Prof. Ralph Müller

The indispensable role of the skeletal system in enabling motion and providing structural support to vital organs underscores the imperative for efficacious interventions in the context of substantial bone defects. Notably, Bone Graft Substitutes (BGS) have emerged as a viable therapeutic avenue, exhibiting success in promoting bone formation through their inherent osteoinductive potential. Despite the considerable promise associated with BGSs, the intricate mechanisms governing material-induced osteoinduction remain elusive. Bohner et al. have recently proposed a novel mechanism termed Sustained Local Ionic Homeostatic Imbalance (SLIHI) as a potential instigator of material-induced osteoinduction. The hypothesis posits that localized depletion of calcium and phosphate during the calcification process may lead to SLIHI, thereby modulating inflammation and instigating an osteoinductive response. Calcium signaling is pivotal in diverse biological and cellular processes, with a particular impact on skeleton mineralization. The continuous remodeling of the skeleton, involving bone resorption and new bone deposition, results in fluctuating extracellular calcium levels corresponding to different phases of remodeling. To comprehensively explore SLIHI, it becomes imperative to scrutinize the spatial distribution and concentration of calcium. This investigation can be effectively conducted through quantitative imaging techniques utilizing calcium-sensitive ratiometric probes. Ratiometric imaging is a scientific approach that utilizes specialized probes to assess variations in calcium concentrations outside cells quantitatively. This technique relies on the simultaneous acquisition of signals at two distinct wavelengths, allowing for the calculation of a ratio that is sensitive to changes in extracellular calcium levels. Here, we aim to assess the impact of ion-regulating BGS on extracellular calcium concentrations and the concurrent influence on adjacent immune cells.

This project seeks to implement ratiometric imaging both in controlled laboratory conditions (in vitro) and within living organisms (in vivo) to examine how bone graft substitutes influence extracellular calcium levels and interact with immune cells. The ultimate goal is to enhance our understanding of these processes to optimize osteoinduction. Candidates with prior familiarity with cell culture and microscopy will be preferably considered. The project goals could be tailored to the student`s interests, expertise, and project requirements.

Dr. Stefanie S. Wissmann, Postdoc (email:stefanie.wissmann@hest.ethz.ch), ETH Zürich / Laboratory for Bone Biomechanics (https://www.bone.ethz.ch/) For application, please provide your CV and transcripts of B.Sc. and M.Sc.

Project 1 – Establishing a 3D In Vitro Co-culture of Osteoclasts and Osteoblasts Bone cells such as osteoclasts, and osteoblasts have significant roles in bone remodeling. So far in our models, we have established long-term culture of osteoblasts and osteocytes. However, bone-resorbing cells such as osteoclasts were not incorporated into the system. In this project, human osteoclast precursors will be introduced, and functional and molecular biology assays will be established along with the time-lapsed microCT scans. Furthermore, the interplay of cell-mediated resorption and formation of extracellular matrix will be evaluated with time-lapsed CT scans and molecular biology assays. This project can be tailored to suit a Bachelor’s Thesis, semester Project or research internship. The students will receive hands-on experience in handling primary cells, bioreactors, 3D printing and conducting molecular biology assays. Project 2 – Optimization of Fibrin as A Bioink for 3D Bioprinting Fibrin-based bioink formulations have garnered research interest due to their excellent biological properties. It is widely used in clinics as a hemostatic agent and surgical sealant. Fibrin and fibrinogen-based proteins are known to have diverse roles in different stages of tissue repair such as cell adhesion, inflammation, angiogenesis, and remodeling. The goal of this project is to develop a 3D printable bio-ink formulation of fibrin and mechanical properties will be tailored to support human mesenchymal stem cell differentiation. This project can be tailored to suit a Masters’ Thesis, Semester Project or Research Internship. The students will receive hands-on experience in primary cell cultivation, inkjet printing, extrusion 3D printing and molecular biology assays.

