Development of collagen/hydroxyapatite composites with angiogenic properties

Die beiden Schlüsselkomponenten für die Entwicklung von Knochengewebe sind osteogene Stamm- oder Vorläuferzellen und ein dreidimensionales Gerüstmaterial. In der vorliegenden Arbeit wurden zum einen das Gerüst und zum anderen spezifische Stammzellen hinsichtlich ihrer angiogenen Eigenschaften untersucht. Da die beiden Prozesse Angiogenese und Knochenbildung miteinander gekoppelt sind, ist es für gewebezüchtete Konstrukte von entscheidender Bedeutung, dass sie eine Gefäßdurchblutung aus dem umgebenden Gewebe der Implantationsstelle ermöglichen und induzieren. Auf der Grundlage der experimentellen Ergebnisse lassen sich die wichtigsten Erkenntnisse der vorliegenden Arbeit wie folgt zusammenfassen: 1) Das Ziel der ersten Arbeit war es, die angiogenen Eigenschaften von Kollagen/Hydroxylapatit-Gerüsten zu verbessern. Um dieses Ziel zu erreichen, wurde ein VEGF-Mimicry Peptid an Hydroxylapatitpartikel gekoppelt, bevor die Gerüste hergestellt wurden. Es konnte bestätigt werden, dass das VEGF-mimetische Peptid QK, das mit einem Heptaglutamat-Rest (E7-Tag) modifiziert wurde, eine deutlich höhere Bindungsaffinität zu Hydroxylapatit-Partikeln aufweist als das unmodifizierte QK-Peptid. Die Quantifizierung von Tube Formation Assays ergab eine signifikant höhere Anzahl an Tubes, die von HUVECs in Gegenwart von E7-QK-Peptiden gebildet wurden, als in Gegenwart des unmodifizierten QK-Peptids. Darüber hinaus wurde gezeigt, dass E7-QK-Peptide signifikant pro-angiogene Gene in HUVECs induzieren, verglichen mit den Expressionsniveaus, die in der Kontrollgruppe, die mit unmodifizierten Peptiden kultiviert wurde, festgestellt wurden, und verglichen mit der negativen Kontrollgruppe, die ohne Peptide oder ohne Wachstumsfaktorzusätze kultiviert wurde. In der 3D-Zellkultur zeigten die durchgeführten Sprouting Assays eine signifikant höhere Sprossenbildung von HUVEC-Sphäroiden in Kollagen/Hydroxylapatit-Verbundgerüsten, die die E7-QK-Peptide enthielten, im Vergleich zur detektierten Sprossenbildung in Gerüsten mit unmodifizierten QK-Peptiden. Wir kamen zu dem Schluss, dass E7-QK-Peptide ausgezeichnete angiogene Wirkungen aufweisen und sich sehr gut für die Herstellung von Kollagen/Hydroxylapatit-Verbundwerkstoffen mit angiogenen Eigenschaften eignen. 2) Das Ziel der zweiten Arbeit war die Analyse der angiogenen Eigenschaften von Sekretomen, die aus vorkonditionierten iMSCs gewonnen wurden. Nachdem bestätigt wurde, dass iMSCs auf die IFN-γ-Stimulation unter normoxischen und hypoxischen Bedingungen mit einer Hochregulierung der Expression des HLA-II-Moleküls reagieren, wurden die angiogenen Effekte der isolierten iMSC-Sekretome auf HUVECs analysiert. Tube Formation und Wound Healing Assays zeigten in der 2D-Kultur, dass HUVECs, die mit dem Sekretom von iMSCs behandelt wurden, die unter Hypoxie mit IFN-γ konditioniert wurden, eine signifikant höhere Anzahl von Tubes bildeten und die Wunde schneller schlossen als die unbehandelte HUVEC-Gruppe. HUVECs, die in Gegenwart von Sekretomen aus vorkonditionierten iMSCs (mit IFN-γ und Hypoxie) kultiviert wurden, zeigten im Vergleich zu allen anderen Gruppen eine Hochregulierung von sieben pro-angiogenen Genen und eine Herunterregulierung von drei anti-angiogenen Faktoren. HUVEC-Sphäroide, die auf Kollagen/Hydroxylapatit-Scaffolds ausgesät wurden und in Gegenwart des Sekretoms von vorkonditionierten iMSCs (mit IFN-γ und Hypoxie) kultiviert wurden, zeigten eine signifikant höhere Sprossenbildung im Vergleich zu den Sprossen der unbehandelten Gruppe. Wir kamen zu dem Schluss, dass das Sekretom, das von IFN-γ-vorstimulierten iMSCs unter hypoxischen Bedingungen produziert wird, unter allen analysierten Bedingungen die stärkere angiogene Wirkung hervorruft. In einem nächsten Schritt sollte ein geeigneter Ansatz für die Konservierung und den Einbau der Sekretom-Komponenten in die Gerüste entwickelt werden, und beide in dieser Arbeit aufgezeigten Strategien müssen in vivo auf ihre bessere Funktionalität getestet werden

