Rabbit Articular Chondrocytes Research Articles

Retracted] Protective effects of baicalin on rabbit articular chondrocytes invitro.

[This retracts the article DOI: 10.3892/etm.2017.4116.].

E3 Ubiquitin Ligase Constitutive Photomorphogenic 1 Regulates Differentiation and Inflammation via MAPK Signaling Pathway in Rabbit Articular Chondrocytes

Constitutive photomorphogenic 1 (COP1), is an E3 ubiquitin ligase that plays a role in the regulation of various cellular processes including cell growth, differentiation, and survival in mammals. In certain conditions such as overexpression or loss of function, COP1 acts either as an oncogenic protein or as a tumor suppressor by targeting specific proteins for ubiquitination-mediated degradation. However, the precise role of COP1 has not been well studied in primary articular chondrocytes. In this study, we investigated the role of COP1 in chondrocyte differentiation. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that COP1 overexpression reduced type II collagen expression, promoted cyclooxygenase 2 (COX-2) expression, and reduced sulfated proteoglycan synthesis, as detected by Alcian blue staining. Upon siRNA treatment, revived type II collagen, sulfated proteoglycan production, and decreased COX-2 expression. Phosphorylation of p38 kinase and ERK-1/-2 signaling pathways was regulated by COP1 upon cDNA and siRNA transfection in chondrocytes. The inhibition of the p38 kinase and ERK-1/-2 signaling pathways with SB203580 and PD98059 ameliorated the expression of type II collagen and COX-2 in transfected chondrocytes, thus suggesting that COP1 regulates differentiation and inflammation in rabbit articular chondrocytes via the p38 kinase and ERK-1/-2 signaling pathway.

Zhuifeng tougu capsules inhibit the TLR4/MyD88/NF-κB signaling pathway and alleviate knee osteoarthritis: In vitro and in vivo experiments.

Background: Knee osteoarthritis (KOA), a chronic degenerative disease, is mainly characterized by destruction of articular cartilage and inflammatory reactions. At present, there is a lack of economical and effective clinical treatment. Zhuifeng Tougu (ZFTG) capsules have been clinically approved for treatment of OA as they relieve joint pain and inflammatory manifestations. However, the mechanism of ZFTG in KOA remains unknown. Purpose: This study aimed to investigate the effect of ZFTG on the TLR4/MyD88/NF-κB signaling pathway and its therapeutic effect on rabbits with KOA. Study design: In vivo, we established a rabbit KOA model using the modified Videman method. In vitro, we treated chondrocytes with IL-1β to induce a pro-inflammatory phenotype and then intervened with different concentrations of ZFTG. Levels of IL-1β, IL-6, TNF-α, and IFN-γ were assessed with histological observations and ELISA data. The effect of ZFTG on the viability of chondrocytes was detected using a Cell Counting Kit-8 and flow cytometry. The protein and mRNA expressions of TLR2, TLR4, MyD88, and NF-κB were detected using Western blot and RT-qPCR and immunofluorescence observation of NF-κB p65 protein expression, respectively, to investigate the mechanism of ZFTG in inhibiting inflammatory injury of rabbit articular chondrocytes and alleviating cartilage degeneration. Results: The TLR4/MyD88/NF-κB signaling pathway in rabbits with KOA was inhibited, and the levels of IL-1β, IL-6, TNF-α, and IFN-γ in blood and cell were significantly downregulated, consistent with histological results. Both the protein and mRNA expressions of TLR2, TLR4, MyD88, NF-κB, and NF-κB p65 proteins in that nucleus decreased in the ZFTG groups. Moreover, ZFTG promotes the survival of chondrocytes and inhibits the apoptosis of inflammatory chondrocytes. Conclusion: ZFTG alleviates the degeneration of rabbit knee joint cartilage, inhibits the apoptosis of inflammatory chondrocytes, and promotes the survival of chondrocytes. The underlying mechanism may be inhibition of the TLR4/MyD88/NF-kB signaling pathway and secretion of inflammatory factors.

Increase in NO causes osteoarthritis and chondrocyte apoptosis and chondrocyte ERK plays a protective role in the process.

