Spatial Transcriptomics Approaches to Study Mechanobiology and Tissue Crosstalk During Fracture Healing

Fracture healing is a mechanical and spatially controlled process involving crosstalk of multiple tissues. To precisely capture and understand molecular mechanism during fracture healing, there is a need to integrate spatially-resolved molecular analyses into mechanically-controlled preclinical fracture healing models. In my presentation, I will provide an overview of our multimodal preclinical set-up in mice. We combine spatial transcriptomics (ST) approaches with mechanically-controlled fracture healing models, which provide options for in vivo monitoring of healing progression and individualized application of mechanical loading during the healing process. Using our ST workflow for formalin-fixed paraffin-embedded (FFPE) musculoskeletal samples from femur defect studies in mice, we observed gene expression clusters indicative of specific tissues as seen by spatial overlay with histology. The distinct molecular sub-clusters seen within the different tissues further indicate the importance of spatially-resolved gene expression data for understanding tissue crosstalk during fracture healing. In further studies, we performed ST on FFPE samples obtained at multiple timepoints during the fracture healing process in skeletally mature C57BL/6J mice ranging from post-operative day 3 to post-operative day 21 (GR/22/2022). Via this approach, characteristic spatiotemporal expression patterns of genes involved in endochondral ossification, mineralization and remodeling were seen. Further, distinct patterns of myokines, osteokines as well as mechanically-regulated markers indicate a potential for ST-based identification of potential treatment targets considering tissue crosstalk and mechanobiology during fracture healing. To conclude, the applicability of the developed ST approach will be shown for capturing the intersection and crosstalk between multiple tissues, highlighting the integration into multimodal approaches to study the mechanobiology during fracture healing.