Cartilage tissue engineering: controlled release of growth factors. Effects of GDF-5, sexual steroid hormons and oxygen

URN to cite this document:: urn:nbn:de:bvb:355-opus-7179
DOI to cite this document:: 10.5283/epub.10510

Appel, Bernhard

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Date of publication of this fulltext: 11 Apr 2007 08:12

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Item type:	Thesis of the University of Regensburg (PhD)
Open Access Type:	Primary Publication
Date:	10 April 2007
Referee:	Achim (Prof. Dr.) Göpferich
Date of exam:	20 September 2006
Institutions:	Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical Technology (Prof. Göpferich)
Keywords:	Tissue Engineering , Wachstumsfaktor , Dedifferenzierung , Knorpelzelle , Knorpel , Kontrollierte Wirkstofffreisetzung , Knorpel , Controlled Release , tissue engineering , cartilage , dedifferentation , controlled release , GDF-5
Dewey Decimal Classification:	500 Science > 540 Chemistry & allied sciences
Status:	Published
Refereed:	Yes, this version has been refereed
Created at the University of Regensburg:	Yes
Item ID:	10510

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Abstract (English)

Abstract (English)

Articular cartilage is likely a major candidate to be successfully regenerated by tissue engineering methods; first commercial products have already reached the market. However, to fully regenerate articular cartilage tissue, further optimization appears mandatory with regard to the application of morphogens, the maintenance of chondrocyte phenotype by applied morphogens and improved culture conditions, and the composition of engineered cartilage constructs.
In this thesis, it was demonstrated that cartilage-effective proteins such as insulin can be released from cylindrical lipid matrices, preserving the bioactivity of incorporated and released proteins. In addition, it was demonstrated that the employed cartilage engineering in vitro assay facilitates the testing of controlled release devices with regard to their biological efficacy in a complex 3-D system without the need for the expense of laboratory animals. As a second controlled release device the suitability of solid lipid templated macroporous scaffolds as protein release system and at the same time as artificial cell carrier was proven. The embedded insulin was released from those smart scaffolds over an extended period of time under retention of its bioactivity facilitating the generation of cartilaginous constructs.
In the area of morphogens, experiments with GDF-5 alone and in combination with insulin on expanded chondrocytes resulted in a clear shift of the cells towards the cartilaginous phenotype. Especially in combination with insulin, GDF-5 appeared to enable the redifferentiation of expanded chondrocytes and the concurrent generation of relevant cartilaginous constructs exhibiting substantial fractions of the extracellular matrix components glycosaminoglycans and collagen. With regard to the synergistic effects of GDF-5 and insulin observed, further research is required to elucidate the mechanism on the molecular level. The fact that GDF-5 was demonstrated to decisively modulate the response to another cartilage-effective protein contributes to the emerging picture of the role GDF-5 apparently plays in chondrogenesis and cartilage physiology.
Further experiments were conducted investigating the effects of sexual steroid hormones on tissue engineered cartilage. In contrast to the previous chapters, the effects of testosterone, estrogen and dehydroepiandrosterone on extracellular matrix development and increase of wet weight of cartilaginous constructs were generally small and remained distinctly behind the anabolic efficacy of protein factors like insulin and GDF-5. From these preliminary experiments, no definite recommendation could be made for the application of steroid hormones in in-vitro cartilage engineering. Thus, further research needs to be conducted to get a more comprehensive view of the potential of steroid hormones in growing engineered tissues.
Despite the oxygen partial pressure of 10% and lower in native articular cartilage, the majority of in vitro investigations including cartilage engineering approaches are conducted using an oxygen concentration of 21%. As a third aspect of this thesis, the effects of differential oxygen partial pressure (5% and 21% O2) on cartilaginous constructs were investigated. Taking together both the controversial results from many studies in the literature and the results obtained here, still specific standard oxygen conditions for cartilage engineering cannot be defined. Nevertheless, in our cell culture model it could be demonstrated that oxygen can be utilized as a supporting tool which modulates the response to an applied growth factor.

Translation of the abstract (German)

Artikulärer Knorpel ist sicher einer der wichtigsten Kandidaten für die erfolgreiche Wiederherstellung von Geweben durch Tissue Engineering. Erste kommerzielle Produkte haben ja auch schon die Marktreife erreicht. Um jedoch eine vollständige Regeneration artikulären Knorpelgewebes zu erreichen sind noch weitere Optimisierung notwendig. Hierbei wurde die Aufmerksamkeit auf die gezielte Applikation von Faktoren über einen verlängerten Zeitraum gelegt. Bei der Auswahl der Faktoren wurde der Erhalt des Phänotyps der Knorpelzellen durch die zugegebenen Faktoren besonders berücksichtigt. Weiterhin wurden verbesserte Kulturbedingungen und die Zusammensetzung der gezüchteten Gewebestücke untersucht.
In dieser Doktorarbeit konnte gezeigt werden, dass Knorpel-aktive Faktoren wie z.B. Insulin aus zylinderförmigen Lipidkörpern unter Erhalt der Bioaktivität des eingebetteten Proteins freigesetzt werden können. Zusätzlich konnte gezeigt werden, dass die 3D Knorpelzellkultur ein geeigneter in-vitro Versuchsansatz auf Bioaktivität für komplexe Protein-Freigabeformen ist. Dieser in-vitro Assay zeigt sich daher auch geeignet, Tierversuche einzusparen.
Aus dem Bereich möglicher Wachstumsfaktoren erwies sich GDF-5 allein oder in der Kombination mit Insulin gegeben als Redifferentierungsagens für vorkultivierte und expandierte Chondrozyten. Die dabei beobachteten synergistischen Effekte von GDF-5 und Insulin werden noch weitere Versuche notwendig machen, um ihr molekulares Zusammenspiel aufzuklären.
Weitere Experimente beschäftigten sich mit den Effekten von Sexualhormonen auf gezüchtetem Knorpelgewebe. Im Gegensatz zu den vorherigen Kapiteln zeigten Testosteron und Dehydroepiandrosteron nur kleine Effekte auf die Entwicklung der Extrazellularmatrix und der Steigerung des Nassgewichts.
Weitere Experimente wurden unter verringertem Sauerstoffpartialdruck durchgeführt. In der Mehrzahl aller Kulturbedingungen wird Sauerstoff mit 21% Partialdruck verwendet. Im natürlichen Knorpelgewebe sind jedoch Partialdrücke von 10% oder weniger zu finden. In diesen Versuchen konnte gezeigt werden, dass Sauerstoff ein interessantes und wirksames Werkzeug in der Knorpelneuzüchtung sein kann.

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