LPS at low concentration promotes the fracture healing through regulating the autophagy of osteoblasts via NF-κB signal pathway.

OBJECTIVE: To investigate the effect of low-concentration lipopolysaccharide (LPS) on proliferation and apoptosis of osteoblasts and to discover the mechanism of low-concentration LPS in facilitating the proliferation of osteoblasts. MATERIALS AND METHODS: MC3T3-E1 osteoblasts were treated with LPS, 3-methyladenine (3-MA, autophagy inhibitor), and BAY11-7082 (inhibitor of nuclear factor-kappa b, NF-κB), respectively. The cell cycles were detected using a flow cytometer. Cell proliferation and activity of MC3T3-E1 osteoblasts were explored by cell counting kit-8. Western blotting and immunofluorescence assay were performed to detect the protein level. RNA expression was measured through polymerase chain reaction (PCR) and immunofluorescence assay. RESULTS: At the third day after cell culture, cell infusion reached 80%, and cells were taken as the subjects. At 6 h after treatment with low-concentration LPS, the proliferation and activity of cells were higher than those at 1 h and 12 h after treatment, and the apoptotic level was significantly lower than that in cells at 12 h after treatment. The proliferation and activity of cells in the low-concentration LPS group were significantly higher than those in the control group, 3-MA group and BAY11-7082 group, and the apoptotic level was lower than those in these groups. Compared with those of cells in control group and BAY11-7082 group, the messenger RNA (mRNA) and protein expressions and nuclear transfer of cells in low-concentration LPS group were significantly elevated, but there were no statistically significant differences in comparisons with the 3-MA group. In the experiment of cell autophagy, the autophagic level in cells in low-concentration LPS group was higher than those in the control group, 3-MA group and BAY11-7082 group. CONCLUSIONS: Through the NF-κB signaling pathway in osteoblasts, low-concentration LPS can activate the autophagy and promote cell proliferation, thereby inhibiting cell apoptosis and accelerating the fracture healing.

MicroRNA-30c suppressed giant-cell tumor of bone cell metastasis and growth via targeting HOXA1.

OBJECTIVE: To dissect the functioning mode of miR-30c on giant cell tumor of bone cell metastasis and growth and provide therapeutic targets for giant cell tumor of bone. PATIENTS AND METHODS: By quantitative Real-time polymerase chain reaction (qRT-PCR), miR-30c expression level in 62 pairs of giant cell tumor of bone cells tissue samples and five breast cancer-derived cell lines. Using miR-30c mimics and inhibitors, we analyzed the effects of miR-30c over-expression and knockdown on cell proliferation, invasion, and migration. Dual-luciferase activity assay was recruited to examine the potential target gene HOXA1, which predicted by several databases. Protein level was studied using Western blot. RESULTS: MiR-30c expressed significantly lower in giant cell tumor of bone tissue samples and cell lines. Over-expression miR-30c in giant cell tumor of bone cells decreased the cell proliferation, invasion, and migration abilities while down-regulation miR-30c in giant cell tumor of bone cells increased these abilities oppositely. Dual-luciferase and Western blot confirmed HOXA1 as a target gene of miR-30c. Furthermore, up-regulation of HOXA1 reserved the suppressive effect of miR-30c over-expression on cell growth and progression. CONCLUSIONS: miR-30c could suppress giant cell tumor of bone cell proliferation and progression via HOXA1, which might provide a new target for giant cell tumor of bone diagnosis and therapy.

Development of In situ hybridization and real-time PCR assays for the detection of Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis.

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real-time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue-specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.

Effect of melatonin supplementation on cryopreserved sperm quality in mouse.

BACKGROUND: Antioxidants protect spermatozoa against cell damage during cryopreservation. OBJECTIVE: To investigate whether melatonin supplement in the extender may improve the quality of cryopreserved mouse sperm. METHODS: Kunming mice sperm frozen in extender R18S3 (18% (w/v) raffinose and 3% (w/v) skim milk) supplemented with melatonin were thawed and evaluated. RESULTS: Mouse spermatozoa were cryopreserved in the freezing extender R18S3 that contained melatonin at 0, 0.125, 0.25 and 0.5 mg/mL melatonin. The extender without melatonin supplement was associated with increased formation of reactive oxygen species (ROS) and decreased sperm motility. Melatonin supplement at 0.125 mg/mL significantly increased the progressive motility of sperm in comparison to other melatonin concentration or control. The percentage of thawed viable sperm with ROS was lower in the melatonin-treated groups than in untreated group. Melatonin supplement also increased antiapoptotic gene Bcl-xl expression in the thawed sperm. CONCLUSION: Supplement of 0.125 mg/mL melatonin could reduce oxidative damage and apoptosis.

Acute toxic effects of sonodynamic therapy on hypertrophic scar fibroblasts of rabbit ears.