Nitric oxide (NO) and reactive oxygen species (ROS) play an important role in the pathology of human osteoarthritis (OA). Ankylosing spondylitis (AS) and atypical OA have similar clinical manifestations and often require differential diagnosis. The mechanism is however not totally clear yet. This study aims to investigate the effects of excessive NO-ROS in OA patients and the effects of extracellular signal-regulated kinases (ERK) pathway in NO-induced apoptosis of chondrocytes during OA progress. Serum samples from OA or AS as pathological control patients and healthy controls were collected for NO and related chemical measurements. The rabbit articular chondrocytes were cultured in vitro, and NO was applied by Sodium Nitroprusside (SNP) in culture medium to mimic OA condition in patients. The level of SNP-evoked chondrocyte apoptosis with or without PD98059 (ERK-specific inhibitor) was evaluated by TUNEL assay, Annexin V flow cytometry and Western blotting. The activity and mRNA expression of caspase-3 in chondrocytes were measured by assay kits and RT-PCR. The levels of NO and malondialdehyde (MDA) in serum were significantly higher in OA patients, while only MDA was significantly higher in AS patients. However, the level of superoxide dismutase (SOD) was lower in both OA and AS patients. SNP induced chondrocyte apoptosis was enhanced by PD98059 with increased protein expression and functional activity of caspase-3. The increase in nitric oxide occurs specifically in OA patients. ERK pathway may play a protective role on the NO-induced chondrocyte apoptosis, and inhibition of ERK pathway enhances the NO-induced apoptosis.

Effects of Androgen Receptor Overexpression on Chondrogenic Ability of Rabbit Articular Chondrocytes.

The role of sex hormones and their receptors has drawn much attention in the process of cartilage regeneration. This study aimed to investigate the effect of androgen receptor (AR) on the chondrogenic ability of articular chondrocytes and the related mechanism. Articular chondrocytes were isolated, cultured, identified by toluidine blue staining and then transduced with lentivirus carrying the AR gene. The cell viability was determined using Cell Counting Kit-8, and cell apoptosis was assessed by flow cytometry analysis. The effects of AR overexpression on the expression of cartilage-specific proteins and some signalling molecules were evaluated by real-time PCR and Western blotting. Using 24 New Zealand rabbits, the regeneration of rabbit articular cartilage defects was further investigated in vivo and evaluated histologically. The overexpression of AR significantly reduced the apoptosis rate of chondrocytes but did not affect their proliferation. The overexpression of AR also promoted the expression of Sry-related HMG box 9, collagen II and aggrecan, decreased the expression of matrix metalloproteinase-13, and downregulated p-S6 and RICTOR. The experimental group with AR-overexpressing chondrocytes exhibited superior regeneration of cartilage defects. AR overexpression can maintain the phenotype of chondrocytes and promote chondrogenesis in vitro and in vivo. mTOR-related signalling was inhibited.

Expression of Concern on "Andrographolide Enhances Proliferation and Prevents Dedifferentiation of Rabbit Articular Chondrocytes: An In Vitro Study".

Expression of Concern on "Andrographolide Enhances Proliferation and Prevents Dedifferentiation of Rabbit Articular Chondrocytes: An In Vitro Study".

Iksan526 Rice Callus Extract Induces Dedifferentiation of Rabbit Articular Chondrocytes via ERK1/2 and PI-3K/Akt Pathways

The resveratrol-enriched transgenic rice line Iksan526 (IS526), first developed by the Rural Development Administration of Korea using genetic engineering techniques, shows beneficial health effects in mitigating metabolic syndrome and obesity. However, the effects of IS526 on the differentiation of chondrocytes and the underlying mechanism have not been investigated in detail. In this study, the effects and cellular regulatory mechanisms of IS526 on rabbit articular chondrocytes were examined. Following IS526 callus extract treatment, the expression levels of differentiation-related proteins were detected via western blotting, Alcian blue staining and immune-luorescence staining. IS526 decreased the type II collagen and proteoglycan levels in dose- and time-dependent manners. We further analyzed the effects of IS526 on skeleton genesis in zebrafish larvae using Alcian blue staining, which showed a reduction in cartilage formation along with increased production of matrix metalloproteinase (MMP)-13. IS526 also increased the phosphorylation of ERK1/2 and p38 kinase but inhibited the phosphorylation of Akt. Pharmacological inhibition of MMP-13 blocked the IS526-induced decrease in type II collagen levels. Inhibition of p38 kinase or PI-3K/Akt with SB203580 and LY294002 enhanced the suppression of type II collagen, but the blockage of ERK-1/2 by PD98059 rescued IS526-induced dedifferentiation. These results suggested that IS526 regulates type II collagen and MMP-13 expression via the ERK1/2 and PI-3K/Akt pathways in rabbit articular chondrocytes.

