【作者】 崔巍；

【导师】 郭晓东；

【作者基本信息】 华中科技大学 ， 外科学（骨科）， 2017， 博士

【摘要】 第一部分 BMP-2小分子活性多肽P28体外诱导MC3T3-E1细胞增殖及成骨分化能力的评价目的 通过比较P28和rhBMP-2对小鼠成骨细胞前体细胞MC3T3-E1增殖及成骨分化活力的影响,评价P28发挥诱导成骨分化的能力。方法 分别使用P28或rhBMP-2对MC3T3-E1细胞进行干预,不添加任何生长因子的 ·空白组作为对照,培养1、3、5天后使用钙黄绿素对细胞进行染色,荧光显微镜观察各组细胞活力及增殖趋势;培养1、3、5、7天后采用MTT法检测各组细胞的增殖活力;培养7、14天后使用ALP染色液对细胞进行染色,显微镜下定性观察各组细胞ALP活性,同时采用ALP定量检测试剂盒定量评价各组细胞ALP活性;培养14、21天后使用茜素红染色液对钙结节进行染色,显微镜下观察各组细胞钙结节生成情况;培养1、3、7天后,Real-Time PCR检测Runx2、OCN、ColⅠ基因的表达情况,Western blot检测Runx2、p-Smad1/5/8、OCN、ColⅠ蛋白表达量,观察各组细胞成骨分化活性。结果钙黄绿素染色后在荧光显微镜下观察结果和MTT法检测结果显示P28组和rhBMP-2组细胞活力和增殖趋势明显高于空白组,差异有统计学意义(p<0.05),P28组与rhBMP-2组之间无显著差异(p>0.05);ALP染色和ALP定量检测结果显示P28组和rhBMP-2组细胞ALP活性明显高于空白组,差异有统计学意义(P<0.05),P28组与rhBMP-2组之间无显著差异(P>0.05);茜素红染液钙结节染色结果显示P28组和rhBMP-2组的钙结节明显多于空白组,P28组与rhBMP-2组之间无明显区别;Real-Time PCR检测和Western blot检测结果显示P28组与rhBMP-2组较空白组相比,成骨分化相关基因Runx2、OCN、ColⅠ表达明显增高,成骨分化相关蛋白Runx2、p-Smad1/5/8、OCN、ColⅠ表达量明显增高,rhBMP-2组略高于P28组,但无显著统计学差异。结论P28可以通过BMP-2信号通路中的Smad信号通路来实现对MC3T3-E1细胞增殖和成骨分化的促进与调控,同时P28具有与rhBMP-2接近的诱导成骨分化的良好生物活性。第二部分 煅烧骨/介孔二氧化硅纳米颗粒/P28复合材料的制备、理化性质及其体外控释的实验研究目的 探讨煅烧骨/介孔二氧化硅纳米颗粒/P28复合材料的制备方法及理化性质,并评价其体外释放动力学。方法 制备介孔二氧化硅纳米颗粒(MSN),采用透射电子显微镜(TEM)、Brunauer-Emmett-Teller(BET)法及 Barrett-Joyner-Halenda(BJH)法对制备的 MSN进行鉴定;按照不同的P28:MSN质量比例(1:1.5、1:2、1:2.5、1:3、1:3.5、1:4)确定P28与MSN之间适宜的投药比例;制备煅烧骨(TBC)和TBC/MSN支架材料,通过三维视频显微镜、场发射扫描电子显微镜(SEM)及能谱仪对两种材料的理化性质进行比较,以确定MSN是否能够良好的与TBC材料复合;用TBC和TBC/MSN两种材料分别负载3 mg P28,计算TBC/P28和TBC/MSN/P28两种复合材料所能负载的P28量,随后将两种复合材料置于恒温水浴摇床,按照第1、2、3、5、7、10、12、14、18、21和30天的时间点提取释放液测量P28释放量,比较两种支架材料负载P28的体外释放对力学。结果 制备的MSN粒径主要分布在180 ±30 nm,比表面积高达702.7 m2/g,表面孔径主要为4 nm,孔容积为0.8 cm3/g,具有良好的介孔结构和充足的载药空间;选取质量比为P28:MSN = 1:3作为较为适宜的投药比例进行药物负载,包封率达到81.88±4.44%;TBC和TBC/MSN材料的三维视频显微镜、SEM及能谱结果显示MSN较为理想的附着在TBC材料内部,分布较为均匀,并且不会对TBC良好的孔隙结构产生较大的影响;TBC/P28和TBC/MSN/P28两种复合材料对P28的负载量分别可以达到80%和98%左右,同时TBC/MSN/P28较TBC/P28释放更为缓慢和持久(p<0.05)。结论 MSN可以作为P28良好的载体材料,并且可以较为理想的与TBC支架材料相结合,并在体外环境实现对P28的长期、缓慢的释放。第三部分 煅烧骨/介孔二氧化硅纳米颗粒负载P28体外诱导MC3T3-E1细胞粘附、增殖及成骨分化的实验研究目的 通过比较 MC3T3-E1 细胞在 TBC、TBC/MSN、TBC/P28 和 TBC/MSN/P28四组材料上的粘附、增殖以及成骨分化情况,评价TBC/MSN/P28对MC3T3-E1细胞发挥诱导成骨分化的能力。方法 TBC和TBC/MSN两种材料分别负载2 mg P28,然后将实验分为TBC组、TBC/MSN组、TBC/P28组和TBC/MSN/P28组,将MC3T3-E1细胞分别种植在四组材料上,培养24小时后通过间接的细胞计数法检测四组材料上的细胞粘附率;培养3天后使用钙黄绿素和碘化丙啶对细胞染色,通过激光共聚焦显微镜观察四组材料上细胞的活力及分布趋势;培养2、4、6、8天后通过MTT法检测各组材料上细胞的增殖活力;培养5、10、15天后采用ALP定量检测试剂盒检测四组材料上细胞向成骨细胞分化的情况。结果 MC3T3-E1细胞在TBC/P28组和TBC/MSN/P28组的粘附率要明显高于TBC组和TBC/MSN组,差异有统计学意义(p<0.05),TBC/P28组与TBC/MSN/P28组间无显著统计学意义(p>0.05);钙黄绿素和碘化丙啶染色后通过激光共聚焦显微镜观察显示TBC/P28组和TBC/MSN/P28组支架材料上的细胞数量明显多于TBC组和TBC/MSN组;MTT法检测结果显示整个培养周期中TBC/P28组和TBC/MSN/P28组的细胞数量明显高于TBC组和TBC/MSN组,差异具有统计学意义(P<0.05),当培养第6和8天时,TBC/MSN/P28组细胞数量开始高于TBC/P28组,差异具有统计学差异(p<0.05);ALP定量检测试剂盒检测结果显示整个培养周期中TBC/P28组和TBC/MSN/P28组的ALP活性均明显高于TBC组和TBC/MSN组,差异具有统计学意义(p<0.05),培养第15天时,TBC/MSN/P28组明显高于TBC/P28组,差异具有统计学意义(P<0.05)。结论 四组材料均具有良好的生物相容性,TBC/P28和TBC/MSN/P28相比TBC和TBC/MSN进一步表现出了对细胞的趋化、促进增殖和分化的能力。TBC/MSN/P28复合材料所具备的缓释性能可以更好地促进P28发挥成骨诱导活性。第四部分 煅烧骨/介孔二氧化硅纳米颗粒负载P28修复兔桡骨临界性骨缺损能力的实验研究目的 评价 TBC/MSN/P28复合材料修复兔桡骨临界性骨缺损的能力,进一步探讨该复合材料在体内环境修复大范围骨缺损的能力。