Greetje Renders

Vincent Jalkarese

In a healthy joint, articular cartilage and subchondral bone act together in transmitting load pressure through joints. Therefore, the integrity of both tissues is necessary for adequate joint function. When one element falls out of balance, it inevitably triggers multiple physiological, biochemical, and biomechanical responses from the other components as the joint system seek to rebalance itself. The close interaction between the two tissues is evident from their roots during development. Bone and cartilage were originally one tissue, and jointly differentiated into two distinct tissues to be found in the adult. It is generally accepted that the mutual feedback between the two tissues is required as they specialized and adapted to their unique roles and functions within the joint organ. As such, bone and cartilage in the mature joint are interdependent, and are only capable of fulfilling their biomechanical functions with their mutual support from a macro-structural to a micro-structural level. Furthermore, the intimate relationship between bone and cartilage involves multiple pathways of interaction; many of which were shown to regulate the metabolic and remodeling mechanisms of the tissues.

Our main research questions were: Is µCT a useful technique for quantitative bone research in the jaw joint? Is a µCT-based biomechanical model suitable for osteoarthritis (OA) research in the jaw joint? Is µCT usable for cartilage assessment in the jaw joint of a human and murine model? With obtaining answers to these questions, we hope to contribute to finding new methods and ways to improve our understanding of OA development and the creation of effective treatments. One of the scopes in future OA research could be, for instance, to examine the complex interactions of different tissues in OA progression using methods provided in this thesis, especially focusing on the importance of subchondral bone in the early stages of disease development.

Chapter 2 describes the application of conventional µCT to the mandibular component of the human jaw joint for quantitative measurements of bone structural (e.g., cortical and trabecular morphology) and material parameters (e.g., tissue mineral density and distribution). The dimension of the jaw joint is very suitable for this kind of research and was therefore our main joint of interest. Our study was the first one that examined the tissue mineral density (TMD) and its distribution in cortical and trabecular bone of the human mandibular condyle (Chapter 2a). The TMD in the mandibular condyle may reflect age and remodeling rate of the bone tissue. Quantification of the TMD facilitates a better understanding of possible effect on adaptive remodeling on mineralization of the condyle in the healthy and diseased joint and its possible consequences for mechanical properties. A conventional µCT system was used to measure TMD values. Mean TMD was higher in cortical than in trabecular bone and differed significantly between different cortical regions. The variation in TMD distribution was significantly larger in the anterior cortex than the posterior and subchondral cortex, indicating a larger amount of heterogeneity of mineralization anteriorly. Within the cortical cortex, TMD increased with distance from the cortical canals to the periphery. Similarly, TMD of the trabecular bone increased with distance from the surface of the trabeculae to their cores. On a biomechanical level, the difference in TMD between condylar trabecular and cortical bone suggests a large difference in Young’s moduli.

Another achievement of this thesis was high-resolution visualization of the cortical porosity network in 3D and providing further quantitative information via the conventional µCT technique (Chapter 2b). We were able to examine cortical porosity in relation to TMD, in order to get more information about the principal directions of stress and strains during loading. Quantification of porosity and tissue mineral density of bone facilitates a better understanding of possible effects of adaptive bone remodeling and possible consequences for its mechanical properties. The cortical canals in the subchondral cortex of the condyle were oriented in mediolateral direction, and in the anterior and posterior cortex in superoinferior direction. Cortical porosity (average 3.5%) did not differ significantly between the cortical regions. It correlated significantly with the diameter and number of cortical canals, but not with cortical tissue mineral density. In trabecular bone (average porosity 79.3%) a significant negative correlation was found between surface area of the trabeculae and tissue mineral density; such a correlation was not present between the surface area of the cortical canals and the tissue mineral density of cortical bone. No relationship between trabecular and cortical porosity or between trabecular tissue mineral density and cortical tissue mineral density was found, suggesting that adaptive remodeling is independent and different between trabecular and cortical bone. We conclude that the principal directions of stress and strains are primarily directed mediolaterally in the subchondral cortex and superoinferiorly in the anterior and posterior cortex, and that the amount of remodeling is larger in trabecular than in cortical bone of the mandibular condyle; in trabecular bone variation in the level of remodeling is related to the available surface area of the trabeculae.

