融合肽minTBP-1-PRGDN涂层影响钛表面成骨细胞粘附、增殖及分化功能的体外研究
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摘要
金属钛因其良好的耐腐蚀性、生物相容性和骨整合性,在临床上已广泛用作于牙科修复种植体、正畸支抗种植体或人工关节。钛种植体稳定性是其植入成功的关键,多种活性肽和蛋白已被用于修饰钛种植体表面,以促进骨与种植体之间的达到稳定的骨性整合。RGD (Arg-Gly-Asp)肽作为大部分细胞外基质蛋白(包括一些矿化相关蛋白)的共有序列,是目前最有效和应用广泛和的促进生物材料表面细胞粘附的肽,而如何将肽固定于生物材料的表面是提高细胞粘附的前提。学者们试图借助于羟基、氨基或羧基等功能基团,将RGD通过共价结合的方式固定在生物材料的表面。然而,这些偶联方式存在一些缺点：除了复杂的处理过程外,偶联剂的毒性或者活性基团因水解而失活等现象均不容忽视。TBP-1 (RKLPDAPGMHTW)是一种新的从12肽噬菌体库中分离出来的肽适体,其具有与金属钛特异性相互作用的能力,其N端的RKLPDA (minTBP-1)序列是钛结合的重要位点。鉴于其对钛强大的特异性识别能力,minTBP-1常以与其他基序相结合形成融合肽,使得这些融合肽被赋予了同样的对钛特异性识别的能力。鉴于此,本研究合成一种含有RGD和minTBP-1序列的融合—RKLPDAPRGDN,探讨其与钛表面的相互作用；以及用该融合肽作为钛表面涂层,探讨对成骨细胞附着、伸展、增殖、分化及矿化功能方面的影响。
第一部分融合肽minTBP-1-PRGDN与钛表面相互作用的研究
目的探讨融合肽minTBP-1-PRGDN、minTBP-1及PRGDN在不同浓度时对钛亲和性的影响；氧化和未氧化钛表面对三种肽结合能力的影响；FITC标记对肽结合能力影响。
方法实验一：钛片经顺序打磨、清洗、消毒备用,三种肽以不同浓度(1μg/ml,10μg/ml, 100μg/ml, 1000μg/ml)孵育钛片过夜,双蒸水洗去未结合的肽。实验二：钛片经顺序打磨、清洗、一部分经30%硝酸氧化(另一部分不氧化)、消毒备用,XPS检测氧化与未氧化钛片表面元素成分；三种肽以100μg/ml浓度分别孵育氧化和未氧化的钛片过夜,双蒸水洗去未结合的肽。实验三：钛片经顺序打磨、清洗、氧化、消毒备用,三种肽以100μg/ml浓度按照未标记与标记比例为0：1(None)、10:1、100:1分别孵育钛片过夜,双蒸水洗去未结合的肽。荧光显微镜拍照,IPP图像分析软件采集荧光照片的像素点、以平均每个钛片上的荧光像素点的数量作为结合到钛片表面的肽数量的间接反映。
结果实验一：三种肽随着浓度的增加其结合到钛片上的数量均逐渐增加,100μg/ml时各种肽在钛片的结合接近饱和。实验二：经氧化后的钛片表面氧元素含量增加；minTBP-1-PRGDN和minTBP-1在氧化的钛片表面的结合数量增加,而PRGDN肽数昊减少。实验三：随着未标记肽掺入到标记肽中的比例增加,钛片表面荧光数量减少。
第二部分融合肽minTBP-1-PRGDN对成骨细胞粘附的影响
目的探讨不同肽涂层的钛表面对成骨细胞附着数量、伸展形态及面积、粘附相关基因表达的影响。
方法钛片经顺序打磨、清洗、氧化、消毒备用,三种肽以100μg/ml浓度孵育钛片表面过夜,未有肽孵育组作为空白对照；PBS洗去未结合的肽；成骨细胞在钛片表面生长一定时间后(实验四、五为1h,实验六为24h),PBS洗去未粘附细胞。实验四：Alamar Blue比色法检测附着细胞的数量;实验五：FITC-鬼笔环肽染色粘附细胞,荧光显微镜照相,IPP软件分析细胞伸展面积；实验六：RT-PCR荧光定量检测Integrinβ1和Cadherin-11基因表达。
结果实验四：minTBP-1-PRGDN涂层钛片上附着的成骨细胞数量最多、PRGDN涂层组的细胞数量最少：实验五：minTBP-1-PRGDN融合肽涂层的钛片表面的成骨细胞伸展充分、面积最大,但细胞伪足少；PRGDN涂层的钛片表面成骨细胞伸展最不充分,多接近于立方形,伸展面积与其他各组相比也是最小；实验六：Integrinβ1和Cadherin-11基因表达量在PRGDN组均为最高。
第三部分融合肽minTBP-1-PRGDN对成骨细胞增殖的影响
目的探讨不同肽涂层的钛表面对成骨细胞增殖数量、形态及相关基因表达的影响。
方法实验七：MTT比色法检测四组成骨细胞在24h、48h、72h时的增殖数量；细胞经Giemsa's染色后金相显微镜观察在增殖72h时的形态；实验八：RT-PCR荧光定量检测Collagen la 1和Cyclin D1基因表达。
结果实验七：各组细胞随时间延长数量逐渐增加,24h时minTBP-1-PRGDN涂层钛片表面的成骨细胞数量最多；72h时各组细胞大部分呈长梭形,伸展充分,并沿着钛片表面打磨的痕迹方向排列；实验八：cyclin D1:对照组外,其余各组表达量随时间延长而增加;达到72h时,minTBP-1-PRGDN组的cyclin D1表达量最大。CollagenⅠα1：各组表达量随时间延长降低后又有所增高；24 h时,PRGDN组的表达量最高；48h时,空白对照组表达量最低；72h时,minTBP-1组表达量最高。
第四部分融合肽minTBP-1-PRGDN对成骨细胞分化及功能的影响
目的探讨不同肽涂层的钛表面对成骨细胞分化过程中ALP酶活性、矿化相关基因表达、矿化结节形成数量及面积大小的影响。