The projects are multidisciplinary, and students from biology, bioengineering and mechanical engineering backgrounds are welcome. The project goals could be tailored to the student’s interests, expertise, and project requirements.

Dr Chris Steffi (e-mail: chris.steffi@hest.ethz.ch), Postdoc, Laboratory for Bone Biomechanics, ETH Zurich

Postmenopausal osteoporosis is a prevalent bone disease leading to decreased bone integrity and an increased risk of fractures. Bone as an organ is able to continuously change its internal trabecular microstructure to adapt to changing mechanical and metabolic demands [1]. Bone remodelling is executed by bone forming cells called osteoblasts and resorbing cells called osteoclasts. Bone sensing cells called osteocytes are believed to pass on the local micromechanical conditions to the executing cells. During postmenopausal osteoporosis, these cellular mechanisms are disturbed towards increased bone resorption [2]. The exact mechanism of action of the disease is not well understood to date. Agent-based in silico models have the potential to investigate research areas, which are to date not accessible with experimental data. In silico models can simulate changes to bone structure allowing testing of the mechanisms of action of drug treatments and providing insight into individualised estimations of treatment effects [3]. This project is part of a wider effort to create a comprehensive in silico multi-physics agent-based model for the investigation of osteoporotic bone mechanophysiology and the bone response to treatments. The proposed work will implement and evaluate the bone response to estrogen depletion. This work will form the basis for subsequent in silico investigation of potential osteoporosis treatments. The chosen mechanism of action of estrogen will be validated against available experimental reference data. The validation criterion will be the resulting bone morphometry that the in silico model produced. Visual representations of the resulting three-dimensional bone structures will be generated. Finally, the project will be prepared and presented. Literature: [1] Burr DB. Targeted and nontargeted remodeling. Bone 2002;30:2–4. [2] Riggs BL. et al., Overview of osteoporosis. Western Journal of Medicine 1991;154:63. [3] Ruiz-Lozano R. et al., An in silico approach to elucidate the pathways leading to primary osteoporosis: age-related vs. postmenopausal. Biomech Model Mechanobiol. 2024.

- Prepare a timetable of the work to be performed - Perform a short literature review (3 papers) regarding the role of estrogen for the bone and the effects of estrogen depletion leading to postmenopausal osteoporosis - Implement a potential way of action for estrogen depletion into an existing micro-MPA-model (Python) - Assess the micro-MPA model accuracy (differences in bone morphometry from existing experimental validation data (Python, JupyterNotebook) - Generate visual representations of the simulated bone response to estrogen depletion (ParaView, Python) - Write a detailed report of the project • Prepare a presentation summarizing the project (MS PowerPoint)

Dr. Friederike Schulte, Laboratory for Bone Biomechanics, ETH Zurich, Switzerland, friederike.schulte@hest.ethz.ch

To address this century-old problem, the Laboratory for Bone Biomechanics has established a novel approach termed “spatial mechanomics”. Spatial mechanomics is defined as the integration of spatially-resolved “omics” with in silico models of the mechanical environment to investigate the interactions between local mechanical environments and cellular / molecular responses (Figure 1). This is a highly cross-disciplinary area of research combining in vivo models, imaging, “omics” techniques and computational modelling / analyses. There are opportunities for student projects within this area of research that can be tailored to suitable candidates. Ideally applicants will be candidates with an interest or experience in musculoskeletal research, “omics” techniques, computational analyses, histology and/or image processing.

We are interested in the following: • To develop a molecular-based understanding of bone healing mechanobiology. • To investigate how this mechano-sensitivity is compromised with age.

