Single-Cell RNA-Seq Reveals Transcriptomic Heterogeneity and Post-Traumatic Osteoarthritis-Associated Early Molecular Changes in Mouse Articular Chondrocytes

Articular cartilage is a connective tissue lining the surfaces of synovial joints. When the cartilage severely wears down, it leads to osteoarthritis (OA), a debilitating disease that affects millions of people globally. The articular cartilage is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes with varying morphology and potentially different functions. Elucidating the molecular and functional profiles of various chondrocyte subtypes and understanding the interplay between these chondrocyte subtypes and other cell types in the joint will greatly expand our understanding of joint biology and OA pathology. Although recent advances in high-throughput OMICS technologies have enabled molecular-level characterization of tissues and organs at an unprecedented resolution, thorough molecular profiling of articular chondrocytes has not yet been undertaken, which may be in part due to the technical difficulties in isolating chondrocytes from dense cartilage ECM. In this study, we profiled articular cartilage from healthy and injured mouse knee joints at a single-cell resolution and identified nine chondrocyte subtypes with distinct molecular profiles and injury-induced early molecular changes in these chondrocytes. We also compared mouse chondrocyte subpopulations to human chondrocytes and evaluated the extent of molecular similarity between mice and humans. This work expands our view of chondrocyte heterogeneity and rapid molecular changes in chondrocyte populations in response to joint trauma and highlights potential mechanisms that trigger cartilage degeneration.

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Signaling and transcriptional networks governing late synovial joint development

10.1101/2021.08.25.457383 ◽

2021 ◽

Author(s):

Qin Bian ◽

Yu-Hao Cheng ◽

Emily Y. Su ◽

Yuqi Tan ◽

Dong Won Kim ◽

...

Keyword(s):

Articular Cartilage ◽

Single Cell ◽

Articular Chondrocytes ◽

Cruciate Ligament ◽

Fibrous Tissue ◽

Cell Types ◽

Transcriptional Networks ◽

Synovial Joint ◽

Joint Development ◽

Cell Type Specific

Background: During synovial joint development, cavitation marks the end of the emergence of new cell types and the onset of the consolidation of cell type specific programs. However, the transcriptional programs that regulate this crucial stage prior to joint maturation are incompletely characterized. Gdf5-lineage cells give rise to the majority of joint constituents such as articular cartilage, meniscus, ligament, and tendon. Therefore, to explore pre-maturation of the synovial joint, we performed single cell RNA-Seq analysis of 1,306 Gdf5-lineage cells from the murine knee joint at E17.5. Results: Using computational analytics and in situ hybridization, we identified nine sub-states contributing to articular cartilage, meniscus, cruciate ligament, synovium, lining, and surrounding fibrous tissue. We identified a common progenitor population that is predicted to give rise to ligamentaocytes, articular chondrocytes, and lining cells. We further found that while a large number of signaling pathways orchestrate the differentiation of this progenitor to either ligamentocytes or to lining cells, only continued FGF signaling guides these cells to a default chondrocyte fate. Conclusions: Our single cell transcriptional atlas is a resource that can be used to better understand and further study synovial joint development and the reactivation of embryonic programs in diseases such as osteoarthritis.

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

Disease Models & Mechanisms ◽

10.1242/dmm.044719 ◽

2020 ◽

Vol 13 (7) ◽

pp. dmm044719 ◽

Cited By ~ 1

Author(s):

Craig M. Keenan ◽

Lorenzo Ramos-Mucci ◽

Ioannis Kanakis ◽

Peter I. Milner ◽

Andrew Leask ◽

...

Keyword(s):

Articular Chondrocytes ◽

Cartilage Degeneration ◽

Research Society ◽

Knee Joints ◽

Matricellular Protein ◽

Micro Computed Tomography ◽

Non Invasive ◽

Post Surgery ◽

Osteoarthritis Research Society ◽

Post Traumatic

ABSTRACTCCN2 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.

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Post-traumatic osteoarthritis development is not modified by postnatal chondrocyte deletion of CCN2

10.1101/2020.02.28.969105 ◽

2020 ◽

Author(s):

Craig M Keenan ◽

Lorenzo Ramos-Mucci ◽

Ioannis Kanakis ◽

Peter I Milner ◽

Andrew Leask ◽

...

