RGD短肽修饰壳聚糖作为钛表面hBMP-2cDNA基因载体表达效率的研究
摘要
随着社会经济发展、人民生活水平提高以及口腔修复意识的进步,越来越多牙列缺损或牙列缺失的患者选择种植义齿修复缺失牙。种植义齿成功关键在于种植体植入后与骨形成良好的骨整合。20世纪60年代Branemark教授创立的骨结合理论奠定了现代口腔种植学的生物学基础,骨结合理论是指：有生命的骨组织(bone)与种植体(implant)之间的直接结合,无纤维组织围绕种植体,这种结合必须而且能够承受负重。
目前口腔种植体常用的材料主要是纯钛、钛合金以及一些复合材料。纯金属钛具有质轻,对震动减幅力大,低弹性模量等优点,同时具有较高的硬度、极限抗拉(张)强度、屈服强度、疲劳强度以及抗腐蚀性疲劳极限等特点,因此纯钛种植体的应用比较广泛。不仅如此,由于钛及钛合金具有优点,近年来,钛合金在骨科临床方面得到了广泛的应用。已成为口腔医学及矫形外科领域广泛应用的材料。
但不足之处是钛属于生物惰性材料,在临床应用中所需愈合时间较长,且植入机体后组织反应表现为非特异性和随机性。因此,努力探索新的种植体表面改性方法获得新型的种植体,在保证高种植成功率的同时显著缩短种植体与牙槽骨发生骨整合的时间,减轻患者的痛苦,不仅有较大的科学意义而且有实际的临床应用前景。
在近二十年的研究当中,钛表面处理方法,诱导成骨因子,成骨因子相关基因以及包裹基因的载体,载体的修饰,构建成功的复合体与钛表面的结合方法这几个方面成为了研究的热点与重点。大量的研究表明,经过物理化学处理后的钛基种植体的初期稳定性和长期成功率都明显高于没有进行表面粗化处理的种植体。原因可能有两方面,一是经过物理化学处理后的种植体表面的无机成分可能有利于成骨细胞的粘附、增殖和分化,从而促进骨整合；二是粗化表面大大增加了种植体的表面积,提高机械嵌合力,增加种植体的初期稳定性。
随着研究的深入,将功能明确的活性物质引入种植体表面的生物化学改性研究已逐渐成为种植体研究的热点。相对于其他物理化学方法,这种方法直接利用对成骨细胞的功能表达有促进作用的活性分子来影响组织反应,促进新骨形成因而这种方法较传统的物理化学方法更为直接有效,有望获得比无机表面的种植体更快的骨整合速度。研究显示功能性蛋白(纤维粘连蛋白、骨形态发生蛋白、细胞粘附短肽RGD等)可以被引入到种植体表面。
其中hBMP2是已知的具有强成骨诱导能力的一类生长因子。hBMP2作为骨生长促进因子,广泛存在于人的硬组织中,主要分布在具有成骨倾向的细胞浆中,包括成骨、成釉及成牙本质细胞和成软骨细胞等,具有诱导成骨作用,能够诱发未分化的间充质细胞转化为成骨细胞,hBMP2在可以克隆的7个hBMP成员基因中诱导骨形成活性最强。但单纯的]hBMP2在体内代谢较快,难以形成持续作用,且蛋白本身仍较基因体积大,影响种植体表面厚度。由此可见,选择一种合适的载体有效而可控的将活性分子基因引入到种植体表面并保持其良好的生物活性；同时控制种植体表面活性分子的释放,使其在合适的时间段对细胞增殖分化和组织重构保持合适的浓度,是我们应该关注的种植体表面生物活性化的根本问题。
基因载体可分为病毒载体和非病毒载体,病毒类载体有较高的转染效率,但具有一些安全性问题,如病毒重组、免疫原性等,因此利用非病毒载体系统携带靶基因进行基因治疗较病毒类载体更为安全。在众多非病毒载体系统中,阳离子聚合物是一种非常有前景的载体。壳聚糖是存在于自然界中的唯一种带阳离子的能生物降解的纳米粒载体材料,具有良好的生物相容性、可降解性和粘膜粘附性,特别适合包载蛋白质、多肽、疫苗和基因等生物活性大分子[18]。现壳聚糖主要作为代替脂质体及病毒类载体作为药物、蛋白、基因等的载体被人们广泛研究。壳聚糖可以通过分子表面的正电荷与带负电荷的DNA相结合,阻止DNA酶(DNasel)对DNA的降解,有效浓缩DNA[19],此外还具有黏附作用强、毒副作用小、来源丰富、价格低廉等优势。但壳聚糖的低靶向性和低转染效率这一局限性往往会限制了它的应用。为提高其靶向性以及转染效率,许多研究在壳聚糖分子上进行短肽修饰,以达到提高其转染效率的目的。
RGD肽是一类含精氨酸—甘氨酸—天冬氨酸(Arg-Gly-Asp)序列在生物体内广泛存在的短肽,是整合素(Intedrin)和其配体之间相互作用的识别位点,能与11种整合素特异性结合,能促进成骨细胞的生长,促使骨组织再生,抑制破骨细胞之间及其与基质之间的黏附。RGD小分子靶向肽修饰壳聚糖作为基因表达载体,其转染效率较单纯壳聚糖作为载体有明显的提高。同时,还有研究[26-27]发现,将RGD短肽固定在种植体表面,能促使种植体四周新骨生长,使种植体的抗拉力增强。因此,将RGD与壳聚糖接枝,即改善了壳聚糖基因载体的性能,亦可改善种植体表面性能,从基因和肽水平同时促进钛种植体表面的成骨。
目前在钛种植体表面固定和/或释放生物大分子的方法主要包括物理吸附法、化学固定法、层层自组装法及载体法。其中层层自组装技术(Layer-by-Layer self-assemble technique, LbL)是应用较多也较为成熟的方法。概括而言,就是在荷电的基材表面通过静电相互作用,交替地吸附上带相反电荷的聚电解质,形成自组装多层聚电解质复合物(polyelectrolyte complex, PEC)薄膜,也叫静电自组装。该技术由Decher G.等在20世纪90年代最先提出和应用。LbL技术制备条件简单、温和,不受基材形状的限制,薄膜具有较高的热力学稳定性,可以控制涂层循环的次数达到控制涂层的量,并可以装载细胞因子实现缓释功能,然而层层自组装是通过静电作用实现的,由于RGD短肽分子量较小,所带有的电荷很少,所以单纯依靠自组装来固定RGD,得到的种植体表面RGD量较少,有学者对此进行了改进[30-31]。将层层自组装引入到种植体表面RGD组装方法中,并和化学偶联相结合来组装RGD,能明显促进RGD在种植体表面的结合力和数量,这是RGD在种植体表面组装方法的重要进展。
