Journal of Veterinary Clinics (한국임상수의학회지)

The Korean Society of Veterinary Clinics (한국임상수의학회)
권호 표지

Effects of Ascorbic Acid on the Proliferation of Subcultured Canine Chondocytes in Monolayer and Alginate Beads Culture

단순배양과 알긴산배양에서 개 연골세포의 증식에 있어 계대배양에 따른 아스코빅산의 영향

  • Kim Gon-Hyung (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Park Jin-Uk (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Hossain Mohammad Alamgir (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Cho Ki-Rae (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Kim Joong-Hyun (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University) ;
  • Choi Seok-Hwa (Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University)
  • Published : 2006.12.30
Download PDF
⟨ Previous Next ⟩

Abstract

Ascorbic acid has been used widely as a medium supplement to stimulate cell proliferation, but its effects on cell proliferation have not yet been elucidated, and no reports have analyzed effects on subcultured chondrocytes. Subcultured canine chondrocytes of passage one, two and four were cultured in monolayer and alginate beads with and without ascorbic acid. Cell proliferation was examined by 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) (XTT) colormetric assay. Ascorbic acid stimulated cell proliferation significantly in both culture methods (p<0.05). The increased cell numbers by stimulation with ascorbic acid were significantly high in passage one cells compared to that of other passages. Differences in cell proliferative capacity by subculturing were not determined. These results suggest that ascorbic acid stimulated the proliferation of subcultured canine chondrocytes and enhanced it more in low-passage cells than in the other cells tested.

아스코빅산은 세포 증식촉진을 위해 배지에 첨가하여 사용되나, 증식 촉진 작용기전에 대해서는 구체적으로 밝혀져 있지 않으며 연골세포의 계대배양에 따른 효과에 대해서는 보고된바 없다. 제 1, 2그리고 4계대배양한 개 연골세포의 단순배양과 알긴산 배양을 아스코빅산 첨가 배지와 무첨가 배지에서 실시하였다. 세포증식은 2,3-Bis(2-methoxy-4-nitro-5- sulfophenyl )-2H-tetrazolium - 5-carboxanilide inner salt)(XTT)검사법을 이용하였다. 아스코빅산 첨가에 의해 세포증식 촉진효과가 모든 계대배양 세포에서 관찰되었으나, 제 1계대배양 연골세포에서 아스코빅산의 세포능식 촉진 효과가 제 2, 4계대배양 연골세포보다 유의차 있게 높게 나타났다 (p<0.05). 아스코빅산 무첨가 배지에서 계대배양에 따른 세포 증식에 차이가 관찰되지 않았다. 따라서 아스코빅산은 낮은 계대배양단계의 연골세포에 있어 더욱 효과적인 세포증식 촉진효과가 있는 것으로 판단된다.

