Low-Molecular-Weight Fish Collagen Peptide (Valine-Glycine-Proline-Hydroxyproline-Glycine-Proline-Alanine-Glycine) Prevents Osteoarthritis Symptoms in Chondrocytes and Monoiodoacetate-Injected Rats.

The objective of this study was to investigate the effect of low-molecular-weight fish collagen (valine-glycine-proline-hydroxyproline-glycine-proline-alanine-glycine; LMWCP) on H2O2- or LPS-treated primary chondrocytes and monoiodoacetate (MIA)-induced osteoarthritis rat models. Our findings indicated that LMWCP treatment exhibited protective effects by preventing chondrocyte death and reducing matrix degradation in both H2O2-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. This was achieved by increasing the levels of aggrecan, collagen type I, collagen type II, TIMP-1, and TIMP-3, while simultaneously decreasing catabolic factors such as phosphorylation of Smad, MMP-3, and MMP-13. Additionally, LMWCP treatment effectively suppressed the activation of inflammation and apoptosis pathways in both LPS-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. These results suggest that LMWCP supplementation ameliorates the progression of osteoarthritis through its direct impact on inflammation and apoptosis in chondrocytes.

Native Collagen II Relieves Bone Impairment through Improving Inflammation and Oxidative Stress in Ageing db/db Mice.

Ageing-related bone impairment due to exposure to hyperglycemic environment is scarcely researched. The aim was to confirm the improvement effects of undenatured type II collagen (UC II) on bone impairment in ageing db/db mice, and the ageing model was established by normal feeding for 48-week-old. Then, the ageing db/db mice were randomly assigned to UC II intervention, the ageing model, and the chondroitin sulfate + glucosamine hydrochloride control groups. After 12 weeks of treatment, femoral microarchitecture and biomechanical parameters were observed, biomarkers including bone metabolism, inflammatory cytokines, and oxidative stress were measured, and the gastrocnemius function and expressions of interleukin (IL) 1ß, receptor activator of nuclear factor (NF)-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) were analyzed. The results showed that the mice in the UC II intervention group showed significantly superior bone and gastrocnemius properties than those in the ageing model group, including bone mineral density (287.65 ± 72.77 vs. 186.97 ± 32.2 mg/cm3), gastrocnemius index (0.46 ± 0.07 vs. 0.18 ± 0.01%), muscle fiber diameter (0.0415 ± 0.005 vs. 0.0330 ± 0.002 mm), and cross-sectional area (0.0011 ± 0.00007 vs. 0.00038 ± 0.00004 mm2). The UC II intervention elevated bone mineralization and formation and decreased bone resorption, inflammatory cytokines, and the oxidative stress. In addition, lower protein expression of IL-1ß, RANKL, and TRAP in the UC II intervention group was observed. These findings suggested that UC II improved bones impaired by T2DM during ageing, and the likely mechanism was partly due to inhibition of inflammation and oxidative stress.

Type II Collagen from Cartilage of Acipenser baerii Promotes Wound Healing in Human Dermal Fibroblasts and in Mouse Skin.

Type II collagen is an important component of cartilage; however, little is known about its effect on skin wound healing. In this study, type II collagen was extracted from the cartilage of Acipenser baerii and its effect on in vitro and in vivo wound healing was compared to type I collagen derived from tilapia skin. Sturgeon cartilage collagen (SCC) was composed of α1 chains and with a thermal denaturation (Td) at 22.5 and melting temperature (Tm) at 72.5 °C. Coating SCC potentiated proliferation, migration, and invasion of human dermal fibroblast adult (HDFa) cells. Furthermore, SCC upregulated the gene expression of extracellular matrix (ECM) components (col Iα1, col IIIα1, elastin, and Has2) and epithelial-mesenchymal transition (EMT) molecules (N-cadherin, Snail, and MMP-1) in HDFa. Pretreatment with Akt and mitogen-activated protein kinase (MAPK) inhibitors significantly attenuated the HDFa invasion caused by SCC. In mice, the application of SCC on dorsal wounds effectively facilitated wound healing as evidenced by 40-59% wound contraction, whereas the untreated wounds were 18%. We observed that SCC reduced inflammation, promoted granulation, tissue formation, and ECM deposition, as well as re-epithelialization in skin wounds. In addition, SCC markedly upregulated the production of growth factors in the dermis, and dermal and subcutaneous white adipose tissue; in contrast, the administration of tilapia skin collagen (TSC) characterized by typical type I collagen was mainly expressed in the epidermis. Collectively, these findings indicate SCC accelerated wound healing by targeting fibroblast in vitro and in vivo.

