Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells <i>in vitro</i> _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LU Yanling ¹ , LIN Tian ¹ , LIU Yuying ¹ , BAO Lirong ¹ ,  WU Yu ²

1. Guangxi Medical University, Nanning Guangxi, 530021, P.R.China;
2. Pediatric Dentistry Department, College of Stomatology, Guangxi Medical University, Nanning Guangxi, 530021, P.R.China;

Corresponding author：

WU Yu, Email: wuyu0912@163.com

Keywords：

Bone morphogenetic protein 2; dexamethason; dental pulp cells; proliferation and differentiation

DOI：

10.7507/1002-1892.201701072

Video：

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Objective To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cellsin vitro. Methods Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10^–8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10^–8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance (A) value at 562 nm by alizarin red staining at 21 days after culture. Results Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D (P<0.05), in group C than group A (P<0.05), and in group A than group B (P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D (P<0.05), and in group C than groups A and B (P<0.05), but no significant difference was found between groups A and B (P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D (P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days (P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D (P<0.05), and in groups A and B than group D (P<0.05), but there was no significant difference between groups A and B (P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. TheA values of mineralized nodules showed significant difference between groups (P<0.05). Conclusion BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.

Citation： LU Yanling, LIN Tian, LIU Yuying, BAO Lirong, WU Yu. Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells in vitro . Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(6): 738-744. doi: 10.7507/1002-1892.201701072 Copy

1.	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, 2001, 97(25): 13625-13630.
2.	Kim BC, Bae H, Kwon IK, et al. Osteoblastic/cementoblastic and neural differentiation of dental stem cells and their applications to tissue engineering and regenerative medicine. Tissue Eng Part B Rev, 2012, 18(3): 235-244.
3.	Yang X, van der Kraan PM, Bian Z, et al. Mineralized tissue formation by BMP2-transfected pulp stem cells. J Dental Res, 2009, 88(11): 1020-1025.
4.	秦伟, 高现灵, 林正梅. 人骨形成蛋白-2 对人牙髓细胞增殖和分化的影响. 牙体牙髓牙周病学杂志, 2014, 24(10): 568-571.
5.	高晓增, 张柳, 李任增, 等. 地塞米松在人骨髓基质细胞诱导成骨细胞中的作用. 中国骨质疏松杂志, 2006, 12(1): 50-53.