Keyword(s):

Articular Chondrocytes ◽

Cartilage Degeneration ◽

Knee Joints ◽

Matricellular Protein ◽

Non Invasive ◽

Post Surgery ◽

Surgical Model ◽

Summary Statement ◽

Post Traumatic

AbstractCCN2 is a matricellular protein involved in several critical 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-CT, and processed for histology. Sections were stained with toluidine blue and scored using the OARSI grading system. In the non-invasive model cartilage lesions were present in the lateral femur but no significant differences were observed between wildtype (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 did not modify the development of PTOA in mice, suggesting that chondrocyte expression of CCN2 in adults is not a critical player in protecting cartilage from the degeneration associated with PTOA.Summary StatementPost-natal deletion of CCN2 in chondrocytes does not affect the development of post-traumatic osteoarthritis in mice.

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Genetic Deletion of Interleukin-15 Is Not Associated with Major Structural Changes Following Experimental Post-Traumatic Knee Osteoarthritis in Rats

Applied Sciences ◽

10.3390/app11157118 ◽

2021 ◽

Vol 11 (15) ◽

pp. 7118

Author(s):

Ermina Hadzic ◽

Garth Blackler ◽

Holly Dupuis ◽

Stephen James Renaud ◽

Christopher Thomas Appleton ◽

...

Keyword(s):

Articular Cartilage ◽

Subchondral Bone ◽

Structural Changes ◽

Cartilage Damage ◽

Wild Type ◽

Significant Difference ◽

Post Traumatic ◽

Interleukin 15 ◽

Joint Trauma ◽

Surgically Induced

Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease, leading to articular cartilage breakdown, osteophyte formation, and synovitis, caused by an initial joint trauma. Pro-inflammatory cytokines increase catabolic activity and may perpetuate inflammation following joint trauma. Interleukin-15 (IL-15), a pro-inflammatory cytokine, is increased in OA patients, although its roles in PTOA pathophysiology are not well characterized. Here, we utilized Il15 deficient rats to examine the role of IL-15 in PTOA pathogenesis in an injury-induced model. OA was surgically induced in Il15 deficient Holtzman Sprague-Dawley rats and control wild-type rats to compare PTOA progression. Semi-quantitative scoring of the articular cartilage, subchondral bone, osteophyte size, and synovium was performed by two blinded observers. There was no significant difference between Il15 deficient rats and wild-type rats following PTOA-induction across articular cartilage damage, subchondral bone damage, and osteophyte scoring. Similarly, synovitis scoring across six parameters found no significant difference between genetic variants. Overall, IL-15 does not appear to play a key role in the development of structural changes in this surgically-induced rat model of PTOA.

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Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.4.7897185 ◽

1995 ◽

Vol 43 (4) ◽

pp. 447-457 ◽

Cited By ~ 57

Author(s):

D M Salter ◽

J L Godolphin ◽

M S Gourlay

Keyword(s):

Articular Cartilage ◽

Growth Plate ◽

Articular Chondrocytes ◽

Knee Joints ◽

Matrix Composition ◽

Epiphyseal Growth Plate ◽

Surface Receptors ◽

Cartilage Differentiation ◽

Health And Disease ◽

And Function

During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

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Single-Cell RNA Sequencing With Combined Use of Bulk RNA Sequencing to Reveal Cell Heterogeneity and Molecular Changes at Acute Stage of Ischemic Stroke in Mouse Cortex Penumbra Area

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.624711 ◽

2021 ◽

Vol 9 ◽

Author(s):

Kang Guo ◽

Jianing Luo ◽

Dayun Feng ◽

Lin Wu ◽

Xin Wang ◽

...

Keyword(s):

Ischemic Stroke ◽

Single Cell ◽

Rna Sequencing ◽

Therapeutic Targets ◽

Cell Types ◽

Acute Stage ◽

Gene Markers ◽

Molecular Changes ◽

Early Stages ◽

The Impact

Stroke has been the leading cause of adult morbidity and mortality over the past several years. After an ischemic stroke attack, many dormant or reversibly injured brain cells exist in the penumbra area. However, the pathological processes and unique cell information in the penumbra area of an acute ischemic stroke remain elusive. We applied unbiased single cell sequencing in combination with bulk RNA-seq analysis to investigate the heterogeneity of each cell type in the early stages of ischemic stroke and to detect early possible therapeutic targets to help cell survival. We used these analyses to study the mouse brain penumbra during this phase. Our results reveal the impact of ischemic stroke on specific genes and pathways of different cell types and the alterations of cell differentiation trajectories, suggesting potential pathological mechanisms and therapeutic targets. In addition to classical gene markers, single-cell genomics demonstrates unique information on subclusters of several cell types and metabolism changes in an ischemic stroke. These findings suggest that Gadd45b in microglia, Cyr61 in astrocytes, and Sgk3 in oligodendrocytes may play a subcluster-specific role in cell death or survival in the early stages of ischemic stroke. Moreover, RNA-scope multiplex in situ hybridization and immunofluorescence staining were applied to selected target gene markers to validate and confirm the existence of these cell subtypes and molecular changes during acute stage of ischemic stroke.