方法实验九：PNPP法检测成骨细胞诱导分化第3,7,10,14,21,28d时的ALP酶活性;实验十：RT-PCR荧光定量检测ALP、OPN、BSP、OC基因表达；实验十一：细胞诱导矿化28d后,茜素红染色矿化结节,金相显微镜照相,IPP软件分析各组结节数量及平均面积。
结果实验九：各组成骨细胞诱导3-10d内,ALP活性值缓慢递增,从14d开始,ALP量的增加开始加速,28d时达到峰值；实验十：minTBP-1-PRGDN涂层表面成骨细胞分泌的OPN、OC、BSP及ALP四种基因的表达趋势均为：随着时间的延长,表达量先上升后降低,且在第14天时达到高峰。而minTBP-1涂层表面成骨细胞分泌的这四种基因的表达趋势均为随着时间的延长,表达量逐渐上升。空白对照组中,除了OC表达先降低后增高以外,其余三种基因的表达均为逐渐升高。而PRGDN组中除了BSP的表达先增加后减少外,其他三种基因的表达均为先降低升高。四种基因在第7d时,PRGDN组的表达均为最高；第14d时minTBP-1-PRGDN组表达量最高；实验十一：minTBP-1-PRGDN组的结节面积最大而minTBP-1组的平均面积最小；结节数量在PRGDN组最多而minTBP-1组最少。
Titanium (Ti) has been widely used as an embedding biomaterial in dental implants, artificial joints and so on, owing to its good biocompatibility, osseointegration, resistance to corrosion and low allergenicity. The key point of successful implant is the achievement of fine stability between Ti and bone tissue. Various kinds of natural or artificial peptides and proteins have been widely investigated to promote osseointegration. The RGD (Arg-Gly-Asp) sequence, which can be found in most extracellular matrix (ECM) proteins, including some mineralized-related proteins, is the most effective and most often employed peptide sequence for improving cell adhesion on biomaterial surfaces. Stable immobilization of peptides on the surface of biomaterials is essential to promote strong cell adhesion. RGD peptides commonly covalently bind to the surface of biomaterials via functional moieties such as hydroxyl, amino, and carboxyl radicals. However, many problems may arise in these coupling methods:besides the complex treatment procedures, the coupling reagent may be cytotoxic or the active group may be deactivated quickly by hydrolysis. A new peptide aptamer, named TBP-1 (RKLPDAPGMHTW) which is isolated from a linear 12-mer peptide phage library, has been proved to have the character of specifically interacting with the surface of Ti. Its N-terminal, RKLPDA (minTBP-1), is sufficient for Ti binding. Considering its specific and strong binding ability, minTBP-1 has been used in the form of combination with other motifs, which endowed the chimeric peptides with specific binding ability to Ti. In this study, we synthesized a chimeric peptide with double motifs containing a RGD and a minTBP-1 sequence, and investigated its binding ability to Ti and its effect to osteoblast on attachment, spreading, proliferation, differentiation and mineralization ability as well.
