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~(-13)mol·L~(-1)-10~(-7)mol·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~(-9)M和10~(-8)M浓度的ADM的促增殖能力最强,而对于HUVEC来说,10~(-8)M 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~(-8)M、10~(-7)M 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。
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 HUVEC
Objective: 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~(-13)mol·L~(-1)-10~(-7)mol·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~(-9)M and 10~(-8)M while in HUVEC the density is 10~(-8)M. 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~(-8)M and 10~(-7)M. 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–adrenomedullin
Objective: 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 cells
Objective: 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 cells
4. The influence of ADM-loading scaffold material on the related functional gene and protein of MG63 and HUVEC cells
Objective: 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.
引文
[1]K. Kitamura, K. Kangawa, M. Kawamoto, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma[J].Biochem Biophys Res Commun,1993,192(2): 553-560.
[2]K. Kitamura, J. Sakata, K. Kangawa, et al. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin[J].Biochem Biophys Res Commun,1993,194(2): 720-725.
[3]J. Sakata, T. Shimokubo, K. Kitamura, et al. Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide[J].Biochem Biophys Res Commun,1993,195(2): 921-927.
[4]S. Kapas, K. J. Catt,A. J. Clark. Cloning and expression of cDNA encoding a rat adrenomedullin receptor[J].J Biol Chem,1995,270(43): 25344-25347.
[5]K. Kangawa, K. Kitamura, N. Minamino, et al. Adrenomedullin: a new hypotensive peptide[J].J Hypertens Suppl,1996,14(5): S105-110.
[6]R. Muff, W. Born,J. A. Fischer. Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions[J].Eur J Endocrinol,1995,133(1): 17-20.
[7]A. M. Richards, M. G. Nicholls, L. Lewis, et al. Adrenomedullin[J].Clin Sci (Lond),1996,91(1): 3-16.
[8]W. K. Samson. Adrenomedullin and the control of fluid and electrolyte homeostasis[J].Annu Rev Physiol,1999,61(363-389.
[9]S. J. Wimalawansa. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily[J].Crit Rev Neurobiol,1997,11(2-3): 167-239.
[10]T. Ishimitsu, M. Kojima, K. Kangawa, et al. Genomic structure of human adrenomedullin gene[J].Biochem Biophys Res Commun,1994,203(1): 631-639.
[11]D. L. Hay,D. M. Smith. Adrenomedullin receptors: molecular identity and function[J].Peptides,2001,22(11): 1753-1763.
[12]S. Sugo, N. Minamino, K. Kangawa, et al. Endothelial cells actively synthesize and secrete adrenomedullin[J].Biochem Biophys Res Commun,1994,201(3): 1160-1166.
[13]李光金.肾上腺髓质素[J].国外医学生理、病理科学与临床分册,1997,17(2): 1721.
[14]张丽丽.肾上腺髓质素与妊娠关系的研究进展[J].国外医学妇产科学分册,2002,29(3): 1311.
[15]T. Hata, K. Miyazaki,K. Matsui. Decreased circulating adrenomedullin in pre-eclampsia[J].Lancet,1997,350(9091): 1600.
[16]E. Fabrega, F. Casafont, J. Crespo, et al. Plasma adrenomedullin levels in patients with hepatic cirrhosis[J].Am J Gastroenterol,1997,92(10): 1901-1904.
[17]K. Yamamoto, U. Ikeda, H. Sekiguchi, et al. Plasma levels of adrenomedullin in patients with mitral stenosis[J].Am Heart J,1998,135(3): 542-549.
[18]K. Ehlenz, B. Koch, P. Preuss, et al. High levels of circulating adrenomedullin in severe illness: correlation with C-reactive protein and evidence against the adrenal medulla as site of origin[J].Exp Clin Endocrinol Diabetes,1997,105(3): 156-162.
[19]Y. Hirata, C. Mitaka, K. Sato, et al. Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis[J].J Clin Endocrinol Metab,1996,81(4): 1449-1453.
[20]J. Hanze, K. Dittrich, J. Dotsch, et al. Molecular cloning of a novel human receptor gene with homology to the rat adrenomedullin receptor and high expression in heart and immune system[J].Biochem Biophys Res Commun,1997,240(1): 183-188.
[21]S. M. Foord,F. H. Marshall. RAMPs: accessory proteins for seven transmembrane domain receptors[J].Trends Pharmacol Sci,1999,20(5): 184-187.
[22]S. P. Kennedy, D. Sun, J. J. Oleynek, et al. Expression of the rat adrenomedullin receptor or a putative human adrenomedullin receptor does not correlate with adrenomedullin binding or functional response[J].Biochem Biophys Res Commun,1998,244(3): 832-837.
[23]L. M. McLatchie, N. J. Fraser, M. J. Main, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor[J].Nature,1998,393(6683): 333-339.
[24]S. Kapas,A. J. Clark. Identification of an orphan receptor gene as a type 1 calcitonin gene-related peptide receptor[J].Biochem Biophys Res Commun,1995,217(3): 832-838.
[25]A. Martinez, S. Kapas, M. J. Miller, et al. Coexpression of receptors for adrenomedullin, calcitonin gene-related peptide, and amylin in pancreatic beta-cells[J].Endocrinology,2000,141(1): 406-411.