方法 TBC和TBC/MSN两种材料分别负载7.5 mg P28,然后将实验分为四组:TBC、TBC/MSN、TBC/P28和TBC/MSN/P28,20新西兰大白兔随机分为4组,制备双侧桡骨中段15 mm临界性骨缺损,并分别植入上述材料。术后6周和12周,各组均随机取材5个桡骨标本,行大体观、X线检查、CT三维重建观察缺损愈合情况,随后行组织学切片HE、Masson染色,观察新生骨组织生长情况,并借助Image-Pro Plus v6.0对新生骨组织量化,进一步评价骨缺损修复情况。结果大体观、X线检查及CT三维重建结果显示TBC组6周时材料形状清楚,无明显骨痂生成,12周时材料有所降解,仍无明显骨痂形成,材料与宿主骨间无明显融合;TBC/MSN组6周和12周所见与TBC组相似;TBC/P28组6周时材料密度有所增高,材料与宿主骨间可见少量新生骨组织生成,12周时材料形状消失,表面有大量骨痂包裹,材料与宿主骨间有新生骨组织连接,缺损区基本消失;TBC/MSN/P28组6周时材料密度增高,表面有少量骨挪包裹,与宿主骨间有新生骨组织形成,12周时材料完全被骨痂包裹,与宿主骨间大量新生骨组织连接,融合良好,缺损区完全消失。组织学切片HE及Masson染色可见TBC组6周时无新生骨组织形成,12周时仅有极少量新生骨组织生产;TBC/MSN组所见与TBC组相似;TBC/P28组和TBC/MSN/P28组6周已有大量新生骨组织生成,明显多于TBC/MSN组和TBC组12周所见,12周时新生骨组织继续增多并充满材料孔隙内部,骨组织较6周时进一步成熟。TBC/P28组和TBC/MSN/P28组明显高于TBC/MSN组和TBC组,有显著统计学差异(p<0.05),6周时TBC/MSN/P28组与TBC/P28组无明显统计学差异(p>0.05),但12周时TBC/MSN/P28组高于TBC/P28组,有显著统计学差异(p<0.05)。结论TBC/MSN/P28复合材料能够缓慢释放具有骨诱导活性的生长因子P28,发挥稳定长效的成骨作用,是一种较为理想的骨组织工程修复材料。更多还原

【Abstract】 PartⅠThe assessment on proliferation and osteogenic differentiation abilities of MC3T3-E1 cells in vitro using BMP-2-related peptide P28Objective By comparing the effects of P28 and rhBMP-2 on the proliferation and osteogenic differentiation of MC3T3-E1 cells,to assess the osteogenic activity of P28.Methods The MC3T3-E1 cells were cultured in medium containing P28 or rhBMP-2,and the Control without any growth factor.Cell proliferation ability was measured by Calcein-AM staining after 1,3 and 5 days of culture,and MTT assay after 1,3,5 and 7 days of culture.ALP activity was measured by ALP staining and ALP kit after 7 and 14 days of culture.Calcium nodules were observed by Alizarin red staining after 14 and 21 days of culture.The osteogenic differentiation related genes of Runx2,OCN and Col I were measured by Real-Time PCR,and the osteogenic differentiation related proteins of Runx2,p-Smad 1/5/8,OCN and Col I were measured by Western blot after 1,3 and 7 days of culture.Results The Calcein-AM staining and MTT assay results showed that the MC3T3-E1 cells proliferation ability of P28 group and rhBMP-2 group were significantly higher than Control(P<0.05),and there was no significant difference between P28 group and rhBMP-2 group(P>0.05).The results of ALP activity and Alizarin red staining showed that the P28 had similar osteoinductive to rhBMP-2,and both of P28 group and rhBMP-2 group had significant difference between with Control(P<0.05).The Real-Time PCR and Western blot results also showed that P28 and rhBMP-2 played similar osteoinductive by regulating the Smad pathway.Conclusion P28 had similar osteoinductive to rhBMP-2 in promoting proliferation and osteogenic differentiation of MC3T3-E1 cells.Part ⅡA study on the preparation and physic-chemical property of true bone ceramics/mesoporous silica nanoparticles/P28,and its controlled release in vitroObjective To investigate the preparing method and physic-chemical property of true bone ceramics/mesoporous silica nanoparticles/P28,and evaluated its release kinetics