The objective of this study was to observe the acute cytotoxic effects of hematoporphyrin monomethyl ether sonodynamic therapy (HMME-SDT) on hypertrophic scar fibroblasts of rabbit ears. We first assessed the effects of different irradiation times and HMME concentrations on the survival of hypertrophic scar fibroblasts using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the optimum irradiation time and HMME concentration. The hypertrophic scar fibroblast cell suspensions of the rabbit ears were divided into four groups, the survival rates were detected using the MTT assay, and the type of cell death was detected by Annexin V/propidium iodide (PI) double staining flow cytometry. Our results showed that HMME-SDT significantly reduced the viability of hypertrophic scar fibroblasts of rabbit ears at ultrasonic irradiation times of 30, 60, and 90 s, but not 10 s (P < 0.05). HMME alone had no significant effect on the cell survival rate at any irradiation time (P > 0.05). In contrast, the cell survival rate was significantly decreased at an irradiation time of 10 s and HMME concentrations of 20 and 50 µg/mL (P < 0.05). Furthermore, Annexin V/PI double staining showed necrosis and apoptosis of the hypertrophic scar fibroblasts. Given our results, HMME might be an effective sound-sensitive agent for SDT as it has a significant lethal effect on hypertrophic scar fibroblasts of rabbit ear cultured in vitro. HMME-SDT may therefore provide a new method for the treatment of hypertrophic scar formation.

Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization.

Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.

Research of arginylglycylaspartic to promote osteogenesis of bone marrow mesenchymal cells on chitosan/hydroxyapatite scaffolds.

A high degree of cell adhesion to a scaffold at the initial stage of cell inoculation is essential to bone tissue engineering. In realising high cell adhesion rate on a scaffold within a few hours, a chitosan/hydroxyapatite (CS/HA) scaffold with a channel/sphere pore was prepared via in situ hybridisation in combination with lyophilisation, in which the HA nanoparticles were dispersed in the CS uniformly. The size of the channel pore and the sphere pore of the CS/HA scaffold was 150 µm to 650 µm and 3 µm to 15 µm, respectively. The compression strength and porosity of the CS/HA scaffold were 3.54 ± 0.32 MPa and 88.4%, respectively. The nitrogen content increased by 7.5% compared with the CS/HA scaffold without Arg-Gly-Asp (RGD) modification. More than 67% of the RGD in the PBS solution diffused into the CS/HA scaffold spontaneously. The effect of the RGD peptide on bone marrow mesenchymal stem cells (MSCs) on the CS/HA scaffold was investigated through cell adhesion rate, alkaline phosphatase (ALP) activity, and mineralised calcium nodules. The cell adhesion rates of the CS/HA scaffold with different RGD concentrations (50, 100 mg/L) were 71.6% and 80.7%, respectively, after 4 hours of culture; the rates were 30.9% and 47.5% higher than that of the CS/HA group (54.7%), respectively. The expressed ALP content of the CS/HA scaffold with RGD (191 ± 6 U/g protein) was 107.7% higher than that (92 ± 9U/g protein) of CS/HA (p<0.01). Furthermore, a higher amount of mineralised calcium nodules with red brown appeared in the CS/HA scaffold with RGD as opposed to that in the CS/HA group. The RGD peptide in the CS/HA scaffold not only achieved high cell adhesion in a short period of time, but also enhanced cell adhesion ability and promoted the MSCs to differentiate from osteoblasts.

Result of a randomized clinical trial comparing different types of anesthesia on the immune function of patients with osteosarcoma undergoing radical resection.

AIM: The purpose of this article was to explore the effects of different anesthesia drugs and techniques on the immune function of patients with osteosarcoma around the knee undergoing radical resection. METHODS: Forty-five ASA (American Society of Anesthesiologists) I-II patients were randomized and divided into three groups: the epidural anesthesia group (Group A), the general anesthesia group (Group B), and the combination of epidural anesthesia and general anesthesia group (Group C). The populations of T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+/CD8+) and a possible association between these variables were investigated 2 h before anesthesia, before and after skin incision, and on the 1st, 3rd and 5th days after operation. RESULTS: The serum sIL-2 levels and T lymphocyte subset populations did not show significant differences among the three groups before anesthesia and skin incision. Serum sIL-2R increased 2 h after skin incision and on the 1st and 3rd day after operation in groups A and B (P < 0.01), and was higher than that of group C 2 h after skin incision and on the 1st day after operation (P < 0.01). Serum sIL-2R increased on the 1st postoperative day in group C. The CD3+, CD4+ and CD4+/CD8+ populations decreased significantly in all groups 2 h after skin incision, and on the 1st and 3rd days after operation (P < 0.05). However, in group C, CD4+/CD8+ levels had almost returned to baseline values on the 3rd day after operation (P > 0.05), and were significantly higher than those of groups A and B (P < 0.05). On the 5th day after operation, CD3+, CD4+ and CD4+/CD8+ levels had returned to baseline values before anesthesia in group C (P > 0.05), and were significantly higher than those of groups A and B (P < 0.05). CONCLUSION: Epidural anesthesia combined with general anesthesia might reduce the stress reaction and the effect of anesthetic drugs on sIL-2 levels and T lymphocyte subsets, contributing to the restoration of immune function in cancer patients.