Idea Response and Adoption in Open Innovation Communities: The Signaling Role of Linguistic Style

Organizations are increasingly using open innovation communities to gain external ideas and foster user innovation. However, mass user generated content is making idea selection a tricky and time-consuming work. From the perspective of linguistic styles, this article explores the effect of writing style cues in the content of ideas (i.e., negative emotionality, self-interest oriented, cognitive oriented, future oriented) on idea response and adoption. 1,579 ideas collected from the Fantasy Westward Journey Online Ⅱ forum are used to extract writing style cues. The hypothesis tests, via logistic regression models, demonstrate that the linguistic styles of ideas can indeed function as a signaling role and deliver novel insights into the relationship between these linguistic cues and the likelihood of idea responses and adoption. Specifically, a community member’s use of self-interest oriented, cognitive oriented and future oriented writing styles has a positive effect on idea response; negative emotionality and cognitive oriented writing styles signal more possibility of idea adoption. This study offers theoretical implications by extending the innovation management research stream to the big data era and also provides managerial implications that can lead to more effective exploitation of open innovation communities

Retinoic Acid Plays Critical Roles in the Late Development of the Heart

Vitamin A, via its active metabolite retinoic acid (RA), actively participates in many biological processes including cardiogenesis. Yet RA was found to be highly teratogenic as both excess and deficiency of this critical morphogen result in congenital heart defects. Studies presented here aims to gain insight in mechanisms regulating RA metabolism during embryogenesis and enrich our knowledge of the critical roles of RA during late heart development. Previously, our lab reported that a short-chain dehydrogenase/reductase, i.e. DHRS3, is required for preventing excessive accumulation of RA and thus safeguarding mouse embryogenesis during mid-gestation. The current study expanded the investigation and studied the physiological importance of DHRS3 in RA metabolism at multiple embryonic stages. Consistent with the elevated RA synthesis and signaling at E14.5, genetic ablation of Dhrs3 results in an expansion of RA signaling at E10.5 and E12.5 globally; as well as in the fetal hearts at E10.5, 12.5 and 13.5. Dhrs3-null mutants display a spectrum of congenital defects, including defects in the heart, skeleton and cranial nerves, which collectively result in mid-gestational lethality in Dhrs3-/- embryos. Reduction of maternal intake of vitamin A successfully rescued the Dhrs3-/- fetuses and allows them to survive into full-size adults with normal growth rate. These data jointly demonstrated the indispensability of DHRS3 in reducing the accumulation of RA in various developmental stages and in the formation of fetal organs. With the advancement of knowledge in RA metabolism gained in the first part of this dissertation, the critical roles of RA in cardiogenesis was further explored in the current work. During late cardiogenic stages, the major source of RA is the epicardium. Epicardium contributes greatly to the formation of coronary vessels and the myocardium via 1) giving rise to migratory epicardial cells that differentiate into perivascular cells, and 2) secreting cardiogenic factors. This dissertation employed multiple in vivo and in vitro models to determine the influence of RA on epicardial behaviors and the subsequent epicardial-regulated cardiogenic events. In vitro studies demonstrated that inhibition of RA synthesis in epicardial cell disrupted cytoskeletal reorganization and preserved epithelial characteristics, which resulted in a reduction of migration of epicardial cells. On the contrary, addition of RAR agonist to activate RA signaling strongly induced remodeling of cytoskeleton represented by the formation of stress fibers as well as filopodia marked by polymerized F-actin. RA signaling also abolished the membranous distribution of epithelial markers and induces expression of numerous metalloproteases to pave the way for cell migration. Data from in vivo models showed consistent observation in the regulation of epicardial migration by RA: excess RA in Dhrs3-/- embryos enhances the intramyocardial invasion of epicardial cells whereas deficiency of RA largely retained epicardial cells in the intact epicardium on the surface of the myocardium. To further understand the molecular mechanisms underlying the regulation of epicardial EMT by RA, we employed transcriptomic analysis, which in combination with further molecular assays clearly demonstrated that RhoA pathway is activated by RA and proves to be critical for RA-induced morphological changes in epicardial cells. Furthermore, potentially as a consequence of altered epicardial behavior and functions in response to aberrant RA signaling, both deficiency and excess RA led to compromised coronary vessel formation and hypoplastic ventricular myocardium in vivo. Vascular hierarchy was severely impaired and density of intramyocardial vessels was drastically diminished by altered RA signaling. Lack of proper recruitment and differentiation of vascular smooth muscle cells further acerbated the malformations in coronary vessels in response to abnormal RA signaling. These observations provided novel evidence of the teratogenic nature of RA and collectively demonstrated that RA signaling is not only actively involved but also critically important in the late heart development, which may shed light on the discovery of methods in preventing congenital heart diseases as well as the identification of novel targets for treating cardiovascular diseases

