【作者】 王林；

【导师】 周延民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2011， 博士

【摘要】 种植义齿被誉为人类的“第三副牙齿”,他有着传统义齿不可代替的优点,但不是所有病例都是种植义齿修复的适应症,现在存在的最大问题就是骨量不足——局部或大范围的骨缺损。造成骨缺损的原因很多,可以是牙槽嵴的废用性萎缩,也可以是外伤引起的。无论哪种情况都可能使种植体无法很好的存留于牙槽骨内并获得良好的初期稳定性。植骨技术的应用解决了这一难题,但是自体骨源要开辟第二术区,且创伤大患者难以接受,如何得到理想的人工骨支架材料是目前研究的热点。骨支架材料是新生骨组织生长的支架,在骨组织工程中占有重要地位。但是高分子支架材料生物活性较低,力学性能较差,容易引发无菌性炎症;而无机支架材料的生物活性高,力学性能也相对较好,但是如何促进骨细胞迅速的长入支架材料是一个难题。肾上腺髓质素是日本学者从嗜铬细胞瘤中分离出的一种具有广泛生物学效应的肽类物质。他是成骨细胞有效的有丝分裂原,同时又在血管发生中起到关键作用,还参与体内许多其他功能。关于ADM在骨代谢中的研究,国内外尚无定论,有学者认为ADM在体内、体外都对成骨细胞、软骨细胞具有促有丝分裂作用,在体内还具有抑制骨吸收作用,目前尚未发现对破骨细胞有直接影响。同时它是一种新的内皮细胞生长因子,起到促血管生成的作用,是一种有效的血管生成因子。ADM可刺激小鼠后肢缺血模型中肢体血流量的恢复,增加的血流量反映了增加的继发毛细血管密度。因而,在软硬组织的重建中,ADM的促进血管生成特性应发挥了重要作用。将ADM和人工骨支架材料复合后是否会使人工骨支架材料既具有促进骨形成作用又有血管化活性国内外尚无研究。本研究从体外实验的角度探讨了载ADM的PLGA/纳米羟基磷灰石支架材料对成骨样细胞MG63、HUVEC的增殖、分化的影响,Real time PCR探讨支架材料本身及缓释的ADM对成骨及血管化相关基因的影响,Western Blot探讨他们对CollagenⅠ、Runx2、VEGF蛋白表达的影响,为其应用于临床增加骨量提供理论依据。1.不同浓度肾上腺髓质素对MG63、HUVEC增殖及分化的影响目的:采用人成骨样细胞MG63作为成骨细胞模型,HUVEC作为血管内皮细胞模型,评价ADM对其生物学特性的影响。方法:96孔板中成骨样细胞MG63细胞接种于含10%胎牛血清的高糖DMEM中,HUVEC细胞接种于含10%胎牛血清的IMDM中。加入梯度浓度的药物1μL,终浓度为10^-13mol·L^-1-10^-7mol·L^-1对照组加入等量的PBS,在加药后1、2、3、4、5天终止。培养通过MTT比色法检测ADM对MG63、HUVEC细胞的增殖活性的影响、通过ALP活性测试检测ADM对MG63细胞的分化活性的影响,找出对两种细胞增殖分化最好的浓度。结果:对于MG63细胞10^-9M和10^-8M浓度的ADM的促增殖能力最强,而对于HUVEC来说,10^-8M ADM促增殖能力最强。我们选择4个高浓度的ADM对MG63的ALP表达进行检测,10^-8M、10^-7M ADM促成骨细胞分化能力最强。在后续的工作中我们选择10^-8 M作为ADM的最优工作浓度。2.载肾上腺髓质素微球复合PLGA/纳米羟基磷灰石支架材料的制备、表征及其降解研究目的:探讨ADM载入壳聚糖微球的可能性,并且制备包裹微球的支架材料,探讨微球与支架材料的表征、机械性能及其体内外降解的能力。方法:以TPP为交联剂采用离子乳化交联法制备壳聚糖微球。并在此过程中将ADM载入其中。应用热致相分离法制备PLGA/nHA支架材料并将载药微球包裹其中。通过扫描电镜表征材料表面性能,动物体内及体外12周的监测材料的降解,通过压缩试验测试材料的机械性能,来判断微球的载入是否给材料本身的性能造成了影响。结果:微球直径均匀,成正态分布,平均直径为42.69μm。利用热致相分离法制备PLGA/nHA支架材料具有典型的多聚物孔状结构,孔径约在50-220μm,且孔与孔之间相互穿通联接。HPLC测定后计算出药物的载药率约为0.058%,包封率79.4%。载药微球前三天突释达到50.7%,从第四天开始缓慢释放,并在第十天达到68.3%。PLGA/nHA/CMs支架材料第1周ADM的释放达到21.0%,2-4周呈线性缓慢释放,第4周末累计释放量达到43.7%,到12周的累计释放量达到51.8%。空白支架材料的孔隙率达到90.8%,而载入30%壳聚糖微球后的孔隙率也可以达到88.9%,两组数据未见明显的统计学差异（P>0.05）。但是空白支架材料的密度（0.045±0.017 ）g/mL却显著的低于载30%微球的支架材料的密度（0.083±0.020）g/mL（P<0.05）。PLGA/nHA/CMs支架材料在前三周代谢稍快,以后达到线性代谢模式,在12周末代谢达到12.23%,而PLGA/nHA空白支架材料到12周末的失重率为8.27%。12周末PLGA/nHA/CMs支架材料组的PBS的PH为6.88±0.12,而PLGA/nHA空白支架材料为6.49±0.09。吸水率方面:PLGA/nHA/CMs支架材料在第一周吸水率即达到66.9%,且逐渐缓慢上升,到第6周达到82.15%后趋于稳定,在12周时达到88.34%。体内降解的失重率:PLGA/nHA/CMs支架材料在前4周代谢较快,第四周末失重率达到10.7%。4-8周趋于平缓,8-12周又处于上升期,到12周时达到21.4%。而PLGA/nHA空白支架材料开始降解较慢,到第8周达到10%,后成直线上升趋势,在12周时达到20.1%。这个数据和12周末PLGA/nHA/CMs空白支架材料的失重率21.4%是基本一致的。压缩试验测试结果显示载入30%微球后材料的抗压强度得到了显著的提升,PLGA/nHA/CMs支架材料的抗压强度为（1.54±0.20）MPa, PLGA/nHA/CMs支架材料抗压模量为（7.24±0.42）MPa。3.载药复合支架材料对MG63、HUVEC增殖及分化的影响目的:采用人成骨样细胞MG63作为成骨细胞模型,HUVEC作为血管内皮细胞模型,评价载药支架材料及空白支架材料对其生物学特性的影响。方法:MG63、HUVEC常规培养,接种于细胞培养板,以PLGA/nHA/CMs/ADM为实验组,PLGA/nHA/CMs为对照组,空细胞为空白对照组。通过材料溶血行为,ALP活性的测定,支架材料表面细胞荧光染色,MTT等检测手段综合评价载药支架材料的溶血性能及生物活性。结果:复合空白微球的支架材料溶血率为0.171%,远远小于5%,接近阴性对照。说明本研究中复合微球的支架材料是安全的,无急性溶血活性。载药支架材料以及支架材料本身对成骨细胞以及血管内皮细胞的增殖以及成骨细胞的分化均有一定的促进作用。4.载ADM复合支架材料对MG63、HUVEC相关功能基因及蛋白的影响目的:采用人成骨样细胞MG63作为成骨细胞模型,HUVEC作为血管内皮细胞模型,探讨载药支架材料对两种细胞内成骨及血管化相关基因和蛋白表达的影响,方法:MG63、HUVEC常规培养,接种于材料表面,以PLGA/nHA/CMs/ADM为实验组,PLGA/nHA/CMs为对照组,空细胞为空白对照组。