In situ ornamenting poly(ε-caprolactone) electrospun fibers with different fiber diameters using chondrocyte-derived extracellular matrix for chondrogenesis of mesenchymal stem cells

Biomimetic instructive tissue engineering scaffolds are critical for achieving successful tissue regeneration. In the present study, we developed a novel scaffold via ornamenting poly(ε-caprolactone) (PCL) electrospun fibers with a chondrocyte-derived extracellular matrix (ECM)-coating, which was applied for chondrogenesis of mesenchymal stem cells (MSCs). PCL fibrous films with different fiber diameters (1282±121 nm, 549±61 nm and 285±38 nm) were first prepared via electrospinning. Rabbit articular chondrocytes (rACs) were cultured on PCL fibrous scaffolds, followed by a decellularization treatment to generate decellularized ECM (dECM)-coated PCL scaffolds (dECM/PCL). Rabbit bone marrow-derived MSCs (rMSCs) were then seeded onto these scaffolds and adhesion, proliferation and chondrogenic differentiation were evaluated. dECM/PCL scaffolds displayed distinct surface microstructural features with varying fiber diameters and fibrous mesh-like ECM with more developed collagen fibers was observed on nanofibers. On dECM/PCL scaffolds, rMSCs tended to spread more at 24 h post-seeding and proliferated better within 7 d compared to those on uncoated PCL scaffolds. Based on analysis of gene expression, rMSCs underwent the best chondrogenic differentiation on dECM/PCL scaffolds of 549-nm fibers. Collectively, such dECM/PCL composite scaffolds are very promising for cartilage tissue regeneration.

Simvastatin abolishes nitric oxide- and reactive oxygen species-induced cyclooxygenase-2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes.

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO- or ROS-induced cyclooxygenase-2 (COX-2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)- and interleukin 1 beta (IL-1β)-induced COX-2 production. In addition, simvastatin attenuated SNP-induced NO production and IL-1β-induced ROS generation. Treatment with simvastatin prevented SNP- and IL-1β-induced nuclear factor kappa B (NF-κB) activity. Inhibiting NO production and ROS generation using N-acetylcysteine (NAC) and NG-monomethyl- l-arginine ( l-NMMA), respectively, accelerated the influence of simvastatin on NF-κB activity. In addition, NAC blocked SNP and simvastatin-mediated COX-2 production and NF-κB activity but did not alter IL-1β and simvastatin-mediated COX-2 expression. l-NMMA treatment also abolished IL-1β-mediated COX-2 expression and NF-κB activation, whereas SNP and simvastatin-mediated COX-2 expression were not altered compared with the levels in the SNP and simvastatin-treated cells. Our findings suggested that simvastatin blocks COX-2 expression by inhibiting SNP-induced NO production and IL-1β-induced ROS generation by blocking the NF-κB pathway.

Synovium stem cell-derived matrix enhances anti-inflammatory properties of rabbit articular chondrocytes via the SIRT1 pathway.

