Intermittent cyclic mechanical compression promotes endplate chondrocytes degeneration by disturbing Nrf2/PINK1 signaling pathway-dependent mitophagy.

An abnormal mechanical load is a pivotal inducer of endplate cartilage degeneration, which subsequently promotes intervertebral disc degeneration. Our previous study indicated that intermittent cyclic mechanical compression (ICMC) promotes endplate chondrocyte degeneration, but the mechanism underlying this effect is unclear. In this study, we investigated PTEN-induced kinase 1(PINK1) dependent mitophagy during ICMC-induced endplate chondrocyte degeneration. Furthermore, we determined whether NF-E2-related factor 2 (Nrf2) activation correlated with PINK1-dependent mitophagy regulation and increased oxidation resistance of endplate chondrocytes under ICMC application. First, we generated a mechanical compression-induced endplate chondrocyte degeneration model in vitro and in vivo. ICMC was found to promote endplate chondrocyte extracellular matrix degradation. PINK1-mediated mitophagy was suppressed in the ICMC-stimulated endplate chondrocytes, while increased mitochondrial reactive oxygen species generation suggested that mitophagy is involved in the protective effect of mechanical strain on endplate chondrocytes. Moreover, Nrf2 expression, interaction with Kelch-like ECH-associated protein (Keap1), and nuclear translocation were inhibited by ICMC. Nrf2 overexpression inhibited reactive oxygen species production and reversed ICMC-induced endplate chondrocyte degeneration. Transfection with PINK1 shRNA abolished this effect and partially blocked Nrf2-induced mitophagy. Our findings suggested that ICMC could inhibit the Nrf2/PINK1 signaling pathway to reduce the mitophagy levels which significantly promote oxidative stress and thereby endplate chondrocyte degeneration. Therapeutic regulation of the Nrf2/PINK1 signaling pathway may be an efficient anabolic strategy for inhibiting this process.

Chordin-Like 1 Improves Osteogenesis of Bone Marrow Mesenchymal Stem Cells Through Enhancing BMP4-SMAD Pathway.

Chordin-like 1 (CHRDL1) is a secreted glycoprotein with repeated cysteine-rich domains, which can bind to BMPs family ligands. Although it has been reported to play important roles in several systems, the exact roles of CHRDL1 on human bone mesenchymal stem cells (hBMSCs) osteogenesis remain to be explored. The present study aimed to investigate the roles of CHRDL1 on the osteogenic differentiation of hBMSCs and the underlying molecular mechanisms. We found that CHRDL1 was upregulated during hBMSCs osteogenesis, and rhBMP-4 administration could enhance CHRDL1 mRNA expression in a dose and time dependent manner. Knockdown of CHRDL1 did not affect hBMSCs proliferation, but inhibited the BMP-4-dependent osteogenic differentiation, showing decreased mRNA expression levels of osteogenic markers and reduced mineralization. On the contrary, overexpression of CHRDL1 enhanced BMP-4 induced osteogenic differentiation of hBMSCs. Moreover, in vivo experiments by transplanting CHRDL1 gene modified hBMSCs into nude mice defective femur models displayed higher new bone formation in CHRDL1 overexpression groups, but lower new bone formation in CHRDL1 knockdown groups, compared with control groups. In consistent with the bone formation rate, there were increased CHRDL1 protein expression in new bone formation regions of defective femur in CHRDL1 overexpression groups, while reduced CHRDL1 protein expression in CHRDL1 knockdown groups compared with control groups. These indicate that CHRDL1 can promote osteoblast differentiation in vivo. Furthermore, the mechanisms study showed that CHRDL1 improved BMP-4 induced phosphorylation of SMAD1/5/9 during osteogenic differentiation of hBMSCs. Besides, promotion of osteogenic differentiation and the activation of SMAD phosphorylation by CHRDL1 can be blocked by BMP receptor type I inhibitor LDN-193189. In conclusion, our results suggested that CHRDL1 can promote hBMSCs osteogenic differentiation through enhancing the activation of BMP-4-SMAD1/5/9 pathway.

