Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla

Zhang,Wen; Zhang,Xiaolei; Ling,Junqi; Wei,Xi; Jian,Yutao

doi:10.3892/mmr.2016.4993

Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla

Authors:
- Wen Zhang
- Xiaolei Zhang
- Junqi Ling
- Xi Wei
- Yutao Jian
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Affiliations: Guangdong Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
Published online on: March 16, 2016 https://doi.org/10.3892/mmr.2016.4993
Pages: 3747-3754
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Stem cells from apical papilla (SCAP) possess clear osteo‑/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo‑/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo‑/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector‑mediated gene transfection was conducted with SCAP in order to construct blank vector‑transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene‑transfected SCAP (SCAP‑VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP‑VEGF was demonstrated to exhibit increased proliferation, and SCAP‑BMP2‑VEGF exhibited reduced proliferation during eight days observation. SCAP‑BMP2‑VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP‑BMP2‑VEGF group exhibited a significantly greater number of ALP‑positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo‑/odontogenic differentiation of human SCAP.

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1	Huang GT, Sonoyama W, Liu Y, Liu H, Wang S and Shi S: The hidden treasure in apical papilla: The potential role in pulp/dentin regeneration and bioroot engineering. J Endod. 34:645–651. 2008. View Article : Google Scholar : PubMed/NCBI
2	Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Wang S and Shi S: Mesenchymal stem cell-mediated functional tooth regeneration in swine. PloS One. 1:e792006. View Article : Google Scholar : PubMed/NCBI
3	Huang GT, Gronthos S and Shi S: Mesenchymal stem cells derived from dental tissues vs. those from other sources: Their biology and role in regenerative medicine. J Dent Res. 88:792–806. 2009. View Article : Google Scholar : PubMed/NCBI
4	Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P and Geurtsen W: Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP). Arch Oral Biol. 56:709–721. 2011. View Article : Google Scholar : PubMed/NCBI
5	Tziafas D and Kodonas K: Differentiation potential of dental papilla, dental pulp and apical papilla progenitor cells. J Endod. 36:781–789. 2010. View Article : Google Scholar : PubMed/NCBI
6	Lin CS, Xin ZC, Dai J and Lue TF: Commonly used mesenchymal stem cell markers and tracking labels: Limitations and challenges. Histol Histopathol. 28:1109–1116. 2013.PubMed/NCBI
7	Bakopoulou A, Leyhausen G, Volk J, Koidis P and Geurtsen W: Comparative characterization of STRO-1 (neg)/CD146 (pos) and STRO-1 (pos)/CD146 (pos) apical papilla stem cells enriched with flow cytometry. Arch Oral Biol. 58:1556–1568. 2013. View Article : Google Scholar : PubMed/NCBI
8	Friedlander LT, Cullinan MP and Love RM: Dental stem cells and their potential role in apexogenesis and apexification. Int Endod J. 42:955–962. 2009. View Article : Google Scholar : PubMed/NCBI
9	Wang J, Zhang H, Zhang W, Huang E, Wang N, Wu N, Wen S, Chen X, Liao Z, Deng F, et al: Bone morphogenetic protein-9 effectively induces osteo/odontoblastic differentiation of the reversibly immortalized stem cells of dental apical papilla. Stem Cells Dev. 23:1405–1416. 2014. View Article : Google Scholar : PubMed/NCBI
10	Lin ZM, Qin W, Zhang NH, Xiao L and Ling JQ: Adenovirus-mediated recombinant human bone morphogenetic protein-7 expression promotes differentiation of human dental pulp cells. J Endod. 33:930–935. 2007. View Article : Google Scholar : PubMed/NCBI
11	Zhang W, Zhang X, Ling J, Liu W, Ma J and Zheng J: Proliferation and odontogenic differentiation of BMP2 genetransfected stem cells from human tooth apical papilla: An in vitro study. Int J Mol Med. 34:1004–1012. 2014.PubMed/NCBI