Autologous chondrocyte implantation (ACI) is a promising approach to repair cartilage defects; however, the cartilage trauma-induced inflammatory environment compromises its clinical outcomes. Cell-derived decellularized extracellular matrix (DECM) has been used as a culture substrate for mesenchymal stem cells (MSCs) to improve the cell proliferation and lineage-specific differentiation. In this study, DECM deposited by synovium-derived MSCs was used as an in vitro expansion system for rabbit articular chondrocytes and the response of DECM-expanded chondrocytes to pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was evaluated. Compared with those grown on tissue culture polystyrene (TCPS), the proliferation rate was significantly improved in DECM-expanded chondrocytes. TCPS- and DECM-expanded chondrocytes were isolated and induced to redifferentiation in a high-density pellet culture. DECM-expanded chondrocytes exerted a stronger resistance to 1 ng/mL of IL-1β than TCPS-expanded cells, but the production of cartilage matrix in both groups was inhibited by 5 ng/mL of IL-1β. When exposed to 1 or 5 ng/mL of TNF-α, DECM-expanded chondrocytes showed higher levels of cartilage matrix synthesis than TCPS-expanded cells. In addition, the gene expression of IL-1β- or TNF-α-induced matrix degrading enzymes (MMP3, MMP9, MMP13, and ADAMTS5) was significantly lower in DECM-expanded chondrocytes than TCPS-expanded cells. Furthermore, we found that SIRT1 inhibition by nicotinamide completely counteracted the protective effect of DECM on chondrocytes in the presence of IL-1β or TNF-α, indicating that the SIRT1 signaling pathway was involved in the DECM-mediated enhancement of anti-inflammatory properties of chondrocytes. Taken together, this work suggests that stem cell-derived DECM is a superior culture substrate for in vitro chondrocyte expansion by improving proliferation and enhancing the anti-inflammatory properties of chondrocytes. DECM-expanded chondrocytes with enhanced anti-inflammatory properties hold great potential in clinically ACI-based cartilage repair.

Simvastatin induces differentiation in rabbit articular chondrocytes via Wnt/β-catenin pathway

Simvastatin is widely used as a specific inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase to reduce the levels of low-density lipoprotein cholesterol. However, its regulatory mechanism in chondrocyte differentiation is unclear. This study was conducted to evaluate the effects and signalling pathway of simvastatin on chondrocyte differentiation. We found that simvastatin induced chondrocyte differentiation, as confirmed by increased type II collagen expression and induced sulphated proteoglycan synthesis. Western blotting results showed that expression of type II collagen increased 6-fold in a dose-dependent manner compared with that in the control. Further, nuclear/cytosol fraction analysis revealed that simvastatin reduced the expression and translocation of β-catenin into the nucleus from the cytoplasm by approximately 50% compared with that in the control. A luciferase assay using a T cell factor/lymphoid enhancer factor reporter construct was performed to test the transcriptional activity of β-catenin. Simvastatin-induced differentiation was dependent on inactivation of β-catenin, as simvastatin inhibited accumulation of β-catenin, which was characterized by translocation of β-catenin to the nucleus as shown by immunofluorescence staining and the luciferase assay. Prevention of β-catenin degradation by inhibition of the proteasome with z-Leu-Leu-Leu-CHO blocked the increase in type II collagen expression. Simvastatin treatment reduced chondrocyte dedifferentiation induced by retinoic acid or serial monolayer culturing by 50% compared to that in the non-treated cells. Our findings demonstrate that simvastatin increases differentiation of rabbit articular chondrocytes via the β-catenin pathway.

Silk fibroin/carboxymethyl chitosan hydrogel with tunable biomechanical properties has application potential as cartilage scaffold

Tissue engineering is a promising strategy for cartilage repair and regeneration. However, an ideal scaffolding material that not only mimics the biomechanical properties of the native cartilage, but also supports the chondrogenic phenotype of the seeding cells is in need. In this study, we developed a silk fibroin (SF) and carboxymethyl chitosan (CMCS) composite hydrogel with enzymatic cross-links (horseradish peroxidase and hydrogen peroxide) and β-sheet cross-links (ethanol treatment). Results of Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), and X-ray diffraction (XRD) verified that SF/CMCS composite hydrogels had a tunable β-sheet structure. Therefore, by increasing the time of ethanol treatment from 0 h to 8 h, a series of parameters including pore size (from 50 to 300 μm), equilibrium swelling (from 78.1 ± 2.6% to 91.9 ± 0.9%), degradation (from 100% to 9% reduction in mass over 56 days), rheological properties (storage modulus from 177 Pa to 88,904 Pa), and mechanical properties (compressive modulus from 13 to 829 kPa) of the hydrogels were adjusted. In particular, the material parameters of the hydrogels with 2 h ethanol treatment appeared most suitable for engineered cartilage. Furthermore, the in vitro cellular experiments showed that the hydrogels supported the adhesion, proliferation, glycosaminoglycan synthesis, and chondrogenic phenotype of rabbit articular chondrocytes. Finally, subcutaneous implantation of the hydrogels in mice showed no infections or local inflammatory responses, indicating a good biocompatibility in vivo. In conclusion, the chemical-physical cross-linking SF/CMCS composite hydrogels, with tunable material properties and degradation rate, good biocompatibility, are promising scaffolds for cartilage tissue engineering.

