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Objective:To investigate whether the antibacterial copper sulfide (CuS)/graphene oxide (GO) nanosheets composite film can promote angiogenesis and osteogenesis in vitro. Methods:GO and CuS/GO nanosheets were synthesized and mixed into polyvinyl alcohol (PVA)/carboxymethyl cellulose (CMC) hydrogel films. The study was conducted in 4 groups: PVA/CMC/GO, PVA/CMC/CuS/GO, PVA/CMC (only PVA/CMC-based film) and blank control (no material). The PVA/CMC, PVA/CMC/GO and PVA/CMC/CuS/GO films were characterized by electron scanning microscopy and energy dispersive spectrometer. The biocompatibility of different films (PVA/CMC/CuS/GO films with concentrations of CuS/GO nanotablets of 0, 50, 100, 200, 400, and 800 μ g/mL) was evaluated by CCK-8, live/dead cell staining, and hemolysis test. The angiogenesis was evaluated by cell migration and tube forming test in vitro. Alkaline phosphatase and alizarin red staining were used to evaluate osteogenesis in vitro, and the expression of osteogenic genes was measured by immunofluorescence staining and RT-qPCR. In addition, the bacterial plate counting method and bacteriostatic circle method were used to evaluate the antibacterial activity of films. Results:In the PVA/CMC/GO and PVA/CMC/CuS/GO groups, the surface of the PVA/CMC-based film was smooth and flat whereas the nanosheets composite films were irregularly flaky and convex. The biosafety experiments showed that the PVA/CMC-based film composited with GO or CuS/GO nanosheets at the concentration of 100 μg/mL had good biocompatibility. The results of angiogenesis in vitro showed that the migration ratio of HUVEC cells in the PVA/CMC/CuS/GO group was significantly better than those in the PVA/CMC/GO, PVA/CMC and control groups ( P<0.001). In the experiment of tube forming area and length, the PVA/CMC/CuS/GO group was significantly better than the PVA/CMC/GO, PVA/CMC and control groups ( P<0.001). The osteogenic differentiation in vitro displayed that the alkaline phosphatase and alizarin red staining of MC3T3-E1 cells in the PVA/CMC/CuS/GO group were significantly better than those in the PVA/CMC/GO, PVA/CMC and control groups ( P<0.001). In addition, the fluorescence intensity of immunofluorescence staining in alkaline phosphatase and type Ⅰcollagen on MC3T3-E1 cells, and the mRNA expression levels of osteogenic related genes including alkaline phosphatase, bone morphogenetic protein 2, osteocalcin and osteopontin in the PVA/CMC/CuS/GO group were significantly higher than those in the PVA/CMC/GO, PVA/CMC and control groups ( P<0.001). The antibacterial assay showed that the PVA/CMC/CuS/GO group had a significantly greater antibacterial activity and a significantly larger inhibition zone against Gram-positive bacteria and Gram-negative bacteria than the PVA/CMC/GO, PVA/CMC and control groups ( P< 0.001). Conclusions:PVA/CMC films composited with GO or CuS/GO nanosheets demonstrate ideal biocompatibility and antibacterial properties which promote angiogenesis and osteogenic differentiation in vitro. In particular, antibacterial PVA/CMC/CuS/GO composite films with the coupling function of angiogenesis and osteogenesis are expected to provide a new strategy for infectious bone defects.
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Objective@#To compare the hepatobiliary injury difference of newborn BALB/c mice infected by different titers of rhesus rotavirus(RRV).@*Methods@#Neonatal mice(n=80) were randomly separated into 4 groups and were intraperitoneally inoculated with different titers of rotavirus: High titer group(1×107 PFU/ml); Medium titer group(1×106 PFU/ml); Low titer group(2.5×105 PFU/ml); Control group (only culture medium) within the first 24 hours after birth. All mice were sacrificed at day 12 after RRV inoculation then the liver and blood samples were collected. Meanwhile, mice were observed daily for at least 12 days, including their weight, skin color and survival situation. Liver functions were examined by serum biochemical test and morphologic changes in the biliary tract were observed. Tissue sections underwent H&E staining and immunohistochemically analysis for the presence of CK19.@*Results@#Compared with the normal mice, the mice in the experimental group had different degrees of skin jaundice, weight lost, survival rate decreased, liver function damage. In the experimental group, the symptom of low titer group was light, and could be restored to normal, however, when compared with the low titer group, the mice in the high titer group were serious, their skin jaundice was more obvious, weight was significantly reduced and irreversible, survival rate was lower(50%), liver function of TBIL, DBIL, TBA, ALT, ALP were significantly increased.Further analysis showed that the high titer group had high bile duct obstruction rate (80%), with no case of obstruction in the low titer group. Histologic analysis also showed intrahepatic bile duct atresia in the high titer group, a large number of inflammatory cell infiltrated around the portal area, while the morphology of intrahepatic bile duct was almost normal and just a small amount of inflammatory cell infiltrated around the portal area in the low titer group.@*Conclusions@#Different titers of rotavirus had different effects on the newborn mice hepatobiliary system: high titer was easy to cause biliary atresia, and low titer caused hepatitis.
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Objective To investigate the osteogenic ability of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan (DS/rhBMP2/CS) nano microspheres combined with coralline hydroxyapatite (CHA) in repair of segmental bone defects.Methods DS/rhBMP2/CS microspheres prepared by ionic crosslinking method were adsorbed into CHA by lyophilization.Seventy-two New Zealand rabbits were randomly divided into 3 equal groups after they had been made into models of bone defect at the right radius.The defects in the 3 groups were implanted respectively with CHA,rhBMP2/CHA and DS/rhBMP2/ CS/CHA.Another 18 animals served as a blank control group.Blood and bone samples were obtained at 4,8 and 12 weeks after implantation.The serum BGP was detected,and the bone grafts were scanned by micro-CT for calculation of volume ratio of the new bone.Hematoxylin and eosin (HE) staining was performed after bone decalcification.Results All the 72 animals recovered well without any infection or graft exposure.Gross observation at postoperative 12 weeks showed that the DS/rhBMP2/CS/CHA group was the best in the quality,quantity and strength of the new bone,as well as in the healing of bone defects.The serum levels of bone gamma-carboxyglutamic-acid-containing protein at all time points in the DS/rhBMP2/CS/CHA group were significantly higher than those in the CHA and rhBMP2/CHA groups (P ＜ 0.05).Micro-CT scanning demonstrated obvious progress in bone formation,cortical bone and marrow cavity at all time points in the DS/rhBMP2/CS/CHA group which showed significantly faster bone reconstruction synchronized with material degradation and significantly higher volume ratio of the new bone than the other 2 groups (P ＜ 0.05).Histological examinations showed better morphology of mature cortical bone and new marrow cavity at all time points in the DS/rhBMP2/CS/CHA group than in the other 2 groups.Conclusion Since DS/rhBMP2/CS/CHA possesses a better mechanism of sustained-releasing rhBMP2 to induce bone formation because of its reticular and hole-hole-connected structure,it may perform better in repairing segmental bone defects than simple CHA or rhBMP2/CHA.
