Experimental Study Of LIPUS Regulating The Release Of SDF-1α From Nanobubbles In Early Angiogenesis Of Bone Regeneration

Objective:Large and irregular bone defects caused by various factors such as congenital malformation,inflammation,trauma or surgery are difficult to repair by natural healing,which has been a difficult problem in clinical treatment.In the process of bone repair,blood vessel formation precedes bone formation,and early blood vessel formation plays an important role in bone regeneration.Endothelial Progenitor Cells(EPCs)have the ability to differentiate into mature endothelial cells,and adsorption of EPCs near bone defects can overcome the defects in vitro culture and accelerate endothelialization.Stromal cell-derived factor-1α(SDF-1α)is an important progenitor Cell homing Factor.Studies have shown that appropriate concentrations of SDF-1α are necessary to attract and capture endothelial progenitor cells involved in blood vessel formation.How to load SDF-1α to maintain its internal stability and appropriate effective concentration is the key to its stable function.Nanometer ultrasonic microvesicle(nanovesicle)drug delivery system has been highly valued.Poly Lactic Acid Co-Glycolic acid(PLGA)is commonly used as a nanoparticle carrier,which can carry drugs and growth factors.The slow release rate of simple drug-loaded nanovesicle is unstable,and it is difficult to accurately control the drug release rate at different stages.Low Intensity Pulse Ultrasound(LIPUS)is proving to be a promising gene and drug delivery method with unique advantages in targeted drug delivery and controlled release.Based on the above theories,in order to seek a better way to promote early angiogenesis,this study prepared SDF-1α nanovesicles and combined them with LIPUS,in order to provide a new strategy for bone tissue engineering therapy.This study is divided into three parts: In the first part,PLGA nanobubble system loaded with bioactive factor SDF-1 α was constructed and its physicochemical properties were characterized;The second part was in vitro cell experiment to explore the chemotaxis,proliferation and migration ability of SDF-1 α-loaded PLGA nanovesicles in Human Umbilical Vein Endothelial Cells(HUVECs)under LIPUS controlled release.In the third part,the skull defect model of SD rats was established to investigate the ability of SDF-1αnanovesicles under LIPUS controlled release to recruit endothelial progenitor cells and induce vascularization at the bone defect site to promote bone formation.Methods:In the first part,PLGA nanobubbles loaded with SDF-1α were prepared by double emulsification/solvent evaporation method,EDC/NHS method and vacuum freeze-drying technology.The appearance,distribution and structure of the nanobubbles were observed by optical microscope and scanning electron microscope.The particle size and surface potential of the nanobubbles were detected by Malvern laser particle size analyzer.The encapsulation rate and carrying capacity of the nanobubbles were determined by ELISA,and the cumulative release of SDF-1α nanovesicles under LIPUS irradiation was detected.In the second part,the migration,chemotaxis and proliferation of human umbilical vein endothelial cells(HUVECs)induced by SDF-1α-loaded PLGA nanobubbles under LIPUS irradiation were evaluated by scratch test,Transwell test and CCK-8 test.The third part evaluated the effect of LIPUS regulation of SDF-1α-loaded PLGA nanobubbles on angiogenesis and osteogenesis in vivo.The skull defect model of SD rats was constructed,and the silk fibroin/GO hydrogel prepared by our research group was injected into the defect site.Control group,SDF-1α group,SDF-1α nanobubble group and SDF-1α nanobubble +LIPUS group were set up.The cranial specimens at 2w,4w and 8w after surgery were evaluated by H&E staining and immunohistochemistry.The cranial specimens at 4w,8w and 12 w were evaluated by Micro-CT,H&E staining and Masson staining for bone regeneration.Finally,the toxicity of nanobubbles on important organs of rats was detected by H&E staining.Results:(1)In this study,PLGA nanobubbles containing SDF-1α were successfully prepared.The nanobubbles were white powdery and dissolved in water as milky white suspension.Under the optical microscope,it is spherical,uniform in size,uniform in distribution and good dispersion.Fluorescence microscopy showed that SDF-1α loaded on the surface of PLGA nanobubbles showed bright green fluorescence,indicating that FITC-modified SDF-1α was bound to the PLGA shell.The particle size of SDF-1αnanobubbles measured by Malvern laser particle size analyzer is about 673.6±0.200 nm.The smaller particle size has strong stability in vivo and good anti-sound pressure ability,which enhances the ability of slow release and controlled release.The results of scanning electron microscopy showed that the SDF-1α nanobubble was spherical and contained a large cavity,which made it possessed of ultrasonic controllability.ELISA showed that the nanobubbles had good encapsulation rate and carrying capacity.The cumulative release curve of SDF-1α showed that SDF-1α nanobubbles could be released slowly for 7 days,and LIPUS could promote the sudden release of SDF-1α nanobubbles and increase the local concentration,and then sustainably release the SDF-1α nanobubbles at a stable rate to maintain the effective therapeutic concentration.