因人成骨肉瘤MG-63细胞株为具有人成骨细胞表型特征的成骨细胞模型,因此本研究通过对比修饰前后的纯钛表面对人成骨肉瘤细胞MG63初期黏附增殖影响,对比钛片经喷砂与烧结两种处理后,壳聚糖载体携带质粒DNA在纯钛表面人成骨肉瘤细胞MG63中的转染、表达效率差异。
目的：
1、利用钛片模拟临床种植体,探索新的种植体表面改性方法,在保证高种植成功率的同时,缩短种植体与牙槽骨发生骨整合的时间,减轻患者的痛苦,获得快速、有序、可控骨整合。
2、利用壳聚糖作为非病毒载体,在钛片表面引入骨形态发生蛋白、细胞粘附短肽RGD,提高骨整合速度。
3、比较钛片经喷砂与烧结两种处理后,其表面携带的hBMP-2cDNA基因在人成骨肉瘤细胞MG63内的转染效率与表达差异。方法：
1、采用烧结和喷砂后进行酸碱处理的方法对纯钛片表面进行处理,并检测其表征。
2、选择hBMP-2cDNA作为活化因子,设计合成小分子靶向肽RGD短肽,选择具有良好骨诱导性的生物大分子壳聚糖,将RGD(Arg-Gly-Asp)短肽与壳聚糖通过酰化反应发生偶联形成RGD/CS复合体,通过元素分析仪对复合体进行元素含量检测,采用红外光谱仪对RGD/CS复合体进行验证。
3、采用复凝聚法制备RGD/CS/hBMP-2cDNA复合体,利用原子力显微镜对RGD/CS/hBMP-2cDNA复合体进行表征检测。
4、采用层层自组装以及化学偶联技术将RGD/CS/hBMP-2cDNA复合体接枝到事先处理好的钛表面,对钛表面采用EB染色检验。
5、在接枝有RGD/CS/hBMP-2cDNA复合体的钛片表面培养人成骨肉瘤MG-63细胞,通过扫描电镜检测细胞在喷砂与烧结两种不同处理钛片表面生长状况；利用荧光显微镜观察RGD/CS/hBMP-2cDNA复合体在MG-63细胞内的转染效率。
6、通过Real-Time PCR与Western blotting实验检测不同处理钛片表面hBMP-2cDNA基因在MG-63细胞内的表达水平。
结果：
1、红外光谱分析表明,壳聚糖与RGD肽偶联后的产物RGD/CS在1630cm-1附近的酰胺Ⅰ带震动显著增加,表明偶联过程中有大量碳氧双键生成,证明壳聚糖和RGD肽成功偶联；通过元素分析表明：在偶联了RGD肽后,样本IGD/CS的N原子含量比例,明显较单纯的壳聚糖为高,进一步表明：壳聚糖接枝RGD成功。
2、酶切与电泳实验表明：]hBMP-2cDNA基因电泳条带大小符合预期；质粒测序结果表明：载体构建过程中,hBMP-2cDNA未发生基因突变,载体构建成功。
3、电泳实验表明：RGD/CS/hBMP-2cDNA在N/P≥2时,RGD/CS与hBMP-2cDNA质粒完全复合；原子力显微镜检测表明：N/P=2-50的RGD/CS/hBMP-2cDNA复合体粒径为137-259nm,Zeta电位在+25mV～+30mV之间,且原子力显微镜结果表明复合物为类球形且分布良好。
4、纯钛片经烧结处理后,经扫描电镜检测发现,其表面均匀分布有“Ω”状洞穴。
5、EB染色实验表明：接枝有RGD/CS/hBMP-2cDNA复合体的钛片,能被EB染色,说明RGD/C2/hBMP-2cDNA复合体接枝钛片实验成功。
6、经壳聚糖酶消化,钛片表面接枝的RGD/CS/hBMP-2cDNA复合体释放出的hBMP-2cDNA质粒,经测序表明：RGD/CS/hBMP-2cDNA复合体接枝钛后hBMP-2cDNA基因序列稳定性良好,未发生基因突变。
7、细胞转化实验表明：壳聚糖偶联RGD后再包裹hBMP-2cDNA质粒,其在人成骨肉瘤细胞MG63内的转染效率明显较单纯的壳聚糖包裹hBMP-2cDNA质粒转染效率为高。
8、细胞扫描电镜检测表明：表面经氧化性混酸溶液处理形成“Ω”状的纯钛片,有利于细胞在其表面贴壁生长；培养在接枝有RGD/CS/hBMP-2cDNA复合体的钛片上的人成骨肉瘤细胞MG63,部分镶嵌在“Ω”形状的凹洞中,大大提高了细胞对钛表面的吸附力。
9、荧光显微镜观察发现,培养在接枝有RGD/CS/hBMP-2cDNA复合体的钛片上的人成骨肉瘤细胞MG63,钛表面BMP2质粒能有效转化到人成骨肉瘤细胞MG63内。
10、Real-Time PCR与Western blotting实验表明：在接枝有RGD/CS/hBMP-2cDNA复合体的钛片上的人成骨肉瘤细胞MG63内超量表达了BMP2蛋白,且其表达量显著高于培养在未经修饰的钛片上的人成骨肉瘤细胞MG63。
结论：
本研究表明：RGD能有效提高壳聚糖的细胞转染效率；经RGD短肽修饰的壳聚糖可以作为hBMP-2cDNA质粒包裹载体；钛表面hBMP-2cDNA表达载体能有效转化到人成骨肉瘤细胞MG63内；表面经过RGD/CS/hBMP-2cDNA复合体修饰后的钛种植体有利于人成骨肉瘤细胞MG63在其表面贴壁生长并超量表达BMP2蛋白。
As the social development and the progress of people'realization in mouth rehabilitation,more and more people comes to plant or restore their missing teeth. The key to success of implant denture is the formation of good bone after implantation. Professor Branemark founded in the1960s Bone combined with theory laid the foundation of modern oral planting of biology, The theory of bone combined is as following:living bone tissue combined with implant directly, without the fibrous tissue around the implant, and this combination must be able to withstand weight.
