Loss of periostin function impairs ligament fibroblast activity and facilitates ROS-mediated cellular senescence.

Anterior cruciate ligament (ACL) injuries pose a significant challenge due to their limited healing potential, often resulting in premature arthritis. The factors and mechanisms contributing to this inadequate healing process remain elusive. During the acute phase of injury, ACL tissues express elevated periostin levels that decline over time. The functional significance of periostin in ligament biology remains understudied. In this study, we investigated the functional and mechanistic implications of periostin deficiency in ACL biology, utilizing ligament fibroblasts derived from patients and a murine model of ACL rupture. Our investigations unveiled that periostin knockdown compromised fibroblast growth characteristics, hindered the egress of progenitor cells from explants, and arrested cell-cycle progression, resulting in the accumulation of cells in the G0/G1 phase and moderate apoptosis. Concurrently, a significant reduction in the expression of cell-cycle and matrix-related genes was observed. Moreover, periostin deficiency triggered apoptosis through STAT3Y705/p38MAPK signaling and induced cellular senescence through increased production of reactive oxygen species (ROS). Mechanistically, inhibition of ROS production mitigated cell senescence in these cells. Notably, in vivo data revealed that ACL in Postn-/- mice exhibited a higher tearing frequency than wild-type mice under equivalent loading conditions. Furthermore, injured ACL with silenced periostin expression, achieved through nanoparticle-siRNA complex delivery, displayed an elevated propensity for apoptosis and senescence compared to intact ACL in C57BL/6 mice. Together, our findings underscore the pivotal role of periostin in ACL health, injury, and potential for healing.

Osteoarthriris year in review 2023: genetics, genomics, and epigenetics.

OBJECTIVE: To elucidate the scientific advances made in the last 12 months within the realm of osteoarthritis genetics, genomics, and epigenetics. This review paper highlights major research publications that enhance our current understanding of the role of genetics, genomics, and epigenetics in osteoarthritis. METHODS: A systematic literature search was conducted on pubmed.ncbi.nlm.nih.gov on "March 17, 2023", using the following keywords: "osteoarthritis" in combination with any of these terms: "genetic(s)", "mutation(s)", "genomic(s)", "epigenetic(s)", "DNA methylation", "noncoding RNA", "lncRNA", "circular RNA", "microRNA", "transcriptomic(s)", "RNA sequencing", "single cell RNA sequencing", or "single nucleus RNA sequencing". The selection comprised original research articles published in the English language between the OARSI congresses of 2022 and 2023. RESULTS: A total of 2178 research articles were identified, which subsequently reduced to 67 unique articles relevant to the field. Current trends in osteoarthritis genetics research involve meta-analyses of various cohorts to explore the impact of gene variants on osteoarthritis-related outcomes, such as pain. Early developmental changes within the joint were also found to influence osteoarthritis through genetic variations. Researchers also prioritize testing the mechanisms and functions of miRNAs, circRNAs, and lncRNAs. Potential drug targets began to emerge; however, independent validation studies are lacking. Single cell RNA sequencing studies revealed unique immune cell populations in the knee; however, no study reported single nucleus RNA sequencing analysis. CONCLUSIONS: This review focused on recent advances in the above-mentioned themes within the field of osteoarthritis. These advances improve our understanding of the disease's complexity and guide us toward functional assessments of genetic/epigenetic outcomes and toward their translational and clinical applications.

Three decades of advancements in osteoarthritis research: insights from transcriptomic, proteomic, and metabolomic studies.