(2)The chemotactic ability of HUVECs in each group was detected by single-layer cell scratch test in vitro,and the results showed that the experimental group had certain chemotactic effect on HUVECs,which verified the ability of SDF-1α to chemotactic HUVECs.SDF-1α nanobubbles +LIPUS group had the strongest chemotaxis,followed by SDF-1α nanobubbles group,indicating that the slow release of nanobubbles was conducive to the long-term function of active factors.Under LIPUS irradiation,the release of nanobubbles was enhanced,thus enhancing the chemotaxis of active factors on cells.Transwell cell migration experiment showed that the cell coverage area in SDF-1α group,SDF-1α nanobubbles group and SDF-1α nanobubbles +LIPUS group was significantly increased by crystal violet staining.Cells in the SDF-1α nanobubbles +LIPUS group basically covered the entire field of vision,and had the highest mobility.LIPUS stimulation enhanced the recruitment effect of SDF-1α on HUVECs.In order to verify cell survival and proliferation,CCK-8 method was used to detect the effects of different groups on the growth of HUVECs.The results showed that no obvious growth inhibition and cytotoxicity were observed in HUVECs cultured in vitro,indicating that was safe and biocompatible,meeting the basic requirements of in vivo drug delivery system.The cell proliferation rate of SDF-1α nanobubbles +LIPUS group was significantly higher than that of other groups,which was suitable for in vivo regeneration and repair experiments.(3)In vivo H&E staining showed more angiogenesis in the SDF-1α nanobubbles group,indicating that SDF-1α sustained-release nanobubbles can effectively recruit endothelial progenitor cells.The angiogenesis ability of SDF-1α nanobubbles +LIPUS group was the strongest,which may be that LIPUS regulates the release of SDF-1αnanobubbles,forming high concentrations locally,and recruiting more circulating and local endothelial progenitor cells to participate in angiogenesis,repair and regeneration.Immunohistochemical staining showed that the positive expression of endothelial progenitor cell markers CD133,CD31 and Emcn(endothelial cell markers),VEGF was the most obvious in SDF-1α nanobubbles +LIPUS group,which was consistent with the H&E staining results.Micro-CT showed that the early repair of bone defects first occurred at the edge of the defect.The SDF-1α nanobubbles +LIPUS group had the largest amount of new bone and relatively dense bone density.Further H&E staining and Masson staining were performed to quantitatively analyze bone formation.The results also showed that the quality and quantity of bone formation in SDF-1α nanobubbles +LIPUS group were significantly better than those in other groups,suggesting that bone regeneration was more active.The above experimental results further demonstrated in vivo that SDF-1α loaded with nanobubbles could sustainably release and maintain the concentration of SDF-1α,while LIPUS could further regulate the release of SDF-1α nanobubbles,improve the chemotaxis effect of SDF-1α on EPCs,and more effectively enhance angiogenesis,thus repairing bone defects.Conclusion:(1)In this study,PLGA nanobubbles loaded with SDF-1α were successfully prepared and their basic physicochemical properties were determined.PLGA nanobubbles can be used as SDF-1α protein carrier and sustainably release SDF-1α for more than 7 days in vitro.LIPUS can achieve the release of SDF-1α loaded with PLGA nanobubbles and improve the local concentration.(2)SDF-1α promoted migration,chemotaxis and proliferation of HUVECs in vitro;The sustained release of PLGA nanobubbles containing SDF-1α can promote the migration,recruitment,chemotaxis and proliferation of HUVECs in a long time.LIPUS can accurately regulate the release of SDF-1α-loaded PLGA nanobubbles,meeting the local effective concentration,and realizing the control of drug release rate requirements at different stages.(3)The skull defect model of male SD rats was successfully constructed.This study verified the ability of LIPUS to regulate the release of SDF-1α nanobubbles to recruit endothelial progenitor cells,promote angiogenesis and further promote osteogenesis.