The current commonly oral implant materials is mainly pure titanium, titanium alloys, and some composite materials. The advantages of pure titanium is qualitative light, for vibration damping force is large, low modulus of elasticity, simultaneously has characteristics about high hardness and ultimate tensile (zhang) strength, yield strength, fatigue strength and corrosion resistance of fatigue limit. so the application of pure titanium implant very extensive. Not only that, because of the advantages of titanium and titanium alloy, in recent years, the titanium alloy has been widely used in orthopaedic clinic, and widely used in the field of orthopaedic surgery oral medicine.
But the titanium is the biological inert materials, require for a long time to heal in the clinical application, and tissue response performance for nonspecific and randomness after implanting into body.Therefore, trying to explore a new implant surface modification methods for new implants, There are great scientific significance and practical prospect of clinical application that to make sure high success rate of planting significantly and shorten the implant and the alveolar bone bone integration time and relieve the suffering of the patient.
In nearly20years, titanium surface treatment method, factor inducing osteogenesis, bone morphogenic factor related genes and gene carrier, the carrier of the modifications, complex and titanium surface of building a successful method of combining became the research hot spot and focus. A large number of studies have shown that the initial stability of the titanium implant and the long-term success rate were significantly higher than no implant surface roughening treatment after dealing with the physical chemistry.The reason may have two aspects, firstly, the inorganic composition is plant on the surface of the body surface after dealing with the physical chemistry could promote the adhesion, proliferation and differentiation of osteoblast, so as to promote the integration of bone;Secondly, coarsening surface can increase the surface area of the implant greatly, improve mechanical embedded together, and increase the initial stability of the implant.
With the deepening of the research, The body surface biological chemical modification research that introduce the function clear active substances to grow on the surface become hot topics in the study of implant gradually. Relative to other physical and chemical methods, this method directly using the function expression of osteoblasts promote the activity of molecular reaction to influence the organization, to promote the new bone formation and thus this method is compared with the traditional physical and chemical method is more direct and effective, is expected to get faster than inorganic surface of implant bone integration speed. Studies have shown that functional protein (protein fiber adhesion, BMP, cell adhesion short peptide RGD, etc.) can be introduced into the implant surface.
HBMP2were known as growth factors because of the strong induced osteogenesis ability. HBMP2exists in human hard tissues widely as bone growth promoting factor. They mainly distribute in cytoplasm with osteogenesis tendency, including osteogenesis, into glaze and odontoblasts and into cartilage cells, etc. and they can induce osteogenesis, and induce undifferentiated mesenchymal cells into osteoblast. The activity of gene hBMP2is strongest in7hBMP members that can be clonedis. But hBMP2metabolise is so fast by the body that it is difficult to form a continuous function, despite the volume of protein is so big that affect the thickness of the implant surface. So, to choose a suitable carrier effective and controllable reactive molecules gene is introduced into the implant surface and keep its good biological activity,and control the release of the implant surface active molecules at the same time, in the proper period of cell proliferation differentiation and tissue reconstruction maintain the appropriate concentration, is that we should focus on the fundamental problem of implant surface bioactivity.
Gene carrier can be divided into viral vector and non viral vector. The transfection efficiency of viral vector is higher than non viral vector, but there are some security problems, such as virus recombination, immunogenicity, and therefore the non viral vector systems for gene therapy with virus carrier is more secure.In the numerous non viral vector system, cationic polymer is a very promising carrier. Chitosan is a kind of biodegradable nanoparticles only with cationic carrier materials existing in the nature, with good biocompatibility, biodegradable and mucosal adhesive, especially suitable for package carrier proteins, peptides, such as vaccines and gene bioactive molecules. Now instead of liposomes and virus main kinds of carrier chitosan as a drug, protein and gene carrier has been widely studied.Chitosan can combine with the negatively charged DNA through the positive charge on the surface of molecules, prevent DNA enzyme (DNaseI) on the degradation of DNA, the effective concentration of DNA. Moreover, there are some advantage about the strong adhesion effect, side effects, rich source, and a small price.But the dis advantage about low targeting and low transfection efficiency of chitosan often limits its application.In order to improve its targeting and transfection efficiency, much research on the chitosan molecular peptide modified, in order to reach the purpose of improving its transfection efficiency.
RGD peptide is a kind of short peptide that contains1-arginine glycine-aspartate (Arg Gly Asp) sequence in the widespread, it is recognition sites of the interaction between integrin and the ligand, and it can combine with11kinds of integrin specificity, and promote the growth of osteoblasts and bone tissue regeneration, and inhibit adhesion between substrate and osteoclast. RGD targeting peptide modified chitosan as a gene expression vector, its transfection efficiency is higher than pure chitosan as carrier obviously.At the same time, the implant surface with fixing short RGD peptides, can cause bone growth around implant, make the implant enhancement of tensile resistance.Therefore, the graft copolymerization between RGD and chitosan, can improve the performance of the chitosan carrier, and improve the implant surface properties, and promote the osteogenesis of titanium implants surface from the level of gene and peptide.
