Mechanobiology and Mineralization Dynamics in Bone Health and Disease: Insights from Computational Modeling, Organotypic Models, and In Vivo Studies

Abstract

Bone is a fascinating material which is able to functionally adapt to mechanical demands by remodeling itself. Discussions about bone health and disease, typically revolve around increased bone fracture risk and strategies aimed at mitigation or prevention of such fractures. Animal models, particularly mice, have played a significant role in advancing bone disease research, elucidating phenomena such as the influence of local mechanical stimuli on bone remodeling, known as mechanoregulation. They have been crucial in the advancement of pharmaceutical treatments of osteoporosis, a prevalent bone disease characterized by decreased bone mass, particularly affecting the elder population. However, for some diseases such as osteogenesis imperfecta (OI), a rare heterogeneous genetic disorder characterized by increased bone fragility and deformities, animal models reach their limitations. In vitro bone models are an emerging technology for understanding disease mechanisms and developing targeted therapies. Using micro-computed tomography (micro-CT) has enabled a range of non-destructive image-based analysis in vivo such as investigating mineralization dynamics or mechanoregulation using micro finite element (micro-FE) models based on these images. Tailoring such methods to in vitro approaches will lead to additional insights from bone tissue engineering and may enable advances in the development and optimization of in vitro bone models. The address this, the present thesis has been divided into three aims: (i) developing a volumetric method to analyze mechanoregulation in in vitro bone scaffold, (ii) analyze tissue mineralization and mechanoregulation in a FKBP10-related OI organotypic bone model, and (iii) investigate the influence of osteoporosis treatments on mechanoregulation in vivo. To address the first aim of the thesis we aimed to develop and implement a volumetric method for quantifying mechanoregulation of bone formation in tissue-engineered constructs, utilizing micro-CT images and micro-FE analysis. We first established a conversion function correlating tissue mineral density and Young’s modulus for in vitro applications, enabling micro-FE modelling in bone tissue engineering. Secondly, we examined hydroxyapatite scaffolds seeded with human mesenchymal stem cells, incubated over 8 weeks with one mechanically loaded and a control group from a previous study. Significantly higher mechanoregulation of bone formation was observed in the loaded samples compared to non-loaded controls during culture in osteogenic medium. Subsequently, the method was applied to an in vivo mouse study investigating the effect of loading frequencies on bone adaptation, exploring the applicability for in vivo studies. Differences in mechanoregulation of bone formation between loading conditions were detected. Notably, mechanoregulation in bone formation was more pronounced compared to the surface-based method. In the second part of the thesis, we used and extended computational methods to investigate tissue mineralization in an organotypic bone model for FKBP10-related OI. First, we created mechanically loaded 3D-bioprinted patient-specific organotypic bone models for FKBP10-related OI and healthy controls from patient derived osteoblasts. Using time-lapsed micro-CT, hypermineralization was observed in FKBP10-related OI samples compared to healthy controls. Additionally, we observed a decoupling of tissue mineral density and experimental stiffness in OI samples. Employing sample-specific micro-FE analysis allowed us to replicate experimental stiffness, and to detect similar mechanoregulation in both FKBP10-related OI organotypic bone models and healthy controls. Regional analysis revealed heterogeneous mineralization, microarchitectural irregularities, and scaffold microporosity in OI samples. Our findings suggest that the dysregulated mineralization observed is a primary contributor to the altered mineral-mechanical properties observed in FKBP10-related organotypic bone models, whereas mechanoregulation appears to be consistent with that of healthy controls. To address the third aim of the thesis, we investigated the effects of combining mechanical loading with pharmaceutical treatments (bisphosphonates, parathyroid hormone, or sclerostin antibodies) on mechanoregulation in trabecular bone in ovariectomized C57Bl/6J mice. Mechanical loading synergistically enhanced trabecular bone mass under parathyroid hormone or sclerostin antibody therapy. Mechanoregulation analysis revealed bone remodeling was targeted to mechanical demands in all treatments, albeit to varying degrees. Bisphosphonate treatment decreased mechanosensitivity compared to anabolic treatments, which could not be recovered by mechanical loading. Mechanical loading enhanced the mechanoregulatory response of sclerostin antibody treatment and parathyroid hormone treatment, suggesting that incorporating physical therapy into anabolic pharmaceutical regimens may improve therapeutic outcomes in osteoporosis management. In summary, this thesis explored two key areas in bone research: mechanoregulation and mineralization in tissue-engineered bone constructs, and the influence of combination therapies on mechanoregulation for postmenopausal osteoporosis. Firstly, we were able to quantify mechanoregulation of bone formation in tissue engineered bone constructs. Secondly, we analyzed mineralization dynamics and mechanoregulation in organotypic bone models of OI, shedding light on how dysregulated mineralization impairs stiffness. Finally, we demonstrated that combining mechanical loading and anabolic pharmaceutical treatments in a mouse model of osteoporosis can increase therapy efficacy and decrease fracture risk. Ultimately, we have shown that by tailoring computational methods to in vitro applications, we can fully leverage non-destructive image-based analysis to maximize research insights. Our results highlight the importance of mechanoregulation and suggest that in the future more resources should be dedicated to understanding the potential of physical therapy in bone disease management.