In situ forming hydrogels based on polyethylene glycol itaconate for tissue engineering application

Novel strategies have been proposed to enhance the quality of surgery by scheming noninvasive methods. For this reason, photo-curable in situ forming hydrogels have been well developed during advancements in the regenerative medicine. In this study, polyethylene glycol itaconate (PEGI) was synthesized by reacting polyethylene glycol (PEG) with different molecular weights (1000, 4000 and 8000 $$\hbox {g mol}^{-1}$$) and itaconyl chloride. The synthesized PEGIs were fully characterized and employed as a macromonomer for the preparation of in situ forming hydrogels using a combination of camphorquinone and dimethylaminoethyl methacrylate as a reactive photoinitiator system, and hydroxyethyl methacrylate as a reactive diluent. The physical properties of the hydrogels including gel yield, equilibrium swelling and compressive strength were determined. The hydrogel based on PEG 4000 with a gel yield of 86%, a water uptake of 103%, a compressive modulus of 11.2 MPa, an elongation at break of 9% and a curing time of 4 min was selected for the encapsulation of rabbit articular chondrocyte cells. The cytocompatibility of the in situ formed hydrogels was evaluated using 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, live-dead fluorescence assays and optical microscopy observations. Glycosaminoglycans were quantified by dimethylmethylene blue staining from the encapsulated chondrocytes after 14 days. The proposed in situ forming hydrogel can be considered as an injectable and photocurable carrier for cell delivery in cartilage tissue engineering.

Effects of Clematis chinensis Osbeck mediated by low-intensity pulsed ultrasound on transforming growth factor-β/Smad signaling in rabbit articular chondrocytes.

Clematis chinensis Osbeck (CCO) is an essential herb that has been shown to promote the biological functions of cartilage cells. In this study, we aimed to explore whether and how low-intensity pulsed ultrasound (LIPUS) enhanced CCO delivery into chondrocytes and stimulated biological activity in vitro. Chondrocytes were isolated from knee articular cartilage of 2-week-old rabbits and treated with LIPUS plus CCO or recombinant transforming growth factor beta 1 (TGF-β1; 0.5ng/mL), with or without anti-TGF-β1 antibodies (10μg/mL), for 3days. Cell proliferation was assessed by Cell-Counting Kit-8 assays. Immunocytochemistry, western blotting, and quantitative polymerase chain reaction were applied to detect the expression of type II collagen and some molecules in the TGF-β1 signal pathway. LIPUS plus 0.1mg/mL CCO solution promoted chondrocyte proliferation and type II collagen and TGF-β1 expression synergistically in vitro (P < 0.05). In addition, treatment with anti-TGF-β1 antibodies blocked this effect (P < 0.01), but not completely. CCO plus LIPUS also showed more enhanced effects on promoting TGF-β receptor II and Smad2 signaling and reducing Smad7 signaling than either intervention separately (P < 0.05). CCO plus LIPUS promoted extracellular matrix deposition by accelerating the TGF-β/Smad-signaling pathway in chondrocytes.

Erratum for: "Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro" [Braz J Med Biol Res (2014) 47(8): e3935

[This corrects the article doi: 10.1590/1414-431X20143935].

Andrographolide-releasing collagen scaffold enhance the ability of chondrocytes to maintain their specific phenotype under inflammatory environment in vitro

The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 ( TIMP-1), collagen II ( COL II), aggrecan ( Aggrecan) and the ratio of COL II/ collagen I( COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 ( MMP-1) and matrix metalloproteinase-13 ( MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.

Inhibition of the Expression of Matrix Metalloproteinases in Articular Chondrocytes by Resveratrol through Affecting Nuclear Factor-Kappa B Signaling Pathway.