Currently, the methods to fix or release of biological macromolecules including physical adsorption method, chemical method, layer upon layer self-assembly method and carrier method.The Layer-by-Layer self-assemble technique is applied maturely more and more, on the surface of the base material that charged by electrostatic interaction, alternately with opposite charge on the adsorption of polyelectrolyte, form self-assembled multilayer polyelectrolyte complexes thin film. This technology proposed and used firstly by Decher G in the1990s. LbL technology preparation conditions is simple and gentle, and it is not subject to the restrictions by base material. The film has high thermodynamic stability, the amount of coating can be control by changing the number of coating cycle, and can be loaded cytokines with slow-release function. However, the Layer-by-Layer self-assemble technique work through the electrostatic effect. Because of the small molecular weight of RGD peptide, the charge is very few, so the RGD on the surface of implant is less. The Layer-by-Layer self-assemble technique is introduced to implant surface RGD assembly method, and combined with chemical coupling to assemble RGD, it can obviously promote the RGD on the growing surface binding force and quantity, and this is the important progress about RGD assembly method on implant surface.
The osteogenesis sarcoma cell line MG63is recognized as people osteoblast phenotype characteristics of osteoblast model. So this study have compared effect on MG63cells proliferation before and after the modification of titanium surface, and compared the plasmid DNA transfection and expression efficiency in the surface of pure titanium MG63the titanium plate between sandblasting and sintering treatment.
Objective:
1, To explore new implant surface modification method, and shorten the bone integration time between implant and the alveolar bone and reduce the patient's pain.
2, The chitosan is used as a viral vector, and BMP, cell adhesion peptide RGD is introduced into the titanium plate surface, and to increase the speed of bone integration.
3, To compare transfection efficiency and expression differences in the titanium plate surface between sandblasting and sintering treatment with BMP-2gene expression vector in MG63cell.
Methods:
1, The pure titanium surface treat with acid and alkali after sand blasting or sintering, and detect its characterization.
2, Choose hBMP-2cdna as activation factor, design and synthesis of small molecules targeting peptide RGD peptide, choose biological macromolecular chitosan with good bone induced, the RGD (Arg Gly Asp) short peptide and chitosan through acylation reaction form RGD/CS complex coupling, evaluate the element content with the composite element analyzer, authenticate RGD/CS complex with infrared spectrometer.
3, Compound condensing RGD/CS/BMP-2cdna complex, the RGD/CS/BMP-2cdna complexes were characterized using atomic force microscopy.
4, The RGD/CS/hBMP-2cDNA complex has been grafted to titanium surface with Layer-by-Layer self-assemble technique and chemical coupling technology, and the surface of titanium has been dyeing test with EB.
5, Cultivate the osteogenesis sarcoma MG-63cells titanium surface grafting with RGD/CS/BMP-2cdna complex, and test the growth condition of cells in sand blasting and sintering of two different processing titanium surface; and the transfection efficiency RGD/CS/BMP-2cdna complex in MG-63cell has been tested with fluorescent microscope.
6, Test BMP-2gene expression level in MG-63cells of different processing titanium surface through the Real-Time PCR and Western blotting experiment.
Results:
1, Ehrough infrared spectrum detection showed that after coupling the RGD peptides, CS-RGD near1630cm-1amide I bring increased significantly, in the process of coupling generates more carbon oxygen double bond, confirmed the amidation of successful CS and RGD peptide coupling;Through the element analysis shows that after coupling the RGD peptides, sample N atom content proportion of CS-RGD, than pure CS is high, it further shows that CS graft RGD success.
2, Enzyme digestion and electrophoresis experiment showed that the BMP-2gene electrophoresis stripe size in line with expectations;Plasmid sequencing results show that the process of building a carrier, BMP-2gene mutation, carrier build is successful.
3, Electrophoresis experiment showed that the CS-RGD/bmp2cDNA in the N/P=2, CS-RGD and bmp2cDNA completely compound;Atomic force microscope detection showed that the N/P=2～50RGD-CS/bmp2cDNA complex particle size is137-259nm, Zeta potential between+25mV～+30mV, and atomic force microscopy results showed that the compound for the class, spherical and well distributed;
4, Pure titanium after sintering treatment, detected by scanning electron microscopy (sem) test, it is uniformly distributed over the surface of "Ω cave."
5, EB dyeing experiments showed that the grafting with RGD-CS/bmp2cDNA complex titanium plate, can be EB stain, illustrating RGD-CS/bmp2cDNA complex graft titanium plate experiment success;
6, After enzyme digestion, RGD/CS/BMP-2cdna complex grafted in titanium surface has been released from chitosan,and the sequencing experiment showed that there is no gene mutation about RGD/CS/BMP-2cdna complex after grafting.
7, Cell transformation experiment shows that the parcel again after CS coupling RGD BMP2plasmid, the transfection efficiency in MG63cells obviously a pure CS parcel BMP2plasmid transfection efficiency is high;
8, Scanning electron microscopy (sem) test show that the surface treated by oxidation of mixed acid solution form "Ω" in the shape of a pure titanium plate, is advantageous to the growth of cells on the surface to stick wall;Training in graft RGD-CS/bmp2cDNA complex titanium plate on the MG63cells, part of the Mosaic in "Ω" in the shape of a concave hole, greatly improving the cells of the titanium surface adsorption force;
9, Fluorescence microscope observation found that training in graft RGD-CS/bmp2cDNA complex MG63cells on titanium plate, titanium surface BMP2plasmid effectively into MG63cells;
10, Real-Time PCR and Western blotting experiment showed that MG63cells growing in titanium plate with graft RGD/CS/Bmp-2cdna complex expressed BMP2proteins, and the expression level is higher than the MG63cells training in titanium plate without modification significantly.
