Bone morphogenetic protein 9-induced osteogenic differentiation of dental follicle cells in vitro

doi:10.3969/j.issn.2095-4344.2015.14.021

Abstract

Abstract:

BACKGROUND: Bone morphogenetic protein 9 is proved to promote the osteogenic differentiation of various kinds of stem cells, but whether it can induce the osteogenic differentiation of dental follicle cells in vitro is yet unclear.

OBJECTIVE: To investigate whether bone morphogenetic protein 9 can induce the osteogenic differentiation of rat dental follicle cells in vitro.

METHODS: Purified rat dental follicle cells at passage 3 were transfected with bone morphogenetic protein 9 adenovirus. Then, alkaline phosphatase activity, calcium deposition and expression of osteogenesis-related factors at mRNA and protein levels were detected in the dental follicle cells.

RESULTS AND CONCLUSION: After transfection with bone morphogenetic protein 9, the dental follicle cells showed continuously enhanced alkaline phosphatase activities and obviously enhanced calcium deposition. Real-time PCR results demonstrated that the mRNA expressions of alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin and core binding factor were increased significantly. The western blot assay showed that the expression of osteopontin enhanced in the dental follicle cells after transfection with bone morphogenetic protein 9. In summary, bone morphogenetic protein 9 can induce the osteogenic differentiation of dental follicle cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Dental Sac, Stem Cells, Bone Morphogenetic Proteins, Cell Differentiation

CLC Number:

R394.2

Wu Yan, Huang Lan . Bone morphogenetic protein 9-induced osteogenic differentiation of dental follicle cells in vitro [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(14): 2255-2260.

Figures/Tables 3

2.1 大鼠牙囊细胞的形态组织块贴壁后8 h可见细胞从组织块中爬出并贴壁生长(图1A)。贴壁的原代细胞主要有两种形态：一种为梭形细胞，为主要成分，呈放射状排列，可见细胞中央有卵圆形核，表现为典型的成纤维细胞；另一种呈扁平不规则形态，连接成团，细胞中央可见圆形核，表现为上皮型细胞(图1B)。原代细胞经过传代，至第3代时，得到纯化的牙囊细胞，细胞呈梭形(图1C)。荧光显微镜下观察牙囊细胞中绿色荧光蛋白表达情况：结果显示Ad-BMP9和Ad-GFP感染牙囊细胞24 h后，感染率约50%，说明Ad-BMP9感染牙囊细胞成功(图2)。 2.2 骨形态发生蛋白9诱导牙囊细胞成骨向分化后碱性磷酸酶的表达牙囊细胞经过分离、培养后，纯化的第3代细胞用于后续实验。碱性磷酸酶通常被认为是细胞成骨向分化的早期指标，因此在感染后3，5，7 d分别进行碱性磷酸酶活性定量检测，并于感染后第7天进行碱性磷酸酶活性染色。结果显示，实验组可见大量蓝色沉淀(图3A)，而对照组仅可见少量蓝色沉淀物(图3B)。实验组碱性磷酸酶活性在感染后第3天即有表达增强，第5天出现大幅增强，第7天相较第5天时碱性磷酸酶活性更强，表现出明显的时间效应趋势，实验组与对照组结果差异有显著性意义(P < 0.05，图3C)。 2.3 骨形态发生蛋白9诱导牙囊细胞成骨向分化后细胞钙盐沉积钙盐沉积被用于进一步检测骨形态发生蛋白9对牙囊细胞的诱导成骨功能，这是除了碱性磷酸酶活性检测之外的另一个经典检测成骨活性的指标。由于该指标晚于碱性磷酸酶出现，因此感染骨形态发生蛋白9之后继续培养至第21天再进行钙盐沉积检测。茜素红S染色显示在骨形态发生蛋白作用下，牙囊细胞有非常显著的钙盐沉积，红色的钙盐沉积已连接成片(图4A)；而对照组只有散在的红色斑点沉积(图4B)。这提示骨形态发生蛋白诱导牙囊细胞成骨分化后，早晚期的成骨指标均有明显变化。 2.4 骨形态发生蛋白9诱导牙囊细胞表达成骨相关基因为了分析有哪些基因参与了骨形态发生蛋白9诱导牙囊细胞成骨向分化的过程，采用Real time PCR检测细胞分别感染Ad-BMP9和Ad-GFP 1 d后成骨相关基因的表达，其中碱性磷酸酶是早期成骨指标，骨涎蛋白、骨钙素和骨桥蛋白为晚期成骨指标，而核心结合因子2则是位于上游的成骨关键控制基因。结果显示，与对照组相比，Ad-BMP9感染的牙囊细胞表现出增强的基因表达，差异均有显著性意义 (P < 0.05)，见图5。 2.5 骨形态发生蛋白9诱导大鼠牙囊细胞表达骨桥蛋白细胞分别感染Ad-BMP9和Ad-GFP 14 d后，采用Western blot检测大鼠牙囊细胞分泌骨桥蛋白的情况。结果表明，实验组和对照组内参均表达稳定，实验组可见骨桥蛋白的表达，而对照组几乎没有骨桥蛋白的表达(图6)。"

