Construction of PAMAM-PQQ functional layer on titanium surface and its effect on biological behavior of endocardial cells
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摘要: 植/介入手术是心血管疾病最主要的治疗手段。器械植入时与血液直接接触及对植入部位造成损伤，从而引起血栓和炎症等并发症。对材料进行表面改性是提高材料生物相容性的主要方法之一。本文通过在碱活化Ti表面固定树状大分子聚酰胺胺(Polyamido amine，PAMAM)，利用PAMAM的氨基基团接枝抗炎、抗氧化分子吡咯喹啉醌(Pyrroloquinoline quinone， PQQ)，构建了PAMAM-PQQ功能层，并探究了不同浓度PQQ对材料表面生物相容性的影响。FTIR、XPS和氨基定量证明了PQQ成功接枝到材料表面。SEM、水接触角检测证明了材料表面具有多孔网状结构和高度亲水性。血液实验显示功能层降低了血小板的粘附数量和激活程度，表现出较好的抗凝血能力，其中PQQ浓度为300 nmol/mL时效果最优。细胞静态培养结果显示功能层均提高了内皮细胞和心肌细胞的活性及促进两种细胞的增殖与迁移。氧化损伤实验显示功能层能降低H2O2对内皮细胞和心肌细胞损伤，当PQQ浓度为300 nmol/mL时对两种细胞保护效果最佳。综上所述，在钛表面成功构建了PAMAM-PQQ功能层，该功能层具有良好的血液相容性、细胞相容性及抗氧化损伤能力，有望用于心内植入器械表面改性的研发及应用。Abstract: Implantation/interventional surgery is the main treatment for cardiovascular diseases. Direct contact with blood during device implantation and damage to the implantation site can cause complications such as thrombosis and inflammation. Surface modification of materials is one of the main methods to improve the biocompatibility of materials. In this study, the dendrimer polyamide amine (PAMAM) was immobilized on the surface of alkaline activated Ti, and pyrroloquinoline quinone (PQQ), an anti-inflammatory and anti-oxidative molecule, was grafted on the amino group of PAMAM to construct the PAMAM-PQQ functional layer. The effects of different concentrations of PQQ on the surface biocompatibility of the materials were also investigated. FTIR, XPS and amino quantification demonstrated that PQQ was successfully grafted to the surface of the material. SEM and water contact angle detection proved that the surface of the material has porous mesh structure and high hydrophilicity. Blood tests showed that the functional layer reduced the number of platelet adhesion and activation degree, showing good anticoagulant ability, and the best effect was when the PQQ concentration was 300 nmol/mL. Static cell culture results showed that the functional layer improved the activity of endothelial cells and myocardial cells, and promoted the proliferation and migration of the two kinds of cells. Oxidative damage experiment showed that the functional layer could reduce the damage of H2O2 on endothelial cells and myocardial cells, and the best protective effect was achieved when the concentration of PQQ was 300 nmol/mL. In summary, the functional layer of PAMAM-PQQ was successfully constructed on the Titanium surface, which has good blood compatibility, cell compatibility and antioxidant damage ability, and is expected to be used for the development and application of surface modification of cardiac implantable devices.
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Key words:
- surface modification /
- pyrroloquinoline quinone /
- anti-coagulation /
- cell compatibility /
- antioxidation
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图 1 聚酰胺胺(PAMAM)-吡咯喹啉醌(PQQ)功能层的制备过程
EDC—1-ethyl-(3-dimethylaminopropyl)carbamide diimide; NHS—N-hydroxysuccinimide
Figure 1. Preparation process of polyamido amine (PAMAM)-pyrroloquinoline quinone (PQQ) functional layer
图 4 样品表面氨基量
**—Probability p<0.01; ***—p<0.001
Figure 4. Amino content on sample surface
图 7 改性后样品TiPP300表面PQQ释放量
Figure 7. Release of PQQ from the modified TiPP300 sample surface
图 8 各样品表面血小板荧光及数量统计结果
Figure 8. Results of platelet fluorescence and number on the sample surface
图 10 样品表面30 min和45 min凝血结果
Figure 10. Coagulation results at 30 min and 45 min on the sample surface
OD—Optical density
图 11 各样品内皮细胞静态培养结果
CCK-8—Cell counting kit-8; ECs—Endothelial cells
Figure 11. Results of endothelial cell culture of samples
图 12 各样品心肌细胞静态培养结果
MCs—Myocardial cells
Figure 12. Results of myocardial cells culture of samples
图 13 各样品内皮细胞活死染色结果
Figure 13. Results of live and dead staining of endothelial cells of samples
图 14 各样品内皮细胞内活性氧(ROS)含量检测结果
DCFH-DA—2, 7-dichlorodihydrofluorescein diacetate
Figure 14. Results of reactive oxide species (ROS) content in endothelial cells of samples
图 15 各样品H2O2损伤后心肌细胞荧光图片
Figure 15. Fluorescence images of myocardial cells of samples after H2O2 injury
图 16 各样品H2O2损伤下心肌细胞检测结果
LDH—Lactate dehydrogenase; SOD—Superoxide dismutase; MDA—Malonaldehyde
Figure 16. Results of myocardial cells detection of samples under H2O2 injury
表 1 各样品编号
Table 1. Marking of each sample
Sample PAMAM/(mg·mL−1) PQQ/(mg·mL−1) TiPP200 1 200 TiPP250 1 250 TiPP300 1 300 
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表 2 各样品表面元素原子含量
Table 2. Atomic proportion of each elementon the sample surface
Sample C/% N/% O/% C/N TiOH 45.2 2.2 52.6 20.4 TiP 52.4 12.2 35.4 4.3 TiPP 60.4 12.4 27.3 4.9
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