In order to investigate the effects of hyperbaric oxygen (HBO) on the proliferation and differentiation of human osteoblasts isolated from alveolar bone under various pressure and exposure time, osteoblasts from human alveolar bone were seeded in 24 well plates at a cell density of 2 500 cells per well. There are four treatment groups which were 2.4ATA for 90 min, 2.4ATA for 30 min, 1.5ATA for 90 min and 1.5ATA for 30 min. Osteoblasts culture were treated one time everyday for up to 10 days in a temperature and humidity controlled custom-made seven-litre hyperbaric unit. Control samples were incubated in a standard humidified incubator at 37℃ containing 5% CO2 and 95% atmospheric air. Proliferation of osteoblasts were evaluated by WST-1 assay before and 16 h after HBO on day 1,2,3,4,6,8,10. The cytotoxic effect of HBO on osteoblasts was assessed by a toxicology assay kit. For differentiation study, osteoblasts were seeded in 96 well plates at a cell density of 10 000 cells per well. After 3 days normal culture, medium was changed to osteogenic medium. Subsequently, cultures were exposed daily to HBO of 2.4ATA for 90 min and 1.5ATA for 90 min up to 13 days. Mineralization was evaluated by calcium deposition assay, alkaline phosphatase (ALP) activity and von Kossa staining. To assess the effect of pressure on cell proliferation and differentiation, hyperbaric air treatment was observed in this study. It showed that HBO treatment promotes proliferation of osteoblast in the presence of 10% foetal calf serum (FCS). No significant change in extracellular LDH activity before and after HBO treatment. The study of differentiation demonstrated that HBO enhanced differentiation associated with increased bone nodule formation, calcium deposition and alkaline phosphatase activity. These result suggests that HBO treatment significantly stimulated osteogentic differentiation, which implies a potential application of HBO in bone tissue engineering.

Objective To detect nucleos(t)ide-resistance mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) isolated from hepatocytes of patients with chronic HBV infection and to analyze the correlation between the mutations found in cccDNA and relaxed circular DNA (rcDNA). MethodsForty patients with chronic HBV infection were investigated. Preoperation serum samples and non-tumor liver tissues were collected.Intrahepatic HBV cccDNA and rcDNA were selectively extracted by co-precipitation of sodium dodecyl sulphate-protein and QIAamp DNA Mini Kit, and further purification with plasmid-safe ATP-dependent DNase (PSAD).Thereafter,cccDNA were amplified by selective polymerase chain reaction (PCR) or nested PCR using the primers spanning both the two gaps in HBV genome and covering the common mutations associated with nucleoside analogues resistance (rt169- rt250).Intrahepatic HBV rcDNA and pre-operation serum HBV rcDNA were also extracted and then amplified by PCR.The PCR products were then purified and sequenced.Results Among the 40 patients,intrahepatic HBV cccDNA were detected in 31 patients,and HBV rcDNA were detected in liver samples of 35 patients and pre-operation serum samples of 21 patients. The PCR products amplified from these samples were all successfully sequenced.rtM204Ⅰ mutation was detected in intracellular HBV cccDNA,rcDNA and serum HBV rcDNA in 2 patients.Both rtM204Ⅰ and rtQ215H were detected in intrahepatic HBV cccDNA and rcDNA in 2 patients,while no corresponding mutation was observed in serum HBV rcDNA of these two patients.Three variants including rtM204V,rtM204V and rtV173L-rtL180M-rtM204V were detected in serum HBV rcDNA in 3 patients,while corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA of these patients.Condsions The results suggest that antiviral nucleos(t) ide resistance mutations can be found in HBV cccDNA in chronic hepatitis B patients. The dominant resistant mutation found in intrahepatic HBV cccDNA/rcDNA may be different from that in serum HBV rcDNA.