In conclusion, this study showed that RGD can effectively improve the cell transfection efficiency, CS coating with short peptide RGD, can be used as a carrier of titanium implant surface plasmid packaging;Surface after RGD-CS/bmp2cDNA complex modification of titanium implant, is advantageous to the MG63cells adherent growth on the surface, to improve cells on the surface of titanium NianZhaoLi;Titanium surface BMP2protein expression vector into a MG63cells effectively, cultivate in graft RGD-CS/bmp2cDNA complex titanium plate on the excess of MG63cells can express BMP2protein.
目前口腔种植体常用的材料主要是纯钛、钛合金以及一些复合材料。纯金属钛具有质轻,对震动减幅力大,低弹性模量等优点,同时具有较高的硬度、极限抗拉(张)强度、屈服强度、疲劳强度以及抗腐蚀性疲劳极限等特点,因此纯钛种植体的应用比较广泛。不仅如此,由于钛及钛合金具有优点,近年来,钛合金在骨科临床方面得到了广泛的应用。已成为口腔医学及矫形外科领域广泛应用的材料。
但不足之处是钛属于生物惰性材料,在临床应用中所需愈合时间较长,且植入机体后组织反应表现为非特异性和随机性。因此,努力探索新的种植体表面改性方法获得新型的种植体,在保证高种植成功率的同时显著缩短种植体与牙槽骨发生骨整合的时间,减轻患者的痛苦,不仅有较大的科学意义而且有实际的临床应用前景。
在近二十年的研究当中,钛表面处理方法,诱导成骨因子,成骨因子相关基因以及包裹基因的载体,载体的修饰,构建成功的复合体与钛表面的结合方法这几个方面成为了研究的热点与重点。大量的研究表明,经过物理化学处理后的钛基种植体的初期稳定性和长期成功率都明显高于没有进行表面粗化处理的种植体。原因可能有两方面,一是经过物理化学处理后的种植体表面的无机成分可能有利于成骨细胞的粘附、增殖和分化,从而促进骨整合；二是粗化表面大大增加了种植体的表面积,提高机械嵌合力,增加种植体的初期稳定性。
随着研究的深入,将功能明确的活性物质引入种植体表面的生物化学改性研究已逐渐成为种植体研究的热点。相对于其他物理化学方法,这种方法直接利用对成骨细胞的功能表达有促进作用的活性分子来影响组织反应,促进新骨形成因而这种方法较传统的物理化学方法更为直接有效,有望获得比无机表面的种植体更快的骨整合速度。研究显示功能性蛋白(纤维粘连蛋白、骨形态发生蛋白、细胞粘附短肽RGD等)可以被引入到种植体表面。
其中hBMP2是已知的具有强成骨诱导能力的一类生长因子。hBMP2作为骨生长促进因子,广泛存在于人的硬组织中,主要分布在具有成骨倾向的细胞浆中,包括成骨、成釉及成牙本质细胞和成软骨细胞等,具有诱导成骨作用,能够诱发未分化的间充质细胞转化为成骨细胞,hBMP2在可以克隆的7个hBMP成员基因中诱导骨形成活性最强。但单纯的]hBMP2在体内代谢较快,难以形成持续作用,且蛋白本身仍较基因体积大,影响种植体表面厚度。由此可见,选择一种合适的载体有效而可控的将活性分子基因引入到种植体表面并保持其良好的生物活性；同时控制种植体表面活性分子的释放,使其在合适的时间段对细胞增殖分化和组织重构保持合适的浓度,是我们应该关注的种植体表面生物活性化的根本问题。
基因载体可分为病毒载体和非病毒载体,病毒类载体有较高的转染效率,但具有一些安全性问题,如病毒重组、免疫原性等,因此利用非病毒载体系统携带靶基因进行基因治疗较病毒类载体更为安全。在众多非病毒载体系统中,阳离子聚合物是一种非常有前景的载体。壳聚糖是存在于自然界中的唯一种带阳离子的能生物降解的纳米粒载体材料,具有良好的生物相容性、可降解性和粘膜粘附性,特别适合包载蛋白质、多肽、疫苗和基因等生物活性大分子[18]。现壳聚糖主要作为代替脂质体及病毒类载体作为药物、蛋白、基因等的载体被人们广泛研究。壳聚糖可以通过分子表面的正电荷与带负电荷的DNA相结合,阻止DNA酶(DNasel)对DNA的降解,有效浓缩DNA[19],此外还具有黏附作用强、毒副作用小、来源丰富、价格低廉等优势。但壳聚糖的低靶向性和低转染效率这一局限性往往会限制了它的应用。为提高其靶向性以及转染效率,许多研究在壳聚糖分子上进行短肽修饰,以达到提高其转染效率的目的。
RGD肽是一类含精氨酸—甘氨酸—天冬氨酸(Arg-Gly-Asp)序列在生物体内广泛存在的短肽,是整合素(Intedrin)和其配体之间相互作用的识别位点,能与11种整合素特异性结合,能促进成骨细胞的生长,促使骨组织再生,抑制破骨细胞之间及其与基质之间的黏附。RGD小分子靶向肽修饰壳聚糖作为基因表达载体,其转染效率较单纯壳聚糖作为载体有明显的提高。同时,还有研究[26-27]发现,将RGD短肽固定在种植体表面,能促使种植体四周新骨生长,使种植体的抗拉力增强。因此,将RGD与壳聚糖接枝,即改善了壳聚糖基因载体的性能,亦可改善种植体表面性能,从基因和肽水平同时促进钛种植体表面的成骨。
目前在钛种植体表面固定和/或释放生物大分子的方法主要包括物理吸附法、化学固定法、层层自组装法及载体法。其中层层自组装技术(Layer-by-Layer self-assemble technique, LbL)是应用较多也较为成熟的方法。概括而言,就是在荷电的基材表面通过静电相互作用,交替地吸附上带相反电荷的聚电解质,形成自组装多层聚电解质复合物(polyelectrolyte complex, PEC)薄膜,也叫静电自组装。该技术由Decher G.等在20世纪90年代最先提出和应用。LbL技术制备条件简单、温和,不受基材形状的限制,薄膜具有较高的热力学稳定性,可以控制涂层循环的次数达到控制涂层的量,并可以装载细胞因子实现缓释功能,然而层层自组装是通过静电作用实现的,由于RGD短肽分子量较小,所带有的电荷很少,所以单纯依靠自组装来固定RGD,得到的种植体表面RGD量较少,有学者对此进行了改进[30-31]。将层层自组装引入到种植体表面RGD组装方法中,并和化学偶联相结合来组装RGD,能明显促进RGD在种植体表面的结合力和数量,这是RGD在种植体表面组装方法的重要进展。