6.	杨林, 陶天遵, 刘枫晨, 等. 地塞米松对成人成骨细胞增殖和分化影响的实验研究. 中华骨科杂志, 2001, 21(8): 493-497.
7.	Rodríguez-Lozano FJ, Bueno C, Insausti CL, et al. Mesenchymal stem cells derived from dental tissues. Int Endod J, 2011, 44(9): 800-806.
8.	Vainio S, Karavanoval I, Jowett A, et al. Identification of BMP2, 4 as a signal mediating secon dary induetion between epithelial and mesenehymal tissues during early tooth development. Cell, 1993, 75(1): 45-58.
9.	张悦, 胡杨, 沈玉凤, 等. bFGF/BMP-2 转染大鼠骨髓干细胞体内成牙的研究. 口腔医学研究, 2016, 32(3): 239-243.
10.	林颖, 秦伟, 邹瑞, 等. 促丝裂原激活蛋白激酶在牙髓干细胞向成牙本质细胞分化和牙髓损伤修复中的作用. 国际口腔医学杂志, 2016, 43(3): 343-347.
11.	Yang J, Ye L, Hui TQ, et al. Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway. Int J Oral Sci, 2015, 7(2): 95-102.
12.	周俊, 王兆晶, 陈文瑨, 等. 血管内皮生长因子和骨形成蛋白 2 诱导犬恒牙原位牙髓再生. 中华口腔医学研究杂志(电子版), 2016, 10(1): 5-10.
13.	刘彩宏, 王军强. 转化生长因子 β ₁ 在骨形态发生蛋白 2 诱导的人牙周膜干细胞成骨分化中的作用. 牙体牙髓牙周病学杂志, 2016, 26(6): 332-337.
14.	Monteiro N, Martins A, Ribeiro D, et al. On the use of dexamethasone-loaded liposomes to induce the osteogenic differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med, 2013, 9(9): 1056-1066.
15.	Rimando MG, Wu H, Lee O, et al. Bimodal Effects of Dexamethasone on Osteogenic Differentiation of Mesenchymal Stromal Cells. Cytotherapy, 2016, 18(6): S77-S78.
16.	董平, 肖苒. 糖皮质激素促间充质干细胞的成骨效应. 中国组织工程研究, 2013, 17(14): 2625-2632.
17.	汪建样, 殷嫦嫦, 邬亚华, 等. GDF-5 和 Dex 通过 Wnt/β-catenin 信号通路促进 rBMSCs 体外成软骨分化. 基础医学与临床, 2016, 36(12): 1624-1629.
18.	郭冕, 王宏伟, 谢春成, 等. 骨形态发生蛋白质 2 介导 Smad 和丝裂原活化蛋白激酶通路促进人神经胶质瘤细胞增殖的实验研究. 中华神经外科杂志, 2015, 31(4): 386-391.
19.	谢小伟, 裴福兴, 康鹏德, 等. 不同剂量、多疗程地塞米松对大鼠成骨细胞生物活性及 Wnt 信号通路的影响. 中国骨质疏松杂志, 2013, 19(8): 823-828.
20.	范锲, 赵劲民, 苏伟, 等. 地塞米松对大鼠骨髓间充质干细胞凋亡的影响. 中国组织工程研究与临床康复, 2011, 15(14): 2477-2481.
21.	Ji YM, Jeon SH, Park JY, et al. Dental stem cell thrapy with calcium hydroxidein dental pulp capping. Tissue Eng Part A, 2010, 16(6): 1823-1833.
22.	Saito T, Ogawa M, Hata Y, et al. Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts. J Endod, 2004, 30(4): 205-208.
23.	Fujiwara N, Sakakura Y, Nawa T. Short-term mineralization of dentin and enamelin the mouse embryonic molars cultured in serum-free, chemically defined medium. Arch Histol Cytol, 1991, 54(4): 411-418.
24.	逄键梁, 吴补领, 张亚庆, 等. 牙本质基质蛋白 1 在人牙胚发育过程中的表达和意义. 牙体牙髓牙周病学杂志, 2003, 13(1): 18-20.
25.	Qin C, D’Souza R, Feng JQ. Dentin matrix protein 1 (DMP1): new and important roles for biomineralization and phosphate homeostasis. J Dental Res, 2008, 86(12): 1134-1141.
26.	宋扬, 秦青, 高菊荣, 等. BMP-2、bFGF 和 Dex 联合应用对犬牙周膜细胞增殖及矿化结节形成能力的影响. 中华老年口腔医学杂志, 2014, 12(2): 80-84.
27.	徐璐璐, 鄂玲玲, 吴婷, 等. BMP-2 和地塞米松对体外培养大鼠牙囊细胞矿化能力的影响. 口腔颌面修复学杂志, 2013, 14(3): 129-132.