分别于1、3、5天终止培养,消化细胞,提取RNA和蛋白。用Real time PCR和Western Blot的方法检测COL1α1,Runx2,SP7,OPN,VEGF,RAMP2基因的表达及VEGF、CollagenⅠ、RUNX2蛋白的表达水平。结果:Real-time PCR结果显示实验组MG63细胞内Runx2 mRNA的表达在第1天略高于对照组,显著的高于空白对照组;第3天,实验组、对照组Runx2表达水平均显著上调;第5天三组mRNA水平均有所下调,实验组mRNA水平略高于空白对照组。而第1天实验组SP7 mRNA表达水平略低于对照组,和空白对照组基本处于同一水平,第3天,实验组SP7表达显著上调,高于空白支架组和空白对照组;第5天时实验组的SP7表达更高,是对照组的2.7倍,是空白对照组的4.68倍。且对照组显著的高于空白对照组,是其1.73倍。对照组OPN mRNA表达水平在第1天就显著增高,第3天趋势与第1天相似,第5天三组未见明显的区别。实验组MG63细胞内COL1α1 mRNA的表达在第3天和第5天显著的高于对照组和空白对照组,空白对照组和对照组的COL1α1 mRNA一直处于同一水平。实验组材料表面HUVEC细胞内RAMP2基因的表达在第1天略低于空白对照组但略高于空白支架组。第3天,实验组RAMP2基因的表达量显著增高,是空白支架组的2.3倍,是空白对照组的2.4倍;第5天,载药支架组RAMP2基因的表达量是空白支架组的2.06倍,是空白对照组的2.57倍。而VEGF基因的表达在第1、3、5天均显著的高于对照组和空白对照组,第1天VEGF基因表达量是对照组的1.78倍,是空白对照组的1.68倍。第3天VEGF基因表达量是对照组的1.55倍,是空白对照组的2.1倍。第五天VEGF基因表达量是对照组的1.88倍,是空白对照组的2.32倍。对照组和空白对照组在第1天未见显著差别,第3、5天对照组VEGF基因表达量略低于空白对照组。Western Blot检测第1天实验组Runx2的蛋白表达量显著高于空白对照组和对照组。第3天,实验组Runx2的蛋白表达量显著高于空白对照组,而对照组Runx2的蛋白表达量显著低于空白对照组。第5天,对照组和实验组的Runx2的蛋白表达量无显著性差异,而空白对照组Runx2的蛋白表达量显著低于对照组和实验组。Western Blot显示第1天,实验组和对照组的CollagenⅠ的蛋白表达量显著的高于空白对照组。第3天,实验组CollagenⅠ的蛋白表达量与空白对照组无明显差异,同mRNA水平的变化基本一致。Western Blot检测VEGF的蛋白表达量的结果显示第1天,实验组和对照组VEGF的蛋白表达量显著高于空白对照组。第3天,三组间未见显著性差异,第5天,实验组VEGF蛋白表达量显著高于对照组与空白对照组。综上所述,ADM对成骨细胞、血管内皮细胞有着显著的促增殖和分化作用,制备的ADM-壳聚糖微球载入PLGA/nHA支架材料对成骨细胞、血管内皮细胞也有着促增殖和分化作用,并在早期可以促进成骨及血管化相关基因及蛋白的表达,本研究为缓释ADM的支架材料应用于口腔种植领域提供了理论基础。更多还原

【Abstract】 Implant denture is regarded as“the third set of teeth”and has more irreplaceable advantages than traditional dental prosthesis. Not all the dentition defect cases suitable for implant restoration since some of them have the big problem of bone defect. There are many reasons that can cause bone defect, such as disuse atrophy of alveolar bone or external injury. Any of the factors can affect the initial stability of the implant by influencing the osteointegration. This problem can be solved by bone graft technical. But, to obtain the autogenous bone, we need to develop a second operation area, which patients can hardly accept since the extra trauma. At present, the researchers are focusing on gaining ideal synthetic bone scaffold materials, which provide support for the growth of new bone and occupy an important position in bone tissue engineering. Polymer scaffold materials have a lower biological activity and mechanical properties than inorganic scaffold materials and easy to cause aseptic inflammation. But inorganic scaffold materials also have a problem of how to promote bone cells to grow into them.Adrenomedullin （ADM） is a potent peptide with extensive biological effects that was originally isolated from pheochromocytoma by Japanese scholars. It is an effective mitogen of osteoblast, and also has a key role in vasculogenesis and takes part in many other functions in vivo. The role of ADM in bone metabolism is uncertain and some researchers considered that ADM has the mitosis promotion role on osteoblast and cartilage cell both in vitro and in vivo. Also, ADM has the role of inhibiting bone resorption in vivo, while its direct influence