Tanshinone IIA alleviates IL-1ß-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.

OBJECTIVE: This study explored the pharmacological mechanism of Tanshinone IIA (TAN IIA) in the treatment of Osteoarthritis (OA), which provided a certain reference for further research and clinical application of Tan IIA in OA. METHODS: CHON-001 cells were stimulated with 10 µg/mL IL-1ß for 48 h and treated with 10 µM TAN IIA for 48 h. Cellular viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, and Cleaved caspase-3 was measured by Immunoblot assay and RT-qPCR. TNF-α, IL-6, and iNOS in CHON-001 cells were determined by RT-qPCR and ELISA. To further verify the effect of TAN IIA on OA, a rat model of OA in vivo was established by right anterior cruciate ligament transection. TAN IIA was administered at 50 mg/kg or 150 mg/kg for 7 weeks. The degree of cartilage destruction in OA rats was observed by TUNEL and HE staining. Cleaved caspase-3 and FBXO11 were measured by immunohistochemical staining, RT-qPCR, and Immunoblot. TNF-α, IL-6, and iNOS in chondrocytes of OA rats were detected by ELISA. RESULTS: IL-1ß stimulated CHON-001 cell apoptosis and inflammation, and TAN IIA had anti-apoptosis and anti-inflammatory effects on IL-1ß-regulated CHON-001 cells. TAN IIA down-regulated FBXO11 and inhibited PI3K/AKT and NF-κB pathways, thereby alleviating apoptotic and inflammatory reactions in CHON-001 cells under IL-1ß treatment. Moreover, TAN IIA treatment improved chondrocyte apoptosis and inflammations in OA rats. CONCLUSION: TAN IIA inhibits PI3K/Akt and NF-κB pathways by down-regulating FBXO11 expression, alleviates chondrocyte apoptosis and inflammation, and delays the progression of OA.

Phenotype and synthesis activity of joint chondrocytes extracted from newborn rats with prenatal ethanol exposure.

Thirteen female Wistar rats were divided into two groups: one treated with ethanol and the other of untreated. Four newborns from each mother were selected and weighed, measured, and evaluated for physical characteristics. From these neonates, chondrocytes were extracted from the articular cartilages of the femur and tibia, and cultivated in a chondrogenic medium at 37oC and 5% CO2. At 7, 14, and 21 days of cultivation, alkaline phosphatase activity tests, MTT conversion to formazan, and percentage area covered by cells per field were performed. At 21 days, the percentage of PAS+ areas in 3D cultures was performed, as well as the evaluation of gene transcript expression for aggrecan, SOX-9, collagen type II, collagen X, Runx-2, and VEGF by real-time RT-PCR. The means were compared by Student's t-test. The weight of the ethanol group neonates was significantly lower than that of the controls. Chondrocyte cultures from the ethanol group showed significantly higher AP activity, MTT conversion, and cell percentage. There was higher expression of collagen type II and lower expression of SOX-9 in the ethanol group. There was no difference in the percentage of PAS+ areas in pellets and in expression of aggrecan, collagen X, Runx-2, or VEGF between groups. In conclusion, prenatal exposure to ethanol alters the phenotype and activity of offspring chondrocytes, which may be mechanisms by which endochondral bone formation is compromised by maternal ethanol consumption.

In vivo effect of UV-photofunctionalization of CoCrMo in processes of guided bone regeneration and tissue engineering.

Photofunctionalization of implant materials with ultraviolet (UV) radiation have been subject of study in the last two decades, and previous research on CoCrMo discs have showed good results in terms of bioactivity and the findings of apatite-like crystals in vitro. In the current study, CoCrMo domes were photofunctionalized with UV radiation of 254 nm on their internal faces during 24 hr; they were implanted in rabbit tibia and remained for 3, 4, and 6 weeks. The potential to induce bone formation beneath the dome-shaped membranes was evaluated through morphometric, histologic, and density measurements; and the results were compared with those obtained under control untreated domes. Higher density values were observed for irradiated domes at 3 weeks, whereas higher volumes were obtained under photofunctionalized domes for longer periods (4 and 6 weeks). Histologically, woven bone was formed by endochondral ossification in all cases; differences in the architecture and size of the trabeculae and in the number of osteoblasts were noted between irradiated and non-irradiated samples. The UV radiation of 254 nm generated a larger bone volume fraction compared to that found in the absence of UVC radiation and induced an increase of density in the early stages of healing, leading to a better initial bone quality and improved osseointegration.

