Preliminary study of TRPV4 affects chondrocyte degeneration / 中国骨伤

OBJECTIVE@#To explore and verify that transient receptor potential vanilloid 4(TRPV4) affects chondrocyte degeneration.@*METHODS@#Neonatal SD rats were selected, primary chondrocytes were extracted, and identified by toluidine blue staining and alcian blue staining;an in vitro chondrocyte inflammation model was constructed by IL-1β, and TRPV4 inhibitor was used to treat chondrocytes under inflammatory conditions, and the chondrocytes were treated by RT-PCR method was used to detect matrix metallopeptidase 13(MMP-13), a disintegrin and metalloproteinase with thrombospondin 5, (ADAMTS-5)、nitric oxide synthase 2(NOS2)、Collagen, type II alpha 1(Col2α1)and aggrecan (Acan) mRNA in chondrocytes; primary chondrocytes were treated with different concentrations of TRPV4 overexpression plasmid, and the optimal overexpression dose was screened. The mRNA expressions of TRPV4, MMP-13, ADAMTS-5, NOS2, Col2α1 and Acan in chondrocytes under the optimal TRPV4 overexpression dose were detected.@*RESULTS@#Toluidine blue staining and Alcian blue staining identified the extracted cells as primary chondrocytes;RT-PCR showed that TRPV4, MMP-13, ADAMTS-5, NOS2 mRNA in chondrocytes treated with TRPV4 inhibitor under inflammatory conditions. The expression of Col2α1 mRNA was significantly decreased (P<0.05), and the expression of Col2α1 mRNA was increased (P<0.05). Although there was no significant difference in the expression of Acan mRNA, the overall trend was also increasing. The expression of Col2α1 and Acan mRNA in chondrocytes was significantly decreased (P<0.05), and the expression of NOS2 mRNA was increased(P<0.05), but there was no significant difference in MMP-13 and ADAMTS-5 (P>0.05).@*CONCLUSION@#Inhibiting the expression of TRPV4 can down-regulate the expression of genes related to chondrocyte degeneration.

Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway / 中国中药杂志

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-β-galactosidase(SA-β-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1β and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1β and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Kindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular joint in mice / 国际口腔科学杂志·英文版

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint (TMJ) osteoarthritis (OA); however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4 (Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2 and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore, Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

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.

Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Differentiation of mesenchymal stem cells into nucleus pulposus cells in vitro / 华中科技大学学报（医学）（英德文版）

To find a new source of seed cells for constructing tissue-engineered intervertebral disc, nucleus pulposus (NP) cells and mesenchymal stem cells (MSCs) were isolated from New Zealand white rabbits. The nucleus pulposus cells population was fluorescence-laelled and co-cultured with MSCs with or without direct contact. Morphological changes were observed every 12 h. Semi-quantitative reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9, aggreacan and type II collagen every 24 h after the co-culture. MSCs treated with direct contact rounded up and presented a ring-like appearance. The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h. No significant change was found after coculture without direct contact. Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.

Avaliação do metabolismo celular decorrente da aplicação de força nos condrócitos do côndilo mandibular e do joelho de suínos / Comparison of in vitro desponse to mechanical loading between the porcine mandibular condyle and ankle articular cartilages

Funcionalmente, a cartilagem da articulação têmporo-mandibular assemelha-se à cartilagem da articulação do joelho por possuírem lubrificação para resistir à fricção e fornecerem proteção às forças mecânicas externas. Entretanto, o efeito das forças de tensão sobre as cartilagens dessas duas articulações ainda permanece obscura. O objetivo desse estudo foi avaliar, in vitro, as alterações metabólicas nos condrócitos extraídos do tecido cartilaginoso do côndilo mandibular e do joelho de suínos, decorrentes da aplicação de forças mecânicas, em relação à síntese de DNA e de proteoglicanos (PTG). Além disso, foi verificada a expressão de colágeno tipo II e de agrecanos no RNAm dos condrócitos dessas duas articulações, tempo-dependente do cultivo celular, utilizando-se a análise quantitativa de PCR em tempo real. Os condrócitos foram submetidos às forças mecânicas de tração de 2 kPa (3 porcento de alongamento), 5 kPa (7 porcento de alongamento) e 10 kPa (12 porcento de alongamento), em uma freqüência de 30 ciclos/min. durante 12 e 24 horas. Os resultados demonstraram que os condrócitos do côndilo mandibular quando submetidos às forças de 2 kPa e de 5 kPa, apresentaram um aumento estatisticamente significativo da síntese de DNA e de PTG, em 12 h. (p < 0,01) e em 24 h. (p < 0,05). Exceto o aumento da síntese de DNA do grupo submetido à força de 5 kPa que durante 24h. não foi estatisticamente significativo (p > 0,05). A força de 10 kPa causou uma diminuição estatisticamente significativa na síntese de DNA e de PTG nos condrócitos do côndilo mandibular, em ambos os tempos de ensaio mecânico (p < 0,01). Por outro lado, os condrócitos do joelho apresentaram um aumento na síntese de DNA e de PTG quando submetidos à todas as magnitudes de força de tração...

Functionally, the mandibular condylar cartilage is similar to the ankle articular cartilage, both provides lubrication to resist friction and offers protection against external mechanical loading. However, the effect of tension loadings on these two articular cartilages remains unclear. The purpose of this study was to evaluate in vitro, the metabolism of the chondrocytes isolated from the cartilage tissues of porcine mandibular condyle and ankle, in response to the tension mechanical forces, related to the syntheses of DNA and proteoglycan (PTG). It was also verified the expression of mRNA type II collagen and aggrecan on the condrocytes of these two joints on culture time-dependent, using a quantitative real-time PCR analysis. The chondrocytes were submitted to tensile mechanical strains of 2 kPa (3 percent elongation), 5 kPa (7 percent elongation) and 10 kPa (12 percent elongation), with a frequency of 30 cycles/min for 12 and 24 hours. The results showed that the condrocytes from mandibular condyle, when submitted to tension forces of 2 kPa and 5 kPa, demonstrated a statistically significant enhancement of DNA and PTG, in 12 h. (p < 0.01) and in 24 h. (p < 0.05). Except the increase of DNA synthesis of the group submitted to the force of 5 kPa during 24 h. that was not statistically significant (p > 0.05). The force of 10 kPa caused a statistically significant decrease of DNA and PTG syntheses on the condrocytes of mandibular condyle, in both periods of mechanical stimulation (p < 0.01). On the other side, the condrocytes of ankle showed an increase of DNA and PTG syntheses when subjected to all the magnitudes of tension forces...