[26]G. Christopoulos, K. J. Perry, M. Morfis, et al. Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product[J].Mol Pharmacol,1999,56(1): 235-242.
[27]R. Muff, N. Buhlmann, J. A. Fischer, et al. An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3[J].Endocrinology,1999,140(6): 2924-2927.
[28]H. Kinoshita, K. Kato, M. Kuroki, et al. Plasma adrenomedullin levels in patients with diabetes[J].Diabetes Care,2000,23(2): 253-254.
[29]K. Sakai, K. Saito,N. Ishizuka. Adrenomedullin synergistically interacts with endogenous vasodilators in rats: a possible role of K(ATP) channels[J].Eur J Pharmacol,1998,359(2-3): 151-159.
[30]T. W. Moody, M. J. Miller, A. Martinez, et al. Adrenomedullin binds with high affinity, elevates cyclic AMP, and stimulates c-fos mRNA in C6 glioma cells[J].Peptides,1997,18(8): 1111-1115.
[31]A. Sato,D. J. Autelitano. Adrenomedullin induces expression of c-fos and AP-1 activity in rat vascular smooth muscle cells and cardiomyocytes[J].Biochem Biophys Res Commun,1995,217(1): 211-216.
[32]K. Meeran, D. O'Shea, P. D. Upton, et al. Circulating adrenomedullin does not regulate systemic blood pressure but increases plasma prolactin after intravenous infusion in humans: a pharmacokinetic study[J].J Clin Endocrinol Metab,1997,82(1): 95-100.
[33]T. Nishikimi, K. Kitamura, Y. Saito, et al. Clinical studies on the sites of production and clearance of circulating adrenomedullin in human subjects[J].Hypertension,1994,24(5): 600-604.
[34]K. Sato, Y. Hirata, T. Imai, et al. Characterization of immunoreactive adrenomedullin in human plasma and urine[J].Life Sci,1995,57(2): 189-194.
[35]L. M. Montuenga, J. M. Mariano, M. A. Prentice, et al. Coordinate expression of transforming growth factor-beta1 and adrenomedullin in rodent embryogenesis[J].Endocrinology,1998,139(9): 3946-3957.
[36]L. M. Montuenga, A. Martinez, M. J. Miller, et al. Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action[J].Endocrinology,1997,138(1): 440-451.
[37]J. Cornish, K. E. Callon, D. H. Coy, et al. Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo[J].Am J Physiol,1997,273(6 Pt 1): E1113-1120.
[38]M. Taniyama, K. Kitamura, Y. Ban, et al. Elevated plasma adrenomedullin level in hyperthyroidism[J].Eur J Clin Invest,1996,26(6): 454-456.
[39]R. Di Iorio, E. Marinoni, D. Scavo, et al. Adrenomedullin in pregnancy[J].Lancet,1997,349(9048): 328.
[40]J. Cornish, A. Grey, K. E. Callon, et al. Shared pathways of osteoblast mitogenesis induced by amylin, adrenomedullin, and IGF-1[J].Biochem Biophys ResCommun,2004,318(1): 240-246.
[41]G. Mazzocchi, G. Albertin, P. G. Andreis, et al. Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland[J].Peptides,1999,20(12): 1479-1487.
[42]H. A. Coppock, A. A. Owji, C. Austin, et al. Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity[J].Biochem J,1999,338 ( Pt 1)(15-22.
[43]M. Kohno, K. Yasunari, M. Minami, et al. Regulation of rat mesangial cell migration by platelet-derived growth factor, angiotensin II, and adrenomedullin[J].J Am Soc Nephrol,1999,10(12): 2495-2502.
[44]N. Parameswaran, P. Nambi, D. P. Brooks, et al. Regulation of glomerular mesangial cell proliferation in culture by adrenomedullin[J].Eur J Pharmacol,1999,372(1): 85-95.
[45]林景荣.肾上腺髓质素刺激成骨细胞增殖的机制[J].北京大学学报(医学版),2002,34(4): 709-712.
[46]K. Kitamura, E. Matsui, J. Kato, et al. Adrenomedullin (11-26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla[J].Peptides,2001,22(11): 1713-1718.
[47]H. Kano, M. Kohno, K. Yasunari, et al. Adrenomedullin as a novel antiproliferative factor of vascular smooth muscle cells[J].J Hypertens,1996,14(2): 209-213.
[48]I. R. Reid, R. Civitelli, L. V. Avioli, et al. Parathyroid hormone depresses cytosolic pH and DNA synthesis in osteoblast-like cells[J].Am J Physiol,1988,255(1 Pt 1): E9-15.
[49]J. Cornish, K. E. Callon, C. Q. Lin, et al. Comparison of the effects of calcitonin gene-related peptide and amylin on osteoblasts[J].J Bone Miner Res,1999,14(8): 1302-1309.
[50]袁亚兵.肾上腺髓质素与骨代谢研究进展[J].国外医学·生理、病理科学与临床分册,2004,24(6): 516-519.
[51]J. Cornish, D. Naot,I. R. Reid. Adrenomedullin--a regulator of bone formation[J].Regul Pept,2003,112(1-3): 79-86.