Effect of cadmium on the viability on monolayer cultures of synoviocytes, chondrocytes, and Hoffa: A preliminary study.

Osteoarthritis (OA) is the gradual loss of articular cartilage and involves several tissues, such as the synovial membrane, meniscus, ligaments, and adipose tissue known as Hoffa fat pad. There are largely unexplored factors that lead to OA development, such as the impact of exposure to heavy metals like cadmium (Cd) on the viability of cells in the knee joint tissue. The objective of this report was to identify the cell type with the highest susceptibility to Cd toxicity with respect to cell viability and death. Our findings showed that a concentration as low as 3 µM cadmium chloride for 12 h affects the viability of synovial cells, and a concentration of 10 µM affects Hoffa cells. Our results suggest that Cd can affect the viability of synovial and chondral cells primarily. In contrast, Hoffa cells were less susceptible, likely because Cd favors the production of pro-inflammatory cytokines before triggering their death as part of its damage mechanism at the articular level.

Effect of cadmium on the concentration of essential metals in a human chondrocyte micromass culture.

BACKGROUND: An essential element imbalance in the joint might favor gradual degeneration of the articular cartilage. It has been reported that cadmium (Cd) plays an antagonistic role with regards to the presence of essential elements, such as zinc (Zn), iron (Fe), and manganese (Mn), which may favor the development of disabling diseases, like osteoarthritis (OA) and osteoporosis. METHODS: 3D cultures of human chondrocytes were phenotyped with the Western blot technique and structurally evaluated with histological staining. The samples were exposed to 1, 5, and 10 µM of CdCl2 for 12 h, with a non-exposed culture as control. The concentration of Cd, Fe, Mn, Zn, chromium (Cr), and nickel (Ni) was quantified through plasma mass spectrometry (ICP-MS). The data were analyzed with a Kruskal Wallis test, a Kendall's Tau test and Spearman's correlation coefficient with the Stata program, version 14. RESULTS: Our results suggest that Cd exposure affects the structure of micromass cultures and plays an antagonistic role on the concentration of essential metals, such as Zn, Ni, Fe, Mn, and Cr. CONCLUSION: Cd exposure may be a risk factor for developing joint diseases like OA, as it can interfere with cartilage absorption of other essential elements that maintain cartilage homeostasis.

Bushenhuoxue formula promotes osteogenic differentiation of growth plate chondrocytes through ß-catenin-dependent manner during osteoporosis.

BACKGROUND: Bushenhuoxue formula (BSHXF) has shown excellent clinical effects on the treatment of osteoporosis in China. The aim of this study is to determine the anti-osteoporosis effects and precise molecular mechanisms of BSHXF on mouse models. METHODS: Ten-week-old female C57BL/6 J mice were subjected to ovariectomy and provided a daily treatment of BSHXF. At 8 weeks post-surgery, the femurs were harvested for tissue analyses including µCT, histology, qRT-PCR and immunohistochemical (IHC) staining of ß-catenin, ALP and FABP4. To investigate the role of ß-catenin in the anti-osteoporosis effects of BSHXF, relative experiments mentioned above were performed in ß-catenin conditional knockout mice. RESULTS: Ovariectomized (OVX) mice presented severe bone loss and excessive fat accumulation in the chondro-osseous junction underneath the growth plate, with decreased expression of ALP and increased expression of FABP4. BSHXF significantly recovered the OVX-induced abnormal osteogenesis and adipogenesis with the activation of ß-catenin in growth plate chondrocytes. Further, we generated growth plate chondrocyte-specific ß-catenin knockout (ß-cateninGli1ER) mice that exhibited bone loss and fat accumulation in the chondro-osseous junction, similar to the OVX mice. However, BSHXF failed to rescue the osteoporosis-like phenotype in ß-cateninGli1ER mice, indicating the anti-osteoporosis effects of BSHXF act mainly through ß-catenin signaling. No significant restoration of ALP and FABP4 was observed in ß-cateninGli1ER mice after the treatment of BSHXF. CONCLUSIONS: BSHXF attenuates osteoporosis by promoting osteogenic differentiation of growth plate chondrocytes mainly in ß-catenin-dependent manner. BSHXF is considered as a new candidate for the treatment of osteoporosis.

Evaluation of the articular cartilage in the knees of rats with induced arthritis treated with curcumin.

