Development Of Core-shell Structured Microtissues And Its Application In Bone Defect Regeneration

Purpose:To improve the mechanical property and osteoinductivity of microtissues,core-shell structured microtissues with good osteogenic property was developed.And to further expand the application of microtissues,a magnetic structured liquid droplet was fabricated,which realizes the fine manipulation and assembly of microstissues through magnetic moment programming and droplet welding.Methods:1.Micro-stencil array chip was used to fabricate core-shell structured microgels.The gross structure was characterized by light microscopy and SEM,and the elastic modulus,BMP-2release efficiency and cell inoculation rate were measured.The mechanical properties of core-shell microgels were tested.Subsequently,BMSCs were seeded on core-shell structured microgels,and then transfered into the bioreactor for cell culture.After culture,cell viability was detected by means of live dead cells staining,MTT and ds DNA quantification at the corresponding time points.After changing the osteo-induction medium,the formation of microtissues was detected by measuring the expression of osteogenic related genes and VON KOSSA staining.2.SD rats were selected as the experimental animals.Firstly,the ectopic subcutaneous model was established in the back of rats,and core-shell structured microtissues were implanted.1month later,the samples were taken,and the amount of new bone and bone mineral density were evaluated by micro-CT.The amount of new bone and the number of new blood vessels were analyzed by H&E and CD31.After that,core-shell microtissues were implanted into the 5 mm cranial defect.Samples were taken after 4 weeks and 12 weeks.The in situ new bone mass and bone mineral density were analyzed by micro CT.The repair of microtissues were analyzed by H & E,Masson,COL-1 and OCN.3.The magnetic structured droplets were synthesized and molded by mold,and their magnetic properties and rheological properties were characterized.Then the direction of the magnetic moment of the droplets was controlled by magnetization and re-magnetization,and the external magnetic field was used to control and weld them.After improving the magnetic manipulation and welding method of droplets,cells were added into droplets,and the magnetic structured droplets were used for fine manipulation and assembly of microtissues.Results:1.SEM showed that the microgel had obvious core-shell structure,and the Young's modulus of core-shell microgels was about 3 times of that of gelatin microgel.During 26 days,the core-shell microgels could achieve sustained release of BMP-2.Compared with gelatin microtissues,live/dead assay and SEM showed that the cells on the core-shell microtissues survived better,and secreted more extracellular matrix.MTT and ds DNA quantitative results showed that the cell activity of core-shell microtissues was better.Von Kossa and calcium quantitative results showed that there were more mineralized nodules in the core-shell microstructure.2.Four weeks after implantation of ectopic subcutaneous model in rat,the results of micro CT and histology showed that the core-shell microstructure had the most amount of new bone tissue,and the results of CD31 immunohistochemistry showed that the core-shell microtissues could promote the formation of new blood vessels.After 12 weeks of in situ repair of rat skull defect,the new bone mass of core-shell structure microstructure was the largest in the defect area,and the defect area was basically filled up.The osteogenic activity of core-shell structure microtissues in vivo was significantly better than that of other groups.3.Non-spherical droplets can be prepared by mold or glass capillary,and adjacent droplets can be welded by heated needle.After adding iron oxide nanoparticles,the droplets have ferromagnetism,which can magnetize and re magnetize the droplets,and use the external magnetic field to make the droplets move or rotate.Through the magnetic moment programming,different kinds of droplets can be arranged according to the design,welding droplets and cross-linking,which can realize the fine control and assembly of microtissues by magnetic control of structured droplets.Conclusions:1.The core-shell microgels showed good mechanical and osteoinductive properties.As the core,DBM microparticles can provide good mechanical support for the microgel,and BMP-2 loaded on DBM core can promote the osteoinductive activity of the microgels.Compared with gelatin,BMSCs with core-shell microgels have better proliferation activity and osteogenic effect.2.The core-shell structure can be used for ectopic bone formation in the back of rats,and can promote the repair of critical cranial defect in situ.This may be related to the good mechanical support and osteoinductive activity provided by DBM microparticles;in addition,gelatin shell with open porous structure can also provide protection for BMSCs,thus improve the repair in vivo.3.Through magnetization and re-magnetization,the move and turn of droplets can be controlled by external magnetic field.On this basis,the multi-level assembly of microtissues can be realized by magnetic moment programming.This kind of microtissues assembly method based on magnetically controlled structured droplets can not only design each step of assembly,but also realize the assembly of different kinds of microtissues.