Keywords

References

  1. Aulthouse AL, Beck M, Griffey E, Sanford J, Arden K, Machado MA, Horton WA. Expression of the human chondrocyte phenotype in vitro. In Vitro Cell Dev Biol 1989; 25: 659-668 https://doi.org/10.1007/BF02623638
  2. Benya PD. Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982; 30: 215-224 https://doi.org/10.1016/0092-8674(82)90027-7
  3. Bonaventure J, Kadhom N, Cohen-Solal L, Ng KH, Bourguignon J, Lasselin C, Freisinger P. Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads. Exp Cell Res 1994; 212: 97-104 https://doi.org/10.1006/excr.1994.1123
  4. Bounelis P, Daniel JC. The effect of ascorbate on embryonic chick sternal chondrocytes cultured in agarose. Tissue Cell 1983; 15: 683-693 https://doi.org/10.1016/0040-8166(83)90043-5
  5. Brittberg M, Nilsson A, Lindahl A, Ohlsson C, Peterson L. Rabbit articular cartilage defects treated with autologous cultured chondrocytes. Clin Orthop 1996; 326: 270-283 https://doi.org/10.1097/00003086-199605000-00034
  6. Daniel JC, Pauli BU, Kuettner KE. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate. J Cell Biol 1984; 99: 1960-1969 https://doi.org/10.1083/jcb.99.6.1960
  7. de Haart M, Marijnissen WJ, van Osch GJ, Verhaar JA. Optimization of chondrocyte expansion in culture. Effect of TGF -2, bFGF and L-ascorbic acid on bovine articular chondrocytes. Acta Orthop Scand 1999; 70: 55-61 https://doi.org/10.3109/17453679909000959
  8. Dozin B, Quarto R, Campanile G, Cancedda R. In vitro differentiation of mouse embryo chondrocytes: requirement for ascorbic acid. Eur J Cell Biol 1992; 58: 390-394
  9. Fragonas E, Valente M, Pozzi-Mucelli M, Toffanin R, Rizzo R, Silvestri F, Vittur F. Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate. Biomaterials 2000; 21: 795-801 https://doi.org/10.1016/S0142-9612(99)00241-0
  10. Freyria AM, Ronziere MC, Roche S, Rousseau CF, Herbage D. Regulation of growth, protein synthesis, and maturation of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B. J Cell Biochem 1999; 76: 84-98
  11. Grande DA, Singh IJ, Pugh J. Healing of experimentally produced lesions in articular cartilage following chondrocyte transplantation. Anat Rec 1987; 218: 142-148 https://doi.org/10.1002/ar.1092180208
  12. Guo JF, Jourdian GW, MacCallum DK. Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect Tissue Res 1989; 19: 277-297 https://doi.org/10.3109/03008208909043901
  13. Hauselmann HJ, Fernandes RJ, Mok SS, Schmid TM, Block JA, Aydelotte MB, Kuettner KE, Thonar EJ. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J Cell Sci 1994; 107: 17-27
  14. Hering TM, Kollar J, Huynh TD, Varelas JB, Sandell LJ. Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension. Arch Biochem Biophys 1994; 314: 90-98 https://doi.org/10.1006/abbi.1994.1415
  15. Ivanov VO, Ivanova SV, Niedzwiecki A. Ascorbate affects proliferation of guinea-pig vascular smooth muscle cells by direct and extracellular matrix-mediated effects. J Mol Cell Cardiol 1997; 29: 3293-3303 https://doi.org/10.1006/jmcc.1997.0555
  16. Kim G, Okumura M, Bosnakovski D, Ishiguro T, Park CH, Kadosawa T, Fujinaga T. Effects of ascorbic acid on proliferation and biological properties of bovine chondrocytes in alginate beads. Jpn J Vet Res 2003; 51: 83-94
  17. Lemare F, Steimberg N, Le Griel C, Demignot S, Adolphe M. Dedifferentiated chondrocytes cultured in alginate beads: restoration of the differentiated phenotype and of the metabolic responses to interleukin-1. J Cell Physiol 1998; 176: 303-313 https://doi.org/10.1002/(SICI)1097-4652(199808)176:2<303::AID-JCP8>3.0.CO;2-S
  18. Marijnissen WJ. van Osch GJ, Aigner J, Verwoerd-Verhoef HL, Verhaar JA. Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM). Biomaterials 2000; 21: 571-580 https://doi.org/10.1016/S0142-9612(99)00218-5
  19. McAlindon TE, Jacques P, Zhang Y, Hannan MT, Aliabadi P, Weissman B, Rush D, Levy D, Felson DT. Do antioxidant micronutrients protect against the development and progression of knee osteoarthritis? Arthritis Rheum 1996; 39: 648-656 https://doi.org/10.1002/art.1780390417
  20. McDevitt CA, Lipman JM, Ruemer RJ, Sokoloff, L. Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 2. Characterization of proteoglycans. J Orthop Res 1988; 6: 518-524 https://doi.org/10.1002/jor.1100060407
  21. Meacock SC, Bodmer JL, Billingham ME. Experimental osteoarthritis in guinea-pigs. J Exp Pathol 1990; 71: 279-293
  22. Paige KT, Cima LG, Yaremchuk MJ, Vacanti JP, Vacanti CA. Injectable cartilage. Plast Reconstr Surg 1995; 96: 1390-1398 https://doi.org/10.1097/00006534-199511000-00024
  23. Ronziere MC, Roche S, Gouttenoire J, Demarteau O, Herbage D, Freyria AM. Ascorbate modulation of bovine chondrocyte growth, matrix protein gene expression and synthesis in three-dimensional collagen sponges. Biomaterials 2003; 24: 851-861 https://doi.org/10.1016/S0142-9612(02)00418-0
  24. Schwartz ER, Oh WH, Leveille CR. Experimentally induced osteoarthritis in guinea pigs: metabolic responses in articular cartilage to developing pathology. Arthritis Rheum 1981; 24: 1345-1355 https://doi.org/10.1002/art.1780241103