In the present study, we tried to examine whether resveratrol regulates the expression of matrix metalloproteinases (MMPs) through affecting nuclear factor-kappa B (NF-κB) in articular chondrocytes. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of resveratrol on IL-1β-induced secretion of MMP-3 was investigated in rabbit articular chondrocytes using western blot analysis. To elucidate the action mechanism of resveratrol, effect of resveratrol on IL-1β-induced NF-κB signaling pathway was investigated in SW1353, a human chondrosarcoma cell line, by western blot analysis. The results were as follows: (1) resveratrol inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) resveratrol reduced the secretion of MMP-3; (3) resveratrol inhibited IL-1β-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Bα (IκBα); (4) resveratrol inhibited IL-1β-induced phosphorylation and nuclear translocation of NF-κB p65. This, in turn, led to the down-regulation of gene expression of MMPs in SW1353 cells. These results suggest that resveratrol can regulate the expression of MMPs through affecting NF-κB by directly acting on articular chondrocytes.

Effect of prednisolone on rabbit articular chondrocytes treated with sodium nitroprusside

This study was carried out to investigate the protective effect of prednisolone in rabbit primary cultured articular chondrocytes treated with sodium nitroprusside (SNP), a nitric oxide donor. After a cell phenotype was determined, the MTT assay and Western blot analysis of type II collagen, cylooxygenase-2 (COX-2) and phosphorylated extracellular regulated kinase (pERK) were performed in the control, SNP (298 μg/ml) alone or SNP plus prednisolone (0.05-50 μg/ml)-treated rabbit articular chondrocytes. Immunofluorescence staining of type II collagen was also performed. Cell morphology indicated that SNP treatment induced cytotoxicity, and that the SNP-induced cytotoxicity was inhibited by prednisolone treatment. MTT assay showed that the SNP treatment resulted in a significant decrease in the level of cell viability compared with that of control (p&lt;0.01), and that the prednisolone treatment resulted in a decrease in the SNP-induced cytotoxicity. SNP treatment resulted in a decrease in the level of type II collagen, compared with the control chondrocytes. The prednisolone treatment recovered the down-regulated expression of type II collagen induced by SNP, showing a significant level in 5 μg/ml of the prednisolone treatment group compared to the SNP treatment group (p&lt;0.05). A significant increase in COX-2 was significantly induced by the SNP treatment compared to control chondrocytes (p&lt;0.01). The COX-2 expression was decreased by the prednisolone treatment, showing a significant level in 50 μg/ml of the prednisolone treatment group compared to the SNP treatment group (p&lt;0.05). These phenomena was confirmed by immunofluorescence staining. Furthermore, the SNP treatment significantly induced a decrease of pERK expression compared to the control chondrocytes (p&lt;0.01). The prednisolone treatment recovered its expression, showing a significant level in 0.5 μg/ml of the prednisolone treatment group compared to the SNP treatment group (p&lt;0.05). Taken the above results together, prednisolone is considered to inhibit SNP-induced cell death and dedifferentiation, and modulated expression of COX-2 and pERK in rabbit articular chondrocytes.

Comparison of rabbit bone marrow mesenchymal stem cells and chondrocytes co-cultured in vitro for the establishment of four kinds of cartilage without scaffold

