99Tc-MDP-induced human osteoblast proliferation, differentiation and expression of osteoprotegerin

Chen,Jie; Lan,Youyu; He,Yue; He,Chengsong; Xu,Fen; Zhang,Yugao; Zhao,Yi; Liu,Yi

doi:10.3892/mmr.2017.6839

99Tc-MDP-induced human osteoblast proliferation, differentiation and expression of osteoprotegerin

Authors:
- Jie Chen
- Youyu Lan
- Yue He
- Chengsong He
- Fen Xu
- Yugao Zhang
- Yi Zhao
- Yi Liu
View Affiliations

Affiliations: Department of Rheumatology and Immunology, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, P.R. China, Department of Rheumatology and Immunology, The First Affiliated Hospital of Sichuan Medical University, Luzhou, Sichuan 646000, P.R. China, Department of Ophthalmology, The First Affiliated Hospital of Sichuan Medical University, Luzhou, Sichuan 646000, P.R. China
Published online on: June 22, 2017 https://doi.org/10.3892/mmr.2017.6839
Pages: 1801-1809
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The aim of the present study was to examine the influence of technetium methylenediphosphonate (99Tc-MDP) on the proliferation and differentiation of human osteoblasts. Human iliac cancellous bone was isolated and cultured with either 99Tc‑MDP, β fibroblast growth factor (as a positive control) or medium only (as a negative control). Proliferation was assessed by direct cell counting, CCK‑8 assay and bromodeoxyuridine staining. The cell cycle and rate of apoptosis was assessed by propidium iodide staining and flow cytometry. Alkaline phosphatase (ALP) activity was assessed by the p‑nitrophenyl phosphate method and mineralized nodules were stained with Alizarin Red. Expression of osteocalcin (OCN) and bone morphogenetic protein‑2 (BMP‑2) was assessed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and expression levels of osteoprotegerin (OPG) and receptor activator of NF‑κB ligand (RANKL) were assessed by RT‑qPCR and ELISA. Isolated human osteoblasts stained positively for ALP and developed mineralized nodules. Treatment with 10‑5‑10‑10 M 99Tc‑MDP enhanced proliferation and 48 h incubation with 10‑8 M 99Tc‑MDP increased the proportion of cells in S‑phase, decreased the proportion in G0/G1 phase, and increased the cell proliferation index. The rate of apoptosis also increased, but the increase was not significant. Cells incubated with 10‑6‑10‑9 M 99Tc‑MDP for 3‑9 days exhibited increased ALP activity and mineralized nodule development. 10‑8 M 99Tc‑MDP increased BMP‑2 and OPG expression levels and OPG secretion, but OCN mRNA expression levels and RANKL secretion were not significantly altered at 72 h. 99Tc‑MDP treatment induced osteoblast proliferation and differentiation without affecting apoptosis. These findings provide proof of concept for the future use of 99Tc‑MDP in the treatment of bone‑destructive diseases.

