Spatial Transcriptomics in Bone Mechanomics: Exploring the Mechanoregulation of Fracture Healing in the Era of Spatial Omics.

In recent decades, the field of bone mechanobiology has sought experimental techniques to unravel the molecular mechanisms governing the phenomenon of mechanically-regulated fracture healing. Each cell within a fracture site resides within different local micro-environments characterized by different levels of mechanical strain - thus, preserving the spatial location of each cell is critical in relating cellular responses to mechanical stimuli. Our spatial transcriptomics based “mechanomics” platform facilitates spatially-resolved analysis of the molecular profiles of cells with respect to their local in vivo mechanical environment by integrating time-lapsed in vivo micro-computed tomography, spatial transcriptomics, and micro-finite element analysis. We investigate the transcriptomic responses of cells as a function of the local strain magnitude by identifying the differential expression of genes in regions of high and low strain within a fracture site. Our platform thus has the potential to address fundamental open questions within the field and to discover mechano-responsive targets to enhance fracture healing. Female 12-week-old mice (n = 4) received mid-diaphyseal femoral defects (0.68 ± 0.04 mm) using an established osteotomy surgical protocol. In vivo micro-CT imaging is performed weekly at the fracture site. Mice which exhibit bridging at 3 weeks post-surgery are then subdivided into Control and Loaded groups. Between weeks 3 – 5, mice receive either cyclic mechanical loading or sham-loading three times per week. All mice are euthanized at 5 weeks post-surgery. Spatial transcriptomics analyses are performed on the explanted femurs (n = 1 Control, n = 1 Loaded). Micro-finite element models - based upon in vivo micro-CT images - are used to generate tissue-scale 3D maps of the mechanical environment. We then correlated the molecular responses of individual cells to their local mechanical environment.