on osteoclast hasn’t yet found. As a new found endothelial cell growth factor, ADM can promote vasculogenesis. ADM can stimulate the recovery of the hindlimb blood flow after ischemia-reperfusion in mice, which reflect the augmentation of secondary capillary density. So, ADM plays a significant role by promoting vasculogenesis in the reconstruction of soft and hard tissue.Whether the synthetic bone scaffold materials have both promoting bone formation effect and vasculogenesis activity after combine with ADM is uncertain from the report of domestic and international researchers.In our research we evaluated the effect of PLGA/nano-hydroxyapatite scaffold containing chitosan microspheres for controlled delivery of ADM on the proliferation and differentiation of MG-63 and HUVEC cells in vitro. We used real time PCR to detect the characteristic of the synthetic scaffold and the influence of ADM on bone formation and vasculogenesis related genes. And then, their influence on Collagen I, Runx2 and VEGF protein was detected by Western Blot. Our results provide theoretical basis for clinical application in the future.1. The influence of different density ADM on the proliferation and differentiation of MG63 and HUVECObjective: Osteoblast-like cells MG63 was used as the model of human osteoblast and HUVEC was used as the model of vascular endothelial cell to evaluate the influence of ADM on their biological characteristic.Methods: MG63 and HUVEC cells were cultured in high goucose DMEM and IMDM respectively in 96 well plates and both medium contain 10% fetal bovine serum. Put 1μL gradient concentration ADM in and make the final concentration to 10^-13mol·L^-1-10^-7mol·L^-1. Put equal amount of PBS in the control group and check them in the 1^st day, 2nd day, 3^rd day, 4th day and 5^th day. To find the optimal density of ADM that is conducive to the proliferation and differentiation of MG63 and HUVEC, we detected the proliferation activity of MG63 and HUVEC cells by MTT and the differentiation activity of MG63 cells by ALP activity test.Results: ADM has the strongest ability to promote MG63 proliferation when the density is 10^-9M and 10^-8M while in HUVEC the density is 10^-8M. In the test of ALP activity, the strongest promotion ability on the differentiation activity of MG63 cells was appeared when the density of ADM was 10^-8M and 10^-7M. Thus, we will choose 10^-8 M as the optimal ADM density in our following study.2. The preparation, characterization and degradation of PLGA/nano-hydroxyapatite scaffold containing chitosan microspheres for controlled delivery of mutifucational peptide–adrenomedullinObjective: To investigate the possibility of adrenomedullin loaded into chitosan microspheres and prepare the scaffold that containing microspheres. Study the characterization, mechanical function and degradation ability of the microspheres and the scaffold in vitro and in vivo.Methods: In the presence of tripolyphosphate （TPP）, chitosan microspheres （CMs） loaded with adrenomedullin （ADM） were prepared