In work package 1, CRISPR/Cas-mediated genome editing was used to generate fluorescent reporter mice for osteoblasts and osteoclasts. Specifically, osteoblast (Ibsp) and osteoclast-specific targets (Acp-5, Calcr) were labeled with fluorescent proteins (eGFP, mCherry). The phenotyping of the dual-fluorescent reporter mice for bone cells and the prematurely aged PolgA mice was finalized with preparation and submission of manuscripts (Yilmaz et al. submitted to Cell Reports; Singh et al., to be submitted). In order to allow single cell transcriptomics of bone cells considering their local 3D mechanical micro-environment (LivE), both isolation of single cells via laser-capture-microdissection (LCM) and Visium spatial transcriptomics have been used. To enable the integration of organ- and tissue-level data from micro-computed tomography with sub-cellular information from histological sections, a novel correlative multimodal imaging (CMI) pipeline has been developed also integrating Visium spatial transcriptomics data. To study the effect of aging on bone adaptation, we applied cyclic mechanical loading to vertebrae of premature aging PolgA(D257A/D257A) mice, showing prematurely aged PolgA mice to be not mechano-responsive, whereas younger PolgA mice showed similar anabolic bone adaptation compared to wildtype animals. To study trabecular bone regeneration, we established a femur defect loading model and assessed the regeneration potential of biomaterials in this model showing again that aging negatively affected fracture healing in prematurely aged PolgA mice.
In work package 2, computational tools which interface with experimental studies and data were developed. In the mechanically loaded femoral defect studies, it was critical that the healed bone could always support the applied load, and the tissue scale mechanical loading was consistent across the groups. To achieve this, a novel method termed “real-time Finite Element (rtFE)” was developed to determine suitable loading parameters in vivo. In order to accurately understand the local mechanical in vivo environment (LivE), an instrumented external fixator crossbar was developed. Combined with a multiscale FE model it was possible to determine the load transfer through the femoral defect and the external fixator during mouse locomotion, which improved the estimations for LivE mechanics and provided an important input parameter for the in silico models, where a novel hybrid model was developed combining micro-multi-physics with agent based modelling to model the cells. The reactions cause them to modify their environment by releasing chemical signals, or altering the local bone matrix. The mechanical signal sensed by cells is calculated by micro-FE while the chemical signaling is a reaction-diffusion of molecules and binding sites. The novel 3D micro-MPA models for bone adaptation and regeneration were characterized by high fidelity and flexibility for simulating biological processes on the cellular and organ scale. The micro-MPA models for bone adaptation and regeneration have been applied to study the effect of ageing by adapting the behavior and their mechanosensitivity using the in vivo data obtained for the prematurely aging PolgA mice.
