Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

DeepsmirUD: Prediction of Regulatory Effects on microRNA Expression Mediated by Small Molecules Using Deep Learning.

Aberrant miRNA expression has been associated with a large number of human diseases. Therefore, targeting miRNAs to regulate their expression levels has become an important therapy against diseases that stem from the dysfunction of pathways regulated by miRNAs. In recent years, small molecules have demonstrated enormous potential as drugs to regulate miRNA expression (i.e., SM-miR). A clear understanding of the mechanism of action of small molecules on the upregulation and downregulation of miRNA expression allows precise diagnosis and treatment of oncogenic pathways. However, outside of a slow and costly process of experimental determination, computational strategies to assist this on an ad hoc basis have yet to be formulated. In this work, we developed, to the best of our knowledge, the first cross-platform prediction tool, DeepsmirUD, to infer small-molecule-mediated regulatory effects on miRNA expression (i.e., upregulation or downregulation). This method is powered by 12 cutting-edge deep-learning frameworks and achieved AUC values of 0.843/0.984 and AUCPR values of 0.866/0.992 on two independent test datasets. With a complementarily constructed network inference approach based on similarity, we report a significantly improved accuracy of 0.813 in determining the regulatory effects of nearly 650 associated SM-miR relations, each formed with either novel small molecule or novel miRNA. By further integrating miRNA-cancer relationships, we established a database of potential pharmaceutical drugs from 1343 small molecules for 107 cancer diseases to understand the drug mechanisms of action and offer novel insight into drug repositioning. Furthermore, we have employed DeepsmirUD to predict the regulatory effects of a large number of high-confidence associated SM-miR relations. Taken together, our method shows promise to accelerate the development of potential miRNA targets and small molecule drugs.

Cellular characterisation of advanced osteoarthritis knee synovium.

OBJECTIVES: Osteoarthritis (OA) is increasingly recognised as a whole joint disease, with an important role for synovium. However, the repertoire of immune cells and fibroblasts that constitute OA synovium remains understudied. This study aims to characterise the cellular composition of advanced OA synovium and to explore potential correlations between different cell types and patient demographics or clinical scores. METHODS: Synovium, collected from 10 patients with advanced OA during total knee replacement surgery, was collagenase-digested, and cells were stained for flow cytometry analysis. Formalin-fixed paraffin-embedded synovium was sectioned, stained with immunofluorescence, and imaged using the multiplex Cell DIVE platform. Patient demographics and clinical scores were also collected. RESULTS: The proportion of immune cells in OA synovium varied between patients (8-38% of all cells). Macrophages and T cells were the dominant immune cell populations, together representing 76% of immune cells. Age positively correlated with the proportion of macrophages, and negatively correlated with T cells. CCR6+ T cells were found in 6/10 patients; these patients had a higher mean Kellgren-Lawrence grade across the three knee compartments. Immunofluorescence staining showed that macrophages were present in the lining as well as distributed throughout the sublining, while T and B cells were mainly localised near vessels in the sublining. Fibroblast subsets (CD45-PDPN+) based on the expression of CD34/CD90 or FAP/CD90 were identified in all patient samples, and some populations correlate with the percentage of immune cells or clinical scores. Immunofluorescence staining showed that FAP expression was particularly strong in the lining layer, but also present throughout the sublining layer. CD90 expression was exclusively found around vessels in the sublining, while CD34 was mostly found in the sublining but also occasionally in the lining layer. CONCLUSIONS: There are significant differences in the relative proportions and subsets of immune cells in OA synovium; exploratory correlative analyses suggest that these differences might be correlated with age, clinical scores, or fibroblast subsets. Additional studies are required to understand how different cell types affect OA pathobiology, and if the presence or proportion of cell subsets relates to disease phenotypes.

Research perspectives-Pipelines to human tendon transcriptomics.

