【作者】 王婷；

【导师】 郭澍；

【作者基本信息】 中国医科大学 ， 整形外科学， 2018， 博士

【摘要】 目的:口腔颌面位于人体的显露部位,具有特殊的解剖结构和功能,颌面部骨骼遭受损伤后对容貌和发音、进食、咀嚼等功能都有较大影响,因此在颌面部骨缺损的临床治疗中,恢复面部正常解剖结构、功能及外观一直是口腔颌面外科及整形外科医师面临的一大难题。组织工程骨具有来源渠道多样、低免疫原性、可根据缺损组织的大小形态进行设计和具有生命力等优点,应用骨组织工程技术治疗骨缺损还可以克服自体骨移植供体不足和异体骨移植免疫排斥等缺点。近年来,随着科学技术的进步,骨组织工程研究取得了极大的飞跃,将组织工程三要素种子细胞、支架材料和生长因子联合起来制备的活性人工骨成为克服骨缺损修复难题中的关键点和研究热点。本研究制备可控释BMP-2和VEGF的nHAC/PLGAs三维多孔支架,并与ADSCs复合,检测该活性人工骨的体内及体外成骨活性,并初步探讨BMP-2和VEGF共同作用增强ADSCs成骨分化的相关分子机制,更切实地模拟骨缺损修复的微环境,以期为组织工程骨支架的设计、骨缺损修复治疗的创新和相关机制研究的深化提供理论和实验依据。实验方法:1、提取鼠尾胶原,制备负载BMP-2及VEGF的PLGA微球,检测BMP-2及VEGF的释放动力学特性和活性。2、制备nHAC/PLGAs三维多孔支架,检测支架的孔径、孔隙率、吸水率、力学特性和降解特性。3、将hADSCs接种于nHAC/PLGAs支架内部,检测其增殖活性,观察细胞在支架表面的生长状况。4、在BMP-2与VEGF单独及共同作用下,检测hADSCs于nHAC/PLGAs支架内部对成骨标志物col1的表达、ALP活性和矿化结节形成情况,以及对成骨转录因子RUNX-2、Dlx-5、osterix的表达情况,并检测成骨相关信号通路BMP/Smad、PI3K/Akt、p38 MAPK的激活情况。5、检测在Akt抑制剂LY294002、p38抑制剂SB203580或携带osterix shRNA的慢病毒的处理下,各组hADSCs成骨相关指标的表达情况。6、分离提取兔ADSCs,检测其增殖活性、表面分子标记表达情况及成脂、成骨和成软骨分化的能力。7、制备兔下颌骨临界骨缺损模型,并移植复合rADSCs的nHAC/PLGAs-BMP-2/VEGF支架,于不同时间点对缺损处进行三维CT和X光检查,并对下颌骨标本进行组织学染色观察。结果:1、PLGAs微球中BMP-2和VEGF的包封率分别为68.94%±2.00%和53.94%±3.29%。前7天为突释期,BMP-2的释放量约为包封总量的58.94%,VEGF约为55.17%;第8至17天为匀速释放期,约28.74%的BMP-2和27.95%的VEGF释放;后11天为慢速释放期,仅有6.19%的BMP-2和9.85%的VEGF释放。所释放的BMP-2增强了rat BMSCs的ALP活性,VEGF增强了Eahy926细胞的增殖活性。2、nHAC/PLGAs支架为三维多孔结构,孔径大小从数十μm到近300μm不等,PLGA微球直径约为9.95μm。支架的孔隙率为79.46%,吸水率为561.51%。支架的弹性模量为0.72±0.17 MPa,抗压强度为2.85±0.49 MPa。在体外降解过程中,支架在最初2周降解迅速,之后降解速度略减慢;支架的三维多孔结构约维持28天,至第56天基本完全坍缩,PLGA微球在六周内基本完全降解。3、hADSCs在支架上的增殖活性随时间呈上升趋势,SEM观察细胞在支架表面表现出正常的形态和生物学行为。4、VEGF组hADSCs的增殖活性增强,col1表达、ALP活性和矿化结节形成及RUNX-2、Dlx-5和osterix的表达无明显变化;BMP-2组hADSCs增殖活性无明显变化,但col1表达、ALP活性和矿化结节形成及RUNX-2、Dlx-5和osterix的表达均明显增强;除RUNX-2的表达无明显差异外,BMP-2/VEGF组各指标表达较BMP-2组进一步增强。5、VEGF组pAkt和p-p38表达增强,pSamd表达无变化;BMP-2组pSamd和p-p38表达增强,pAkt表达无变化;BMP-2/VEGF组pAkt和p-p38的表达较两者单独作用时增强。6、LY294002对各组col 1蛋白表达和矿化结节无明显影响;SB203580显著抑制了col 1蛋白的表达和矿化结节的形成,而对Dlx-5及osterix蛋白表达无显著影响,但可使osterix蛋白核/浆分布比例明显下降。osterix稳定敲减后各组col 1表达明显减弱,几乎无矿化结节形成。7、rADSCs增殖迅速,表达基质干细胞标志物CD90,CD44和CD29;成脂诱导14天后,胞内可见多个脂滴;成骨诱导21天后,细胞外基质可见多个钙结节。成软骨诱导28天后,细胞分泌大量硫酸黏蛋白,可被Alcian染色液染为淡蓝色。8、在兔下颌骨临界骨缺损修复过程中,影像学提示空白对照组骨缺损腔未见缩小,VEGF组的骨缺损腔直至术后12周才见少量新生骨;BMP-2和BMP-2/VEGF组自第4周起便可见明显新生骨质,至术后12周可达完全修复,但BMP-2组新生骨的数量和修复速度均不及较BMP-2/VEGF组。大体标本观察提示:空白对照组骨缺损腔内仅有肉芽组织填充,VEGF组骨缺损腔内支架逐渐降解,为纤维结缔组织替代,至12周可见少量薄层新生骨痂;BMP-2和BMP-2/VEGF组骨缺损腔随时间推移变窄,支架体积逐渐缩小,与周围组织紧密融合,可见大量新生骨痂长入材料内部,至术后12周BMP-2/VEGF组原骨缺损区位置难辨,骨痂改建良好。组织学染色观察提示:术后4周,对照组骨缺损腔为大量纤维素和红细胞填充,VEGF组支架内可见大量红细胞,至术后12周支架几乎消失,缺损腔为大量纤维结缔组织和少许新生骨和血管填充。术后4周,BMP-2和BMP-2/VEGF组的支架边缘与缺损腔边缘略融合,可见新生骨质和新生血管,至术后12周支架基本完全降解,由大量新生骨质及新生血管取代,BMP-2/VEGF组以板层骨和骨细胞形成规则的骨小梁为主。结论:1、应用改良的复乳化法可成功制备PLGA微球,且可实现其对BMP-2及VEGF的控释,并可保持这两种生长因子的活性。2、nHAC/PLGAs支架材料具有良好的物理性能,其降解特性可初步满足骨缺损修复的要求,还具有良好的生物相容性和低毒性。3、体外实验中,VEGF对hADSCs在nHAC/PLGAs支架内的增殖有明显促进作用,BMP-2对其成骨分化有明显促进作用;VEGF与BMP-2共同作用的骨诱导效应更佳,这可能主要是通过p38 MAPK通路的激活从而引起osterix蛋白核内转位增加导致的。4、缓释VEGF和/或BMP-2的nHAC/PLGAs支架材料负载rADSCs所形成的活性人工骨在体内具有不同程度的成骨效应,VEGF和BMP-2同时应用对兔下颌骨临界骨缺损处的骨化及血管化具有极大的促进作用。更多还原

