Structural Variations in Articular Cartilage Matrix Are Associated with Early-Onset Osteoarthritis in the Spondyloepiphyseal Dysplasia Congenita (Sedc) Mouse

Heterozgyous spondyloepiphyseal dysplasia congenita (sedc/+) mice expressing a missense mutation in col2a1 exhibit a normal skeletal morphology but early-onset osteoarthritis (OA). We have recently examined knee articular cartilage obtained from homozygous (sedc/sedc) mice, which express a Stickler-like phenotype including dwarfism. We examined sedc/sedc mice at various levels to better understand the mechanistic process resulting in OA. Mutant sedc/sedc, and control (+/+) cartilages were compared at two, six and nine months of age. Tissues were fixed, decalcified, processed to paraffin sections, and stained with hematoxylin/eosin and safranin O/fast green. Samples were analyzed under the light microscope and the modified Mankin and OARSI scoring system was used to quantify the OA-like changes. Knees were stained with 1C10 antibody to detect the presence and distribution of type II collagen. Electron microscopy was used to study chondrocyte morphology and collagen fibril diameter. Compared with controls, mutant articular cartilage displayed decreased fibril diameter concomitant with increases in size of the pericellular space, Mankin and OARSI scores, cartilage thickness, chondrocyte clustering, proteoglycan staining and horizontal fissuring. In conclusion, homozygous sedc mice are subject to early-onset knee OA. We conclude that collagen in the mutant’s articular cartilage (both heterozygote and homozygote) fails to provide the normal meshwork required for matrix integrity and overall cartilage stability.     

Identifying Novel Osteoarthritis-Associated Genes in Human Cartilage Using a Systematic Meta-Analysis and a Multi-Source Integrated Network

Osteoarthritis, the most common joint disorder, is characterised by deterioration of the articular cartilage. Many studies have identified potential therapeutic targets, yet no effective treatment has been determined. The aim of this study was to identify and rank osteoarthritis-associated genes and micro-RNAs to prioritise those most integral to the disease. A systematic meta-analysis of differentially expressed mRNA and micro-RNAs in human osteoarthritic cartilage was conducted. Ingenuity pathway analysis identified cellular senescence as an enriched pathway, confirmed by a significant overlap (p < 0.01) with cellular senescence drivers (CellAge Database). A co-expression network was built using genes from the meta-analysis as seed nodes and combined with micro-RNA targets and SNP datasets to construct a multi-source information network. This accumulated and connected 1689 genes which were ranked based on node and edge aggregated scores. These bioinformatic analyses were confirmed at the protein level by mass spectrometry of the different zones of human osteoarthritic cartilage (superficial, middle, and deep) compared to normal controls. This analysis, and subsequent experimental confirmation, revealed five novel osteoarthritis-associated proteins (PPIB, ASS1, LHDB, TPI1, and ARPC4-TTLL3). Focusing future studies on these novel targets may lead to new therapies for osteoarthritis.     

Circulating YKL-40 Level,but not CHI3L1 Gene Variants,Is Associated with Atherosclerosis-Related Quantitative Traits and the Risk of Peripheral Artery Disease

YKL-40, a pleotropic cytokine, is emerging as a risk factor and a prognostic predictor of atherosclerotic cardiovascular disease. We attempted to elucidate the genetic, clinical and biochemical correlates of circulating YKL-40 level and, by combining it with CHI3L1 gene variants, with the risk and long-term mortality of peripheral artery disease (PAD). Plasma YKL-40 concentrations were measured in 612 Taiwanese individuals who had no clinically overt systemic disease. Clinical parameters, CHI3L1 gene promoter variants and 18 biomarker levels were analyzed. Eighty-six PAD patients were further enrolled for analysis. Significant associations were found between CHI3L1 genotypes/haplotypes and YKL-40 levels for the health examination subjects (smallest p = 8.36 × 10⁻⁷ for rs4950928 and smallest p = 1.72 × 10⁻¹⁰ for haplotype TGG) and also for PAD patients. For the health examination subjects, circulating YKL-40 level, but not CHI3L1 gene variants, were positively associated with age, smoking, and circulating levels of triglyceride, lipocalin 2 and multiple inflammatory biomarkers and negatively associated with low-density-lipoprotein cholesterol levels. Circulating YKL-40 level is also significantly associated with the risk of PAD (p = 3.3 × 10⁻²³). Circulating YKL40 level, but not CHI3L1 gene promoter variants, is associated with the risk of PAD in Taiwanese. The association of YKL-40 levels with multiple quantitative traits relating to the risk of PAD may provide a molecular basis linking YKL-40 to atherosclerotic cardiovascular disease.     

