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新型培养基促进牙乳头细胞成骨分化及其在牙周骨组织再生中的应用

王涛 王世恺 陈国庆 田卫东

王涛, 王世恺, 陈国庆, 等. 新型培养基促进牙乳头细胞成骨分化及其在牙周骨组织再生中的应用[J]. 四川大学学报(医学版), 2020, 51(6): 735-741. doi: 10.12182/20201160101
引用本文: 王涛, 王世恺, 陈国庆, 等. 新型培养基促进牙乳头细胞成骨分化及其在牙周骨组织再生中的应用[J]. 四川大学学报(医学版), 2020, 51(6): 735-741. doi: 10.12182/20201160101
WANG Tao, WANG Shi-kai, CHEN Guo-qing, et al. A Novel Chemically Defined Medium Enhanced the Osteogenic Potential and Periodontal Bone Regeneration of Dental Papilla Cells[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2020, 51(6): 735-741. doi: 10.12182/20201160101
Citation: WANG Tao, WANG Shi-kai, CHEN Guo-qing, et al. A Novel Chemically Defined Medium Enhanced the Osteogenic Potential and Periodontal Bone Regeneration of Dental Papilla Cells[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2020, 51(6): 735-741. doi: 10.12182/20201160101

栏目: 口腔医学进展

新型培养基促进牙乳头细胞成骨分化及其在牙周骨组织再生中的应用

doi: 10.12182/20201160101
基金项目: 国家重点研发计划(No.2017YFA0104800)资助
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    E-mail:drtwd@sina.com

A Novel Chemically Defined Medium Enhanced the Osteogenic Potential and Periodontal Bone Regeneration of Dental Papilla Cells

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  • 摘要:   目的  探讨新型化学成分明确培养基(chemically defined medium, CDM)对牙乳头细胞(dental papilla cells, DPCs)成骨分化潜能及体内牙周骨再生的影响。  方法  分离大鼠 DPCs,按培养基不同分为传统培养基(conventional medium, CM)组和CDM组,分别在CM和新型CDM中培养,评估两组DPCs细胞表面标记、增殖、迁移、成骨分化潜能;制备SD大鼠牙周骨缺损模型,CM和CDM两组DPCs分别复合胶原凝胶植入缺损区域内,评估两组DPCs在牙周骨缺损中的骨再生修复能力。  结果  在CM和CDM组中,DPCs细胞均显示相似的细胞表面标记;与CM相比,CDM可促进DPCs增殖、克隆形成及细胞迁移(P<0.05);定量PCR显示CDM组DPCs成骨相关基因Runx2、AlpOpn上调(P<0.05);碱性磷酸酶(alkaline phosphatase, ALP)染色显示CDM组DPCs细胞 ALP活性高于CM组(P<0.05);细胞经成骨诱导后,茜素红染色显示CDM组DPCs细胞成骨分化能力高于CM组(P<0.05)。在大鼠体内牙周骨缺损模型中,移植CDM组DPCs细胞8周后,HE染色显示缺损区域皮质骨连续的新生骨,而CM组新生骨较少,骨皮质仍未愈合,microCT扫描定量分析显示CDM组骨体积分数(BV/TV)高于CM组(P<0.05)。  结论  CDM培养基可促进DPCs增殖和成骨分化能力,为未来细胞治疗中干细胞培养基的选择提供了新的方案。
  • 图  1  DPCs的形态及表面标记鉴定

    A: Cell morphology of DPCs in the CM and CDM groups (×400); B: Surface markers of DPCs detected by flow cytometry.

    Figure  1.  Morphology and surface marker of rat dental papilla cells (DPCs)

    图  2  两种培养基中DPCs的特性

    A: Growth curve of DPCs (n=5); B: Crystal violet staining and quantitative analysis of colony-forming unit of DPCs (CFU) (n=3); C: Immunofluorescence staining of Oct4 and Sox2 in DPCs; D: Cell migration assay and quantitative analysis (×100, n=3). *P<0.05, vs. CM.

    Figure  2.  Characterization of DPCs

    图  3  两种培养基中DPCs成骨分化能力比较

    A: Relative expression of Runx2, Alp and Opn of DPCs cultured in CM and CDM (n=3); B: The ALP staining of DPCs cultured in CM and CDM; C: Alizarin Red S staining and quantitative analysis of mineralized bone nodules (×100, n=3). *P<0.05, vs. CM.

    Figure  3.  Comparison of osteogenic differentiation capability of DPCs cultured in CM and CDM

    图  4  动物体内移植DPCs用于牙周骨缺损的修复

    A: MicroCT analysis the regeneration of periodontal bone defect; B: HE staining showed the new formed bone tissue in bone defect area (×40).

    Figure  4.  Regeneration of periodontal bone defect by transplantation of DPCs

    表  1  定量PCR引物序

    Table  1.   Primer sequence used for real-time PCR analysis

    GeneForward (5′ to 3′)Reverse (5′ to 3′)Product length/bp
    AlpCGTTGACTGTGGTTACTGCTGACTTCTTGTCCGTGTCGCTCAC113
    Runx2TCATTTGCACTGGGTCACATTCTCAGCCATGTTTGTGCTC142
    OpnAAGCGTGGAAACACACAGCCTTTGGAACTCGCCTGACTG161
    β-actinACGGTCAGGTCATCACTATCGGGCATAGAGGTCTTTACGGATG155
    Alp:Alkaline phosphatase; Runx2: Runt-related transcription factor 2; Opn: Osteopontin.
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出版历程
  • 收稿日期:  2020-02-12
  • 修回日期:  2020-05-29
  • 刊出日期:  2020-11-20

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