Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: “Why, Who, What, Where, When, and How” (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

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

Flexor tendon injuries are common and heal poorly owing to both the deposition of function- limiting peritendinous scar tissue and insufficient healing of the tendon itself. Therapeutic options are limited due to a lack of understanding of the cell populations that contribute to these processes. Here, we identified a bi-fated progenitor cell population that originates from the epitenon and goes on to contribute to both peritendinous fibrosis and regenerative tendon healing following acute tendon injury. Using a combination of genetic lineage tracing and single cell RNA-sequencing (scRNA-seq), we profiled the behavior and contributions of each cell fate to the healing process in a spatio-temporal manner. Branched pseudotime trajectory analysis identified distinct transcription factors responsible for regulation of each fate. Finally, integrated scRNA-seq analysis of mouse healing with human peritendinous scar tissue revealed remarkable transcriptional similarity between mouse epitenon- derived cells and fibroblasts present in human peritendinous scar tissue, which was further validated by immunofluorescent staining for conserved markers. Combined, these results clearly identify the epitenon as the cellular origin of an important progenitor cell population that could be leveraged to improve tendon healing.

/pdf/epitenon-derived-cells-comprise-a-distinct-progenitor-1g6tfw94.pdf

Epitenon-derived cells comprise a distinct progenitor population that contributes to both tendon fibrosis and regeneration following acute injury

Resident macrophages exist in a variety of tissues, including tendon, and play context-specific roles in their tissue of residence. In this study, we define the spatiotemporal distribution and phenotypic profile of tendon resident macrophages and their crosstalk with neighboring tendon fibroblasts and the extracellular matrix (ECM) during murine tendon development, growth, and homeostasis. Fluorescent imaging of cryosections revealed that F4/80+ tendon resident macrophages reside adjacent to Col1a1-CFP+ Scx-GFP+ fibroblasts within the tendon fascicle from embryonic development (E15.5) into adulthood (P56). Through flow cytometry and qPCR, we found that these tendon resident macrophages express several well-known macrophage markers, including Adgre1 (F4/80), Mrc1 (CD206), Lyve1, and Folr2, but not Ly-6C, and express the Csf1r-EGFP (“MacGreen”) reporter. The proportion of Csf1r-EGFP+ resident macrophages in relation to the total cell number increases markedly during early postnatal growth, while the density of macrophages per mm2 remains constant during this same time frame. Interestingly, proliferation of resident macrophages is higher than adjacent fibroblasts, which likely contributes to this increase in macrophage proportion. The expression profile of tendon resident macrophages also changes with age, with increased pro-inflammatory and anti-inflammatory cytokine expression in P56 compared to P14 macrophages. In addition, the expression profile of limb tendon resident macrophages diverges from that of tail tendon resident macrophages, suggesting differential phenotypes across anatomically and functionally different tendons. As macrophages are known to communicate with adjacent fibroblasts in other tissues, we conducted ligand-receptor analysis and found potential two-way signaling between tendon fibroblasts and resident macrophages. Tendon fibroblasts express high levels of Csf1, which encodes macrophage colony stimulating factor (M-CSF) that acts on the CSF1 receptor (CSF1R) on macrophages. Importantly, Csf1r-expressing resident macrophages preferentially localize to Csf1-expressing fibroblasts, supporting the “nurturing scaffold” model for tendon macrophage patterning. Lastly, we found that tendon resident macrophages express high levels of ECM-related genes, including Mrc1 (mannose receptor), Lyve1 (hyaluronan receptor), Lair1 (type I collagen receptor), Ctss (elastase), and Mmp13 (collagenase), and internalize DQ Collagen in explant cultures. Overall, our study provides insights into the potential roles of tendon resident macrophages in regulating fibroblast phenotype and the ECM during tendon growth.