Antiosteoarthritic Effect of Morroniside in Chondrocyte Inflammation and Destabilization of Medial Meniscus-Induced Mouse Model

Osteoarthritis (OA) is a common degenerative disease that results in joint inflammation as well as pain and stiffness. A previous study has reported that Cornus officinalis (CO) extract inhibits oxidant activities and oxidative stress in RAW 264.7 cells. In the present study, we isolated bioactive compound(s) by fractionating the CO extract to elucidate its antiosteoarthritic effects. A single bioactive component, morroniside, was identified as a potential candidate. The CO extract and morroniside exhibited antiosteoarthritic effects by downregulating factors associated with cartilage degradation, including cyclooxygenase-2 (Cox-2), matrix metalloproteinase 3 (Mmp-3), and matrix metalloproteinase 13 (Mmp-13), in interleukin-1 beta (IL-1β)-induced chondrocytes. Furthermore, morroniside prevented prostaglandin E2 (PGE2) and collagenase secretion in IL-1β-induced chondrocytes. In the destabilization of the medial meniscus (DMM)-induced mouse osteoarthritic model, morroniside administration attenuated cartilage destruction by decreasing expression of inflammatory mediators, such as Cox-2, Mmp3, and Mmp13, in the articular cartilage. Transverse microcomputed tomography analysis revealed that morroniside reduced DMM-induced sclerosis in the subchondral bone plate. These findings suggest that morroniside may be a potential protective bioactive compound against OA pathogenesis.     

Ameliorative Effects of PACAP against Cartilage Degeneration. Morphological,Immunohistochemical and Biochemical Evidence from in Vivo and in Vitro Models of Rat Osteoarthritis

Osteoarthritis (OA); the most common form of degenerative joint disease, is associated with variations in pro-inflammatory growth factor levels, inflammation and hypocellularity resulting from chondrocyte apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide endowed with a range of trophic effects in several cell types; including chondrocytes. However; its role in OA has not been studied. To address this issue, we investigated whether PACAP expression is affected in OA cartilage obtained from experimentally-induced OA rat models, and then studied the effects of PACAP in isolated chondrocytes exposed to IL-1β in vitro to mimic the inflammatory milieu of OA cartilage. OA induction was established by histomorphometric and histochemical analyses. Changes in PACAP distribution in cartilage, or its concentration in synovial fluid (SF), were assessed by immunohistochemistry and ELISA. Results showed that PACAP abundance in cartilage tissue and SF was high in healthy controls. OA induction decreased PACAP levels both in affected cartilage and SF. In vitro, PACAP prevented IL-1β-induced chondrocyte apoptosis, as determined by MTT assay; Hoechst staining and western blots of apoptotic-related proteins. These changes were also accompanied by decreased i-NOS and COX-2 levels, suggesting an anti-inflammatory effect. Altogether, these findings support a potential role for PACAP as a chondroprotective agent for the treatment of OA.     

