Study On Injectable Microtissue Construction Of Nanoclay-PHA And Repair Of Skull Defect In Rats

Objective:1.The feasibility of constructing Nanoclay-PHA(NP)microgel was studied,and the material science and biocompatibility were characterized.2.The injectable bone microtissue with good biomimetic properties and osteogenic activity was constructed based on NP microgel,and the cell proliferation and osteogenic properties were characterized.3.To verify the effect of injectable bone microtissue on the repair of critical skull defect in rats,and to study the internal mechanism of promoting bone repair.Methods:1.NP microgels were prepared by improved double emulsification method.According to the concentration of Nanoclay,five concentrations of PHA,1%NP,3%NP,5%NP and 7%NP microgels were prepared.Based on the difference of preparation conditions of double emulsification method,microgel groups with different homogenizer speed,different magnetic agitator speed and different diameter were set up.The morphology and porosity of microgels were detected by optical microscope,laser confocal microscope and environmental scanning electron microscope(ESEM)to explore the best preparation conditions and parameters.2.The bone marrow mesenchymal stem cells(BMSCs)of 5-day-old male SD rats were extracted by washing method,and then expanded and passaged in vitro.P2 generation BMSCs was implanted on different concentrations of NP microgel and simple PHA microgel to construct injectable bone microtissue.Cultured in vitro.On the 1st,4th,7th and10 th day,the cell activity in microtissue was compared by living cell staining,nuclear and cytoskeleton staining and CCK8 method.After 7 days of amplification,the osteogenic induction solution was replaced to induce osteogenesis of the microtissue.The osteogenic effect of injectable bone microtissue was compared with alizarin red and calcium on the14 th and 21 st,respectively.The expression of osteogenic genes OCN,Runx2,BMP-2 and Smad1 was quantitatively detected by q RT-PCR.3.The 6-week-old male SD rats were selected.Firstly,under anesthesia,electric drill was used to drill holes in the skull to establish a critical bone defect model with 5mm diameter,and close the wound layer by layer.Two weeks later,the rats were anesthetized again,and PHA microgel,3%NP microgel,injectable bone microtissue and PHA microtissue with osteogenic activity were injected into the skull defect site respectively.At the 4th and 12 th week,the bone repair effect of injectable bone microtissue in rat skull defect was evaluated by microcomputer tomography(Micro CT),Hype,Masson,OCN staining,Col-1 and gross specimen morphology.Results:1.The spherical NP microgel can be constructed by the improved double emulsification method.The surface morphology and porosity of the microgel can be adjusted by adjusting the Nanoclay concentration,the speed of the homogenizer and the speed of the magnetic agitator.It is found that the microgel with 3%Nanoclay concentration of 350 μm-300 μm in diameter has good porosity and small heteromorphism when the homogenizer is 8000 rpm for 2 min and the magnetic mixer is 400 rpm for 6h.In the follow-up experiments,this concentration was selected to construct microgel.2.The staining of living dead cells showed that there were more living cells and fewer dead cells in the injectable bone microtissue than in the control group,and the quantitative analysis of cytoskeleton area and cell count also showed that the number of cells in the injectable bone microtissue was larger than that in the control group.the cytoskeleton area was significantly larger than that in the control group.CCK-8 quantitative analysis showed that the proliferation of injectable bone microtissue cells increased gradually during the 10-day culture cycle,and the cell activity was better than that of PHA microtissue.Quantitative detection of osteogenic genes indicates that injectable bone microtissue is superior to PHA microtissue in osteogenesis.3.The micro-CT analysis showed that the area of skull defect in injectable bone microtissue group was better than that in other groups at 4 and 12 weeks.At 12 weeks,the PHA microtissue group and injectable bone micro-tissue formed a bony connection with the surrounding in situ skull,and Hype,Masson,OCN staining also showed that 3% of the micro-tissue group new bone tissue,collagen-1,neovascularization were better than the control group.Conclusion:1.The NP microgel with uniform pore size and porosity can be constructed by double emulsification.2.The injectable bone microtissue can promote the proliferation and osteogenic differentiation of rat BMSCs and show good osteogenic activity in vitro.3.The injectable bone microtissue can promote the repair of critical skull defect in rats.