骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化

doi:10.19439/j.sjos.2020.03.004

上海口腔医学 ›› 2020, Vol. 29 ›› Issue (3): 242-249.doi: 10.19439/j.sjos.2020.03.004

骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化

李静^1,2,3, 张艺^1,2,3, 王金华^1,2,3

1.重庆医科大学附属口腔医院儿童口腔科,重庆 400015;
2.口腔疾病与生物医学重庆市重点实验室,重庆 401120;
3.重庆市高校市级口腔生物医学工程重点实验室,重庆 401120

收稿日期:2020-02-20 修回日期:2020-03-24 出版日期:2020-06-25 发布日期:2020-07-29
通讯作者: 王金华,E-mail: 500190@hospital.cqmu.edu.cn
作者简介:李静(1989-),女,硕士,E-mail: 2017110892@stu.cqmu.edu.cn
基金资助:
重庆市基础研究和前沿技术研究计划（cstc2018jcyjAX0731）; 重庆市自然科学基金（cstc2018jcyjAX0731）; 2016年重庆高校创新团队建设计划资助项目（CXTDG201602006）

Bone morphogenetic protein-2, -4, -6, -7 and -9 differentially mediated osteogenic differentiation of immortalized odontoblasts

LI Jing^1,2,3, ZHANG Yi^1,2,3, WANG Jin-hua^1,2,3

1. Stomatological Hospital of Chongqing Medical University. Chongqing 400015;
2. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences. Chongqing 401120;
3. Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education. Chongqing 401120, China

Received:2020-02-20 Revised:2020-03-24 Online:2020-06-25 Published:2020-07-29

摘要/Abstract

摘要： 目的研究永生化成牙本质细胞（immortalized odontoblasts,iODs）的间充质干细胞表型和骨形态发生蛋白（bone morphogenetic protein, BMP）-2、-4、-6、-7和-9差异介导iODs成骨分化的作用。方法使用SV40 T抗原永生化成牙本质细胞（odontoblasts）,建立iOD细胞株,并连续检测BMP的内源性表达。使用Ad-Easy技术合成表达BMP和GFP的重组腺病毒,使用MTT试剂盒检测iOD的增殖能力。通过免疫荧光检测iOD的间充质干细胞表面标志物。在体外,使用半定量RT-PCR、碱性磷酸酶活性、茜素红染色及油红O染色检测iOD的成骨和成脂分化能力。最后,使用micro-CT和组织学分析评估体内形成的异位矿化组织的体积和密度。采用SPSS 21.0软件包中的单因素方差分析和Scheffe的多重比较检验对结果进行分析。结果 SV40 T抗原有效地永生化OD。iOD细胞株保持良好的增殖活性,表达间充质干细胞表面标志物并具有多向分化能力。相比BMP-4、-6和-7,BMP-2和BMP-9具有更强的介导iOD的成骨分化作用。结论 ODs和成骨生长因子（如BMP-2和BMP-9）可用作骨组织生物工程的有效策略。

关键词: 骨形态发生蛋白, 成牙本质细胞, 永生化, 成骨分化

Abstract: PURPOSE: This study was aimed to investigate the mesenchymal stem cell (MSC) phenotypes of immortalized odontoblasts(iODs) and bone morphogenetic protein -2, -4, -6, -7, and -9 (BMPs) differentially regulate the mineralization of iODs. METHODS: ODs were immortalized by SV40 T antigen to establish iOD lineages, and the endogenous expression of BMPs was successively examined. Recombinant adenoviruses expressing BMPs and GFP were generated using Ad-Easy technology. The proliferation capability of iODs was examined using an MTT kit. MSC markers of iODs were examined by immunofluorescence. In vitro, semiquantitative RT-PCR, alkaline phosphatase(ALP) activity assay, matrix mineralization assay and oil red O staining assay were used to examine the osteogenic and adipogenic differentiation capabilities of iODs. Statistical significance among groups was analyzed by one-way analysis of variance and Scheffe's multiple comparison test was SPSS 21.0 software package. Finally, the volume and density of ectopic mineralized tissues formed in vivo were assessed by micro-CT and histological analysis. RESULTS: ODs can be efficiently immortalized by SV40 T antigen, and the resulting iODs maintained an excellent proliferative activity, expressed certain MSC markers and possessed multiple differentiation capabilities. BMP-2 and BMP-9 regulated iODs osteogenic differentiation better than BMP-4, -6, and -7. CONCLUSIONS: Our findings suggest that ODs and osteogenic growth factors such as BMP-2 and BMP-9 can be used as an efficacious strategy for bone tissue engineering.