Indian Hedgehog in Synovial Fluid Is a Novel Marker for Early Cartilage Lesions in Human Knee Joint

To determine whether there is a correlation between the concentration of Indian hedgehog (Ihh) in synovial fluid (SF) and the severity of cartilage damage in the human knee joints, the knee cartilages from patients were classified using the Outer-bridge scoring system and graded using the Modified Mankin score. Expression of Ihh in cartilage and SF samples were analyzed with immunohistochemistry (IHC), western blot, and enzyme-linked immunosorbent assay (ELISA). Furthermore, we detected and compared Ihh protein levels in rat and mice cartilages between normal control and surgery-induced osteoarthritis (OA) group by IHC and fluorescence molecular tomography in vivo respectively. Ihh expression was increased 5.2-fold in OA cartilage, 3.1-fold in relative normal OA cartilage, and 1.71-fold in OA SF compared to normal control samples. The concentrations of Ihh in cartilage and SF samples was significantly increased in early-stage OA samples when compared to normal samples (r = 0.556; p < 0.001); however, there were no significant differences between normal samples and late-stage OA samples. Up-regulation of Ihh protein was also an early event in the surgery-induced OA models. Increased Ihh is associated with the severity of OA cartilage damage. Elevated Ihh content in human knee joint synovial fluid correlates with early cartilage lesions.     

Osteoarthritis in the XXIst Century: Risk Factors and Behaviours that Influence Disease Onset and Progression

Osteoarthritis (OA) is a growing public health problem across the globe, affecting more than half of the over 65 population. In the past, OA was considered a wear and tear disease, leading to the loss of articular cartilage and joint disability. Nowadays, thanks to advancements in molecular biology, OA is believed to be a very complex multifactorial disease. OA is a degenerative disease characterized by “low-grade inflammation” in cartilage and synovium, resulting in the loss of joint structure and progressive deterioration of cartilage. Although the disease can be dependent on genetic and epigenetic factors, sex, ethnicity, and age (cellular senescence, apoptosis and lubricin), it is also associated with obesity and overweight, dietary factors, sedentary lifestyle and sport injuries. The aim of this review is to highlight how certain behaviors, habits and lifestyles may be involved in the onset and progression of OA and to summarize the principal risk factors involved in the development of this complicated joint disorder.     

Remodeling Osteoarthritic Articular Cartilage under Hypoxic Conditions

Osteoarthritis (OA) is one of the leading joint diseases induced by abnormalities or inflammation in the synovial membrane and articular cartilage, causing severe pain and disability. Along with the cartilage malfunction, imbalanced oxygen uptake occurs, changing chondrocytes into type I collagen- and type X collagen-producing dedifferentiated cells, contributing to OA progression. However, mounting evidence suggests treating OA by inducing a hypoxic environment in the articular cartilage, targeting the inhibition of several OA-related pathways to bring chondrocytes into a normal state. This review discusses the implications of OA-diseased articular cartilage on chondrocyte phenotypes and turnover and debates the hypoxic mechanism of action. Furthermore, this review highlights the new understanding of OA, provided by tissue engineering and a regenerative medicine experimental design, modeling the disease into diverse 2D and 3D structures and investigating hypoxia and hypoxia-inducing biomolecules and potential cell therapies. This review also reports the mechanism of hypoxic regulation and highlights the importance of activating and stabilizing the hypoxia-inducible factor and related molecules to protect chondrocytes from mitochondrial dysfunction and apoptosis occurring under the influence of OA.     

Increased Chondrocyte Apoptosis Is Associated with Progression of Osteoarthritis in Spontaneous Guinea Pig Models of the Disease

Osteoarthritis (OA) is the most common joint disease characterised by degradation of articular cartilage and bone remodelling. For almost a decade chondrocyte apoptosis has been investigated as a possible mechanism of cartilage damage in OA, but its precise role in initiation and/or progression of OA remains to the determined. The aim of this study is to determine the role of chondrocyte apoptosis in spontaneous animal models of OA. Right tibias from six male Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs were collected at 10, 16, 24 and 30 weeks of age. Fresh-frozen sections of tibial epiphysis were microscopically scored for OA, and immunostained with caspase-3 and TUNEL for apoptotic chondrocytes. The DH strain had more pronounced cartilage damage than BS2, especially at 30 weeks. At this time point, the apoptotic chondrocytes were largely confined to the deep zone of articular cartilage (AC) with a greater percentage in the medial side of DH than BS2 (DH: 5.7%, 95% CI: 4.2–7.2), BS2: 4.8%, 95% CI: 3.8–5.8), p > 0.05). DH had a significant progression of chondrocyte death between 24 to 30 weeks during which time significant changes were observed in AC fibrillation, proteoglycan depletion and overall microscopic OA score. A strong correlation (p ≤ 0.01) was found between chondrocyte apoptosis and AC fibrillation (r = 0.3), cellularity (r = 0.4) and overall microscopic OA scores (r = 0.4). Overall, the rate of progression in OA and apoptosis over the study period was greater in the DH (versus BS2) and the medial AC (versus lateral). Chondrocyte apoptosis was higher at the later stage of OA development when the cartilage matrix was hypocellular and highly fibrillated, suggesting that chondrocyte apoptosis is a late event in OA.     