by an emulsion-ionic cross-linking method. Then, PLGA/ nano-hydroxyapatite scaffold that containing microspheres was developed by thermally induced phase separation. Scanning electron microscope was used to analyze the characterization of the microspheres and scaffold. The material degradation have been tested within 12 weeks in vitro and in vivo. To study whether there has any changes in the nature performance of the material after loaded with microspheres, we detected the mechanical function by compress experiment.Results: The diameter of the microspheres was well-distributed and has the Gaussian distribution with the average diameter of 42.69μm. PLGA/nHA scaffold material has the typical polymerizer poriform structure with the pore size of 50-220μm and the pores were interconnected. The drug-loading rate was 0.058% and the encapsulation efficiency was 79.4% after calculated by HPLC determination. The initial burst release of microspheres loading with adrenomedullin in the first three days was 50.7% and slowed down in the forth day, then reached 68.3% in the 10th day. The release of ADM from PLGA/nHA/CMs scaffold material was 21.0% in the first week and shown a linearity slow release in 2-4 weeks, then reach the total release amount of 51.8% in the 12th week. The interval porosity of the blank scaffold material was 90.8% and the interval porosity after loaded 30% chitosan microspheres can also reach 88.9%. The density of the scaffold with no microsphere was （0.045±0.017）g/mL, which was significantly lower than the density of scaffold containing 30% chitosan microspheres （0.083±0.020）g/mL （P<0.05）. The degradation of PLGA/nHA/CMs scaffold material was slightly faster in the first three weeks and then reaches a linearity mode by in vitro weight loss testing. The degradation of PLGA/nHA/CMs scaffold material was 12.23% at the end of 12 weeks while the weight loss of PLGA/nHA scaffold material was 8.27%. The PH of incubation buffer for PLGA/nHA/CMs scaffold material was 6.88±0.12 and for PLGA/nHA scaffold material was 6.49±0.09 at the end of 12 weeks. As for the absorption rate, the result shown that the rate of PLGA/nHA/CMs scaffold material was 66.9% in the first week and gradually rises till stable in 82.15% in the 6th week and reach 88.34% in the 12th week. The weight loss rate of PLGA/nHA/CMs scaffold material after degradation in vivo was faster in the first four weeks and reach 10.7% at the end of the forth week. The rate was tended to be mild in 4-8 weeks and shown a rise period in 8-12 weeks, and then reached 21.4% in the 12th week. The compress experiment shown the compressive strength of the scaffold loaded 30% chitosan microspheres have been improved significantly. The compressive strength of PLGA/nHA/CMs scaffold material was （1.54±0.20）MPa and the compressive modulus was rised to （7.24±0.42）MPa. 3. The influence of drug-loading scaffold material on the proliferation and differentiation of MG-63 and HUVEC cellsObjective: Osteoblast-like cells MG63 was used as the model of osteoblast