Key words: Bone morphogenetic protein, Odontoblasts, Immortalized odontoblasts, Osteogenic differentiation

中图分类号:

R781.3

李静, 张艺, 王金华. 骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化[J]. 上海口腔医学, 2020, 29(3): 242-249.

LI Jing, ZHANG Yi, WANG Jin-hua. Bone morphogenetic protein-2, -4, -6, -7 and -9 differentially mediated osteogenic differentiation of immortalized odontoblasts[J]. Shanghai Journal of Stomatology, 2020, 29(3): 242-249.

参考文献

[1] Kim HK, Lee JS, Kim JH, et al.Bone-forming peptide-2 derived from BMP-7 enhances osteoblast differentiation from multipotent bone marrow stromal cells and bone formation[J]. Exp Mol Med, 2017, 49(5): e328.
[2] Fu T, Liang P, Song J, et al.Matrigel scaffolding enhances bMP9-induced bone formation in dental follicle stem/precursor cells[J]. Int J Med Sci, 2019, 16(4): 567-575.
[3] Ruan W, Xue Y, Zong Y, et al.Effect of BMPs and Wnt3a co-expression on the osteogenetic capacity of osteoblasts[J]. Mol Med Rep, 2016, 14(5): 4328-4334.
[4] Hakki SS, Bozkurt B, Hakki EE, et al.Bone morphogenetic protein-2, -6, and -7 differently regulate osteogenic differentiation of human periodontal ligament stem cells[J]. J Biomed Mater Res B Appl Biomater, 2014, 102(1): 119-130.
[5] Dominici M, Le Blanc K, Mueller I, et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement[J]. Cytotherapy, 2006, 8(4): 315-317.
[6] Wang J, Zhang H, Zhang W, et al.Bone morphogenetic protein-9 effectively induces osteo/odontoblastic differentiation of the reversibly immortalized stem cells of dental apical papilla[J]. Stem Cells Dev, 2014, 23(12): 1405-1416.
[7] Gronthos S, Mankani M, Brahim J, et al.Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo[J]. Proc Natl Acad Sci USA, 2000, 97(25): 13625-13630.
[8] Kang KJ, Ryu CJ, Jang YJ.Identification of dentinogenic cell-specific surface antigens in odontoblast-like cells derived from adult dental pulp[J]. Stem Cell Res Ther, 2019, 10(1): 128.
[9] Hao J, Ramachandran A, George A.Temporal and spatial localization of the dentin matrix proteins during dentin biomineralization[J]. J Histochem Cytochem, 2009, 57(3): 227-237.
[10] Liao C, Ou Y, Wu Y, et al.Sclerostin inhibits odontogenic differentiation of human pulp-derived odontoblast-like cells under mechanical stress[J]. J Histochem Cytochem, 2019, 234(11): 20779-20789.
[11] Kim JY, Kim DS, Auh QS, et al.Role of protein phosphatase 1 in angiogenesis and odontoblastic differentiation of human dental pulp cells[J]. J Endod, 2017, 43(3): 417-424.
[12] Liu Y, Ma X, Guo J, et al.All-trans retinoic acid can antagonize osteoblastogenesis induced by different BMPs irrespective of their dimerization types and dose-efficiencies[J]. Drug Des Devel Ther, 2018, 12: 3419-3430.
[13] Miyagi M, Mikawa S, Sato T, et al.BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII are differentially expressed in the adult rat spinal cord[J]. Neuroscience, 2012, 203: 12-26.
[14] Tantilertanant Y, Niyompanich J, Everts V, et al.Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells[J]. J Cell Physiol, 2019, 234(4): 4528-4539.
[15] van Baardewijk LJ, van der Ende J, Lissenberg-Thunnissen S, et al. Circulating bone morphogenetic protein levels and delayed fracture healing[J]. Int Orthop, 2013, 37(3): 523-527.
[16] Beederman M, Lamplot JD, Nan G, et al.BMP signaling in mesenchymal stem cell differentiation and bone formation[J]. J Biomed Sci Eng, 2013, 6(8A): 32-52.
[17] Kamiya N.The role of BMPs in bone anabolism and their potential targets SOST and DKK1[J]. Curr Mol Pharmacol, 2012, 5(2): 153-163.