Chapter 3 describes and discusses the use of µCT-based Finite Element (FE) analyses to determine the influence of the intratrabecular mineralization on several mechanical output parameters (e.g., apparent elastic modulus and intratrabecular stress/strain patterns) of trabecular bone in the human mandibular condyle. Due to daily loading, trabecular bone is subjected to deformations (i.e., strain), which lead to stress in the bone tissue. When stress and/or strain deviate from the normal range, the remodeling process leads to adaptation of the bone architecture and its tissue mineral density to effectively withstand the sustained altered loading. As the apparent mechanical properties of bone are assumed to depend on the degree and distribution of mineralization, the goal of our study in Chapter 3a was to examine the influences of mineral heterogeneity on the biomechanical properties of trabecular bone in the human mandibular condyle. Two different sets of Finite Element (FE) models of trabecular samples were constructed; tissue stiffness was either scaled to the local tissue mineral density of bone as measured with µCT (heterogeneous) or tissue stiffness was assumed to be homogeneous. Compression and shear tests were simulated to determine the apparent elastic moduli in both model types (homogeneous vs. heterogeneous). The incorporation of mineralization variation decreased the apparent Young’s and shear moduli by maximally 21% in comparison to the homogeneous model. The heterogeneous model apparent moduli correlated significantly with bone volume fraction and tissue mineral density of bone. It was concluded that disregarding mineral heterogeneity may lead to considerable overestimation of apparent elastic moduli in FE models.

Knowledge of the influence of mineral variations (i.e., mineral heterogeneity) on biomechanical bone behavior at the trabecular level is limited. The aim of Chapter 3b was to investigate how this material property affects the intratrabecular distributions of stress and strain in human adult trabecular bone. The influence of intratrabecular mineral heterogeneity was analyzed by comparing the same models as constructed in Chapter 3a. Interesting effects were seen regarding intratrabecular stress and strain distributions. In the homogeneous model, the highest stresses were found at the surface with a significant decrease towards the core. Higher superficial stresses could indicate higher predicted fracture risk in the trabeculae. In the heterogeneous model this pattern was different. A significant increase in stress with increasing distance from the trabecular surface was found followed by a significant decrease towards the trabecular core. This suggests trabecular bending during a compression. In both models a decrease in strain values from surface to core was predicted, which is consistent with trabecular bending. When mineral heterogeneity was taken into account, the predicted intratrabecular patterns of stress and strain are more consistent with expected biomechanical behavior as based on mineral variations in trabeculae. Our findings indicate that mineral heterogeneity should not be neglected when performing biomechanical studies on topics such as the (long-term or dose dependent) effects of antiresorptive treatments.

Chapter 4 describes the different implications of high-dosage bisphosphonate treatment on bone tissue parameters (e.g., mineral density, trabecular and cortical thickness) in the knee vs. jaw joint and mandible vs. humerus in a mouse model via µCT assessment and histological techniques. Bisphosphonates are bone antiresorptive agents traditionally used on a relatively large scale for treatment of bone metabolic diseases and on a smaller scale for bone metastasis treatment. A study on the effects of bisphosphonate treatment on healthy instead of diseased animals will give more insight into the basic mechanisms of bisphosphonates and their effects on different bone sites. In Chapter 4a we aimed to assess the effect of BP on the mouse knee and jaw joint. At baseline and after treatment with zoledronic acid (ZA) for one, three or six months, we combined bone assessment via µCT and histology. Our results showed that, in the knee joint, ZA treatment increased TMD, bone volume, trabecular thickness but did not influence cortical thickness. In both control and the ZA group, a higher trabecular TMD compared to cortical TMD was seen. Unseen in the knee joint, ZA treatment in the jaw joint resulted in bone-site specific changes in mineralization; a significant time-dependent higher TMD was evident in the subchondral bone compared to the most distal region of the condyle. MicroCT images revealed the presence of mineral in this region and histology showed that this region did not contain mature bone tissue but cartilage-like tissue. Our data indicates the possibility of site-specific negative side effects, i.e. disturbing normal mandibular development under the influence of bisphosphonate therapy.