【Abstract】 Objective:Because of the exposed location and special anatomical structure and function,damages to oral and maxillofacial bones severely disrupt appearance,pronunciation,ingress,chewing and other functions.Therefore,repairing maxillofacial bone defect and restoring facial anatomical structure,function as well as appearance have been a conundrum for maxillofacial surgeon and orthopedics.Tissue engineereing bone takes advantages of unlimited source,low antigenicity,pre-controlled shape and potent vitality over autografts and allografts.Preparing tissue engineering artificial bone by integrating seeding cells,scaffold materials and growth factors has achieved great progress to realize bone defect repairing.The present study prepared VEGF/BMP-2sustainably releasing-nHAC/PLGAs scaffolds and inoculated ADSCs to detect osteogenic activity of this active artificial bone in vitro and in vivo,and further investigated the molecular mechanism of crosstalk between BMP-2 and VEGF enhancing ADSCs osteodifferentiation,which more realisticly simulated the microenvironment of bone defect repair.Hoping to provide theoretical and experimental basis for designing tissue engineering scaffolds,innovating bone defect repairing treatments and deepening related mechanism research.Methods:1.Rat tail collagen was extracted and BMP-2/VEGF loaded PLGA microspheres were prepared to detect the release characteristics and activity of BMP-2and VEGF from microspheres.2.Three-dimensional porous nHAC/PLGAs scaffold was prepared to detect its pore size,porosity,water absorption,mechanical properties and degradation characteristics.3.hADSCs were inoculated into the nHAC/PLGAs scaffold to detect their proliferative activity and observe their morphology on the scaffold surface.4.hADSCs were inoculated into the BMP-2 and VEGF respectively or simultaneously releasing-nHAC/PLGAs scaffold to detect the expression of osteogenic indicators col 1,ALP activity and mineralized nodule formation,and osteogenic differentiation factors RUNX-2,Dlx-5 and osterix,and phosphoration of signaling pathway related protein Smad,Akt and p38.5.hADSCs were inoculated in the BMP-2 and VEGF respectively or simultaneously releasing-nHAC/PLGAs scaffold under the treatment of Akt inhibitor LY294002,p38 inhibitor SB203580 or lentivirus containing osterix shRNA to detect the expression of osteogenic indicators.6.Rabbit ADSCs were isolated and cultured to detect their proliferative characteristics,surface molecular markers expression and multi-differentiative ability.7.Rabbit mandibular critical bone defect model was prepared and rADSCs loaded nHAC/PLGAs-BMP-2/VEGF scaffold were implanted,then mageological examination,general observation and histological observation were conducted at 4^th,8^thh and 12^thh week post-operatively.Results:1.The encapsulation efficiency of BMP-2 and VEGF in PLGAs microspheres was 68.94%±2.00%and 