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

项目6涉及证明先前掺入植入物中以诱导细胞分化的生长因子的成功空间分级及其释放的时间控制。此外，将研究在加工和释放过程中蛋白质活性的保存。为此，建立了合适的方法来测定相关培养基中生长因子的含量和活性。为了快速和成本有效地半定量测定释放的蛋白质的量以及控制其分子完整性（特别是关于降解），采用蛋白质电泳（变性和天然）与蛋白质印迹的组合。特别是关于可视化调查与荧光检测将进行。将通过酶联免疫吸附试验（ELISA）和选择反应监测（将作为替代定量方法建立）对释放的蛋白量进行高级定量分析。报告基因测试（TGF-β/BMP信号通路靶基因的启动子元件）和人间充质干细胞的分化分析（基因表达）可得出蛋白质生物活性的结论。此外，还将研究储存和运输或释放介质中添加剂的使用对结果的影响程度以及靶蛋白之间是否存在交叉反应。将开发并优化用于分级植入物及其组件释放研究的实验装置和合适介质，以确定其在质量控制方面的适用性以及后期体内性能。除了使用液体释放介质之外，水凝胶还将用作蛋白质释放的受体系统。在这种情况下，使用微透析和/或开放流微灌注的释放生长因子的收集将进行评估和特殊的释放细胞将被设计。这些方法将用于研究生长因子从分级植入物及其从P5获得的组分中的释放以及释放的蛋白质的活性。基于这些实验的结果，将进一步开发释放系统。将与P5一起评价使用专门制备的BMP-2聚集体作为植入物贮库的潜力。在第二个供资期，将特别关注建立体外-体内相关性（IVIVC）。此外，在与其他项目的合作中，P6将详细阐述无菌植入物生产的概念。

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.