Publikationsserver der Universitätsbibliothek Marburg

Titel:Funktionalisierung von PLLA-Nanofasern mittels integrinbindender RGD-Sequenzen im Rahmen des Tissue Engineering
Autor:Bockelmann, Sarah Kristin
Weitere Beteiligte: Schofer, Markus (Prof. Dr.)
Veröffentlicht:2017
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0156
DOI: https://doi.org/10.17192/z2017.0156
URN: urn:nbn:de:hebis:04-z2017-01568
DDC: Medizin, Gesundheit
Titel(trans.):RGD-functionalisation of PLLA-Nanofibers for Tissue Engineering
Publikationsdatum:2017-03-06
Lizenz:https://creativecommons.org/licenses/by-nc-sa/4.0

Dokument

Schlagwörter:
PLLA Tissue Engineering, Tissue Engineering, Regenerative Medizin

Zusammenfassung:
Im klinischen Alltag stellt die Versorgung von Knochendefekten bei nur geringer Verfügbarkeit des körpereigenen Materials eine große Herausforderung dar. Das Tissue Engineering bietet eine geeignete Methode zur Produktion von Knochenersatzmaterial. Hierbei wird körpereigenenes Material unter Laborbedingungen (in vitro) gezüchtet und dem Organismus replantiert. Vorausgehende Arbeiten konnten in Elektrospinnprozessen biokompatible Scaffolds aus Kollagenfasern herstellen, welche ein gutes Angehen und Differenzieren von humanen mesenchymalen Stammzellen (hMSC) ermöglichten. Der Nachteil dieser Kollagen-Scaffolds lag jedoch in der geringen mechanischen Stabilität. In den Forschungsergebnissen zeigte sich, dass die osteoinduktive Wirkung von Kollagen auf eine Aminosäuresequenz Arginin-Glycin-Asparaginacid (RGD) zurückzuführen ist. Ziel dieser Arbeit war es die osteoinduktive Wirkung des Kollagens mit Hilfe von RGD-Sequenzen mit den mechanisch stabileren Poly(L-lactid)Nanofasern (PLLA) zu verbinden. Hierzu erfolgte die Zellkultivierung von hMSC über 22 Tage unter osteoinduktiven Bedingungen und Wachstumsbedingungen auf PLLANanofaserscaffolds. Es wurden quantitative Bestimmungen der hMSCDifferenzierungsmarker Osteocalcin, Kollagen und Alkalischer Phosphatase über RealtimePCR und Fluoreszensmikroskopie durchgeführt. Es wurden eine lineare und eine zyklische RGD-Sequenz miteinander verglichen sowie unterschiedliche Einbringungsverfahren der Sequenzen in die Faser untersucht. Die RGD-Sequenzen wurden mittels Suspension und mittels Emulsion in die Faser eingebracht sowie nach Plasmabehandlung des PLLAScaffolds und Kopplungen von EDC (1-Ethyl-3(3dimethylaminopropyl) carbodiimid) und NHS (N-Hydroxysulfosuccinimid) an die Oberfläche der Faser gekoppelt. Die strukturellen Veränderungen auf die Fasereigenschaften der unterschiedlichen Behandlungsmethoden wurden mittels Elektronenmikroskopie (Beurteilung Faserdurchmesser und Kontaktwinkel) und Zugdehnungsmessgerät (Beurteilung der Reißfestigkeit) beurteilt. Die vorliegende Arbeit zeigt, dass die zyklisch angeordnete RGD-Sequenz eine signifikant höhere Zelldifferenzierung (p<0,016) gegenüber der linearen Variante bei jedoch gleicher Zellzahl aufweist. Die mittels Emulsion eingebrachten Sequenzen boten keinen signifikanten Vorteil gegenüber der mittels Suspension eingebrachten Fasern (p>0,05). Das Kopplungsverfahren mittels Plasmabehandlung erbrachte eine Tendenz zur vermehrten Osteoinduktivität (Steigerung der Genprodukte aus der RealtimePCR der Alkalischen Phosphatase, Osteocalcin und Kollagen) gegenüber der unbehandelten PLLA-Faser. Die Fasereigenschaften änderten sich durch die vorgenommenen Einbringungs- oder Kopplungsverfahren nicht signifikant. Diese Arbeit konnte zeigen, dass alle aufgeführten Verfahren für die Einbringung der osteoinduktiven RGD-Sequenzen geeignet sind. Die zyklische RGD Variante zeigt sich induktiver als die lineare und steigert die Zelldifferenzierung insbesondere als Oberflächenkontakt nach Plasmabehandlung der Faser. Ein Nachweisverfahren welches die Quantität der RGD-Sequenzen an der Faseroberfläche darstellen kann, um die osteoinduktive Wirkung zu optimieren und es zu einem möglichen Träger für das Tissue Engineering zu machen, sollte entwickelt werden.

Summary:
In clinical practice the adressing of bone defects with less availability of the autologous material are major challenges. The Tissue Engineering offers a sufficient method in order to produce bone substitutes. In this case autologous material from the patient is removed, cultured in laboratory conditions (in vitro) and replanted to the human organism. In previous studies biocompatible scaffolds made of collagen fibre during electrospinning processes were developed and enabled a good approach and differentiation of human mesenchymale stem cells (hMSC). The disadvantage of those Collagen-Scaffolds is a lesser mechanical stability. The results of current research showed that the osteoinductive effect of collagen is due to an amino acid sequence Arginin-Glycin-Asparaginsäure (RGD). The aim of this study was to relate the osteoinductive effect of the collagen with the influence of RGD-sequences on mechanical more stable Poly(lactid)Nanofibres (PLLA). For this, the cell culturing of hMSCs was carried out over a period of 22 days under osteoinductive terms and growth conditions. In this study quantitative determinations of hMSC differentiation markers such as osteocalcin, collagen and alkaline phosphatase over real time PCR and fluorescence microscopy were performed. We compared a linear and cyclic RGD-sequence with each other and investigated various methods of introducing sequences into the fiber. The RGD sequences were introduced by suspension and emulsion into the fiber as well as after plasma treatment of the PLLA scaffolds and linking of EDC (1-ethyl-3 (3dimethylaminopropyl) carbodiimide) and NHS (Nhydroxysulfosuccinimide) to the surface of the fiber. The structural changes within the fiber characteristics of different procedures were evaluated by electron microscopy (assessment of the fiber diameter and contact angle) and by a tensile testing machine (assessing the tear strength). In our study we were able to display that the cyclically arranged RGD sequence has significant higher cell differentiation (p <0.016) compared with the linear variation yet same cell number. The sequences which were introduced by emulsion offered no significant advantage towards the fibers introduced by suspension (p> 0.05). The coupling method using plasma treatment resulted in increased osteoinductivity (increase of gene products from the real time PCR of alkaline phosphatase, osteocalcin and collagen) compared to the PLLA fiber. Moreover, there were no significant changes within the fiber characteristics by different incorporation and coupling methods. Taking all aspects into consideration we could show in this study that all performed methods are suitable for the incorporation of osteoinductive RGD sequences. The cyclic variation of RGD is more inductive than the linear one and increases cell differentiation in particular as a surface contact by plasma treatment of the fiber. However, it is necessary to find a verification procedure which is able to measure the quantity of the RGD sequences in the fiber surface. So the osteoinductive effect will be optimized and the scaffolds will be a possible medium for tissue engineering.

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