Fig. 1

Neurovascular ingrowth and transcriptional profile after soft tissue trauma. Traumatic injury (complete Achilles tenotomy with dorsal burn to induce heterotopic ossification) was performed on 6–8 weeks old, male and female C57BL/6 J mice, with analyses at 7 and 21 days. Day 0 indicates uninjured control. a–c Representative pan-neuronal Beta III Tubulin (TUBB3) immunofluorescent staining within sagittal sections of tendon injury site, day 0, 7 and 21 post-tenotomy. d Quantification of TUBB3⁺ nerves within the injury site over time, including overall TUBB3 staining intensity, number of nerve junctions, and number of nerve branches. e–g Representative immunofluorescent staining of the endothelial marker CD31 over the 21-day time course post-injury. h Quantification of CD31⁺ blood vessels within the injury site over time, including overall CD31 staining intensity and vessel diameter. i–m Single cell RNA sequencing analysis of pooled cells from the injury site, obtained at day 0, 7 and 21 post-injury. i UMAP visualization of endothelial and pericyte clusters at all timepoints, and (j) UMAP visualization indicating cells at day 0, 7 and 21 post-injury. k Percentage of endothelial cells and pericytes across time as a function of both cell types combined. l, m GO term analysis of endothelial cells (red) and pericytes (blue) at day 7 and day 21 post injury in relation to uninjured control. n Modular index scoring of cell proliferation, vessel growth, and vessel maturation in endothelial cells across time points. Gene lists for modular scores are shown in File S1. Dashed white lines indicate edges of Achilles tendon. Scale bars: 100 μm. For histology, n = 4 animals per timepoint. For scRNA-Seq, n = 3 animals per timepoint. Individual dots in scatterplots represent values from single animal measurements, while mean and one SD are indicated by crosshairs and whiskers. *P < 0.05; **P < 0.01 in relation to D 0 timepoint. Two-tailed student’s t test was used for two-group comparisons (b, d). For more than two groups, one-way ANOVA followed by post hoc analysis was performed (n). Non-parametric data was analyzed using the Kruskal–wallis with Dunn’s multiple comparison test (n)