The Gut Bacterial Community Composition of Wild Cervus albirostris (White-Lipped Deer) Detected by the 16S Ribosomal RNA Gene Sequencing.

Cervus albirostris (white-lipped deer) is an endemic species in China. As the name implies, C. albirostris has a characteristic pure white marking around their mouth and on the underside of the throat. The animal is a typical alpine species normally living at the height of 3500-4300 m. In this study, by pyrosequencing the 16S rRNA gene sequences, we for the first time analyzed the gut bacterial community composition in eight feces samples of wild C. albirostris. From a total of 243,634 high-quality sequences, we identified 186 genera, included in 17 prokaryotic phyla in the feces. The relative proportions of Firmicutes and Bacteroidetes were highly consistent in each individual sample. The most frequently detected genus was Ruminococcaceae UCG-005, ranging from 6.70 to 21.00%, displaying positively connections with the Rikenellaceae RC9 gut group. The bacterial communities associated with C. albirostris provide the basic knowledge for further microbiological studies and facilitates the conservation efforts of this vulnerable deer species.

Anti-inflammatory and anti-osteoarthritis effects of tectorigenin.

Osteoarthritis (OA) is a common and dynamic disease of the joints, including the articular cartilage, underlying bones and synovium. In particular, OA is considered as the degeneration of the cartilage. Tectorigenin (Tec) is known to affect many biological processes; however, its effects on articular chondrocytes remain unclear. This study aimed to assess the effects of Tec on articular cartilage. In vitro, Tec inhibited the expression levels of type X collagen, cyclooxigenase-2, matrix metalloproteinase (MMP)-3 and MMP-13, but enhanced the expression of Runx1, type II collagen and aggrecan in the presence of IL-1ß. Meanwhile, Tec inhibited apoptosis through the Bax/Bcl-2/caspase-3 pathway, upregulating p-Bad, downregulating the Bax/Bcl-2 ratio, and activating caspase-3 compared with IL-1ß treatment only. Moreover, this process was partially regulated by NF-κB P65. In vivo, the chondroprotective effects of Tec were assessed by establishing a model of surgically induced OA. Tec-treated joints exhibited fewer osteoarthritic changes than saline-treated joints. Meanwhile, 1.5 µg/kg Tec treatment produced a greater protective effect than 0.75 µg/kg Tec. The Osteoarthritis Research Society International (OARSI) scoring system, employed to assess histopathological grading of the models, as well immunohistochemistry for Aggrecan Neoepitope and MMP-3, further confirmed the results. In conclusion, this study showed that Tec plays a chondroprotective role in the OA process by preventing articular cartilage degeneration and chondrocyte apoptosis via the NF-κB P65 pathway.

Cyclic compressive stress-induced scinderin regulates progress of developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a common musculoskeletal disorder characterized by a mismatch between acetabulum and femoral head. Mechanical force plays an important role during the occurrence and development of abnormities in acetabulum and femoral head. In this study, we established a mechanical force model named cyclic compressive stress (Ccs). To analyze the effect of Ccs on DDH, we detected special genes in chondrocytes and osteoblasts. Results showed that Ccs downregulated chondrogenesis of ADTC5 in a concentration-dependent manner. Moreover, the mRNA level of Scinderin (Scin) considerably increased. We established lentivirus-SCIN(GV144-SCIN) to transfect hBMSCs, which were treated with different Ccs levels (0.25 Hz*5 cm, 0.5 Hz*5 cm, and 1 Hz*10 cm); the result showed that overexpression of Scin upregulated osteogenesis and osteoclastogenesis. By contrast, expression of chondrocyte-specific genes, including ACAN, COL-2A, and Sox9, decreased. Further molecular investigation demonstrated that Scin promoted osteogenesis and osteoclastogenesis through activation of the p-Smad1/5/8, NF-κB, and MAPK P38 signaling pathways, as well as stimulated the expression of key osteoclast transcriptional factors NFATc1 and c-Fos. Moreover, Scin-induced osteogenesis outweighed osteoclastogenesis in defective femur in vivo. The results of the analysis of Micro-CT confirmed these findings. Overall, Ccs influenced the development of DDH by promoting osteogenesis and cartilage degradation. In addition, Scin played a vital role in the development of DDH.

