Osteoarthritis (OA) is a chronic condition caused by cartilage degradation, and there are currently no effective methods for preventing the progression of this disease; gene therapy is a relatively novel method for treating arthritis. Decreased collagen type II (Col2) expression within the cartilage matrix is an important factor for the development of OA, and Wnt3a serves a significant role in cartilage homeostasis. The present study assessed whether Wnt3a knockdown promoted Col2 expression in chondrocytes. Lentivirus-introduced small interfering RNA was used to knock down the expression of Wnt3a in primary rat chondrocytes, and then IL-1β treatment was used to establish an OA chondrocyte model. The expression of target genes (Wnt3a, Col2, MMP-13 and β-catenin) was analyzed using reverse transcription-quantitative PCR, western blotting and immunocytochemistry. There was significantly less MMP-13 and β-catenin expression in the Wnt3a knockdown cells compared with the other controls. Col2 expression was significantly higher in the Wnt3a-knockdown cells compared with the control cells, indicating that knockdown of Wnt3a may promote Col2 expression. Consequently, Wnt3a was indicated to be an important factor in cartilage homeostasis, and Wnt3a knockdown may serve as a novel method for OA therapy.

Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259-273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.

Epicutaneous immunization is a potential non-invasive technique for antigen-specific immune-modulation. Topical application of protein antigens to barrier-disrupted skin induces potent antigen-specific immunity with a strong Th2-bias. In this study, we investigate whether the autoimmune inflammatory response of chronic collagen-induced arthritis (CCIA) in DBA/1-TCR-beta Tg mice can be modified by epicutaneous immunization. We show that epicutaneous immunization with type II collagen (CII) inhibited development and progression of CCIA and, importantly, also ameliorated ongoing disease as indicated by clinical scores of disease severity, paw swelling and joints histology. Treated mice show reduced CII-driven T cell proliferation and IFN-gamma production, as well as significantly lower levels of CII-specific IgG2a serum antibodies. In contrast, CII-driven IL-4 production and IgE antibody levels were increased consistent with skewing of the CII response from Th1 to Th2 in treated mice. IL-4 production in treated mice was inversely correlated with disease severity. Moreover, T cells from treated mice inhibited proliferation and IFN-gamma production by T cells from CCIA mice, suggesting induction of regulatory T cells that actively inhibit effector responses in arthritic mice. The levels of CD4(+)CD25(+) T cells were however not increased following epicutaneous CII treatment. Together, these results suggest that epicutaneous immunization may be used as an immune-modulating procedure to actively re-programme pathogenic Th1 responses, and could have potential as a novel specific and simple treatment for chronic autoimmune inflammatory diseases such as rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA).

High mobility group box chromosomal protein 1 (HMGB1) is a nuclear protein that acts as a pro-inflammatory mediator following extracellular release. The protein is aberrantly expressed extracellularly in the settings of clinical and experimental synovitis. Therapy based on HMGB1 antagonists has shown encouraging results in experimental arthritis and warrants further scientific exploration using independent methods. In the present study we asked whether nuclear sequestration of HMGB1 preventing HMGB1 release would be beneficial for synovitis treatment.

Full‑thickness and large area defects of articular cartilage are unable to completely repair themselves and require surgical intervention, including microfracture, autologous or allogeneic osteochondral grafts, and autologous chondrocyte implantation. A large proportion of regenerative cartilage exists as fibrocartilage, which is unable to withstand impacts in the same way as native hyaline cartilage, owing to excess synthesis of type I collagen in the matrix. The present study demonstrated that low‑dose halofuginone (HF), a plant alkaloid isolated from Dichroa febrifuga, may inhibit the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. In addition, HF was revealed to inhibit the phosphorylation of mothers against decapentaplegic homolog (Smad)2/3 and promoted Smad7 expression, as well as decrease the synthesis of type I collagen synthesis. Results from the present study indicated that HF treatment suppressed the synthesis of type I collagen by inhibiting the transforming growth factor‑β signaling pathway in chondrocytes. These results may provide an alternative solution to the problems associated with fibrocartilage, and convert fibrocartilage into hyaline cartilage at the mid‑early stages of cartilage regeneration. HF may additionally be used to improve monolayer expansion or 3D cultures of seed cells for the tissue engineering of cartilage.

The tissue engineering approach in osteoarthritic cell therapy often requires the delivery of a substantially high cell number due to the low engraftment efficiency as a result of low affinity binding of implanted cells to the targeted tissue. A modification towards the cell membrane that provides specific epitope for antibody binding to a target tissue may be a plausible solution to increase engraftment. In this study, we intercalated palmitated protein G (PPG) with mesenchymal stem cells (MSCs) and antibody, and evaluated their effects on the properties of MSCs either in monolayer state or in a 3D culture state (gelatin microsphere, GM). Bone marrow MSCs were intercalated with PPG (PPG-MSCs), followed by coating with type II collagen antibody (PPG-MSC-Ab). The effect of PPG and antibody conjugation on the MSC proliferation and multilineage differentiation capabilities both in monolayer and GM cultures was evaluated. PPG did not affect MSC proliferation and differentiation either in monolayer or 3D culture. The PPG-MSCs were successfully conjugated with the type II collagen antibody. Both PPG-MSCs with and without antibody conjugation did not alter MSC proliferation, stemness, and the collagen, aggrecan, and sGAG expression profiles. Assessment of the osteochondral defect explant revealed that the PPG-MSC-Ab micromass was able to attach within 48 h onto the osteochondral surface. Antibody-conjugated MSCs in GM culture is a potential method for targeted delivery of MSCs in future therapy of cartilage defects and osteoarthritis.

