Introduction 6 Fig. 2: Schematic illustration of bone hierarchy from macroscopic to nanoscale level 35 . 22.214.171.124. Mechanical characteristics of bone Besides dental enamel, bone represents one of the stiffest and most dense tissues within the human body. Healthy bone has a mean mass density of 2-3 g/cm 3 which depends strongly on cell, blood vessel and nerve content 23 . The two main types of adult bone – cortical and cancellous bone – can easily be distinguished by their porosity and density 36,37 . Classical mechanical characterisation is normally ascertained with macroscopic pressure test. Lewis and Nyman 38 collected a brought overview of elastic modulus and hardness, ranging from 15 GPa to 32 GPa for the Young’s modulus of human trabecular bone. The wide variability of force values is caused by the dynamic turnover of long bones, which ends up in a trabecular orientation along the lines of force within the bone. Another critical step is the preparation before force measurement, such as fixation, boiling for protein denaturation or simple drying. All these mechanical properties rely on the nanoscopic structure and chemical environment within the trabecular bone.
Introduction 7 126.96.36.199. Biophysical characteristics of bone In the last years, a lot of effort was put into analysing the bone structures below the micrometre scale. In this context, the combination of atomic force microscopy and nanoindentation offers a potent technique, which allows post-hoc analysis and a more precise insight into the bone ultrastructure 39 . Recent studies could show that the molecular level has a great influence onto the overall stiffness of bone 40 , which were previously predicted by mathematical models 41 . So called ‘sacrificial bonds’ within collagen molecules showed a distinct calcium ion dependency and supposed to be the main factor for energy dissipation on the nanoscale of bone and consequently for the whole macroscopic bone. This calcium sensitivity could also be proven for other ECM proteins of bone, like osteopontin 42 . Continuative studies encircled the location of these sacrificial bonds between mineralized collagen fibrils 43 . Furthermore, nanoscale topography and therewith ECM composition and stiffness seems to have a great influence on gene expression, cell morphology, integrin mediated signalling and migration 44-47 . Besides the cellular effects, mineralised constituents like bone crystals reveal a dissolve resistance within the scale of several hundred nanometre 48,49 . This shows the tremendous influence of nanoscale elements on the cellular and mechanical function of bone. 1.2.3. Bone biology The dynamic response to mechanical loading is a close interplay of cells and their chemical compounds. Three completely differentiated cell types, namely matrix producing osteoblast, matrix resorbing osteoclasts and orchestrating osteocytes (over 90% of bone cells), provide the base for living bones 50 . These cells rely extensively on cell-matrix and cell-cell contacts, which are mediated by transmembranous proteins like integrins, connexions, cadherins and specific surface receptors for cytokines, hormones and growth factors. Due the difficulty to obtain osteocytes, there are only few studies which tried to evaluate the physiological function of these cells. Up to now, it is clear whether the mechanosensing and therewith cell-cell signalling of osteocytes is procured by fluid flow between dendritic connected cells and thereafter coordinating further bone remodelling cascades 51-55 . This dynamic turnover is provided by osteoblast and osteocytes, which have no mechanosensing function, but are responsible for either matrix deposition or degradation by osteoblast mediated
Supplementary data 57 Supp.Fig. 1:
Appendix 59 vacuum pump for cell cu
Abbreviations 61 List of abbreviati
List of figures 63 List of figures
List of figures 65 Fig. 18: Compari
Acknowledgments 67 Acknowledgments