Tendon transcriptomics is a rapidly growing field in musculoskeletal biology. The ultimate aim of many current tendon transcriptomic studies is characterization of in vitro, ex vivo, or in vivo, healthy, and diseased tendon microenvironments to identify the underlying pathways driving human tendon pathology. The transcriptome interfaces between genomic, proteomic, and metabolomic signatures of the tendon cellular niche and the response of this niche to stimuli. Some of the greatest bottlenecks in tendon transcriptomics relate to the availability and quality of human tendon tissue, hence animal tissues are frequently used even though human tissue is most translationally relevant. Here, we review the variability associated with human donor and procurement factors, such as whether the tendon is cadaveric or a clinical remnant, and how these variables affect the quality and relevance of the transcriptomes obtained. Moreover, age, sex, and health demographic variables impact the human tendon transcriptome. Tendons present tissue-specific challenges for cell, nuclei, and RNA extraction that include a dense extracellular matrix, low cellularity, and therefore low RNA yield of variable quality. Consideration of these factors is particularly important for single-cell and single-nuclei resolution transcriptomics due to the necessity for unbiased and representative cell or nuclei populations. Different cell, nuclei, and RNA extraction methods, library preparation, and quality control methods are used by the tendon research community and attention should be paid to these when designing and reporting studies. We discuss the different components and challenges of human tendon transcriptomics, and propose pipelines, quality control, and reporting guidelines for future work in the field.

Oral antimicrobial peptide-EGCG nanomedicines for synergistic treatment of ulcerative colitis.

The pathogenesis of ulcerative colitis (UC) is associated with severe inflammation, damaged colonic barriers, increased oxidative stress, and intestinal dysbiosis. The majority of current medications strive to alleviate inflammation but fail to target additional disease pathologies. Addressing multiple symptoms using a single 'magic bullet' remains a challenge. To overcome this, a smart epigallocatechin-3-gallate (EGCG)-loaded silk fibroin-based nanoparticle (NP) with the surface functionalization of antimicrobial peptides (Cathelicidin-BF, CBF) was constructed, which could be internalized by Colon-26 cells and RAW 264.7 macrophages with high efficiencies. The resulting CBF-EGCG-NPs efficiently restored colonic epithelial barriers by relieving oxidative stress and promoting epithelium migration. They also alleviated immune responses through downregulation of pro-inflammatory factors, upregulation of anti-inflammatory factors, M2 macrophage polarization, and lipopolysaccharide (LPS) elimination. Interestingly, oral administration of hydrogel (chitosan/alginate)-embedding CBF-EGCG-NPs could not only retard progression and treat UC, but also modulate intestinal microbiota by increasing their overall diversity and richness and augmenting the abundance of beneficial bacteria (e.g., Firmicutes and Lactobacillaceae). Our work provides a "many birds with one stone" strategy for addressing UC symptoms using a single NP-based oral platform that targets immune microenvironment modulation, LPS clearance, and microbial remodeling.

The anterior cruciate ligament in murine post-traumatic osteoarthritis: markers and mechanics.

BACKGROUND: Knee joint injuries, common in athletes, have a high risk of developing post-traumatic osteoarthritis (PTOA). Ligaments, matrix-rich connective tissues, play important mechanical functions stabilising the knee joint, and yet their role post-trauma is not understood. Recent studies have shown that ligament extracellular matrix structure is compromised in the early stages of spontaneous osteoarthritis (OA) and PTOA, but it remains unclear how ligament matrix pathology affects ligament mechanical function. In this study, we aim to investigate both structural and mechanical changes in the anterior cruciate ligament (ACL) in a mouse model of knee trauma. METHODS: Knee joints were analysed following non-invasive mechanical loading in male C57BL/6 J mice (10-week-old). Knee joints were analysed for joint space mineralisation to evaluate OA progression, and the ACLs were assessed with histology and mechanical testing. RESULTS: Joints with PTOA had a 33-46% increase in joint space mineralisation, indicating OA progression. Post-trauma ACLs exhibited extracellular matrix modifications, including COL2 and proteoglycan deposition. Additional changes included cells expressing chondrogenic markers (SOX9 and RUNX2) expanding from the ACL tibial enthesis to the mid-substance. Viscoelastic and mechanical changes in the ACLs from post-trauma knee joints included a 20-21% decrease in tangent modulus at 2 MPa of stress, a decrease in strain rate sensitivity at higher strain rates and an increase in relaxation during stress-relaxation, but no changes to hysteresis and ultimate load to failure were observed. CONCLUSIONS: These results demonstrate that ACL pathology and viscoelastic function are compromised in the post-trauma knee joint and reveal an important role of viscoelastic mechanical properties for ligament and potentially knee joint health.

Cartilage-Specific Cre Recombinase Transgenes/Alleles in the Mouse.

