氨等离子体锚定短肽的消旋聚乳酸骨支架研究

STUDIES ON POLY-D,L-LACTIDE ACID SCAFFOLDS MODIFIED BY CONJUGATION OF BIOACTIVE PEPTIDES VIA AMMONIA PLASMA TREATMENT

目的运用氨等离子体辅助接枝技术,在消旋聚乳酸(poly-D,L-lactide acid,PDLLA)表面以酰胺键接枝甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(Gly-Arg-Gly-Asp-Ser,GRGDS)短肽,探讨制备活性骨替代材料的可行性。方法使用溶剂浇铸/颗粒沥析法制备圆片状直径8mm、厚1mm的PDLLA三维骨支架,行氨等离子体改性处理制备表面氨基化PDLLA(aminated PDLLA,A/PDLLA),测量未改性(0min对照组)及改性5、10、20min组支架的孔径、孔隙率和表面水接触角。以上各组A/PDLLA放入1mg/mLFITC标记的GRGDS肽溶液[1-(3-二甲基氨基丙基)-3-乙基碳化二亚胺盐酸盐、N-羟基琥珀酰亚胺、活性肽的摩尔比值为1.5:1.5:1.0],室温下低速震荡24h行表面接枝处理,得到接枝肽处理材料(peptides conjugated A/PDLLA,PA/PDLLA)。对未改性(0min对照组)、改性5、10、20min及其后接枝肽处理的材料行X射线光电子能谱(X-rayphotoelectron spectroscopy,XPS)仪检测;对PDLLA和改性0、5、10、20min后接枝肽处理的材料行激光共聚焦显微镜观察及高效液相色谱(high performance liquid chromatography,HPLC)定量分析。全骨髓贴壁法分离、培养SD大鼠BMSCs,取第3～6代细胞接种于以下3组材料:20min氨等离子体改性组(A/PDLLA组)、20min氨等离子体改性后接枝肽组(PA/PDLLA组)及未经处理的PDLLA对照组(PDLLA组)。各组BMSCs-材料复合物培养16h后测定细胞上架数及上架率;培养4、8d行扫描电镜观察。结果 PDLLA孔径和孔隙率不随氨等离子体改性时间变化而变化(P>0.05)。随着氨等离子体改性时间延长,A/PDLLA支架表面水接触角逐渐减小,材料亲水性能逐步改善,各组间比较差异均有统计学意义(P<0.001)。XPS检测示,A/PDLLA支架表面出现N元素,且随改性时间延长,N1s峰渐趋明显,N/C逐渐加大;PA/PDLLA支架N/C相应加大,表面出现S2p峰。激光共聚焦显微镜观察示PA/PDLLA支架荧光强度随改性时间延长而明显增强。HPLC成功测得经10min和20min氨等离子体改性后接枝肽处理的PA/PDLLA接枝肽量,两者比较差异有统计学意义(P<0.001);而PDLLA及经0、5min氨等离子体改性后接枝肽处理的PA/PDLLA接枝肽量未检出。与BMSCs复合培养16h,A/PDLLA组和PA/PDLLA组细胞上架数和上架率均高于PDLLA组,3组间两两比较差异均有统计学意义(P<0.01)。扫描电镜观察示,A/PDLLA组和PA/PDLLA组比PDLLA组更能促进BMSCs贴附增殖,尤以PA/PDLLA组最明显。结论氨等离子体改性能明显促进PDLLA表面以酰胺键接枝FITC-GRGDS短肽;此仿生人工骨材料生物活性稳定,能明显促进BMSCs贴附和增殖。

Objective To study the feasibility of preparation of the poly-D,L-lactide acid（PDLLA） scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser（GRGDS） peptides via amide linkage formation.Methods PDLLA scaffolds（8 mm diameter,1 mm thickness） were prepared by solvent casting/particulate leaching procedure and then treated by ammonia plasma.The consequent scaffolds were labeled as aminated PDLLA（A/PDLLA）.The pore size,porosity,and surface water contact angle of groups 0（un-treated control）,5,10,and 20 minutes A/PDLLA were measured.A/PDLLA scaffolds in groups above were immersed into the FITC labelled GRGDS aqueous solution which contain 1-[3-（dimethylamino） propyl]-3-ethylcarbodiimide hydrochloride（EDC.HCl） and N-hydroxysuccinimide（NHS）,the molar ratio of peptides/EDC.HCL /NHS was 1.5：1.5：1.0,then brachytely sloshed for 24 hours in room temperature.The consequent scaffolds were labelled as peptides conjugated A/PDLLA（PA/PDLLA）.The scaffolds in groups 0,5,10,and 20 minutes A/PDLLA and groups correspondingly conjugation of peptides were detected using X-ray photoelectron spectroscopy（XPS）.The scaffolds in groups of conjugation of peptides were measured by confocal laser scanning microscope and high performance liquid chromatography（HPLC）,un-treated and un-conjugated scaffolds employed as control.Bone marrow mesenchymal stem cells（BMSCs） from SD rats were isolated and cultured by whole bone marrow adherent culture method.BMSCs at the 3rd-6th passages were seeded to the scaffolds as follows：20 minutes ammonia plasma treatment（group A/PDLLA）,20 minutes ammonia plasma treatment and conjugation of GRGDS（group PA/PDLLA）,and untreated PDLLA control（group PDLLA）.After 16 hours of culture,the adhesive cells on scaffolds and the adhesive rate were calculated.After 4 and 8 days of culture,the BMSCs/scaffold composites was observed by scanning electron micorscope（SEM）.Results No significant difference in pore size and porosity of PDLLA were observed between before and after ammonia plasma treatments（P 0.05）.With increased time of ammonia plasma treatment,the water contact angle of A/PDLLA scaffolds surface was decreased,and the hydrophilicity in the treated scaffolds was improved gradually,showing significant differences when these groups were compared with each other（P 0.001）.XPS results indicated that element nitrogen appeared on the surface of PDLLA treated by ammonia plasma.With time passing,the peak N1s became more visible,and the ratio of N/C increased more obviously.After PDLLA scaffolds treated for 0,5,10,and 20 minutes with ammonia plasma and subsequent conjugation of peptides,the ratio of N/C increased and the peak of S2p appeared on the surface.The confocal laser scanning microscope observation showed that the fluorescence intensity of PA/PDLLA scaffolds increased obviously with treatment time.The amount of peptides conjugated for 10 minutes and 20 minutes PA/PDLLA was detected by HPLC successfully,showing significant differences between 10 minutes and 20 minutes groups（P 0.001）.However,the amount of peptides conjugated in un-treated control and 0,5 minutes PA/PDLLA scaffolds was too small to detect.After 16 hours co-culture of BMSCs/scaffolds,the adhesive cells and the adhesive rates of A/PDLLA and PA/PDLLA scaffolds were higher than those of PDLLA scaffolds,showing significant difference between every 2 groups（P 0.01）.Also,SEM observation confirmed that BMSCs proliferation in A/PDLLA and PA/PDLLA groups was more detectable than that in PDLLA group,especially in PA/PDLLA group.Conclusion Ammonia plasma treatment will significantly increase the amount of FITC-GRGDS peptides conjugated to surface of PDLLA via amide linkage formation.This new type of biomimetic bone has stablized bioactivities and has proved to promote the adhesion and proliferation of BMSCs in PDLLA.