Shared cellular characteristics of dysfunctional stromal cell differentiation underpin impaired healing in two models of perturbed fracture healing

10% of the fifteen million bone fractures each year that occur in the US fail to heal, which leads to significant loss of function, morbidity, and mortality. However, very little is known about the cellular and molecular mechanisms underpinning inferior healing. Two perturbed fracture models, surgically induced ischemia and the genetic deletion of the self-antigen CD47, were studied to identify unifying cellular and molecular mechanisms. C57Bl/6 wild-type (WT) mice with intact vasculature (WT Intact), WT mice with induced ischemia (WT Ischemic), and CD47-null mice with intact vasculature (CD47-KO) underwent transverse tibial fractures. Single-cell RNA-sequencing (scRNAseq) was performed during the inflammatory stage (4 days post-fracture) and the stromal stage (7 days post fracture) to assess transcriptomic alterations in perturbed fracture calluses. Overall design: Tibial fractures were performed at 18(±2) weeks of age in C57 Bl/6 mice. A hypodermic needle was first placed as an intramedullary nail followed by tibial fracture using a three-point bend apparatus. Alternatively, mice underwent ischemic fracture by isolation and ligation of the left femoral artery prior to fracture described above. We additionally fractured CD47-null mice on a Bl/6 background. Fracture calluses were harvested at 4 and 7 days post fracture. Periosteum was harvested as well from uninjured WT and CD47-null mice. This represents 8 conditions (1) WT intact vasculature 4 days post-fracture, (2) WT Ischemic 4 days post-fracture, (3) CD47-null intact vasculature 4 days post-fracture, (4) WT intact vasculature 7 days post-fracture, (5) WT Ischemic 7 days post-fracture, (6) CD47-null intact vasculature 7 days post-fracture, (7) WT periosteum, and (8) CD47-null periosteum. For each fracture condition, we sequenced a single biological replicate made up of 8 pooled calluses (equal male/female).