Part One:The interaction between minTBP-1-PRGDN and titanium
Object To explore the affinities of minTBP-1-PRGDN, minTBP-1and PRGDN to Ti at different concentrations; the different affinities of three peprtides to unoxidized and oxidized Ti; the influence of FITC labling on peptide affinity.
Methods Experiment one:Ti disks were wet-polished, washed and sterilized sequentially, and incubated overnight with three peptides separativly at four different concentrations of 1μg/ml,10μg/ml,100μg/ml and 1000μg/ml, then washed with ddH2O to remove unbound peptides. Experiment two:Ti disks were wet-polished, washed and sterilized sequentially. Half numbers of disks were oxidized with 30% HNO3 and the others were not oxidized. The elements on the surfaces of oxidized and unoxidized Ti disks were detected by XPS. Then all disks were incubated overnight with three peptides separativly at concentration of 100μg/ml, and then washed with ddH2O to remove unbound peptides. Experiment three:Ti disks were wet-polished, washed, oxidized and sterilized sequentially, and then incubated overnight with the combination of unlabeled and FITC-labeled peptides in the proportion of 0:1,10:1, and 100:1, washed with ddH2O to remove unbound peptides. Photos of coated disks were taken by fluorescence microscope and analyzed by Image-Pro(?) Plus software package by measuring the average number of fluorescent pixels per disk.
Results Experiment one:The numbers of binding prptides rose with the concentrations increasing, and the binding of three peptides were close to saturation at 100μg/ml. Experiment two:The propotion of oxygen on oxidized disks increased. The affinity of minTBP-1-PRGDN and minTBP-1 to oxidized Ti disks increased while that of PRGDN dramatically decreased. Experiment three:With the increasing proportion of unlabeled peptide mixing into labeled peptide, fluorescent pixels in three groups all decreased, the FITC labling did not affect the peptide.binding ability.
Part Two:The effect of minTBP-1-PRGDN on osteoblast adhension
Object To explore the effects of different Ti coatings with minTBP-1-PRGDN, minTBP-land PRGDN on osteoblast attachment, spreading and the expression of adhension-relative gene. Methods Ti disks were wet-polished, washed, oxidized and sterilized sequentially, and then incubated overnight with three kinds of unlabled peptides separativly, washed with ddH2O to remove unbound peptides. Osteoblast were seeded on Ti disks and adhered for some time(1h in experiment 4 and 5,24h in experiment 6), and then non-adhesive cells were washed with PBS. Experiment four:The quantification of adhesive cells was colorimetry using Alamar Blue. Experiment five:The morphous of adhered adhesive cells were stained with phalloidin-FITC and taken photos with fluorescence microscope. And then the extent of cell spreading on Ti disks was measured quantitatively by measuring the cell areas with Image-Pro? Plus software package. Experiment six:The adhension-relative gene integrinβ1 and cadherin-11 were detected by RT-PCR.
Results Experiment four:The number of attached cells was most on minTBP-1-PRGDN pre-coated disks and least on PRGDN pre-coated disks. Experiment five:Cells adhering on minTBP-1-PRGDN pre-coated disks exhibited active spreading and mainly oblate or round shape, lacking in pseudopodia. Cells in PRGDN pre-coated group were almost cuboidal with a few pseudopodias and had the least spreading area. Experiment six:The adhension-relative gene Integrinβ1 and Cadherin-11 were expressed most obviously in PRGDN group.