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Rapamycin microparticles induce autophagy, prevent senescence and are effective in treatment of Osteoarthritis

10.1101/2021.07.20.453073 ◽

2021 ◽

Author(s):

Kaamini M Dhanabalan ◽

Ameya A Dravid ◽

Smriti Agarwal ◽

Ramanath K Sharath ◽

Ashok K Padmanabhan ◽

...

Keyword(s):

Articular Chondrocytes ◽

Symptomatic Relief ◽

Cartilage Damage ◽

Treatment Strategies ◽

Standard Of Care ◽

Cartilage Degeneration ◽

Current Standard ◽

Disease Modifying Drugs ◽

Post Traumatic ◽

Sulphated Glycosaminoglycans

Trauma to the knee joint is associated with significant cartilage degeneration and erosion of subchondral bone, which eventually leads to osteoarthritis (OA), resulting in substantial morbidity and healthcare burden. With no disease-modifying drugs in clinics, the current standard of care focuses on symptomatic relief and viscosupplementation. Modulation of autophagy and targeting senescence pathways are emerging as potential treatment strategies. Rapamycin has shown promise in OA disease amelioration by autophagy upregulation, yet its clinical use is hindered by difficulties in achieving therapeutic concentrations, necessitating multiple weekly injections. Here, we have synthesized rapamycin - loaded poly (lactic-co-glycolic acid) microparticles (RMPs) that induced autophagy, prevented senescence and sustained sulphated glycosaminoglycans(sGAG) production in primary human articular chondrocytes from OA patients. RMPs were potent, nontoxic, and exhibited high retention time (up to 35 days) in mice joints. Intra-articular delivery of RMPs effectively mitigated cartilage damage and inflammation in surgery-induced OA when administered as a prophylactic or therapeutic regimen. Together, our studies demonstrate the feasibility of using RMPs as a potential clinically translatable therapy to prevent and treat post-traumatic osteoarthritis.

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Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis

Osteoarthritis and Cartilage ◽

10.1016/j.joca.2019.03.001 ◽

2019 ◽

Vol 27 (7) ◽

pp. 1084-1093 ◽

Cited By ~ 3

Author(s):

I. Takahashi ◽

T. Matsuzaki ◽

H. Kuroki ◽

M. Hoso

Keyword(s):

Articular Cartilage ◽

Rat Model ◽

Cartilage Degeneration ◽

Knee Joints ◽

Monosodium Iodoacetate ◽

Articular Cartilage Degeneration

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Global Gene Expression Analysis Identifies Age-Related Differences in Knee Joint Transcriptome during the Development of Post-Traumatic Osteoarthritis in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms21010364 ◽

2020 ◽

Vol 21 (1) ◽

pp. 364 ◽

Cited By ~ 3

Author(s):

Aimy Sebastian ◽

Deepa K. Murugesh ◽

Melanie E. Mendez ◽

Nicholas R. Hum ◽

Naiomy D. Rios-Arce ◽

...

Keyword(s):

Knee Joint ◽

Age Groups ◽

Knee Joints ◽

Compression Injury ◽

Post Injury ◽

Transcriptional Responses ◽

Molecular Changes ◽

Age Related ◽

Post Traumatic ◽

Old Mice

Aging and injury are two major risk factors for osteoarthritis (OA). Yet, very little is known about how aging and injury interact and contribute to OA pathogenesis. In the present study, we examined age- and injury-related molecular changes in mouse knee joints that could contribute to OA. Using RNA-seq, first we profiled the knee joint transcriptome of 10-week-old, 62-week-old, and 95-week-old mice and found that the expression of several inflammatory-response related genes increased as a result of aging, whereas the expression of several genes involved in cartilage metabolism decreased with age. To determine how aging impacts post-traumatic arthritis (PTOA) development, the right knee joints of 10-week-old and 62-week-old mice were injured using a non-invasive tibial compression injury model and injury-induced structural and molecular changes were assessed. At six-week post-injury, 62-week-old mice displayed significantly more cartilage degeneration and osteophyte formation compared with young mice. Although both age groups elicited similar transcriptional responses to injury, 62-week-old mice had higher activation of inflammatory cytokines than 10-week-old mice, whereas cartilage/bone metabolism genes had higher expression in 10-week-old mice, suggesting that the differential expression of these genes might contribute to the differences in PTOA severity observed between these age groups.

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