53.94%±3.29%respectively.The amount of BMP-2 and VEGF released during the first 7 days was about 58.94%and 55.17%of the total encapsulation amount respectively,that was 28.74%and 27.95%respectively during 8^thto 17^thday,and that was only 6.19%and 9.85%respectively during the next 11 days.The released BMP-2 enhanced the ALP activity of rat BMSCs,and released VEGF promoted the proliferation of Eahy926 cells.2.The nHAC/PLGAs scaffold occupied a three-dimensional porous structure with a diameter ranging from tens ofμm to nearly300μm,and the diameter of PLGA microspheres was about 9.95μm.The porosity of the scaffold was 79.46%and the water absorption rate was 561.51%.The elasticity modulus of the scaffold was 0.72±0.17 MPa,and compressive strength was 2.85±0.49 MPa.In vitro degradation process,the degradation rate of the scaffold was rapid in the first 2weeks,then slightly slowed down;the three-dimensional porous structure of scaffold maintained within 28 days,then completely collapsed within 56 days;PLGA microspheres completely degraded at 6^thh week.3.Cellular proliferative activity of hADSCs in the scaffold increased with time and was similar to that of control group.hADSCs showed normal morphology and biological behavior on the scaffold surface under SEM observation.4.VEGF alone can promote hADSCs proliferation,but had no significant effects on collgen I expression,ALP activity and mineralized nodule formation,and RUNX-2,Dlx-5 and osterix expression;BMP-2 alone has no obvious effect on the proliferation of hADSCs,but increased collgen I expression,ALP activity and mineralized nodule formation,and RUNX-2,Dlx-5 and osterix expression;Except for RUNX-2,combination of BMP-2 and VEGF further enhanced the expression of osteogenic indicators.5.VEGF alone increased expression of pAkt and p-p38,but had no effect on pSamd.BMP-2 alone increased expression of pSamd and p-p38,but had no effect on pAkt.Enhancement of simultaneous BMP-2 and VEGF treatment on the pAkt and p-p38 expression was higher than that of BMP-2 or VEGF treatment alone.6.LY294002 treatment had no significant effect on collgen I expression and mineralized nodules formation of hADSCs.SB203580 treatment significantly inhibited collgen I expression and mineralization nodules formation,had no significant effect on the expression of Dlx-5 and osterix,but decreased nucleus/cytoplasm ratio of osterix.Collgen I expression and mineralized nodules formation in each group remarkably weakened after stable osterix knockout.7.rADSCs proliferated rapidly and expressed stromal stem cell markers CD90,CD44 and CD29.Multiple lipid droplets were observed in the enlarged cell after adipogenic induction,and numberous calcium nodules were observed in the extracellular matrix after osteogenic induction.rADSCs secreted a large number of sulphate mucoprotein that can be stained light blue by Alcian dye after chondrogenic induction.8.In the repairing