ABSTRACT: The healing of ligaments is a complex and highly regulated process, including an early stage, a repair stage, and a remodeling stage. Increasing concerns have been attached to the concept of cell heterogeneity which is of great significance for understanding the complicated biological functions of tissues. Meanwhile, it also provides important insights for the development of novel therapies for disease management. Single-cell RNA sequencing (scRNAseq) is a technique employed to identify the molecular heterogeneity of a single cell in tissue by detecting its transcriptome. The current paper conducted in-depth research on the anterior cruciate ligament tissues of three patients and three healthy individuals who were injured one week, three weeks, and six months later using the scRNAseq technique. A profound and unbiased analysis was performed on the gene expression of 83 195 single cells in vitro collected from the 6 cases, to identify potential cells or genes that may be related to tissue recovery. Additionally, immunohistochemistry was adopted to verify the findings of scRNAseq approach. In this research, we formally distinguish between tenocytes and fibroblasts for the first time. In addition to fibroblasts, macrophages, endothelial cells, stromal cells, epithelial cells, T cells, B cells, chondrocytes, and monocytes, the anterior cruciate ligament tissues have at least additional 10 types of tendon cell populations, including tenocytes A, B, C, D, and E, fibroblasts, astrocytes, monocytes, myeloid cells, and stromal cells. These findings indicated the existence of a wide variety of specialized tenocytes and opened up a new way for subsequent investigations on cell therapy of tendon diseases.