OBJECTIVE: Osteoarthritis (OA) is a complex disease involving contributions from both local joint tissues and systemic sources. Patient characteristics, encompassing sociodemographic and clinical variables, are intricately linked with OA rendering its understanding challenging. Technological advancements have allowed for a comprehensive analysis of transcripts, proteomes and metabolomes in OA tissues/fluids through omic analyses. The objective of this review is to highlight the advancements achieved by omic studies in enhancing our understanding of OA pathogenesis over the last three decades. DESIGN: We conducted an extensive literature search focusing on transcriptomics, proteomics and metabolomics within the context of OA. Specifically, we explore how these technologies have identified individual transcripts, proteins, and metabolites, as well as distinctive endotype signatures from various body tissues or fluids of OA patients, including insights at the single-cell level, to advance our understanding of this highly complex disease. RESULTS: Omic studies reveal the description of numerous individual molecules and molecular patterns within OA-associated tissues and fluids. This includes the identification of specific cell (sub)types and associated pathways that contribute to disease mechanisms. However, there remains a necessity to further advance these technologies to delineate the spatial organization of cellular subtypes and molecular patterns within OA-afflicted tissues. CONCLUSIONS: Leveraging a multi-omics approach that integrates datasets from diverse molecular detection technologies, combined with patients' clinical and sociodemographic features, and molecular and regulatory networks, holds promise for identifying unique patient endophenotypes. This holistic approach can illuminate the heterogeneity among OA patients and, in turn, facilitate the development of tailored therapeutic interventions.

Evolution and advancements in genomics and epigenomics in OA research: How far we have come.

OBJECTIVE: Osteoarthritis (OA) is the most prevalent musculoskeletal disease affecting articulating joint tissues, resulting in local and systemic changes that contribute to increased pain and reduced function. Diverse technological advancements have culminated in the advent of high throughput "omic" technologies, enabling identification of comprehensive changes in molecular mediators associated with the disease. Amongst these technologies, genomics and epigenomics - including methylomics and miRNomics, have emerged as important tools to aid our biological understanding of disease. DESIGN: In this narrative review, we selected articles discussing advancements and applications of these technologies to OA biology and pathology. We discuss how genomics, deoxyribonucleic acid (DNA) methylomics, and miRNomics have uncovered disease-related molecular markers in the local and systemic tissues or fluids of OA patients. RESULTS: Genomics investigations into the genetic links of OA, including using genome-wide association studies, have evolved to identify 100+ genetic susceptibility markers of OA. Epigenomic investigations of gene methylation status have identified the importance of methylation to OA-related catabolic gene expression. Furthermore, miRNomic studies have identified key microRNA signatures in various tissues and fluids related to OA disease. CONCLUSIONS: Sharing of standardized, well-annotated omic datasets in curated repositories will be key to enhancing statistical power to detect smaller and targetable changes in the biological signatures underlying OA pathogenesis. Additionally, continued technological developments and analysis methods, including using computational molecular and regulatory networks, are likely to facilitate improved detection of disease-relevant targets, in-turn, supporting precision medicine approaches and new treatment strategies for OA.

Novel mechanistic role of Kif26b in adipogenic differentiation of murine multipotent stromal cells.

Emerging evidence delineates that obesity, a complex metabolic disorder, impairs the structure and function of stromal cells residing in various tissues. The exuberant adipose tissue mass observed in obesity is, in part, associated with hyperplasia of adipocytes resulting from recruitment of multipotent stromal cells within the stromal vascular fraction of adipose tissues. However, a clear understanding of the causal role of stromal cells and biological factors in obesity is lacking. In our quest to understanding the role of kinesin family member 26B (KIF26B), we found that KIF26B regulates osteogenic and chondrogenic differentiation of stromal/progenitor cells. In this study, we sought to examine the effects of Kif26b loss-of-function on adipogenic differentiation of murine C3H10T1/2 multipotent stromal cells. In vitro loss-of-function studies demonstrated that Kif26b knockdown by lentivirus mediated shRNA markedly dampened the differentiation potential of C3H10T1/2 cells to adipocytes and suppressed the expression of adipogenesis-related genes e.g., Pparg, C/ebpα, Fabp4 and Adipoq. Analysis of cell cycle revealed that Kif26b knockdown resulted in elevated expression of cyclins (Ccnd1, Ccnb1, Ccna2) along with rapid cell cycle progression from G0/G1 to S and G2 phases. Mechanistically, reduced adipogenic differentiation of Kif26b-deficient cells was partly dependent on PPARγ, a key transcription factor implicated in adipogenesis. This observation was experimentally supported as loss of adipogenesis was partially rescued by the addition of PPARγ agonist, rosiglitazone in Kif26b-deficient cells. We further found that silencing of Kif26b lessened the protein levels of phospho-AKT(Ser473), phospho-S6(Ser235/236), and phospho-mTOR(Ser2448), the major component of AKT/mTOR complex 1 (mTORC1) signaling at the basal level. Together, these data define a novel role of Kif26b in regulating the commitment of C3H10T1/2 multipotent stromal cells to the adipocyte lineage and provide a practical framework for further experiments to establish its therapeutic potential for the treatment of problems associated with adipogenesis such as obesity at the cellular and molecular level.