Image Matching and Recognition Techniques for Mobile Multimedia Applications

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Pre-conditioning with IFN-γ and hypoxia enhances the angiogenic potential of iPSC-Derived MSC secretome

Induced pluripotent stem cell (iPSC) derived mesenchymal stem cells (iMSCs) represent a promising source of progenitor cells for approaches in the field of bone regeneration. Bone formation is a multi-step process in which osteogenesis and angiogenesis are both involved. Many reports show that the secretome of mesenchymal stromal stem cells (MSCs) influences the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair of the damaged area. However, the effects of iPSC-derived MSCs secretome on angiogenesis have seldom been investigated. In the present study, the angiogenic properties of IFN-γ pre-conditioned iMSC secretomes were analyzed. We detected a higher expression of the pro-angiogenic genes and proteins of iMSCs and their secretome under IFN-γ and hypoxic stimulation (IFN-H). Tube formation and wound healing assays revealed a higher angiogenic potential of HUVECs in the presence of IFN-γ conditioned iMSC secretome. Sprouting assays demonstrated that within Coll/HA scaffolds, HUVECs spheroids formed significantly more and longer sprouts in the presence of IFN-γ conditioned iMSC secretome. Through gene expression analyses, pro-angiogenic genes (FLT-1, KDR, MET, TIMP-1, HIF-1α, IL-8, and VCAM-1) in HUVECs showed a significant up-regulation and down-regulation of two anti-angiogenic genes (TIMP-4 and IGFBP-1) compared to the data obtained in the other groups. Our results demonstrate that the iMSC secretome, pre-conditioned under inflammatory and hypoxic conditions, induced the highest angiogenic properties of HUVECs. We conclude that pre-activated iMSCs enhance their efficacy and represent a suitable cell source for collagen/hydroxyapatite with angiogenic properties.S.W. was financed by the China Scholarship Council (CSC) (grant 201908080045)

Identification of long non-protein coding RNAs in chicken skeletal muscle using next generation sequencing

AbstractVertebrate genomes encode thousands of non-coding RNAs including short non-coding RNAs (such as microRNAs) and long non-coding RNAs (lncRNAs). Chicken (Gallus gallus) is an important model organism for developmental biology, and the recently assembled genome sequences for chicken will facilitate the understanding of the functional roles of non-coding RNA genes during development. The present study concerns the first systematic identification of lncRNAs using RNA-Seq to sample the transcriptome during chicken muscle development. A computational approach was used to identify 281 new intergenic lncRNAs in the chicken genome. Novel lncRNAs in general are less conserved than protein-coding genes and slightly more conserved than random non-coding sequences. The present study has provided an initial chicken lncRNA catalog and greatly increased the number of chicken ncRNAs in the non-protein coding RNA database. Furthermore, the computational pipeline presented in the current work will be useful for characterizing lncRNAs obtained from deep sequencing data