Investigations into mechanotransduction in connective tissue extracellular matrix (ECM) have demonstrated that collagen networks show cell-independent mechanosensitive behavior. It has been suggested that mechanical strain could lead to conformational changes in the molecular structure of collagen, thereby influencing the susceptibility to other molecules. In the process of normal aging, the collagen fibrils in cartilage undergo a non-enzymatic process known as glycation. It involves the accumulation of advanced glycation end products (AGEs) after exposure to sugars, resulting in the formation of cross-links between the collagen fibrils. This process is correlated with increased stiffness and brittleness of the cartilage, making it more prone to mechanical damage. The goal of this thesis was to assess whether mechanical compression has any effects on the formation of non-enzymatic cross-links during the aging of articular cartilage. Two different models have been developed to mimic aging knees that undergo static and dynamic compression. Healthy cartilage explants were exposed to L-threose sugar to induce artificial aging. During incubation, these explants were submitted to either static or dynamic unconfined compression. Treatment with static compression consisted of a 5, 10 or 15\% strain throughout the whole incubation period, using a custom-made bioreactor. Treatment with dynamic compression consisted of multiple loading cycles at a frequency of either 0.01 Hertz (Hz) or 1 Hz, using a Dynamic Mechanical Analyzer (DMA). We conducted cartilage surface color analyses, micro‐indentation tests, dynamic mechanical analyses and biochemical measurements of pentosidine cross‐links to assess the effects of advanced glycation cross‐linking under these different conditions. Dynamic compression at a frequency of 1 Hz was found to affect the formation of non-enzymatic cross-links. Biomechanical and biochemical data showed a similar trend, namely, the average values for equilibrium modulus, dynamic moduli, phase shifts and pentosidine per collagen level were noticeably higher (or lower in case of phase shift) for the 1 Hz treated samples compared to samples of other treatment groups. The results of these studies suggest that compression at the physiological frequency of walking does affect the formation of cross-links in the articular cartilage during aging. These findings contribute to a better understanding of the mechanochemistry of collagen fibrils, which is necessary to develop future strategies against cartilage aging and deterioration.

Knowledge of the influence of loading directions on trabecular bone remodeling in spine is of significant value in understanding the development of spine deformities and vertebral bone quality across different scales. Information on the constitution of a preferred trabecular orientation and mechanical properties of trabecular bone are important indicators in this respect. The current thesis aimed at exploring these aspects across multiple length scales in the spine. The thesis is divided in two parts. The influence of loadings less dominant than compression, i.e. shear, on the constitution of a preferred trabecular orientation in the spine on the macro-tissue level (>10 mm) was investigated in the first part (Part I). This influence was related to mechanical characteristics of trabecular structures on the micro-tissue scale (1-10 mm) in the second part (Part II). In Part I, primary trabecular orientations (PTOsmacro) near the superior and inferior vertebral endplates of L1 and L5 of 6 human spine cadavers were determined on the macro level using micro computed tomography imaging (voxel size = 120 m3), by calculating the dominant fabric principal vector. Their relative deviations to the axial compression vectors in the spines, quantified by the normals to the endplate (NEs), were determined afterwards. The average deviation between the PTOmacro and NEs was 6.24⁰ (±4.34⁰). The PTOsmacro did not show a preference towards the anterior or posterior direction relative to the NE. From the deviations, it was concluded that trabecular bone in the spine predominantly adapts to compression loads. However, secondary loading directions, such as shear, are of additional influence. In Part II, 13 small cubes (6.0x6.0 mm) from the volumes of interest in Part I were analysed on the micro level with regard to elasticity. Components, component ratios and primary elastic orientations (PEOmicro) of elasticity tensors, computed by the simulation of mechanical tests in finite element (FE) models, were calculated. PTOs of the cubes (PTOsmicro) were compared to the PEOsmicro and related to the PTOsmacro and NEs (Part I) qualitatively. Elasticity tensor components were within a reasonable range (approximately 1-250 MPa, excluding outliers) and no material symmetry was found, i.e. the structures were mechanically anisotropic. PTOsmicro deviated 13.90⁰ (±8.04⁰) with respect to the PEOsmicro on average. 10 out of 13 PEOsmicro had similar anterior or posterior tendencies as the PTOsmacro with respect to the NEs. 11 out of 13 PTOsmicro had similar anterior or posterior tendencies as PTOsmacro with respect to the NEs. Elastic properties of typical trabecular structures in the vertebral bodies were successfully determined. Due to a relatively low resolution, PEOsmicro deviated strongly with the PTOsmicro. Such deviations could function as indicators for bone quality in skeletal disease diagnostics using low resolution imaging. PTOsmicro and PEOsmicro agreed relatively well to the PTOsmacro on the macro-tissue level, in terms of anteriorly or posterior tendencies relative to axial loading in the spine. This outcome shows promise for multi-scalar biomechanical analysis of trabecular bone.

Osteogenesis imperfecta (OI), also known as brittle bone disease, affects 1 in 10.000 births. This disease is characterized by skeletal dysplasias, bone fragility and many secondary health issues. It is a heritable disorder affecting collagen type I, which is an essential protein in bone tissue. Post-translational overmodifications in collagen biosynthesis disturb the collagen packing and alter the mineralization process. This results in bone with poor structural parameters and inferior mechanical properties. In the current study, we have tested the hypothesis that the structural and mechanical properties in bone samples from OI patients are related to altered post-translational modifications in the collagen molecules.Hydroxylysine (Hyl) and the crosslinks hydroxylysyl-pyridinoline (HP) and lysyl-pyridinoline (LP) in bone samples from OI patients were measured with liquid chromatography. Bone quantity, in terms of volumetric bone mineral density (vBMD) and volumetric tissue mineral density (vTMD), was measured with micro-computed tomography. Hardness and Young’s moduli were derived from indentation experiments. Lysine was overhydroxylated in OI bone (p < 0.001), which led to increased HP values (p < 0.001) and higher HP/LP ratios (p = 0.013) compared to controls. OI tissue mineral density was more heterogeneously distributed, although average vTMD values were similar in both groups. The vBMD was significantly higher in controls compared to OI bone samples. Elevated Hyl levels were significantly related to decreased vBMD and vTMD in bone samples from OI patients. In both OI and control samples, an increase in LP crosslinks was associated with elevated vTMD values. Indentation with a spherical tip (r = 0.25mm) showed no altered mechanical properties in OI compared with controls. Unfortunately, we could not determine if impaired overmodifications in collagen are related to the poor mechanical properties of OI bone. Young’s moduli and hardness were not related to HP levels or HP/LP ratios. In control bone samples a negative correlation between Hyl residues and mechanical properties is observed. More mechanical experiments are necessary to address the hypothesis appropriately.