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven Gaussia luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young's tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.

The extracellular matrix protein nephronectin (Npnt) is known to be critical for kidney development, but its function in inflammatory diseases is unknown. Here, we developed a new enzyme-linked immunosorbent assay system to detect Npnt in various autoimmune diseases, which revealed that plasma Npnt levels are increased in various mouse autoimmune models. We also report that antibodies against the α8β1 integrin-binding region of Npnt protect mice from anti-type II collagen-induced arthritis, suggesting that Npnt may be a potential therapeutic target molecule for the prevention of autoimmune arthritis.

Local inflammation in the joint is considered to contribute to osteoarthritis (OA) progression. Here, we describe an immunomodulating nanoparticle for OA treatment. Intradermal injection of lipid nanoparticles (LNPs) loaded with type II collagen (Col II) and rapamycin (LNP-Col II-R) into OA mice effectively induced Col II-specific anti-inflammatory regulatory T cells, substantially increased anti-inflammatory cytokine expression, and reduced inflammatory immune cells and proinflammatory cytokine expression in the joints. Consequently, LNP-Col II-R injection inhibited chondrocyte apoptosis and cartilage matrix degradation and relieved pain, while injection of LNPs loaded with a control peptide and rapamycin did not induce these events. Adoptive transfer of CD4+CD25+ T cells isolated from LNP-Col II-R-injected mice suggested that Tregs induced by LNP-Col II-R injection were likely responsible for the therapeutic effects. Collectively, this study suggests nanoparticle-mediated immunomodulation in the joint as a simple and effective treatment for OA.

This study investigated the effect of type II collagen extract on SD rats with deteriorated immunity caused by methotrexate. The test samples were dosed once a day for 28 days by gastric gavage at dosage 250 mg/kg and 500 mg/kg after methotrexate treatment, and the changes on body weight, total blood leukocyte numbers, the percentages of B-cells, CD4+ T-cells and CD8+ T-cells in the blood and spleen were observed. The changes on body weight, the total blood leukocyte numbers, the total lymphocyte numbers in the spleen, the ratio of CD4+ and CD8+ T-cells in the blood and spleen were increased significantly in type II collagen extract groups as compared with the control group. According to the above results, type II collagen extract has an effect of increasing immune responses on rats with deteriorated immunity caused by methotrexate.

Genetic tools such as the Cre-Lox reporter system are powerful aids for tissue-specific cell tracking. For example, it would be useful in examining intervertebral disc (IVD) cell populations in normal and diseased states. A Cre recombinase and its recognition site, loxP have been adapted from the bacteriophage for use in genetic manipulation. The reporter mice used here express the red fluorescent protein, tdTomato with flanking LoxP sites (Rosa26 TdTomato mice). We compared two different Collagen type II (Col2) promoter constructs that drive Cre-recombinase expression in mice: (a) Col2-Cre, which allows constitutive Cre-recombinase expression under the control of the Col2 promoter/enhancer and (b) Col2-CreER, which contains a shorter promoter/enhancer region than Col2-Cre, but has human estrogen binding elements that bind tamoxifen, resulting in Cre-recombinase expression. The goal of the study is to characterize Cre-recombinase distribution pattern in Col2-Cre and Col2-CreER mice using tdTomato as reporter in the spine. The expression patterns of these two mice were further compared with Col2 gene expression in the native mouse NP and AF tissues by real-time PCR. We crossed Col2-Cre mice or Col2-CreER mice with the tdTomato reporter mice, and compared the tdTomato expression patterns. Col2-CreER/tdTomato mice were injected with tamoxifen at postnatal day 7 to activate the Cre-recombinase. TdTomato in the constitutively active Col2-Cre mice was detected in the nucleus pulposus (NP), the entire annulus fibrosus (AF), and in cartilaginous endplate and growth plate cells in the lower lumbar and coccygeal spine. In contrast, when Col2-CreER activity was induced by tamoxifen at P7, tdTomato was limited to the inner AF, and was absent from the NP. We have described the differences in Col2 reporter gene expression, in Col2-Cre/tdTomato and Col2-Cre-ER/tdTomato mouse IVD. The information provided here will help to guide future investigations of IVD biology.

Osteoarthritis is the most widespread joint-affecting disease. Patients with osteoarthritis experience pain and impaired mobility resulting in marked reduction of quality of life. A progressive cartilage loss is responsible of an evolving disease difficult to treat. The characteristic of chronicity determines the need of new active disease modifying drugs. Aim of the present research is to evaluate the role of low doses of native type II collagen in the rat model of osteoarthritis induced by sodium monoiodoacetate (MIA).

