Human bone-on-a-chip system for preclinical investigations of new therapeutic approaches for autosomal recessive osteopetrosis

This project introduces a human Bone-on-a-Chip system to connect fundamental research and translational applications. Utilizing the HUMIMIC Chip2 platform from TissUse, our approach is based on a decellularized human bone scaffold and sequential colonization allowing for long-term cultivation of 6 weeks. Within this cultivation period, we observe the maintenance and self-organization of primary bone forming cells, bone resorbing cells and immune cells, which enable us to assess the dynamics of the bone matrix and immune responses within the bone marrow microenvironment. In parallel, a system was developed utilizing induced pluripotent stem cells (iPSCs). These cells offer the advantage that genetic diseases can be mimicked and the phenotype and (patho)physiology of gene-corrected cells can be studied. In this thesis, the rare genetic disease autosomal recessive osteopetrosis (ARO) was examined as an example using the developed system. ARO is characterized by impaired bone resorption, for which the only treatment available is allogeneic stem cell transplantation. This therapy bears the risks of infections during patient conditioning and a graft-versus-host reaction. Autologous therapies offer a promising treatment strategy that can be developed and tested with our animal-free system. During long-term cultivation, the system is monitored by analyzing soluble factors and validating immune cell populations by fluorescence-activated cell sorting (FACS). By co-registering the scaffold before and after cultivation, it is possible to visualize very pronounced bone resorption and formation and to reflect a potential imbalance, as it is the case for ARO. To validate the physiological relevance of our in vitro bone model, we performed a single nuclei ribonucleic acid (snRNA) sequencing analysis to offer insights into the transcriptional landscape of individual cells within the bone construct. Additionally, histological stainings provide a structural comparison between the in vitro and ex vivo tissues. Currently, our Bone-on-a-Chip system is being used to preclinically test two gene therapy strategies for targeting ARO. In conclusion, our Bone-on-a-Chip system is a versatile translational research tool. Long-term culture allows us to observe orchestrated cell behavior, enabling detailed investigation of bone dynamics and immune responses. The integration of iPSCs improves the modeling of genetic diseases and expands applications in both fundamental and translational research.