覆盖富血小板血浆3D打印聚己内酯支架对牙髓细胞体外生物学行为的影响

The influence of 3D-printed polycaprolactone scaffolds coated with platelet-rich plasma on the biological functions of dental pulp cells

目的探讨覆盖不同处理方法富血小板血浆(PRP)的3D打印聚己内酯(PCL)支架对牙髓细胞黏附、增殖及分化的影响。方法用冻干法和凝胶法制备PRP并覆盖在3D打印的聚己内酯支架上,免疫荧光染色观察各组支架牙髓细胞的早期黏附情况,细胞计数试剂盒(CCK-8)检测细胞增殖情况,Transwell小室检测细胞迁移情况,碱性磷酸酶(ALP)试剂盒测定ALP活性,实时荧光定量聚合酶链反应(PCR)检测细胞成骨相关基因的表达。采用单因素方差分析进行统计分析,LSD-t检验进行两两比较。结果免疫荧光结果显示黏附在冻干组的细胞最多(1999.33个/总视野),凝胶组次之(1043.33个/总视野),单纯支架组最少(843.00个/总视野),而且冻干组和凝胶组相比,差异有统计学意义(F=19.36,P<0.01),冻干组和单纯支架组对比,差异有统计学意义(F=23.42,P<0.01)。细胞增殖实验显示,3组支架的细胞都呈增长趋势,但是在各个测定时间点,任何2组之间的细胞增殖都没有显著差异。细胞迁移结果显示,冻干组(279.75个/视野)和凝胶组(167.00个/视野)的细胞数目显著高于单纯支架组(134.75个/视野),差异有统计意义(F_(冻单组)=7.45、F_(凝单组)=1.88,P<0.01),而冻干组和凝胶组相比则差异无统计学意义。ALP活性结果显示,3个组别中冻干组活性最高(ALP_(7d)=12.57 U/gprot、ALP_(14d)=23.20 U/gprot、ALP_(21d)=58.98 U/gprot),在第7天凝胶组次之(6.65 U/gprot),单纯支架组最低(5.93 U/gprot),而在第14和21天,单纯支架组次之(ALP_(14d)=15.56 U/gprot、ALP_(21d)=53.74 U/gprot),凝胶组最低(ALP_(14d)=13.35 U/gprot、ALP_(21d)=47.83 U/gprot)。在第7、14和21天,冻干组的ALP活性显著高于凝胶组(F_(7d)=3.20,P_(7 d))<0.01;F_(14 d)=5.34,P_(14 d)<0.01;F_(21 d)=6.04,P_(21 d)<0.01)和单纯支架组(F_(7 d)=3.60,P_(7 d))=0.04;F_(14 d)=4.14,P_(14 d)<0.01;F_(21 d)=2.84,P_(21 d)=0.01)。在第7和14天,凝胶组和单纯支架的细胞ALP活性相比差异没有统计学意义。成骨相关基因表达中,在第7和14天的RUNX2和OCN表达中,除了第14天的凝胶组的OCN表达外,冻干组(OCN_(7 d)=4.67、RUNX2_(7 d)=2.32、RUNX2_(14 d)=5.88)的表达高于单纯支架组(OCN_(7 d)=1.00、RUNX2_(7 d)=1.00、RUNX214 d=1.00),差异具有统计意义(F_(7 d)=11.1,P_(7 d))<0.01;F_(7 d)=3.20,P_(7 d))=0.04;F_(14 d)=11.80,P_(14 d)<0.01),凝胶组(OCN_(7 d)=2.60、RUNX2_(7 d)=2.23、RUNX214 d=4.67)的表达显著高于单纯支架组,差异有统计学意义F_(7 d)=4.85,P_(7 d))<0.01;F_(7 d)=2.98,P_(_(7 d))=0.03;F_(14 d)=8.87,P_(14 d)<0.01)。在第7天的OCN和第14天的RUNX2的表达中,冻干组(OCN_(7 d)=4.67)的表达水平高于凝胶组(OCN_(7 d)=2.60),差异有统计学意义(F=6.26,P<0.01);而在第14天的OCN中,凝胶组表达和冻干组无明显差异。结论覆盖PRP的3D打印PCL支架比单纯的支架更有利于牙髓细胞的黏附、增殖和成骨分化,且冻干法优于凝胶法。