Quadruped Gait and Regulation of Apoptotic Factors in Tibiofemoral Joints following Intra-Articular rhPRG4 Injection in Prg4 Null Mice

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome leads to diarthrodial joint arthropathy and is caused by the absence of lubricin (proteoglycan 4—PRG4), a surface-active mucinous glycoprotein responsible for lubricating articular cartilage. In this study, mice lacking the orthologous gene Prg4 served as a model that recapitulates the destructive arthrosis that involves biofouling of cartilage by serum proteins in lieu of Prg4. This study hypothesized that Prg4-deficient mice would demonstrate a quadruped gait change and decreased markers of mitochondrial dyscrasia, following intra-articular injection of both hindlimbs with recombinant human PRG4 (rhPRG4). Prg4^−/− (N = 44) mice of both sexes were injected with rhPRG4 and gait alterations were studied at post-injection day 3 and 6, before joints were harvested for immunohistochemistry for caspase-3 activation. Increased stance and propulsion was shown at 3 days post-injection in male mice. There were significantly fewer caspase-3-positive chondrocytes in tibiofemoral cartilage from rhPRG4-injected mice. The mitochondrial gene Mt-tn, and myosin heavy (Myh7) and light chains (Myl2 and Myl3), known to play a cytoskeletal stabilizing role, were significantly upregulated in both sexes (RNA-Seq) following IA rhPRG4. Chondrocyte mitochondrial dyscrasias attributable to the arthrosis in CACP may be mitigated by IA rhPRG4. In a supporting in vitro crystal microbalance experiment, molecular fouling by albumin did not block the surface activity of rhPRG4.     

Expression and Localization of Thrombospondins,Plastin 3, and STIM1 in Different Cartilage Compartments of the Osteoarthritic Varus Knee

Osteoarthritis (OA) is a multifactorial disease which is characterized by a change in the homeostasis of the extracellular matrix (ECM). The ECM is essential for the function of the articular cartilage and plays an important role in cartilage mechanotransduction. To provide a better understanding of the interaction between the ECM and the actin cytoskeleton, we investigated the localization and expression of the Ca²⁺-dependent proteins cartilage oligomeric matrix protein (COMP), thrombospondin-1 (TSP-1), plastin 3 (PLS3) and stromal interaction molecule 1 (STIM1). We investigated 16 patients who suffered from varus knee OA and performed a topographical analysis of the cartilage from the medial and lateral compartment of the proximal tibial plateau. In a varus knee, OA is more pronounced in the medial compared to the lateral compartment as a result of an overloading due to the malalignment. We detected a location-dependent staining of PLS3 and STIM1 in the articular cartilage tissue. The staining intensity for both proteins correlated with the degree of cartilage degeneration. The staining intensity of TSP-1 was clearly reduced in the cartilage of the more affected medial compartment, an observation that was confirmed in cartilage extracts by immunoblotting. The total amount of COMP was unchanged; however, slight changes were detected in the localization of the protein. Our results provide novel information on alterations in OA cartilage suggesting that Ca²⁺-dependent mechanotransduction between the ECM and the actin cytoskeleton might play an essential role in the pathomechanism of OA.     

