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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (1): 28-34. doi: 10.19723/j.issn.1671-167X.2019.01.006

• 论著 • 上一篇    下一篇

胶原静电纺纳米纤维膜对人牙髓细胞生物学行为的影响

张倩莉1,袁重阳1,刘力2,温世鹏2,△(),王晓燕1,△()   

  1. 1. 北京大学口腔医学院·口腔医院,牙体牙髓科　国家口腔疾病临床医学研究中心　口腔数字化医疗技术和材料国家工程实验室　口腔数字医学北京市重点实验室,北京　100081
    2. 北京化工大学,北京市先进弹性体工程技术研究中心, 北京　100029
  • 收稿日期:2018-10-10 出版日期:2019-02-18 发布日期:2019-02-26
  • 通讯作者: 温世鹏,王晓燕 E-mail:wensp@mail.buct.edu.cn;wangxiaoyan@pkuss.bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(51503004)

Effects of electrospun collagen nanofibrous matrix on the biological behavior of human dental pulp cells

Qian-li ZHANG1,Chong-yang YUAN1,Li LIU2,Shi-peng WEN2,△(),Xiao-yan WANG1,△()   

  1. 1. Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
    2. Beijing Engineering Research Centre of Advanced Elastomers, Beijing University of Chemical Technology, Beijing 100029, China
  • Received:2018-10-10 Online:2019-02-18 Published:2019-02-26
  • Contact: Shi-peng WEN,Xiao-yan WANG E-mail:wensp@mail.buct.edu.cn;wangxiaoyan@pkuss.bjmu.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(51503004)

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摘要：

目的:比较人牙髓细胞(human dental pulp cells,hDPCs)在胶原静电纺纳米纤维膜(collagen nanofibrous matrix,Col_NFM)与直接沉积胶原膜(collagen flat film,Col_FF)上的黏附、增殖和分化情况,探究胶原纳米纤维支架对hDPCs生物学行为的影响。方法:采用扫描电镜(scanning electron microscopy, SEM)观察两种胶原膜的表面形貌,并比较其表面接触角和溶胀性能。将hDPCs分别接种于两种胶原膜表面共培养,SEM和激光共聚焦显微镜(laser scanning microscope,LSM)观察hDPCs在支架表面的生长形态,并用CCK-8法测定hDPCs的增殖情况。在诱导14 d后,比较成牙本质分化相关基因的表达变化,茜素红染色观察矿化结节的形成情况。结果:SEM图可见Col_NFM组纤维直径为(884±159) nm,纤维之间存在大量三维连通的孔隙结构,而Col_FF组表面平坦,未见孔隙结构。Col_NFM组瞬间表面接触角为85.03°±4.45°,溶胀度为3,Col_FF组瞬间表面接触角为98.98°±5.81°,溶胀度为1,Col_NFM组的亲水性和溶胀性能更佳。SEM和LSM结果显示,Col_NFM组hDPCs表现为不规则多角形,呈三维生长,Col_FF组细胞在二维平面上呈纺锤形生长。CCK-8结果显示,hDPCs在Col_NFM支架上增殖活性更高。在诱导14 d后,Col_NFM组成牙本质分化相关基因表达水平较Col_FF组显著升高(P<0.05),茜素红染色也更深。结论:Col_NFM具有纳米尺度的微观结构,并具备良好的亲水性和溶胀性能,相较于Col_FF,hDPCs在Col_NFM表面表现出更好的黏附、增殖和分化性能。

关键词: 胶原, 静电纺丝, 牙髓再生, 纳米纤维, 支架

Abstract:

Objective: To compare cell adhesion, proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs) on electrospun collagen nanofibrous matrix (Col_NFM) with that on collagen flat film (Col-FF), to investigate the biological effect of collagen nanofibrous matrix on hDPCs. Methods: The surface morphology of the two different collagen scaffold was analyzed by scanning electron microscopy (SEM), and the contact angle and the swelling ratio were also measured. Then hDPCs were implanted on the two different collagen scaffolds, the cell morphology was observed using SEM and laser scanning microscope (LSM), and cell proliferation was evaluated by the CCK-8 assay. After hDPCs cultured on the two different collagen scaffold with odontoblastic medium for 14 days, the expression of odontoblastic differentiation related genes was detected by real-time PCR, and alizarin red staining was used to test the formation of mineralized nodules. Results: From the SEM figures, the fibers’ diameter of Col_NFM was (884±159) nm, and there were abundant three dimensional connected pore structures between the fibers of Col_NFM, while the surface of Col_FF was completely flat without pore structure. The contact angle at 0 s of Col_NFM was 85.03°±4.45°, and that of Col_FF was 98.98°±5.81°. The swelling ratio of Col_NFM was approximately 3 folds compared with dry weight sample, while that of Col_FF was just 1 fold. Thus Col_NFM indicated better hydrophilicity and swelling property. SEM and LSM showed that hDPCs on Col_NFM presented an irregular and highly branched phenotype, and could penetrate into the nanofibrous scaffold. In contrast, the cells were spread only on the surface of Col_FF with a spindle-shaped morphology. CCK-8 assays showed that hDPCs on Col_NFM showed higher proliferation rate than on Col_FF. After hDPCs were cultured on the two different collagen scaffolds with odontoblastic medium for 14 days, more expressions of odontoblastic differentiation related genes, such as dentin sialophosphoprotein (DSPP) and dentin matrix proten-1 (DMP1) were determined in Col_NFM group (P<0.05), and more mineralization depositions were also observed in Col_NFM group according to the results of alizarin red staining. Conclusion: Col_NFM with nanoscale microstructure achieves better hydrophilic and swelling properties than Col_FF, and hDPCs cultured with Col_NFM present higher activity on cell adhesion, proliferation and odontoblastic differentiation.

Key words: Collagen, Electrospinning, Pulp regeneration, Nanofibers, Scaffold

中图分类号: 

  • R781.3

表1

引物序列"

Gene Gene sequence (5'-3')
DSPP Forward: ATATTGAGGGCTGGAATGGGGA
Reverse: TTTGTGGCTCCAGCATTGTCA
DMP1 Forward: AGGAAGTCTCGCATCTCAGAG
Reverse: TGGAGTTGCTGTTTTCTGTAGAG
GAPDH Forward: GAAGGTGAAGGTCGGAGTC
Reverse: GAGATGGTGATGGGATTTC

图1

支架的扫描电镜图片"

图2

支架表面接触角"

图3

支架溶胀度"

图4

扫描电镜观察不同支架表面hDPCs的生长形态"

图5

激光共聚焦显微镜观察不同支架表面hDPCs的生长形态"

图6

hDPCs在不同支架上的增殖情况"

图7

实时荧光定量PCR检测hDPCs诱导14天后DSPP、DMP1基因相对表达情况"

图8

诱导第14天后茜素红染色结果"

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ISSN 1671-167X
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