[18] Vukicevic S, Grgurevic L.BMP-6 and mesenchymal stem cell differentiation[J]. Cytokine Growth Factor Rev, 2009, 20(5-6): 441-448.
[19] Song R, Wang D, Zeng R, et al.Synergistic effects of fibroblast growth factor-2 and bone morphogenetic protein-2 on bone induction[J]. Mol Med Rep, 2017, 16(4): 4483-4492.
[20] Yan F, Luo S, Jiao Y, et al.Molecular characterization of the BMP7 gene and its potential role in shell formation in Pinctada martensii[J]. Int J Mol Sci, 2014, 15(11): 21215-21228.
[21] Cui J, Zhang W, Huang E, et al.BMP9-induced osteoblastic differentiation requires functional Notch signaling in mesenchymal stem cells[J]. Lab Invest, 2019, 99(1): 58-71.
[22] Yamada Y, Ito K, Nakamura S, et al.Promising cell-based therapy for bone regeneration using stem cells from deciduous teeth, dental pulp, and bone marrow[J]. Cell Transplant, 2011, 20(7): 1003-1013.
[23] Lee YC, Chan YH, Hsieh SC, et al.Comparing the osteogenic potentials and bone regeneration capacities of bone marrow and dental pulp mesenchymal stem cells in a rabbit calvarial bone defect model[J]. Int J Mol Sci, 2019, 20(20): 5015.
[24] Zhou T, Pan J, Wu P, et al.Dental follicle cells: roles in development and beyond[J]. Stem Cells Int, 2019: 9159605.
[25] vom Brocke J, Schmeiser HH, Reinbold M, et al. MEF immortalization to investigate the ins and outs of mutagenesis[J]. Carcinogenesis, 2006, 27(11): 2141-2147.
[26] Garulli C, Kalogris C, Pietrella L, et al.Dorsomorphin reverses the mesenchymal phenotype of breast cancer initiating cells by inhibition of bone morphogenetic protein signaling[J]. Cell Signal, 2014, 26(2): 352-362.
[27] Carreira AC, Lojudice FH, Halcsik E, et al.Bone morphogenetic proteins: facts, challenges, and future perspectives[J]. J Dent Res, 2014, 93(4): 335-345.
[28] Dong X, Shen B, Ruan N, et al.Expression patterns of genes critical for BMP signaling pathway in developing human primary tooth germs[J]. Histochem Cell Biol, 2014, 142(6): 657-665.
[29] Huang X, Wang F, Zhao C, et al.BMP9/GDF2 dentinogenesis and tooth-alveolar bone complex defects in knockout mice[J]. Stem Cells Dev, 2019, 28(10): 683-694.
[30] Wise GE, He H, Gutierrez DL, et al.Requirement of alveolar bone formation for eruption of rat molars[J]. Eur J Oral Sci, 2011, 119(5): 333-338.
[31] Toyono T, Nakashima M, Kuhara S, et al.Expression of TGF-beta superfamily receptors in dental pulp[J]. J Dent Res, 1997, 76(9): 1555-1560.
[32] TMiyazono K, Maeda S, Imamura T.BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk[J]. Cytokine Growth Factor Rev, 2005, 16(3): 251-263.
[33] Oryan A, Alidadi S, Moshiri A, et al.Bone morphogenetic proteins: a powerful osteoinductive compound with non-negligible side effects and limitations[J]. Biofactors, 2014, 40(5): 459-481.
[34] Zheng W, Chen Q, Zhang Y, et al.BMP9 promotes osteogenic differentiation of SMSCs by activating the JNK/Smad2/3 signaling pathway[J]. J Cell Biochem, 2020, 121(4): 2851-2863.
[35] Papanna MC, Al-Hadithy N, Somanchi BV, et al.The use of bone morphogenic protein-7 (OP-1) in the management of resistant non-unions in the upper and lower limb[J]. Injury, 2012, 43(7): 1135-1140.
[36] Luu HH, Song WX, Luo X, et al.Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells[J]. J Orthop Re, 2007, 25(5): 665-677.

骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化

Bone morphogenetic protein-2, -4, -6, -7 and -9 differentially mediated osteogenic differentiation of immortalized odontoblasts

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