Single Cell Omics for Musculoskeletal Research.

PURPOSE OF REVIEW: The ability to analyze the molecular events occurring within individual cells as opposed to populations of cells is revolutionizing our understanding of musculoskeletal tissue development and disease. Single cell studies have the great potential of identifying cellular subpopulations that work in a synchronized fashion to regenerate and repair damaged tissues during normal homeostasis. In addition, such studies can elucidate how these processes break down in disease as well as identify cellular subpopulations that drive the disease. This review highlights three emerging technologies: single cell RNA sequencing (scRNA-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and Cytometry by Time-Of-Flight (CyTOF) mass cytometry. RECENT FINDINGS: Technological and bioinformatic tools to analyze the transcriptome, epigenome, and proteome at the individual cell level have advanced rapidly making data collection relatively easy; however, understanding how to access and interpret the data remains a challenge for many scientists. It is, therefore, of paramount significance to educate the musculoskeletal community on how single cell technologies can be used to answer research questions and advance translation. This article summarizes talks given during a workshop on "Single Cell Omics" at the 2020 annual meeting of the Orthopedic Research Society. Studies that applied scRNA-seq, ATAC-seq, and CyTOF mass cytometry to cartilage development and osteoarthritis are reviewed. This body of work shows how these cutting-edge tools can advance our understanding of the cellular heterogeneity and trajectories of lineage specification during development and disease.

Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

Periostin (POSTN), a secretory matricellular matrix protein, plays a multitude of biologic functions. Various splice variants of POSTN have been described; however, their expression pattern and functional implications are not completely understood. This study was undertaken to decipher the differential expression pattern of POSTN and its splice variants in various tissues and cell types. We show that POSTN was more highly expressed in anterior cruciate ligament (ACL) remnants compared with articular cartilage at the cellular and tissue level. Isoforms 1 and 8 were highly expressed only in articular chondrocytes, suggesting their splice-specific regulation in chondrocytes. To discern the role of total POSTN and full-length human POSTN isoform 1 (hPOSTN-001), we stably transfected human chondrosarcoma 1 (hCh-1) cell line with hPOSTN-001 using a pcDNA3.1-hPOSTN-001 construct. RNA-sequencing analysis of hCh-1 cells identified differentially expressed genes with a known role in chondrocyte function and osteoarthritis. Similar expression of a subset of candidate genes was revealed in ACL progenitor cells and chondrocytes as well as in ACL progenitor cells in which POSTN activity was altered by overexpression and by small interfering RNA gene knockdown. Cells expressing total POSTN, not isoform 1, exhibited increased cell adhesion potential. These findings suggest an important role for POSTN in the knee.-Cai, L., Brophy, R. H., Tycksen, E. D., Duan, X., Nunley, R. M., Rai, M. F. Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

Osteoarthritis following meniscus and ligament injury: insights from translational studies and animal models.

PURPOSE OF REVIEW: The interaction between joint injuries and posttraumatic osteoarthritis (PTOA) is generally thought to be mechanical in nature, however, surgical intervention has little effect on the development of PTOA. Little is known about the biological underpinning of how meniscus and anterior cruciate ligament (ACL) tears lead to cartilage degeneration. This review summarizes the latest findings regarding biological factors that influence how the knee responds to meniscus and ligament injuries, how meniscus and/or ACL tears turn the joint in the direction of PTOA and whether patient risk for PTOA after meniscus/ACL injury can be predicted. RECENT FINDINGS: Literature indicates that numerous intrinsic and extrinsic factors are associated with the biological response of the knee to injuries associated with PTOA. Gene/protein biomarkers provide insight into the biologic response of the knee to meniscus/ACL tears and the relationship to osteoarthritis in at-risk patients. Animal studies detail the time-course of disease pathogenesis and inform about the molecules that potentially alter the course of disease. SUMMARY: The molecular metabolic state of the meniscus/ACL after injury is associated with several biological factors. The limited studies to date provide initial evidence on the early molecular manifestations of injury, suggesting possible mechanisms for further study.

