EFFECT OF TITANIUM PARTICLES ON PROLIFERATION, DIFFERENTIATION, AND cytoMORPHOLOGY OF OSTEOBLASTS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LINMianhui , SHIYupeng , WANFLei ,  YEJunqiang

Department of Osteoarthropathy, Shenzhen Longgang Central Hospital, Shenzhen Guangdong, 518116, P. R. China;

Corresponding author：

YEJunqiang, Email: xysteven2008@gmail.com

Keywords：

Titanium particle; Osteoblasts; Proliferation; Differentiation; Cytomorphology; Rat

DOI：

10.7507/1002-1892.20140130

Video：

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Objective To study the effect of titanium particles on the proliferation, differentiation, and cytomorphology of osteoblasts, and to explore the possible internal relations and mechanism. Methods Calvarial osteoblasts were separated from 10 newborn Sprague Dawley rats by repeated enzyme digestion, and were cultured in vitro. The cells were identified by alkaline phosphatase (ALP) staining and alizarin red staining. The cells at passage 3 were cultured with titanium particles culture medium at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/mL (0.01, 0.05, 0.1, 0.5, and 1 mg/mL groups). The absorbance (A) values were detected by cell counting kit 8 at 7 days after cultured to compare the effect of titanium particles at different concentrations on proliferation, and median lethal concentration was screened out. The expression of collagen type I was detected by ELISA to observe the effect of titanium particles on differentiation. The osteoblasts co-cultured with titanium particles of median lethal concentration (experimental group) for 7 days, and double fluorescence staining with FITC-phalloidine and propidium iodide was performed. The cytomorphology variation of osteoblasts after swallowing titanium particles was observed under laser scanning confocal microscope. The osteoblasts at passage 3 cultured with culture medium without titanium particles served as control group. Results The cultured cells were identified as osteoblasts by ALP staining and alizarin red staining. Different concentrations of titanium particles could inhibit osteoblasts proliferation and differentiation in varying degrees, showing significant difference when compared with the control group at 7 days after culture (P<0.05). The cell proliferation and differentiation were decreased with increased titanium particles concentration; significant differences were found between the other groups (P<0.05) except 0.01 and 0.05 mg/mL groups (P>0.05). The median lethal concentration of titanium particles was 0.5 mg/mL. Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts that swallowed titanium particles in the experimental group. Conclusion Titanium particles can inhibit proliferation and differentiation of osteoblasts. The effect may be related to variation of cytomorphology after swallowing titanium particles.

Citation： LINMianhui, SHIYupeng, WANFLei, YEJunqiang. EFFECT OF TITANIUM PARTICLES ON PROLIFERATION, DIFFERENTIATION, AND cytoMORPHOLOGY OF OSTEOBLASTS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(5): 581-585. doi: 10.7507/1002-1892.20140130 Copy