Please contact Neashan Mathavan via: nmathavan@ethz.ch

**Who We are:** Laboratory for Bone Biomechanics (Müller Group) works on musculoskeletal systems. The group develops a quantitative model of bone at the cellular, molecular, tissue, and organ levels. We seek a highly motivated Bachelor's or master’s student dedicated to creating a multimodal transcriptome analysis framework to deepen bone fracture healing. This project offers an opportunity to gain valuable work experience within a highly interdisciplinary and international team. **Project Description** Fracture healing is a complex process that involves a series of biological events, including inflammation, angiogenesis, and bone remodelling. This remodelling process helps maintain bone density, repair micro-damage that occurs due to everyday activities, and adapt bones to the specific needs of an individual's body. In essence, bone remodelling during fracture repair is a sophisticated and dynamic orchestration of cellular activities that reabsorb excess bone and revitalize the bone tissue to its former strength and functionality, ultimately facilitating a seamless and enduring restoration of the injured bone. The healing process is physiologically complex, involving both biological and mechanical aspects. Mechanical loading is a crucial factor in the regulation of fracture healing. The forces and strains experienced by the bone during everyday activities influence the cellular responses, callus formation, bone deposition, remodelling, and, ultimately, the successful recovery of the fractured bone. Effective control of mechanical loading is pivotal in treating and recovering fractures, ensuring the best possible healing and functional results. However, the mechanisms underlying spatial cell reorganization during loading, which contributes to fracture healing, remain unclear. Therefore, the project aims to investigate and explore the fracture healing process of mice using spatial transcriptome changes in response to mechanical loading. By shedding light on this aspect, we aim to contribute to the broader understanding of fracture healing and potentially pave the way for more effective treatment strategies in the future.

Developing a Robust R/Python Framework for Seamless Integration of Multimodal Data, Subcellular Spatial Transcriptomics, Cell Segmentation, and Advanced Analysis Leveraging the Power of High-Performance Cluster Computing with a Focus on the 10X Visium Sequencing Platform. **Requirements:** • B.Sc or M.Sc. in Bioinformatics, Data science, computational biology, Physics, Mathematics or a related field. • Proficiency in R/Bioconductor, Python, or a similar programming language • Familiarity with tools for the analysis of single-cell or spatial transcriptomics methods (Seurat, Scanpy), Image analysis (Napari, QuPath, ImageJ), Bash scripting, Python packaging, Parallel computing

Please get in touch with me to get more detailed information about the project by email or reach out in person at Gloriastrasse 37/39, 8092 Zürich, Switzerland. Email: amit.singh@hest.ethz.ch LinkedIn: https://www.linkedin.com/in/amit-singh-63373417

Bone tissue undergoes continuous and finely tuned remodeling processes orchestrated by osteocytes, bone-resorbing osteoclasts, and bone-forming osteoblasts. Disruptions in the balance of these cellular activities, whether due to cancer or genetic mutations such as osteogenesis imperfecta, lead to systemic health conditions that are clinically challenging to treat. Traditional treatments often overlook the significant heterogeneity within patient populations, and current research and development predominantly rely on animal models that fail to accurately represent human bone biology. To address these limitations, our research group has pioneered the development of organotypic 3D bioprinted bone organoids that closely mimic the native bone microenvironment. These advanced models provide a more accurate platform for studying bone diseases, evaluating drug responses, and testing therapeutic interventions. A critical component of advancing these models is the ability to monitor their microstructural development and mechanical properties over time using micro-computed tomography (micro-CT). This project aims to develop and integrate an automated image processing and analysis pipeline tailored for our extensive micro-CT datasets of 3D bioprinted bone organoids. The student will be tasked with running the pipeline on existing micro-CT data, incorporating our established volumetric mechanoregulation analysis, and performing FE analysis to assess the mechanical properties of the bone organoids. Additionally, the student will explore correlations between stiffness parameters monitored during culture and the results obtained from FE analysis, thereby enhancing our understanding of bone biomechanics and improving the reliability of diagnostic assays and drug testing platforms.

The primary goal of this project is to develop and implement an automated image processing and analysis pipeline for micro-CT images of 3D bioprinted bone organoids, integrating our volumetric mechanoregulation analysis method and finite element (FE) analysis to evaluate bone biomechanics.

