【作者】 罗超；

【导师】 孙家明；

【作者基本信息】 华中科技大学 ， 外科学（整形外科）， 2021， 博士

【摘要】 目的:1.构建DBM-明胶的核壳结构微组织,在体外评价其力学仿生性能和促BMSCs成骨诱导活性;2.建立大鼠皮下异位成骨模型和原位颅骨缺损模型,评价核壳结构微组织的体内骨缺损修复再生能力;3.建立一种磁控结构化液滴技术,通过磁矩编程和液滴焊接实现骨组织工程微组织的精细操控及组装。方法:1.在体外利用微阵列芯片制备DBM-明胶的核壳结构微凝胶,通过光镜和SEM表征其大体结构,并测量其弹性模量、BMP-2缓释效率、细胞接种率,初步验证核壳结构微凝胶的力学仿生性能。随后接种BMSCs于核壳结构微凝胶,并置入生物反应器一体化扩增诱导制备核壳结构微组织,接种后于相应时间点通过活死细胞染色、MTT、ds DNA定量等方法检测微组织上细胞活性;更换成骨诱导液以后,通过测量成骨相关基因的表达、Von Kossa染色等方法检测微组织的成骨诱导能力。2.选取SD大鼠作为实验对象,首先构建大鼠背部皮下异位成骨模型,并植入核壳结构微组织,1个月后取材,通过micro-CT评估新生骨量和骨密度,通过H&E和CD31分析其新生骨组织量及新生血管数量。随后构建大鼠临界颅骨缺损模型,并植入核壳结构微组织,分别于1月和3月取材,通过Micro-CT分析其原位新生骨量,通过H&E、Masson、Col-1、OCN组织学分析其原位新生骨组织量。3.利用海藻酸钠和POSS-NH₂在水-油交界面的电荷互补作用形成结构化液滴,通过模具塑形,通过磁滞回线测量液滴磁学特性,通过流变测试检验液滴铁磁性与溶液粘弹性之间的关系。通过磁化和重新磁化的方式控制液滴运动,并在液滴连接处高温处理焊接液滴,通过紫外光照使Gelma交联成凝胶,并加入Ca Cl₂使海藻酸钠化学交联,最终完成微组织的组装。结果:1.光镜和SEM下观察到明显的核壳结构,核壳结构微凝胶的弹性模量（2.7±1.2 MPa）比单纯明胶微凝胶（1±0.3 Mpa）高,在26天内核壳结构微凝胶能实现BMP-2的持续释放（累计释放量达175 pg/10μL）。相比于单纯明胶微组织,活/死细胞染色和SEM显示核壳结构微组织上细胞存活的更好,且细胞外基质分泌更多。体外培养7 d,MTT（OD值:核壳组:1.8±0.5;明胶组:1.4±0.1）和ds DNA定量（ds DNA含量:核壳组:36±0.2 ng/mg;明胶组:24±0.1 ng/mg）的结果显示核壳结构微组织上细胞活性更好。成骨诱导21 d,Von Kossa和钙定量（钙含量:核壳组0.62 mol/L,明胶组0.21mol/L）的结果表明核壳结构微组织上累积更多矿化结节。2.植入大鼠背部异位成骨模型4周后,micro-CT（骨量:核壳组:1.2±0.4 mm³;明胶组:0.25±0.15 mm³）和组织学的结果表明核壳结构微组织新生骨组织量更多,CD31免疫组化（微血管数:核壳组:62±5个/视野;明胶组:35±3个/视野）的结果表明核壳结构微组织新生血管数量更多。对大鼠原位颅骨缺损修复12周后,micro-CT（骨量:核壳组:2.8±0.5 mm³;明胶组:1.5±0.2mm³）和组织学（H&E,Masson,Col-1和OCN）的结果表明核壳结构微组织对原位临界颅骨缺损的修复效果更好。3.由10%Gelma、1%海藻酸钠和2.5 mg/ml纳米氧化铁组成的预混液具备铁磁性（Mr/Ms:0.172,矫顽力Hc:5.978 k A/m）,单个及多个液滴均可被外磁场（20 m T）磁化,磁化后施加更强的磁场（23 m T）可使液滴重新磁化。通过磁化及重新磁化可实现混合BMSCs液滴的磁矩编程,焊接液滴,交联,完成骨组织工程微组织精细操控及组装。结论:1.核壳结构微组织具有良好的力学和骨诱导活性双仿生性能。DBM微颗粒作为内核,可以为微组织提供良好的力学支撑,其上负载的BMP-2可促进微组织的成骨诱导活性。相比于单纯明胶微组织,核壳结构微组织上BMSCs增殖活性更好,且细胞成骨效果更好。2.核壳结构微组织能在大鼠背部皮下异位成骨,且能促进大鼠原位临界颅骨缺损的修复。这可能与DBM微颗粒提供的良好力学支撑和骨诱导活性相关;此外,具有开放式多孔结构的明胶壳层也可以BMSCs提供保护,使BMSCs在体内更好的发挥修复作用。3.通过磁化和重新磁化的方式可以利用外磁场操控液滴的运动和移动,在此基础上可通过磁矩编程实现骨组织工程微组织的多级组装。这种基于磁控结构化液滴的微组织组装方式不仅可以精细设计组装的每个步骤,也可以实现不同种类微组织的组装。更多还原

【Abstract】 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.更多还原