1.	Bozic KJ, Kurtz SM, Lau E, et al. The epidemiology of revision total knee arthroplasty in the United States. Clin Orthop Relat Res, 2010, 468(1):45-51.
2.	Bozic KJ, Kurtz SM, Lau E, et al. The epidemiology of revision total hip arthroplasty in the United States. J Bone Joint Surg (Am), 2009, 91(1):128-133.
3.	Drees P, Eckardt A, Gay RE, et al. Mechanisms of disease:molecular insights into aseptic loosening of orthopedic implants. Nat Clin Pract Rheumatol, 2007, 3(3):165-171.
4.	Yang SY, Zhang K, Bai L, et al. Polymethylmethacrylate and titanium alloy particles activate peripheral monocytes during periprosthetic inflammation and osteolysis. J Orthop Res, 2011, 29(5):781-786.
5.	Ollivere B, Wimhurst JA, Clark IM, et al. Current concepts in osteolysis. J Bone Joint Surg (Br), 2012, 94(1):10-15.
6.	Hundri-Haspl Z, Pecina M, Haspl M, et al. Plasma cytokines as markers of aseptic prosthesis loosening. Clin Orthop Relat Res, 2006, (453):299-304.
7.	Pioletti DP, Kottelat A. The influence of wear particles in the expression of osteoclastogenesis factors by osteoblasts. Biomaterials, 2004, 25(27):5803-5808.
8.	魏均强, 蔡谞, 王岩, 等. 人工关节无菌性松动的发生和防治. 中国修复重建外科杂志, 2010, 24(3):296-300.
9.	Jiang Y, Jia T, Gong W, et al. Titanium particle-challenged osteoblasts promote osteoclastogenesis and osteolysis in a murine model of periprosthestic osteolysis. Acta Biomater, 2013, 9(7):7564-7572.
10.	Millett PJ, Sabokbar A, Allen MJ, et al. Osteoblast activity around failed total hip replacements:synovial fluid levels of osteocalcin and alkaline phosphatase. Hip international, 1995, 5(1):8-14.
11.	Vermes C, Chandrasekaran R, Jacobs JJ, et al. The effects of particulate wear debris, cytokines, and growth factors on the functions of MG-63 osteoblasts. J Bone Joint Surg (Am), 2001, 83-A(2):201-211.
12.	Haleem-Smith H, Argintar E, Bush C, et al. Biological responses of human mesenchymal stem cells to titanium wear debris particles. J Orthop Res, 2012, 30(6):853-863.
13.	Chiu R, Smith KE, Ma GK, et al. Polymethylmethacrylate particles impair osteoprogenitor viability and expression of osteogenic transcription factors Runx2, osterix, and Dlx5. J Orthop Res, 2010, 28(5):571-577.
14.	Yamamoto N, Furuya K, Hanada K. Progressive development of the osteoblast phenotype during differentiation of osteoprogenitor cells derived from fetal rat calvaria:model for in vitro bone formation. Biol Pharm Bull, 2002, 25(4):509-515.
15.	蔡贤华, 陈安民, 闫玉华, 等. 实验用细胞可吞噬钛微粒的制备与表征分析. 中华实验外科杂志, 2005, 22(9):1098-1099.
16.	Pioletti DP, Takei H, Kwon SY, et al. The cytotoxic effect of titanium particles phagocytosed by osteoblast. J Biomed Mater Res, 1999, 46(3):399-407.
17.	Lenz R, Mittelmeier W, Hansmann D, et al. Response of human osteoblasts exposed to wear particles generated at the interface of total hip stems and bone cement. J Biomed Mater Res A, 2008, 89(2):370-378.
18.	Maoqiang L, Zhenan Z, Fengxiang L, et al. Enhancement of osteoblast differentiation that is inhibited by titanium particles through inactivation of NFATc1 by VIVIT peptide. J Biomed Mater Res A, 2010, 95(3):727-734.
19.	Lochner K, Fritsche A, Jonitz A, et al. The potential role of human osteoblasts for periprosthetic osteolysis following exposure to wear particles. Int J Mol Med, 2011, 28(6):1055-1063.
20.	戴闵, 周通华, 熊皞, 等. 金属离子对成骨细胞凋亡、细胞周期分布及ALP分泌的影响. 中国修复重建外科杂志, 2011, 25(1):56-60.
21.	史玉朋, 蒋绍艳, 林绵辉. 淫羊藿苷防治人工关节无菌性松动的实验研究. 实用医学杂志, 2013, 29(增刊):33-34.
22.	Saldaña L, Vilaboa N. Effects of micrometric titanium particles on osteoblast attachment and cytoskeleton architecture. Acta Biomater, 2010, 6(4):1649-1660.
23.	Kwon SY, Takei H, Pioletti DP, et al. Titanium paticles inhibit osteoblast adhesion to fibronectin-coated substrates. J Orthop Res, 2000, 18(2):203-211.
24.	Kwon SY, Lin T, Takei H, et al. Alterations in the adhesion behavior of osteoblasts by titanium particle loading:Inhibition of cell function and gene expression. Biorheology, 2001, 38(2-3):161-183.
25.	曹慧, 陈槐卿, 吴江, 等. 钛颗粒对诱骨条件下骨髓间充质干细胞增殖能力的影响. 四川大学学报:医学版, 2004, 35(3):323-326.