Dr. sc. Gian Nutal Schädli Senior Postdoc and PHRT Fellow Institute for Biomechanics, ETH Zürich GLC H 20.2, Gloriastrasse 37/39, 8092 Zürich https://www.bone.ethz.ch/ Tel: +41 44 633 41 75 CEO and Co-founder CompagOs AG, ETH Zürich Spin-off c/o MAS Solutions GmbH, Rigistrasse 15c, 6331 Hünenberg https://www.compagos.ch/

Osteoporosis is the most common musculoskeletal disease occurring during aging and is characterized by a reduction in bone strength and increased risk of fracture due to low bone mass and impaired bone microarchitecture. [1] The main therapeutic interventions for osteoporosis are antiresorptive (BIS, SERM, Denosumab, Estrogen) and anabolic drugs (PTH). A change of lifestyle, such as to quit smoking or increase physical exercise, can be supportive of the above mentioned pharmacological interventions. Osteocytes are the most abundant cells in bone tissue and are involved in mechanosensing and mechanotransduction through their lacunocanalicular network (LCN). During aging and the onset of osteoporosis, the LCN is of particular interest due to its role in bone remodeling and adaptation to mechanical load [2]. Multiple studies have investigated lacunar density in the aging human population [3-8] and in osteoporotic patients [4,9-11] using histomorphometric techniques, however, these techniques don’t allow for an accurate 3D representation of the whole LCN. MicroCT can be considered the gold standard in hierarchical 3D imaging of bone structure and function [12] and delivers the key images relevant in this project of assessing the influence of osteoporosis and the effect of different treatments on osteocyte lacunar properties. The 3D-3D registration of high resolution (1.2 µm) to low resolution (10.5 µm) microCT images (see image, courtesy of Li and Eskesen, 2016/2019) of the same murine vertebra samples gives insight into the hierarchical and structural organization of trabecular bone. The time-lapsed information of the _in vivo_ micro-CT images furthermore allows to investigate specific sites of bone formation and resorption and their influence on osteocyte lacunar morphology. References: 1. Sambrook P,et al. 2006. doi:10.1016/s0140-6736(06)68891-0 2. Miyauchi A, et al. J Biol Chem. 2000. doi:10.1074/jbc.275.5.3335 3. Mori S, et al. Bone. 1997. doi:10.1016/s8756-3282(97)00200-7 4. Mullender MG, et al. Bone. 1996. doi:10.1016/8756-3282(95)00444-0 5. Qiu S, et al. Bone. 2002. doi:10.1016/s8756-3282(02)00819-0 6. Torres-Lagares D, et al. J Craniomaxillofac Surg. 2010. doi:10.1016/j.jcms.2009.07.012 7. Vashishth D, et al. Anat Rec A Discov Mol Cell Evol Biol. 2005. doi:10.1002/ar.a.20146 8. Vashishth D, et al. Bone. 2000. doi:10.1016/s8756-3282(00)00236-2 9. Soicher MA, et al. Bone. 2011. doi:10.1016/j.bone.2010.11.009 10. Mullender MG, et al. Calcif Tissue Int. 2005. doi:10.1007/s00223-005-0043-6 11. Qiu S, et al. J Bone Miner Res. 2003. doi:10.1359/jbmr.2003.18.9.1657 12. Müller R. Nat Rev Rheumatol. 2009. doi:10.1038/nrrheum.2009.107

Expanding a computational framework (Python) for the successful registration of high to low resolution microCT images of murine vertebras for 3D morphometric and hierarchical analysis of osteocyte lacunar properties at different sites of the vertebral bone (sites of bone formation and resorption). Previous knowledge of python programming and advanced image analysis are of advantage but not a requirement. Any motivated student with a passion for bone and image analysis techniques is welcome to broaden their knowledge and learn new skills. The project goals will be tailored to the student’s interests, expertise, and project requirements.

Sara Lindenmann, doctoral student (e-mail: sara.lindenmann@hest.ethz.ch), ETH Zürich / Laboratory for Bone Biomechanics (https://www.bone.ethz.ch/) For an application, please provide your CV and transcripts of B.Sc. and M.Sc.