In Chapter 4b we investigated whether in vivo exposure to bisphosphonates has a different effect on long bone and jaw osteoclasts, and on the turnover of these different bones. The same animal experiment was used as described in Chapter 4a. The effects on the number of osteoclasts, bone mineralization, and bone formation were measured in the long bones and in the jaw. Long-term treatment with zoledronic acid reduced the number of jaw bone marrow cells, without affecting the number of long-bone marrow cells. Zoledronic acid treatment did not affect the number of osteoclasts in vivo. Yet, the bisphosphonate increased bone volume and mineral density of both long bone and jaw. Interestingly, six months of treatment suppressed bone formation in the long bones without affecting the jaw. Unexpectedly, we showed that bisphosphonates can cause molar root resorption, mediated by active osteoclasts. Our findings provide more insight into bone-site-specific effects of bisphosphonates and into the aetiology of osteonecrosis of the jaw. We demonstrated that bisphosphonates can stimulate osteoclast activity at the molar roots.

Chapter 5 describes the introduction of a novel µCT technique called EPIC-µCT. Both conventional µCT and EPIC-µCT are applied ex vivo to both human and mouse jaw joint for 3D assessment of bone and cartilage morphology. The temporomandibular joint (TMJ) is susceptible to the development of osteoarthritis (OA). More detailed knowledge of its development is essential to improve our insight into TMJ-OA. It is imperative to have a standardized, reliable 3-D imaging method that allows for detailed assessment of both bone and cartilage in healthy and diseased joints. We aimed to determine the applicability of a contrast-enhanced µCT technique for ex vivo research of mouse and human TMJ. Equilibrium Partitioning of an Ionic Contrast agent via µCT (EPIC-µCT) was previous applied for cartilage assessment in the knee joint. The method was ex vivo applied to the mouse TMJ and adapted for the human TMJ. EPIC-µCT (30’ immersion time) was applied to mouse mandibular condyles and 3-D imaging revealed average cartilage thickness measurements via EPIC-µCT comparable to histomorphometric measurements. For human healthy and OA-affected TMJ samples the protocol was adjusted to an immersion time of one hour. 3-D imaging revealed a significant thicker cartilage layer in joints with early signs of OA compared to healthy joints. A subsequent significant thinner layer was found in human joints with late signs of OA. The EPIC-µCT technique is effective for the ex vivo assessment of 3-D cartilage morphology in the mouse as well as human TMJ and allows bone-cartilage interaction research in TMJ-OA.

In this thesis, conventional and contrast-enhanced µCT techniques were applied ex vivo to the complete human jaw joint. The combination of complimentary imaging techniques is promising for gaining more fundamental understanding of healthy and osteoarthritic joints as it permits the visualization of both bone and cartilage tissue. The combination of conventional and EPIC-µCT methodology could furthermore allow future possibilities to study the cartilage-bone complex as a whole. The mechanical interaction between its different constituents and the dynamical loading patterns of the joint during habitual function and prolonged heavy loading conditions can be the central issues. The dimension of the joint at all its stages of development enables to assess the fine architecture of both the subchondral bone and trabecular bone by µCT techniques without fully disrupting its integrity. One goal of this approach could be to predict the changes in the subchondral and trabecular bone in the joint as a consequence of overloading conditions and compare these changes with the abnormal bone structure observed in OA. A second goal could be to predict changes in the loading history of the articular cartilage in the relation to the prerequisites for cartilage maintenance.

With the µCT-based biomechanical computer models that can be produced, it is possible to predict the biomechanical interaction of bone and cartilage tissue and the consequence of this interaction in a healthy and osteoarthritic human joint. Furthermore, an animal experiment can be used to induce bone changes allowing for detailed investigation into the influence of specific bone changes on the overlying articular cartilage in the animal jaw vs. knee joint. Combining both µCT techniques in an animal experiment will allow us to further investigate the influence of specific bone changes on the overlying cartilage ex vivo. Our data can be useful to find answers to the hypothesis: Bone changes precede cartilage changes and/or damage in the development of osteoarthritis. With the results of this thesis, we hope to contribute to the implementation of new methods and ways to understanding of OA development and the creation of effective treatments.

The results described add up to the following general conclusions:
• The range of daily loading patterns influences variations in tissue mineral density and cortical porosity, and therefore remodeling provides bone tissue a useful tool to adapt to changes in these loading pattern.
• A more specific input for a biomechanical model provides a more specific output and thus outcome. Depending on the research question a choice should be made which variable should be used.
• Responses to high-dosage bisphosphonate treatment are bone-sites-specific (jaw bone vs. long bones) and can additionally cause a possible detrimental and unknown effect on the jaw joint.
• The EPIC-µCT technique is effective for ex vivo assessment of 3D bone and cartilage morphology in the mouse as well as human jaw joint plausible useful for bone-cartilage interaction research in jaw joint osteoarthritis.