Treatment of Type II Collagen-Induced Rat Rheumatoid Arthritis Model by Interleukin 10 (IL10)-Mesenchymal Stem Cells (BMSCs).

BACKGROUND Rheumatoid arthritis model (CIA) rats were treated by tail vein injection of IL-10-modified bone marrow mesenchymal stem cells (BMSCs) to investigate its feasibility and intrinsic molecular mechanism. MATERIAL AND METHODS The CIA rat model was established by induction type II collagen, and IL-10-modified BMSCs was established by transfecting BMSCs with adenovirus. IL-10-modified BMSCs were used to treat the CIA rats. The therapeutic effect was evaluated by measuring the changes in body weight, ankle swelling, and forced swimming time, as well as observation of synovial hyperplasia and cartilage tissue repair by HE staining. Western blot analysis and ELISA were used to detect gene expression. RESULTS After 4 weeks and 8 weeks of treatment with IL10-BMSCs, the body weight, swelling value, resting time, and forced swimming struggle time of CIA rats were significantly higher than those of BMSCs-treated and -untreated CIA rats (P<0.05). Compared to BMSCs-treated CIA model rats, after treatment with IL10-BMSCs, the repair rate of osteoarticular cartilage was higher and the inhibition of synovial proliferation was better, and serum IL-17, IL-1ß, and TNF-alpha levels were lower. We found that the protein level of SIRT1 in peripheral blood mononuclear cells was lower, the protein level in spleen was higher, and phosphorylation of p65 protein in peripheral blood mononuclear cells was reduced. CONCLUSIONS The efficacy of tail vein injection of IL-10-modified BMSCs in treatment of CIA rats was superior to that of BMSCs alone, which may be related to the more pronounced suppression of IL-10-modified BMSCs in peripheral blood inflammation and spleen immune response.

Novel Hydrolyzed Chicken Sternal Cartilage Extract Improves Facial Epidermis and Connective Tissue in Healthy Adult Females: A Randomized, Double-Blind, Placebo-Controlled Trial.

CONTEXT: Dietary supplement manufacturers claim cutaneous anti-aging properties for their products; however, research supporting these claims remains sparse. OBJECTIVES: The study intended to determine if a correlation existed between the effects of a collagen dietary supplement and changes associated with skin aging. DESIGN: The study was a 12-week, double-blind, placebo-controlled trial. SETTING: The study took place at a clinical facility specializing in dermatological testing that could perform biophysical, instrumental analysis on the effects of proprietary supplement on human skin. PARTICIPANTS: Participants were 128 females, aged 39-59 (50.57 ± 5.55). INTERVENTION: Participants were randomly assigned to an intervention or a placebo. The intervention consisted of twice daily oral administration of a supplement containing 500 mg BioCell Collagen, a chicken sternal cartilage derived dietary ingredient composed of a naturally-occurring matrix of hydrolyzed collagen type-II (≥300 mg), chondroitin sulfate (≥100 mg), hyaluronic acid (≥50 mg). OUTCOME MEASURES: The primary parameters included transepidermal water loss, viscoelasticity, hydration, (indirect) collagen content, chromophore (melanin) content and hemoglobin level, and photographic analysis. An expert visually graded participants' skin to determine the intervention's efficacy, measuring facial lines and wrinkles, crow's feet lines and wrinkles, skin texture and smoothness, and skin tone. The presence of erythema and/or dryness determined tolerance. Secondary outcome measures were tolerance and incidence of adverse events, and the participant's perception of the supplement's value. RESULTS: For the 113 participants completing the study, the dietary supplementation compared to a placebo: (1) significantly reduced facial lines and wrinkles (P = .019) and crow's feet lines and wrinkles (P = .05), (2) increased skin elasticity (P = .008) and cutaneous collagen content (P < .001) by 12%, (3) improved indicators associated with a more youthful skin appearance based on visual grading and wrinkle width (P = .046), and (4) decreased skin dryness and erythema. No difference existed between the supplement and the placebo for skin-surface water content or retention. The supplement was well tolerated, with no reported adverse reactions. CONCLUSIONS: Dietary supplementation with chicken, sternal cartilage extract supports the accumulation of types-I/III collagen in skin to promote increased elasticity and reduced skin wrinkling.

