Summary

This study identifies sex-based differences in ACL tissue at the cellular and transcriptomic level, revealing a unique PDGFRA+/TPPP3+ ligament progenitor cell population with potential implications for understanding and addressing the higher ACL tear rates seen in female athletes.

Abstract

Introduction

ACL tear rates have increased significantly over the past several decades. One discrepancy not well understood is the differences in tear rate between sexes. Women have been found to experience tear rates that are 2-8 times higher than men. However, the statistically significant difference in terms of tear rate is lost between sexes following ACL reconstruction (ACLR), suggesting an intrinsic difference between male and female ACL tissue.

Methods

ACL samples were collected from four male and five female patients (n = 9) following ACL reconstruction surgery (under IRB and ethics board approval). Live cells were collected through flow cytometry and sent for single-cell RNA-sequencing utilizing 10x Genomics 3’ kits. The datasets were analyzed using Cellranger, Freemuxlet, and Seurat, to perform quality control, cluster cells, and determine differential gene expression between male and female groups. Significant expression was set at greater than a 25% increase in expression level (log2FC > 0.32). Subpopulation characterization was performed with in situ and in vitro immunofluorescence and flow cytometry analysis. Live PDGFRA+/ TPPP3+ cells were isolated from tissue digests, collected through flow cytometry, cultured in expansion media, and differentiated using adipogenic, fibrogenic, and chondrogenic differentiation kits.

Results

We identified distinct cell types by gene expression, including fibroblasts (PDGFRa), pericytes (RGS5), endothelial cells (PECAM1), macrophages (CD86/MRC1), and T-cells (CD3E). We demonstrate five distinct fibroblast subpopulations and a unique PDGFRA+/ TPPP3+ ligament progenitor cell population (LPCs) that share several genotypic similarities with muscle fibroadipogenic progenitor stem cells (FAPs). Protein expression, both in situ and in vitro, confirmed the presence of this population in human ACL tissue. Differential gene expression analysis found upregulation of MMP14, an estrogen-sensitive protein that can cleave type 1 collagen and influence tissue stiffness, in female fibroblasts and upregulated expression of collagen genes associated with dysregulation and degradation in female LPCs. Differentiation assays identified the fibrogenic, adipogenic, and chondrogenic differentiation potential of LPCs.

Discussion

These results demonstrate that sex differences exist between male and female ACL at the cellular and transcriptomic levels. Of the five distinct fibroblast subpopulations, we identified a CD55+/PRG4+ subpopulation which may play an important role in the maintenance of intra-articular joint health, a CHI3L2+ subpopulation expressing genes associated with adipogenesis, an ACAN+ subpopulation expressing genes associated with chondrogenesis, and a unique PDGFRA+/ TPPP3+ ligament progenitor cell population (LPCs) that can differentiate into fibroblasts, adipocytes, and chondrocytes. Given that these LPCs resemble FAPs, a highly modifiable cell type in muscle, and exhibit a distinct differentiation capacity, this population could be leveraged in future studies to explore ligament regeneration and the role of different LPC pathways on ACL biomechanical properties and gain a better understanding of how to reduce ACL rupture rates, particularly in female athletes.

DISCLOSURES
The authors have no relevant disclosures.

ACKNOWLEDGMENTS
The authors would like to thank the following funding sources: UCSF Department of Orthopaedic Surgery Seed Grant 5014.