View Figures

View References

1	Thommesen L, Stunes AK, Monjo M, Grøsvik K, Tamburstuen MV, Kjøbli E, Lyngstadaas SP, Reseland JE and Syversen U: Expression and regulation of resistin in osteoblasts and osteoclasts indicate a role in bone metabolism. J Cell Biochem. 99:824–834. 2006. View Article : Google Scholar : PubMed/NCBI
2	Stein GS and Lian JB: Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev. 14:424–442. 1993. View Article : Google Scholar : PubMed/NCBI
3	Pockwinse SM, Stein JL, Lian JB and Stein GS: Developmental stage-specific cellular responses to vitamin D and glucocorticoids during differentiation of the osteoblast phenotype: Interrelationship of morphology and gene expression by in situ hybridization. Exp Cell Res. 216:244–260. 1995. View Article : Google Scholar : PubMed/NCBI
4	Quarto N, Li S, Renda A and Longaker MT: Exogenous activation of BMP-2 signaling overcomes TGFβ-mediated inhibition of osteogenesis in Marfan embryonic stem cells and Marfan patient-specific induced pluripotent stem cells. Stem Cells. 30:2709–2719. 2012. View Article : Google Scholar : PubMed/NCBI
5	Teitelbaum SL: Bone resorption by osteoclasts. Science. 289:1504–1508. 2000. View Article : Google Scholar : PubMed/NCBI
6	Takahashi N, Udagawa N and Suda T: A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun. 256:449–455. 1999. View Article : Google Scholar : PubMed/NCBI
7	Hofbauer LC and Heufelder AE: Role of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in bone cell biology. J Mol Med (Berl). 79:243–253. 2001. View Article : Google Scholar : PubMed/NCBI
8	Khosla S: Minireview: The OPG/RANKL/RANK system. Endocrinology. 142:5050–5055. 2001. View Article : Google Scholar : PubMed/NCBI
9	Lai K, Jin C, Tu S, Xiong Y, Huang R and Ge J: Intravitreal injection of (99)Tc-MDP inhibits the development of laser-induced choroidal neovascularization in rhesus monkeys. Graefes Arch Clin Exp Ophthalmol. 252:1049–1057. 2014. View Article : Google Scholar : PubMed/NCBI
10	Lai K, Xu L, Jin C, Wu K, Tian Z, Huang C, Zhong X and Ye H: Technetium-99 conjugated with methylene diphosphonate (99Tc-MDP) inhibits experimental choroidal neovascularization in vivo and VEGF-induced cell migration and tube formation in vitro. Invest Ophthalmol Vis Sci. 52:5702–5712. 2011. View Article : Google Scholar : PubMed/NCBI
11	Zhao Y, Wang L, Liu Y, Akiyama K, Chen C, Atsuta I, Zhou T, Duan X, Jin Y and Shi S: Technetium-99 conjugated with methylene diphosphonate ameliorates ovariectomy-induced osteoporotic phenotype without causing osteonecrosis in the jaw. Calcif Tissue Int. 91:400–408. 2012. View Article : Google Scholar : PubMed/NCBI
12	Chen J, He Y and He CS: The development of Technetium [99Tc] Methylenediphosphonate. China Pharmacy. 6:553–555. 2010.
13	Wang L, Gu Q, Xu Y, Li S, Gui J, Yang J, Yao Q and Ji Y: Effects of Yunke (technetium-99 conjugated with methylene diphosphonate; (99)Tc-MDP) and/or colloidal chromic phosphate phosphonium-32, alone and in combination, in rats with adjuvant arthritis. Clin Exp Pharmacol Physiol. 35:23–28. 2008. View Article : Google Scholar : PubMed/NCBI
14	Su D, Shen M, Gu B, Wang X, Wang D, Li X and Sun L: (99)Tc-methylene diphosphonate improves rheumatoid arthritis disease activity by increasing the frequency of peripheral γδ T cells and CD4(+) CD25 Foxp3(+) Tregs. Int J Rheum Dis. 19:586–593. 2016. View Article : Google Scholar : PubMed/NCBI
15	Mu R, Chen S and Li ZG: Therapeutic effect of 99mTc-MDP and its role in proinflammatory cytokines in rheumatoid arthritis. Chinese Journal of Rheumatology. 1:39–41. 2004.
16	Kanno S, Hirano S and Kayama F: Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells. Toxicology. 196:137–145. 2004. View Article : Google Scholar : PubMed/NCBI
17	Im GI, Qureshi SA, Kenney J, Rubash HE and Shanbhag AS: Osteoblast proliferation and maturation by bisphosphonates. Biomaterials. 25:4105–4115. 2004. View Article : Google Scholar : PubMed/NCBI
18	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
19	Diduch DR, Coe MR, Joyner C, Owen ME and Balian G: Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization. J Bone Joint Surg Am. 75:92–105. 1993. View Article : Google Scholar : PubMed/NCBI
20	Bianco P, Riminucci M, Bonucci E, Termine JD and Robey PG: Bone sialoprotein (BSP) secretion and osteoblast differentiation: Relationship to bromodeoxyuridine incorporation, alkaline phosphatase, and matrix deposition. J Histochem Cytochem. 41:183–191. 1993. View Article : Google Scholar : PubMed/NCBI
21	Gratzner HG: Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science. 218:474–475. 1982. View Article : Google Scholar : PubMed/NCBI
22	Saito Y, Yoshizawa T, Takizawa F, Ikegame M, Ishibashi O, Okuda K, Hara K, Ishibashi K, Obinata M and Kawashima H: A cell line with characteristics of the periodontal ligament fibroblasts is negatively regulated for mineralization and Runx2/Cbfa1/Osf2 activity, part of which can be overcome by bone morphogenetic protein-2. J Cell Sci. 115:4191–4200. 2002. View Article : Google Scholar : PubMed/NCBI
23	Gori F, Thomas T, Hicok KC, Spelsberg TC and Riggs BL: Differentiation of human marrow stromal precursor cells: Bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation. J Bone Miner Res. 14:1522–1535. 1999. View Article : Google Scholar : PubMed/NCBI
24	Rudkin GH, Yamaguchi DT, Ishida K, Peterson WJ, Bahadosingh F, Thye D and Miller TA: Transforming growth factor-beta, osteogenin, and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells. J Cell Physiol. 168:433–441. 1996. View Article : Google Scholar : PubMed/NCBI
25	Geusens PP, Landewé RB, Garnero P, Chen D, Dunstan CR, Lems WF, Stinissen P, van der Heijde DM, van der Linden S and Boers M: The ratio of circulating osteoprotegerin to RANKL in early rheumatoid arthritis predicts later joint destruction. Arthritis Rheum. 54:1772–1777. 2006. View Article : Google Scholar : PubMed/NCBI
26	Kim HR, Lee SH and Kim HY: Elevated serum levels of soluble receptor activator of nuclear factors-kappaB ligand (sRANKL) and reduced bone mineral density in patients with ankylosing spondylitis (AS). Rheumatology (Oxford). 45:1197–1200. 2006. View Article : Google Scholar : PubMed/NCBI
27	Hamdy NA: Targeting the RANK/RANKL/OPG signaling pathway: A novel approach in the management of osteoporosis. Curr Opin Investig Drugs. 8:299–303. 2007.PubMed/NCBI
28	Gong W, Dou H, Liu X, Sun L and Hou Y: Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis. Clin Exp Pharmacol Physiol. 39:886–893. 2012. View Article : Google Scholar : PubMed/NCBI