Oral administration of undenatured native chicken type II collagen (UC-II) diminished deterioration of articular cartilage in a rat model of osteoarthritis (OA).

OBJECTIVE: The aim of this study was to determine the ability of undenatured native chicken type II collagen (UC-II) to prevent excessive articular cartilage deterioration in a rat model of osteoarthritis (OA). METHODS: Twenty male rats were subjected to partial medial meniscectomy tear (PMMT) surgery to induce OA. Immediately after the surgery 10 rats received vehicle and another 10 rats oral daily dose of UC-II at 0.66 mg/kg for a period of 8 weeks. In addition 10 naïve rats were used as an intact control and another 10 rats received sham surgery. Study endpoints included a weight-bearing capacity of front and hind legs, serum biomarkers of bone and cartilage metabolism, analyses of subchondral and cancellous bone at the tibial epiphysis and metaphysis, and cartilage pathology at the medial tibial plateau using histological methods. RESULTS: PMMT surgery produced moderate OA at the medial tibial plateau. Specifically, the deterioration of articular cartilage negatively impacted the weight bearing capacity of the operated limb. Immediate treatment with the UC-II preserved the weight-bearing capacity of the injured leg, preserved integrity of the cancellous bone at tibial metaphysis and limited the excessive osteophyte formation and deterioration of articular cartilage. CONCLUSION: Study results demonstrate that a clinically relevant daily dose of UC-II when applied immediately after injury can improve the mechanical function of the injured knee and prevent excessive deterioration of articular cartilage.

Zaocys type II collagen regulates the balance of Treg/Th17 cells in mice with collagen-induced arthritis.

OBJECTIVES: Zaocys type II collagen is an active collagen extracted from Zaocys that has been used to treat rheumatoid arthritis in China for over 1000 years. However, the mechanism still remains unknown. Therefore, we set out to investigate the inhibitory effect and possible mechanism of action of zaocys type II collagen on collagen-induced arthritis. METHODS: Collagen-induced arthritis was induced in C57BL/6 mice by immunisation with type II collagen. After immunisation, the mice were treated with Zaocys type II collagen. Clinical and histological scores were assessed and the cytokine levels in the serum and lymphocytes supernatant from the spleen and mesenteric lymph node were determined by enzyme-linked immune sorbent assay. The T-helper 17 cell and regulatory-T cell frequencies were analysed by flow cytometry and the expression of interest markers was examined by direct immuno-fluorescence. RESULTS: The arthritis score and severity of histological inflammation and cartilage destruction were dose-dependently reduced after treatment. The analysis results indicated that Zaocys type II collagen significantly increased the proportion of regulatory-T cells and lowered the T-helper 17 cells, it also increased the number of regulatory-T cells and conversely decreased the T-helper 17 cells in synovial tissue compared with the model group. Treatment also caused a higher level of transforming growth factor-ß and a decreased production of interleukin -17A. CONCLUSIONS: The oral administration of Zaocys type II collagen potently suppressed the severity of collagen-induced arthritis by repairing the imbalance between regulatory-T cells and T-helper 17 cells, suggesting that it might be a promising candidate for the treatment of rheumatoid arthritis.

