Sex-specific effects of injury and beta-adrenergic activation on metabolic and inflammatory mediators in a murine model of post-traumatic osteoarthritis.

OBJECTIVE: Metabolic processes are intricately linked to the resolution of innate inflammation and tissue repair, two critical steps for treating post-traumatic osteoarthritis (PTOA). Based on lipolytic and immunoregulatory actions of norepinephrine, we hypothesized that intra-articular ß-adrenergic receptor (ßAR) stimulation would suppress PTOA-associated inflammation in the infrapatellar fat pad (IFP) and synovium. DESIGN: We used the ßAR agonist isoproterenol to perturb intra-articular metabolism 3.5 weeks after applying a non-invasive single-load compression injury to knees of 12-week-old male and female mice. We examined the acute effects of intra-articular isoproterenol treatment relative to saline on IFP histology, multiplex gene expression of synovium-IFP tissue, synovial fluid metabolomics, and mechanical allodynia. RESULTS: Injured knees developed PTOA pathology characterized by heterotopic ossification, articular cartilage loss, and IFP atrophy and fibrosis. Isoproterenol suppressed the upregulation of pro-fibrotic genes and downregulated the expression of adipose genes and pro-inflammatory genes (Adam17, Cd14, Icam1, Csf1r, and Casp1) in injured joints of female (but not male) mice. Analysis of published single-cell RNA-seq data identified elevated catecholamine-associated gene expression in resident-like synovial-IFP macrophages after injury. Injury substantially altered synovial fluid metabolites by increasing amino acids, peptides, sphingolipids, phospholipids, bile acids, and dicarboxylic acids, but these changes were not appreciably altered by isoproterenol. Intra-articular injection of either isoproterenol or saline increased mechanical allodynia in female mice, whereas neither substance affected male mice. CONCLUSIONS: Acute ßAR activation altered synovial-IFP transcription in a sex and injury-dependent manner, suggesting that women with PTOA may be more sensitive than men to treatments targeting sympathetic neural signaling pathways.

Effect of biological sex and short-term high-fat diet on cellular proliferation, ribosomal biogenesis, and targeted protein abundance in murine articular cartilage.

Objective: To identify factors contributing to sex-differences in OA risk by evaluating the short-term effect of high-fat (HF) diet on sex-specific changes in cartilage cell proliferation, ribosomal biogenesis, and targeted extra-cellular and cellular protein abundance. Materials and methods: Knee cartilage was harvested to the subchondral bone from 20-week-old female and male C57BL/6J mice fed a low-fat or HF diet for 4 weeks and labeled with deuterium oxide for 1, 3, 5, 7, 15, or 21 days. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Protein concentration was measured using targeted high resolution accurate mass spectrometry. Results: HF diet increased the maximal deuterium incorporation into DNA from approximately 40 to 50%, albeit at a slower rate. These findings, which were magnified in female versus male mice, indicate a greater number of proliferating cells with longer half-lives under HF diet conditions. HF diet caused distinct sex-dependent effects on deuterium incorporation into RNA, increasing the fraction of ribosomes undergoing biogenesis in male mice and doubling the rate of ribosome biogenesis in female mice. HF diet altered cartilage protein abundance similarly in both sexes, except for matrilin-3, which was more abundant in HF versus LF conditions in female mice only. Overall, HF diet treatment had a stronger effect than sex on cartilage protein abundance, with most changes involving extracellular matrix and matrix-associated proteins. Conclusions: Short-term HF diet broadly altered cartilage matrix protein abundance, while sex-dependent effects primarily involved differences in cell proliferation and ribosomal biogenesis.

Teaming up to overcome challenges toward translation of new therapeutics for osteoarthritis.

As a leading global cause of musculoskeletal-related disability, osteoarthritis (OA) represents a public health urgency. Understanding of disease pathogenesis has advanced substantially in the past decade, yet no disease-modifying therapeutics have advanced to the clinic. To address this challenge, the CARE-AP (Cartilage Repair strategies to alleviate Arthritis Pain) collaborative research team was convened to bring together relevant multidisciplinary expertise and perspectives from across the VA research community nationwide. The first CARE-AP Annual Research Symposium took place (virtually) in February 2022 with roughly 90 participants. A number of innovative and therapeutic strategies were discussed, including siRNA approaches coupled with novel nanoparticle-based delivery systems, cellular engineering approaches to develop reparative cells that can probe the joint environment and respond to disease-specific cues, and novel biofabrication techniques to improve tissue engineering and effect "biological joint replacement." In addition, challenges and advances in rehabilitation approaches, imaging outcomes, and clinical studies were presented, which were integrated into a framework of recommendations for running "preclinical trials" to improve successful clinical translation.

Imaging mass cytometry reveals tissue-specific cellular immune phenotypes in the mouse knee following ACL injury.

Objective: To develop an imaging mass cytometry method for identifying complex cell phenotypes, inter-cellular interactions, and population changes in the synovium and infrapatellar fat pad (IFP) of the mouse knee following a non-invasive compression injury. Design: Fifteen male C57BL/6 mice were fed a high-fat diet for 8 weeks prior to random assignment to sham, 0.88 â€‹mm, or 1.7 â€‹mm knee compression displacement at 24 weeks of age. 2-weeks after loading, limbs were prepared for histologic and imaging mass cytometry analysis, focusing on myeloid immune cell populations in the synovium and IFP. Results: 1.7 â€‹mm compression caused anterior cruciate ligament (ACL) rupture, development of post-traumatic osteoarthritis, and a 2- to 3-fold increase in cellularity of synovium and IFP tissues compared to sham or 0.88 â€‹mm compression. Imaging mass cytometry identified 11 myeloid cell subpopulations in synovium and 7 in IFP, of which approximately half were elevated 2 weeks after ACL injury in association with the vasculature. Notably, two monocyte/macrophage subpopulations and an MHC IIhi population were elevated 2-weeks post-injury in the synovium but not IFP. Vascular and immune cell interactions were particularly diverse in the synovium, incorporating 8 unique combinations of 5 myeloid cell populations, including a monocyte/macrophage population, an MHC IIhi population, and 3 different undefined F4/80+ myeloid populations. Conclusions: Developing an imaging mass cytometry method for the mouse enabled us to identify a diverse array of synovial and IFP vascular-associated myeloid cell subpopulations. These subpopulations were differentially elevated in synovial and IFP tissues 2-weeks post injury, providing new details on tissue-specific immune regulation.