因人成骨肉瘤MG-63细胞株为具有人成骨细胞表型特征的成骨细胞模型,因此本研究通过对比修饰前后的纯钛表面对人成骨肉瘤细胞MG63初期黏附增殖影响,对比钛片经喷砂与烧结两种处理后,壳聚糖载体携带质粒DNA在纯钛表面人成骨肉瘤细胞MG63中的转染、表达效率差异。
目的：
1、利用钛片模拟临床种植体,探索新的种植体表面改性方法,在保证高种植成功率的同时,缩短种植体与牙槽骨发生骨整合的时间,减轻患者的痛苦,获得快速、有序、可控骨整合。
2、利用壳聚糖作为非病毒载体,在钛片表面引入骨形态发生蛋白、细胞粘附短肽RGD,提高骨整合速度。
3、比较钛片经喷砂与烧结两种处理后,其表面携带的hBMP-2cDNA基因在人成骨肉瘤细胞MG63内的转染效率与表达差异。方法：
1、采用烧结和喷砂后进行酸碱处理的方法对纯钛片表面进行处理,并检测其表征。
2、选择hBMP-2cDNA作为活化因子,设计合成小分子靶向肽RGD短肽,选择具有良好骨诱导性的生物大分子壳聚糖,将RGD(Arg-Gly-Asp)短肽与壳聚糖通过酰化反应发生偶联形成RGD/CS复合体,通过元素分析仪对复合体进行元素含量检测,采用红外光谱仪对RGD/CS复合体进行验证。
3、采用复凝聚法制备RGD/CS/hBMP-2cDNA复合体,利用原子力显微镜对RGD/CS/hBMP-2cDNA复合体进行表征检测。
4、采用层层自组装以及化学偶联技术将RGD/CS/hBMP-2cDNA复合体接枝到事先处理好的钛表面,对钛表面采用EB染色检验。
5、在接枝有RGD/CS/hBMP-2cDNA复合体的钛片表面培养人成骨肉瘤MG-63细胞,通过扫描电镜检测细胞在喷砂与烧结两种不同处理钛片表面生长状况；利用荧光显微镜观察RGD/CS/hBMP-2cDNA复合体在MG-63细胞内的转染效率。
6、通过Real-Time PCR与Western blotting实验检测不同处理钛片表面hBMP-2cDNA基因在MG-63细胞内的表达水平。
结果：
1、红外光谱分析表明,壳聚糖与RGD肽偶联后的产物RGD/CS在1630cm-1附近的酰胺Ⅰ带震动显著增加,表明偶联过程中有大量碳氧双键生成,证明壳聚糖和RGD肽成功偶联；通过元素分析表明：在偶联了RGD肽后,样本IGD/CS的N原子含量比例,明显较单纯的壳聚糖为高,进一步表明：壳聚糖接枝RGD成功。
2、酶切与电泳实验表明：]hBMP-2cDNA基因电泳条带大小符合预期；质粒测序结果表明：载体构建过程中,hBMP-2cDNA未发生基因突变,载体构建成功。
3、电泳实验表明：RGD/CS/hBMP-2cDNA在N/P≥2时,RGD/CS与hBMP-2cDNA质粒完全复合；原子力显微镜检测表明：N/P=2-50的RGD/CS/hBMP-2cDNA复合体粒径为137-259nm,Zeta电位在+25mV～+30mV之间,且原子力显微镜结果表明复合物为类球形且分布良好。
4、纯钛片经烧结处理后,经扫描电镜检测发现,其表面均匀分布有“Ω”状洞穴。
5、EB染色实验表明：接枝有RGD/CS/hBMP-2cDNA复合体的钛片,能被EB染色,说明RGD/C2/hBMP-2cDNA复合体接枝钛片实验成功。
6、经壳聚糖酶消化,钛片表面接枝的RGD/CS/hBMP-2cDNA复合体释放出的hBMP-2cDNA质粒,经测序表明：RGD/CS/hBMP-2cDNA复合体接枝钛后hBMP-2cDNA基因序列稳定性良好,未发生基因突变。
7、细胞转化实验表明：壳聚糖偶联RGD后再包裹hBMP-2cDNA质粒,其在人成骨肉瘤细胞MG63内的转染效率明显较单纯的壳聚糖包裹hBMP-2cDNA质粒转染效率为高。
8、细胞扫描电镜检测表明：表面经氧化性混酸溶液处理形成“Ω”状的纯钛片,有利于细胞在其表面贴壁生长；培养在接枝有RGD/CS/hBMP-2cDNA复合体的钛片上的人成骨肉瘤细胞MG63,部分镶嵌在“Ω”形状的凹洞中,大大提高了细胞对钛表面的吸附力。
9、荧光显微镜观察发现,培养在接枝有RGD/CS/hBMP-2cDNA复合体的钛片上的人成骨肉瘤细胞MG63,钛表面BMP2质粒能有效转化到人成骨肉瘤细胞MG63内。
10、Real-Time PCR与Western blotting实验表明：在接枝有RGD/CS/hBMP-2cDNA复合体的钛片上的人成骨肉瘤细胞MG63内超量表达了BMP2蛋白,且其表达量显著高于培养在未经修饰的钛片上的人成骨肉瘤细胞MG63。
结论：
本研究表明：RGD能有效提高壳聚糖的细胞转染效率；经RGD短肽修饰的壳聚糖可以作为hBMP-2cDNA质粒包裹载体；钛表面hBMP-2cDNA表达载体能有效转化到人成骨肉瘤细胞MG63内；表面经过RGD/CS/hBMP-2cDNA复合体修饰后的钛种植体有利于人成骨肉瘤细胞MG63在其表面贴壁生长并超量表达BMP2蛋白。
As the social development and the progress of people'realization in mouth rehabilitation,more and more people comes to plant or restore their missing teeth. The key to success of implant denture is the formation of good bone after implantation. Professor Branemark founded in the1960s Bone combined with theory laid the foundation of modern oral planting of biology, The theory of bone combined is as following:living bone tissue combined with implant directly, without the fibrous tissue around the implant, and this combination must be able to withstand weight.