[52]R. Muff, W. Born,J. A. Fischer. Adrenomedullin and related peptides: receptors and accessory proteins[J].Peptides,2001,22(11): 1765-1772.
[53]D. Naot, K. E. Callon, A. Grey, et al. A potential role for adrenomedullin as a local regulator of bone growth[J].Endocrinology,2001,142(5): 1849-1857.
[54]S. M. Forrest, K. W. Ng, D. M. Findlay, et al. Characterization of an osteoblast-likeclonal cell line which responds to both parathyroid hormone and calcitonin[J].Calcif Tissue Int,1985,37(1): 51-56.
[55]N. Buhlmann, K. Leuthauser, R. Muff, et al. A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1[J].Endocrinology,1999,140(6): 2883-2890.
[56]K. M. Caron,O. Smithies. Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional Adrenomedullin gene[J].Proc Natl Acad Sci U S A,2001,98(2): 615-619.
[57]A. Kubo, N. Minamino, Y. Isumi, et al. Production of adrenomedullin in macrophage cell line and peritoneal macrophage[J].J Biol Chem,1998,273(27): 16730-16738.
[58]W. Risau. Mechanisms of angiogenesis[J].Nature,1997,386(6626): 671-674.
[59]D. Ribatti, B. Nico, R. Spinazzi, et al. The role of adrenomedullin in angiogenesis[J].Peptides,2005,26(9): 1670-1675.
[60]T. Eto. A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides[J].Peptides,2001,22(11): 1693-1711.
[61]J. P. Hinson, S. Kapas,D. M. Smith. Adrenomedullin, a multifunctional regulatory peptide[J].Endocr Rev,2000,21(2): 138-167.
[62]J. Lopez,A. Martinez. Cell and molecular biology of the multifunctional peptide, adrenomedullin[J].Int Rev Cytol,2002,221(1-92.
[63]Y. Zhao, S. Hague, S. Manek, et al. PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non estrogenic mechanism in the human endometrium[J].Oncogene,1998,16(3): 409-415.
[64]S. Iimuro, T. Shindo, N. Moriyama, et al. Angiogenic effects of adrenomedullin in ischemia and tumor growth[J].Circ Res,2004,95(4): 415-423.
[65]T. Shindo, Y. Kurihara, H. Nishimatsu, et al. Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene[J].Circulation,2001,104(16): 1964-1971.
[66]S. Fernandez-Sauze, C. Delfino, K. Mabrouk, et al. Effects of adrenomedullin on endothelial cells in the multistep process of angiogenesis: involvement of CRLR/RAMP2 and CRLR/RAMP3 receptors[J].Int J Cancer,2004,108(6): 797-804.
[67]W. Kim, S. O. Moon, M. J. Sung, et al. Angiogenic role of adrenomedullin through activation of Akt, mitogen-activated protein kinase, and focal adhesion kinase in endothelial cells[J].Faseb J,2003,17(13): 1937-1939.
[68]P. Kinnunen, I. Szokodi, M. G. Nicholls, et al. Impact of NO on ET-1- and AM-induced inotropic responses: potentiation by combined administration[J].Am J Physiol Regul Integr Comp Physiol,2000,279(2): R569-575.
[69]W. Kim, S. O. Moon, S. Lee, et al. Adrenomedullin reduces VEGF-induced endothelial adhesion molecules and adhesiveness through a phosphatidylinositol 3'-kinase pathway[J].Arterioscler Thromb Vasc Biol,2003,23(8): 1377-1383.
[70]N. Tokunaga, N. Nagaya, M. Shirai, et al. Adrenomedullin gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia: benefits of a novel nonviral vector, gelatin[J].Circulation,2004,109(4): 526-531.
[71]M. Abe, M. Sata, H. Nishimatsu, et al. Adrenomedullin augments collateral development in response to acute ischemia[J].Biochem Biophys Res Commun,2003,306(1): 10-15.
[72]R. Nakamura, J. Kato, K. Kitamura, et al. Adrenomedullin administration immediately after myocardial infarction ameliorates progression of heart failure in rats[J].Circulation,2004,110(4): 426-431.
[73]H. F. Dvorak. Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy[J].J Clin Oncol,2002,20(21): 4368-4380.
[74]S. Hague, L. Zhang, M. K. Oehler, et al. Expression of the hypoxically regulated angiogenic factor adrenomedullin correlates with uterine leiomyoma vascular density[J].Clin Cancer Res,2000,6(7): 2808-2814.
[75]S. Kamitani, M. Asakawa, Y. Shimekake, et al. The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells[J].FEBS Lett,1999,448(1): 111-114.
[76]S. Frayon, C. Cueille, S. Gnidehou, et al. Dexamethasone increases RAMP1 and CRLR mRNA expressions in human vascular smooth muscle cells[J].Biochem Biophys Res Commun,2000,270(3): 1063-1067.
[77]任海霞.微球制剂的应用研究进展[J].药学进展,2007,31(2): 59-63.
[78]韩敏.载药微球制剂的研究进展[J].应用化工,2007,36(5): 493-495.
[79]K. G. Desai,H. J. Park. Preparation of cross-linked chitosan microspheres by spray drying: effect of cross-linking agent on the properties of spray dried microspheres[J].J Microencapsul,2005,22(4): 377-395.