in vitro.Methods The mesoporous silica nanoparticles(MSN)were prepared,and MSN were detected by transmission electron microscope(TEM),Brunauer-Emmett-Teller(BET)method and Barrett-Joyner-Halenda(BJH)mothod.According to the different ratio of MSN:P28 quality(1:1.5,1:2,1:2.5,1:3,1:3.5,1:4)to determine the optimum dosage ratio between P28 and MSN.True bone ceramics(TBC)and TBC/MSN scaffold materials were prepared,and the surface morphology and physic-chemical property of them were investigated by three-dimensional video microscope,scanning electron microscope(SEM)and energy dispersive spectrometer(EDS).TBC and TBC/MSN were loaded with 3 mg P28,and the release kinetics of TBC/P28 and TBC/MSN/P28 were measured and compared at 1,2,3,5,7,10,12,14,18,and 30 days.Results The particle size of MSN was mainly distributed in 180 ± 30 nm,the specific surface area was 702.7 m2/g,the pore size was about 4 nm,and the pore volume was about 0.8 cm3/g.Thus,MSN had good mesoporous structure and sufficient drug loading space.P28:MSN = 1:3 as a suitable dosage proportion of drug loading,encapsulation efficiency reached 81.88 ± 4.44%.The results of three-dimensional video microscope,SEM and EDS showed that MSNs were successfully attached to the interior of the TBC,and had a uniform distribution.Moreover,MSNs did not have a greater impact on the pore structure of TBC.The drug loading rate of P28 in TBC and TBC/MSN was about 80%and 98%respectively,and the release kinetics of P28 in TBC/MSN was slower than that in TBC(P<0.05).Conclusion MSN is a kind of satisfactory carrier material for P28,which can be combined satisfactory with TBC scaffold,and TBC/MSN had slower release of P28 in vitro.Part ⅢAdhesion,proliferation and osteogenic differentiation of MC3T3-E1 cells on the TBC/MSN loaded with P28Objective By comparing the adhesion,proliferation and osteogenic differentiation of MC3T3-E1 cells on TBC,TBC/MSN,TBC/P28 and TBC/MSN/P28 in vitro,to evaluate the ability of TBC/MSN/P28 to induce osteogenic differentiation.Methods TBC and TBC/MSN were loaded with 3 mg P28 respectively,and the study included four kinds of material:TBC,TBC/MSN,TBC/P28 and TBC/MSN/P28.The MC3T3-E1 cells were cultured on the TBC,TBC/MSN,TBC/P28 and TBC/MSN/P28 scaffold materials,and the adhesion rate of MC3T3-E1 cells were measured after 24 hours of culture.The cells were stained with Calcein-AM and PI after 3 days of culture,and the viability and distribution were observed by confocal laser scanning microscope.The proliferative ability was measured by MTT assay after 2,4,6 and 8 days of culture.The ALP activity was measured by ALP kit after 5,10 and 15 days of culture.Results The adhesion rate of TBC/P28 and TBC/MSN/P28 group was higher than the TBC and TBC/FMSN group(P<0.05),and there was no significant difference between TBCfP28 and TBC/MSN/P28 group(P>0.05).The confocal laser scanning microscope showed that the viability and distribution of TBC/P28 and TBC/MSN/P28 group were better