Objective A large number of cells in four groups were cultured without scaffold in centrifuge tube, observing size and biological characteristics of aggregation co-culture system of cartilage tissue, and evaluate co-culture technology without support by centrifugal tube, which provides methodological guidance and experimental basis for the clinical repair of articular cartilage defect. Methods New Zealand white rabbits underwent puncture of bone marrow. Rabbit articular cartilage chondrocytes were separated, obtain relatively pure by type Ⅱ trypsin and collagenase, and then cultured. Isolation and culture of bone marrow mesenchymal stem cells by density gradient centrifugation to obtain the 3 passage of purified Bone marrow mesenchymal stem cells (BMSCs). Using alcian blue staining, safranin O-light green staining and type Ⅱ collagen immunohistochemical staining of Articular Chontrocytes (ACs) were identified. CD34, CD44, CD45 and CD105 molecular surface markers by immunofluorescence identification of BMSCs. BMSCs and ACs were mixed into co-culture (CO) groups according to the ratio of 1∶3 and 2∶2, the total number of cells was 4×107, 1 500 r/min centrifugation for 5 minutes, and the cell aggregation was co cultured in 15 ml centrifuge tube. The equivalent chondrocytes were taken as AC group, and the same amount of BMSCs was induced into the BMSCs induction group (transforming growth factor-beta 1, TGF-β1), and cultured in the centrifuge tube as comparative research. Four groups of high number of cells were cultured for 1 weeks, 2 weeks, 3 weeks and 4 weeks after paraffin tissue sections were dewaxed by alcian blue staining, saffron O -light green staining and immunohistochemical staining for type Ⅱ collagen. HE staining was used to observe the morphology and structure of cartilage tissue in each group under the condition of in vitro culture. Using alcian blue colorimetric method for quantitative detection of liquid supernatant of glycosaminoglycan content, enzyme-linked immunosorbent adsorption method was used to detect the liquid supernatant of type Ⅱ collagen content. Results ACs alcian blue, safranin O-light green and type Ⅱ collagen immunohistochemical staining were positive, the adherent subculture could obtain satisfactory purity of BMSCs and ACs. BMSCs immunofluorescence staining of CD44 (+ ), CD105 (+ ), CD45 (-), CD34 (-). AC group, 2∶2 and 1∶3 co-culture group, alcian blue staining, safranin O-light green staining and type Ⅱ collagen immunohistochemical staining were positive. The staining of BMSC induced group was weakly positive. HE showed that AC group and 1∶3 group were gradually fused into initial cartilage tissue. Gradually the integration of group cells induced by 2∶2 And BM, but no cartilage formed after four weeks. After 4 weeks, group AC [(179.43±59.19) μg/ml] and 2∶2 [(158.06±23.77) μg/ml], 1∶3 group [(160.06±30.58) μg/ml] GAG content had no significant difference (F=0.210, P=0.812). After 4 weeks, group AC [(185.13±35.96) ng/ml] and 2∶2 [(169.72±32.56) ng/ml], 1∶3 [(148.91±33.16) ng/ml] type Ⅱ collagen content had no significant difference (F=0.680, P=0.515); AC group [(179.43±59.19) μg/ml] and BM induced group [(70.74±40.17) μg/ml] GAG content was statistically significant (t=3.980, P=0.001); AC group [(185.13±35.96) ng/ml] and BM induced group [(116.83±40.17) ng/ml] type Ⅱ collagen content was statistically significant (t=3.434, P=0.003). Conclusion BMSCs and ACs were cultured in centrifuge tube by aggregation can induce differentiation of BMSCs to ACs, and get the biological characteristics of the AC. The AC group and 1∶3 co-culture group formed the rudiment of cartilage tissue after four weeks with HE staining, but BM induced group and 2∶2 group did not. Key words: Articular chondrocytes; Mesenchymal stem cells; Co-culture; No-scaffold; Tissue engineering cartilage

Simvastatin-dependent actin cytoskeleton rearrangement regulates differentiation via the extracellular signal-regulated kinase-1/2 and p38 kinase pathways in rabbit articular chondrocytes

Alterations in cell morphology involve changes in the actin cytoskeleton and play crucial roles in determining chondrocyte phenotypes. Although the effects of simvastatin (SV) have been demonstrated in various cell types, the mechanisms and effects of SV on chondrocyte differentiation and actin cytoskeletal rearrangement are still unclear. Here, we investigated the roles of actin filament rearrangement on SV-induced differentiation of rabbit articular chondrocytes. Treatment with SV caused actin remodeling in comparison with that in untreated chondrocytes, as determined by immunofluorescence staining. Moreover, treatment with cytochalasin D (CD) and jasplakinolide (JAS), which modulate actin filament formation, resulted in reorganization of the actin cytoskeleton compared with that induced by SV in chondrocytes. In addition, CD synergistically enhanced the SV-induced increase in type II collagen expression, whereas JAS dramatically inhibited SV-induced differentiation. We also found that differentiation via SV-dependent actin cytoskeleton changes was regulated by the extracellular signal-regulated kinase (ERK)−1/2 and p38 kinase pathways. These results demonstrated that actin cytoskeletal rearrangement by SV regulated type II collagen expression and suggested that ERK-1/2 and p38 kinase pathways may play important roles in SV-induced type II collagen expression by altering actin cytoskeletal reorganization in rabbit articular chondrocytes.