Citrullination of collagen II affects integrin-mediated cell adhesion in a receptor-specific manner.

Citrullinated collagen II (CII) is a well-known autoantigen in rheumatoid arthritis (RA). However, the direct effects of CII citrullination on cell behavior have not been described. To study whether citrullination of CII could affect cellular functions, we measured the adhesion of 3 different cell types (human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal stem cells) with impedance-based technology. The binding of different collagen receptor integrins to citrullinated collagen was studied by CHO cell lines, each overexpressing 1 of the 4 human collagen receptors on the cell surface, and with solid-phase binding assays, using the recombinant human integrin α1I, α2I, α10I, and α11I domains. Collagen citrullination decreased the adhesion of synovial fibroblasts ∼50% (P<0.05) and mesenchymal stem cells ∼40% (P<0.05) by specifically decreasing the binding of integrins α10ß1 and α11ß1 to arginine-containing motifs, such as GFOGER. In contrast, citrullination had only a minor effect on the function of α1ß1 and α2ß1 integrins, which have been reported to play a critical role in regulating leukocyte function. Molecular modeling was used to explain the detected functional differences at the structural level. Given that the integrins regulate cell metabolism, proliferation, and migration, we suggest that collagen citrullination modifies the pathogenesis of RA. Here, CII citrullination was shown to decrease the survival of mesenchymal stem cells.

The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs.

During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably employ non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To increase the success of translating these strategies for clinical application, this study aimed to delineate whether NC-secreted factors of different species have a regenerative effect on human CLCs. Human, canine and porcine NC-rich NP tissue and NC-conditioned medium (NCCM) were analysed biochemically and histologically. Human CLC micro-aggregates from degenerated IVDs were cultured in human, canine or porcine NCCM. Collagen, glycosaminoglycan (GAG) and DNA content was determined and histology was performed. Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than that of human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human CLC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human CLCs, indicating a cross-species effect. Bioactive compound(s) are present in NCCM of different species that may reverse human IVD degeneration, supporting further research into strategies based on NC-technology employing canine or porcine models for their translation into humans.

Hyperleptinemia suppresses aggravation of arthritis of collagen-antibody-induced arthritis in mice.

BACKGROUND: Leptin is an adipocytokine produced by adipocytes and controlling body weight. It is unclear whether leptin works as a proinflammatory or an anti-inflammatory cytokine. We investigated the effects of hyperleptinemia on leptin transgenic (LepTg) mice in terms of cartilage destruction, bone destruction, joint synovitis, and serum cytokine levels by using a mouse model of collagen-antibody-induced arthritis (CAIA). METHODS: CAIA was induced for female age-matched 6- to 8-week-old C57BL/6 J control mice and LepTg mice. Mice were injected intraperitoneally with 5 mg of a combination of monoclonal antibody specific for type II collagen on day 0 and 12.5 mg of lipopolysaccharide (LPS) on day 3. Clinical evaluation of arthritis was monitored for 14 days, and hind paws were examined clinically and histologically. Serum cytokine levels of interleukin (IL)-1ß, IL-6, IL-10, and IL-17 and tumor necrosis factor alpha (TNF-α) were also analyzed on days 0 and 5. Moreover, THP-1 cells, which are human monocytic cell line derived from an acute monocytic leukemia patient, were cultured and differentiated into macrophages. The effects of leptin on messenger RNA (mRNA) expression of IL-6 were examined by real-time quantitative polymerase chain reaction (RT-PCR). RESULTS: Serum leptin concentrations were approximately ninefold higher in LepTg mice (62.0 ± 20.7 ng/ml) than in control mice (7.2 ± 0.5 ng/ml). Severity of clinical paw swelling, arthritis score, synovial hyperplasia, and cartilage damage were suppressed in LepTg mice with CAIA. Although serum cytokine levels of IL-1ß, IL-17, and IL-10 and TNF-α showed no significant changes in two mice, serum levels of IL-6 in LepTg mice were suppressed at day 5. Moreover, in vitro study showed that IL-6 elevation following LPS exposure in THP-1 cells was suppressed with high leptin concentrations. CONCLUSION: Our finding suggests that hyperleptinemia suppress IL-6 responses and progression of joint inflammation. Leptin may play an anti-inflammatory role under hyperleptinemia.