The proinflammatory cytokine tumor necrosis factor-α (TNF-α)-induced degradation of extracellular matrix (ECM), such as type II collagen in chondrocytes, plays an important role in the development of osteoarthritis (OA). Telmisartan, an angiotensin II (Ang-II) receptor blocker, is a licensed drug used for the treatment of hypertension. However, the effects of Telmisartan in tumor necrosis factor-α (TNF-α)-induced damage to chondrocytes and the progression of OA are unknown. In this study, we found that treatment with Telmisartan attenuated TNF-α-induced oxidative stress by reducing the levels of mitochondrial reactive oxygen species (ROS) and the production of protein carbonyl in human C28/I2 chondrocytes. Interestingly, Telmisartan inhibited TNF-α-induced expression and secretions of proinflammatory mediators such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and monocyte chemotactic protein 1 (MCP-1). Notably, stimulation with TNF-α reduced the levels of type II collagen at both the mRNA and the protein levels, which was rescued by the treatment with Telmisartan. Mechanistically, we found that Telmisartan restored TNF-α-induced reduction of SOX-9. Silencing of SOX-9 blocked the inhibitory effects of Telmisartan against TNF-α-induced degradation of type II collagen. These findings suggest that Telmisartan might be a potential and promising agent for the treatment of OA.

To investigate the occurrence and frequency of anti-citrullinated protein antibodies (ACPA) to cyclic citrullinated type II collagen (COL2) epitope with a capacity to bind joint cartilage.

In murine collagen-induced arthritis (CIA), self-reactive T cells can recognize peptide antigens derived from type-II collagen (CII). Activation of T cells is an important mediator of autoimmune diseases. Thus, T cells have become a focal point of study to treat autoimmune diseases. In this study, we evaluated the efficacy of recombinant MHC class II molecules in the regulation of antigen-specific T cells by using a self peptide derived from CII (CII260-274; IAGFKGEQGPKGEPG) linked to mouse I-A(q) in a murine CIA model. We found that recombinant I-A(q)/CII260-274 molecules could be recognized by CII-specific T cells and inhibit the same T cells in vitro. Furthermore, the development of CIA in mice was successfully prevented by in vivo injection of recombinant I-A(q)/CII260-274 molecules. Thus, treatment with recombinant soluble MHC class II molecules in complex with an immunodominant self-peptide might offer a potential therapeutic for chronic inflammation in autoimmune disease such as rheumatoid arthritis. [BMB Reports 2016; 49(6): 331-336].

The migration of chondrocytes from healthy to injured tissues is one of the most important challenges during cartilage repair. Additionally, maintenance of the chondrogenic phenotype remains another limitation, especially during monolayer culture in vitro. Using both the differentiated and undifferentiated chondrogenic ATDC5 cell line, we showed that extracellular nucleotides are able to increase the migration rate of chondrocytes without affecting their chondrogenic phenotype. We checked the potency of natural nucleotides (ATP, ADP, UTP, and UDP) as well as their stable phosphorothioate analogs, containing a sulfur atom in the place of one nonbridging oxygen atom in a phosphate group. We also detected P2y1, P2y2, P2y4, P2y6, P2y12, P2y13, and P2y14 mRNA transcripts for nucleotide receptors, demonstrating that P2y1 and P2y13 are highly upregulated in differentiated ATDC5 cells. We showed that ADPβS, UDPβS, and ADP are the best stimulators of migration of differentiated chondrocytes. Additionally, ADP and ADPβS positively affected the expression of type II collagen, a structural component of the cartilage matrix.

Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration.

Pulmonary hypoplasia and respiratory failure are primary causes of death in patients with osteogenesis imperfecta (OI) type II. OI is a genetic skeletal disorder caused by pathogenic variants in genes encoding collagen type I. It is still unknown if the collagen defect also affects lung development and structure, causing lung hypoplasia in OI type II. The aim of this study was to investigate the intrinsic characteristics of OI embryonic lung parenchyma and to determine whether altered collagen type I may compromise airway development and lung structure. Lung tissue from nine fetuses with OI type II and six control fetuses, matched by gestational age, was analyzed for TTF-1 and collagen type I expression by immunohistochemistry, to evaluate the state of lung development and amount of collagen. The differentiation of epithelium into type 2 pneumocytes during embryonic development was premature in OI type II fetuses compared to controls (p < 0.05). Collagen type I showed no significant differences between the two groups. However, the amount of alpha2(I) chains was higher in fetuses with OI and the ratio of alpha1(I) to alpha2(I) lower in OI compared to controls. Cell differentiation during lung embryonic development in patients with OI type II is premature and impaired. This may be the underlying cause of pulmonary hypoplasia. Altered cell differentiation can be secondary to mechanical chest factors or a consequence of disrupted type I collagen synthesis. Our findings suggest that collagen type I is a biochemical regulator of pulmonary cell differentiation, influencing lung development.