Impact of surface topographies on osteoclast resorption

Throughout our lifetime, bone is continuously removed and new bone is deposited in the process called "bone remodelling". This has two purposes, firstly, to maintain the structural integrity of the skeleton, and secondly, to fulfil its functional adaptation to the requirements of an individual. Two cell populations are responsible for this process: the osteoclasts, which remove old bone, and the osteoblasts, which deposit new bone. In order to understand bone remodelling, it is helpful to understand the processes of bone resorption separately from that of bone formation. In previous work, we have investigated the process of tissue formation by osteoblasts in vitro in three-dimensional scaffolds with controlled architecture. We have shown that osteoblasts respond to their local geometrical environment without any external biochemical signalling, following rules of curvature driven growth determined by surface tension. This project extends and complements our previous work on osteoblasts by looking at the other side of the bone-remodelling process, to osteoclast, with respect to environmental geometry. As osteoblasts respond to their local geometrical environment this immediately suggests that osteoclasts may also do so, which would have fundamental consequences on the understanding of targeted bone remodelling. The aim of this project is to answer following questions: Do osteoclasts recognize topographical features? Do the cells actively react to it? How does surface topography in the form of microcracks and resorption lacunae influence osteoclast behaviour? Does the filling of resorption lacunae with new bone tissue depend on curvature? To answer these questions we are going to use osteoclasts cultured in vitro on bone slices, which enables us to decouple the influence of two key players: biochemistry and geometry. We are interested in the impact of local geometries (microcracks and resorption lacunae) upon the osteoclast resorption behaviour. Firstly, by using bone slices with introduced microcracks we want to investigate the role of microcracks on the initiation of osteoclastic bone resorption. Since it is speculated, that microcracks, which arise during normal activities, are initiation sites for bone resorption, thus causing site specific remodelling. Secondly, we want to determine how the resorption activity of single osteoclasts is guided by existing resorption lacunae. Since one patho-mechanism for osteoporosis is increased bone resorption, it is important to understand if osteoclasts recognize the existence of lacunae and adapt their resorption position, which may get important for (thin) trabeculae. Then, osteoclast resorption behaviour will be described in relation to their topographical environment in a time dependent manner and the kinetics of the development of marker proteins will be displayed. Thirdly, new tissue formation in lacunae will be studied. This part will be realized by isolated primary osteoblasts and analyzed with respect to local curvature of lacunae. Furthermore, experimental data will then be introduced into theoretical models applied to bone remodelling algorithms. The combination of in vitro and in silico data can help to illuminate what is going on in vivo. This project will investigate the contribution of geometric coupling osteoclastic bone resorption to topography. This has important consequences in understanding the complex process of bone resorption, and will perhaps highlight a new mechanism for bone remodelling.

Bone is a living tissue which is continuously renewed through the process of remodeling. This remodeling is crucial for maintaining skeletal structure and function and the adaptation of the skeleton to specific mechanical needs. Osteoclasts play a crucial role in this process. The main physiological function of these osteoclasts in vivo is the resorption of bone matrix, which precedes the formation of new bone. We investigated osteoclast to find out their motivation and mechanisms of their action. Even normal physical activities during life cause the rise of microcracks in our skeleton, which then may reduce bone strength. Due to maintenance of architectural stability of the bone, these microcracks need to be removed, which is done by targeted bone resorption realized by the osteoclasts. We found out that osteoclasts per se do own toposensitivity, which would be a prerequisite to recognize microcracks and o react with targeted resorption of that area to repair this damage. Thus, in case of targeted bone resorption to remove microcracks, osteoclasts are guided by other cells in bone (e.g. osteoblasts or osteocytes). Furthermore, osteoclasts show a different resorption behavior on two important mineralized materials: bone and dentin, since dentin is widely used as a mineralised substrate for in vitro investigations of osteoclast resorption and data obtained from such experiments are often extrapolated to osteoclastic behaviour on bone. Dentin promotes the generation of resorbing osteoclasts, but once resorption has started, it proceeds independently of the material in a similar way on bone and dentin. This demonstrates that dentin is a suitable model substrate to study osteoclastic resorption in vitro and allows extrapolation of the data to osteoclastic behaviour on bone, as long as cell-material-dependent interactions and osteoclast generation processes are not part of the analyses. These resorption traces of osteoclasts appear either as round/elliptic traces, so-called pits or as longitudinal traces, so-called trails. By confocal laser scanning microscopy we elucidated the mechanism how, such a trail is formed by an osteoclast: the cell attaches with one side of the resorption organelle outside the trails and with the other side inside the trail, thus sealing that narrow part to be resorbed next. This 3D-configuration of the resorption organelle just allows vertical resorption layer by layer from the surface to a depth in combination with horizontal cell movement. Thus, trails are not just traces of a simple horizontal translation of osteoclasts during resorption in a worm-like style.In this resorption process the serine-threonin kinase MK2 (MAPK-activated protein