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.
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