Distinct Inflammatory Macrophage Populations Sequentially Infiltrate Bone‐to‐Tendon Interface Tissue After Anterior Cruciate Ligament (ACL) Reconstruction Surgery in Mice

Abstract

Macrophages are important for repair of injured tissues, but their role in healing after surgical repair of musculoskeletal tissues is not well understood. We used single-cell RNA sequencing (RNA-seq), flow cytometry, and transcriptomics to characterize functional phenotypes of macrophages in a mouse anterior cruciate ligament reconstruction (ACLR) model that involves bone injury followed by a healing phase of bone and fibrovascular interface tissue formation that results in bone-to-tendon attachment. We identified a novel "surgery-induced" highly inflammatory CD9+ IL1+ macrophage population that expresses neutrophil-related genes, peaks 1 day after surgery, and slowly resolves while transitioning to a more homeostatic phenotype. In contrast, CX3CR1+ CCR2+ macrophages accumulated more slowly and unexpectedly expressed an interferon signature, which can suppress bone formation. Deletion of Ccr2 resulted in an increased amount of bone in the surgical bone tunnel at the tendon interface, suggestive of improved healing. The "surgery-induced macrophages" identify a new cell type in the early phase of inflammation related to bone injury, which in other tissues is dominated by blood-derived neutrophils. The complex patterns of macrophage and inflammatory pathway activation after ACLR set the stage for developing therapeutic strategies to target specific cell populations and inflammatory pathways to improve surgical outcomes.

Responding to ACL Injury and its Treatments: Comparative Gene Expression between Articular Cartilage and Synovium

“…However, immune activation can take several forms, and not all forms of inflammation are equivalent. Diversity of immune function is well characterized in wound healing in tissues such as skin and lung, but far less is known about the diversity of immune mechanisms that regulate healing in load-bearing musculoskeletal tissues after injury [31][32][33]. The current study showed that following every form of surgical intervention, the cartilage responded with greater immune activation than the synovium in mid-term osteoarthritis after adjusting for baseline differences between the tissues.…”

“…Beyond modeling the joint response to ACL injury, previous animal models of ACL transection followed by ligament reconstruction have featured RNA sequencing of several knee joint tissues (e.g., bone, tendon, cartilage, synovium) [33,41,42]. characterized cartilage and synovium transcriptomes in a porcine model at one and four weeks following ACL transection, with and without reconstruction [41,42].…”

Effect of CCR2 Knockout on Tendon Biomechanical Properties in a Mouse Model of Delayed Rotator Cuff Repair

“…Finally, H2-Aa is a histocompatibility class-II antigen A, which enables peptide antigen binding activity and is involved in the response to interferon gamma. Although further studies are necessary to fully elucidate the roles of these individual genes, addressing the interferon response in particular could be promising for further investigation, as interferons have previously been reported to promote injury-associated inflammation, suppress collagen synthesis, and inhibit fibroblast proliferation [45][46][47][48] .…”

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how)

“…ACL injury models fall into three categories: (i) surgical ACL transection, (ii) noninvasive ACL rupture, and (iii) ACL transection and reconstruction. Due to the limited size, direct surgical repair of the ACL is less commonly performed in rodents 14,15,18–28,38 . ACL transection procedures offer great control over the extent of the injury while minimizing damage to adjacent tissues.…”