1. Bozic KJ, Kurtz SM, Lau E, et al. The epidemiology of revision total knee arthroplasty in the United States. Clin Orthop Relat Res, 2010, 468(1):45-51.
2. Bozic KJ, Kurtz SM, Lau E, et al. The epidemiology of revision total hip arthroplasty in the United States. J Bone Joint Surg (Am), 2009, 91(1):128-133.
3. Drees P, Eckardt A, Gay RE, et al. Mechanisms of disease:molecular insights into aseptic loosening of orthopedic implants. Nat Clin Pract Rheumatol, 2007, 3(3):165-171.
4. Yang SY, Zhang K, Bai L, et al. Polymethylmethacrylate and titanium alloy particles activate peripheral monocytes during periprosthetic inflammation and osteolysis. J Orthop Res, 2011, 29(5):781-786.
5. Ollivere B, Wimhurst JA, Clark IM, et al. Current concepts in osteolysis. J Bone Joint Surg (Br), 2012, 94(1):10-15.
6. Hundri-Haspl Z, Pecina M, Haspl M, et al. Plasma cytokines as markers of aseptic prosthesis loosening. Clin Orthop Relat Res, 2006, (453):299-304.
7. Pioletti DP, Kottelat A. The influence of wear particles in the expression of osteoclastogenesis factors by osteoblasts. Biomaterials, 2004, 25(27):5803-5808.
8. 魏均强, 蔡谞, 王岩, 等. 人工关节无菌性松动的发生和防治. 中国修复重建外科杂志, 2010, 24(3):296-300.
9. Jiang Y, Jia T, Gong W, et al. Titanium particle-challenged osteoblasts promote osteoclastogenesis and osteolysis in a murine model of periprosthestic osteolysis. Acta Biomater, 2013, 9(7):7564-7572.
10. Millett PJ, Sabokbar A, Allen MJ, et al. Osteoblast activity around failed total hip replacements:synovial fluid levels of osteocalcin and alkaline phosphatase. Hip international, 1995, 5(1):8-14.
11. Vermes C, Chandrasekaran R, Jacobs JJ, et al. The effects of particulate wear debris, cytokines, and growth factors on the functions of MG-63 osteoblasts. J Bone Joint Surg (Am), 2001, 83-A(2):201-211.
12. Haleem-Smith H, Argintar E, Bush C, et al. Biological responses of human mesenchymal stem cells to titanium wear debris particles. J Orthop Res, 2012, 30(6):853-863.
13. Chiu R, Smith KE, Ma GK, et al. Polymethylmethacrylate particles impair osteoprogenitor viability and expression of osteogenic transcription factors Runx2, osterix, and Dlx5. J Orthop Res, 2010, 28(5):571-577.
14. Yamamoto N, Furuya K, Hanada K. Progressive development of the osteoblast phenotype during differentiation of osteoprogenitor cells derived from fetal rat calvaria:model for in vitro bone formation. Biol Pharm Bull, 2002, 25(4):509-515.
15. 蔡贤华, 陈安民, 闫玉华, 等. 实验用细胞可吞噬钛微粒的制备与表征分析. 中华实验外科杂志, 2005, 22(9):1098-1099.
16. Pioletti DP, Takei H, Kwon SY, et al. The cytotoxic effect of titanium particles phagocytosed by osteoblast. J Biomed Mater Res, 1999, 46(3):399-407.
17. Lenz R, Mittelmeier W, Hansmann D, et al. Response of human osteoblasts exposed to wear particles generated at the interface of total hip stems and bone cement. J Biomed Mater Res A, 2008, 89(2):370-378.
18. Maoqiang L, Zhenan Z, Fengxiang L, et al. Enhancement of osteoblast differentiation that is inhibited by titanium particles through inactivation of NFATc1 by VIVIT peptide. J Biomed Mater Res A, 2010, 95(3):727-734.
19. Lochner K, Fritsche A, Jonitz A, et al. The potential role of human osteoblasts for periprosthetic osteolysis following exposure to wear particles. Int J Mol Med, 2011, 28(6):1055-1063.
20. 戴闵, 周通华, 熊皞, 等. 金属离子对成骨细胞凋亡、细胞周期分布及ALP分泌的影响. 中国修复重建外科杂志, 2011, 25(1):56-60.
21. 史玉朋, 蒋绍艳, 林绵辉. 淫羊藿苷防治人工关节无菌性松动的实验研究. 实用医学杂志, 2013, 29(增刊):33-34.
22. Saldaña L, Vilaboa N. Effects of micrometric titanium particles on osteoblast attachment and cytoskeleton architecture. Acta Biomater, 2010, 6(4):1649-1660.
23. Kwon SY, Takei H, Pioletti DP, et al. Titanium paticles inhibit osteoblast adhesion to fibronectin-coated substrates. J Orthop Res, 2000, 18(2):203-211.
24. Kwon SY, Lin T, Takei H, et al. Alterations in the adhesion behavior of osteoblasts by titanium particle loading:Inhibition of cell function and gene expression. Biorheology, 2001, 38(2-3):161-183.
25. 曹慧, 陈槐卿, 吴江, 等. 钛颗粒对诱骨条件下骨髓间充质干细胞增殖能力的影响. 四川大学学报:医学版, 2004, 35(3):323-326.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF TITANIUM PARTICLES ON PROLIFERATION, DIFFERENTIATION, AND cytoMORPHOLOGY OF OSTEOBLASTS

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