Inhibitory Effect of Conditioned Medium of Silk Fibroin-Treated Osteoblasts in Osteoclast Differentiation

실크피브로인을 처리한 MC3T3-E1 조골세포 조건배양액의 파골세포 분화억제효과

  • Yeo, Joo-Hong (Dept. of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Park, Kyung-Ho (Dept. of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University) ;
  • Ju, Won-Chul (Dept. of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University) ;
  • Lee, Jin-Ah (Dept. of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University) ;
  • Lee, Kwang-Gill (Dept. of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Woo, Soon-Ok (Dept. of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Han, Sang-Mi (Dept. of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Kweon, Hae-Yong (Dept. of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Kim, Sung-Su (Dept. of Anatomy, Chung-Ang University) ;
  • Cho, Yun-Hi (Dept. of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University)
  • 여주홍 (농촌진흥청 농업생물부) ;
  • 박경호 (경희대학교 동서의학대학원 의학영양학과) ;
  • 주원철 (경희대학교 동서의학대학원 의학영양학과) ;
  • 이진아 (경희대학교 동서의학대학원 의학영양학과) ;
  • 이광길 (농촌진흥청 농업생물부) ;
  • 우순옥 (농촌진흥청 농업생물부) ;
  • 한상미 (농촌진흥청 농업생물부) ;
  • 권해용 (농촌진흥청 농업생물부) ;
  • 김성수 (중앙대학교 의과대학 해부학교실) ;
  • 조윤희 (경희대학교 동서의학대학원 의학영양학과)
  • Published : 2008.08.30


In this study, we investigated the indirect effect of silk-fibroin on osteoclastic differentiation of RAW264.7 cells. The conditioned medium were collected from MC3T3-E1 osbeoblasts treated with $0.001\;mg/mL{\sim}0.1\;mg/mL$ silk fibroin for 6 days, mixed in 1:1 ratio with osteoclast medium, and then added into RAW264.7 cells with receptor activator of nuclear factor kappa B ligand (RANKL), a differentiation inducer for 3 days. Of osteoclastic cytokines in the conditioned medium, the protein expression of osteoprotegerin (OPG) with silk-fibroin was not significantly different. However, the protein expression of interleukin (IL)-$1{\beta}$ was specifically lower in a dose dependent manner. In RAW264.7 cells, the conditioned medium with silk-fibroin inhibited RANKL induced osteoclastic differentiation as total number of multinucleated tartrate-resistant alkaline phosphatase (TRAP)-positive osteoclasts in a dose dependent manner. Taken together, we demonstrated that the conditioned medium of silk-fibroin treated osteoblasts inhibits RANKL induced differentiation of osteoclasts with inhibiting selective expression of IL-$1{\beta}$.