[80]E. E. Hassan, R. C. Parish,J. M. Gallo. Optimized formulation of magnetic chitosan microspheres containing the anticancer agent, oxantrazole[J].Pharm Res,1992,9(3): 390-397.
[81]L. Illum, N. F. Farraj,S. S. Davis. Chitosan as a novel nasal delivery system for peptide drugs[J].Pharm Res,1994,11(8): 1186-1189.
[82]S. A. Agnihotri, N. N. Mallikarjuna,T. M. Aminabhavi. Recent advances on chitosan-based micro- and nanoparticles in drug delivery[J].J Control Release,2004,100(1): 5-28.
[83]G. Durmaz J. Akbuga. Preparation and evaluation of cross-linked chitosan microspheres containing furosemide[J].Int. J. Pharm.,1994,111(3): 217-222.
[84]K. Nishimura, S. Nishimura, H. Seo, et al. Macrophage activation with multi-porous beads prepared from partially deacetylated chitin[J].J Biomed Mater Res,1986,20(9): 1359-1372.
[85]S. Ozbas-Turan, J. Akbuga,C. Aral. Controlled release of interleukin-2 from chitosan microspheres[J].J Pharm Sci,2002,91(5): 1245-1251.
[86]H. Q. Mao, K. Roy, V. L. Troung-Le, et al. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency[J].J Control Release,2001,70(3): 399-421.
[87]P. He, S. S. Davis,L. Illum. Sustained release chitosan microspheres prepared by novel spray drying methods[J].J Microencapsul,1999,16(3): 343-355.
[88]P. He, S. S. Davis,L. Illum. Chitosan microspheres prepared by spray drying[J].Int J Pharm,1999,187(1): 53-65.
[89]A. Ganza-Gonzalez, S. Anguiano-Igea, F. J. Otero-Espinar, et al. Chitosan and chondroitin microspheres for oral-administration controlled release of metoclopramide[J].Eur J Pharm Biopharm,1999,48(2): 149-155.
[90]P. Giunchedi, I. Genta, B. Conti, et al. Preparation and characterization of ampicillin loaded methylpyrrolidinone chitosan and chitosan microspheres[J].Biomaterials,1998,19(1-3): 157-161.
[91]H. Tokumitsu, H. Ichikawa,Y. Fukumori. Chitosan-gadopentetic acid complex nanoparticles for gadolinium neutron-capture therapy of cancer: preparation by novel emulsion-droplet coalescence technique and characterization[J].Pharm Res,1999,16(12): 1830-1835.
[92]Y. Kawashima, T. Handa, A. Kasai, et al. Novel method for the preparation of controlled-release theophylline granules coated with a polyelectrolyte complex of sodium polyphosphate-chitosan[J].J Pharm Sci,1985,74(3): 264-268.
[93]Y. Pan, Y. J. Li, H. Y. Zhao, et al. Bioadhesive polysaccharide in protein delivery system:chitosan nanoparticles improve the intestinal absorption of insulin in vivo[J].Int J Pharm,2002,249(1-2): 139-147.
[94]Y. Xu,Y. Du. Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles[J].Int J Pharm,2003,250(1): 215-226.
[95]J. A. Ko, H. J. Park, S. J. Hwang, et al. Preparation and characterization of chitosan microparticles intended for controlled drug delivery[J].Int J Pharm,2002,249(1-2): 165-174.
[96]S. A. Agnihotri,T. M. Aminabhavi. Controlled release of clozapine through chitosan microparticles prepared by a novel method[J].J Control Release,2004,96(2): 245-259.
[97]J. Akbuga,N. Bergisadi. Effect of formulation variables on cis-platin loaded chitosan microsphere properties[J].J Microencapsul,1999,16(6): 697-703.
[98]S. G. Kumbar, A. R. Kulkarni,M. Aminabhavi. Crosslinked chitosan microspheres for encapsulation of diclofenac sodium: effect of crosslinking agent[J].J Microencapsul,2002,19(2): 173-180.
[99]S. R. Jameela, A. Misra,A. Jayakrishnan. Cross-linked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs[J].J Biomater Sci Polym Ed,1994,6(7): 621-632.
[100]R. Hejazi,M. Amiji. Stomach-specific anti-H. pylori therapy. I: Preparation and characterization of tetracyline-loaded chitosan microspheres[J].Int J Pharm,2002,235(1-2): 87-94.
[101]H. Zhang, I. A. Alsarra,S. H. Neau. An in vitro evaluation of a chitosan-containing multiparticulate system for macromolecule delivery to the colon[J].Int J Pharm,2002,239(1-2): 197-205.
[102]M. L. Lorenzo-Lamosa, C. Remunan-Lopez, J. L. Vila-Jato, et al. Design of microencapsulated chitosan microspheres for colonic drug delivery[J].J Control Release,1998,52(1-2): 109-118.
[103]F. Shikata, H. Tokumitsu, H. Ichikawa, et al. In vitro cellular accumulation of gadolinium incorporated into chitosan nanoparticles designed for neutron-capture therapy of cancer[J].Eur J Pharm Biopharm,2002,53(1): 57-63.