Chaenomeles Fructus (CF), the Fruit of Chaenomeles sinensis Alleviates IL-1β Induced Cartilage Degradation in Rat Articular Chondrocytes

Osteoarthritis (OA) causes persistent pain, joint dysfunction, and physical disability. It is the most prevalent type of degenerative arthritis, affecting millions of people worldwide. OA is currently treated with a focus on pain relief, inflammation control, and artificial joint surgery. Hence, a therapeutic agent capable of preventing or delaying the progression of OA is needed. OA is strongly associated with the degeneration of the articular cartilage and changes in the ECM, which are primarily associated with a decrease in proteoglycan and collagen. In the progress of articular cartilage degradation, catabolic enzymes, such as matrix metalloproteinases (MMPs), are activated by IL-1β stimulation. Given the tight relationship between IL-1β and ECM (extra-cellular matrix) degradation, this study examined the effects of Chaenomeles Fructus (CF) on IL-1β-induced OA in rat chondrocytes. The CF treatment reduced IL-1β-induced MMP3/13 and ADAMTS-5 production at the mRNA and protein levels. Similarly, CF enhanced col2a and aggrecan accumulation and chondrocyte proliferation. CF inhibited NF-κB (nuclear factor kappa B) activation, nuclear translocation induced by IL-1β, reactive oxygen species (ROS) production, and ERK phosphorylation. CF demonstrated anti-OA and articular regeneration effects on rat chondrocytes, thus, suggesting that CF is a viable and fundamental therapeutic option for OA.     

Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage

Mesenchymal stem cells (MSCs) are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA). Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs) promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT) and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA) or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE) and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I) but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals.     

Cartilage Homeostasis and Osteoarthritis

Healthy limb joints are important for maintaining health and attaining longevity. Endochondral ossification (the replacement of cartilage with bone, occurring during skeletal development) is essential for bone formation, especially in long-axis bones. In contrast to endochondral ossification, chondrocyte populations in articular cartilage persist and maintain joint tissue into adulthood. Articular cartilage, a connective tissue consisting of chondrocytes and their surrounding extracellular matrices, plays an essential role in the mechanical cushioning of joints in postnatal locomotion. Osteoarthritis (OA) pathology relates to disruptions in the balance between anabolic and catabolic signals, that is, the loss of chondrocyte homeostasis due to aging or overuse of cartilages. The onset of OA increases with age, shortening a person’s healthy life expectancy. Although many people with OA experience pain, the mainstay of treatment is symptomatic therapy, and no fundamental treatment has yet been established. To establish regenerative or preventative therapies for cartilage diseases, further understanding of the mechanisms of cartilage development, morphosis, and homeostasis is required. In this review, we describe the general development of cartilage and OA pathology, followed by a discussion on anabolic and catabolic signals in cartilage homeostasis, mainly microRNAs.     

Histone Extraction from Human Articular Cartilage for the Study of Epigenetic Regulation in Osteoarthritis

Osteoarthritis (OA) is a chronic disease that affects articular cartilage, causing its degeneration. Although OA is one of the most prevalent pathologies globally, there are no definitive treatments available. Recently, research has focused on elucidating the complex interplay that takes place between inflammatory processes and epigenetic regulation, showing that histone post-translational modifications (PTMs) can exert a pronounced effect on the expression of OA-related genes. OA chondrocytes enhance the production of interleukin 1β (IL-1β) and interleukin 8 (IL-8), which are epigenetically regulated. These cytokines upregulate the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, which promote the extracellular matrix (ECM) destruction. This motivates the study of histone PTMs to investigate the epigenetic regulation of proinflammatory molecules, but the absence of specific protocols to extract histones from human articular cartilage has complicated this task. The lack of effective methods can be explained by the structural complexity and low cellularity of this tissue, which are responsible for the biomechanical properties that allow the movement of the joint but also complicate histone isolation. Here, we provide a histone extraction procedure specifically adapted for cryopreserved human articular cartilage that can be useful to understand epigenetic regulation in OA and accelerate the search for novel strategies.     

Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint,Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells

The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC.     

Isolation and Heterologous Expression of a Polygalacturonase Produced by Fusarium oxysporum f. sp. cubense Race 1 and 4

Fusarium wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The K_m and V_max values of PGC3 from FOC4 were determined to be 0.70 mg·mL⁻¹ and 101.01 Units·mg·protein⁻¹·min⁻¹, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3–7 and >50% activity in 10–50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris.