Objective To develop an asymmetric porous guided bone regeneration(GBR)membrane made from antibacterial zinc oxide nanoparticle(nZnO)/poly(3-hydroxybutyrate-co-4-hydroxy butyrate)copolymer(P34HB),and detect its osteogenic performance.Methods Ultrasonic dispersion technique,solvent displacement method and freeze-drying technique were used to prepare P34HB composite GBR membrane loaded with nZnO at different doses(0％,0.5％,1.0％and 1.5％).Scanning electron microscopy(SEM)and energy spectrometry(EDS)were conducted to determine the load of nZnO and observe the structure of the prepared membrane.Then MC3T3-E1 cells were implanted in a 24-well culture plate with the prepared GBR membrane pre-laid in the well.Thus the experiment included blank control group(no membrane)and groups with membranes composing 0％,0.5％,1.0％and 1.5％nZnO,respectively.CCK-8 and cell scratch assays were used to detect cell proliferation capacity and migration ability.The protein adsorption capacity of GBR membrane was detected by protein adsorption experiment.The in vitro degradation of GBR membrane was tested;the activity of alkaline phosphatase(ALP)was detected with chemical test.And calcified nodules were observed with alizzarin red staining;the antibacterial ability of GBR membranes was tested with in vitro antibacterial experiments.Results Four groups of nZnO/P34HB composite GBR membranes with 0％,0.5％,1.0％and 1.5％nZnO were successfully prepared.SEM results showed that the composite membrane was porous on one side and dense on the other side.EDS indicated the incorporation of zinc elements in the composite membrane.The results of cell proliferation assay showed that 0.5％and 1.0％nZnO/P34HB group promoted the proliferation of MC3T3-E1 cells,when compared with the blank control group and 0％and 1.5％nZnO/P34HB groups(P＜0.05).The results of cell scratch assay showed that MC3T3-E1 cells in the 0.5％nZnO/P34HB group migrated significantly over the scratch edge,with the cell mobility higher than that of the other 3 membrane groups in 48 h after culture(P＜0.01).Protein adsorption experiment displayed there was no significant difference in optical density of the 4 group of membrane,indicating that the addition of nZnO had no effect on the protein adsorption capacity of the material itself.The results of enzymatic experiment showed that though the quality of the 4 membranes was continuously decreased with elapse of time,the degradation rate was still relatively lower after 28 d of treatment.ALP staining and alizarin red staining displayed that the 0.5％nZnO/P34HB group obtained the strongest ALP activity and the largest amount of calcified nodules when compared with the other 3 groups(P＜0.01).Higher mineralization was observed in the 0％and 0.5％nZnO/P 34 HB groups than the other 2 groups(P＜0.01).The results of in vitro antibacterial experiments showed that the number of colonies in the 0.5％nZnO/P 34 HB group was significantly less than that in the blank control group and the 0％nZnO/P34HB group(P＜0.01).Conclusion Our prepared P34HB composite GBR membrane loaded with nZnO is in asymmetric porous structure,and can promote cell proliferation,facilitate cell osteogenesis and have bacteriostatic ability.

Objective:To observe the expression of deleted in malignant brain tumor protein 1 (DMBT1) in rat acute respiratory distress syndrome (ARDS) model induced by sepsis and its relationship with ARDS related biomarkers.Methods:Forty-eight healthy male rats were randomly divided into sham operation group (Sham group) and ARDS model group, and the rats in each group were further divided into three subgroups at 6, 12 and 24 hours after operation, with 8 rats in each subgroup. The rats in the Sham group were exposed to the cecum only, and sepsis induced ARDS model was reproduced by cecal ligation and puncture (CLP) in the ARDS model group. The general performance was observed at 6, 12, 24 hours after operation. Abdominal aortic blood of rats was collected, and the levels of DMBT1, surfactant-associated protein D (SP-D), vascular endothelial growth factor (VEGF), interleukins (IL-6, IL-10) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The lung tissues were collected, and the lung wet/dry weight (W/D) ratio was determined. The lung tissue pathological changes were observed under light microscope after hematoxylin-eosin (HE) staining, and the lung tissue injury score was evaluated. The expression of DMBT1 protein in lung tissue was determined by Western blotting. The relationship between the serum DMBT1 and SP-D, VEGF, IL-6, IL-10, lung tissue injury score were analyzed by Pearson correlation analysis.Results:Rats in the ARDS model group showed obvious pathological manifestations after operation. The alveolar structure destruction, inflammatory cell infiltration, and alveolar hemorrhage were observed under microscope. Compared with the Sham group, the lung tissue injury score and the lung W/D ratio at 12 hours after operation in the ARDS model group were significantly increased (lung tissue injury score: 3.35±0.13 vs. 1.16±0.07, lung W/D ratio: 5.36±0.44 vs. 4.38±0.35, both P < 0.05), and pulmonary edema was present, which suggested that the ARDS model caused by CLP was successfully reproduced. The results of ELISA and Western blotting showed that the levels of serum DMBT1, SP-D, VEGF and IL-6 in the ARDS model group increased gradually with time, while the level of IL-10 increased first and then decreased. Compared with the Sham group, the levels of DMBT1 in serum and the expressions of DMBT1 protein in lung tissue in the ARDS model group were significantly increased from 6 hours after operation [serum (ng/L) : 231.96±19.17 vs. 187.44±10.19, lung tissue (DMBT1/β-actin): 2.05±0.19 vs. 0.93±0.25, both P < 0.05], and the levels of SP-D, VEGF, IL-6 and IL-10 in serum were significantly increased from 12 hours after operation [SP-D (ng/L): 73.35±8.05 vs. 43.28±5.77, VEGF (ng/L): 89.85±8.47 vs. 43.19±5.11, IL-6 (ng/L): 36.01±2.48 vs. 17.49±1.77, IL-10 (ng/L): 84.55±8.41 vs. 39.83±5.02, all P < 0.05]. Pearson correlation analysis showed that serum DMBT1 was positively correlated with serum SP-D, VEGF, IL-6, IL-10 and lung injury score at 12 hours and 24 hours in the ARDS model group (12 hours: r values were 0.946, 0.942, 0.931, 0.936, 0.748, respectively; 24 hours: r values were 0.892, 0.945, 0.951, 0.918, 0.973, respectively; all P < 0.05). Conclusion:DMBT1 is a novel early biomarker of ARDS by affecting alveolar epithelial cell, alveolar capillary permeability and inflammatory response.