  1. Parfitt AM. 1994. Osteonal and hemi-osteonal remodeling: The spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem 55: 273-286
  2. Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC. 1998. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Miner Res 13: 793-802
  3. Udagawa N, Takakashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T. 1990. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepaared by bone marrow-derived stromal cells. Proc Natl Acad Sci USA 87: 7260-7264
  4. Kong YY, Boyle WJ, Penninger JM. 2001. Osteoprotegerin and receptor activator of nuclear factor $_KB$ ligand in the pathogenesis and treatment of rheumatoid arthritis. Arthritis Rheum 44: 253-259<253::AID-ANR41>3.0.CO;2-S
  5. Kurolouchi K, Kambe F, Yasukawa K, Izumi R, Ishiguro N, Iwata H. 1998. TNF-$\alpha$ increases expression of IL-6 and ICAM-1 genes through activation of NK-$_KB$ in osteoblast-like ROS17/2.8 cells. J Bone Miner Res 13: 1290-1299
  6. Chaudhary LR, Spelsber TC, Riffs BL. 1992. Production of various cytokines by normal human osteoblast-like cells in response to interleukin 1$\beta$ and tumor necrosis factor-$\alpha$: lack of regulation by 17$\beta$-estradiol. Endocrinology 130: 2528-2534
  7. Smith DD, Gowen M, Mundy GR. 1987. Effects of interferon-gamma and other cytokines in collagen synthesis in fetal rat bone cultures. Endocrinology 120: 2494-2499
  8. Jilka RL. 1998. Cytokines, bone remodeling, and estrogen deficiency: a 1998 update. Bone 23: 75-81
  9. Keeting PE, Rifas L, Harris SA, Colvard DS, Spelsberg TC, Peck WA. 1991. Evidence for interleukin-1 production by cultured normal human osteoblast-like cells. J Bone Miner Res 6: 827-833
  10. Lorenz CH, Armin EH. 1997. Osteoprotegerin: a novel local player in bone metabolism. Eur J Endocrinol 137: 345-346
  11. Tobias JH, Compston JE. 1999. Does estrogen stimulate osteoblast function in postmenopausal women? Bone 24: 121-124
  12. Rogers J. 1967. Estrogens in the menopause and postmenopause. N Engl J Med 280: 364-367
  13. Baker VL, Leitman D, Jaffe RB. 2000. Selective estrogen receptor modulators in reproductive medicine and biology. Obstet Gynecol Surv 55: 21-47
  14. Gray TK, Frynn TC, Gray KM, Nabell LM. 1987. 17$\beta$-estradiol acts directly on the clonal osteoblast cell line UMR106. Proc Natl Acad Sci 84: 6267-6271
  15. Aldercreutz H, Mazur W. 1996. Phyto-oestrogens in relation to cancer and other human health risks. Proc Nutr Soc 55: 399-417
  16. Gregory HA, Frank D, Caroline J, Tara C, Rebecca LH, Jingsong C, Helen L, John R, David LK. 2003. Silk-based biomaterials. Biomaterials 24: 401-416
  17. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL. 2006. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 27: 3115-3124
  18. Yeo JH, Lee KG, Kim HC, Oh YL, Kim AJ, Kim SY. 2000. The effect of PVA/chitosan/fibroin (PCF)-blended sponge on wound healing in rats. Biol Pharm Bull 23: 1220-1223
  19. Yeo JH, Lee KG, Kweon HY, Woo SO, Han SM, Kim SS, Demura M. 2006. Fractionation of a silk fibroin hydrolysate and its protective function of hydrogen peroxide toxicity. J Appl Polym Sci 102: 772-776
  20. Kim DK, Kang YK, Lee MY, Lee KG, Yeo JH, Lee WB, Kim YS, Kim SS. 2005. Neuroprotection and enhancement of learning and memory by BF-7. J Health Sci 51: 317-324
  21. Susan S, Mary BM, Gloria G, David LK. 2001. Functionalized silk-based biomaterials for bone formation. J Biomed Mater Res 54: 139-148<139::AID-JBM17>3.0.CO;2-7
  22. Madyarov S, Lee KG, Yeo JH, Nam J, Lee YW. 2000. Improved method for the preparation of silk fibroin hydrolysates. Korean J Seric Sci 41: 102-110
  23. Fanti P, Monier-Faugere MC, Geng Z, Schmidt J, Morris PE, Cohen D, Malluche HH. 1998. The phytoestrogen genistein reduces bone loss in short-term ovariectomized rats. Osteoporosis Int 8: 274-281
  24. Sugimoto E, Yamaguchi M. 2000. Anabolic effect of genistein in osteoblastic MC3T3-E1 cells. Int J Mol Med 5: 515-520
  25. Chambers TJ. 2000. Regulation of the differentiation and function of osteoclasts. J Pathol 192: 4-13<::AID-PATH645>3.0.CO;2-Q
  26. Hotokezaka H, Sakai E, Kanaoka K, Saito K, Matsuo KI, Kitaura H, Yoshida N, Nakayama K. 2002. U0126 and PD98059, specific inhibition of MEK, accelerate differentiation of RAW264.7 cells into osteoclast-like cells. J Biol Chem 277: 47366-47372
  27. Kang SA, Jang KH, Cho YH, Hong KH, Kong SH, Choue RW. 2001. High performance liquid chromatographic analysis of isoflavones in soybean and blackbean. J Arahe 8: 44-48
  28. Arnett TR. 1990. Update on bone cell biology. Eur J Orthod 12: 81-90
  29. Kameda T, Mano H, Yuasa T, Mori Y, Miyazawa K, Shiokawa M, Nakamaru Y, Hiroi E, Hiura K, Kameda A, Yang NN, Hakeda Y, Kumegawa M. 1997. Estrogen inhibits bone resorption by directly inducing apoptosis of the bone resorbing osteoclasts. J Exp Med 186: 489-495
  30. Mok SK, Shin LLS. 1996. The effects of prostaglandine and dibutylryl cAMP on osteoblastic cell activity and osteoclast generation. J Wonkwang Dental Res Int 6: 43-62
  31. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T. 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95: 3597-3602
  32. Khosla S. 2001. The OPG/RANKL/RANK system. Endocrinology 142: 5050-5055
  33. Thomas C, Spelsberg M, Subramaniam B, Lawrence R, Sundeep K. 2005. The actions and interactions of sex steroids and growth factors/cytokines on the skeleton. Mol Endo 13: 819-828
  34. Evans DM, Ralston SH. 1996. Nitric oxide and bone. J Bone Miner Res 11: 300-305
  35. Helfrich MH, Evans DE, Grabowski PS, Pollock JS, Ohshima H, Ralston SH. 1997. Expression of nitric oxide synthase isoforms in bone and bone cell cultures. J Bone Miner Res 12: 1108-1115

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