Objective To study the influence of different 3D-printed polycaprolactone scaffolds coated with platelet-rich plasma on the adhesion,proliferation and differentiation of dental pulp cells （DPCs）. Methods DPCs were seeded on the scaffold of three groups. Cell attachment,proliferation and ALP activity were evaluated with immunofluorescence staining,CCK-8 assay and ALP kit,respectively. The expression of osteogenic genes was determined with RT-PCR. Results More cell attachment was found on the freeze-dried PRP-PCL scaffold（1999.33/field）than the rest（1043.33 and 843.00/field,F=19.36,P〈0.01）. In the cell proliferation test,the amount of cells was increased in all groups but there was no statistically significant difference between groups of the same day. The number of migrated cells at the bottom of the transwell chamber in the freeze-dried PRP-PCL scaffold group（279.75/field） was significantly higher than that of the gelatinous PRP-PCL scaffold group（167.00/field）and the bare PRP-PCL scaffold group（134.75/field）（Ff-b=7.45,Fg-b=1.88,P〈0.01）. There was no significant difference in the migrated cells between the gelatinous PRP-PCL scaffold and bare PRP-PCL scaffold. The ALP activity of the freeze-dried PRP-PCL scaffold（ALP7 d=12.57 U/gprot,ALP14 d=23.20 U/gprot,ALP21 d=58.98 U/gprot）was significantly higher than that of the gelatinous PRP-PCL scaffold（ALP7 d=6.65 U/gprot, ALP14 d=13.35 U/gprot,ALP21 d=47.83 U/gprot）and the bare PCL scaffold（ALP7 d=5.93 U/gprot、ALP14 d=15.56 U/gprot、ALP21 d=53.74 U/gprot）on 7,14 and 21 d（F7 d=3.20,P7 d〈0.01;F14 d=5.34,P14 d〈0.01;F21 d=6.04,P21 d〈0.01）（F7 d=3.60,P7 d=0.04;F14 d=4.14,P14 d〈0.01;F21 d=2.84,P21 d=0.01）. However,no statistically significant difference was found in the ALP activity between the gelatinous PRP-PCL scaffold and the bare PCL scaffold except for that on 21 d. Except for the expression of OCN by DPSCs on the gelatinous PRP-PCL scaffold on 14 d,the expression of RUNX2 and OCN by DPSCs on freeze-dried PRP-PCL scaffold（OCN7 d=4.67,RUNX27 d=2.32,RUNX214 d=5.88）and gelatinous PRP-PCL scaffold （OCN7 d=2.60,RUNX27 d=2.23,RUNX214 d=4.67）on 7 and 14 d were significantly higher than that on the bare scaffold（OCN7 d=1.00,RUNX27 d=1.00,RUNX214 d=1.00）（F7 d=11.1,P7 d〈0.01;F7 d=3.20, P7 d=0.04;F14 d=11.80,P14 d〈0.01）. The expression of OCN on7 d and RUNX2 on 14 d on the freeze-dried PRP-PCL scaffold（OCN7 d=4.67）was significantly higher than that of gelatinous PRP-PCL scaffold （OCN7 d=2.60,F=6.26,P〈0.01）. However,no statistical significance was found in the expression of OCN on 14 d between the freeze-dried PRP-PCL scaffolds and the gelatinous PRP-PCL scaffolds. Conclusion The freeze-dried PRP-PCL scaffolds were more favorable for the adhesion,proliferation and differentiation of DPCs compared with the gelatinous PRP-PCL scaffolds.