/pdf/cd206-tendon-resident-macrophages-and-their-potential-3qx9spb9.pdf

CD206+ tendon resident macrophages and their potential crosstalk with fibroblasts and the ECM during tendon growth and maturation

The high incidence of incomplete or failed healing after rotator cuff repair (RCR) has led to an increased focus on the biologic factors that affect tendon-to-bone healing. Inflammation plays a critical role in the initial tendon-healing response. C-C chemokine receptor type 2 (CCR2) is a chemokine receptor linked to the recruitment of monocytes in early inflammatory stages and is associated with an increase in pro-inflammatory macrophages. The purpose of this study was to evaluate the role of CCR2 in tendon healing following RCR in C57BL/6J wildtype (WT) and CCR2-/- knockout (CCR2KO) mice in a delayed RCR model.Fifty-two 12-week-old, male mice were allocated to 2 groups (WT and CCR2KO). All mice underwent unilateral supraspinatus tendon (SST) detachment at the initial surgical procedure, followed by a delayed repair 2 weeks later. The primary outcome measure was biomechanical testing. Secondary measures included histology, gene expression analysis, flow cytometry, and gait analysis.The mean load-to-failure was 1.64 ± 0.41 N in the WT group and 2.50 ± 0.42 N in the CCR2KO group (p = 0.030). The mean stiffness was 1.43 ± 0.66 N/mm in the WT group and 3.00 ± 0.95 N/mm in the CCR2KO group (p = 0.008). Transcriptional profiling demonstrated 7 differentially expressed genes (DEGs) when comparing the CCR2KO and WT groups (p < 0.05) and significant differences in Type-I and Type-II interferon pathway scores (p < 0.01). Flow cytometry demonstrated significant differences between groups for the percentage of macrophages present (8.1% for the WT group compared with 5.8% for the CCR2KO group; p = 0.035). Gait analysis demonstrated no significant differences between groups.CCR2KO may potentially improve tendon biomechanical properties by decreasing macrophage infiltration and/or by suppressing inflammatory mediator pathways in the setting of delayed RCR.CCR2 may be a promising target for novel therapeutics that aim to decrease failure rates following RCR. 

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

Abstract Orthopedic conditions have emerged as global health concerns, impacting approximately 1.7 billion individuals worldwide. However, the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders. The advent of single-cell RNA sequencing (scRNA-seq) technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity. Nevertheless, investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges. In this comprehensive review, we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines. By utilizing these methodologies, crucial insights into the developmental dynamics, maintenance of homeostasis, and pathological processes involved in spine, joint, bone, muscle, and tendon disorders have been uncovered. Specifically focusing on the joint diseases of degenerative disc disease, osteoarthritis, and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension. These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders. 

pdf/advancing-skeletal-health-and-disease-research-with-single-1ztxsrxwou.pdf

Advancing skeletal health and disease research with single-cell RNA sequencing

During tendon healing, macrophages are thought to be a key mediator of scar tissue formation, which prevents successful functional restoration of the tendon. However, macrophages are critical for successful tendon healing as they aid in wound debridement, extracellular matrix deposition, and promote fibroblast proliferation. Recent work has sought to better define the multi-faceted functions of macrophages using depletion studies, while other studies have identified a tendon resident macrophage population. To begin to delineate the functions of tendon-resident versus circulation-derived macrophages, we examined the tendon healing phenotype in Chemokine Receptor 2 (CCR2) reporter (CCR2GFP/+), and knockout mice. CCR2 is a chemokine receptor primarily found on the surface of circulating bone marrow-derived monocytes, with CCR2 being an important mediator of macrophage recruitment to wound environments. Surprisingly, CCR2GFP/+ cells were present in the tendon during adult homeostasis, and single-cell RNA sequencing identified these cells as tendon-resident macrophages and T cells. During both homeostasis and healing, CCR2 knockout resulted in a substantial decrease in CCR2GFP+ cells and pan-macrophages. Additionally, loss of CCR2 resulted in reduced numbers of myofibroblasts and impeded functional recovery during late healing. This study highlights the heterogeneity of tendon-resident and recruited immune cells and their contributions following injury, and establishes an important role for CCR2 in modulating both the adult tendon cell environment and tendon healing process. 

<scp>CCR2</scp> is expressed by tendon resident macrophage and T cells, while <scp>CCR2</scp> deficiency impairs tendon healing via blunted involvement of tendon‐resident and circulating monocytes/macrophages

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<scp>CCR2</scp> is expressed by tendon resident macrophage and T cells, while <scp>CCR2</scp> deficiency impairs tendon healing via blunted involvement of tendon‐resident and circulating monocytes/macrophages