新型活性修饰聚乳酸组织工程骨的体外成骨研究

Osteogenic ability of poly-D,I-lactide acid(PDLLA) scaffolds affected by bioactive modification in vitro

[目的]探讨氨等离子体改性、酰胺键接枝甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(GRGDS)短肽的活性修饰方法对消旋聚乳酸(PDLLA)组织工程骨体外成骨能力的影响。[方法]制备圆片状直径8 mm、厚1 mm的PDLLA三维多孔支架,分为三组:表面氨基化PDLLA(aminated PDLLA,A/PDLLA,A组),接枝肽A/PDLLA(peptides conjugated A/PDLLA,PA/PDLLA,PA组),以未经处理的PDLLA(P组)作为对照组。实时荧光定量聚合酶链式反应(RT-qPCR)检测各组材料上骨髓间充质干细胞(BMSCs)、骨钙素(OCN)、Ⅰ型胶原(Col-Ⅰ)、碱性磷酸酶(ALP)、骨形态发生蛋白-2(Bmp-2)和骨桥蛋白(OPN)mRNA的表达变化并进行ALP活性测定和钙黄绿素矿化荧光染色。[结果]qPCR结果示:第3 d,PA组各基因表达倍数均较P组高;除OCN外的各基因表达也均较A组高。第7 d,A组OCN(13.13±1.28)、Col-Ⅰ(23.71±6.51)和OPN(27.4±7.17)mRNA表达倍数为最高,PA组次之。第14 d,多数基因表达上调,A组和PA组表达倍数较对照组明显增高。PA组OCN(49.21±7.03)、ALP(24.26±3.41)和BMP-2(11.82±2.38)mRNA的表达较A组高,差异有统计学意义(P<0.05)。ALP活性检测结果示:A组和PA组ALP活性持续升高,P组活性第21 d较第14 d时略有下降。第7 d,A组和PA组之间ALP活性比较无统计学意义(P>0.05),但均高于P组(P<0.01);第14 d和第21 d,三组间ALP活性两两比较均有统计学意义,ALP活性PA组>A组>P组。钙黄绿素矿化荧光染色观察示,骨支架上钙盐沉积与ALP的活性变化趋势相平行。[结论]氨等离子体改性能够促进聚乳酸组织工程骨支架上BMSCs早期向成骨细胞分化,改性后接枝GRGDS肽的新型活性修饰PDLLA具有更好的促BMSCs体外成骨能力。

[Objective]To study the effects of bioactive modification of ammonia plasma and conjugated Gly-Arg-Gly-Asp-Ser（GRGDS） to Poly-D,L-lactide acid（PDLLA） in vitro. [Methods]PDLLA scaffolds were made in a diameter of 8 mm-circle with 1 mm-thickness and they were divided into 3 groups： Group P（untreated PDLLA） as control group,Group A（aminated PDLLA,A/PDLLA）and Group PA（peptides conjugated A/PDLLA,PA/PDLLA）.Real time quantitative PCR（RT-qPCR） was used to detect the osteocalcin（OCN）,collagen Ⅰ（Col-Ⅰ）,alkaline phosphatase（ALP）,bone morphogenetic protein 2（BMP-2） and osteopontin（OPN） mRNA expression of bone marrow mesenchymal stem cells（BMSCs） in scaffolds in each group.Activity of alkaline phosphatase and staining of the calcification were studied by calcein fluorescent dye as well.[Results]The results from qPCR showed that bone-related gene expression in Group PA was higher than Group P at 3 days.Also,it was higher than Group A except OCN mRNA on the same day.The fold changes of gene expression of OCN（13.13±1.28）,Col-Ⅰ（23.71±6.51）and OPN（27.4±7.17） in Group A were the highest among the three groups at 7 days,and it was taken second place in Group PA.Most of the gene expressions were elevated in all groups at 14 days.The fold changes of gene expression in Group A and Group PA were higher than the control.OCN（49.21±7.03）,ALP（24.26±3.41） and BMP-2（11.82±2.38） mRNA in Group PA was significantly higher than Group A（P0.05）.ALP activity rose continuously in groups A and PA.It was slightly declined in Group P at 21 days.There were no statistical significances in ALP between groups A and PA（P0.05）.But,both were higher than the controls（P0.01）.In groups P,A,and PA,there were statistical significances in ALP between each pair of groups at 14 and 21 days.ALP activity in Group PA was the highest among the three groups.Group P was the lowest one.Calcein fluorescent staining in vitro showed parallel relation between mineralization and ALP activity.[Conclusion]The results indicate that the treatment of ammonia plasma will promote early osteo-differentiation of BMSCs in PDLLA scaffolds,and bioactive modification of PDLLA by ammonia treatment and subsequent conjugation of GRGDS peptides may have better ossification in vitro.