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

• Applicant: Dr. Thomas Hanke
• Subject Area: Biomaterials
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269395506

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.

• DFG Programme: Research Grants
• Cooperation Partners: Professor Dr.-Ing. Chokri Cherif; Professor Dr. Michael Gelinsky; Professorin Dr. Sabine Wenisch