This study was designed to evaluate the anti-inflammatory effects of a curcumin treatment on the knee of rats with induced osteoarthritis. Fifteen adult rats were used and divided in three groups: the osteoarthritis group (OAG), control group (CG-without induction of osteoarthritis), and curcumin-treated osteoarthritis group (COAG). Osteoarthritis was induced in the right knee of rats in the OAG and COAG by administering an intra-articular injection of 1 mg of zymosan. Fourteen days after induction, 50 mg/kg curcumin was administered by gavage daily for 60 days to the COAG. After the treatment period, rats from all groups were euthanized. Medial femoral condyles were collected for light microscopy and immunohistochemical staining. The expression of SOX-5, IHH, MMP-8, MMP-13, and collagen 2 (Col2) was analyzed. The COAG exhibited an increase in the number of chondrocytes in the surface and middle layers compared with that of the OAG and CG, respectively. The COAG also showed a decrease in the thicknesses of the middle and deep layers compared with those of the OAG, and an increase in Col2 expression was observed in all articular layers (surface, middle, and deep) in the COAG compared with that in the OAG. SOX-5 expression was increased in the surface and deep layers of the COAG compared with those in the OAG and CG. Based on the results of this study, the curcumin treatment appeared to exert a protective effect on cartilage, as it did not result in an increase in cartilage thickness or in MMP-8 and MMP-13 expression but led to increased IHH, Col2, and SOX-5 expression and the number of chondrocytes.

Electrospraying Oxygen-Generating Microparticles for Tissue Engineering Applications.

BACKGROUND: The facile preparation of oxygen-generating microparticles (M) consisting of Polycaprolactone (PCL), Pluronic F-127, and calcium peroxide (CPO) (PCL-F-CPO-M) fabricated through an electrospraying process is disclosed. The biological study confirmed the positive impact from the oxygen-generating microparticles on the cell growth with high viability. The presented technology could work as a prominent tool for various tissue engineering and biomedical applications. METHODS: The oxygen-generated microparticles fabricated through electrospraying processes were thoroughly characterization through various methods such as X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) analysis, and scanning electron microscopy (SEM)/SEM-Energy Dispersive Spectroscopy (EDS) analysis. RESULTS: The analyses confirmed the presence of the various components and the porous structure of the microparticles. Spherical shape with spongy characteristic microparticles were obtained with negative charge surface (ζ = -16.9) and a size of 17.00 ± 0.34 µm. Furthermore, the biological study performed on rat chondrocytes demonstrated good cell viability and the positive impact of increasing the amount of CPO in the PCL-F-CPO-M. CONCLUSION: This technological platform could work as an important tool for tissue engineering due to the ability of the microparticles to release oxygen in a sustained manner for up to 7 days with high cell viability.

Assessment of Cell Viability of Fresh Osteochondral Allografts in N-Acetylcysteine-Enriched Medium.

OBJECTIVE: The purpose of this study was to evaluate the effect of N-acetylcysteine (NAC)-enriched storage medium on fresh osteochondral viability at 4°C. Our hypothesis was that the cell viability of chondrocytes obtained from human osteochondral tissue and stored at 4°C significantly improves in the presence of NAC. DESIGN: Controlled laboratory study. For this study, 8 samples of femoral condyle osteochondral tissue were obtained from patients undergoing total knee replacement. The samples were stored at either 4°C in phosphate-buffered saline (PBS) or at 3 different concentrations of NAC (NAC 1, 2, and 5 mM). Cell viability was analyzed at time 0 and 4 weeks by flow cytometry. The results of cell viability (median) were analyzed statistically using analysis of variance and Tukey's post hoc test. P values <0.05 were considered statistically significant. RESULTS: The viability at time 0 was 95.5% ± 3.7%. At 4 weeks, the cell viability was 56.8% ± 20.1% in the control group (PBS), 83.8% ± 11.9% in the group stored with NAC 1 mM, 73.4% ± 13.6% in the group stored with NAC 2 mM, and 66.4% ± 27.7% in the group stored with NAC 5 mM. A statistically significant difference from the baseline viability (time 0) was observed in the PBS control group (P = 0.0018) but not in the other groups. A statistically significant difference was observed in the NAC 1 mM group compared with the PBS group (P = 0.0255). CONCLUSION: The use of NAC at 1 mM concentration improves cell viability after 4 weeks of storage in chondrocytes obtained from human osteochondral tissue.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1ß, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1ß by suppressing the increase in IL-1ß, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1ß-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1ß to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Propolis Reduces the Expression of Autophagy-Related Proteins in Chondrocytes under Interleukin-1ß Stimulus.

