construction-evaluation-murine-calvarial-osteolysis-model-exposure-to

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

Nanjing University

Polar interactions such as electrostatic forces and hydrogen bonds play an essential role in biological molecular recognition. On a protein surface, polar interactions occur mostly in a hydrophobic environment because nonpolar amino acid residues cover ~75% of the protein surface. We report that ionic interactions on a hydrophobic surface are modulated by their subnanoscale distance to the surface. We developed a series of ionic head groups-appended self-assembled monolayers with C2, C6, C8, and C12 space-filling alkyl chains, which capture a dendritic guest via the formation of multiple salt bridges. The guest release upon protonolysis is progressively suppressed when its distance from the background hydrophobe changes from 1.2 (C2) to 0.2 (C12) nanometers, with an increase in salt bridge strength of ~3.9 kilocalories per mole.

Ionic interactions. Subnanoscale hydrophobic modulation of salt bridges in aqueous media.

Mesenchymal stem cells (MSCs) have several features that make them an attractive option for potentiating cartilage repair. Synovium-derived (SMSCs) have been recently recognized as an excellent source. SRY-related HMG-box (Sox) family plays an important role in the proliferation and differentiation of SMSCs. However, the role of Sox4 in human SMSCs remains elusive. In the present study, we investigated the role of Sox4 in SMSCs through gain-of-function studies and found that Sox4 promoted cell proliferation and chondrogenesis. Furthermore, Sox4 could directly bind to the promoter of long noncoding RNA DANCR and increased its expression. Finally, knockdown of DANCR could reverse the stimulative effect of Sox4 on the proliferation and chondrogenesis of SMSCs. Taken together, our data highlights the pivotal role of Sox4 in the proliferation and differentiation of SMSCs.

Sox4 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cell via activation of long noncoding RNA DANCR.

The bradykinin B2 receptor (BDKRB2) plays a key role in the inflammation process of osteoarthritis. Nitric oxide has also long been considered to be a catabolic factor that contributes to inflammatory response and the osteoarthritis disease pathology. Several studies have reported that the BDKRB2 +9/-9 bp polymorphisms are associated with transcription of the receptor. However, the roles of BDKRB2 polymorphisms in inflammation in osteoarthritis remain unclear. This study enrolled 156 subjects with primary knee osteoarthritis and 58 healthy volunteers. BDKRB2 polymorphisms were genotyped, and the mRNA and protein levels of BDKRB2 in synovial tissues from osteoarthritis patients were measured by quantitative real-time polymerase chain reaction and western blot analysis, respectively. Nitric oxide production in serum from patients with osteoarthritis was measured using a nitric oxide assay kit. We found that the mean BDKRB2 mRNA levels were significantly higher in Kallgren-Lawrence grade-4 osteoarthritis patients than patients with lower grade osteoarthritis. The +9/-9 bp polymorphisms significantly affected the BDKRB2 mRNA and protein expression levels in synovial tissues from osteoarthritis subjects. Osteoarthritis patients with +9/-9 and -9/-9 genotypes had higher BDKRB2 expression levels in synovial tissue and nitric oxide production in serum. Moreover, positive correlation was found between the BDKRB2 levels in synovial tissue and nitric oxide production. Compared with health controls, significant increases of nitric oxide production in osteoarthritis were detected which were associated with increasing severity of osteoarthritis. Multiple linear regression analysis (adjusted for gender and age) showed serum nitric oxide level was positively associated with BDKRB2 polymorphism and Kallgren-Lawrence grade and was inversely associated with obesity. Our findings showed that the BDKRB2 +9/-9 bp polymorphisms affected the gene expression and nitric oxide production, which were associated with radiographic severity of osteoarthritis, suggesting that the BDKRB2 +9/ -9 bp polymorphisms may act as a genetic modulator of osteoarthritis, and play an essential role in inflammatory process in osteoarthritis.

BDKRB2 +9/-9 bp polymorphisms influence BDKRB2 expression levels and NO production in knee osteoarthritis.

Platelet-rich plasma (PRP) is now widely used as a promising treatment for patients with tendinopathy. However, the efficacy of PRP treatment for tendinopathy is controversial mainly because of inconsistent results from human clinical trials and particularly because the concentration and effect of leukocytes in PRP remain largely unknown.

Harmful Effects of Leukocyte-Rich Platelet-Rich Plasma on Rabbit Tendon Stem Cells In Vitro.

The aim of the study was to investigate the mechanisms of human β-defensin 3 (HBD-3) regulation of the immune response and the lipopolysaccharide/Toll-like receptor-4 (LPS/TLR4)-mediated signaling pathway. A TLR4 extracellular gene fragment was cloned into the pET32a plasmid to determine its expression inand purification. A dialysis labeling method was used to stain HBD-3 with fluorescein isothiocyanate (FITC). FITC-HBD-3 was used to induce the differentiation of human peripheral blood mononuclear cells (MNC) into immature dendritic cells (imDC). Binding reactions were established using FITC-HBD-3 and sTLR4 into cell suspensions. Flow cytometry (FCM) was used to analyze the results. Western blot analysis confirmed the identity of nuclear factor-κB (NF-κB) and was used to quantify its nuclear translocation. The results showed that, HBD-3 bound to imDC in a Ca-dependent manner, and sTLR4 and LPS competitively inhibited the binding. HBD-3 competitively blocked the binding of LPS and imDC by binding to imDC. HBD-3 significantly decreased the translocation of LPS-induced NF-κB into the nucleus. In conclusion, HBD-3 can competitively inhibit the binding of LPS and imDC through its binding to TLR4 molecules, which are expressed in imDC, thereby preventing LPS from inducing the maturity of the imDCs.