Increased expression of Siglec-1 on peripheral blood monocytes and its role in mononuclear cell reactivity to autoantigen in rheumatoid arthritis.

OBJECTIVES: Elevated expression of Siglec-1 on circulating monocytes has been reported in some inflammatory and autoimmune diseases, but its expression and role in RA has not been elucidated. The aims of this study were to determine the expression of Siglec-1 in peripheral blood and to explore its role in mononuclear cell reactivity to autoantigen in RA. METHODS: Siglec-1 protein and mRNA levels in 42 RA patients, 39 OA patients, 28 SLE patients and 42 normal controls were determined by flow cytometry and quantitative RT-PCR, respectively. In addition, 10 patients with active RA received DMARDs for 12 weeks and the frequencies of Siglec-1-positive cells and the 28-joint DAS (DAS28) were assessed before and after therapy. Furthermore, TNF-α, IFN-γ and type II collagen were used to up-regulate Siglec-1. Peripheral blood mononuclear cells (PBMCs) from different groups were stimulated with mitogens or antigens and cell proliferation and cytokine production were determined. RESULTS: The protein and mRNA levels of Siglec-1 on PBMCs and monocytes in RA patients were significantly higher than those in OA patients and healthy controls. Moreover, the expression of Siglec-1 protein on PBMCs was positively correlated with DAS28, ESR, high-sensitivity CRP and IgM-RF, but not with anti-CCP antibody. Interestingly, Siglec-1 expression was decreased in parallel with the decrease in the DAS28 after 12 weeks of anti-rheumatic treatment. Furthermore, TNF-α, IFN-γ and type II collagen can up-regulate Siglec-1 in PBMCs. Elevated PBMC proliferation and proinflammatory cytokine production to collagen stimulation in RA patients decreased when Siglec-1 was inhibited by anti-Siglec-1 antibodies. CONCLUSION: Elevated Siglec-1 expression in PBMCs and monocytes can potentially serve as a biomarker for monitoring disease activity in RA. Siglec-1 may also play a proinflammatory role in stimulating lymphocyte proliferation and activation in RA.

Novel functions for type II procollagen.

Cartilage is unique in being established as an avascular tissue during development. Cartilage also has the property of being resistant to tumor invasion with tumors arising on the periphery of cartilage and in bone, but sparing the cartilage. These properties have been investigated for many years beginning in the 1970's. Many anti-angiogenic molecules have been isolated from cartilage in small amounts. Portions of molecules from cartilage also possess anti-angiogenic properties when released from the parent protein by degradative extracellular enzymes. This review highlights a new anti-angiogenic and anti-tumor moiety from cartilage, the NH2-propeptide of type IIB collagen. When released from the procollagen during synthesis, the propeptide has the capacity to act on its own to protect the cartilage by killing of endothelial cell, osteoclasts and tumor cells.

Type II collagen and gelatin from silvertip shark (Carcharhinus albimarginatus) cartilage: isolation, purification, physicochemical and antioxidant properties.