Part Three:The effect of minTBP-1-PRGDN on osteoblat proliferation
Object To explore the effects of different Ti coatings with minTBP-1-PRGDN, minTBP-1and PRGDN on osteoblast proliferation, including cell numbers and the expression of proliferation-relative gene.
Methods Experiment seven:MC3T3-E1 osteoblasts proliferated on different pre-coated disks for 24h,48h and 72h, and then cell numbers were evaluated by MTT assay, and cell shape and density were observed with metallurgical microscope after Giemsa's staining. Experiment eight:The proliferation-relative gene collagen Iα1 and cyclin Dl were detected by RT-PCR.
Results Experiment seven:Cell numbers in all groups were increasing with the time in four groups. At 24h, cell number of minTBP-1-PRGDN group was the maximum and significant difference was found by comparing with control group. Osteoblasts presented fusiform shape and lined according with the orientation of Ti disk burnishing. Experiment eight:The expression of cyclin D1 gene were increasing with the time in all groups except control group. At 72h, the gene expression of cyclin Dl was most conspicuous. For the expression of collagen Iα1 gene, PRGDN group was the most at 24h, and control group was the least at 48h, and minTBP-1 group was the most at 72h.
Part Four:The effect of minTBP-1-PRGDN on osteoblat differentiation and function
Object To explore the effects of different Ti coatings with minTBP-1-PRGDN. minTBP-1and PRGDN on osteoblast differentiation and function, including ALP activity, expression of differentiation-relative gene, and formation of mineralized nodules.
Methods Experiment nine:Osteoblasts were incubated, ALP activity was determined as the rate of p-nitrophenol release from p-nitrophenylphosphate substrate at day 3,7,10,14, 21 and 28. Experiment ten:The differentiation-relative gene were detected by RT-PCR, including ALP, OPN, BSP and OC. Experiment eleven:After being incubated with differentiation medium for 28d, osteoblasts were stained with Alizarin red-S for displaying mineralized nodules. And then nodules were taken photos with metallurgical microscope and their numbers and size were measured quantitatively with Image-Pro? Plus software package.
Results Experiment nine:ALP activity increased slowly from day 3 to 10, and increased acceleratively from day 14 and reached the peak value at day 28. Experiment ten: OPN、OC、BSP and ALP genes in minTBP-1-PRGDN group all increased firstly and dropped afterwards. However, the four genes in minTBP-1 group increased sustainedly with time. In control group, OPN、BSP and ALP genes increased sustainedly, but OC gene decreased firstly and raised then. In PRGDN group, OPN、OC and ALP genes decreased firstly and raised then, but BSP increased firstly and dropped afterwards. The expression of four genes highestly in PRGDN group on day 7, and in minTBP-1-PRGDN group on day 14. Experiment eleven:The average area of mineralized nodules was the biggest in minTBP-1-PRGDN group and the smallest in minTBP-1-group. The numbers of nodules were the most in PRGDN group and the least in minTBP-1-group.