HBD-3 regulation of the immune response and the LPS/TLR4-mediated signaling pathway.

Cartilage tissues have limited capacity for repair after damage and then cause osteoarthritis, so finding alternative treatment is ongoing. Mesenchymal stem cells (MSCs) have become a promising therapy for cartilage damage and diseases due to the advantages of easy separation, high proliferative potentiality, and genetic stability. Synovium-derived MSCs (SMSCs) have been recognized as an ideal source for cartilage repair. In our previous study, we found that Sox4 promoted proliferation and chondrogenesis of SMSCs through upregulation of long noncoding RNA (lncRNA) DANCR. However, the exact molecular mechanism by which DANCR promotes proliferation and chondrogenesis of SMSCs remains unknown. In the present study, we investigated the effect of lncRNA DANCR on the proliferation and chondrogenesis of SMSCs. We found that overexpression of DANCR could promote proliferation and chondrogenesis of SMSCs, while knockdown of DANCR had the opposite effect. Moreover, our data demonstrated that DANCR directly interacted with myc, Smad3, and STAT3 mRNA to regulate their stability. Finally, we found that the promotion of SMSC proliferation induced by DANCR depended on myc. Also, DANCR activated chondrogenesis of SMSCs via upregulation of Smad3 and STAT3 expression. Our growing knowledge of the role of DANCR is pointing toward its potential use as a novel therapeutic approach for cartilage damage and diseases.

Long Noncoding RNA DANCR Is a Positive Regulator of Proliferation and Chondrogenic Differentiation in Human Synovium-Derived Stem Cells.

The ethanol extract of the aerial parts of Alstonia yunnanensis Diels afforded five new monoterpenoid indole alkaloids, alstiyunnanenines A-E (1-5), along with one known compound, alstoniascholarine I (6). The structures of the isolated compounds were established based on 1D and 2D (H-H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic potential using eight tumor cell lines. As a result, alkaloids 4-6 exhibited cytotoxicities against all tested tumor cell lines, especially against osteosarcoma cell lines (SOSP-9607, MG-63, Saos-2, M663) with ICvalues<6μM.

Cytotoxic monoterpenoid indole alkaloids from Alstonia yunnanensis Diels.

The mechanism of human β-defensin 3 (HBD-3) in methicillin-resistant(MRSA-induced infection of implant drug-resistant bacteria biofilm in the mouse tibial bone marrow was studied. Healthy adult male Sprague-Dawley rats with average weight of 230 g were selected to construct the infection model of MRSA-induced implant drug-resistant bacteria biofilm in the mouse left tibial bone marrow. The drugs were intraperitoneally injected after 24 h medullary cavity infection, and the experimental groups included the model group, HBD-3 group, and vancomycin group (20 rats in each group). The model group was injected with 10 ml saline, HBD-3 group was injected with 10 ml of 8 µg/ml (1 MIC) and vancomycin group was injected with 10 ml of 0.5 µg/ml (1 MIC), five animals in each group were sacrificed on the 1, 7, 14 and 21 days, respectively. Observation was carried out on whether there was swelling and purulent secretion on the local wound; 1 ml venous sinus blood of eye socket was collected for blood routine examination and blood culture, and the laser scanning confocal microscopy was used to observe the morphology of the biofilm on the implant surface and the number of viable bacteria. Immunohistochemical staining was adopted to test the expression of nuclear factor-κB (NF-κB) and toll-like receptor 4 (TLR-4), and ELISA method was used to test interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), IL-1α and interferon-γ (INF-γ)-inducible protein-10 (IP-10) expression levels. There was no death due to infection in the HBD-3 group or vancomycin group, 1 case with significant wound swelling was found, respectively, in each group, but there was no purulent secretion. The percentage of the total white blood cells and neutrophil granulocytes as well as the biofilm morphology and the number of viable bacteria in the model group was gradually increased with time, while those in the HBD-3 group and vancomycin group were decreased with time. The comparative difference among groups was statistically significant (P<0.05); those in the HBD-3 group and vancomycin group at each time-point was decreased significantly compared with the model group, and the difference among groups was statistically significant (P<0.05), but in terms of the comparison between the HBD-3 group and vancomycin group, the difference was not significantly different (P>0.05). The NF-κB and TLR-4 expressions in the model group and vancomycin group were not significantly changed at each time-point, those in the HBD-3 group began to increase on the 1st day, and reached the peak on the 7th day and began to decline on the 14th day, and the comparative difference at each time-point was statistically significant (P<0.05); those in the HBD-3 group were significantly higher than the model group and vancomycin group at each time-point and the difference was statistically significant (P<0.05). The IL-10, TNF-α, IL-1α, and IP-10 expressions in the model group at each time were significantly higher than the other two groups and the difference was statistically significant (P<0.05); in terms of the comparison between the HBD-3 group and vancomycin group, the difference was not statistically significant (P>0.05). In conclusion, β-defensin 3 can inhibit the bacterial growth by regulating inflammation and immune responses in the MRSA-induced implant drug-resistant bacteria biofilm infection in the mouse tibial bone marrow.