12	Yang X, van der Kraan PM, Bian Z, Fan M, Walboomers XF and Jansen JA: Mineralized tissue formation by BMP2-transfected pulp stem cells. J Dent Res. 88:1020–1025. 2009. View Article : Google Scholar : PubMed/NCBI
13	Hiltunen MO, Ruuskanen M, Huuskonen J, Mähönen AJ, Ahonen M, Rutanen J, Kosma VM, Mahonen A, Kröger H and Ylä-Herttuala S: Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo. FASEB J. 17:1147–1149. 2003.PubMed/NCBI
14	Jacobsen KA, Al-Aql ZS, Wan C, Fitch JL, Stapleton SN, Mason ZD, Cole RM, Gilbert SR, Clemens TL, Morgan EF, et al: Bone formation during distraction osteogenesis is dependent on both VEGFR1 and VEGFR2 signaling. J Bone Miner Res. 23:596–609. 2008. View Article : Google Scholar : PubMed/NCBI
15	Dvorak HF: Angiogenesis: Update 2005. J Thromb Haemost. 3:1835–1842. 2005. View Article : Google Scholar : PubMed/NCBI
16	Zhang W, Liu W, Ling J, Lin Z, Gao Y, Mao X and Jian Y: Odontogenic differentiation of vascular endothelial growth factor-transfected human dental pulp stem cells in vitro. Mol Med Rep. 10:1899–1906. 2014.PubMed/NCBI
17	Lee JH, Um S, Jang JH and Seo BM: Effects of VEGF and FGF-2 on proliferation and differentiation of human periodontal ligament stem cells. Cell Tissue Res. 348:475–484. 2012. View Article : Google Scholar : PubMed/NCBI
18	Matsushita K, Motani R, Sakuta T, Yamaguchi N, Koga T, Matsuo K, Nagaoka S, Abeyama K, Maruyama I and Torii M: The role of vascular endothelial growth factor in human dental pulp cells: Induction of chemotaxis, proliferation and differentiation and activation of the AP-1-dependent signaling pathway. J Dent Res. 79:1596–1603. 2000. View Article : Google Scholar : PubMed/NCBI
19	Kanczler JM, Ginty PJ, White L, Clarke NM, Howdle SM, Shakesheff KM and Oreffo RO: The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation. Biomaterials. 31:1242–1250. 2010. View Article : Google Scholar
20	Kumar S, Wan C, Ramaswamy G, Clemens TL and Ponnazhagan S: Mesenchymal stem cells expressing osteogenic and angiogenic factors synergistically enhance bone formation in a mouse model of segmental bone defect. Mol Ther. 18:1026–1034. 2010. View Article : Google Scholar : PubMed/NCBI
21	Lin CY, Chang YH, Lin KJ, Yen TC, Tai CL, Chen CY, Lo WH, Hsiao IT and Hu YC: The healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells. Biomaterials. 31:3222–3230. 2010. View Article : Google Scholar : PubMed/NCBI
22	Lin Z, Wang JS, Lin L, Zhang J, Liu Y, Shuai M and Li Q: Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Exp Ther Med. 7:625–629. 2014.PubMed/NCBI
23	Schönmeyr BH, Soares M, Avraham T, Clavin NW, Gewalli F and Mehrara BJ: Vascular endothelial growth factor inhibits bone morphogenetic protein 2 expression in rat mesenchymal stem cells. Tissue Eng Part A. 16:653–662. 2010. View Article : Google Scholar :
24	Chen K, Xiong H, Huang Y and Liu C: Comparative analysis of in vitro periodontal characteristics of stem cells from apical papilla (SCAP) and periodontal ligament stem cells (PDLSCs). Arch Oral Biol. 58:997–1006. 2013. View Article : Google Scholar : PubMed/NCBI
25	Wang J, Liu B, Gu S and Liang J: Effects of Wnt/β-catenin signalling on proliferation and differentiation of apical papilla stem cells. Cell Prolif. 45:121–131. 2012. View Article : Google Scholar : PubMed/NCBI
26	Qu B, Liu O, Fang X, Zhang H, Wang Y, Quan H, Zhang J, Zhou J, Zuo J, Tang J and Tang Z: Distal-less homeobox 2 promotes the osteogenic differentiation potential of stem cells from apical papilla. Cell Tissue Res. 357:133–143. 2014. View Article : Google Scholar : PubMed/NCBI
27	Dai J and Rabie AB: VEGF: An essential mediator of both angiogenesis and endochondral ossification. J Dent Res. 86:937–950. 2007. View Article : Google Scholar : PubMed/NCBI
28	Ryoo HM, Lee MH and Kim YJ: Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells. Gene. 366:51–57. 2006. View Article : Google Scholar
29	Uchida S, Sakai A, Kudo H, Otomo H, Watanuki M, Tanaka M, Nagashima M and Nakamura T: Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats. Bone. 32:491–501. 2003. View Article : Google Scholar : PubMed/NCBI