The current commonly oral implant materials is mainly pure titanium, titanium alloys, and some composite materials. The advantages of pure titanium is qualitative light, for vibration damping force is large, low modulus of elasticity, simultaneously has characteristics about high hardness and ultimate tensile (zhang) strength, yield strength, fatigue strength and corrosion resistance of fatigue limit. so the application of pure titanium implant very extensive. Not only that, because of the advantages of titanium and titanium alloy, in recent years, the titanium alloy has been widely used in orthopaedic clinic, and widely used in the field of orthopaedic surgery oral medicine.
But the titanium is the biological inert materials, require for a long time to heal in the clinical application, and tissue response performance for nonspecific and randomness after implanting into body.Therefore, trying to explore a new implant surface modification methods for new implants, There are great scientific significance and practical prospect of clinical application that to make sure high success rate of planting significantly and shorten the implant and the alveolar bone bone integration time and relieve the suffering of the patient.
In nearly20years, titanium surface treatment method, factor inducing osteogenesis, bone morphogenic factor related genes and gene carrier, the carrier of the modifications, complex and titanium surface of building a successful method of combining became the research hot spot and focus. A large number of studies have shown that the initial stability of the titanium implant and the long-term success rate were significantly higher than no implant surface roughening treatment after dealing with the physical chemistry.The reason may have two aspects, firstly, the inorganic composition is plant on the surface of the body surface after dealing with the physical chemistry could promote the adhesion, proliferation and differentiation of osteoblast, so as to promote the integration of bone;Secondly, coarsening surface can increase the surface area of the implant greatly, improve mechanical embedded together, and increase the initial stability of the implant.
With the deepening of the research, The body surface biological chemical modification research that introduce the function clear active substances to grow on the surface become hot topics in the study of implant gradually. Relative to other physical and chemical methods, this method directly using the function expression of osteoblasts promote the activity of molecular reaction to influence the organization, to promote the new bone formation and thus this method is compared with the traditional physical and chemical method is more direct and effective, is expected to get faster than inorganic surface of implant bone integration speed. Studies have shown that functional protein (protein fiber adhesion, BMP, cell adhesion short peptide RGD, etc.) can be introduced into the implant surface.
HBMP2were known as growth factors because of the strong induced osteogenesis ability. HBMP2exists in human hard tissues widely as bone growth promoting factor. They mainly distribute in cytoplasm with osteogenesis tendency, including osteogenesis, into glaze and odontoblasts and into cartilage cells, etc. and they can induce osteogenesis, and induce undifferentiated mesenchymal cells into osteoblast. The activity of gene hBMP2is strongest in7hBMP members that can be clonedis. But hBMP2metabolise is so fast by the body that it is difficult to form a continuous function, despite the volume of protein is so big that affect the thickness of the implant surface. So, to choose a suitable carrier effective and controllable reactive molecules gene is introduced into the implant surface and keep its good biological activity,and control the release of the implant surface active molecules at the same time, in the proper period of cell proliferation differentiation and tissue reconstruction maintain the appropriate concentration, is that we should focus on the fundamental problem of implant surface bioactivity.
Gene carrier can be divided into viral vector and non viral vector. The transfection efficiency of viral vector is higher than non viral vector, but there are some security problems, such as virus recombination, immunogenicity, and therefore the non viral vector systems for gene therapy with virus carrier is more secure.In the numerous non viral vector system, cationic polymer is a very promising carrier. Chitosan is a kind of biodegradable nanoparticles only with cationic carrier materials existing in the nature, with good biocompatibility, biodegradable and mucosal adhesive, especially suitable for package carrier proteins, peptides, such as vaccines and gene bioactive molecules. Now instead of liposomes and virus main kinds of carrier chitosan as a drug, protein and gene carrier has been widely studied.Chitosan can combine with the negatively charged DNA through the positive charge on the surface of molecules, prevent DNA enzyme (DNaseI) on the degradation of DNA, the effective concentration of DNA. Moreover, there are some advantage about the strong adhesion effect, side effects, rich source, and a small price.But the dis advantage about low targeting and low transfection efficiency of chitosan often limits its application.In order to improve its targeting and transfection efficiency, much research on the chitosan molecular peptide modified, in order to reach the purpose of improving its transfection efficiency.
RGD peptide is a kind of short peptide that contains1-arginine glycine-aspartate (Arg Gly Asp) sequence in the widespread, it is recognition sites of the interaction between integrin and the ligand, and it can combine with11kinds of integrin specificity, and promote the growth of osteoblasts and bone tissue regeneration, and inhibit adhesion between substrate and osteoclast. RGD targeting peptide modified chitosan as a gene expression vector, its transfection efficiency is higher than pure chitosan as carrier obviously.At the same time, the implant surface with fixing short RGD peptides, can cause bone growth around implant, make the implant enhancement of tensile resistance.Therefore, the graft copolymerization between RGD and chitosan, can improve the performance of the chitosan carrier, and improve the implant surface properties, and promote the osteogenesis of titanium implants surface from the level of gene and peptide.