[104]A. M. De Campos, A. Sanchez,M. J. Alonso. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A[J].Int J Pharm,2001,224(1-2): 159-168.
[105]T. Chandy, R. F. Wilson, G. H. Rao, et al. Changes in cisplatin delivery due to surface-coated poly (lactic acid)-poly(epsilon-caprolactone) microspheres[J].J BiomaterAppl,2002,16(4): 275-291.
[106]A. S. Greenwald, S. D. Boden, V. M. Goldberg, et al. Bone-graft substitutes: facts, fictions, and applications[J].J Bone Joint Surg Am,2001,83-A Suppl 2 Pt 2(98-103.
[107]M. C. Gupta,S. N. Khan. Application of bone morphogenetic proteins in spinal fusion[J].Cytokine Growth Factor Rev,2005,16(3): 347-355.
[108]T. Kokubo, H. M. Kim,M. Kawashita. Novel bioactive materials with different mechanical properties[J].Biomaterials,2003,24(13): 2161-2175.
[109]S. Li, J. R. De Wijn, J. Li, et al. Macroporous biphasic calcium phosphate scaffold with high permeability/porosity ratio[J].Tissue Eng,2003,9(3): 535-548.
[110]A. A. Deschamps, M. B. Claase, W. J. Sleijster, et al. Design of segmented poly(ether ester) materials and structures for the tissue engineering of bone[J].J Control Release,2002,78(1-3): 175-186.
[111]P. Pelissier, F. Villars, S. Mathoulin-Pelissier, et al. Influences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats[J].Plast Reconstr Surg,2003,111(6): 1932-1941.
[112]W. Sun, B. Starly, A. Darling, et al. Computer-aided tissue engineering: application to biomimetic modelling and design of tissue scaffolds[J].Biotechnol Appl Biochem,2004,39(Pt 1): 49-58.
[113]P. V. Giannoudis, H. Dinopoulos,E. Tsiridis. Bone substitutes: an update[J].Injury,2005,36 Suppl 3(S20-27.
[114]P. D. Costantino,C. D. Friedman. Synthetic bone graft substitutes[J].Otolaryngol Clin North Am,1994,27(5): 1037-1074.
[115]A. Ogose, N. Kondo, H. Umezu, et al. Histological assessment in grafts of highly purified beta-tricalcium phosphate (OSferion) in human bones[J].Biomaterials,2006,27(8): 1542-1549.
[116]R. W. Bucholz. Nonallograft osteoconductive bone graft substitutes[J].Clin Orthop Relat Res,2002,395): 44-52.
[117]R. Z. LeGeros. Properties of osteoconductive biomaterials: calcium phosphates[J].Clin Orthop Relat Res,2002,395): 81-98.
[118]J. C. Keller, J. G. Collins, G. G. Niederauer, et al. In vitro attachment of osteoblast-like cells to osteoceramic materials[J].Dent Mater,1997,13(1): 62-68.
[119]T. Yuasa, Y. Miyamoto, M. Kon, et al. Proliferation and differentiation of cultured MC3T3-E1 osteoblasts on surface-layer modified hydroxyapatite ceramic with acid and heattreatments[J].Dent Mater J,2005,24(2): 207-212.
[120]B. S. Kim, C. E. Baez,A. Atala. Biomaterials for tissue engineering[J].World J Urol,2000,18(1): 2-9.
[121]M. Geiger, R. H. Li,W. Friess. Collagen sponges for bone regeneration with rhBMP-2[J].Adv Drug Deliv Rev,2003,55(12): 1613-1629.
[122]M. Jarcho. Calcium phosphate ceramics as hard tissue prosthetics[J].Clin Orthop Relat Res,1981,157): 259-278.
[123]M. Chazono, T. Tanaka, H. Komaki, et al. Bone formation and bioresorption after implantation of injectable beta-tricalcium phosphate granules-hyaluronate complex in rabbit bone defects[J].J Biomed Mater Res A,2004,70(4): 542-549.
[124]N. Kondo, A. Ogose, K. Tokunaga, et al. Bone formation and resorption of highly purified beta-tricalcium phosphate in the rat femoral condyle[J].Biomaterials,2005,26(28): 5600-5608.
[125]P. N. De Aza,Z. B. Luklinska. Effect of glass-ceramic microstructure on its in vitro bioactivity[J].J Mater Sci Mater Med,2003,14(10): 891-898.
[126]Yu XZ Shu C, Xiao ZY. Synthesis and sintering of nanocrystalline hydroxyapatite powders by gelatin-based precipitation method [J].Ceramics International,2007,33(2): 193-196.
[127]C. Du, F. Z. Cui, Q. L. Feng, et al. Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity[J].J Biomed Mater Res,1998,42(4): 540-548.
[128]Y. D. Halvorsen, D. Franklin, A. L. Bond, et al. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells[J].Tissue Eng,2001,7(6): 729-741.
[129]王银龙,谢红军.纳米羟基磷灰石复合骨髓基质细胞修复兔股骨缺损实验研究[J].口腔医学,2008,28(3): 154-156.
[130]M. Kikuchi, S. Itoh, S. Ichinose, et al. Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo[J].Biomaterials,2001,22(13): 1705-1711.
