Preparation and properties of asymmetric porous antimicrobial nZnO/P34HB composite guided bone regeneration membrane

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.

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.

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).

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.