1. 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, 2001, 97(25): 13625-13630.
2. Kim BC, Bae H, Kwon IK, et al. Osteoblastic/cementoblastic and neural differentiation of dental stem cells and their applications to tissue engineering and regenerative medicine. Tissue Eng Part B Rev, 2012, 18(3): 235-244.
3. Yang X, van der Kraan PM, Bian Z, et al. Mineralized tissue formation by BMP2-transfected pulp stem cells. J Dental Res, 2009, 88(11): 1020-1025.
4. 秦伟, 高现灵, 林正梅. 人骨形成蛋白-2 对人牙髓细胞增殖和分化的影响. 牙体牙髓牙周病学杂志, 2014, 24(10): 568-571.
5. 高晓增, 张柳, 李任增, 等. 地塞米松在人骨髓基质细胞诱导成骨细胞中的作用. 中国骨质疏松杂志, 2006, 12(1): 50-53.
6. 杨林, 陶天遵, 刘枫晨, 等. 地塞米松对成人成骨细胞增殖和分化影响的实验研究. 中华骨科杂志, 2001, 21(8): 493-497.
7. Rodríguez-Lozano FJ, Bueno C, Insausti CL, et al. Mesenchymal stem cells derived from dental tissues. Int Endod J, 2011, 44(9): 800-806.
8. Vainio S, Karavanoval I, Jowett A, et al. Identification of BMP2, 4 as a signal mediating secon dary induetion between epithelial and mesenehymal tissues during early tooth development. Cell, 1993, 75(1): 45-58.
9. 张悦, 胡杨, 沈玉凤, 等. bFGF/BMP-2 转染大鼠骨髓干细胞体内成牙的研究. 口腔医学研究, 2016, 32(3): 239-243.
10. 林颖, 秦伟, 邹瑞, 等. 促丝裂原激活蛋白激酶在牙髓干细胞向成牙本质细胞分化和牙髓损伤修复中的作用. 国际口腔医学杂志, 2016, 43(3): 343-347.
11. Yang J, Ye L, Hui TQ, et al. Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway. Int J Oral Sci, 2015, 7(2): 95-102.
12. 周俊, 王兆晶, 陈文瑨, 等. 血管内皮生长因子和骨形成蛋白 2 诱导犬恒牙原位牙髓再生. 中华口腔医学研究杂志(电子版), 2016, 10(1): 5-10.
13. 刘彩宏, 王军强. 转化生长因子 β ₁ 在骨形态发生蛋白 2 诱导的人牙周膜干细胞成骨分化中的作用. 牙体牙髓牙周病学杂志, 2016, 26(6): 332-337.
14. Monteiro N, Martins A, Ribeiro D, et al. On the use of dexamethasone-loaded liposomes to induce the osteogenic differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med, 2013, 9(9): 1056-1066.
15. Rimando MG, Wu H, Lee O, et al. Bimodal Effects of Dexamethasone on Osteogenic Differentiation of Mesenchymal Stromal Cells. Cytotherapy, 2016, 18(6): S77-S78.
16. 董平, 肖苒. 糖皮质激素促间充质干细胞的成骨效应. 中国组织工程研究, 2013, 17(14): 2625-2632.
17. 汪建样, 殷嫦嫦, 邬亚华, 等. GDF-5 和 Dex 通过 Wnt/β-catenin 信号通路促进 rBMSCs 体外成软骨分化. 基础医学与临床, 2016, 36(12): 1624-1629.
18. 郭冕, 王宏伟, 谢春成, 等. 骨形态发生蛋白质 2 介导 Smad 和丝裂原活化蛋白激酶通路促进人神经胶质瘤细胞增殖的实验研究. 中华神经外科杂志, 2015, 31(4): 386-391.
19. 谢小伟, 裴福兴, 康鹏德, 等. 不同剂量、多疗程地塞米松对大鼠成骨细胞生物活性及 Wnt 信号通路的影响. 中国骨质疏松杂志, 2013, 19(8): 823-828.
20. 范锲, 赵劲民, 苏伟, 等. 地塞米松对大鼠骨髓间充质干细胞凋亡的影响. 中国组织工程研究与临床康复, 2011, 15(14): 2477-2481.
21. Ji YM, Jeon SH, Park JY, et al. Dental stem cell thrapy with calcium hydroxidein dental pulp capping. Tissue Eng Part A, 2010, 16(6): 1823-1833.
22. Saito T, Ogawa M, Hata Y, et al. Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts. J Endod, 2004, 30(4): 205-208.
23. Fujiwara N, Sakakura Y, Nawa T. Short-term mineralization of dentin and enamelin the mouse embryonic molars cultured in serum-free, chemically defined medium. Arch Histol Cytol, 1991, 54(4): 411-418.
24. 逄键梁, 吴补领, 张亚庆, 等. 牙本质基质蛋白 1 在人牙胚发育过程中的表达和意义. 牙体牙髓牙周病学杂志, 2003, 13(1): 18-20.
25. Qin C, D’Souza R, Feng JQ. Dentin matrix protein 1 (DMP1): new and important roles for biomineralization and phosphate homeostasis. J Dental Res, 2008, 86(12): 1134-1141.
26. 宋扬, 秦青, 高菊荣, 等. BMP-2、bFGF 和 Dex 联合应用对犬牙周膜细胞增殖及矿化结节形成能力的影响. 中华老年口腔医学杂志, 2014, 12(2): 80-84.
27. 徐璐璐, 鄂玲玲, 吴婷, 等. BMP-2 和地塞米松对体外培养大鼠牙囊细胞矿化能力的影响. 口腔颌面修复学杂志, 2013, 14(3): 129-132.

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Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells in vitro

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