process of rabbit mandibular critical bone defect,mageological examination indicated no narrowing of bone defect cavity of control group,only a small amount of new bone were observed in defect cavity of VEGF group until 12^thweek after operation;Obvious new bone formation was observed in BMP-2 and BMP-2/VEGF group at 4^thh week after operation,and the bone defect healed completely at 12^thh week,but the amount of new bone in BMP-2 group were less and the healing rate was slower than that of BMP-2/VEGF group.General observation of the samples indicated bone defect cavity of control group was filled with granulation tissue,scaffold of VEGF group degraded gradually and was replaced with fibrous connective tissue,a few thin callus in bone defect cavity were observed until 12^thweek after operation;Bone defect cavity of BMP-2 and BMP-2/VEGF group narrowed along with time,the scaffolds gradually degraded and infiltrated tightly into the surrounding tissues,a large amount of new bone callus grew into the material,and callus of BMP-2/VEGF group remodeled well to hardly distinguish the original defect location at 12^thh week after operation.Histological observation indicated bone defect cavity of control group was filled with cellulose and erythrocytes,numerous erythrocytes grew into the scaffold of VEGF group at 4^thh week after operation,the scaffold almost disappeared and was replaced with a large number of fibrous connective tissue,a few new bone and blood vessels at 12^thh week after operation.At 4^thh week after operation,scaffolds of BMP-2 and BMP-2/VEGF group infiltrated into the edge of defect cavity,new bone and blood vessels can be observed in the“fusion area”.At 12^thh week after operation,scaffolds of BMP-2 and BMP-2/VEGF group almost completely degraded and were replaced with a large number of new bone and blood vessels,and lamellar bone and regular bone trabecula dominated in BMP-2/VEGF group.Conclusion:1.PLGA microspheres can be successfully prepared by the modified double emulsification method to achieve sustainable release of BMP-2 and VEGF and maintain their activity.2.nHAC/PLGAs scaffold possessed excellent physical properties,favorable degradation characteristics,satisfactory biocompatibility,low toxicity and potent osteoinductive effect.3.In vitro experiments,VEGF alone significantly promoted hADSCs proliferation,BMP-2 alone significantly promoted hADSCs osteogenic differentiation in nHAC/PLGAs scaffold,osteoinductive effect of simultaneous VEGF and BMP-2 treatment further enhanced,which may be mainly through increasing nuclear translocation of osterix protein by activation of the p38 MAPK pathway.4.In vivo experiments,rADSCs loaded BMP-2/VEGF sustainably releasing-nHAC/PLGAs scaffold displayed different osteogenic effect,simutaneous application of VEGF and BMP-2 can greatly promote ossification and vascularization in rabbit mandibular critical bone defect.更多还原