[131]Y. S. Pek, S. Gao, M. S. Arshad, et al. Porous collagen-apatite nanocomposite foams as bone regeneration scaffolds[J].Biomaterials,2008,29(32): 4300-4305.
[132]C. Liu, Z. Han,J. T. Czernuszka. Gradient collagen/nanohydroxyapatite composite scaffold: development and characterization[J].Acta Biomater,2009,5(2): 661-669.
[133]H. Tsuchida, J. Hashimoto, E. Crawford, et al. Engineered allogeneic mesenchymalstem cells repair femoral segmental defect in rats[J].J Orthop Res,2003,21(1): 44-53.
[134]Y. Zhang, J. Song, B. Shi, et al. Combination of scaffold and adenovirus vectors expressing bone morphogenetic protein-7 for alveolar bone regeneration at dental implant defects[J].Biomaterials,2007,28(31): 4635-4642.
[135]Y. Wang, D. D. Rudym, A. Walsh, et al. In vivo degradation of three-dimensional silk fibroin scaffolds[J].Biomaterials,2008,29(24-25): 3415-3428.
[136]H. J. Kim, U. J. Kim, H. S. Kim, et al. Bone tissue engineering with premineralized silk scaffolds[J].Bone,2008,42(6): 1226-1234.
[137]Li YB Cheng XM, Zuo Y. Properties and in vitro biological evaluation of nano hydroxapatite/chitosan membranes for bone guided regeneration[J]. Materials Science and Engineering: C,2009,29(1): 29-35.
[138]H. Ohsumi, H. Hirata, T. Nagakura, et al. Enhancement of perineurial repair and inhibition of nerve adhesion by viscous injectable pure alginate sol[J].Plast Reconstr Surg,2005,116(3): 823-830.
[139]J. M. Kanczler, P. J. Ginty, J. J. Barry, et al. The effect of mesenchymal populations and vascular endothelial growth factor delivered from biodegradable polymer scaffolds on bone formation[J].Biomaterials,2008,29(12): 1892-1900.
[140]肖德明,胡波,等.血管内皮生长因子复合纳米羟基磷灰石人工骨修复免桡骨缺损[J].中国组织工程研究与临床康复杂志,2008,23(12): 6209-6212.
[141]H. Wang, Y. Li, Y. Zuo, et al. Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering[J].Biomaterials,2007,28(22): 3338-3348.
[142]Moonb LS Luonga ND, Lee DS, et al. Surface modification of poly(l-lactide) electrospun fibers with nanocrystal hydroxyapatite for engineered scaffold applications [J].Materials Science and Engineering: C,2008,28(8): 1242-1249.
[143]Firouzdor V Nejati E, Eslaminejad MB. Needle-like nano hydroxyapatite/poly(l-lactide acid) composite scaffold for bone tissue engineering application [J]. Materials Science and Engineering: C,2008,29(3): 942-949.
[144]Das G Sinha A, Sharma BK,et al. Poly(vinyl alcohol)–hydroxyapatite biomimetic scaffold for tissue regeneration [J].Materials Science and Engineering: C,2007,27(70-74.
[145]毛天球,刘冰,等.胶原基纳米骨复合rhBMP-2及钛膜修复即刻钛种植体周围骨缺损[J]..稀有金属材料与工程,2006,35(2): 331-334.
[146]S. Kim, S. S. Kim, S. H. Lee, et al. In vivo bone formation from human embryonicstem cell-derived osteogenic cells in poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffolds[J].Biomaterials,2008,29(8): 1043-1053.
[147]P. Zhang, Z. Hong, T. Yu, et al. In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with poly(L-lactide)[J].Biomaterials,2009,30(1): 58-70.
[148]H. Ueda, L. Hong, M. Yamamoto, et al. Use of collagen sponge incorporating transforming growth factor-beta1 to promote bone repair in skull defects in rabbits[J].Biomaterials,2002,23(4): 1003-1010.
[149]J. O. Hollinger, J. Brekke, E. Gruskin, et al. Role of bone substitutes[J].Clin Orthop Relat Res,1996,324): 55-65.
[150]S. L. Ishaug-Riley, L. E. Okun, G. Prado, et al. Human articular chondrocyte adhesion and proliferation on synthetic biodegradable polymer films[J].Biomaterials,1999,20(23-24): 2245-2256.
[151]N. A. Peppas,R. Langer. New challenges in biomaterials[J].Science,1994,263(5154): 1715-1720.
[152]C. M. Agrawal,K. A. Athanasiou. Technique to control pH in vicinity of biodegrading PLA-PGA implants[J].J Biomed Mater Res,1997,38(2): 105-114.
[153]J. E. Devin, M. A. Attawia,C. T. Laurencin. Three-dimensional degradable porous polymer-ceramic matrices for use in bone repair[J].J Biomater Sci Polym Ed,1996,7(8): 661-669.
[154]廖二元,罗湘杭,周后德,等.成骨肉瘤MG263细胞分化特性及分化过程中的基因表达[J].湖南医科大学学报,2001,26(2): 701-711.
[155]D. Guidolin, G. Albertin, R. Spinazzi, et al. Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2[J].Peptides,2008,29(11): 2013-2023.
