Single-Cell Analyses of Heterotopic Ossification: Characteristics of Injury-Related Senescent Fibroblasts

INTRODUCTION: Injury-related cellular senescence may be involved in heterotopic ossification, and no research has been performed about this before. METHODS AND RESULTS: The study utilized integrated single-cell RNA-sequencing (scRNA-seq) data from heterotopic ossification samples. The number of senescent cells increased from day 3 and reached the highest level at day 21. However, the expression level of Cyclin Dependent Kinase Inhibitor 2A (Cdkn2a) has no such tendency as the change of cell amount, indicating that the expression level of Cdkn2a may be different in different types of senescent cells or the same time of senescent cell at different time points. The expression level of SASPs (senescence associated secret phenotypes) was also different in different types of senescent cells or at different time points. The GO (gene ontology) analysis revealed that the senescent cells were significantly correlated with the ossification processes, like ECM organization, cell adhesion, ossification, cartilage development, etc. Trajectory analysis showed that injury-related senescent fibroblasts (day 7 and 21) and age-related senescent fibroblasts (day 0 and 42) were in different branches. GO analysis demonstrated that injury-related senescent fibroblasts were mainly related to ossification and ECM remodeling. The KEGG (Kyoto Encyclopedia of Genes and Genomes) results revealed that the ossification was significantly corrected with protein processing in PI3K-Akt signaling, MAPK signaling, focal adhesion, etc. CONCLUSION: Consequently, we demonstrated that, unlike age-related senescence, the injury-related senescence demonstrated significantly different SASP phenotypes. The injury-related senescence of fibroblasts is associated with heterotopic ossification formation and may act through PI3K/Akt-induced SASPs.