and use HUVEC as the model of vascular endothelial cell to evaluate the influence of drug-loading scaffold material and blank scaffold material on their biological characteristic.Methods: MG63 and HUVEC cells were conventional cultured and put into cell culture plate. PLGA/nHA/CMs/ADM was the experimental group, PLGA/nHA/CMs were the control group and the cells were the blank group. Evaluate the hemolysis characteristic and the biological activity of the drug-loading scaffold material by hemolysis test, ALP activity test, fluorescein stain of scaffold surface cell and MTT.Results: The hemolysis rate of the PLGA/nHA/CMs was 0.171% and far less than 5%, which close to the blank group. The results shown that the PLGA/nHA/CMs scaffold was safe to be used and had no acute hemolysis activity. The drug-loading scaffold and scaffold itself could accelerate the proliferation of osteoblast-like cells and vascular endothelial cells and the differentiation of osteoblast-like cells4. The influence of ADM-loading scaffold material on the related functional gene and protein of MG63 and HUVEC cellsObjective: an Osteoblast-like cell MG63 was used as the model of osteoblast and use HUVEC as the model of vascular endothelial cell to evaluate the influence of drug-loading scaffold material on their bone formation and vascularize related gene and protein expression.Methods: MG63 and HUVEC cells were conventional cultured and put onto the surface of the scaffold material. PLGA/nHA/CMs/ADM was the experimental group, PLGA/nHA/CMs were the control group and the cells were the blank group. Extract RNA and protein after cellula digestivum in the 1^st day, 3^rd day and 5^th day. Detect the expression of COL1α1, Runx2, SP7, OPN, VEGF and RAMP2 gene and the expression of VEGF, Collagen I and RUNX2 protein.Results: The results of Real-time PCR showed that the expression of Runx2 mRNA in MG63 cells in experimental group was slightly higher than the control group and significantly higher than the blank group in the 1^st day. The expression of Runx2 mRNA was significantly up-regulated in the experimental group and the control group in the 3^rd day. The mRNA expression of the three groups was all down-regulated in the 5^th day and was slightly higher in the experimental group than in the blank group. The expression of SP7 mRNA in the experimental group was a little lower than the control group and at the same level of the blank group in the 1^st day. While in the 3^rd day, the SP7 mRNA expression was significantly up-regulated in the experimental group and higher than the other two groups. The SP7 mRNA expression was even higher in the experimental group in the 5^th day, which was 2.7 times higher than the control group and 4.68 times higher than the blank group. The control group was 1.73 times higher than the blank group. The expression of OPN mRNA in the control group was obviously increased in the 1^st day and the same result was got in the 3^rd day, while in the 5^th day there’s no evident difference in the three