Monitoring of osteoclast formation and activity on different scaffolds for bone replacement by means of microscopy

本研究的目的是研究支架结构对体外破骨细胞形成的影响，以及由此产生的破骨细胞的性质。显微镜主要用于研究。同时，将通过生物化学和分子生物学试验对这一问题进行研究。它必须证明，在多大程度上体外监测破骨细胞的发生和各种骨替代材料支架上的活性是有意义的。该项目的中心是对各种建筑的各种支架进行体外研究。这项研究的目的是评估支架支持破骨细胞生成的能力，以及它们基于破骨细胞的可吸收性。将研究三种类型的支架。将对三种类型的支架进行研究，首先是静电纺纺织品聚己内酯支架，其次是绘制的磷酸钙支架，第三是含有磷酸钙颗粒的大孔胶原二氧化硅支架。这些支架已经由提议者进行调查，或者将由合作伙伴提供。在细胞培养实验中，首先在研究的支架上产生一系列不同调节的破骨细胞，其次分析各种支架结构对特定破骨细胞群体的影响。为了实现不同的调节，将使用形成PBMC（外周血单个核细胞）的不同分数的破骨细胞以及两种不同的细胞来源（PBMC和RAW 264.7）和包括hMSC/成骨细胞的共培养。刺激细胞因子M-CSF和RANK-L分别添加不同程度或完全不添加。目的是检查不同的调节在多大程度上改变了破骨细胞对支架的反应，以及最低限度调节的破骨细胞群体是否使物理支架参数（如结构）对破骨细胞形成和活性的作用可见。在这里，通过直接添加高效细胞因子，这些参数的叠加性较弱。为此，贴壁细胞的数量和分布、它们的长入、肌动蛋白细胞骨架的组织、窝状体模式、局灶黏附、细胞形态、细胞间的直接相互作用将通过扫描电镜进行研究，并在特定的荧光标记后，通过cLSM进行研究。融合的发生、囊体分布的动态、肌动蛋白环、封闭区和局灶粘连将通过延时clsm可视化。这个问题，在多大程度上支架作用于破骨细胞导致细胞的变化和随后形成的亚群，也将得到回答。为此，将确定破骨细胞群体的特征差异，例如关于酸化和组织蛋白酶K和MMP活性之间的比例。

Goal of the proposal is to investigate the influence of scaffold architecture on the in vitro-osteoclastogenesis as well as the nature of the resulting osteoclasts. Mainly, microscopy is designated for investigation. At the same time, this issue will be investigated by means of biochemical and molecular biological tests. It has to be shown, to what extend an in vitro-monitoring of osteoclastogenesis and activity on various scaffolds from bone replacement material can be operated meaningfully. The in vitro-investigation of a variety of scaffolds of various architectures is in the center of this project. The study will be performed in order to evaluate the ability of the scaffolds to support osteoclastogenesis as well as their osteoclast-based resorbability. Three types of scaffolds will be investigated. The investigation of three scaffold types will be conducted, at first an electrospun textile poly-caprolacton scaffold, at second a plotted calciumphosphate scaffold, and at third a macroporous collagen silica scaffold containing calcium phosphate particles. These scaffolds are already under investigation by the proposer or will be provided by co-operation partners. In the cell culture experiments, firstly a series of differently modulated osteoclasts will be generated on the scaffolds under investigation, and secondly the impact of the various scaffold architectures on the particular osteoclast populations will be analyzed. In order to achieve the different modulations, different fractions of osteoclast forming PBMC (peripheral blood mononuclear cells) as well as two different cell sources (PBMC and RAW 264.7) and a co-culture including hMSC/osteoblasts will be used. The stimulating cytokines M-CSF and RANK-L are added in different extend or their addition is completely spared, respectively. The aim is to examine, to what extend the different modulations change the response of the osteoclasts to the scaffolds, as well as, whether minimally modulated osteoclast populations made visible the action of physical scaffold parameters, like architecture, on osteoclast formation and activity. Here, these parameters are less strongly superimposed by the direct addition of high-potency cytokines. For that purpose, numbers and distribution of the adherent cells, their ingrowth, organization of the actin cytoskeleton, podosom pattern, focal adhesions, cell morphology, direct cell-cell interactions will be investigated by SEM and, after specific fluorescence labelling, by cLSM. The fusion occurence, dynamics of podosom distribution, actin ring, sealed zone, and focal adhesions will be visualised by time lapse-cLSM. The question, how far the scaffold action on the osteoclasts causes a change of the cells and a subsequent formation of subpopulations, will be answered, too. For that purpose, characteristic differences of the osteoclast populations, e.g. concerning the acidification and the ratio between cathepsin K and MMP activities will be identified.