Quantification of release and activity of the signaling factors BMP-2, TGF-ß3, Smad8 L+MH2

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Project 6 is concerned with the demonstration of a successful spatial gradation of the growth factors previously incorporated into the implant in order to induce cell differentiation and of the temporal control of their release. Moreover, the preservation of protein activity during processing and release is going to be investigated. For this purpose, suitable methods are established that allow the determination of amount and activity of the growth factors in relevant media. For a rapid and cost-efficient semiquantitative determination of the amount of released proteins as well as for a control for their molecular integrity (in particular, with regard to degradation), protein electrophoresis (denaturing and also native) in combination with Western Blots is employed. In particular with regard to visualization investigations with fluorescence detection will be carried out. Advanced quantitative analysis of the amount of released protein will be performed by enzyme-linked immunosorbent assay (ELISA) and Selected Reaction Monitoring which is going to be established as an alternative quantification method. Reporter tests (promotor elements of target genes of the TGF-β/BMP signaling pathway) and differentation analysis (gene expression) of human mesenchymal stem cells allow conclusions on the biological activity of the proteins. Further, it will be investigated how far storage and transport or the use of additives in release media may influence results and whether there are cross-reactions between the target proteins. Experimental setups and suitable media for release studies on the graded implants and their components will be developed and optimized with respect to their suitability regarding quality control as well as a later performance in vivo. In addition to the use of liquid release media hydrogels are going to be used as acceptor systems for protein release. In this context, the use of microdialysis and/or open flow microperfusion for the collection of released growth factors is going to be evaluated and special release cells will be designed. These methods will be used to investigate the release of growth factors from graded implants and their components obtained from P5 and the activity of the released proteins. Based on the results from these experiments, the release systems will be further developed. Together with P5 the potential of using specifically prepared aggregates of BMP-2 as a depot on the implants will be evaluated. In the second funding period, there will be a special focus on the establishment of an in vitro-in vivo-correlation (IVIVC). Moreover, in a combined effort with the other projects, P6 will elaborate concepts for the production of a sterile implant.