The mechanism of human β-defensin 3 in MRSA-induced infection of implant drug-resistant bacteria biofilm in the mouse tibial bone marrow.

miRNAs have been reported to regulate cellular differentiation by modulating multiple signaling pathways. Long noncoding RNA (lnc RNA) DANCR was previously identified to be critical for the chondrogenesis of human synovium-derived mesenchymal stem cells (SMSC), however, the underlying molecular mechanism requires better understanding. Here, miRNA expression profiling in DANCR overexpressed in SMSCs identified significant down-regulation of, which serves as a downstream target of DANCR. Notably,overexpression reversed DANCR-induced cell proliferation and chondrogenic differentiation of SMSCs, which suggested thatantagonized the function of DANCR. Mechanistically, highly expressedresulted in the decreased expression of the TGF-β pathway member Smad4, and inhibition ofenhanced the expression level of Smad4. Depletion of Smad4 suppressed the promotion of DANCR in cell proliferation and chondrogenesis of SMSCs. Collectively, our results characterized-Smad4 axis as a major downstream functional mechanism of lncRNA DANCR in promoting the chondrogenesis in SMSCs.

Long noncoding RNA DANCR regulates-Smad 4 axis to promote chondrogenic differentiation of human synovium-derived mesenchymal stem cells.

Dioscin, a typical steroid saponin, has been reported to promote osteoblastic cell differentiation. However, the underling mechanisms remain to be elucidated. In the present study, it was identified that dioscin (0.5, 1, 5, 10 and 25 µg/ml) promoted MC3T3‑E1 cell proliferation and differentiation in a dose‑dependent manner. Western blot analysis showed that dioscin regulated autophagy‑associated protein expression in MC3T3‑E1 cells; it promoted the expression of apoptosis stimulated protein of p53‑2 (ASPP2), and inhibited the expression of nuclear factor (NF)‑κβ and microtubule‑associated protein 1 light chain 3β, in a concentration‑dependent manner. Caffeic acid phenethyl ester (CAPE) was used to inhibit the activation of NF‑κB and examine the effect of the ASPP2/NF‑κβ pathway on osteoblastic cell differentiation, proliferation and autophagy. It was identified that CAPE reversed the regulation of dioscin on osteoblastic cell differentiation, proliferation and autophagy. In conclusion, the present study revealed that dioscin promoted osteoblast proliferation and differentiation by inhibiting cell autophagy via the ASPP2/NF‑κβ pathway. These results are the first, to the best of our knowledge, to reveal the involvement of autophagy in the effects of dioscin on the prevention and therapy of osteoporosis.

Article	Year
Ionic interactions. Subnanoscale hydrophobic modulation of salt bridges in aqueous media. Science (New York, N.Y.)\| PubMed ID: 25931555	2015
Sox4 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cell via activation of long noncoding RNA DANCR. Journal of molecular histology\| PubMed ID: 26514989	2015
BDKRB2 +9/-9 bp polymorphisms influence BDKRB2 expression levels and NO production in knee osteoarthritis. Experimental biology and medicine (Maywood, N.J.)\| PubMed ID: 26764266	2017
Harmful Effects of Leukocyte-Rich Platelet-Rich Plasma on Rabbit Tendon Stem Cells In Vitro. The American journal of sports medicine\| PubMed ID: 27184544	2016
HBD-3 regulation of the immune response and the LPS/TLR4-mediated signaling pathway. Experimental and therapeutic medicine\| PubMed ID: 27703496	2016
Long Noncoding RNA DANCR Is a Positive Regulator of Proliferation and Chondrogenic Differentiation in Human Synovium-Derived Stem Cells. DNA and cell biology\| PubMed ID: 27982693	2017
Cytotoxic monoterpenoid indole alkaloids from Alstonia yunnanensis Diels. Fitoterapia\| PubMed ID: 28040532	2017
The mechanism of human β-defensin 3 in MRSA-induced infection of implant drug-resistant bacteria biofilm in the mouse tibial bone marrow. Experimental and therapeutic medicine\| PubMed ID: 28413476	2017
Long noncoding RNA DANCR regulates-Smad 4 axis to promote chondrogenic differentiation of human synovium-derived mesenchymal stem cells. Bioscience reports\| PubMed ID: 28674107	2017
Dioscin enhances osteoblastic cell differentiation and proliferation by inhibiting cell autophagy via the ASPP2/NF-κβ pathway. Molecular medicine reports\| PubMed ID: 28849197	2017

Shuo Chen

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