第一部分融合肽minTBP-1-PRGDN与钛表面相互作用的研究
目的探讨融合肽minTBP-1-PRGDN、minTBP-1及PRGDN在不同浓度时对钛亲和性的影响；氧化和未氧化钛表面对三种肽结合能力的影响；FITC标记对肽结合能力影响。
方法实验一：钛片经顺序打磨、清洗、消毒备用,三种肽以不同浓度(1μg/ml,10μg/ml, 100μg/ml, 1000μg/ml)孵育钛片过夜,双蒸水洗去未结合的肽。实验二：钛片经顺序打磨、清洗、一部分经30%硝酸氧化(另一部分不氧化)、消毒备用,XPS检测氧化与未氧化钛片表面元素成分；三种肽以100μg/ml浓度分别孵育氧化和未氧化的钛片过夜,双蒸水洗去未结合的肽。实验三：钛片经顺序打磨、清洗、氧化、消毒备用,三种肽以100μg/ml浓度按照未标记与标记比例为0：1(None)、10:1、100:1分别孵育钛片过夜,双蒸水洗去未结合的肽。荧光显微镜拍照,IPP图像分析软件采集荧光照片的像素点、以平均每个钛片上的荧光像素点的数量作为结合到钛片表面的肽数量的间接反映。
结果实验一：三种肽随着浓度的增加其结合到钛片上的数量均逐渐增加,100μg/ml时各种肽在钛片的结合接近饱和。实验二：经氧化后的钛片表面氧元素含量增加；minTBP-1-PRGDN和minTBP-1在氧化的钛片表面的结合数量增加,而PRGDN肽数昊减少。实验三：随着未标记肽掺入到标记肽中的比例增加,钛片表面荧光数量减少。
第二部分融合肽minTBP-1-PRGDN对成骨细胞粘附的影响
目的探讨不同肽涂层的钛表面对成骨细胞附着数量、伸展形态及面积、粘附相关基因表达的影响。
方法钛片经顺序打磨、清洗、氧化、消毒备用,三种肽以100μg/ml浓度孵育钛片表面过夜,未有肽孵育组作为空白对照；PBS洗去未结合的肽；成骨细胞在钛片表面生长一定时间后(实验四、五为1h,实验六为24h),PBS洗去未粘附细胞。实验四：Alamar Blue比色法检测附着细胞的数量;实验五：FITC-鬼笔环肽染色粘附细胞,荧光显微镜照相,IPP软件分析细胞伸展面积；实验六：RT-PCR荧光定量检测Integrinβ1和Cadherin-11基因表达。
结果实验四：minTBP-1-PRGDN涂层钛片上附着的成骨细胞数量最多、PRGDN涂层组的细胞数量最少：实验五：minTBP-1-PRGDN融合肽涂层的钛片表面的成骨细胞伸展充分、面积最大,但细胞伪足少；PRGDN涂层的钛片表面成骨细胞伸展最不充分,多接近于立方形,伸展面积与其他各组相比也是最小；实验六：Integrinβ1和Cadherin-11基因表达量在PRGDN组均为最高。
第三部分融合肽minTBP-1-PRGDN对成骨细胞增殖的影响
目的探讨不同肽涂层的钛表面对成骨细胞增殖数量、形态及相关基因表达的影响。
方法实验七：MTT比色法检测四组成骨细胞在24h、48h、72h时的增殖数量；细胞经Giemsa's染色后金相显微镜观察在增殖72h时的形态；实验八：RT-PCR荧光定量检测Collagen la 1和Cyclin D1基因表达。
结果实验七：各组细胞随时间延长数量逐渐增加,24h时minTBP-1-PRGDN涂层钛片表面的成骨细胞数量最多；72h时各组细胞大部分呈长梭形,伸展充分,并沿着钛片表面打磨的痕迹方向排列；实验八：cyclin D1:对照组外,其余各组表达量随时间延长而增加;达到72h时,minTBP-1-PRGDN组的cyclin D1表达量最大。CollagenⅠα1：各组表达量随时间延长降低后又有所增高；24 h时,PRGDN组的表达量最高；48h时,空白对照组表达量最低；72h时,minTBP-1组表达量最高。
第四部分融合肽minTBP-1-PRGDN对成骨细胞分化及功能的影响
目的探讨不同肽涂层的钛表面对成骨细胞分化过程中ALP酶活性、矿化相关基因表达、矿化结节形成数量及面积大小的影响。
方法实验九：PNPP法检测成骨细胞诱导分化第3,7,10,14,21,28d时的ALP酶活性;实验十：RT-PCR荧光定量检测ALP、OPN、BSP、OC基因表达；实验十一：细胞诱导矿化28d后,茜素红染色矿化结节,金相显微镜照相,IPP软件分析各组结节数量及平均面积。
结果实验九：各组成骨细胞诱导3-10d内,ALP活性值缓慢递增,从14d开始,ALP量的增加开始加速,28d时达到峰值；实验十：minTBP-1-PRGDN涂层表面成骨细胞分泌的OPN、OC、BSP及ALP四种基因的表达趋势均为：随着时间的延长,表达量先上升后降低,且在第14天时达到高峰。而minTBP-1涂层表面成骨细胞分泌的这四种基因的表达趋势均为随着时间的延长,表达量逐渐上升。空白对照组中,除了OC表达先降低后增高以外,其余三种基因的表达均为逐渐升高。而PRGDN组中除了BSP的表达先增加后减少外,其他三种基因的表达均为先降低升高。四种基因在第7d时,PRGDN组的表达均为最高；第14d时minTBP-1-PRGDN组表达量最高；实验十一：minTBP-1-PRGDN组的结节面积最大而minTBP-1组的平均面积最小；结节数量在PRGDN组最多而minTBP-1组最少。
Titanium (Ti) has been widely used as an embedding biomaterial in dental implants, artificial joints and so on, owing to its good biocompatibility, osseointegration, resistance to corrosion and low allergenicity. The key point of successful implant is the achievement of fine stability between Ti and bone tissue. Various kinds of natural or artificial peptides and proteins have been widely investigated to promote osseointegration. The RGD (Arg-Gly-Asp) sequence, which can be found in most extracellular matrix (ECM) proteins, including some mineralized-related proteins, is the most effective and most often employed peptide sequence for improving cell adhesion on biomaterial surfaces. Stable immobilization of peptides on the surface of biomaterials is essential to promote strong cell adhesion. RGD peptides commonly covalently