Type II acid soluble collagen (CIIA), pepsin soluble collagen (CIIP) and type II gelatin (GII) were isolated from silvertip shark (Carcharhinus albimarginatus) cartilage and examined for their physicochemical and antioxidant properties. GII had a higher hydroxyproline content (173 mg/g) than the collagens and cartilage. CIIA, CIIP and GII were composed of two identical α1 and ß chains and were characterized as type II. Amino acid analysis of CIIA, CIIP and GII indicated imino acid contents of 150, 156 and 153 amino acid residues per 1000 residues, respectively. Differing Fourier transform infrared (FTIR) spectra of CIIA, CIIP and GII were observed, which suggested that the isolation process affected the secondary structure and molecular order of collagen, particularly the triple-helical structure. The denaturation temperature of GII (32.5 °C) was higher than that of CIIA and CIIP. The antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl radicals and the reducing power of CIIP was greater than that of CIIA and GII. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. Accordingly, the physicochemical and antioxidant properties of type II collagens (CIIA, CIIP) and GII isolated from shark cartilage were found to be suitable for biomedical applications.

The mechanistic role of curcumin on matrix metalloproteinases in osteoarthritis.

A systematic mechanistic review was performed to determine mechanistic evidence for curcumin on pro-inflammatory matrix metalloproteinases and Osteoarthritis to understand the underlying pathophysiology, and to evaluate available human intervention evidence to inform clinical decision making. The systematic literature search was performed in 3 tranches (reviews, mechanistic, intervention studies) using PubMed, with no date limitations and using specific search terms. 65 out of 393 screened papers were accepted based on detailed inclusion and exclusion criteria. The mechanistic search was divided into three searches and the intervention searches were subdivided into four searches. Curcumin demonstrated significant inhibition of matrix metalloproteinases linked to cartilage degradation in Osteoarthritis through reduced activation of the nuclear factor kappa-B signaling pathway via suppressing phosphorylation of Iκßa and p65 nuclear translocation. Mechanistic evidence implicated matrix metalloproteinases in Osteoarthritis by decreasing Type II collagen, leading to cartilage damage. As a potential nutritional intervention for Osteoarthritis, curcumin could reduce inflammatory markers and improve pain and function scores. The evidence indicates most formulations of turmeric extract and curcumin extract, bio-enhanced and non-bio-enhanced, are effective at improving inflammatory markers and pain and function to a greater or lesser extent. Due to the high heterogeneity of the formulations, dosage, and duration of the studies, further research is needed to fully understand curcumin's potential as a promising non-pharmaceutical intervention for Osteoarthritis. This mechanism review identifies a gap in current research for the mechanism by which Type II collagen is mediated.

Molecular basis for T cell response induced by altered peptide ligand of type II collagen.

Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9·I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-ζ and signal via FcRγ and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcRγ, or CD3-ζ, we demonstrate that A9·I-A(q) preferentially activates FcRγ/Syk but not CD3-ζ/Zap-70. Moreover, a genetic absence of Syk or FcRγ significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcRγ and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.

Mixed type I and type II collagen scaffold for cartilage repair: ultrastructural study of synovial membrane response and healing potential versus microfractures (a pilot study).