Gremlin2 Suppression Increases the BMP-2-Induced Osteogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells Via the BMP-2/Smad/Runx2 Signaling Pathway.

Osteoblasts are essential for maintaining skeletal architecture and modulating bone microenvironment homeostasis. From numerous associated investigations, the BMP-2 pathway has been well-defined as a vital positive modulator of bone homeostasis. Gremlin2 (Grem2) is a bone morphogenetic protein (BMP) antagonists. However, the effect of Grem2 on the BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs) remains ambiguous. This study aimed to analyze the procedure in vitro and in vivo. The differentiation of hBMSCs was assessed by determining the expression levels of several osteoblastic genes, as well as the enzymatic activity and calcification of alkaline phosphatase. We found that Grem2 expression was upregulated by BMP-2 within the range of 0-1 µg/mL, and significant increases were evident at 48, 72, and 96 h after BMP-2 treatment. Si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs, whereas overexpression of Grem2 had the opposite trend. The result was confirmed using a defective femur model. We also discovered that the BMP-2/Smad/Runx2 pathway played an important role in the process. This study showed that si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs via the BMP-2/Smad/Runx2 pathway. J. Cell. Biochem. 118: 286-297, 2017. © 2016 Wiley Periodicals, Inc.

P120-Catenin Protects Endplate Chondrocytes From Intermittent Cyclic Mechanical Tension Induced Degeneration by Inhibiting the Expression of RhoA/ROCK-1 Signaling Pathway.

STUDY DESIGN: The changes of endplate chondrocytes induced by intermittent cyclic mechanical tension (ICMT) were observed by realtime reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis. OBJECTIVE: To investigate the role of RhoA/ROCK-1 signaling pathway and E-cadherin/P120-catenin complex in endplate chondrocytes degeneration induced by ICMT. SUMMARY OF BACKGROUND DATA: ICMT can induce the endplate chondrocyte degeneration. However, the relationship between P120-catenin or RhoA/ROCK-1 signaling pathway and endplate chondrocytes degeneration induced by ICMT is not clear. METHODS: ICMT (strain at 0.5 Hz sinusoidal curve at 8% elongation) was applied to rat endplate chondrocytes for 6 days, 16 hours a day. The cell viability and apoptosis were examined by the LIVE/DEAD assay and flow cytometry. Histological staining was used to examine the lumbar disc tissue morphology and extracellular matrix. To regulate RhoA/ROCK-1 signaling pathway and the expression of E-cadherin and P120-catenin, RhoA/ROCK-1 pathway-specific inhibitors, E-cadherin, and p120-catenin plasmid were applied. Coimmunoprecipitation was employed to examine the interaction between E-cadherin and P120-catenin, P120-catenin, and RhoA. The related gene expression and protein location was examined by realtime reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. RESULTS: There was no change of viability verified by LIVE/DEAD assay and flow cytometry after ICMT loading. ICMT loading led to RhoA/ROCK-1 signaling activation and the loss of the chondrogenic phenotype of endplate chondrocytes. Inhibition of RhoA/ROCK-1 signaling pathway significantly ameliorated the degeneration induced by ICMT. The expression of P120-catenin and E-cadherin were inhibited by ICMT. ICMT reduced the interaction between P120-catenin and E-cadherin. Furthermore, over-expression of P120-catenin and E-cadherin can suppress the expression of chondrogenic gene, over-expression of P120-catenin can suppress the RhoA/ROCK-1 signaling pathway, but over-expression of E-cadherin cannot do it. CONCLUSION: P120-catenin protects endplate chondrocytes from ICMT Induced degeneration by inhibiting the expression of RhoA/ROCK-1 signaling pathway. LEVEL OF EVIDENCE: N/A.

Clostridium luticellarii sp. nov., isolated from a mud cellar used for producing strong aromatic liquors.