Suppression of NF-κB activity via nanoparticle-based siRNA delivery alters early cartilage responses to injury.

Osteoarthritis (OA) is a major cause of disability and morbidity in the aging population. Joint injury leads to cartilage damage, a known determinant for subsequent development of posttraumatic OA, which accounts for 12% of all OA. Understanding the early molecular and cellular responses postinjury may provide targets for therapeutic interventions that limit articular degeneration. Using a murine model of controlled knee joint impact injury that allows the examination of cartilage responses to injury at specific time points, we show that intraarticular delivery of a peptidic nanoparticle complexed to NF-κB siRNA significantly reduces early chondrocyte apoptosis and reactive synovitis. Our data suggest that NF-κB siRNA nanotherapy maintains cartilage homeostasis by enhancing AMPK signaling while suppressing mTORC1 and Wnt/ß-catenin activity. These findings delineate an extensive crosstalk between NF-κB and signaling pathways that govern cartilage responses postinjury and suggest that delivery of NF-κB siRNA nanotherapy to attenuate early inflammation may limit the chronic consequences of joint injury. Therapeutic benefits of siRNA nanotherapy may also apply to primary OA in which NF-κB activation mediates chondrocyte catabolic responses. Additionally, a critical barrier to the successful development of OA treatment includes ineffective delivery of therapeutic agents to the resident chondrocytes in the avascular cartilage. Here, we show that the peptide-siRNA nanocomplexes are nonimmunogenic, are freely and deeply penetrant to human OA cartilage, and persist in chondrocyte lacunae for at least 2 wk. The peptide-siRNA platform thus provides a clinically relevant and promising approach to overcoming the obstacles of drug delivery to the highly inaccessible chondrocytes.

Gene expression in human meniscal tears has limited association with early degenerative changes in knee articular cartilage.

Purpose/Aim: Meniscus tears are a common injury to the knee associated with the development of osteoarthritis. Gene expression in the injured meniscus may be associated with early degeneration in the articular cartilage. The purpose of this study was to test the hypothesis that gene expression in meniscus tears is associated with early degenerative changes in the articular cartilage at the time of partial meniscectomy. MATERIALS AND METHODS: Torn meniscus was removed at the time of partial meniscectomy in 68 patients without radiographic osteoarthritis. Meniscal mRNA expression was measured by quantitative PCR for multiple molecular markers of osteoarthritis and cartilage homeostasis. The presence of early degenerative changes in the knee was recorded by X-ray (N = 63), magnetic resonance imaging (MRI, N = 48), and arthroscopy (N = 63). Gene expression was tested for correlation with the presence/absence of degenerative changes after adjusting for age, sex, and body mass index. RESULTS: Overall gene expression varied significantly with degenerative changes based on X-ray (P = 0.047) and MRI (P = 0.018). The linear combination of gene variation was also significant. However, only adiponectin (ADIPOQ) (P = 0.015) was expressed at a significantly lower level in patients with chondrosis on MRI, while the expression of ADIPOQ (P = 0.035) and resistin (RETN) (P = 0.017) was higher in patients with early degenerative changes on X-ray. None of the genes varied significantly with presence/absence of chondrosis as measured by arthroscopy. CONCLUSIONS: There is an overall association of gene expression in meniscal tears to early degenerative changes in the knee, but only a limited number of specific genes demonstrate this relationship. The roles of adiponectin and resistin in knee injury and osteoarthritis deserve further study.

Transcriptome analysis of injured human meniscus reveals a distinct phenotype of meniscus degeneration with aging.

OBJECTIVE: Meniscus tears are associated with a heightened risk of osteoarthritis. This study aimed to advance our understanding of the metabolic state of injured human meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to the patient's age and degree of cartilage chondrosis. METHODS: The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (n = 12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. Variations in the relative changes in gene expression with age and chondrosis were analyzed, and integrated biologic processes were investigated computationally. RESULTS: We identified a set of genes in torn menisci that were differentially expressed with age and chondrosis. There were 866 genes that were differentially regulated (≥1.5-fold difference and P < 0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed, while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed, and those involved in T cell differentiation and apoptosis were elevated. CONCLUSION: Differences in age-related gene expression suggest that in older adults, meniscal cells might dedifferentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but they maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis.

