RGD多肽表面修饰同种异体骨的方法研究
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摘要
目的：1．探讨应用紫外辐射与化学耦和的方法，将含ROD序列的多肽共价接枝到同种异体骨表面，以提高材料对细胞的粘附能力，2．采用正交试验方案，选择将含RGD序列的多肽共价结合于同种异体骨片上最佳的试验因素及水平的组合。
方法：采用紫外辐射活化同种异体骨表面的羧基，EDC作为耦和剂，将人工合成的含RGD序列的八肽(谷氨酸-脯氨酸-精氨酸-甘氨酸-天冬氨酸-天冬酰胺-酪氨酸-精氨酸，Glu-Pro-Arg-Gly-Asp-Asn-Tyr-Arg，EPRGDNYR)共价接枝在骨片表面。以空白骨片为对照，用环境扫描电子显微镜观察骨片表面形貌变化，通过X线光电子能谱分析修饰表面的元素含量变化检测RGD多肽接枝情况。采取正交试验拟定RGD浓度为0.1mg／ml、0.5mg／ml和1mg／ml，紫外辐射时间为30、60和90分钟，EDC浓度为1％、2％和3％3个水平，进行正交试验。以氮元素含量为考核指标，对9次试验结果进行直观分析，确定试验因素对结果影响的主次关系以及每个因素的最佳水平，找出最佳组合。
结果：经环境扫描电子显微镜观察发现固定RGD多肽后的骨片表面粗糙程度较空白骨片均有不同程度降低，骨盐支架结构稀疏，孔隙内被大量基质成分所充填，孔隙密度减小。接枝多肽后的骨片表面形貌均有所改变。X线光电子能谱分析仪(XPS)检测表明接枝多肽后的骨片其氮原子含量均较空白骨片有不同程度地增加，骨片上的酰胺键增强，证明多肽成功地固定于同种异体骨片表面。正交实验结果为：A因素中1、2、3水平和分别为12、16.7、10.3，极差为6.4，水平2较优；B因素中1、2、3水平和分别为12.5、14.8、11.7，极差为3.1，水平2较优：C因素中1、2、3水平和分别为11.7、10.6、16.7，极差为6.1，水平3较优。由此得出RGD浓度在三者中影响最重要，EDC浓度其次，紫外辐射时间再次之，最佳组合为A_2B_2C_3。
结论：经紫外辐射活化羧基，EDC耦合接枝RGD的方法，可成功将RGD多肽共价结合于同种异体骨片表面。由正交分析可知影响本实验因素的主次关系依次为RGD浓度，EDC浓度，紫外辐射时间。最佳试验方案为RGD浓度为0.5mg／ml，紫外辐射60分钟，EDC浓度为3％。
Objective: 1. To investigate the method with application ofultraviolet radiation and the chemical coupling to graftpolypeptide containing ROD series on allograft bone throughcovalent bond in order to enhance the adhesion ability of thematerial. 2. To choose the best combination of experimentalfactor and level to affect the graft the polypeptide containingROD series on allograft bone fragment by orthogonal experimentdesign.
Methods: The carboxyl group on the surface of allograft bonewas activated by ultraviolet radiation. Using EDC as thecoupling agent, the synthetic RGD containing octapeptide(Glu-Pro-irg-Gly-isp-Asn-Tyr-Arg, EPRGDNYR) was bonded to thesurface of allograft bone through covalent bond. Blank bonefragment was used as control, morphological change on theallograft bone surface was observed by the environmentalscanning electronic microscope. The grafting of RGDpolypeptide was inspected by the change of element content onthe surface analyzed by X-ray photoelectronspectroscopy(XPS). By orthogonal tests, the concentration ofRGD was subjected to be 0.1mg/ml, 0.5mg/ml and 1.0mg/ml, theduration for ultraviolet radiation, 30, 60 and 90 minutes, andthe concentration of EDC, 1％, 2％and 3％respectively, and theorthogonal experiment was conducted using above factors. Theresults of 9 experiments were analyzed, using the content of nitrogen as the assessment indicator so as to determine theeffect of experimental factors to the results and the optimumlevels of each factorsto find out the best combination of thesefactors.
Results: By observing under the environmental scanningelectronic microscope, we found out that the surface asperityof allograft bone fragments bonded with RGD polypeptide waslower than the blank ones. The frame structure of bone saltwas also sparse, with the holes filled with numerous groundsubstances. Hole separation density was decreased. Changeswere seen on the morphology of surface of polypeptide bondedbone fragments. Examined by XPS, the content of nitrogen atomsin polypeptide bonded bone fragments was higher than the blankcounterparts in various degrees. The amido bond in the bonefragment was strengthened, showing that polypeptide wassuccessfully bonded to the surface of allograft bone. Theresults of orthogonal tests are: the sum of level 1,2 and 3of factor A are 12,16.7 and 10.3 respectively, with level 2wasbetter. The sum of level 1,2 and 3 of factor B are 12.5,14.8and 11.7 respectively, with level 2 was better. The sum of level1,2 and 3 of factor C are 11.7,10.6 and 16,7 respectively, withlevel 3 was better. From the results, it can be seen that theconcentration of RGD is the most important among the threefactors, seconded by concentration of EDC, with the durationof ultraviolet radiation being the least important. The bestcombination is then A_2B_2C_3.
Conclusion: By activating the carboxyl group with ultravioletradiation, and coupling EDC and RGD, we successfully adherethe RGD polypeptide onto the surface of allograft bone bycovalent bond. From the orthogonal analyses, it can be seen that the factors affecting this experiment are, ranking fromthe most important one, RGD concentration, EDC concentration,and ultraviolet radiation time. The best experiment solutionwould be: RGD concentration, 0.5mg/ml, EDC concentration, 3%and ultraviolet radiation time, 60 minutes.