30	Casagrande L, Demarco FF, Zhang Z, Araujo FB, Shi S and Nör JE: Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res. 89:603–608. 2010. View Article : Google Scholar : PubMed/NCBI
31	Mastrangelo F, Piccirilli M, Dolci M, Teté S, Speranza L, Patruno A, Gizzi F, Felaco M, Artese L and De Lutiis MA: Vascular endothelial growth factor (VEGF) in human tooth germ center. Int J Immunopathol Pharmacol. 18:587–594. 2005.PubMed/NCBI
32	Scheven BA, Man J, Millard JL, Cooper PR, Lea SC, Walmsley AD and Smith AJ: VEGF and odontoblast-like cells: Stimulation by low frequency ultrasound. Arch Oral Biol. 54:185–191. 2009. View Article : Google Scholar
33	Virtej A, Løes S, Iden O, Bletsa A and Berggreen E: Vascular endothelial growth factors signalling in normal human dental pulp: A study of gene and protein expression. Eur J Oral Sci. 121:92–100. 2013. View Article : Google Scholar : PubMed/NCBI
34	Wu J, Huang GT, He W, Wang P, Tong Z, Jia Q, Dong L, Niu Z and Ni L: Basic fibroblast growth factor enhances stemness of human stem cells from the apical papilla. J Endod. 38:614–622. 2012. View Article : Google Scholar : PubMed/NCBI
35	Choi MH, Noh WC, Park JW, Lee JM and Suh JY: Gene expression pattern during osteogenic differentiation of human periodontal ligament cells in vitro. J Periodontal Implant Sci. 41:167–175. 2011. View Article : Google Scholar : PubMed/NCBI
36	MacDougall M, Gu TT and Simmons D: Dentin matrix protein-1, a candidate gene for dentinogenesis imperfecta. Connect Tissue Res. 35:267–272. 1996. View Article : Google Scholar : PubMed/NCBI
37	Lee SY, Kim SY, Park SH, Kim JJ, Jang JH and Kim EC: Effects of recombinant dentin sialoprotein in dental pulp cells. J Dent Res. 91:407–412. 2012. View Article : Google Scholar : PubMed/NCBI
38	Zhang W, Zhu C, Ye D, Xu L, Zhang X, Wu Q, Zhang X, Kaplan DL and Jiang X: Porous silk scaffolds for delivery of growth factors and stem cells to enhance bone regeneration. PloS One. 9:e1023712014. View Article : Google Scholar : PubMed/NCBI
39	Zhang W, Zhu C, Wu Y, Ye D, Wang S, Zou D, Zhang X, Kaplan DL and Jiang X: VEGF and BMP-2 promote bone regeneration by facilitating bone marrow stem cell homing and differentiation. Eur Cell Mater. 27:1–11; discussion 11–12. 2014. View Article : Google Scholar : PubMed/NCBI
40	Bai Y, Li P, Yin G, Huang Z, Liao X, Chen X and Yao Y: BMP-2, VEGF and bFGF synergistically promote the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Biotechnol Lett. 35:301–308. 2013. View Article : Google Scholar
41	Xiao C, Zhou H, Liu G, Zhang P, Fu Y, Gu P, Hou H, Tang T and Fan X: Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration. Biomed Mater. 6:0150132011. View Article : Google Scholar : PubMed/NCBI
42	Tian XB, Sun L, Yang SH, Fu RY, Wang L, Lu TS, Zhang YK and Fu DH: Ectopic osteogenesis of mouse bone marrow stromal cells transfected with BMP 2/VEGF (165) genes in vivo. Orthop Surg. 1:322–325. 2009. View Article : Google Scholar : PubMed/NCBI
43	Hou H, Zhang X, Tang T, Dai K and Ge R: Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1. Biotechnol Lett. 31:1183–1189. 2009. View Article : Google Scholar : PubMed/NCBI
44	Behr B, Sorkin M, Lehnhardt M, Renda A, Longaker MT and Quarto N: A comparative analysis of the osteogenic effects of BMP-2, FGF-2 and VEGFA in a calvarial defect model. Tissue Eng Part A. 18:1079–1086. 2012. View Article : Google Scholar :
45	Li P, Bai Y, Yin G, Pu X, Huang Z, Liao X, Chen X and Yao Y: Synergistic and sequential effects of BMP-2, bFGF and VEGF on osteogenic differentiation of rat osteoblasts. J Bone Miner Metab. 32:627–635. 2014. View Article : Google Scholar
46	Akeel S, El-Awady A, Hussein K, El-Refaey M, Elsalanty M, Sharawy M and Al-Shabrawey M: Recombinant bone morphogenetic protein-2 induces up-regulation of vascular endothelial growth factor and interleukin 6 in human pre-osteoblasts: Role of reactive oxygen species. Arch Oral Biol. 57:445–452. 2012. View Article : Google Scholar