A strictly anaerobic, Gram-stain-positive bacterium, designated FW431T, was isolated from a mud cellar used for producing strong aromatic Chinese liquors. The strain was able to produce butanoic acid, an important component of the aroma style of Chinese liquors. Cells of strain FW431T were straight or slightly curved rods with a polar endospore and peritrichous flagella. The major cellular fatty acids (>10 % of the total) were C16 : 0, C18 : 1ω9c and C18 : 0. Biolog assays indicated that the strain preferably metabolizes palatinose, l-fucose, ß-hydroxybutyric acid, l-rhamnose and α-ketobutyric acid among 95 carbon sources tested. FW431T was related most closely to Clostridium ljungdahlii DSM 13528T and Clostridium kluyveri DSM 555T based on 16S rRNA gene sequence similarities of 95.0 and 94.2 %, respectively. The DNA G+C content of the genomic DNA was 44.4 mol%. Based on the evidence presented here, FW431T ( = CGMCC 1.5201T = KCTC 15519T) is proposed as the type strain of a novel species, Clostridium luticellarii sp. nov.

Bactericidal properties and biocompatibility of a gentamicin-loaded Fe3O4/carbonated hydroxyapatite coating.

Postoperative implant-associated infection remains a serious complication in total joint arthroplasty (TJA) surgery. The addition of antibiotics to bone cement is used as an antimicrobial prophylaxis in cemented joint arthroplasty; however, in cementless arthroplasty, there are no comparable measures for the local delivery of antibiotics. In this study, a gentamicin-loaded Fe3O4/carbonated hydroxyapatite coating (Gent-MCHC) was fabricated according to the following steps: (i) deposition of Fe3O4/CaCO3 particles on Ti6Al4V substrates by electrophoretic deposition; (ii) conversions of MCHC from Fe3O4/CaCO3 coatings by chemical treatment; and (iii) formation of Gent-MCHC by loading gentamicin into MCHC. MCHC possessed mesoporous structure with a pore size of about 3.8 nm and magnetic property with the saturation magnetization strength of about 4.03 emu/g. Gent-MCHC had higher drug loading efficiency and drug release capacity, and superior biocompatibility and mitogenic activity than Ti6Al4V. Moreover, Gent-MCHC deterred bacterial adhesion and prevented biofilm formation. These results demonstrate that Gent-MCHC can be used as a local drug delivery system to prevent implant-associated infection in TJA surgery.

Technical feasibility and histopathologic studies of poly (N-isopropylacrylamide) as a non-adhesive embolic agent in swine rete mirabile.

BACKGROUND: Non-adhesive liquid embolic agents are increasingly gaining importance in the embolization of cerebral arteriovenous malformations (AVMs). We investigated the use of poly (N-isopropylacrylamide) (PNIPAM) as a non-adhesive embolic agent in swine rete mirabile. METHODS: The PNIPAM hydrogel was mixed with iohexol and embolization was performed in swine rete mirabile in 30 animals. The microcatheter was examined after embolization. Follow-up angiography was performed for embolic efficacy after embolization. Embolized retia were examined histopathologically, and the alterations of inside rete and surrounding tissue were observed. RESULTS: The copolymer hydrogel was used for rete embolization in 30 swine, 28 swine survived the procedure, 2 swine died, 1 swine died of cerebrum infarction and the other died of embolic agent reflux into the occipital artery. The inside wall of the microcatheter was smooth, without copolymer adhering to it. Follow-up angiography was performed in 22 swine, there was no rete recanalization in 20 swine and partial rete recanalization in 2 swine because of the trunk embolization of ascending pharyngeal arteries. Histopathologically, the copolymer was found diffused into vessels of 100 - 150 microm in diameter. In acute group, neutrophils scattered surrounding the copolymer and endothelial integrity was observed, without endothelial denuding and necrosis. In subacute and chronic groups, the copolymer was found inside retia, a few mononuclear cells and eosinocytes scattered inside and surrounding it. The muscular layer was loosened with most muscular nuclei degraded. CONCLUSION: Experimental rete embolization with PNIPAM, made radiopaque with iohexol, is technically feasible in swine. Because of its properties, PNIPAM has great potential as a therapeutic non-adhesive embolic agent.