Distinct patterns of cytokines, chemokines, and growth factors in synovial fluid after ACL injury in comparison to osteoarthritis.

This study analyzed knee synovial fluid after anterior cruciate ligament (ACL) tear and in osteoarthritis (OA) to test the hypotheses that concentrations of cytokines, chemokines, and growth factors differ (a) by diagnosis and (b) after ACL tear by time from injury and presence/absence of concomitant meniscus tear. Synovial fluid samples were collected from two groups, ACL tears (with or without meniscus tear) (N = 13) and Kellgren-Lawrence grade 3 and 4 OA (N = 16), undergoing clinically indicated aspiration of the knee joint. Multiple cytokines, chemokines, and growth factors were assessed using a multiplexed 45-protein panel. Comparisons were made for the concentrations of all molecules between ACL tear and OA patients, isolated versus combined ACL and meniscus tears, and categorized by time from injury: acute or early subacute (<15 days, N = 8) versus late subacute or chronic (>15 days and <3 months, N = 5). ACL tear patients have higher levels of six molecules (IL-4, IL-5, IL-13, PlGF-1, bNGF, TNF-α) in knee synovial fluid compared to OA patients. Isolated ACL tears express higher levels of IL-4, IL-13 and IFN-γ and lower levels of IL-7 than ACL tears with a concomitant meniscus tear. SDF-1α, PlGF-1, IL-1RA, HGF, bNGF, and BDNF levels are elevated immediately after injury and drop off significantly in the late subacute phase (after 15 days). Synovial fluid from knees with ACL tears have elevated metabolic activity compared to knees with OA. The cytokine profiles after ACL tears are influenced by the time from injury and the presence of meniscus tears. These findings offer valuable insights into the levels of cytokines, chemokines, and growth factors in the knee after ACL injury, information which may have important implications for the diagnosis, prognosis and treatment of this common pathology.

Heritability of articular cartilage regeneration and its association with ear wound healing in mice.

OBJECTIVE: Emerging evidence suggests that genetic components contribute significantly to cartilage degeneration in osteoarthritis pathophysiology, but little information is available on the genetics of cartilage regeneration. Therefore, this study was undertaken to investigate cartilage regeneration in genetic murine models using common inbred strains and a set of recombinant inbred (RI) lines generated from LG/J (healer of ear wounds) and SM/J (nonhealer) inbred mouse strains. METHODS: An acute full-thickness cartilage injury was introduced in the trochlear groove of 8-week-old mice (n=265) through microsurgery. Mouse knee joints were sagittally sectioned and stained with toluidine blue to evaluate regeneration. For the ear wound phenotype, a bilateral 2-mm through-and-through puncture was created in 6-week-old mice (n=229), and healing outcomes were measured after 30 days. Broad-sense heritability and genetic correlations were calculated for both phenotypes. RESULTS: Time-course analysis of the RI mouse lines showed no significant regeneration until 16 weeks after surgery; at that time, the strains could be segregated into 3 categories: good, intermediate, and poor healers. Analysis of heritability (H2) showed that both cartilage regeneration (H2=26%; P=0.006) and ear wound closure (H2=53%; P<0.00001) were significantly heritable. The genetic correlations between the two healing phenotypes for common inbred mouse strains (r=0.92) and RI mouse lines (r=0.86) were found to be extremely high. CONCLUSION: Our findings indicate that articular cartilage regeneration in mice is heritable, the differences between the mouse lines are due to genetic differences, and a strong genetic correlation between the two phenotypes exists, indicating that they plausibly share a common genetic basis. We therefore surmise that LG/J by SM/J intercross mice can be used to dissect the genetic basis of variation in cartilage regeneration.

Gene Expression in Glenoid Articular Cartilage Varies Across Acute Instability, Chronic Instability, and Osteoarthritis.