Currently, the methods to fix or release of biological macromolecules including physical adsorption method, chemical method, layer upon layer self-assembly method and carrier method.The Layer-by-Layer self-assemble technique is applied maturely more and more, on the surface of the base material that charged by electrostatic interaction, alternately with opposite charge on the adsorption of polyelectrolyte, form self-assembled multilayer polyelectrolyte complexes thin film. This technology proposed and used firstly by Decher G in the1990s. LbL technology preparation conditions is simple and gentle, and it is not subject to the restrictions by base material. The film has high thermodynamic stability, the amount of coating can be control by changing the number of coating cycle, and can be loaded cytokines with slow-release function. However, the Layer-by-Layer self-assemble technique work through the electrostatic effect. Because of the small molecular weight of RGD peptide, the charge is very few, so the RGD on the surface of implant is less. The Layer-by-Layer self-assemble technique is introduced to implant surface RGD assembly method, and combined with chemical coupling to assemble RGD, it can obviously promote the RGD on the growing surface binding force and quantity, and this is the important progress about RGD assembly method on implant surface.
The osteogenesis sarcoma cell line MG63is recognized as people osteoblast phenotype characteristics of osteoblast model. So this study have compared effect on MG63cells proliferation before and after the modification of titanium surface, and compared the plasmid DNA transfection and expression efficiency in the surface of pure titanium MG63the titanium plate between sandblasting and sintering treatment.
Objective:
1, To explore new implant surface modification method, and shorten the bone integration time between implant and the alveolar bone and reduce the patient's pain.
2, The chitosan is used as a viral vector, and BMP, cell adhesion peptide RGD is introduced into the titanium plate surface, and to increase the speed of bone integration.
3, To compare transfection efficiency and expression differences in the titanium plate surface between sandblasting and sintering treatment with BMP-2gene expression vector in MG63cell.
Methods:
1, The pure titanium surface treat with acid and alkali after sand blasting or sintering, and detect its characterization.
2, Choose hBMP-2cdna as activation factor, design and synthesis of small molecules targeting peptide RGD peptide, choose biological macromolecular chitosan with good bone induced, the RGD (Arg Gly Asp) short peptide and chitosan through acylation reaction form RGD/CS complex coupling, evaluate the element content with the composite element analyzer, authenticate RGD/CS complex with infrared spectrometer.
3, Compound condensing RGD/CS/BMP-2cdna complex, the RGD/CS/BMP-2cdna complexes were characterized using atomic force microscopy.
4, The RGD/CS/hBMP-2cDNA complex has been grafted to titanium surface with Layer-by-Layer self-assemble technique and chemical coupling technology, and the surface of titanium has been dyeing test with EB.
5, Cultivate the osteogenesis sarcoma MG-63cells titanium surface grafting with RGD/CS/BMP-2cdna complex, and test the growth condition of cells in sand blasting and sintering of two different processing titanium surface; and the transfection efficiency RGD/CS/BMP-2cdna complex in MG-63cell has been tested with fluorescent microscope.
6, Test BMP-2gene expression level in MG-63cells of different processing titanium surface through the Real-Time PCR and Western blotting experiment.
Results:
1, Ehrough infrared spectrum detection showed that after coupling the RGD peptides, CS-RGD near1630cm-1amide I bring increased significantly, in the process of coupling generates more carbon oxygen double bond, confirmed the amidation of successful CS and RGD peptide coupling;Through the element analysis shows that after coupling the RGD peptides, sample N atom content proportion of CS-RGD, than pure CS is high, it further shows that CS graft RGD success.
2, Enzyme digestion and electrophoresis experiment showed that the BMP-2gene electrophoresis stripe size in line with expectations;Plasmid sequencing results show that the process of building a carrier, BMP-2gene mutation, carrier build is successful.
3, Electrophoresis experiment showed that the CS-RGD/bmp2cDNA in the N/P=2, CS-RGD and bmp2cDNA completely compound;Atomic force microscope detection showed that the N/P=2～50RGD-CS/bmp2cDNA complex particle size is137-259nm, Zeta potential between+25mV～+30mV, and atomic force microscopy results showed that the compound for the class, spherical and well distributed;
4, Pure titanium after sintering treatment, detected by scanning electron microscopy (sem) test, it is uniformly distributed over the surface of "Ω cave."
5, EB dyeing experiments showed that the grafting with RGD-CS/bmp2cDNA complex titanium plate, can be EB stain, illustrating RGD-CS/bmp2cDNA complex graft titanium plate experiment success;
6, After enzyme digestion, RGD/CS/BMP-2cdna complex grafted in titanium surface has been released from chitosan,and the sequencing experiment showed that there is no gene mutation about RGD/CS/BMP-2cdna complex after grafting.
7, Cell transformation experiment shows that the parcel again after CS coupling RGD BMP2plasmid, the transfection efficiency in MG63cells obviously a pure CS parcel BMP2plasmid transfection efficiency is high;
8, Scanning electron microscopy (sem) test show that the surface treated by oxidation of mixed acid solution form "Ω" in the shape of a pure titanium plate, is advantageous to the growth of cells on the surface to stick wall;Training in graft RGD-CS/bmp2cDNA complex titanium plate on the MG63cells, part of the Mosaic in "Ω" in the shape of a concave hole, greatly improving the cells of the titanium surface adsorption force;
9, Fluorescence microscope observation found that training in graft RGD-CS/bmp2cDNA complex MG63cells on titanium plate, titanium surface BMP2plasmid effectively into MG63cells;
10, Real-Time PCR and Western blotting experiment showed that MG63cells growing in titanium plate with graft RGD/CS/Bmp-2cdna complex expressed BMP2proteins, and the expression level is higher than the MG63cells training in titanium plate without modification significantly.
In conclusion, this study showed that RGD can effectively improve the cell transfection efficiency, CS coating with short peptide RGD, can be used as a carrier of titanium implant surface plasmid packaging;Surface after RGD-CS/bmp2cDNA complex modification of titanium implant, is advantageous to the MG63cells adherent growth on the surface, to improve cells on the surface of titanium NianZhaoLi;Titanium surface BMP2protein expression vector into a MG63cells effectively, cultivate in graft RGD-CS/bmp2cDNA complex titanium plate on the excess of MG63cells can express BMP2protein.
引文
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