[156]Y. Zhang, O. Andrukhov, S. Berner, et al. Osteogenic properties of hydrophilic and hydrophobic titanium surfaces evaluated with osteoblast-like cells (MG63) in coculture with human umbilical vein endothelial cells (HUVEC)[J].Dent Mater,26(11): 1043-1051.
[157]Y. X. Huang, J. Ren, C. Chen, et al. Preparation and properties of poly(lactide-co-glycolide) (PLGA)/ nano-hydroxyapatite (NHA) scaffolds by thermally induced phase separation and rabbit MSCs culture on scaffolds[J].J Biomater Appl,2008,22(5): 409-432.
[158]K. G. Desai,H. J. Park. Encapsulation of vitamin C in tripolyphosphate cross-linkedchitosan microspheres by spray drying[J].J Microencapsul,2005,22(2): 179-192.
[159]J. Varshosaz. The promise of chitosan microspheres in drug delivery systems[J].Expert Opin Drug Deliv,2007,4(3): 263-273.
[160]Li YL Xu YX, Chen LQ,et al. Preparation and releasing behavior of chitosan microspheres/nano-hydroxyapatite/PLGA scaffolds:Compared to nano-hydroxyapatite/PLGA scaffolds and chitosan microspheres[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2010,14(3): 452-456.
[161]Colonna C. Dorati R., Genta I,,et al. Effect of porogen on the physico-chemical properties and degradation performance of PLGA scaffolds[J].Polymer Degradation and Stability,2010,95(1): 694-701.
[162]X. Niu, Q. Feng, M. Wang, et al. Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2[J].J Control Release,2009,134(2): 111-117.
[163]Zhang L Li JF, Li JF, et al. Properties of Drug-loaded Chitosan Microspheres Crosslinked by Vanillin and Analysis of Microsphere-forming Mechanism[J].Chem. J. Chinese Universities,2008,29(9): 1874-1879
[164]S. E. Kim, J. H. Park, Y. W. Cho, et al. Porous chitosan scaffold containing microspheres loaded with transforming growth factor-beta1: implications for cartilage tissue engineering[J].J Control Release,2003,91(3): 365-374.
[165]X. Z. Shu,K. J. Zhu. Controlled drug release properties of ionically cross-linked chitosan beads: the influence of anion structure[J].Int J Pharm,2002,233(1-2): 217-225.
[166]D. Z. Cai, C. Zeng, D. P. Quan, et al. Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-beta1 delivery for cartilage tissue engineering[J].Chin Med J (Engl),2007,120(3): 197-203.
[167]K. G. Desai, C. Liu,H. J. Park. Characteristics of vitamin C encapsulated tripolyphosphate-chitosan microspheres as affected by chitosan molecular weight[J].J Microencapsul,2006,23(1): 79-90.
[168]C. Y. Lin, R. M. Schek, A. S. Mistry, et al. Functional bone engineering using ex vivo gene therapy and topology-optimized, biodegradable polymer composite scaffolds[J].Tissue Eng,2005,11(9-10): 1589-1598.
[169]M. A. Attawia, K. M. Herbert, K. E. Uhrich, et al. Proliferation, morphology, and protein expression by osteoblasts cultured on poly(anhydride-co-imides)[J].J Biomed Mater Res,1999,48(3): 322-327.
[170]QL Feng XF Niu, MB Wang,et al. Preparation and characterization of chitosan microspheres for controlled release of synthetic oligopeptide derived from BMP-2[J].Journal of Microencapsulation,2009,26(4): 297-305.
[171]F. M. Chen, Z. F. Wu, H. H. Sun, et al. Release of bioactive BMP from dextran-derived microspheres: a novel delivery concept[J].Int J Pharm,2006,307(1): 23-32.
[172]QL Feng XFNiu, MB Wang, et al. In vitro degradation and release behavior of porous poly(lactic acid) scaffolds containing chitosan microspheres as a carrier for BMP-2-derived synthetic peptide[J].Polymer Degradation and Stability,2009,94(2): 176-182.
[173]H. J. Sung, C. Meredith, C. Johnson, et al. The effect of scaffold degradation rate on three-dimensional cell growth and angiogenesis[J].Biomaterials,2004,25(26): 5735-5742.
[174]A. S. Shanbhag, J. J. Jacobs, J. Black, et al. Macrophage/particle interactions: effect of size, composition and surface area[J].J Biomed Mater Res,1994,28(1): 81-90.
[175]M. C. Wake, P. D. Gerecht, L. Lu, et al. Effects of biodegradable polymer particles on rat marrow-derived stromal osteoblasts in vitro[J].Biomaterials,1998,19(14): 1255-1268.
[176]J. H. Lee, G. Khang, J. W. Lee, et al. Interaction of Different Types of Cells on Polymer Surfaces with Wettability Gradient[J].J Colloid Interface Sci,1998,205(2): 323-330.
[177]U. J. Kim, J. Park, H. J. Kim, et al. Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin[J].Biomaterials,2005,26(15): 2775-2785.
[178]U. Meyer, A. Buchter, H. P. Wiesmann, et al. Basic reactions of osteoblasts on structured material surfaces[J].Eur Cell Mater,2005,9(39-49.
[179]C. K. Colton. Implantable biohybrid artificial organs[J].Cell Transplant,1995,4(4): 415-436.