BACKGROUND: Shoulder instability is a common pathology associated with an elevated risk of osteoarthritis (OA). Little is known about gene expression in the cartilage of the glenohumeral joint after dislocation events, particularly as it relates to the risk of posttraumatic OA. This study tested the hypothesis that gene expression in glenoid cartilage varies among acute instability (<3 dislocations), chronic instability (≥3 dislocations), and OA. METHODS: Articular cartilage was collected from the anteroinferior glenoid of consenting patients undergoing shoulder stabilization surgery (n = 17) or total shoulder arthroplasty (n = 16). Digital quantitative polymerase chain reaction was used to assess the relative expression of 57 genes (36 genes from OA risk allele studies, 21 genes from differential expression studies), comparing (1) OA versus instability (acute and chronic combined), (2) acute versus chronic instability, (3) OA versus acute instability, and (4) OA versus chronic instability. RESULTS: The expression of 11 genes from OA risk allele studies and 9 genes from differential expression studies was significantly different between cartilage from patients with instability and those with OA. Pro-inflammatory genes from differential expression studies and genes from OA risk allele studies were more highly expressed in cartilage in the OA group compared with the instability group, which expressed higher levels of extracellular matrix and pro-anabolic genes. The expression of 14 genes from OA risk allele studies and 4 genes from differential expression studies, including pro-inflammatory genes, anti-anabolic genes, and multiple genes from OA risk allele studies, was higher in the acute instability group compared with the chronic instability group. Cartilage in the OA group displayed higher expression of CCL3, CHST11, GPR22, PRKAR2B, and PTGS2 than cartilage in the group with acute or chronic instability. Whereas cartilage in both the acute and chronic instability groups had higher expression of collagen genes, cartilage in the OA group had expression of a subset of genes from OA risk allele studies or from differential expression studies that was lower than in the acute group and higher than in the chronic group. CONCLUSIONS: Glenoid cartilage has an inflammatory and catabolic phenotype in shoulders with OA but an anabolic phenotype in shoulders with instability. Cartilage from shoulders with acute instability displayed greater (cellular) metabolic activity compared with shoulders with chronic instability. CLINICAL RELEVANCE: This exploratory study identified genes of interest, such as CCL3, CHST11, GPR22, PRKAR2B, and PTGS2, that have elevated expression in osteoarthritic glenoid cartilage. These findings provide new biological insight into the relationship between shoulder instability and OA, which could lead to strategies to predict and potentially modify patients' risk of degenerative arthritis due to shoulder instability.

Synovial Fluid Proteomics From Serial Aspirations of ACL-Injured Knees Identifies Candidate Biomarkers.

BACKGROUND: Anterior cruciate ligament (ACL) tears often result in knee effusion and an increased risk for developing knee osteoarthritis (OA) in the long run. The molecular profile of these effusions could be informative regarding initial steps in the development of posttraumatic OA after an ACL tear. HYPOTHESIS: The proteomics of knee synovial fluid changes over time after ACL injury. STUDY DESIGN: Descriptive laboratory study. METHODS: Synovial fluid was collected from patients with an acute traumatic ACL tear presenting to the office for evaluation (18.31 ± 19.07 days from injury) (aspiration 1) and again at the time of surgery (35.41 ± 58.15 days after aspiration 1 (aspiration 2). High-resolution liquid chromatography mass spectrometry was used to assess the quantitative protein profile of synovial fluid, and differences in protein profile between the 2 aspirations were determined computationally. RESULTS: A total of 58 synovial fluid samples collected from 29 patients (12 male, 17 female; 12 isolated ACL tear, 17 combined ACL and meniscal tear) with a mean age and body mass index of 27.01 ± 12.78 years and 26.30 ± 4.93, respectively, underwent unbiased proteomics analysis. The levels of 130 proteins in the synovial fluid changed over time (87 high, 43 low). Proteins of interest that were significantly higher in aspiration 2 included CRIP1, S100A11, PLS3, POSTN, and VIM, which represent catabolic/inflammatory activities in the joint. Proteins with a known role in chondroprotection and joint homeostasis such as CHI3L2 (YKL-39), TNFAIP6/TSG6, DEFA1, SPP1, and CILP were lower in aspiration 2. CONCLUSION: Synovial fluid from knees with ACL tears exhibits an increased burden of inflammatory (catabolic) proteins relevant to OA with reduced levels of chondroprotective (anabolic) proteins. CLINICAL RELEVANCE: This study identified a set of novel proteins that provide new biological insights into the aftermath of ACL tears. Elevated inflammation and decreased chondroprotection could represent initial disruption of homeostasis, potentially initiating the development of OA. Longitudinal follow-up and mechanistic studies are necessary to assess the functional role of these proteins in the joint. Ultimately, these investigations could lead to better approaches to predict and possibly improve patient outcomes.

