesis and angiogenesis. In this study, supplementation of hydrogels containing either Matrigel™ and rat tail collagen I (Matrigel™/rCOL) or human collagen (hCOL) with SeaPlaque™ agarose were analyzed with regard to construct thickness and formation and characteristics of endothelial cell (EC) networks compared to constructs without agarose. Additionally, the effect of increased rCOL content in Matrigel™/rCOL constructs was studied. An increase of rCOL content from 1 mg/mL to 3 mg/mL resulted in an increase of construct thickness by approximately 160%. The high rCOL content, however, impaired the formation of an EC network. The supplementation of Matrigel™/rCOL with agarose increased the thickness of the hydrogel construct by approximately 100% while supporting the formation of a stable EC network. The use of hCOL/agarose composite hydrogels led to a slight increase in the thickness of the 3D hydrogel construct and supported the formation of a multi-layered EC network compared to control constructs. Our findings suggest that agarose/collagen-based composite hydrogels are promising candidates for tissue engineering of vascularized constructs as cell viability is maintained and the formation of a stable and multi-layered EC network is supported. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Chick CFC Controls Lefty1 Expression in the Embryonic Midline and Nodal Expression in the Lateral Plate

AbstractMembers of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left–right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger–Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left–right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory

Decelularizarea de succes a aortei porcine pentru generarea scaffoldului acelular necesar în obținerea grefelor vasculare inginerești

Laboratory of Tissue Engineering and Cell Cultures, Nicolae Anestiadi Department of Surgery no. 1, Nicolae Testemitanu SUMPh, LEBAO, Hannover Medical School, GermanyBackground. Based on limited availability of autologous vascular grafts, the development of tissueengineered blood vessels (TEBVs) has reached significant research interest in the last couples of years. Fabrication of TEBVs from decellulazed scaffolds seems to be an attractive research strategy. Objective of the study. To develop a method for the generation of cell-free vascular scaffolds by combining a detergent-based decellularization (DC) strategy with subsequent nuclease treatment. Material and Methods. Cryopreserved porcine aortae were treated with detergents for 48h under rotation (24h exposure to 0.5% SDS and 0.5% SDC, followed by 24h exposure to 1% TritonX-100) with/without following enzymatic digestion (48h exposure to 300 U/ml DNase I solution). The efficacy of DC was evaluated by 4’,6-Diamidino-2-phenylindole (DAPI) and hematoxylin and eosin (H&E) stainings. Results. H&E staining revealed no persisting cells in all groups, including the samples which were not treated with DNase. The DAPI stain of those specimens, however, uncovered substantial amounts of residual DNA. Additional enzymatic treatment with DNase led to such a reduction of residual DNA that DAPI positive structures could not be detected. Conclusion. A direct stain of DNA, e.g. DAPI, is much more sensitive measure for the presence of cell debris than the H&E stain. In addition, detergent-based decellularization technique per se is not sufficient for complete cell and debris removal and has to be combined with a DNase digestion.Introducere. Din cauza deficiențelor funcționale a grefelor vasculare (GV) existente, dezvoltarea substituenților sanguini, prin tehnicile de inginerie tisulară, a avansat semnificativ în ultimii ani. Producerea GV în baza matrixului acelular pare a fi o strategie atractivă. Scopul studiului: Dezvoltarea unei metode de generare a scaffoldului decelularizat, prin combinarea unei tehnici bazate pe detergenți, suplimentată cu prelucrarea cu nuclează. Material și Metode. Aorta porcină crioprezervată a fost tratată cu detergenți timp de 48h (expunerea la 0.5% SDS și 0.5% SDC pe 24h, urmată de spălarea cu soluție de 1% TX-100) cu/fără următoarea digestie enzimatică (prelucrarea cu soluție 300 U/ml DNază). Eficacitatea decelularizării (DC) a fost evaluată prin colorația 4’,6-Diamidino-2-fenilindol(DAPI) și Hematoxilină-Eozină (H&E). Rezultate. Colorația H&E nu a evidențiat celule persistente în nici un grup, inclusiv în probele tratate fără nuclează. Cu toate acestea, colorația DAPI a acelorași specimene a descoperit cantități substanțiale de ADN rezidual. Prelucrarea suplimentară cu DNază a dus la o reducere semnificativă a ADN-ului subzistent, încât structuri DAPI pozitive nu au fost detectate. Concluzii. Colorațiile cu afinitatea pentru ADN, d.e. DAPI, sunt mai sensibile pentru evaluarea prezenței resturilor celulare, decât H&E. Tehnica DC bazată pe detergenți nu este suficientă pentru îndepărtarea completă a celulelor și a detritului, și trebuie combinată cu prelucrarea enzimatică