The association between microfracture of the subchondral plate and a coverage scaffold has emerged as a promising strategy to treat cartilage lesions in a one-step procedure. Between different types of scaffolds (e.g. collagen, hyaluronic acid, polyglycolic acid) currently studied, type I collagen scaffold is the most used for this purpose, and is currently adopted for humans. The aim of this study was to test a novel scaffold made of mixed type I and II collagen (I-IICS) in order to define the immunological reaction of the synovial tissue and the repair capabilities induced by the collagen membrane when associated with microfracture. Eight New Zealand White rabbits, aged 180 days, were operated on bilaterally on the medial femoral condyle. A circular cartilage lesion was performed up to the calcified layer of the medial femoral condyle, and the centre of the lesion was microfractured. Randomly, one of the two lesions was covered with the I-IICS (treated), and the other was left uncovered (control). The synovial membrane reaction and the quality of the cartilage tissue repair were investigated at 2, 90, 180 and 270 days macroscopically, histomorphologically and ultrastructurally. Expression of tumor necrosis factor-alpha (TNF-alpha) in synovial tissue by immunocytochemistry analyses was also investigated. In the control group, at 2 days gold particles were localized mainly on synoviocyte type A, less on synoviocytes type B and on collagen bundles; in the treated group the reaction is more intense in cells in the matrix, but at 180 days controls and treated joints were very similar. The synovial membranes of the joints receiving the I-IICS did not reveal significant changes compared to the age-matched controls. Signs of inflammation were present at the 90-day time-point, and became less evident at afterwards. The degradation of the scaffolds was already evident at the 90-day time-point. The quality of the cartilage repair of the rabbits treated with the I-IICS was slightly better in 5 cases out of 6 in comparison to the controls. However, a statistically significant difference was not detected (p=0.06). Scaffolds made of mixed type I and II collagen exhibited good biocompatibility properties in vivo and favoured cartilage restoration when associated with microfracture, as shown in this pilot study.

Mucosal-associated invariant T cells promote inflammation and exacerbate disease in murine models of arthritis.

OBJECTIVE: The function of mucosal-associated invariant T (MAIT) cells remains largely unknown. We previously reported an immunoregulatory role of MAIT cells in an animal model of multiple sclerosis. The aim of this study was to use animal models to determine whether MAIT cells are involved in the pathogenesis of arthritis. METHODS: MR1-/- and MR1+/+ DBA/1J mice were immunized with bovine type II collagen (CII) in complete Freund's adjuvant to trigger collagen-induced arthritis (CIA). To assess CII-specific T cell recall responses, lymph node cells from mice with CIA were challenged with CII ex vivo, and cytokine production and proliferation were evaluated. Serum levels of CII-specific antibodies were measured by enzyme-linked immunosorbent assay. Collagen antibody-induced arthritis (CAIA) was induced in MR1-/- and MR1+/+ C57BL/6 mice by injection of anti-CII antibodies followed by injection of lipopolysaccharide. To demonstrate the involvement of MAIT cells in arthritis, we induced CAIA in MR1-/- C57BL/6 mice that had been reconstituted with adoptively transferred MAIT cells. MAIT cell activation in response to cytokine stimulation was investigated. RESULTS: The severity of CIA was reduced in MR1-/- DBA/1J mice. However, T and B cell responses to CII were comparable in MR1-/- and MR1+/+ DBA/1J mice. MR1-/- C57BL/6 mice were less susceptible to CAIA, and reconstitution with MAIT cells induced severe arthritis in MR1-/- C57BL/6 mice, demonstrating an effector role of MAIT cells in arthritis. MAIT cells became activated upon stimulation with interleukin-23 (IL-23) or IL-1ß in the absence of T cell receptor stimuli. CONCLUSION: These results indicate that MAIT cells exacerbate arthritis by enhancing the inflammation.

Dexamethasone preconditioning improves the response of collagen-induced arthritis to treatment with short-term lipopolysaccharide-stimulated collagen-loaded dendritic cells.

