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

该提案的目的是研究支架结构对体外破骨细胞生成的影响以及由此产生的破骨细胞的性质。显微镜主要是用来进行研究的。同时，将通过生化和分子生物学试验对这一问题进行调查。必须证明，体外监测骨替代材料在各种支架上的破骨细胞生成和活性的程度可以有意义地进行。各种建筑的各种支架的体外研究是本项目的中心。这项研究将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吸收能力。将对三种类型的支架进行调查。将进行三种支架类型的研究，第一种是电纺纺织聚己内酯支架，第二种是带图案的磷酸钙支架，第三种是含有磷酸钙颗粒的大孔胶原二氧化硅支架。这些脚手架已由建议者进行调查，或将由合作伙伴提供。在细胞培养实验中，首先在被研究的支架上产生一系列不同调制的破骨细胞，然后分析不同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，将使用不同比例的破骨细胞形成外周血单核细胞，以及两种不同的细胞来源(PBMC和RAW 264.7)以及包括hMSC/成骨细胞的共培养。刺激性细胞因子M-CSF和RANK-L分别不同程度加入或完全不加入。其目的是研究不同的调节在多大程度上改变破骨细胞对支架的反应，以及最小限度地调节破骨细胞种群是否使物理支架参数(如结构)对破骨细胞的形成和活性的作用可见。在这里，这些参数不那么强烈地叠加在直接添加的高效力细胞因子上。为此，贴附细胞的数量和分布、它们的生长、肌动蛋白细胞骨架的组织、足形态、焦点粘连、细胞形态、细胞与细胞的直接相互作用将通过扫描电子显微镜和特定荧光标记后的激光共聚焦显微镜进行研究。时间推移-CLSM将显示融合的发生、足体分布的动态、肌动蛋白环、密封区和局灶性粘连。支架对破骨细胞的作用在多大程度上导致了细胞的变化和随后的亚群的形成，这个问题也将得到回答。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和基质金属蛋白酶活性之间的比率。

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.