Reduction in xenogeneic epitopes on porcine endothelial cells by periodate oxidation

Background: Patterns of humoral immune responses represent a major hurdle in terms of pig-to-human xenotransplantation approaches. The best-known xenogeneic glycan antigens present in pigs are the αGal (Galili antigen) and the non-human sialic acid Neu5Gc. As there are further differences between porcine and human cellular surface glycosylation, a much broader range of glycan epitopes with xeno-reactive relevance can be anticipated. Therefore, we set out to chemically modify porcine cellular surface glycans in a global approach by applying sodium periodate (NaIO4) oxidation. Methods: Porcine endothelial cells were exposed to oxidation with 1 to 5 mM NaIO4 for different time periods at 37 °C or 4 °C and under static or dynamic conditions. The impact on cellular survival was determined by applying live/dead assays. Oxidation of αGal-epitopes was assessed by fluorescence microscopy-based quantification of isolectin-B4 (IL-B4) staining. Overall immunogenicity of porcine cells was determined by human serum antibody binding. Results: Treatment of porcine endothelial cells and tissues with NaIO4 led to reduced binding of the αGal-specific IL-B4 and/or human serum antibodies. NaIO4 was revealed to be cytotoxic when performed at elevated temperatures and for a prolonged time. However, by applying 2 mM NaIO4 for 60 min at 4 °C, a high extent of cellular viability and a relevant reduction in detectable αGal epitope were observed. No differences were detected irrespectively on whether the cells were oxidized under static or flow conditions. Conclusions: Glycan epitopes on living cells can be oxidized with NaIO4 while maintaining their viability. Accordingly, this strategy holds promise to prevent immune reactions mediated by preformed anti-glycan antibodies

Om utveckling av elevers förmåga att resonera om friktion i de tidiga skolåren

Studien har undersökt hur elever i de tidiga skolåren kan utveckla förmåga att resonera om fenomenet friktion. Studien bygger på en intervention i årskurs 1 där elever har arbetat med begrepp för friktion och rörelse på lekplatsen. I artikeln analyseras elevfilmer där eleverna visar och berättar om friktion på en lekplats. Filmerna har analyserats fenomenografiskt. Resultaten visar på tre kvalitativt skilda sätt att erfara friktion bland eleverna: A) Friktion som relaterat till hastighet, B) Friktion som egenskap som påverkar hastighet, C) Friktion som materialegenskap som påverkar hastighet. Studien visar att elever redan i grundskolans tidiga år kan urskilja innebörder av begreppet friktion och visa differentierade skillnader i att hantera begreppet. Studien bidrar på så sätt till en precisering av vilket kunnande som kan utvecklas i tidig fysikundervisning.Stockholm Teaching & Learning Studie

Formation of three-dimensional tubular endothelial cell networks under defined serum-free cell culture conditions in human collagen hydrogels

Abstract Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering of constructs with clinically relevant size as nourishment of cells is challenged by the diffusion limit. In vitro generation of 3D networks is often achieved under conditions using serum containing cell culture medium and/or animal derived matrices. Here, 3D endothelial cell networks were generated by using human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) employing human collagen I as hydrogel and decellularized porcine small intestinal submucosa as starter matrix. Matrigel/rat tail collagen I hydrogel was used as control. Resulting constructs were cultivated either in serum-free medium or in endothelial growth medium-2 serving as control. Endothelial cell networks were quantified, tested for lumen formation, and interaction of HUVECs and hASCs. Tube diameter was slightly larger in constructs containing human collagen I compared to Matrigel/rat tail collagen I constructs under serum-free conditions. All other network parameters were mostly similar. Thereby, the feasibility of generating 3D endothelial cell networks under serum-free culture conditions in human collagen I as hydrogel was demonstrated. In summary, the presented achievements pave the way for the generation of clinical applicable constructs