Cartilage is a specialized skeletal tissue with a unique extracellular matrix elaborated by its resident cells, chondrocytes. The tissue presents in several forms, including growth plate and articular cartilage, wherein chondrocytes follow a differential differentiation program and have different fates. The induction of gene modifications in cartilage specifically relies on mouse transgenes and knockin alleles taking advantages of transcriptional elements primarily active in chondrocytes at a specific differentiation stage or in a specific cartilage type. These transgenes/alleles have been widely used to study the roles of specific genes in cartilage development, adult homeostasis, and pathology. As cartilage formation is critical for postnatal life, the inactivation or significant alteration of key cartilaginous genes is often neonatally lethal and therefore hampers postnatal studies. Gold standard approaches to induce postnatal chondrocyte-specific gene modifications include the Cre-loxP and Tet-ON/OFF systems. Selecting the appropriate promoter/enhancer sequences to drive Cre expression is of crucial importance and determines the specificity of conditional gain- or loss-of-function models. In this chapter, we discuss a series of transgenes and knockin alleles that have been developed for gene manipulation in cartilage and we compare their expression patterns and efficiencies.

Meniscal and ligament modifications in spontaneous and post-traumatic mouse models of osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a whole joint disease that affects all joint tissues, with changes in the articular cartilage (AC), subchondral bone and synovium. Pathologies in menisci and ligaments, however, are rarely analysed, although both are known to play vital roles in the mechanical stability of the joint. The aim of our study was to describe the pathological changes in menisci and ligament during disease development in murine spontaneous and post-traumatic surgically induced OA and to quantify tissue mineralisation in the joint space using micro-computed tomography (µCT) imaging during OA progression. METHODS: Knees of Str/ort mice (spontaneous OA model; 26-40 weeks) and C57CBA F1 mice following destabilisation of medial meniscus (DMM) surgery (post-traumatic OA model; 8 weeks after DMM), were used to assess histological meniscal and ligament pathologies. Joint space mineralised tissue volume was quantified by µCT. RESULTS: Meniscal pathological changes in Str/ort mouse knees were associated with articular cartilage lesion severity. These meniscal changes included ossification, hyperplasia, cell hypertrophy, collagen type II deposition and Sox9 expression in the fibrous region near the attachment to the knee joint capsule. Anterior cruciate ligaments exhibited extracellular matrix changes and chondrogenesis particularly at the tibial attachment site, and ossification was seen in collateral ligaments. Similar changes were confirmed in the post-traumatic DMM model. µCT analysis showed increased joint space mineralised tissue volume with OA progression in both the post-traumatic and spontaneous OA models. CONCLUSIONS: Modifications in meniscal and ligament mineralisation and chondrogenesis are seen with overt AC degeneration in murine OA. Although the aetiology and the consequences of such changes remain unknown, they will influence stability and load transmission of the joint and may therefore contribute to OA progression. In addition, these changes may have important roles in movement restriction and pain, which represent major human clinical symptoms of OA. Description of such soft tissue changes, in addition to AC degradation, should be an important aspect of future studies in mouse models in order to furnish a more complete understanding of OA pathogenesis.

Post-traumatic osteoarthritis development is not modified by postnatal chondrocyte deletion of Ccn2.

CCN2 is a matricellular protein involved in several crucial biological processes. In particular, CCN2 is involved in cartilage development and in osteoarthritis. Ccn2 null mice exhibit a range of skeletal dysmorphisms, highlighting its importance in regulating matrix formation during development; however, its role in adult cartilage remains unclear. The aim of this study was to determine the role of CCN2 in postnatal chondrocytes in models of post-traumatic osteoarthritis (PTOA). Ccn2 deletion was induced in articular chondrocytes of male transgenic mice at 8 weeks of age. PTOA was induced in knees either surgically or non-invasively by repetitive mechanical loading at 10 weeks of age. Knee joints were harvested, scanned with micro-computed tomography and processed for histology. Sections were stained with Toluidine Blue and scored using the Osteoarthritis Research Society International (OARSI) grading system. In the non-invasive model, cartilage lesions were present in the lateral femur, but no significant differences were observed between wild-type (WT) and Ccn2 knockout (KO) mice 6 weeks post-loading. In the surgical model, severe cartilage degeneration was observed in the medial compartments, but no significant differences were observed between WT and Ccn2 KO mice at 2, 4 and 8 weeks post-surgery. We conclude that Ccn2 deletion in chondrocytes does not modify the development of PTOA in mice, suggesting that chondrocyte expression of CCN2 in adults is not a crucial factor in protecting cartilage from the degeneration associated with PTOA.This article has an associated First Person interview with the first author of the paper.