RNA-Seq analysis reveals sex-dependent transcriptomic profiles of human subacromial bursa stratified by tear etiology.

Rotator cuff tendinopathy, a major cause of shoulder disability, occurs due to trauma or degeneration. Our molecular understanding of traumatic and degenerative tears remains elusive. Here, we probed transcript level differences between traumatic and degenerative tears. Subacromial bursa tissues were collected from patients with traumatic or degenerative tears during arthroscopy (N = 32). Transcripts differentially expressed by tear etiology were detected by RNA-seq. RNA-seq results were validated by real-time quantitative polymerase chain reaction. We identified 334 protein-coding transcripts differentially expressed between traumatic and degenerative tears in females and 167 in males at a fold-change greater than 2. In females, XIRP2, MYL1, MYBPC1, TNNT1, and LMOD2, were highly expressed in traumatic tears whereas TPSD1, CDSN, RCVRN, LTBP4, and PTGS1 were elevated in degen tears. Transcripts elevated in traumatic tears represented muscle cell differentiation and development, and muscle contraction whereas those elevated in degenerative tears represented cell activation and immune response. In males, AZGP1, CNTFR, COL9A1, ZNF98, and EREG were highly elevated in traumatic tears whereas MYL2, HOXD11, SLC6A7, CADM1, and MMP17 were highly expressed in degenerative tears. Transcripts elevated in traumatic tears represented metabolic/catabolic processes, and transmembrane protein transport while processes related to cell cycle were mainly enriched in degenerative tears. Numerous long noncoding RNAs were differentially expressed between traumatic and degenerative tears in both sexes. In summary, this study provides insights into molecular biology of bursa in patients with rotator cuff tendon disease based on tear acuity and novel sex-based transcript differences that could inform clinical decision making in treating patients with traumatic or degenerative shoulder injuries.

Proteomic Profile Analysis of Synovial Fluid in Patients With Anterior Cruciate Ligament Tears.

BACKGROUND: Anterior cruciate ligament (ACL) tears are associated with posttraumatic osteoarthritis, but the early biological changes that initiate joint degeneration after this injury are not well characterized. ACL tears typically result in effusion in the knee, which may provide insight into the initial response of the joint to injuries. HYPOTHESIS: Patient- and injury-specific factors are associated with the proteomics of synovial fluid in knees with ACL tears. STUDY DESIGN: Descriptive laboratory study. METHODS: Synovial fluid was collected from 105 patients (38 male, 67 female) with an acute traumatic ACL tear. Patient- and injury-specific factors such as age, sex, body mass index, time from injury, presence/absence of concomitant meniscal tears, and location of concomitant bone bruises (if present) were recorded. The protein concentration of synovial fluid was measured, followed by benchmarking of samples for multi-affinity high-abundance protein depletion. An isotropically labeled high-resolution nano-liquid chromatography with tandem mass spectrometry-based proteomic approach was used to determine the synovial fluid protein profile. Data were processed, quality controlled, and analyzed computationally for each patient and injury factor. RESULTS: The proteomics of synovial fluid from ACL tears was associated with patient sex, injury pattern, and location of bone bruises but not with patient age, body mass index, or time from injury. Knees with an isolated ACL tear had higher glutathione peroxidase 1 (GPX1) and plastin 3 levels than knees with an ACL tear and meniscal tear. A bone bruise on the lateral femoral condyle was associated with elevated leptin and glucose-6-phosphate dehydrogenase (G6PD) levels. A bone bruise on the lateral tibial plateau was associated with decreased GPX1 levels. Male patients had higher matrix metalloproteinase 9 and lower G6PD levels than female patients. CONCLUSION: Patient sex, injury pattern, and bone bruise location were important determinants of the proteomic profile of effusion resulting from ACL tears. CLINICAL RELEVANCE: Longitudinal follow-ups to see if and how proteomic differences relate to clinical outcomes and mechanistic studies to assess the role that specific proteins play in the joint are warranted. Ultimately, these investigations could lead to better approaches to predict clinical outcomes and identify possible interventions to optimize outcomes in these patients.