bind to the surface of biomaterials via functional moieties such as hydroxyl, amino, and carboxyl radicals. However, many problems may arise in these coupling methods:besides the complex treatment procedures, the coupling reagent may be cytotoxic or the active group may be deactivated quickly by hydrolysis. A new peptide aptamer, named TBP-1 (RKLPDAPGMHTW) which is isolated from a linear 12-mer peptide phage library, has been proved to have the character of specifically interacting with the surface of Ti. Its N-terminal, RKLPDA (minTBP-1), is sufficient for Ti binding. Considering its specific and strong binding ability, minTBP-1 has been used in the form of combination with other motifs, which endowed the chimeric peptides with specific binding ability to Ti. In this study, we synthesized a chimeric peptide with double motifs containing a RGD and a minTBP-1 sequence, and investigated its binding ability to Ti and its effect to osteoblast on attachment, spreading, proliferation, differentiation and mineralization ability as well.
Part One:The interaction between minTBP-1-PRGDN and titanium
Object To explore the affinities of minTBP-1-PRGDN, minTBP-1and PRGDN to Ti at different concentrations; the different affinities of three peprtides to unoxidized and oxidized Ti; the influence of FITC labling on peptide affinity.
Methods Experiment one:Ti disks were wet-polished, washed and sterilized sequentially, and incubated overnight with three peptides separativly at four different concentrations of 1μg/ml,10μg/ml,100μg/ml and 1000μg/ml, then washed with ddH2O to remove unbound peptides. Experiment two:Ti disks were wet-polished, washed and sterilized sequentially. Half numbers of disks were oxidized with 30% HNO3 and the others were not oxidized. The elements on the surfaces of oxidized and unoxidized Ti disks were detected by XPS. Then all disks were incubated overnight with three peptides separativly at concentration of 100μg/ml, and then washed with ddH2O to remove unbound peptides. Experiment three:Ti disks were wet-polished, washed, oxidized and sterilized sequentially, and then incubated overnight with the combination of unlabeled and FITC-labeled peptides in the proportion of 0:1,10:1, and 100:1, washed with ddH2O to remove unbound peptides. Photos of coated disks were taken by fluorescence microscope and analyzed by Image-Pro(?) Plus software package by measuring the average number of fluorescent pixels per disk.
Results Experiment one:The numbers of binding prptides rose with the concentrations increasing, and the binding of three peptides were close to saturation at 100μg/ml. Experiment two:The propotion of oxygen on oxidized disks increased. The affinity of minTBP-1-PRGDN and minTBP-1 to oxidized Ti disks increased while that of PRGDN dramatically decreased. Experiment three:With the increasing proportion of unlabeled peptide mixing into labeled peptide, fluorescent pixels in three groups all decreased, the FITC labling did not affect the peptide.binding ability.
