Experimental Study Of Gelatin-CaO2/SAPNS/PLGA Oxygen Release Composites Scaffold Combined With Peripheral Blood Mesenchymal Stem Cells For The Reconstruction Of Critical-sized Calvarial Bone Defect

BackgroundBone tissue engineering can provide a new source of material for repairing large bone defects.It has become a hot topic in bone tissue engineering research in recent years.Although a lot of progress has been made in the field of bone tissue engineering in recent years,there are still some problems which need to be well solved.Among them,the survival of seed cells on scaffold is one of most important factors that restrict the research and application of bone tissue engineering in clinical.There are many factors that affect the survival of seed cells in the host.More and more studies have shown that hypoxia is one of the most important factors.Therefore,the preparation of a controllable,slow,continuous oxygen release material to improve the survival rate of seed cells in bone tissue engineering scaffolds will provide a new strategy for tissue engineering to repair bone defects,especially large bone defects.There is little report on the use of oxygen release materials in bone tissue engineering at present.ObjectiveGelatin and CaO2 were used to make gelatin-CaO2 oxygen release microspheres,composited with self-assembling peptide nanofiber scaffold(SAPNS)to form controlled slow continuous oxygen-release composites material.Then the effect of the composite material on the survival of the seed cells under the condition of hypoxia in vitro and the osteogenesis in vivo was detected.Method1.Preparation of Gelatin/CaO2 controlled slow oxygen microspheresGelatin dissolved in PBS solution with heating,then added CaO2 powder by Gelatin:CaO2=2:1,after been full suspension,the suspension was rapidly added into the liquid paraffin which was on the magnetic stirrer with heating and in high speed stirring,50%glutaraldehyde was added into it,then under low temperature stirring condition until solidification.Remove the paraffin and glutaraldehyde on the surface of microspheres by using isopropanol,then the microspheres were freezing dried.2.The oxygen release efficiency of Gelatin-CaO2 oxygen release microspheres Detection of oxygen release in vitro,75mg oxygen release microspheres was put into 15ml centrifuge tube containing 3 ml PBS solution,at 1D 3D 5D 7D 10D 14D 17D 21D 28D,the oxygen content of dissolved oxygen in PBS was detected.Oxygen release test in vivo,the Gelatin-CaO2 oxygen releasing microspheres were placed in the lateral femoral condyle of New Zealand white rabbits.They were removed at 1W,2W,3W and 4W respectively,and their oxygen release effects were detected.3.Gelatin-CaO2 oxygen release microspheres promoted cell survival under low oxygen conditions in vitro Experimental group:Gelatin-CaO2 group,control group:blank group,Gelatin microsphere group,pure CaO2 group.The cell suspension was added to the round culture dish with a diameter of 10 cm.After 3h,the original culture medium was abandoned and the new medium after anoxic treatment was added,and the treatment factors were added to the experimental group.The flow cytometry was carried out after the culture of 3D under low oxygen conditions.4.Preparation and transplantation of Gelatin-CaO2/SAPNS/PBMSC/PLGA composite scaffoldThe Gelatin-CaO2/SAPNS/PBMSC/PLGA composite material was prepared as following method:the cell suspension of PBMSCs suspension in 15ml DMEM/F12 medium was rapidly mixtured with 100 ?l SAPNS,Gelatin-CaO2 microspheresat at low temperature mixture,then placed on the PLGA membrane,and covered with another piece of PLGA film.The prepared Gelatin-CaO2/SAPNS/PBMSC/PLGA composite system was implanted in the critical-sized calvarial bone defect model.In the control group,the PBMSC/SAPNS/PLGA group,the Gelatin/SAPNS/PBMSC/PLGA group and the CaO2/SAPNS/PBMSC/PLGA group were the same as those in the experimental group.5.the effect of Gelatin-CaO2 oxygen release microspheres promotion the survival of PBMSCs in the scaffold in vivoAfter 2W,3 rats in each group were randomly selected for perfusion,and then prepared into frozen sections with thickness of 10?m.The sections were stained with BrdU and DAPI immunofluorescence.The specific methods were referenced to the instructions of BrdU and DAPI staining kit,and the positive rate of BrdU was calculated.6.imaging and histology were used to detect bone tissue regenerationAfter been transplanted for 10W,the bone defect specimens were Micro-CT scaned and 3D reconstructed for the analysis of Bone Mineral Density(BMD)and bone volume(BV).The specimens were then tested for histology.7.statistical analysisThe data is processed with SPSS20.0,and the value is represented by mean standard deviation.The data were compared with t test,and the difference was significant when p<0.05.Results1.Gelatin-CaO2 oxygen release microspheres with slow and continuous controlled oxygen release efficiency.In vitro release oxygen detection showed that the Gelatin-CaO2/SAPNS composite oxygen release material with slow nd continuous controlled oxygen releasing efficiency for more than 3W;the in vivo release oxygen detection showed that the Gelatin-CaO2/SAPNS composite oxygen release oxygen release effect were significantly higher than those in vitro at the same time points;two kinds of oxygen detection methods were not found obvious burst release phenomenon.2.Gelatin-CaO2 oxygen release microspheres obviously promoted cell survival under hypoxic conditions.The oxygen release material was cultured in vitro with PBMSCs for 3 days,and the flow cytometry was detected.We found that the cell in the experimental group was significantly higher than that in the control group.The results of cell survival test in vivo showed that the rate of BrdU staining positive cells in the experimental group was also much higher than that of the control group.3.Gelatin-CaO2/PBMSC/SAPNS/PLGA composite scaffold materials are used to promote the repair of extreme skull defects.The postoperative 10W imaging test results showed that the bone defects in the experimental group largely repaired,while in PBMSC/SAPNS/PLGA group,Gelatin/PBMSC/SAPNS/PLGA Group repair effect is roughly the same,but still most of the defects were not repaired,group CaO2/PBMSC/SAPNS/PLGA bone defect repair effect is the worst,only a little new bone in bone defect edge.Conclusion1.In this experiment,the Gelatin-CaO2 microspheres with controllable slow and sustained oxygen release characteristics were successfully prepared.The microspheres could obviously promote the survival of seed cells under the condition of hypoxia in vitro and in vivo.2.The Gelatin-CaO2/SAPNS/PLGA oxygen release composite scaffold improved the survival of the seed cells and significant improved the reconstruction of the bone defect.It provides a new strategy for the repair of bone defect,and has a good prospect of application.