BACKGROUND: Osteoarthritis (OA) is a progressive and multifactorial disease that is associated with aging. A number of changes occur in aged cartilage, such as increased oxidative stress, decreased markers of healthy cartilage, and alterations in the autophagy pathway. Propolis extracts contain a mixture of polyphenols and it has been proved that they have high antioxidant capacity and could regulate the autophagic pathway. Our objective was to evaluate the effect of ethanolic extract of propolis (EEP) on chondrocytes that were stimulated with IL-1ß. METHODS: Rabbit chondrocytes were isolated and stimulated with IL-1ß and treated with EEP. We evaluated cell viability, nitric oxide production, healthy cartilage, and OA markers, and the expression of three proteins associated with the autophagy pathway LC3, ATG5, and AKT1. RESULTS: The EEP treatment reduces the expression of LC3, ATG5, and AKT1, reduces the production of nitric oxide, increases the expression of healthy markers, and reduces OA markers. CONCLUSIONS: These results suggest that treatment with EEP in chondrocytes that were stimulated with IL-1ß has beneficial effects, such as a decrease in the expression of proteins associated with autophagy, MMP13, and production of nitric oxide, and also increased collagen II.

Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats / Efeito inibidor direto da cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos da cartilagem articular de ratos

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)

O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)

Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats / Efeito inibidor direto da cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos da cartilagem articular de ratos

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)

O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)

No effect of topical application of tranexamic acid on articular cartilage.

PURPOSE: The objective of this study was to evaluate potential cytotoxicity of TXA on articular cartilage by assessing chondrocyte viability of osteochondral explants after exposure to different concentrations and durations of TXA. METHODS: Thirty-nine osteochondral plugs (OCPs) were harvested from three adult Yucatan minipigs immediately after their death. OCPs were divided into 13 groups exposed to different concentrations of TXA (1, 2 and 4 mg/ml in saline solution) for 1, 3 and 6 h. Negative controls were exposed to saline solution for 0, 1, 3 and 6 h. Chondrocyte viability was assessed by Live/Dead cell assay and calculated as the ratio of live cells (green fluorescence) to overall cells (green + red cells) for each concentration of TXA and time point in a 50-µm scanned image. RESULTS: No correlation was found between chondrocyte viability, and TXA concentration and time of exposure. Overall, chondrocyte viability ranged from 90 to 99%. There was no statistical difference among control group, 1, 2 and 4 mg/ml TXA solutions at each time point [1 h (n.s.), 3 h (n.s.), 6 h (n.s.)]. Similarly, no statistical difference among groups was observed when comparing cell viability at 1, 3 and 6 h of TXA exposure, (Fig. 2) [1 mg/ml (n.s.), 2 mg/ml (n.s.), and 4 mg/ml (n.s.)]. CONCLUSIONS: In conclusion, doses of TXA approximating the current clinical protocols for topical use did not demonstrate any cytotoxic effects on cartilage explants in a Yucatan mini pig model. Thus, supporting the topical application for procedures with intact cartilage, such as partial knee replacement surgery and cartilage repair procedures.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Priming stem cells with protein kinase C activator enhances early stem cell-chondrocyte interaction by increasing adhesion molecules.

BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)-induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.

A phase I-II controlled randomized trial using a promising novel cell-free formulation for articular cartilage regeneration as treatment of severe osteoarthritis of the knee.