[180]L. G. Griffith, G. Naughton. Tissue engineering-current challenges and expanding opportunities[J]. Science,2002,295(5557): 1009-1014.
[181]Liu Y Yu T, Wang Y, et al. Preparation and Bioactivity of the Composite of PLGA and Hydroxyapatite Nanocrystals Surface-grafted with L-lactic Acid Oligomer[J]. Chem. J. Chinese Universities,2009,30(7): 1439-1444.
[182]L. L. Nikitenko, D. M. Smith, S. Hague, et al. Adrenomedullin and the microvasculature[J].Trends Pharmacol Sci,2002,23(3): 101-103.
[183]K. Anselme. Osteoblast adhesion on biomaterials[J].Biomaterials,2000,21(7): 667-681.
[184]B. D. Boyan, T. W. Hummert, D. D. Dean, et al. Role of material surfaces in regulating bone and cartilage cell response[J].Biomaterials,1996,17(2): 137-146.
[185]I. Watanabe, M. McBride, P. Newton, et al. Laser surface treatment to improvemechanical properties of cast titanium[J].Dent Mater,2009,25(5): 629-633.
[186]K. J. Livak,T. D. Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J].Methods,2001,25(4): 402-408.
[187]Q. Kang, M. H. Sun, H. Cheng, et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery[J].Gene Ther,2004,11(17): 1312-1320.
[188]D. Chen, M. Zhao,G. R. Mundy. Bone morphogenetic proteins[J].Growth Factors,2004,22(4): 233-241.
[189]A. Javed, G. L. Barnes, B. O. Jasanya, et al. runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins[J].Mol Cell Biol,2001,21(8): 2891-2905.
[190]K. Miyazono, S. Maeda,T. Imamura. Coordinate regulation of cell growth and differentiation by TGF-beta superfamily and Runx proteins[J].Oncogene,2004,23(24): 4232-4237.
[191]K. Nakashima, X. Zhou, G. Kunkel, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation[J].Cell,2002,108(1): 17-29.
[192]Y. Gao, A. Jheon, H. Nourkeyhani, et al. Molecular cloning, structure, expression, and chromosomal localization of the human Osterix (SP7) gene[J].Gene,2004,341(101-110.
[193]M. A. Milona, J. E. Gough,A. J. Edgar. Expression of alternatively spliced isoforms of human Sp7 in osteoblast-like cells[J].BMC Genomics,2003,4(43.
[194]M. H. Lee, Y. J. Kim, W. J. Yoon, et al. Dlx5 specifically regulates Runx2 type II expression by binding to homeodomain-response elements in the Runx2 distal promoter[J].J Biol Chem,2005,280(42): 35579-35587.
[195]Q. Tu, P. Valverde, J. Chen. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells[J].Biochem Biophys Res Commun,2006,341(4): 1257-1265.
[196]Y. Ohyama, A. Nifuji, Y. Maeda, et al. Spaciotemporal association and bone morphogenetic protein regulation of sclerostin and osterix expression during embryonic osteogenesis[J].Endocrinology,2004,145(10): 4685-4692.
[197]M. H. Lee, T. G. Kwon, H. S. Park, et al. BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2[J].Biochem Biophys Res Commun,2003,309(3): 689-694.
[198]杨志明,余希杰,马骏荣.成骨细胞的细胞社会学特征[J].中国修复重建外科学杂志,1998,12(6): 350-354.
[199]S. F. El-Amin, H. H. Lu, Y. Khan, et al. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering[J].Biomaterials,2003,24(7): 1213-1221.
[200]M. Karlsson, E. Palsgard, P. R. Wilshaw, et al. Initial in vitro interaction of osteoblasts with nano-porous alumina[J].Biomaterials,2003,24(18): 3039-3046.
[201]T. J. Webster, C. Ergun, R. H. Doremus, et al. Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics[J].J Biomed Mater Res,2000,51(3): 475-483.
[202]李亦文,顾国珍,孙皎,等.皮肤再生膜的生物相容性系列研究[J].中国修复重建外科杂志,2000,14(1): 44-48.
[203]E. J. Mackie. Osteoblasts: novel roles in orchestration of skeletal architecture[J].Int J Biochem Cell Biol,2003,35(9): 1301-1305.
[204]马纪,潘秋辉,于永春,等.转录因子Osterix对成骨细胞Col1 a1基因的反式激活作用[J].中国生物化学与分子生物学报,2007,23(11): 921-925.
[205]N. Ferrara,W. J. Henzel. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J].Biochem Biophys Res Commun,1989,161(2): 851-858.
[206]F. Shalaby, J. Rossant, T. P. Yamaguchi, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice[J].Nature,1995,376(6535): 62-66.
[207]M. Clauss. Molecular biology of the VEGF and the VEGF receptor family[J].Semin Thromb Hemost,2000,26(5): 561-569.
[208]G. Neufeld, T. Cohen, S. Gengrinovitch, et al. Vascular endothelial growth factor (VEGF) and its receptors[J].Faseb J,1999,13(1): 9-22.
[209]付小兵,刘文忠,张宝林.血管内皮细胞生长因子及其受体与血管形成的研究进展[J].中国危重病急救医学,2003,15(4): 251-253.