RNA-Seq reveals distinct transcriptomic differences in rotator cuff tendon based on tear etiology and patient sex.

Rotator cuff tears are a common pathology in the shoulder and generally have two underlying etiologies: traumatic and degenerative. Little is known about the molecular underpinning of these etiologies. Here we queried transcript level differences in tear etiology stratified by sex in 31 patients with rotator cuff tears. Tendon tissues were isolated from females (N = 16) and males (N = 15) with traumatic (N = 16) or degenerative (N = 15) tears during arthroscopy. Differentially expressed transcripts were identified by RNA-seq and biological processes were probed computationally. Expression of some transcripts was validated by real-time quantitative polymerase chain reaction (qPCR). We identified 339 and 336 transcripts differentially expressed by tear etiology in females and males, respectively, at a fold-change greater than |2|. In females, GSTM1, MT1G, S1008A, ACSM3, DSC, FAM110C, and VNN2 were elevated in traumatic tears representing metabolic/catabolic processes, and immune response whereas CHAD, CLEC3A, IBSP, TNMD, APLNR, and CPA3 were elevated in degenerative tears representing tissue morphogenesis and developmental processes, angiogenesis, and extracellular matrix organization. In males, ELOA3B, CXCL8, ADM, TNS4, and SPOCK1 were elevated in traumatic tears representing localization of endoplasmic reticulum, chromosome organization, leukocyte/neutrophil degranulation, and protein transport whereas MYL2, TNNC1, MB, CPA3, APLNR, and CA3 were highly upregulated in degenerative tears representing muscle cell differentiation and development and angiogenesis. Numerous novel lncRNAs were identified to be differentially expressed by tear etiology in both sexes. Real-time qPCR confirmed RNA-seq data. This study improves our understanding of tendon biology based on underlying etiology (trauma or degeneration) in a sex-specific manner. These findings may help drive clinical decision-making in females and males with traumatic and degenerative shoulder injuries.

KIF26B Silencing Prevents Osseous Transdifferentiation of Progenitor/Stem Cells and Attenuates Ectopic Calcification in a Murine Model.

Ectopic calcification is an osteogenic process that leads to the formation of inappropriate bone within intra-articular soft tissues, often in response to injury or surgery. The molecular mechanisms governing this phenotype have yet to be determined. Using a population genetics approach, we identified an association of the kinesin superfamily member 26b (Kif26b) with injury-induced ectopic calcification through quantitative trait locus analysis of recombinant inbred mouse strains, consistent with a genomewide association study that identified KIF26B as a severity locus for ectopic calcification in patients with hip osteoarthritis. Despite these associations of KIF26B with ectopic calcification, its mechanistic role and functional implications have not yet been fully elucidated. Here, we aim to decipher the functional role of KIF26B in osseous and chondrogenic transdifferentiation of human and murine progenitor/stem cells and in a murine model of non-invasive injury-induced intra-articular ectopic calcification. We found that KIF26B ablation via lentivirus-mediated shRNA significantly arrested osteogenesis of progenitor/stem cells and suppressed the expression of typical osteogenic marker genes. Conversely, KIF26B loss-of-function increased chondrogenesis as demonstrated by enhanced Safranin-O staining and by the elevated expression of chondrogenic marker genes. Furthermore, cell function analysis revealed that KIF26B knockdown significantly decreased cell viability and proliferation and induced cellular apoptosis. Mechanistically, loss of osteogenesis was reverted by the addition of a Wnt agonist, SKL2001, demonstrating a role of KIF26B in canonical Wnt/ß-catenin signaling. Finally, intra-articular delivery of Kif26b shRNA in B6-129SF2/J mice significantly hampered the development of intra-articular ectopic calcification at 8 weeks after injury compared with mice treated with non-target scrambled shRNA. In summary, these observations highlight that KIF26B plays a crucial role in ectopic bone formation by repressing osteogenesis, but not chondrogenesis, potentially via modulating Wnt/ß-catenin signaling. These findings establish KIF26B as a critical determinant of the osteogenic process in pathologic endochondral bone formation and an actionable target for pharmacotherapy to mitigate ectopic calcification (and heterotopic ossification). © 2021 American Society for Bone and Mineral Research (ASBMR).