than TBC and TBC/MSN group,and there were few dead cells in four groups.The MTT assay results showed that the proliferation ability of TBC/P28 and TBC/MSN/P28 group was higher than the TBC and TBC/MSN group in the whole culture period(P<0.05),and TBC/MSN/P28 group was higher than TBC/P28 group after 6 and 8 days of culture(P<0.05).The results of ALP activity showed that TBC/P28 and TBC/MSN/P28 group was higher than the TBC and TBC/MSN group in the whole culture period(P<0.05),and TBC/MSN/P28 group was higher than TBC/P28 group after 15 days of culture(P<0.05).Conclusion The four materials had good biocompatibility,and the adhesion,proliferation and osteogenic differentiation abilities of TBC/P28 and TBC/MSN/P28 were better than TBC and TBC/MSN.The sustained release properties of TBC/MSN/P28 can promote the P28 to play biological activity.PartⅣRepair of rabbit radial critical bone defect using TBC/MSN loaded with P28Objective To evaluate the ability of TBC/MSN/P28 to repair the radial critical bone defect in rabbits,and explored the ability of the composite material to repair large bone defects in vivo.Methods 20 New Zealand rabbits were randomly divided into four groups:TBC group,TBC/MSN group,TBC/P28 group and TBC/MSN/P28 group.A 15mm length critical bone defect was prepared in bilateral radius of rabbits,and the corresponding material was filled in the defect.After 6 and 12 weeks of operation,to evaluate the bone defect repair of four groups by the X ray,three-dimensional CT(3D-CT)and histological examination(HE and Masson staining).Results The results of gross observation,X ray and 3D-CT showed that there was no obvious new bone formation in TBC group after 6 and 12 weeks of operation.The findings of TBC/MSN group were similar to TBC group.In TBC/P28 and TBC/MSN/P28 group,there was small amount of callus covered the surface of the material at 6 weeks after operation.At 12 weeks,there was a large number of callus covered the surface of the TBC/P28 and TBC/MSN/P28 materials,and the materials were firmly connected with the host bone.In addition,the material had a certain degree of degradation.The results of histological examination showed that TBC group had no obvious new bone tissue at 6 weeks,and only a small amount of new bone tissue was observed at 12 weeks.The findings of TBC/MSN group were similar to TBC group.At 6 weeks,there were a large number of new bone tissues in TBC/P28 and TBC/MSN/P28 group,and the area of new bone tissue was significantly more than that of TBC and TBC/MSN groups at 6 and 12 weeks(P<0.05).At 12 weeks,the new bone tissues of TBC/P28 and TBC/MSN/P28 group were further increased,and further matured into lamellar bone.And the area of new bone tissue in TBC/MSN/P28 group was more than that in TBC/P28 group(P<0.05).Conclusion TBC/MSN/P28 could sustain release the BMP-2-related peptide P28 which has the ability of osteoinductive,and it is an ideal repair complex material for bone tissue engineering.更多还原