Part Two:The effect of minTBP-1-PRGDN on osteoblast adhension
Object To explore the effects of different Ti coatings with minTBP-1-PRGDN, minTBP-land PRGDN on osteoblast attachment, spreading and the expression of adhension-relative gene. Methods Ti disks were wet-polished, washed, oxidized and sterilized sequentially, and then incubated overnight with three kinds of unlabled peptides separativly, washed with ddH2O to remove unbound peptides. Osteoblast were seeded on Ti disks and adhered for some time(1h in experiment 4 and 5,24h in experiment 6), and then non-adhesive cells were washed with PBS. Experiment four:The quantification of adhesive cells was colorimetry using Alamar Blue. Experiment five:The morphous of adhered adhesive cells were stained with phalloidin-FITC and taken photos with fluorescence microscope. And then the extent of cell spreading on Ti disks was measured quantitatively by measuring the cell areas with Image-Pro? Plus software package. Experiment six:The adhension-relative gene integrinβ1 and cadherin-11 were detected by RT-PCR.
Results Experiment four:The number of attached cells was most on minTBP-1-PRGDN pre-coated disks and least on PRGDN pre-coated disks. Experiment five:Cells adhering on minTBP-1-PRGDN pre-coated disks exhibited active spreading and mainly oblate or round shape, lacking in pseudopodia. Cells in PRGDN pre-coated group were almost cuboidal with a few pseudopodias and had the least spreading area. Experiment six:The adhension-relative gene Integrinβ1 and Cadherin-11 were expressed most obviously in PRGDN group.
Part Three:The effect of minTBP-1-PRGDN on osteoblat proliferation
Object To explore the effects of different Ti coatings with minTBP-1-PRGDN, minTBP-1and PRGDN on osteoblast proliferation, including cell numbers and the expression of proliferation-relative gene.
Methods Experiment seven:MC3T3-E1 osteoblasts proliferated on different pre-coated disks for 24h,48h and 72h, and then cell numbers were evaluated by MTT assay, and cell shape and density were observed with metallurgical microscope after Giemsa's staining. Experiment eight:The proliferation-relative gene collagen Iα1 and cyclin Dl were detected by RT-PCR.
Results Experiment seven:Cell numbers in all groups were increasing with the time in four groups. At 24h, cell number of minTBP-1-PRGDN group was the maximum and significant difference was found by comparing with control group. Osteoblasts presented fusiform shape and lined according with the orientation of Ti disk burnishing. Experiment eight:The expression of cyclin D1 gene were increasing with the time in all groups except control group. At 72h, the gene expression of cyclin Dl was most conspicuous. For the expression of collagen Iα1 gene, PRGDN group was the most at 24h, and control group was the least at 48h, and minTBP-1 group was the most at 72h.
Part Four:The effect of minTBP-1-PRGDN on osteoblat differentiation and function
Object To explore the effects of different Ti coatings with minTBP-1-PRGDN. minTBP-1and PRGDN on osteoblast differentiation and function, including ALP activity, expression of differentiation-relative gene, and formation of mineralized nodules.
Methods Experiment nine:Osteoblasts were incubated, ALP activity was determined as the rate of p-nitrophenol release from p-nitrophenylphosphate substrate at day 3,7,10,14, 21 and 28. Experiment ten:The differentiation-relative gene were detected by RT-PCR, including ALP, OPN, BSP and OC. Experiment eleven:After being incubated with differentiation medium for 28d, osteoblasts were stained with Alizarin red-S for displaying mineralized nodules. And then nodules were taken photos with metallurgical microscope and their numbers and size were measured quantitatively with Image-Pro? Plus software package.
Results Experiment nine:ALP activity increased slowly from day 3 to 10, and increased acceleratively from day 14 and reached the peak value at day 28. Experiment ten: OPN、OC、BSP and ALP genes in minTBP-1-PRGDN group all increased firstly and dropped afterwards. However, the four genes in minTBP-1 group increased sustainedly with time. In control group, OPN、BSP and ALP genes increased sustainedly, but OC gene decreased firstly and raised then. In PRGDN group, OPN、OC and ALP genes decreased firstly and raised then, but BSP increased firstly and dropped afterwards. The expression of four genes highestly in PRGDN group on day 7, and in minTBP-1-PRGDN group on day 14. Experiment eleven:The average area of mineralized nodules was the biggest in minTBP-1-PRGDN group and the smallest in minTBP-1-group. The numbers of nodules were the most in PRGDN group and the least in minTBP-1-group.
引文
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