Immunotolerance reaction for allograft-limb in rats induced by gene-modified cell transfusion.

The therapeutic value of the transforming growth factor beta 1 (TGF-b) in transplantation has been reported; However, cell-mediated gene therapy using TGF-b is not applied to the organ transplantation widely. This study was to evaluate whether TGF-b-modified donor spleen cell specific transfusion in rat heterotopic allo-limb transplantation could induce tolerance tolerogenicity and prolong allograft's survival time. The Splenic T-cell in Wistar rats responsing to donor spleen cells which received TGF-b-transduced were severely impaired.The Survival time of Sprague-Dawley Allograft-limb in Wistar rats given TGF-b-modified donor spleen cells (5¥106 cells/well, administration of donor TGF-b-transduced donor spleen cells 7 days before transplantation) was extended modestly but significantly.

Experimental study of COX-2 selective and traditional non-steroidal anti-inflammatory drugs in total hip replacement.

This study investigated the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on peri-operative blood loss during elective total hip replacement. Patients were randomized to receive enteric-coated diclofenac 50 mg (n = 18), rofecoxib 12.5 mg (n = 17) or placebo (n = 16) administered orally three times daily for 2 weeks prior to surgery. Severe adverse effects resulting in discontinuation of trial participation occurred in six patients in the diclofenac group, five patients in the rofecoxib group and two patients in the placebo group; all drop-outs occurred at various times after surgery. Compared with placebo, peri-operative blood loss increased by 32% in the diclofenac group and by 7% in the rofecoxib group. Total mean +/- SD blood loss was 1040 +/- 136 ml in the diclofenac group, 844 +/- 83 ml in the rofecoxib group and 789 +/- 82 ml in the placebo group. Thus, administering a non-selective NSAID 2 weeks prior to elective total hip replacement significantly increases peri-operative blood loss.

Role of NF-kappaB in TNF-alpha-induced COX-2 expression in synovial fibroblasts from human TMJ.

In the temporomandibular joint (TMJ) synovium, cyclo-oxygenase-2 (COX-2) expression has been believed to be directly related to joint pain and synovitis. Here we investigated the role of Nuclear Factor kappaB (NF-kappaB) in the regulation of COX-2 expression in synovial fibroblasts from human TMJ induced by tumor necrosis factor-alpha (TNF-alpha). By reverse-transcriptase/polymerase chain-reaction (RT-PCR) and Western blotting analysis, TNF-alpha induced a dose- and time-dependent increase in COX-2 expression. Electrophoretic mobility shift assay (EMSA) revealed that transient NF-kappaB activation in the COX-2 promoter was triggered by TNF-alpha. In parallel with transient NF-kappaB activation, the rapid translocation of NF-kappaB, particularly the p65 subunit, from the cytoplasm into the nucleus was demonstrated. Pre-treatment with pyrolidine dithiocarbamate (PDTC), one of the NF-kappaB inhibitors, prevented binding to the COX-2 promoter and expression of COX-2 protein in response to TNF-alpha. These findings indicate that activation of NF-kappaB is responsible for TNF-alpha-induced COX-2 expression in synovial fibroblasts from the TMJ.

[Study on in vitro culture and cryopreservation by vitrification of blastocysts derived from single mouse 2-cell embryos' blastomeres].

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.

Cloned piglets born after nuclear transplantation of embryonic blastomeres into porcine oocytes matured in vivo.

Nuclear transplantation in the pig is more difficult than in other domestic animals and only one embryonic nuclear transplantation (NT) pig has been born to date. In this study, reconstituted porcine embryos were produced by electrofusion of blastomeres from in vivo four-cell embryos to enucleated in vivo or in vitro matured (IVM) oocytes. Nuclear transfer using cumulus cells as nuclear donors was also conducted. When blastomeres were used as donors, the electrofusion rate was significantly higher in oocytes matured in vivo (91.5%) than in those matured in vitro (66.1%) (p < 0.01). After fusion, the NT embryos reconstituted from in vivo matured oocytes developed to blastocysts at a rate of 10.3% after culture in rabbit oviducts for up to 5 days, while only 5.9% of the NT embryos reconstructed from in vitro matured oocytes developed to blastocyst stage. Electrofusion rate of cumulus cell nuclei with enucleated IVM oocytes was lower (47.6%) and only 1.5% (2/136) of the reconstituted eggs developed in vitro to morula stage, and 1.9% developed to blastocysts when cultured in the ligated rabbit oviducts. Transfer of 94 embryos reconstructed by blastomere NT with in vivo matured oocytes to five synchronous recipients resulted in the birth of two cloned piglets. No piglet was born following transfer to two recipients of embryos (n = 39) derived from NT with in vitro matured oocytes. The results demonstrate that in vivo matured oocytes are better recipients than those matured in vitro for pig cloning.