Background. Pharmacologically modulated dendritic cells (DCs) have been shown to restore tolerance in type II collagen-(CII-) induced arthritis (CIA). We examined the effect of dexamethasone (DXM) administration as a preconditioning agent, followed by an injection of lipopolysaccharide-(LPS-) stimulated and CII-loaded DCs on the CIA course. Methods. After CIA induction, mice pretreated with DXM were injected with 4-hour LPS-stimulated DCs loaded with CII (DXM/4hLPS/CII/DCs). Results. Mice injected with DXM/4hLPS/CII/DCs displayed significantly less severe clinical disease compared to animals receiving 4hLPS/CII/DCs alone or those in which only DXM was administered. Cytokine profile evaluation showed that CD4+ T cells from DXM/4hLPS/CII/DCs and 4hLPS/CII/DCs groups release higher IL-10 levels than those from mice receiving DXM alone or CIA mice. CD4+ T cells from all DC-treated groups showed less IL-17 release when compared to the CIA group. On the contrary, CD4+ T cells from DXM/4hLPS/CII/DCs and 4hLPS/CII/DCs groups released higher IFN- γ levels than those from CIA group. Conclusion. A combined treatment, including DXM preconditioning followed by an inoculation of short-term LPS-stimulated CII-loaded DCs, provides an improved strategy for attenuating CIA severity. Our results suggest that this benefit is driven by a modulation in the cytokine profile secreted by CD4+ T cells.

Nuclear factor-κB activation by type II collagen peptide in articular chondrocytes: its inhibition by hyaluronan via the receptors.

OBJECTIVE: This study aimed to examine nuclear factor-κB (NF-κB) activation by a synthetic peptide from type II collagen fragment (CB12-II) and its inhibition by hyaluronan (HA) via its receptors, CD44, and intercellular adhesion molecule-1 (ICAM-1) in chondrocytes. METHODS: Osteoarthritic cartilage explants or chondrocytes in monolayer were cultured with CB12-II. Secreted levels of matrix metalloproteinase (MMP)-13 in conditioned media and NF-κB activation in chondrocytes were determined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Cultures were pretreated with HA to evaluate the inhibitory effect on CB12-II action, and the role of HA receptors in HA effect was investigated using antibodies to CD44 and ICAM-1. RESULTS: CB12-II stimulated phosphorylation and nuclear translocation of NF-κB, leading to increased MMP-13 production. HA suppressed NF-κB activation and MMP-13 induction by CB12-II. The individual antibody to CD44 or ICAM-1 partially reversed HA effect on CB12-II action, and both antibodies in combination completely blocked the HA effect. CONCLUSIONS: This study clearly demonstrates that CB12-II activates NF-κB for MMP-13 induction and that HA inhibits CB12-II action through interaction with CD44 and ICAM-1 in chondrocytes. HA administration into osteoarthritic joints could suppress the catabolic action of matrix degradation products such as CB12-II as a potent NF-κB inhibitor.

Psoralidin Induced Differentiation from Adipose-derived Stem Cells to Nucleus Pulposus-like Cells by TGF-ß/Smad Signaling.

BACKGROUND: Psoralidin (PL) could affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). The role of PL is still unclear in adipose-derived stem cells (ADSCs). AIMS: This study aimed to investigate the effects of PL on ADSCs differentiation into nucleus pulposus-like cells and the TGF-ß/Smad signaling pathway. METHODS: The proliferation and apoptosis of ADSCs were detected. The nucleus pulposus cell-related markers (CD24, BASP1, KRT19, and Aggrecan) and TGF-ß/Smad signaling pathway indexes were analyzed. RESULTS: The results showed that compared to the control group, the cell activity was increased in the PL group, and the apoptosis rate was decreased. The mRNA and protein levels of nucleus pulposus cells markers (CD24, BASP1, KRT19, Aggrecan, and Collagen Type II) and TGF-ß/Smad signaling pathway-related indexes (TGF-ß, SMAD2, and SMAD3) were increased in PL group. After treatment with PL and TGF-ß silencing, the TGF-ß/Smad signaling pathway-related indicators (TGF-ß, SMAD2, and SMAD3) and nucleus pulposus cells markers (CD24, BASP1, KRT19, Aggrecan, and Collagen Type II) were found to be higher in the sh-TGF-ß +PL group than in the sh-TGF-ß group. CONCLUSION: In conclusion, our study showed that PL might induce the differentiation of ADSCs to nucleus pulposus cells through the TGF-ß/Smad signaling pathway. It might have the potential application value in the treatment of intervertebral disc degeneration.