References

[1] Harada H, Kettunen P, Jung HS, et al. Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling.J Cell Biol. 1999;147(1):105-120.
[2] Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009; 88(9):792-806.
[3] Morsczeck C, Götz W, Schierholz J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol. 2005;24(2):155-165.
[4] Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004;364(9429):149-155. ?
[5] Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97(25):13625-13630.
[6] Honda MJ, Nakashima F, Satomura K, et al. Side population cells expressing ABCG2 in human adult dental pulp tissue. Int Endod J. 2007;40(12):949-958.
[7] Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100(10):5807-5812.
[8] Sonoyama W, Liu Y, Yamaza T, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod. 2008;34(2): 166-171.
[9] Yao S, Pan F, Prpic V, et al. Differentiation of stem cells in the dental follicle.J Dent Res. 2008;87(8):767-771.
[10] Ten Cate AR. The development of the periodontium--a largely ectomesenchymally derived unit. Periodontol 2000. 1997; 13:9-19.
[11] Saygin NE, Giannobile WV, Somerman MJ. Molecular and cell biology of cementum. Periodontol. 2000;24:73-98.
[12] Chai Y, Jiang X, Ito Y, et al. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000;127(8):1671-1679.
[13] Morsczeck C, Petersen J, Völlner F, et al. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells. Electrophoresis. 2009;30(7):1175-1184.
[14] Morsczeck C, Schmalz G, Reichert TE, et al. Gene expression profiles of dental follicle cells before and after osteogenic differentiation in vitro. Clin Oral Investig. 2009; 13(4):383-391.
[15] Morsczeck C. Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro. Calcif Tissue Int. 2006; 78(2):98-102.
[16] Luu HH, Song WX, Luo X, et al. Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res. 2007;25(5):665-677.
[17] Kang Q, Song WX, Luo Q, et al. A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. Stem Cells Dev. 2009;18(4):545-559.
[18] Luther G, Wagner ER, Zhu G, et al. BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential. Curr Gene Ther. 2011;11(3):229-240.
[19] Zhang H, Wang J, Deng F, et al. Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs). Biomaterials. 2015;39:145-154.
[20] Cho MI, Garant PR. Development and general structure of the periodontium. Periodontol 2000. 2000;24:9-27.
[21] Handa K, Saito M, Yamauchi M, et al. Cementum matrix formation in vivo by cultured dental follicle cells. Bone. 2002; 31(5):606-611.
[22] Hou LT, Liu CM, Chen YJ, et al. Characterization of dental follicle cells in developing mouse molar. Arch Oral Biol. 1999; 44(9):759-770.
[23] D'Errico JA, MacNeil RL, Takata T, et al. Expression of bone associated markers by tooth root lining cells, in situ and in vitro. Bone. 1997;20(2):117-126.
[24] Deng ZL, Sharff KA, Tang N, et al. Regulation of osteogenic differentiation during skeletal development. Front Biosci. 2008; 13:2001-2021.
[25] Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am. 2003;85-A(8):1544-1552.
[26] Huang J, Yuan SX, Wang DX, et al. The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts. Biomaterials. 2014;35(36):9649-9659.
[27] Kang Q, Sun MH, Cheng H, et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther. 2004;11(17):1312-1320.
[28] Luther G, Wagner ER, Zhu G, et al. BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential. Curr Gene Ther. 2011;11(3):229-240.
[29] Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev. 1996; 10(13):1580-1594.
[30] Zhao GQ. Consequences of knocking out BMP signaling in the mouse. Genesis. 2003;35(1):43-56.