BACKGROUND: A promising novel cell-free bioactive formulation for articular cartilage regeneration, called BIOF2, has recently been tested in pre-clinical trials. The aim of the present study was to evaluate the efficacy and safety of BIOF2 for intra-articular application in patients with severe osteoarthritis of the knee. METHODS: A prospective, randomized, 3-arm, parallel group clinical trial was conducted. It included 24 patients with severe osteoarthritis of the knee (WOMAC score 65.9 ± 17). Before they entered the study, all the patients were under osteoarthritis control through the standard treatment with nonsteroidal anti-inflammatory drugs (NSAIDs), prescribed by their family physician. Patients were distributed into three groups of 8 patients each (intra-articular BIOF2, total joint arthroplasty, or conservative treatment with NSAIDs alone). The WOMAC score, RAPID3 score, and Rasmussen clinical score were evaluated before treatment and at months 3, 6, and 12. BIOF2 was applied at months 0, 3, and 6. Complete blood count and blood chemistry parameters were determined in the BIOF2 group before treatment, at 72 h, and at months 1, 3, 6, and 12. In addition, articular cartilage volume was evaluated (according to MRI) at the beginning of the study and at month 12. RESULTS: The NSAID group showed no improvement at follow-up. Arthroplasty and BIOF2 treatments showed significant improvement in all the scoring scales starting at month 3. There were no statistically significant differences between the BIOF2 group and the arthroplasty group at month 6 (WOMAC score: 19.3 ± 18 vs 4.3 ± 5; P = 0.24) or month 12 (WOMAC score: 15.6 ± 15 vs 15.7 ± 17; P = 1.0). Arthroplasty and BIOF2 were successful at month 12 (according to a WOMAC score: ≤ 16) in 75% of the patients and the daily use of NSAIDs was reduced, compared with the group treated exclusively with NSAIDs (RR = 0.33, 95% CI 0.12-0.87, P = 0.02. This result was the same for BIOF2 vs NSAIDs and arthroplasty vs NSAIDs). BIOF2 significantly increased the articular cartilage by 22% (26.1 ± 10 vs 31.9 ± 10 cm2, P < 0.001) and produced a significant reduction in serum lipids. BIOF2 was well tolerated, causing slight-to-moderate pain only upon application. CONCLUSIONS: The intra-articular application of the new bioactive cell-free formulation (BIOF2) was well tolerated and showed no significative differences with arthroplasty for the treatment of severe osteoarthritis of the knee. BIOF2 can regenerate articular cartilage and is an easily implemented alternative therapy for the treatment of osteoarthritis. Trial registration Cuban Public Registry of Clinical Trials (RPCEC) Database RPCEC00000250. Registered 08/15/2017-Retrospectively registered, http://rpcec.sld.cu/en/trials/RPCEC00000250-En .

Chondroitin sulfate and glucosamine sulfate associated to photobiomodulation prevents degenerative morphological changes in an experimental model of osteoarthritis in rats.

The aim of this study was to compare the effects of combined treatment with chondroitin sulfate and glucosamine sulfate (CS/Gl) and photobiomodulation (PBM) on the degenerative process related to osteoarthritis (OA) in the articular cartilage in rats. Forty male Wistar rats were randomly divided into four groups: OA control group (CG); OA animals submitted to PBM treatment (PBM); OA animals submitted to CS/Gl treatment (CS/Gl); OA submitted to CS/GS associated with PBM treatments (GS/Gl + PBM). The CS/Gl started 48 h after the surgery, and they were performed for 29 consecutive days. Moreover, PBM was performed after the CS/Gl administration on the left joint. Morphological characteristics and immunoexpression of interleukin 10 (IL-10) and 1 beta (IL-1ß) and collagen type II (Col II) of the articular cartilage were evaluated. The results showed that all treated groups (CS/Gl and PBM) presented attenuation signs of degenerative process (measured by histopathological analysis) and lower density chondrocytes [PBM (p = 0.0017); CS/Gl (p = 0.0153) and CS/Gl + PBM (p = 0.002)]. Additionally, CS/Gl [associated (p = 0.0089) or not with PBM (p = 0.0059)] showed significative lower values for OARSI grade evaluation. Furthermore, CS/GS + PBM decreased IL-1ß protein expression (p = 0.0359) and increased IL-10 (p = 0.028) and Col II imunoexpression (p = 0.0204) compared to CG. This study showed that CS/Gl associated with PBM was effective in modulating inflammatory process and preventing the articular tissue degradation in the knees OA rats.

Diacerein, its beneficial impact on chondrocytes and notable new clinical applications

Diacerein (DCN) was obtained by diacetylation of an anthraquinone derivative rhein and was approved by FDA in 2008, in the treatment of osteoarthritis due to its inhibitory effect on proinflammatory cytokines, including IL-6 and IL-1ß. It was synthesized in 1980s and marketed as a tablet in some European Union and Asian countries from 1994. Along with its great potential in the treatment of osteoarthritis, its other applications are also being explored day by day, such as in the treatment of psoriasis, epidermolysis bullosa, breast cancer, type 2 diabetes and periodontitis. The main aim of this review is to explore mechanism of action, various applications and side effects associated with DCN. This has been reviewed that apart from the risk of diarrhea on long-term administration of DCN, various clinical studies has also shown its modest benefits in treatment of various pathological conditions. Hence, DCN is emerging as a new and potentially safe derivative with maximum therapeutic efficacies and minimum side effects which can results in improving the living status of patients suffering from various inflammatory diseases

Priming stem cells with protein kinase C activator enhances early stem cell-chondrocyte interaction by increasing adhesion molecules

BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)- induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.