groups. The COL1α1 mRNA expression in the experimental group was obviously higher than the other two groups in the 3^rd and 5^th day and the expression was in the same level in the control group and the blank group.The expression of RAMP2 gene in HUVEC cells in the experimental group was slightly lower than the control group but higher than the blank group in the 1^st day. The expression was obviously increased in the experimental group, which was 2.3 times higher than the control group and 2.4 times higher than the blank group in the 3^rd day. In the 5^th day, the gene expression in experimental group was 2.06 times higher than the control group and 2.57 times higher than the blank group. The expression of VEGF gene in the experimental group was significantly higher than the other two groups in the 1^st day, 3^rd day and 5^th day. It’s 1.78 times than the control group and 1.68 times higher than the blank group in the 1^st day; 1.55 times than the control group and 2.1 times higher than the blank group in the 3^rd day; 1.88 times than the control group and 2.32 times higher than the blank group in the 5^th day. There’s no obvious difference between the control group and the blank group in the 1^st day and the VEGF expression in the control group was slightly lower than the blank group.The expression of Runx2 protein was obviously higher in experimental group than the other two groups in the 1^st day after detected by Western Blot. The protein expression was significantly higher in the experimental group than the blank group, while was significantly lower in the control group than the blank group in the 3^rd day. In the 5^th day, the protein expression has no significantly difference between the experimental group and the control group, while the blank group was significantly lower than the other two groups.Detected by Western Blot, the Collagen I protein expression in the experimental group and the control group was significantly higher than the blank group in the 1^st day. There’s no obvious difference in the experimental group and the blank group in the 3^rd day, which was the same with their mRNA level. The expression of VEGF protein in the experimental group and the control group was significantly higher than the blank group in the 1^st day after detected by Western Blot. There’s no significant difference among the three groups in the 3^rd day and the protein expression in the experimental group was significantly higher than the other two groups.In conclusion, ADM has a noticeable promoting effect on the proliferation and differentiation of osteoblast and vascular endothelial cell. PLGA/nano-hydroxyapatite scaffold containing chitosan microspheres for controlled delivery of adrenomedullin also has the promoting effect on these two kinds of cells and can promote the bone formation and vascularization related gene and protein expression in the early stage. This research provide theoretical basis for the dental implant clinical application of controlled delivery of adrenomedullin scaffold.更多还原