方法：采用紫外辐射活化同种异体骨表面的羧基，EDC作为耦和剂，将人工合成的含RGD序列的八肽(谷氨酸-脯氨酸-精氨酸-甘氨酸-天冬氨酸-天冬酰胺-酪氨酸-精氨酸，Glu-Pro-Arg-Gly-Asp-Asn-Tyr-Arg，EPRGDNYR)共价接枝在骨片表面。以空白骨片为对照，用环境扫描电子显微镜观察骨片表面形貌变化，通过X线光电子能谱分析修饰表面的元素含量变化检测RGD多肽接枝情况。采取正交试验拟定RGD浓度为0.1mg／ml、0.5mg／ml和1mg／ml，紫外辐射时间为30、60和90分钟，EDC浓度为1％、2％和3％3个水平，进行正交试验。以氮元素含量为考核指标，对9次试验结果进行直观分析，确定试验因素对结果影响的主次关系以及每个因素的最佳水平，找出最佳组合。
结果：经环境扫描电子显微镜观察发现固定RGD多肽后的骨片表面粗糙程度较空白骨片均有不同程度降低，骨盐支架结构稀疏，孔隙内被大量基质成分所充填，孔隙密度减小。接枝多肽后的骨片表面形貌均有所改变。X线光电子能谱分析仪(XPS)检测表明接枝多肽后的骨片其氮原子含量均较空白骨片有不同程度地增加，骨片上的酰胺键增强，证明多肽成功地固定于同种异体骨片表面。正交实验结果为：A因素中1、2、3水平和分别为12、16.7、10.3，极差为6.4，水平2较优；B因素中1、2、3水平和分别为12.5、14.8、11.7，极差为3.1，水平2较优：C因素中1、2、3水平和分别为11.7、10.6、16.7，极差为6.1，水平3较优。由此得出RGD浓度在三者中影响最重要，EDC浓度其次，紫外辐射时间再次之，最佳组合为A_2B_2C_3。
结论：经紫外辐射活化羧基，EDC耦合接枝RGD的方法，可成功将RGD多肽共价结合于同种异体骨片表面。由正交分析可知影响本实验因素的主次关系依次为RGD浓度，EDC浓度，紫外辐射时间。最佳试验方案为RGD浓度为0.5mg／ml，紫外辐射60分钟，EDC浓度为3％。
Objective: 1. To investigate the method with application ofultraviolet radiation and the chemical coupling to graftpolypeptide containing ROD series on allograft bone throughcovalent bond in order to enhance the adhesion ability of thematerial. 2. To choose the best combination of experimentalfactor and level to affect the graft the polypeptide containingROD series on allograft bone fragment by orthogonal experimentdesign.
Methods: The carboxyl group on the surface of allograft bonewas activated by ultraviolet radiation. Using EDC as thecoupling agent, the synthetic RGD containing octapeptide(Glu-Pro-irg-Gly-isp-Asn-Tyr-Arg, EPRGDNYR) was bonded to thesurface of allograft bone through covalent bond. Blank bonefragment was used as control, morphological change on theallograft bone surface was observed by the environmentalscanning electronic microscope. The grafting of RGDpolypeptide was inspected by the change of element content onthe surface analyzed by X-ray photoelectronspectroscopy(XPS). By orthogonal tests, the concentration ofRGD was subjected to be 0.1mg/ml, 0.5mg/ml and 1.0mg/ml, theduration for ultraviolet radiation, 30, 60 and 90 minutes, andthe concentration of EDC, 1％, 2％and 3％respectively, and theorthogonal experiment was conducted using above factors. Theresults of 9 experiments were analyzed, using the content of nitrogen as the assessment indicator so as to determine theeffect of experimental factors to the results and the optimumlevels of each factorsto find out the best combination of thesefactors.
Results: By observing under the environmental scanningelectronic microscope, we found out that the surface asperityof allograft bone fragments bonded with RGD polypeptide waslower than the blank ones. The frame structure of bone saltwas also sparse, with the holes filled with numerous groundsubstances. Hole separation density was decreased. Changeswere seen on the morphology of surface of polypeptide bondedbone fragments. Examined by XPS, the content of nitrogen atomsin polypeptide bonded bone fragments was higher than the blankcounterparts in various degrees. The amido bond in the bonefragment was strengthened, showing that polypeptide wassuccessfully bonded to the surface of allograft bone. Theresults of orthogonal tests are: the sum of level 1,2 and 3of factor A are 12,16.7 and 10.3 respectively, with level 2wasbetter. The sum of level 1,2 and 3 of factor B are 12.5,14.8and 11.7 respectively, with level 2 was better. The sum of level1,2 and 3 of factor C are 11.7,10.6 and 16,7 respectively, withlevel 3 was better. From the results, it can be seen that theconcentration of RGD is the most important among the threefactors, seconded by concentration of EDC, with the durationof ultraviolet radiation being the least important. The bestcombination is then A_2B_2C_3.
Conclusion: By activating the carboxyl group with ultravioletradiation, and coupling EDC and RGD, we successfully adherethe RGD polypeptide onto the surface of allograft bone bycovalent bond. From the orthogonal analyses, it can be seen that the factors affecting this experiment are, ranking fromthe most important one, RGD concentration, EDC concentration,and ultraviolet radiation time. The best experiment solutionwould be: RGD concentration, 0.5mg/ml, EDC concentration, 3%and ultraviolet radiation time, 60 minutes.
引文
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