Synovial Fluid Immune Cell Composition Following Intraarticular Fracture May Contribute to Posttraumatic Osteoarthritis

Abstract

Intra-articular ankle fracture (IAF) often leads to post-traumatic osteoarthritis (PTOA), resulting in significant long-term morbidity. While previous research has focused on the inflammatory cytokines and matrix metalloproteinases within the synovial fluid fracture hematoma (SFFH), the immune cell populations within SFFH that contribute to PTOA development remain underexplored. This study aimed to characterize the immune cell populations in SFFH to better understand their role in the inflammatory response and potential for inducing lasting cartilage damage. Twenty-four patients with IAF underwent surgical ankle aspiration to collect SFFH, which was analyzed using polychromatic flow cytometry. The analysis revealed that 72.8% of the CD45+ cells were lymphocytes, predominantly CD3+ T cells (76.5%), with 42.1% being CD4+ and 39.2% CD8+ T cells. Additionally, monocytes accounted for 21.2% of CD45+ cells, with small populations of natural killer cells and myeloid-derived suppressor cells also present. These findings emphasize the predominance of T cells, particularly CD4+ subsets, in the immune response following IAF. Understanding these dynamics is essential for developing targeted interventions to prevent PTOA. Future research should focus on elucidating the specific roles of these immune cell populations in PTOA progression and exploring potential therapeutic strategies.

Keywords: T cell subsets; adaptive immune response; cartilage damage; cytokines; flow cytometry; immune cell profiling; inflammatory response; intra-articular ankle fracture (IAF); post-traumatic osteoarthritis (PTOA); synovial fluid fracture hematoma (SFFH).

Figure 1

Polychromatic Flow Cytometry. Representative density plots and corresponding gating strategy used for cell population identification. Example obtained from one patient sample of SFFH. Sequential 2-dimensional hierarchical gating used to access subset of Monocytes, Lymphocytes, Myeloid-Derived Suppressor, and Dendritic cells. Sequential 2-dimensional gates are drawn using black text on the axes labels, bold black text above each plot indicates parent population (gate), red arrows indicate parent and child gates, bold red text is used to label populations identified by each respective plot. (A) Gates used in Pre-processing analysis include Time (Time versus FSC) to remove clogs/air bubbles, FSC and SSC singlets (FSC-W versus FSC-H and SSC-W versus SSC-H) to remove aggregates, Dead Cell exclusion using Zombie gating, CD235a used to remove red blood cells contamination, followed by identifying immune cells using CD45+ and Scatter gates to identify Lymphocytes (low FSC & SSC) and Monocytes (high FSC & SSC). (B) Lymphocytes were identified using scatter gating (FSC versus SSC), subsets of Lymphocytes were identified as follows: T cells (CD3+, CD3 versus CD19), B cells (CD3-CD19+), NK cells (CD3-CD19-CD16+56+), and NKT cells (CD3+CD56+). Cytotoxic T cells (Tc) and Helper T cells (Th) subsets of T Lymphocytes were identified as CD4-CD8+ (Tc) and CD4+CD8- (Th) using CD4 versus CD8 gated on T cells. Subsets of Th and Tc were identified as Naïve (N) (CD45RA+CCR7+); Central Memory (CM)(CD45RA-CCR7+); Effector Memory (EM) (CD45RA-CCR7-); and TEMRA (E) (CD45RA+CCR7-). Activation of Th (ATh) and Tc (ATc) was determined by HLA-DR+. (C) Monocytes were identified using scatter gating (FSC versus SSC), CD14 versus CD16 was used to identify subsets of Monocytes: CD14+CD16- (Classical Monocytes, M1), CD14-CD16+ (Non-Classical Monocytes, M2), CD14+CD16+ (Intermediate Monocytes). Myeloid-Derived Suppressor cells (MDSCs) were identified as Lin-(CD3-CD19-CD16-CD56-)HLA-DRlowCD14+ [31]. Dendritic Cells (DC) were identified as Lin-(CD3-CD19-CD16-CD56-) HLA-DR+.

Figure 2

Immune Profile of SFFH. Bar charts with dots representing percent presence of CD45+ gated subgroups (left) and CD3+ gated (right) subgroups within individual samples of SFFH. Error bars represent standard error of the mean.

Figure 3

Time dependent immune profile of SFFH. Scatter plots showing trends in percent of CD45+ (left) and CD3+ (right) subtypes depending on time from injury to SFFH aspiration. NS; not significant.